Detection Of Golgi Protein 73 Based On Manganese Oxide-based Nanozyme And Aptamer Sensing

Golgi protein 73（GP73）is a novel liver cancer marker for early diagnosis and screening of hepatocellular carcinoma（HCC）.In this paper,for the purpose of trace detection of GP73,using aptamer as the recognition molecule,combining the advantages of the simple and intuitive colorimetric method and rapid response of the electrochemical method,introducing multifunctional probes and combining the signal amplification method of enzyme-like catalytic amplification.An electrochemical/colorimetric dual-mode aptamer sensing platform based on a manganese oxide-based nanoenzyme（hemin-reduced graphene oxide-trimanganese tetroxide,H-rGO-Mn₃O₄）was developed to achieve accurate and sensitive analysis of GP73 in serum.The main research contents of this paper are divided into the following aspects:（1）Synthesis and properties study of H-rGO-Mn₃O₄ nanoenzymes:H-rGO-Mn₃O₄nanoenzymes were prepared by a two-step synthesis method,Fourier transform infrared spectroscopy（FI-IR）,X-ray diffraction（XRD）,Ultraviolet–visible spectroscopy spectroscopy（UV-vis）,X-ray photoelectron spectroscopy（XPS）,scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,particle size and Zeta potential characterization analysis to verify that the synthesis was successful,and the performance of H-rGO-Mn₃O₄ nanoenzymes were also analyzed.（2）Research on the detection of GP73 by colorimetric aptasensor:Two GP73 aptamers（Apt1,Apt2）were selected by circular dichroism,and the amino-modified Apt1 was immobilized on the surface of H-rGO-Mn₃O₄ nanoenzymes byπ-πadsorption as the detection probe;meanwhile,Apt2 was used as the capture probe to construct a GP73colorimetric aptasensor based on the H-rGO-Mn₃O₄ nanoenzymes.The H-rGO-Mn₃O₄nanoenzymes catalyze the oxidation of the substrate 3,3’,5,5’-tetramethylbenzidine（TMB）by H₂O₂,which changes the system solution from colorless to blue,thus realizing the visual detection of GP73.Under the optimal conditions,the logarithm of GP73 concentration（0.05-50.0 ng/mL）showed a good linear relationship with the absorbance,the calibration equation was Y=-0.0384 lgC_GP73+0.4835,R²=0.9964,and the lowest detection was 36.94 pg/mL.The content of GP73 in actual serum samples was directly detected,the RSD was in the range of0.49%-4.91%,and the relative error compared with the enzyme-linked immunosorbent assay（ELISA）method was in the range of 0.23%-3.61%.The sensor has excellent specificity,stability,and reproducibility.（3）Dual-signal sandwich-type electrochemical aptasensor for detection of GP73:gold nanoparticles@poly-o-phenylenediamine（Au@POPD）was modified on the surface of the screen-printed electrode（SPE）to immobilize Apt2.H-rGO-Mn₃O₄-Apt1 was used as a detection probe to successfully design a sandwich-type electrochemical aptasensor for the detection of GP73.The reversible redox reaction between hemin（Fe（Ⅲ））/hemin（Fe（Ⅱ））in H-rGO-Mn₃O₄ can be used as an in situ signaling indicator and TMB as another signaling probe,two kinds of electrical signals were detected and recorded by square wave voltammetry（SWV）.Under the optimal conditions,the difference between the oxidation current signal of TMB and the oxidation current signal of hemin showed a linear relationship with the logarithm of GP73 concentration（0.01-100.0 ng/mL）.The calibration equation for the current response of the TMB signal isΔI_TMB=6.2723 lgC+14.6864,R²=0.9937,and the calibration equation for the current response of the hemin signal isΔI_hemin=1.8634lgC+5.7833,R²=0.9956,and the LOD is 7.1 pg/mL（S/N=3）with a sensitivity of 2.442μA/μM/cm².Meanwhile,the sensor has good specificity,reproducibility,and stability.The content of GP73 in actual serum was detected by the standard addition method,and the recovery rate was between 98.66%-121.11%.（4）Study on the detection of GP73 by electrochemical/colorimetric dual-mode aptasensor:AuNPs were used to modify SPE,and the complementary strand（cDNA）of Apt1 was immobilized on the surface of the AuNPs/SPE;Apt1 was immobilized on the surface of the H-rGO-Mn₃O₄ nanoenzymes as a detection probe;using the competition between cDNA and GP73 and the detection probe,TMB acts as an electrochemical and colorimetric dual-function probe,and the signal amplification method of enzyme-like catalytic amplification to construct a GP73 electrochemical/colorimetric dual-mode aptasensor based on the H-rGO-Mn₃O₄ nanoenzymes.Under the optimal conditions,the logarithm of GP73 concentration（0.0001-100.0 ng/mL）has a good linear relationship with the SWV current change value and absorbance,and the calibration equation of the electrochemical mode was Y=3.1696 lgC+24.9896,R²=0.9963.The calibration equation of the colorimetricthe model is Y=0.3386-0.0417 lgC,R²=0.9931.The minimum detection limit was 0.1 pg/mL.The constructed dual-mode sensor has the good anti-interference ability as well as excellent stability and reproducibility.The concentration of GP73 in serum samples was detected by direct measurement,and the RSD value of the electrochemical method was in the range of 1.43%-5.93%,and compared with the ELISA method,the relative error of was in the range of 0.39%-4.07%;while the RSD value of the colorimetric method was in the range of 2.58%-6.79%,and compared with the ELISA method,the relative error of was in the range of 0.22%-5.48%.Thus,the applicability of the method is verified.（5）Comparison of three aptasensors:Compared with colorimetric aptasensor that are simple to prepare,low-cost,and can detect targets with naked eyes,electrochemical aptasensor has higher sensitivity and wider linear range,and lower detection limit,and achieves the dual-signal detection of the target,reducing the possibility of misdiagnosis and missed diagnosis.Compared with the single electrochemical or colorimetric method,the electrochemical/colorimetric dual-mode aptasensor not only has a wider detection range,and lower detection limit but also has higher precision and accuracy.