# Lignocellulosic biomass and its main structural polymers as sustainable materials for (bio)sensing applications

Arnaud Kamdem Tamo, Ingo Doench, Gullit Deffo, Sherman Lesly Zambou Jiokeng, Giscard Doungmo, Cyrille Ghislain Fotsop, Ranil Clément Tonleu Temgoua, Evangéline Njanja, Ignas Kenfack Tonle, Anatoli Serghei, et al.

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## ► To cite this version:

Arnaud Kamdem Tamo, Ingo Doench, Gullit Deffo, Sherman Lesly Zambou Jiokeng, Giscard Doungmo, et al.. Lignocellulosic biomass and its main structural polymers as sustainable materials for (bio)sensing applications. Journal of Materials Chemistry A, 2025, 13 (30), pp.24185-24253. 10.1039/D5TA02900G. hal-05399261

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Submitted on 4 Dec 2025

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Lignocellulosic Biomass and its Main Structural Biomacromolecules Including Cellulose, Hemicellulose and Lignin as Renewable Functional Polymers for (Bio)Sensing Applications

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Arnaud Kamdem Tamo a,*, Ingo Doench b,c, Gullit Deffo d, Sherman Lesly Zambou Jiokeng e, Giscard Doungmo f, Cyrille Ghislain Fotsop g, Ranil Clément Tonleu Temgoua h, Evangéline Njanja i, Ignas Kenfack Tonle j, Anatoli Serghei a, Alexandra Montembault a, Laurent David a, Anayancy Osorio-Madrazo b,c,*

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a Ingénierie des Matériaux Polymères (IMP), Université Claude Bernard Lyon 1, INSA de Lyon, Université Jean Monnet, CNRS, UMR 5223, 69622 Villeurbanne CEDEX, France;

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b Laboratory of Organic and Macromolecular Chemistry (IOMC), Jena Center for Soft Matter (JCSM), and Center for Energy and Environmental Chemistry Jena (CEEC), Friedrich Schiller University of Jena, 07743 Jena, Germany;

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c Laboratory of Organ Printing, University of Bayreuth, 95447 Bayreuth, Germany;

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d Faculty of Chemistry and Mineralogy, Leipzig University, 04103 Leipzig, Germany;

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e Institute of Analytical and Bioanalytical Chemistry, University of Ulm, 89081 Ulm, Germany;

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f Institut für Anorganische Chemie, Christian-Albrechts-Universität zu Kiel, Max-Eyth-Str. 2, 24118 Kiel, Germany;

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g Otto-von-guericke-University Magdeburg, Chemical Institute Industrial Chemistry, Universitätsplatz 2, 39106 Magdeburg, Germany;

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h Federal Institute for Materials Research and Testing, BAM Richard-Willstätter-Straße 11, 12489 Berlin, Germany;

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i Electrochemistry and Chemistry of Materials, Department of Chemistry, University of Dschang, P.O. Box 67, Dschang, Cameroon.

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* To whom correspondence may be addressed

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Abstract

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(Bio)sensors are integral to various aspects of daily life, serving functions related to safety, security, surveillance, monitoring, and awareness. In modern sensor devices, polymers play a crucial role with increasing interest in natural variants, especially in biosensing applications, due to their exceptional properties. Biopolymers, unlike synthetic counterparts, are abundant in nature, making them ideal candidates for renewable, biodegradable, and biocompatible materials in sensor. Enhancing sensor performance to achieve rapid response times to stimuli is a key objective in sensor development. Lignocellulosic compounds from plants hold promise in meeting such requirements due to their ease of processing, substantial surface area, diverse pore sizes and morphologies, modifiable surface characteristics, flexibility, printability, low density, and favorable physicochemical, mechanical, electrical, and thermal properties. Despite their potential, challenges remain in developing sensors and biosensors from Lignocellulosic biomass and their structural polymers, including addressing structural heterogeneity, optimizing processing techniques, ensuring stability and durability, understanding sensing mechanisms, and overcoming scalability and cost-effectiveness limitations. Growing research in the past decades has focused on lignocellulosic composite materials due to their economical and environmentally friendly attributes. This review focused on the valorization of Lignocellulosic biomass, particularly cellulose, hemicellulose, and lignin, in (bio)sensors development. It explores the macromolecular structure, sources and inherent properties of Lignocellulosic biomass, recent advancements in lignocellulose-based sensor
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technology, including emerging trends and innovative designs, and extraction processes of the three main lignocellulosic polymer constituents and their applications in sensor development, which emphasis on biosensors and biopolymers as electrode materials. Future directions involve fundamental research on structure-property relationships, process optimization for scalability and cost-effectiveness, multidisciplinary collaboration to address interdisciplinary challenges, and application-driven design to meet real-world needs in various fields.

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**Keywords:** Lignocellulosic biomass, cellulose, lignin, hemicellulose, modified electrodes, sensors, biosensors.

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# 1. Introduction

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Sensors serve as ubiquitous analytical platforms utilized across diverse environments to detect events or changes with high precision ¹⁻³. These platforms commonly integrate an active device, known as a transducer, which is capable of acquiring a physical quantity and converting it into a signal suitable for subsequent processing. They are employed for measuring various physical or chemical properties, such as concentration, temperature, activity, resistance, capacitance, conduction and heat transfer ⁴,⁵. The pivotal characteristics of (bio)sensors encompass their sensitivity to input changes and their selectivity in discriminating the response to specific targets from adjacent inputs. Enhancements in sensor performance are continually pursued through the integration of biological receptors or additional materials that amplify the transducer response ³,⁶. These receptors must exhibit excellent target recognition capabilities and efficient interaction to ensure effective signal transduction to the output system ⁷,⁸. Accordingly, sensor technologies exploit novel materials to streamline processes and enhance key attributes, including sensitivity, specificity, and reproducibility ³,⁶,⁹,¹⁰. A plethora of (bio)sensors, including analytical ¹¹⁻¹³, humidity ¹⁴,¹⁵, strain ¹⁶⁻¹⁸ and gas sensors ¹⁹,²⁰ are documented in the literature for diverse applications. Sensor technology offers myriad benefits, such as predictive and preventive maintenance, facilitating faster transmission of measurement data, enhancing accuracy, improving process control, and bolstering asset health ³,²¹. Moreover, the development of efficient (bio)sensors for sensitive determination of target element levels in various environments holds significant importance. Sensors confer numerous advantages, including straightforward operation, high reliability, simplicity, time and cost efficiency, portability, and direct applicability in point-of-care assays. These attributes highlight the importance of sensor technology in modern analytical and environmental monitoring endeavors ²²,²³.

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The development of new sensors has focused on the development of novel materials for electrode modification ²⁴⁻²⁸. Traditionally, electroactive materials employed for supporting surfaces or electrodes exhibit inert characteristics devoid of electroactive species, while maintaining conductivity across a broad potential range ⁸,²⁹,³⁰. Carbon-based electrodes have emerged as a focal point for enhancing electroanalytical performance, particularly in terms of sensitivity and specificity. Lignocellulosic biomass (LCB), primarily composed of carbon-rich biopolymers such as cellulose, hemicellulose, and lignin, have garnered increasing attention as surface modifiers to augment the selectivity and sensitivity of (bio)sensors ¹¹,¹³,³¹⁻³³. These bio-based materials offer a plethora of advantageous properties, including high surface area, porosity, hierarchical structure, permselective coating capabilities, and suitability for use as immobilization matrices ³⁴,³⁵. As constituents of plant cell walls, LCB are abundantly available in nature and serve as the primary source of the most prevalent biopolymer globally, cellulose. Consequently, LCB are regarded as promising renewable resources, characterized by their affordability
and non-toxic nature. These macromolecular materials exhibit diverse chemical functionalities such as hydroxyl, carboxyl, amine, amide, and thiol, enabling self-assembly and the ability to chemically or physically bind numerous molecules and biological entities, as well as form chelates with metal ions ³⁶,³⁷. The exceptional mechanical, thermal, physicochemical, and electrical properties of LCB, coupled with their eco-friendly attributes such as renewability, biocompatibility, and biodegradability, position the major components cellulose, hemicellulose, and lignin as prime candidates for substituting current sensing and biosensing technologies ¹⁴,³⁸,³⁹. The significant annual production of biomass waste, estimated at approximately 140 Gt globally, presents a substantial management challenge due to its potential negative environmental impact ⁴⁰. Often, biomass waste is incinerated by local populations for heating purposes, leading to the release of CO₂ into the environment and contributing to the exacerbation of global warming. However, numerous pathways for valorizing biomass and its by-products have been documented in the literature, spanning various fields such as renewable energy production, construction, furnishing, biofuel synthesis, and chemical industries ⁴⁰,⁴¹. Concurrently, an increasing number of research endeavors are directed towards the valorization of these bio-based materials in sensor and biosensor technologies.

Lignocellulosic biomass undergoes a biorefinery process to yield value-added products, which find numerous applications in (bio)sensing. The components of lignocellulosic biomass, namely cellulose, lignin, and hemicellulose, offer diverse utility as byproducts, yielding sugars, ethanol, and various chemicals ⁴²,⁴³. Despite the abundance and low cost of Lignocellulosic biomass, the primary challenge lies in achieving high selectivity and yield in the production of value-added chemicals at an economically viable scale ⁴⁴. This review focuses on recent advancements demonstrating the utilization of lignocellulosic biomass in sensor applications. It presents recent research showcasing the utilization of Lignocellulosic biomass, along with their major chemical biopolymers, in both sensing and biosensing realms. The review is structured into three main sections. Firstly, a comprehensive description of the intricate and complex macromolecular structure of Lignocellulosic biomass is provided, along with insights into their sources, properties, and various types. Additionally, a brief overview of their chemical composition is presented, providing foundational knowledge for subsequent discussions. Subsequently, the process of fractionation is examined, which involves various pretreatment methods aimed at isolating different polymers such as cellulose, hemicellulose, and lignin. The review includes a thorough discussion on the properties and characteristics of each by-product obtained through fractionation, thereby elucidating their potential applications in (bio)sensor technologies. Finally, the review summarizes the existing applications of Lignocellulosic biomass, cellulose, hemicelluloses, and lignin in the fields of (bio)sensing. By collating recent research findings, this section provides insights into the diverse range of (bio)sensor applications facilitated by these biomaterials.

## 2. From hierarchical structure of plant biomass to their application in the development of (bio)sensor technologies

### 2.1. Plant biomass and their potential as bio-based materials

Lignocellulosic biomass is primarily derived from various sources, including agricultural residues such as rice straw, rice husk, coconut shells, wheat straw, and sugarcane bagasse ⁴⁵,⁴⁶. Additionally, woody waste residues like wood chips, branches, and sawdust from saw mills and paper mills contribute to this biomass pool. These materials are often considered low-value byproducts of industrial sectors, including forestry. Furthermore, energy crops such as miscanthus, energycane, and grasses are cultivated specifically for their high yield of lignocellulosic biomass, which serves as a crucial raw material
for the production of second-generation biofuels ⁴⁷,⁴⁸. The structure and composition of lignocellulosic biomass vary depending on factors such as the plant species, soil composition, and climate. Nonetheless, there are commonalities across these materials ⁴⁹. Lignocellulosic biomass typically comprises approximately 90% organic macromolecules, namely cellulose, hemicelluloses, and lignin, which form the structural backbone of plant cell walls. In addition to these macromolecules, lignocellulosic biomass contains 2 to 10% of low molecular weight compounds referred to as extractibles, along with mineral components ⁴⁴,⁵⁰. In their review, Isikgora and Remzi Becer ⁴⁴ provided a comprehensive summary of various types of biomass, emphasizing the diversity in cellulose, hemicellulose, and lignin content across different plant types. Lignocellulosic biomass, derived from various plant sources, shows diverse chemical compositions, particularly in terms of cellulose, hemicellulose, and lignin content ⁴⁴. Hardwood species such as poplar, oak, and eucalyptus have relatively high cellulose levels (40.4–54.1%), with oak displaying the highest hemicellulose content (35.9%), but lower lignin percentages compared to softwoods. Softwood varieties like pine and Douglas fir typically exhibit moderate cellulose (42.0–50.0%) and higher lignin content (20.0–27.9%), reflecting their sturdier structure. Agricultural wastes, including wheat straw, barley hulls, and rice husks, exhibit more variability in composition ⁴⁴. For instance, wheat straw and barley straw have moderate cellulose (35.0–43.0%), while their hemicellulose content can be as high as 36.0%, though lignin remains lower (6.3–19.0%). Rice husks show a wider range of hemicellulose (12.0–29.3%) and lignin (15.4–20.0%), indicative of structural differences in these wastes. Grasses, including switchgrass, generally have moderate cellulose content (25.0–40.0%) and a wide range of hemicellulose (25.0–50.0%) and lignin (10.0–30.0%). This variability in composition among biomass types is key to determining their suitability for biofuel production and other industrial applications, with lignin content particularly influencing biomass recalcitrance and conversion efficiency ⁴⁴.

Lignocellulosic biomass, comprising cellulose, hemicellulose, and lignin, stands as an abundant and renewable resource with immense potential in the realm of bio-based materials (Figure 1). This diverse array of organic compounds, predominantly sourced from plant cell walls, holds promise as a sustainable alternative to traditional materials derived from fossil fuels ⁵¹. With growing concerns over environmental sustainability and the finite nature of fossil resources, the exploration and utilization of lignocellulosic biomass represent a pivotal avenue in the quest for greener and more sustainable material solutions. At the forefront of this exploration is cellulose, a polysaccharide polymer consisting of glucose units interconnected through β-1,4-glycosidic bonds ⁴³,⁴⁶. As the most abundant organic compound on Earth, cellulose serves as the primary structural component in plant cell walls, providing rigidity, strength, and resilience. The inherent properties of cellulose, including its biodegradability, low toxicity, and exceptional mechanical strength, render it an attractive candidate for a wide range of applications in various industries ⁵².

Hemicellulose, offers complementary properties that further enhance the versatility of lignocellulosic biomass. Unlike cellulose, hemicellulose exhibits greater heterogeneity in its composition, comprising various monosaccharides such as xylose, arabinose, and mannose, among others ⁵³,⁵⁴. This diversity lends hemicellulose its amorphous nature and water-soluble characteristics, making it suitable for applications requiring adhesion, film formation, and hydrogel formation ⁵⁴. Lignin, although often considered a byproduct of biomass processing, plays a crucial role in providing structural support and hydrophobicity to plant cell walls. As a complex aromatic polymer composed of phenylpropanoid units, lignin confers rigidity and impermeability to plant tissues, contributing to their resistance against microbial degradation and environmental stresses ⁵⁵,⁵⁶. Despite its inherent complexity and challenges associated
167 with its extraction and processing, lignin exhibits immense potential as a renewable feedstock for the
168 synthesis of value-added products, including adhesives, thermoplastics, and carbon fibers ⁵⁶,⁵⁷. Celluloses
169 and hemicelluloses are carbohydrates which are tightly linked to the lignin - aromatic polymer - through
170 covalent and hydrogen bonds, making the structure highly complex and robust (Figure 1) ⁴⁴.

171 The utilization of lignocellulosic biomass and its constituent components extends beyond
172 traditional applications in the pulp and paper industry. With advancements in biorefinery technologies and
173 interdisciplinary research efforts, novel approaches have emerged for the valorization of biomass-derived
174 sugars, polymers, and chemicals ⁵⁸,⁵⁹. Through processes such as enzymatic hydrolysis, fermentation,
175 and thermochemical conversion, lignocellulosic feedstocks can be transformed into biofuels,
176 biochemicals, and biomaterials, thus reducing reliance on fossil resources and mitigating environmental
177 impacts associated with conventional manufacturing processes ⁶⁰,⁶¹. In addition to their environmental
178 benefits, bio-based materials derived from lignocellulosic biomass offer unique advantages in terms of
179 performance, functionality, and sustainability. These materials can exhibit tailored properties through
180 modification and blending, allowing for customization to meet specific application requirements in sectors
181 ranging from construction and automotive to packaging and biomedical engineering ⁶¹. Furthermore, the
182 abundance and renewability of lignocellulosic feedstocks provide a reliable and cost-effective source of
183 raw materials, mitigating supply chain risks and promoting economic resilience in a rapidly evolving global
184 landscape ⁶². Despite the considerable progress made in harnessing the potential of lignocellulosic
185 biomass, several challenges persist in realizing its full commercial viability. Issues such as feedstock
186 variability, pretreatment efficiency, and product diversification necessitate continued research and
187 development efforts to optimize processes and overcome technical barriers ⁴⁹,⁶². Moreover, the
188 establishment of robust infrastructure and supportive policy frameworks is essential to incentivize
189 investment and foster market adoption of bio-based materials on a larger scale ⁶³.

<figure><img src="image_3.png" /><figcaption>**Figure 1:** Scheme of main components and arrangement of lignocellulosic biomass. Reproduced with permission from Ref.⁶⁴, Copyright © 2020 Springer Nature.</figcaption></figure>

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Copyright © 2020 Springer Nature.

## 2.2. Pretreatment of Lignocellulosic biomass and polymer extraction

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LCB and their derivatives exhibit compelling properties and significant potential, rendering them
valuable for the production of various chemicals and biopolymers, such as cellulose, hemicellulose, and
196 lignin. These biopolymers can subsequently undergo conversion into isolated components, leading to the 197 generation of high-value-added products, including hydrocarbons, chemical materials, and biofuels 65. The 198 intricate and densely intertwined structure of LCB poses a significant challenge due to its impediment to 199 chemical accessibility, thereby impeding the digestibility of biomass and the extraction of its constituent 200 components 66. To address this challenge, effective biomass enhancement strategies often require a 201 pretreatment step, strategically designed to disrupt the intricate structure of LCB. This disruption aims to 202 facilitate the degradation or release of constituent components, consequently improving both chemical 203 and biological accessibility to these valuable materials. This pretreatment step is essential for unlocking 204 the full potential of LCB in various applications, ranging from biofuels to high-value chemicals and 205 biopolymers 66,67. Following pretreatment, LCB undergo a series of transformations resulting in the 206 extraction of lignin, hemicellulose, and cellulose, which leads to the production of simpler components. 207 This process significantly enhances the versatility and applicability of LCB-derived biopolymers across a 208 wide range of fields and industries 42,66. These applications span across various fields, including 209 engineering, where they are used in structural materials, and biomedicine, for drug delivery systems and 210 tissue engineering scaffolds 43,68. In the energy sector, they play a role in batteries and solar cells, while 211 membrane technologies leverage them for water purification and gas separation 69. They also find use in 212 optoelectronic devices for electronic applications and in polymer nanocomposites to enhance material 213 properties 70. Other applications include textiles for sustainable fabric production, the food industry for 214 packaging and additives 71, and the medical sector for wound dressings 72 and implants 43,68. Additionally, 215 they are utilized in the cosmetic industry for natural formulations 73 and in the pharmaceutical sector for 216 drug encapsulation and controlled release systems 74,75. Finally, the packaging industry benefits from these 217 materials to create eco-friendly solutions with improved barrier properties and sustainability 9,47. 218

219 Effective pretreatment strategies must satisfy multiple criteria to optimize their utility in the 220 processing of Lignocellulosic biomass. Firstly, they should aim to enhance sugar formation or the potential 221 for subsequent hydrolysis, thereby facilitating the conversion of biomass into fermentable sugars 76. 222 Additionally, these strategies must prevent the degradation or loss of carbohydrates, ensuring the 223 preservation of valuable biomass constituents 77. Furthermore, it is crucial to avoid the generation of 224 inhibitory byproducts during pretreatment, which could impede subsequent hydrolysis and fermentation 225 processes, thereby maximizing the overall efficiency of biomass conversion 78. Finally, cost-effectiveness 226 is paramount, necessitating the development of pretreatment methods that are economically viable and 227 scalable for industrial applications. These criteria collectively guide the design and implementation of 228 pretreatment techniques, ensuring their efficacy in enabling the efficient utilization of lignocellulosic 229 resources for the production of biofuels and other value-added products.35 A diverse array of pretreatment 230 methods is utilized for Lignocellulosic biomass (LCB), encompassing physical techniques such as milling, 231 grinding, and irradiation; mechanical approaches including steam explosion, extrusion, and cavitation; 232 chemical treatments like acid hydrolysis, alkaline hydrolysis, and organosolv processes; biological 233 methods such as enzymatic hydrolysis and microbial fermentation; and occasionally, combinations of 234 these techniques 79,80. These varied strategies are employed to address the intricate structural 235 complexities of LCB, aiming to enhance their utility by promoting accessibility to constituent components 236 for subsequent processing and application in various industries, including biorefineries, bioenergy 237 production, and biopolymer synthesis 34,79,80. 238

