Construction Of Optical DNA Nanosensor And Its Application In The Detection Of Ribonuclease H

Ribonuclease H（RNase H）can specifically hydrolyze the RNA strands in DNA/RNA duplex to release DNA strands,and plays an important role in important cellular processes such as DNA replication,repair and transcription.Therefore,it is very important to develop a method for detecting RNase H activity with high sensitivity.In this paper,two optical DNA nanosensors are constructed,and their multiple advantages in the process of signal recognition,signal conversion and signal amplification have been used to achieve high-sensitivity detection of RNase H.Firstly,the DNA walker driven by DNAzyme is used to assemble a fluorescent nanoprobe,which is used to detect RNase H,which realizes high detection performance of the target.The operation process is simple and it shows excellent specificity.Then use the DNA strand displacement reaction and use Surface Enhanced Raman Scattering（SERS）,which make use of non-destructive,good response,and fast detection speed.Combined with the good point of easy separation of magnetic beads,it further realizes the colorimetric and SERS dual-mode multi-signal rapid detection of RNase H.This work has reference significance for the subsequent screening of anti-AIDS drugs and the research of biological drugs.The main research contents and achievements are as follows:1.The DNA walker driven by DNAzyme for sensitive detection of ribonuclease HA signal amplification strategy driven by DNAzyme on the surface of nanoparticles was proposed to detect the activity of RNase H,the detection range is 0 U/m L-5 U/m L,and the detection limit is as low as 0.0085 U/m L.Firstly,the blocked enzyme(Mn²⁺-DNAzyme)chain and substrate chain are modified on the surface of 13 nm gold nanoparticles（Au NPs）to make them a DNA walker probe.One end of the substrate chain is modified with FAM fluorophore,when RNase H is not present,the fluorescence of the system is very weak due to the good fluorescence quenching effect of Au NPs.In the presence of RNase H,since Au NPs quench the fluorescence of FAM,the observed fluorescence signal is extremely small.With the addition of Mn²⁺,the activated enzyme chain cleaves from the RNA bases on the substrate chain,the released FAM restores its fluorescence signal due to its departure from Au NPs.Finally,the enzyme chain modified on the surface of Au NPs moves on the Au NPs like a machine and continuously cuts the substrate chain to release the fluorescent group,which acts as a signal amplifying function,thereby indirectly detecting the activity of RNase H.This method has been proved by experiments that it has good performance in detecting RNase H in cell lysates and can be used for inhibitor screening.2.Colorimetric and SERS dual-mode magnetic separation DNA nanosensor for sensitive detection of ribonuclease HThe Raman probe Au@4-MBA@Ag NPs with SERS signal was synthesized,and the functional DNA strand was modified.Similarly,after the magnetic beads are incubated with gold particles,they modify the functional DNA strands to become capture probes.When RNase H is present,the RNA in DNA/RNA is hydrolyzed and then released DNA single strands.Once the released DNA single strands are complementary to the S₂-DNA on the Raman probe,the SERS probe will not interact with S₁-DNA which on the surface of Fe₃O₄@Au NPs so that they will not be captured.On the contrary,the SERS probe will be captured,thus forming a satellite structure of Fe₃O₄@Au NPs-Au@4-MBA@Ag NPs.After magnetic adsorption,the SERS signal of the supernatant can be measured to qualitatively RNase H and quantitative analysis.At the same time,since the SERS probe has its own color and G-quadruplex on the surface,after adding hemin for incubation,adding TMB color reagent and H₂O₂ can also be used to visually observe the reaction results.This experiment solves the shortcomings of the previous working,such as signal single output and long detection time.It has the advantage of simple operation,the detection range is 0 U/m L-2.5 U/m L and its detection limit as low as 0.0018 U/mL.