Studies On Chemiluminescence And Imaging Immunoassay Methods

Chemiluminescence (CL) immunoassay, combing the specificity of immunoassay and the sensitivity of CL, possesses the advantages of high sensitivity, high selectivity, low cost and pollution free, and has been widely used in clinical diagnosis, biological analysis, environmental monitoring, food security and other fields. Compared with the conventional single-analyte immunoassay methods, the spatial-resolved multiplex immunoassay based on sensor arrays has much higher throughput and enhanced assay performance. By coupling with nanomaterials-based or DNA-based signal amplification strategy, the multiplex immunoassay can achieve the perfect unification of high throughput and high sensitivity. With the use of CL imaging detector, multiple tumor markers at low levels can be simultaneously detected, which is of significance for early cancer screening and diagnosis. In addition to mutiplex analytes and high sensitivity, the simplication of operation procedures and fast speed are all-time goals of immunoassay. This thesis focuses on multiplex CL imaging, high sensitive immunoassay, and fast and simple bioanalysis to carry out some researches as follows:1. Chemiluminescence Imaging Immunoassay of Multiple Tumor Markers for Cancer ScreeningAn ultrasensitive multiplexed CL immunoassay method was developed for the detection of four tumor markers by combining a horseradish peroxidase (HRP) and signal antibodies labeled gold nanoparticles (AuNPs) as trace tag and a disposable immunosensor array. The method was then applied to screen different cancers. The array was prepared by immobilizing capture antibodies on the screen-printed silanized glass slide in a 4×12 format. With a sandwich-type immunoassay format, ultrahigh sensitivity was obtained by the enzymatic signal amplification using HRP as the catalyst of the luminol and hydrogen peroxide (H2O2). The CL signals were collected by a charge coupled detector (CCD) camera simultaneously. Using a-fetoprotein (AFP), carcinoma antigen 125 (CA 125), carcinoma antigen 153 (CA 153) and carcinoembryonic antigen (CEA) as model analytes, this method showed wide linear ranges with the detection limits down to 7×10-6 ng/mL,1.7×10-5 U/mL,8.6×10-6 U/mL and 1.6×10-5 ng/mL, respectively. The immunosensor array exhibited acceptable reproducibility and stability. The assay results of tumor markers in serum samples were in acceptable agreement with the reference values. When applied into cancer screening, it also has exellent accuracy. This method showed potential application for clinical diagnosis and early cancer screening.2. Multilayer Hemin/G-Quadruplex Wrapped Gold Nanoparticles as Tag for Ultrasensitive Multiplex Immunoassay by Chemiluminescence ImagingA multilayer DNAzyme wrapped AuNPs was designed as tag for ultrasensitive chemiluminescence imaging immunoassay of multiple tumor markers. G-quadruplex DNA (G4 DNA) can bind hemin to form HRP mimicking enzyme and catalysis the CL of luminol and H2O2, thus a long DNA sequences called as signal DNA containing multiple and repeated G4 structure was synthetized. The preparation of multilayer hemin/G-quadruplex wrapped AuNPs was performed by assembling high ratio of the signal DNAs and a longer biotinylated DNA on the AuNPs, and then binding hemin with the G4 structure. The as-prepared tracer was then used to carry the signal antibodies for ultrasensitive multiplex immunoassay with a disposable 4 X 12 immunosensor array. The array was prepared by coating capture antibodies on screen-printed glass slides. After the formation of immunocomplex, the tracer could be captured on the sensor array via avidin-biotin system. After the addition of CL substrate, multilayer DNAzyme on the tracer could enhance the CL signals of luminol. The novel tracer and avidin-biotin system could both amplify the detectable signal. Moreover, one tracer here could be employed to detect four tumor markers, thus simplified the preparation of tracers. Using AFP, human chorionic gonadotropin-beta (β-hCG), CA 125 and CEA as model analytes, the simultaneous multiplexed immunoassay method showed linear ranges of 5 orders of magnitude with the detection limits down to 2.7 X 10-5 ng/mL,1.1×10-5 IU/L,1.7×10-5 U/mL and 2.0×10-5 ng/mL, respectively. The assay results of tumor markers in serum samples with the proposed method were in a acceptable agreement with the reference values, showing promising application in earl cancer diagnosis and bioanalysis.3. Proximity Hybridization-Triggered Signal Switch for Homogeneous Chemiluminescent BioanalysisA proximity hybridization-triggered signal switch was presented for convenient