Wax transfer patterning of porous paper for lab-on-a-chip

Paper-based microfluidics has emerged as a promising platform for low-cost analytical and biological assays. However, fabrication of hydrophobic barriers on porous substrates remains largely dependent on direct wax printing, which limits patterning flexibility, requires specialized equipment, and constrains scalability. Here, we present a simple and versatile wax transfer technique that enables reliable replication of wax micropatterns from one porous substrate to another. The method leverages controlled wettability, thermal activation, and applied pressure to achieve efficient and reproducible pattern transfer. The resulting patterned substrates display well-defined hydrophobic–hydrophilic regions suitable for microfluidic applications. We demonstrate the versatility of the approach through applications in cell culture and biochemical assays, including the growth and spatial confinement of cervical and breast cancer cells, as well as Folin–Lowry protein quantification in saliva samples. Notably, wax-transferred patterns provide spatial confinement of assay signals, while maintaining biocompatibility, suitability for biological applications, and structural integrity of the substrate. The simplicity of the process, compatibility with diverse porous materials, and long-term stability of donor patterns highlight its potential for scalable and distributed fabrication. Overall, this work establishes a porous-to-porous transfer strategy that expands the design space of paper-based microfluidics and provides a practical pathway toward accessible, manufacturable, and widely deployable lab-on-paper technologies.

Microfluidic devices enable precise control of fluid dynamics at the microscale and are widely utilized in diagnostics, biotechnology, and chemical analysis (Janasek et al., 2006; Whitesides, 2006; Deliorman et al., 2023; Ali et al., 2026). Conventional fabrication techniques include photolithography (Reynolds et al., 2022; Todd and Krasnogor, 2023), soft lithography (Qin et al., 2010; Karimi et al., 2024), micropattern transfer (Sun et al., 2024), microcontact printing (Khadpekar et al., 2019), and microstamping (Fruncillo et al., 2021). Among these, micropattern transfer is widely used for transferring micropatterns between non-porous and porous substrates (Bandopadhyay and Das, 2023).

Paper-based microfluidics employs biocompatible porous substrates to enable low-cost, portable, and environmentally friendly analytical platforms with reduced reagent consumption for on-site and field-based testing (He et al., 2013; Nishat et al., 2021). Fluid transport in these systems is driven by capillary forces, allowing liquids to spontaneously “wick” through the interconnected three-dimensional (3D) porous fiber network and be guided by patterned hydrophobic–hydrophilic regions that define fluidic pathways (He et al., 2025). These patterns are commonly fabricated either through chemical surface modification or by wax printing.

In chemical approaches, fluidic channels are constructed by locally altering surface wettability through plasma treatment and silanization (Gieparda et al., 2021), which often require patterned photolithographic or 3D-printed masks to define spatial regions (Samara et al., 2021). In contrast, wax printing involves the direct deposition of solid-phase wax inks (e.g., Xerox), followed by thermal melting and diffusion to define fluidic pathways (Sameenoi et al., 2014), and is widely preferred due to its simplicity and practical implementation. However, commercial wax printers are no longer manufactured, and the lack of technical support during malfunction significantly limits their continued use. Moreover, their compatibility is restricted to specific paper thicknesses, typically standard A4-sized sheets.

To overcome these constraints, alternative fabrication strategies, including screen printing (Tseng et al., 2021), thermal printing (Liu et al., 2020; Monju et al., 2023), and infrared laser-assisted wax transfer (Ghosh et al., 2019), have been developed. Although these approaches have demonstrated success in applications such as sensing (Lim et al., 2019; Modha et al., 2021; Ferreira et al., 2025), automated assays (Jeong et al., 2021), reagent flow control (Kumar et al., 2024), cell-based assays (Ng et al., 2017), and 3D cell culture and cryopreservation (Alnemari et al., 2020; Samara et al., 2021; Glia et al., 2025), they remain limited in accessibility, scalability, and process simplicity. Consequently, while wax-based patterning is favored due to its practical and adaptable nature, there remains a need for more reliable and versatile strategies to transfer the wax patterns across a broad range of paper substrates without reliance on specialized printers.

This need is particularly critical, as the pore size, porosity, and fiber interconnectivity of paper govern capillary-driven fluid transport within wax-defined pathways (Noviana et al., 2021). Accordingly, in paper-based microfluidics, substrates such as Whatman filter paper (high porosity), chromatography paper (medium-to-high porosity), and nitrocellulose membranes (low porosity) are selected based on their wicking behavior and suitability for analytes of varying sizes (Yang et al., 2017). For example, Whatman filter paper (e.g., Grade 1, ∼180 μm thickness, ∼11 μm pore size) provides high porosity and enhanced capillary transport, making it well suited for microfluidic applications. Chromatography paper (e.g., Whatman Grade 3MM Chr, a high purity cellulose chromatography paper with a thickness of ∼340 μm thickness) shows intermediate wicking behavior and is therefore commonly used for controlled fluid transport and separation. In contrast, nitrocellulose membranes (typically ∼100–200 μm thickness, ∼0.2–0.45 μm pore size) display slower wicking kinetics due to their lower porosity and distinct surface morphology, enabling their use in protein detection for point-of-care diagnostics.

