Joint Detection Of GP73 & AFP Based On Electrochemical Aptamer

Golgi protein 73(GP73)has emerged as a novel blood-based biomarker for the early diagnosis of hepatocellular carcinoma(HCC).Due to its high sensitivity,specificity,and rapid response,the electrochemical aptasensor has been widely applied to serum biomarkers for HCC.At first,a competitive electrochemical aptasensor using GP73 aptamers as recognition molecules was constructed.Then,an electrochemical dual-aptasensor was constructed to accurately analyze GP73 and AFP jointly,which would reduce the false positive probability of detecting a single marker.The whole manuscript encompasses the following facets:(1)Synthesis and performance of r GO-Fc-PANi and Nr GO-Cu2O nanocomposites:Two nanocomposites,reduced graphene oxide-ferrocene-polyaniline(r GO-Fc-PANi)and nitrogen-doped reduced graphene oxide-cuprous oxide(Nr GO-Cu2O),were prepared by covalent binding,physical adsorption,and chemical reduction.The two nanocomposites were verified by scanning electron microscopy(SEM),transmission electron microscopy(TEM),ultraviolet-visible light spectroscopy(UV-vis),Fourier infrared spectroscopy(FT-IR),X-ray photoelectron spectroscopy(XPS),zeta potential and particle size,and proved that the nanocomposites were synthesized successfully.At the same time,differential pulsed voltammetry(DPV)was used to analyze the electrochemical activity of the two nanocomposites.(2)Detection GP73 by the competitive electrochemical aptasensor:The competitive electrochemical aptamer sensor based on r GO-Fc-PANi nanocomposites modified screen printed electrodes(SPE)was constructed by using r GO-Fc-PANi nanocomposites to form signal probes,and introducing aptamer complement chains(c DNA)to form a double-stranded structure with aptamers on the probe,which achieved high sensitivity detection of GP73.GP73 concentration(0.001 ng/m L～100.0 ng/m L)showed a positive log-linear relationship with the DPV signal,and the working curves were:Y=1.271×lg X+4.6719,R~2=0.9912,and the Limit of detection(LOD)was 0.15 pg/m L(S/N=3).The content of GP73in human serum was detected by direct measurement.The relative standard deviation(RSD)was 1.08%～6.96%,and the relative error was 0.11%～6.89%.In addition,the sensor has excellent specificity,stability,and reproducibility.Therefore,this competitive electrochemical aptamer sensor has a certain clinical application value.(3)Detection of GP73 and AFP by the electrochemical dual-aptasensor:The r GO-Fc-PANi and Nr GO-Cu2O nanocomposites were combined with GP73Apt and AFPApt to form r GO-Fc-PANi-GP73Apt and Nr GO-Cu2O-AFPApt signal probes,and c DNA was introduced into the r GO-Fc-PANi-GP73Apt probe to form a double-stranded structure.Using BSA solution as the solvent,the two probes were mixed in equal volumes to construct a mixed-signal probe(c MSP),and the c MSP was modified on the surface of the SPE where Au NPs were deposited,and an electrochemical dual-aptasensor that could jointly detect GP73 and AFP was constructed.In the range of 0.001ng/m L～100.0ng/m L,the DPV signal change generated by the dual-aptasensor showed a good log-linear relationship with different GP73concentrations,the linear regression equation wasΔIGP73=0.4137×lg X+1.7387,R~2 was0.9906,LOD was 0.48 pg/m L,and the DPV current signal change generated by the dual-aptasensor showed a good log-linear relationship with different AFP concentrations.The linear regression equation isΔIAFP=0.4729×lg X+2.2321,R~2 is 0.9706,and LOD is 1.77pg/m L.At the same time,the sensor has good specificity,stability,and reproducibility.The actual human serum sample was detected by direct measurement method.The RSD value of GP73 content was between 1.15%～3.52%,and the detected AFP content RSD was between3.96%～5.97%,and the test results were relatively accurate.