Unmodified gold nanoparticles (AuNPs)-based aptasensing (uGA) assay has been widely implemented in the determination of many different targets, but there are few reports on protein detection using uGA. Here, we designed a uGA assay for protein detection including the elimination of interfering proteins. Positively charged protein can be absorbed directly on the surface of AuNPs to form “protein corona”, which results in the aggregation of AuNPs even without salt addition, thereby preventing target protein detection. To overcome this problem, we systematically investigated the effect of modifying the pH of the solution during the uGA assay. A probe solution with a pH slightly higher than the isoelectric points (pI) of the target protein was optimal for protein detection in the uGA assay, allowing the aptamer to selectively detect the target protein. Three proteins (beta-lactoglobulin, lactoferrin, and lysozyme) with different pI were chosen as model proteins to validate our method. Positively charged interfering proteins (with pIs higher than the optimal pH) were removed by centrifugation of protein corona/AuNPs aggregates before the implementation of actual sample detection. Most importantly, the limit of detection (LOD) for all three model proteins was comparable to that of other methods, indicating the significance of modulating the pH. Moreover, choosing a suitable pH for a particular target protein was validated as a universal method, which is significant for developing a novel, simple, cost-effective uGA assay for protein detection.
Selective Target Protein Detection Using a Decorated Nanopore into a Microfluidic Device
