Development of a Thiolated PAMAM-Modified Aptamer-Based Impedimetric Biosensor for Zonulin Measurement as a Biomarker...

Altered intestinal permeability is closely associated with gastrointestinal inflammation, and zonulin/pre-haptoglobin-2 has been investigated as a mediator and biomarker of tight junction regulation. This study describes the development of an aptamer-based electrochemical impedance spectroscopy (EIS) biosensor for zonulin measurement. A 40-nucleotide DNA aptamer candidate, ZON-Apt-40 (5'-GGTTGCGTACGATGGTAGCGTTGACCTGCGTACCAAGTTC-3'), was selected based on its length, balanced GC content, absence of long homopolymeric regions, predicted stem-loop formation, and compatibility with thiol-mediated immobilization. Mfold analysis predicted two low-energy secondary structures; the most stable had a Gibbs free energy of -5.63 kcal/mol and contained an exposed loop/bulge region spanning nucleotides 17-23. A DNA-like three-dimensional model of the aptamer was docked against a human zonulin model, and the predicted binding pose suggested that the exposed loop region interacted with polar and positively charged protein residues. Experimentally, a gold microelectrode was modified with thiolated poly(amidoamine) dendrimer (PAMAM-SH), followed by immobilization of AuNP-conjugated thiolated ZON-Apt-40 and blocking with human serum albumin. FTIR confirmed PAMAM-SH functionalization, whereas cyclic voltammetry and EIS verified stepwise electrode modification. Chronoimpedance measurements identified 200 s as a practical readout time. The sensor exhibited a concentration-dependent EIS response to zonulin, with the calibration equation y = 12.276x + 1509 and R² = 0.9981 ± 0.0012. The calculated limits of detection and quantification were 0.949 and 2.877 ng/mL, respectively. Zonulin-spiked serum samples yielded coefficients of variation of 5.1% and 8.3% at 10 and 50 ng/mL, respectively. Overall, the platform demonstrates preliminary feasibility as a rapid, low-volume, label-free impedimetric aptasensor for zonulin detection; however, further validation using larger clinical sample sets and orthogonal binding assays is required.