Construction Of Biosensor By DNA Precisely Regulating Fluorescence Signal Transformation Of Silver Nanoclusters

High sensitivity detection of disease-related nucleic acid sequences is of great significance for early diagnosis,clinical treatment and prevention of virus transmission.At present,most of the common fluorescent biosensors for nucleic acid sequence detection use commercial organic dyes or semiconductor quantum dots as the fluorescence signal output,but they are limited by their high biological toxicity and low photostability.As a new type of fluorescent nanomaterials,silver nanoclusters(AgNCs)have the advantages of simple synthesis,high fluorescence quantum yield,adjustable excitation and emission wavelengths,good photobleaching resistance and biocompatibility.In this paper,we use fluorescent agncs as the signal output,and construct a biosensor with high sensitivity and universality based on Y-type DNA conformation conversion and exonuclease assisted target cycle signal amplification strategy to realize the detection and analysis of different target DNA.Specifically,we designed the whole sensor as two parts:fluorescence probe system and target cycle signal amplification system.The fluorescent probe system is a Y-type DNA three strand structure to regulate the signal change of AgNCs fluorescent probe,in which two DNA strands are used as the probe strand,and the third strand interacts with the trigger strand generated by the signal amplification system to transform the Y-type DNA structure and realize the regulation of the fluorescence of the probe strand.The signal amplification system is composed of a hairpin DNA structure and an exonuclease,which can respond to the target with high sensitivity and release the initiation chain.The characteristic of this strategy is that it does not need to change the design of fluorescent probe DNA for target transformation,so the design is simple and universal.Its universality has been successfully verified in the following two DNA detection systems.(1)In the first part of our work,we synthesized AgNCs in situ as fluorescence signal,and used gold nanoparticles(AuNPs)with the same excellent biocompatibility as the quenching group to design two probe chains bound to Y-type DNA fluorescence probe system.Y-type DNA(Y-DNA)is formed by hybridization of the third strand of L-DNA(partially complementary to the two probes respectively),which leads to fluorescence resonance energy transfer between AgNCs and AuNPs and turns off the fluorescence signal of AgNCs.After the addition of target T-DNA,it hybridizes with the hairpin HP in the target cyclic signal amplification system to form HP/T-DNA double strand,which is recognized by Exo Ⅲ and hydrolyzed to form trigger strand.The released T-DNA can combine with the next HP,and a large number of trigger strand can be obtained through multiple cycles.Then,the trigger strand was combined with L-DNA to form a trigger/L-DNA double strand by strand replacement reaction,and the two probe strands were de helix into a single strand to restore the yellow fluorescence signal of AgNCs and achieve high sensitivity detection of target T-DNA.There was a good linear relationship between fluorescence intensity and T-DNA concentration in the range of 0.0150 nm,and the detection limit was 28 pM.(2)In the second part,in order to improve the detection performance and verify its universality,we constructed a ratio fluorescent probe sensor to detect HIV gene sequence of human immunodeficiency virus.First,the original sequence of Y-type DNA triple strand structure was unchanged,but only the AgNCs template was added to construct ratio probe.In situ reduction,Y1/AgNCs,Y2/AgNCs and Y3/AgNCs were obtained.Y1/AgNCs used the original third chain sequence to respond to trigger chain.Because we don’t need to introduce AuNPs as quenching group,the experiment is more convenient.When the target HIV is not present,the three probes can cross to form Y-DNA,and at this time,AgNCs forms dimer structure and emits red fluorescence.When the target HIV exists,the hairpin HP in the target response system responds to it and circulates to get a large number of trigger chains under the action of ExoⅢ.Then,the trigger/Y1-DNA is formed by the combination of chain replacement and Y1-DNA,which leads to the helix of all probe chains to single strand,and the agncs fluorescence signal is restored to yellow.Unlike the previous on off signal switch,the high sensitivity detection of target HIV gene sequence is achieved by monitoring the ratio of yellow fluorescence of AgNCs monomer and red fluorescence of AgNCs dimer.The results showed that the two fluorescence intensity ratios and HIV concentration had a good linear relationship in the range of 0-0.02 nm and 1-500 nm.The detection limit was 2.2 pM,which was 13 times higher than the previous work.