Piezoelectric Electrochemical Studies On Nanobiosensing Of Tumor Cells And Proteins

Researches on novel cytosensors and biocompatible interfaces are of great significance for advances of biomedical engineering,biotechnology,and tissue engineering.The development of new and effective electrochemical biosensors for tumor relevant antigens is important for tumor's early diagnosis in practice. Nanomaterials of special nanostructure possess interesting physical and chemical properties,making them highly promising in bioanalysis and biosensing applications.In this dissertation,the recent progress of cell-based biosensing and electrochemical immunoassay,application of nanomaterials and quartz crystal microbalance(QCM)in bio/chemo fields are reviewed,and a series of studies on cell adhesion,detection of cell viability,adsorption of proteins and immunosensor fabrication were carried out with QCM and electrochemical techniques.The studies are summarized as follows.1.The gold-electrode QCM was used to monitor the one-day incubation of human breast cancer cells(MCF-7).In combination with an optical microscope simulation experiment that shows the cell-population pictures at various stages,the QCM responses to the cells' adhesion,spreading and proliferation on the electrode surface are discussed.The resonant frequency(â–³f₀)and the motional resistance(â–³R₁)responses were found mainly from mixed effects of mass,viscodensity and surface stress,and in proportion to the cell coverage.2.The QCM was used to monitor the adhesion of mammalian cells on a chitosan(CS)/multiwalled carbon nanotubes(MWCNTs)composite modified gold electrode.The human breast cancer cells(MCF-7)were adhered to and grown on the CS/MWCNTs film modified gold surface or a net CS film modified gold surface,and the process of which was continuously monitored and displayed by changes of theâ–³f₀ andâ–³R₁ of the QCM.The attachment rate and viability of the cells when proliferating on the two surfaces were detected by the MTT assay.The presence and state of cells on the electrode surface were confirmed by the fluorescent microscopy.Cyclic voltammetry and electrochemical impedance spectroscopy of the ferricyanide/ferrocyanide couple were examined before and after the cell adhesion.3.A simple protocol was proposed for rapid magnetic isolation,immobilization and electrochemical detection of leukemia K562 cells via their decoration with cationic magnetic nanoparticles(CMNPs).The CMNPs modified living cells could be rapidly isolated from the medium and immobilized firmly on the electrode surface via a magnetic field.The cyclic voltammetric experiments of the cells immobilized at glassy carbon electrode exhibited an irreversible anodic current peak whose height is proportional to cell viability, and the 5-fluorouracil cytotoxicity results obtained from the proposed magnetic-electrochemical method were supported by conventional MTT assays. 4.A new biocompatible interface made up of hydroxyapitite-multiwalled carbon nanotubes(HA-MWCNTs)nanocomposite was developed for adhesion and electrochemical detection of human osteoblast-like cells (MG-63).The HA-MWCNTs nanocomposite was synthesized by self-assembling nano-hydroxyapatite onto multiwalled carbon nanotubes in solution.The QCM was used to monitor the adhesion of MG-63 cells on HA-MWCNTs modified gold electrode.The nanocomposites interface showed improved immobilization capacity for cells and good biocompatibility for preserving the activity of immobilized living cells.The living cells immobilized on an HA-MWCNTs modified glassy carbon electrode exhibited an irreversible anodic peak response being positively associated with the density and viability of cells,which was used to describe the cells growth and evaluate the effectiveness of antitumor drug 5-fluorouracil(5-FU)on the cells.The obtained 5-FU cytotoxicity results agreed well with those from conventional MTT assays.5.The QCM,in combination with electrochemical impedance spectroscopy (EIS),has been utilized to monitor in situ anti-human IgG adsorption on several Au-based surfaces,bare Au,nanogold/4-aminothiophenol(4AT)/Au, and multi-walled carbon nanotubes(MWCNT)/Au,and succeeding human IgG reactions.The resonant frequency(f₀)and the motional resistance(R₁)of the piezoelectric quartz crystal(PQC)as well as electrochemical impedance parameters were measured and discussed.Cyclic voltammetry(CV)and electrochemical impedance spectroscopy(EIS)of the ferricyanide/ferrocyanide couple were examined before and after electrode modification,antibody adsorption and antibody-antigen reactions.We found that the amount for antibody adsorption was the greatest on the colloid Au modified surface,and that at MWCNT ranked the second,while the specific bioactivity was almost identical on the four kinds of surfaces.Two parameters simultaneously obtained at the colloid Au modified surface,â–³f₀ andâ–³C_s(interfacial capacitance),have been used to estimate the association constant of the immunoreaction.6.An ultrasensitive impedance immunosensor for the detection of human alpha fetoprotein(AFP)has been fabricated based on Au microelectrode and gold nanoparticle(nanoAu).A nanoAu monolayer was assembled on the Au microelectrode(d=100μm)to immobilize AFP antibody(anti-AFP),the electrode before and after anti-AFP modification was characterized by cyclic voltammetry(CV)and atom force microscopy(AFM).The factors influencing the performance of the immunosensor were studied in detail. Different concentrations of AFP antigen were determined and the results were compared with the normal Au disk electrode.The Rot response was proportional to logarithm of AFP concentration ranging from 0.005-50 ng mL^-1and the detection limit was 0.76 pg mL^-1.The presented immunosensor is ultrasensitive,selective,and promising for clinical immunoassays.7.A novel lable-free amperometric immunosensor for the determination of carcinoembryonic antigen(CEA)based on nanocomposite containing electron mediator has been developed.Firstly gold nanoparticle(nanoAu) and toluidine blue doped-silica nanoparticle(TB-silica)were synthesized, after mixing with chitosan(CS),the nanoAu/CS/TB-silica nanocomposite was cast on the electrode surface to prepare immuno electrode.The factors influencing the performance of the amperometric immunosensor were studied in detail.Different concentrations of CEA antigen were determined and the current response was proportional to CEA concentration ranging from 1 to 80 ng mL^-1and the detection limit was 0.65 pg mL^-1.The nanocomposite modified electrode exhibited excellent redox reversibility and sensitivity due to synergistic effect of nanoparticles.Tests performed with this immunosensor showed high selectivity and stability.