A Label-free Fluorescence Assay for Trypsin Based on the Electron Transfer between Oligonucleotide-stabilized Ag Nanoclusters and Cytochrome c

Abstract

Herein we attempt to develop a simple, specific and costeffective fluorescent method for trypsin activity assay through the combination of Cyt c and Ag NCs. The assay principle is elaborated in Scheme 1. We firstly synthesize oligonucleotide-2014 © The Japan Society for Analytical Chemistry † To whom correspondence should be addressed. A label-free fluorescent assay for the detection of trypsin by using oligonucleotide-templated silver nanoclusters (Ag NCs) and cytochrome c (Cyt c) has been demonstrated. When negatively charged Ag NCs and positively charged Cyt c are mixed, they tend to form a hybrid complex, and then lead the fluorescence of Ag NCs to be quenched significantly due to electron transfer between Ag NCs and the heme cofactor of Cyt c. In the presence of trypsin, it catalyzes the hydrolytic cleavage of Cyt c to small peptide fragments, and releases the heme moiety from the Ag NCs/Cyt c complex; the quenched fluorescence restores therewith. By virtue of this specific response, the fluorescent biosensor has a linear range of from 0.7 to 4 μg mL -1 and from 9 to 120 μg mL -1 with a detection limit of 58.7 ng mL -1. Aside from the easy manufacture aspect, our method also possesses a high signal-to-background ratio (~11), excellent selectivity and good biocompatibility, which makes it a promising bioanalysis for a trypsin activity assay. Keywords

Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

“…Trypsin can be determined with a linear range of 0.0–50.0 ng/mL (inset in Figure 3C). The detection limit of trypsin was calculated as 1 ng/mL according to the rule of three times the standard deviation over the blank response, which was lower than that of some fluorescence assays reported recently [9,12,13,41,42,43]. The label-free properties of the method can offer the advantages of easy preparation of DNA-AgNCs, low cost, and operation convenience.…”

“…However, these methods require fluorescence labeling and require sophisticated equipment and laborious experiment procedures. Therefore, several label-free assays for trypsin activity have been developed recently [12,13]. However, simple, sensitive, and label-free assays for trypsin are still lacking [14,15].…”

A sensitive assay for trypsin using poly(thymine)-templated copper nanoparticles as fluorescent probes

“…This proposed method provides desirable sensitivity, with a 30 detection limit of 42 ng/mL, and also excellent selectivity, with no interference from other proteins. Additionally, compared with other reported approaches for trypsin assay, this biosensor also has potential advantages, such as easy operation without tedious synthesis processes for conjugated polymers or quantum dots; 35 low-cost without any labels or costly peptide. Therefore, this sensing technique may hold great promise for protease activity related biochemical applications.…”

“…35 According to previous studies, 23 reaction at a higher concentration of trypsin with an increased reaction rate.…”

“…Recently, peptide has been extensively utilized to construct various sensitive, labelfree biosensors for trypsin assay by fluorimetry, 10-18 35 colorimetry, 19,20 electrochemistry 21 or SERS. 22 However, most of these reported methods are based on conjugated polyelectrolytes, 10,11 quantum dots (QDs), 12-14 gold/silver nanoparticles, 15,20 which require sophisticated synthesis processes, many types of reagents and the toxicity of QDs.…”