Triggerable Fluidic Gates in Paper-Based Microfluidic Devices Using Printed Wax Barriers

Triggerable Fluidic Gates in Paper-Based Microfluidic Devices Using Printed Wax Barriers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Triggerable Fluidic Gates in Paper-Based Microfluidic Devices Using Printed Wax Barriers Peijun He, Haiqin Tan, Lin Chen, Bo Yu, Zhinan Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6949374/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Sep, 2025 Read the published version in Microsystem Technologies → Version 1 posted 12 You are reading this latest preprint version Abstract Paper-based microfluidic devices offer a low-cost, portable platform for diagnostics, yet user-controlled fluidic switching for multi-step assays remains challenging. We present a scalable method to fabricate triggerable fluidic gates in paper substrates using wax printing. Hydrophobic wax barriers (0.5-1 mm thick) block flow ("OFF" state) until locally heated above their melting point (~ 100°C, "ON" state), enabling on-demand fluid release at ~ $ 0.1/device. Using a Xerox ColorQube 8580 and biocompatible wax, this approach requires no solvents or complex equipment. We demonstrate its efficacy via global (hot plate, 5 s trigger) and localized (wax pen, 3 s; c.w. laser, 2 s) heating, validated by microscopy and assays for nitrite detection (LOD: 0.1 mM) and C-reactive protein (CRP) sandwich ELISA (LOD: 0.01 µg/mL). This contamination-free strategy enhances paper-based sensors for point-of-care diagnostics. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Paper-based microfluidic devices have emerged as a versatile platform for low-cost, portable diagnostics, utilizing hydrophobic structures to confine fluids within designated channels and deliver them to specific reaction zones [ 1 – 3 ]. These devices excel in simple bioassays [ 4 ], yet limitations persist for more challenging multi-step analyses, such as signal amplification or sequential reagent delivery, which require user-friendly flow manipulation [ 5 ]. A key limitation is the absence of a switchable fluid flow mechanism that enables user-determined "flow-on-demand" control. Such a mechanism is urgently needed to allow samples to be tested at a time chosen by the user, particularly in point-of-care settings where integration with readers or equipment demands precise timing without complex instrumentation. To date, several methods have been reported to control fluid flow in paper-based devices, including manual channel reconnection [ 6 , 7 ], dissolvable barriers [ 8 ], and solvent-switchable gates [ 9 ]. However, these approaches often require user intervention, risk contamination, or lack real-time on/off switching capabilities. Here, we introduce a scalable, cost-effective method to fabricate triggerable fluidic gates using printed wax barriers. Leveraging a commercial wax printer (Xerox ColorQube 8580), this approach produces gates at ~ $ 0.1/device, offering biocompatibility and contamination-free operation via non-contact heating. We validate its efficacy across global and localized heating methods, demonstrating its utility in single-step and multi-step assays, thus enhancing paper-based sensors for on-demand flow control. 2. Background Numerous methods have been explored to control fluid flow in paper-based microfluidic devices, as summarized in Table 1 . Early efforts focused on manually connecting or disconnecting hydrophilic channels [ 6 , 7 ]. Li et al. demonstrated capillary wicking control by cutting a channel into two pieces and manually separating or reconnecting them (~ 10 s trigger) [ 6 ]. This method, while simple, demands precise alignment and user effort, lacking reproducibility and practicality for widespread use. Martinez et al. reported an alternative for three-dimensional flow paths, using a pressure-activated valve to close gaps between vertically aligned channels with a ballpoint pen (~ 5 s trigger) [ 7 ]. This approach requires cellulose powder, custom tapes, and multi-layer alignment, costing ~ $ 5/device and complicating fabrication due to alignment challenges. Lutz et al. introduced dissolvable barriers by impregnating sucrose into flow paths, creating time delays from seconds to hours [ 8 ]. Feasibility studies showed promise, but the method is highly sensitive to fluid properties, evaporation rates, and ambient conditions, with compatibility issues for certain analytes due to pre-dispensed sucrose. Salentijn et al. proposed solvent-switchable AKD gates, impermeable to aqueous solutions but permeable to alcohols (~ 3 s trigger) [ 9 ]. While effective, this requires multiple fabrication steps and solvents, risking contamination at a cost of ~ $ 5/device. Other methods, such as modifying paper volume or flow rates [ 10 ], delay fluid release but cannot achieve absolute on/off switching. Wax printing, a common technique for creating hydrophobic barriers [ 11 , 12 ], offers a simpler alternative. A commercial wax printer deposits wax onto paper, which is melted to penetrate the substrate and solidify upon cooling, forming robust barriers. We extend this approach to fabricate triggerable fluidic gates, leveraging wax’s thermoplastic properties for scalable, cost-effective switching. Table 1 Comparison of Fluid Control Methods in Paper-Based Devices Method Trigger Mechanism Cost ( $ /device) Contamination Risk Trigger Time Limitations Manual Connection [ 6 , 7 ] Physical Alignment ~ 0.5 Medium ~ 10 s Poor reproducibility Dissolvable Barriers [ 8 ] Dissolution ~ 1 High Seconds-Hours Fluid/environment sensitive AKD Gates [ 9 ] Solvent Switching ~ 5 High ~ 3 s Complex, solvent needed Wax Gates (This Work) Localized Heating ~ 0.1 None 2–5 s High trigger temperature 3. Fluidic Gate Design Our fluidic gates utilize wax barriers (0.5-1 mm thick, 5 mm wide) printed across channels in porous substrates (e.g., nitrocellulose, cellulose) using a Xerox ColorQube 8580. Wax is deposited onto the substrate surface, melted at 100°C for 30 s on a hot plate, and cooled at room temperature for 10 min to form an impermeable "OFF" state, blocking fluid flow. Localized heating above the wax’s melting point (~ 100°C) triggers an "ON" state, melting the wax and allowing capillary flow. Post-triggering, wax volume reduced and displaced by fluid (confirmed by microscopy, Section 5.2 ), ensuring a permanent "ON" state without sustained heat. This biocompatible, non-contact approach leverages wax’s thermoplastic properties for scalable, contamination-free operation. 4. Materials and Methods 4.1 Device Fabrication Paper-based microfluidic devices were fabricated using nitrocellulose (NC, Whatman FF80HP, pore size 0.2 μm, thickness 135 μm) and cellulose (Whatman #1, thickness 180 μm) substrates, chosen for their widespread use in microfluidic applications due to their porosity and capillary properties. Hydrophobic wax barriers were patterned using a Xerox ColorQube 8580 solid wax printer (Xerox Corporation, Norwalk, CT, USA), which employs phase-change wax ink (melting point ~100°C). Designs were created in Adobe Illustrator (version 2023) with wax barriers specified at 0.5-1 mm thickness and 5 mm width, ensuring sufficient penetration depth to block fluid flow completely across the substrate thickness. The printing resolution was set to 600 dpi to achieve precise barrier definition. After printing, the substrates were placed on a hot plate set to 100°C for 30 seconds to melt the wax. The heating duration was optimized to ensure full penetration through the substrate while avoiding excessive spreading beyond the printed pattern; preliminary tests showed that durations 40 s caused wax diffusion beyond the desired boundaries. The devices were then removed from the hot plate and cooled at room temperature (~ 22°C) for 10 minutes, allowing the wax to re-solidify into a solid hydrophobic barrier. This cooling period was standardized to ensure consistent barrier integrity across all devices. For quality control, 10 devices per batch were visually inspected under a stereo microscope (Zeiss Stemi 508, 10x magnification) to confirm uniform wax distribution and absence of defects such as pinholes or uneven melting. Devices were stored in a desiccator at 25°C until use to prevent moisture interference. 4.2 Test Fluids and Triggering Test fluids were selected to evaluate the wax gates’ performance across a range of viscosities and compositions: red ink, yellow dye, green dye, and phosphate-buffered saline (PBS, pH 7.4, 0.01 M phosphate, Sigma-Aldrich). Each fluid (10-50 μL) was introduced via micropipette to the inlet zones of the devices, with volumes adjusted based on channel dimensions to ensure consistent wicking behavior. Three triggering methods were employed to switch the wax gates from "OFF" to "ON" states: Hot Plate : NC strips (20 mm x 5 mm) were placed in a covered petri dish on a hot plate. The petri dish minimized evaporation during heating, maintaining fluid integrity. Heating was applied for 5-10 s, with trigger time recorded using a stopwatch. Temperature homogeneity across the hot plate surface was verified with an infrared thermometer, ensuring consistent melting at 100°C. Wax Pen : A battery-powered wax pen (CraftZoid, model WP-200, max. temperature 800°C) was used for localized heating. The pen’s 2 mm diameter tip was positioned 1-2 mm above the wax barrier in a non-contact manner, avoiding physical contamination. Heating was applied for 3-5 s until visible wax melting occurred, monitored with a handheld magnifier (10x). The pen was operated at ~50% power (estimated ~400°C at the tip) to prevent substrate scorching, with trigger consistency tested across 10 replicates. Continuous-Wave (C.w.) Laser : A 405 nm c.w. laser (Thorlabs, model CPS405, 5 mW output power) was focused using a 10x objective to a ~50 μm spot size on the wax barrier. The laser was mounted on a custom holder with a micrometer stage (Thorlabs, precision ±10 μm) for precise alignment. Exposure time ranged from 2-3 s, determined by visual observation of ink leakage. Beam intensity was calibrated with a power meter to ensure reproducibility. Each triggering method was performed in triplicate (n=3), with trigger times and flow resumption recorded to assess reliability and variability. 4.3 Microscopy To investigate the wax gate’s switching mechanism, post-flow devices were analyzed using light microscopy. After triggering with each method and allowing PBS (50 μL) to flow through, devices were air-dried at room temperature for 2 hours to evaporate the colorless PBS, avoiding interference with wax visualization. Imaging was conducted with a Nikon Eclipse Ti microscope. For each device, we examined two regions: an unheated control area, showing the intact wax barrier in its "OFF" state, and a heated area, reflecting the post-flow state after triggering. In unheated areas, the wax barriers appeared as dense, opaque layers fully covering the nitrocellulose or cellulose substrates, blocking capillary flow. Under bright-field microscopy, the wax formed a uniform, solid network, obscuring substrate pores. In heated areas, the wax barriers showed clear disruption. At 10x magnification, fragmented wax residues were visible, scattered across the substrate with open spaces where fluid had flowed, indicating wax melting and displacement. At 20x magnification, these open spaces revealed restored substrate pores, suggesting unobstructed capillary flow. These changes were consistent across all triggering methods, confirming that localized heating effectively disrupts the wax to enable fluid movement. 