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Article

In Situ Fabrication of Controlled Porous Manifold Coupled with Non-Planar Microelectrodes for Microfluidic Biosensors

by
Najamuddin Naveed Khaja
1,
Sushma Yadav
1,
Niranjan Haridas Menon
1,
Sreerag Kaaliveetil
1,
Guangliang Liu
1,
Yu-Hsuan Cheng
1,*,
Kathleen McEnnis
1 and
Sagnik Basuray
1,2,*
1
Department of Chemical and Materials Engineering, New Jersey Institute of Technology, Newark, NJ 07102, USA
2
Department of Biomedical Engineering, New Jersey Institute of Technology, Newark, NJ 07102, USA
*
Authors to whom correspondence should be addressed.
Chemosensors 2026, 14(8), 171; https://doi.org/10.3390/chemosensors14080171
Submission received: 10 June 2026 / Revised: 21 July 2026 / Accepted: 22 July 2026 / Published: 25 July 2026
(This article belongs to the Section (Bio)chemical Sensing)
Browse Figures
Versions Notes

Abstract

The demand for a versatile and portable point-of-use (POU) sensor platform has surged due to the pandemic, especially in countries with limited medical laboratory facilities. We recently unveiled a portable, non-planar, interdigitated, flow-through, porous electrode platform that automatically measures electrochemical impedance spectroscopy (EIS) signals from various biomarkers. However, the packed powder exhibited a loss of performance over time due to displacement, leaching, and poor stability. Herein, we modified the packing strategy by synthesizing the sensing material within the channel, thereby improving adhesion, structural integrity, and stability. Leveraging the exceptional thermal stability, mechanical strength, and chemical resistance of polyimide (PI), we developed a novel fabrication approach that combines liquid-phase inversion and breath-figure techniques to create a porous PI manifold with single-walled carbon nanotubes (SWCNTs) under varying humidity conditions. Scanning electron microscope (SEM) analysis revealed that lower relative humidity (RH) conditions yield larger but less uniformly distributed pores, leading to increased channel pressure. The manifold demonstrated exceptional stability under rigorous flow conditions, withstanding a high flow rate of 30 µL/min while maintaining consistent pressure-EIS responses. The device produced a measurable proof-of-concept impedance response following exposure to a femtomolar concentration of complementary target ssDNA in 1× PBS within 15 min. A formal limit of detection was not determined in the present study. We developed a mechanically stable sensor design with improved durability under repeated flow conditions by systematically optimizing synthesis conditions and manifold configuration. This innovative fabrication strategy demonstrates the importance of packing methodology in sensor design and paves the way for robust, scalable, and efficient diagnostic solutions in resource-limited settings.

1. Introduction

Developing portable, multiple-purpose, point-of-use (POU) sensor platforms is essential, particularly for regions with limited access to advanced laboratory equipment. These sensors offer a rapid, economical, and reliable means of detecting contaminants, pathogens, or biomarkers [1,2,3]. By integrating innovative materials, miniaturized detection systems, and user-friendly interfaces, POU sensors provide real-time on-site analysis without requiring complex instrumentation and specialized personnel [4,5,6,7]. Recently, we introduced a novel modular, non-planar, interdigitated, flow-through, porous, electrode-based microfluidic electrochemical sensor for detecting emerging contaminants such as perfluorooctanesulfonate and biomolecules [8,9]. That platform technology was highly adaptable and could support a wide range of nanostructured materials, combined with capture molecules targeting diverse chemical and biological analytes, thereby significantly enhancing its sensitivity and selectivity [10]. Moreover, it offers modularity and multiplexing capabilities, making it a versatile tool for a range of applications [11,12,13]. Alternatives to glass and polymer walls have been shown to work [14,15]. Recently, Cheng et al. provided a pathway to transition the lab-based electrochemical methodology from an expensive benchtop analyzer to a significantly more portable, lower-cost USB analyzer with similar sensitivity [16]. However, a key limitation of previously packed microfluidic platforms is the potential displacement, leaching, or redistribution of the packed sensing material during flow. These effects may compromise material retention and contribute to variability in sensor performance. To address this limitation, the present study introduces an in situ fabricated porous PI manifold designed to improve the structural retention and operational stability of the sensing material within the microchannel. A systematic multi-chip reproducibility assessment was not performed in the present study; therefore, reduced chip-to-chip variation is not claimed.
Here, we propose a novel in situ fabrication methodology incorporating a low-cost PI porous manifold integrated into the microfluidic channel, which can be dotted with CNTs for biosensor applications. The proposed manifold is designed to address the limitations of simply packing a chip with nanomaterials by providing a stable structure that improves material retention within the microchannel without interfering with the electrochemical response. Additionally, the manifold is engineered using a two-step process with sufficient porosity to operate under flow-through conditions at high flow rates and low fluidic pressure, while maintaining chemical and electrochemical stability in the electrolytes and electrode materials and facilitating analyte transport. Additionally, CNTs’ properties, such as their high surface area, excellent electronic properties, and strong interaction with DNA, make them promising materials for DNA sensing [17]. Polyimide (PI) was selected as the supporting matrix for the porous manifold because of its high thermal stability, mechanical robustness, excellent dielectric properties, low coefficient of thermal expansion, and resistance to chemical degradation [18]. These polymers are synthesized via polycondensation of a dianhydride monomer and a diamine [19,20,21]. Additionally, the nitrogen (N) bonds within their carbon skeleton introduce pseudo-capacitance, enhance the wettability of carbon-based materials, and contribute to their superior mechanical toughness and chemical resistance [22]. Among the various methods, such as phase inversion, the breath figure method can be more valuable for creating porous polymer films due to its environmental friendliness, low cost, scalability, and highly ordered porous structure [23,24,25,26,27]. Hence, acetone, carbon disulfide (CS2), and chloroform cannot be used [26]. Acetone or similar solvents are readily vaporized and quickly weaken the microfluidic channel, causing the device to separate. CS2 and chloroform cannot be used because they are too fluid and tend to adsorb onto the tape channel layer before the molecules can reach the polymer solvent. A highly concentrated chloroform/PI solution (higher than 40 wt%) cannot form a porous structure when exposed to moisture. Most solvents that dissolve PI also adversely affect the channel-layer polymers. The original precursor, poly(amic acid) (PAA), in N-methyl-2-pyrrolidone (NMP), provides higher viscosity, is not affected by the channel wall as quickly, and is also used in the phase inversion method. However, NMP is more challenging to remove, and the water vaporizes faster than NMP. This results in the porous structure disappearing after moisture removal, as the remaining NMP redissolves the PAA. Hence, we modified the traditional method by combining these methods: the polymer solvent was first exposed to moisture, forming a permanently porous structure; after that, the structure was immersed in water that maintained the nonsolvent and extracted NMP from the polymer. We have successfully mitigated this issue by introducing a PAA porous manifold with different packing materials to form a non-planar, interdigitated electrode. To the best of our knowledge, this is the first report showing the in situ fabrication of a PI porous structure with CNTs using the breath figure technique, which is used to detect ssDNA. This work will advance cost-effective diagnostic solutions and address the steep demand for portable, cost-effective, and automated biosensors in global healthcare.

