DEVELOPING A NO-WASH ELECTROCHEMICAL IMMUNOASSAY FOR 25- HYDROXYVITAMIN D3 DETECTION

Candidate: Aaliya Pathan
Supervisor: Dr. Darius Rackus

Abstract:

Vitamin D3 deficiency is highly prevalent, making it one of the biomarkers for several chronic diseases. As a result, there has been a demand for portable testing platforms to enhance disease management and monitoring accessibility. Currently, determination of vitamin D3 levels relies on laboratory-based immunoassays which face challenges such as interference through non-specific adsorption and the need for washing to remove unbound labels, making it complex and time-consuming. To address this challenge, we report a wash-less electrochemical competitive immunoassay to measure free 25-hydroxyvitamin D3.  Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to monitor each modification step and quantify analyte binding. The assay exhibited strong selectivity for 25(OH)D3 in the presence of interferents such as bovine serum albumin (BSA), cholecalciferol, and complex matrices like infant formula, confirming the assay's potential for reliable POC diagnostics. Optimization of antibody orientation via protein A and VDBA14 significantly improved sensitivity and specificity from the no-wash physisorbed approach which generated a limit of detection (LOD) of 16.52 ng/mL compared to the LODs of the protein A assay being 5.22 ng/mL and the VDBA14 aptamer assay reporting 5.91 ng/mL. All three approaches generated a lower LOD than the reported levels of Vitamin D3 deficiency (20 ng/mL) showcasing that this work represents a significant advancement toward accessible, low-cost, and efficient vitamin D3 detection. By eliminating washing steps and utilizing electrochemical techniques, this work paves the way for broader implementation of equitable diagnostic tools in both clinical and remote settings.