Microfluidic paper-based analytical devices (µPADs) are revolutionizing point-of-care (POC) diagnostics, offering low-cost, rapid, and user-friendly platforms for various bioassays. Among the innovative fabrication techniques for µPADs, the C-µPAD method stands out for its simplicity and efficiency in creating hydrophobic barriers on chromatography paper. This article delves into the characterization of C-µPAD, highlighting its fabrication process, performance, and applications in POC diagnostics, particularly for glucose monitoring, immunoassays, and heavy metal detection.
The C-µPAD fabrication process leverages the chemical vapor deposition (CVD) of trichlorosilane (TCS) to selectively modify chromatography paper, creating well-defined hydrophobic barriers. As illustrated in Figure 1(a), this method involves applying a vinyl mask onto the paper, followed by exposure to TCS vapor in a low-pressure chamber. The TCS molecules react with the hydroxyl groups on the cellulose fibers of the paper, forming stable covalent bonds and rendering the exposed areas hydrophobic. This process is highly reproducible and allows for the creation of intricate microfluidic patterns.
Figure 1: Development of paper-based microfluidic platform using C-µPAD technique. (a) Schematic illustration of fabrication process using CVD and TCS. (b) and (c) Examples of 2D channel patterns on C-µPAD showcasing positive and negative features. (d) Demonstration of a multi-layered C-µPAD with top and bottom layer channels.
Hydrophobic Barrier Characterization and Optimization
The effectiveness of the hydrophobic barriers is crucial for the performance of C-µPADs. Contact angle measurements are employed to assess the hydrophobicity achieved through the CVD process. The study revealed a direct relationship between CVD duration and contact angle, reaching a saturation point at approximately 125° after 10 minutes of CVD. A contact angle of 115° was found to be sufficient for robust bioassays. The silanized hydrophobic patterns are invisible and maintain the paper’s flexibility, making C-µPADs versatile for various applications. Figure 1(b–d) showcases diverse C-µPAD patterns with food dyes, demonstrating the sharp hydrophobic borders and uniform wicking within the hydrophilic channels. This CVD method is adaptable to different paper types, broadening its applicability.
Resolution and Channel Dimension Control
To optimize the C-µPAD fabrication technique, the resolution was characterized by varying CVD duration and channel dimensions. Figure 2 illustrates the impact of CVD duration on channel area. A 30-second CVD duration at 53 °C was identified as optimal, providing the best balance between hydrophilic and hydrophobic areas on both sides of the paper (Figure 2(a, b)). Longer durations led to a decrease in hydrophilic area due to over-penetration of the hydrophobic reagent, while shorter durations resulted in incomplete hydrophobic barrier formation, causing spreading. With the optimized 30-second CVD duration, C-µPAD fabrication achieved a channel width resolution down to 500 μm. This limit is attributed to the aspect ratio of channel width and paper thickness relative to the TCS vapor penetration rate.
Figure 2: Characterization of C-µPAD technique by controlling CVD duration and channel area. (a) Front side and (b) Back side of patterned chromatography paper after 30 seconds CVD. (c) Relationship between channel area and CVD duration for different channel sizes, indicating optimal duration at 30 seconds for balanced hydrophobicity.
Fluid Flow Velocity in C-µPADs
Fluid transport properties are crucial for microfluidic devices. The flow velocity in C-µPADs was compared to that of normal chromatography paper. Figure 3(a–d) visually contrasts the fluid velocities, while Figure 3(e) quantifies a minimal ~5% variation in overall velocities. This slight variation is considered negligible and likely arises from the inherent heterogeneity of chromatography paper structure, including fiber direction, pore size, and distribution. This demonstrates that the C-µPAD fabrication process does not significantly alter the intrinsic wicking properties of the paper, ensuring reliable fluid transport for assays.
Figure 3: Distance vs. time analysis on normal chromatography paper and C-µPAD for fluid flow rate comparison. (a–f) Visual time-lapse comparison of red dye solution flow in untreated and treated paper. (g) Quantitative comparison of distance traveled by red dye in C-µPAD channels versus normal chromatography paper over time, showing minimal velocity variation.
