The Label-free Electrochemical Immunosensor Based On Modified Mesoporous Silica For Simultaneous Detection

In this paper, a series of nanohybrids have been prepared, such as goldnanoparticles, ionic liquids and mesoporous silica. The novel label-freeelectrochemical immunoassay strategy was proposed for the simultaneous detection oftwo tumor markers. The finished major works are as follows,1. The functional mesoporous silica (MPS) of different pore size and three kind ofmodified mesoporous silica materials were synthesized in this paper. The functionalMPS of different pore size were prepared by changing the dosage of pore-enlargingagent of trimethylbenzene (TMB) in the process of synthesis, and then decorated bothinside and outside the pore walls of mesoporous silica. The modificatory mesoporoussilica GNPs/MPS were prepared by fixing the gold nanoparticles on the internal porewalls using sodium borohydride reduction method. Besides, we have synthesized CdSquantum dots (QDs) modified with mercaptoacetic acid and CdSe QDs, and thefunctional mesoporous silica materials were prepared by embedding CdS QDs andCdSe QDs into the channels respectively. The application of mesoporous materialscould be broaden by using the channels of the functional mesoporous silica materialsprepared in this paper to construct a label free channel immunosensor, greatly reducedthe difficulty and cost in preparation of nanopores in traditional ion channels sensor,and also improved the reproducibility of the preparation of channels immunosensor.2. A label-free electrochemical immunoassay strategy was proposed for thesimultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) andα-fetoprotein (AFP). The functional mesoporous silica was synthesized for theconstruction of the label-free immunosensor. The Si-OH groups on the external surfaceof the mesoporous silica were terminated by trimethylchlorosilane (TMCS), while theSi-OH groups on the internal pore walls were modified with amino groups. Theelectrochemical substrates of methyleneblue (MB) and6-(Ferrocenyl) hexanethiol(FeC) were embedded into the channels of gold nanoparticles(GNPs) modifiedmesoporous silica (MPS), respectively. The monoclonal antibodies of CEA (anti-CEA) and of AFP (anti-AFP) were immobilized into the channels of MB-GNPs-MPS andFeC-GNPs-MPS. Thus, the immunological probes of anti-CEA/MB-GNPs-MPS andanti-AFP/FeC-GNPs-MPS were obtained. The double-analyte immunosensor wasconstructed by coating the probes respectively onto the different areas of indium-tinoxide electrode. When the immunosensor was incubated with sample solution, CEAand AFP were introduced into the mesopores after the immunological reaction. Thenonconductive immunoconjugates blocked the electron transfer through the channels.The simultaneous detection of CEA and AFP could be achieved by monitoring thecurrent change before and after the immunological reaction. GNPs confined inside themesopores could promote the electron transportation through the pore channels to theelectrode surface. Under the optimal conditions, the linear ranges of CEA and AFPwere0.05-50ng mL-1and0.1-100ng mL-1with the detection limits of0.1ng mL-1and0.1ng mL-1(S/N=3), respectively.3. A label-free electrochemical immunoassay strategy was proposed for thesimultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) andα-fetoprotein (AFP). The electrochemical substrates of ferrocenecarboxylic acid(FCA) and methylene blue (MB) were co-immobilized inside the channels of the ionicliquids (ILs) modified mesoporous silica (MPS), respectively. The monoclonalantibody of CEA (anti-CEA) and the monoclonal antibody of AFP (anti-AFP) wererespectively co-immobilized inside the materials of FCA-IL-MPS and MB-IL-MPS.The ITO slide was separated lengthways into two uniform parts by insulation glue soas to avoid the cross-talk of the two portions. Finally, the suspension solutions werecoated respectively onto the different areas of indium-tin oxide (ITO) electrode. Thedouble-analyte immunosensor was constructed by the probes of CEA and AFP ontothe different areas of ITO. When the double-analyte immunosensor was dipped into thesample solution, the antigens of CEA and AFP reacted with their correspondingmonoclonal antibodies on the different area of the modified ITO electrode. After theimmunological reaction, the nonconductive immunoconjugates formed inside the MPSchannels. With the increasing concentrations of CEA and AFP antigens, the spatialblocking and impedance on the sensor surface increased, thus the electric responsetransfer from the solution to the electrode surface was blocked and the DPV currentsdecreased. The electrochemical signals for CEA were detected by using FCA as theelectron mediator. The electrochemical signals for AFP were detected by using MB asthe electrochemical substrates. Then, the simultaneous detection of CEA and AFPcould be achieved. The electrode modification process was further characterized bycyclic voltammetric measurements and electrochemical impedance spectroscopy. ILunits inside the mesopores could promote the electron transportation through the porechannel. To clarify the adsorption of FCA and MB into the mesopores of ILs-modified MPS, the IR spectra were recorded. The linear ranges of CEA and AFP were0.5～80ng mL-1and0.5～100ng mL-1with the detection limits of0.1ng mL-1and0.1ngmL-1(S/N＝3), respectively. The fabricated immunosensor shows appropriatesensitivity because of the good electric conductivity of ILs. It is an alternative to themultianalyte detection of antigens or other bioactive molecules.