Parylene Double-Layer Coated Screen-Printed Carbon Electrode for Label-Free and Reagentless Capacitive Aptasensing of Gliadin

Celiac patients are required to strictly adhere to a gluten-free diet because even trace amounts of gluten can damage their small intestine and leading to serious complications. Despite increased awareness, gluten can still be present in products due to cross-contamination or hidden ingredients, making regular monitoring essential. With the goal of guaranteeing food safety for consuming labeled gluten-free products, a capacitive aptasensor was constructed to target gliadin, the main allergic gluten protein for celiac disease. The success of capacitive aptasensing was primarily realized by coating a Parylene double-layer (1000 nm Parylene C at the bottom with 400 nm Parylene AM on top) on the electrode surface to ensure both high insulation quality and abundant reactive amino functionalities. Under the optimal concentration of aptamer (5 μM) used for immobilization, a strong linear relationship exists between the amount of gliadin (0.01–1.0 mg/mL) and the corresponding ΔC response (total capacitance decrease during a 20 min monitoring period after sample introduction), with an R2 of 0.9843. The detection limit is 0.007 mg/mL (S/N > 5), equivalent to 0.014 mg/mL (14 ppm) of gluten content. Spike recovery tests identified this system is free from interferences in corn and cassava flour matrices. The analytical results of 24 commercial wheat flour samples correlated well with a gliadin ELISA assay (R2 = 0.9754). The proposed label-free and reagentless capacitive aptasensor offers advantages of simplicity, cost-effectiveness, ease of production, and speediness, making it a promising tool for verifying products labeled as gluten-free (gluten content <20 ppm).

