# Fluid transport in cellulose-based composite microfluidic paper-based analytical devices

Erwan Troussel, Julien Gomez-Martinez, Aurore Denneulin, Jean-Charles

Brès, Davide Beneventi

► To cite this version:

Erwan Troussel, Julien Gomez-Martinez, Aurore Denneulin, Jean-Charles Brès, Davide Beneventi. Fluid transport in cellulose-based composite microfluidic paper-based analytical devices. Cellulose, 2025, 32 (7), pp.4199-4211. 10.1007/s10570-025-06509-8. hal-05362290

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Submitted on 13 Nov 2025

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# Fluid transport in cellulose-based composite microfluidic paper-based analytical devices

Erwan Troussel<fnref n="1" /> • Julien Gomez-Martinez<fnref n="2" /> • Aurore Denneulin<fnref n="1" /> • Jean-Charles Brès<fnref n="2" /> • Davide Beneventi<fnref n="1" />

Corresponding author: Erwan Troussel erwan.troussel@lgp2.grenoble-inp.fr and Davide Beneventi davide.beneventi@lgp2.grenoble-inp.fr

## Abstract

Point-of-care (POC) devices offer a considerable opportunity to tackle epidemics through rapid, sensitive and specific screening. Microporous nitrocellulose membranes are the most widely used substrate for POC devices due to their high affinity with biological reagents such as proteins and oligonucleotides. However, nitrocellulose has several disadvantages: it is produced through an environmentally unfriendly chemical process, and it is converted with a subtractive process. In addition, nitrocellulose hydrophobicity requires surfactants to increase membrane wettability. The aim of this work is to manufacture a microfluidic paper-based analytical device (µPAD) composite of a microfibrillated cellulose matrix (MFC) embedded with microcrystalline cellulose (MCC) and silicon oxide particles (SiO₂) to manage water uptake and fluid flow kinetics within the fluidic strip. Several composite inks were formulated by varying the MFC, MCC and SiO₂ mass fractions, and the impact of particle size and mass fraction of the composite on water uptake was compared. An additive manufacturing technique was assessed using a fluid dispenser mounted on a 6-axis robot to manufacture the cellulose-based strips. One key objective was to develop a µPAD as an alternative to nitrocellulose-based POC devices. The µPAD composite was assessed with a migration test in a lateral flow configuration. Promising results were obtained by producing strips with a water capillary rise of 4 cm in 5 minutes, comparable to the performance of nitrocellulose. Finally, the µPAD strips were spotted with proteins and sealed with MFC in order to produce a functional cellulose-based POC proof of concept

## Keywords

Paper-based analytic devices (PAD) • cellulosic composite • microfluidic • Functional materials

Contact e-mail: erwan.troussel@grenoble-inp.fr

## Introduction

Successful containment of an epidemic outbreak requires rapid, sensitive and specific screening (Yang et al. 2022). Point of care (POC) devices, which allow tests to be performed in the patient's proximity, present a significant potential to address this challenge (Drain et al. 2014; Vashist et al. 2015). To provide a framework for these devices, the World Health Organization (WHO) established the ASSURED criteria, which comprise eight key values: Affordable, Sensitive, Specific, User friendly, Rapid and Robust, Equipment-free and Deliverable to end users (Smith et al. 2018; Naseri et al. 2022).

Microfluidic paper-based analytical devices (µPADs) effectively address these criteria. In fact, paper is a low-cost and widespread raw material, which can be utilized in fluidic devices without any external system or pump due to the action of capillary forces (Tong et al. 2021). Paper is both a biocompatible and

<footnote n="1">¹Univ. Grenoble Alpes, CNRS, Grenoble INP¹, LGP2, F-38000 Grenoble, France</footnote>

<footnote n="2">²Pathogenesis and Control of Chronic Infections, University of Montpellier, INSERM, EFS, F-34184 Montpellier, France</footnote>

<footnote n="1">¹ Institute of Engineering Univ. Grenoble Alpes</footnote>
biodegradable material, requiring a minimal number of reagents and allowing the grafting of biological reagents such as protein, enzymes, DNA, or other macromolecules (Hou et al. 2022).

Current POC devices utilize nitrocellulose as the substrate for µPADs. Nitrocellulose is produced by the esterification of cellulose, such as cotton linters, with a solution of sulfuric acid and nitric acid (Sullivan et al. 2020). The resulting nitrate groups are strong dipoles that can directly bind to peptide bond dipoles (Tang et al. 2022). Nitrocellulose is manufactured in the form of a roll which then needs to be cut, transformed and modified to fit the µPAD. To manage the fluid pathway, several unit operations, such as cutting and thermoforming (Kabir et al. 2020) modify the physical barriers of the structure, or convert it to become hydrophobic using plasma treatment (Singh et al. 2018) or alkyl ketene dimer treatment (AKD) (Li et al. 2010). However, nitrocellulose has several drawbacks such as its complex and polluting manufacturing process, the requirement of surfactant to increase wettability and the subtractive process required for µPAD production.

