Bifunctionalized Carbon Nanotubes-Based Electrochemical Immunosensing And Application For Tumor Marker Detection

Objective:Multiwalled carbon nanotubes（MWCNT）have received intense interest in the field of chemical biosensing due to their unique electrical,optical,thermal,and mechanical properties.However,because of the low reactivity of MWCNT,it is challenging to precisely control the amount of the electrochemical probe and bio-conjugator on the MWCNT surface with sequential reactions for biosensing,which is usually achieved by a step-by-step reaction due to different reaction conditions.Herein,we developed a one-pot electrografting procedure based on free-radical polymerization to attach both an electrochemical probe and a biomolecular coupling agent on MWCNT in a highly controlledmanner.A vinyl ferrocene（VFc）and N-hydroxy succinimide acrylate（NSA）were selected as the electrochemical probe and biomolecular coupling agent,respectively.Because of their similar reaction type,the electrografting of different ratios of NSA and VFc onto MWCNT can be achieved by a one-pot reaction.By taking alpha-fetoprotein（AFP）as a model analyte,an immunosensor based on bifunctionalized MWCNT was fabricated.Method:First,pristine MWCNT was treated with the mixture of sulfuric acid and nitric acid,forming carboxylic acid functionalized MWCNT with enhanced water dispersibility.Carboxylic acid functionalized MWCNT was modified on the glassy carbon electrode（GCE）surface（MWCNT/GCE）,and the mixture of VFc and NSA was dissolved in room temperature ionic liquid（IL）by grinding.The VFc and NSA were co-electrografted to the MWCNT surface（poly（NSA-VFc）/MWCNT）by using MWCNT/GCE as a working electrode via a cyclic voltammetry（CV）technique.Then the molar ratio of funtionalizing agents（NSA:VFc）on MWCNT was optimized.The electrochemical performance of the one-pot electrografted composite（poly（NSA-VFc）/MWCNT）was compared with those by multi-step electropolymerization via differential pulse voltammetry（DPV）.The optimized bifunctionalized MWCNT was characterized by scanning electron microscope（SEM）,transmission electron microscope（TEM）and Raman analysis.With the optimized bifunctionalized MWCNT as both redox probes and substrate for capture antibody（Ab₁）,an electrochemical immunosensor for AFP was fabricated by assembling Ab₁,BSA and AFP onto the MWCNT-modified electrode.The fabrication process was monitored with cyclic voltammetry（CV）,and DPV was applied for quantitative detection of AFP with the as-fabricated immunosensor.The related linear regression equation was obtained according to the relationship of the electrochemical signal and the AFP concentration,and the linear range and limit of detection were achieved accordingly.Finally,the specificity,reproducibility,stability and reliability of the as-fabricated immunosesnor were assessed.Result:TEM and SEM analysis showed the MWCNT after electropolymerization of NSA and VFc exhibited a rough edge and a larger diameter,which could be due to the electrografted polymer on the sidewall of the MWCNT.DPV was applied to assess the electrochemical performance of bifunctionalized MWCNT with different VFc:NSA ratio of 1:1,1:2,1:3,and a VFc:NSA ratio of 1:2 showed the best performance,and a comparison study showed that the bifunctionalized MWCNT prepared by one-pot electropolymerizaton had higher electrochemical activity than the one by two-step electropolymerization.The successful functionalization of MWCNT was confirmed by TEM,SEM,EDS and Raman analysis.By taking AFP as a model analyte,an electrochemical immunosensor was constructed by using the bifunctionalized MWCNT as both the bioconjugator and redox probes.CV results confirmed the successful construction of the immunosensor.From the DPV results,a linear regression equation could be described asΔI/I₀=0.11792 lg c _AFP(ng·m L^-1)+0.0631,and the correlation coefficient was 99.56%.The electrochemical immunosensor showed a wide linear range from 10 ng·m L^-1 to 50μg·m L^-1 with a limit of detection（LOD）of 1.14 ng·m L^-1.In addition,the relative standard deviation（RSD）of constructed immunosensor was 6.14%.The immunosensor retained more than 80%of the initial signal after a storage for 14 d,suggesting a satisfactory stability of the immunosensor.The recovery study of the immunosensor showed a good recovery in the range of 91.47%–105.92%and a relative standard deviation（RSD）of less than 6.20%.The above results suggested that the electrochemical immunosensor was reliable for AFP sensing in clinical diagnosis.Conclusion:In summary,we prepared bifunctionalized MWCNT by one-pot electrografting method for highly sensitive electrochemical immunoassay.Using VFc and NSA as an electrochemical probe and a coupling agent,respectively,the bifunctionalized MWCNT prepared in one-pot electrografting exhibited more electrochemical activity compared to those prepared with two steps.Moreover,the electrografting of the VFc and NSA led to the electropolymerization of the monomers on the MWCNT sidewalls,enhancing the attachment of the ferrocene and succinimide groups,which promoted the redox signal and capture antibodies,two critical factors for enhanced biosensing sensitivity.By taking AFP as a model analyte,the electrochemical immunosensor using bifunctionalized MWCNT as both the probe and coupling agent for Ab₁ demonstrated a much wider linear range compared to those reported before.This work opens new avenues for a one-pot bifunctionalization of carbon nanotubes and other carbon nanomaterials with promising electrochemical activity and high reactivity for biomolecule conjugation that can be used in electrochemical biosensing for clinical diagnosis.