2. Materials and Methods

2.1. Chemicals and Materials

The NMP solution containing 15 wt% poly(amic acid) (PAA) [poly(pyromellitic dianhydride-co-4,4′-oxydianiline)] gel was purchased from Sigma-Aldrich (St. Louis, MO, USA). Standard glass slides (1304G) were obtained from Globe Scientific Inc. (Mahwah, NJ, USA), while deionized (DI) water was sourced from a Milli-Q Direct Water Purification System(MilliporeSigma, Burlington, MA, USA). Double-sided tapes (ARcare® 90106NB) featuring a polyester film and MA-69 medical-grade acrylic pressure-sensitive adhesives on both sides, with a total thickness of 140 µm (including the polyester layer and adhesive), were supplied by Adhesives Research Inc. (Glen Rock, PA, USA). The 4294A Precision Impedance Analyzer from Keysight Technologies (Santa Rosa, CA, USA) was utilized for EIS measurements. Carboxylic acid-functionalized short single-walled carbon nanotubes (SWCNTs, 98%+) were acquired from US Research Nanomaterials Inc. (Houston, TX, USA). The fabrication of top and bottom microelectrode glass slides was performed at the Nanofabrication Facility of the CUNY Advanced Science Research Center (New York, NY, USA). Channel tapes were cut using a Cricut® Maker™ (Cricut Inc., South Jordan, UT, USA). Probe-DNA (pDNA) and target-DNA (tDNA) sequences with oligonucleotide configurations of 5′-/5AmMC6/CGTCCAAGCGGGCTGACTCATCAAG-3′ and 5′-CTTGATGAGTCAGCCCGCTTGGACG-3′, respectively, were purchased from Integrated DNA Technologies (Coralville, IA, USA). The 4294A Precision Impedance Analyzer from Keysight Technologies ensured consistent electrochemical detection limits, meeting standards established in previous studies. A custom chip clipper was 3D-printed on a Formlabs Form 3B, and contact gold pins were sourced from Digi-Key. The fully automatic fluidic control system was purchased from LabSmith Inc. All 1× PBS solutions used in this study were prepared at pH 7.4. The fully automated fluidic control system, composed of LabSmith components including syringe pumps, selector valves, pressure sensors, and control interfaces, is shown in Figure S1. This setup enables precise, programmable fluid handling for device operation. The pressure drop studies were done using a pressure sensor connected to both the inlet and outlet of the chip.

2.2. Breath Figure Instrument

In Figure 1, we observe the initial segment of the breath-figure instrument. This setup uses two regulators to control the air in the system. One air pipeline leads to a DI water moisture chamber, producing a saturated vapor bubble. The other air pipeline produces dry air for a silica moisture absorber. Both air streams then converge in a mixing chamber. Here, the saturated and dry air mix, and the moist air exits the chamber and is regulated by a flow controller into the breathing chamber. A moisture meter is positioned next to the electrode chip inside the breathing chamber to ensure the appropriate moisture level. Throughout the process, the proportions of saturated vapor and dry air are carefully controlled to prevent moisture accumulation from droplets on pipeline walls.