A new manufacturing approach is currently under investigation, based on the fabrication of a cellulose-
based composite using conventional additive manufacturing processes (Liu et al. 2020; Hilber and Jakoby
2023; Solin et al. 2021). This method allows the production of µPADs with customizable designs without
consuming additional raw materials. The fluidic pathways consist of a polymer matrix that reinforces the
structural and mechanical properties (Scaffaro et al. 2018; Wang et al. 2019) and micro- and nanoparticles
that promote fluid flow. Recent studies (Doherty et al. 2014; Hilber and Jakoby 2023) highlighted that water
uptake significantly depends on the polymer ratio due to the modification of the composite structure and
surface energy. The research conducted by Hassanin and colleagues (Hassanin et al. 2013) further
illustrates that the fluctuations in porosity and density are contingent upon the quantity of particles
incorporated within the matrix. Indeed, the addition of TiO₂ particles reduces the composite density by
increasing its intrinsic porosity. The possibility to print the composite enables the formation of more
complex fluidic systems and more tunable channel designs compared to the traditional paper-based fluidic
systems.

This work aims to fabricate a cellulosic composite µPAD based on the breakthrough strategy developed by Hilber and Jakoby and Solin and colleagues (Hilber and Jakoby 2023; Solin et al. 2021) using mainly non-chemically transformed cellulosic matrix and particles. Microfibrillated cellulose (MFC) was used as the matrix and serves as a binder and structural reinforcement, while microcrystalline cellulose (MCC) and silicon dioxide (SiO₂) were used as particles to control water uptake within the strip. MCC was selected among several cellulosic powders for its high crystallinity and minimal deformation during drying, while SiO₂ particles were chosen for their spherical shape, size homogeneity and low surface energy. The composite was printed by a dispensing system coupled to a 6-axis robot arm on a cellulose substrate to produce a microfluidic device that is nearly 100% composed of cellulose. The composite was also evaluated for the detection of proteins using a migration buffer employed in nitrocellulose-based lateral flow assays (Gomez-Martinez et al. 2018).

**Materials and methods**

Chemicals and materials

MFC was purchased from Weidmann (Celova M250R-G) in the form of an aqueous suspension at 3% weight concentration. These MFCs are obtained by mechanical processing, and the nominal mean length of the microfibrils was 9.4 µm. The secondary components used in the formulation of the cellulose microparticle aqueous inks were MCC, a ground cellulose powder with particle size ranging from +60 mesh (250 µm) to +200 mesh (74 µm), and spherical SiO₂ particles with an average particle size of 4 µm (as determined by preliminary laser diffraction measurements). Particles were purchased from Sigma-Aldrich. Nitrocellulose microfluidics strips referenced as High Flow 120 and 135 (HF120 and HF135), immersion pads, glass-fiber conjugate pads, and absorbent pads were purchased from Merck Millipore (Darmstadt, Germany). The HF120 and HF135 nitrocellulose membranes had a nominal 4 cm-water uptake of 120 s and 135 s, respectively. Sodium chloride-sodium citrate (SSC) buffer 20× concentrate (pH 7.0) and biotinylated bovine
serum albumin (BSA) were purchased from Thermo Fisher Scientific, sodium dodecyl sulfate (SDS), Tween 20 and Urea from Sigma-Aldrich, and 40nm anti-biotin antibody-conjugated gold nanoparticles at 9 × 10¹⁰ particles/mL (BBIsolutions, Cardiff, UK) were purchased from Tebubio (France). Alkyl ketene dimer (AKD) used to hydrophobize MFC films (Aquapel J220 Alkaline Size) was purchased from Solenis (DE, USA).

## Cellulose microparticle aqueous inks

To create microfluidic channels, two formulations of cellulose-based ink were prepared using MFC as binder and either MCC or SiO₂ at different mass ratios in order to study the impact of each component on the capillary flow kinetics.

For each formulation, different ratios of MFC and secondary components were evaluated. The slurry was composed of 10-30% MFC by mass (w/w) and 70-90% of secondary components by mass of the final mass formulation. For each formulation, 5 g of MFC slurry at a 3% concentration was used. The 5 g MFC slurry was poured in a beaker and secondary components were subsequently added and stirred for 2 minutes with a spatula.

## Strip fabrication

Two different processes were used to manufacture sample strips: molding (Fig 1 a) and direct ink writing using a pneumatic dispenser (Fig 1 b) and Fig 1 c).

<figure><img src="image_2.png" /><figcaption>**Fig 1** Pictures of the two processes used to manufacture sample strips a) molding b) direct ink writing using a 6-axis robot and a dispensing device, c) schema of the pneumatic dispensing device</figcaption></figure>

### Sample molding process

Molded sample strips were studied first. The mold was manufactured in ABS (acrylonitrile butadiene styrene) because of its stability during the drying step **Fig 1 a**. It consisted of six channels, each measuring 56 mm in length, 7 mm in width and 0.7 mm in thickness, with a total volume of 275 mm³ per strip. A PET film was applied to the bottom of the mold to create a flat surface, unstructured by the roughness of the mold. The formulations were spread with a spatula into each mold.

This straightforward and user-friendly manufacturing process enables the production of reproducible samples, which were then used for initial characterizations.