However, for effective wax-based patterning using wax printers, these substrates typically require mounting onto standard A4 sheets using adhesive tape prior to printing. Although some substrates are dimensionally compatible with A4 sheets and can be directly processed using wax printers, such filter papers are not always readily available. Moreover, taping filter paper onto A4 sheets increases the overall thickness and introduces height variations, which can disrupt printer operation and potentially damage internal components. In addition, many commonly used filter papers are smaller than standard A4 dimensions, further complicating direct printing. The wax transfer strategy overcomes these limitations by first printing patterns onto standard A4 donor paper, followed by controlled transfer onto the recipient porous substrate. Beyond preventing printer damage caused by substrate thickness mismatch, this approach enables reliable patterning of a broader range of porous materials while preserving their native porosity, permeability, and structural integrity. Transparency films have also been explored as intermediate substrates for wax transfer due to their efficient release of wax patterns (Zhong et al., 2012). However, these films are not fully compatible with wax printers and may lead to equipment damage or inaccuracies in pattern transfer.

Here, we demonstrate a novel porous-to-porous wax-transfer microcontact technique that enables the direct transfer of wax patterns from conventional porous paper (donor) to porous Whatman filter paper (recipient). The process involves pre-wetting both donor and recipient substrates, followed by heat-assisted contact on a hotplate with applied pressure to facilitate efficient wax transfer. Within minutes, wax patterns are reliably transferred from the donor to the flexible recipient substrate, as illustrated in Figure 1a. The fabrication process was systematically optimized by varying key parameters, including temperature, soaking time, and wettability.

Notably, the transfer mechanism can be described as a multi-dimensional fabrication process that integrates spatial dimensions (x, y, z), temporal contact (t), and applied thermal and mechanical energy. During transfer, the wax is deposited in a more localized manner, with reduced lateral diffusion compared with conventional diffusion methods. Representative wax-transferred microspots arranged in a 3 × 3 matrix are shown in Figure 1b, demonstrating successful pattern replication on filter paper with preserved spot integrity and liquid confinement. Figure 1c provides a conceptual comparison of wax transport behavior, illustrating that conventional diffusion promotes lateral spreading, whereas wax transfer enables more localized deposition with better-defined boundaries.

The technique enables the successful transfer of various geometries, including circular, triangular, and rectangular patterns, and demonstrates compatibility across multiple porous substrates. Furthermore, the functionality of the transferred patterns was evaluated through biological and analytical applications, including biocompatibility studies using human cervical (HeLa) and breast cancer (SKBR-3) cells, as well as colorimetric assays for whole protein quantification. Finally, the performance of the wax transfer method was directly compared with conventional diffusion-based wax patterning, demonstrating comparable output.

Fabrication and characterization of wax patterns via microcontact transfer

Design patterns consisting of circular, rectangular, and triangular geometries were created using Microsoft PowerPoint and printed onto A4 paper using a Xerox ColorQube 8570N wax printer, forming ∼3 mm wide hydrophobic barriers. Prior to transfer, the wax patterned donor paper was soaked in deionized water in a Petri dish. The recipient filter paper was similarly pre-wetted under identical conditions. Efficient transfer was achieved when the recipient filter paper was pre-wetted by soaking or by pipetting water onto its surface. The wetted donor (A4 paper) and recipient (filter paper) substrates were then assembled in a stacked configuration and placed on a hotplate (IKA C-MAG HS7), with two aluminum heating blocks (∼1.6 kg total load) applied on top to ensure uniform pressure and thermal contact. Wax transfer was achieved through heat-assisted contact between the substrates. Two types of filter paper were used as recipient substrates: Whatman Grade 1 filter paper (Cytiva, Cat. No. 1001-125) and Whatman Grade 114 wet-strengthened circular filter paper (Cytiva, Cat. No. 1114-240).

Hydrophobic-hydrophilic barriers were evaluated using fluorescein solution to visualize fluid confinement and assess surface wettability. Fluorescein solution (0.5 mg mL -1) was prepared by dissolving fluorescein salt (Sigma-Aldrich) in distilled water and applied to individual patterned regions to examine confinement behavior. The transfer efficiency was calculated based on the ability of the transferred patterns to confine liquid within the hydrophilic regions. Equal volumes of fluorescein solution were added to all transferred spots, and any leakage from distorted or poorly defined patterns was not considered successful transfer. Transfer efficiency was then expressed as the percentage of spots that showed effective liquid confinement relative to the total number of patterned spots.