2.3. Microfluidic Chip Assembly Protocol

The assembly process for the chip is illustrated in Figure 2. The fabrication of the chip begins with depositing a gold electrode on a glass substrate (Figure 2A). A gold electrode was fabricated on a standard glass slide using a standard lithography technique, as described previously [8]. To form the microfluidic channel, a layer of pressure-sensitive double-sided tape (Arcare) is carefully aligned and applied over the gold electrode (Figure 2B). The tape is precisely cut using a Cricut Maker, a computer-controlled cutting machine, ensuring accurate dimensions and alignment of the channel design. A pressure-sensitive, double-sided tape (Arcare) was aligned with the microelectrode and adhered to the electrode’s glass slide. Next, a mixed solution is prepared by sonication of SWCNTs and PAA to achieve a homogeneous dispersion. This mixture is cast into the microfluidic channel (Figure 2C). The porous structure of the chip is developed using the modified breath figure method (Figure 2D). Then, the device is exposed to a controlled high-humidity environment (70–90% RH) for 20 min, during which water droplets condense on the surface of the polymer solution. These droplets act as templates for forming pores, resulting in a highly porous polymer matrix. A phase inversion process stabilizes the porous structure by immersing the glass slide in deionized water overnight (Figure 2E). This step removes residual solvents from the polymer matrix, enhancing the integrity of the porous structure and ensuring uniform pore distribution. The polymer is then subjected to a slow imidization process (Figure 2F) to convert the polyamic acid into PI. This transformation is achieved by gradually heating the device from 60 °C to 180 °C overnight. The imidization process solidifies the porous PI matrix, creating a mechanically durable structure with uniform pores. After forming the porous PI structure (Figure 2G), the device is assembled by sealing it with a top glass slide (Figure 2H). The sealing process incorporates the gold electrode within the microfluidic channel, ensuring a leak-proof assembly and maintaining the structural integrity of the porous matrix. The final completed device (Figure 2I) forms a porous flow-through electrode integrated within a microfluidic system. In situ SWCNT-polymer composite fabrication of the device is detailed in Figure S2, and the microscopic images highlight critical steps, including polymer casting, imidization, and porous flow-through electrode formation.

2.4. On-Chip EDAC Protocol

The assembled chip is cleaned with 100 mM MES buffer at a flow rate of 5 µL/min for 16 min. After the initial washing, an activation solution containing 100 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and 100 mM N-hydroxysulfosuccinimide (sulfo-NHS) in 100 mM MES buffer flows into the chip at 5 µL/min for 16 min. After activation, the chip is washed with 80 µL of 1× PBS at 30 µL/min. After washing the chip, 80 µL of 10 µM ssDNA flows through the channel; the flow is then stopped, and the chip is left to rest overnight with the solution still inside the channel. The chip is again washed with 1× PBS for 1 h at 5 µL/min.

2.5. Chip Protocol

The clean chip was tested with PBS concentrations ranging from 0.01× to 1× at a flow rate of 5 µL/min. Stabilization was performed using 1× PBS and monitored through pressure measurements and impedimetric signals. The EIS settings included a frequency range of 40 Hz to 100 MHz, an amplitude of 500 mV, and no signal averaging. The excitation amplitude of 500 mV was selected based on our previously published characterization of the same non-planar, flow-through microelectrode platform. In that study, EIS spectra collected at excitation amplitudes of 100, 200, and 500 mV showed comparable Nyquist profiles for the evaluated electrode configurations, with no measurable amplitude-dependent distortion of the characteristic impedance response. The 500 mV amplitude provided improved signal quality, particularly for the high-impedance working-electrode configuration, and was therefore used in the present study to ensure stable and reproducible measurements. The DNA testing followed a protocol similar to our previous work, but with an optimized duration of 15 min. The chip was first flushed with 1× PBS to establish a baseline (wash) EIS signal. Next, the target analytes were introduced for biorecognition and capture. Finally, the chip was rinsed with the same buffer to obtain the post-capture EIS signal. The difference between the wash and rinse EIS signals was used to estimate the amount of ssDNA captured on the chip.

3. Results

3.1. Breath Figure Self-Assembly of Membranes

The breath figure method utilizes moisture in the air to condense on the polymer solution, inducing small local phase inversions that produce an ordered honeycomb-structured membrane. In general, the breath figure method includes the following steps [26]: (a) The liquid surface becomes cold with the solvent evaporation; (b) Water condenses on the cold surface to become water droplets (nucleation); (c) Water droplets form a close-packed hexagonal array because of the convective currents of the evaporation and moist airflow; (d) Water droplets sink into the polymer solution; (e) New nucleation and growth of the moist air induce the formation of another water droplet layer; (f) New layers of close-packed hexagonal water droplet arrays are generated; (g) The solution surface cools back to room temperature after the solvent evaporates, creating the honeycomb film after water evaporation; (h) Repeating the process from (d) to (f), another layer of the porous membrane forms. Hence, the polymer and solvent pair selection becomes crucial for the overall process. The phase behavior studies were conducted to elucidate the formation of porous structures, and the ternary phase diagram for the PAA-NMP-water system is shown in Figure S3.