### Fluid dispenser printing process

The printed sample strips were manufactured using a pneumatic on/off needle valve DV-5425 purchased from Vieweg coupled to a stainless-steel needle with ellipsoidal section (4.5 x 0.5 mm) and connected to a 6-axis robotic arm (TX2 60, Staübli) **Fig 1 b**. The pneumatic valve was synchronized with the robot movement in order to control the printed flow of the formulation. The composite paste was placed in a plastic pressurized syringe connected to the valve. The pressure applied inside the syringe was 4.6 bar, the
needle lift height over the printing bed was set to 2 mm and the printed tracks length to 80 mm (Fig 1 c). Track width and thickness depended on the robotic arm speed, which was varied between 150 and 350 mm.s⁻¹, and the extrusion flow rate which was set to 1.6 g.s⁻¹. As a function of the printing parameters, the width and the thickness of the dry track varied between 5 and 3 mm and 0.8 and 0.4 mm, respectively.

The samples were initially printed on the same PET film as the molded samples and were used to characterize water migration kinetics.

Subsequently, in order to produce strips with variable geometry, trials were conducted to directly print composite strips onto a cellulosic substrate (Tenhunen et al. 2018). The samples undergo printing on a layer of wet MFC treated with (X% w/w) AKD, with a basis weight of 15 g/m², which is obtained through spraying. Then, they were encapsulated by a second MFC layer with a basis weight of 15 g/m².

## Two-step drying

Each sample strip was dried in a two-step procedure. The first step consisted in drying the sample in an oven at 90°C for 5–10 minutes, depending on the formulation, to evaporate most of the water present in the aqueous ink. The second step involved drying under light pressure using a Rapid Köthen hand sheet former from Savoie Maintenance Service at 85 °C for 30 minutes to evaporate the remaining water.

## Structural and physical characterization

### Porosity

Two types of porosity were evaluated: the apparent porosity and the open porosity. The apparent porosity considers the entire material's porosity, including closed, open and surface porosity. Whereas the open porosity is the porosity accessible to the test liquid, thus contributing to liquid uptake during capillary-induced flow.

The apparent porosity was calculated based on the sample strip's dimensions and mass, measured with an electronic caliper and a precision balance, respectively. It was calculated as follow:

$$ \varepsilon_{app} = 1 - \frac{\rho_{mes}}{\rho_{bulk}} $$

**Equation 1 apparent porosity**

where ρ mes the composite geometric density and ρ bulk is the composite density calculated from the nominal density and mass fractions of the matrix and particle (g.m⁻³).

The open porosity was determined by calculating the difference between the air fractions derived from geometric and gravimetric densities. Gravimetric density was calculated by weighing the sample under water using a force tensiometer (Biolin Sigma 700) and Equation 2:

$$ \rho_{gravimetric} = \frac{m_{air}}{m_{water} - m_{air}} * (\rho_{water} - \rho_{air}) + \rho_{air} $$

**Equation 2 gravimetric density**

where m air is the mass of the sample in air (g), m water is the mass of the sample in water (g), ρ air is the density of air (g.m⁻³) and ρ water is the density of water (g.m⁻³).
Finally, the air fraction was calculated as:

$$ \Phi_{air} = \omega_{cs} * \left(1 - \frac{\rho_{mes}}{\rho_{cs}}\right) + \omega_{MFC} * \left(1 - \frac{\rho_{mes}}{\rho_{cellulose}}\right) $$

### Equation 3 air fraction

Where $\omega$ is the weight fraction of secondary components or MFC, $\rho_{mes}$ is the geometric or gravimetric density of the sample (g.m⁻³), $\rho_{cellulose}$ is the density of the cellulose (g.m⁻³) and $\rho_{cs}$ is the density of the secondary components (g.m⁻³).

Internal structure imaging was done by scanning electron microscope (SEM) observation (quanta 200, FEI, USA) at ×200–800 magnification on a transverse slice of strip. The samples were cut with a scalpel blade. A layer of gold a few nanometers thick was then deposited on the section to metalize the sample. The samples were observed under an electric field of 15 kV, maintained at a working distance of 9 mm.

Microtomography measurements were conducted by Novitom (Grenoble, France) at ESRF (European Synchrotron Radiation Facility). The pixel size of the measurements corresponded to 0.72 µm, the beam energy was set to 25 keV, and the field of view was configured to 2.63 x 2.63 x 1.47 mm³ for 5,000 projections. A set of measurements was obtained from two distinct regions of the same sample.

### Liquid uptake

To assess the fluidic properties of the composite, the time taken for 4 cm of water uptake by vertical strips was measured and compared with that of commercial nitrocellulose membranes (Yahaya et al. 2019). The liquid uptake was analyzed using the Lucas-Washburn law (Washburn 1921), which describes fluid front advances as a function of time:

$$ L_{[t]} = \sqrt{\frac{r * \gamma * \cos(\theta)}{2 * \mu}} * t $$

### Equation 4 Lucas Washburn law

where $L_{[t]}$ (m) is the front advances, r (m) is the pore radius, $\gamma$ (N.m⁻¹) is the liquid surface tension, $\mu$ (Pa.s) is the liquid dynamic viscosity, $\theta$ (°) is the contact angle between the liquid and the solid phase and t (s) is the time.