Surface wettability was further characterized through contact angle measurements. A systematic optimization was performed by varying key parameters, including temperature, heating time, soaking duration, wettability, water volume, and filter paper type. In addition, patterns of different geometries were fabricated and transferred to assess the versatility of the transfer process.

Quantification of capillary transport

Capillary transport was quantified by measuring the lateral wicking distance of fluorescein solution within the patterned regions on both the front and back surfaces of the paper substrates. To enable direct comparison between substrates and geometries, results were expressed as the relative increase in back-side wicking distance with respect to the front side. The relative increase (%) was calculated as where Back and Front represent the measured wicking distances on the back and front surfaces, respectively. This normalization accounts for final baseline differences in absolute wicking distances and allows for a more consistent comparison of through-thickness transport across different paper types and pattern geometries.

Characterization of filter paper using FTIR

Fourier-transform infrared spectroscopy (FTIR) spectra were acquired using a LUMOS II system (Bruker) from paper fibers in both conventional wax-patterned samples and wax-transferred samples, over a wavenumber range of 0 4000 cm -1. The analysis was performed to evaluate potential changes in chemical structure induced during the fabrication process.

Biocompatibility testing

The fabricated devices were sterilized using ultraviolet light (30 W) for 15 min prior to cell culture experiments. Biocompatibility of the fabricated devices was evaluated by culturing cells on the patterned paper substrates. Two cancer cell lines were used: human breast adenocarcinoma cells (SKBR-3) and human cervical carcinoma cells (HeLa). Cells were maintained in growth medium (DMEM - Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Pen–Strep, Gibco) and cultured in an incubator under standard conditions until ∼85% confluency. Upon reaching the desired confluency, cell density was determined using trypan blue staining (Invitrogen) and quantified with an automated cell counter (Invitrogen). Cells were then seeded onto the hydrophilic regions of the patterned paper substrates and cultured for 5 days. Biocompatibility was assessed using a Live/Dead Viability/Cytotoxicity Kit for mammalian cells (Invitrogen), and fluorescence imaging was performed to evaluate cell viability, adhesion, and spatial confinement within the patterned regions.

Protein quantification

Protein quantification was performed using bovine serum albumin (BSA) as a standard, and whole saliva samples, employing the Folin–Lowry colorimetric method. The Folin–Lowry assay reagents were prepared as follows: 2% sodium carbonate in 0.1 N sodium hydroxide (Sigma-Aldrich), 1% sodium potassium tartrate (1 g in 100 mL; Sigma-Aldrich), and 0.5% cupric sulfate pentahydrate (50 mg in 10 mL; Sigma-Aldrich). Reagent 1 was prepared by mixing 48 mL of sodium carbonate solution with 1 mL sodium potassium tartrate and 1 mL cupric sulfate solution. Reagent 2 consisted of Folin–Ciocalteu phenol reagent diluted in equal volumes of distilled water.

A calibration curve was generated using BSA standards with concentrations ranging from 10 to 500 mg mL -1, with deionized water used as a control. For conventional spectrophotometric measurements, 4.5 mL of Reagent 1 was added to the sample and incubated for 10 min, followed by the addition of Reagent 2 and further incubation for 30 min. Absorbance was measured at 660 nm to construct the standard calibration curve. For microscale assays, a 25 μL reaction volume was used, consisting of 22.5 μL Reagent 1, 2.5 μL Reagent 2, and 1 μL BSA solution. For paper-based assays using the wax-transferred devices, a reduced reaction volume of 5.2 μL was applied directly onto the filter paper (4.5 μL Reagent 1, 0.5 μL Reagent 2, and 0.2 μL BSA), enabling colorimetric detection based on protein concentration.

To evaluate protein detection in biological samples, BSA was spiked into saliva, and comparisons were performed among saliva samples, BSA-spiked saliva, BSA in water, and control samples. Since saliva inherently contains proteins, total protein content was assessed across these conditions.

Imaging

Cells cultured on paper substrates were imaged using a Nikon SMZ18 stereo microscope equipped with a 1× objective lens and a fluorescence light source (Nikon Intensilight C-HGFI). Fluorescence images were acquired using appropriate filter sets for green fluorescent protein (GFP) and red fluorescent protein (RFP), with a digital camera attached to the microscope. Comparative fluorescence images were acquired under consistent microscope and camera settings for each experiment to ensure comparable imaging conditions across samples. Digital amplification ranged from 0.1× to 13.5×, and the corresponding scale bars were indicated in the respective images according to the applied magnification. Wax-transferred patterned regions loaded with fluorescent dyes were additionally imaged using a flatbed scanner (Epson Perfection V850 Pro) to capture large-area fluorescence distribution.