3.2. Effect of Relative Humidity on the Porous PI Manifold

Relative humidity strongly influenced the morphology of the porous PI manifold. As shown in Figure 3, the control sample prepared without the breath-figure step exhibited no clearly defined surface microporosity, whereas samples fabricated at 70%, 80%, and 95% RH developed distinct porous structures. At 70% RH, a more organized honeycomb-like morphology was observed, while increasing RH produced smaller, more densely distributed, and less regularly arranged pores. The controlled breath-figure conditions evaluated in this study were 70%, 80%, and 95% RH, with 70% RH representing the lowest tested condition. The sample labeled as 0% RH was prepared without the breath-figure step and was included only as a phase-inversion control.
Microscopic observations confirmed that the fabrication conditions strongly influenced the morphology of the porous PI/SWCNT manifold. The control sample prepared without the breath-figure step showed no clearly defined surface macroporosity. In contrast, the sample fabricated at 70% RH developed a structured porous architecture. As shown in Figure 4, the top-view SEM image revealed two distinct pore regions: a first layer containing pores approximately 10 µm or larger and a second underlying layer containing pores smaller than 10 µm. The larger pores are associated with the initial condensation of water droplets on the PAA/NMP surface, whereas the smaller secondary pores likely result from subsequent condensation, coalescence, and sedimentation of droplets within the polymer solution [28]. The side and cross-sectional views in Figure 4 further revealed multiple interconnected channels with dimensions below 10 µm, providing continuous pathways for electrolyte transport through the manifold. SWCNTs were visibly embedded within the pore walls and distributed throughout the porous matrix. The top surface exhibited a relatively organized pore arrangement, whereas the bottom region showed more discontinuous and irregular pore-wall structures. Enlarged SEM images also showed SWCNT-COOH aggregates distributed along the internal pore walls, confirming their incorporation throughout the three-dimensional porous network. At higher RH conditions, the porous structure became smaller, denser, and less organized, likely due to increased water-droplet condensation and coalescence, which compressed or ruptured the walls between neighboring pores. Overall, these observations demonstrate that RH affects pore size, pore-wall integrity, internal connectivity, and the three-dimensional architecture of the PI/SWCNT manifold.
Figure 5 compares the porous PI structures formed under different RH conditions. The control sample prepared without the breath-figure step showed no significant microscale surface pores. At 70% RH, a hierarchical architecture was observed, consisting of larger surface pores of approximately 20 µm and a second layer of smaller pores below 10 µm. At higher RH, the pore structure became smaller, denser, and more disrupted, particularly near the bottom of the manifold. This morphology may result from increased condensation, coalescence, and migration of water droplets through the PAA/NMP solution under capillary forces [18]. Because NMP evaporates more slowly than water, the subsequent water-immersion phase-inversion step is necessary to extract residual solvent and stabilize the temporary porous structure. Together, these observations demonstrate that RH influences pore size, organization, and structural continuity throughout the manifold [26,29].
SEM images and the corresponding pore size distributions shown in Figure 6 illustrate how RH influences pore size. At 70% RH, the average pore diameter is approximately 17 µm, characterized by larger, well-defined pores due to material swelling from moderate moisture absorption. At 80% RH, the average pore diameter decreases to approximately 10 µm, indicating reduced material expansion with increasing moisture absorption. At 95% RH, the pores become densely packed, with an average diameter of roughly 5 µm, reflecting maximum moisture absorption and a more compact structure. Interestingly, at 70% RH, the first condensed droplet layer forms larger pores than those observed at 80% or 95% RH. Compared to the 0% RH condition, the second phase inversion step facilitates the formation of larger pores. During the initial breath-figure step, insufficient droplets sink to the bottom at 70% RH, are subsequently removed, and are immersed in water. This results in a structure observed at 0% RH, as shown in Figure 4. The tunnel-like features are formed by rapid mixing of the solvent and nonsolvent, with the porous structure predominantly localized in the bottom layer. At the same time, the tunnel remains confined to the top layer. The controlled breath-figure conditions evaluated in this study were 70%, 80%, and 95% RH, with 70% RH representing the lowest humidity condition investigated. RH values below 70% were not systematically evaluated. The sample labeled as 0% RH was prepared without the breath-figure step and was included only as a phase-inversion control.

3.3. Characterization of PAA and PI

FTIR spectra assessed the presence of different functional groups in the porous manifold and PAA. Figure 7 presents the FTIR spectra for the slow, low-temperature imidization process, providing insight into the chemical transition from PAA to PI. The spectrum reveals several characteristic peaks associated with the formation of imide groups, confirming the progress of imidization [30,31,32]. The peak at 1777 cm−1 corresponds to the C=O asymmetric stretching vibration, while the peak at 1724 cm−1 is attributed to the C=O symmetric stretching vibration, both characteristic indicators of imide functionality. Additionally, the spectrum displays the C−N asymmetric stretching vibration at 1378 cm−1 and the C=O bending vibration at 737 cm−1, confirming the successful formation of imide groups in the polymer matrix. Although the formation of imide groups is evident, residual features of the PAA precursor remain, as the imidization was carried out at a relatively low temperature of 180 °C. A broad absorption band in the 2500–3300 cm1 range, associated with COOH and NH2 groups, indicates incomplete conversion of PAA to PI under these conditions. This suggests that while low-temperature imidization initiates the chemical transition effectively, it does not eliminate the functional groups characteristic of PAA, thereby partially retaining the precursor material.
The residual amic-acid functional groups resulting from incomplete imidization may influence the performance of the porous manifold. These polar groups may increase surface hydrophilicity and facilitate electrolyte penetration into the porous structure. They may also provide additional chemically active sites that interact with the carboxylated SWCNTs and biomolecules during surface functionalization. However, incomplete imidization may also increase solvent uptake, swelling, and variability in the polymer’s dielectric and interfacial properties, which could affect the baseline impedance and long-term stability of the sensor. Therefore, the relatively low imidization temperature used in this study represents a compromise between promoting PI formation and preserving the microfluidic materials and porous architecture. Further optimization of the imidization temperature and duration will be required to determine the relationships among degree of conversion, mechanical stability, and electrochemical sensing performance. The spectrum thus underscores the balance between imidization progress and the limitations imposed by the chosen reaction conditions, offering a detailed view of the chemical transformation dynamics under slow, low-temperature processing. This analysis is critical for understanding the interplay of temperature and reaction time in achieving optimal imidization in polymeric systems.

