A Novel Method For Detecting Adenosine And Theophylline By Single Molecule Photobleaching Counting Based On Split Aptamer

With the development of society and the improvement of people’s living standard,people have a deeper understanding of health care,and the market demand for health food is also increasing.However,the production and adulteration of health food,illegal addition and other problems emerge one after another.To solve these problems,researchers have developed a series of detection methods.Among them,Single molecule photobleaching(SMPB)based biosensor is an efficient,sensitive and new method for the operation of complex instrument without the need for professional technicians,which has received wide attention.Aptamer is a kind of oligonucleotide that can specifically recognize the target.Split aptamer is to split a complete nucleic acid aptamer into two at the appropriate position to jointly identify the target object,which is conducive to the design of sensing method.In this paper,Adenosine and Theophylline are identified by multiple repeat appositive probes based on the split aptamer appositive of Adenosine and Theophylline,and the Degree of aggregation(DOA)of the fluorescence probe is examined by single fluorescence molecular photobleaching,so as to develop a new method for detecting Adenosine and Theophylline with high sensitivity.The main results are as follows:(1)A new method for the detection of adenosine by photobleaching of monochromatic fluorescent moleculesIn this work,we designed an improved split aptamer probe to identify a single adenosine molecule in solution,and constructed a hypersensitive detection method for adenosine based on single molecule photobleaching.A short strand of DNA in the split aptamer was labeled with a molecular dye,and another aptamer fragment was extended to have multiple recognizable sites and labeled with a molecular dye.In the presence of adenosine,the short chain aptamer probe specifically bound to the long chain aptamer,resulted in a concentration dependent self-aggregation process.On the basis of single molecule photobleaching experiment,the DOA of short DNA probe on long chain aptamer was measured accurately.Through statistical analysis of DOA at different target concentrations,a clear curve relationship between DOA and target molecule concentration(such as adenosine)was established.The detection Limit(LOD)was 44.5 pM,lower than the fluorescence analysis results reported recently.Due to its high sensitivity and good selectivity,this sensing strategy will be widely used in biomolecular analysis in complex environments.(2)A new co-localization method for the detection of theophylline by photobleaching of two-color fluorescent moleculesDue to the fact that RNA nucleic acid aptamers are easy to be enzymatic hydrolyzed,it is not suitable to construct fragments with multiple recognition sites(>3),and the "streptavidin-biotin" reaction model can solve this problem.First,the fluorescent dye cy5 was modified at one terminal of a split aptamer(cy5-probe1).The biotin and the fluorescent dye(FAM)were modified at both terminal of the other probe having two recognition sites of theophylline.The modified probe bound to streptavidin to form an aptamer probe complex(SA-probe2-fam)with 8 recognizable sites.When theophylline was present,cy5-probe1 bound to SA-probe2-fam,caused aggregation of the two-color fluorescent dye.We found the specifically recognized fluorescent spot(cy5)by colocalization,and the DOA of cy5 spot represented the concentration of the target.The relationship between the DOA of cy5 and theophylline concentration was statistically analyzed.The detection limit was 92.1 pM,which could meet the needs of highly sensitive detection of theophylline.At the same time,the recovery rates of the method in solution and serum were investigated,which ranged from 99.43~100.36% and 96.86~98.75%,respectively.