A New Sensing Platform Based in CNF-TiO2NPs-Wax on Polyimide Substrate for Celiac Disease Diagnostic

Abstract

Celiac disease (CD), a human leukocyte antigen-associated disorder, is caused by gluten sensitivity and is characterized by mucosal alterations in the small intestine. Currently, its diagnosis involves the determination of serological markers. The traditional method for clinically determining these markers is the enzyme-linked immunosorbent assay. However, immunosensors offer sensitivity and facilitate the development of miniaturized and portable analytical systems. This work focuses on developing an amperometric immunosensor for the quantification of IgA antibodies against tissue transglutaminase (IgA anti-TGA) in human serum samples, providing information on a critical biomarker for CD diagnosis. The electrochemical device was designed on a polyimide substrate using a novel solid ink of wax and carbon nanofibers (CNFs). The working electrode microzone was defined by incorporating aminofunctionalized TiO₂ nanoparticles (TiO₂NPs). The interactions and morphology of CNFs/wax and TiO₂NPs/CNFs/wax electrodes were assessed through different characterization techniques. Furthermore, the device was electrochemically characterized, demonstrating that the incorporation of CNFs into the wax matrix significantly enhanced its conductivity and increased the active surface area of the electrode, while TiO₂NPs contributed to the immunoreaction area. The developed device exhibited remarkable sensitivity, selectivity, and reproducibility. These results indicate that the fabricated device is a robust and reliable tool for the precise serological diagnosis of CD.

Keywords: celiac disease diagnosis; electrochemical; immunosensor; nanomaterials.

Figure 1

Design and fabrication of the three-electrode system using wax-printed hydrophobic barriers and selective application of CNFs/wax and TiO₂NPs/CNFs/wax inks. Electrochemical activation was carried out in a buffer solution at pH 4.5 at −9 V for 10 s to prepare the working electrode.

Figure 2

Schematic representation of the immobilization of TG onto TiO₂NPs via glutaraldehyde crosslinking and subsequent IgA anti-TGA antibody detection. The electrochemical signal, generated by HRP-catalyzed oxidation of catechol, was proportional to the antibody concentration in serum samples.

Figure 3

(a) shows the corresponding CVs of blank for CNFs/wax/polyimide electrode recorded in PBS solution 0.1 mol L⁻¹ pH 6.50 (green line), CNFs/wax/polyimide electrode (black line), and TiO₂NPs/CNFs/wax/polyimide electrode (red line) in a 1 mmol [Fe(CN)6]^4−/3− redox couple solution in PBS 0.1 mol L⁻¹ pH 6.5; (b) exhibits CVs for the same electrode platforms in 1 mm L⁻¹ of Q solution in a 1 mm L⁻¹ citrate-phosphate buffer at pH 5.0; (c) shows the influence of scan rate on the peak current obtention; and (d) EIS measurements for the electrode platform before and after TiO₂NPs incorporation.

Figure 4

SEM images show the morphological changes on the wax/polyimide electrode surface after incorporating CNFs and TiO₂NPs at different magnifications ((a) wax surface image, (b) TiO₂NPs/CNFs/wax at 400×, (c) TiO₂NPs/CNFs/wax at 800×) and (d) EDS spectrum, which confirms the presence of carbon and titanium, indicating successful integration of the nanocomposite components.

Figure 5

(a) shows XRD patterns of wax (black line) and TiO₂NPs/CNFs/wax (blue line); and (b) shows the FTIR spectrum of wax, CNFs/wax and TiO₂NPs/CNFs/wax.

Figure 6

Electrochemical characterization of the nanocomposite inks shows that increasing CNF to a concentration of 35% enhances conductivity (a). Similarly, TiO₂NPs reached maximum electrochemical response at 20 mg (b).

Figure 7

(a) shows the optimization of reaction time and (b) exposes the correlation between the proposed method and the spectrophotometric ELISA reference method.

Figure 8

Cross reactivity evaluation: IgA anti-TGA (50 U mL⁻¹) detection in presence of albumin (110,000 mg dL⁻¹) (A), creatinine (3 mg dL⁻¹) (C), immunoglobulin A (700 mg dL⁻¹) (IgA), glucose (200 mg dL⁻¹) (G), and immunoglobulin G (4000 mg dL⁻¹) (IgG).