3.4. Manifold Stability in the Fabricated Microfluidic Chip

The stability of the manifold within the microfluidic channel was tested by measuring the pressure response across various flow rates (5–30 μL/min). Figure 8 demonstrates the stability of the pressure response across varying humidity conditions, specifically at 95%, 80%, and 70% RH. The results reveal that the pressure drop increases with increasing flow rate, highlighting the relationship between these parameters. Pressure drop depends on the overall permeability of the porous manifold, not on surface pore size alone. Although 70% RH produced larger surface pores, the underlying smaller pores and constricted, tortuous pathways may have increased hydraulic resistance. Since connectivity and permeability were not directly measured, this interpretation is based on the observed SEM morphology.
Notably, the data for 70% RH closely resemble those for 80% RH, indicating similar pressure behavior across these conditions. The slight fluctuations in the pressure response, referred to as crinkles, are attributed to the syringe pump’s piston mechanism, an inherent characteristic of its mechanical design. Despite these minor fluctuations, the system maintains stability. The flow rate reaches up to 30 µL/min, with signal stability achieved after 90 s, an improvement over the previous sensor chip design, which was limited to a maximum flow rate of 5 µL/min. The pressure response remains stable between 90 and 180 s after the initial flow adjustment, demonstrating the chip’s ability to handle higher flow rates without compromising stability. Although noise from the flow pump setup is observed, it does not affect the system’s overall reliability. These findings underscore the chip’s enhanced operational capacity and robust performance across varying humidity and flow-rate conditions.
Figure 9 shows the impedimetric results from changes in background buffers (A) and long-term tests (B) at different flow rates and with automated switching processes. Figure 9A shows the chip’s EIS response with a PI-manifold, measured at different PBS concentrations at 5 µL/min. The inset of Figure 9 shows the EIS spectra through the full range of frequencies, and the inset of Figure S4 shows the circuit diagram used in this analysis. The chip response closely follows the decrease in electrolyte concentration, demonstrating its sensitivity to environmental changes within the channel and variations in the spiked buffer concentration. Long-term stability tests were conducted by performing five sequential 80 µL injections at varying flow rates on the same chip, amounting to a total runtime of 23.3 min under harsh conditions. These tests confirmed the chip’s stability, with no interference observed in the EIS signals due to platform operation or background buffer accumulation. The detection protocol is efficient, requiring only 15 min to complete.
The EIS semicircles in the Nyquist plot display responses to PBS buffer concentrations ranging from high to low. At lower buffer concentrations, the semicircles become disproportionately large, indicating a significant increase in resistance, which complicates comparisons of surface charge resistance changes. Conversely, the capacitance value diminishes at higher buffer concentrations, and the charge transfer resistance (Rct) becomes challenging to fit. In our previous study, the relatively large gap between the counter and working electrodes necessitated the use of 10X PBS. However, the current manifold reduces the resistance between the electrodes without altering their spatial arrangement, thereby enabling stable operation across a range of buffer concentrations from 0.01× PBS to 1× PBS.
Figure 9B highlights the chip’s stability by showing pressure changes at flow rates from 10 µL/min to 30 µL/min. Each test involved injecting 80 µL of 1× PBS buffer at various flow rates using the same chip. The EIS signal remained consistent throughout the process, even during pump loading, valve rotations, and buffer reloading. Furthermore, no accumulation of PBS molecules was observed within the manifold or chip channels, ensuring that the EIS signal was unaffected by physical adhesion within the chip. Minor spectral fluctuations were observed at specific frequencies and may arise from variations in the electrical contact resistance of the spring-loaded connector, cabling, or other instrumental factors. Because the individual contributions of these components were not independently isolated, the fluctuations cannot be assigned exclusively to the spring connector. These fluctuations did not alter the overall impedance trends or the conclusions of this study.