−9 Over the past decade, aptamer-based biosensors (aptasensors) have garnered significant attention across diverse applications in clinical diagnostics, environmental monitoring, food safety, and bioanalytical research due to a multitude of advantages.Aptamers are novel biorecognition elements that can be selected to bind specifically to a wide range of target molecules of interest, including ions, toxins, proteins, viruses, bacteria, and even whole cells.This ensures accurate and sensitive detection, as well as ease of customization.Aptamers can be designed to have minimal cross-reactivity with similar molecules, reducing the chances of false positives in detection assays.Their rapid target recognition ability also makes them suitable for real-time screening uses.The ease of versatile modification, such as the incorporation of functional groups (e.g., thiol, amino, hydroxyl) and conjugation of labels (e.g., fluorescent dyes, enzymes), enhances adaptability to different experimental setups.Additionally, aptamers exhibit stability under a variety of conditions, including changes in temperature and pH, contributing to their robustness for use in diverse environments.−12 Recently, an aptamer sequence called Gli4 was successfully selected against the immunotoxic 33-mer gliadin peptide.It was applied in a competitive electrochemical magneto assay that enabled gluten quantification down to 0.5 ppm, with no cross-reactivity to the matrices of nongluten containing grains. 13Later, a simpler architecture of a competitive electrochemical assay was developed with the Gli4 aptamer, achieving a lower detection limit of 0.38 ppm for gluten. 14However, despite meeting the requirements for gluten-free product monitoring in terms of both selectivity and sensitivity, those system designs still rely on the strategies of enzyme labeling and the addition of electrochemical active redox reagents.The lack of simplicity did hamper its implementation as a point of care testing device.
Capacitive biosensors achieve real-time and ultrasensitive detection by measuring minute alterations in the properties of the dielectric layer at the electrode−electrolyte interface when specific target molecules bind to the biorecognition elements immobilized on the sensing surface.The capacitance C of this dielectric layer between two electrodes can be determined using the basic equation as follows where ε 0 represents the dielectric constant in vacuum (8.85 pF/m), ε r stands for its dielectric constant, A indicates its area, and δ is the thickness of dielectric layer.This nonfaradaic biosensing technique often operates in a label-free and reagentless mode, requiring only simple readout electronics.This simplicity is beneficial for miniaturization and incorporation into portable devices, offering a promising solution for low-cost, convenient, and on-site diagnostics in point of care testing applications. 15,16Various capacitive aptasensing platforms have been developed and successfully applied to detect small molecules such as organophosphorus pesticides 17 and bisphenol A, 18 proteins like thrombin 19 and human epidermal growth factor receptor, 20 as well as cells such as Escherichia coli 21 and lung carcinoma cells. 22The fabrication of an insulation layer covering on the electrode surface is considered to be the most important issue for the success of capacitive aptasensing.This layer must be well-insulated and hole-free to avoid charge leakage to the electrode, preventing dramatic fluctuations in the baseline capacitance level.Besides, the layer should possess reactive functionalities for the subsequent chemical conjugation of the biorecognition elements.To maintain good sensitivity, this layer also needs to be as thin as possible. 23arylene is the trade name referring to a family of polymers known as poly(p-xylylene).These polymers are typically produced through the chemical vapor deposition (CVD) process, free from solvents, catalysts, and plasticizers.During CVD, the granular dimer precursor (di-p-xylylene) vaporizes and forms a uniform, hole-free pure polymeric coating onto the substrate, creating a conformal layer with customizable thickness ranging from tens of nanometers to tens of micrometers.Parylene coatings offer several desirable properties, including high chemical resistance, low moisture permeability, and electrical insulation.The use of Parylene as an insulation layer is advantageous because it provides a protective barrier without altering the dimensions or features of the underlying substrates.This conformal coating ensures that even complex and intricate structures are adequately insulated, contributing to the reliability and longevity of electronic devices.To date, there are over 10 commercially accessible variants of Parylene.The unsubstituted, monochloro-substituted, and dichloro-substituted versions, named Parylene N, Parylene C, and Parylene D, respectively, are the most commonly used industrial coatings.Among them, Parylene C is an FDA-approved class IV biocompatible polymer due to its excellent water and gas barrier properties and has been widely applied to implantable devices.Other versions of functionalized Parylene, such as Parylene A (monoamino-substituted), Parylene AM (monoaminomethylsubstituted), and Parylene H (monoaldehyde-substituted) coatings, can also serve as chemical anchors, providing reactive functional groups for the immobilization of biomolecules.