A Rapid Electrochemical DNA Biosensor for Serum Uric Acid Using the Pre-equilibrium Binding Mechanism

The precise and rapid detection of uric acid (UA) in human serum is pivotal for diagnosing and managing metabolic disorders such as gout, kidney disease, and hyperuricemia. However, current equilibrium-based assays suffer from long incubation time, interference from serum components, and a narrow dynamic range. Herein, we present a robust electrochemical DNA assay (EC-DNA) that can respond rapidly (within 1 min) to serum UA under pre-equilibrium conditions, with an extended dynamic range (1-1000 μM). Our assay employs a Mg²⁺-dependent UA aptamer that can form a metastable hairpin-like structure, allowing for rapid UA binding. The reaction mechanism was validated using electron transfer rate measurements, electrochemical melting experiments, and urea-induced denaturation assays. This pre-equilibrium approach minimizes nonspecific protein adsorption and signal decay, ensuring reliable detection in complex biofluids such as serum and saliva. Our sensor demonstrated a strong correlation with commercial colorimetric assays (Pearson r = 0.947) and successfully differentiated UA levels between healthy individuals and gout patients. Overall, our EC-DNA assay not only presents a rapid and robust method to detect UA in complex biological samples but also provides valuable insights into DNA binding assays under pre-equilibrium conditions.