Capillary uptake was experimentally measured by recording the mass variation of strips immersed 2 mm vertically in liquid at 25°C using a force tensiometer (Biolin Sigma 700). The liquid uptake was monitored for 5 minutes with a 2 Hz sampling frequency. The height of capillary uptake was calculated as:

$$ h_{[t]} = \frac{m_{[t]}}{\varepsilon * S * \rho_{l}} $$

### Equation 5 liquid uptake

where $h_{[t]}$ (m) is the height of capillary rise, $m_{[t]}$ (g) the measured mass, $\varepsilon$ the open porosity, S (m²) the cross-sectional area of the strip and $\rho_{l}$ (g.m⁻³) the liquid density.

All experiments were conducted using deionized water. Additionally, trials were performed using a standard buffer used in lateral flow assays (Gomez-Martinez et al. 2018). Three samples, produced independently, were tested. The value presented is the mean of the three samples tested.

### Density and surface tension characterization

Liquid density and surface tension were characterized using a force tensiometer (Biolin Sigma 700). The density module used was a spherical glass probe with a volume of 0.918 cm³ calibrated with water. Whereas the mean surface tension was measured using De Nouy ring and averaged from ten measurements.
## Strip integration

As application, the optimized strips were integrated into more complex devices to evaluate theirability to detect protein targets.

A µPAD cellulose-composite strip replaced the standard nitrocellulose membrane in a previously described lateral flow test (Gomez-Martinez et al. 2018). Two spots of biotinylated BSA (0.1 µl, 25 µg/ml) were applied at the top of the strip. After spotting, the membranes were dried at 80°C for 40 minutes. Anti-biotin antibody-conjugated gold nanoparticles (1.5 × 10⁸ particles) were loaded onto the conjugate pad and dried at 60°C for 15 minutes. These gold nanoparticles served as visual tracers. The buffer used in this application consisted of 1× SSC, 0.5% SDS, and 1M urea. In standard nitrocellulose devices, after 1 mm immersion into 200 µL of buffer, the capillary migration induces the rehydration and release of dried gold nanoparticles. The nanoparticles travel through the membrane and are captured at the control zone by immobilized biotinylated-BSA, forming red spots. This test was used with the composite strips in order to qualitatively evaluate the reagent flow.

## Results and discussion

### Structural characterization of the cellulose-based printed composites

As outlined in the materials and methods section, the first test samples were prepared by paste molding (Fig 2 a). All samples had consistent dimensions, with a length of 50.7 ± 1.3 mm and a width of 6 ± 0.2 mm. However, as shown in Fig 2 b), the thickness ranged between 250 and 650 µm as a function of the MCC and SiO₂ mass fraction. During drying, the strips underwent anisotropic shrinkage. This shrinkage progressively decreased from approximately 68% to 13%, corresponding to the increasing mass fraction of rigid particles and the reduction of water introduced via the MFC slurry (Fig 2 b). It can be observed that composites including SiO₂ exhibit greater thickness and volume than those containing MCC at equivalent weight fractions. This phenomenon can be attributed to the fact that SiO₂ does not absorb water, in contrast to MCC, and consequently MFC-SiO₂ shows less shrinkage during the drying process. Moreover, MFC-SiO₂ composites exhibit higher brittleness compared to MFC-MCC.

<figure><img src="image_3.png" /><figcaption>Fig 2 a) Dried molded strips and b) effect of particle weight fractions on dry strip volume and thickness. The strip volume before drying was 275 mm³</figcaption></figure>

Fig 3 illustrates that the apparent porosity of the strips is consistently higher than the open porosity due to the presence of both open and closed pores (i.e. inaccessible to water during gravimetric tests). Strips composed of MFC and SiO₂ particles exhibit higher porosity compared with those made from MFC and MCC. This is likely due to the uniform distribution of rigid, hydrophilic SiO₂ particles and the disruption of the MFC network leading to reduced particle packing during the drying process (Krol et al. 2015). Notably, as the particle weight fraction increases, SiO₂ and MCC display distinct behaviors: higher MCC fractions resulted in decreased porosity due to structural stratification and pore closure, while higher SiO₂ fractions increased open porosity.
<figure><img src="image_4.png" /><figcaption>Fig 3 Apparent and open porosity in function of the mass ratio of secondary components</figcaption></figure>

SEM images of the strips with 80% particle weight fraction (Fig 4), further support the earlier observations. As illustrated in Fig 4 a) and Fig 4 c), a clear distinction in structural organization is evident between the MFC-MCC composite and the MFC-SiO₂ composite. The MFC-MCC exhibits a heterogeneous structure, characterized by the arrangement of MCC particles with larger pores, while the MFC-SiO₂ displays a more homogeneous structure with a layered arrangement. Higher magnification SEM images corroborate these observations. As illustrated in Fig 4 b), the MCC particles have a disordered configuration, with the MFCs acting as binder. Fig 4 d) shows the layering of the composite, interspersed with SiO₂ particles. The SiO₂ particles, being smaller than the MCC particles, induce the formation of a greater number of pores smaller than pores in the MFC-MCC composite.

<figure><img src="image_5.png" /><figcaption>Fig 4 SEM pictures of 20% MFC + 80% MCC at a) 200X magnification and b) 800X magnification and 20% MFC + 80% SiO2 at c) 200X magnification and d) at 800X magnification</figcaption></figure>
Water uptake by the manufactured strips

**Fig 5** shows the comparison of the capillary rise of water in porous strips of different compositions with high-flow nitrocellulose references. Curves display a stepwise shape which was attributed to the measurement accuracy limits of the utilized equipment. A similar trend was observed in the curves obtained with the nitrocellulose references.

