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

Abstract

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).

Keywords: Parylene AM; Parylene C; aptamer; gluten; insulation layer.

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′-NH₂-modified 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

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

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

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

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

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 7

Fluorescence image of the 1400 nm Parylene double-layer coating after immobilizing 5 μM of the 3′-FAM-labeled and 5′-NH₂-modified Gli4 aptamer through glutaraldehyde.

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.

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.