Construction Of Biosensors Based On Radio Frequency Signal And Isothermal Amplification And The Application In Nucleic Acid Detection

Background:Radio frequency(RF)biosensors refer to sensors with a working rate between 300 k Hz and 300 GHz,including common acoustic sensors and microwave sensors.The working frequency of common acoustic sensors is generally less than 10 GHz,including quartz crystal microbalance(QCM),surface acoustic wave sensor(SAW)and film bulk acoustic resonator(FBAR)sensor,etc.Microwave sensors include microstrip line microwave biosensor,waveguide cavity microwave biosensor and Microwave metamaterial biosensor.Leaky Surface Acoustic Wave(LSAW)is a kind of surface acoustic wave excited by a special tangential piezoelectric crystal.The research team conducted a lot of exploration in the research of surface leaky acoustic wave in the early stage,and constructed a series of surface leaky acoustic wave biosensors,which realized the detection of various biological macromolecules such as protein and nucleic acid.However,the LSAW biosensor constructed in the early stage has limited sensitivity,and the sensor is easily affected by evaporation and vibration when exposed to the air during the reaction.It is necessary to design and prepare a new type of LSAW biosensor to improve its fundamental frequency to improve its sensitivity,and improve the detection system to reduce the negative impact of environment on the test.In the early stage of research on metamaterial sensors,the research group found that the resonance ring of the metamaterial sensor is sensitive to the dielectric constant of the surrounding materials,and the type and quantity of materials around the resonance ring can be judged by sensing the dielectric constant.If the resonant ring of the metamaterial sensor is integrated with the strip,qualitative analysis can be made through the color change of the strip on the one hand,and quantitative analysis can be made through the resonant state of the metamaterial sensor on the product of the strip on the other hand.It should be noted that in biological reactions,since the concentration of the target to be tested is relatively low in many cases and exists in the form of solution,the dielectric constant of water affects the detection ability of the metamaterial sensor towards the target.The change of resonant frequency cannot directly reflect the type of substance,that is,simple metamaterial sensor cannot guarantee the specificity of detection.It is necessary to achieve specific detection by biological means.Recombinase aid amplification(RAA)can be used for thermostatic expansion of nucleic acid at 37-42℃,with simple primer design,greatly simplifying the requirements of the instrument,and better meeting the requirement of fast and simple.The organic combination of metamaterial side-flow immunochromatography strip and RAA technology is expected to construct a rapid,simple,accurate and quantitative nucleic acid detection technology,and can be applied to the rapid detection of Staphylococcus aureus waiting target nucleic acid,which can provide a new technology for the identification and detection of Staphylococcus aureus.Nucleic acid testing is still the preferred laboratory test method for screening and confirming severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection.In order to solve the problem of false negatives and improve the accuracy of diagnosis,on the one hand,it is necessary to strictly implement specimen collection requirements,on the other hand,it is necessary to improve existing detection methods or develop new detection methods.The QCM sensor is highly sensitive to the surface mass load and is suitable for liquid phase detection.The detection of viral RNA through the QCM biosensor may be an effective solution for the detection of SARS-CoV-2.In order to effectively detect the RNA sequence of SARS-CoV-2,it is necessary to achieve high sensitivity and specificity of the detection method on QCM.To enlarge the mass load caused by the target to improve the detection sensitivity is a key problem to be solved in constructing QCM biosensors.As nanomaterials,Y-shaped DNA(Y-shaped DNA)has the advantages of easy modification,simple synthesis and high stability,and is more easily combined with the gold surface of sensors.It is theoretically feasible to increase the surface mass load of QCM sensor by Y-DNA mediated nucleic acid amplification reaction.Another key issue is the high specificity of detection through target identification on QCM.Y-DNA side-strand terminal hybridization is not sufficient to achieve high specificity for detection.hybrid chain reaction(HCR)can identify the target by using stem ring probes and trigger the mutual opening of stem rings,which can specifically recognize and amplify signals.At the same time,to ensure the accuracy of SARS-CoV-2 detection,the two arms of Y-DNA can be used to detect the conserved sequence of ORF1 ab gene and N gene of SARS-CoV-2.In conventional enzyme-participated isothermal amplification technology,nucleic acid is generally used as a core component to control the occurrence of the reaction,and enzymes are usually designed with complex nucleic acid to achieve target detection.Most of these detection methods use the entropy-driven strand displacement reaction as the controller to initiate the next reaction,and realize the analysis and detection of the target target by regulating the sequence,secondary structure and spatial arrangement of the nucleic acid.However,the nucleic acid sequences in the reaction system are interlocking,and the reaction system is particularly complicated and easy to be contaminated;when targeting multiple detection targets,the over-reliance on base complementation and nucleic acid structure will cause the design difficulty to increase exponentially.Suppose from another perspective,taking