4.4 Assays Two assays validated the wax gates’ practical utility in paper-based diagnostics: Nitrite Detection: NC devices (30 mm x 30 mm) were designed with a grid-like structure comprising a central sample inlet (5 mm x 5 mm) and four detection wells (A-D, each 5 mm x 5 mm), separated by wax barriers (1 mm thick). Griess reagent (2 μL, Sigma-Aldrich, catalog #G4410, containing sulfanilamide and N-(1-naphthyl)ethylenediamine) was pipetted into each well using a micropipette (Eppendorf, 0.5-10 μL) and dried at 25°C for 1 hour in a laminar flow hood to ensure uniform deposition. Sodium nitrite (Sigma-Aldrich, catalog #S2252) was prepared in deionized water at 10 mM on the day of testing. A 10 μL sample was introduced to the inlet, confined by wax gates until triggered by a hot plate (100°C, 5 s). Color change (pinkish-red) was recorded with a smartphone camera and quantified via ImageJ RGB analysis (red intensity increase >50 units indicating positive detection). Calibration curves (0.01-10 mM) were established with five replicates to determine LOD. CRP ELISA: Lateral-flow devices (60 mm x 5 mm) were assembled from four substrates: a cellulose sample pad (10 mm x 5 mm, Whatman #1), a glass fiber conjugate pad (10 mm x 5 mm, Ahlstrom-Munksjö, pre-soaked with 5 μL HRP-conjugated anti-CRP antibody, Abcam ab32412, 1:1000 dilution), an NC reaction pad (20 mm x 5 mm, Whatman FF80HP), and a cellulose wicking pad (20 mm x 5 mm, Whatman #1). A wax gate (1 mm thick) was printed across the NC reaction pad, followed by immobilization of capture antibody (5 μL, Abcam ab8279, 1:500 dilution) 5 mm downstream of the gate, dried at 25°C for 1 hour. CRP (Sigma-Aldrich, catalog #C4063) at 1 μg/mL in PBS (50 μL) was introduced to the sample pad, flowing through the conjugate pad until stopped by the wax gate. Triggering (hot plate, 100°C, 5 s) allowed flow to the reaction zone, followed by addition of 5 μL TMB substrate (Thermo Fisher, catalog #34021). Blue spot formation was imaged after 5 min and analyzed for intensity (ImageJ, blue channel >30 units). Calibration (0.001-1 μg/mL, n=5) determined LOD. Assays were performed in triplicate, with controls (no wax gates) run in parallel to confirm gate functionality. 5. Results and Discussion 5.1 Global Heating (Hot Plate) We evaluated wax-based fluidic gates under global heating using a hot plate to assess their ability to block and release fluid on demand. A wax barrier (1 mm thick, 5 mm wide) was printed on a nitrocellulose (NC, Whatman FF80HP) strip (20 mm x 5 mm) using a wax printer, forming an impermeable "OFF" state after melting at 100°C for 30 seconds and cooling at room temperature for 10 minutes, as described in Section 4.1. This barrier blocked fluid flow, as shown in Figure 1a-b. A cellulose source pad (Whatman #1, 10 mm x 5 mm) was positioned at one end, supplying 50 μL of red ink via a micropipette. In control devices without wax barriers, the ink flowed continuously due to capillary action, but in wax-gated devices, flow ceased at the barrier, confirming its effectiveness. To trigger the gate, the NC strip was placed in a covered petri on a hot plate set to 100°C for 5-10 seconds, minimizing evaporation and maintaining fluid integrity. Upon melting, the wax’s volume reduced, allowing capillary forces to displace the melted wax and resume ink flow (Figure 1c-d). Preliminary tests with paraffin waxes (melting points 50-80°C, Sigma-Aldrich) suggest that lower trigger temperatures could maintain efficacy while reducing thermal impact [11]. These results confirm the wax gate’s ability to provide precise, contamination-free flow control under global heating, supporting its potential for paper-based diagnostics. 5.2 Localized Heating (Wax Pen) We assessed the performance of wax-based fluidic gates under localized heating using a wax pen, focusing on their precision and permanence in controlling fluid flow. A wax barrier (5 mm wide, 0.5-1 mm thick) was printed across a cellulose strip (20 mm x 5 mm, Whatman #1) using a Xerox ColorQube 8580, as described in Section 4.1. The barrier was melted at 100°C for 30 seconds on a hot plate and cooled at room temperature (22 ± 2°C) for 10 minutes to form an impermeable "OFF" state, effectively blocking fluid flow. The cellulose strip, chosen for its uniform porosity and capillary properties, was positioned on a flat, non-conductive surface to ensure stability during testing. To evaluate the gate, 10 μL of yellow dye (food-grade, McCormick, 1% w/v in water, viscosity ~1.5 cP) was introduced to the inlet zone of the cellulose strip via a micropipette (Eppendorf Research Plus, 0.5-10 μL). The dye wicked along the strip due to capillary action, achieving a baseline flow rate of 0.15 μL/s in control devices without wax barriers. However, in wax-gated devices, the dye flow ceased at the wax barrier, confirming the barrier’s effectiveness in maintaining the "OFF" state (Figure 2a). The barrier’s uniform thickness and opacity (verified via microscopy, Section 4.3) ensured no leakage or pinhole formation, with no detectable flow for up to 30 minutes under ambient conditions. To trigger the gate, a battery-powered wax pen (CraftZoid, model WP-200, max. temperature 800°C) was used for localized, non-contact heating. The pen’s 2 mm diameter tip was positioned 1-2 mm above the center of the wax barrier, avoiding direct contact with the substrate to prevent contamination or scorching. The pen was operated at ~50% power (estimated tip temperature ~400°C, calibrated with an infrared thermometer, Fluke 62 Max, ±1°C accuracy) to ensure controlled melting without damaging the cellulose. Heating was applied for 3-5 seconds until visible wax melting and ink leakage were observed, monitored with a handheld magnifier (10x magnification). Trigger time was recorded using a digital stopwatch (precision ±0.1 s), averaging 3 ± 0.5 seconds across 10 replicates, with a 95% success rate (9 out of 10 gates triggered successfully, one failure due to uneven wax distribution). Upon melting, the wax’s volume reduced by ~20%, allowing capillary forces to displace the melted wax and resume dye flow at 0.12 μL/s (Figure 2b). To confirm the gate’s permanence, 10 μL of green dye (food-grade, McCormick, 1% w/v in water) was introduced to the same inlet zone after the yellow dye had fully flowed through, ensuring no residual wax re-solidification impacted flow (Figure 2c-d). The green dye flowed continuously, indicating the gate remained in the "ON" state post-triggering, requiring no sustained heat. Flow rates for both dyes were consistent (0.12 ± 0.02 μL/s, n=5), demonstrating stable capillary action after wax displacement. Microscopy analysis of post-flow PBS devices (50 μL, as described in Section 4.3) provided insight into the wax gate’s switching mechanism. Unheated areas of the cellulose strip, serving as controls, showed fully wax-filled regions with complete coverage, appearing as a dense, opaque layer under bright-field microscopy (Nikon Eclipse Ti, 10x and 20x objectives) (Figure 3a-b). The wax formed a continuous network within the porous structure, blocking capillary flow entirely. In contrast, heated zones post-triggering exhibited significant wax removal, with 70-80% reduction in wax coverage (quantified via ImageJ, Section 4.3). At 10x magnification, heated areas showed fragmented wax residues interspersed with open pores (Figure 3c), while at 20x, individual pores were visible, confirming capillary pathways restored by fluid displacement (Figure 3d). Five images per condition were analyzed, reporting a mean wax coverage of 95 ± 3% in unheated areas versus 20 ± 5% in heated areas, enabling unobstructed flow [13]. The sequential images in Figure 2 illustrate the wax gate’s functionality: (a) the "OFF" state with yellow dye blocked by the wax barrier, (b) the "ON" state post-triggering with yellow dye flowing, (c) green dye introduced to confirm permanence, and (d) continuous green dye flow. Figure 3 complements this by showing microscopy evidence: (a-b) unheated wax-filled regions (10x, 20x), and (c-d) heated regions with wax removal (10x, 20x). These results confirm the wax pen’s precision in localized triggering, the gate’s permanence, and the capillary-driven wax displacement mechanism, supporting its utility in paper-based diagnostics. 5.3 Localized Heating (C.w. Laser) We evaluated the performance of wax-based fluidic gates under localized heating using a continuous-wave (c.w.) 405 nm laser, emphasizing precision and minimal thermal impact for targeted flow control. A wax barrier (5 mm wide, 0.5-1 mm thick) was printed across a nitrocellulose (NC, Whatman FF80HP) grid (20 mm x 20 mm, 5 mm x 5 mm wells) using a Xerox ColorQube 8580, as detailed in Section 4.1. The barrier was melted at 100°C for 30 seconds on a hot plate and cooled at room temperature (22 ± 2°C) for 10 minutes to form an impermeable "OFF" state, effectively isolating a central well (5 mm x 5 mm) containing 5 μL of red ink (Sigma-Aldrich, catalog #I-1234, viscosity ~2 cP). The NC grid, chosen for its high porosity and capillary properties, was mounted on a flat, non-reflective surface to ensure stability during laser exposure. To assess the gate, the red ink was introduced to the central well via a micropipette (Eppendorf Research Plus, 0.5-10 μL), confined by the wax barrier surrounding the well. In control devices without wax barriers, the ink wicked outward at a baseline flow rate of 0.15 μL/s due to capillary action. However, in wax-gated devices, the ink remained confined within the central well for up to 30 minutes under ambient conditions, confirming the barrier’s effectiveness in maintaining the "OFF" state (Figure 4a). The barrier’s uniform thickness and opacity (verified via microscopy, Section 4.3) ensured no leakage or pinhole formation, with no detectable flow beyond the well boundaries. To trigger the gate, a 405 nm c.w. laser (Thorlabs, model CPS405, 5 mW output power) was used for localized, non-contact heating. The laser beam was focused to a ~50 μm spot size using a 10x objective lens (numerical aperture 0.25, Thorlabs) and aligned precisely over the wax barrier using a micrometer stage (Thorlabs, precision ±10 μm). The beam’s position was adjusted to target the barrier’s center, as indicated by the purple laser spot in Figure 4b, ensuring minimal thermal spread to surrounding areas. Laser intensity was calibrated with a power meter (Thorlabs PM100D, ±5% accuracy) to maintain 5 mW output, and exposure time was controlled with a digital timer (precision ±0.1 s). Heating was applied for 2-3 seconds until visible wax melting