The conversion of lignocellulosic biomass into valuable polymers requires an essential 237 pretreatment step to break down the complex structure of the biomass 48,66. This overview (Table 1) 238
presents a detailed examination of various pretreatment methods, categorized into physical<sup>81,82</sup>, chemical
<sup>83-85</sup>, physico-chemical<sup>86,87</sup>, and biological<sup>86,88</sup> approaches. Each method is outlined with its working
principle, highlighting how it alters the biomass structure to enhance polymer extraction efficiency.
Furthermore, the advantages and limitations of each technique are discussed, offering insights into their
effectiveness, scalability, and environmental impact. The optimization of polymer extraction from
lignocellulosic biomass necessitates the integration of multiple pretreatment methods due to the complex
nature of lignin and cellulose interactions<sup>80,86</sup>. Physical pretreatments such as pyrolysis, milling, and
ultrasonication offer valuable preliminary steps by breaking down biomass and increasing surface area,
but they are often limited by high energy requirements and incomplete decomposition, which necessitates
further processing to obtain desired polymers<sup>79,80,86</sup>. Chemical methods, such as alkali and acid
pretreatments, are effective in breaking down lignin and hemicellulose but present challenges like
managing chemical waste and the risk of cellulose degradation. This requires careful control and
monitoring of these processes, which are commonly employed for biomass fractionation and biopolymer
extraction, to minimize negative impacts and ensure efficient outcomes<sup>34,61</sup>. Organosolv and ionic liquid
treatments provide high efficiency in lignin removal but come with high costs and environmental concerns
<sup>89-91</sup>. Physico-chemical methods like steam explosion<sup>92,93</sup> and supercritical CO<sub>2</sub> <sup>94</sup> treatment combine
effective disruption of the lignocellulosic matrix with lower toxicity, yet they require sophisticated
equipment and may still fall short in selectively targeting all biomass components. Biological methods,
such as bacterial<sup>95</sup> and enzymatic<sup>96,97</sup> treatments, offer environmentally friendly and specific degradation
but are slow and require precise conditions to be effective. To achieve optimal polymer extraction, a
combination of these pretreatment methods is often necessary. For instance, combining physical and
chemical pretreatments can enhance biomass disruption and improve cellulose accessibility while
mitigating some limitations of individual methods<sup>87,98</sup>. Physical methods, such as mechanical grinding or
milling, break down the biomass into smaller particles, increasing the surface area, while chemical
pretreatments, like alkali or acid treatments, effectively remove lignin and hemicellulose. This combination
allows for more efficient cellulose extraction by making it more accessible and reducing processing time
<sup>87,98</sup>. Similarly, integrating biological treatments with physico-chemical approaches can balance the
benefits of high specificity and environmental friendliness with the need for more efficient biomass
processing<sup>99,100</sup>. Biological treatments, such as the use of enzymes or microorganisms, offer high
specificity by targeting particular components of the biomass, like lignin or hemicellulose. These methods
are environmentally friendly, as they generate minimal chemical waste and are generally carried out under
mild conditions<sup>101</sup>. However, biological treatments can be time-consuming and less efficient for large-
scale biomass processing. To overcome these limitations, physico-chemical approaches, such as alkali,
acid, or thermal treatments, are often combined with biological methods. These approaches enhance the
overall efficiency by breaking down the rigid structures of lignin and hemicellulose more rapidly,
facilitating better access for biological agents to complete the process<sup>99,100</sup>. The integration of both
methods not only speeds up the biomass fractionation process but also ensures that polymer extraction
is carried out in a more sustainable and effective manner, minimizing environmental impact while
maximizing yield and quality. This hybrid approach is particularly important for large-scale industrial
applications, where efficiency and sustainability are critical factors<sup>99,100</sup>.
<table><caption>280 Table 1: Overview of pretreatment methods for lignocellulosic biomass in polymer extraction: principles, advantages, and limitations</caption><thead><tr><th>Category</th><th>Pretreatment methods</th><th>Working principle</th><th>Advantages</th><th>Limitations</th><th>Ref.</th></tr></thead><tbody><tr><td rowspan="7">Physical methods</td><td>Pyrolysis</td><td>Thermal decomposition at high temperatures (300-900°C) in the absence of oxygen, leading to the breakdown of biomass.</td><td><ul><li>Produces biochar, bio-oil, and syngas</li><li>Reduces biomass volume</li><li>Can be used as a preliminary step for polymer extraction</li></ul></td><td><ul><li>High energy requirement</li><li>Complex product separation</li><li>May lead to incomplete decomposition</li><li>Requires further treatment to obtain desired polymers</li><li>Complex process control</li><li>Potential formation of tar and char residues</li></ul></td><td>82,102</td></tr><tr><td>Milling</td><td>Mechanical size reduction by using grinding mills to break down biomass into smaller particles, increasing the surface area for further processing.</td><td><ul><li>Increases surface area of biomass</li><li>Facilitates further chemical or biological treatment.</li><li>Does not require chemicals</li></ul></td><td><ul><li>High energy consumption</li><li>Wear and tear of milling equipment</li></ul></td><td>103,104</td></tr><tr><td>Grinding</td><td>Similar to milling, it involves mechanical force to reduce particle size, but typically refers to more aggressive size reduction.</td><td><ul><li>Increases surface area</li><li>Improves enzyme accessibility</li><li>Simple and scalable</li></ul></td><td><ul><li>Energy-intensive</li><li>May not sufficiently disrupt lignocellulose structure</li><li>Can cause excessive dust generation</li></ul></td><td>105,106</td></tr><tr><td>Ultrasonication</td><td>Application of high-frequency sound waves to create cavitation bubbles that disrupt the biomass structure, facilitating polymer extraction.</td><td><ul><li>Increases porosity and enzyme accessibility</li><li>Can be combined with other treatments</li><li>Improves penetration of chemicals in further treatment</li></ul></td><td><ul><li>Limited to small-scale applications</li><li>Requires specialized equipment</li><li>Equipment cost</li></ul></td><td>107-110</td></tr><tr><td>Chipping</td><td>Cutting large biomass into small chips, facilitating easier handling and increasing the surface area for subsequent treatments.</td><td><ul><li>Simple process</li><li>Low-cost method for reducing biomass size</li><li>Reduces biomass size for easier transport and handling</li></ul></td><td><ul><li>Does not significantly alter biomass structure</li><li>Limited effect on lignin disruption</li><li>Not effective alone for polymer extraction</li></ul></td><td>111,112</td></tr><tr><td>Pelletization</td><td>Compressing biomass into pellets, increasing its density, and improving the handling and storage efficiency.</td><td><ul><li>Improves biomass density</li><li>Facilitates transport and storage</li><li>Improves uniformity for further processing</li></ul></td><td><ul><li>Energy-intensive</li><li>Mainly useful for logistic purposes</li><li>Does not alter biomass structure significantly.</li></ul></td><td>81,113</td></tr><tr><td>Torrefaction</td><td>Mild pyrolysis (200-300°C) in the absence of oxygen, leading to partial decomposition and removal of moisture and volatiles, making the biomass more brittle and easier to grind.</td><td><ul><li>Produces a hydrophobic, energy-dense product</li><li>Improves grindability</li><li>Reduces moisture content.</li></ul></td><td><ul><li>Loss of some volatile components</li><li>Moderate energy requirement</li></ul></td><td>114-117</td></tr></tbody></table>
<table><tr><td></td><td>Microwave-assisted pretreatment</td><td>Application of microwave radiation to rapidly heat the biomass, causing thermal and chemical disruption of the cell wall structure.</td><td><ul><li>Efficient heating</li><li>Can be combined with chemical pretreatment for enhanced effect</li></ul></td><td><ul><li>Requires specialized equipment</li><li>Uneven heating can occur</li></ul></td><td>69,90</td></tr><tr><td></td><td>Extrusion pretreatment</td><td>Forcing biomass through a screw orifice at high temperature and pressure, causing shear and friction that break down the biomass structure.</td><td><ul><li>Continuous process</li><li>Enhances mixing with other reagents</li><li>Reduces particle size and disrupts lignocellulosic structure.</li></ul></td><td><ul><li>High energy consumption</li><li>Equipment wear and tear</li><li>Requires optimization of operating conditions</li></ul></td><td>118,119</td></tr><tr><td rowspan="5">Chemical methods</td><td>Alkali pretreatment</td><td>Use of alkaline substances (e.g., NaOH, KOH) to break down lignin and hemicellulose, making cellulose more accessible.</td><td><ul><li>Effective lignin removal</li><li>Improves cellulose accessibility</li><li>Relatively mild conditions.</li></ul></td><td><ul><li>Can result in chemical waste</li><li>Requires neutralization post-treatment</li><li>High chemical consumption</li><li>Potential environmental issues with waste disposal</li><li>Can lead to cellulose degradation if not controlled</li></ul></td><td>83,120,121</td></tr><tr><td>Acid pretreatment</td><td>Use of dilute or concentrated acids (e.g., H<sub>2</sub>SO<sub>4</sub>, HCl) to hydrolyze hemicellulose and disrupt the lignocellulosic structure.</td><td><ul><li>Highly effective at breaking down hemicellulose</li><li>Enhances cellulose digestibility</li><li>Relatively fast reaction times</li></ul></td><td><ul><li>Corrosive leading to equipment degradation</li><li>Requires neutralization and disposal of acidic waste</li></ul></td><td>122-124</td></tr><tr><td>Organosolv pretreatment</td><td>Use of organic solvents (e.g., ethanol, methanol) often in combination with water and acid catalysts to solubilize lignin and hemicellulose.</td><td><ul><li>High lignin removal efficiency</li><li>Solvents can be recovered and reused</li><li>Produces high-purity cellulose.</li></ul></td><td><ul><li>Requires complex solvent recovery systems</li><li>Potential toxicity of solvents</li></ul></td><td>89,125,126</td></tr><tr><td>Ionic Liquids (ILs)</td><td>Use of ILs to dissolve lignin and disrupt the crystalline structure of cellulose, making it more accessible for subsequent enzymatic hydrolysis.</td><td><ul><li>High efficiency in lignin removal</li><li>Tunable properties of ILs</li><li>Can be tailored to specific biomass types</li></ul></td><td><ul><li>High cost of ILs</li><li>Potential environmental impact if not recovered</li><li>Potential toxicity of ILs</li></ul></td><td>91,110,112,127</td></tr><tr><td>Oxidative pretreatment</td><td>Use of oxidative agents (e.g., H<sub>2</sub>O<sub>2</sub>, O<sub>3</sub>) to break down lignin and other components by oxidation, enhancing cellulose accessibility.</td><td><ul><li>Effective lignin removal</li><li>Can be combined with other treatments</li><li>Can improve the efficiency of subsequent enzymatic hydrolysis</li></ul></td><td><ul><li>Potential formation of inhibitory by-products</li><li>Requires safe handling of oxidants</li><li>May require extensive washing of products</li></ul></td><td>128,129</td></tr><tr><td>Physico-chemical methods</td><td>Steam explosion</td><td>Rapid heating of biomass with high-pressure steam followed by sudden decompression, causing the biomass to rupture and disrupt its structure.</td><td><ul><li>Disrupts lignin-carbohydrate complex</li><li>Effective hemicellulose removal when combined with other methods</li><li>Improves enzyme accessibility</li></ul></td><td><ul><li>May produce inhibitory compounds (e.g., furfural)</li><li>Requires high-pressure equipment. Partial lignin removal</li><li>Requires high energy for steam generation</li></ul></td><td>130,131</td></tr></table>
<table><tr><td></td><td>Supercritical CO<sub>2</sub> (scCO<sub>2</sub>) treatment</td><td>Use of CO<sub>2</sub> above its critical point (31°C, 7.4 MPa) to penetrate and disrupt the biomass structure, often combined with co-solvents for enhanced effect.</td><td><ul><li>Non-toxic, recyclable solvent</li><li>Mild operating conditions</li><li>Can selectively extract certain components</li></ul></td><td><ul><li>High-pressure equipment required</li><li>Limited lignin disruption alone</li><li>Cost of scCO<sub>2</sub> process setup.</li></ul></td><td>94,132,133</td></tr><tr><td></td><td>Ammonia fiber explosion (AFEX)</td><td>Treatment of biomass with liquid ammonia under high pressure, followed by rapid release of pressure, causing the biomass to swell and disrupt its structure.</td><td><ul><li>Effective lignin and hemicellulose removal</li><li>No significant sugar degradation</li><li>Ammonia can be recovered</li></ul></td><td><ul><li>High cost and recovery of ammonia</li><li>Safety concerns with handling ammonia</li></ul></td><td>134,135</td></tr><tr><td rowspan="3">Biological methods</td><td>Bacterial treatment</td><td>Use of bacteria (e.g., cellulolytic bacteria) to degrade lignin and hemicellulose, leaving cellulose accessible for polymer extraction.</td><td><ul><li>Environmentally friendly</li><li>Low energy requirement</li><li>Selective degradation</li></ul></td><td><ul><li>Slow process</li><li>Requires careful control of microbial conditions</li><li>Requires specific conditions for bacterial growth</li><li>Limited control over degradation rates</li></ul></td><td>95,97</td></tr><tr><td>Enzymatic treatment</td><td>Use of specific enzymes (e.g., cellulases, hemicellulases) to break down cellulose and hemicellulose into fermentable sugars for polymer extraction.</td><td><ul><li>High specificity</li><li>Operates under mild conditions</li><li>Minimal by-product formation</li></ul></td><td><ul><li>High cost of enzymes</li><li>Requires precise conditions</li><li>Requires pretreatment to make cellulose accessible</li></ul></td><td>96,97,101,128</td></tr><tr><td>Fungi pretreatment</td><td>Use of ligninolytic fungi (e.g., white-rot fungi) to selectively degrade lignin, enhancing the accessibility of cellulose for polymer extraction.</td><td><ul><li>Environmentally friendly</li><li>Selective lignin degradation</li><li>Can improve the efficiency of subsequent enzymatic treatments</li></ul></td><td><ul><li>Slow process</li><li>Requires precise environmental control (e.g., temperature, moisture)</li><li>May not fully remove lignin</li><li>May be difficult to scale up</li></ul></td><td>88,136</td></tr></table>
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### 2.2.1. Physical methods

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Physical methods for the pretreatment of biomass encompass a diverse range of techniques aimed at altering the physical characteristics of biomass feedstocks to enhance their accessibility and reactivity for subsequent conversion processes. These methods, which do not involve chemical reactions, typically focus on reducing the size of biomass particles, disrupting the structural integrity of biomass components, and increasing the surface area available for enzymatic or chemical reactions. Among the physical pretreatment methods commonly employed are pyrolysis, milling, grinding, ultrasonication, chipping, pelletization, torrefaction, microwave-assisted pretreatment, and extrusion pretreatment.

(i) Pyrolysis involves the thermal decomposition of biomass at high temperatures (300°C to 900°C) without oxygen, breaking it down into biochar, bio-oil, and syngas<sup>82,137</sup>. Biochar, a carbon-rich solid, improves soil fertility, water retention, and carbon sequestration, and can be used to produce activated carbon for water purification, air filtration, and energy storage<sup>137</sup>. Bio-oil, a complex mixture of oxygenated hydrocarbons, can be refined into biofuels like biodiesel and renewable diesel, as well as platform chemicals for plastics, resins, and pharmaceuticals<sup>138</sup>. Syngas, composed of carbon monoxide, hydrogen, and carbon dioxide, can be used for power generation or as a precursor for chemicals such as methanol, ammonia, and synthetic natural gas<sup>82</sup>.

(ii) Milling is a key physical pretreatment method that mechanically reduces biomass into smaller particles using equipment like hammer mills, ball mills, or attrition mills<sup>86,103,104</sup>. This increases the surface area, enhancing accessibility for enzymes or catalysts during hydrolysis or fermentation. By reducing particle size, milling exposes internal biomass structures, particularly cellulose and hemicellulose, facilitating enzyme penetration and accelerating the conversion of complex carbohydrates into fermentable sugars or valuable intermediates<sup>104,139</sup>. However, excessive milling can be energy-inefficient and costly. Therefore, balancing milling degree with desired particle size reduction is essential to optimize efficiency and minimize costs. Meticulous control and optimization of milling parameters are crucial for effective biomass conversion<sup>103</sup>.

(iii) Grinding, akin to milling, constitutes a fundamental step in biomass pretreatment that reduces biomass particle size using equipment like grinders or crushers. It is particularly effective for Lignocellulosic biomass such as wood chips or agricultural residues<sup>105</sup>. Grinding causes structural changes by fragmenting and pulverizing the biomass, increasing surface area exposure and making cellulose and hemicellulose more accessible for enzymatic action during hydrolysis or fermentation. This process breaks down the lignin, cellulose, and hemicellulose matrix, enhancing enzymatic digestibility and reducing the need for excessive enzymes, thus optimizing process economics and sustainability<sup>140</sup>. Additionally, grinding equipment can be customized to achieve specific particle size distributions, maximizing efficiency in subsequent bioconversion steps. As a key physical pretreatment method, grinding facilitates the use of lignocellulosic biomass as a renewable feedstock for biofuels and biochemicals production<sup>106,141</sup>.

(iv) Ultrasonication at high intensity causes biomass to break down by applying high-frequency sound waves to biomass slurries, creating cavitation. This results in the rapid formation and collapse of microbubbles, generating intense localized forces that mechanically disrupt cell walls and break down the complex biomass structure. This process creates pores and fractures in the biomass, enhancing mass transfer and facilitating enzyme or catalyst penetration. Ultrasonication also increases the surface area of cellulose and hemicellulose, improving their accessibility for hydrolysis and boosting saccharification efficiency<sup>108,142</sup>. Additionally, it effectively disrupts Lignocellulosic biomass, particularly those high in
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lignin, which is a major barrier to enzymatic degradation. By pretreating lignocellulosic feedstocks rich in lignin, ultrasonication enhances the efficiency of subsequent fermentation processes for biofuel or biochemical production <sup>143,144</sup>.

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(v) **Chipping**, a fundamental physical pretreatment method, entails the mechanical reduction of biomass into uniformly sized chips utilizing dedicated machinery such as chippers or shredders <sup>145</sup>. This technique is widely used for woody biomass, improving handling, storage, and transportation logistics. By breaking down the biomass into smaller, uniform chips, chipping significantly increases the surface area available for interaction with subsequent pretreatment and conversion processes, thereby improving overall efficiency <sup>146</sup>. The uniformity of chipped biomass ensures consistent material properties, facilitating predictable and controllable downstream operations. Smaller particle sizes improve the penetration of solvents, enzymes, or catalysts during processing, enhancing yield and product quality <sup>87</sup>. Additionally, chipped biomass has better flow characteristics, reducing the risk of clogging in handling and feeding systems <sup>87</sup>.

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(vi) **Pelletization**, a fundamental physical pretreatment method, compresses biomass into pellets using equipment like pellet mills or extruders <sup>113</sup>. This process subjects the biomass to high pressure and temperature, altering its composition and structure. Mechanical compression can partially disrupt the lignocellulosic structure, breaking down complex components and increasing enzyme or catalyst accessibility during conversion processes like enzymatic hydrolysis or thermochemical conversion <sup>81,113</sup>. This enhances conversion efficiency and yields. Pellets, with their uniform shape and size, streamline handling, transportation, and storage, facilitating automated feeding systems in industrial settings and reducing labor requirements. Their dense nature also saves storage space and lowers logistical costs <sup>147</sup>.

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(vii) **Torrefaction** is a key thermal pretreatment method in biomass processing, typically conducted at 200°C to 300°C under an inert atmosphere <sup>115,116</sup>. This process expels moisture and volatile compounds, reducing biomass weight and increasing energy density, which improves combustion properties and energy efficiency. Additionally, torrefaction enhances biomass stability, reducing degradation and spoilage during storage <sup>148</sup>. It alters the physical and chemical properties, making Lignocellulosic biomass more brittle and easier to grind, which aids downstream processes like enzymatic hydrolysis or thermochemical conversion <sup>149,150</sup>. Torrefied biomass also resists biological degradation, extending shelf life and potential applications. It is particularly useful in co-firing with coal in power plants, serving as a renewable energy source that reduces greenhouse gas emissions. Furthermore, torrefied biomass is suitable for gasification, producing syngas for various industrial uses, thus promoting energy diversification and sustainability <sup>151</sup>.

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(viii) **Microwave-assisted pretreatment** is a sophisticated method heats and modifies biomass using microwave irradiation <sup>69</sup>. Unlike traditional thermal methods, such as pyrolysis or torrefaction, microwave pretreatment has distinct advantages. It rapidly and uniformly heats biomass, minimizing processing time and energy consumption. Microwaves thermal degradation <sup>69,90</sup>. This enhances enzymatic hydrolysis efficiency and overall conversion yields. Tailoring microwave pretreatment to specific feedstocks and conditions makes it a promising technology for sustainable biofuel and biochemical production <sup>69,152</sup>.

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(ix) **Extrusion pretreatment** subjects biomass to intense mechanical forces by forcing it through a narrow orifice under high pressure and temperature <sup>118</sup>. This process causes mechanical shearing, breaking down lignocellulosic bonds and reducing particle size, which increases the biomass surface area for better chemical and enzyme accessibility during hydrolysis <sup>118,119</sup>. Additionally, extrusion helps remove
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extractives and impurities, enhancing feedstock purity for downstream processing. This method improves enzymatic digestibility, reduces particle size, increases surface area, and purifies biomass, making it a valuable option for biomass conversion and biofuel production, advancing sustainable and efficient biorefinery processes ¹¹⁸,¹¹⁹.

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Overall, physical pretreatment methods offer diverse approaches to modify the structure and composition of biomass, thereby improving its suitability for bioconversion processes. These methods play a crucial role in unlocking the potential of biomass as a renewable and sustainable feedstock for the production of biofuels, biochemicals, and bioproducts, contributing to the transition towards a bio-based economy.

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### 2.2.2. Chemical methods

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Chemical pretreatment of biomass is essential for converting it into valuable products like biofuels, biochemicals, and biomaterials. This process uses various chemicals to modify biomass structure, enhancing its readiness for enzymatic hydrolysis or fermentation. The primary goal is to disrupt the complex biomass composition, mainly cellulose, hemicellulose, and lignin, to improve accessibility for conversion ³⁴. The chemical pretreatment includes different methods:

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(i) Alkali pretreatment plays a pivotal role for lignocellulosic biomass biorefinery of and component fractionation. It involves solvation and hydrolytic decomposition (saponification) of lignocellulose particles, primarily targeting lignin. This method reduces lignin content, increasing the rate and yield of enzymatic hydrolysis of carbohydrates ¹⁵³,¹⁵⁴. Alkali chemicals react with lignin and carbohydrates, dissolving lignin and facilitating enzymatic hydrolysis of cellulose and hemicellulose into sugars ⁸³. This treatment enhances cellulose digestibility by solubilizing lignin and decreasing cellulose crystallinity. Common alkali agents like sodium hydroxide, potash, and ammonia efficiently break ester bonds linking lignin and xylan ⁸³,¹²¹. Through this method disrupts lignin structure and partially hydrolyzes hemicellulose. The alkaline conditions cause biomass swelling, increasing porosity and cellulose accessibility, essential for subsequent biochemical conversion steps ¹²¹. This method is particularly effective for high-lignin biomass, as it removes lignin, a complex polymer encasing cellulose fibers. By removing lignin, alkali pretreatment improves enzymatic cellulose digestion, boosting the efficiency of downstream processes for producing biofuels, biochemicals, and other value-added products from lignocellulosic biomass ¹²¹.

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(ii) Acid pretreatment: It hydrolyzes hemicellulose into its monomers, enhancing cellulose availability, and biodegradability. Typically applied after alkali pretreatment and lignin extraction, it aims to solubilize hemicellulosic portions of lignocellulosic material (LCB) ¹²²,¹²³. This method uses mineral acids like sulfuric acid ($H_2SO_4$), hydrochloric acid (HCl), or organic acids like acetic acid, to break down hemicellulose into fermentable sugars and solubilize lignin, improving cellulose accessibility for enzymatic hydrolysis ¹²². Acid pretreatment can be conducted under various conditions, optimizing sugar release and minimizing by-products. Dilute acid pretreatment (0.5-1.0% sulfuric acid) at moderate temperatures (140-190 °C) effectively removes hemicellulose, increasing glucose yields from cellulose to nearly 100%, and enhancing enzymatic hydrolysis susceptibility ¹²²,¹⁵⁵. The flowchart in Figure 2A depicts the steps for obtaining solvent extraction cellulose nanocrystals (SE-CNC) and without solvent extraction cellulose nanocrystals (WSE-CNC). Before scanning electron microscopy (SEM) analysis (Figure 2B), images after each isolation step showed color changes from brown to pale-yellow post-purification. SE-B1 bleaching was insufficient, leaving residual lignin. SE-cellulose exhibited finer particles compared to WSE-cellulose due to waxes ¹⁵⁶. SEM images (Figure 2C) revealed cleaner surfaces and smaller cellulose particles from solvent extraction, suggesting extractable compounds contribute to WSE-cellulose agglomeration ¹⁵⁶.
Previous studies described the extraction procedure of cellulose from the biomass in two major steps: (1) pretreatment and removal of the amorphous phase using alkali solution (NaOH) to partially or completely remove lignin, wax, and oils, depolymerizing native cellulose structure, and exposing crystallites; and 2) sulfuric acid hydrolysis under dynamic stirring to prepare nanocellulose from pretreated and bleached biomaterial<sup>84,85,156-158</sup>.