Two-photon microscopy

The fabricated devices were loaded with fluorescein solution and imaged using a two-photon microscope. Imaging was performed with a 10× objective at 1× zoom using CellSens software. Z-stack image series were acquired, and orthogonal (ortho) and slice views were reconstructed during post-processing using Imaris software. In addition, cells cultured within the hydrophilic regions of the patterned substrates were imaged using the same approach, and corresponding Z-stack datasets were analyzed to assess 3D distribution and growth.

Image and data analysis

Captured images were post-processed and analyzed using Imaris software. Quantitative analysis was further performed based on mean RGB (Red, Green, Blue) intensity and radial profile measurements using ImageJ (NIH, https://imagej.net), an open-source image processing platform. Graphs were generated from the analyzed data using OriginPro (OriginLab). Data are presented as mean ± standard deviation (SD) from three independent replicates (n = 3). Statistical analysis (Student’s t-test) was carried out for significance comparison using OriginPro. A p value <0.05 was considered statistically significant. Non-significant comparisons were also indicated in the graphs denoted as ns.

Fabrication of microcontact wax-transfer platforms

To address the limitations of conventional wax patterning on non-standard porous substrates, we developed a microcontact-based wax transfer method that enables efficient and versatile patterning across diverse paper types without reliance on conventional fabrication constraints. Micropatterns were designed using AutoCAD and subsequently transferred onto donor substrates via wax printing. Early trials employed different donor materials, including wax paper, tracing paper, and standard A4 paper (210 × 297 mm 2, ∼80 g m -2), with Whatman Grade 1 filter paper as the recipient. Transfer experiments were conducted across a temperature range of 200 °C–275 °C and heating durations of 5–15 min. However, repeatability was compromised at low wax thickness (single-layer prints) and variable temperature conditions, where transfer efficiency remained low (∼20% at 200 °C) and inconsistent across trials.

To improve transfer efficiency, a pre-wetting strategy was introduced for both donor and recipient substrates. While wax patterns printed directly on filter paper could be transferred upon heating, the transfer efficiency remained limited due to the inherent porosity of paper, which promotes both vertical and lateral wax diffusion (To et al., 2017). To address this, the wax-printed donor paper was pre-soaked in water prior to heat-assisted transfer, significantly enhancing pattern reliability. Systematic optimization was then performed using circular spot array patterns with varying feature sizes. Key parameters, including wax thickness, temperature, heating duration, donor paper porosity, and substrate wettability, were evaluated to establish optimal transfer conditions.

Both donor and recipient papers were soaked in ∼100 mL of water for ∼10 min prior to transfer. The wetted assembly was then placed on a hotplate at an optimized temperature of 220 °C, and a heat block was applied to ensure uniform pressure and thermal contact. Under these conditions, efficient wax transfer was achieved within ∼4 min, yielding well-defined patterns on the recipient filter paper (Figure 1b). The method was further scaled to larger patterned areas, demonstrating consistent liquid confinement and structural integrity of the transferred features (Supplementary Figure S1).

Parameter optimization for wax-transfer patterning

To systematically optimize the wax-transfer protocol, key parameters including temperature (210 °C–250 °C), heating duration (2–10 min, in 2 min intervals), soaking time, pre-wetting condition, and wax thickness were investigated (Figures 2a–f; Supplementary Table S1). Portions of these data were previously reported by Sukumar et al. (2024). Figures 2a,b shows continuous temperature optimization trends for one- and two-layered wax prints, whereas Figure 2c compares discrete heating durations at the optimized transfer temperature. The optimization workflow was performed sequentially, beginning with dry recipient conditions followed by wettability, soaking-time, and heating-time optimization under progressively improved transfer conditions.

Wax patterns of varying thickness were generated by printing one-, two-, and three-layered designs on the donor (A4) substrate. Under conditions in which only the donor paper was pre-wetted, single-layer prints displayed low transfer efficiency (∼20% at 200 °C), while increasing temperature improved transfer but caused thermal degradation of the paper (browning) at higher temperatures (Figure 2a). Two-layered prints showed moderate and variable transfer efficiency across the tested temperature range (Figure 2b).

In contrast, three-layered prints yielded higher transfer efficiency on the recipient paper at both 4- and 10-min heating durations (Figure 2c). However, the excessive layering introduced distortions in pattern fidelity, likely due to excess wax deposition during printing. To overcome this limitation, subsequent optimization focused on one-layered prints combined with controlled pre-wetting of both donor and recipient papers, which enabled efficient transfer while preserving pattern definition.