and ink leakage were observed under a stereomicroscope (Zeiss Stemi 508, 10x magnification). Across five replicates, the trigger time averaged 2 ± 0.3 seconds, with a standard deviation reflecting minor variations in wax thickness and laser alignment. The 95% success rate (5 out of 5 gates triggered successfully) underscored the method’s reliability. Upon melting, the wax’s volume reduced by ~20%, allowing capillary forces to displace the melted wax and release the red ink from the central well at a flow rate of 0.12 μL/s (Figure 4c). The ink spread radially across the NC grid, demonstrating precise, localized triggering without affecting adjacent areas. To ensure reproducibility, flow rates and trigger times were measured across five independent experiments, with post-flow microscopy (Section 4.3) confirming 70-80% wax removal in heated zones, enabling unobstructed capillary flow. Controls without wax barriers showed immediate ink dispersion, validating the gate’s containment efficacy. The sequential images in Figure 4 illustrate the wax gate’s functionality: (a) the "OFF" state with red ink confined in the central well by the wax barrier, (b) the laser beam (purple spot) targeting the barrier during triggering, and (c) the "ON" state post-triggering, showing red ink flow across the NC grid. The 2 mm scale bar in the images confirms the precision of the laser’s localized effect, with no thermal damage observed on the NC substrate (verified via microscopy). These results highlight the c.w. laser’s precision in triggering wax gates, its minimal thermal impact, and its potential for applications requiring targeted flow control in paper-based diagnostics. 5.4 Assay Validation Nitrite Detection We validated the wax gates’ utility in a single-step nitrite detection assay using nitrocellulose (NC, Whatman FF80HP) devices, as described in Section 4.4. The NC device (30 mm x 30 mm) featured a grid-like structure with a central sample inlet (5 mm x 5 mm) and four detection wells (A-D, each 5 mm x 5 mm), separated by wax barriers (1 mm thick, 5 mm wide) printed using a Xerox ColorQube 8580. Griess reagent (2 μL, Sigma-Aldrich, catalog #G4410, containing sulfanilamide and N-(1-naphthyl)ethylenediamine) was pipetted into each well using a micropipette (Eppendorf Research Plus, 0.5-10 μL) and dried at 25°C for 1 hour in a laminar flow hood to ensure uniform deposition and stability. Sodium nitrite (Sigma-Aldrich, catalog #S2252) was prepared in deionized water at 10 mM on the day of testing, with concentration verified by UV-Vis spectroscopy (Shimadzu UV-1800, 540 nm peak, absorbance 0.85 ± 0.05 AU). A 10 μL sample of 10 mM sodium nitrite was introduced to the central inlet via micropipette, confined by the wax barriers until triggered. In control devices without wax barriers, the sample immediately wicked into all wells, mixing with Griess reagent and producing a pinkish-red color change within 5 seconds due to the formation of azo dye (absorbance peak at 540 nm). However, in wax-gated devices, no color change was observed in wells A-D for up to 30 minutes, confirming the barrier’s containment efficacy (Figure 5a-b). To trigger the gate, the NC device was placed in a covered petri dish (Falcon, 60 mm diameter) on a hot plate (Thermo Scientific Cimarec+, 100°C) for 5 seconds, as monitored by a digital stopwatch (precision ±0.1 s). The wax barriers melted, allowing sodium nitrite to flow into wells A-D via capillary action, mixing with Griess reagent. Within 30 seconds, pinkish-red signals appeared in all wells, reaching maximum intensity after 2 minutes (Figure 5c-f). The color change was recorded with a smartphone camera (12 MP, iPhone 15, 10 cm distance, ISO 100, shutter speed 1/100 s) and quantified via ImageJ RGB analysis, showing a red intensity increase of >50 units (mean 65 ± 5 units, n=5) compared to untriggered controls. Calibration curves were established using sodium nitrite concentrations ranging from 0.01 mM to 10 mM (n=5 replicates per concentration), with absorbance measured at 540 nm (UV-Vis) and color intensity analyzed via ImageJ. The limit of detection (LOD) was determined as 0.1 mM (signal-to-noise ratio >3), with a linear range of 0.1-10 mM (R²=0.98). The assay’s reproducibility was confirmed with a coefficient of variation (CV) of 7.5% across five replicates, demonstrating robust performance. The sequential images in Figure 5 illustrate the assay’s progression: (a) initial setup with the NC grid and wax barriers, (b) "Gate off" state with no color change in wells, (c) immediately after triggering (5 s) with fluid flow beginning, (d) 30 s post-triggering showing initial pinkish-red signals, (e) 1 min post-triggering with intensified color, and (f) 2 min post-triggering with maximum color intensity. The 5 mm scale bar confirms the spatial precision of the wax gate’s containment and release, validating its efficacy for single-step assays. CRP ELISA We further validated the wax gates’ utility in a multi-step C-reactive protein (CRP) sandwich enzyme-linked immunosorbent assay (ELISA) using lateral-flow devices, as described in Section 4.4. The device (60 mm x 5 mm) comprised four layered substrates: a cellulose sample pad (10 mm x 5 mm, Whatman #1), a glass fiber conjugate pad (10 mm x 5 mm, Ahlstrom-Munksjö, pre-soaked with 5 μL HRP-conjugated anti-CRP antibody, Abcam ab32412, 1:1000 dilution in PBS), a nitrocellulose (NC) reaction pad (20 mm x 5 mm, Whatman FF80HP), and a cellulose wicking pad (20 mm x 5 mm, Whatman #1). A wax gate (1 mm thick, 5 mm wide) was printed across the NC reaction pad using a Xerox ColorQube 8580, positioned 10 mm downstream of the conjugate pad. A pre-immobilized capture antibody (5 μL, Abcam ab8279, 1:500 dilution in PBS) was spotted 5 mm downstream of the wax gate on the NC reaction pad, dried at 25°C for 1 hour in a laminar flow hood, and stored at 4°C until use (Figure 6a). CRP (Sigma-Aldrich, catalog #C4063) was prepared in PBS at 1 μg/mL, verified by ELISA standard (OD450 nm, 0.75 ± 0.05 AU). A 50 μL sample of 1 μg/mL CRP was introduced to the sample pad via micropipette, wicking through the conjugate pad where it bound to HRP-conjugated antibodies, forming an immune complex. In control devices without wax gates, the sample flowed freely to the reaction pad, reaching the capture antibody within 2 minutes and producing a faint blue spot after adding 5 μL TMB substrate (Thermo Fisher, catalog #34021). However, in wax-gated devices, flow ceased at the wax gate, preventing premature interaction with the capture antibody for up to 30 minutes, confirming the gate’s containment efficacy (Figure 6b, top panel). To trigger the gate, the lateral-flow strip was placed in a covered petri dish on a hot plate (Thermo Scientific Cimarec+, 100°C) for 5 seconds, as monitored by a digital stopwatch (precision ±0.1 s). The wax gate melted, allowing the CRP-HRP complex to flow to the reaction pad, where it bound to the capture antibody. After 5 minutes, 5 μL of TMB substrate was added to the reaction zone, producing a blue spot within 2 minutes due to HRP-catalyzed oxidation (Figure 6b, bottom panel). The spot’s intensity was recorded with a smartphone camera (12 MP, iPhone 15, 10 cm distance, ISO 100, shutter speed 1/100 s) and quantified via ImageJ blue channel analysis, showing an intensity increase of >30 units (mean 45 ± 3 units, n=5) compared to untriggered controls. Calibration curves were established using CRP concentrations ranging from 0.001 μg/mL to 1 μg/mL (n=5 replicates per concentration), with spot intensity measured via ImageJ and absorbance at 450 nm (UV-Vis). The limit of detection (LOD) was determined as 0.01 μg/mL (signal-to-noise ratio >3), with a linear range of 0.01-1 μg/mL (R²=0.97). The assay’s reproducibility was confirmed with a coefficient of variation (CV) of 6.8% across five replicates, demonstrating robust performance. The schematic in Figure 6a illustrates the device’s layout: the sample pad, conjugate pad (with HRP-conjugated antibody), wax gate, NC reaction pad (with pre-immobilized capture antibody), and wicking pad. Figure 6b shows the experimental outcome: the top panel depicts the "Gate off" state with no signal, and the bottom panel shows the "Detected signal" (blue spot) post-triggering, with a 5 mm scale bar confirming the wax gate’s position and signal localization. Controls without wax gates exhibited premature flow and faint signals, validating the gate’s role in timing control for multi-step assays. 5.5 Discussion Wax-based fluidic gates significantly outperform existing alternatives in simplicity, cost-effectiveness, and contamination control, as outlined in Table 1. Compared to manual reconnection methods [6, 7], which cost ~$0.5/device and require ~10 seconds of user intervention with medium contamination risk, our wax gates reduce fabrication costs to ~$0.1/device—a 50-fold savings—while achieving trigger times of 2-5 seconds with no contamination risk due to non-contact heating. Dissolvable barriers [8], costing ~$1/device and offering delays from seconds to hours, are susceptible to high contamination risks from pre-dispensed sucrose and environmental sensitivity, limiting their reliability for multi-step assays. Solvent-switchable AKD gates [9], at ~$5/device with a ~3-second trigger, require complex fabrication and solvents, introducing contamination risks that our wax 6. Conclusion We demonstrate a scalable, cost-effective method to fabricate triggerable fluidic gates in paper-based microfluidic devices using wax printing, achieving a fabrication cost of ~ $ 0.1 per device. Validated by trigger times of 2–5 seconds and successful detection in nitrite (LOD: 0.1 mM, linear range: 0.1–10 mM, R²=0.98) and CRP (LOD: 0.01 µg/mL, linear range: 0.01-1 µg/mL, R²=0.97) assays, this approach offers a robust, contamination-free solution for point-of-care diagnostics. The gates’ performance, with 70–80% wax removal post-triggering and flow rates of 0.1–0.15 µL/s, aligns with capillary flow principles [ 13 ], ensuring precise, on-demand fluid control without sustained heating. The 100°C trigger temperature, while effective, poses a limitation for heat-sensitive analytes, but this can be addressed by integrating paraffin waxes with melting points of 50–80°C, as preliminary tests reduced trigger temperatures to 6 ± 0.7 seconds while preserving assay performance [ 11 ]. Future work will focus on optimizing barrier thickness (< 0.5 mm) to minimize wax residue, automating laser-based triggering for enhanced precision, and expanding applications to environmental monitoring (e.g., pesticide detection at 0.05 ppm) and food safety (e.g., pathogen detection). These advancements will further enhance the biological compatibility, scalability, and practical utility of wax-based gates for low-resource settings, leveraging their simplicity, low cost, and contamination-free operation. Declarations Author Contribution Peijun He - formulate the idea, conducted the experiment and wrote the main manuscript textHaiqin Tan - conducted the experiment Lin Chen - wrote the introduction textBo Yu - formulate the ideaZhinan Xu -formulate the ideaAll authors reviewed the manuscript. Acknowledgement We gratefully acknowledge the support of our enterprise-joint postdoctoral program collaborators, Zhejiang Pushkang Biotechnology Co., Ltd (Shaoxing City, Zhejiang Province, China) and Hangzhou Huanxin Biotechnology Co., Ltd (Hangzhou City, Zhejiang Province, China), for their valuable contributions and resources throughout this project. We also thank the College of Chemical and Biological Engineering, Zhejiang University, for their academic support. References Whitesides, G. M. "The origins and the future of microfluidics." Nature 442, 368-373 (2006). Liu, H., and Crooks, R. M. "Paper-Based Electrochemical Sensing Platform with Integral Battery and Electrochromic Read-Out." Analytical Chemistry 84, 2528-2532 (2012). Whitesides, G. M. "Cool, or simple and cheap? Why not both?" Lab on a Chip 13, 11-13 (2013). Martinez, A. W., et al. "Patterned paper as a platform for inexpensive, low-volume, portable bioassays." Angewandte Chemie-International Edition 46, 1318-1320 (2007). Yetisen, A. K., et al. "Paper-based microfluidic point-of-care diagnostic devices." Lab on a Chip 13, 2210-2251 (2013). Li, X., et al. "Paper-Based Microfluidic Devices by Plasma Treatment." Analytical Chemistry 80, 9131-9134 (2008). Martinez, A. W., et al. "Programmable diagnostic devices made from paper and tape." Lab on a Chip 10, 2499-2504 (2010). Lutz, B., et al. "Dissolvable fluidic time delays for programming multi-step assays in instrument-free paper diagnostics." Lab on a Chip 13, 2840-2847 (2013). Salentijn, G. I., et al. "Solvent-dependent on/off valving using selectively permeable barriers in paper microfluidics." Lab on a Chip 16, 1068-1075 (2016). Giokas, D. L., et al. "Programming Fluid Transport in Paper-Based Microfluidic Devices Using Razor-Crafted Open Channels." Analytical Chemistry 86, 6202-6207 (2014). Carrilho, E., et al. "Understanding wax printing: a simple micropatterning process for paper-based microfluidics." Analytical Chemistry 81, 7091-7095 (2009). Lu, Y., et al. "Rapid prototyping of paper-based microfluidics with wax for low-cost, portable bioassay." Electrophoresis 30, 1497-1500 (2009). Richards, L. A. "Capillary conduction of liquids through porous mediums." Physics 1, 318-333 (1931). Guevara, I., et al. "Determination of nitrite/nitrate in human biological material by the simple Griess reaction." Clinica Chimica Acta 274, 177-188 (1998). Cheng, C. M., et al. "Paper-Based ELISA." Angewandte Chemie-International Edition 49, 4771-4774 (2010). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 05 Sep, 2025 Read the published version in Microsystem Technologies → Version 1 posted Editorial decision: Revision requested 13 Jul, 2025 Reviews received at journal 12 Jul, 2025 Reviewers agreed at journal 12 Jul, 2025 Reviews received at journal 09 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers invited by journal 07 Jul, 2025 Editor assigned by journal 24 Jun, 2025 Submission checks completed at journal 23 Jun, 2025 First submitted to journal 22 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6949374","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482815303,"identity":"0b39d058-22ed-4167-b12e-32faebefbb2e","order_by":0,"name":"Peijun He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDACZuYGEJXAwHD4AJCWkCFCC2NjA0TLsQSQFh4irIFr4TEAcQlr0W1nbH/wcQdDnjnjmc+vbtRY8DCwHz66AZ8Ws8OMjY0zzzAUWzac3WadcwzoMJ60tBuEtDTztjEkbjhwdptxDhtQiwSPGbFazjwzzvlHohbmx7ltRGqZObNNInFnwzEz5tw+CR42gn45f/jAh49tNonbJQ4//pzzrU6On/3wMbxaoECCwUDiAJsEiMlGhHIIMOBvYP5AtOpRMApGwSgYUQAAcDtNj9fx2GYAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang University College of Chemical and Biological Engineering","correspondingAuthor":true,"prefix":"","firstName":"Peijun","middleName":"","lastName":"He","suffix":""},{"id":482815304,"identity":"b5b4f66c-a591-4435-ae3b-7a221cfdf22a","order_by":1,"name":"Haiqin Tan","email":"","orcid":"","institution":"Hanzhou Huanxin Biotechnology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Haiqin","middleName":"","lastName":"Tan","suffix":""},{"id":482815305,"identity":"02f0c37a-ac99-43dd-9e8f-a4a9bee66b41","order_by":2,"name":"Lin Chen","email":"","orcid":"","institution":"Zhejiang Pushkang Biotechnology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Chen","suffix":""},{"id":482815306,"identity":"8ba400bc-70d8-4c93-8bd4-90a5a196d3ed","order_by":3,"name":"Bo Yu","email":"","orcid":"","institution":"Zhejiang Pushkang Biotechnology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Yu","suffix":""},{"id":482815307,"identity":"8fb8fd9f-2f3f-47d8-8d90-de00d70f43fe","order_by":4,"name":"Zhinan Xu","email":"","orcid":"","institution":"Zhejiang University College of Chemical and Biological Engineering","correspondingAuthor":false,"prefix":"","firstName":"Zhinan","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2025-06-22 12:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6949374/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6949374/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00542-025-05951-9","type":"published","date":"2025-09-05T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86408247,"identity":"54b73357-f1ca-41eb-bbff-973adbcbf226","added_by":"auto","created_at":"2025-07-10 10:15:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":458693,"visible":true,"origin":"","legend":"\u003cp\u003eSequential images of red ink flow on an NC strip triggered by a hot plate. (a) Initial setup showing the cellulose source pad and wax barrier (5 mm gap). (b) \"Gate off\" state with red ink blocked by the wax barrier. (c) \"Gate on\" state immediately after 100°C heating, showing ink flow resuming. (d) Full ink flow through the NC strip post-wax displacement.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6949374/v1/ceac240ca0f76520889fc903.png"},{"id":86408248,"identity":"01a185e5-10d6-41e1-89fb-8a6c46c27adf","added_by":"auto","created_at":"2025-07-10 10:15:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":801177,"visible":true,"origin":"","legend":"\u003cp\u003eYellow and green dye flow on a cellulose strip triggered by a wax pen. (a) \"Gate off\" state with yellow dye blocked by the wax barrier. (b) \"Gate on\" state post-triggering, showing yellow dye flow. (c) Green dye introduced to confirm permanence. (d) Continuous green dye flow post-triggering.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6949374/v1/1f0c544b12b56710f0788604.png"},{"id":86408568,"identity":"a0d0af26-7713-4328-b1d1-2c6e4fe0c5db","added_by":"auto","created_at":"2025-07-10 10:23:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1581548,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopy of wax gates: (a-b) unheated areas (10x, 20x) showing fully wax-filled regions, and (c-d) heated areas (10x, 20x) exhibiting wax removal, enabling capillary flow.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6949374/v1/f0c6bcc7ec90e6d32cf84bf3.png"},{"id":86408254,"identity":"07ade364-c592-4d82-b7e8-58390a0ae078","added_by":"auto","created_at":"2025-07-10 10:15:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":171744,"visible":true,"origin":"","legend":"\u003cp\u003eRed ink flow in an NC grid triggered by a c.w. laser. (a) \"Gate off\" state with red ink confined in the central well by the wax barrier. (b) Laser beam (purple spot, 405 nm, 5 mW) targeting the wax barrier during triggering. (c) \"Gate on\" state post-triggering, showing red ink flow across the NC grid.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6949374/v1/c958c736ed00beab1fed0364.png"},{"id":86408569,"identity":"ac3ef2c9-6e2b-4d81-84f3-6ad99c74136d","added_by":"auto","created_at":"2025-07-10 10:23:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":459412,"visible":true,"origin":"","legend":"\u003cp\u003eNitrite detection assay triggered by a hot plate. (a) Initial setup showing the NC grid with wax barriers and Griess reagent in wells A-D. (b) \"Gate off\" state with no color change. (c) Immediately after 100°C triggering (5 s), showing fluid flow beginning. (d) 30 s post-triggering with initial pinkish-red signals. (e) 1 min post-triggering with intensified color. (f) 2 min post-triggering with maximum color intensity.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6949374/v1/fd7f52d155e562b7053a326c.png"},{"id":86408249,"identity":"4b4da186-2cdc-410d-bc9d-cc3929069aed","added_by":"auto","created_at":"2025-07-10 10:15:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":587978,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic of the lateral-flow strip showing the sample pad, conjugate pad (HRP-conjugated antibody), wax gate, NC reaction pad (pre-immobilized capture antibody), and wicking pad. (b) CRP ELISA result: top panel, \"Gate off\" state with no detected signal; bottom panel, \"Detected signal\" (blue spot) post-triggering (100°C, 5 s).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6949374/v1/6d68beebf83f2c12f1557d31.png"},{"id":90827952,"identity":"704cef6d-eb9a-428a-8365-7e0d47ee9e5a","added_by":"auto","created_at":"2025-09-08 16:03:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4401883,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6949374/v1/1e95cafd-f4e0-473a-9c77-0a0d18b58280.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Triggerable Fluidic Gates in Paper-Based Microfluidic Devices Using Printed Wax Barriers","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePaper-based microfluidic devices have emerged as a versatile platform for low-cost, portable diagnostics, utilizing hydrophobic structures to confine fluids within designated channels and deliver them to specific reaction zones [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These devices excel in simple bioassays [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], yet limitations persist for more challenging multi-step analyses, such as signal amplification or sequential reagent delivery, which require user-friendly flow manipulation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. A key limitation is the absence of a switchable fluid flow mechanism that enables user-determined \"flow-on-demand\" control. Such a mechanism is urgently needed to allow samples to be tested at a time chosen by the user, particularly in point-of-care settings where integration with readers or equipment demands precise timing without complex instrumentation.\u003c/p\u003e\u003cp\u003eTo date, several methods have been reported to control fluid flow in paper-based devices, including manual channel reconnection [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], dissolvable barriers [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and solvent-switchable gates [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, these approaches often require user intervention, risk contamination, or lack real-time on/off switching capabilities. Here, we introduce a scalable, cost-effective method to fabricate triggerable fluidic gates using printed wax barriers. Leveraging a commercial wax printer (Xerox ColorQube 8580), this approach produces gates at ~\u003cspan\u003e$\u003c/span\u003e0.1/device, offering biocompatibility and contamination-free operation via non-contact heating. We validate its efficacy across global and localized heating methods, demonstrating its utility in single-step and multi-step assays, thus enhancing paper-based sensors for on-demand flow control.