| As is known to all protein is critical to our life. Disease biomarkers are some specific protein and it is of great importance to quantify their concentrations in ultralow levels for early diagnosis and treatment monitoring. Thus it is highly desired to develop the immunoassay technique for accurate, sensitive and rapid analysis of biomarkers in the bioassay field. The electrochemical (EC) immunosensor is the right technique that combined the advantages of electrochemical biosensor and immunoassay thus to improve the sensitivity. Based on the immunoreactions about antigen and antibody, EC immunosensors have been very attractive in the protein assay for their portable and low-cost detection. The rapid development and extensive application of nanotechnology has provided much more effective route to make further improvement on the detection sensitivity and selectivity owing to the good property of nanomaterials. In this work, we report on the preparation of a new class of signal amplification tags and develop some sensitive sandwich-type EC immunosensors to achieve the ultralow level detection of phosphorylated protein and nitrated protein which are effective disease biomarkers. Coupling of the new nanomaterials, the easily fabricated immunosensors show better signal amplification and excellent promise for future medical treatments.Our work consists of three parts, as follows:1. Sensitivity detection of phospho-p53(S15) using a nanogold-coated silica nanocomposites/enzymes based electrochemical immunosensorSilica nanospheres which are of higher specific surface area are widely used as nanocarriers to load much biomacromolecules for bioassays and nanogold has gained tremendous attention for biosensor systems by virtue of the unique optical, structural and catalytic properties. Thus we firstly synthesized the nanogold-coated silica nanocomposites as multienzymes (HRP) and detection antibodies carriers, then cast single walled carbon nanotubes/chitosan composites on the electrodes to attach the capture antibodies. Based on the sandwich-type immunosensors, we could achieve the sensitive quantification of phopho-p53(S15) antigen. We also demonstrated that the measurements of phospho-p53(S15) antigen gave excellent correlations with standard ELISA assays what could be of potential applications in real bioanalysis.2. Disposable electrochemical immunosensor for ultrasensitive detection of OP-AChE adducts using graphene/TiO2/chitosan nanocompositesAs is reported by many researches, TiO2nanoparticles have strong affinity to the phosphoric group of phosphopeptides and other organophosphorus. Graphene, a monolayer of carbon atoms, has gained tremendous attention for biosensor systems due to the fascinating optical, structural and catalytic properties. Herein we synthesized the graphene/TiO2/chitosan nanocomposites by chemical reduction not only to enhance the binding of OP-AChE adducts, but also to accelerate the electric transmittion, which are also to amplify the detection signal. Then we synthesized a new material, gold nanorods, to use as ferritin (labels) and anti-AChE antibodies carriers. After immunoreactions, the SWV signal could be obtained by dropping hydrochloric acid to release the ferric iron. On the optimal condition, the immunosensor shows the linear response range and detection limit of0.07nM-60nM and0.03nM. We also demonstrate that the measurement of OP-AChE adducts in human serum so that the easily fabricated system shows potential applications in clinical bioanalysis.3. Ultrasensitive electrochemical immunosensor for nitrated ceruloplasmin protein determination using graphene/nanogold/chitosan nanocomposites and lead phosphate-encoded apoferritinWe firstly synthesized the graphene/nanogold/chitosan nanocomposites by chemical reduction to immobilize nitrated protein capture antibodies. Then we prepared a new material, lead phosphate-encoded apoferritin which contained thousands of lead phosphate nanoparticles in the hollow inner cavity and used multiple walled carbon nanotubes as labels and detection antibodies carriers. After immunoreactions, drop some microliters of acetic acid buffer to release lead ion and electrochemical signal could be obtained by SWV. On the optimal condition, using nitrated ceruloplasmin protein as model analyte, the immunosensor shows the linear response range and detection limit of10ngmL-1～5μgmL-1and5ngmL-1(S/N=3). It is pointed that the proposed immunosensor array showed low-cost, acceptable reproducibility and stability. |