3.5. ssDNA Detection Using a New PI Manifold Microfluidic Chip

Figure 10 shows the chip’s impedimetric response during the EDAC coupling reaction and target ssDNA detection. The inset in the graph shows the full-range frequency spectra of the measurements. The chip is first cleaned with 0.1 M MES buffer, then the EDAC coupling reagents are injected to activate the carboxylic groups. After activation, the chip is quickly washed with 80 µL of 1× PBS at 30 µL/min to clean the channel. It is noted that the manifold volume in the channel is only 0.07 µL. Afterward, the chip is treated overnight with 80 µL of 10 µM ssDNA probe to ensure the coupling reactions. During each reaction step, the semicircle size in the impedance spectrum decreases as the RH setting decreases from 95% to 70%. Figure S4 shows the fitted Rct values under varying RH conditions and highlights the humidity-dependent impedance behavior of the PI/SWCNT platform. The observed response at the tested femtomolar target-DNA concentration serves as proof of concept for sensing and should not be interpreted as a formal limit of detection.
SEM cross-sectional images in Figure 4 reveal that most of the CNTs are concentrated along the walls between the pores of the manifold. At 70% RH, the thicker pore walls retain more CNT compared to 80% and 95% RH. This higher CNT retention enhances the manifold’s conductivity, as the thicker walls provide more pathways for efficient charge transfer. The RH-controlled pore morphology may therefore influence sensing performance by affecting pore-wall thickness, CNT retention, and the continuity of the conductive network. At 70% RH, the comparatively thicker pore walls appeared to retain more SWCNTs, which may support more continuous CNT-to-CNT pathways and facilitate charge transport between the electrodes. The carboxylated SWCNTs also provide functional sites for EDAC/NHS-mediated probe-DNA immobilization, thereby connecting the conductive network with the biorecognition interface. In contrast, the thinner or locally disrupted pore walls observed at higher RH may reduce CNT retention and interrupt conductive pathways. Following target-DNA capture, changes in interfacial charge distribution, steric blocking, and charge-transfer behavior contribute to the observed impedance response. Because CNT loading, network percolation, and local conductivity were not quantified independently, this mechanism is proposed based on combined SEM morphology and EIS observations.
Figure 10D–F illustrate the target ssDNA experiments conducted at fM target DNA concentrations using 1× PBS as the background buffer without a redox probe. The changes in the EIS signal are compared to the initial values to evaluate the detection performance. The MES and EDAC measurements were used as process controls to monitor the surface-cleaning and activation steps, respectively. However, these controls do not distinguish complementary target hybridization from nonspecific DNA adsorption. Because mismatched and non-complementary DNA controls were not evaluated in the present study, the results should be interpreted as proof-of-concept detection of the complementary target rather than a complete demonstration of sequence selectivity. Future studies will include mismatched and non-target DNA controls to assess nonspecific adsorption and establish the platform’s analytical specificity.
The present biosensing measurements were conducted to demonstrate the proof-of-concept capability of the porous PI/SWCNT platform for detecting complementary target ssDNA. A comprehensive analytical validation, including replicate measurements across independently fabricated devices, statistical comparison, calibration curve construction, and quantitative determination of the limit of detection, was not performed in this study. Therefore, the observed impedance changes should be interpreted as preliminary evidence of sensing feasibility rather than as a complete quantitative assessment of analytical performance.
Several factors have been proposed to explain the electronic properties after DNA bonding to the electrode in EIS sensors, such as the ssDNA probe potentially falling or blocking the electrode surface after bonding [33,34] and increasing the resistance of the electrode, or tunnel effects of ssDNA, which increase the electrode conductivity [35,36,37] after the bonding, and the base properties of each amino acid, the conductivity [37]. Hence, each impedimetric DNA biosensor usually depends on its biomodification to determine the direction of the change. The combination of the CNT-enriched porous PI matrix and the controlled RH environment plays a critical role in the chip’s performance. The ability to modulate structural features, such as pore wall thickness and CNT retention, through humidity control directly affects the chip’s conductivity and sensing efficiency. These findings underscore the importance of optimizing fabrication conditions to achieve superior performance. The observed impedance response following exposure to femtomolar concentrations of complementary target ssDNA provides proof of concept that the platform can detect nucleic acids. Further replicate measurements, multi-chip studies, and quantitative calibration are required to establish the platform’s sensitivity, detection limit, and reproducibility. This innovative design, which integrates a porous flow-through electrode with a microfluidic system, shows potential for rapid, flow-through, and versatile biosensing applications.

