Research On Optical Nano-biosensor Based On Exonuclease-assisted Sgnal Amplification

The detection of ultra-low concentration samples is of great significance in many fields such as disease diagnosis,pathogen detection,gene diagnosis and treatment,and the stoichiometric ratio of traditional detection methods is 1:1,which greatly limits its sensitivity and application range.In order to achieve ultra-sensitive detection of samples,people have successively proposed some signal amplification strategies based on biomolecules.Currently,signal amplification optical biosensing strategies have disadvantages such as multiple labels and cumbersome processes.How to improve and develop new signal amplification optical sensing methods is a difficult problem faced by biomedical and chemists.Based on a variety of tool enzymes as an auxiliary signal amplification technology,it has many advantages such as low equipment cost,simple operation,high sensitivity,strong specificity,short reaction time,and relatively mild reaction conditions.It is widely used in biochemistry,environment,and food.And so on in the analysis and testing,and has achieved rapid development.Based on the above problems,a label free biosensor system was constructed with kanamycin and DNA methyltransferase（DNMT）as the analysis objects,aptamer as the recognition element,nucleic acid tool enzyme,nano materials,hybridization chain reaction and other signal amplification technologies,and applied to the analysis and detection of milk and serum samples.This paper is divided into four chaptersChapter 1 Introduction summarizes the principles and characteristics of nucleic acid aptamers and biosensing,expounds the classification and sensing mechanism of optical biosensing,and focuses on the recent research progress of signal amplification optical biosensing.The purpose and significance of the research are briefly described.Chapter 2 Preparation of Nanomaterials:With the rapid development of new materials,nanomaterials have a place in this field with higher strength,toughness and sensitivity.Optical sensing technology combines nanomaterials as specific identification elements to form nano-optical sensors with high sensitivity,which are widely used in biomedicine,fine chemicals,gas sensing and other fields.Because the biggest problem restricting the development of nanomaterials is the preparation of nanomaterials,so nanomaterials are prepared by various methods to solve this problem.First,Au NPs are obtained by seed growth method and sol method,and nanospheres and Au NSTs of different particle sizes are prepared according to the selected surfactant and particle material.The synthesized nanomaterials were characterized by ultraviolet-visible spectroscopy,surface-enhanced Raman spectroscopy,transmission electron microscopy and scanning electron microscopy.Chapter 3 In this study,double-stranded DNA（ds DNA）probes were synthesized by manipulating the aptamer and its complementary DNA（c DNA）to ensure the detection of targets with high selectivity and excellent sensitivity.Here,protamine can not only bind to negatively charged gold nanoparticles,but also interact with polyanionic DNA.After adding the target kanamycin,a target-aptamer complex is formed and c DNA is released.Therefore,both the aptamer and the c DNA can be digested by Exo I,and the captured kanamycin is released to trigger the target cycle and signal amplification.Under optimized conditions,the proposed colorimetry method achieves a low detection limit of 2.8×10^-14 M,a wide linear range and excellent selectivity.Our strategy demonstrates the great potential of manufacturing various biosensors based on target-induced aptamer configuration changes.Chapter 4 This chapter uses DNMT to specifically recognize and catalyze the conversion of5’-GATC-3’base sequence generation 5’-G-Am-TC-3’,restriction endonuclease（Dpn I）selectively recognizes and cleaves 5’-G-Am-TC-3’to release the principle of single strands,using the highly efficient hydrolysis properties of Exo III（That is,it can only hydrolyze the double-stranded DNA containing the flat head and the DNA at the 3’end of the recess and release the remaining DNA.If the 3’end protrudes more than 4 bases,it will not be decomposed）,by regulating the recognition probe HP and signal amplification DNA（C1DNA,C2DNA）base composition and structure,to achieve the double amplification of the system,combined with the separation characteristics of magnetic nanomaterials and the SERS signal enhancement mechanism on the surface of nanomaterials,to construct a suitable SERS sensing interface for the analysis and detection of DNMT.Under optimized conditions,the proposed method achieves a low detection limit of 0.15 U,a wide linear range and excellent selectivity.In addition,this new type of sensing system also has potential versatility.By changing the substrate,it is convenient to design the recognition element DNase for other target analytes.