High sensitivity detection of biotinylated molecules using a high-resolution resistive pulse sensor

Biotinylation is a widely used technique for tagging molecules to enable their detection, isolation, or immobilization. Reliable detection of biotinylated molecules is critical for maintaining the integrity of downstream processes and ensuring the accuracy of analytical and diagnostic assays. Here, we present a novel strategy for a highly sensitive analysis of biotinylated targets based on gold nanoparticle counting. This approach integrates a high-resolution microfluidic resistive pulse sensor with a competitive nanodimer formation assay. In this method, biotinylated targets inhibit nanodimer formation between biotin- and streptavidin-modified gold nanoparticles, resulting in fewer nanodimers being produced. The change in nanodimer quantity is then measured by the resistive pulse sensor, allowing both the presence and concentration of the biotinylated target to be quantified. Using biotinylated BSA as a model target, we demonstrated that changes as small as 0.7606 pg mL^-1 produced a significantly detectable shift in dimer ratio. Leveraging its single-particle detection capability, this strategy provides ultra-sensitive quantification with minimal calibration and sample preparation. The ability of our approach to universally detect biotinylated molecules holds great potential to advance a wide range of biotinylation applications in biotechnology, diagnostics and tissue engineering.