4. Conclusions

In this study, we presented a novel approach combining liquid phase inversion and breath figure methods to overcome the limitations of solvent selection inherent in the traditional breath figure method. Using this protocol, we successfully fabricated a porous PI manifold within a 500 µm microfluidic channel under mild conditions. This study demonstrated the design, fabrication, and evaluation of the chip, a microfluidic biosensing platform integrated with a porous PI flow-through electrode enriched with CNTs. SEM analysis revealed that lower RH conditions resulted in larger, but less uniformly distributed, pores in the PI matrix. These structural changes increased channel pressure, highlighting RH’s influence on the pore architecture and overall chip performance. SEM analysis also revealed a dual-layer pore structure at lower RH, with the distinction between layers diminishing at higher RH levels. The manifold demonstrated exceptional stability under rigorous flow conditions, withstanding a harsh flow rate of 30 µL/min while maintaining consistent pressure and EIS responses. Furthermore, the EIS signals remained stable, suggesting that the manifold is well-suited for high-shear-rate applications. The device incorporates an on-chip EDAC protocol for functionalization with ssDNA probes and produces a measurable proof-of-concept impedance response following exposure to femtomolar concentrations of complementary target ssDNA in 1× PBS within 15 min. Additional replicate, multi-chip, and concentration-dependent studies are required to assess the platform’s analytical performance quantitatively. Although the device produced a measurable response to the complementary target DNA, further studies using mismatched, non-complementary, and potentially interfering species are required to establish sequence selectivity and evaluate nonspecific adsorption. These results highlight the manifold’s significant potential to enhance future microfluidic flow-through applications, particularly in high-performance sensing and analysis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/chemosensors14080171/s1, Figure S1: Fully automated fluidic control system from Labsmith Inc., featuring key components for precise fluid handling and control. The system includes the uProcess Syringe Pump (SPS01) for accurate fluid dispensing and the uProcess Automation Interface (EIB200) for system control and automation; Figure S2: Schematic depicting the fabrication process and characterization of the chip. (A) Mixed solution preparation: (I) PAA resin and (II) Addition of SWCNTs (III) the mixture is sonicated to produce a homogeneous mixed solution ready for use. (B) Polymer casting: The prepared mixed solution is cast into the microfluidic channel created by the double-sided tape adhered to the gold electrode substrate. (C) Final device and microscopic images: The fully assembled chip is shown in the top panel, with the microscopic images highlighting various structural features. (i) Shows a microscope image of the polymer solvent cast and blue color refers to flow of dye through the manifold channel, (ii) the color change of polyimide from white to brown during the imidization process, (iii) the process with SWCNT incorporated into the polymer, and (iv) demonstrates the flow of food dye through the porous structure; Figure S3: Ternary phase diagram of the polymer solution system: The diagram represents the composition of the PAA, NMP (N-methyl-2-pyrrolidone), and water, expressed in weight percentages (wt%). The gray data points indicate the composition pathway for the PAA solution during the phase separation process. The plotted line highlights the regions of interest where the balance between polymer, solvent (NMP), and nonsolvent (water) influences the formation of a porous structure in the microfluidic channel; Figure S4: The fitting results for surface resistance components under relative humidity (RH) levels of 70%, 80%, and 90% highlight the impact of humidity on the material’s electrochemical behavior.

Author Contributions

N.N.K.: Methodology, Investigation, Conceptualization, Visualization, Writing—original draft. S.Y.: Conceptualization, Visualization, Writing, and Reviewing. N.H.M.: Formal Analysis, Writing—Review & Editing. S.K.: Visualization, Writing—Review & Editing. G.L.: Validation, Writing—Review & Editing. Y.-H.C.: Methodology, Investigation, Conceptualization, Visualization, Writing—original draft. K.M.: Methodology, Writing—Review & Editing. S.B.: Writing—Review & Editing, Conceptualization, Methodology, Resources. All authors have read and agreed to the published version of the manuscript.