<figure><img src="image_6.png" /><figcaption>Fig 5 Water uptake for the six formulations tested and the two nitrocellulose references</figcaption></figure>

The kinetics of water uptake highlights that particle composition notably affects capillary rise. The highest liquid uptake rate was achieved with a formulation consisting of 30% MFC and 70% MCC. In addition, all formulations containing MCC demonstrated higher capillary rise than those containing SiO₂ particles. The observed phenomenon can be attributed to the different pore size of the MFC-MCC composites as compared to the MFC-SiO₂ composites. This difference is evident in the SEM images shown in **Fig 4**. This trend is in line with the Lucas-Washburn law where pore size is the predominant factor influencing the liquid migration kinetics.

Although the capillary rise in the cellulose-composite strips was generally lower than in the HF120 and HF135 nitrocellulose references. The strips exhibited a capillary flow comparable to the nitrocellulose references. Initially, the capillary rise was rapid, occurring within the first tens of seconds. However, the kinetic rate subsequently decelerated. The 5 cm length nitrocellulose strips saturated in 200 seconds subsequently reaching a plateau. In contrast, the strips did not attain this plateau at 5 minutes, as they had not reached saturation. It is important to note that the high capillary rise in slightly hydrophilic nitrocellulose strips (Zhdanov et al. 2003) was enhanced by the presence of surfactants on the HF120 and HF135 surfaces, as indicated by the low surface tension of 53.7 mN/m after washing the nitrocellulose strips in 200 mL of deionized water. After washing, the contact angle rose from <20° of the unwashed membrane to 112°.

The capillary rise speed and height progressively decreased as the mass fraction of MCC increased. This was attributed to the high concentration of large particles, which cause the strip's structure to break down and generate fractures that impair the capillary rise. Conversely, SiO₂ particles improved capillary rise, as supported by porosity measurements and SEM observations.

Based on these findings, three new formulations with 20% MFC by mass and varying amounts of the two secondary components (20/60/20, 20/40/40, and 20/20/60 of MFC/MCC/SiO₂, respectively) were tested. The aim was to leverage MCC to enhance capillary rise and SiO₂ to maintain the structural integrity of the composite strip.

20% MFC is the medium between 30% and 10% MFC, which represent the optimum formulations for MCC and SiO₂ particles respectively. The results are presented in **Fig 6**.
<figure><img src="image_7.png" /><figcaption>**Fig 6** Water uptake for the five formulations with 20% MFC tested and the two nitrocellulose references</figcaption></figure>

The results presented in **Fig 6** highlight the influence of the secondary components on water uptake. Formulations with higher MCC content displayed increased water uptake, whereas those with higher SiO₂ content had reduced water uptake. The formulation containing 60% MCC and 20% SiO₂, as well as that with 40% MCC and 40% SiO₂, exhibited water uptake similar to the 80% MCC formulation. However, the use of SiO₂ results in a more fluid formulation due to its inability to absorb water, compared to MCC. Consequently, the processing of the formulation through either the molding or direct printing method is more straightforward. Nevertheless, the 20% MFC, 60% MCC, and 20% SiO₂ formulation exhibited enhanced homogeneity and ease of printing. Additionally, this formulation comprises the highest proportion of cellulose among the two.

To enhance the formulation, dilution of the initial paste to reduce the dry mass content, and water exchange with a lower surface tension solvent to reduce capillary forces during drying were considered (Bisson et al. 2003). Two new formulations were developed based on these insights:

- 20% MFC, 60% MCC, 20% SiO₂, with an additional 20% of the total mass as distilled water

- 20% MFC, 60% MCC, 20% SiO₂, with an additional 20% of the total mass as ethanol. The final liquid phase consisted of 50% ethanol and 50% distilled water by mass.

The water uptake results for these new formulations and 20% MFC, 60% MCC, 20% SiO₂ as a comparative are shown in **Fig 7**.

<figure><img src="image_8.png" /><figcaption>**Fig 7** Water uptake of the optimized formulation of 20% MFC, 60% MCC, 20% SiO₂, with 20% water dilution, and with solvent exchange and 20% ethanol dilution and the two nitrocellulose references</figcaption></figure>

The optimized formulations show a higher water uptake than the initial three-component formulation (20% MFC, 60% MCC, 20% SiO₂). Diluting with water and using solvent exchange improved pore connectivity, leading to increased open porosity. Specifically, the open porosity increased from 0.42 in the non-optimized formulation to 0.54 with 20% water dilution and further increased to 0.66 with solvent exchange and 20% ethanol dilution. This improvement is attributed to the lower surface tension of ethanol compared with that
of water, resulting in fewer hydrogen bonds with cellulose and less structural collapse during drying (Sterlyagov et al. 2020).