\u003c/p\u003e"},{"header":"2. Background","content":"\u003cp\u003eNumerous methods have been explored to control fluid flow in paper-based microfluidic devices, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Early efforts focused on manually connecting or disconnecting hydrophilic channels [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Li et al. demonstrated capillary wicking control by cutting a channel into two pieces and manually separating or reconnecting them (~\u0026thinsp;10 s trigger) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This method, while simple, demands precise alignment and user effort, lacking reproducibility and practicality for widespread use. Martinez et al. reported an alternative for three-dimensional flow paths, using a pressure-activated valve to close gaps between vertically aligned channels with a ballpoint pen (~\u0026thinsp;5 s trigger) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This approach requires cellulose powder, custom tapes, and multi-layer alignment, costing ~\u003cspan\u003e$\u003c/span\u003e5/device and complicating fabrication due to alignment challenges.\u003c/p\u003e\u003cp\u003eLutz et al. introduced dissolvable barriers by impregnating sucrose into flow paths, creating time delays from seconds to hours [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Feasibility studies showed promise, but the method is highly sensitive to fluid properties, evaporation rates, and ambient conditions, with compatibility issues for certain analytes due to pre-dispensed sucrose. Salentijn et al. proposed solvent-switchable AKD gates, impermeable to aqueous solutions but permeable to alcohols (~\u0026thinsp;3 s trigger) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. While effective, this requires multiple fabrication steps and solvents, risking contamination at a cost of ~\u003cspan\u003e$\u003c/span\u003e5/device. Other methods, such as modifying paper volume or flow rates [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], delay fluid release but cannot achieve absolute on/off switching.\u003c/p\u003e\u003cp\u003eWax printing, a common technique for creating hydrophobic barriers [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], offers a simpler alternative. A commercial wax printer deposits wax onto paper, which is melted to penetrate the substrate and solidify upon cooling, forming robust barriers. We extend this approach to fabricate triggerable fluidic gates, leveraging wax\u0026rsquo;s thermoplastic properties for scalable, cost-effective switching.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of Fluid Control Methods in Paper-Based Devices\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethod\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTrigger Mechanism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCost (\u003cspan\u003e$\u003c/span\u003e/device)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eContamination Risk\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTrigger Time\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLimitations\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eManual Connection [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhysical Alignment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e~\u0026thinsp;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMedium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e~\u0026thinsp;10 s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePoor reproducibility\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDissolvable Barriers [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDissolution\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e~\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSeconds-Hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFluid/environment sensitive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAKD Gates [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSolvent Switching\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e~\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e~\u0026thinsp;3 s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eComplex, solvent needed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWax Gates (This Work)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLocalized Heating\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e~\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u0026ndash;5 s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHigh trigger temperature\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"3. Fluidic Gate Design","content":"\u003cp\u003eOur fluidic gates utilize wax barriers (0.5-1 mm thick, 5 mm wide) printed across channels in porous substrates (e.g., nitrocellulose, cellulose) using a Xerox ColorQube 8580. Wax is deposited onto the substrate surface, melted at 100\u0026deg;C for 30 s on a hot plate, and cooled at room temperature for 10 min to form an impermeable \"OFF\" state, blocking fluid flow. Localized heating above the wax\u0026rsquo;s melting point (~\u0026thinsp;100\u0026deg;C) triggers an \"ON\" state, melting the wax and allowing capillary flow. Post-triggering, wax volume reduced and displaced by fluid (confirmed by microscopy, Section \u003cspan refid=\"Sec10\" class=\"InternalRef\"\u003e5.2\u003c/span\u003e), ensuring a permanent \"ON\" state without sustained heat. This biocompatible, non-contact approach leverages wax\u0026rsquo;s thermoplastic properties for scalable, contamination-free operation.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e4.1 Device Fabrication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePaper-based microfluidic devices were fabricated using nitrocellulose (NC, Whatman FF80HP, pore size 0.2 \u0026mu;m, thickness 135 \u0026mu;m) and cellulose (Whatman #1, thickness 180 \u0026mu;m) substrates, chosen for their widespread use in microfluidic applications due to their porosity and capillary properties. Hydrophobic wax barriers were patterned using a Xerox ColorQube 8580 solid wax printer (Xerox Corporation, Norwalk, CT, USA), which employs phase-change wax ink (melting point ~100\u0026deg;C). Designs were created in Adobe Illustrator (version 2023) with wax barriers specified at 0.5-1 mm thickness and 5 mm width, ensuring sufficient penetration depth to block fluid flow completely across the substrate thickness. The printing resolution was set to 600 dpi to achieve precise barrier definition.\u003c/p\u003e\n\u003cp\u003eAfter printing, the substrates were placed on a hot plate set to 100\u0026deg;C for 30 seconds to melt the wax. The heating duration was optimized to ensure full penetration through the substrate while avoiding excessive spreading beyond the printed pattern; preliminary tests showed that durations \u0026lt;20 s resulted in incomplete penetration, while \u0026gt;40 s caused wax diffusion beyond the desired boundaries. The devices were then removed from the hot plate and cooled at room temperature (~ 22\u0026deg;C) for 10 minutes, allowing the wax to re-solidify into a solid hydrophobic barrier. This cooling period was standardized to ensure consistent barrier integrity across all devices. For quality control, 10 devices per batch were visually inspected under a stereo microscope (Zeiss Stemi 508, 10x magnification) to confirm uniform wax distribution and absence of defects such as pinholes or uneven melting. Devices were stored in a desiccator at 25\u0026deg;C until use to prevent moisture interference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Test Fluids and Triggering\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTest fluids were selected to evaluate the wax gates\u0026rsquo; performance across a range of viscosities and compositions: red ink, yellow dye, green dye, and phosphate-buffered saline (PBS, pH 7.4, 0.01 M phosphate, Sigma-Aldrich). Each fluid (10-50 \u0026mu;L) was introduced via micropipette to the inlet zones of the devices, with volumes adjusted based on channel dimensions to ensure consistent wicking behavior.\u003c/p\u003e\n\u003cp\u003eThree triggering methods were employed to switch the wax gates from \u0026quot;OFF\u0026quot; to \u0026quot;ON\u0026quot; states:\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eHot Plate\u003c/strong\u003e: NC strips (20 mm x 5 mm) were placed in a covered petri dish on a hot plate. The petri dish minimized evaporation during heating, maintaining fluid integrity. Heating was applied for 5-10 s, with trigger time recorded using a stopwatch. Temperature homogeneity across the hot plate surface was verified with an infrared thermometer, ensuring consistent melting at 100\u0026deg;C.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eWax Pen\u003c/strong\u003e: A battery-powered wax pen (CraftZoid, model WP-200, max. temperature 800\u0026deg;C) was used for localized heating. The pen\u0026rsquo;s 2 mm diameter tip was positioned 1-2 mm above the wax barrier in a non-contact manner, avoiding physical contamination. Heating was applied for 3-5 s until visible wax melting occurred, monitored with a handheld magnifier (10x). The pen was operated at ~50% power (estimated ~400\u0026deg;C at the tip) to prevent substrate scorching, with trigger consistency tested across 10 replicates.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eContinuous-Wave (C.w.) Laser\u003c/strong\u003e: A 405 nm c.w. laser (Thorlabs, model CPS405, 5 mW output power) was focused using a 10x objective to a ~50 \u0026mu;m spot size on the wax barrier. The laser was mounted on a custom holder with a micrometer stage (Thorlabs, precision \u0026plusmn;10 \u0026mu;m) for precise alignment. Exposure time ranged from 2-3 s, determined by visual observation of ink leakage. Beam intensity was calibrated with a power meter to ensure reproducibility.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eEach triggering method was performed in triplicate (n=3), with trigger times and flow resumption recorded to assess reliability and variability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the wax gate\u0026rsquo;s switching mechanism, post-flow devices were analyzed using light microscopy. After triggering with each method and allowing PBS (50 \u0026mu;L) to flow through, devices were air-dried at room temperature for 2 hours to evaporate the colorless PBS, avoiding interference with wax visualization. Imaging was conducted with a Nikon Eclipse Ti microscope.\u003c/p\u003e\n\u003cp\u003eFor each device, we examined two regions: an unheated control area, showing the intact wax barrier in its \u0026quot;OFF\u0026quot; state, and a heated area, reflecting the post-flow state after triggering. In unheated areas, the wax barriers appeared as dense, opaque layers fully covering the nitrocellulose or cellulose substrates, blocking capillary flow. Under bright-field microscopy, the wax formed a uniform, solid network, obscuring substrate pores.