Gold Nanoparticles-based Engineering Of The Biosensing Interface For Biodiagnosis

The early diagnosis of diseases is a great challenge in clinical medicine,and it is urgent to develop highly sensitive,specific,simple and portable biological diagnosis methods.In recent years,the rapid development of DNA nanotechnology and inorganic nanomaterials has brought new opportunities for the exploration of new biological diagnostic methods.Among many nanosensors,DNA-AuNPs has been widely used due to its excellent ability of biomolecular recognition,unique optical properties and good biocompatibility.In the construction of sensing interface,the heterogeneous self-assembly layer of DNA probes on the interface will seriously affect the biometrics and performance of the sensors.How to construct a biorecognition interface with high activity and high efficiency,reasonably regulate the uniformity and order of DNA probes on the nanoscale,make DNA fully exert its biological activity,and expand the application of DNA-AuNPs probes in biological diagnosis is the key scientific problem that this paper mainly solves.We modified the DNA probes with polyadenine block(polyA)tail on the surface of gold nanoparticles.By adjusting the length of polyA block,the density of DNA probes on the surface of gold nanoparticles can be controlled in a reasonable and orderly manner,which can effectively reduce the energy barrier for molecular recognition and improve the detection rate and sensitivity of sensors.The research work in this paper is based on accurate interface regulation,orderly regulation and improved the response performance of DNA-AuNPs probe in biomarker diagnosis.The main contents are as follows:1.Based on the method of preparing polyadenine block(polyA)DNA-AuNPs conjugation developed by the research group,polyA-mediated "nanoflare" probes based on fluorescence resonance energy transfer(FRET)were prepared for the detection and imaging of ATP in living cells.Compared with traditional single-dye probes,FRET nanoflare probes have better anti-interference performance in complex environments such as nucleases and glutathione.To obtain the best biological signal,the density of the probes on the AuNPs interface was reasonably adjusted to avoid the spatial obstruction caused by the over-densified probes.After optimization,the detection limit of the probes for ATP in the solution was 12.9 μM.Then,we delivered the probes into the cells,which can realize the imaging analysis of the intracellular ATP.The results showed that polyA-mediated FRET nanoflare probes responded quickly to intracellular ATP.The probes have broad application prospects in the detection of biomarkers in living cells.2.Based on the polyA-mediated DNA-AuNPs probes,we constructed a two-color "nanoflare" probes with sticky ends for detection and in situ imaging of two tumor-related m RNAs in living cells.The sequence of nanoflares with sticky ends was designed to be complementary to the target m RNA,which will be adhered to the target m RNA after recognizition,and then it will achieve the effect of in-situ analysis of m RNA expression quantification.The detection of dual targets can avoid the generation of false positive signals and improve the accuracy of diagnostic results.The detection limit of c-myc target gene and Gal Nac-T target gene were 1.45 n M and 2.32 n M.Compared with the thiol probes without interface regulation,the detection limit of the probe regulated by polyA was reduced by ～ 20 times.More importantly,the nanoprobes can not only distinguish cancer cells from normal cells,but also can reflect the different expression levels of specific tumor m RNA in cells.This strategy enables real-time in situ imaging of endogenous related m RNA in tumor cells,provides new tools for studying the function of endogenous m RNA,and has potential application value in biological system monitoring and drug delivery.3.We proposed the DNA-AuNPs colorimetric sensing strategy based on molybdenum disulfide,which was applied to the rapid visualization detection of target nucleic acids.The presence of the target nucleic acid will trigger the exonuclease III(EXO III)-mediated cyclic shearing reaction,effectively realizing the amplification of the target signal.We used the adsorption and aggregation phenomenon between the molybdenum disulfide nanosheet and the single cohesive end of the DNA-gold nanoprobe as the signal output,and achieved the in situ detection of target nucleic acid by naked eye observation or ultraviolet absorption spectrum.By orderly regulating the assembly density of the capture probes on the gold nanoparticles interface,we greatly improved the working activity of exonuclease III and the hybridization efficiency of DNA on the interface,and programmed and regulated the dynamic response range and detection sensitivity of the sensor.This strategy has a broad application prospect in medical diagnosis and portable detection.