−27 Since the deposition of Parylene is a low-cost, wellestablished industrial manufacturing technique that enables finely controlled conformal coating along with mass production capability, it can also offer a series of advantages such as being ultrathin, chemically inert, providing high electrical insulation, and possessing reactive functionalities.Therefore, it may be an ideal insulation layer for constructing capacitive biosensors and further implementation as a point of care testing device.To the best of our knowledge, this study represents the first attempt to apply Parylene C (bottom layer) and Parylene AM (top layer) as the functionalized insulation double-layer for capacitive aptasensing.The double-layer was coated onto the surface of a screen-printed carbon electrode (SPCE), followed by immobilizing the 5′-NH 2 -modified Gli4 aptamer using the cross-linker glutaraldehyde, and ultimately for the detection of gliadin in commercial wheat flours (Figure 1).These analytical results were further compared with those obtained from a commercial gliadin ELISA assay.Although false negatives may occur with the ELISA assay when gliadins are denatured during certain food processing procedures, it remains the most recommended method for providing acceptable results regarding the raw materials used in glutenfree food production by the AOAC International. 7,9Therefore, the AOAC-approved ELISA assay was chosen as the reference method in the current study.Additionally, interferences from other nongluten containing flour matrices were also evaluated.
Parylene Coating.The Parylene C coating service was provided by Taiwan Innova Efficiency (Taipei, Taiwan).The Parylene AM coating was achieved using a self-designed CVD polymerization system. 28The CVD conditions involved initially sublimating the Parylene AM dimer precursor at 100−120 °C under reduced pressure (0.2−0.3 Torr).Subsequently, it was transferred into the pyrolysis zone, kept at 670 °C, through a constant argon flow (5 cc/min).The material vapor finally reached a temperature-controlled sample holder (15 °C), where it spontaneously polymerized onto the surface of SPCEs (from Delta Electronics, Taipei, Taiwan).The surface topography of the Parylene-coated SPCE and the coating thickness were acquired by a three-dimensional profile confocal laser microscope (VK-9500, Keyence Corporation, Osaka, Japan).
Aptamer Immobilization.Aliquots of the aptamer solution were diluted with a 50 mM Tris−HCl buffer, pH 7.4, containing 250 mM NaCl and 5 mM MgCl 2 to the desired concentration.The mixture was heated at 98 °C for 5 min, cooled to 4 °C for 5 min, and then conditioned at 25 °C for 30 min to ensure the proper folding of its specific three-dimensional conformation.Onto the sensing area of the Parylene-coated SPCE, 40 μL of a 5% glutaraldehyde solution was initially dropped and allowed to react for 90 min.The electrode was then washed with double-distilled water.Subsequently, it was treated with 40 μL of the aforementioned aptamer solution and kept in a moisture-saturated box at 4 °C overnight to implement the cross-linking reaction between Parylene AM and the 5′-NH 2 -modified aptamer through glutaraldehyde.After washing out the unbound aptamers, 40 μL of a 100 mM glycine solution was added for 90 min to block the free aldehyde groups of glutaraldehyde, preventing nonspecific binding of other proteins during the real sample aptasensing.To examine the success of aptamer immobilization, a fluorescence microscope (Zeiss Axio Vert.A1, M&T Optics, Taipei, Taiwan) was used for observation.
Capacitance and Dissipation Factor Measurements.The Parylene-coated SPCE or the subsequently aptamer-immobilized SPCE was connected to the LCR meter probes (LCR-6300, Good Will Instrument, Taipei, Taiwan) and immersed in an Eppendorf tube containing 2 mL of phosphate-buffered saline (PBS) + solution (0.1 M PBS, pH 7.0, containing 1 mM MgCl 2 ).The capacitance and dissipation factor (D value) were simultaneously examined using the series equivalent circuit with a measurement frequency of 10 kHz and a bias voltage of 0.5 V.The lower the D value, the less power loss when AC is applied through the capacitor, indicating better insulation quality of the Parylene coating.
Capacitive Aptasensing of Gliadin.To ensure the insulation quality of aptamer-immobilized SPCE for the success of capacitive aptasensing, a 30 min capacitance measurement was initially conducted in a 2 mL PBS + solution.Upon observing a stable baseline capacitance level, 0.2 mL of PBS + solution was extracted and replaced with 0.2 mL of gliadin sample solution (0.01−1.0 mg/mL, dissolved in 70% methanol).The total decrease in capacitive response resulting from the affinity binding between the aptamer and gliadin during the immediately subsequent 20 min measurement was monitored and calculated for further regression analyses.
Analysis of Gliadin in Real Samples.In a test tube, a total of 1 g of commercial flour sample was mixed with 10 mL of 70% methanol and ultrasonicated for 20 min.The extraction mixture was then centrifuged at 10,000g for 15 min.The supernatant solution from the wheat flour sample was used to replace 10% of the volume of PBS + solution for capacitive aptasensing.This supernatant was directly used for gliadin ELISA assay quantification.The supernatant solutions from corn flour and cassava flour were spiked with aliquots of the gliadin standard solution to achieve the desired concentration, and then used to replace 10% of the volume of PBS + solution for recovery tests by capacitive aptasensing.

