Construction Of Ratiometric Electrochemiluminescence Biosensor And Its Application In Detection Of MCF-7 Cell,Methylase And Carcinoembryonic Antigen

As a new type of electrochemical technology,electrochemiluminescence?ECL?is widely used in bioassays.So far,most methods only analyze a single ECL signal marker,and the detection signal is easy to be interfered and the stability is poor.In order to meet the clinical analysis needs of disease diagnosis,it is necessary to develop some new,simple and sensitive ECL methods to help improve the diagnosis of clinical diseases.In this paper,different kinds of nano-probes are prepared,and combined with the characteristics of anti-fouling and excellent biocompatibility of sensing interface,a ratio ECL biosensor is constructed to realize the detection of MCF-7 cells,methylase,and carcinoembryonic antigen.The specific details is as follows:1.A dual-signal electrochemiluminescence?ECL?detection system based on conductive polymer hydrogel?CPH?is constructed by using CdTe quantum dots?QDs?and luminol as double luminophores.Firstly,aniline,N,N'-methylenebisacrylamide?MBAA?and acrylic acid?AA?is electropolymerized on the electrode surface to prepare CPH to improve the biocompatibility and conductivity of the sensing interface.The internal standard molecule luminol and coreactant potassium persulfate?K₂S₂O₈?is immobilized in the CPH in the process of polymerization.Since the hydrogel is fixed on the electrode surface,electron transfer is probably much easier when coreactant potassium persulfate?K₂S₂O₈?immobilized in the hydrogel compared to that in solution.Secondly,Cancer cells are captured to the electrode surface by another aptamer linked to the Au nanoparticles immobilized on the CPH through Au-S bonds.Thirdly,another aptamer of tumor cells tagged with CdTe QDs?CdTe-Apt 2?signal molecules connected on the sensing interface to realize ECL detection.A good linear relationship is found based on the?ECL_CdTe/?ECL_luminoluminol against the concentration of cancer cells under optimized conditions,the precise detection of tumor cells can be realized.The ECL internal standard method can provide more accurate detection results in complex environments and eliminate interference in the system by self-calibration of two emission spectra.Therefore,the ECL cell sensor constructed in this experiment can significantly improve the accuracy and reliability of cell detection in complex biological media.This method have broad application prospects in health care monitoring and clinical diagnosis.2.An anti-fouling electrochemiluminescent?ECL?biosensor is designed for the detection of DNA methyltransferase?Dam MTase?activity based on a dual-signaling strategy.Briefly,the ITO electrode is modified with PANI,AuNPs and peptide to construct an anti-fouling interface.Then,a hairpin DNA containing the symmetric sequence of 5?-CATC-3?is connected on the anti-fouling interface.In the presence of Dam MTase and DpnI,the specific sequence of hairpin DNA H₁ is cut off.The Au@luminol labeled on H₁ will be removed away from the sensing interface,and the ECL signal intensity of luminol will be reduced.The residual DNA and two hairpin DNA could produce HCR reaction,and leads to the formation of extended dsDNA polymers which cause the insertion of numerous PTC-NH₂ into the dsDNA grooves,resulting in significantly amplified ECL signal.Methylase activity is detected by a decrease in the intensity of the anodic luminescence and an increase in the intensity of the cathode luminescence.This method has great potential to be further applied in early clinical diagnosis.3.The system uses Au@luminol and CdS quantum dots?CdS QDs?as signal probes to construct a ratiometric electrochemiluminescence?ECL?system for detecting carcinoembryonic antigens?CEA?.And the ITO electrode is modified with PANI,AuNPs and peptide.Polyaniline?PANI?and gold nanoparticles?AuNPs?greatly enhance the conductivity and specific surface area of the electrode,and improve the detection sensitivity.CdS QDs functionalized DNA strand as capture probe and cathode ECL emitters,CEA aptamer modified with anode ECL emitters Au@luminol and quencher cyanine dye?Cy5?fluorophore.After the DNA capture probe combined with CEA aptamer,the cathode ECL of the QDs is quenched by ECL resonance energy transfer?ERET?between CdS QDs and Cy5 molecule,while the anode ECL from Au@luminol can still be detected.In the presence of CEA,binding to the substrate strand,and then releases the Cy5 and Au@luminol,which results in the recovery of the cathode ECL of the QDs and the decrease of the anode ECL simultaneously.Based on the ratio of ECL intensity at two excitation potentials to sensitively analyze the concentration of CEA,this ratio-type ECL sensor will provide a reliable and sensitive detection method for biosensing and clinical diagnosis.