Immune-independent and label-free fluorescent assay for Cystatin C detection based on protein-stabilized Au nanoclusters

Cystatin C (Cys C) is a significant cysteine protease inhibitor in human bodies, and is proposed as a fascinating novel marker of glomerular filtration rate for kidney injury detection. Almost all traditional methods for Cys C measurement are immunoassays. In this article, we report a simple, immune-independent (no need to rely on immunoassay) and label-free method for Cystatin C detection using BSA-stabilized Au nanoclusters (Au NCs) as a fluorescent probe. This method relies on the BSA scaffold degradation caused by the cysteine protease activity of papain and the specific inhibition of papain activity by Cys C. The fluorescence of BSA-Au NCs can be effectively quenched by papain, and restored by the coexistence of Cys C. Under optimized conditions, this method enables sensitive and selective measurement of Cys C concentration in the range of 25ng/mL–2.0μg/mL with the detection limit of 4.0ng/mL, which is above 40 fold lower than that of commercial immune-based methods. SDS-PAGE, the absorption spectroscopy, transmission electron microscope, dynamic light scattering, and X-ray photoelectron spectroscopy were performed to discuss the quenching mechanism. In addition, percentage recoveries of Cys C in the spiked urine samples were ranged from 102.2% to 114.9% with the relative standard deviation ranging from 0.9–1.8%, demonstrating the applicability of the developed method in clinical samples. Furthermore, the present approach would be potentially extended to other proteases and their inhibitors detection with different protein-stabilized Au NCs. ► A simple, immune-independent and label-free method for Cystatin C detection was developed. ► Cys C can be selective and sensitive detected using BSA-Au NCs as a fluorescent probe. ► The quenching mechanism of Au-NCs was primarily elucidated.