<figure><img src="image_4.png" /><figcaption>**Figure 2:** (A) Schematic representation of the cellulose nanocrystals (CNC) extraction process. (B) Images depicting (a) crude acacia bark, (b) ground acacia bark, (c) SE-B1, (d) SE-cellulose, and (e) WSE-cellulose. (C) Scanning electron microscopy (SEM) images showing (a) SE-NaOH, (b) WSE-NaOH, (c) SE-B1, (d) WSE-B1, (e, f) SE-cellulose at different magnifications, and (g, h) WSE-cellulose at different magnifications. Reproduced with permission from Ref. <sup>156</sup>, Copyright © 2017 Elsevier.</figcaption></figure>

(iii) *Organosolv pretreatment:* It involves a chemical reaction between LCB and an aqueous organic solvent at temperatures of 140 to 220°C. This process selectively extracts lignin and hemicellulose preserving cellulose-rich residues. The selective organic solvents, with the help of acid or alkaline catalysts, breaks down the complex lignocellulosic structure, solubilizing lignin and hemicellulose, while leaving cellulose fibers intact <sup>126</sup>. This result in high-purity lignin and hemicellulose streams, which can be further processed into biofuels, biochemicals, or industrial materials <sup>125,126</sup>. Organosolv pretreatment offers the flexibility to optimize extraction efficiency and component purity and is more environmentally friendly than traditional chemical methods due to the use of more benign organic solvents that can be reclaimed and recycled. Common solvents include ketones, alcohols, and weak acids, typically at 40 to 80% concentrations in water. Ethanol is often used for its cost-effectiveness and ease of recovery, while butanol, although more efficient in lignin removal, is limited by its high cost <sup>159,160</sup>. Advantages of organosolv pretreatment include the separation of high-purity cellulose with minimal degradation, high-
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quality lignin isolation, efficient fractionation of hemicellulose, and high recovery efficiency of organic
solvents post-process¹²⁵,¹⁵⁹,¹⁶⁰.

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(iv) Ionic Liquids (ILs): Characterized as organic salts with low melting points, ILs have emerged as promising solvents for biomass pretreatment due to their exceptional solvent properties tailored for lignocellulosic biomass ⁹⁰,⁹¹,¹²⁷. These unique properties enable efficient removal of lignin and disruption of the biomass structure, rendering ILs valuable tools in biomass processing. They interact with and solvate the aromatic moieties of lignin through π-π and n-π interactions, rendering them highly effective in extracting lignin from Lignocellulosic biomass, resulting in cellulose-rich residues ¹⁶¹. These solvents, comprising cations or anions, belong to a class of highly stable and polar solvents characterized by their low melting points and negligible vapor pressures ¹⁶². The ionic liquid pretreatment offers remarkable thermal and chemical stability, creating safer processing conditions. It features solvents with low vapor pressures that remain in a liquid state across a broad temperature range ¹⁶¹,¹⁶². Two primary approaches for utilizing ionic liquids in lignocellulosic biomass fractionation have emerged: hydrolyzing carbohydrate polymers using acidic ionic liquids and chemically disrupting the lignocellulosic structure without achieving complete dissolution of the biomass. Despite their potential, challenges such as ionic liquid recovery and coast remain barriers to widespread adoption in large-scale applications ¹⁶¹.

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(v) *Oxidative pretreatment*: Employing oxidizing agents such as hydrogen peroxide (H₂O₂), ozone (O₃), or peracetic acid (CH₃CO₃H) oxidative pretreatment represents a significant step in biomass conversion processes. These agents break down lignin and enhance the digestibility of cellulose, a key constituent of biomass, by inducing lignin bonds cleavage and generating reactive oxygen species ¹²⁸,¹⁶³. This process reduces lignin content and improves sugar yields from biomass, enhancing the efficiency of subsequent chemical and enzymatic hydrolysis processes and contributing to the valorization of lignocellulosic biomass ¹²⁹. The versatility of oxidative pretreatment lies in its ability to be tailored to specific biomass feedstocks and process conditions, offering researchers and engineers a valuable tool for optimizing biomass conversion processes and maximizing product yields. Additionally, oxidative pretreatment holds promise for advancing the development of sustainable biorefinery technologies, contributing to the transition towards a more environmentally friendly and resource-efficient bioeconomy ¹²⁸,¹⁶³.

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Pretreatment methods affecting the physical parameters as well as their chemical bonding (bond cleavage) and intermolecular interactions of LCB are called physicochemical techniques ⁹²,⁹⁸. Below are listed and briefly described several of these techniques.

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(i) *Steam explosion*: Developed and patented in 1926 by Mason, this process treats LCB with high-pressure steam for short periods of time, followed by sudden decompression (explosion), achieving defibration by a combination of chemical and mechanical action ⁹³. The treatment alters the lignocellulosic structure by disrupting bonds between cellulose, hemicellulose, and lignin. Hemicellulose is partially hydrolyzed and solubilized during the process, rendering the biomass more porous and accessible to enzymatic hydrolysis ⁹²,⁹³. Steam explosion is versatile, applicable to various biomass feedstocks, and widely used in second-generation biofuels production, offering enhanced digestibility and sustainability ⁹³.

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(ii) *Supercritical carbon dioxide (scCO₂) treatment*: Initially investigated by Duan and Sun, biomass pretreatment with carbon dioxide involves subjecting it to high pressures (above 20 MPa) and
temperatures (above 160°C). The utilization of scCO₂ offers numerous advantages, including non-toxicity, cost-effectiveness, and widespread availability, rendering it a green solvent ¹⁶⁴. The process of biomass pretreatment with CO₂ typically involves introducing CO₂ liquid from a storage tank into a reactor containing biomass, with subsequent vacuum application to remove air from the reactor. As pressure in the reactor increases to match that of the CO₂ tank, the reactor is immersed in an ice bath to facilitate the influx of additional CO₂. Heating the reactor using a heating tape or water bath brings it to the desired temperature for achieving the supercritical state. The presence of water in the CO₂ mixture promotes the formation of carbonic acid (H₂CO₃), which aids in the penetration of biomass and facilitates the hydrolysis of hemicelluloses ¹⁶⁵,¹⁶⁶. The subsequent decompression of the reaction system induces structural alterations and fragmentation of the biomass, leading to an increase in its specific surface area ¹²⁰. The pretreated biomass can then undergo enzymatic hydrolysis to extract sugars. The resulting hydrolysate, enriched in sugars, can be transported to production facilities for further fermentation. CO₂ is currently integrated into enzymatic processes to enhance the accessibility of biomass to chemicals and the hydrolysis of polymer components in Lignocellulosic biomass, thereby yielding sugars. These isolated sugars can be fermented into value-added chemicals ¹⁶⁷. Achieving high enzymatic hydrolysis rates hinges significantly on enzyme stability, influenced by factors such as pressure, temperature, pH, the number of depressurization steps, water content, carbonic acid formation, solid content, enzyme species, and substrate and end-product types. Enzymatic processes may be hindered by the blocking of enzyme active sites, underscoring the importance of carefully controlling these parameters ¹⁶⁸.

(iii) *Ammonia fiber explosion (AFEX)*: AFEX treats biomass with liquid ammonia under high pressure and moderate temperatures, breaking down lignocellulose and improving accessibility for enzymatic or chemical conversion ¹³⁴,¹³⁵. It selectively removes lignin while preserving cellulose and hemicellulose fractions, improving fermentable sugars yields. AFEX operates under mild conditions, reducing energy consumption and environmental impact, and is adaptable to Lignocellulosic biomass ¹³⁵.

### 2.2.4. Biological methods

Biological pretreatment of Lignocellulosic biomass is a crucial step in the production of biofuels and biochemicals from biomass. This process involves the use of microorganisms or enzymes to degrade lignin, hemicellulose, and other complex polymers present in plant cell walls, making the cellulose more accessible for subsequent hydrolysis into fermentable sugars. Several biological methods have been developed and employed for this purpose, each with its own advantages and limitations ¹⁶⁹,¹⁷⁰.

(i) *Bacterial treatment*: Bacterial species exhibit rapid growth and ease of genetic manipulation leading to biomass pretreatment using ligninolytic strains to further delignify and generate purer carbohydrates for saccharification, resulting in a more environmentally friendly and energy-efficient process ⁹⁷. Microorganisms such as brown-, white-, and soft-rot fungi are used in biological treatment to degrade lignin and hemicellulose in waste materials, with white-rot fungi being particularly effective due to their production of lignin-degrading enzymes and others enzymes essential for pretreating lignocellulosic feedstocks. The following reactions are generally involved: oxidative, demethylation (or demethoxylation), side-chain oxidation, and propyl side-chain cleavage ¹⁷¹,¹⁷². In contrast to white rot fungi, brown-rot fungi are a group of wood-degrading fungi with a mechanism relevant for industrial bioconversion. These fungi remove nearly 100% of carbohydrates from wood, but they usually remove little lignin ¹³⁶. The efficient degradation of LCB is mainly distributed among fungi and bacteria. They degrade LCB by modifying its structure and degrading it into simpler substrates for ligninolytic enzyme
digestion. Cellulose and hemicellulose are degraded in biological pretreatment to monomeric sugars using cellulolytic and hemicellulolytic microorganisms ¹⁷³. Cellulolytic bacteria can be found in different genera such as *Clostridium*, *Ruminococcus*, *Caldicellulosiruptor*, *Butyrivibrio*, *Acetivibrio*, *Cellulomonas*, *Erwinia*, *Thermobifida*, *Fibrobacter*, *Cytophaga*, and *Sporocytophaga* ¹⁷⁴. This mechanism involves the action of bioagents capable of producing enzymes that degrade lignin, hemicellulose, and cellulose. For example, white-rot fungi secrete Echinodontium taxodii 2538, which is involved in the delignification of Chinese willow (hardwood) and China-fir (softwood) for varied residence times up to 120 days ¹⁰¹. Nine bacterial strains identified by 16S rRNA gene sequencing as species of Streptomyces, Bacillus, and Paenibacillus isolated from the lagoon were used as seed organisms to degrade lignin in sawdust lignocellulosic biomass up to 83% at the rate of 1.80 × 10⁻⁵ g d⁻¹ cm⁻³ ¹⁷⁵. Su et al. ⁸⁸ evaluated lignin-degrading fungi for the biological pretreatment of corn stover. Sixty-three strains were screened out, and *Myrothecium verrucaria* secreted three lignin-degrading enzymes simultaneously during the bio-pretreatment process. The activity levels of laccase, lignin peroxidase, and manganese peroxidase were 6.61, 0.78, and 1.31 U g⁻¹ dry biomass. Tsegaye et al.¹⁷⁶ studied the biodelignification and hydrolysis of rice straw by novel bacteria isolated from wood-feeding termites. Their results showed that maximum lignin removal was achieved in separate biodelignification and hydrolysis processes after the 14th day of treatment by *Ochrobactrum oryzae* BMP03 (53.74% lignin removal). Hydrolysis of the biodelignified rice straw released 69.96% of total reducing sugars after the 14th day of hydrolysis by Bacillus sp. BMP01.

(ii) Enzymatic treatment: In nature, lignocellulosic biomass undergoes hydrolysis through a plethora of enzymes mainly produced by fungi and bacteria which synergistically degrade cellulose, hemicellulose, and lignin ¹⁷⁷. It is crucial to select the appropriate enzyme capable of hydrolyzing the target biopolymer in the biomass matrix according to the goal of the investigation. Cellulose, hemicellulose, and lignin conversion are catalyzed respectively by cellulases, hemicellulases, and ligninases. For instance, cellulases, primarily produced by fungi, bacteria, and protozoans, include endocellulases, cellobiohydrolase, and β-glucosidase, which catalyze the hydrolysis of the β-(1-4) linkages of cellulose to soluble sugars. Cellulases isolated from the filamentous fungus *Trichoderma reesei*, an anamorph of the fungus *Hypocrea jecorina*, are currently the main cellulases used in the industrial development for the conversion of cellulose into glucose ¹⁷⁸,¹⁷⁹. *Trichoderma reesei*, having the capacity to produce large amounts of cellulolytic enzymes, secretes three main types of cellulose-degrading enzymes for catalyzing this enzymatic hydrolysis process: (1) endoglucanases (1,4-β-D-glucanohydrolase, EC 3.2.1.4) catalyze random cleavage of the internal β-1,4 bonds in the cellulosic polymers; (2) exoglucanases or cellobiohydrolases (1,4-β-D-glucan cellobiohydrolase, EC 3.2.1.91) catalyze hydrolysis of the second outermost β-1,4 bonds by attacking the cellulose from the ends only, releasing mainly cellobiose; and (3) β-glucosidases (β-d-glucoside glucohydrolase, EC 3.2.1.21) are only active on cello-oligosaccharides and cellobiose, releasing glucose monomer units from the cellobiose, for instance ¹⁷⁹⁻¹⁸². Hemicellulases comprise enzymes like endoxylanases, β-xylosidase, endo-mannanases, endo-α-L-arabinanase, α-L arabinofuranosidases, α-d-glucuronidases, α-galactosidases, and many other supplementary enzymes, which hydrolyse hemicelluloses into xylo-oligosaccharides and D-xylose sugar ¹⁸³. Lignin-modifying enzymes (LMEs) are various types of enzymes produced by fungi and bacteria that catalyze the breakdown of lignin. In general, bacteria that grow in lignin secrete various oxidative enzymes that assist in lignin depolymerization or modification. There are extracellular fungal oxidoreductases that catalyze the degradation of lignin by oxidation. LMEs include peroxidases, such as lignin peroxidase (EC 1.11.1.14, LiP), manganese peroxidase (EC 1.11.1.13, MnP), versatile peroxidase (EC 1.11.1.16, VP), and
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many phenoloxidases (laccases). Laccases and peroxidase enzymes can also cause lignin degradation through low molecular weight free radicals such as OH, depolymerizing the phenolic and non-phenolic lignin polymers, and mineralizing the insoluble lignin<sup>57,184</sup>.

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(iii) *Fungi Pretreatment*: White-rot fungi, exemplified by species like *Phanerochaete chrysosporium*, *Trametes versicolor*, and *Pleurotus ostreatus*, are renowned for their lignin-degrading capabilities. They secrete ligninolytic enzymes like lignin peroxidases, manganese peroxidases, and laccases, which break down lignin into smaller aromatic compounds facilitating lignocellulose degradation<sup>136</sup>. Unlike chemical or physical methods that often result in indiscriminate biomass degradation, white-rot fungi preserve the cellulose and hemicellulose fractions, maintaining the structural integrity of the biomass. This selective lignin degradation not only enhances the accessibility of cellulose to enzymatic hydrolysis but also generates valuable lignin-derived compounds that can be utilized for various downstream applications, including the production of value-added chemicals and materials<sup>136,172</sup>. In contrast to white-rot fungi, brown-rot fungi such as *Postia placenta* and *Gloeophyllum trabeum*, focus on cellulose degradation through oxidative mechanism, inducing cellulose depolymerization without significant lignin degradation<sup>185</sup>. Brown-rot fungi pretreatments alters the crystalline structure of cellulose, enhancing its accessibility to cellulolytic enzymes during hydrolysis<sup>186</sup>. Both white-rot or brown-rot fungi pretreatment methods offer promising avenues for improving lignocellulosic biomass conversion efficiency and sustainability, contributing to the development of bio-based industries and renewable resources utilization for a greener future<sup>185,186</sup>.

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## 2.3. Properties and (bio)sensing applications of Lignocellulosic biomass

Lignocellulosic biomass, comprising cellulose, hemicellulose, and lignin is a complex biomaterial with diverse properties and applications in the life sciences.

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### 2.3.1. LCB properties

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The physical properties of including particle size, density, grindability, moisture sorption, and thermal properties play a crucial role in its functional attributes<sup>35</sup>. Biomass particles typically exhibit irregular shapes, posing challenges in accurately measuring their length, width, and thickness. Experimental investigations conducted by Guo et al.<sup>187</sup> elucidated the anisotropic nature of biomass particles, wherein their optical, physical, and mechanical properties exhibited directional dependence. This anisotropy resulted in biomass particles having a needle-like shape and a high aspect ratio<sup>187</sup>. Furthermore, the aspect ratio of various biomass particles varied significantly with particle size, with larger particles exhibiting distinct aspect ratios. Additionally, the particle size distributions of LCB exhibit linear dependence on different sizes of biomass particles, which, in turn, are linked to variations in the mechanisms governing particle break-up, primarily influenced by the structural characteristics of the material<sup>187</sup>. According to Gera et al.<sup>188</sup> the aspect ratio of biomass particles significantly impacted heat transfer during their thermal conversion processes. Similarly, Wang et al.<sup>189</sup> demonstrated that an increase in particle size resulted in greater weight loss of biomass particles during torrefaction. Moreover, biomass particle size plays a crucial role in biomass conversion yield and energy consumption. It influences mass and heat transfer processes and directly affects the rate of enzymatic bioconversion of biomass particles<sup>190</sup>. In the scientific literature, two types of density are commonly discussed in relation to lignocellulosic biomass particles. Bulk density, defined as the mass of numerous biomass particles divided by the total volume they occupy, and particle density (also known as true density), which refers
to the density of the particles constituting the material excluding the volume occupied by pore spaces <sup>191</sup>. Bulk density is a critical physical property that plays a significant role in the design of logistic systems for biomass handling. Several factors, including size, shape, moisture content, individual particle density, and surface characteristics, influence bulk density <sup>192</sup>. Studies conducted by Rezaei et al. <sup>193</sup> have highlighted the importance of size, shape, and density of biomass particles in their transportation, fluidization, rates of drying, and thermal decomposition. Pelleting of wood particles has been shown to increase particle density and reduce variability in physical properties among biomass particles. This increase in bulk density of Lignocellulosic biomass enhanced energy production efficiency <sup>193</sup>. For example, Liu et al. <sup>194</sup> demonstrated improvements in the bulk density of bamboo pellets for energy production. They found that incorporating 40% pine particles into bamboo particles increased the bulk density of pellets from 0.54 g/cm<sup>3</sup> to 0.60 g/cm<sup>3</sup>. Furthermore, significant interactions were observed between bulk density, inorganic ash content, net calorific value, combustion rate and heat release rate <sup>194</sup>.

In addition, the physical and mechanical pretreatment of Lignocellulosic biomass (LCB) considers processes such as particle size reduction and associated energy costs, which are closely linked to a physical property known as grindability. Grindability, quantify by the Hardgrove grindability index (HGI), impacts grinding efficiency and combustion performance <sup>195</sup>. Biomass presents challenges in grinding due to its fibrous and resilient nature, making it difficult to achieve uniform particle size reduction, consequently leading to diminished combustion efficiency <sup>115</sup>. Grinding and torrefaction processes applied to Lignocellulosic biomass result in a reduction in material toughness and stiffness, primarily attributed to the breakdown of the hemicellulose matrix and depolymerization of cellulose, consequently altering the aspect ratio of resulting fibers. Grinding operations reduce both the particle size and length, as well as the thickness of the material <sup>116,148</sup>. It has been observed that there is a significant reduction of approximately 90% in energy consumption for torrefied biomass chips compared to non-torrefied wood chips <sup>117</sup>. Moreover, moisture sorption is another crucial parameter to consider when assessing the characteristics and properties of biomass. This parameter can be mathematically estimated at equilibrium by examining the relationship between water content and equilibrium relative humidity of a material, which is graphically represented by a curve known as a moisture sorption isotherm. This isotherm provides insight into the water content of biomass material at a specific temperature. Various mathematical models have been developed to describe sorption isotherms <sup>196</sup>. The quality of lignocellulosic feedstock relies on factors such as moisture content and uptake, which are directly correlated with intrinsic properties of biomass materials, including porosity, surface area, and surface chemistry. The sorption or desorption of moisture by lignocellulosic biomass is contingent upon the vapor pressure of water within the material and its surroundings <sup>197</sup>.

The mechanical properties of Lignocellulosic biomass, vital for various applications, are influenced by their hierarchical structure and chemical composition. Cellulose, the primary load-bearing component, determines stiffness and tensile strength. Highly ordered cellulose fibrils provide structural integrity and resist tensile forces, while crystalline regions contribute to high tensile strength, preventing deformation or failure <sup>43,198,199</sup>. Hemicellulose, amorphous and surrounding cellulose fibrils, adds flexibility and toughness, absorbing energy and dampening mechanical stresses <sup>53,54</sup>. Lignin, despite its minor weight, significantly enhances mechanical properties by acting as a binder. Its amorphous, cross-linked structure reinforces the cellulose-hemicellulose matrix, enhancing compression strength and impact resistance. The presence of lignin reduces the susceptibility of Lignocellulosic biomass to deformation under compressive loads and increases their ability to absorb energy during impact events <sup>88,153,200</sup>.
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The thermal properties of LCB, including thermal conductivity and specific heat are vital for understanding biomass combustion behavior and large-scale power generation modeling ¹⁹⁷. Mason et al. ²⁰¹ conducted evaluations, wood pellets and exhibited thermal conductivity values ranging between 0.11 and 0.12 W/mK, while miscanthus and willow were slightly lower about 0.11 and 0.10 W/mK, respectively. In contrast, wheat straw and rape straw may have significantly lower thermal conductivity values, potentially as low as 0.05 W/mK. Specific heat quantifies the heat required to raise the temperature of a substance by one degree Celsius per unit mass, crucial for understanding heat transport in biomass particles and their size-related implications for processing and utilization ¹⁰².