homogeneous chemiluminescent detection of a wide range of affinity target biomolecules, combined with the nicking enzyme recycling strategy to achieve high sensitivity. The target promoted the formation of a proximate complex via the proximity hybridization of two help DNA strands and the DNA strands labeled to affinity ligands, which subsequently unfolded the self-reporting molecular beacon to switch on the chemiluminescence signal. The response could be further amplified with a in situ enzymatic recycling strategy for highly sensitive chemiluminescence detection. By using antibody as the affinity ligand, this simple protocol could sensitively and fast detect CEA in one step with a detection limit down to 24 pg/mL. The proposed methods could also be adapt for the detection of DNA and thrombin. The one-step and wash-free assay possessed the advantages of high sensitivity, acceptable accuracy, and satisfactory versatility of analytes, leading to various applications in bioanalysis.4. Chemiluminescence Imaging for a Protein Assay via Proximity-Dependent DNAzyme FormationA novel chemiluminescence immunoassay method was developed for simple and ultrasensitive detection of tumor markers or thrombin, based on target triggerd formation of DNAzyme and a disposable 4 X 12 immunosensor array. The immunosensor array was prepared by covalently immobilizing capture antibodies on an aldehyde-functionalized disposable glass chip. The assay was composed of two affinity probes both of which were protein affinity ligands such as antibody and aptamer conjugated with one fragment of G-quadruplex. On the sensing surfaces the target protein triggered the formation of sandwich complex between two affinity probes and subsequently the close proximity of two G-quadruplex fragments and hemin to in situ form the peroxidase-mimicking hemin-G-quadruplex DNAzyme. After the addition of CL substrate, the DNAzyme could catalyze luminol CL. Using CEA and thrombin as model analytes, this proposed array-based immunoassay method showed wide linear ranges of 4-5 orders of magnitude with the detection limits down to 0.15 ng/mL and 0.49 pM, respectively. Due to the in situ generation of DNAzyme, the proposed method showed high selectivity and low background, thus could achieve high sensitivity and wide linear range without signal amplification. Its easy operation, short assay time, good sensitivity and nice versatility made the proposed array-based assay a promising candidate for point-of-care testing.5. High-Throughput Imaging Assay of Multiple Proteins via Target-Induced DNA Assembly and CleavageA target-induced DNA assembly and cleavage method was presented for highly sensitive imaging assay of multiple protein biomarkers on a DNA chip. The DNA chip was prepared by immobilizing DNA1-FITC on a screen-printed disposable aldehyde-modified glass slide in a 6 rows X 16 columns format. The target protein could be recognized by DNA3/2-antibody and DNA4-antibody together, thus produced the immunocomplex, led to the proximity of DNA3 and DNA4, and released the DNA2 to hybridize with DNA1 on the DNA chip to trigger the cleavage recycling of DNA 1 by nicking enzyme. Due to the cleavage removal of DNA1-FITC, the fluorescence (FL) intensity of FITC decreased. Meanwhile, less HRP labeled anti-FITC could be captured on the sensing sites, leading to the "switch off" of the CL intensity. For detecting multiple proteins, corresponding affinity probes should be prepared. Using AFP, CA 125, carcinoma antigen 199 (CA 199) and CEA as model analytes, this proposed multiplexed immunoassay method showed acceptable precision with the detection limits down to 0.029 pg/mL、0.016 U/mL、0.011 U/mL and 0.06 ng/mL (FL),0.022 pg/mL,0.012 U/mL,0.010 U/mL and 0.048 ng/mL (CL), respectively. The assay using normal DNA array was simple, sensitive, high throughput, and universal for various analytes, showing good applicability in clinical diagnosis.6. Quantum Dots (QDs) Chemiluminescence and Its Application in Immunoassay of Cancer BiomarkerA novel chemiluminesence system was presented by integrating the unique optical properties of CdTe QDs and peroxyoxalate chemiluminescence. The CL mechanism of CdTe-TCPO-H2O2 system was investigated and applied in immunoassay of CEA with a disposable protein chip. Avidin modified polystyrene sphere and biotin modified CdTes were prepared to perform the signal amplification and improve the detection sensitivity. The research on QDs CL was critical to the understanding of the characteristics of QDs itself, and also extended the nanomaterial-based CL system. Additionally, the proposed CL system and method had great potential in clinical diagnosis.