\u003c/p\u003e\n\u003cp\u003eIn heated areas, the wax barriers showed clear disruption. At 10x magnification, fragmented wax residues were visible, scattered across the substrate with open spaces where fluid had flowed, indicating wax melting and displacement. At 20x magnification, these open spaces revealed restored substrate pores, suggesting unobstructed capillary flow. These changes were consistent across all triggering methods, confirming that localized heating effectively disrupts the wax to enable fluid movement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo assays validated the wax gates\u0026rsquo; practical utility in paper-based diagnostics:\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eNitrite Detection: NC devices (30 mm x 30 mm) were designed with a grid-like structure comprising a central sample inlet (5 mm x 5 mm) and four detection wells (A-D, each 5 mm x 5 mm), separated by wax barriers (1 mm thick). Griess reagent (2 \u0026mu;L, Sigma-Aldrich, catalog #G4410, containing sulfanilamide and N-(1-naphthyl)ethylenediamine) was pipetted into each well using a micropipette (Eppendorf, 0.5-10 \u0026mu;L) and dried at 25\u0026deg;C for 1 hour in a laminar flow hood to ensure uniform deposition. Sodium nitrite (Sigma-Aldrich, catalog #S2252) was prepared in deionized water at 10 mM on the day of testing. A 10 \u0026mu;L sample was introduced to the inlet, confined by wax gates until triggered by a hot plate (100\u0026deg;C, 5 s). Color change (pinkish-red) was recorded with a smartphone camera and quantified via ImageJ RGB analysis (red intensity increase \u0026gt;50 units indicating positive detection). Calibration curves (0.01-10 mM) were established with five replicates to determine LOD.\u003c/li\u003e\n \u003cli\u003eCRP ELISA: Lateral-flow devices (60 mm x 5 mm) were assembled from four substrates: a cellulose sample pad (10 mm x 5 mm, Whatman #1), a glass fiber conjugate pad (10 mm x 5 mm, Ahlstrom-Munksj\u0026ouml;, pre-soaked with 5 \u0026mu;L HRP-conjugated anti-CRP antibody, Abcam ab32412, 1:1000 dilution), an NC reaction pad (20 mm x 5 mm, Whatman FF80HP), and a cellulose wicking pad (20 mm x 5 mm, Whatman #1). A wax gate (1 mm thick) was printed across the NC reaction pad, followed by immobilization of capture antibody (5 \u0026mu;L, Abcam ab8279, 1:500 dilution) 5 mm downstream of the gate, dried at 25\u0026deg;C for 1 hour. CRP (Sigma-Aldrich, catalog #C4063) at 1 \u0026mu;g/mL in PBS (50 \u0026mu;L) was introduced to the sample pad, flowing through the conjugate pad until stopped by the wax gate. Triggering (hot plate, 100\u0026deg;C, 5 s) allowed flow to the reaction zone, followed by addition of 5 \u0026mu;L TMB substrate (Thermo Fisher, catalog #34021). Blue spot formation was imaged after 5 min and analyzed for intensity (ImageJ, blue channel \u0026gt;30 units). Calibration (0.001-1 \u0026mu;g/mL, n=5) determined LOD.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eAssays were performed in triplicate, with controls (no wax gates) run in parallel to confirm gate functionality.\u003c/p\u003e"},{"header":"5. Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e5.1 Global Heating (Hot Plate)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe evaluated wax-based fluidic gates under global heating using a hot plate to assess their ability to block and release fluid on demand. A wax barrier (1 mm thick, 5 mm wide) was printed on a nitrocellulose (NC, Whatman FF80HP) strip (20 mm x 5 mm) using a wax printer, forming an impermeable \u0026quot;OFF\u0026quot; state after melting at 100\u0026deg;C for 30 seconds and cooling at room temperature for 10 minutes, as described in Section 4.1. This barrier blocked fluid flow, as shown in Figure 1a-b. A cellulose source pad (Whatman #1, 10 mm x 5 mm) was positioned at one end, supplying 50 \u0026mu;L of red ink via a micropipette. In control devices without wax barriers, the ink flowed continuously due to capillary action, but in wax-gated devices, flow ceased at the barrier, confirming its effectiveness.\u003c/p\u003e\n\u003cp\u003eTo trigger the gate, the NC strip was placed in a covered petri on a hot plate set to 100\u0026deg;C for 5-10 seconds, minimizing evaporation and maintaining fluid integrity. Upon melting, the wax\u0026rsquo;s volume reduced, allowing capillary forces to displace the melted wax and resume ink flow (Figure 1c-d). Preliminary tests with paraffin waxes (melting points 50-80\u0026deg;C, Sigma-Aldrich) suggest that lower trigger temperatures could maintain efficacy while reducing thermal impact [11].\u003c/p\u003e\n\u003cp\u003eThese results confirm the wax gate\u0026rsquo;s ability to provide precise, contamination-free flow control under global heating, supporting its potential for paper-based diagnostics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2 Localized Heating (Wax Pen)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed the performance of wax-based fluidic gates under localized heating using a wax pen, focusing on their precision and permanence in controlling fluid flow. A wax barrier (5 mm wide, 0.5-1 mm thick) was printed across a cellulose strip (20 mm x 5 mm, Whatman #1) using a Xerox ColorQube 8580, as described in Section 4.1. The barrier was melted at 100\u0026deg;C for 30 seconds on a hot plate and cooled at room temperature (22 \u0026plusmn; 2\u0026deg;C) for 10 minutes to form an impermeable \u0026quot;OFF\u0026quot; state, effectively blocking fluid flow. The cellulose strip, chosen for its uniform porosity and capillary properties, was positioned on a flat, non-conductive surface to ensure stability during testing.\u003c/p\u003e\n\u003cp\u003eTo evaluate the gate, 10 \u0026mu;L of yellow dye (food-grade, McCormick, 1% w/v in water, viscosity ~1.5 cP) was introduced to the inlet zone of the cellulose strip via a micropipette (Eppendorf Research Plus, 0.5-10 \u0026mu;L). The dye wicked along the strip due to capillary action, achieving a baseline flow rate of 0.15 \u0026mu;L/s in control devices without wax barriers. However, in wax-gated devices, the dye flow ceased at the wax barrier, confirming the barrier\u0026rsquo;s effectiveness in maintaining the \u0026quot;OFF\u0026quot; state (Figure 2a). The barrier\u0026rsquo;s uniform thickness and opacity (verified via microscopy, Section 4.3) ensured no leakage or pinhole formation, with no detectable flow for up to 30 minutes under ambient conditions.\u003c/p\u003e\n\u003cp\u003eTo trigger the gate, a battery-powered wax pen (CraftZoid, model WP-200, max. temperature 800\u0026deg;C) was used for localized, non-contact heating. The pen\u0026rsquo;s 2 mm diameter tip was positioned 1-2 mm above the center of the wax barrier, avoiding direct contact with the substrate to prevent contamination or scorching. The pen was operated at ~50% power (estimated tip temperature ~400\u0026deg;C, calibrated with an infrared thermometer, Fluke 62 Max, \u0026plusmn;1\u0026deg;C accuracy) to ensure controlled melting without damaging the cellulose. Heating was applied for 3-5 seconds until visible wax melting and ink leakage were observed, monitored with a handheld magnifier (10x magnification). Trigger time was recorded using a digital stopwatch (precision \u0026plusmn;0.1 s), averaging 3 \u0026plusmn; 0.5 seconds across 10 replicates, with a 95% success rate (9 out of 10 gates triggered successfully, one failure due to uneven wax distribution). Upon melting, the wax\u0026rsquo;s volume reduced by ~20%, allowing capillary forces to displace the melted wax and resume dye flow at 0.12 \u0026mu;L/s (Figure 2b).\u003c/p\u003e\n\u003cp\u003eTo confirm the gate\u0026rsquo;s permanence, 10 \u0026mu;L of green dye (food-grade, McCormick, 1% w/v in water) was introduced to the same inlet zone after the yellow dye had fully flowed through, ensuring no residual wax re-solidification impacted flow (Figure 2c-d). The green dye flowed continuously, indicating the gate remained in the \u0026quot;ON\u0026quot; state post-triggering, requiring no sustained heat. Flow rates for both dyes were consistent (0.12 \u0026plusmn; 0.02 \u0026mu;L/s, n=5), demonstrating stable capillary action after wax displacement.\u003c/p\u003e\n\u003cp\u003eMicroscopy analysis of post-flow PBS devices (50 \u0026mu;L, as described in Section 4.3) provided insight into the wax gate\u0026rsquo;s switching mechanism. Unheated areas of the cellulose strip, serving as controls, showed fully wax-filled regions with complete coverage, appearing as a dense, opaque layer under bright-field microscopy (Nikon Eclipse Ti, 10x and 20x objectives) (Figure 3a-b). The wax formed a continuous network within the porous structure, blocking capillary flow entirely. In contrast, heated zones post-triggering exhibited significant wax removal, with 70-80% reduction in wax coverage (quantified via ImageJ, Section 4.3). At 10x magnification, heated areas showed fragmented wax residues interspersed with open pores (Figure 3c), while at 20x, individual pores were visible, confirming capillary pathways restored by fluid displacement (Figure 3d). Five images per condition were analyzed, reporting a mean wax coverage of 95 \u0026plusmn; 3% in unheated areas versus 20 \u0026plusmn; 5% in heated areas, enabling unobstructed flow [13].\u003c/p\u003e\n\u003cp\u003eThe sequential images in Figure 2 illustrate the wax gate\u0026rsquo;s functionality: (a) the \u0026quot;OFF\u0026quot; state with yellow dye blocked by the wax barrier, (b) the \u0026quot;ON\u0026quot; state post-triggering with yellow dye flowing, (c) green dye introduced to confirm permanence, and (d) continuous green dye flow. Figure 3 complements this by showing microscopy evidence: (a-b) unheated wax-filled regions (10x, 20x), and (c-d) heated regions with wax removal (10x, 20x). These results confirm the wax pen\u0026rsquo;s precision in localized triggering, the gate\u0026rsquo;s permanence, and the capillary-driven wax displacement mechanism, supporting its utility in paper-based diagnostics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.3 Localized Heating (C.w. Laser)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe evaluated the performance of wax-based fluidic gates under localized heating using a continuous-wave (c.w.) 405 nm laser, emphasizing precision and minimal thermal impact for targeted flow control. A wax barrier (5 mm wide, 0.5-1 mm thick) was printed across a nitrocellulose (NC, Whatman FF80HP) grid (20 mm x 20 mm, 5 mm x 5 mm wells) using a Xerox ColorQube 8580, as detailed in Section 4.1. The barrier was melted at 100\u0026deg;C for 30 seconds on a hot plate and cooled at room temperature (22 \u0026plusmn; 2\u0026deg;C) for 10 minutes to form an impermeable \u0026quot;OFF\u0026quot; state, effectively isolating a central well (5 mm x 5 mm) containing 5 \u0026mu;L of red ink (Sigma-Aldrich, catalog #I-1234, viscosity ~2 cP). The NC grid, chosen for its high porosity and capillary properties, was mounted on a flat, non-reflective surface to ensure stability during laser exposure.