Preparation And Application Of Novel Fluorescent Biosensors Based On Ploydopamine Nanoparticles And Functional Nucleic Acids

Objective:Biosensing technology is a new type of analysis technology that has been rapidly developed through cross-disciplinary integration.Thanks to its unique advantages of high sensitivity,good selectivity,convenient detection process and time saving,the technology is widely used in food safety,medical diagnosis,environment monitoring and other fields.With the continuous progress and rapid development of life sciences,it is of great significance to realize the highly sensitive and high-throughput detection of different biomolecules.However,the detection of trace biomolecules by some biosensors is still not accurate enough,and false positive signals occur from time to time.Therefore,this paper is based on the high fluorescence quenching performance of polydopamine nanoparticles and the specific recognition ability of functional nucleic acids to achieve accurate detection of trace amounts of different biomolecules.1.In the first part,we designed a fluorescent sensor with high sensitivity and good selectivity for ochratoxin(OTA)based on the superior fluorescence quenching performance of polydopamine nanoparticles and the specific recognition ability of nucleic acid aptamers for the target detection.2.In the second part,based on the fact that Ag~+can adjust the catalytic activity of Mgzyme and the high affinity of cysteine with Ag~+,we constructed a highly sensitive fluorescent biosensor for the detection of cysteine(Cys).Methods:1.Polydopamine nanoparticles(PDANS)with excellent fluorescence quenching properties were synthesized using dopamine as raw materials,and fluorescein(FAM)was labeled at the 5'end of the nucleic acid aptamer that can specifically recognize OTA.In the absence of OTA,the nucleic acid aptamer is adsorbed on PDANS by?-?stacking.After the addition of OTA,the nucleic acid aptamer specifically recognizes and binds OTA,thereby inducing the nucleic acid aptamer to fold from a random state into a G-quadruplex structure,and the adsorption force of the structure to PDANS is weakened.2.A catalytic molecular beacon system(CAMB)was designed by using molecular beacons labeled with a fluorescent group(FAM)and a quenching group(BHQ1)at both ends as the substrate chain and combining the cycle amplification characteristics of DNAzyme.Since Ag~+can combine with the C base of the Mgzyme catalytic active center to form an Ag~+-Mgzyme complex,the catalytic activity of Mgzyme is suppressed.After adding Cys,the strong binding force of Cys and Ag~+destroys the C-Ag~+-C base pair in the DNAzyme-Ag~+complex,and the catalytic activity of Mgzyme is restored.Results:1.Due to OTA and its nucleic acid aptamers form a G-quadruplex structure,which greatly weakens the quenching effect of PDANS on FAM,the fluorescence intensity of the sensing system is significantly enhanced.2.When Cys is not added,the catalytic activity of Mgzyme is inhibited by Ag~+.Therefore,the Mgzyme-Ag~+complex cannot catalyze MB,so the sensor has a very low background fluorescence intensity.The addition of the target Cys weakens the effect of Ag~+on the catalytic activity of Mgzyme.The activated DNAzyme hybridizes with MB and initiates multiple cyclic cleavage reactions.The fluorescence signal of the sensing system is significantly enhanced.Conclusion:1.The fluorescence sensor constructed in the first part has good performance and can quickly and efficiently realize the quantitative detection of OTA,with a detection limit of 20nM.The sensor is used for the determination of the recovery rate of OTA in red wine,the result is satisfactory.2.In the second part,MB's low background fluorescence and Mgzyme's multiple signal amplification make the sensor highly sensitive,with a detection limit of 2nM.In addition,the high affinity of Cys and Ag~+makes the sensor show good selectivity.Finally,we used the sensor to determine the recovery rate of Cys in actual urine,and the results were satisfactory.