Novel Analytical Methods For Proteases Detection Based On Fluorescent Nanomaterials

Proteases play a crucial role in the physiological process of cellsignaling, cell growth， maintenance of morphology， and neural activity.Over-expression or activity imbalance of proteases is associated with manydiseases. Study on the structure of proteases and their function is a hot spotin life science. Proteases are also widely used in the field of diseasediagnosis and therapy, food processing and leather industry. Therefore,sensitive detection of proteases has valuble significance. Fulin colorimetricmethod is the most commonly used method for proteases activitydetermination. However, the relative poor repeatability and the comp licatedmanipulation of this method limitits application in rapid detection of a largenumber of actual samples. Therefore, it’s urgent to develop simple, accurateand sensitive methods to analyze the activity of proteases. Fluorescentnanomaterials, such as gold clusters, quantum dots, have great promise inbioanalysis due to their high fluorescence intensity, good light stability,large stokes shift and excellent biocompatibility.Based on the considerations above, we developed several methods forlable-free detection of proteases and cysteine by taking advantage of uniqueoptical properties of nano-materials (gold clusters and quantum dots). Thespecific works are summarized as follows:(1) A fluorescent analytical method for papain and cystatin C detectonwas constructed, based on a new fluorescence quenching mechanism ofBSA-Au Clusters (BSA-Au NCs). With the cysteine protease activity,papain can digest the BSA scaffold which coated on the surface of Au NCsandinduce the aggregation of Au NCs, which results in the fluorescencequenching of BSA-Au NCs. The fluorescence of BSA-Au NCs responsesvery well to the concentration of papain. Under optimized conditions, thismethod enables sensitive detection of papain concentration with thedetection limit of0.07μg/mL. This method was further extended to thedetermination of Cys C, the specific inhibition of papain, where thefluorescence of BSA-Au NCs can be effectively restored by the coexistenceof Cys C and papain. Compared to well-known immunoassay for cystatin C measurement, this immune-independent method has many advantages, suchas high selectivity, good stability, low cost, simple operation, norequirement for synthesis of expensive antibodies and comp licatedmodification process, and a low detection limit (4ng/mL). This methodhas been successfully applied for the detection of cystatin C in the practicalurine sample, and satisfactory recoveries for the spiked urine samplesanalysis were obtained (between102.2%and114.9%). Furthermore, thepresent approach would be potentially extended to other proteases and theirinhibitors detection with protein-stabilized Au NCs.(2) Since papain can be effectively activated by cysteine, a newcysteine analytical method was developed based on the papain fluorescentsensor constructed above. In this method, the fluorescence of BSA-Au NCscan sensitively response to cysteine concentration through the quantitiveregulation of papain activity by cysteine. This simple, lable-free cysteinesensor has good selectivity, low detection limit (15nM), and wide linearrange (0.02-10.0μM).(3) A novel label-free fluorescent assay for thrombin measurement wasdeveloped, which is based on the aggregation behavior change of theunmodified CdTe quantum dots. Through binding to the surface of CdTequantum dots, the positively charged substrate peptide (GGLVPRGSCC) canbalance the the negatively charge covered on the surface of the negativelycharge, and induce the aggregation of thelatter,, which results in thefluorescence quenching of CdTe quantum dots. After hydrolysis of thesubstrate peptide by thrombin, not the positive charge product (GGLVPR),but the uncharged fragment (GSCC) binds to the surface of CdTe quantu mdots. Hence, the CdTe quantum dots keep stability in the solution, and theirfluorescence is maintained. The activity of thrombin was achieved bydetecting the fluorescence intensity change of CdTe quantum dots. Thismethod for thrombin activity detection has low detection limit (1.5μU/mL)and the satisfactory recoveriesin the siked serum sa mples (between94.3%and104.7%). Compared with the previous methods, this method does notrequire complicated process for quantum dot modification, and thedetection is very fast (in1-2min). With the help of microplate-reader, theassay offers the possibility to achieve a high-throughput screening ofthrombin drugs.