■ RESULTS AND DISCUSSION
Characterization of Parylene Coating.Our initial attempt was to coat a single layer of Parylene AM to assess whether the simplest design could achieve the desired insulation quality, along with its inherent reactive functionalities for the subsequent aptamer immobilization.As depicted in Figure 2, the thickness of Parylene AM clearly increased with CVD time from 0.5 h (206 nm) to 2 h (400 nm).Subsequently, the rate of thickness increase appeared to slow down, with thicknesses of 428 nm for 3 h coating and 446 nm for 4 h coating.This finding indicates that the deposition of Parylene AM through CVD can hardly create a thicker layer over 500 nm within a few hours.Furthermore, there is a tendency for capacitance to decrease with the increase in Parylene AM thickness.This occurs because the covering of thicker Parylene AM at the electrode−electrolyte interface leads to a gradual increase in the thickness of the dielectric layer, as stated in eq 1.However, all conditions of Parylene AM coating exhibited poor insulation quality, not only as indicated by D values exceeding 1.0, but also by the capacitance responses remaining in the tens of nF range with significant deviations.This phenomenon may be attributed to the Parylene AM coating not providing sufficient compactness to cover the rough, undulatory electrode surface, thus failing to achieve effective electrical insulation.This deficiency leads to noticeable charge leakage, consequently resulting in the With a fixed Parylene AM coating thickness of 400 nm on top of Parylene C (400 ± 23.4 nm, N = 5), the measured capacitance (see the circles in Figure 4) further declined compared to Figure 3, which is consistent with the description in eq 1.To verify long-term water resistance, these Parylene double-layer coated electrodes were immersed in the buffer solution overnight (12 h).However, undesirable fluctuations in capacitance and elevated capacitance levels were observed for all groups following immersion treatment, except for the 1400 nm thickness group (see the triangles in Figure 4).This circumstance proves that only the Parylene double-layer coating thicker than 1400 nm can provide preferable insulation quality for the subsequent aptamer immobilization and capacitive gliadin detection in an aqueous environment.
Although self-assembled monolayers (SAMs) are the most commonly used insulating strategy for capacitive biosensing applications, 17,19,20,22 they still have limitations that need to be overcome.For instance, they are sensitive to experimental conditions, and gradual degradation may destroy the insulation quality during biosensing.The type of headgroup of SAMs is limited, restricting their versatility beyond metallic electrode surfaces.Additionally, the formation of a uniform coverage is time-consuming, often requiring an overnight process.In contrast, Parylene is chemically inert with high electrical insulation and can be deposited onto all kinds of underlying substrates within a few hours, making it superior for constructing capacitive biosensing platforms over SAMs.
To assess the reactivity of inherent amino functional groups on the Parylene double-layer surface, a green fluorescent dye, FITC, was employed to specifically label its free amino groups.Figure 5 reveals a high density of FITC response, suggesting abundant reactive amino functionalities on the surface of the Parylene double-layer.The fluorescence coverage was estimated to be 72.2 ± 4.1% (N = 3).This finding is beneficial for the subsequent chemical conjugation of biorecognition elements.
For ultrasensitive capacitive sensing, it is essential to preserve the cavity space between an electrode pair as much as possible after Parylene coating.This is because the majority of electric field lines concentrate in this region.Even a minute change in dielectric properties within the cavity space can result in the most sensitive capacitive response.As depicted in Figure 6A, the surface profile of a bare SPCE displays a height difference of approximately 15 μm between the lateral carbon paste electrodes and the central underlying substrate.This height difference remains almost unchanged when a 1400 nm Parylene double-layer is applied afterward (Figure 6B).This phenomenon is in accordance with the described feature of   Parylene coating through CVD, which is its ability to form a uniform and conformal layer without altering the dimensions of the underlying substrate.However, there is still plenty of cavity space remaining while covering on the Parylene doublelayer, which means the ultrasensitive capacitive aptasensing may be implemented as expected.
Characterization of Aptamer Immobilization.To confirm the success of aptamer immobilization, 5 μM of 3′-FAM-labeled and 5′-NH 2 -modified Gli4 aptamer was cross-linked onto the surface of Parylene double-layer using glutaraldehyde.The fluorescence microscopic observation in Figure 7 clearly reveals the high intensity of green fluorescence emitted by the successfully immobilized aptamer.The fluorescence coverage was determined to be 58.2 ± 4.8% (N = 3).