Regarding the chemical characteristics of LCB, proximate and ultimate analyses are common methods employed to chemically characterize such biomass ²⁰². Proximate analysis provides an overview of biomass composition, yielding information on easily measured parameters. This analysis typically determines volatile matter, ash content, moisture content, and fixed carbon content using standardized methods, such as E 872 and D 1102 for wood fuels, and E 871 for wood fuels, as well as by difference ¹¹⁴. Volatile matter refers to the gaseous phase formed during thermal degradation, comprising both condensable and non-condensable vapors released upon heating ¹¹⁴. Ash content represents the residue left behind after combustion, typically containing primary components like silica, calcium, and iron oxides ²⁰³. Moisture content indicates the water content present in a biomass sample and is indicative of the mineralized aqueous solution containing various ions or species ²⁰⁴. Fixed carbon is the residual carbon content remaining after volatiles are expelled, calculated by subtracting the sum of moisture and ash content from the total mass ²⁰⁵. Conversely, ultimate (elemental) analysis provides a breakdown of the elemental composition of biomass, including carbon, hydrogen, oxygen, nitrogen, and sulfur contents of the organic coal substance ²⁰⁶. This analysis is typically performed using specialized laboratory equipment such as carbon, hydrogen, nitrogen, sulfur (CHNS) analyzers ²⁰⁶. Additionally, the macromolecular content of biomass can be determined using a biomass parietal composition analysis, commonly assessed using the Van Soest protocol. This method involves sequential partitioning of cell walls, extracting soluble components with various solvents to evaluate parietal polymer content ²⁰⁷. In addition, LCB and their derivatives possess a plethora of intriguing properties, including high surface area, diverse chemical functionalities, intricate macromolecular structure, biodegradability, high porosity, ultrafine network structure, favorable mechanical properties, tunable biocompatibility, and suitability for permselective coating and immobilization matrices. These materials also exhibit high adsorption capacity and numerous surface-active functional groups such as hydroxyl (OH), amino (NH₂), ether (C-O), carbonyl (C=O), and carboxyl (COOH) groups ¹²,²⁰⁸⁻²¹¹. Furthermore, LCB exhibit diverse chemical properties impacting their reactivity and susceptibility to degradation processes. Cellulose, the dominant structural component, resists chemical attack due to its crystalline structure and strong intermolecular hydrogen bonding, while hemicellulose, although a polysaccharide like cellulose, is more susceptibile to hydrolysis owing to its amorphous nature and accessible glycosidic bonds ¹⁴,⁴³,⁵³. Lignin, the complex aromatic polymer, presents challenges due to its highly cross-linked structure and chemical composition, necessitating specialized processes for removal or modifications such as alkaline delignification or enzymatic breakdown, to effectively depolymerize and extract it from lignocellulosic biomass ²¹²,²¹³.

LCB interacts intricately and dynamic with microorganisms and enzymes in various biological processes, from natural decomposition to controlled biotechnological applications such as composting and enzymatic hydrolysis ⁹⁶,¹⁵⁰,¹⁷⁴. These interactions are pivotal for converting lignocellulosic biomass into valuable industrial and environmental products. Microorganisms, including fungi, bacteria, and microbial
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consortia, produce lignocellulases to breakdown cellulose, hemicellulose, and lignin<sup>174</sup>. Cellulases break the β-1,4-glycosidic bonds in cellulose<sup>178</sup>, while hemicellulases act on the diverse polysaccharide structures of hemicellulose<sup>214</sup>. Ligninases, like lignin peroxidases, manganese peroxidases, and laccases, depolymerize lignin, releasing aromatic compounds and facilitating lignin degradation<sup>215</sup>. Factors like lignin content, composition, and structure influence microbial and enzymatic degradation accessibility<sup>213</sup>.

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### 2.3.2. Lignocellulosic biomass for (bio)sensor applications

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LCB sourced from plants, composed primarily of cellulose, hemicellulose, and lignin, hold immense potential for (bio)sensing technologies due to their renewable nature and unique properties<sup>12,13</sup>. These materials offer biocompatibility, sustainability, and versatility, making them attractive for diverse sensing platforms. LCB are inherently biocompatible and non-toxic, ideal for use in biological sensing, with minimal risk of adverse reactions or contamination<sup>12,216</sup>. They are renewable and biodegradable, offering environmental sustainability compared to traditional sensing materials derived from fossil fuels or non-renewable materials<sup>210,217</sup>. Moreover, the surface chemistry of LCB can be easily modified by chemical modification, surface grafting, or bioconjugation, to introduce functional groups or biomolecules, enabling tailored sensing applications. In addition, the optical properties of biomass, such as birefringence and transparency, enable its use in developing optical sensors based on changes in light transmission or polarization<sup>218,219</sup>. LCB can also exhibit photoluminescence or fluorescence properties, enabling fluorescence-based sensing approaches for analyte detection<sup>220,221</sup>. Recent studies have demonstrated methods to impart electrical conductivity or semiconducting behavior to LCB, offering opportunities for electrochemical or field-effect transistor-based sensing platforms<sup>14,31,222</sup>. Additionally, these materials have shown promise for applications such as wearable sensors<sup>223</sup>, point-of-care diagnostics<sup>224</sup>, or environmental monitoring<sup>12,225</sup>. Additionally, LCB can be processed into various forms, including films<sup>14</sup>, fibers<sup>12,226</sup>, membranes<sup>227,228</sup>, and aerogels<sup>217,223</sup>, offering flexibility and adaptability for sensing applications. Flexible and lightweight lignocellulosic sensors can conform to irregular surfaces or integrate into wearable devices for continuous monitoring. Their porous nature allows for enhanced sensitivity and selectivity by encapsulation or immobilization of sensing elements<sup>229-231</sup>.

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(Bio)sensor are analytical tools that employs sensing element (catalysts) or s biologically derived recognition systems, coupled with a transducer, to measure target analyte concentrations by converting biochemical reactions into a quantifiable physical signal. Electrochemical methods and (bio)sensors are highly sought after due to their simplicity, high sensitivity, selectivity, rapid response time, and versatility for detecting various chemical compounds<sup>11,232</sup>. Sensors selectivity and sensitivity rely on the properties of the receptor elements where the analyte interacts. Achieving high sensitivity requires effective analyte recognition and efficient signal transduction<sup>233,234</sup>. Sensors are categorized based on the physical quantity or analyte they measure, including energy source (active/passive), physical contact (contact/non-contact), comparability (absolute/relative), analog/digital, and signal detection (physical, chemical, thermal, biological) (Figure 3A)<sup>8</sup>. (Bio)sensors integrate biological elements with electronic components include analytes, bioreceptors, transducers, electronics, and displays, to detect physiological changes or chemical/biological materials. They can detect low concentrations of specific pathogens, toxic chemicals, or pH levels finding applications in medical diagnosis, environmental monitoring, and food/drug analysis (Figure 3B)<sup>8</sup>.

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LCB could enhance the selectivity and sensitivity of electrochemical sensors or biosensors as chemical surface modifiers. This is attributed to their high surface area, chemical functionalities,
732 macromolecular structure, biodegradability, high porosity, ultrafine network structure, renewability, good
733 mechanical properties, and permselective coating properties <sup>29,35,235</sup>. Additionally, LCB could serve as
734 suitable supports for biological molecules like enzymes <sup>229,230</sup> and as biotemplates/scaffolds for
735 immobilizing guest materials such as nanoparticles and fluorescence-labeled compounds <sup>220,231</sup>. They are
736 also used in the fabrication of bio-based organic/inorganic hybrid nanocomposites, exhibiting exceptional
737 sensing properties <sup>224,231</sup>. Despite their attractive properties, the hybrid complex microstructure of native
738 LCB remains underutilized, particularly in thin film electrode device development.

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 3:** (A) Sensors classification based on measurand, energy source, physical contact, signal conversion, output, comparability, sensor material, specifications, and applications. (B) Schematic representation of a typical biosensor.
Reproduced with permission from Ref. ⁸, Copyright © 2021 MDPI.</figcaption></figure>

Recent advancements in scientific literature highlighted the utilization of Lignocellulosic biomass for electrochemical sensing applications. For instance, Njine-Bememba et al. ²³⁶ employed aminated pretreated Ayous sawdust functionalized with two organosilanes for the analysis of electrochemical probes ([Fe(CN)₆]³⁻ and [Ru(NH₃)₆]³⁺) via cyclic voltammetry (CV). The presence of silanes facilitated the

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gradual accumulation of anionic species, while repelling cationic species were repelled as the solution was acidified (Figures 4A and 4B) ²³⁶. Kenne et al. ³² fabricated thin film electrodes using native and NaOH-treated Ayous (*Triplochiton scleroxylon*) sawdusts, resulting in enhanced selective detection and electroanalysis of paraquat compared to bare glassy carbon electrodes (GCE) (Figure 4C) ³². Following optimization, detection limits (based on signal-to-noise (S/N) ratio of 3) for GCE modified by natural and NaOH-treated Ayous sawdusts were 5.49×10⁻⁹ mol/L and 3.02×10⁻⁹ mol/L, respectively ³². Furthermore, alkaloid extracts from *Rauvolfia macrophylla Stapf* and *Tabernaemontana contorta* were investigated for their corrosion inhibition properties on C38 steel in 1 M HCl and 0.5 M H₂SO₄ using electrochemical techniques such as electrochemical impedance, potentiodynamic polarization, and mass loss measurements ²³⁷,²³⁸. Field-emission scanning electron microscopy (FESEM) images showed a smooth surface before immersion (Figure 4Da). After immersion in 1 M HCl and 0.5 M H₂SO₄ without inhibitor, severe damage occurred, with iron clusters and punctures (Figure 4Db,c), due to iron oxidation. In the presence of alkaloid extract from *Rauvolfia macrophylla* Stapf (AERMS) as shown in Figure 4Dc-e, the steel surface remained rust-free, indicating effective corrosion prevention. These studies demonstrated the effective corrosion inhibition properties of steel surfaces in the presence of alkaloid extracts, attributed to their spontaneous chemical adsorption on the electrode surface, forming a stable layer that enhanced the sustainability of C38 steel in acidic environments ²³⁷,²³⁸. Two recent additional studies have explored coffee husks as lignocellulosic matrices to enhance sensor sensitivity for the electrochemical quantification of organic substances. Njanja et al. ²²⁵ elaborated a lignocellulose-based amperometric sensor, employing coffee husks as modifiers to prepare a composite carbon paste electrode (CPE) for methylene blue dye electroanalysis. The sensor exhibited a linear concentration range for methylene blue dye between 1 and 125 µmol/L, with a detection limit of 3 µM ²²⁵. Mbokou and coworkers ²³⁹ evaluated the electroanalytical performance of a CPE-modified with coffee husks for acetaminophen quantification in commercialized pharmaceutical tablets. The developed sensor exhibited higher sensitivity with a linear response from 6.6 µM to 0.5 mM and a detection limit of 0.66 µM (S/N=3) ²³⁹. The proposed sensor successfully determined the amount of acetaminophen in commercialized tablets (Doliprane 500 and Doliprane 1000), yielding a recovery rate ranging from 98% to 103% ²³⁹. Additionally, Radotić et al. ²⁴⁰ investigated the use of maize cell wall as modifiers of a CPE for heavy metals electroanalysis, showing good performance under analytical conditions. Kuan et al. ²⁴¹ developed lignocellulose-based analytical devices for bioanalysis, focusing on applications in food and water safety, and urinalysis. They used bamboo stirrers as fluidic channels to transport analytes and a small piece of wood stirrer as a reaction zone. This approach facilitated the creation of point-of-care diagnostics based on bamboo, achieving sensitivity and providing on-site, rapid detection of Escherichia coli (E. coli) in water ²⁴¹. Furthermore, Zhang et al. ²⁴² doped lignocellulose with nitrogen to prepare binary composites modified with metal sulfides for the non-enzymatic sensing of ascorbic acid, dopamine, and nitrite. The fabricated electrodes demonstrated excellent electrocatalytic activity toward ascorbic acid (AA), dopamine (DA), and nitrite, along with good sensitivity, a wide linear range, and low detection limits of 3.0 µM for AA, 0.25 µM for DA, and 0.20 µM for nitrite ²⁴².
<figure><img src="image_7.png" /><figcaption>B)</figcaption></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>D)</figcaption></figure>

<figure><img src="image_12.png" /></figure>

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786 **Figure 4:** (A) Cyclic voltammograms at equilibrium of [Ru(NH<sub>3</sub>)<sub>6</sub>]<sup>3+</sup> at a concentration of 2×10<sup>-4</sup> mol L<sup>-1</sup> recorded at various pH levels (a) 5.5, (b) 3, (c) 2, and (d) 1 on both nude and modified glassy carbon electrodes (GCE) in 0.1 mol L<sup>-1</sup> KCl at a scan rate of 50 mV s<sup>-1</sup> <sup>236</sup>, Copyright © 2015 De Gruyter. (B) Cyclic voltammograms at equilibrium of [Fe(CN)<sub>6</sub>]<sup>3-</sup> at a concentration of 10<sup>-3</sup> mol L<sup>-1</sup> recorded at various pH levels (a) 5.5, (b) 3, (c) 2, and (d) 1 on both nude and modified GCE electrodes in 0.1 mol L<sup>-1</sup> KCl at a scan rate of 50 mV s<sup>-1</sup>. Reproduced with permission from Ref. <sup>236</sup>, Copyright © 2015 De Gruyter. (C) Cyclic voltammograms of paraquat at a concentration of 5×10<sup>-5</sup> mol L<sup>-1</sup> recorded in 0.1 mol L<sup>-1</sup> NaCl at a scan rate of 100 mV s<sup>-1</sup> on (a) bare glassy carbon (BGC), (b) the pristine sawdust thin film electrode (PSTFE), and (c) that issued from sodium hydroxide treated sawdust (SSTFE) electrodes. Reproduced with permission from Ref. <sup>32</sup>, Copyright © 2012 Elsevier. (D) Field-emission scanning electron microscopy (FESEM) micrographs of C38 steel: (a) freshly polished surface, (b) after 3 h immersion in 1 M HCl, (c) after 3 h immersion in 1 M HCl with 100 mg/L of alkaloid extract from *Rauvolfia macrophylla* stapf (AERMS), (d) after 3 h immersion in 0.5 M H<sub>2</sub>SO<sub>4</sub>, and (e) after 3 h immersion in 0.5 M H<sub>2</sub>SO<sub>4</sub> with 100 mg/L AERMS. Reproduced with permission from Ref. <sup>238</sup>, Copyright © 2019 American Chemical Society.

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Several studies have explored biomass calcination and activated carbon for sensing applications. Veeramani et al <sup>222</sup>. utilized *Bougainvillea spectabilis* flowers as precursors to fabricate porous activated carbon (AC), later combined with graphene sheets to form graphene sheet-porous activated carbon (GPAC). GPAC showed remarkable properties for supercapacitors and sensitivity (7.2 µA/µM/cm<sup>2</sup>) in detecting catechin (Figure 5A), with a broad linear range (4–368 µM), and a low limit of detection (0.67 µM) (Figure 5B) <sup>222</sup>.
<figure><img src="image_13.png" /><figcaption>**Figure 5:** (A) Schematic illustration of GPAC synthesis and its applications as an electrode material for supercapacitors and catechin sensor. (B) (a) Cyclic voltammetry (CV) profiles for bare (a'), AC-700 (b'), GPAC (c'), and AC-900 (d') GCEs with 240 mM CA, alongside a blank measurement (e'), at a scan rate of 50 mV s⁻¹. (b) CV curves at varying scan rates (50–500 mV s⁻¹) for GPAC-modified GCE, with the inset depicting peak current vs. square root of the scan rate. (c) pH studies ranging from pH 4 to 10 for the GPAC electrode. Inset (i): peak current vs. pH, and inset (ii): correlation between peak potential (E₀) and pH. (d) DPV curves for various CA concentrations over GPAC-modified GCE, with the inset showing anodic oxidation peak current (Ipa) vs. CA concentration (mM). Electrolyte solution: 0.05 M PBS (pH 7.0); scan rate: 50 mV s⁻¹. Reproduced with permission from Ref. ²², Copyright © 2017 Royal Society of Chemistry.</figcaption></figure>

Kim et al. ²¹⁶ developed a two-step procedure to prepare biomass-derived carbon for modifying GCEto accurately quantify acetaminophen. Using CV and differential pulse voltammetry (DPV), the modified electrode exhibited high sensitivity, selectivity, and a low detection limit of 0.004 µM for acetaminophen. Evaluation of these sensors in a real samples confirmed its superior electrochemical performance ²¹⁶. Similarly, Chen et al. ²⁴³ synthesized biomass-derived N-doped porous carbon-based sensors for the simultaneous determination of dihydroxybenzene isomers using pig lung as a biomass
precursor, achieving detection limits of 0.078, 0.057, and 0.371 µmol/L for catechol, resorcinol, and hydroquinone respectively<sup>243</sup>. In addition, Somba et al.<sup>13</sup> prepared alkali activated carbon based theobroma cocoa pods LCB as sensing plateform for the electrochemical determination of Xanthine (Xa) in fresh fish sample. The electrodes were used through DPV to optimize the different parameters that can affect the determination of Xa and a calibration curve has been plotted in the range from 1.0 to 12.0 µM, leading to a detection limit of 0.264 µM. The interference, reproducibility, and stability were evaluated and the sensor was successfully applied in fresh fish sample <sup>13</sup>. In addition, Biomass has proven to be an effective material for enzyme immobilization in electrochemical sensors, offering a sustainable and efficient platform for biosensor development <sup>11,244</sup>. Deffo et al.<sup>11</sup> leveraged this potential by developing a second-generation uric acid biosensor, chemically immobilizing the enzyme uricase on functionalized multiwalled carbon nanotubes grafted onto lignocellulosic material (LCB) derived from palm oil fiber, using a ferrocene mediator <sup>11</sup>. Differential pulse voltammetry (DPV) results demonstrated a rapid response of the bioelectrode toward uric acid (0.185 V), with high sensitivity (41.14 µA/mM) and a low detection limit (19 µM) within a linear range of 10–1000 µM. The low Michaelis–Menten constant (Km = 31.364 µM) indicated a high affinity between uric acid and the enzyme at the electrode surface. Furthermore, the biosensor detected uric acid in just 2 s, a fast response attributed to the advantageous characteristics of the LCB <sup>11</sup>. Moreover, numerous studies have explored the calcination of LCB and the successful use of resulting activated carbons as electrode modifiers for sensitive pollutant detection in various matrices <sup>245-249</sup>.

LCB are also employed as enzymes sources for electrochemical catalysis of organic molecules in enzymatic biosensors. Fatibello-Filho et al. <sup>250</sup> used chronoamperometry to determine paracetamol using an avocado tissue (*Persea americana*) biosensor. The dry avocado tissue powder provided polyphenol oxidase (E.C.1.14.18.1), catalyzing the oxidation of paracetamol to N-acetyl-p-benzoquinoneimine. The resulting currents obtained at 70 s were proportional to the paracetamol concentration (1.2×10<sup>-4</sup> to 5.8×10<sup>-3</sup> mol/L) with a detection limit of 8.8×10<sup>-5</sup> mol/L <sup>250</sup>. Similarly, Cruz et al. <sup>251</sup> used sweet potato tissue peroxidase to determine hydroquinone in cosmetic creams. The enzyme catalyzed the oxidation of hydroquinone to quinone, reduced electrochemically back to hydroquinone at-0.22 V <sup>251</sup>. Another biosensor based on stearic acid-graphite powder and sweet potato (*Ipomoea batatas* (L.) Lam.) tissue peroxidase showed high catalytic performanceand compatibility with non-aqueous solvents and long lifetime <sup>252</sup>. A summary of recent bio(sensing) research studies based on LCB is provided in Table 2. In recent years, research on Lignocellulosic biomass for electrochemical applications has demonstrated their potential in developing various sensors with notable performance characteristics. For instance, the pristine sawdust thin film electrode (PSTFE) was employed to detect paraquat using square wave voltammetry (SWV), achieving a linear range of 0.1 to 0.725 µM and a detection limit of 0.005 µM, showcasing its high sensitivity <sup>32</sup>. Meanwhile, sodium hydroxide treated sawdust thin film electrode (SSTFE) extended the applications of Lignocellulosic biomass by detecting similar analytes with improved performance <sup>32</sup>. Coffee husk-based carbon paste electrodes (CHs/CPE) were successfully used to detect methylene blue and acetaminophen, with the former being detected in river water with a linear range of 1 to 125 µM and a detection limit of 3 µM <sup>225</sup>, while the latter was detectable in commercial tablets with a linear range of 6.6 to 500 µM and a detection limit of 0.66 µM <sup>239</sup>. Additionally, cellulose-based carbon paste electrodes (C/CPE) were utilized for lead ion (Pb<sup>2+</sup>) detection in tap water with a remarkable detection limit of 0.02 µM within a linear range of 0.1 to 100 µM <sup>240</sup>. On the other hand, the nitrogen-doped lignocellulose with CoS<sub>2</sub>-MoS<sub>2</sub> composite (N-LC/CoS<sub>2</sub>-MoS<sub>2</sub>/GCE) displayed a broad range for
detecting ascorbic acid, dopamine, and nitrite, underscoring its versatile sensing capability<sup>242</sup>. Moreover, graphene sheet-like porous activated carbon (GPAC/GCE) showed effective detection of catechin from green tea leaves, with a detection limit of 0.67 µM within a range of 4 to 368 µM<sup>222</sup>. In contrast, carbon nanospheres (CNSs/GCE) demonstrated superior nitrite detection in lake and sea water with an exceptionally low detection limit of 0.0146 µM<sup>246</sup>. The advancements in electrode materials, including carbon activated with ZnCl<sub>2</sub> (CA-Zn/GCE) and its iron-composite variant (CA-ZnFe/GCE), have also expanded the range of detectable substances such as caffeine and other analytes, illustrating their applicability in diverse matrices<sup>247</sup>. The application of different electrochemical methods, including differential pulse voltammetry (DPV) and chronoamperometry (CA), highlights the evolution of lignocellulosic-based sensors from basic to highly sophisticated detection platforms across various environmental and commercial samples.

