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# Advances in Microtechnology for Improved Cytotoxicity Assessment

Maite Garcia-Hernando¹,², Fernando Benito-Lopez²* and Lourdes Basabe-Desmonts¹,³*

¹BIOMICS Microfluidics Group, Microfluidics Cluster UPV/EHU, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Spain, ²Analytical Microsystems & Materials for Lab-on-a-Chip (AMMa-LOAC) Group, Microfluidics Cluster UPV/EHU, Analytical Chemistry Department, University of the Basque Country UPV/EHU, Leioa, Spain, ³IKERBASQUE, Basque Foundation of Science, Bilbao, Spain

In vitro cytotoxicity testing is essential in the pharmaceutical and environmental industry to study the effects of potential harmful compounds for human health. Classical assays present several disadvantages: they are commonly based on live-death labelling, are highly time consuming and/or require skilled personnel to be performed. The current trend is to reduce the number of required cells and the time during the analysis, while increasing the screening capability and the accuracy and sensitivity of the assays, aiming single cell resolution. Microfabrication and surface engineering are enabling novel approaches for cytotoxicity assessment, offering high sensitivity and the possibility of automation in order to minimize user intervention. This review aims to overview the different microtechnology approaches available in this field, focusing on the novel developments for high-throughput, dynamic and real time screening of cytotoxic compounds.

**Keywords:** microtechnology, lab-on-a-chip, cytotoxicity, surface engineering, microfluidics, Sensors, Biosensors, single cell

## INTRODUCTION

In the last decades, remarkable advancements have been made in the field of microtechnology to improve analytical processes in biology, through miniaturization, for biosensing DNA (Bulyk et al., 1999; Zhang et al., 2010) and protein arrays (He et al., 2008; Ramachandran et al., 2008; Lopez-Alonso et al., 2013; Gonzalez-Pujana et al., 2019), on-chip electrophoresis (Fritzsche et al., 2010; Ou, et al., 2019), microimmunoassays (Riahi et al., 2016; Hu et al., 2017), microfluidic cell sorting (Shields et al., 2015; Vaidyanathan et al., 2018) and for cellular membrane modelling (Hirano-Iwata et al., 2010; Strulson and Maurer, 2011; Galvez et al., 2020), among others (Beebe, et al., 2002; Sackmann, et al., 2014). In fact, microtechnology enables the precise control of the topography and the surface chemistry, leading to engineered platforms for the study of cellular processes or biosensing and, at the same time, bringing advantages such as time saving, reduced costs and working space, automation of the processes, increased sensitivity and reduced volumes of the required reagents (Wurm et al., 2010; Azuaje-Hualde et al., 2017).

Cytotoxicity assays are crucial in basic research, material science, environmental analysis, pharmaceutical industry and also, in the development of anticancer therapies, where the understanding of the resistance of cancer cells to new therapies is of major importance. Fluorescent and colorimetric assays are the most widely used methods for cytotoxicity assessment (Stoddart, 2011). There are many fluorescent dyes in the market for the measurement of cell viability, whose working principle varies from dye exclusion assays (penetrating the cellular membrane of death cells, staining them), DNA condensation-based assays (which emit fluorescence when they bind nucleic acid molecules) and assays monitoring

**OPEN ACCESS**

**Edited by:**
Patricia Krawczak,
IMT Lille Douai, France

**Reviewed by:**
M. Gabriella Santonicola,
Sapienza University of Rome, Italy
Huaiyu Wang,
Chinese Academy of Sciences (CAS),
China

*Correspondence:
Lourdes Basabe-Desmonts
lourdes.basabe@ehu.eus
Fernando Benito-Lopez
fernando.benito@ehu.eus

**Specialty section:**
This article was submitted to Colloidal Materials and Interfaces, a section of the journal Frontiers in Materials

Received: 14 July 2020
Accepted: 26 October 2020
Published: 20 November 2020

**Citation:**
Garcia-Hernando M, Benito-Lopez F, Basabe-Desmonts L (2020) Advances in Microtechnology for Improved Cytotoxicity Assessment. *Front. Mater.* 7:582030
doi: 10.3389/fmats.2020.582030
a metabolic function (Ramirez et al., 2010; Stoddart, 2011). Fluorescent dyes are popular because they are sensitive and provide an easy readout by fluorescence or optical microscopy. Besides, flow cytometry is a well established technique for the assessment of cellular toxicity, which combined with some of the previously mentioned alive/dead staining, enables the performance of highly sensitive analysis of cellular viability with single cell resolution (Adan et al., 2017). Since fluorescence gives a semi-quantitative analogic signal and many fluorescent dyes are compatible with single cell analysis, they are often combined with flow cytometry to obtain absolute and real quantification of alive and dead cells within a population. Besides the noteworthy resolution and efficacy of flow cytometry, it requires trained personnel to perform it, as well as staining procedures and manual handling, which take time and increase the possibility of human errors. Furthermore, flow cytometry does not permit real-time monitoring (Gelles and Chipuk, 2016). Therefore, miniaturized systems that reduce manual handling procedures and facilitate fast and easy measurements of cellular viability are promising alternatives to fight the current drawbacks in cytotoxicity assessment, such as the manual intervention, use of labels and the lack of real time information or monitoring.

Nowadays, the large variety of microfabrication techniques leads to the manufacturing of microsystems of many different natures. All the miniaturized platforms for cytotoxicity testing can be usually combined with traditional cellular viability assays, resulting on assays with the same working principle of the traditional staining protocols for the signalling of alive and dead cells, but with improved performance. This is due to the improvements that microtechnology brings in terms of real-time monitoring capability, increased sensitivity, reduced times for the cytotoxicity assessment, decreased volumes of the required reagents and thus, produces a lower cost per analysis. Also, thanks to their small dimensions, it opens the door to multiplexed and multisampling analysis, increasing the yield in the performed protocols (Wang et al., 2007; Sugiura et al., 2008). Furthermore, alternative working principles for the assessment of cytotoxicity are now available due to the sensitivity gained by working at the microscale.

This review aims to summarize the state of the art of the microtechnology used in cytotoxicity tests, and to discuss the novelty that microfabrication techniques and materials bring to this field of research. In this work, we aspire to build a bridge between microfabrication, material science and cytotoxicity, highlighting the advantages brought to the field. In order to do that, the most common microfabrication techniques, microsystems and materials for cytotoxicity will be first explained. Then, the different types of micro analytical platforms existing for cytotoxicity assessment will be comprehensively reviewed (Figure 1).

## MICROFABRICATION TECHNIQUES USED IN CYTOTOXICITY

Microfabrication techniques were originally developed for the microelectronics industry, specifically for the manufacturing of

microelectromechanical systems (MEMs), making possible the integration of sensors to develop fully functional microdevices. Due to the high precision and resolution of the features achieved by these techniques, they soon became useful for the control of tissues and cells at the microscale, becoming attractive for biosensing, leading to the development of BioMEMs (Eribol et al., 2016). The biocompatibility of the materials used in bio-applications is crucial, e.g., the microdevices, in order to avoid any cellular toxic response. Therefore, micromachining for BioMEMs requires adaptations of the fabrication processes and/or the materials used for device fabrication in order to guarantee cellular viability (Fischer et al., 2011).

At the microscale, any small perturbation on the flow or a high density of adhered cells can cause huge stress to them, resulting on exhaustion of nutrients or even migration, which can be misunderstood as a cellular response to the toxic (Wang et al., 2007). These phenomena can be avoided through microfabrication; on one hand, inducing physical cell adhesion control by patterning small microwells on polymeric materials (Vajrala et al., 2019; Zhang et al., 2019) or on C- (Lee et al., 2006) and on U-shaped (Wang et al., 2007) microsieves in the culturing chambers. On the other hand, cell adhesion can be chemically controlled with micropatterns of proteins (Liu et al., 2013; Hamon et al., 2016) or hydrophilic-hydrophobic sites (Nath et al., 2004; Zhang et al., 2007), modulating cell adhesion by enhancing cell affinity for specific areas, keeping them comfortably located. These methods control cell adhesion density and homogeneity of the pattern. Moreover, in the case of C-shaped microsieves, the flow within the chamber is also slowed down, reducing shear stress in cells (Wang et al., 2007), making the device more appropriate for cell culture.

## Sotf-Lithography

Photolithography, a lithographic process that uses optical methods for the printing of features, is the most used technique to make integrated circuits, traditionally on rigid surfaces (Levinson, 2005). In particular, the development of soft-lithography, which derives from photolithography and serves to replicate structures from rigid molds -like the ones made by photolithography- on elastomeric or flexible materials, made a huge breakthrough in the manufacturing of BioMEMs. It opened a range of possibilities to use microstructured elastomeric materials, more suitable to keep cellular comfort and to be closer to the mimicking of cellular environment, (Xia and Whitesides, 1998).

The most common material in soft-lithography is polydimethylsiloxane (PDMS), which allows for the fabrication of devices by fast molding techniques, featuring resolutions down to nanometer scale. Also, due to the deformability of this elastomeric material, it facilitates leak-proof fluidic connections as well as integration of valves and other fluidic components. It can be covalently bonded to another PDMS piece or glass surface by a simple plasma oxidation step, resulting on the generation of leaking free, well-sealed devices. Moreover, PDMS is biocompatible, transparent (Bélanger and Marois, 2001) and non fluorescent (Piruska et al., 2005) therefore, suitable for cell applications.
<figure><img src="image_97.png" /><figcaption>**FIGURE 1** | Classification of microtechnology based cytotoxicity techniques in literature.</figcaption></figure>

Due to the high resolution of photolithography, the location of cells can be accurately controlled, and the pattern can be precisely transferred to PDMS. The cell positioning capability of PDMS molds has been demonstrated and used for cytotoxicity assessment using U-shaped microsieves, with minimum separation of 8 µm, patterned within culturing chambers (Figures 2A). These features induced low flow regions and a good distribution of cells along the chamber to obtain cellular monolayers with adequate microenvironment (Wang et al., 2007). In the mentioned work, the percentage of cellular death in response to five toxins (digitonin, saponin, CoCl₂, NiCl₂ and acrolein) was obtained when using Live/Death staining thanks to an adequate cell positioning. Similar idea was used by Mo et al., who combined a micropillar array for uniform cell positioning with a precisely controlled, low flow-rate gradient to reduce the shear stress to human induced pluripotent stem cells (HiPSC) derived neurospheres, and obtain reliable responses against riluzole drug (Mo et al., 2020).

Soft-lithography is the most popular technique to fabricate
gradient generators in microdevices. They generate a gradient in
the composition of a solution, like, in this case, the toxic
compound to be tested. Thanks to this PDMS gradient
generating devices, several works have demonstrated that
High-Throughput (HTP) toxicity analysis of specific
compounds can be simultaneously carried out, at different
concentrations, on chip, for cytotoxicity assessment
(Hosokawa et al., 2011; McCormick et al., 2017; Li et al., 2018)

PDMS has been the most commonly used material for the
fabrication of prototypes during the last decade but it presents
some drawbacks such as absorption of small molecules and high

gas permeability, thus there is a demand to fabricate microfluidic
devices made of other polymers. As an example, Nguyen *et al.*
reported a hybrid polymeric microfluidic device for cytotoxicity
testing, made of poly(methyl methacrylate) with integrated
polyethylene terephthalate (Nguyen *et al.*, 2019). The hybrid
device was fabricated by milling machine, and was successfully
used for cytotoxicity assessment, showing more reliable results
than conventional PDMS devices, due to the poly(methyl
methacrylate) natural impermeability to small molecules.

Electrode Patterning

Electrode patterning has been one of the most used
microfabrication techniques for the monitoring of cytotoxicity
responses from the emergence of microtechnology in this field,
and it is usually based on additive (metal deposition, insulation),
semi-additive (photolithography) and subtractive (etching)
processes to fabricate the whole functional electric circuit. The
integration or deposition of electrodes on PDMS is challenging
(Lim et al., 2006; Guo et al., 2012), so rigid materials such as glass
or silicon are commonly used as substrates for electrode
patterning, which enables the adequate adhesion of the
electrodes on the substrate.

For instance, there are a family of cytotoxicity tests based on
gold microelectrode arrays named as xCELLigence Real Time Cell
Analyzer (RTCA), which perform continuous monitoring of
cytotoxicity by electrical impedance (Ke et al., 2011). This
technology relies on the covering of 70-80% of the surface of
a well in a 96 well plate with an array of microelectrodes, being
this whole area able to measure impedance under a constant
<figure><img src="image_98.png" /><figcaption>**FIGURE 2** | (A) Schematic of a cytotoxicity testing microsystem with U-shaped sieves for cell adhesion control, fabricated by soft photolithography. Reproduced from (Wang et al., 2007) with permission from The Royal Society of Chemistry. (B) Design of an electrochemical impedance surface microelectrode array and zoomed picture of the electrodes (yellow) and the cells cultured on top (red), fabricated by electrode patterning. Reprinted with permission from (Xing et al., 2005). (C) 3D printed, reversibly pluggable microfluidic for toxicity screening. Reprinted with permission from (Ding et al., 2015) Copyright © 2015 American Chemical Society.</figcaption></figure>

voltage (Figures 2B). A variation on the impedance measurements can be attributed to the natural proliferation of cells, to cell detachment and/or to changes in the cytoskeleton-surface interaction due to the response of the cells to external stimuli such as the presence of a toxic compound.

In another work, gold Interdigitated Electrode Structures were fabricated on glass by lithographic techniques, sealed with a PDMS upper part to create a cell culture chamber (Ceriotti et al., 2007). According to the results, the system based on Interdigitated Electrode Structures detected and quantified impedance changes caused by the modulation of cellular adhesion in response to sodium arsenite, like in previously mentioned microelectrode arrays.

Furthermore, due to the resolution of photolithography, surface gold microsensors can be patterned even for the positioning of single cells. For instance Asphahani et al. (2012), used single cell surface electrodes with a diameter of 30 µm to dynamically measure the adhesion of single cells to the electrode substrate in the presence of a hydrogen peroxide (H₂O₂), sodium arsenate (NaAsO₂) or disodium hydrogen arsenate (Na₂HAsO₄). This system requires the precise position of single cells on the electrodes, but the avoidance of cell adhesion out of the electrodes is crucial too, to help decreasing the noise that can affect the measurement of impedance of single cells. To address this challenge, the authors functionalized the gold electrodes with RGD peptide to induce ligand-mediated natural cell adhesion. As just

mentioned, electrode patterning can be complemented with surface modification techniques to modify the gold electrode surface and provide specific capabilities to the sensors. For instance, Nery and co-workers integrated a microarray of gold sensors on glass through common Printed Circuit Board technology. Then, seven different selective membranes were deposited on the sensors, resulting on four ion selective electrodes for K⁺, Na⁺, H⁺, and Ca²⁺, and three partially selective membrane electrodes (amine-selective, cation-selective and anion- selective), as well as two unmodified gold electrodes for the sensing of red-ox reactions (Nery et al., 2014). The manufacturing and modification of these electrodes lead to the continuous monitoring of cellular death through potentiometric flow-through sensing, correlating changes in the media of the analytes with cellular cytotoxicity.

On the other hand, electrobiofabrication implies the use of electrode signals to specifically pattern electroactive polymers on the electrodes, which in comparison to traditional electrode functionalization strategies simplifies the fabrication of the devices, as demonstrated by Shang et al. (2018). Authors functionalized gold microelectrodes with a bio-based redox capacitor film, which enabled the *in situ* polymerization of chitosan, a redox-active polymer that amplifies the signal from an electron transfer. The system transduced molecular signals into electronic outputs, which allowed the quantification of cell viability of Caco-2 cells in presence of Triton X-100. Results were compared to the ones obtained by a live/dead fluorescent viability kit. The platform showed high sensitivity to measure molecular interactions by chronocoulometry and correlate them with cellular death. Carbon nanotubes are also promising materials for electrode manufacturing due to their high electrical conductivity, chemical stability and good mechanical properties. Aligned carbon nanotubes have been combined with surface electrode patterning techniques to fabricate sensitive floating surface electrodes. These novel electrodes became efficient tools for the electrophysiological monitoring of cells for cytotoxicity assessment (Ta et al., 2014; Ba et al., 2017). According to the authors, the system is in addition reusable, which avoids device-to-device repeatability issues.

## 3D Printing

3D printing technology was initially developed for rapid prototyping of devices, but soon, due to the improvements provided in precision, resolution, and repeatability, it became a promising technique for industrial production. Usually, traditional bio-MEM fabrication techniques, e.g., electrode patterning and soft lithography (Ho et al., 2015; Capel et al., 2018), require different types of bulky equipment and imply multiple steps and long fabrication times, in order to get the final product. Techniques like soft-lithography are fine for laboratory scale, but when industrial scale is required, simpler fabrication processes such as 3D printing, are on demand. Furthermore, 3D printing offers a range of biocompatible materials that can be printed with good resolution and in a single step, facilitating large-scale production of devices (Hart et al., 2016; Capel et al., 2018). 3D printing technology is still in its infancy when it comes to cytotoxicity assessing microdevices, but some recently
developed platforms suggest that it is a promising tool that may facilitate the manufacturing of cytotoxicity testing microdevices in the near future.

For instance, ultra-high resolution Multijet 3D printing uses UV light to polymerize the photoresist, which is deposited in tiny droplets by the printer to obtain highly complex and well-defined features. Sweet *et al.* used this technique to fabricate a microdevice, which integrated a network of interconnected 3D enclosed microchannels of 750 µm diameter, for the screening of bacterial cells' resistance (Sweet *et al.*, 2017). Due to the design and translucency of the material, UV spectroscopy based absorbance measurements were used to *in situ* determine bacterial cytotoxic response to antibiotics.