Demonstrating Bioassay Capabilities for Point-of-Care Diagnostics
Glucose Assay for Diabetes Monitoring
The C-µPAD platform’s potential for POC diagnostics was validated through glucose assays, crucial for diabetes management. Both well-spot and lateral flow C-µPAD designs were tested using standard glucose samples. In well-spot C-µPADs (Figure 4(a)), color intensity increased proportionally with glucose concentration (0 to 160 mg/dL), achieving a limit of detection (LOD) of 13 mg/dL, comparable to commercial glucose meters. Lateral flow glucose assays (Figure 4(b, c)) also demonstrated a clear color gradient with varying glucose concentrations, achieving a LOD of 23 mg/dL. The results from lateral flow C-µPADs showed good correlation with standard 96-well plate assays, confirming their reliability (Supplementary Figure S2). Furthermore, human blood glucose assays were successfully performed on well-spot C-µPADs with a plasma separation membrane (Figure 4(d–f)), demonstrating the platform’s applicability in complex biological samples for POC glucose monitoring.
Figure 4: Demonstration of glucose assay on well-spot and lateral flow C-µPAD. (a) Color intensity variation with glucose concentration in well-spot C-µPAD. (b) Lateral flow C-µPAD glucose assays showing color gradient with varying concentrations. (c) Linear relationship between glucose concentration and color intensity in lateral flow C-µPAD. (d) Plasma separation from human blood on C-µPAD. (e) Well-spot glucose assays on C-µPAD using glucose-spiked blood samples. (f) Comparison of glucose assay results using standard glucose and glucose-spiked whole blood samples on well-spot C-µPAD, showing strong linear relationship between glucose concentration and gray intensity.
Immunoassay for TNFα Quantification
Beyond glucose, C-µPADs were also applied to sandwich immunoassays for quantifying TNFα, a key inflammatory biomarker. Well-spot C-µPADs were used to perform immunoassays following the procedure in Figure 5(a). Magnetic particles functionalized with anti-TNFα capture antibodies were employed to capture TNFα. The formation of the immune complex was indicated by blue color development, which turned yellow after adding a stop solution (Figure 5(b)). The yellow color intensity correlated with TNFα concentration (1~1000 ng/mL), exhibiting a log-linear relationship (Figure 5(c)) and achieving a LOD of 3 ng/mL. While less sensitive than conventional ELISA, the C-µPAD immunoassay significantly reduced reaction time and reagent volume, highlighting its advantages for rapid POC diagnostics.
Figure 5: Immunoassay for TNFα quantification on C-µPAD. (a) Immunoassay procedure on C-µPAD. (b) Color gradient after TNFα assay with varying concentrations. (c) Log-linear relationship between TNFα concentrations and color intensity, demonstrating quantitative immunoassay capability.
Heavy Metal Detection for Environmental Monitoring
The versatility of C-µPADs extends to environmental monitoring, as demonstrated by heavy metal detection assays. Nickel (Ni) detection was performed using both well-spot and patterned C-µPADs. Amine functionalization of C-µPADs using APTES enabled selective immobilization of dimethylglyoxime (DMG), a colorimetric reagent for Ni detection (Figure 6(a, b)). The C-µPAD maintained its hydrophobic barriers even at elevated temperatures, unlike wax-printed µPADs. The intensity of the DMG-Ni complex color was proportional to Ni concentration (Figure 6(c)), with a detection limit of 150 μg/L (Figure 6(d)), showing improved sensitivity compared to previous reports. This highlights C-µPAD’s suitability for silane functionalization and robust performance in heavy metal detection.
Figure 6: Heavy metal detection on C-µPAD. (a) Amine functionalization on “Ni” symbol patterned C-µPAD. (b) Ni detection on “Ni” symbol patterned C-µPAD using DMG. (c) Color variation with different Ni concentrations on well-spot C-µPAD. (d) Standard curve for Ni detection, showing log-linear relationship between Ni concentration and color intensity.
Conclusion: C-µPAD for Advanced Point-of-Care Diagnostics
The C-µPAD fabrication technique offers a robust and versatile platform for developing Microfluidic Paper-based Analytical Devices For Point-of-care Diagnosis. Its simple CVD-based fabrication, excellent hydrophobic barrier properties, and compatibility with various bioassays, including glucose monitoring, immunoassays, and heavy metal detection, make it a promising tool for decentralized diagnostics. The demonstrated capabilities of C-µPADs align with the critical needs for POC diagnostics: low reagent volume, rapid analysis, portability, potential for multiplex detection, and low cost. The long shelf-life and stable performance further enhance its practical utility, positioning C-µPADs as a significant advancement in paper-based microfluidics for global healthcare applications.