<table><caption>**Table 2** : Summary of research studies based on Lignocellulosic biomass as (bio)sensing components in electrochemical applications</caption><thead><tr><th>Electrodes</th><th>Electrochemical methods</th><th>Analytes</th><th>Linear range (µM)</th><th>Detection limit (µM)</th><th>Real medium</th><th>Ref.</th></tr></thead><tbody><tr><td>PSTFE/GCE</td><td rowspan="2">SWV</td><td rowspan="2">Paraquat</td><td>0.1 - 0.725</td><td>0.005</td><td rowspan="2">/</td><td rowspan="2">32</td></tr><tr><td>SSTFE/GCE</td><td>0.05 - 0.6</td><td>0.003</td></tr><tr><td>CHs/CPE</td><td>SWV</td><td>Methylene blue</td><td>1 - 125</td><td>3</td><td>River water</td><td>225</td></tr><tr><td>CHs/CPE</td><td>SWV</td><td>Acetaminophen</td><td>6.6 - 500</td><td>0.66</td><td>Commercialized tablets (Doliprane® 500 and Doliprane 1000)</td><td>239</td></tr><tr><td>C/CPE</td><td rowspan="4">SWV</td><td rowspan="4">Pb<sup>2+</sup></td><td>0.1 - 100</td><td>0.02</td><td rowspan="4">Tap water</td><td rowspan="4">240</td></tr><tr><td rowspan="3">X/CPE</td><td>0.1 - 50</td><td>/</td></tr><tr><td>0.1 - 100</td><td>/</td></tr><tr><td>0.1 - 100</td><td>/</td></tr><tr><td>L/CPE</td><td rowspan="4">Cu<sup>2+</sup></td><td>0.1 - 20</td><td>0.08</td><td rowspan="4"></td><td rowspan="4"></td></tr><tr><td rowspan="3">CW/CPE</td><td>0.1 - 20</td><td>/</td></tr><tr><td>0.1 - 20</td><td>/</td></tr><tr><td>0.1 - 20</td><td>/</td></tr><tr><td rowspan="3">N-LC/CoS<sub>2</sub>-MoS<sub>2</sub>/GCE</td><td rowspan="3">CA</td><td>Ascorbic acid,</td><td>9.9 - 6582</td><td>3.0</td><td rowspan="3">Urine</td><td rowspan="3">242</td></tr><tr><td>Dopamine,</td><td>0.99 - 261.7</td><td>0.25</td></tr><tr><td>Nitrite</td><td>0.5 - 5160</td><td>0.20</td></tr><tr><td>GPAC/GCE</td><td>DPV</td><td>Catechin</td><td>4 - 368</td><td>0.67</td><td>Green tea leaves</td><td>222</td></tr><tr><td>ZKAKC/GCE</td><td>DPV</td><td>Acetaminophe n</td><td>0.01 - 20</td><td>0.004</td><td>Urine</td><td>216</td></tr><tr><td rowspan="3">HPCs/GCE</td><td rowspan="3">DPV</td><td>Catechol</td><td>0.5 - 320</td><td>0.078</td><td rowspan="3">River water</td><td rowspan="3">243</td></tr><tr><td>Resorcinol</td><td>0.5 - 340</td><td>0.057</td></tr><tr><td>Hydroquinone</td><td>1 - 360</td><td>0.371</td></tr><tr><td>N,P-MMC/GCE</td><td>CA</td><td>H<sub>2</sub>O<sub>2</sub></td><td>100 - 10000</td><td>6.8</td><td>/</td><td>253</td></tr><tr><td>CNANAs/GCE</td><td>CA</td><td>H<sub>2</sub>O<sub>2</sub></td><td>5 - 1760</td><td>3.53</td><td>Human urine and serum</td><td>217</td></tr><tr><td>CNSs/GCE</td><td>CA</td><td>Nitrite</td><td>0.2 - 350</td><td>0.0146</td><td>Lake and sea water</td><td>246</td></tr><tr><td>CA-Zn/GCE</td><td rowspan="2">DPV</td><td rowspan="2">Caffeine</td><td>39.8 - 458</td><td>28.5</td><td rowspan="2">Coca-Cola and Pepsi</td><td rowspan="2">247</td></tr><tr><td>CA-ZnFe/GCE</td><td>39.8 - 637</td><td>16.0</td></tr><tr><td>GCE/ACCP</td><td>DPV</td><td>Xanthine</td><td>1.0 - 12.0</td><td>0.264</td><td>Fresh fish</td><td>13</td></tr></tbody></table>
<table><tr><td>VCPTE</td><td>CA</td><td>Acetaminophe n</td><td>120 - 5800</td><td>88</td><td>Resprin, Resfenol, Tylenol, Vick Pyrena.</td><td>250</td></tr><tr><td>PCPET</td><td>CA</td><td>Hydroquinone</td><td>75 - 1600</td><td>8.1</td><td>Cosmetic creams (Clariderm, Claripel)</td><td>251</td></tr><tr><td>StCPET</td><td>CA</td><td>Hydroquinone</td><td>62 - 1500</td><td>8.5</td><td>Cosmetic creams (Clariderm, Claripel)</td><td>252</td></tr><tr><td>UOx/Fc/FMWCNT-POF/Pt</td><td>DPV</td><td>Uric acid</td><td>10 - 1000</td><td>19</td><td>Urine</td><td>11</td></tr></table>

877 GCE: Glassy carbon electrode; PSTFE: pristine sawdust thin film electrode; SSTFE: sodium hydroxide treated sawdust thin film electrode; 878 SWV: square wave voltammetry; CHs: coffee husks; CPE: carbon paste electrode; C: cellulose, X: xylan, L: lignin; CW: cell wall; CoS₂-MoS₂: 879 binary metal sulfide composite; N-LC: N-doped lignocellulose; CA: chronoamperometry; GPAC: graphene sheet-like porous activated carbon; 880 DPV: differential pulse voltammetry; ZKAKC: ZnCl₂-KOH activated kelp carbon; HPCs: Nitrogen doped hierarchical porous carbon materials; 881 N,P-MMC: Nitrogen and phosphorus co-doped hierarchical micro/mesoporous carbon; CNANAs: carbon nanoballs aggregation networks- 882 based aerogels; CNSs: carbon nanospheres; CA-Zn: carbon activated with ZnCl₂; CA-ZnFe: carbon activated with ZnCl₂:FeCl₃; ACCP: activated 883 carbon cacao pods; VCPTE: vaseline carbon paste tissue electrode; PCPET: paraffingraphite powder modified with sweet potato tissue; 884 StCPET: stearic acid-graphite powder modified with sweet potato tissue; UOx/Fc/FMWCNT-POF/Pt: Uricase coated ferrocene, functionalized 885 multiwalled carbon nanotube and palm oil fiber on bare platinum.

886 Despite significant progress in utilizing LCB for sensing applications, several challenges hinder 887 their widespread. One key issue is optimizing material properties and fabrication techniques to improve 888 sensor performance metrics such as sensitivity, selectivity, and stability. Achieving optimal sensor 889 performance demands meticulous refinement of Lignocellulosic biomass, including their composition, 890 morphology, and surface chemistry, to ensure enhanced sensing capabilities and reliable detection of 891 target analytes. Additionally, standardizing sensing protocols and validation procedures in real-world 892 environments is crucial to ensure the efficacy and reliability of lignocellulosic-based sensors. Robust 893 validation protocols are indispensable for establishing the accuracy, reproducibility, and consistency of 894 sensor responses under diverse operating conditions and environmental contexts, thereby bolstering 895 confidence in their utility for practical applications. Scalability and cost-effectiveness in sensor fabrication 896 also pose significant challenges, particularly for large-scale deployment in industrial and commercial 897 settings. Overcoming these barriers necessitates the developing scalable manufacturing processes and 898 cost-efficient production methodologies to meet mass production demands while maintaining 899 performance and quality standards. Addressing these challenges requires interdisciplinary collaboration 900 across materials science, chemistry, biology, and engineering. By fostering such collaborations, scientists 901 and researchers can drive innovation and develop advanced sensor platforms that harness the unique 902 properties of LCB, leading to significant advancements in sensing technologies with applications in 903 healthcare, environmental monitoring, and beyond.

## 2.4. Nanocellulose based sensing platforms

### 2.4.1. Nanocellulose microstructure and preparation

906 Cellulose, the most abundant renewable polymer on Earth, was first isolated from plant material 907 by Anselme Payen in 1838. Payen discovered that cellulose comprises approximately 40 to 45% carbon, 908 6 to 6.5% hydrogen, and the remainder oxygen. Its annual production is estimated at 5 × 10¹¹ metric tons 909 ²⁵⁴. Cellulose can be sourced from plants, animals, algae, and fungi, but most comes from plant fibers
43,68. Nanocelluloses are nanoscale rod-like or fibrillar elements of cellulose, measuring 100 nm or less in one dimension. Cellulose nanofibers (CNFs) are the smallest cellulose building blocks, forming the elementary fibril composed of numerous cellulose chains. CNFs typically exhibit diameters ranging from 5 to 50 nm and lengths of several micrometers, characterized by a high aspect ratio 255,256. Cellulose, a ubiquitous polysaccharide in plant cell walls, consists of repeating glucose units linked by β-1,4-glycosidic bonds, with the chemical formula (C6H10O5)n (Figure 6a). Its exhibits a hierarchical structure with both crystalline and amorphous regions. The crystalline regions are characterized by highly ordered arrangements of cellulose chains, stabilized by extensive hydrogen bonding between adjacent chains 43,257. These regions contribute to the exceptional mechanical strength and stiffness of cellulose. These regions form microfibrils or elementary fibrils with parallel cellulose chains 43,258. The amorphous regions, lacking long-range order, are more accessible to solvents and chemical reagents due to their less rigid structure. Amorphous regions play a crucial role in cellulose overall flexibility and susceptibility to degradation processes 178,198. The ratio of crystalline to amorphous regions varies depending on factors such as the source of cellulose, processing methods, and environmental conditions (Figure 6b) 46. Cellulose from different plant sources or subjected to different treatments may exhibit varying degrees of crystallinity, which in turn impacts its properties and applications 46,156,158.

<figure><img src="image_14.png" /><figcaption>**Figure 6:** (a) Schematic representation of the linear structure of cellulose, highlighting the presence of three hydroxyl (–OH) groups located at the C2, C3, and C6 atoms of each β-D-glucopyranose unit. (b) Composite depiction of lignocellulosic biomass, illustrating the coexistence of amorphous and crystalline regions within its structure. Reproduced with permission from Ref. 259, Copyright © 2017 Elsevier.</figcaption></figure>

Understanding cellulose structure across multiple scales is crucial for elucidating its properties and applications. At the molecular level, cellulose molecules consist of glucose units linked by β-1,4-glycosidic bonds, resulting in linear chains with extended, rigid structures. These chains can adopt different conformations, such as extended or folded, influencing the overall properties of cellulose (Figure 7A) 260. Intermolecularly, cellulose molecules interact primarily through hydrogen bonding, Van der Waals forces,
and hydrophobic interactions, forming highly organized arrays within fibrils. These fibrils exhibit high tensile strength and stiffness. Interfacial interactions between cellulose fibrils and other molecules, such as hemicelluloses and lignin, influence composite materials properties <sup>261</sup>. Additionally, interfibril interactions involve fibrils aggregation twist and bundle into fibers, (Figure 7A), contributing to the hierarchical organization in materials like paper. Understanding these interactions is crucial for designing and engineering cellulose-based materials with tailored mechanical, optical, and chemical properties <sup>262</sup>.

The crystal structure of cellulose is defined by glucose monomers linked by β-1,4-glycosidic bonds. In cellulose I and cellulose II, these glucose units form extended linear chains, with each glucose unit rotated approximately 180 degrees relative to its neighboring, allowing hydrogen bonds between adjacent hydroxyl (-OH) groups <sup>261</sup>. This hydrogen bonding imparts stability and rigidity to the cellulose structure. In the solid state, AGU units undergo a 180° rotation due to β-linkages. Each AGU unit has three hydroxyl (OH) groups at positions C-2, C-3, and C-6. The terminal groups of the cellulose molecule differ: the C-1 OH group at one end acts as an aldehyde group with reducing activity, while, the C-4 OH group at the opposite end serves as an alcohol group, termed the non-reducing end <sup>43,257,263</sup>. The cellobiose monomer, AGU, has three -OH groups that form strong hydrogen bonds with adjacent glucose units within the same chain and different chains, creating intramolecular and intermolecular hydrogen bonding networks, respectively. These structural features contribute to the remarkable properties and functionalities of cellulose and its nanoscale derivatives <sup>264,265</sup>.

Hydrogen bonding in cellulose is fundamental to its structural integrity and properties. In cellulose I, hydrogen bonds form between hydroxyl groups (-OH) on neighboring glucose units along the polymer chain occurring between the oxygen atom of one glucose unit and the hydrogen atom attached to the hydroxyl group of an adjacent glucose unit <sup>261,266</sup>. The orientation of hydroxymethyl groups (-CH₂OH) alternates along the polymer chain, creating distinct hydrogen bonding patterns <sup>261-263</sup>. In cellulose II, the hydrogen bonding arrangement differs slightly. While the basic principle of hydrogen bonding between hydroxyl groups remains, hydroxyl groups at positions 2 (OH2) and 6 (OH6) can interchangeably act as hydrogen bond donors or acceptors, depending on the orientation of neighboring glucose units (Figure 7B) <sup>262</sup>.
<figure><img src="image_15.png" /><figcaption>**Figure 7:** (A) Representation of the structure of cellulose across multiple length scales and organizational levels. Dihedral angles $\Phi$ and $\Psi$ are defined by the atomic sequences O5'-C1'-O4-C4 and C1'-O4-C4-C3, respectively. (B) Depiction of the hydrogen bonding system in two cellulose allomorphs, cellulose I and cellulose II. The orientation of hydroxymethyl groups is emphasized, referred to as *tg* in cellulose I and *gt* in cellulose II. Reproduced with permission from Ref. ²⁶², Copyright © 2022 Springer Nature.</figcaption></figure>

The hierarchical organization of cellulose within LCB spans multiple length scales, from the molecular to the macroscopic level, each contributing distinct properties. At the nanoscale, cellulose molecules form microfibrils, which are long thread-like structures composed of linear chains, typically a few to tens of nanometers wide and extending over several micrometers ⁴³,²⁶⁷. These microfibrils have crystalline and amorphous regions, aggregating into larger fibrils with diameters from tens to hundreds of nanometers. Fibrils further organize into cellulose fibers, the building blocks of LCB such as wood, plant stems, and fibers ²⁶⁷,²⁶⁸. At higher levels, cellulose fibers form intricate networks within the lignocellulosic matrix, reinforced by interactions with hemicellulose and lignin, which provide structural
support and stability. The precise arrangement varies with plant species, tissue type, and growth conditions⁴⁶. The hierarchical organization is crucial for the mechanical strength, rigidity, and overall properties of LCB, allowing, researchers to tailor LCB for applications in construction materials, textiles, biofuels, and biomedical devices. Within cellulose fibrils, there exist regions where the cellulose chains exhibit a highly organized (crystalline) structure, alongside regions characterized by disorderliness (amorphous-like) ²⁵⁷,²⁶⁹. This arrangement is depicted in Figure 8. The structure of nanocellulose is governed by intra- and inter-chain hydrogen bonding, as well as Van der Waals forces. These interactions contribute to the linear-chain configuration of cellulose, the stability of glycosidic linkage motifs, and its limited solubility in polar solvents ²⁵⁷,²⁷⁰.

<figure><img src="image_16.png" /><figcaption>**Figure 8:** Schematics of single cellulose chain repeat unit, showing the directionality of the 1→4 linkage and inter-/intrachain hydrogen bonding showing configurations of the crystalline and amorphous regions. The scheme also exhibits schematic procedure (from the top to down) of dilute acid pretreatment process to isolate the crystalline (ordered) domains of cellulose from amorphous regions. Reproduced with permission from Ref. ²⁷⁰, Copyright © 2019 Elsevier.</figcaption></figure>

In LCB, cellulose polymers adopt an elongated, rigid rod-like conformation, facilitated by the equatorial arrangement of glucose residues. These residues interact through hydrogen bonds, forming microfibrils that give cellulose exceptional tensile strength ⁷⁶. The properties of cellulose are largely dependent on its degree of polymerization. Cellulose from wood pulp typically exhibits chain lengths of 300 to 1700 units, while cotton, other plant fibers, and bacterial cellulose can range from 800 to 10,000 units ²⁷¹. Within plant cell walls, cellulose fibers form aggregate of microfibrils, with intermolecular hydrogen bonds between adjacent molecules leading to robust adhesion and the formation of overlapping parallel arrays of about 40 cellulose chains with the same polarity ²⁷².

Nanocellulose, including cellulose nanofibrils (CNF), cellulose nanocrystals (CNC), and bacterial nanocellulose (BNC), exhibits remarkable structural characteristics and diverse applications ⁴³. CNF consist of fibrous nanoparticles with diameters typically ranging from a few to tens of nanometers and lengths extending into the micrometer scale. Derived through mechanical or enzymatic disintegration of cellulose fibers, CNF is a hierarchical network with high surface area-to-volume ratios, providing exceptional mechanical properties like high tensile strength and modulus, flexibility, and transparency
1005 178,198. CNC comprises rod-shaped nanoparticles with diameters of 5 to 20 nm and lengths of hundreds of nm. These are obtained through acid hydrolysis or enzymatic treatment of cellulose fibers, which selectively removes amorphous regions, isolating crystalline cellulose domains 273,274. The surface of CNC is adorned with hydroxyl groups, rendering them highly hydrophilic and facilitating functionalization for various applications 274. BNC produced by bacteria, such as Acetobacter xylinum, has distinct microstructural features. Synthesized through the extracellular polymerization of glucose units, BNC consists of ultrafine nanofibers with diameters of 20 to 100 nanometers and lengths of several micrometers 275. BNC forms a highly porous and interconnected network of nanofibrils, forming a three-dimensional scaffold with exceptional mechanical properties, high surface area, and water-holding capacity. BNC exhibits biocompatibility, biodegradability, and transparency, making it well-suited for applications in biomedical engineering and food packaging 275-277.

1016 Each type of nanocellulose, with its unique features, plays a crucial role in various applications. CNC, also known as cellulose whiskers, cellulose nanowhiskers, cellulose microfibrils, microcrystalline cellulose, and nanocrystalline cellulose, exhibits a needle-shaped morphology with dimensions typically ranging from 100 to 200 nm in length and 5 to 20 nm in diameter 158,258,273. These nanocrystals are characterized by high crystallinity and exceptional strength, with a modulus of elasticity (E) up to 150 GPa. CNCs can form liquid crystal suspensions, making them suitable for applications in materials science, such as reinforcement agents in nanocomposites 278. Additionally, microfibrillated cellulose (MFC) refers to cellulose fibrils obtained by mechanically shredding wood fibers into much smaller components than the original fibers. For context, while manufacturing pulp from a tree with a trunk diameter of approximately 40 cm, the resulting pulp fiber is around 40 µm 279-281. Wood consists of a cellulose matrix with fibers forming this matrix made up of fibril bundles that consist of smaller elements known as microfibrils. Through fibrillation, cellulose fibers are separated into a three-dimensional network of microfibrils, significantly increasing the surface area for interactions 267,268. These entangled fibrils constitute MFC, suitable for a wide range of applications, in papermaking, rheology modifiers, and biocompatible materials 272,280.

1031 Wood generally serves as a representative type of biomass providing a detailed example of the structural and hierarchical organization found in biological materials as well as their macromolecular composition 282. The hierarchical and porous structure of wood spans multiple length scales, from the molecular level to the macroscopic scale of the wood stem (Figure 9a). At the microscale, wood exhibits a hierarchical porous structure characterized by hollow channels aligned longitudinally. These channels are surrounded by the secondary walls of individual wood cells, which are composed of three distinct layers: S1, S2, and S3 283,284. The interior lumen of the cell, or the void space at the center, is encased by a rigid cell wall formed from three primary biopolymers namely cellulose, hemicellulose and lignin. These components combine to create a natural fibril-based composite with properties heavily influenced by the orientation of aligned cellulose fibrils embedded within the lignin and hemicellulose matrix (Figure 9b) 283,284.

1042 The cell wall structure consists of bundles of cellulose microfibrils that include both crystalline and amorphous regions. These microfibrils are comprised of elementary fibrils with diameters of approximately 3 nm 285. The cellulose chains are formed by covalent bonding of repeating D-glucose units, which are stabilized by intrachain and interchain hydrogen bonds, resulting in stiff and linear cellulose chains 43. At the cellular and tissue levels, the composition of wood varies depending on the tree species. In softwoods, over 90% of the cells are tracheids, which differ in diameter and wall thickness according to
1048 to their function. Larger-diameter tracheids with thinner walls are adapted for water transport (referred
to as ‘earlywood’), while smaller-diameter tracheids with thicker walls provide mechanical strength
1049 (known as ‘latewood’) 283,284,286,287. Conversely, hardwoods, which evolved later, have specialized cells such
1050 as vessels for water transport and fibers for mechanical strength, giving them a competitive edge in
1051 various climates 284,288.
1052

1053 The hierarchical porous structure of wood is further defined by its lumina (the open interiors of
1054 the cells), which vary in diameter from a few micrometers to about half a millimeter. These lumina, along
1055 with the micrometer-sized pits traversing the cell walls and the nanometer-sized pores between cellulose
1056 fibrils within the walls, contribute to the intricate porous network of wood (Figure 9) 283,284. The structural
1057 anisotropy of wood is marked by the directionality of its channels and cellulose fibrils across different
1058 scales (Figure 9c) 284. Microscale features include the alignment of lumina in vessels, tracheids, and fibers
1059 along the longitudinal direction. At the nanoscale, cellulose nanofibrils within the cell walls are oriented
1060 parallel to each other at specific angles relative to the cell axis, typically ranging from 0 to 60 degrees,
1061 depending on the tissue type and layer 284,289. The gaps between these nanofibrils form nanopores with
1062 similar alignments. At the molecular scale, cellulose chains are linearly aligned, contributing to a
1063 hierarchical anisotropic structure with abundant multi-scale pores. This organization facilitates efficient
1064 multiphase transport of ions, molecules, gases, solid particles, and liquids, as well as multispectral energy
1065 transfer, including photons, phonons, microwaves, and acoustic waves. Such structural complexity
1066 endows wood with a range of intriguing properties that are advantageous for various applications 43,284,289.
<figure><img src="image_17.png" /></figure>

## b Composition in cross section and the longitudinal direction

<figure><img src="image_18.png" /></figure>

## c Anisotropy of wood at multiple length scales

<figure><img src="image_19.png" /><figcaption>**Figure 9:** The hierarchical and porous structure of wood across various length scales. (a) Wood exhibits a hierarchical porous architecture with longitudinally aligned hollow channels. The secondary walls of individual wood cells are composed of three</figcaption></figure>
distinct layers (S1, S2, and S3) encasing an interior lumen or void space. The rigid cell walls are primarily made up of cellulose, hemicelluloses, and lignin. (b) Cross-sectional and longitudinal views of the wood cell wall structure show that cellulose microfibrils, embedded within the lignin and hemicellulose matrix, display both crystalline and amorphous regions. (c) The anisotropic nature of wood structure is evident at multiple scales, including the macroscale and microscale orientation of wood cells, the microscale organization of cell walls, the nanoscale alignment of cellulose fibrils, and the molecular-scale arrangement of cellulose chains. Reproduced with permission from Ref. ²⁸⁴, Copyright © 2020 Springer Nature.

The extraction of nanocellulose from LCB is a rapidly growing field, driven by its remarkable properties and vast potential for applications across various industries. Nanocellulose, with its high aspect ratio, biocompatibility, renewability, and remarkable mechanical strength, has garnered significant attention. The extraction process typically involves two steps: pretreatment of biomass and extraction of nanocellulose ¹²⁴,¹⁵⁷,²⁹⁰,²⁹¹. Biomass pretreatment removes non-cellulosic components like lignin, hemicellulose, and various extractives, and enhancing cellulose fiber accessibility. Various methods, including chemical, physical, and biological, are employed ⁶⁶,²⁹². Nanocellulose extraction isolates cellulose nanofibrils or nanocrystals from the cellulose-rich fraction using mechanical, chemical, and biological methods, depending on desired properties ²⁹²,²⁹³. Mechanical methods desintegrate cellulose fibers through high-pressure homogenization, microfluidization, or ultrasonication, yielding CNF ¹²⁴,¹⁷⁸,²⁷⁹. Chemical methods, like acid hydrolysis or enzymatic hydrolysis, remove amorphous regions to produce CNC ²⁹¹,²⁹⁴. Biological methods, such as bacterial fermentation, also yield BNC ²⁹⁵,²⁹⁶.

