| Immunosensor array is an immunologic chip on which hundreds of thousands of immunosensors were immobilized on a basic plate. It has been a hot topic in the field of the study of immunoassay due to it can be detected easily and was high throughout. Optical immunosensor array means that an immunoassay which based on the detection of the light signal emitted from the sensors, such as fluorescence, chemiluminescence, surface resonance plasma, surface-enhanced roman scattering and so on. Using fluorescent labels has been a mature technology in immunoassay; therefore, the immunosensor array based on the detection of the fluorescence is a hot topic in the immunologic field. Confocal laser microscopy was used generally in the study of immunology for the achievement of the fluorescent image not only qualitatively, but also quantitatively. In the detection which is qualitatively, such results can meet the need, but if the results should be quantitatively, then the image should be transformed into data by using some softs, this is difficult and time consuming, and the result can not be very exactly. Therefore, in this study, we constructed a new laser induced fluorescence millimeter sensor array detection platform to measure the fluorescent signal of the sensor array, and use it in the new fluorescent immunoassay, through this way, the detection becomes more easily and more sensitive.The thesis consists of two chapters:Chapter one:The review In this part, a review about immunology, immunosensor array based on optical detection method, and several kinds of detection method of the sensor array was given, and the advantages and disadvantages of these detection methods were shown.Chapter two:Research reports which are composed of five components as follows:1. Construction of the laser induced fluorescence device and its application evaluation.A new laser induced fluorescence detection device was constructed firstly by us. The sensitivity of the device using laser as light source was 500 higher than using Xe lamp as light source.Therefore, it will meet the need of the fluorescent immunoassay which need high sensitivity.2. Synthesis of the core-shell fluorescent silica nanoparticles and character of its fluorescence.Core-shell fluorescent silica nanoparticles were synthesized and we measured their size, fluorescence. The TEM image of the fluorescent nanoparticles showed that the nanoparticles were 30+5 nm in diameter and uniform without aggregation. Spectrofluorometric measurements were also used to characterize the nanoparticles. Compared with the pure Rubpy dye, the complete spectrum of the nanoparticle suspensions was only shifted towards the longer wavelength by 3 nm, indicating that the spectral characterization of the fluorescence dye was not changed greatly when it was doped inside the nanoparticles and modified with biomolecules. Photo bleaching experiment and the leakage of the dye also were detected, and the results demonstrated that our nanopaticles were suited to be used as immunologic labels.3. A novel immunoassay for carcinoembryonic antigen (CEA) using laser-induced fluorescence millimeter sensor array detection platformA method employing FFCSNs as fluorescent labels in fluoroimmunoassay of CEA model was established, and a laser-induced fluorescence millimeter sensor array detection platform. High specificity and sensitivity were obtained based on this method just using 1-2μL samples. The Calibration curve for the CEA was△I=3.607c+0.8696 with r=0.9933, the linear range was 1 pg-80 pg. The detection limit of cea was 0.3 pg. The relative standard deviation (RSD) for 5 parallel measurements of CEA (40 pg) was 5.5%.The comparison of the analytical character for CEA between ELISA and laser induced fluorescence immunoassay demenstraed that our new method was credible.4. An ultrasensitive immunosensor array for determination of staphylococcal enterotoxin BA method employing FFCSNs as fluorescent labels in fluoroimmunoassay of SEB model was established using two mAbs recognizing different epitopes of SEB, and a laser-induced fluorescence millimeter sensor array detection platform. High specificity and sensitivity were obtained based on this method. In this study, a typical "sandwich" method was adopted, and we used a new device for the measurement of fluorescence. The calibration curve for the SEB was I F=66.14[SEB]+149.8 with R2=0.9904, the linear range was 0.05 pg-5 pg. The results of sample analysis were also compared between ELISA and our laser induced fluorescence immunoassay.5. A novel immunoassay for residual bovine serum albumin (BSA) in vaccines using laser-induced fluorescence millimeter sensor array detection platformA method employing FFCSNs as fluorescent labels in fluoroimmunoassay of BSA model was established using two mAbs recognizing different epitopes of BSA and a laser-induced fluorescence millimeter sensor array detection platform. Thus, the platform constructed by us made the immunoassay highly sensitive, specific, and simple for high-throughput detection of BSA. Moreover, it needs fewer samples than most immunoassays. Under the conditions selected, the signal for BSA was a linear function of concentration over the range 1.0～100 ng/mL with the regression equation of IF=1.079[BSA]+19.56 (r=0.9880), and the detection limit of our method was 0.3 ng/mL (3σ). We also use ELISA to detect BSA, the calibration curve of the colorimetric ELISA was△I=0.00238[BSA]+0.3715 (r=0.9907), and the linear range was 0.5～40 ng/mL. Compared with colorimetric ELISA, the immunoassay constructed by us has a broader linear range and a higher sensitivity. |
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