Some cytotoxicity platforms reported single 3D printed components such as a modular microfluidic cartridge to regulate reagent volumes during the analysis (Ding *et al.*, 2015) (Figures 2C) or a 3D printed gradient generator to create multi-drug combinations (Chen *et al.*, 2018). In both cases, 3D printing was combined with traditional fluorescent and absorbance cytotoxicity assays. Although just a piece of the system was fabricated by 3D printing, the versatility of the platform was increased, facilitating any modification in the design.

# PLATFORMS FOR CYTOTOXICITY ASSESSMENT BY MICROTECHNOLOGY

## Microfluidics Combined With Traditional Assays

Cytotoxicity assays are typically based on the colorimetric or fluorescent staining of cells for the determination of cell viability or for metabolite detection in order to identify specific events (Ramirez *et al.*, 2010; Stoddart, 2011). These assays are known to be sensitive and effective but, when combined with microtechnology, multiplexing and reduced manual handling and analysis times, among others, become a reality.

### Droplet Microfluidics

Droplet microfluidics provides HTP analysis in cell biology by the generation of microscale droplets through immiscible multiphase flows, resulting on picoliter-scale and monodisperse emulsion droplets. This technique leads to the miniaturization of the bioassays, avoiding capillary effects and evaporation, working as individual bio-microreactors. Moreover, the possibility of using this droplets with non-adherent cells is a reality (Mashaghi *et al.*, 2016). The number of cells encapsulated in each droplet can be slightly modulated following Poisson distribution (Mazutis *et al.*, 2013). Moreover, microdroplets are usually compatible with standard screening microscopies and readouts, enabling the combination with traditional colorimetric and fluorometric cytotoxicity assays. As an example of this combination, Du *et al.* (2013) measured two live/death fluorescent dyes within each droplet containing cells, when exposed to different concentrations of paclitaxel and 5-fluorouracil anticancer drugs. This was also demonstrated in a

previous work, in which the process to make the droplets was consciously optimized to get a good Poisson distribution, allocating 25 cells per droplet with high probability (Yu *et al.*, 2010). Cell viability was investigated in the presence of doxorubicin anticancer drug.

Boedicker *et al.* generated monodispersed aqueous droplets on chip, and tested the viability of bacterial cells in response to different antibiotics (Boedicker *et al.*, 2008). Every droplet had an integrated fluorescent viability indicator. The encapsulation was carried out in a “stochastic confinement” in order to have higher cell density per droplet, looking for a reduced detection time. In this work, cellular viability was determined in response to different concentrations of seven antibiotics. Sarkar and coworkers have extensive expertise in generating droplets on chip, including droplet formation for cell viability assessment used for the development of anticancer therapies (Sarkar *et al.*, 2015; Sarkar *et al.*, 2020). They first reported a microfluidic droplet generator for the encapsulation of single cancer cells and for the monitoring of the individual cells in presence of doxorubicin anticancer drug with live/dead staining. They evaluated the sensitivity of cells to the drug, by monitoring their viability (Sarkar *et al.*, 2015). More recently, they used the same droplet technology to compare immunotherapy with Natural Killer cells with different cell types, and determined that the treatment based on the combination of herceptin and CD16⁺ Natural Killer cells was more effective for SKOV3 cells than for HER2 (Sarkar *et al.*, 2020).

The HTP formation of droplets has also been reported on a PDMS nanowell array fabricated by soft lithography. Coupled with another PDMS holding layer for the containment of the oil, the droplets were formed when a cell suspension was added through a capillary, creating the droplets with the cells encapsulated (Du *et al.*, 2013). Ethidium bromide and calcein staining were then used to assess cell viability in the presence of 5-fluorouracil, in the droplets, by fluorescence microscopy.

On the other hand, surface engineering techniques are tools that enable patterning of droplets on surface, at specific locations. Geyer *et al.* used UV-initiated photografting of a hydrophilic polymer whose hydrophilicity was inverted by UV through a photomask to create superhydrophilic-superhydrophobic micropatterns on nanoporous polymer films, with drastic difference in wettability to spontaneously create an array of separated aqueous microdroplets (Geyer *et al.*, 2011). These patterns were then used by Popova *et al.* for cytotoxicity testing (Popova *et al.*, 2015). Each of these droplets worked as individual microreactors for cell culturing, and by a sandwiching technique, the doxorubicin anticancer drug was specifically added to the droplets. Using fluorescent viability staining, the alive and dead cells, in the presence of the doxorubicin, were observed in a HTP and pipetting-free way (Figures 3A).

### Gradient Based Microsystems

The formation of gradients in microfluidic devices has applications in the field of cytotoxicity testing since gradient generators enable the screening of different concentrations of a
<figure><img src="image_99.png" /></figure>

certain toxic compound at a single step (Dai et al., 2010) (Figures 3B). Ruan et al. (2009) reported a hybrid glass-PDMS microfluidic device with a multi-serpentine based gradient generator made by soft lithography. Cellular viability in response to different concentrations of AsO₃ and buthionine sulfoximine chemicals was investigated using acridine orange/ethidium bromide viability assay by fluorescence. When a multi-serpentine based gradient generator is combined with a high-density single-cell array, it results in a microsystem for simultaneous cytotoxicity assessment of numerous concentrations of KCN in this case, with single-cell resolution (Hosokawa et al., 2011). However, the flow-rates commonly used in serpentine gradient generators can be harmful for some cell types such as neurons, so in a recent work, an asymmetric microfluidic device was reported for the formation of drug gradients, which requires lower flows to generate gradients, as mentioned in the section above (Mo et al., 2020).

A multiple dilution method was used in a PDMS microfluidic device to improve the creation of a concentration gradient solution, avoiding distinct parts of the cell experiencing different drug concentrations during the test (Dai et al., 2010). Using Fluorescence Resonance Energy Transfer authors were able to measure cellular death by means of CFP-YFP fluorophores, performing simultaneous cytotoxicity testing of multiple concentrations of etoposide anticancer drug in parallel. The use of hydrogels is another approach to generate gradients in microfluidics. Hydrogels are able to form gradients due to their porous internal structure, which can be interestingly modulated to fulfill specific concentration requirements (Ahadian et al., 2014; Cosson and Lutolf, 2014). As an example, an agarose gel on a microelectrode patterned device allowed the cytotoxic electrochemical monitoring under different concentrations of cisplatin and paclitaxel chemotherapy drugs, which were created through the hydrogel (Tran et al., 2013).
3D printing technology opens the possibility of designing all kind of microfluidic circuitry, including the fabrication of advanced gradients (Sochol et al., 2016). Microsystems capable of generating a gradient of three (nitrofurantoin, tetracycline and trimethoprim) (Sweet et al., 2017) or four (doxorubicin, celecoxib, 5-fluorouracil, and cyclophosphamide) (Chen et al., 2018) toxic compounds allow the monitoring of the cytotoxic response against many combinations of compounds in a single run.

## Multiplexing

Miniaturization offers multiplex analysis on chip and it has been reported for many bio-applications, increasing the number of analysis with single cell resolution and so cytotoxicity studies (Kersten et al., 2005; Zhao et al., 2015). Wang and co-workers developed a microfluidic device for multiplex cytotoxicity testing (Wang et al., 2007). The device had several independent cell culturing chambers with U-shaped micro sieves designed to allocate about ten cells per sieve, guaranteeing cell-cell contact, adequate stimulation and comfort. That design enabled the HTP cytotoxicity testing of three cell lines and two concentrations of five toxins (digitonin, saponin, $CoCl_2$, $NiCl_2$ and acrolein) simultaneously.

Droplet microfluidics provides with a different approach to perform simultaneous cell viability assessment, testing different concentrations of doxorubicin and daunorubicin drugs. Based on a hydrophilic/hydrophobic patterning, droplet arrays, working as independent bioreactors, can be individually loaded with the toxic compound of interest or concentrations of the toxic, which leads to HTP and even multiplexing analysis of cytotoxicity using fluorescent live/death assays (Popova et al., 2015; Popova et al., 2017).

Another multiplexing approach relies on the analysis of two different events in parallel, instead of measuring the cellular response to different toxic compounds or concentrations. A microsystem was reported for the characterisation of drug metabolites and for the analysis of their cytotoxic response (Ma et al., 2009). The microsystem had a PDMS bottom layer with three microwells with sol-gel containing encapsulated cells. On top, a quartz layer with a microfluidic network allowed the flow of metabolites (Figures 3C). Due to the multilayer design of the device, the sol-gel acted as a filter to separate drug metabolite detection and cytotoxicity testing, successfully fulfilling both analysis with no cross contamination.

Multifunctional materials deserve special attention for multiplexing. In particular, hydrogels are widely applied as nanocarriers for drug delivery purposes due to their stimuliresponsive behavior. Nanocarriers are easily captured by cells, so, their combination with a sensing functional material could lead to the simultaneous drug release and monitoring of the process (Wu et al., 2010). For instance, Wang and co-workers developed hybrid microspheres made of pH responsive acrylic acid and temperature responsive poly-*N*-isopropilacrylamide, combined with $SiO_2$ photonic crystal microspheres, which are dielectric structures with an energy band that favors the movement of photons (Wang et al., 2018). Temperature and pH responsiveness of these hybrid and inverse opal hydrogel photonic crystal

microspheres, enabled the controllable drug loading/release, whereas due to the photonic crystal component, the cellular viability was dynamically monitored at every point during drug release. Actually, when cells were alive, the microenvironment surrounding them was acidic due to their secretion, green, but when the drug was released and apoptosis was triggered, the pH of the microenvironment was neutralized, changing the color of the spheres to red, which could be observed even by naked eye.

## Combinatorial Drug Screening

As mentioned above, cytotoxicity tests are crucial in environmental and pharmacological industries, especially when the toxicity of a compound has to be assessed. Toxicology studies are not simple since the effect of a toxic compound can vary pretty much when in contact with other chemical compounds, sometimes causing an additive effect. However, the combination of more than one chemical or drug causes sometimes synergy (greater effect) or antagonism (lower effect), which may be considered in order to address their toxicity, or their effectiveness in case of therapeutic studies (Foucquier and Guedj, 2015). Therefore, combinatorial-drug-testing can decrease toxicity, reduce the development of drug resistance and even increase the effectiveness of therapies.

Combinatorial tests can be either based on the sequential or simultaneous addition of more than one toxic compound. Considering the first situation, a microsystem based on droplet arrays was reported for sequential toxic combinatorial testing in where the cells in the droplets were sequentially stimulated by two drugs, doxorubicin and daunorubicin (Popova et al., 2017). The microsystem enabled the visualization of cellular viability by fluorescence, combining live/death fluorescent staining. The results showed variations in cell viability when using a single drug or the sequential addition of the two drugs.

Sweet *et al.* developed a platform for simultaneous cytotoxicity analysis of more than one toxic, generating a gradient with the combination of three different compounds (Sweet *et al.*, 2017). In addition, the platform enabled the identification of the relationship between the toxics. Binary combinations between the toxics correlated well with the results previously published in literature (Bollenbach, 2015). On the other hand, the combination of three toxics identified antagonistic (less effective), synergistic (more effective) and suppressive (least effective) regions during drug screening.

Curved microchannels for fluid mixing driven by Dean flow dynamics -the study of flow in curved channels- were integrated in cytotoxicity screening microsystems to generate gradients and mixing of three compounds, two anticancer drugs (doxorubicin and cisplatin) and the cell media, leading to highly efficient fluid mixing and single-step screening of twelve different combinations (Shen et al., 2020). The twelve combinations were simultaneously investigated for cell viability using a fluorescent live/death assay.

Moreover, a microdevice capable of performing combinatorial screening of two drugs (doxorubicin and mitoxantrone) was reported by Kim *et al.* (2012), either simultaneously or sequentially. The platform contains 64 individual cell-culture
chambers, avoiding the metabolites to be transferred from one chamber to another (Figures 3D). The design of the device allowed the sequential combination of compounds using an accurate gradual valve-opening process, first with one of the toxics, and 24 h after, with the second one. The system showed high screening capability and, with the integrated valve-system, the common drawbacks of existing microdevices on metabolite transference to different cell culturing chambers was sorted out.

Moreover, synergistic effect can be also obtained combining photo-therapy with chemotherapy, so Flont *et al.* evaluated the effect of doxorubicin drug and nano-encapsulated meso-tetraphenylporphyrin photosensitiser on chip (Flont *et al.*, 2020). Using a microfluidic device mimicking the fluid flows, photodynamic therapy with a photosensitiser and chemotherapy with doxorubicin were sequentially applied to non-malignant and cancer ovarian cells simultaneously, demonstrating the synergistic effect of both therapies, leading to a higher rate of death cancer cells according to MTT viability assay.

## Label-Free Microsystems

Common cytotoxicity assays based on enzymatic activity or membrane penetration processes require cell labelling for viability studies. Their widely spread use relies on their elevated effectiveness, reliability and sensitivity, which do not compromise cellular functions such as growth and proliferation. Many efforts are being done to enhance their signal and durability during continuous observation under excitation, e.g., conjugating them with quantum dots to avoid photobleaching over long time exposure to light (Zhao *et al.*, 2009; Zhao *et al.*, 2013). Dye labelling, is time consuming, implies multiple steps and often only detects end points, not allowing continuous monitoring, without showing kinetic or dynamic data. Generally, dyes do not drastically affect essential cellular functions such as growth and proliferation, but there are evidences claiming that cell tracing dyes considerably increase living cell stiffness, deeply increasing the Young's modulus of the cellular membrane and therefore enhance cellular rigidity and adhesion (Lulevich *et al.*, 2009). Hence, there is a current demand on label-free cytotoxicity monitoring systems for the dynamic analysis of cells upon toxic exposure. Microtechnology brings new approaches to assess cytotoxicity, correlating alternative parameters with cellular death, such as cell adhesion or mechanics, which can be hardly measured without microtechnology.

## Microsystems Based on Cell-Substrate Interaction

Cell-substrate interactions are key in most of the vital cellular processes, such as proliferation, differentiation and migration. Regardless of the cell death mechanism, when cells are in the presence of a toxic, undergo morphological changes or disturbances in their membrane, compromising its integrity, which may affect cell adhesion to the substrate (Cho *et al.*, 2008). In the past decades, electrochemical impedance spectroscopy has settled as one of the most popular techniques for the dynamic analysis of cell adhesion to a substrate (Ke *et al.*, 2011), named as electric cell-substrate impedance sensing. The microsystems following this working principle, are based on an array of surface gold electrodes manufactured by lithographic

techniques. Cells act as insulating particles, when they adhere to the electrodes, the area in contact with the electrolyte coming from the cell culturing medium is reduced, resulting on an increase on the electric resistance and a decrease in the capacitance values (Hug, 2003). Higher resistance and/or lower capacitance imply a higher density of cells covering the electrode. Based on this technology xCELLigence RTCA emerged for the dynamic monitoring of cellular adhesion on a microarray of gold sensors. In general, when cells are in the presence of a toxic compound, cell death is preceded by a number of morphological and biochemical pathways that at the end lead to death. Impedance based cell adhesion biosensors, enable continuous monitoring of cellular responses against a toxic, such as morphological changes, migration and death (Ke *et al.*, 2011). Using this technology, several studies reported applications on cytotoxicity assessment and showed real-time and dynamic monitoring of cellular responses to different toxic compounds (Xing *et al.*, 2005; Urcan *et al.*, 2010; Irelan *et al.*, 2011; Menotti *et al.*, 2017). Even though electrochemical impedance continuously decreases in the presence of the drug, this value cannot be directly correlated with cellular detachment until signal reaches 0. Electrochemical impedance has been proven sensitive for the monitoring of cellular response to toxics. Nevertheless, until signal does not reach 0 (when there is no cell on the electrodes), due to that sensitivity, the platform may be probably detecting not only cellular detachment, but also changes in morphology caused by the presence of the toxic, and not just detachment or death (Atienza *et al.*, 2005; Xing *et al.*, 2005; Abassi *et al.*, 2009; Susloparova *et al.*, 2015). In order to confirm the correlation between change in impedance signal and quantification of cellular death, xCELLigence RTCA can be coupled with common fluorescent assays for a deeper understanding of the cytotoxic response.

Surface chemistry modification at the micro-scale enables the precise positioning of cells, which following specific adhesion patterns, can lead to new functionalities. In a recent work, microcontact-printing, which creates defined patterns of molecules on surfaces by stamping a micropatterned PDMS, was used to create single cell adhesion dots arrays to address cell viability by optical microscopy (Garcia-Hernando *et al.*, 2020). Cell adhesion spots made by fibronectin protein were designed to allocate single cells, attributing an individual value to each cell. Due to this individuality, the technique enables the counting of adhered cells at every point, so, cellular detachment in the presence of $K_2CrO_4$ and DMSO chemical compounds was dynamically monitored for several hours, giving real time and quantitative information of cellular death (Figures 4A).

Piezoelectric quartz systems have also been proven useful for live cell biosensing, due to their excellent mass sensing capabilities, leading to the development of mass nanosensors called quartz crystal microbalances (Wang *et al.*, 2011). A quartz crystal microbalances based benchtop platform, comprised of a microplate with a 2 × 2 mm resonant waveguide grating sensor per well was used to monitor cytotoxicity, based on the changes in the dynamic mass redistribution (Farkas *et al.*, 2018). The employed microplate design allowed 384 simultaneous and high-resolution measurements every 3 s, per well. Using the
<figure><img src="image_100.png" /></figure>

**FIGURE 4** | Microsystems for label-free cytotoxicity testing. (A) Quantification of cell single-cell adhesion for cytotoxicity assessment by optical microscopy. Adapted from (Garcia-Hernando et al., 2020) Copyright © 2020 American Chemical Society. (B) Working principle of the electrophysiological monitoring of cells for cytotoxicity assessment on aligned carbon nanotubes based gold floating electrodes Adapted with permission from (Ta et al., 2014) Copyright © 2014 American Chemical Society. (C) Schematic of the Photonic crystal microspheres based cytotoxicity platform, showing the control variation under external stimuli for the detection of cell viability. Adapted from (Wang et al., 2018) with permission from The Royal Society of Chemistry. (D) Cytotoxicity monitoring system based on nanomotion sensing of cellular processes. Adapted with permission from (Ruggeri et al., 2017) Copyright © 2017, Springer Nature.

mentioned mass nanosensor, cell death was detected as a negative shift in the dynamic mass redistribution in the presence of toxic compounds. The cytotoxic response is represented in terms of cell size reduction, weakening the adhesion strength and promoting cellular detachment. Therefore, the platform was able to perform non-invasive and continuous measurements for real-time and dynamic monitoring of cytotoxic effects of Roundup Classic, polyethoxylated tallow amine and glyphosate herbicides.

