The Fluorescence-based Biosensors For Nucleic Acid Rapid Detection And Its Applications

In recent years,biosensing technology has provided new technical support for the prevention and control of infectious diseases and early aid diagnosis of major diseases,and promoted the development of rapid nucleic acid detection technology.The fluorescence-based biosensing technology has received much attention in the fields of research and practical application due to its advantages of fast response time,low detection cost and high sensitivity.However,most fluorescence-based nucleic acid biosensors have own bottlenecks,such as high preparation costs,long detection time,high requirements for testing equipment,and the challenge of aggregation-caused quenching.To address these problems,this thesis constructs the fluorescence-based biosensors based on reduced graphene oxide using the fluorescence resonance energy transfer principle and the aggregation-induced emission principle for the detection and analysis of SARS-CoV-2 RNA and breast cancer-associated DNA,respectively.The main research contains of this thesis is as follows.(1)Based on the fluorescence resonance energy transfer(FRET)principle,the rapid detection of SARS-CoV-2 RNA was achieved at low cost and high sensitivity by partially reducing graphene oxide and optimising the detection strategy and detection conditions.The rGO modified by alkaline hydrothermal method retains the advantages of GO’s large surface area and biocompatibility while offering superior single-strand adsorption and fluorescence quenching capabilities,which results in the enhancement of the sensing performance.By optimising the assay protocol and reaction parameters,the detection time was reduced to less than 15 minutes and the detection limit was lowered to 0.684 pM.The fluorescence signal of the assay system remained stable for 7 days.It’s demonstrated statistical accuracy in the simultaneous detection of multiple samples.The practicality of the assay was validated by detection of SARS-CoV-2 pseudovirus RNA.The developed rGO-based fluorescence biosensing assay has the advantages of rapid response,simplified interaction,low cost,and good prospects for application in the rapid diagnosis of SARS-CoV-2 and other infectious diseases.(2)Based on the principle of aggregation-induced emission(AIE),azide-functionalised tetraphenylene(TPE-N3)was used as a fluorescent molecule for rapid turn-on biosensing of nucleic acids with the aid of copper-free click chemistry(SPAAC).The TPE-DNA probe with AIE properties was constructed by the spontaneous coupling of the azide group of the TPE-N3 molecule and the cyclic alkyne group DBCO of the DNA probe.The AIE property probe was constructed by attaching TPE molecules to the 3’ and 5’ of the DNA probe,respectively.A dualprobe sensing solution has been adopted.The fluorescence of TPE-DNA probe itself was at turn-off state.When the double strand was formed by pairing with the target DNA strand,the TPE molecules at the ends of the probe accumulated and stacked in the middle of the double strand inducing fluorescence with good specificity.The whole detection process of the assay was about 10 minutes.The limit of the detection of the assay was 39 pM.The AIE-based dualprobe fluorescence assay is versatile and flexible,allowing the attachment of AIE molecules with different emission wavelengths to the probe,promising multi-signal or multi-location detection.In conclusion,this thesis utilized the fluorescence-based biosensing technology constructed a rapid detection system for two representative nucleic acids,viral RN A and cancer marker ctDNA fragments.We proposed a rapid detection platform for SARS-CoV-2 viral RNA based on the FRET principle and a cancer ctDNA detection assay based on the AIE principle.The results showed that the detection methods help solving the problems of long detection time,high cost and ACQ problem.Combining the above fluorescence-based sensing technology with intelligent information technology will present a wide range of applications in rapid screening of diseases.