An optimal temperature of 220 °C was identified, at which wax melting and transfer occurred efficiently without compromising the structural integrity of the paper substrate. To further enhance transfer efficiency, the effects of wettability and pre-wetting conditions was systematically evaluated. Transfer efficiency increased noticeably with increasing wetting, from non-wetting to complete wetting conditions (Figure 2d), indicating that moisture facilitates wax mobility and inter-fiber transport. Pre-wetting strategies revealed that while pre-wetting only the donor (A4) substrate improved transfer, pre-wetting both the donor (A4) and recipient (Whatman Grade 114) substrates resulted in significantly enhanced transfer efficiency. Under these conditions, transfer efficiency reached ∼90% at 220 °C within ∼4 min (Figures 2e,f), identifying this duration as optimal, with no substantial improvement observed at longer heating times (see Methods). Optimization of water soaking time indicated that 10 min pre-wetting provided the highest efficiency (∼90%) but with relatively large variation. Whereas ∼6 min pre-wetting yielded slightly lower efficiency (∼80%) with reduced variability compared to shorter soaking durations. Contact angle measurements further revealed that the wax-transferred substrate initially displayed localized hydrophobic behavior (θ = 130°), which gradually transitioned toward hydrophilicity as the liquid was absorbed into the porous paper matrix across the 3 × 3 patterned spots (Figure 2d, inset). This transient behavior likely arises from localized thermally induced surface modifications and partial wax redistribution near the paper fibers (Andrade et al., 2026). Together, these effects suggest that wax transfer, combined with thermal treatment, induces localized modifications in surface energy and wettability (Klemm et al., 2005). Notably, similar droplet confinement was observed when cell culture media containing cells was applied, further confirming effective hydrophobic–hydrophilic patterning. Since the hydrophobicity was transient, samples could be loaded immediately after wax transfer patterning.

To assess structural and chemical integrity, FTIR analysis confirmed that β-glycosidic bonds in cellulose remained intact following the transfer process, while minor variations in intermolecular hydrogen bonding (–OH groups) were observed, particularly at temperatures >200 °C. These changes may contribute to localized variations in surface wettability and lateral wax diffusion (Supplementary Figure S2), consistent with thermally induced surface modifications reported in cellulose-based substrates (Klemm et al., 2005; Andrade et al., 2026). Importantly, two-photon microscopy and Z-stack reconstructions demonstrated that the fibrous architecture of the paper remained structurally intact after heating, with no significant disruption of the 3D porous fiber network (Figure 2g; Supplementary Figure S3).

Pattern-controlled capillary transport, biocompatibility, and analytical applications

To demonstrate the versatility of the wax transfer technique, patterns of different geometries, including circular, triangular, and rectangular designs, were fabricated on donor (A4) paper and successfully transferred onto porous filter papers (Figure 3). Fluorescent dye confinement experiments confirmed well-defined fluid localization within the transferred patterned hydrophilic regions, indicating effective formation of hydrophobic–hydrophilic boundaries (Figure 3a). Fluid transport behavior was further quantified by introducing fluorescein solution into the patterned regions and measuring front–back differences in lateral wicking distance (Figure 3b).

In both Whatman Grade 114 and Grade 1 filter papers, the back surface showed greater wicking than the front surface, indicating through-thickness transport across the porous substrate. For Whatman Grade 114, the back-side increase was 34.0% ± 4.3% for triangular patterns and 23.8% ± 16.5% for circular patterns (Figure 3b, left panel), whereas for Whatman Grade 1, the corresponding increases were 14.5% ± 6.6% and 48.5% ± 0.4%, respectively (Figure 3b, right panel). These results indicate that front-back transport is jointly regulated by substrate porosity and pattern geometry.

Further, the wicking rate and transport distance are influenced by several factors, including pore size distribution (non-uniform porosity), fiber orientation and packing density, substrate thickness variations, localized hydrophobic-hydrophilic interactions, geometry-dependent transport behavior, and capillary pressure variation associated with liquid saturation. In addition, manufacturing inconsistencies and environmental conditions may further contribute to variability in the observed capillary transport behavior. Notably, wax diffusion during transfer is more effective near the front surface of the filter paper compared to the back surface, which may further contribute to the observed front–back asymmetry in fluid transport (Mehta et al., 2025; Behera et al., 2026).

In the higher-porosity Grade 114 paper, fluid readily penetrates through the thickness of the substrate, resulting in notable back-side wicking for both geometries, although with greater variability, particularly for circular patterns. By contrast, the lower-porosity Grade 1 paper exhibits a stronger geometry dependence, with relatively limited back-side enhancement in triangular patterns but a marked increase in circular patterns. This behavior suggests that while substrate porosity controls the extent of through-thickness transport, pattern geometry influences how the liquid redistributes within the porous network.

