Research On Detection Of Renal Cell Carcinoma Based On The Specific Interaction Between 3-aminophenyl Boronic Acid And Sialic Acid

Objective:Renal cell carcinoma(RCC)is a malignant urinary neoplasm with an adult prevalence of 2%to 3%.It has been increasing in recent years and has a high mortality rate.Due to lack of significant early symptoms,the existing detection technology can not meet the requirements for early diagnosis of the disease susceptible to delays.Therefore,to study and establish a new method of detection has important clinical value for early diagnosis of renal cell carcinoma,treatment and prognosis.Biosensors as a class of fast,accurate and ultra-sensitive analytical components have become a focus of attention in the field of biomedical analysis in recent years.In particular,cell sensors with living cells as their sensitive components have attracted the attention of clinical diagnostic researchers.Studies have shown that changes in the expression of sialic acid(SA)molecules on the cell surface are closely related to the occurrence and development of malignant tumors and can be used as a tumor marker.When malignant tumors occur,the expression of sialic acid molecules is significantly increased,and the cell adhesion is reduced,which facilitates the entrance of cancer cells into blood or urine.Therefore,based on the good biocompatibility,ultra-sensitivity,high specificity,and stability of cell sensors and SA marker,this study will construct a novel cell sensing technique for the detection of renal cancer cellsusing the surface-specific sialic acid molecules of renal cancer cells,polypyrrole(PPy)and silver submicron complexes(Ag@BSA)as a signal amplification material and3-aminophenylboronic acid(APBA)as a molecular probe.Methods:(1)Ag@BSA submicron particles were prepared by using the conductivity of silver particles and the functionality of bovine serum albumin(BSA).The electron microscopy(SEM and TEM),FT-IR and energy diffraction electron spectroscopy(EDS)were used to characterize the synthesized material,while using the CCK-8 assay to investigate the cytotoxicity of Ag@BSA;(2)PPy has a large specific surface area and good conductivity,and it is applied with Ag@BSA micro-particlesone-step co-deposition onto the surface of the gold electrode to form a PPy-Ag@BSAcompositefilmbycyclicvoltammetry.SEM,three-dimensional laser scanning and cyclic voltammetry were used to verify the successful co-deposition of PPy-Ag@BSA composite films.(3)Differential pulse voltammetry(DPV)and electrochemical impedance spectroscopy(EIS)were applied to describe the cytosensor construction process.(4)Cyclic voltammetry was used to optimize the number of PPy-Ag@BSA composite membrane co-deposition cycles,APBA fixed incubation time,and capture cell time to improve the specificity and sensitivity of the sensor.(5)The proposedsensor was used to detect different concentrations of renal cancer cell lines 786-O,and the detection range and detection limit of the sensor were obtained by EIS.(6)EIS was used to verify the specificity,stability,and reproducibility of the cell sensor.(7)Cytosensors were used to detect clinicalurine samples from kidney cancer patients.RESULTS:Electron microscopy,FT-IR and EDS all results confirmed the successful synthesis of Ag@BSA.At the same time,CCK-8test showed that Ag@BSA has good biocompatibility.SEM,three-dimensional laser scanning and cyclic voltammetry results showed that PPy-Ag@BSA was successfully deposited on the surface of the gold electrode,increasing the surface area and conductivity of the electrode,indicating that the PPy-Ag@BSA complex was successfully synthesized for the first time.After electrochemical validation of the successful construction of the sensor,the resultsof optimization showed that the PPy-Ag@BSA co-deposition cycle was 15 cycles,the APBA incubation time was 60 minutes,and the cell blinding with APBA time was 60 minutes.Under the optimal experimental conditions,the sensor detection range is 17~1.7×10~6 cells/mL,and the low detection limit is(S/N=3)6 cells/mL.The cytosensor shows good specificity,stability and reproducibility.The results of the 3 cases of RCC showed that the sensor can quickly and specifically identify RCC in urine samples,which initially showed that it has potential clinical application value.Conclusion:In this study,the impedance-based cell sensor was successfully constructed by using the PPy-Ag@BSA complex and the specific connection between APBA and sialic acid.The sensor has low limit of detection,high sensitivity and good stability and can be used for detection of renal cancer cells in urine.The study provides a scientific basis for the early diagnosis and clinical application of renal cell carcinoma.