Studies On Novel Label-Free Chemiluminescence Immunoassay Methods

Label-free immunoassay takes the advantage of no label step during the measurements, thus it shows distinct superiorities such as low detection cost, less sample consumption, rapid assay speed, and simple manipulation in comparison with label immunoassay. Chemiluminescence (CL) immunoassay, combing the specificity of immunoassay and the sensitivity of CL, has become one of reseach hotspots in the field of immunoassay. Mimic enzymes possess the intrinsic advantages such as simple prepration, stable nature, easy modification, flexible and convenient application, high catalytic activity and specificity selectivity of substrate. Moreover, The spatial resolution of multiplex immunoassay based on immune sensor arrays can achieve high throughput and simultaneous detection of several analytes. This thesis proposed novel label-free CL immunoassay strategy for the first time. Based on this facile strategy, mimic enzymes replace nature enzymes and developing multiplex immunoassay method from conventional flow-injection single-analyte immunoassay, realized repid, sensitive and low-cost detection of proteins. The detail research was carried out as follows:(1) In this work, a new label-free CL immunoassay method is proposed for the cheap, fast and convenient detection of proteins. This novel strategy can be facilely achieved according to the following principle:the capture antibody and horseradish peroxidase (HRP) are co-immobilized on the Au nanoparticles-chitosan composite interface. The specific immunoreaction between the capture antibody and antigen samples effectively hinders the diffusion of the CL substrate into HRP on the sensing interface and further inhibits the enzymatic CL reaction, thus causing the decrease in CL signals. Compared with the conventional label CL immunoassay, this mode has a lower cost, less consumption, faster speed and simpler operation. Using human immunoglobulin G (HIgG) as a model analyte, the CL signals decrease linearly as the increasing concentration of HIgG ranged from 0.10 to 80 ng/mL with a correlation coefficient of 0.9980. The detection limit was calculated to be 0.068 ng/mL at a signal/noise ratio of 3. The label-free assay method shows high sensitivity, excellent specificity, acceptable reproducibility and accuracy. This work provides a new approach for the development of a promising label-free CL immunoassay method that will result in the innovation of the CL immunoassay technique and play an important role in clinical disease diagnosis.(2) In this work, a novel label-free CL immunoassay method was designed based on smart CuS nanoparticles (CuSNPs) as peroxidase mimetics. The CuSNPs were synthesized by a facile coprecipitation method, and showed high catalytic activity and stability. This efficient label-free CL immunoassay could be easily achieved through a simple strategy. Firstly, CuSNPs dispersed in chitosan were dropped on the epoxy-activated glass slide to form a solid CL signal interface. Streptavidin was then used to functionalize CuSNPs to capture the biotinylated antibody, further producing a sensing interface. After on-line incubation with antigen molecules, the formed immunocomplex on the sensing interface effectively blocked the diffusion of CL substrate into the signal interface and further inhibited the mimic enzymatic CL reaction, thus leading to the decrease in CL signals. Compared to the conventional label CL immunoassay, the proposed label-free assay mode is more simple, cheap and fast. Using alpha-fetoprotein as model analyte, the proposed label-free CL immunoassay method showed a wide linear range from 0.10 to 60 ng/mL with a low detection limit of 0.066 ng/mL. Moreover, the peroxidase mimetics based label-free CL immunoassay system showed high sensitivity, excellent specificity, acceptable reproducibility and good accuracy. This research provided a new and promising strategy for the development of highly efficient label-free CL immunoassay system.(3) In this work, a novel label-free CL immunoassay based on CuO nanorods (CuONRs) as peroxidase mimetics was proposed for highly efficient bioanalysis. The use of CuO nanorods improved the shortcomings of nature enzymes such as poor stability and their catalutic activity can be easily denatured by environment changes. Moreover, CuONRs were reported as excellent peroxidase mimetics which possessed high catalytic activity and better stability to exposure CL intensity of luminol-H2O2-CuONRs. Here, we firstly dropped CuONRs on the epoxy silanized glass slide to form a solid CL "signal surface". After streptavidin fictionalized the CuONRs, the biotinylated antibody was assembled by the biotin and streptavidin system to form a "sensing surface". Then different concentrations of the antigen were introduced into a micro immunoreactor for performing the on-line incubation. The formed immunocomplex on the sensing surface effectively blocked the diffusion of CL substrate into the signal surface, and further inhibited the mimic enzymatic CL reaction. Based on the fact that CL signals decreased linearly with the increase of antigen concentrations, a new label-free CL immunoassay strategy based on CuONRs was proposed for the quantitative detection of proteins. Using carcinoembryonic antigen (CEA) as the model target, the proposed label-free CL immunoassay method showed a wide linear range from 0.10 to 60 ng/mL with a detection limit of 0.048 ng/mL at a signal/noise ratio of 3. Moreover the label-free assay method based on peroxidase mimetics shows high sensitivity, excellent specificity, acceptable reproducibility and accuracy, showing a promising potential in bioanalysis and other relative fields.(4) In this work, a novel label-free CL imaging immunoassay based on CuSNPs as peroxidase mimetics was developed for simultaneous detection of two tumor markers. The array with 4×12 immunosensing cells was prepared by the disposable epoxy silanized glass substrate through the screen-printing technology. Then CuSNPs dispersed in chitosan were dropped in the immunosensing cells. Antibodies were immobilized on the CuSNPS-chitosan with the biotin-streptavidin system. After incubation with antigen molucules, the formed immunocomplex can block the diffusion of CL substrate into the signal interface and further inhibited the mimic enzymatic CL reaction. The decreaseing CL signals was collected by CCD camera. Using alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as the model targets, this method showed wide linear ranges 0.10-70 and 0.10-60 ng/mL with the detection limits 0.028 and 0.029 ng/mL (S/N=3), respectively. The proposed immosensor realized the cheap, fast, high throuput and sensitivity detection of tumor markers, showing potential application for clinical diagnosis and early cancer screening.