Capacitive Aptasensing of Gliadin.Before introducing the gliadin sample into the test solution, a 30 min capacitance baseline measurement was conducted in a 2 mL PBS + solution (black curve in Figure 8).For a well-insulated aptamer-immobilized sensing electrode, a stable capacitance level below 10 pF can be observed, with a standard deviation lower than 0.004 pF (0.0024−0.0039 pF, N = 5) for consecutive data points during the last 10 min of monitoring.The flow disturbance attributed to the action of 10% sample solution volume replacement immediately led to an increase in capacitance.Following the replacement with 70% ethanol (the solvent of gliadin, green curve), the capacitance returned to a stable level within the subsequent 10 min (the standard    deviation for consecutive data points during the period of 10− 20 min after sample replacement ranged from 0.0021 to 0.0034 pF, N = 5).This suggests that the steady-state dielectric layer thickness can be achieved within a 10 min period after the operational disturbance.Other overlaid curves, depicting the presence of gliadin (in blue, yellow, and orange), further illustrate the dynamic binding behavior of Gli4 aptamer− gliadin interactions.The addition of more gliadin corresponds to a greater capacitive decrease, attributable to the synergistic effect of forming a thickened dielectric layer and replacing high dielectric constant water molecules (ε r = 80) with lower dielectric constant protein molecules (ε r = 20), such as gliadin, as described in eq 1.Additionally, the gradual decrease in capacitance seems to stabilize after 20 min of sample introduction, the total decrease in capacitance during the 20 min consecutive monitoring was therefore used for gliadin quantification.
To achieve optimal analytical performance in capacitive aptasensing, we investigated the immobilized dosage of the aptamer ranging from 1 to 10 μM.As shown in Table 1, a strong linear relationship was observed between the capacitive response and gliadin concentration within the dynamic range of 0.01−1.0mg/mL for all aptamer dosages used during immobilization.The sensitivity of gliadin aptasensing can be clearly enhanced with an elevated aptamer dosage from 1 to 5 μM (see the slopes in Table 1).However, it worsens when the dosage is increased to 10 μM.This occurrence may result from the denser immobilization of aptamers on the surface of the Parylene double-layer coated electrode.When gliadin molecules are captured by the immobilized aptamers, the conformational change in the aptamers results in steric hindrance to neighboring aptamers, creating insufficient space to conjugate with target gliadin molecules.Therefore, 5 μM aptamer appears to be an optimal concentration for the present work.The relative standard deviations were less than 10.5% for all concentration points (N = 5), and gliadin concentrations lower than 0.007 mg/mL (7 ppm) could be determined with an S/N ratio higher than 5. Since approximately 50% of wheat gluten proteins consist of monomeric gliadins, 29 our LOD value for gluten should be calculated by multiplying it by a factor of 2, 30 resulting in 14 ppm.Although this LOD for gluten is higher than that obtained by the competitive electrochemical magneto assay 13 and the competitive electrochemical assay, 14 both developed with the same Gli4 aptamer, it still fulfills the European legislated threshold of 20 ppm required for verifying food labeling as gluten-free.The storage stability of the constructed aptasensor was preliminarily tested by storing the aptamerimmobilized SPCEs in PBS + solution at 4 °C.After a oneweek storage period, a negligible loss in the capacitive aptasensing response was observed.However, further longterm systematic evaluation is needed.
Although other label-free and reagentless biosensing alternatives, such as the quartz crystal microbalance-based immunosensor, 31 the localized surface plasmon resonancebased immunological assay, 32 and the gold nanoparticle aggregation-based colorimetric aptasensor, 33 have demonstrated more sensitive performance in gliadin detection than the proposed capacitive aptasensing platform, our method remains superior in terms of cost-effectiveness, robustness under environmental conditions, and simplicity in the design of readout electronics.These advantages make it particularly suitable for developing point-of-care testing devices.
Analysis of Gliadin in Real Samples.Twenty-four commercial wheat flour samples were employed to evaluate the reliability of the proposed capacitive aptasensor by comparing its analytical results with those obtained from a gliadin ELISA assay.By introducing the supernatant extract into the test solution, the corresponding capacitance decrease was recorded and then interpolated into the calibration curve obtained with gliadin standards to determine the gliadin content in real samples.As depicted in Figure 9, a strong positive linear correlation (R 2 = 0.9754) was observed, indicating that the proposed method offers accurate detection of gliadin in real gluten-containing samples.Moreover, two commonly used gluten-free grain samples were spiked with varying concentrations of gliadin.The recovery values, ranging from 93.9 to 107.1% (Table 2), suggest that the developed system is interference-free from the matrices of corn and cassava flours, further proving its potential for practical uses.

