Research On The Method And Application Of Ratiometric FRET Analysis Based On Aptamer And CDNA

Fluorescence Resonance Energy Transfer(FRET)refers to the process of non-radiative energy transfer between a fluorescent donor and an acceptor within a certain distance range(10-100?).This process causes the intensity of the donor’s fluorescence to weaken,and the acceptor is excited or not excited to fluoresce.This technology has been widely used in analytical detection,biosensing,optical imaging and other fields because of its fast response,simple operation and no need to clean.In the development process of the FRET method based on c DNA(Complementary Deoxyribonucleic Acid),the substance is detected either indirectly by competing with the c DNA or by c DNA-triggered strand displacement reaction.Therefore,the optimal design of c DNA is very necessary,which plays a very important role in improving the sensitivity of the method.However,few researchers have carried out more comprehensive optimization design on it.In addition,the general method mostly uses singlepeak off-on or on-off fluorescence signal to detect the substance to be tested.However,the fluorescence signal is susceptible to many factors,which is unfavorable for quantitative detection of the target substance.In order to solve these problems and facilitate the establishment of related methods,the study has established a ratiometric FRET detection method based on aptamer and c DNA.Ochratoxin A(OTA)is a mycotoxin mainly produced by Aspergillus and Penicillium,which easily contaminates a variety of foods and Chinese medicinal materials,such as cereals,wine,coffee,glycyrrhiza,nutmeg,etc.Studies have shown that the OTA has strong carcinogenic,teratogenic and hepatorenal toxicity on the human body,and is classified as a potential 2B carcinogen,which seriously threatens human life and health.Therefore,it’s very necessary to develop a rapid,simple,highly sensitive,selective and reliable OTA analytical detection method.Aptamers are single-stranded nucleic acid sequences selected in vitro by Systematic Evolution of Ligands by Exponential Enrichment(SELEX),which can specifically recognize target molecules.In recent years,aptamers have been used as capture probes to analyze analytes because of their high affinity,strong specificity,easy synthesis and modification,and low cost.It can be compatible with a variety of signal modes,such as fluorescence,electrochemistry,mass spectrometry,colorimetry,etc.As a result,it can develop a variety of detection methods to achieve the detection of the targets.Based on this,the study established a ratiometric FRET detection method based on OTA-aptamer(OTA-Apt)and c DNA.During the establishment of this method,the establishment of FRET detection method based on c DNA was systematically improved.Fluorescence spectra were used to determine the fluorescence response of FRET system,and CD spectrum and molecular dynamics simulation were used to verify part of the experiment.The c DNA was designed and studied comprehensively from the aspects of position,length and base sequence,and finally a c DNA sequence was optimized.The fluorescence response ratio of the c DNA sequence was higher than that of other c DNA in the optimization process,and the detection limit was as low as 3.9 n M.This paper is mainly divided into the following three parts.The first chapter is the preface.It introduced the principle of fluorescence resonance energy transfer technology,the conditions to be met,classification and development.The application of c DNA in the establishment of FRET method is described.The characteristics and applications of aptamers were described.The harm,distribution and detection methods of ochratoxin A were reviewed.The second chapter is the design and construction of a ratiometric FRET fluorescence method based on aptamer and c DNA.Firstly,the OTA-Apt and c DNA modified with fluorescent groups were determined by ultraviolet and fluorescence spectra and compared with relevant calculations.FAM(6-carboxyfluorescein)and ROX(6-Carboxyl-XRhodamine)were selected to modify OTA-Apt and c DNA,respectively.It constitutes a ratio-type fluorescent probe.Secondly,from the view of of the position,length,base sequence,a series of c DNA sequence were comprehensively designed and introduced into FRET system for fluorescence spectrometry.CD spectra and molecular dynamics simulations were used to determine the conformation and binding sites of OTA-Apt for the related auxiliary validation,and finally the c DNA sequence with the highest fluorescence response ratio is determined.It is believed that this sequence makes the detection sensitivity of the method the highest.The third chapter describes optimization of ratiometric FRET fluorescence method based on OTA-Apt and c DNA and the application of OTA samples.It has optimized the buffer conditions including p H value and metal cation,the incubation temperature and time with FRET system,the proportion and concentration of aptamer and c DNA and a series of methodological verification,such as selectivity,linearity and recovery rate.