**Fig 8** shows the micro-tomography analysis of sample 20% MFC, 60% MCC, 20% SiO₂ composite, with an additional 20% of the total mass in ethanol. **Fig 8 a)** is the cross-section of the sample obtained by micro-tomography and **Fig 8 c)** is the corresponding SEM image. The two images corroborate each other and show that the strip is composed of pores with heterogeneous sizes. **Fig 8 b)** shows the volume-weighted average size distribution of closed pores. The calculated average diameter is 13.35 µm for the first zone of the sample and 10.91 µm for the second zone. This difference can be explained by the heterogeneity of the pores in the membrane. Those pore sizes are comparable to that of nitrocellulose membranes, whose pore sizes range from 0.5 to 15 µm. **Fig 8 d)** shows the pore volume fraction in the sample. It is 70% in the first zone and 80% in the second zone. The calculated apparent porosity for this formulation is 79%. The differences can be explained by the fact that the apparent porosity is calculated over the entire strip, unlike the results obtained by micro-tomography.

<figure><img src="image_9.png" /><figcaption>**Fig 8** Section of the of 20% MFC, 60% MCC, 20% SiO₂, with solvent exchange and 20% ethanol dilution a) micro-tomography view, b) SEM image and c) volume-weighted average pore size and d) the pore volume fraction</figcaption></figure>

## Lateral flow buffer characterization

In order to test capillary flow in cellulose strips with test fluids optimized for nitrocellulose lateral flow assays, a dedicated migration buffer (1X SSC, 10% Tween-20, 0.5% sodium dodecyl sulfate, and 1 M urea) was used as carrier of anti-biotin antibody-conjugated gold nanoparticles instead of water. According to the Lucas-Washburn law, the physico-chemical properties of the fluid can significantly influence capillary rise kinetics, impacting the design of the test. The density, surface tension, and viscosity of the migration buffer are summarized in Table 1.
<table><caption>**Table 1** Comparison of migration buffer and water properties</caption><thead><tr><th></th><th>Migration buffer</th><th>Water</th></tr></thead><tbody><tr><th>Density (g.cm<sup>-3</sup>)</th><td>1.029</td><td>0.997</td></tr><tr><th>Surface tension (mN.m<sup>-1</sup>)</th><td>38.9 ± 0.2</td><td>72.8</td></tr><tr><th>Viscosity (mPa.s)</th><td>2.630 ± 0.276</td><td>1.005</td></tr></tbody></table>

The migration buffer has the same density as water but shows substantial differences in surface tension and viscosity. While the buffer is mainly water-based, the presence of surfactant (Tween 20 and SDS) at concentrations higher than the critical micelle concentration explains its lower surface tension and higher viscosity (Ziani et al. 2008). Tween 20 is typically used to enhance capillary flow by mitigating the hydrophobic forces in nitrocellulose (Wong and Tse 2009, p. 10). Whereas the role of SDS is to stabilize gold nanoparticles in suspension to avoid their agglomeration (Mendoza-Cachú et al. 2018). However, since the cellulose based composite strips are hydrophilic, the use of Tween 20 is unnecessary.

Buffer uptake was tested with the optimized formula (20% MFC, 60% MCC, 20% SiO₂ + 20% of the total mass as ethanol).

<figure><img src="image_10.png" /><figcaption>**Fig 9** Comparison of water and migration buffer with 0%, 4% and 10% Tween 20 uptake using optimized formula strips of 20% MFC, 60% MCC, 60% SiO₂ + 20% of the total mass of ethanol</figcaption></figure>

The results, shown in **Fig 9**, indicate that after 5 minutes, the buffer uptake was approximately half that of water. This difference is attributed to: the higher viscosity of the buffer, which impairs fluid flow through the pores, and the lower surface tension of the buffer, which reduces capillary flow in hydrophilic materials (Wijnhorst et al. 2020). Similar investigations be conducted for other applications involving different migration fluids to ensure comprehensive understanding of the underlying mechanisms.”

To mitigate this effect, the buffer formulation for cellulose-based composite strips should have the surfactant omitted to better align with the hydrophilic nature of the composite. During the application tests, Tween 20 was not added to the migration buffer.

## Direct ink writing of the composite strips

In order to produce strips with variable geometry, trials were conducted to directly print composite strips onto a PET film, to observe a similar bottom surface as mold sample. Two formulations — 20% MFC, 60% MCC, 20% SiO₂ and 20% MFC, 60% MCC, 20% SiO₂ with 20% distilled water — were evaluated.
<figure><img src="image_11.png" /><figcaption>**Fig 10** Water uptake of samples obtained by dispenser printing 20% MFC 60% MCC 20% SiO₂ and 20% MFC 60% MCC 60% SiO₂ + 20% of the total mass of distilled water, the reference 20% MFC 60% MCC 20% SiO₂ obtained by molding and the two nitrocellulose references</figcaption></figure>

As shown in **Fig 10**, all printed sample strips exhibited lower water uptake than molded sample strips, which is attributed to a difference in geometric density and in open porosity caused by the manufacturing process. The geometric density measurement is 550 ± 30 kg/m³ for the printed samples versus 466 ± 40 kg/m³ for the molded ones. Furthermore, the open porosity of printed strips was 0.39, while that of molded strips was 0.55. Dispenser printing requires a threshold pressure to extrude cellulose-based inks (Gauss et al. 2021). The nozzle's dimensions (in this case, 4.5 x 0.5 mm) generate shear stress during extrusion, compacting the formulation and resulting in densification in the wet state. This compaction translates into lower apparent and open porosity after drying. A similar trend was observed with undiluted printed samples. To mitigate this, increasing the nozzle radius could help reduce shear stress, though it would lead to thicker strips in both the wet and dry states.