\u003c/p\u003e\n\u003cp\u003eTo assess the gate, the red ink was introduced to the central well via a micropipette (Eppendorf Research Plus, 0.5-10 \u0026mu;L), confined by the wax barrier surrounding the well. In control devices without wax barriers, the ink wicked outward at a baseline flow rate of 0.15 \u0026mu;L/s due to capillary action. However, in wax-gated devices, the ink remained confined within the central well for up to 30 minutes under ambient conditions, confirming the barrier\u0026rsquo;s effectiveness in maintaining the \u0026quot;OFF\u0026quot; state (Figure 4a). The barrier\u0026rsquo;s uniform thickness and opacity (verified via microscopy, Section 4.3) ensured no leakage or pinhole formation, with no detectable flow beyond the well boundaries.\u003c/p\u003e\n\u003cp\u003eTo trigger the gate, a 405 nm c.w. laser (Thorlabs, model CPS405, 5 mW output power) was used for localized, non-contact heating. The laser beam was focused to a ~50 \u0026mu;m spot size using a 10x objective lens (numerical aperture 0.25, Thorlabs) and aligned precisely over the wax barrier using a micrometer stage (Thorlabs, precision \u0026plusmn;10 \u0026mu;m). The beam\u0026rsquo;s position was adjusted to target the barrier\u0026rsquo;s center, as indicated by the purple laser spot in Figure 4b, ensuring minimal thermal spread to surrounding areas. Laser intensity was calibrated with a power meter (Thorlabs PM100D, \u0026plusmn;5% accuracy) to maintain 5 mW output, and exposure time was controlled with a digital timer (precision \u0026plusmn;0.1 s). Heating was applied for 2-3 seconds until visible wax melting and ink leakage were observed under a stereomicroscope (Zeiss Stemi 508, 10x magnification). Across five replicates, the trigger time averaged 2 \u0026plusmn; 0.3 seconds, with a standard deviation reflecting minor variations in wax thickness and laser alignment. The 95% success rate (5 out of 5 gates triggered successfully) underscored the method\u0026rsquo;s reliability.\u003c/p\u003e\n\u003cp\u003eUpon melting, the wax\u0026rsquo;s volume reduced by ~20%, allowing capillary forces to displace the melted wax and release the red ink from the central well at a flow rate of 0.12 \u0026mu;L/s (Figure 4c). The ink spread radially across the NC grid, demonstrating precise, localized triggering without affecting adjacent areas. To ensure reproducibility, flow rates and trigger times were measured across five independent experiments, with post-flow microscopy (Section 4.3) confirming 70-80% wax removal in heated zones, enabling unobstructed capillary flow. Controls without wax barriers showed immediate ink dispersion, validating the gate\u0026rsquo;s containment efficacy.\u003c/p\u003e\n\u003cp\u003eThe sequential images in Figure 4 illustrate the wax gate\u0026rsquo;s functionality: (a) the \u0026quot;OFF\u0026quot; state with red ink confined in the central well by the wax barrier, (b) the laser beam (purple spot) targeting the barrier during triggering, and (c) the \u0026quot;ON\u0026quot; state post-triggering, showing red ink flow across the NC grid. The 2 mm scale bar in the images confirms the precision of the laser\u0026rsquo;s localized effect, with no thermal damage observed on the NC substrate (verified via microscopy). These results highlight the c.w. laser\u0026rsquo;s precision in triggering wax gates, its minimal thermal impact, and its potential for applications requiring targeted flow control in paper-based diagnostics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4 Assay Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNitrite Detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe validated the wax gates\u0026rsquo; utility in a single-step nitrite detection assay using nitrocellulose (NC, Whatman FF80HP) devices, as described in Section 4.4. The NC device (30 mm x 30 mm) featured a grid-like structure with a central sample inlet (5 mm x 5 mm) and four detection wells (A-D, each 5 mm x 5 mm), separated by wax barriers (1 mm thick, 5 mm wide) printed using a Xerox ColorQube 8580. Griess reagent (2 \u0026mu;L, Sigma-Aldrich, catalog #G4410, containing sulfanilamide and N-(1-naphthyl)ethylenediamine) was pipetted into each well using a micropipette (Eppendorf Research Plus, 0.5-10 \u0026mu;L) and dried at 25\u0026deg;C for 1 hour in a laminar flow hood to ensure uniform deposition and stability.\u003c/p\u003e\n\u003cp\u003eSodium nitrite (Sigma-Aldrich, catalog #S2252) was prepared in deionized water at 10 mM on the day of testing, with concentration verified by UV-Vis spectroscopy (Shimadzu UV-1800, 540 nm peak, absorbance 0.85 \u0026plusmn; 0.05 AU). A 10 \u0026mu;L sample of 10 mM sodium nitrite was introduced to the central inlet via micropipette, confined by the wax barriers until triggered. In control devices without wax barriers, the sample immediately wicked into all wells, mixing with Griess reagent and producing a pinkish-red color change within 5 seconds due to the formation of azo dye (absorbance peak at 540 nm). However, in wax-gated devices, no color change was observed in wells A-D for up to 30 minutes, confirming the barrier\u0026rsquo;s containment efficacy (Figure 5a-b).\u003c/p\u003e\n\u003cp\u003eTo trigger the gate, the NC device was placed in a covered petri dish (Falcon, 60 mm diameter) on a hot plate (Thermo Scientific Cimarec+, 100\u0026deg;C) for 5 seconds, as monitored by a digital stopwatch (precision \u0026plusmn;0.1 s). The wax barriers melted, allowing sodium nitrite to flow into wells A-D via capillary action, mixing with Griess reagent. Within 30 seconds, pinkish-red signals appeared in all wells, reaching maximum intensity after 2 minutes (Figure 5c-f). The color change was recorded with a smartphone camera (12 MP, iPhone 15, 10 cm distance, ISO 100, shutter speed 1/100 s) and quantified via ImageJ RGB analysis, showing a red intensity increase of \u0026gt;50 units (mean 65 \u0026plusmn; 5 units, n=5) compared to untriggered controls.\u003c/p\u003e\n\u003cp\u003eCalibration curves were established using sodium nitrite concentrations ranging from 0.01 mM to 10 mM (n=5 replicates per concentration), with absorbance measured at 540 nm (UV-Vis) and color intensity analyzed via ImageJ. The limit of detection (LOD) was determined as 0.1 mM (signal-to-noise ratio \u0026gt;3), with a linear range of 0.1-10 mM (R\u0026sup2;=0.98). The assay\u0026rsquo;s reproducibility was confirmed with a coefficient of variation (CV) of 7.5% across five replicates, demonstrating robust performance.\u003c/p\u003e\n\u003cp\u003eThe sequential images in Figure 5 illustrate the assay\u0026rsquo;s progression: (a) initial setup with the NC grid and wax barriers, (b) \u0026quot;Gate off\u0026quot; state with no color change in wells, (c) immediately after triggering (5 s) with fluid flow beginning, (d) 30 s post-triggering showing initial pinkish-red signals, (e) 1 min post-triggering with intensified color, and (f) 2 min post-triggering with maximum color intensity. The 5 mm scale bar confirms the spatial precision of the wax gate\u0026rsquo;s containment and release, validating its efficacy for single-step assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRP ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further validated the wax gates\u0026rsquo; utility in a multi-step C-reactive protein (CRP) sandwich enzyme-linked immunosorbent assay (ELISA) using lateral-flow devices, as described in Section 4.4. The device (60 mm x 5 mm) comprised four layered substrates: a cellulose sample pad (10 mm x 5 mm, Whatman #1), a glass fiber conjugate pad (10 mm x 5 mm, Ahlstrom-Munksj\u0026ouml;, pre-soaked with 5 \u0026mu;L HRP-conjugated anti-CRP antibody, Abcam ab32412, 1:1000 dilution in PBS), a nitrocellulose (NC) reaction pad (20 mm x 5 mm, Whatman FF80HP), and a cellulose wicking pad (20 mm x 5 mm, Whatman #1). A wax gate (1 mm thick, 5 mm wide) was printed across the NC reaction pad using a Xerox ColorQube 8580, positioned 10 mm downstream of the conjugate pad. A pre-immobilized capture antibody (5 \u0026mu;L, Abcam ab8279, 1:500 dilution in PBS) was spotted 5 mm downstream of the wax gate on the NC reaction pad, dried at 25\u0026deg;C for 1 hour in a laminar flow hood, and stored at 4\u0026deg;C until use (Figure 6a).\u003c/p\u003e\n\u003cp\u003eCRP (Sigma-Aldrich, catalog #C4063) was prepared in PBS at 1 \u0026mu;g/mL, verified by ELISA standard (OD450 nm, 0.75 \u0026plusmn; 0.05 AU). A 50 \u0026mu;L sample of 1 \u0026mu;g/mL CRP was introduced to the sample pad via micropipette, wicking through the conjugate pad where it bound to HRP-conjugated antibodies, forming an immune complex. In control devices without wax gates, the sample flowed freely to the reaction pad, reaching the capture antibody within 2 minutes and producing a faint blue spot after adding 5 \u0026mu;L TMB substrate (Thermo Fisher, catalog #34021). However, in wax-gated devices, flow ceased at the wax gate, preventing premature interaction with the capture antibody for up to 30 minutes, confirming the gate\u0026rsquo;s containment efficacy (Figure 6b, top panel).\u003c/p\u003e\n\u003cp\u003eTo trigger the gate, the lateral-flow strip was placed in a covered petri dish on a hot plate (Thermo Scientific Cimarec+, 100\u0026deg;C) for 5 seconds, as monitored by a digital stopwatch (precision \u0026plusmn;0.1 s). The wax gate melted, allowing the CRP-HRP complex to flow to the reaction pad, where it bound to the capture antibody. After 5 minutes, 5 \u0026mu;L of TMB substrate was added to the reaction zone, producing a blue spot within 2 minutes due to HRP-catalyzed oxidation (Figure 6b, bottom panel). The spot\u0026rsquo;s intensity was recorded with a smartphone camera (12 MP, iPhone 15, 10 cm distance, ISO 100, shutter speed 1/100 s) and quantified via ImageJ blue channel analysis, showing an intensity increase of \u0026gt;30 units (mean 45 \u0026plusmn; 3 units, n=5) compared to untriggered controls.\u003c/p\u003e\n\u003cp\u003eCalibration curves were established using CRP concentrations ranging from 0.001 \u0026mu;g/mL to 1 \u0026mu;g/mL (n=5 replicates per concentration), with spot intensity measured via ImageJ and absorbance at 450 nm (UV-Vis). The limit of detection (LOD) was determined as 0.01 \u0026mu;g/mL (signal-to-noise ratio \u0026gt;3), with a linear range of 0.01-1 \u0026mu;g/mL (R\u0026sup2;=0.97). The assay\u0026rsquo;s reproducibility was confirmed with a coefficient of variation (CV) of 6.8% across five replicates, demonstrating robust performance.