Constructions Of Electrochemical Biosensors Based On Magnetic Fe₃O₄/α-Fe₂O₃ Hetero-nanorods And Their Sensitive Detections Of Tumor Markers

“Precision medicine”is the most advanced concept of disease treatment in recent years,which has become a research hotspot in the medical field.As an important part of precision medicine,the detection of tumor markers provides strong support for its clinical application,and it has been widely used in the clinical diagnosis and classification of tumors.In the last decade,advances in genomics and proteomics have brought new challenges to existing tumor marker detection methods,so it is essential to develop more efficient detection tools.Among many bioanalysis devices,electrochemical biosensors have shown outstanding performance due to their high sensitivity,specificity,rapid detection,and potential for point-of-care testing（POCT）.In addition,studies have proved that the introduction of magnetic nanomaterials into the electrochemical biosensing interface can enhance electrochemical response,which promotes the development and manufacture of various electrochemical sensing devices.In this paper,a novel magnetic Fe₃O₄/α-Fe₂O₃ hetero-nanorod was prepared by a combination of hydrothermal and calcined reduction methods.The controllable construction of the magnetic nanorod was achieved by optimizing the preparation conditions,and magnetic Fe₃O₄/α-Fe₂O₃@Au nanocomposite was successfully prepared by loading it with gold.Using the magnetic nanocomposite as a signal amplifier and a substrate for the immobilization of recognition molecules,combined with magnetically induced self-assembly and magnetic separation techniques,novel electrochemical biosensors were successfully constructed for the sensitive detection of tumor markers HE4 protein,TP53 gene,and HER2 protein,respectively.The main results of the research are as follows:（1）Construction of magnetic Fe₃O₄/α-Fe₂O₃@Au nanocomposites.Using FeCl₃ and NH₄H₂PO₄ as raw materials,the precursorα-Fe₂O₃ nanorods were prepared by a hydrothermal method.Using glucose as a reducing agent,magnetic Fe₃O₄/α-Fe₂O₃ hetero-nanorods were prepared by calcination and reduction of the precursor nanorods.Finally,the magnetic Fe₃O₄/α-Fe₂O₃@Au nanocomposites were successfully prepared by an improved method of reducing chloroauric acid with sodium borohydride.The effects of reaction conditions on the size,morphology,phase composition,and magnetic properties of the products were investigated by TEM,SEM,XRD,and VSM.The optimum reaction conditions were as follows:the concentration of FeCl₃ was 0.2 M,the concentration of NH₄H₂PO₄ was 9.6×10^-4 M,the solution volume was 60 m L,and the hydrothermal temperature was 180℃,the hydrothermal time was 12h,the mass ratio of the precursorα-Fe₂O₃ nanorods to glucose was 1:20,the calcination temperature was 600℃,and the calcination time was 3 h.The magnetic Fe₃O₄/α-Fe₂O₃ hetero-nanorods prepared using the selected conditions had the highest Ms value(78.2 emu·g^-1)and the smallest size,which was conducive to their biosensing applications.The magnetic hetero-nanorods were uniformly dispersed spindle rod structures with an average length and diameter of215.0 nm and 79.2 nm,respectively.Finally,the magnetic Fe₃O₄/α-Fe₂O₃@Au nanocomposites were successfully constructed with a Ms value of 65.1 emu·g^-1,and the surface of the magnetic nanocomposites was coated with dark black Au particles with an average diameter of 6.2 nm.（2）Construction of a novel label-free HE4 electrochemical biosensor.This sensor was developed by introducing the magnetic Fe₃O₄/α-Fe₂O₃@Au nanocomposites as a substrate for molecules immobilization and signal amplification,and DNA aptamers（Apt）were employed as specific recognition probes of the sensor.Cyclic voltammetry（CV）and electrochemical impedance spectroscopy（EIS）results confirmed that the sensor was successfully constructed.The current variation of differential pulse voltammetry（DPV）under different conditions was investigated to obtain optimal sensing performance.The results indicated that under the optimal conditions(the concentration of Fe₃O₄/α-Fe₂O₃@Au was 25 mg·m L^-1,the concentration of ssDNA/Apt was 20μM,the incubation temperature of HE4 was 37℃,the incubation time of HE4 was 75 min),the electrochemical response of the sensor was positively correlated with the logarithm of HE4 concentration（R²=0.996）.The linear range was 0.1 pg·m L^-1-1 ng·m L^-1,the limit of detection（LOD）was 27.5 fg·m L^-1,and the limit of quantification（LOQ）was 91.7fg·m L^-1.The analytical performance studies demonstrated that the sensor had excellent selectivity for HE4 and could detect trace HE4 in complex sample matrices.The relative standard deviation（RSD）of five parallel electrodes was only 2.7%,which revealed that the sensor had good reproducibility.After 7 days of storage at 4℃,the current response was about102.6%of the initial value,proving that the sensor had promising stability.The feasibility of detecting HE4 in diluted human serum samples was verified,and the recovery rate was 99.3-104.1%（RSD≤4.4%）.The incubation of biomolecules could be accomplished in a liquid phase due to the favorable magnetic properties of Fe₃O₄/α-Fe₂O₃,which was more efficient than common sensors that incubated on the solid surface of the electrodes.（3）Construction of a novel electrochemical biosensor for the detection of the TP53 gene.Using peptide nucleic acid（PNA）as a specific recognition probe,a novel electrochemical biosensor for the TP53 gene was successfully constructed based on the principle of complementary base pairing.The influence of different reaction conditions on the sensor performance was discussed to obtain the optimal detection conditions,which were as follows:the concentration of PNA was 3μM,the incubation temperature of tDNA was 60℃,the incubation time of tDNA was 20 min.Using these conditions,the LOD of the biosensor was 0.26p M,the LOQ was 0.85 p M,and the linear range was 1 p M-1μM.The biosensor had excellent selectivity,reproducibility（RSD=1.1%）,and stability,it can distinguish single base mismatched DNA without enzymes and additional DNA amplification procedures.After 7 days of storage at4℃,the current value was still 94.4%of the initial value.Besides,the recovery rate was between97.5%and 104.2%（RSD≤4.1%）.Therefore,the ability to detect real samples was verified.（4）Construction of a novel electrochemical biosensor for the detection of HER2.A novel electrochemical HER2 sensor was constructed by cooperating with two specific recognition probes:DNA aptamer and PNA.The sensor detected HER2 based on two principles.The first principle was that Apt captured the macromolecular HER2 protein,and ssDNA chains were simultaneously released,causing changes in the electrochemical signal.The other principle was that PNA captured the released ssDNA chains,which converted the electrochemical signal changes caused by HER2 to those caused by the number of short strand ssDNA.The optimal detection conditions of the sensor were as follows:the concentration of ssDNA/Apt was 12μM,the incubation time of HER2 was 75 min,the concentration of PNA was 4μm,the incubation time of ssDNA was 1.5 h,and the incubation time of supernatant was 1.5 h.Using the optimal conditions,the LOD of the sensor for HER2 detection was down to 4.1 fg·m L^-1,and the detection range was 0.01 pg·m L^-1-5 ng·m L^-1,demonstrating that the ultrasensitive detection of HER2 was achieved.The HER2 sensor exhibited excellent selectivity,reproducibility（RSD≤1.8%）,and stability（the current response was between 105.2%and 111.6%of the initial value after 7 days of storage at 4℃）.The recovery rate of real samples was 95.9-115.7%（RSD≤5.1%）.