Figure 10a provides a detailed representation of the structural and macromolecular composition of wood biomass, illustrating the arrangement of key components such as cellulose, hemicellulose, and lignin within the biomass. Figure 10b outlines the commonly employed processes for extracting nanocellulose from cellulosic fibers derived from biomass ²⁹⁷,²⁹⁸. This process begins with the pretreatment of biomass, aimed at removing non-cellulosic components like lignin and hemicellulose, which is essential for isolating the cellulose-rich fibers. After this initial purification step, the cellulosic fibers undergo mechanical treatment to produce cellulose nanofibers (CNFs), which are long, flexible, and highly entangled structures. Following this, acid hydrolysis is applied to the fibers to break them down further and extract cellulose nanocrystals (CNC), which are shorter, rod-like structures with highly crystalline regions. Finally, a chemical treatment is performed to isolate hairy cellulose nanocrystalloids, which are a more chemically complex form of nanocellulose, featuring surface-bound functional groups that can enhance their reactivity and performance in various applications ²⁹⁷,²⁹⁸. Hairy cellulose nanocrystalloids (HCNC) are a novel class of nanocellulose. These nanoparticles possess a unique structure, featuring a crystalline core similar to conventional cellulose nanocrystals (CNC), but with polymer chains protruding from their ends, giving them a “hairy” appearance ²⁹⁹,³⁰⁰. HCNC are produced through a process called periodate oxidation, which creates amorphous regions at the ends of the crystalline core. These amorphous regions can be functionalized with various agents to impart specific properties. This distinct structure allows HCNC to be used in a wide range of applications, such as improving filler retention in papermaking ²⁹⁹,³⁰⁰.
<figure><img src="image_20.png" /><figcaption>**Figure 10:** (a) A detailed depiction of the structural and biomacromolecular composition of wood biomass, highlighting the organization of key components, including cellulose, hemicellulose, and lignin. Reproduced with permission from Ref. ²⁹⁷, Copyright © 2023 American Chemical Society. (b) An overview of the typical processes used to extract nanocellulose from cellulosic fibers in biomass. Reproduced with permission from Ref. ²⁹⁸, Copyright © 2022 MDPI.</figcaption></figure>

CNC and CNF are two types of cellulose nanomaterials, each with unique features and properties. CNC consists of highly crystalline cellulose segments with diameters of 2 to 20 nanometers and length of 100 to 500 nm, giving it a whisker shape and exceptional tensile strength. Despite its remarkable strength, CNC possesses a remarkably low density and high degree of thermal stability, due to the strong intermolecular hydrogen bonding within the crystalline lattice, which imparts structural integrity to the nanocrystals under heat stress (Figure 11A and 11B) ²⁷³,²⁷⁸,²⁹¹. In contrast, CNF exhibit a more fibrous morphology with diameters ranging from a few to tens of nanometers and lengths extending into the micrometer range ¹⁷⁸,¹⁹⁸. While CNF lacks the same level of crystallinity as CNC, it still exhibits considerable
1120 tensile strength and shares its low density and high thermal stability ³⁰¹. Both materials have diverse applications in biomedicine, electronics, composites, and sustainable packaging thanks to their unique properties such as high tensile strength, low density, high thermal stability, and crystallinity (Figures 11A and 11B) ²⁶⁹.
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<figure><img src="image_21.png" /><figcaption>**Figure 11:** (A) Schematic depiction of the production process of cellulose nanofibers (CNF) and cellulose nanocrystals (CNC) derived from fiber cell walls, highlighting key features of CNF and CNC. Reproduced with permission from Ref. ³⁰², Copyright © 2018 Royal Society of Chemistry. (B) Illustration outlining the production pathways of CNFs and CNCs, accompanied by microscopic views showcasing their distinct structures. Reproduced with permission from Ref. ³⁰³, Copyright © 2014 Elsevier.</figcaption></figure>

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1134 BNC stands out as a promising nanomaterial, mainly synthesized by various fermentation bacteria ²⁷⁶,²⁹⁵. While it finds applications in textiles, cosmetics, and food products, its exceptional properties make it particularly promising for medical uses such as wound dressing, tissue engineering, and drug delivery systems. Various bacterial genera, including *Acetobacter*, *Achromobacter*, *Bacillus*, *Sarcina*, *Aerobacter*, *Agrobacterium*, *Escherichia*, *Azotobacter*, *Rhizobium*, *Enterobacter*, *Klebsiella*, *Salmonella*, are capable of producing cellulose, broadening the scope of BNC production ³⁰⁴. The synthesis of bacterial cellulose
typically unfolds through four distinct stages within bacterial cells: activation of monosaccharides, polymerization, addition of acyl groups, and excretion of BNC from the cell ³⁰⁵,³⁰⁶. Two common methods for BNC synthesis involve static culture, preferring for uniform and well-defined BNC structures, ²⁰⁷,²⁰⁸ and stirred culture, offering scalability and versatility but resulting in variable morphology and properties ²⁹⁶,³⁰⁷. The biosynthetic pathway of cellulose involves intricate biochemical reactions and cellular mechanisms, illustrates in Figure 12a.

Bacterial cellulose, synthesized by *Acetobacter xylinus* bacteria, exhibits a unique morphology under SEM, appearing as a dense network nanofiber (Figure 12b). The production process of bacterial cellulose by Acetobacter xylinus involves the bacterial fermentation of glucose, extrusion of cellulose fibrils into the surrounding medium, where they self-assemble and formation of a gel-like pellicle at the air-liquid interface ³⁰⁸. Over time, this pellicle undergoes further cellulose synthesis and maturation, leading to the formation of a robust and highly pure bacterial cellulose membrane (Figures 12a and 12b). This process is influenced by various factors such as temperature, pH, oxygen availability, and nutrient composition, all of which can impact the final morphology and properties of the produced bacterial cellulose ³⁰⁶,³⁰⁸.

Bacterial cellulose possesses properties ideal for biomedical applications, including high purity, tensile strength, and biocompatibility. Its water-holding capacity and permeability make it ideal for absorbent materials like wound dressings, while its nanofibrous structure enhances delivery and efficacy of bioactive compounds (Figures 12c and 12d). Additionally, it can be tailored for specific mechanical, optical, and barrier properties, suitable for food packaging, filtration membranes, and electronic devices. With its biodegradability and eco-friendly production process, bacterial cellulose offers sustainable solutions across industries, promising future innovations (Figures 12c and 12d).

<figure><img src="image_22.png" /></figure>
**Figure 12:** (a) Biosynthetic pathway of cellulose in the cells of microorganisms. Reproduced with permission from Ref. ³⁰⁹, Copyright © 2017 MDPI. (b) Scanning electron microscopy images capturing *Acetobacter xylinus* and the process of bacterial cellulose formation. Reproduced with permission from Ref. ³⁰⁸, Copyright © 2014 Elsevier. (c) Highlights of properties of bacterial cellulose. Reproduced with permission from Ref. ³¹⁰, Copyright © 2023 Elsevier. (d) Schematic of application areas of bacterial cellulose. Reproduced with permission from Ref. ³¹¹, Copyright © 2021 Elsevier.

The comparison of various cellulose extraction methods and resulting products across different biomass sources reveals distinct trends and insights into the efficiency and applicability of cellulose-based materials. Table 3 highlights several studies focused on the isolation and characterization of cellulose materials with notable properties derived from a diverse range of lignocellulosic biomasses, demonstrating their potential for various applications ²⁷³,²⁹⁰,²⁹¹,³¹². The studies collectively explored a diverse array of biomass sources, which included agricultural residues such as sugarcane bagasse ²⁹⁰ and coconut fibers ²⁹⁰, as well as date palm biomass ³¹³, jackfruit rind ¹²⁴, and oil palm mesocarp fiber ²⁹¹. The extraction methods employed varied widely, incorporating multiple pre-treatments such as Soxhlet extraction with organic solvents ²⁹⁰ and alkaline/acid treatments ¹²⁴,³¹³. Following these initial treatments, the biomass underwent additional processes including bleaching, delignification, and further purification steps like centrifugation and sonication. This diverse array of methods was designed to achieve a balance between processing efficiency and the quality of the resultant cellulose. For instance, the use of alkali and acid treatments was a common practice across many studies, aimed at removing non-cellulosic components and enhancing the purity of the cellulose ¹²⁴,³¹³. However, the studies also revealed significant variability in pre-treatment conditions and the specific chemicals used, such as sodium chlorite/sulfuric acid ²⁹⁰, which underscored the need for optimizing extraction protocols tailored to each type of biomass. This variability highlights the complexity of achieving optimal cellulose extraction and the necessity for further refinement of extraction techniques to improve yield based on the characteristics of the biomass used.

The studies yielded various forms of cellulose, including microcrystalline cellulose (MCC) ²⁹⁰, α-cellulose ³¹³, cellulose nanofibrils (CNFs) ³¹⁴, and cellulose nanocrystals (CNCs) ²⁷³,³¹⁴, each with distinct properties influenced by the extraction and pre-treatment processes. The cellulose extracted from sugarcane bagasse and corn cob demonstrated high crystallinity, reaching up to 89%, and exhibited significant thermal stability, which rendered it particularly suitable for applications in biocomposites nanoreinforcement ²⁹⁰. This high level of crystallinity and thermal stability was attributed to the effective removal of non-cellulosic components and the optimization of extraction conditions ²⁹⁰. Additionally, cellulose extracted from date palm biomass and jackfruit rind exhibited high crystallinity (above 80%) and significant thermal stability, highlighting the effectiveness of the methodologies used in these studies to produce nanocellulose with desirable properties. The crystallinity index and thermal degradation temperatures were key indicators of structural order and thermal resilience, respectively. These attributes directly impacted the suitability of the cellulose for various applications, with higher crystallinity and thermal stability generally leading to improved performance in advanced uses ¹²⁴,³¹³.

In addition, FTIR spectroscopy, X-ray diffraction (XRD), and thermogravimetric analysis (TGA) were routinely employed to evaluate the structural and thermal characteristics of the extracted cellulose across various studies ²⁷³,²⁹⁰,³¹²,³¹³. FTIR spectroscopy provided insights into the chemical functional groups and confirmed the removal of non-cellulosic materials ²⁹¹,³¹². XRD analysis revealed that increased crystallinity was a common finding, which generally improved the material mechanical properties and thermal stability ²⁷³,²⁹⁰. For instance, cellulose nanocrystals extracted from Ensete ventricosum and oil palm mesocarp fiber demonstrated high crystallinity and thermal stability, attributes that were
advantageous for advanced material applications, including biocomposites and green technologies<sup>291,312</sup>. Conversely, some studies identified a reduction in crystallinity or thermal stability linked to specific processing conditions. Notably, the oxidation treatment applied to traditional Chinese medicine residues and the carboxylation process for cellulose nanocrystals resulted in decreased crystallinity and altered thermal properties<sup>315</sup>. These changes were attributed to the chemical modifications and structural disruptions introduced during the extraction and modification processes, highlighting the sensitivity of cellulose properties to processing conditions. The extracted cellulose and its derivatives had been utilized across a variety of fields. High-crystallinity cellulose had been applied effectively as a reinforcement agent in biocomposites, eco-friendly packaging materials, and textiles due to its enhanced structural properties and stability<sup>312-315</sup>. CNC in particular, had proven to be highly valuable in the development of advanced materials, including nanocomposites, biofuels, and technologies for environmental remediation<sup>131,273,315</sup>. CNF are characterized by their high strength, flexibility, and large surface area, which endow them with exceptional mechanical and thermal properties<sup>43,301</sup>. These features make CNF highly versatile for a range of applications, including reinforcing materials in composites, enhancing the mechanical properties of paper<sup>316</sup> and textiles<sup>317</sup>, and serving as a bio-based material for sustainable packaging<sup>318</sup> and tissue engineering<sup>198,199,319</sup>. Additionally, CNF high surface area allows for effective incorporation of functional additives, making it suitable for advanced applications in electronics<sup>30,320</sup>, sensors<sup>226,316</sup>, and medical devices<sup>321,322</sup>.
<table><caption>1220 Table 3: Summary of some research studies on extraction and characterization of cellulose from various biomass sources</caption><thead><tr><th>Biomass used</th><th>Extraction methods involved</th><th>Type of extracted cellulose</th><th>Properties of extracted cellulose</th><th>Ref.</th></tr></thead><tbody><tr><td>■ Sugarcane bagasse (SB)<br>■ Corn cob (CC)<br>■ Coconut fibers (CF)<br>■ Hybrid combinations : SB-CC and SB - CF</td><td>■ Dewaxing using Soxhlet extraction with benzene and methanol.<br>■ Delignification involved sodium chlorite, glacial acetic acid, washing, and filtering.<br>■ Alkaline treatment removed hemicellulose and lignin using sodium hydroxide.<br>■ Acid hydrolysis extracted microcrystalline cellulose with 2 wt% sulfuric acid.<br>■ Purification included dialysis, centrifugation, and sonication for stable cellulose suspension.</td><td>Microcrystalline cellulose (MCC)</td><td>■ SB-CC had 89% while SB-CF had 84.39% crystallinity.<br>■ SB-CC had 0.8903 mm size and 0.272 PDI; SB-CF had 0.2297 mm size and 0.289 PDI.<br>■ Degradation of samples started at ~111.67°C, significant decomposition at ~350°C, lignin degraded at ~379°C.<br>■ FTIR showed higher cellulose content; X-ray diffraction confirmed improved crystallinity in hybrid biomasses.</td><td>290</td></tr><tr><td>■ Date palm trees (10-15 years old) from Al-Ain, UAE; Parts investigated : Rachis, leaflet, and fiber</td><td>■ Samples were washed with deionized water and dried in a shaded area.<br>■ Soxhlet extraction was used to remove resins and waxes.<br>■ Extract-free biomass was leached with 0.1 M HCl at 100°C for 2 h.<br>■ Residue was treated with 0.1 M NaOH at 100°C with stirring for 2 h.<br>■ Dried cellulose was bleached with acetic acid, hydrogen peroxide, and sulfuric acid.<br>■ α-Cellulose was determined using 17.4% NaOH and washing in 20% acetic acid.</td><td>■ Isolated cellulose<br>■ α-cellulose</td><td>■ Cellulose content varied: rachis 78.63%, leaflet 75.64%, fiber 70.40%; crystallinity index was 52.27%.<br>■ Decomposition of cellulose started at 200-250°C; major degradation occurred between 180-350°C.<br>■ Functional groups were similar; key peaks included OH, CH, C-O-C, with residual lignin peaks.<br>■ Rachis and fiber celluloses had porous rod-like structures; leaflet cellulose was less defined.<br>■ Major crystalline peaks were at 2θ = 22°; crystallinity indexes were high, indicating well-ordered structures.</td><td>313</td></tr><tr><td>■ Bleached hardwood (Birch, Betula) Kraft pulp</td><td>■ Hardwood pulp was treated with imidazole/water mixtures at different water contents.<br>■ Treatment occurred at 120°C for 24 hours, followed by agitation and water quenching.<br>■ Treated pulp was sonicated, yielding a turbid supernatant, which was then purified and characterized.</td><td>■ Cellulose nanofibrils (CNFs)<br>■ Cellulose nanocrystals (CNCs)</td><td>■ CNFs had width of 8 ± 4 nm and length several micrometers.<br>■ CNCs measured 4 ± 2 nm wide and 156 ± 37 nm long.<br>■ CNFs and CNCs had a crystallinity index of ~67% and ~73%.<br>■ Both CNFs and CNCs retained 9-10% of residual xylan content.</td><td>314</td></tr><tr><td>■ Jackfruit rind (Artocarpus heterophyllus)</td><td>■ Dewaxing, moderate alkali delignification, and bleaching with alkaline hydrogen peroxide.<br>■ Extraction of CNFs using organic-inorganic acid hydrolysis using acetic acid-HCl mixture under mild hydrothermal conditions.<br>■ Homogenization : Post-hydrolysis to produce CNFs.</td><td>■ Cellulose nanofibers (CNFs)</td><td>■ CNFs had an average diameter of ~28 nm, as shown by TEM.<br>■ PXRD confirmed cellulose I structure and 87.36% crystallinity.<br>■ FTIR confirmed removal of hemicellulose and lignin.<br>■ TGA/DTA showed initial degradation at 100°C and peak at 324-340°C.<br>■ SEM and EDX verified fiber purity and elemental composition.<br>■ Chemical composition : 48.02% carbon and 51.98% oxygen.</td><td>124</td></tr></tbody></table>
<table><tr><td>■ Waste date leaves obtained from a UAE farm</td><td>■ Date leaves were washed, dried, and crushed to a particle size of 120 µm.<br/>■ The leaves were soaked in brine solution for 24 h.<br/>■ The soaked material was treated with brine at pH 3-4 and 80°C for 6 h.<br/>■ The resulting cellulose was filtered, washed, and dried.</td><td>■ Needle-shaped cellulose fibers</td><td>■ DLS provided size distribution data; CNFs had a narrower range.<br/>■ Mean zeta potential was 24.1 mV.<br/>■ Needle-shaped cellulose fibers had a high crystallinity index of 64.75%.<br/>■ Proximate analysis showed higher volatile matter, lower ash, and moisture.<br/>■ Ultimate analysis of cellulose fibers showed higher carbon and hydrogen content.<br/>■ Higher thermal degradation temperature noted in treated fibers.<br/>■ Crystallinity index increased from 53.58% to 64.75% after treatment.<br/>■ Pretreatment method yielded long, needle-shaped fibers with high thermal stability.</td><td>323</td></tr><tr><td>■ Non-edible parts of jackfruit (Artocarpus heterophyllus) peel</td><td>■ Sodium chlorite method: Dewaxed jackfruit peel was treated with sodium chlorite, bleached, and alkali-treated.<br/>■ Acetic acid and nitric acid: Biomass was treated with NaOH, bleached, and purified with acids and ethanol.<br/>■ Formic acid treatment: Hemicellulose removed, then cellulose was bleached with formic acid and hydrogen peroxide.<br/>■ SCNCs production: Cellulose was hydrolyzed with H<sub>2</sub>SO<sub>4</sub>, then processed to obtain spherical cellulose nanocrystals.</td><td>■ Isolated cellulose<br/>■ Spherical cellulose nanocrystals (SCNCs)</td><td>■ The isolated cellulose demonstrated high crystallinity (83.42%) as shown by XRD analysis, with a sharp endothermal peak at 323°C in DSC and a stable thermal profile in TGA.<br/>■ The SCNCs had an average diameter of 130 nm and exhibited a zeta potential of -11.6 mV, indicating their stability in suspension.</td><td>324</td></tr><tr><td>■ Traditional Chinese medicine residues, primarily plant rhizomes</td><td>■ Carboxylated cellulose nanocrystals (CNCs) were prepared using sequential periodate-chlorite oxidation.<br/>■ The process involved soaking the cellulose in water, reacting it with sodium periodate (NaIO<sub>4</sub>) and sodium chlorite (NaClO<sub>2</sub>), and then purifying the oxidized fibers to obtain dialdehyde cellulose (DAC).<br/>■ These DACs were subsequently treated to produce CNCs with high carboxyl content.</td><td>■ Carboxylated cellulose nanocrystals (CNCs)</td><td>■ The CNCs had an average length of 139.3 nm and a width of 10 nm.<br/>■ The crystallinity index of the CNCs decreased following oxidation, but the cellulose I structure was preserved.<br/>■ The thermal stability of CNCs decreased with reduced particle size and crystallinity, as revealed by TGA.<br/>■ FTIR analysis indicated changes in hydrogen bonding with decreasing particle size</td><td>315</td></tr><tr><td>■ Pomegranate peels, both outer and inner layers, as the biomass source for cellulose extraction</td><td>■ The extraction process involved a multi-step approach starting with delignification using sodium hydroxide to remove lignin, followed by mercerization to produce holocellulose.<br/>■ Subsequent bleaching with sodium chlorite and acid treatment was used to purify and further process the cellulose.</td><td>■ Holocellulose<br/>■ α-cellulose.</td><td>■ Extracted cellulose exhibited enhanced crystallinity and reduced particle size.<br/>■ FTIR confirmed the presence of characteristic cellulose peaks.<br/>■ XRD analysis showed increased cellulose crystallinity, particularly in the outer layer compared to the inner layer.<br/>■ The chemical revealed a notable increase in cellulose content to 71% in treated samples from an initial 60% in untreated peels.</td><td>325</td></tr></table>
<table><tr><td>Cocoa pod husks (CPH) from the Colombian cocoa industry.</td><td><ul><li>CPH was pre-treated with 2% sodium chlorite at 80°C, then hydrolyzed with sodium hydroxide.</li><li>The fibers were bleached with 5% sodium hypochlorite and dried to produce tCPH.</li><li>A control sample (tCPHnpe) was treated similarly without high pressure.</li></ul></td><td><ul><li>Cellulose microfibers</li></ul></td><td><ul><li>The treatment reduced lignin by 14% and increased holocellulose content.</li><li>FTIR spectra revealed higher cellulose in tCPH, with less hemicellulose and lignin.</li><li>tCPH had lower moisture content at 5.91% compared to 6.72% of CPH.</li><li>XRD showed tCPH crystallinity increased to 42.84%, while that of CPH was 28.83%.</li><li>SEM images showed tCPH with well-defined cellulose microfibers, while tCPHnpe had agglomerated fibers.</li></ul></td><td>326</td></tr><tr><td>Date palm stem waste</td><td><ul><li>Date palm stem waste was first bleached using acidified sodium chlorite (NaClO<sub>2</sub>) to remove non-cellulosic components.</li><li>The biomass was then treated with sodium hydroxide (NaOH) to further remove lignin.</li><li>Cellulose was subjected to hydrolysis using 62 wt% sulfuric acid (H<sub>2</sub>SO<sub>4</sub>) at 45 °C for 45 minutes to produce CNCs.</li></ul></td><td><ul><li>Cellulose nanocrystals (CNCs)</li></ul></td><td><ul><li>CNCs exhibited high thermal stability with degradation temperatures of 249.5 °C and thermal analysis temperatures ranging from 290–297 °C.</li><li>CNCs had a high activation energy (125.6 kJ/mol), reflecting a robust crystalline structure with strong hydrogen bonding.</li><li>CNCs demonstrated good physical stability with a ζ potential of -29.3 mV and a narrow particle size distribution, indicating stable suspension.</li></ul></td><td>327</td></tr><tr><td>Oil palm mesocarp fiber (OPMF) sourced from FELDA Serting Hilir Palm Oil Mill, Kuala Lumpur, Malaysia</td><td><ul><li>The extraction process involved multiple steps. First, OPMF was treated with NaOH solution to remove lignin and hemicelluloses.</li><li>Following alkali treatment, the fiber was bleached using a combination of acetate buffer, aqueous chlorite, and distilled water to purify it.</li><li>Finally, CNCs were extracted through acid hydrolysis with H<sub>2</sub>SO<sub>4</sub> followed by centrifugation, dialysis, and sonication.</li></ul></td><td><ul><li>Cellulose nanocrystals (CNCs)</li></ul></td><td><ul><li>Extracted CNCs exhibited increased crystallinity (77.80%) compared to the original OPMF (44.61%).</li><li>This enhancement in crystallinity was attributed to the removal of amorphous regions and non-cellulosic components.</li><li>CNCs showed improved thermal stability and a rod-like morphology with an average diameter of 4.52 nm, as confirmed by SEM and TEM.</li><li>CNCs exhibited rod-like shapes with diameters ranging from 1 to 6 nm.</li></ul></td><td>291</td></tr><tr><td>pseudo stem fiber of Ensete ventricosum (commonly known as ensete fiber), a type of banana plant fiber</td><td><ul><li>The extraction process involved a two-step pretreatment.</li><li>Initially, the fiber was subjected to alkali treatment to remove hemicellulose, followed by an alkalinized hydrogen peroxide delignification process to further purify the cellulose.</li><li>Optimal conditions for the extraction of CNCs were determined using response surface methodology.</li></ul></td><td><ul><li>Cellulose nanocrystals (CNCs)</li></ul></td><td><ul><li>The cellulose exhibited an increased crystallinity index from 65% to 75%, indicating enhanced structural order.</li><li>CNCs demonstrated improved thermal stability, as revealed by TGA.</li><li>The chemical structure was modified compared to the raw fiber, showing alterations due to the pretreatment processes.</li><li>Morphological analysis via SEM confirmed a rod-like shape of CNCs.</li></ul></td><td>312</td></tr></table>

1221 SB stands for Sugarcane bagasse, CC is Corn cob, and CF represents Coconut fibers. MCC denotes Microcrystalline cellulose, and FTIR refers to Fourier-transform infrared spectroscopy.