enzymes as the core of the reaction system and combining enzymes with different functions can simplify nucleic acid sequence and structure design,so as to reduce interference and realize more efficient detection.Because the object of each module is nucleic acid,so different modules can rely on nucleic acid to connect each other,three different enzyme base modules in series to form enzyme circuit,it is expected to establish a new thermostatic amplification method and greatly improve the detection sensitivity.Objectives:1.Design and prepare a new type of LSAW biosensor with high fundamental frequency and high sensitivity,and build a stable,reliable,and anti-interference radio frequency signal control and detection platform;2.Integrate lateral flow immunochromatographic test strips with metamaterial biosensors,and use recombinase aid amplification to achieve rapid detection of Staphylococcus aureus nucleic acid;3.Simultaneously detect the conservative sequence of the ORF1 ab gene and N gene of SARS-CoV-2 through Y-shaped DNA(Y-DNA)and hybrid chain reaction(HCR)on the QCM sensor,achieving high sensitivity and high Specific SARS-CoV-2 detection;4.Using the high selectivity and specificity of the enzyme,the enzyme is used as the core of the reaction system to construct an enzyme-based circuit,and a new isothermal amplification method is established based on the EXPAR reaction.Sequences such as micro RNA and ts RNA are detected.Methods:1.The construction and verification of new surface acoustic wave biosensors and the establishment of radio frequency signal control and detection platforms1)Use COMSOL Multiphysics 5.2 software to simulate and design a new type of LSAW biosensor2)Preparation of the novel LSAW biosensors using micro/nano processing techniques using different substrate materials and design processes3)Design and process a testing fixture for the novel LSAW biosensor using microfluidic pipelines and different materials4)Write software for the novel LSAW biosensor based on Lab VIEW and C # Languagea and build a test platform5)Verify the performance of the novel LSAW biosensor and complete the detection of the target Pseudomonas aeruginosa nucleic acid on it2.Detection of Staphylococcus aureus based on metamaterial-lateral flow immunochromatographic test strips and RAA1)Design and manufacture : Preparation of metamaterials-lateral flow immunochromatographic test strips by simulation design,wax spray printing and other processes2)The metamaterial-lateral flow immunochromatographic test strip was characterized by electron microscope,atomic force microscope and other detection methods,and the performance of the strip was verified by glucose solution of different concentrations3)Establish a fluorescent quantitative PCR method for Staphylococcus aureus nucleic acid based on RAA4)Complete the detection of Staphylococcus aureus DNA on the metamaterial-lateral flow immunochromatographic test strip and compare it with other methods3.Research on the detection of SARS-CoV-2 based on quartz crystal microbalance and Y-DNA-HCR1)Build a detection system that can be used for 5 MHz fundamental frequency QCM chips2)Design Y-DNA probes and HCR probes for the ORF1 ab and N genes of SARS-CoV-23)Complete the assembly of Y-DNA probe and validation of HCR in a liquid phase environment4)Complete the detection of viral nucleic acid on QCM sensors and conduct methodological analysis4.Establishment of a new constant temperature nucleic acid amplification method RT-NExT(reverse-transcript nicking exponential truncation)and its application in nucleic acid detection1)Establish recognition module by reverse transcription method2)Establish an identification module through exponential amplification reaction3)Establish a signal output module through constant temperature fluorescence signal output4)Connect three modules in series and use them for micro RNA-10 b detection5)Apply RT-NExT to detect ts RNA-66 and ts RNA-86Results and Conclusions:1.The C1 LSAW biosensor with a fundamental frequency of 210 MHz was successfully designed and prepared,and the supporting multi-channel detection fixture LSAW-YJD-6 with the integration of "liquid-mechanics-electricity" was designed.A 16 port RF signal control and detection platform based on the Keysight M9010 A PXIE 10 slot chassis and the M9800 A PXIE vector network analyzer was built.The C1 LSAW biosensor was used to detect the nucleic acid of Pseudomonas aeruginosa,with a linear range of 0.1 n M to 1000 n M and a limit of detection(LOD)of 0.28 n M.It also showed good detection specificity.2.Successfully designed and prepared a metamaterial-lateral flow immunochromatographic test strip with a resonance frequency of 7.0 GHz.The lateral flow immunochromatographic test strip was integrated with the metamaterial biosensor,and the quantitative detection of Staphylococcus aureus DNA was realized by RAA,with a LOD of0.784 ng/m L.The constructed test strip has advantages such as sensitivity,accuracy,and good stability,and is expected to be used in practical detection scenarios such as point of care testing(POCT).3.Through Y-DNA and HCR,the detection of ORF1 ab gene and N gene on the surface of QCM chip was realized.The LOD of ORF1 ab gene,N gene,ORF1 ab gene + N gene were0.15 p M,1.35 p M and 5.56 p M in the linear range of 0.01 n M-1000 n M.High sensitivity and specificity of SARS-CoV-2 detection were achieved,providing a new detection strategy for its infection diagnosis.4.The new thermostatic nucleic acid amplification method RT-NExT based on enzyme circuit was successfully established.Through the organic assembly of target recognition module,amplification module,and signal output module,the detection of micro RNA-10 b with a minimum of 50 a M was achieved.The optimized RT-NExT had high sensitivity and scalability,and the LOD for detecting ts RNA-66 could reach 0.86 a M.