## Microsystems Based on the Detection of Specific Molecules

A potentiometric flow-through sensor was reported for label-free cytotoxicity testing. Instead of monitoring cell-substrate adhesion, gold sensors were specifically manufactured and modified to detect multiple parameters that were indirectly correlated with cellular death, such as ions and amines. The potentiometric selective sensing of cations, anions and amines,

coupled with partial least squares multivariate statistical analysis, led to non-invasive measurements of cellular viability (Nery et al., 2014).

Quantitative electrophysiological determination of cytotoxic responses in the presence of chemical compounds such as histamine, anti-histamine drugs (chlorphenamine and cetirizine) and acetylcholine receptors (nicotine, daidzein, genistin, and tamoxifen) was carried out using carbon nanotube transistors (Ba et al., 2017; Ta et al., 2014) (Figures 4B). For instance, histamine is a neurotransmitter whose presence in the cellular microenvironment leads to the activation of numerous intracellular electrophysiological pathways, including the opening of the $Ca^{2+}$ channel gates and the flow of these ions through the plasma membrane. Therefore, due to the quantitative electrochemical signals detected by the floating microsensors, the evaluation of the effect of anti-histamine drugs on non-excitable HeLa cells was carried out (Ba et al., 2017).
Beyond electrochemistry, pH sensing for the label-free and optical assessment of cytotoxicity has also been reported. Photonic crystals are periodic optical nanostructures with very interesting optical properties, due to their atomic energy gap that enables the movements of photons. Photonic crystal microspheres were reported for the colorimetric assessment of cellular viability even by naked eye (Wang et al., 2018). The microspheres were green when cells were alive, because their secretions made their surrounding microenvironment acidic. However, when cells were in presence of the toxic compound and their death was induced, the photonic crystals turned to red because of the neutralization of the cellular microenvironment (Figures 4C).

## Mechanics to Monitor Cytotoxicity

The study of cellular mechanics offers a new approach to cytotoxicity assessment, based on a better readout at the cellular level. Cell mechanics results from the overall cellular events, so its study permits to observe the state of the general well-being, but also going to individual cellular events that in conventional assays would be hidden within intrinsic variability.

Due to its precision, atomic force microscopy is a powerful tool for nanomotion sensing in single-cell cytotoxicity tests (Zimmer et al., 2014; Ruggeri et al., 2017). Ruggeri et al. (2017) monitored nanometer-scale movements of cells with the nanomotion sensor, while complementary optical microscopy served to monitor cell attachment, cellular healthiness and micrometer scale movements (Figures 4D). Adding a fluorescent live/dead dye, loss of membrane integrity prior cell death was also detected by nanomotion sensing. Moreover, the cytotoxic responses to amyloids were detected and measured real time.

Single-cell compression method by means of atomic force microscopy can also accurately measure single-cell mechanics in nanotoxicity assessment. Zimmer et al. (2014) used single-cell compression following a protocol previously developed by their group (Lulevich et al., 2006), relying on compressing the cell between a spherical probe and a glass substrate. The cytotoxicity test carried out with this method enabled accurate single-cell mechanic profiles (shrinking, swelling and estimation of Young's modulus) leading to highly sensitive monitoring of nanotoxicity.

## SINGLE CELL RESOLUTION

There is a wide range of options for cytotoxicity assessment of cell populations, including microsystems, bringing new insights in cytotoxicity assessment, such as the HTP capability, multiplexing, gradient formations and label-free dynamic monitoring of cellular viability. However, cell populations are highly heterogeneous, and single cell toxicity analysis could make a difference, identifying cell subpopulations that present higher or lower resistance to a certain dose of a toxic compound. There are two main techniques for the isolation of cells as single cells. On one hand, individual cells can be confined in cavities (Hosokawa et al., 2011), and on the other hand, they can be chemically guided to settle at specific locations by protein patterning techniques

(Gonzalez-Pujana et al., 2019), enabling high density of single-cell arrays. Single-cell toxicity testing can provide with quantitative analysis. Fluorescent and colorimetric signals give qualitative data or relatively quantitative values in terms of intensity. However, the identification of single cells as dead or alive provides real numbers and percentages of cellular death, which is actually the basis of the well-stablished flow cytometry technique.

Optical microscopy and label-free monitoring of cytotoxicity is also possible for single-cells. In the recent work from Garcia-Hernando et al., previously mentioned, an array of individual cell adhesion fibronectin islets was used to create single cell arrays for the monitoring of cytotoxicity by optical microscopy (Garcia-Hernando et al., 2020). This system offers two approaches for the assessment of cell viability, depending on the mechanism of action of the toxic compound. The results suggest that when fibronectin-integrin bonding keeps cells attached to the surface, under the effect of some compounds such as $K_2CrO_4$ and DMSO cells detach when they die. In this case, the system allows the quantification of cell detachment correlated with cellular death. Contrarily, other chemical compounds such as $Hg_2SO_4$ inhibit cell detachment when they die, keeping them dead and individually located on a surface. Adding a colorimetric viability staining, the viability of individual cells can be quantified by optical microscopy.

Agarose platforms, with microcavities for single-cell positioning by gravity, have been reported to measure cellular viability in the presence of nimustine DNA-crosslinker (Li et al., 2016). This microsystem enabled fluorescence microscopy based cellular viability assessment, after staining the cells with a fluorescent live/death assay, giving real percentages of death cells within a population. Hosokawa et al. (2011) also developed a microfluidic device with an array of microcavities to obtain high density single-cell arrays. Those cells were incubated in the presence of KCN and stained with a fluorescent live/death dye, identifying each cell as alive or dead, and giving real quantitative information of cellular viability, with the added value of a gradient generator, leading to a HTP screening of individual cells. In this way, cells were cultured as a population but analyzed as individual cells (Figures 5A). In another work, authors reported a single-cell array cytometry on chip for the analysis of tumor cell apoptosis induced by staurosporine anticancer drug. The microdevice was comprised of 440 mechanical traps of 18 µm width for the hydrodynamic positioning and mechanical trapping of single-cells (Wlodkowic et al., 2009). A fluorescent live/death staining was continuously flowed through the microfluidic device, in a low dose, in order to achieve a continuous labelling without deeply compromising cellular viability, due to the presence of the dye. The system was able to analyze the viability of 250 cells every 1 min, giving real percentages of cellular viability due to the single cell counting. In this particular case, authors obtained dynamic and real-time results of toxic induced cellular death thanks to the continuous loading of a low concentration of fluorescent viability tracker.

The study of individual cells is important due to the heterogeneity within populations. Electrochemical impedance demonstrates that the dynamic cytotoxic response of
<figure><img src="image_101.png" /></figure>

**FIGURE 5** | Platforms for cytotoxicity assessment of single-cells and non-adherent cells. (A) Gradient generating microfluidic device based on high density single-cell arrays positioned in microcavities for HTP single-cell cytotoxicity testing. Reproduced with permission from (Hosokawa et al., 2011). Copyright © 2011 American Chemical Society. (B) Digital microfluidics device for single-cell toxicity testing based the entrapment of single non-adherent cells. Adapted from (Kumar et al., 2015) with permission from The Royal Society of Chemistry. (C) Microfluidic platform for the cytotoxicity assessment of single-cells isolated by hydrodynamic forces using integrated, dam cell-trapping structures. Adapted with permission from (Zhao et al., 2009) Copyright © 2009 American Chemical Society.

individual cells varies from one to others (Asphahani et al., 2012). In this work, the authors reported that not all the single cells gave the same impedance curve, meaning that the cytotoxic response was different from one cell to another, as a clear sign of heterogeneity within the population. Furthermore, the differences in the cytotoxic response were more evident at lower concentrations of the toxic compounds (H₂O₂, NaAsO₂ and Na₂HAsO₄), giving curves with higher variability. At higher concentrations of toxic compounds, cytotoxic impedance profiles were more similar between single cells, as a sign of high lethality.

Droplet microfluidics can be used for the encapsulation of isolated single-cells by, for instance, coupling droplet formation with an array of microcavities for the deposition of the encapsulated single-cells. An individual cell containing droplet array coupled with a viability fluorescent assay enabled the quantification of individual dead and alive cells, showing also variable cytotoxic responses to antifungal Amphotericin B, within the studied population (Kumar et al., 2015).

Cellular mechanics provide very specific information at the cellular level, offering an alternative readout of the molecular outputs of cellular events. Nanomotion is a promising technique to monitor the cellular well-being, which due to its sensitivity, can monitor cellular movements at the nano-scale in response to

α-syn presynaptic protein and SiO₂ nanoparticles (Zimmer et al., 2014; Ruggeri et al., 2017).

## NON-ADHERENT CELLS

Most of the current existing microsystems for cytotoxicity assessment are meant to be used for adherent cells, since working with them is less tedious and complex than manipulating non-adherent ones. Cytotoxicity assessment of non-adherent cells is mainly performed by flow cytometry, with accurate and reliable results, but again, involves handling of the sample and a temporary gap from the time the experiment is stopped until the time when analysis takes place (Jahan-Tigh et al., 2012). Advances in non-adherent cell viability systems are on demand, since cytotoxicity testing of, for instance, blood cells and circulating tumor cells are crucial in the biomedical industry.

There are not many options available for the entrapment and subsequent analysis of non-adherent cells, being hydrodynamic forces one of them. A microdevice with integrated dam structures for cell-trapping was used to trap cells by hydrodynamic forces, which were applied by an appendant microfluidic channel (Zhao et al., 2009). Once the non-adherent cells were positioned and
<figure><img src="image_102.png" /><figcaption>**FIGURE 6** | Automation potential of microsystems for cytotoxicity assessment. (A) Automated imaging by fluorescence microscopy. Reproduced with permission from (Li et al., 2018). Copyright © 2018 American Chemical Society. (B) Automatable microfluidic system for combinatorial cytotoxicity screening. Reproduced from (Kim et al., 2012) with permission from The Royal Society of Chemistry.</figcaption></figure>

localized, a live/death fluorescent tracker was used and their intensities were obtained and correlated with the cytotoxic response to the presence of cycloheximide, etoposide and camptothecin antileukemic drugs (Figures 5B).

Droplet microfluidics can be used as tools for the encapsulation and confinement of non-adherent cells. Popova and co-workers demonstrated that an array of droplets made on hydrophilic spots enables the encapsulation and cytotoxicity assessment of T-lymphocytes (Popova et al., 2017). Besides, digital microfluidics permits the confinement of non-adherent individual cells in droplets using an electrowetting-on-dielectric platform. The individually encapsulated cells were positioned as an array of single cells. Then, a stain with cell live/death trackers was used for viability individual cells studies by fluorescence microscopy (Kumar et al., 2015) (Figures 5C).

Floating gold electrode sensors, based on aligned semi-
conducing single walled carbon nanotubes, were used for
dynamic cytotoxicity monitoring of non-adherent small lung
cancer cells in the presence of nicotine and inhibitors of
nicotinic acetylcholine receptors, measuring cell's
electrophysiological responses (Ta et al., 2014). When these
cells are in a nicotine containing environment, the opening of
the Ca²⁺ channels of the plasma membrane is triggered, causing
Ca²⁺ to flow inside of the cell. This results in a depolarization of

the cell, resulting on an increased negative current detected by the
electrodes. The sensitivity of the technique enabled the
cytotoxicity testing of single-cells.

AUTOMATION

Microfabrication enables the integration of several components within the same microsystem, leading to the development of fully integrated devices. They can perform automated processes avoiding handling operations, reducing sample loss and contamination risk, increasing throughput, improving the ease of use and repeatability for, at the end, reduce human errors. Moreover, thanks to automation, remote operation of the microsystems (fluid control and analysis) is possible in case it is required for a particular application (Melin and Quake, 2007).

Many efforts have been made in the automation of microsystems for cytotoxicity testing. Due to the wide use of fluorescent live/dead trackers, automated image processing has been reported in cytotoxicity platforms (Hosokawa et al., 2011; Popova et al., 2017; Li et al., 2018) (Figures 6A). This simplifies data collection, since the platforms can be programmed to take microscopy pictures periodically, for their subsequent analysis, without the need of human supervision.
Protocols based on electrochemical monitoring are also compatible with programming during data acquisition, in a dynamic way, far from end-point measurements. xCELLigence RTCA technology is a good example of that, since the manual sample handling is limited to loading the cells into the platform. From this point on, the system continuously measured impedance over time, collecting automatically dynamic data of cells during cytotoxic response (Gurkan et al., 2011; Ke et al., 2011).

Data interpretation can be automated, for instance, when combinatorial toxic screening is performed. The effect of the combined toxics and their concentrations can be automatically uploaded to a combinational library of chemical compounds, facilitating searching for toxic combinations, speeding up the discovery process of unexpectedly harmful toxics or in contrary, finding the most efficient drug combinations for therapy (Ding et al., 2015). On the other hand, Sarkar et al., used algorithms to assess cytotoxicity mediated by Natural Killer cells. A droplet microfluidics generator was used to trap a target cancer cell with a natural killer cell (1:1 ratio), and the interaction between them was accurately studied by time-lapse images, focusing on interaction duration, frequency and cell viability. With the obtained information, algorithms were developed for automatically interpreting the dynamics of natural killer cells as anticancer therapy (Sarkar et al., 2020).

As an example of automated performance is the microfluidic system for sequential combinatorial therapy testing developed by Flont et al. (2020). Going to more complex automation systems, Kim et al., developed a fully programmable microsystem for drug screening (Kim et al., 2012). The platform is comprised of an upstream concentration generation module coupled to an array of 64 individual chambers for cell culturing. The access to each of the cell culture chambers is individually modulated with a valve-array through a pneumatic channel designed to deliver the desired toxic combination to the chambers, controlled by Labview software (Figures 6B). The synchronized valve-system opens the door for a complete automation of the system. This platform simplifies the screening of toxic compound combinations in the investigation of novel effective therapies against cancer.

## NANOTOXICOLOGY

Nanotoxicology is a subfield of toxicology that focuses on the toxic effects of nano-sized particles, often not caused by the material itself, but by the nano-scale related properties of these particles (Auffan et al., 2009; Catalán et al., 2016; Fadeel, 2019). For instance, the high surface-to-volume ratio of nanoparticles makes them highly reactive when compared to bulk materials (Recordati et al., 2015). Nanoparticles can penetrate cellular membranes, biological barriers and tissues, so they interact with biological systems at the molecular, cellular, organismal and ecosystem levels (Pietroiusti et al., 2018). The increasing interest of using nanoparticles in medicine, opens new possibilities for microtechnology, for instance to mimic whole organs (Organ-on-A-Chip technology), generating devices able

to precisely study the effect of nanomaterials in a similar environment to *in vivo* (Ashammakhi et al., 2020).

Due to the capability of engineered nanomaterials to interact with the cellular membrane—by adsorption, penetration or endocytosis—the study of the interaction between nanomaterials and the cell membrane is important and challenging (Zhang et al., 2012; Churchman et al., 2013). Membrane-on-chip microplatforms emerge for high throughput membrane sensing, giving information of the events happening on a supported cellular membrane. The most used membrane biosensing microtechnology comprises electrochemical sensors fabricated with self-assembled monolayers supported on mercury electrodes that provide information of the interactions or damage on the membrane (Owen et al., 2020; Rashid et al., 2017). Recently, Owen et al. reported an automated, rapid and HTP sensing microfluidic platform for the screening of nanomaterial-biomembrane interactions (Owen et al., 2020). The integrated microfluidic device provided electrochemical information of the interactions of chlorpromazine and citrate coated gold nanomaterial with 1,2-dioleoyl-sn-*glycero*-3- phosphocoline membranes, supposing an easy-to-use interface to assess cytotoxicity based on membrane damage (less than 6 min per assay).

Due to the high toxicity and barrier penetration capability of nanoparticles, the studies of their effects on three-dimensional environments, mimicking natural biosystems, enable more reliable approaches to evaluate the physiological effects of nanomaterials (Ashammakhi et al., 2020). For instance, Arends et al. reported a microfluidic device to study the diffusion of nanoparticles through the basal layer, which is key to control the movement of nanoparticles between blood vessels and the surrounding extravascular space, observing a charge dependant accumulation of the species, similar to the one observed *in vivo* (Arends et al., 2015).

## CONCLUSION

Cytotoxicity assessment is vital in the pharmaceutical and environmental industry for the identification of potentially harmful compounds, as well as for the development of new therapies in medicine. Currently, the most popular techniques are based on labelling cells with fluorescent or colorimetric stains, targeting disrupted membranes of dead cells or specific metabolites. These are very sensitive and effective, but the application of microtechnology in cytotoxicity testing opens the door to more efficient methods that can also be complemented with traditional assays.

Within microtechnology, many techniques have been proven efficient for reliable and sensitive cytotoxicity assessment. It must be highlighted that working at the microscale, HTP approach is possible. Moreover, multiplexed and gradient cytotoxicity assessment systems, capable of collecting higher amounts of data for the correct interpretation of toxicity, become available for general use. Many microdevices for combinatorial toxic screening have been reported, which facilitate research on the
identification of potentially toxic compounds, enabling the
development of more effective therapies in medicine.