■ CONCLUSIONS
The feasibility of utilizing an ultrathin Parylene double-layer as the functionalized insulation conformal coating for capacitive  aptasensing of gliadin has been successfully identified in the present study.This label-free, reagentless, nonfaradaic biosensing approach can be realized through a very simple experimental setup, making it readily available to construct a portable device by incorporating a commercial integrated circuit chip designed for switched capacitance measurement.This advancement is beneficial for performing low-cost, convenient, and real-time field analysis of gluten-containing substances in the near future.Although our LOD value (14 ppm for gluten) still fulfills the European legislated threshold for gluten-free product verification, it is expected to be further lowered by improving the fabrication process of the electrode pair to obtain both a higher aspect ratio and a flattened undulatory electrode surface, which is currently under investigation.

Figure 1 .
Figure 1.Schematic illustration of the capacitive aptasensor for gliadin analysis.Parylene C and Parylene AM were sequentially coated onto an SPCE via CVD, followed by immobilization of the 5′-NH 2modified Gli4 aptamer onto the sensing area using glutaraldehyde.The prepared SPCE was connected to the LCR meter probes and immersed in the testing solution.The change in capacitance resulting from the affinity binding between aptamer and gliadin was monitored and calculated for quantification.

Figure 2 .
Figure 2. Thickness, capacitance (circles), and D value (squares) of Parylene AM coatings with CVD time from 0.5 to 4 h (N = 5).Capacitances and D values were recorded by an LCR meter at 0.5 V, 10 kHz for 10 min in the PBS + solution, and all consecutive data points were used for statistical analysis.The error bars indicate standard deviation of duplicate experiments.

Figure 3 .
Figure 3. Capacitance (circles), and D value (squares) of Parylene C coatings with thickness from 100 to 1000 nm (N = 5).All experimental conditions were the same as described in Figure 2. The error bars indicate standard deviation of duplicate experiments.

Figure 4 .
Figure 4. Effect of overnight immersion (12 h) in PBS + solution on capacitance measurement for Parylene double-layer coatings (N = 5).The double-layer comprised of a Parylene C thickness ranging from 500 to 1000 nm as the bottom layer, and a fixed Parylene AM thickness of 400 nm on top.Circles: without overnight immersion; triangles: with overnight immersion.All experimental conditions were the same as described in Figure 2. The error bars indicate standard deviation of duplicate experiments.

Figure 5 .
Figure 5. Fluorescence image of the 1400 nm Parylene double-layer coating with FITC dye.

Figure 6 .
Figure 6.Top view of surface topography: (A) bare SPCE, and (B) SPCE coated with 1400 nm Parylene double-layer.The black curve illustrates the relative height of the scanned area.The central blue region indicates the underlying SPCE substrate, while the adjacent areas in orange, yellow, and green represent the carbon paste electrode pair.

Figure 8 .
Figure 8.Typical capacitive response of gliadin aptasensing.The black curve represents the baseline in a 2 mL PBS + solution.The other overlaid curves depict the replacement of 0.2 mL of PBS + solution with 0.2 mL of gliadin sample solution.Replacement with 70% ethanol (the solvent of gliadin, shown in green), 0.01 mg/mL gliadin (blue), 0.1 mg/mL gliadin (yellow), and 1.0 mg/mL gliadin (orange).Capacitance was recorded by an LCR meter at 0.5 V, 10 kHz for 60 min.

Table 1 .Figure 9 .
Figure 9.Comparison of the analytical results of 24 commercial wheat flour samples determined by the proposed capacitive aptasensor (N = 3) and by a gliadin ELISA assay (N = 3).The error bars indicate standard deviation of duplicate experiments.

Table 2 .
Recovery Tests of Spiked Corn and Cassava Flour Matrices (N = 3)