## Strip application

To assess the potential of the cellulose-based fluidic strips developed in this study, one preliminary test was performed within the field of POC devices and lateral flow assays: (i) detecting BSA deposits using gold nanoparticles in a lateral flow assay configuration.

**Fig 11 a)** is a visual detection of biotinylated BSA spot interpretation guide. **Fig 11 b)** illustrates protein spot detection through the migration of specific gold nanoparticles in a lateral flow setup. The nanoparticles migrated along the strip, carried by the buffer to the top of the lateral flow test (the purple mark at the top). Upon reaching the protein spots, the anti-biotin antibody-conjugated nanoparticles were selectively captured, allowing them to be visually detected by the appearance of a red dot. This test was used with the composite strips in order to qualitatively evaluate the reagent flow. Migration of nanoparticles represents a first step in the development of real biological tests, including oligonucleotide detection.

<figure><img src="image_12.png" /><figcaption>**Fig 11 a)** visual detection of biotinylated BSA spot interpretation guide, **b)** detectable protein spots (blue arrow) on the cellulose-based strip</figcaption></figure>
Conclusion

This work demonstrates the feasibility of a µPAD primarily composed of cellulose manufactured using additive manufacturing techniques. While the cellulose-based strip shows lower liquid uptake (both with water and migration buffer) compared with microporous nitrocellulose membranes, adjusting the kinetic properties through design modifications can optimize each component of the lateral flow assay.

Protein probes were effectively spotted and visually detected on the strip using anti-biotin-conjugated gold nanoparticles. This demonstrates the potential for nanoparticle migration into the strip. As previously stated, this test marks a preliminary step towards more sophisticated assays. To that end, ongoing oligonucleotide immobilization assays seek to broaden the range of applications to encompass molecular diagnostics. In the perspective of shifting towards a cellulose-based device, trials involving the encapsulation of the strip with cellulose nanopaper would assist in fulfilling this objective. Initial tests have exhibited water migration in an encapsulated strip, indicating the necessity for further investigation. Currently, additional research is being conducted to develop more intricate microfluidic devices.

Looking forward, integrating heaters at the point of µPAD manufacture may further enhance the detection step of biological reagent. This approach aligns with the ASSURED criteria, as it minimizes plastic use, simplifies production compared with conventional µPAD platforms, and requires less handling equipment (Drain et al. 2014).

References

Bisson A, Rigacci A, Lecomte D, et al (2003) Drying of Silica Gels to Obtain Aerogels: Phenomenology and Basic Techniques. DRYING TECHNOLOGY Vol 21 No. 4:593-628. https://doi.org/10.1081/DRT-120019055

Doherty CM, Buso D, Hill AJ, et al (2014) Using Functional Nano- and Microparticles for the Preparation of Metal-Organic Framework Composites with Novel Properties. Acc Chem Res 47:396-405. https://doi.org/10.1021/ar400130a

Drain PK, Hyle EP, Noubary F, et al (2014) Evaluating Diagnostic Point-of-Care Tests in Resource-Limited Settings. Lancet Infect Dis 14:239-249. https://doi.org/10.1016/S1473-3099(13)70250-0

Gauss C, Pickering KL, Muthe LP (2021) The use of cellulose in bio-derived formulations for 3D/4D printing: A review. Composites Part C: Open Access 4:100113. https://doi.org/10.1016/j.jcomc.2021.100113

Gomez-Martinez J, Silvy M, Chiaroni J, et al (2018) Multiplex Lateral Flow Assay for Rapid Visual Blood Group Genotyping. Anal Chem 90:7502-7509. https://doi.org/10.1021/acs.analchem.8b01078

Hilber W, Jakoby B (2023) Solid state fluid transport and sensing based on printed and embedded cellulose-polymer composites: An alternative pathway to paper-based microfluidic devices. Micro and Nano Engineering 19:100183. https://doi.org/10.1016/j.mne.2023.100183

Hou Y, Lv C-C, Guo Y-L, et al (2022) Recent Advances and Applications in Paper-Based Devices for Point-of-Care Testing. J Anal Test. https://doi.org/10.1007/s41664-021-00204-w

Kabir MA, Zilouchian H, Sher M, Asghar W (2020) Development of a Flow-Free Automated Colorimetric Detection Assay Integrated with Smartphone for Zika NS1. Diagnostics 10:42. https://doi.org/10.3390/diagnostics10010042

Krol LF, Beneventi D, Alloin F, Chaussy D (2015) Microfibrillated cellulose-SiO2 composite nanopapers produced by spray deposition. J Mater Sci 50:4095-4103. https://doi.org/10.1007/s10853-015-8965-5

Li X, Tian J, Shen W (2010) Progress in patterned paper sizing for fabrication of paper-based microfluidic sensors. Cellulose 17:649-659. https://doi.org/10.1007/s10570-010-9401-2