Furthermore, the use of microtechnology for cytotoxicity
testing permits the measurement of cellular events such as
cell-substrate adhesion, electrophysiology and mechanics of
cytotoxicity in a label free manner, avoiding the addition of
external labels that even at a low level, could affect cellular
functions. Additionally, label-free sensing allows dynamic and
continuous monitoring of cells during testing, providing to the
user with additional data, e.g., continuous monitoring of cellular
well-being, even before death, as well as kinetic responses against
toxic compounds.

Besides, cytotoxicity assessment at single-cell level is possible
using microtechnology. This enables the study of cytotoxic
responses of cell subpopulations within heterogeneous
populations, which helps investigating cellular resistance, as
well as vulnerabilities of cells to chemical compounds. Single-
cell resolution also enables real quantification of cellular viability,
which would bring cytotoxicity assessment platforms close to the
concept of flow cytometry on a surface. Moreover, the assessment
could be automated using programmable microdevices, which
facilitates their use, leading to more efficient analyses, reducing
human handling and thus, errors.

Nanotoxicology is a recent field within cytotoxicity assessment which is growing fast, because of the emerging interest in nanoparticles for therapy. Reliable information on the effects of engineered nanoparticles on biosystems is crucial, and microtechnology is emerging as a powerful tool for screening nanoparticle—biological system interactions.

Despite the previous advances, microtechnology is still far for
being implemented in daily cytotoxicity assessment. Although the
presented micro-systems have been successfully implemented for
cytotoxicity assessment, their fabrication is still laborious and
long, and it sometimes require specific micromachining
equipment, complicating the mass production of devices. The

use of microplatforms for cytotoxicity testing is very useful for
now when it comes to specific cytotoxic measurements or
information that cannot be obtained with traditional assays,
such as cytotoxicity dynamics, real-time monitoring, gradient
formation and multiplexing; or information that can be obtained
easily and faster than with traditional tools, such as single cell
testing or combinatorial testing. However, microtechnology
seems to have already a place in cytotoxicity assessment, and
it is lately gaining importance and popularity, considering the
increasing number of publications appearing every year with
novel approaches. It can be expected that new trends in this field
will focus on the improvement of the fabrication of the devices,
simplifying processes for potential mass production and looking
for suitable materials with properties that permit a good
performance of the analyses. Therefore, evidence points out
that the future of cytotoxicity assays will be highly conditioned
by the developments made in microtechnology.

AUTHOR CONTRIBUTIONS

MGH: Conceptualization, Ideas, Investigation, Writing - Original Draft, Writing - Review and Editing. FBL and LBD: Conceptualization, Ideas, Resources, Management, Writing - Review and Editing, Supervision, Project administration, Funding acquisition.

ACKNOWLEDGMENTS

Authors would like to acknowledge the funding support from:
University of the Basque Country (PIF16/204), the funding
support from Gobierno de España, Ministerio de Economía y
Competitividad, with Grant No. BIO2016-80417-P (AEI/FEDER,
UE) and Gobierno Vasco under grand IT1271-19.

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Garcia-Hernando, Benito-Lopez and Basabe-Desmonts. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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# Dynamic Wetting of Molten Polymers on Cellulosic Substrates: Model Prediction for Total and Partial Wetting

Monica Francesca Pucci¹*†, Benoît Duchemin², Moussa Gomina¹ and Joël Bréard²

¹CRISMAT, UMR 6508 CNRS, ENSICAEN, Caen, France, ²LOMC, UMR 6294 CNRS, Normandie Univ ULH, Le Havre, France

This work consists in an experimental investigation of forced dynamic wetting of molten polymers on cellulosic substrates and an estimation of models describing this dynamic. A previous work of Pucci et al. (2018) showed that for totally wetting liquids (as paraffin oils), temperature-induced variations in dynamic wetting are included into the capillary number (Ca) and then a master curve of dynamic contact angle ($\theta_d$) as a function of Ca can be obtained. The hydrodynamic theory (HDT) correctly describes the dynamic wetting for $Ca > 2 \cdot 10^{-3}$. For lower Ca, a change in the dynamic wetting behavior was observed. Here, partially wetting liquids (polyethylene glycols, a.k.a. PEGs) at different molecular weight (Mn) were used at temperatures above their melting point to investigate the dynamic wetting behavior on cellulosic substrates for a large range of Ca. It was found that the dynamic curves of $\theta_d$ vs. Ca depend on Mn. Moreover, the HDT correctly describes the experimental measurements for $Ca > 2 \cdot 10^{-3}$. Below this threshold the dynamic contact angle decreases toward the static one. A linear correlation between parameters obtained fitting the HDT and the molecular weight of polymer was found. The prediction of dynamic wetting for low $Ca$ ($Ca < 2 \cdot 10^{-3}$) with the molecular kinetic theory (MKT) was also evaluated and discussed.

**Keywords:** dynamic wetting, capillary effects, molten polymer, molecular weight, cellulosic films

OPEN ACCESS

**Edited by:**

Patricia Krawczak,
IMT Lille Douai, France

**Reviewed by:**

Veronique Michaud,
Federal Institute of Technology in
Lausanne, Switzerland
Christophe Binetruy,
Ecole Centrale de Nantes, France

*Correspondence:

Monica Francesca Pucci
monica.pucci@mines-ales.fr

†Present address:

Monica Francesca Pucci,
LMGC, IMT Mines Alés, UMR 5508
CNRS, Univ Montpellier, Alés, France

**Specialty section:**

This article was submitted to
Colloidal Materials and Interfaces,
a section of the journal
Frontiers in Materials

Received: 17 March 2020

Accepted: 24 April 2020

Published: 19 May 2020

**Citation:**

Pucci MF, Duchemin B, Gomina M and Bréard J (2020) Dynamic Wetting of Molten Polymers on Cellulosic Substrates: Model Prediction for Total and Partial Wetting. *Front. Mater. 7:143.* doi: 10.3389/fmats.2020.00143

## 1. INTRODUCTION

Dynamic wetting occurs in many everyday situations, when water flows on a floor tile or pearl on a plant leaf for example. It concerns different industrial domains and among them, composite manufacturing (Bréard et al., 2003; Verrey et al., 2006; Park et al., 2011; Pucci et al., 2015, 2017b; Caglar et al., 2019). In this case, the flow of a molten polymer in a fibrous preform is governed by a competition of capillary and viscous effects that are included in the dimensionless capillary number $Ca$ (Ruiz et al., 2006):

$$Ca = \frac{\eta\nu}{\gamma_{LV}} \qquad (1)$$

where $\nu$ is the liquid velocity, $\eta$ and $\gamma_{LV}$ are the dynamic viscosity and the liquid surface tension, respectively. Dynamic wetting phenomena are described by a dynamic contact angle $\theta_d$ depending on the Ca. Forced dynamic wetting occurs when the liquid velocity $\nu$ is set via an external force, in
contrast to the spontaneous wetting where only capillary effects drive the flow (Sauer and Kampert, 1998; Mohammad Karim et al., 2016). In all cases, $\theta_d$ is defined as an advancing contact angle ($\theta_a$), when the triple contact line advances on the dry solid surface with a positive convention of v and Ca, or a receding contact angle ($\theta_r$) when it recedes from the wetted surface with a negative convention of v and Ca (De Gennes et al., 2013). For v ≈ 0 m/s (at static conditions) the angle formed between the liquid and the solid is defined by an equilibrium contact angle ($\theta_e$), according to the Young's law, for ideal substrates (Pucci et al., 2017a). In fact, substrates have morphological defects and chemical heterogeneities generating an angle hysteresis and at v ≈ 0 m/s the equilibrium contact angle will be between a static advancing contact angle $\theta_{a,s}$ and a static receding one $\theta_{r,s}$ (Petrov et al., 2003; Bonn et al., 2009; Pucci et al., 2017a). Depending on the equilibrium or static contact angle, liquids can be classified as totally wetting (e.g., paraffin and silicon oils), for which the $\theta_e$ is ≈ 0° and as partially wetting (e.g., polyethylene glycols), when the $\theta_e$ is different from 0°.

In literature, to the best of our knowledge, the majority of experimental works investigated the dynamic wetting using totally wetting liquids and via a visual method, observing the spontaneous spreading of a drop (the speed of the liquid front is not constant), especially at room temperature (Tanner, 1979; Schneemilch et al., 1998; Prevost et al., 1999; Le Grand et al., 2005; Kumar and Deshpande, 2006; Seveno et al., 2009; Duursma et al., 2010; Duvivier et al., 2011). Some works using microfluidic techniques proved that, with partially wetting liquids, established models in literature are no longer valid (Wielhorski et al., 2012; Abdelwahed et al., 2014). A recent work studied molten polymers but in a spontaneous regime of drop spreading (Zhang et al., 2017). This procedure has been applied to rods or fibers with a large diameter (Zhang et al., 2019) but it turns out to be more difficult for thinner fibers (Hansen et al., 2017). Moreover, this method is not suitable to study the forced wetting at a specific range of Ca, since the liquid velocity v is not imposed but it depends on the liquid and on the spontaneous solid/liquid interactions.

In this work, a procedure easily applicable to different types of substrates and fibers and at different conditions of temperature and liquid viscosity was carried out at a large range of Ca, using the weight tensiometric method. This method, based on the Wilhelmy principle, becomes more interesting for studying forced wetting under different conditions for total and partial wetting, imposing the liquid velocity and in both advancing and receding modes (Pucci et al., 2018). Polyethylene glycols, a.k.a. PEGs, at different molecular weight (Mn) were characterized in terms of surface tension and viscosity as a function of temperature and used for tensiometric tests of forced wetting on cellulosic substrates having a very low roughness (<< 100 nm) and with an homogeneous chemical composition of surface (Pucci et al., 2018), in order to validate this protocol. Results are then relevant for a better understanding of dynamic wetting behavior of molten polymers and to estimate the prediction of this behavior by existing models in literature. A discussion about models validity, the obtained physical parameters and their sensitivity deriving from experimental results is then presented.

Moreover, the experimental protocol could be directly applied to study the dynamic wetting of these molten polymers on fibers.

## 2. MODELS OF FORCED WETTING: HDT VS. MKT

Two approaches are mostly used in literature to describe the dynamic wetting. The hydrodynamic theory (HDT) takes into account the viscous dissipation at the core of the liquid, far from the triple contact line. This approach describes forced dynamic wetting for high Ca ($Ca > 10^{-3}$) at a macro-mesoscopic scale (in the order of mm-µm) where the unbalanced driving capillary force is equilibrated by the friction one (De Gennes, 1985). From this viscocapillary balance, the Tanner-De Gennes law was derived and then generalized by Cox and Voinov (C-V), obtaining the following equation (Cox, 1962; Voinov, 1976):

$$ \theta_d^3 - \theta_e^3 = 9\ln\left(\frac{x_{max}}{x_{min}}\right) \frac{\eta v}{\gamma_{LV}} = 9\Gamma Ca \quad (2) $$

where $x_{max}$ and $x_{min}$ are two cutoff lengths employed to integer the energy dissipation. $x_{max}$ was estimated by De Gennes (De Gennes et al., 2013) in the order of the capillary length $k^{-1}$ ($\approx 10^{-3}$ m), beyond which capillary effects are not meaningful, $x_{min}$ is around a characteristic molecular size ($\approx 10^{-9}$ m). The capillary length is defined as follows:

$$ k^{-1} = \sqrt{\frac{\gamma_{LV}}{\rho g}} \quad (3) $$

where $\rho$ is the liquid density and g the acceleration of gravity. The $\Gamma$ value is indeed defined as:

$$ \Gamma = \ln\left(\frac{x_{max}}{x_{min}}\right) \quad (4) $$

Two free parameters have to be identified in this approach (Equation 2): $\Gamma$ and $\theta_e$. $\Gamma$ seems to be exclusively a characteristic of liquid properties according to the hydrodynamic approach, and experimental studies found a range of $\Gamma$ approximately between 10 and 20 for total wetting liquids (De Gennes, 1985; Marsh et al., 1993; Bonn et al., 2009). However, the physical significance of $\Gamma$ and particularly of the $x_{min}$ was questioned, because this value was sometimes found to be less than atomic or molecular dimensions (Schneemilch et al., 1998; Petrov et al., 2003). For total wetting, the equilibrium contact angle can be set to 0 and then $\Gamma$ is the only free parameter in the HDT. It was proven in a previous work (Pucci et al., 2018) with totally wetting liquids that this theory describes well dynamic wetting for $Ca > 2 \cdot 10^{-3}$ at different temperatures. In fact the effect of temperature is included in the Ca and physical parameters of $\Gamma$ were found. Open issues remain about the dynamic behavior of totally wetting liquids at low Ca; the prediction with the MKT will be discussed in this work. For partial wetting, the static contact angles were measured and inserted in models predicting forced dynamic wetting, in order to have also in this case only one free parameter ($\Gamma$). The prediction with HDT and MKT will also be discussed.
The molecular kinetic theory (MKT) investigates dynamic wetting for low Ca at a microscopic scale (in the order of nm). The unbalanced driven capillary force is here equilibrated by a microscopic dissipation in the vicinity of the contact line (Snoeijer and Andreotti, 2013). Contact line motion is a thermally activated process, described by molecular displacements characterized by a jump length λ (of the order of nm) and a quasi-equilibrium displacement frequency k⁰ (of the order of 10⁶ Hz) between adsorption sites of the solid surface (Blake and Haynes, 1969). Liquid velocity v is defined as follows:

$$v = 2k^0\lambda\sinh\left[\frac{\gamma_{LV}\lambda^2(\cos\theta_e - \cos\theta_d)}{2k_B T}\right] \quad (5)$$

where T is the absolute temperature [K] and k_B is the Boltzmann constant, equal to 1.3806 · 10⁻²³ J K⁻¹. From a linear approximation for which sinh(x) ≈ x this equation becomes:

$$v = \frac{k^0\lambda^3}{k_B T} \gamma_{LV}(\cos\theta_e - \cos\theta_d) \quad (6)$$

In this approach the free parameters are: θ_e, λ, and k⁰ (Ranabothu et al., 2005). Another simplified form of this equation can be written considering very small angles, for which cos θ ≈ 1 - θ²/2 and, as a consequence, a polynomial function is obtained:

$$v = \frac{k^0\lambda^3}{k_B T} \gamma_{LV} \left( \frac{\theta_d^2 - \theta_e^2}{2} \right) \quad (7)$$

The term k_B T / k⁰λ³ in the Equations (6) and (7) was defined by Blake and De Coninck (Blake and De Coninck, 2002) as a contact line friction ξ and was related to the work of adhesion, as follows:

$$\xi = \frac{k_B T}{k^0 \lambda^3} = \eta \left(\frac{v_L}{\lambda^3}\right) \exp\left(\frac{\lambda^2 W_a}{k_B T}\right) \quad (8)$$

where v_L is the molecular flow volume and W_a is the work of adhesion:

$$W_a = \gamma_{LV}(1 + \cos\theta_e) \quad (9)$$

Then, if we replace the contact line friction in the linear (Equation 6) and in the simplified (Equation 7) equations, and we want to define the contact angles as a function of the capillary number, the equations become the followings:

$$(\cos\theta_e - \cos\theta_d) = \frac{\xi}{\eta}Ca \quad (10)$$

$$\theta_d^2 - \theta_e^2 = 2\frac{\xi}{\eta}Ca \quad (11)$$

It is interesting to note that, contrarily to the previous expressions (Equations 5–7) where free parameters were three (θ_e, k⁰, and λ) now, if θ_e is known, there will be only one free parameter (ξ), as in the HDT. Moreover, if v_L can be determined, λ could be determined and then k⁰ (Equation 8). Another important observation has to be made about the polynomial form of MKT

for very small angles (Equation 11): this equation is very similar to the HDT law proposed by Cox-Voinov (Equation 2), but with exponent 2 vs. 3 for the HDT.

The predictions of these models for totally and partially wetting liquids at different temperature and viscosity conditions were here evaluated.

# 3. MATERIALS AND METHODS

## 3.1. Materials

### 3.1.1. Cellulosic Films

Cellulosic films, referenced as NatureFlex™ 23 NP and provided by Innovia films®, were used in this study. They were chosen as model substrates to analyse the wetting of plant fibers. Surface characterization of these films has been carried out in a previous work (Pucci et al., 2018). Before wetting tests, cellulosic films were immersed for 1 h in ethanol, to remove the glycerol on surface that had the function of plasticizer, and then they were ambient air-dried (23°C, 50% RH). To be able to carry out dynamic wetting tests (controlled liquid immersion and removal) on these films of thickness 23 µm while avoiding buckling when the sample touches the liquid, films of length 20 mm (along the immersion axis) and width 10 mm were used (Pucci et al., 2018).

### 3.1.2. Polymers

Polymers used to perform partial wetting tests in dynamic mode were three polyethylene glycols (PEGs). PEGs were chosen due to their large availability at different molecular weight and their quite low melting point. The first one with a molecular weight of 1450 g/mol (PEG1450) was provided by ACROS ORGANICS (ref. 418040010), its melting point was at 44 – 48°C. The second one with a molecular weight of 3,350 g/mol (PEG3350) was from DOW (ref. 19760), its melting point was at 53 – 57°C. The third one with a molecular weight of 10,000 g/mol (PEG10000) was from ALFA AESAR (ref. 10200251), its melting point was at 58–63°C. Their surface tension and viscosity beyond the melting point were measured as a function of temperature (sections 3.2.1, 3.2.2). Paraffin oil used to study the total wetting was provided by ACROS ORGANICS (ref. 171400010). Its melting point was at –24°C. Major details are reported in the previous work (Pucci et al., 2018).