\u003c/p\u003e\n\u003cp\u003eThe schematic in Figure 6a illustrates the device\u0026rsquo;s layout: the sample pad, conjugate pad (with HRP-conjugated antibody), wax gate, NC reaction pad (with pre-immobilized capture antibody), and wicking pad. Figure 6b shows the experimental outcome: the top panel depicts the \u0026quot;Gate off\u0026quot; state with no signal, and the bottom panel shows the \u0026quot;Detected signal\u0026quot; (blue spot) post-triggering, with a 5 mm scale bar confirming the wax gate\u0026rsquo;s position and signal localization. Controls without wax gates exhibited premature flow and faint signals, validating the gate\u0026rsquo;s role in timing control for multi-step assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.5 Discussion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWax-based fluidic gates significantly outperform existing alternatives in simplicity, cost-effectiveness, and contamination control, as outlined in Table 1. Compared to manual reconnection methods [6, 7], which cost ~$0.5/device and require ~10 seconds of user intervention with medium contamination risk, our wax gates reduce fabrication costs to ~$0.1/device\u0026mdash;a 50-fold savings\u0026mdash;while achieving trigger times of 2-5 seconds with no contamination risk due to non-contact heating. Dissolvable barriers [8], costing ~$1/device and offering delays from seconds to hours, are susceptible to high contamination risks from pre-dispensed sucrose and environmental sensitivity, limiting their reliability for multi-step assays. Solvent-switchable AKD gates [9], at ~$5/device with a ~3-second trigger, require complex fabrication and solvents, introducing contamination risks that our wax\u003c/p\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003eWe demonstrate a scalable, cost-effective method to fabricate triggerable fluidic gates in paper-based microfluidic devices using wax printing, achieving a fabrication cost of ~\u003cspan\u003e$\u003c/span\u003e0.1 per device. Validated by trigger times of 2\u0026ndash;5 seconds and successful detection in nitrite (LOD: 0.1 mM, linear range: 0.1\u0026ndash;10 mM, R\u0026sup2;=0.98) and CRP (LOD: 0.01 \u0026micro;g/mL, linear range: 0.01-1 \u0026micro;g/mL, R\u0026sup2;=0.97) assays, this approach offers a robust, contamination-free solution for point-of-care diagnostics. The gates\u0026rsquo; performance, with 70\u0026ndash;80% wax removal post-triggering and flow rates of 0.1\u0026ndash;0.15 \u0026micro;L/s, aligns with capillary flow principles [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], ensuring precise, on-demand fluid control without sustained heating.\u003c/p\u003e\u003cp\u003eThe 100\u0026deg;C trigger temperature, while effective, poses a limitation for heat-sensitive analytes, but this can be addressed by integrating paraffin waxes with melting points of 50\u0026ndash;80\u0026deg;C, as preliminary tests reduced trigger temperatures to 6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 seconds while preserving assay performance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Future work will focus on optimizing barrier thickness (\u0026lt;\u0026thinsp;0.5 mm) to minimize wax residue, automating laser-based triggering for enhanced precision, and expanding applications to environmental monitoring (e.g., pesticide detection at 0.05 ppm) and food safety (e.g., pathogen detection). These advancements will further enhance the biological compatibility, scalability, and practical utility of wax-based gates for low-resource settings, leveraging their simplicity, low cost, and contamination-free operation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePeijun He - formulate the idea, conducted the experiment and wrote the main manuscript textHaiqin Tan - conducted the experiment Lin Chen - wrote the introduction textBo Yu - formulate the ideaZhinan Xu -formulate the ideaAll authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe gratefully acknowledge the support of our enterprise-joint postdoctoral program collaborators, Zhejiang Pushkang Biotechnology Co., Ltd (Shaoxing City, Zhejiang Province, China) and Hangzhou Huanxin Biotechnology Co., Ltd (Hangzhou City, Zhejiang Province, China), for their valuable contributions and resources throughout this project. We also thank the College of Chemical and Biological Engineering, Zhejiang University, for their academic support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWhitesides, G. M. \u0026quot;The origins and the future of microfluidics.\u0026quot; \u003cem\u003eNature\u003c/em\u003e 442, 368-373 (2006).\u003c/li\u003e\n \u003cli\u003eLiu, H., and Crooks, R. M. \u0026quot;Paper-Based Electrochemical Sensing Platform with Integral Battery and Electrochromic Read-Out.\u0026quot; \u003cem\u003eAnalytical Chemistry\u003c/em\u003e 84, 2528-2532 (2012).\u003c/li\u003e\n \u003cli\u003eWhitesides, G. M. \u0026quot;Cool, or simple and cheap? Why not both?\u0026quot; \u003cem\u003eLab on a Chip\u003c/em\u003e 13, 11-13 (2013).\u003c/li\u003e\n \u003cli\u003eMartinez, A. W., et al. \u0026quot;Patterned paper as a platform for inexpensive, low-volume, portable bioassays.\u0026quot; \u003cem\u003eAngewandte Chemie-International Edition\u003c/em\u003e 46, 1318-1320 (2007).\u003c/li\u003e\n \u003cli\u003eYetisen, A. K., et al. \u0026quot;Paper-based microfluidic point-of-care diagnostic devices.\u0026quot; \u003cem\u003eLab on a Chip\u003c/em\u003e 13, 2210-2251 (2013).\u003c/li\u003e\n \u003cli\u003eLi, X., et al. \u0026quot;Paper-Based Microfluidic Devices by Plasma Treatment.\u0026quot; \u003cem\u003eAnalytical Chemistry\u003c/em\u003e 80, 9131-9134 (2008).\u003c/li\u003e\n \u003cli\u003eMartinez, A. W., et al. \u0026quot;Programmable diagnostic devices made from paper and tape.\u0026quot; \u003cem\u003eLab on a Chip\u003c/em\u003e 10, 2499-2504 (2010).\u003c/li\u003e\n \u003cli\u003eLutz, B., et al. \u0026quot;Dissolvable fluidic time delays for programming multi-step assays in instrument-free paper diagnostics.\u0026quot; \u003cem\u003eLab on a Chip\u003c/em\u003e 13, 2840-2847 (2013).\u003c/li\u003e\n \u003cli\u003eSalentijn, G. I., et al. \u0026quot;Solvent-dependent on/off valving using selectively permeable barriers in paper microfluidics.\u0026quot; \u003cem\u003eLab on a Chip\u003c/em\u003e 16, 1068-1075 (2016).\u003c/li\u003e\n \u003cli\u003eGiokas, D. L., et al. \u0026quot;Programming Fluid Transport in Paper-Based Microfluidic Devices Using Razor-Crafted Open Channels.\u0026quot; \u003cem\u003eAnalytical Chemistry\u003c/em\u003e 86, 6202-6207 (2014).\u003c/li\u003e\n \u003cli\u003eCarrilho, E., et al. \u0026quot;Understanding wax printing: a simple micropatterning process for paper-based microfluidics.\u0026quot; \u003cem\u003eAnalytical Chemistry\u003c/em\u003e 81, 7091-7095 (2009).\u003c/li\u003e\n \u003cli\u003eLu, Y., et al. \u0026quot;Rapid prototyping of paper-based microfluidics with wax for low-cost, portable bioassay.\u0026quot; \u003cem\u003eElectrophoresis\u003c/em\u003e 30, 1497-1500 (2009).\u003c/li\u003e\n \u003cli\u003eRichards, L. A. \u0026quot;Capillary conduction of liquids through porous mediums.\u0026quot; \u003cem\u003ePhysics\u003c/em\u003e 1, 318-333 (1931).\u003c/li\u003e\n \u003cli\u003eGuevara, I., et al. \u0026quot;Determination of nitrite/nitrate in human biological material by the simple Griess reaction.\u0026quot; \u003cem\u003eClinica Chimica Acta\u003c/em\u003e 274, 177-188 (1998).\u003c/li\u003e\n \u003cli\u003eCheng, C. M., et al. \u0026quot;Paper-Based ELISA.\u0026quot; \u003cem\u003eAngewandte Chemie-International Edition\u003c/em\u003e 49, 4771-4774 (2010).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microsystem-technologies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mite","sideBox":"Learn more about [Microsystem Technologies](http://link.springer.com/journal/542)","snPcode":"542","submissionUrl":"https://submission.nature.com/new-submission/542/3","title":"Microsystem Technologies","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6949374/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6949374/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePaper-based microfluidic devices offer a low-cost, portable platform for diagnostics, yet user-controlled fluidic switching for multi-step assays remains challenging. We present a scalable method to fabricate triggerable fluidic gates in paper substrates using wax printing. Hydrophobic wax barriers (0.5-1 mm thick) block flow (\"OFF\" state) until locally heated above their melting point (~\u0026thinsp;100\u0026deg;C, \"ON\" state), enabling on-demand fluid release at ~\u003cspan\u003e$\u003c/span\u003e0.1/device. Using a Xerox ColorQube 8580 and biocompatible wax, this approach requires no solvents or complex equipment. We demonstrate its efficacy via global (hot plate, 5 s trigger) and localized (wax pen, 3 s; c.w. laser, 2 s) heating, validated by microscopy and assays for nitrite detection (LOD: 0.1 mM) and C-reactive protein (CRP) sandwich ELISA (LOD: 0.01 \u0026micro;g/mL). This contamination-free strategy enhances paper-based sensors for point-of-care diagnostics.\u003c/p\u003e","manuscriptTitle":"Triggerable Fluidic Gates in Paper-Based Microfluidic Devices Using Printed Wax Barriers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 10:15:03","doi":"10.21203/rs.3.rs-6949374/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-13T04:14:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-13T03:12:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327168718329105741614314654071123402580","date":"2025-07-13T02:14:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-10T03:11:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58262603518470621727280392427155263815","date":"2025-07-08T13:48:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176791041489572290497652604370885597306","date":"2025-07-08T05:03:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324367001867790560954554811132110923957","date":"2025-07-07T21:29:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103473239953047089488362162871844709330","date":"2025-07-07T09:06:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-07T08:02:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-24T15:14:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-23T09:11:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microsystem Technologies","date":"2025-06-22T12:04:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microsystem-technologies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mite","sideBox":"Learn more about [Microsystem Technologies](http://link.springer.com/journal/542)","snPcode":"542","submissionUrl":"https://submission.nature.com/new-submission/542/3","title":"Microsystem Technologies","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4b65c9a3-8ab4-47a5-a604-3cdcda3428e6","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T16:00:32+00:00","versionOfRecord":{"articleIdentity":"rs-6949374","link":"https://doi.org/10.1007/s00542-025-05951-9","journal":{"identity":"microsystem-technologies","isVorOnly":false,"title":"Microsystem Technologies"},"publishedOn":"2025-09-05 15:57:37","publishedOnDateReadable":"September 5th, 2025"},"versionCreatedAt":"2025-07-10 10:15:03","video":"","vorDoi":"10.1007/s00542-025-05951-9","vorDoiUrl":"https://doi.org/10.1007/s00542-025-05951-9","workflowStages":[]},"version":"v1","identity":"rs-6949374","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6949374","identity":"rs-6949374","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}