Liu W, Du H, Zhang M, et al (2020) Bacterial Cellulose-Based Composite Scaffolds for Biomedical Applications: A Review. ACS Sustainable Chem Eng 8:7536-7562. https://doi.org/10.1021/acssuschemeng.0c00125
Mendoza-Cachú D, López-Miranda JL, Mercado-Zúñiga C, Rosas G (2018) Functionalization of MWCNTs with Ag-AuNPs by a green method and their catalytic properties. Diamond and Related Materials 84:26-31.
https://doi.org/10.1016/j.diamond.2018.03.004

Naseri M, Ziora ZM, Simon GP, Batchelor W (2022) ASSURED-compliant point-of-care diagnostics for the detection of human viral infections. Reviews in Medical Virology 32:e2263.
https://doi.org/10.1002/rmv.2263

Scaffaro R, Lopresti F, Botta L (2018) PLA based biocomposites reinforced with Posidonia oceanica leaves. Composites Part B: Engineering 139:1-11. https://doi.org/10.1016/j.compositesb.2017.11.048

Singh A, Lantigua D, Meka A, et al (2018) Paper-Based Sensors: Emerging Themes and Applications. Sensors 18:2838. https://doi.org/10.3390/s18092838

Smith S, G. Korvink J, Mager D, Land K (2018) The potential of paper-based diagnostics to meet the ASSURED criteria. RSC Advances 8:34012-34034. https://doi.org/10.1039/C8RA06132G

Solin K, Borghei M, Imani M, et al (2021) Bicomponent Cellulose Fibrils and Minerals Afford Wicking Channels Stencil-Printed on Paper for Rapid and Reliable Fluidic Platforms

Sterlyagov AN, Nizovtsev MI, Borodulin VY, Letushko VN (2020) The effect of air relative humidity on the intensity of evaporating of water-ethanol droplets. J Phys: Conf Ser 1677:012098.
https://doi.org/10.1088/1742-6596/1677/1/012098

Sullivan F, Simon L, Ioannidis N, et al (2020) Chemical reaction modeling of industrial scale nitrocellulose production for military applications. AIChE Journal 66:e16234. https://doi.org/10.1002/aic.16234

Tang R, Xie MY, Li M, et al (2022) Nitrocellulose Membrane for Paper-based Biosensor. Applied Materials Today 26:101305. https://doi.org/10.1016/j.apmt.2021.101305

Tenhunen T-M, Moslemian O, Kammiovirta K, et al (2018) Surface tailoring and design-driven prototyping of fabrics with 3D-printing: An all-cellulose approach. Materials & Design 140:409-419.
https://doi.org/10.1016/j.matdes.2017.12.012

Tong X, Ga L, Zhao R, Ai J (2021) Research progress on the applications of paper chips. RSC Adv 11:8793-8820.
https://doi.org/10.1039/D0RA10470A

Vashist SK, Luppa PB, Yeo LY, et al (2015) Emerging Technologies for Next-Generation Point-of-Care Testing. Trends in Biotechnology 33:692-705. https://doi.org/10.1016/j.tibtech.2015.09.001

Wang G, Zhang D, Wan G, et al (2019) Glass fiber reinforced PLA composite with enhanced mechanical properties, thermal behavior, and foaming ability. Polymer 181:121803.
https://doi.org/10.1016/j.polymer.2019.121803

Washburn EW (1921) The Dynamics of Capillary Flow. Phys Rev 17:273-283.
https://doi.org/10.1103/PhysRev.17.273

Wijnhorst R, de Goede TC, Bonn D, Shahidzadeh N (2020) Surfactant Effects on the Dynamics of Capillary Rise and Finger Formation in Square Capillaries. Langmuir 36:13784-13792.
https://doi.org/10.1021/acs.langmuir.0c01965

Wong R, Tse H (eds) (2009) Lateral Flow Immunoassay. Humana Press, Totowa, NJ

Yahaya ML, Zakaria ND, Noordin R, Razak KA (2019) The Effect of Nitrocellulose Membrane Pore Size of Lateral Flow Immunoassay on Sensitivity for Detection of Shigella sp. in Milk Sample. Materials Today: Proceedings 17:878-883. https://doi.org/10.1016/j.matpr.2019.06.384

Yang S-M, Lv S, Zhang W, Cui Y (2022) Microfluidic Point-of-Care (POC) Devices in Early Diagnosis: A Review of Opportunities and Challenges. Sensors 22:1620. https://doi.org/10.3390/s22041620
Zhdanov SA, Starov VM, Sobolev VD, Velarde MG (2003) Spreading of aqueous SDS solutions over nitrocellulose membranes. Journal of Colloid and Interface Science 264:481-489.
https://doi.org/10.1016/S0021-9797(03)00520-4

Ziani K, Oses J, Coma V, Maté JI (2008) Effect of the presence of glycerol and Tween 20 on the chemical and physical properties of films based on chitosan with different degree of deacetylation. LWT - Food Science and Technology 41:2159-2165. https://doi.org/10.1016/j.lwt.2007.11.023

## Funding

This work was funded by the Agence Nationale de Recherche (ANR, France) via the ANR-21-CE19-0047-02 (CareFab) program.

## Contribution

E.T. wrote the main manuscript. E.T and J.G.M performed the experiments. All authors reviewed the manuscript

## Ethics declarations

### Ethical approval

This paper does not involve any human or animal experiments. Not applicable.

### Conflict of Interest

The authors have no relevant financial or non-financial interests to disclose.