Study On Ultrasensitive Detection Of SARS-CoV-2 Nucleic Acid And Circulating Tumor DNA

Nucleic acids are the main genetic materials of organisms.The structural changes of nucleic acids are closely related to many diseases.Therefore,detection of nucleic acid biomarkers is very important for diagnosis,treatment,and prevention of diseases.Several methods,including high-throughput sequencing,nucleic acid amplification,and Northern blot have been developed to detect these biomarkers.Although these methods have advantages of high specificity and accuracy,they require high cost,time consuming,expensive equipments,and professional manipulations.Hence,it is imperative to develop a novel method for fast and low-cost detection.The biological substances are utilized to develop biosensor that can specifically recognize targets,such as nucleic acid,antibody,cell,or organelle.Biosensor is an advanced monitoring and detection technology with broad application prospect,whichis often developed based on cross-disciplines of chemistry,physics,materials,medicine.Biosensor has advantages of high specificity,high sensitivity,high stability,and low-cost.In thisthesis,electrochemical and fluorescence biosensors have been constructed to detect severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)RNA,circulating tumor(ctDNA)EGFR L858R,and ctDNA EGFR 19Del.The results include the following aspects:(1)Ultrasensitive supersandwich-type electrochemical sensor for SARS-CoV-2 from the infected COVID-19 patients using a smartphone.The recent pandemic outbreak of COVID-19 caused by a novel SARS-CoV-2,poses a threat to public health globally.Thus,developing a rapid,accurate,and easy-toimplement diagnostic system for SARS-CoV-2 is crucial for controlling infection sources and monitoring illness progression.Here,an ultrasensitive electrochemical detection technique was developed to detect SARS-CoV-2 nucleic acid.Au@Fe3O4 and RGO@SCX8-Au were synthesised to enrich the signal materials TB.Based on a supersandwich-type recognition strategy,the technology was confirmed to practicably detect the RNA of SARS-CoV-2 without nucleic acid amplification and reverse-transcription by using a portable electrochemical smartphone.The biosensor showed high specificity and selectivity during in silico analysis and actual testing.A total of 88 RNA extracts from 25 SARS-CoV-2-confirmed patients and eight recovery patients were detected using the biosensor.The detectable ratios(85.5%and 46.2%)were higher than those obtained using RT-qPCR(56.5%and 7.7%).The limit of detection(LOD)of the clinical specimen was 200 copies/mL,which had a low limit of clinical detection.Additionally,only two copies(10 μL)of SARS-CoV-2 were required for per assay.Therefore,we developed an ultrasensitive,accurate,and convenient assay for SARS-CoV-2 detection,providing a potential method for point-of-care testing(POCT).(2)Electrochemical detection of ctDNA mutation in non-small cell lung cancer(NSCLC)based on CRISPR/Cas12a system.Lung cancer is the leading cause of cancer-related death around the world.The circulating tumor DNA(ctDNA)of EGFR L8585R in plasma is crucial for development,targeted drug therapy,and prognosis of non-small cell lung cancer,the main type of lung cancer.Accurately detecting ctDNA using conventional methods is challenging due to its characteristics,such as considerably short size,extremely low level,and short half-life.Thus,developing a rapid,accurate,and cost-effective method for ctDNA EGFR L858R detection is urgently needed.Herein,we developed an electrochemical biosensor of ctDNA EGFR L858R based on the CRISPR/Cas12a system and MB/Fe3O4@COF/PdAu nanocomposites.The CRISPR/Cas12a system played roles in the precise recognition of ctDNA targets and indistinguishable cleavage of single-stranded DNA.Additionally,the MB/Fe3O4@COF/PdAu nanocomposite has good catalytic activity and signal amplification performance.Notably,the LOD of the proposed biosensor was 3.3 aM.The detection results of 25 clinical samples showed that 22 and 20 positive samples were detected by electrochemical detection and droplet digital polymerase chain reaction,respectively.The two samples of uncertain results were identified as positive by sequencing.It is suggested that the electrochemical biosensor has high specificity,stability,and selectivity.Therefore,we established a high-precision,reliable,and convenient method for ctDNA detection,which has a potential application in the diagnosis and prognosis of cancer.(3)Fluorescence detection of ctDNA EGFR 19Del based on CRISPR/Cas12a system.EGFR 19Del is an important therapeutic target for NSCLC.Developing a rapid,accurate,and cost-effective method to detect EGFR 19Del has great significance for cancer patients.In this work,a novel EGFR 19Del fluorescent biosensor was constructed which based on CRISPR/Cas12a system with high specific recognition ability and mesoporous silica(MSNs)enrichment ability.Rhodamine B was enriched by MSNs to synthesise RB@MSNs,and Au NPs was obtained to use as a fluorescence quenching.Under the optimal conditions,the sensor has a good linear relationship between target concentration and fluorescence intensity.The sensor also had a high sensitivity with a LOD of 33 aM.The fluorescent mesoporous silica nanocomposite is easy to synthesize in this work.This method provided a novel idea for the nucleic acid detection based on CRISPR/Cas12a system,which successfully optimized the conventional CRISPR/Cas12a fluorescence detection system.In a word,using interdisciplinary advantages,a series of biosensors have successfully been constructed to detect different types of nucleic acids.These results provided new insights for nucleic acid detection for POCT.