Funding

This manuscript is supported by Sagnik Basuray’s NSF grant #1751795, Career: “ASSURED” electrochemical platform for multiplexed detection of Cancer Biomarker Panel using Shear Enhanced Nanoporous Capacitive Electrodes”. This material is partially based upon work supported by the National Science Foundation under Grant No. (NSF 2331429). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. NSF I-Corps grant #2048361, I-Corps: Point-of-use microfluidics-based electrochemical platform for per- and polyfluoroalkyl substance (PFAS) detection in the source water.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Schematic of the breath-figure apparatus used to control relative humidity during porous-manifold fabrication. Blue arrows indicate the direction of airflow, and the white valve symbols represent the pressure regulators. One air stream passes through the blue DI-water bubbling chamber to generate humidified air, while the second stream passes through the orange silica moisture-absorber chamber to produce dry air. The two air streams are combined at the mixing point and directed into the reaction chamber. The microfluidic device containing the PAA precursor is placed inside the reaction chamber, where the humidified airflow promotes water-droplet condensation. A moisture meter is used to monitor the relative humidity inside the chamber.
Figure 1. Schematic of the breath-figure apparatus used to control relative humidity during porous-manifold fabrication. Blue arrows indicate the direction of airflow, and the white valve symbols represent the pressure regulators. One air stream passes through the blue DI-water bubbling chamber to generate humidified air, while the second stream passes through the orange silica moisture-absorber chamber to produce dry air. The two air streams are combined at the mixing point and directed into the reaction chamber. The microfluidic device containing the PAA precursor is placed inside the reaction chamber, where the humidified airflow promotes water-droplet condensation. A moisture meter is used to monitor the relative humidity inside the chamber.
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Figure 2. Schematic describing the stepwise assembly process of the chip. (A) A gold electrode is fabricated on a glass substrate. (B) A pressure-sensitive double-sided tape (Arcare) is aligned. (C) A mixture of carboxylic SWCNT and PAA solution is sonicated and cast into the microfluidic channel. (D) The modified breath figure method is employed, maintaining 70–90% RH for 20 min to allow the polymer to form a porous structure. (E) Phase inversion is achieved by immersing the glass slide in deionized (DI) water overnight. (F) The imidization process begins by gradually heating the device from 60 °C to 180 °C overnight to transform the PAA into a stable PI porous structure. (G) The resulting porous structure can be observed after the imidization process. (H) The device is assembled by sealing it with a top glass slide. (I) The final completed chip is shown.
Figure 2. Schematic describing the stepwise assembly process of the chip. (A) A gold electrode is fabricated on a glass substrate. (B) A pressure-sensitive double-sided tape (Arcare) is aligned. (C) A mixture of carboxylic SWCNT and PAA solution is sonicated and cast into the microfluidic channel. (D) The modified breath figure method is employed, maintaining 70–90% RH for 20 min to allow the polymer to form a porous structure. (E) Phase inversion is achieved by immersing the glass slide in deionized (DI) water overnight. (F) The imidization process begins by gradually heating the device from 60 °C to 180 °C overnight to transform the PAA into a stable PI porous structure. (G) The resulting porous structure can be observed after the imidization process. (H) The device is assembled by sealing it with a top glass slide. (I) The final completed chip is shown.
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Figure 3. Microscopic images of the polymer surfaces resulting from the two-step breath figure method under varying RH levels, set between 70% and 95%. All images were captured at 100X magnification, illustrating the influence of RH on the formation and morphology of the porous polymer structure.
Figure 3. Microscopic images of the polymer surfaces resulting from the two-step breath figure method under varying RH levels, set between 70% and 95%. All images were captured at 100X magnification, illustrating the influence of RH on the formation and morphology of the porous polymer structure.
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Figure 4. Scanning Electron Microscope (SEM) images of the PI manifold formed using the 2-step breath figure method under 70% RH. The images reveal the polymer’s 3D porous structure from multiple perspectives: top, bottom, and cross-section.
Figure 4. Scanning Electron Microscope (SEM) images of the PI manifold formed using the 2-step breath figure method under 70% RH. The images reveal the polymer’s 3D porous structure from multiple perspectives: top, bottom, and cross-section.
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Figure 5. SEM images of the porous polymer structures formed under varying humidity levels, ranging from 0% to 95%. The images provide views from different perspectives, including top and side views, showcasing the morphology and arrangement of the pores.
Figure 5. SEM images of the porous polymer structures formed under varying humidity levels, ranging from 0% to 95%. The images provide views from different perspectives, including top and side views, showcasing the morphology and arrangement of the pores.
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Figure 6. SEM images and pore size distribution analysis of porous polymer structures formed at RH levels of 70%, 80%, and 95% highlight the effect of humidity on pore morphology and size.
Figure 6. SEM images and pore size distribution analysis of porous polymer structures formed at RH levels of 70%, 80%, and 95% highlight the effect of humidity on pore morphology and size.
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Figure 7. FTIR analysis for the imidization process, showing the transition from PAA to PI, confirming the formation of imide groups. The FTIR spectrum is direct evidence of the chemical transformation during the imidization process.
Figure 7. FTIR analysis for the imidization process, showing the transition from PAA to PI, confirming the formation of imide groups. The FTIR spectrum is direct evidence of the chemical transformation during the imidization process.
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Figure 8. Pressure response stability at different flow rates under varying humidity levels: (A) 95% RH, (B) 80% RH, and (C) 70% RH. The data illustrate the pressure behavior across the flow rates, demonstrating the system’s stability and responsiveness.
Figure 8. Pressure response stability at different flow rates under varying humidity levels: (A) 95% RH, (B) 80% RH, and (C) 70% RH. The data illustrate the pressure behavior across the flow rates, demonstrating the system’s stability and responsiveness.
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Figure 9. The EIS response under two conditions: (A) varying concentrations of background buffers and (B) long-term stability of the chip’s EIS signal.
Figure 9. The EIS response under two conditions: (A) varying concentrations of background buffers and (B) long-term stability of the chip’s EIS signal.
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Figure 10. The impedimetric response during the EDAC reaction (A) 95% RH, (B) 80% RH, and (C) 70% RH. The capture of the target ssDNA is shown in (D) at 95% RH, (E) at 80% RH, and (F) at 70% RH.
Figure 10. The impedimetric response during the EDAC reaction (A) 95% RH, (B) 80% RH, and (C) 70% RH. The capture of the target ssDNA is shown in (D) at 95% RH, (E) at 80% RH, and (F) at 70% RH.
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MDPI and ACS Style

Khaja, N.N.; Yadav, S.; Haridas Menon, N.; Kaaliveetil, S.; Liu, G.; Cheng, Y.-H.; McEnnis, K.; Basuray, S. In Situ Fabrication of Controlled Porous Manifold Coupled with Non-Planar Microelectrodes for Microfluidic Biosensors. Chemosensors 2026, 14, 171. https://doi.org/10.3390/chemosensors14080171

AMA Style

Khaja NN, Yadav S, Haridas Menon N, Kaaliveetil S, Liu G, Cheng Y-H, McEnnis K, Basuray S. In Situ Fabrication of Controlled Porous Manifold Coupled with Non-Planar Microelectrodes for Microfluidic Biosensors. Chemosensors. 2026; 14(8):171. https://doi.org/10.3390/chemosensors14080171

Chicago/Turabian Style

Khaja, Najamuddin Naveed, Sushma Yadav, Niranjan Haridas Menon, Sreerag Kaaliveetil, Guangliang Liu, Yu-Hsuan Cheng, Kathleen McEnnis, and Sagnik Basuray. 2026. "In Situ Fabrication of Controlled Porous Manifold Coupled with Non-Planar Microelectrodes for Microfluidic Biosensors" Chemosensors 14, no. 8: 171. https://doi.org/10.3390/chemosensors14080171

APA Style

Khaja, N. N., Yadav, S., Haridas Menon, N., Kaaliveetil, S., Liu, G., Cheng, Y.-H., McEnnis, K., & Basuray, S. (2026). In Situ Fabrication of Controlled Porous Manifold Coupled with Non-Planar Microelectrodes for Microfluidic Biosensors. Chemosensors, 14(8), 171. https://doi.org/10.3390/chemosensors14080171

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