## 3.2. Methods

### 3.2.1. Rheological Tests

A Physica MCR 501 rheometer from Anton Paar was used to measure viscosity of PEGs as a function of temperature. The used geometry was a standard cone/plate system with a 50 mm diameter, a 1° cone angle and a cone truncation of 50 µm. Preliminary tests were conducted varying the shear rate, assuring that liquids can be considered as Newtonian fluids at the tested conditions of dynamic wetting (Lu et al., 2016). Then, the shear rate was fixed at 100 s⁻¹ and the temperature rate was set at 0.01 °C/s. Two rheological measurements for each liquid were performed in order to verify the repeatability.
### 3.2.2. Pendant and Sessile Drop Tests

A Krüss DSA100 tensiometer equipped with a thermalized syringe (DO3241) and a high temperature chamber (TC21) was used to measure surface tension of PEGs via the pendant drop method. This visual method permits measuring surface tension of liquids at very high temperatures (up to 400°C) (Berry et al., 2015), that is relevant for molten polymers. Knowing the needle diameter (it was of 1.65 mm in this study) and the liquid density, surface tension is determined from the fit of the pendant drop profile (Figure 1A) with the Laplace equation integrated in the Drop Shape Analysis 1.92.1.1 software (Song and Springer, 1996a,b). To determine liquid density at each test temperature, images of sessile drop on a microscope glass slide were acquired (Figure 1B) to obtain the volume of drop (this is also implemented in the software of DSA100) and each drop mass was measured. Test temperatures were 65, 75, 85, 95, and 105 °C. Three sessile drop tests were carried out to measure density and ten pendant drop tests to determine surface tension for each of these temperatures, in order to have reliable trend of surface tension vs. temperature. Supplementary details of this procedure are reported in Pucci et al. (2018).

### 3.2.3. The Wilhelmy Method: Spontaneous and Forced Wetting

A Krüss K100SF tensiometer with a temperature generator and control unit was used to perform dynamic wetting tests with cellulosic films and PEGs. Polymers were heated in the tensiometer up to the highest temperature that can be achieved with this set-up: T = 75°C, i.e., above the melting point.

In order to measure a static contact angle, that is ideally the apparent equilibrium contact angle ($\theta_e$), tensiometric tests of spontaneous wetting were performed. The vessel containing the molten polymer raises at a constant velocity until the detection of the film surface. At this moment, a meniscus is spontaneously formed on the perimeter of the film (the wetted length $p$), resulting in an increase of the liquid mass $m$ that is recorded by the tensiometer. This increase occurs up to a constant value that defines the static condition of meniscus (equilibrium condition). Therefore, this mass can be used in the Wilhelmy equation to

determine a static contact angle:

$$F = mg = p\gamma_{LV} \cos \theta \quad (12)$$

where $F$ is the capillary force.

Figure 2 shows curves recorded by the tensiometer of spontaneous wetting on cellulosic substrates for PEG1450, 3350, and 10000. It is possible to observe that the time to reach the static condition (achieving a constant value of meniscus mass) increases with $Mn$. However, after 120 s static conditions were achieved for all PEGs.

To determine dynamic contact angles $\theta_d$, forced wetting was also performed at the K100SF tensiometer. Different velocities were imposed for immersion and withdrawal of cellulosic film in liquid, from 0.1 to 500 mm/min, in order to obtain experimental measurements in a large range of capillary numbers. Cellulosic film was first immersed in the liquid vessel by the upward motion of the latter (advancing mode), then it was maintained at this position for 120 s (static advancing mode), and finally it was withdrawn from the bath up to the initial position (receding

<figure><img src="image_104.png" /><figcaption>FIGURE 2 | Spontaneous dynamic wetting curves obtained at the tensiometer.</figcaption></figure>

A

1.65 mm

B

1.65 mm

<figure><img src="image_105.png" /></figure>

<figure><img src="image_106.png" /><figcaption>FIGURE 1 | (A) Pendant drop of PEG10000 at T = 95°C. (B) Sessile drop of PEG10000 at 95°C on a microscope glass slide.</figcaption></figure>
motion). Further details of test are described in Pucci et al. (2018). Buoyancy correction was neglected here (Pucci et al., 2017a). At very low velocities it can occur that the force referring to the static contact angle does not differ from the advancing and receding ones (as in Figure 3). Figure 3 shows a forced wetting test for PEG1450 on the cellulosic film at $v = 0.5$ mm/min. This static condition was then used to determine a static contact angle from forced wetting tests. Moreover, since the hysteresis is ≈ 0, this confirms that substrates can be approximated as ideal, that means physico-chemically homogeneous (Schneemilch et al., 1998).

Mean values of capillary forces obtained at the tensiometer in the advancing, static and receding modes were used to find the advancing and receding contact angles by means of the Wilhelmy equation (Equation 12). Static contact angles obtained by the spontaneous wetting and the forced one at the lowest

velocity were compared. These values can be inserted into models predicting dynamic wetting, in order to reduce the number of free parameters to fit.

# 4. RESULTS AND DISCUSSION

## 4.1. Liquid Viscosity and Surface Tension

Viscosity was measured as a function of temperature from $T = 70°C$ (above the melting point) to $90°C$. Figure 4 shows results of experimental semi-logarithmic curves obtained by using the rheometer for the three PEGs as a function of $1/T$. As expected, the higher molecular mass PEG has the highest viscosity. The dependence of viscosity on temperature is exponential and well described by the Arrhenius law.

About surface tension determination, images of pendant and sessile drops were acquired at different temperatures and Figure 5 shows surface tension as a function of temperature. The surface tension decreases linearly as a function of temperature, according to the law of

<figure><img src="image_107.png" /><figcaption>**FIGURE 3** | Forced dynamic wetting curve for PEG1450 on cellulosic film at $v = 0.5$ mm/min.</figcaption></figure>

<figure><img src="image_108.png" /><figcaption>**FIGURE 4** | Viscosity as a function of $1/T$ for PEGs.</figcaption></figure>

<figure><img src="image_109.png" /><figcaption>**FIGURE 5** | Surface tension as a function of temperature for PEGs.</figcaption></figure>

<table><caption>**TABLE 1** | Viscosity, density, and surface tension of polymers at $T = 75°C$.</caption><thead><tr><th><i>T</i> = 75°C</th><th><i>η</i> (Pa s)</th><th><i>ρ</i> (g/cm<sup>3</sup>)</th><th><i>γ</i><sub>LV</sub> (mN/m)</th></tr></thead><tbody><tr><td>PEG1450</td><td>0.056 ± 0.001</td><td>1.090 ± 0.030</td><td>38.9 ± 0.5</td></tr><tr><td>PEG3350</td><td>0.207 ± 0.002</td><td>1.091 ± 0.030</td><td>39.1 ± 0.4</td></tr><tr><td>PEG10000</td><td>3.519 ± 0.030</td><td>1.092 ± 0.030</td><td>39.5 ± 0.4</td></tr><tr><td>Paraffin oil (Pucci et al., 2018)</td><td>0.016 ± 0.006</td><td>0.830 ± 0.030</td><td>21.9 ± 0.7</td></tr></tbody></table>

<table><caption>**TABLE 2** | Comparison of static contact angles obtained in spontaneous and forced wetting modes with PEGs at $T = 75°C$.</caption><thead><tr><th><i>T</i> = 75°C</th><th><i>θ</i><sub>s,spont</sub> (rad)</th><th><i>θ</i><sub>s,forc</sub> (rad)</th></tr></thead><tbody><tr><td>PEG1450</td><td>0.31 ± 0.04</td><td>0.33 ± 0.06</td></tr><tr><td>PEG3350</td><td>0.36 ± 0.05</td><td>0.38 ± 0.09</td></tr><tr><td>PEG10000</td><td>0.45 ± 0.03</td><td>0.49 ± 0.06</td></tr></tbody></table>
Eötvös (Sauer and Dipaolo, 1991; Grundke et al., 1996). Moreover, surface tension is higher for PEG with highest molecular weight. Another important observation is that the difference in surface tension due to *Mn* is more significant as temperature increases (Moreira and Demarquette, 2001).

Viscosities, densities and surface tensions values obtained for the PEGs at T = 75°C are given in **Table 1**. Comparing with paraffin oil used in a previous work at T = 75°C (Pucci et al., 2018), it is possible to observe that the range of viscosity is significantly different for PEGs, due to the molecular weight. The surface tension of PEGs is also higher than for paraffin oil, due to the fact that they are partially wetting liquids.

## 4.2. Static Contact Angle

In order to determine a static contact angle from spontaneous wetting curves, the constant force value achieved at static conditions (beyond a hold for 120 s) was inserted into the Wilhelmy equation (Equation 12). In parallel, tensiometric curves of forced wetting with the lowest velocity were used to obtain the mass of meniscus in the static condition, 120 s after the advancing and just before the receding (as shown in **Figure 3**). The Wilhelmy equation was also used in this case. **Table 2** shows static contact angle values obtained with both methods, from the spontaneous and the forced wetting. Values correspond to an average with a standard deviation over three measurements.

The two sets of values obtained with the two methods are in agreement. The angles obtained for the forced wetting are slightly higher due to the dependence on velocity, that makes them quasi-static contact angles or static advancing contact angles (Pucci et al., 2017b). Values obtained in the spontaneous configuration were then used as $\theta_e$ in models predicting dynamic wetting.

## 4.3. Advancing and Receding Contact Angles

The advancing and receding contact angles were determined according to the Wilhelmy principle as a function of the immersion and withdrawal velocity. **Figure 6** shows the results obtained for PEGs at T = 75°C on cellulosic films at different velocities.

The advancing contact angle increases when the velocity of immersion is increased (Petrov et al., 2003). Moreover, for a given velocity, the larger the *Mn* of PEG, the higher the advancing contact angle. These results also correlate with higher viscosity and higher surface tension for large *Mn*, which implies a higher *Ca* (Equation 1). Therefore, it is relevant to further discuss the results as a function of the *Ca* (sections 4.4, 4.5).

On the other hand, the receding contact angle decreases when the withdrawal velocity increases, and at a given velocity, this parameter also decreases when *Mn* is increased. Moreover, for each polymer, there is a transition from partial to total wetting. This transition was reported in literature (Brochard-Wyart and De Gennes, 1992; Maleki et al., 2007, 2011). In this study, it appears that the transition occurs at lower velocity when *Mn* is higher. Modeling of dewetting was not carried out, but this issue could be addressed in future works. The prediction of advancing contact angle with the HDT and the MKT was then undertaken for PEGs and compared with the behavior of totally wetting liquids at different temperatures (i.e., paraffin oil Pucci et al., 2018).

## 4.4. HDT Prediction

To fit with models the experimental data of advancing contact angle as a function of *Ca*, parameters such as the capillary length $k^{-1}$ and the ratio of viscosity on surface tension ($\eta/\gamma_{LV}$) were determined for each polymer. These parameters are compared with those of paraffin oil (Pucci et al., 2018) in **Table 3**.

<figure><img src="image_110.png" /><figcaption>**FIGURE 6** | Dynamic contact angles as a function of the immersion and withdrawal velocity.</figcaption></figure>
For paraffin oils it has been proven in Pucci et al. (2018) that the HDT fits well the experimentally measured values of contact angles for $Ca > 2 \cdot 10^{-3}$. In the same way, fits of experimental

data for PEGs with Cox-Voinov law were carried out, imposing the measured static contact angle, in order to estimate the free parameter $\Gamma$. In addition, an analysis was undertaken to assess the sensitivity of $\Gamma$ to the parameters determined experimentally. It was carried out in terms of the number of measurements of contact angles (N points in **Figure 7**, referring to the average values over three measurements) and $Ca$ for each PEG, starting from the lowest $Ca$ value and expanding to the higher values, then in the opposite direction. Histograms in **Figure 7** show results of sensitivity analysis: the bars indicate the $\Gamma$ values and the gray triangles represent the coefficients of determination (COD) $R^2$. For all PEGs, we note that when $Ca$ increases (graphs on the left side in **Figure 7**), $\Gamma$ decreases continuously while COD increases from a low value (around 0.6 for $Ca = 7 \cdot 10^{-5}$ in the case of PEG1450) to reach a plateau of around 0.99 beyond

<table><caption>**TABLE 3** | Capillary length and viscosity to surface tension ratio for polymers.</caption><thead><tr><th></th><th>k<sup>-1</sup> (mm)</th><th>(η/γ<sub>LV</sub>) (s/m)</th></tr></thead><tbody><tr><td>PEG1450, 75°C</td><td>1.90 ± 0.09</td><td>1.44 ± 0.01</td></tr><tr><td>PEG3350, 75°C</td><td>1.91 ± 0.07</td><td>5.29 ± 0.01</td></tr><tr><td>PEG10000, 75°C</td><td>1.92 ± 0.07</td><td>89.15 ± 0.25</td></tr><tr><td>Paraffin oil (Pucci et al., 2018), 25°C</td><td>1.80 ± 0.17</td><td>5.38 ± 0.11</td></tr><tr><td>Paraffin oil (Pucci et al., 2018), 50°C</td><td>1.71 ± 0.34</td><td>1.73 ± 0.20</td></tr><tr><td>Paraffin oil (Pucci et al., 2018), 75°C</td><td>1.63 ± 0.23</td><td>0.77 ± 0.20</td></tr></tbody></table>

<figure><img src="image_111.png" /><figcaption>**FIGURE 7** | Sensitivity of $\Gamma$ for PEG1450 (blue), PEG3350 (red), and PEG10000 (green): from the lowest $Ca$ to the highest one (**left**); from the highest $Ca$ to the lowest one (**right**).</figcaption></figure>
<figure><img src="image_112.png" /><figcaption>**FIGURE 8** | $\theta_a$ as a function of Ca for polymers at different temperatures on cellulosic films and prediction of Cox-Voinov law.</figcaption></figure>

$Ca = 2 \cdot 10^{-3}$ (case of PEG1450). Here we find the transition threshold mentioned above for paraffins. This threshold is more difficult to determine for PEG3350 and PEG10000, but the $Ca = 2 \cdot 10^{-3}$ is well at the limit of the zones of strong variation of $R^2$. When Ca is varied from high values to low values (Figure 7—right side), the variation of $\Gamma$ is much less pronounced than in the previous mode. For PEG1450 high and stable values of $R^2$ are observed for $Ca > 2 \cdot 10^{-3}$. The threshold is difficult to detect on the graphs of PEG3350 and PEG10000. However, the $\Gamma$ value is stable and lower in the same range for all Mn values. Therefore, results confirm that this threshold is valid for totally and partially wetting polymers and at different temperatures. The HDT, and particularly Cox-Voinov (C-V) law, fits well forced wetting behavior for $Ca > 2 \cdot 10^{-3}$, in agreement with results found in literature (Petrov et al., 2003).

**Figure 8** shows experimental results of $\theta_d$ vs. Ca and HDT prediction for $Ca > 2 \cdot 10^{-3}$. This graph shows well the difference between total and partial wetting (paraffin oil vs. PEGs). Paraffin oil at different temperatures is well-predicted by Cox-Voinov law for $Ca > 2 \cdot 10^{-3}$ and a master curve was found (Pucci et al., 2018). Below the threshold, at low Ca, a change in slope occurs. For PEGs, fit curves are different as a function of Mn and well predict wetting behavior for $Ca > 2 \cdot 10^{-3}$. At low Ca the $\theta_a$ tends to the static contact angle. Anyway, for all polymers a change of behavior was observed at the threshold of $Ca = 2 \cdot 10^{-3}$.

$\Gamma$ and $R^2$ values derived from the fits with imposed static contact angles for the PEGs are compared to previous results measured for paraffins (Pucci et al., 2018) at different temperatures in Table 4. $\Gamma$ for paraffin oil was found to be consistent with estimated values from the molecular sizes (Pucci et al., 2018) and not dependent on temperature. For PEGs, the sizes of macromolecules depend on Mn and estimating $x_{min}$ (in Equation 4) is a difficult question. However, $\Gamma$ decreases with the increase of Mn, suggesting an increase of $x_{min}$.

<table><caption>**TABLE 4** | Obtained parameters from HDT fit at high Ca for partially and totally wetting polymers.</caption><thead><tr><th></th><th>θ<sub>s,imposed</sub> (rad)</th><th>Γ from fit for Ca > 2 · 10<sup>-3</sup></th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>PEG1450, 75°C</td><td>0.31</td><td>42.5 ± 3.0</td><td>0.975</td></tr><tr><td>PEG3350, 75°C</td><td>0.36</td><td>39.1 ± 2.1</td><td>0.997</td></tr><tr><td>PEG10000, 75°C</td><td>0.45</td><td>28.7 ± 1.3</td><td>0.994</td></tr><tr><td>Paraffin (Pucci et al., 2018), 25°C</td><td>0</td><td>19.0 ± 0.7</td><td>0.984</td></tr><tr><td>Paraffin (Pucci et al., 2018), 50°C</td><td>0</td><td>19.2 ± 1.1</td><td>0.965</td></tr><tr><td>Paraffin (Pucci et al., 2018), 75°C</td><td>0</td><td>19.2 ± 2.4</td><td>0.997</td></tr></tbody></table>

**Figure 9** shows the diminution of $\Gamma$ as a function of Mn, which seems to be well described by a linear law. Further works would focus on the application of HDT in the case of partially wetting polymers as well as the linear dependence of $\Gamma$ to Mn and how to include these results in the models to predict dynamic wetting behavior.

## 4.5. MKT Prediction

At low $Ca$ ($Ca < 2 \cdot 10^{-3}$) the MKT was evaluated for total and partial wetting of polymers on cellulosic substrates. This theory may predict dynamic of wetting at a microscopic (or local) scale, considering microscopic contact angles (Petrov et al., 2003; Bonn et al., 2009).

