A New Method For Detecting Enzyme Activity Based On DNA - AgNCs

Methods for the rapid, accurate and sensitive detections of biomolecules such as nucleic acids, protein and enzyme activity have always been of high interests in analytical chemistry and biomedical research field. Enzymes are produced within living cells with catalytic function, it dominates the metabolism of organisms in various reaction and energy conversion, making the body to maintain normal life activities. Proteases and nucleases play the important roles in organism, such as, heredity, mutation, growth, development, and the body’s metabolism, its structure or content change will cause the body function disorder. Therefore, it is critical to detect the activities of proteases and nucleases.The emergence of new nanomaterials and the development of nanotechnology have provided with new tools for bioanalysis. Recent years, DNA templated silver nanoclusters (DNA-AgNCs) have attracted extensive attention in bioanalysis. The DNA-AgNCs consists of a few silver atoms and possess many excellent fluorescent properties including good quantum yield, tunable fluorescence emission, high photostability, low biological toxicity and excellent biocompatibility. These advantages make DNA-AgNCs widely used in detect metal ions, biomolecules, DNA, RNA, proteins and furthermore bioimaging. Graphene is a single-atom-thick and two-dimensional carbon nanomaterial, its unique DNA absorbing ability and energy-transfer properties have been widely applied in biomolecular detection. Because of high importance on enzyme activity detection and its inhibitor screening, some fluorescent bioanalytical methods was developed for the detection of protease and nuclease activity in combination with the advantages of nanomaterials. Compared with the conventional fluorescence analysis, our proposed methods are simple, no labling, low cost and high sensitivity. In addition, the practicabilities of these methods were also tested. The main contents of this paper as follows:In chapter one, several main nanomaterials in the application of optical analytical methods were briefly introduced. Then, the detection methods of protease and nuclease summarized.In chapter two, based on DNA-templated silver nanoclusters and GO, a new fluorescent biosensing method for assay of trypsin activity and its inhibitor screening has been developed. Peptide probe with six arginine and a cystine has been designed, the probe can bond with DNA-AgNCs through Ag-S bonding, thus making the of the enhancement of the fluorescence of DNA-AgNCs. After the addition of GO, the peptide was adsorbed to the surface of negativity GO, the fluorescence of DNA-AgNCs was quenched by FRET. While trypsin was added, the peptide was degraded into the fragments of amino acids, which resulted that these fragments were released from the surface of GO, FRET disappeared, and the fluorescence of DNA-AgNCs was enhanced. Trypsin with an activity as low as 1 ng/mL can be determined with a linear range of 0-50 ng/mL. This method is simple, sensitive and label-free and can be successfully used for the determination trypsin in serum.In chapter three, a label-free fluorometric method for the detection of S1 nuclease activity and its inhibitor screening has been developed by using DNA-templated silver nanoclusters. Polycytosine oligonucleotide(dC12)can function as both the template for the stabilization of AgNCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without SI nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5×10-8 U μL-1 can be determined with a linear range of 5×10-7 to 1×10-3 U μL-1. The developed method for S1 nuclease is sensitive, facile and does not require any complicated labeling. RPMI 1640 cell medium was used as the complex fluid and spiked with S1 at different concentrations, recovery rate is 94%～98%, which indicates that this method can be used in the analysis of practical samples.