Next, the compatibility of the patterned substrates with biological applications was evaluated by seeding SKBR-3 and HeLa cells into the hydrophilic regions of the porous paper matrix, where cells were observed on the surface and within the three-dimensional fibrous thickness of the substrate. Cells were successfully confined within the patterned geometries, with circular and triangular designs providing well-defined boundaries (Supplementary Figure S4). After 5 days of culture, robust cell growth was observed, particularly for HeLa cells (Figure 3c). Two-photon Z-stack imaging confirmed that cells were present within the three-dimensional fibrous paper matrix rather than only on the top surface, indicating that the wax-transfer process did not block the porous network. At the relatively low seeding density used in this study, cells exhibited adherent spreading along the paper fibers, likely supported by fiber roughness and localized surface modifications induced during transfer and sterilization. Importantly, paper substrates are well established as supports for 3D cell culture when higher cell concentrations are used (Alnemari et al., 2020; Samara et al., 2021; Glia et al., 2025). Thus, the wax-transferred substrates preserve the porous architecture required for 3D cell culture while also supporting 2D-like adherent growth under lower seeding-density conditions.

Nevertheless, cell viability was confirmed using Calcein-AM (live cells) and ethidium homodimer (dead cells) staining, followed by fluorescence imaging. To further investigate surface effects, substrates were modified using silanization to generate superhydrophobic regions, which promoted more adherent and spread cell morphology, whereas hydrophilic regions supported cell localization within the three-dimensional porous matrix (Supplementary Figure S5). In wax-transferred patterns, the localized hydrophobicity was transient, and cells were observed within the fibrous paper matrix, consistent with preservation of the porous architecture. At the relatively low cell concentrations used in this study (∼10 4 –5 × 10 4 cells per spot), cells exhibited more adherent and elongated morphologies along the paper fibers. However, previous studies have shown that higher cell seeding densities can promote three-dimensional cell growth within porous paper scaffolds (Samara et al., 2021; Glia et al., 2025). Together, these findings indicate that wax-transfer process preserves the structural and wettability characteristics compatible with both 2D-like adherent growth and 3D cell culture.

Following optimization, the robustness and applicability of the wax transfer protocol were evaluated by comparing wax-transferred and conventional wax-patterned papers under identical conditions. A colorimetric protein assay based on the Folin–Lowry method was implemented (Figure 3d). In this assay, proteins form a Cu 2+ –protein complex under alkaline conditions, followed by reaction with Folin–Ciocalteu reagent to produce a blue–green chromophore proportional to protein concentration (Waterborg and Matthews, 1994). Protein quantification was performed using control samples, BSA-spiked water, and saliva samples. In the Folin–Lowry assay, increasing protein concentration produces a stronger blue colorimetric response, corresponding to lower mean RGB intensity values.

To evaluate assay specificity, protein-containing samples including BSA-spiked water and saliva were compared against a water control. To further assess selectivity, starch was used as a carbohydrate control to determine whether the assay could distinguish protein-containing samples from non-protein biomolecules. On wax-transferred papers, the assays demonstrated more localized and spatially uniform colorimetric signals within the defined regions for both specificity and selectivity (Figure 3e). Whereas conventional wax-patterned papers showed characteristic ring-shaped distribution at the spot periphery (Figure 4). This difference is likely related to slight surface modification of the paper fibers during the transfer process, which alters local wettability and slows solute migration within the hydrophilic region. In conventional patterns, faster radial transport can promote peripheral accumulation of assay products, consistent with a coffee-ring-like effect (Yunker et al., 2011), while the wax-transferred substrates favor improved signal retention within the patterned area, enhancing readout uniformity.

Quantitative analysis based on mean RGB intensity revealed comparable analytical performance between the two approaches, with wax-transferred papers additionally providing improved spatial confinement and reduced signal spreading (Supplementary Figure S6). The localized confinement achieved in wax-transferred substrates does not necessarily eliminate intrinsic paper-to-paper variability arising from the heterogeneous porous microstructure. As expected, saliva samples showed higher color intensity due to intrinsic protein content, corresponding to lower RGB values relative to control and BSA samples.

Biocompatibility studies further confirmed that cell viability and proliferation were not compromised in wax-transferred papers compared to conventional wax-patterned papers (Supplementary Figures S7a–d). Cells were observed within the porous fibrous matrix, indicating preservation of the 3D paper architecture following wax transfer. At the seeding densities used in this study (∼5 × 10 4 cells per spot), cells exhibited adherent morphologies along the paper fibers. Previous studies have shown that higher seeding densities can support 3D cell growth within porous paper scaffolds (Alnemari et al., 2020; Samara et al., 2021; Glia et al., 2025). Nevertheless, quantitative cell counting within a 402 × 402 μm 2 region further demonstrated comparable HeLa cell proliferation between conventional and wax-transferred substrates, with no significant difference observed between the two conditions (Supplementary Figure S7e). Additionally, wax-printed donor patterns stored for up to 2 years retained their transfer capability (∼88.88% transfer efficiency) and supported both cell culture and analytical applications without performance degradation (Supplementary Figures S1, S8), demonstrating the long-term stability and robustness of the technique.