In order to consider this theory for total wetting and particularly for paraffin, the static contact angle was assumed equal to 0 as in the case of the HDT. Regarding the dynamic contact angles, since the advancing contact angles at low velocity were found to be very small, the polynomial form of MKT (Equation 11) was applied to experimental data for $Ca < 2 \cdot 10^{-3}$. **Figure 10** shows the results, indicating that for paraffin oil this
law fits quite well the experimental data at different temperatures and at $Ca < 2 \cdot 10^{-3}$, allowing to obtain the coefficient of contact line friction $\xi$ (Blake and De Coninck, 2002), shown in **Table 5**. The coefficient of contact line friction decreases as the temperature is increased, and then it is higher when the viscosity is higher, according to the literature (Zhang et al., 2017).

Regarding the partial wetting with PEGs at low $Ca$, the assumption of very small contact angles does not hold, but the linear approximation of MKT (Equation 10) was used. **Figure 8** shows the results of fit, indicating that the model do not predict well the wetting behavior of PEGs at low $Ca$, with aberrant results ($\xi$ and $R^2$ of fit are shown in **Table 5**). The reason could be related to the measurements of static contact angles that do not fulfill the MKT assumption of a microscopic contact angle. Measured values of apparent contact angles are better described by the HDT (Figure 8). However, further works should be carried out on the MKT and the meaning of microscopic contact angles, mainly with partially wetting liquids like molten polymers used for composites processing.

<figure><img src="image_113.png" /><figcaption>**FIGURE 9** | $\Gamma$ obtained from the HDT fit for $Ca > 2 \cdot 10^{-3}$ vs. $Mn$.</figcaption></figure>

## 5. CONCLUSION

An experimental study of the forced dynamic wetting of molten polymers on cellulosic films was carried out, with comparisons to hydrodynamic and molecular kinetic models. The interest here was to test some conditions of wetting (low and high $Ca$ at different $T$ and $Mn$) and to find relevant parameters affecting the manufacturing of composites with a liquid resin, thermoset or thermoplastic. To the best of our knowledge, wetting with molten polymers is poorly known and little documented in literature. An experimental procedure was carried out to measure liquid properties and static and dynamic contact angles at different conditions. The results proved that HDT models can be applied to totally and partially wetting polymers for high $Ca$, but parameters found from models are surprising in the case of PEGs depending on the $Mn$. At low $Ca$, the total wetting can be described by the MKT because the contact angles are small, but this theory do not predict the dynamic behavior of partially wetting polymers. At low $Ca$ for PEGs, the advancing contact angle decreases toward the static one and

<table><caption>**TABLE 5** | Obtained parameters from MKT fit at low $Ca$ for partially and totally wetting polymers.</caption><thead><tr><th></th><th>θ<sub>s,imposed</sub> (rad)</th><th>ξ from fit for Ca &lt; 2 · 10<sup>-3</sup> (Pa s)</th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>Paraffin (Pucci et al., 2018), 25°C</td><td>0</td><td>11.1 ± 1.9</td><td>0.982</td></tr><tr><td>Paraffin (Pucci et al., 2018), 50°C</td><td>0</td><td>3.9 ± 0.6</td><td>0.956</td></tr><tr><td>Paraffin (Pucci et al., 2018), 75°C</td><td>0</td><td>1.0 ± 0.3</td><td>0.906</td></tr><tr><td>PEG1450, 75°C</td><td>0.31</td><td>15.3 ± 1.4</td><td>0.916</td></tr><tr><td>PEG3350, 75°C</td><td>0.36</td><td>73.4 ± 15.2</td><td>0.586</td></tr><tr><td>PEG10000, 75°C</td><td>0.45</td><td>522.1 ± 42.1</td><td>0.842</td></tr></tbody></table>

<figure><img src="image_114.png" /><figcaption>**FIGURE 10** | $\theta_a$ as a function of $Ca$ for polymers at different temperatures on cellulosic films and prediction of MKT.</figcaption></figure>
further analyses have to be developed, in order to establish new
models describing this behavior. Reliable parameters of dynamic
wetting could be then inserted into numerical models predicting
flow processes.

DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the
article/supplementary material.

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AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual
contribution to the work, and approved it for publication.

FUNDING

Authors would thank Bpifrance for the financial support in this
study and the Depestele Group for providing materials.

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**Conflict of Interest:** The authors declare that this study received funding from Bpifrance. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

Copyright © 2020 Pucci, Duchemin, Gomina and Bréard. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Energy Materials

<figure><img src="image_115.png" /><figcaption>Vanessa Fierro</figcaption></figure>

Prof. Vanessa Fierro is a CNRS Research Professor at Institut Jean Lamour, France, a joint laboratory of the University of Lorraine and the CNRS, where she heads the Bio-sourced Materials Research Team. Vanessa has over 300 scientific publications, an h-index of 57 (Google Scholar, accessed February 2021), and an extensive background in porous materials for energy and environmental applications. She received the Charles E. Pettinos Award from the American Carbon Society in 2019 and the CNRS Silver Medal in 2020.

<figure><img src="image_116.png" /><figcaption>Maria Victoria Martínez-Huerta</figcaption></figure>

Dr. Mª Victoria Martínez Huerta is a Senior Scientist from CSIC at the Institute of Catalysis and Petrochemistry (ICP) and has been the Head of the Group Electrocatalysis for Energy and Environment since 2018. Its lines of research are linked to the development of sustainable electrocatalysts for energy conversion and storage and environmental applications that involve a reduction in the environmental impact derived from the use of fossil fuels, mainly in those applications that use hydrogen as an energy carrier, such as fuel cells, electrolyzers, and unitized regenerative fuel cells.

<figure><img src="image_117.png" /><figcaption>Maria Jesus Lazaro</figcaption></figure>
Dr. Mª Jesús Lázaro (Ph.D. in Chemistry) is a Research Professor at the CSIC at the Instituto de Carboquímica (ICB_CSIC). She has more than 250 publications in indexed scientific journals (H Scopus = 49, H WOS = 46). She has carried out more than 90 projects and contracts related to the conversion and storage of clean energy, developing catalysts and materials for fuel cells, electrolyzers, and batteries. She is the Institutional Delegate of CSIC in Aragon. In addition, she is the president of the Spanish Coal Group of the European Carbon and the AMIT-Aragón.
<figure><img src="image_118.png" /></figure>

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updates

# Energy Storage in Supercapacitors: Focus on Tannin-Derived Carbon Electrodes

Jimena Castro-Gutiérrez, Alain Celzard and Vanessa Fierro*

Université de Lorraine, CNRS, IJL, Epinal, France

Supercapacitors (SCs) are energy storage devices that bridge the gap between batteries and conventional capacitors. They can store more energy than capacitors and supply it at higher power outputs than batteries. These features, combined with high cyclability and long-term stability, make SCs attractive devices for energy storage. SCs are already present in many applications, either in combination with other energy storage devices (mainly batteries), or as autonomous energy sources. Porous carbons are presently used in the electrodes of commercial SCs due to their high surface area and their good conductivity. However, new porous materials are continuously being developed. Herein, an outline of the principles of the energy storage mechanism in SCs is presented as a guide to illustrate the research on porous carbons materials for SC applications. Indeed, an overview of these carbons and their synthesis methods is also presented. In the context of an urgent need to progress toward the development of environmentally friendly technologies and methods, the final part of this review focuses on the studies carried out using biosourced carbon precursors, such as tannins, which are natural polyphenolic molecules. In particular, mimosa tannin-derived carbon materials with controlled micro- and mesoporosity can be produced by methods with lower environmental impact and lower health and safety risks because crosslinkers are not needed to produce resins.

**Keywords:** supercapacitors, energy storage, porous carbons, OMCs, tannins

**OPEN ACCESS**

**Edited by:**
Emilia Morallon,
University of Alicante, Spain

**Reviewed by:**
Ramiro Ruiz Rosas,
University of Malaga, Spain
Juan Manuel Sieben,
Universidad Nacional del
Sur, Argentina

*Correspondence:
Vanessa Fierro
vanessa.fierro@univ-lorraine.fr

**Specialty section:**
This article was submitted to *Energy Materials*, a section of the journal *Frontiers in Materials*

**Received:** 08 May 2020
**Accepted:** 12 June 2020
**Published:** 22 July 2020

**Citation:**
Castro-Gutiérrez J, Celzard A and Fierro V (2020) *Energy Storage in Supercapacitors: Focus on Tannin-Derived Carbon Electrodes*. *Front. Mater.* 7:217.
doi: 10.3389/fmats.2020.00217

## INTRODUCTION

World's electricity consumption has increased significantly in recent decades, from ~11,000 TWh in 1990 to ~23,000 TWh in 2016, according to the International Energy Agency (IEA), and there is still a strong dependence on fossil fuels such as coal, oil and gas as energy sources (IEA, 2019). Furthermore, it is generally agreed that the increasing concentration of greenhouse gases in the atmosphere from the 1950s is partly attributable to the burning of fossil fuels, which is causing global climate change (Pachauri et al., 2015). The limits of fossil fuels reserves and the increasing global warming have amplified awareness on the need to phase out the fossil fuel industry. The transition to energy production from renewable sources and environmentally friendly technologies is a public demand. This poses a challenge for the production of electricity based on fluctuating energy sources such as solar or wind, among others. Thus, research aiming to improve the performance of energy storage devices is of great relevance nowadays.
The role of energy storage devices in the electrical system is to collect excess of energy during high production peaks and act as a reservoir, releasing energy when required. **Figure 1A** lists some of the different storage technologies used at different steps of the electrical system (IEA, 2014; Aneke and Wang, 2016). Pumped-storage hydropower and compressed air energy storage are widely used in electric power plants for large-scale production. On the other hand, batteries are suitable for a wider range of mid- and short-term storage applications such as distribution to remote sites, electric vehicles, small-scale production and off-grid storage for houses or buildings, and portable electronic devices also rely on battery use. On the contrary, supercapacitors (SCs) are high-power devices, meaning that they can supply stored energy at fast rates, and they are mostly used in short-term applications where bursts of electricity are needed.

SCs are presently used in a variety of applications, which could be further developed as stand-alone energy suppliers if their energy densities were increased (Sharma and Bhatti, 2010; Aneke and Wang, 2016; González et al., 2016; Wang et al., 2016). Accordingly, the international community has established the pursuit of improving the SCs performances as one of the short-term actions in the context of research and development on energy storage (IEA, 2014). This review is not intended to be exhaustive but to present a global vision of the principles underlying the energy storage mechanism in SCs, and to explain how these principles have guided the research of materials in this field, in particular for the production of carbon electrodes. Given the environmental issues mentioned above, the last part of this review incorporates studies using non-toxic and environmentally friendly carbon precursors, such as tannin extracts, to produce carbon electrodes for SCs.

# SUPERCAPACITORS

## Basic Principles

A SC consists of two parallel electrodes separated by a non-conductive material impregnated with an electrolyte, as illustrated in **Figure 1B**. By applying a potential between the electrodes, the ions in the electrolyte are attracted to the electrode of opposite charge. Derived from charge accumulation and the interaction with the electrode surface, an electrostatic double-layer (EDL) is created on each electrode (González et al., 2016). Hence, there are two contributions to the total capacitance of the device, represented by the two capacitors in series shown in the simplified electric circuit of **Figure 1B**. An equivalent series resistance (ESR) is added to the circuit to account for the resistance of all the components of the device (Xie et al., 2018). The EDL generation process is ideally reversible, resulting in fast responses for charge and discharge, which result in high power in SCs. In addition, high cyclability, i.e., retention of capacitance after repetitive cycles of charge and discharge, and long lifetimes are achieved due to the low degradation of components derived from the reversibility of the EDL formation process.

From a macroscopic point of view, the capacitance C of a SC can be calculated as in a regular capacitor of parallel plates:

$$C = \frac{\epsilon_0 \epsilon A}{d} \qquad (1)$$

where $\epsilon_0$ is the permittivity of vacuum, $\epsilon$ is the permittivity of the electrolyte, A is the area of the electrode, and d is the effective thickness of the EDL. The energy, E, of a capacitor is calculated as follows:

$$E = \int CVdV \qquad (2)$$

where V is the applied voltage to the device, also called potential window. For an ideal EDL SC, C is not a function of V and Equation (2) becomes:

$$E = \frac{1}{2}CV^2 \qquad (3)$$

The power, P, is then calculated through:

$$P = \frac{E}{t_{dis}} \qquad (4)$$

where $t_{dis}$ is the discharge time. **Figure 2** shows a Ragone plot of typical values of energy and power of SCs compared to those of other energy storage devices such as batteries, fuel cells and capacitors.

## Applications

The characteristic E and P-values of SCs bridge the gap between conventional capacitors and batteries, as shown in **Figure 2**. Hence, SCs find their applications either as autonomous energy sources or in combination with other devices, as detailed below (Kötz and Carlen, 2000; Miller and Burke, 2008; Conte, 2010; Sharma and Bhatti, 2010; Gu and Yushin, 2014; Aneke and Wang, 2016; González et al., 2016; Gautham Prasad et al., 2019; IOXUS Inc, 2020; Maxwell Technologies Inc., 2020):

* Memory back-ups. Fast-response SCs are a short-term solution to a brief interruption of power supply to the memory.

* Portable tools. SCs are used as an energy source in camera flashes or portable screwdrivers, since only bursts of energy are required along with rapid and constant recharging.

* Distribution and storage of electrical energy. SCs are able to compensate short-duration voltage fluctuations in the distribution line and to match the variations between production and consumption. In addition, SCs are used to restart collapsed power systems or to supply energy until the primary source is restored. SCs are also used as storage devices in renewable energy production plants, especially in wind power applications, as they require little maintenance.

* Decoupling of power and energy needs. SCs are used to meet the power needs while the principal source of energy is provided by a different system. This type of decoupling is found in hybrid and electric vehicles.
<figure><img src="image_119.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_120.png" /></figure>

**FIGURE 1** | (A) Energy storage technologies used at different scales in the power system (IEA, 2014; Aneke and Wang, 2016). (B) Mechanism of formation of the electrostatic double-layer (EDL) in a SC. In the associated electric circuit, capacitors $C_{e1}$ and $C_{e2}$ represent the contribution to the total capacitance of the EDL formed at the surface of each electrode. The equivalent series resistance (ESR) is also shown.
<figure><img src="image_121.png" /></figure>

* **Regeneration devices.** The energy released by machines with repetitive and constant movements can be recovered by SCs. Thus, SCs are found, for example, in cranes and elevators or in the braking systems of hybrid or electric vehicles like buses, delivery or garbage trucks and trains.

One of the main disadvantages of SCs compared to batteries is that they store less energy, which limits their use as autonomous devices. It is clear from Equation (3) that there are two ways to overcome this drawback: widening the potential window or increasing the capacitance. The former is strongly associated with the choice of electrolyte and the latter with the properties of the electrode. Nevertheless, they are intimately related to each other, because there must be a synergy between all the components of the SC. A clear example of this synergy is the pseudocapacitive behavior that some materials present when used as electrodes, where rapid Faradaic (redox) reactions occur on or near the surface of the electrode. The presence of redox reactions depends on the properties of the electrode material and on the affinity of the electrolyte toward it. SC devices with electrodes made of materials in which both EDL and pseudocapacitance storage mechanisms are present are also referred to as pseudocapacitors (PSCs) and could reach even higher values of capacitance. However, the increased capacitance in PSCs may be accompanied by a loss of power and long-term stability compared to that of SCs (González et al., 2016).

New materials are constantly being developed and their performance as electrodes for SCs must be evaluated. In the following section, the electrochemical testing techniques commonly used on a laboratory scale are briefly described in order to illustrate the expected behavior of a SC. Next, the types and general characteristics of commonly used electrolytes and the advances in the research for porous materials as electrodes for SCs are discussed.

# Electrochemical Testing Techniques
## Cell Configuration

**Figure 3** shows schemes of cell configurations normally used for SC testing. The three-electrode cells (Figure 3A) are useful for estimating the contributions of pseudocapacitance or for better understanding the more fundamental behavior of materials, while the two-electrode cells (Figure 3B) are used to evaluate the performance of materials in operating conditions closer to those of real SC devices (Xie et al., 2018). T-type cells are a combination of two- and three-electrode cell configurations (Figure 3C), which allow studying the performance of each electrode in a SC along with the performance of the whole system operating at the equilibrium potential of both electrodes (Sevilla et al., 2019). Thus, a three-electrode configuration measures the performance of a single electrode (the working electrode in Figure 3A), whereas a two-electrode configuration evaluates the performance of two electrodes as a system (the cell). As shown in Figure 1B, in a symmetric SC, the two electrodes are made of the same material and, based on the associated simplified circuit, its capacitance, C, can be calculated assuming that the contribution of each electrode is identical, i.e., $C_{e1} = C_{e2} = C_e$. Therefore:

$$ \frac{1}{C} = \frac{1}{C_e} + \frac{1}{C_e} \qquad (5) $$

$$ C = \frac{C_e}{2} \qquad (6) $$

Reporting normalized values of capacitance allows for the comparison of results from different studies. Thus, from Equation (6) and considering that the normalization factor of the cell is twice the normalization factor of an electrode, i.e., $N = 2N_e$ (Zuliani et al., 2015):

$$ C_N = \frac{C}{N} = \frac{C_e}{2N_e} = \frac{C_e}{4N_e} = \frac{C_e N_e}{4} \qquad (7) $$

N is generally chosen to be either the mass or the volume of the electrodes. For simplicity, from now on, C and $C_e$ will refer to the capacitances normalized by the mass of the electrode(s), called specific cell capacitance and specific electrode capacitance, respectively.

Many of the materials that can be used as electrodes for SCs are in powder form, so it is necessary to use a binder to agglomerate the powder particles and create a paste, or slurry, from which the electrodes are obtained. Some additives can also be mixed with the active material to improve their electrical conductivity. A correct assessment of the materials performance requires giving the calculations of normalized capacitance as a function of the total mass or the total volume of the electrode, i.e., including binder and/or the additives.

