Construction And Research Of Solution-gated Graphene Transistor Sensor For Prostate Cancer Markers Detection

Solution-gate graphene transistor(SGGT)biosensor is a promising technique for the trace detection of serious disease markers due to its high sensitivity,easy integration,and costeffective.Prostate cancer(PCa)will become the highest incidence of cancer in men in the future,which is a global health problem.Prostate cancer-specific antigen(PSA)screening is the gold standard in PCa diagnosis and has significantly improved patient survival.However,the low specificity and sensitivity of the PSA assay leads to misdiagnosis and overdiagnosis.It is a challenge to find new biomarkers and develop new detection assays in the early accurate diagnosis of PCa.In this thesis,the SGGT biosensor was constructed to detect PCa biomarkers,such as DNA,miRNA-21,PSA,and sarcosine(SAR).The performance of the SGGT biosensor was improved through the bioreceptor selection,the bioreceptor design,and the modification method optimization.A single biomarker,as well as a combination of biomarkers,were detected by the constructed SGGT biosensor to improve the accuracy of early PCa diagnosis.The custom-integrated equipment was developed to realize rapid and portable minimally invasive detection.Carbon quantum dots(CQDs)were used to optimize the immobilization strategy of the probes on the gate of the SGGT biosensor.The CQDs were synthesized by hydrothermal method.The prepared CQDs were immobilized on the surface of the gate through mercaptoacetic acid(MAA).The designed single-stranded DNA(ssDNA)probe was immobilized on CQDs by strong π-π interactions.The ssDNA probe captured the ssDNA target and formed double strands DNA(dsDNA).The dsDNA dropped from the CQDs on the gate electrode.The electronegativity of DNA molecules induces the Dirac voltage(VDirac)shifts of SGGT biosensor transfer curves and channel current(IDS)responses.The limit of detection(LOD)reached 1 aM(1.0 × 10-18 M)which is 2～5 orders of magnitude lower than traditional methods,such as the fluorescent method,electrochemical method,and colorimetric method.The sensor has a good linear range from 1 aM(1.0 × 10-18 M)to 0.1 nM(1.0 × 10-10 M)and good specificity.It effectively distinguished one-base mismatched target DNA and the response time was about 326 s for the 1 aM target DNA molecules.The SGGT biosensor performed rapid and label-free detection of the ultralow concentration of DNA molecules.Spacer biomolecules were used to optimize the arrangement and configuration of the probes on the gate of the SGGT biosensor.The SGGT biosensor performed unamplified and label-free detection of the ultralow concentration of miRNA-21 biomolecules.The designed ssDNA probe with a thiol group at the 5’ end was directly immobilized on the surface of the Au gate electrode.To ensure the orderly arrangement of ssDNA molecular probes on the gate electrode and efficiently capture the target miRNA-21,6-mercapto-1-hexanol(MCH)was used as the spacer biomolecule.The test solution was mixed with PBS and 1 mM MgCl2 solution.Mg2+ ion in the test solution is beneficial to maintain the configuration of ssDNA probes on the gate electrode.It efficiently hybridized with the target miRNA-21 molecule and cause Dirac voltage shifts of the SGGT biosensor transfer characteristic curve.The LOD of the biosensor is 10 zM(1.0 ×10-20 M).The SGGT biosensor detected miRNA-21 without amplification and any chemical or biological markers.The linear range of the biosensor is from 10 zM(1.0 × 10-20 M)to 1 pM(1.0 × 10-12 M),which realizes the real-time detection of miRNA-21 molecules within 5 minutes at the ultra-low concentrations,and well distinguished a mismatched miRNA-21 molecule.Finally,serum samples from patients without RNA extraction and amplification were detected.The results showed that the biosensor well distinguish the cancer patients from the control group and has higher sensitivity(100%)than PSA detection(58.3%).To promote the accuracy of PCa diagnosis,the SGGT biosensor was designed to simultaneously detect PSA and SAR.The SGGT biosensor was integrated with the customhardware platform to realize portable minimally invasive detection.Compared to antibody probes,aptamers have excellent performance,such as binding affinity,excellent chemical stability,good thermal stability,and cost-effective.The aptamers were selected as probes.The aptamers were designed and optimized by a molecular docking tool.The previous work has verified the advantage of the use of an Au-S bond to immobilize the probes.The thiol group is modified at the 5’ end of the aptamer.The aptamers were immobilized on the Au gate electrodes to capture PSA and SAR.The configuration changes of the aptamer can cause variations in the capacitance of the double layers(EDLs)on the surface of the gate.The limit of detection(LOD)of both two biomarkers reached 0.01 fg/mL in testing solutions,3～4 orders of magnitude lower than existing detecting assays.The linear ranges of both two biomarkers were from 0.01 fg/mL to 1 ng/mL.To improve the binding interaction,an electric field pulsed electrode was designed.The fastest response time of PSA and SAR molecules were approximately 4.5 and 13 min,respectively.Whereas,the detection time of other PSA and SAR assay is ≈1～2 h.The smartphone/Pad controlled the custom-built portable integrated platform and directly displayed the testing result.The clinical serum samples tests demonstrated that the biosensor can distinguish PCa patients from the control group.Combined with the PSA and SAR diagnosis,PCa patients can be identified.