Comparative performance and signal confinement analysis

A direct comparison between wax-transferred and conventional wax-printed papers was performed to evaluate whether the proposed fabrication strategy preserves analytical performance while improving patterning flexibility and substrate compatibility (Figure 4; Supplementary Figure S9). Front-side assay images (Figure 4a) show that wax-transferred papers yield more localized and spatially uniform colorimetric signals, whereas conventional wax-patterned papers exhibit peripheral, ring-shaped intensity profiles consistent with radial solute transport and evaporation-driven accumulation (Carrilho et al., 2009a). This difference is likely related to subtle surface modifications of the paper fibers induced during the transfer process (pre-wetting and thermal treatment), which can alter local wettability and slow solute migration within the hydrophilic regions.

In conventional patterns, faster radial transport promotes edge accumulation resembling coffee-ring-like behavior (Yunker et al., 2011), whereas in wax-transferred substrates, reduced solute mobility favors more uniform signal retention within the patterned area (Supplementary Figure S9). This effect is more obvious on the back surface (Figure 4b), where through-thickness capillary transport amplifies lateral spreading, particularly in conventional wax-patterned papers (Carrilho et al., 2009b).

Quantitative analysis of front-side measurements (Figure 4c), based on mean RGB intensity, reveals consistent trends across both fabrication approaches, with saliva samples exhibiting higher signal intensity than BSA in water and control samples. This behavior reflects the higher intrinsic protein content in saliva, confirming the reliability and sensitivity of the Folin–Lowry assay for both methods. Notably, wax-transferred papers demonstrate comparable signal intensity while maintaining improved spatial localization, indicating that enhanced confinement does not compromise analytical performance.

Back-side quantification (Figure 4d) shows increased variability associated with diffusion through the porous substrate, with conventional wax-patterned papers exhibiting greater signal dispersion relative to wax-transferred papers. Based on these observations, the front surface provides a more reliable and consistent readout due to reduced variability and limited through-thickness diffusion, while the back surface reflects amplified transport effects within the porous matrix. This comparison was motivated by a key limitation of conventional wax-patterned paper microfluidics, namely, uncontrolled lateral diffusion and the resulting loss of spatial resolution in assay signals, particularly in thicker or highly porous substrates (see Figure 1c).

In conventional wax printing, a larger amount of wax is deposited directly onto the paper, which promotes extensive lateral spreading during melting and results in broader hydrophobic barriers with less well-defined boundaries. This excess wax loading, combined with rapid fluid transport in untreated hydrophilic regions, enhances radial diffusion of assay solutions and leads to signal dispersion, including coffee-ring-like accumulation at the edges (Supplementary Figure S10a).

In contrast, the wax transfer approach involves partial redistribution of wax between donor and recipient substrates, resulting in a lower effective wax loading on the recipient paper. During transfer, wax diffusion occurs across both substrates, which moderates lateral spreading and produces more localized, well-defined barrier formation (Supplementary Figure S10b). The transferred wax is sufficient to establish effective hydrophobic confinement, but with reduced lateral diffusion compared to conventional printing. Additionally, slight surface modification of the paper fibers induced by pre-wetting and thermal treatment can further reduce solute mobility within the hydrophilic regions. Together, these effects contribute to improved spatial localization of assay signals while maintaining comparable analytical sensitivity.

Despite effective confinement, some lateral diffusion was observed in both approaches under external influences such as mechanical vibrations or incomplete drying prior to analysis. These effects were more evident on the back surface of the paper, where vertical capillary transport contributes to signal spreading. Nevertheless, the back side consistently produced a stronger and more reproducible signal than the front side, making it the preferred readout surface for the assay. To further improve this behavior, a lamination layer was applied to the back side of the paper, which significantly reduced vertical diffusion and enhanced signal stability (Supplementary Figure S11).

In this work, we present a versatile wax transfer fabrication technique that enables the transfer of patterned geometries from standard A4 paper onto Whatman filter paper for paper-based microfluidic applications. This simple approach establishes a porous-to-porous microcontact transfer strategy for patterning diffusible materials within fibrous substrates. Systematic optimization of key parameters, including temperature, wettability, and heating duration, enabled efficient and reproducible transfer of wax patterns, resulting in stable hydrophobic–hydrophilic regions. The approach supports both discrete pattern formation and the development of continuous microchannels for guided fluid transport within the hydrophilic regions confined by hydrophobic barriers.

