Investigation On Functionalized Carbon Nanomaterials And Their Applications In Immune Sensing

Immunoassay is a method developed for antigen determination based on the specificbinding between antigen and antibody. Many techniques including fluorescent spectroscopy,chemiluminiscence, surface plasma resonance, quartz crystal microbalance andelectrochemistry etc. have been used in immunoassay. Electrochemical immunosensorcombined the merits of immunoassay and electrochemical sensing, thus possess manysuperior characteristics such as portability, low cost, fast determination, high sensitivity andin-vivo detection etc. The sensing properties of electrochemical immunosensor mainlydepend on how to load biomaterials to the electrode surface and keep their bioactivities, sothat to improve its sensitivity and selectivity. Recently, carbon nanomaterials have beenwidely used in the field of biosensing. The large specific surface area, good biocompatibilityand superior conductivity of carbon nanomaterials will be advantageous for the loading,marking and accumulation of biomaterials, especially for amplification of detection signals.In the present paper, silk peptide functionalized graphene, poly-L-lysine functionalizedcarbon nanotubes and poly-L-lysine functionalized graphene were prepared and characterizedthoroughly. The as-prepared functionalized carbon nanomaterials were employed as basedsubstrates to loading prostatespecific antigen, α-fetoprotein antibody and IgG antibody toproduce electrochemical immunosensors for the corresponding antigen determination. Themain contents are summarized as follows:1. High-molecular-weight silk peptide was used as a modifier to functionalize grapheneto produce graphene-silk peptide composite nanosheets （Gr-SP）. The obtained graphene-silkpeptide nanosheets were confirmed with transmission electron microscopy （TEM）, x-raydiffraction （XRD）, x-ray photoelectron spectroscopy （XPS）, UV/Vis and FTIR spectra. Gr-SPnanosheets were mixed with prostate specific antibody homogeneously, and coated to glassycarbon electrode surface to fabricate a novel electrochemical immunosensor for prostatespecific antigen determination followed by a cross–linking step with glutaraldehyde. Allunspecific sites were blocked by bovine serum albumin. The as-prepared immunosensor wascharacterized with voltammetry and electrochemical impedance spectroscopy. Usingpotassium ferricyanide as electroactive probe, the experimental conditions for prostatespecific antigen determination were optimized. Under optimal conditions, the difference ofcurrent response before and after being incubated into prostate specific antigen solution waslinearly related to prostate specific antigen concentration in the range of0.1～5.0ngmL^-1and5.0～80.0ng mL^-1with a detection limit of0.053ng mL^-1. The proposedimmunosensor was successfully used for the determination of prostate specific antigen inhuman blood plasma. The results were satisfied with that obtained with ELISA,demonstrating good accuracy of the immunosensor. 2. Single walled carbon nanotubes were functionalized with poly-L-lysine to preparesingle walled carbon nanotubes-poly-L-lysine nanocomposite materials （SWCNs-PLL）. Theas-prepared SWCNs-PLL was characterized with TEM, XPS, FTIR and UV/Vis.SWCNs-PLL were mixed with α-fetoprotein antibody marked by horseradish peroxidase（HRP） homogeneously, and then coated to a glassy carbon electrode surface which wasmodified with prussian blue film to fabricate a novel electrochemical immunosensor forα-fetoprotein determination followed by a cross–linking step with glutaraldehyde. Allunspecific sites were blocked by bovine serum albumin. The as-prepared immunosensor wascharacterized with voltammetry and electrochemical impedance spectroscopy. Based on thecatalytic current response of H2O2, the experimental conditions for α-fetoproteindetermination were optimized. Under optimal conditions, the current response was linearlyrelated to α-fetoprotein concentration in the range of0.05～10.0ng mL^-1and10.0～50.0ng mL^-1with a detection limit of0.011ng mL^-1. The proposed immunosensor wassuccessfully used for the determination of α-fetoprotein in human blood plasma. The resultswere satisfied with that obtained with ELISA, demonstrating good accuracy of theimmunosensor.3. Poly-L-lysine functionalized graphene nanosheets （Gr-PLL） were prepared, andthoroughly characterized with transmission electron microscopy, scanning electronmicroscopy, x-ray diffraction, x-ray photoelectron spectroscopy, UV/Vis and FTIR spectra.Gr-PLL nanosheets were mixed with IgG primary antibodies homogeneously, and coated toglassy carbon electrode surface to fabricate a novel electrochemical immunosensor for IgGdetermination followed by a cross–linking step with glutaraldehyde. All unspecific sites wereblocked by bovine serum albumin. The as-prepared immunosensor was characterized withvoltammetry and electrochemical impedance spectroscopy. After specific binding with IgG,the immunosensor was treated with horseradish peroxidase marked IgG second antibodies.Based on the catalytic current response of H2O2, the experimental conditions for IgGdetermination were optimized. Under optimal conditions, the current response was linearlyrelated to IgG concentration in the range of0.15～160.0ng mL^-1with a detection limit of0.087ng mL^-1. The accuracy of the immunosensor was demonstrated bystandard recovery test.