## Cyclic Voltammetry (CV)

During cyclic voltammetry (CV) tests, a potential (V) is applied and increased at constant rate over time (scan rate) while the current response, I, is measured. An ideal SC would exhibit a rectangular curve when I is plotted as a function of V, see
<figure><img src="image_122.png" /></figure>

<figure><img src="image_123.png" /></figure>

<figure><img src="image_124.png" /><figcaption>**FIGURE 3** | Different configurations for the electrochemical characterization of SCs: (A) three-electrode cell, (B) two-electrode cell, and (C) T-type cell.</figcaption></figure>

**Figure 4A.** During charging, the sharp increase in *I* at the beginning of the cycle is due to the rapid formation of the EDL and it remains constant when *V* increases because no Faradaic reaction occurs. Given the reversible nature of the EDL formation mechanism, the CV curve during discharge obtained when *V* decreases is the exact inverse of the charging curve. Evidently, the CV curves of the real SCs deviate from the ideal one, and those of PSCs also display *I* peaks due to the generation of charge by redox reactions, as shown in **Figure 4A**. In addition, widening the potential window in CV measurements allows identifying the point at which decomposition of the electrolyte occurs, manifesting by an *I* peak at each edge of the CV curve. Then, this information can be used to set the operating voltage of a SC cell. From CV data, *C* is calculated by:

$$C = \frac{\int_{2sVm}^{sVm} dV}{2sVm} \qquad (8)$$

where the numerator is the area delimited by the CV curve, *s* is the scan rate, *V* is the potential window and *m* is the sum of the masses of the two electrodes in the cell. If a three-electrode configuration is used, *C*<sub>e</sub> can be calculated by substituting *m* in Equation (8) for the mass of the working electrode, *m*<sub>e</sub>.

## Galvanostatic Charge-Discharge (GCD)

In the galvanostatic charge-discharge (GCD) technique, a constant *I* is applied to the system while the response *V* is recorded as a function of time. The method is normally carried out with a two-electrode configuration. In an ideal SC, charge and discharge have a linear mirror behavior, resulting in triangular curves. In real SCs and PSCs, the curves deviate from the linear behavior, see **Figure 4B**. Any device will exhibit a sudden drop in potential during the transition from charge to discharge. This is called IR drop and it is directly associated with the ESR of the device. From GCD data, *C* is calculated by:

$$C = \frac{I}{(\Delta V/\Delta t)m} \qquad (9)$$

where *I* is the constant current applied, Δ*V*/Δ*t* is the slope of the discharge curve and *m* is the sum of the masses of the

two electrodes in the SC. *C*<sub>e</sub> can then be approximated using Equation (7). Using this value of *C*, those of *E* and *P* of the SC can be calculated directly from Equations (3) and (4), respectively, keeping in mind that *V* in Equation (3) is the actual operating potential, therefore the IR drop must be subtracted from the maximum potential reached by the cell, i.e., *V* = *V*<sub>max</sub> − *IR*<sub>drop</sub>.

GCD tests performed at increasing values of *I* provide insight into the kinetic behavior of the materials at faster charging rates. Although this can also be evaluated by increasing the scan rate in CV tests, GCD tests are preferred for kinetic analysis because the operating conditions are closer to those of real devices (Beguin and Frackowiak, 2013; Xie et al., 2018).

## Electrochemical Impedance Spectroscopy (EIS)

Electrochemical impedance spectroscopy (EIS) is used to analyze the response of a material to a low-amplitude oscillating potential signal. A Nyquist plot represents the real and imaginary parts of the complex impedance, *Z*<sub>r</sub> and *Z*<sub>i</sub>, respectively. Examples of curves obtained for an ideal SC, a real SC and a PSC are shown in **Figure 4C**. Analysis of Nyquist plots allows associating an equivalent circuit for each type of device, see **Figures 4E-G**, which provides information on the electrochemical system (Bard and Faulkner, 2001; Beguin and Frackowiak, 2013; Xie et al., 2018). For instance, a straight vertical line appears for a material with an ideal SC behavior. Its point of intersection with the *Z*<sub>r</sub> axis represents the value of the ESR of the device, *R*<sub>s</sub>. The pseudocapacitive behavior adds a second charge transfer resistance to the system, *R*<sub>ct</sub>, and a semicircle is observed in the Nyquist plot at high frequencies (HF, > ~10<sup>3</sup> Hz). In porous materials, the limited ion diffusion within the pores is modeled in the equivalent circuit as a constant phase element, *Z*<sub>CPE</sub>, and it manifests itself in the Nyquist plot as a straight line in the HF for a SC and after the semicircle for the PSC (Beguin and Frackowiak, 2013). The longer the line, the more difficult it is for the electrolyte to access the whole surface of the material. Once the surface is completely impregnated, the capacitance no longer depends on the frequency, hence the vertical line appearing at low frequencies (LF, ~10<sup>-3</sup> Hz). In all cases, the intersection with the *Z*<sub>r</sub> axis of the vertical line at LF is defined as the equivalent distributed resistance (EDR) that takes into account the ESR and the charge transfer and ion diffusion resistance of the material.
<figure><img src="image_125.png" /></figure>

<figure><img src="image_126.png" /></figure>

<figure><img src="image_127.png" /></figure>

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<figure><img src="image_129.png" /></figure>

<figure><img src="image_130.png" /></figure>

<figure><img src="image_131.png" /></figure>

**FIGURE 4** | Expected behavior of a supercapacitor (SC) and a pseudocapacitor (PSC) compared to that of an ideal SC during different electrochemical tests: (A) cyclic voltammetry, (B) galvanostatic charge-discharge, and (C,D) electrochemical impedance spectroscopy. Simplified equivalent circuits for: (E) ideal SC, (F) SC, and (G) PSC.

Data obtained from EIS measurements can also be represented in a Bode diagram, see **Figure 4D**, where the phase and/or modulus of the complex impedance are/is plotted as a function of frequency. Bode diagrams are useful tools for easily identifying the relaxation time constant, τ₀, of the device, which is the inverse of the frequency at which the phase angle is 45°. This parameter is related to the power output of the SC. A low τ₀ leads to a rapid charge-discharge resulting in a high power capability (Orazem and Tribollet, 2008; Inamuddin et al., 2018).

## ELECTROLYTES USED IN SCs

The energy stored in a SC can be considerably increased by widening the potential window, see again Equation (3), which is essentially limited by the stability of the electrolyte. Increasing the potential beyond this limit causes the decomposition of the electrolyte, resulting in unwanted reactions that sharply increase the current, reduce the performance and can degrade the materials in the SC (Zhong et al., 2015). Even though the
selection of the electrolyte is mainly defined by its stability, other properties such as conductivity, ion size and mobility, ion-electrode interaction or thermal stability must also be considered. Currently, aqueous and organic liquid electrolytes are the most widely used. **Table 1** shows their general properties.

Aqueous electrolytes are perhaps the most used in academic research because they have a low cost, they are easy to handle, and they present high ionic conductivity and mobility leading to a low ESR (González et al., 2016). Among them, acidic, alkaline, or neutral solutions can be used (Zhong et al., 2015; Wang et al., 2016). Acidic and alkaline electrolytes are likely to provide pseudocapacitance contributions by reacting with oxygen or nitrogen surface functional groups present in carbon electrodes (Zhai et al., 2011; Salinas-Torres et al., 2019). The potential window of aqueous electrolytes is limited to ~1.2 V, beyond which water begins to decompose. Besides, they are not suitable for applications where the temperature is much lower than 0°C or much higher than 100°C (Zhong et al., 2015). Organic electrolytes predominate on the market because they offer the possibility of using larger potential windows. They consist of salts dissolved in organic solvents that can reach potential windows up to 3.5 V and operate at temperatures as low as -40°C (Zhong et al., 2015). However, the larger ion size of typical organic electrolytes limits their mobility, so they have lower conductivities than those of aqueous electrolytes (Wang et al., 2016). In addition, organic electrolytes still face the problems related to their high toxicity, flammability and high costs due to the necessary purification processes and the controlled environment for the assembly of the SCs (Zhong et al., 2015).

## CARBON MATERIALS AS ELECTRODES FOR SUPERCAPACITORS

The use of porous carbon materials as electrodes for SCs has increased over the years. Such materials indeed have high chemical and thermal stability, good conductivity, possibility of adjusting their textural properties, and industrial processes for electrode synthesis and production already established. In addition, carbon cloths or monoliths can be produced and used directly as electrodes, eliminating the need of binders to maintain their shape (Kötz and Carlen, 2000; Sevilla and Mokaya, 2014; Wang et al., 2016). From Equation (1), it can be seen that a material with a high surface area should result in a high-capacitance SC as long as this surface is accessible to electrolyte ions. The latter highlighted the importance of matching the size of the pores to the size of the electrolyte ions. Some studies had also reported that a hierarchical porous structure connecting micro-, meso- and macropores (pore width $w < 2$ nm, $2 < w < 50$ nm, and $w > 50$ nm, respectively) helps improve SC performances by providing interconnected channels that facilitate ion diffusion in the material (Frackowiak, 2007; Inagaki et al., 2010). Porous carbon materials are also of particular interest because the addition of specific functional groups to their surface results in higher capacitances due to improved

conductivity and/or higher pseudocapacitance contributions (Paraknowitsch and Thomas, 2013).

Efforts to increase the energy density in carbon-based SCs focus on these three aspects: development of porous materials with high surface area, tailoring of pore size, and modification of surface chemistry. The following subsections deal with the description of the synthesis processes and the resultant properties of different porous carbon materials for their application as electrodes for SCs.

## Activated Carbons

Most electrodes in SCs are based on powders of activated carbon (AC). There is a predominant use of biomass as raw material for commercial and laboratory production of ACs, given that agricultural, forestry, municipal and animal byproducts are abundant and inexpensive. Coal or coke can also be used to produce ACs (Zhao et al., 2012; Tellez-Juárez et al., 2014; González-García, 2018). **Figure 5** shows schematically the production process by physical or chemical activation methods.

Physical activation is based on the partial and controlled gasification of a carbonaceous material in an atmosphere containing an oxidizing gas, such as steam, $CO_2$, air or their mixes, at temperatures ranging from 350 to 1,000°C. Before physical activation, pyrolysis at temperatures between 400 and 900°C or hydrothermal carbonization (HTC) at lower temperatures (130 to 250°C) is generally required (Cha et al., 2016; Selmi et al., 2018; Mbarki et al., 2019). The final textural and chemical properties of the ACs depend largely on the activation time and temperature. When the same activating agent is used, a higher degree of activation leads to a higher development of porosity and a broadening of the pore size distribution (PSD). Physical activation normally produces materials with surface areas up to ~1,800 $m^2 g^{-1}$ and maximum pore volumes of ~0.9 $cm^3 g^{-1}$ (Sevilla and Mokaya, 2014; González-García, 2018).

Chemical activation is carried out using compounds such as KOH, NaOH, $H_3PO_4$, or $ZnCl_2$, among others. The precursor is mixed with the activating agent and then subjected to pyrolysis at temperatures between 400 and 900°C. Finally, the AC needs to be washed to remove the residues generated during the activation process. Direct chemical activation of biomass is preferred when using $ZnCl_2$ or $H_3PO_4$ because they dehydrate biomass, which reduces the quantity of volatile matter released during pyrolysis, thus increasing the carbon yield (Fierro et al., 2005, 2010). KOH or NaOH can be used to activate either untreated biomass or char (Fierro et al., 2007; Basta et al., 2009; Acosta et al., 2018). Higher amounts of activating agent result in more developed porosity. $H_3PO_4$ and $ZnCl_2$ generate micropores and small mesopores and lead to ACs with surface areas lower than $2,000 m^2 g^{-1}$. On the other hand, KOH can produce higher surface areas, wider PSD and pore volumes of $2.7 cm^3 g^{-1}$ or more. Such further development of porosity is probably due to the generation of $CO_2$ as a by-product of the reaction between the carbon and KOH (Sevilla and Mokaya, 2014). The use of less corrosive or harmless salts for the activation of biomass and biomass-derived precursors, such as potassium carbonate, potassium bicarbonate, potassium oxalate, or sodium thiosulfate, has been explored (Fuertes et al., 2018; Díez et al., 2019). Materials with high surface
<table><caption>**TABLE 1** | Properties of aqueous and organic electrolytes often used in SCs (Lide, 2004; Conte, 2010; Zhong et al., 2015; Lu et al., 2018).</caption><thead><tr><th rowspan="2">Type</th><th rowspan="2">Electrolyte</th><th rowspan="2">Potential window [V]</th><th colspan="2">Solvated ion size [nm]</th><th rowspan="2">Conductivity [S cm<sup>-1</sup>] (concentration)</th><th rowspan="2">Freezing and boiling point [°C]</th></tr><tr><th>Cation</th><th>Anion</th></tr></thead><tbody><tr><td rowspan="3">Aqueous</td><td>H<sub>2</sub>SO<sub>4</sub></td><td>0.7–1.0</td><td>0.28</td><td>0.38</td><td>0.8 (1 M)</td><td></td></tr><tr><td>KOH</td><td>0.9–1.0</td><td>0.33</td><td>0.30</td><td>0.6 (6 M)</td><td></td></tr><tr><td>Na<sub>2</sub>SO<sub>4</sub></td><td>1–1.8</td><td>0.36</td><td>0.38</td><td>0.01 (0.1 M)</td><td></td></tr><tr><td rowspan="2">Organic</td><td>TEABF<sub>4</sub>/ACN</td><td>2.5–2.8</td><td>1.3</td><td>1.16</td><td>0.05 (0.65 M)</td><td>-49 to +82°C</td></tr><tr><td>TEABF<sub>4</sub>/PC</td><td>2.5–3.5</td><td>1.35</td><td>1.4</td><td>0.01 (0.65 M)</td><td>-49 to +242°C</td></tr></tbody></table>

<figure><img src="image_132.png" /><figcaption>**FIGURE 5** | Schematic production process of activated carbons, from biomass and char, by physical or chemical activation.</figcaption></figure>

areas and pore volumes, up to 2,750 m² g⁻¹ and 2.4 cm³ g⁻¹, respectively, have been reported.

The advanced materials described in the following sections can also be activated to improve their textural properties. However, here they differ from ACs materials since their physicochemical properties are mainly related to synthesis rather than the activation process.

## Carbide-Derived Carbons and Zeolite-Templated Carbons

In order to have a higher control of the microporosity, different methods for the synthesis of carbon materials from carbides or zeolites have been developed (Lee et al., 2006; Inagaki et al., 2010). High-temperature chlorination (400–1,200°C) of silicon or titanium carbide is used to remove metal atoms and obtain
microporous materials. Such carbide-derived carbons (CDCs) exhibit surface areas between 1,000 and 2,000 m² g⁻¹ and a uniform and disordered pore structure. The pore size can be adjusted from 0.6 to 1.1 nm by changing the carbide precursor, the chlorination or the heat-treatment temperature (Zhai et al., 2011). On the other hand, zeolite-templated carbons (ZTCs) are produced by incorporating a carbon precursor, e.g., phenol, inside the zeolite structure and carbonizing it. After template removal, a structure with the same periodicity as the original zeolite is obtained. ZTCs reach surface areas higher than 2,000 m² g⁻¹ and present pore volumes of 1 cm³ g⁻¹ or more (Lee et al., 2006; Mostazo-López et al., 2018). For both CDCs and ZTCs, the pore structure consists only of micropores with a minor presence of mesopores. Activation of these materials introduces mesoporosity and broadens the micropores, which can improve ion diffusion in the porous materials when used as electrodes for SCs (Mostazo-López et al., 2018).

## Carbon Gels

Pyrolysis of organic gels prepared from resorcinol-formaldehyde resins has been used to produce carbon gels (CGs) with surface areas between ~500 and ~1,000 m² g⁻¹ and presenting a high fraction of mesopores. In general, the production of CGs involves five steps: polymerization of the precursor to form an organic gel, aging, solvent exchange, drying, and carbonization (Grishechko et al., 2016; Ciszewski et al., 2019). CGs are classified, according to the drying technique, as aerogels, xerogels, or cryogels whether supercritical, subcritical or freeze drying, respectively, has been used (Braghiroli et al., 2019). Each step of the synthesis process has an impact on the porous texture of the resultant CGs. On the one hand, the precursor to catalyst ratio and the pH of the solution modify the polymerization process and lead to materials of different textures (Szczurek et al., 2011a,b). On the other hand, controlling the drying process is of paramount importance to avoid the formation of cracks or the shrinkage of the material (Feinle et al., 2016). For SC applications, it has been found that the mainly mesoporous structure of CGs limits the effectiveness of the EDL mechanism and that activation generally allows developing a narrow porosity not accessible to ions, leading to a moderate improvement in electrochemical performance compared to the increase in surface area (Pandolfo and Hollenkamp, 2006; Zhang and Zhao, 2009).

