Enzymatically-generated Long PolyT Templated-Copper Nanoparticles For Versatile Biosensing Assay Of DNA-related Enzymes Activity

Nanotechnology has given a new insight into the development and application of nanomaterials.In the modern science, combined with chemistry, biology, physics and medicine science, nanotechnology has become one of the most exciting forefront fields in developing the ultra-sensitive detective and imaging analysis. Owing to their small size(normally in the range of 1-100 nm), metal nanoparticles exhibit unique chemical, physical and electronic properties that are different from those of bulk materials, and can be used to construct novel and improved sensing devices; especially, electrochemical sensors and biosensors. Using the metal nanoparticles can improve the sensitivity, lower the detection limit of the sensor, and perform the work that other materials can't finish.In recent years, DNA-templated copper nanoparticles(DNA-CuNPs) have attracted considerable attention in the fields of biosensing and bio-imaging. With the advantages of small size, low biological toxicity and good biocompatibity, DNA-Cu NPs have emerged as promising fluorescent probes for biochemical assays. Terminal deoxynucleotidyl transferase(Td T), a unique DNA polymerase, can catalyze the polymerization without a template to involve in by addition of dNTP to the 3?-OH terminus of primer DNA with a length of three or more nucleotides. Td T is widely used as a molecular biology tool for labeling DNA end, rapid amplification of c DNA ends(RACE), and apoptosis cells detection. In the present study, we have demonstrated a new proof-of-concept of the synthesis of CuNPs based on DNA template generated by DNA polymerization via TdT. And this synthesis strategy can be applied to detection of nuclease activity.(1) We have firstly demonstrated a novel synthesis strategy for silver nanoclusters based on DNA polymerization by Td T. A short oligonucleotide primer could trigger polymerization in a template-free way by Td T to form a long polyT, which could effectively act as the template in the formation of fluorescent CuNPs. In comparison with short T-rich CuNPs, the fluorescence intensity of Td T-generated polyT-CuNPs was greatly improved. Some factors that may influence the synthesis of DNA-Cu NPs were also optimized. Furthermore, transmission electron microscope(TEM) was employed to characterize the DNA-CuNPs. We inferred that the enhanced fluorescene intensity of DNA-Cu NPs template by polyTcould be accounted for the two reasons:(1) Longer DNA sequence could provide better protection for Cu NPs and remove the polar solvent from the surface of the Cu NPs, leading to a fluorescence enhancement.(2) The fluorescence resonance energy transfer(FRET) effect probably occurs and leads to an enhanced fluorescence emission. Finally, the photostability turned out to be quite satisfactory.(2) Based on this novel DNA-CuNPs synthesis strategy, a “turn-on” and label-free method for TdT activity detection was developed with a very low limit of detection(LOD) which was 3.75 U/m L. Additionally, this detection method has a very good reproducibility and recovery. Finally, one common inhibitor of Td T was investigated.(3) Based on the TdT polymerization and synthesis of DNA-CuNPs, a “turn-on” method for nuclease detection was developed universally. Restriction endonuclease(Bam HI) and alkaline phosphatase(ALP) were chosen as the target analytes. The resu ls show that the detection system has very good sensitivity because there is no pre-existing T-rich sequence without cleavage of nuclease and polymerization of TdT. Excellent selectivity was also obtained because of the substrate specificity of nuclease. T his detection system can be extended to detection of other enzymes just with some adjustion of the substrate sequences.(4) Endonuclease V(EndoV), a specific nucleotide excision repair(NER) endonuclease, recognizes deoxyinosine, a deamination product of deoxyadenosine in DNA. Based on the special recognition of deoxyinosine of Endo V and nicking enzyme Nt.BbvCI, the DNA-Cu NPs synthesis strategy was applied to the amplified “turn-on” detection of Endonuclease V. The detection of trace Endonuclease V was a chieved after rational design of three DNA probes-a substrate probe, a complementary probe and an amplification probe. This strategy can be well extended to the assay of other analytes after some changes on the substrate sequence.