Analysis Protein Based On Terminal Protection Of Small-molecule-linked DNA By Combining With Silver Nanoclusters And Gold Nanoparticles

Protein,an important component of cells,tissues and organs in the human body,is the material basis of all life.Protein is bounded up with many physiological activities of the human body,such as maintaining the body’s normal metabolism,ensuring all kinds of substances in the body’s normal delivery and ensuring the normal order of various immunization activities,and so on.Studies have shown that the occurrence of certain diseases in human is closely related to the abnormal expression of proteins.Therefore,the developing of a simple,rapid and sensitive method for proteins assay plays an important meaning in the studies of the protein properties and functions,clinical diagnosis of related diseases,and drug research.The terminal protection of small-molecule-linked DNA is that the DNA terminally labelled with small molecule can be protected from the degradation by exonuclease when the small molecule is specifically bound to its target protein.There is no coding restriction on DNA sequence in the terminal protection.The ends of DNA are easily marked.And the activities of DNA and labeled molecules are not affected by markup process.It is propitious to the combination of several nucleic acid amplifications.Terminal protection provides a new thinking for the simple detection of protein due to the above advantages.Therefore,in recent years,terminal protection has aroused wide interest of researchers in protein assay.In this dissertation,based on terminal protection of small-molecule-linked DNA,we have established two methods for protein detection combining silver nanoclusters(AgNCs)and gold nanoparticles(AuNPs),respectively.The main researches are as follows:1.AgNCs-based fluorescence analysis strategy for protein detection coupled to terminalprotection of small-molecule-linked DNA.In the experiment,a signal strand DNA(ssDNA)probe,whose 3’ terminal is labelled with a biotin molecule is planned.In the absence of target protein streptavidin(STV),the ssDNA probe is degraded from 3’ terminal by Exonuclease I(Exo I).With the addition of AgNCs with strong fluorescence,high fluorescence signal will be detected,when STV is present,it combines with biotin.The ssDNA probe is protected from the degradation by Exo I.The ssDNA probe can hybridize with the exposed DNA sequence of AgNCs,generating fluorescence quenching.The results of the present study indicate that the fluorescence intensities are linearly dependent on the amounts of STV in the range of 2 ~ 40 pmol.For STV,the detection limit is 1.82 pmol.The new method is simple,low-cost and high specificity,but the sensitivity should be improved further.2.AuNPs-based colorimetry strategy for protein detection coupled to terminal protection of small-molecule-linked DNA.In this experiment,we designed a ssDNA probe labelled to a small molecule-biotin.In the presence of STV,STV combines with biotin.The ssDNA probe is protected from the degradation by Exo I.The ssDNA probes adsorb on the surface of AuNPs and stabilize AuNPs against salt-induced aggregation.The red color of the AuNsP will be maintained as a function of STV concentration.In the absence of target protein STV,the ssDNA probe is degraded from 3’ terminal by Exonuclease I(Exo I).Without the protection of ssDNA probe,salt-induced self-aggregation of AuNPs will occur rapidly accompanying with color change from red to blue.The changes of color and absorbance are correlative with the concentration of target protein-STV.the ratios of absorbance at 550 nm and 700 nm are linearly dependent on the amounts of STV in the range of 2 ~ 20 pmol.The detection limit of STV is 1.40 pmol.This strategy is simple and low-cost,which enables visual detection of target protein only by bare eyes.