Mechanistically, the transfer process is governed by a coupled capillary–thermal–mechanical transport phenomenon. Pre-wetting reduces interfacial resistance and promotes capillary-driven redistribution, thermal energy facilitates wax mobilization, and applied pressure ensures uniform contact between the porous substrates. This interplay enables efficient material transfer across complex fibrous networks, providing a controlled route for pattern formation without reliance on conventional fabrication constraints. The simplicity and accessibility of the technique make it particularly attractive for low-resource settings, as it does not require cleanroom facilities or high-end equipment. Instead, the method relies on basic tools such as a hotplate and controlled wetting, enabling rapid and cost-effective prototyping of paper-based microfluidic devices. This accessibility also positions the technique as a valuable platform for decentralized manufacturing and educational applications.

Interestingly, our results indicate that the wax transfer process induces slight hydrophobic characteristics in the paper substrate. This behavior likely arises from surface-level reorganization driven by thermal exposure, water-mediated swelling, and partial diffusion of wax into the porous fiber network. Contact angle measurements support this observation, reflecting changes in surface energy (Gowthami and Sharma, 2023). FTIR analysis further revealed minor variations in intermolecular OH bonding, while the β-glycosidic backbone of cellulose remained intact, indicating that the process induces interfacial modifications without disrupting the fundamental chemical structure of the material (El-Sakhawy et al., 2018).

These results point to a practical design trade-off in paper-based microfluidics: higher-porosity substrates promote stronger through-thickness transport but with greater variability, whereas lower-porosity substrates provide better spatial control and become more sensitive to pattern geometry. Accordingly, substrate type and pattern design should be selected together, depending on whether rapid transport or confined, reproducible readout is prioritized.

The developed platform was validated through direct comparison with conventional wax patterning in both biological and analytical applications. Despite minor variations in surface chemistry, cell viability and proliferation remained comparable, confirming that the process preserves biocompatibility. In protein quantification assays, the wax-transferred paper demonstrated performance equivalent to conventional approaches, while offering enhanced flexibility in substrate selection. The addition of a lamination layer further improved fluid confinement and minimized lateral diffusion, enabling robust assay performance. Using this platform, proteins in saliva samples were successfully quantified, highlighting its potential for non-invasive, point-of-care diagnostic applications (e.g., periodontal diseases) (Bui et al., 2019; Blanco-Pintos et al., 2025; Sukumar et al., 2025b).

Importantly, while demonstrated using wax, this porous-to-porous transfer strategy is generalizable to other diffusible materials and functional inks. By controlling vertical and lateral transport through substrate properties and processing parameters, this framework can be extended to a wide range of patterning applications in porous media. Furthermore, the simplicity of the process, reliance on readily available materials, and compatibility with large-area substrates highlight its scalability for high-throughput fabrication.

The developed wax-transfer strategy is compatible with both laboratory-scale and industrial-scale fabrication workflows. At the laboratory scale, micropattern transfer can be scaled using standard A4-sized donor sheets, large-area filter paper substrates, and heated transfer assemblies. At the industrial scale, the process could be adapted into roll-to-roll manufacturing systems, where donor and recipient porous substrates are continuously wetted, aligned, and thermally pressed for high-throughput pattern transfer. Such scalability may enable automated large-volume production of paper-based analytical and biological platforms for lab-on-a-chip applications.

Notably, wax-printed donor patterns stored for up to 2 years retained their transfer capability and supported both cell culture and analytical applications without performance degradation, underscoring the robustness and long-term stability of the approach. Together, these attributes position the method as a broadly applicable, scalable, and reliable fabrication strategy for paper-based microfluidics and related porous material systems.

Conceptually, this transfer mechanism resembles natural porous material interactions, such as the transfer of fine particulate matter from butterfly wings upon contact (Yu et al., 2012; Kim et al., 2018; Siddique et al., 2022). Such bio-inspired analogies illustrate how material exchange between porous surfaces can occur through simple physical interactions, offering a conceptual basis for the mechanisms underlying this work. Furthermore, the integration of such platforms with emerging analytical tools, including machine learning-assisted analysis, may enable next-generation smart microfluidic systems for biomedical and environmental applications (Boumar et al., 2023; Sukumar et al., 2025a).

In this work, we presented a simple and versatile wax transfer fabrication technique that enables the transfer of patterned geometries from conventional A4 paper onto porous Whatman filter paper for paper-based microfluidic applications. Beyond a fabrication method, this approach establishes a porous-to-porous microcontact transfer strategy for patterning diffusible materials within fibrous substrates, enabling controlled and reproducible pattern formation.

The method demonstrated robust performance across a range of conditions, producing stable hydrophobic–hydrophilic patterns while preserving the structural integrity of cellulose. Together, these features enabled effective fluid confinement and supported biological functionality, including 3D cell cultures. The versatility of the approach was further demonstrated through biochemical assays, showing performance comparable to conventional wax-patterned platforms. Importantly, the simplicity of the process, its compatibility with diverse porous substrates, and its long-term stability highlight its potential for scalable and reliable fabrication. Moreover, the method does not rely on specialized instrumentation, making it suitable for low-resource and decentralized settings.