Construction Of New Antifouling Electrochemical Biosensors And Applications In Detection Of Tumor-related Proteins

Cancer is one of the leading causes of death in the world.On average,2,000people are diagnosed with cancer every hour in the world.Since 1975,the incidence of cancer has gradually increased every year.As a serious non-communicable disease,it is the most important obstacle to the healthy development of every country.Tumor markers are specific substances produced during the growth of tumor cells,and their distribution in different organs in the body is relatively specific.In the past few decades,people have made great efforts to diagnose and treat this disease.And early,accurate and sensitive detection of tumor markers is essential to reduce mortality.Electrochemical methods have received extensive attention due to their high sensitivity,fast response,low background interference,simplicity and low cost.However,human serum is the most popular and intensively studied diagnostic solution for the detection of tumor markers,and its complex environment will inevitably affect electrochemical detection.Therefore,an electrochemical method for detecting tumor markers with high sensitivity,low fouling and high accuracy is urgently needed.Based on this,this article combines high-conductivity electrode surface modification,antifouling materials,signal amplification strategies and ratio detection methods to construct three electrochemical biosensors to realize the detection of thrombin（TB）and carcinoembryonic antigen（CEA）.The specific schemes are as follows:1.An antifouling ratiometric detection method based on antifouling peptides and a new self-enhanced luminescent probe is proposed to achieve the quantitative detection of TB.The electrode is modified with conducting polymer poly（3,4-ethylenedioxythiophene）（PEDOT）and luminol-reduced gold nanoparticles（Au@Luminol）.Then bi-functional peptides anchor to the modified electrode and low-toxicity electrochemiluminescence（ECL）probes are connected to the other end of the bi-functional peptide through amide bonds to form a complete ECL biosensor.The bi-functional peptide includes a new antifouling sequence（DRDRDRDR）and a TB recognizable sequence（SGRPVLG）,while the former can greatly reduce the non-specific adsorption in complex samples and the latter can be cleaved by TB.The new self-enhanced luminescence probe labeled as PAMAM-QDs is composed of low toxic and water-soluble Cu In Zn S/Zn S quantum dots（CIZS/Zn S QDs）and polyamidoamine（PAMAM）via amide bonds.Concentration of TB can be obtained via the ratio of the signals of ECL_QDs/ECL_Au@Luminol accurately and sensitively.The ECL biosensor with the detection line 1.82 f M can work from 10 f M to 1 n M.Thanks to the antifouling property of the designed peptide and the amplification of the new ECL probe,the biosensor can be used for quantifying TB in practical complex samples with high selectivity and sensitivity.2.A ratiometric antifouling electrochemical biosensor was developed based on two-dimensional（2D）nanomaterial MXene loaded with gold nanoparticles（Au NPs）and methylene blue（MB）.The nanocomposite of MXene loaded with Au NPs and MB（MXAu-MB）exhibited excellent conductivity,where the Au NPs were able to capture biomolecules containing sulfhydryl terminus,and the MB molecules were used to generate electrochemical signal.The MXAu-MB was fixed on the electrode surface by Nafion,and the anchored peptide captured the electrochemical signal probe carboxyl-modified ferrocene（Fc）to construct an electrochemical biosensor.The bi-functional peptide containing the anchoring,antifouling and recognizing sequences endowed the sensing surface not only the assaying function,but also the capability to resist nonspecific adsorption from complex samples.In the biosensing system,with the increase of the target concentration,the electrochemical signal of MB remained constant,while the electrochemical signal of Fc gradually decreased,and the ratiometric detection strategy greatly improved the accuracy of the biosensor.In the presence of TB,the recognizing sequence was recognized and cleaved,and the ratiometric signal of Fc and MB indicated the concentration of TB accurately and sensitively,with the detection range from 0.1 p M to 10 n M and a limit of detection of16.1 f M.Electrochemical biosensors based on the MXAu-MB and bi-functional peptides possessed high selectivity,accuracy and sensitivity even in real complex biological samples owing to the excellent antifouling ability of the peptide.3.A novel ratio electrochemical biosensor based on functional 2D nanomaterial MXene was developed.Fe₃O₄ was synthesized in situ on carboxyl functionalized MXene,and then covalently bonded with[Ru（NH₃）₆]³⁺to obtain nanocomposites MXC-Fe₃O₄-Ru.It has high conductivity,hydrophilicity and good antifouling ability.The strong magnetic MXC-Fe₃O₄-Ru can be easily separated and firmly modified on the magnetic gold electrode（MGE）and the DNA double-stranded（ds DNA）containing an Fc-modified CEA aptamer chain can be captured by covalent bond.When CEA is present,CEA binds to the aptamer and leaves the electrode surface,the electrochemical signal of Fc decreases,while the electrochemical signal of[Ru（NH₃）₆]³⁺fixed on the electrode surface remains basically unchanged The ratio of the two is directly proportional to the CEA concentration.The linear range of the sensor is 1 pg/m L to 1μg/m L,and the detection line is 0.62 pg/m L.In addition,MXC-Fe₃O₄-Ru has excellent antifouling performance due to its good hydrophilicity and electric neutrality.It does not need to modify other antifouling materials,which greatly simplifies the electrode construction process,ensures the electron transfer efficiency of the electrode surface.And the ratiometric strategy can reduce the interference of the external environment to the detection signal.Therefore,the sensor can achieve high selectivity,high accuracy and high sensitivity detection of targets in complex samples.