Construction Of Label-free Electrochemical Biosensor Based On Magnetic Nanomaterials For The Detection Of CYP2C9*³ Gene And VEGF₁₆₅ Protein

In recent years,precision medicine and early detection of diseases have attracted much attention,and the demand for detecting biological samples has also increased dramatically.However,the concentrations of biological samples were usually low.Traditional detection methods were often time-consuming and complex operations.Therefore,it is urgent to develop more convenient and efficient new detection methods.Electrochemical biosensors were easy to operate,fast,and sensitive,which could overcome the shortcomings of traditional detection methods and achieve rapid detection of complex samples.Nanomaterials had high electron transfer efficiency,and nanomaterials could be combined with electrochemical sensors to construct sensors with higher sensitivity for the detection of biological samples.In this paper,magnetic Fe₃O₄/Fe₂O₃@Au nanorods and nanoparticles were used as signal amplification media,and the magnetic nanomaterials were combined with magnetic induction self-assembly technology.The two electrochemical nucleic acid sensors were constructed with CYP2C9*3 gene and VEGF₁₆₅ protein as the detection object,and the detection performances of the two electrochemical sensors were evaluated.The main research achievements were as follows:（1）Preparation of magnetic Fe₃O₄/Fe₂O₃@Au nanorods:Fe Cl₃·6H₂O was used as the raw material.The magnetic Fe3O4/Fe2O3 heteroplasmon nanorods were prepared by the heating hydrolysis-combustion calcination method.The concentration of Fe³⁺,hydrolysis time,hydrolysis temperature,amount of absolute ethanol,calcination temperature,and calcination time were optimized.The optimal conditions were hydrolysis of 0.1 M Fe Cl₃ at 90°C for 2 h,and calcination of the precursor at 300 ℃ for2 h（50 m L absolute ethanol）.The average length and diameter of the magnetic Fe₃O₄/Fe₂O₃heteroplasmon nanorods obtained under the optimal conditions were about199 nm and 51 nm,respectively.The saturation magnetization was 13 emu·g^-1,and the Zeta potential was+17.2 m V.The heteroplasmon nanorods were surface modification by the sodium citrate reduction method,and the magnetic Fe₃O₄/Fe₂O₃@Au nanorods were prepared.The mean length of the magnetic Fe₃O₄/Fe₂O₃@Au nanorods was around 210nm,the mean diameter was about 57 nm,the thickness of the gold shell was around 9 nm,the saturation intensity was 7 emu·g^-1,and the Zeta potential was-8.9 m V.（2）Preparation of magnetic Fe₃O₄/Fe₂O₃@Au nanoparticles:Fe（NO₃）₃·9H₂O was used as the raw material.The magnetic Fe3O4/Fe2O3 heteroplasmon nanoparticles were prepared by the heating hydrolysis-combustion calcination method.The effects of the hydrolysis time,hydrolysis temperature,concentration of Fe³⁺,amount of absolute ethanol,calcination temperature,and calcination time were investigated.The optimal preparation conditions were hydrolysis of 0.05 M Fe（NO₃）₃ at 90 ℃ for 8 h,and calcination of the precursor at 200 ℃ for 2 h（20 m L absolute ethanol）.The average diameter of the magnetic Fe₃O₄/Fe₂O₃heteroplasmon nanoparticles prepared under the optimal conditions was about 46 nm.The saturation magnetization was 54 emu·g^-1,the hydrodynamic dimension was 139 nm,and Zeta potential was+25.8 m V.The magnetic Fe₃O₄/Fe₂O₃@Au nanoparticles were successfully prepared by the sodium citrate reduction method.The dimension of the magnetic Fe₃O₄/Fe₂O₃@Au nanoparticles was about 96 nm,the saturation magnetization was 43 emu·g^-1,the hydrodynamic dimension was 363 nm,and Zeta potential was-19.2 m V.（3）Construction of label-free electrochemical biosensor with magnetically induced selfassembly based on magnetic Fe₃O₄/Fe₂O₃@Au nanorods for the detection of CYP2C9*3 gene:the magnetic Fe₃O₄/Fe₂O₃@Au nanorods were self-assembled to the surface of the electrode by magnetic field force,and the thiolated peptide nucleic acids（PNA）were connected to the surface of the nanorods via Au-S bonds.PNA and DNA were hybridized through the base complementary pairing principle to achieve the specific detection of CYP2C9*3 gene,and the electrochemical biosensor exhibited excellent selectivity.The concentration of Fe₃O₄/Fe₂O₃@Au nanorods,PNA concentration,hybridization temperature,and hybridization time were optimized through differential pulse voltammetry（DPV）.The best detection criteria were as follows:the concentration of Fe₃O₄/Fe₂O₃@Au nanorods was 15 mg·m L^-1,the concentration of PNA was 1.6μM,the hybridization time was 30 min,and the hybridization temperature was 70 ℃.Under greatest criteria,the linear range of the biosensor was 1 p M-1μM（R²=0.9941）,the limit of detection（LOD）was 0.95 p M,and the limit of quantitation（LOQ）was 3.18 p M.The relative standard deviation（RSD）of the five electrodes was only 2.6%,which had excellent reproducibility.The sensors stored stably for 15 days in refrigerator,and DPV signal was 82.2%of the original,which had good stability.（4）Construction of label-free electrochemical biosensor with magnetically induced selfassembly based on magnetic Fe₃O₄/Fe₂O₃@Au nanoparticles for the detection of VEGF₁₆₅ tumor marker:the magnetic Fe₃O₄/Fe₂O₃@Au nanorods were self-assembled to the surface of the electrode by magnetic field force,and the thiolated DNA aptamers were connected to the appearance of the nanoparticles via Au-S bonds.The DNA aptamers could specifically recognize the VEGF₁₆₅ protein,and realize the specific detection of the VEGF₁₆₅ tumor marker.The concentration of Fe₃O₄/Fe₂O₃@Au nanoparticles,the concentration of DNA aptamers,the incubation temperature,and the incubation time were majorized by DPV.The best detection conditions were as follows:the concentration of Fe₃O₄/Fe₂O₃@Au nanoparticles was 15 mg·m L^-1,the concentration of DNA aptamers was 1.2μM,the incubation temperature was 37 ℃,and the incubation time was 40 min.Under best conditions,the linear range of the electrochemical sensor was 0.01-10 pg·m L^-1（R²=0.9973）,LOD was 0.01 pg·m L^-1,and LOQ was 0.03 pg·m L^-1.Compared with interfering substances,the electrical signal strength of VEGF₁₆₅ protein was only 35.2%of the other interfering substances,which had good selectivity.RSD of the five electrodes was only 3.7%,which exhibited excellent reproducibility.The electrical signal was still86.7%of the original after storing 15 days at 4 ℃ in refrigerator.The sensor had good stability.