Laser-Induced Detection Of Thrombin Based On Gold Nanoparticle Aptamer Sensor

Objective: A nucleic acid aptamer is a short-chain DNA,RNA,nucleic acid analog or peptide fragment,which can specifically binds to substances such as drugs or proteins.With its high sensitivity and good specificity,it is often used as a carrier for drug delivery,an identifier for gene therapy,and a sensor component for sensitive detection.Thrombin is a biologically active protein that plays an important role in the anticoagulant and procoagulant coagulation cascades of organisms.The drug thrombin can be used for hemostasis of surgery and trauma by local spray,oral or local perfusion.In addition,thrombin has a regulatory effect on the growth of tumor cells,which plays an important role in activating tumorigenic of normal cells and malignant cell metastasis.The detection of thrombin has important clinical diagnostic significance.Methods: A novel,simple and sensitive thrombin detecting method was designed by laser inducting nanogold-modified ds DNA(double-stranded DNA)dehybridization.Then releasing single-strand aptamer and specifically bind to thrombin.Firstly,16 nm gold nanoparticles were prepared by a method of reducing chloroauric acid with sodium citrate.Secondly,TCEP was used to reduce the terminal disulfide bond of the aptamer and to form a double-stranded structure with the complementary strand.After that,the double strands were combined with the gold nanoparticles to complete the assembly of the sensor.Laser-induced release based on nanoparticle composites has been widely used in the fields of gene therapy and drug delivery.Gold nanoparticles(Au NPs)have special physical properties.Local heating around the particles can be achieved by laser irradiation on the surface,so that the temperature around the nano-gold particles increases,and the overall temperature of the solution is almost constant.After different types of laser irradiation,the effects are different.Pulsed lasers tend to release DNA by destroying gold-sulfur bonds(Au-S)for drug delivery,while low-power continuous lasers often release single strands in a dehybridized form.It makes the functionality of single-strands well represented.CW laser-induced DNA release provides a broader perspective for the application of aptamers.Finally,the sensor's purpose for thrombin detection was achieved and optimized through conditional optimization,quantitative studies,selective studies,and recovery experiments.Results: The present syudy summarizes the research progress of nanogold nucleic acid aptamer sensor detection technology,and introduces the purpose,significance,problem solution,innovation and ideas of the experiment.A thrombin detection system based on the Au NPs aptamer sensor was constructed.Au NPs with an average particle size of 16 nm was successfully prepared.The 27-base thrombin single-chain aptamer was binded to its complementary strand,and the fluorescent material and Au NPs were separately modified at both ends of the aptamer to synthesize stable sensor.Through continuous(CW)laser irradiation,the single chain is released to the detection solution and bound to thrombin.The detection of thrombin is achieved by quenching by the Au NPs and fluorescent substances respectively modified on both ends of the aptamer.Optimizing the experimental conditions according to the experimental influencing factors.Conclusion: Quantitative analysis was performed to obtain a concentration-related fitting curve,and the detection limit of the method was 0.11 n M.A selective experiment was performed to confirm that the method was not affected by the interference and the selectivity was good.In human serum,it is verified that the detection method is still effective and has potential value extended to clinical trials.