The Studies Of Graphene Transistor Nucleic Acid Biosensor Based On G-quadruplex Enzyme

In the early diagnosis and screening of diseases,nucleic acid detection has been extensively studied by researchers as an important detection method.However,in the early stage of the disease,the low concentrations of disease biomarkers are detrimental to early screening.Therefore,the development of highly sensitive,fast and portable sensors for nucleic acid molecules is necessary.Field effect transistor（FET）biosensor is one of the emerging popular directions,and has demonstrated excellent performance in the analysis and detection of biomolecules,for example,nucleic acids,cancer cells,proteins,glucose,etc.Because of the principle of transistor,the transistor biosensor is not only a sensor,but also an amplifier.The solution-gated graphene transistor（SGGT）biosensor derived on this basis can not only achieve the sensitivity of the transistor itself,but also has the advantages of miniaturization and portability.SGGT biosensors are more suitable for the analysis and detection of biological substances,and can be used as a potential bedside testing equipment.Currently,the Solution-gated transistor sensing is mainly detected by the interaction of the analyte with the channel.However,the complicated channel modification would make the device less stable.In fact,solution gate transistor sensing can also be studied by the interaction of target with gate,but few researchers have noticed this problem.Moreover,a slight change in the gate electrode can be output by the transistor with a better electrical parameter signal with a higher signal-to-noise ratio.Therefore,the functionalization of the gate is more helpful to improve the sensitivity of the sensor,while reducing the modification of the channel,making the device itself simpler and improving the signal stability.Because of the excellent performance of graphene transistors,we carried out a Solution-gated graphene transistor biosensing study based on the interaction of target with gate for the detection of the nucleic acid markers of disease.The research is mainly divided into two parts:（1）In this chapter,a G-quadruplex-based SGGT sensor is designed and constructed to detect GAA trinucleotide repeat（TNR）sequences.First,polyaniline and a mixed solution of chitosan and streptavidin are sequentially modified on the surface of the grid electrode,and then capture-DNA fixation is performed.When GAA TNR is present,the half of the target can hybridize with the capture-DNA,another half can hybridize with the part sequence of G-quadruplex.Finally,G-quadruplex enzyme is formed on the gate surface in the presence of heme.The G-quadruplex enzyme can catalyze the oxygen reduction reaction of H₂O₂,consequently the GAA TNR can be quantitatively analyzed according to the degree to which the channel current responds.Experiments have also shown that the current response of SGGT is 400 times higher than the traditional electrochemical response.Moreover,the response current changes up to 50μA within the linear concentration range of 100 f M-100 n M,the detection limit is as low as 32.25 f M.There is also an excellent linear relationship between the channel current response and target concentrations:ΔI_DS=8.59 lgc+9.37（R²=0.9918）.Importantly,the sample tests in the serum showed that the recovery rate was91.00-102.5%.These studies have shown that the biosensor has the advantages of high sensitivity,high selectivity,good stability,high reproducibility and strong anti-interference ability.It’s demonstrated that SGGT biosensor is an advantageous platform for manufacturing highly sensitive and simple biosensors,and they provide a feasible method for the early diagnosis of neurodegenerative diseases.（2）In this chapter,the SGGT biosensor based on double amplification is constructed for sensitive detection of miRNA-141.Here we used two amplification enzyme-assisted cycle amplification and rolling circle amplification（RCA）,to construct the biosensor.First,the enzyme-assisted cycle amplification is performed in the solution.In the presence of miRNA-141,the target would compete with ST in ST/HP-1 ds-DNA to form another RNA/DNA hybrid of miRNA-141/HP-1.After the ST is released,the T7 exonuclease（exo）would cut the DNA in the hybrid to release the target that would participate in the next competitive reaction,circularly releasing multiple STs.The released ST will hybridize with the half of HP-3,while another sequence of unhybridized HP-3 would be used as a primer to participate in the amplification of the RCA on the gate.This RCA template is designed and contains a C-rich sequence.After the RCA product was incubated with hemin,a G-quadruplex enzyme was formed on the surface of the gate electrode to catalyze the H₂O₂reaction.The reaction on the gate effectively changes the gate voltage,and the channel current would change accordingly.The results show that the linear range of miRNA detection is 25f M to 25 n M.In this concentration range,the channel current varies up to 90μA,which shows that the device possesses super-high sensitivity.The studies provide a feasible technical platform for the detection of low-concentration analytes such as miRNA,and demonstrate that the transistor biosensor is potential as a bedside detection tool for prostate cancer.