1222 TGA stands for Thermogravimetric analysis, while PXRD is Powder X-ray diffraction. SEM and EDX denote Scanning electron microscopy and Energy-dispersive X-ray spectroscopy,

1223 respectively, and DLS stands for Dynamic light scattering. PDI refers to Polydispersity index. CNCs are Cellulose nanocrystals, CNFs are Cellulose nanofibrils, and HEL signifies Hydrotropic

1224 extracted lignin. IL denotes Ionic liquid, EOL stands for Ethanol organosolv lignin, and H<sub>2</sub>SO<sub>4</sub> is Sulfuric acid. NaOH refers to Sodium hydroxide, and NaClO<sub>2</sub> stands for Sodium chlorite.

1225 TEM is Transmission electron microscopy, and SCNCs are Spherical cellulose nanocrystals. DAC represents Dialdehyde cellulose, CNCs can also refer to Carboxylated cellulose nanocrystals,

1226 OPMF stands for Oil palm mesocarp fiber, DSC is Differential scanning calorimetry, and ζ potential denotes Zeta potential.
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## 2.4.2. Properties of nanocellulose

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Nanocellulose, a versatile nanomaterial derived from cellulose fibers, has garnered significant attention due to its remarkable properties and wide-ranging applications across various industries. Its unique nanostructure, comprising cellulose nanocrystals (CNCs) or cellulose nanofibrils (CNFs), contributes to its remarkable mechanical strength, stiffness, and lightweight properties ⁴³,⁶⁸. As a result, nanocellulose is increasingly used as a reinforcement additive in composite materials, enhancing their mechanical performance while maintaining low weight ²⁹⁷,³⁰³. Additionally, nanocellulose has large surface area-to-volume ratio enables efficient adsorption and absorption processes, making it suitable for applications such as water purification, drug delivery, and wound healing ⁶⁸,³²²,³²⁸. Its biocompatibility and biodegradability, along with easy modification, further expand its utility in biomedical applications like tissue engineering and drug delivery systems ⁴³. Moreover, nanocellulose exhibits excellent thermal stability, optical transparency, and light scattering properties, making it ideal for various applications, including flame-retardant materials, thermal insulators, and optically transparent films ³⁰,³⁰³,³¹⁸.

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The unique properties of nanocellulose stem from its high aspect ratio, large surface area, and abundant hydroxyl groups (-OH) on its surface ⁶⁸,¹⁹⁹,³²⁹. Hydrogen bonding plays a crucial role in stabilizing the nanocellulose structure, contributing to its mechanical strength, thermal stability, and chemical reactivity. Strong hydrogen bonding interactions between cellulose chains result in high tensile strength and stiffness, making nanocellulose suitable for reinforcing materials like polymers and composites ²⁶²,²⁶⁸. Moreover, hydrogen bonding influences nanocellulose crystallinity, which affects its optical, barrier, and rheological properties ³³⁰,³³¹. The presence of exposed hydroxyl groups enables nanocellulose to interact with water molecules and other polar or charged species, making it useful in applications such as water treatment and biosensing. Chemical modification can further enhance nanocellulose properties, allowing for tailored functionalities to meet specific application requirements ²⁶³,²⁷¹,²⁹⁸.

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Nanocellulose manifests extraordinary properties attributed to its allomorphic variations, including cellulose I, II, III, and IV (Figure 13) ³³²,³³³. These allomorphic distinctions intricately influence nanocellulose mechanical, chemical, and thermal properties, thereby dictating its suitability for specific applications ranging from biomedical engineering, where its biocompatibility and tunable surface functionalities are exploited for drug delivery systems, to advanced composite materials, where its high strength-to-weight ratio and reinforcing capabilities are leveraged for enhancing structural integrity in aerospace and automotive industries ⁴³,³³³,³³⁴. Cellulose Iα, the predominant allomorph in natural cellulose, showcases a meticulously organized crystalline structure characterized by parallel cellulose chains arranged in an anti-parallel configuration, fostering robust intermolecular hydrogen bonding. These properties make cellulose Iα ideal for industrial applications requiring high durability and structural integrity, such as papermaking, textiles, and biomedical scaffolds ²⁶²,³³²,³³⁵. Moreover, cellulose Iβ presents a distinct crystalline structure from Iα due to variations in hydrogen bonding configurations, influencing its mechanical strength and susceptibility to enzymatic degradation ³²⁰,³³⁶. Cellulose II, or regenerated cellulose, undergoes a transformative process yielding a structurally more homogeneous material with enhanced accessibility to chemical reagents. It finds applications in textiles, films, and membranes, contributing to fabric strength, flexible packaging, and precise filtration processes ²⁹⁷,³³⁷. Cellulose III emerges through treatment of cellulose I, exhibiting distinctive characteristics and heightened reactivity, suitable for drug delivery systems, coatings, and composite materials ³³⁸,³³⁹. Cellulose IV, the amorphous form, lacks long-range order but maintains the fundamental chemical composition of cellulose. It finds utility in adhesives, flexible films, and packaging materials, offering improved bonding properties,
flexibility, and barrier properties against moisture and gases <sup>340-342</sup>. These various forms of cellulose represent promising avenues for the development of innovative materials tailored to meet specific industrial demands. The crystal structures of cellulose Iα and Iβ project similarly along the chain axes, with notable differences perpendicular to the axis within the hydrogen-bonded sheets. In both, the second sheet (II) is shifted upward by approximately c/4 relative to the first (I) <sup>343</sup>. However, in Iα, the third sheet (III) is also shifted upward by approximately c/4 relative to II, whereas in Iβ, it is shifted downward. This results in a relative displacement of approximately c/2 between III and II in both. The conversion from Iα to Iβ likely involves chains slipping past each other without disrupting hydrogen-bonded sheets, with a slippage of approximately c/2 at the II and III interface (Figure 13B) <sup>343</sup>.

Cellulose, especially at micro- and nanoscales, is prized for its mechanical, chemical, optical, and rheological properties. Acting as a plant structural constituent, cellulose provides mechanical resistance due to its tight intra- and intermolecular bonds, forming microfibrils with remarkable tensile strength <sup>257,344</sup>. The mechanical robustness of cellulose is largely attributed to its highly crystalline regions. For instance, a bundle of stretched crystalline cellulose chains exhibits elastic modulus values ranging between 110 and 220 GPa, a tensile strength between 7.5 and 7.7 GPa, and a density of 1.6 g/cm<sup>3</sup>. However, the transversal modulus of crystalline nanocellulose is considerably lower, typically ranging between 15 and 30 GPa <sup>345-348</sup>. Furthermore, rheological characterization of nanocellulose has revealed intriguing behavior. Dynamical rheology studies of microfibrillated cellulose have demonstrated that aqueous suspensions behaved as gels across the investigated concentration range of 0.125 to 5.9% w/w, with storage modulus ranging from 1.5 Pa to 105 Pa <sup>279</sup>. Cellulose nanocrystals have a relatively lower aspect ratio compared to CNF, while nanofibrils exhibit a much higher aspect ratio due to their small diameter and extended length <sup>257,349</sup>.

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>**Figure 13.** (A) Refined crystal structures depicting cellulose allomorphs Iα, Iβ, II, and III1, with C, O, and H atoms represented by gray, red, and white spheres, respectively. Dashed lines denote hydrogen bonds. Reproduced with permission from Ref. 1294</figcaption></figure>
functionalities ³⁶⁴. By utilizing RAFT, scientists can graft polymers onto cellulose with controlled structures that can further enhance properties like biodegradability and compatibility with various environmental conditions ³⁶⁷,³⁶⁸. These techniques allow the design of cellulose-based materials for various applications from biomedical devices to functional materials, by customizing properties like mechanical strength and responsiveness.

Figure 14C illustrates various methods for modifying cellulose surfaces with macromolecular compounds, each offering tailored solutions for specific applications. The first approach (a) involves attaching polymers with isocyanate groups to cellulose, creating stable urethane linkages that enhance the material properties and compatibility ³⁶²,³⁶⁷. The second method (b) entails reacting polymer amines with preoxidized cellulose aldehyde groups, leading to the formation of imine linkages, which also modify the cellulose surface characteristics ³⁶⁷. The third and fourth methods (c, d) involve the incorporation of epoxy-bearing polymers, resulting in the creation of robust hybrid materials with improved performance and stability. Lastly, the fifth method (e) employs "click" chemistry, where polymers functionalized with azide or alkyne groups react with cellulose aldehyde groups to form covalent bonds, further diversifying the potential uses of modified cellulose ³⁶⁷. These modification techniques enhance cellulose compatibility and functionality, making it suitable for a wide range of applications including biomaterials, coatings, and nanocomposites.
<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /><figcaption>**Figure 14:** (A) Schematic representation illustrating pathways for modifying cellulose using small molecules, including esterification, acylation, silanization, and modification with isocyanates. (B) Examples showcasing cellulose modification through grafting techniques such as ring-opening polymerization (ROP), atom transfer radical polymerization (ATRP), and reversible addition-fragmentation chain transfer (RAFT) polymerization. (C) Various methods explored for modifying the cellulose surface with macromolecular compounds using grafting techniques: (a) attachment of polymers containing isocyanate groups, (b) interaction of polymer amines with aldehyde groups of preoxidized cellulose, (c,d) modification of cellulose with epoxy-bearing polymers, and (e) modification via "click" reactions. Reproduced with permission from Ref. 367, Copyright © 2022 MDPI.</figcaption></figure>
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Nanocellulose offers versatile thermal properties crucial for sensing applications. With high thermal conductivity and stability, it ensures accurate temperature measurements and maintains sensor reliability over time³⁰. Its low thermal expansion minimizes dimensional changes, ensuring precise measurements and long-term durability. Nanocellulose also serves as an effective thermal insulator when incorporated into nanocomposites, offering tailored thermal insulation for various applications³⁶⁹. Additionally, nanocellulose-based sensors exhibit rapid thermal response times, enabling real-time monitoring in dynamic environments²²⁶,²²⁸,³⁷⁰.

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### 2.4.3. Applications of nanocellulose in (bio)sensors

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Nanocellulose, encompassing various types, stands out as a class of environmentally friendly materials with remarkable properties that are gaining prominence in modern technology. Their abundance, low toxicity, large surface area, and renewability make them particularly appealing for a wide range of applications⁴³,⁶⁸,³⁷⁰. Within the context of biosensor materials derived from cellulose, the significance of cellulose and its derivatives, including cellulose-based composites, has been extensively discussed. Nanocelluloses, in particular, have found utility in numerous biosensing applications, facilitating the detection of a diverse array of analytes across different fields²²⁶⁻²²⁸,³⁷⁰. The Table 4 summarizes various recent research studies involving cellulose-based materials as electrode modifiers for electrochemical sensing applications.

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The research presented in the Table 4 highlights the innovative use of cellulose-based materials, often combined with other functional components, to enhance the sensitivity, selectivity, and overall performance of electrochemical sensors²²⁶,²²⁸,³¹⁶,³⁷⁰. Various cellulose nanomaterials, such as nanofibers, nanocrystals, and composites with metal oxides²²⁶, graphene³⁷¹, or polyaniline³⁷², have been integrated into electrodes to detect a range of analytes, including hydrazine²²⁶, 4-chlorophenol³⁷³, dopamine³⁷⁴, hydrogen peroxide³⁷⁵, and tryptophan enantiomers³⁷⁶. The studies demonstrated significant improvements in sensor performance, including lower detection limits, wider linear ranges, and enhanced signal responses compared to unmodified electrodes³⁷¹,³⁷⁴⁻³⁷⁶. These advancements are attributed to the unique properties of cellulose, such as its high surface area, mechanical strength, and biocompatibility, which, when combined with conductive materials like carbon nanotubes or metal nanoparticles, result in superior electrochemical activity³⁷¹,³⁷⁵,³⁷⁷. Additionally, the incorporation of cellulose derivatives in composite films has enabled the development of multifunctional sensors that are responsive not only to chemical analytes but also to environmental factors such as humidity and pH³⁷². The research also emphasized the importance of eco-friendly and cost-effective sensor fabrication techniques, such as the use of plant extracts as reducing agents for nanoparticle synthesis and the application of flexible paper-based electrodes for gas sensing²²⁸,³⁷⁷.
<table><caption>1398 Table 4: Overview of recent studies focused on cellulose-based electrode materials for electrochemical sensing of diverse analytes</caption><thead><tr><td>Electrode materials</td><td>Analytes</td><td>Linear range</td><td>Detection limit</td><td>Main results achieved</td><td>Ref.</td></tr></thead><tbody><tr><td>Cellulose nanofibers/Fe<sub>3</sub>O<sub>4</sub> composite modified glassy carbon electrode (GCE)</td><td>Hydrazine</td><td>0.001 M – 140 M</td><td>0.5 nM</td><td>Enhanced detection of hydrazine with a 2.3x increase in oxidation current and a 60 mV reduction in oxidation potential compared to unmodified GCE. Recovery range : 97.6%-104.9%.</td><td>226</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub> incorporated cellulose nanofiber composite (Fe<sub>3</sub>O<sub>4</sub>/CNF) as part of a modified carbon paste electrode (CPE)</td><td>4-chlorophenol (4-CP)</td><td>1.0 nM – 170 µM</td><td>0.5 nM</td><td><ul><li>Fe<sub>3</sub>O<sub>4</sub>/CNF/CPE demonstrated enhanced sensitivity for detecting 4-CP compared to a bare CPE.</li><li>The oxidation current of 4-CP increased by over 1.67 times and the oxidation potential decreased by approximately 120 mV at optimal conditions.</li><li>The sensor performance was validated with a recovery range of 98.52–103.66% in drinking water and wastewater samples.</li></ul></td><td>373</td></tr><tr><td>Cellulose nanofibers (CNF), graphene oxide (GO), chitosan-graphene oxide composite, acetylcholinesterase (AChE)</td><td>Chlorpyrifos (organophosphate pesticide)</td><td>25–1000 nM</td><td>2.2 nM</td><td><ul><li>The biosensor exhibited high sensitivity for detecting chlorpyrifos with good performance across the linear range of 25–1000 nM.</li><li>The detection and quantification limits were 2.2 nM and 73 nM, respectively.</li><li>The use of CNF and GO improved the sensor sensitivity, making it a precise, quick, and eco-friendly method for monitoring chlorpyrifos contamination in water and juice samples</li></ul></td><td>371</td></tr><tr><td>CelloZIFPaper_In Situ and CelloZIFPaper_Ex Situ (ZIF-8 integrated into cellulose pulp or TEMPO-oxidized cellulose nanofibrils)</td><td>Toxic heavy metals, such as lead ions (Pb<sup>2+</sup>)</td><td>--------</td><td>8 µM for lead ions (Pb<sup>2+</sup>)</td><td><ul><li>The study demonstrated that porous zeolitic imidazolate frameworks (ZIF-8) integrated into cellulose-based papers could be used effectively as adsorbents for heavy metal removal from water, with capacities ranging from 66.2 to 354.0 mg/g.</li><li>These materials served as working electrodes for selective electrochemical sensing of toxic metals, advancing Lab-on-CelloZIFPaper technologies for label-free heavy metal detection.</li></ul></td><td>378</td></tr><tr><td>TEMPO-oxidized cellulose nanofibers (TOC) loaded with silver nanoparticles (AgNPs) and/or graphite (Gr), crosslinked by tannic acid.</td><td>Dopamine (DA)</td><td>0.005–250 µM</td><td>0.0005 µM (S/N = 3)</td><td><ul><li>TOC/AgNPs/Gr composite-modified electrode exhibited enhanced electrochemical performance for dopamine detection.</li><li>It demonstrated a wide linear range, low detection limit, high sensitivity, and excellent anti-interference characteristics.</li><li>Sensors also met clinical criteria for reproducibility, selectivity, stability and recovery, offering a promising approach for dopamine quantification.</li></ul></td><td>374</td></tr><tr><td>Cellulose nanocrystals (CNC)/polyaniline (PANI) composite films</td><td>Humidity, pH, organic solvents</td><td>--------</td><td>--------</td><td><ul><li>The study developed CNC-PANI and CNC-glucose-PANI films with bright structural color through optimized self-assembly.</li><li>These films showed responsiveness to humidity, pH, and organic solvents, with changes in color and conductivity.</li><li>A two-layer CNC-PANI film was incorporated into a circuit, displaying color changes and increased conductivity in response to stimuli.</li></ul></td><td>372</td></tr></tbody></table>
<table><tr><td>Polyaniline/multiwalled carbon nanotubes/carboxymethyl cellulose (PANI/MWCNTs/CMC)</td><td>Ascorbic acid</td><td>0.05 mM – 5 mM</td><td>0.01 mM</td><td><ul><li>Additionally, a PANI electrochromic device with CNC films demonstrated rapid color change from green to blue upon applying up to 1.5 V, with good cyclability and potential applications in information encryption and anti-counterfeiting using the chiral nematic CNC pattern.</li></ul></td><td>379</td></tr><tr><td>MnO<sub>2</sub>/cellulose nanowhiskers hybrid materials</td><td>Hydrogen peroxide</td><td>0.2 – 400 µM</td><td>0.04 µM (S/N = 3)</td><td><ul><li>PANI/MWCNTs/CMC composite was successfully synthesized and characterized, exhibiting enhanced electrical and electrocatalytic properties due to the synergistic effects of its components.</li><li>The composite demonstrated a large surface area, high pore volume, and good dispersion in aqueous solutions.</li><li>As an electrochemical sensor, it showed a linear detection range for ascorbic acid and a high sensitivity of 100.63 µA mM<sup>-1</sup> cm<sup>-2</sup>.</li></ul></td><td>380</td></tr><tr><td>2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanocrystals (TOCNCs) and l-cystines (l-Cys) modified gold (Au) electrode (TOCNC/l-Cys/Au)</td><td>Enantiomers of phenylalanine (Phe), leucine (Leu), and valine (Val)</td><td>--------</td><td>--------</td><td><ul><li>The MnO<sub>2</sub>/cellulose hybrid sensor demonstrated high sensitivity (0.70 µA µM<sup>-1</sup>) and fast electrochemical response.</li><li>The sensor showed excellent performance with a high recovery rate (93-110%) in real sample detection.</li><li>Characterization techniques confirmed the formation and uniform distribution of MnO<sub>2</sub> nanoparticles on cellulose nanowhiskers, enhancing electrocatalytic properties and making it suitable for sensor applications.</li></ul></td><td>381</td></tr><tr><td>Graphene oxide/cellulose nanofibril composite (GO/CNF) modified carbon paste electrode (GO/CNF/CPE)</td><td>p-Nitrophenol</td><td>3.0 nM – 210 µM</td><td>0.8 nM</td><td><ul><li>TOCNC/l-Cys/Au electrode discriminated between enantiomers of the amino acids Phe, Leu, and Val, which are relevant to metabolic diseases.</li><li>The sensor showed significant differences in peak currents for different amino acid enantiomers, confirmed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV).</li><li>The electrode demonstrated effectiveness in distinguishing d- and l-Phe and showed potential for chiral sensing and metabolic disease diagnosis.</li></ul></td><td>382</td></tr><tr><td>Graphite/cellulose nanofibers-silver nanoparticles (Gr/CNF-AgNPs)</td><td>Ascorbic acid (AA), Dopamine (DA), Paracetamol (PA)</td><td>Wide range for all analytes</td><td>0.029 µmol/L for AA, 0.034 µmol/L for DA, 0.019 µmol/L for PA</td><td><ul><li>The GO/CNF/CPE demonstrated high sensitivity, selectivity, and accuracy for detecting p-nitrophenol in water samples.</li><li>The sensor showed a wide linear range and a low detection limit, making it effective for environmental monitoring and analysis.</li></ul></td><td>383</td></tr></table>
<table><tr><td>PANI/D-CMC/ZnO (polyaniline/dialdehyde Carboxymethyl cellulose/ZnO nanoparticles)</td><td>Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>)</td><td>0.50 to 5 µM and 10 to 50 µM</td><td>0.45 µM</td><td><ul><li>The study developed a highly sensitive voltammetric sensor for H<sub>2</sub>O<sub>2</sub> using a hybrid PANI/D-CMC/ZnO material.</li><li>The sensor demonstrated high conductivity and a good performance with a linear range and low detection limit of 0.45 µM for H<sub>2</sub>O<sub>2</sub> under optimal conditions (pH 5.7).</li><li>The sensor effectively measured H<sub>2</sub>O<sub>2</sub> in real samples, such as milk.</li></ul></td><td>375</td></tr><tr><td>PANI/cellulose/WO<sub>3</sub> composite</td><td>Acetone</td><td>0 to 100 ppm</td><td>10 ppm</td><td><ul><li>An electrochemical sensor was developed using a composite of polyaniline (PANI) and tungsten trioxide (WO<sub>3</sub>) doped with cellulose.</li><li>The sensor operates effectively at room temperature, detecting acetone with a high sensitivity and accuracy.</li><li>It demonstrated a linear response with an R<sup>2</sup> value of 0.99415 and a relative standard deviation (RSD) of 5%.</li><li>The sensor achieved a detection limit of 10 ppm, overcoming the challenge of high operating temperatures typically associated with acetone sensing.</li></ul></td><td>384</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@cellulose nanocrystals/Cu nanocomposite (Fe3O4@CNC/Cu) on a graphite screen-printed electrode.</td><td>Venlafaxine</td><td>0.05–600.0 µM</td><td>0.01 µM</td><td><ul><li>The Fe<sub>3</sub>O<sub>4</sub>@CNC/Cu nanocomposite was successfully synthesized using a plant extract as a green reducing agent.</li><li>This nanocomposite was used to modify a graphite screen-printed electrode, enabling sensitive detection of venlafaxine.</li><li>The sensor demonstrated a wide linear range and a low detection limit for venlafaxine.</li><li>It proved to be selective and precise for detecting venlafaxine in urine, water, and pharmaceutical samples.</li></ul></td><td>377</td></tr><tr><td>Quaternized cellulose nanoparticles (QCs)/acetylene black (AB)/enzymes composite electrodes</td><td>Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), glucose</td><td>------</td><td>------</td><td><ul><li>The QCs-AB composite electrodes demonstrated excellent electrocatalytic performance with fast response, high sensitivity, and good stability for detecting H<sub>2</sub>O<sub>2</sub> and glucose.</li><li>The composite material facilitated effective direct electron transfer for glucose oxidase (GOD) and hemoglobin (Hb) immobilized on the electrode.</li><li>This approach offers a promising platform for the development of unmediated enzyme biosensors.</li></ul></td><td>244</td></tr><tr><td>Cellulose microfibers supported reduced graphene oxide (rGO) composite</td><td>Fenitrothion</td><td>Up to 1.134 mM</td><td>8 nM</td><td><ul><li>The cellulose microfibers supported rGO composite electrode demonstrated superior electro-reduction ability and lower reduction potential for fenitrothion compared to other modified electrodes.</li><li>It showed a reduction potential 141 mV lower than a chemically reduced rGO-cellulose microfibers composite.</li></ul></td><td>385</td></tr></table>
<table><tr><td>Composite sensor of cellulose nanocrystals (CNCs) and multiwall carbon nanotubes (MWCNTs)</td><td>Tryptophan (Trp) enantiomers (I-Trp and d-Trp)</td><td>--------</td><td>--------</td><td><ul><li>The electrode achieved a wide linear response range and a low detection limit, and it was successfully used for detecting fenitrothion in various water samples.</li></ul></td><td>386</td></tr><tr><td>Nanoporous gold electrode arrays on cellulose membranes</td><td>Oxygen (O<sub>2</sub>)</td><td>0.054 to 0.177 v/v %</td><td>0.0075%</td><td><ul><li>The chiral sensor demonstrated rapid, high-sensitivity, and stable discrimination of Trp enantiomers, with a 3.5-fold higher peak current ratio for I-Trp compared to d-Trp under optimal conditions (pH 7.0, 25°C).</li><li>The sensor performance varied with CNC type (s-CNCs from H<sub>2</sub>SO<sub>4</sub>-hydrolysis vs. h-CNCs from HCl-hydrolysis), highlighting different affinities and chiral recognition effects.</li><li>The combination of CNC chiral recognition and MWCNTs signal amplification provided an effective method for distinguishing Trp enantiomers.</li></ul></td><td>228</td></tr><tr><td>Assembly of cellulose grafted with hydroxypropyl-β-cyclodextrins (CMC-CD), multi-walled carbon nanotubes (MWCNTs) and copper ions on the surface of a GCE</td><td>Tryptophan (Trp) enantiomers (L-Trp and D-Trp)</td><td>--------</td><td>--------</td><td><ul><li>A novel, cost-effective, and flexible paper-based electrochemical O<sub>2</sub> sensor was developed using nanoporous gold electrode arrays on cellulose membranes and an ionic liquid electrolyte (BMIMPF6).</li><li>The sensor demonstrated high sensitivity, a low detection limit, and a rapid response time of less than 10 s, making it highly promising for environmental monitoring and gas sensing applications.</li></ul></td><td>376</td></tr></table>
**Enhancing gas sensor performance with nanocellulose-based composites.** This material has found extensive application, notably in the advancement of gas sensors. Gas sensors, also referred to as gas detectors, play a crucial role in detecting and identifying various gases for safety and environmental monitoring applications. They are widely utilized for detecting toxic or explosive gases and measuring gas concentrations ³⁸⁷. Emerging approaches for sensing hazardous gases necessitate easy-to-use and cost-effective technologies for widespread adoption ¹⁹,²⁰. In this regard, nanocellulose, with its renewable nature, low cost, abundance, high porosity, mechanical strength, and flexibility, offers promising properties for modifying gas sensor materials. Traditionally, gas sensors have been fabricated using inorganic semiconductor metal oxides such as TiO₂, Fe₂O₃ and ZnO ³⁸⁸⁻³⁹⁰. However, these materials exhibit instability owing to the ionic conducting charge present on their surface. In contrast, organic conducting polymers possess free electrons on their double bonds along the polymer main chain. Additionally, the processing and utilization of semiconductor metal oxides typically require high working temperatures compared to organic conducting polymers, which are effective materials suitable for use at moderate temperatures ³⁸⁷. In contrast, organic conducting polymers like polypyrrole (PPy) and polyaniline (PANI) offer cost-effective synthesis and faster response times at moderate temperatures ³⁹¹. Combining semiconductor metal oxides or carbon materials with conducting polymers and nanocellulose can significantly enhance sensor sensitivity and performance ¹⁹,³⁸²,³⁸³. Several studies have explored this direction, focusing on incorporating nanocelluloses into synthetic polymers to leverage the properties of metal oxides and enhance film formability while providing excellent reinforcement ²⁷⁰,³⁷⁷. For example, composite nanofibers of cellulose/TiO₂/PANI prepared a room temperature showed improved ammonia sensing performance at room temperature (Figure 15A), attributed to enhanced surface area and specific surface characteristics of composites due to PANI presence (Figure 15B) ³⁹². The ammonia sensing performance was assessed using a custom test system at room temperature (Figure 15C). TiO₂ contributed to enhanced response values and sensitivity of developed sensors. Repeat tests demonstrated consistent response and ideal repeatability, indicating high sensor reliability ³⁹². Selectivity tests revealed the sensor high specificity to ammonia over acetone, ethanol, and methanol at 250 ppm concentration, showcasing its practical applicability (Figure 15D) ³⁹². Similarly, Hu et al. ²²⁷ successfully fabricated a novel formaldehyde sensor using nanofibrous polyethyleneimine (PEI)/bacterial cellulose membranes coated on quartz crystal microbalance. This sensor exhibited good selectivity towards formaldehyde and demonstrated excellent reversibility, reproducibility, and linearity with increasing formaldehyde concentrations ²²⁷.
<figure><img src="image_28.png" /><figcaption>**Figure 15:** (A) Diagram depicting the formation of cellulose/TiO₂/PANI composite from cellulose acetate (CA). (B) Scanning Electron Microscopy (SEM) images showcasing cellulose (a), cellulose/TiO₂ (b), cellulose/PANI (c), and cellulose/TiO₂/PANI composite nanofibers (d). (C) Schematic representation of the homemade gas sensing test system. (D) Response to varying concentrations of ammonia (a), repeatability to 250 ppm ammonia (b), and selectivity (c) of cellulose/TiO₂/PANI composite nanofibers. Reproduced with permission from Ref. ³⁹², Copyright © 2016 Elsevier.</figcaption></figure>