## Nanostructured Carbons

Carbon nanotubes, nanofibers and nanospheres (CNTs, CNFs, and CNSs, respectively) have also been used as electrodes of SCs. Although the surface area of CNFs and CNTs is moderate, ~400 m² g⁻¹, they have been considered for SCs electrodes given that their entanglement results in multiple charge storage sites (Gu and Yushin, 2014), as seen in **Figures 6A,B**. CNFs and CNTs obtained by electrospinning techniques or by direct growth on conductive substrates allow flexible mats to be produced, which can be directly used as electrodes in SCs (Ra et al., 2009; Kim et al., 2012; Gu and Yushin, 2014). Likewise, aligned CNFs and CNTs can be grown directly on current collectors (Kim et al., 2003; Gao et al., 2008; Zheng et al., 2017). SCs with electrodes based on aligned CNFs or CNTs could exhibit improved performance

at high charging rates due to a shortening of the conductive path for the charges (Inagaki et al., 2010). However, the performance of CNTs as electrodes in SCs is strongly affected by the presence of impurities (Frackowiak and Beguin, 2001). In addition, activation is generally needed to create micropores on the surface of CNFs and increase the number of charge storage sites (Inagaki et al., 2010; Gu and Yushin, 2014). On the other hand, it is nowadays possible to synthesize a wide variety of CNSs, whether solid, hollow, or onion-like, among others, and with micro-, meso-, and/or macropores, see examples in **Figures 6C,D**. The surface areas vary from low, < 100 m² g⁻¹, to high, ~2,000 m² g⁻¹, and the sphere sizes range from tens of nm to hundreds of µm (Liu et al., 2015). The strategies for synthesizing CNSs include different types of hard- and soft-template techniques, emulsion polymerization, self-assembly or the Stöber method (Liu et al., 2015; Li et al., 2016). Typically, petrochemical precursors are used to produce CNSs, but HTC processes have successfully used glucose, sucrose, cellulose or tannin to obtain CNSs (Braghiroli et al., 2015b; Liu et al., 2015; Li et al., 2016). Uniform CNSs can be packed in high-density electrodes, maintaining the electrolyte accessibility thanks to voids between the particles. Therefore, this type of electrode allows a large amount of energy to be stored in small-volume devices (Ferrero et al., 2015). Despite the advantages of CNSs, the multi-step synthesis process, the use of hazardous substances to etch the hard templates and the low yields associated with soft-template methods limit their extensive use for widespread production (Liu et al., 2015; Li et al., 2016).

## Ordered Mesoporous Carbons

In order to develop materials with a hierarchical pore structure, many studies have been devoted to the design and engineering of ordered mesoporous carbons (OMCs) with tuned morphology and porosity. If necessary, an activation process is carried out to increase their surface area. In this way, OMCs have the advantage of having a hierarchical pore structure to provide interconnected channels for the diffusion of electroactive species, thus making them suitable for application in SCs (González et al., 2016; Lim et al., 2016; Wang et al., 2016). OMCs can be synthesized by hard- or soft-template methods. Both are shown in **Figure 7A**, and further details are given below.

The hard-template method uses an inorganic material that is filled with the carbon precursor. After polymerization, the composite is subjected to carbonization, and the template is then removed. As a result, the OMC obtained is an inverse replica of the template (Lee et al., 2006). Mesoporous silicas are the most used in this kind of techniques, but having to synthesize such templates before sacrificing them makes the procedure expensive, polluting and time-consuming. Thus, efforts to synthesize OMCs from biosourced templates have been made to take advantage of naturally ordered and hierarchical structures found in diatomaceous earth, pollen grains or leaves, among others (Liu et al., 2012; Xia et al., 2012; Gao et al., 2017). However, the procedure still involves the use of hazardous substances to crosslink the precursor and/or to etch the template.

In the soft-template method, an amphiphilic surfactant is used in solution to form micelles. The hydrophilic part of the micelle interacts with the carbon precursor that
<figure><img src="image_133.png" /><figcaption>**FIGURE 6** | (A) Electrospun CNFs from Kraft lignin (Adapted from Vivo-Vilches et al., 2019). SEM (and TEM in the insets) images of: (B) commercial CNTs-Poly(dicyclopentadiene) composite yarn (Adapted from Xin et al., 2020); (C) solid CNSs derived from tannin (Adapted with permission of Elsevier, from Braghiroli et al., 2012; permission conveyed through Copyright Clearance Center, Inc.); (D) ordered mesoporous CNSs from phenol (Adapted from Chang et al., 2015).</figcaption></figure>

polymerizes around the surfactant. Then, the carbonization process eliminates the surfactant, revealing the mesoporosity at the same time (Libbrecht et al., 2017). Different combinations of commercially available block copolymers such as Pluronic® or Vorasurf® as surfactants and phenol, phloroglucinol, resorcinol, or tannin as carbon precursors have been used to successfully synthesize OMCs by soft-template methods (Meng et al., 2006; Wickramaratne and Jaroniec, 2013; Braghiroli et al., 2016; Libbrecht et al., 2017).

In both hard- and soft-templating, the mesopore order is determined by the characteristics of the template. Given the diversity of templates and precursors available, OMCs have surface areas from ~500 to more than 2,000 m² g⁻¹, and various PSDs in the micro—mesopore range. Thus, the versatility of OMCs turns them into an interesting choice of materials to be used as SC electrodes. **Figures 7B–E** shows examples of OMCs with different pore structures.

## Doped Materials

A potential route to increase the energy stored in SCs is heteroatom doping of carbon materials, which promotes fast Faradaic reactions and enhances their performances via pseudocapacitance contributions. All the materials described in the previous sections (Sections Activated Carbons to Ordered Mesoporous Carbons) are subject to surface modification. Although doping with boron, sulfur or phosphorus has been investigated (Paraknowitsch and Thomas, 2013; Bairi et al., 2015; Chen et al., 2015; Elmouwahidi et al., 2018), the introduction of heteroatoms such as oxygen (O) or nitrogen (N) is recurrent because it is relatively easy to perform. In consequence, O- and N-doping have been studied in depth.

**Figure 8A** displays the most common O- and N- surface functional groups associated with improving the performance of carbon-based SCs.

Oxygen functionalities are normally found in porous carbon materials. To further dope the materials with oxygen, activation and/or oxidation treatments in HNO₃, H₂SO₄, or using O₂ can be carried out; electro-oxidation using different basic, neutral or acidic electrolytes has also been explored (Inagaki et al., 2010; Berenguer et al., 2013; Sevilla and Mokaya, 2014). The final amount and the type of functionality on the carbon surface depend on the precursor and the synthesis method. Some of the proposed redox reactions involving oxygen surface groups are shown in **Figures 8B–F** (Chen et al., 2012; Sánchez-Sánchez et al., 2016). In acidic electrolytes, it has been reported that quinone functionalities have the largest contribution to capacitance, **Figure 8B** (Hulicova-Jurcakova et al., 2009). Even though the carboxylic groups could increase the capacitance through the generation of charges, see **Figure 8E**, their high polarity could also produce resistance to ion diffusion, reducing the performance (Sevilla and Mokaya, 2014). Nevertheless, carboxylic and phenolic groups are also likely to react in alkaline solutions, see **Figures 8D,F**, with a positive impact on the SC performance (Zuliani et al., 2018).

N-doping by post-treatment is carried out by HTC and/or annealing of a mixture of the carbon material with a nitrogen precursor, e.g., ammonia or urea (Inagaki et al., 2018). N-doping can also be performed *in situ* by direct pyrolysis of a nitrogen-rich carbon precursor (Raymundo-Piñero et al., 2009) or by multi-step methods to produce CNSs (Braghiroli et al., 2015b; Ferrero et al., 2015; Wang et al., 2018), CNTs (Ayala et al., 2010), or OMCs (Sánchez-Sánchez et al., 2016; Moussa
A

Synthesis of OMCs

Hard
template

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<figure><img src="image_135.png" /></figure>

Filling of template
with precursor

<figure><img src="image_136.png" /></figure>

<figure><img src="image_137.png" /></figure>

Carbonization

<figure><img src="image_138.png" /></figure>

<figure><img src="image_139.png" /></figure>

Removal of
template

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Soft
template

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Precursor polymerizes
around the template

<figure><img src="image_143.png" /></figure>

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Carbonization and
removal of template

<figure><img src="image_145.png" /></figure>

B

<figure><img src="image_146.png" /></figure>

C

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CT2P1W2_60

<figure><img src="image_148.png" /></figure>

D

<figure><img src="image_149.png" /></figure>

E

<figure><img src="image_150.png" /></figure>

**FIGURE 7** | (A) Schematic synthesis of OMCs by hard and soft template methods. (B,C) Hard-templated OMCs using SBA-15 as template and aminobenzoic acid as precursor (Adapted from Sánchez-Sánchez et al., 2016 by permission of The Royal Society of Chemistry). (D,E) Soft-templated OMCs using Pluronic® F127 as template and mimosa tannin as precursor (Adapted from Castro-Gutiérrez et al., 2018 by permission of The Royal Society of Chemistry).

et al., 2018). Isolating the contribution of N-functionalities to the SCs performance is a difficult task since nitrogen is always present with oxygen and can lead to modifications of textural properties. However, a correlation between the SCs performance and the amount and type of nitrogen functionalities has been reported (Frackowiak et al., 2006; Hulicova-Jurcakova et al., 2009; Braghiroli et al., 2015c). N-doped materials can reach high values of capacitance even if their surface areas are relatively low. This have been attributed to pseudocapacitance contributions but also to improved wettability and conductivity of the materials (Inagaki et al., 2010). Most studies agree that the major contribution to pseudocapacitance comes from the pyridinic, pyrrolic, and pyridine groups, see **Figures 8G–K** (Deng et al., 2016). However, some studies have suggested that quaternary groups interact with protons or anions in $H_2SO_4$ or KOH electrolytes, respectively, and could promote electron transfer through the carbon structure (Deng et al., 2016).

TRANSITION TO BIOSOURCED PRECURSORS OF POROUS CARBON MATERIALS

Apart from ACs, which are often produced on an industrial scale from biomass materials, most of the synthesis methods described in section Carbon Materials as Electrodes for Supercapacitors use precursors of petrochemical origin. For example, resorcinol or phenol crosslinked with formaldehyde are widely used to synthesize different types of OMCs (Zhu et al., 2016; Lu et al., 2018; Li et al., 2019). In an effort to research more environmentally friendly technologies and methods, biosourced substitutes for ordinary carbon precursors have been explored. A salt template-assisted, chemical activation process was applied by subjecting gelatin, sucrose, glucose or tannic acid (TA) mixed with KCl and $Na_2S_2O_3$ to carbonization (Fuertes et al., 2018; Sevilla et al., 2019). The resultant micro-mesoporous carbon materials had high surface areas in the range of 1,900–2,720
<figure><img src="image_151.png" /></figure>

$m^2 g^{-1}$. TA has also been used to produce micro-mesoporous CNSs by direct heat-treatment of the precursor mixed with urea and an eutectic salt (NaCl/ZnCl$_2$) (Tiruye et al., 2017) and to synthesize CNFs by carbonization of a glass fabric previously impregnated with TA (Hu et al., 2020). Hard-templating of sucrose with cubic and 2D hexagonal silica templates produced highly porous materials with surface areas as high as 2,000 $m^2 g^{-1}$ (Vix-Guterl et al., 2005). Also based on the use of silica templates, OMCs with surface areas higher than 900 $m^2 g^{-1}$ were synthesized from different types of biomass-derived phenols such as phloroglucinol, gallic acid, catechin, and mimosa tannin (Sanchez-Sanchez et al., 2017a). By soft-template routes, it has been reported that 2D hexagonal OMCs with surface areas ~700 $m^2 g^{-1}$ were obtained by hydrothermal treatment of β-cyclodextrin, extracted from starch, mixed with Pluronic® F127 and P123 (Feng et al., 2014) or by evaporation-induced self-assembly (EISA) using a mixture of lignin, phloroglucinol, glyoxal, and Pluronic® F127 (Herou et al., 2019). Besides, a

phase-separation method was able to produce N-doped 2D hexagonal OMC with a moderate area of 523 $m^2 g^{-1}$ from phloroglucinol, glyoxylic acid, and guanine (Moussa et al., 2018).

## TANNINS AS CARBON PRECURSOR

Tannins are polyphenolic substances present in many plants and trees. They have been used in the leather manufacturing process since the Middle Ages (SilvaTeam, 2018; CAPPEQ, 2019b). Their industrial production increased in the 1970s thanks to the development of tannin adhesives for wood (Fierro et al., 2018). Nowadays, tannins are used not only for tanning leather (hence their name), but also as wine additives, food supplements and cosmetic ingredients (CAPPEQ, 2019c). Although there are several methods for extracting tannins, the solid-liquid method is the one traditionally used on an industrial scale due to its simplicity, efficiency and low cost (de Hoyos-Martínez et al., 2019). The solid-liquid extraction method involves the
dissolution of tannins in water at moderate temperature (60–70°C), after which the solution is concentrated and spray-dried to obtain a red-brown powder (CAPPEQ, 2019a; de Hoyos-Martínez et al., 2019). The chemical composition of the final powder strongly depends on the plant species and the part of the plant from which the tannin has been extracted. However, the similarities in their compositions allow them to be classified into two broad categories, namely hydrolysable or condensed (Tondi and Pettschnigg, 2015; de Hoyos-Martínez et al., 2019). In particular, condensed tannins are of primary interest in the synthesis of carbon materials as they contain more than 70–80% of polyflavonoids. Their aromatic rings with hydroxyl groups give polyflavonoids a reactivity similar to that of phenolic molecules of petrochemical origin such as phenol or resorcinol (Pizzi, 1982; Fierro et al., 2018), as well as a high carbon yield upon pyrolysis, ~50%. More details on the properties of condensed tannins are given in the next subsection.

## Condensed Tannins

The polyflavonoids of condensed tannins are composed of flavan-3-ol moieties made up of two phenolic rings (A and B) linked by a heterocyclic ring. **Figure 9** shows the main four types of flavonoid molecules of condensed tannins as well as with the type of trees in which they are found. Thus, mimosa (*Acacia mearnsii*), quebracho (*Schinopsis balansae* and *Schinopsis lorentzii*), and maritime pine (*Pinus pinaster*) are the most used species for the extraction of condensed tannins, representing 90% of the world's production (Fierro et al., 2018; Shirmohammadli et al., 2018). The reactivity of the flavonoid units depends on the position of the -OH groups and the available reaction sites. In this sense, it has been shown that the A-ring is more reactive than the B-ring (Pizzi, 1982). Indeed, tannins can naturally auto-condense by linking the units at positions 4,6 for profisetinidin and prorobinetinidin or 4,8 for procyanidin and prodelphinidin (see again **Figure 9**), producing oligomers of up to 10 or 11 units (Haslam, 2007). The ability of condensed tannins to auto-condense and react with aldehydes to form phenolic resins makes them an attractive alternative to produce high added-value materials.

In addition to their good chemical reactivity, tannins have the advantage of being non-toxic in the event of exposure to low amounts, and high doses can only cause irritation or have anti-nutritional effects (Schlienger et al., 2012). Compared to the toxicity and hazards of substances like resorcinol, phenol or formaldehyde, as shown in **Table 2**, tannins should be considered as excellent ecological and non-toxic alternative precursor to produce porous carbons.

## Synthesis of Porous Carbons

Condensed tannins have been used to produce a wide variety of high added-value materials. The tannin-derived products can be converted into carbon by high-temperature treatment. The resulting materials are glass-like due to the non-graphitizing nature of the precursor, and present isotropic properties and good electrical conductivity. As for petrochemical precursors, the synthesis of porous materials from tannin usually requires the preparation of a resin by crosslinking with formaldehyde,

hexamine, or glutaraldehyde, among other possible aldehydes. Thus, different materials have been synthesized such as foams (Tondi et al., 2009; Lacoste et al., 2013; Jana et al., 2014; Szczurek et al., 2014; Delgado-Sánchez et al., 2018), gels (Szczurek et al., 2011a,b; Amaral-Labat et al., 2013; Braghiroli et al., 2019), microspheres (Grishechko et al., 2016), or polyHIPEs (Szczurek et al., 2013). Furthermore, tannin autocondensation reactions enable its polymerization without having to use crosslinkers, opening the way for the production of OMCs by self-assembly techniques (Schlienger et al., 2012; Braghiroli et al., 2016). The following section describes some of the methods used to obtain micro-mesoporous carbon materials from tannin, as well as their performance when used as electrodes for SCs, they are summarized in **Table 3**.

### Sol-Gel

CGs were produced from mimosa tannin and formaldehyde (TF) organic cryogels (Amaral-Labat et al., 2012). The pH of mimosa tannin solutions was adjusted with either acetic acid or NaOH diluted in water to obtain a pH ranging from 3.3 to 7.3. Such solutions were placed in an oven at 85°C for 5 days, in which gelation and aging took place. After exchanging water for tert-butanol, the gels were freeze-dried to obtain TF cryogels. Finally, CGs were produced by carbonization of TF cryogels at 900°C. Surface areas from 399 to 1,420 m² g⁻¹ were obtained, with minimum values found for pH 4.3 and 5.3 and attributed to pore collapse during carbonization. The highest surface areas were found for pH > 6. This unusual development of surface area was ascribed to the presence of NaOH, which could act as an activating agent during the carbonization process. The textural characterization revealed that the CGs were mainly micro- and macroporous materials, with an unusually low amount of mesopores.

In order to test their electrochemical performance, the CGs were ground to prepare pellets by mixing the corresponding sample, carbon black and PVDF (75:5:20 wt. %), and then symmetrical SCs were assembled and tested in a T-type cell. CV tests were carried out from 2 to 50 mV s⁻¹ using 4 M H₂SO₄ as electrolyte in a potential window of 1 V. The values of C~e~ of the CGs at 2 mV s⁻¹ ranged from 30 to 109 F g⁻¹, similar to the values found for more expensive carbon aerogels. As expected, the best performance was observed for the materials with the highest surface area. However, as the scan rate increased, the CV curves became narrower and lost their quasi-rectangular shapes, indicating a loss of SC behavior and resulting in a decrease of C~e~. It was concluded that, although these CGs were highly porous, the values of capacitance were generally lower than expected due to the large amount of macropores that do not account for the capacitance. Besides, the sharp drop in C~e~ with the increase in scan rate was related to the absence of mesopores to facilitate the diffusion of electrolyte ions in the micropores.

### Hydrothermal Carbonization

Hydrothermal treatments of aqueous solutions of tannins take advantage of their autocondensation at very acidic or basic pH. By varying pH, tannin concentration and temperature, it is possible to obtain gels or spheres. Besides, it is possible to perform