Ultrasensitive protein concentration detection on a micro/nanofluidic enrichment chip using fluorescence quenching

A micro/nanofluidic enrichment device combined with the Förster resonance energy transfer (FRET) technique has been developed for sensitive detection of trace quantities of protein. In this approach, sample protein is first adsorbed on gold nanoparticles (AuNPs) to occupy part of the AuNP surface. Then, dye-labeled protein is added, which adsorbs to the residual active sites of the AuNP surface, saturating the AuNP surface with protein molecules. The unadsorbed dye-labeled protein remains in a free state in the system. Keeping a fixed amount of dye-labeled protein, a high concentration of sample protein leads to more free dye-labeled protein molecules remaining in the system, and thus a larger photoluminescence signal. Under the action of an electric field, the free dye-labeled protein molecules can be efficiently enriched in front of the nanochannel of a micro/nanofluidic chip, which greatly amplifies the magnitude of the photoluminescence and improves the detection sensitivity. As a demonstration, bovine serum albumin (BSA) and fluorescein isothiocyanate-labeled dog serum albumin (FITC-DSA) are used as sample and fluorescent proteins, respectively. Using the proposed strategy, a detection limit of BSA as low as 2.5 pg/mL can be achieved, which is more than 10³ times lower than the reported minimums in most sensitive commercial protein quantification methods.