Bacterial cellulose, known for its abundant -OH sites and enhanced mechanical properties, has been combined with metal oxides to create gas sensor composite materials. These sensors show favorable responses to various chemical compounds like nitrogen dioxide, acetone, and ethanol at ambient temperatures ²⁰. Leveraging the electrical conductivity and stability of aniline with cellulose nanopaper cross-linked amino graphene has led to the development of CO₂ gas sensors with heightened sensitivity and rapid response characteristics and rapid response characteristics across the range of CO₂ concentrations tested ¹⁹. In industrial applications, carbon-derived materials interacting with cellulose -OH groups form a network, widely used in chemical gas sensing ¹⁸,³⁹³. For instance, Im et al. ³⁹⁴ developed an integrated cellulose polymer concentrator/single-walled carbon nanotubes (SWCNT) sensing system aimed at detecting benzene, toluene, and xylenes (BTX) vapors, as illustrated in Figure 16A. SEM images (Figure 16B) revealed reduced SWCNT network after coating cellulose acetate ³⁹⁴. The system exhibited high sensitivity, with limits of detection for benzene, toluene, and m-xylene vapors recorded at 55 parts per million (ppm), 19 ppm, and 14 ppm, respectively. Additionally, the sensor demonstrated excellent selectivity and fast response times, taking less than 10 seconds to reach equilibrium (Figure 16C). This integration exemplified the potential of composite materials for precise chemical gas sensing applications ³⁹⁴.
<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /><figcaption>**Figure 16:** (A) Illustration of the integrated polymer concentrator/single-walled carbon nanotubes (SWCNT) sensing system. The cellulose concentrating layer absorbed and concentrated analytes, delivering them rapidly to the SWCNT sensing layer. (B) (a) Photographs of the array device with 9 sensors deposited on interdigitated microelectrode array (IDA). SEM images of (b) bare IDA, (c) SWCNTs on IDA, and (d) F5Ph-CA/SWCNT on IDA. (C) (a) Normalized conductance changes [-ΔG/Go (%)] of F5Ph-CA/SWCNT system when exposed to 529 ppm benzene, (b) normalized conductance changes when exposed to benzene, toluene, and m-xylene vapors compared to pristine SWCNT sensor (c). Duplicate sensor responses were consistent, as shown by grey curves representing the second sensor's response. Reproduced with permission from Ref. ³⁹⁴, Copyright © 2016 MDPI.</figcaption></figure>

**Versatile nanocellulose-based chemical sensing platforms.** Studies have explored nanocellulose integration into chemical sensing, highlighting its potential across platforms. Various systems leveraging nanocellulose-based sensing platforms have been developed, showcasing versatility across different dimensionalities. These include 1D fiber-based systems, enhancing sensitivity and specificity. 2D platforms like papers and films offer improved surface area and porosity. 3D matrices, like aerogels and hydrogels, provide precise sensor control and enhanced analyte interactions. Collectively, these advancements underscored the multifaceted nature of nanocellulose-based sensing platforms, paving the way for innovative solutions.

(a) 1D fiber-based sensors offer numerous advantages, including flexibility and good mechanical properties, making them highly desirable for various applications. The degree of orientation of nanofibers within a single fiber is a critical parameter that significantly influences the performance of these materials ³⁹⁵. Nawaz et al. ³⁹⁶ conducted a study wherein they developed an exceptional fluorescent sensor, Phen-MDI-CA, characterized by high selectivity and sensitivity for detecting Fe²⁺. Synthesized Phen-MDI-CA dissolved easily in DMSO and dimethylformamide, enabling fabrication of various materials like fluorescent inks, flexible films, coatings, and microspheres, all emitting bright blue fluorescence (Figure 17A). This sensor was created by chemically bonding 1,10-phenanthroline-5-amine (Phen) onto cellulose
acetate (CA) fibers using 4,4'-methylene diphenyl diisocyanate (MDI) as a crosslinker ³⁹⁶. The process involved homogeneous reaction conditions, resulting in the formation of a robust sensor structure, as illustrated in Figure 17B. This innovative approach demonstrated the potential of 1D fiber-based sensors in achieving high-performance capabilities for detecting specific analytes ³⁹⁶. Fluorescence-based detection mode 1 showed a linear relationship between emission intensity and Fe²⁺ concentration, with a detection limit of 2.6 ppb. Selectivity tests confirmed high Fe²⁺ ion selectivity. UV-Visible absorption exhibited similar selectivity but with a higher detection limit of 4.0 ppb, making fluorescence mode more sensitive and practical (Figure 17C) ³⁹⁶.

<figure><img src="image_32.png" /><figcaption>**Figure 17:** (A) Photos of Phen-MDI-CA in different material forms under visible and 365 nm UV light: (a) fluorescent printing on filter paper, (b) flexible fluorescent film, and (c) coatings on glass and steel. (B) Fe²⁺ ion detection in multimode. Detection</figcaption></figure>
mode 1: fluorescence quenching upon adding Fe²⁺ aqueous solution (50 ppb) into Phen-MDI-CA (7.23 × 10⁻⁶ M in DMSO).

Detection mode 2: visual color change upon adding Phen-MDI-CA into a metal-ion aqueous solution. Detection mode 3: visual color change of test papers by adding metal ions (1 ppm). (C) (a) Fluorescence spectra of Phen-MDI-CA with different Fe²⁺ ion concentrations. Inset: photos of Phen-MDI-CA/DMSO solutions before and after Fe²⁺ ion addition under UV light. (b) Emission intensity vs. Fe²⁺ concentration. (c) Effect of Fe²⁺ concentration on emission intensity change. (d) Fluorescent response of Phen-MDI-CA with various metal aqueous solutions (0.045 µM) using 365 nm excitation wavelength for all measurements. Reproduced with permission from Ref. ³⁹⁶, Copyright © 2018 American Chemical Society.

In another study conducted by Yao et al. ³⁹⁷, a novel multicolor (green, yellow, and orange) macrofiber-based biosensor was developed using moderately oriented bacterial cellulose nanofibers through a wet spinning process ³⁹⁷. This innovative biosensor assembled CdTe-loaded bacterial cellulose nanofibers onto a one-dimensional structure, enabling pH and glucose sensing functionalities. The resulting macrofibers exhibited sigmoidal pH dependence and high sensitivity to glucose concentrations, promising significant advancements in biosensing by providing a versatile platform for precise and efficient analyte detection ³⁹⁷. Moreover, Teodoro et al. ³⁹⁸ synthesized a hybrid material comprising cellulose nanowinkers and silver nanoparticles (AgNPs) for colorimetric detection of H₂O₂. The hybrid sensor demonstrated high sensitivity and selectivity, achieving impressive limits of detection (0.014 µM for concentrations ranging from 0.01–30 µM and 112 µM for concentrations from 60–600 µM) ³⁹⁸. Orelma et al. ³⁹⁹ developed an optical cellulose fiber tailored for water sensor applications, observing over 30 dB reversible attenuation upon immersion in water. Their innovative approach showcases the potential of cellulose-based materials in advancing sensor technologies, particularly for water quality monitoring ³⁹⁹. Furthermore, Kumari et al. ⁴⁰⁰ combined cellulose extracted from pine needles with lysine Schiff base to fabricate a sensor-adsorbent for mercury ions, achieving a maximum adsorption capacity of 258.75 mg/g ⁴⁰⁰. Fu et al. ⁴⁰¹ introduced a facile method for synthesizing and immobilizing polyaniline nanorods onto carboxymethyl cellulose (CMC)-modified cellulose nanofibers, tailored specifically for biosensing applications, yielding a biosensor with a detection limit of 0.374 M and a linear response range of 0.497 M to 2.27 mM ⁴⁰¹. Moreover, Davis and co-workers ⁴⁰² developed a novel approach to encapsulate ionic fluorescent dendrimers (AFD) into cellulose acetate (CA) nanofibers, facilitating the detection of metalloproteins via a quenching mechanism. Figure 18A illustrates the schematic representation of the electrospinning setup, encapsulation of the fluorescent dendrimer, and deacetylation process employed in this study. High-resolution UV confocal microscopy visualized protein detection ⁴⁰². Figure 18B displayed fluorescence images of anionic fluorescent dendrimer (AFD) doped cellulose nanofibers before/after incubation with 10 µM cyt c solution, revealing remarkable quenching effect. This method demonstrated potential for sensitive and selective detection of metalloproteins through distinct responses observed for each protein interacting with the fluorescent fibers, highlighting its applicability in biosensing technologies (Figure 18C) ⁴⁰².
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<figure><img src="image_35.png" /><figcaption>**Figure 18:** (A) Schematic representation of the electrospinning setup, encapsulation of the fluorescent dendrimer, and deacetylation process employed in this study. Five water-soluble fluorescent dendritic compounds (AFD-1, AFD-2, AFD-3, AFD-4, and AFD-5) composed of phenylene-ethynylene repeating units are depicted. (B) Confocal fluorescence images of electrospun nanofibers before (left) and after (right) incubation in a 10 µM cyt c solution for 15 min. (C) Fluorescence emission spectra of AFD-functionalized nanofibers in response to various concentrations of cyt c, Hgb, and BSA (λ<sub>Ex</sub>/λ<sub>Em</sub> = 370/475 nm). Reproduced with permission from Ref. ⁴⁰², Copyright © 2010 American Chemical Society.</figcaption></figure>

In a recent work performed by Chauhan et al. ⁴⁰³, an innovative biosensing platform was developed utilizing electrospun cellulose acetate fiber (CAEFs), on a disposable conducting paper substrate (RCP). (Figure 19A) Immobilizing of antibodies specific to 25-hydroxy vitamin-D3 (AB-25OHD3) and bovine serum albumin (BSA) followed. CV was used to analyze electrode surface changes ⁴⁰³. Comparisons were made between (i) RCP, (ii) CAEF/RCP, (iii) AB-25OHD3/CAEF/RCP, and (iv) BSA/AB-25OHD3/CAEF/RCP electrodes. After modifications, peak currents varied, and Nyquist plots indicated changes in charge-transfer resistance (Rct), confirming modifications. (Figure 19C). The resulting immunoelectrode exhibited a low detection limit of 10.0 ng mL⁻¹ and a sensitivity of 0.16 µA ng⁻¹ mL cm⁻², enabling accurate measurement of 25-OHD3 concentrations spanning from 10 to 100 ng/mL. With reasonable selectivity, reproducibility, and repeatability, this biosensing platform holds potential for clinical diagnostics and biomedical research, offering rapid, sensitive, and precise detection of 25-OHD3 levels in biological samples ⁴⁰³.
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<figure><img src="image_39.png" /><figcaption>**Figure 19:** (A) Production process of the cellulose acetate-based immunosensor. (B) Electrochemical characterization including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of different electrodes: (i) RCP, (ii) CAEF/RCP, (iii) AB-25OHD3/CAEF/RCP, and (iv) BSA/AB-25OHD3/CAEF/RCP immunoelectrode. (C) Evaluation of the BSA/AB-25OHD3/CAEF/RCP immunoelectrode response in phosphate-buffered saline containing [Fe(CN)₆]³⁻/₄⁻ via chronoamperometry, along with the calibration curve relating peak current to 25-OHD3 concentrations. (D) Assessment of immunoelectrode selectivity, reproducibility, and repeatability in PBS containing [Fe(CN)₆]³⁻/₄⁻. Reproduced with permission from Ref. 403, Copyright © 2019 American Chemical Society.</figcaption></figure>

(b) 2D nanocellulose-based substrates offer unique advantages due to their transparency, low thermal expansion, high strength and stiffness, flexibility, and excellent mechanical properties. These characteristics make them highly suitable substrates for flexible electronics applications, particularly in sensors and actuators development ³⁰. Ren et al. ³⁸ fabricated an amperometric glucose biosensor using a composite film comprising gold nanorods and cellulose acetate. Glucose oxidase enzyme was incorporated into the composite film by mixing with gold nanorods and cross-linking with cellulose acetate medium using glutaraldehyde ³⁸. The optimized biosensor showed high sensitivity (8.4 A cm⁻² mM⁻¹), a low detection limit (2 × 10⁻⁵ M), a wide linear range (from 3×10⁻⁵ to 2.2×10⁻³ M), good storage stability, reproducibility, and a strong affinity for glucose ³⁸. Esmaeili et al. ⁴⁰⁴ explored polypyrrole-cellulose nanocrystal (PPy/CNC) composites combined with glucose oxidase (GOx) for biosensing of glucose (Figure 20A). In Figure 20B(a), field emission scanning electron microscopy (FESEM) revealed a globular, dense PPy structure, while PPy/CNC displayed an open, porous structure ⁴⁰⁴. No phase separation was observed, indicating homogeneous polymerization on the CNC surface. The PPy/CNC nanostructure exhibited uniform, fibrous morphology, as previously observed. Aggregates on the screen-printed electrode (SPE) surface were evenly distributed (Figure 20B(b)). CV on SPE/PPy/CNC and SPE/PPy electrodes demonstrated stable redox peaks with CNC enhancing surface area, enzyme immobilization, and electron transfer ⁴⁰⁴. SPE/PPy/CNC electrode exhibited well-defined redox peaks, with a formal
potential estimated at 0.265 V. (Figure 20C). The biosensor showed high sensitivity (approximately 0.73 µA·mM⁻¹) over a dynamic response range from 1.0 to 20 mM glucose, with a low limit of detection (LOD) of (50 ± 10) µM, indicating potential for sensitive glucose detection in various applications (Figure 20D)<sup>404</sup>.

<figure><img src="image_40.png" /><figcaption>**Figure 20:** (A) Schematic representation of an electrochemical glucose biosensor using PPy/CNC membrane. (B) Microstructures of (a) PPy and (b) porous PPy/CNC nanocomposite on SPE surface. (C) Cyclic voltammetry graphs: unmodified bare SPE (a); modified PPy/GOx electrode (b); and PPy/CNC/GOx electrodes with CNC concentrations of 0.3 (c), 0.5 (d), and 0.9 wt% (e). (D) Differential pulse voltammetry (DPV) graph (a') with glucose concentrations of 1 (a), 5 (b), 10 (c), 15 (d), and 20 mM (e); and analytical curve (b') of PPy/CNC/GOx glucose biosensor in 50 mM buffer solution (pH = 7) at 0.5 V/s scan rate versus Ag/AgCl electrode. Reproduced with permission from Ref. <sup>404</sup>, Copyright © 2015 MDPI.</figcaption></figure>

Recently, Koga et al. <sup>316</sup> introduced a novel approach by combining the unique properties of oxide nanowires, cellulose nanofibers, and pencil-drawn electrodes to fabricate a paper-based disposable molecular sensor. This innovative composite structure demonstrated mechanical robustness and served as an efficient bridging network for the nanowire sensor. It facilitated effective access to target molecules while ensuring reliable electrical contact with the electrodes <sup>316</sup>. The sensor developed in this study is illustrated in Figure 21A, showcasing its design and functionality. This integrated sensor offers a cost-effective, portable, and disposable solution for molecular sensing in environmental monitoring, healthcare diagnostics, and food safety <sup>316</sup>.

Several studies have explored the integration of carbon-based materials such as multi-walled carbon nanotubes (MWCNTs), carbon nanotubes (CNTs), and graphene to enhance the mechanical and piezoelectric properties of nanocellulose composites <sup>15,39,405</sup>. For instance, Wan et al. <sup>39</sup> devised a method to fabricate a biocompatible composite of cellulose and carbon nanotubes for cell sensing applications. This composite immobilized leukemia K562 cells on a gold electrode, forming an impedance cell sensor with a detection limit of 2.6 × 10<sup>3</sup> cells mL<sup>-1</sup>. This approach highlights the potential of carbon-nanocellulose composites in developing advanced sensing platforms for biomedical applications <sup>39</sup>. Additionally, Qi et al. <sup>405</sup> introduced a novel humidity sensor using a composite of CNTs and cellulose for water trapping applications. These films demonstrated rapid response times and high sensitivity, with a relative electrical