Determination Of Cancer Marker Based On Surface Plasmon Resonance And Magnetic Microsphere Assited Fluoroimmunoassay

Compared to the traditional methods of determination of cancer marker, surface plasmon resonance(SPR) is one of the powerful analytical tools for directly monitoring molecular interactions, with free label to the target molecules. Due to some advantages, such as fast separation, high efficiency, keep high activity, immonumagnetic beads(IMBs) have been used widely in biological analysis. In this paper, we developed a novel method to detect Carcinoembryonic antigen(CEA) in buffer and human serum spiking samples using a SPR biosensor with gold nanoparticles(GNPs) to enhance signal. Different methods were used to enhance and amplify the signal including sandwich immunoassay and the second signal amplification by GNPs. A novel immunoassay for the determination of CEA was established by combining a fluoroimmunoassay and immunomagnetic separation.In the first chapter, it has been described the principles of SPR technology, tumer marker and IBMs, their advantages, instrument, the detection format and their applications. My mayor work was illustrated. This thesis mainly focuses on development of rapid, sensitive detection methods of CEA by using SPR technology and immonumagnetic beads fluoroimmunoassay.In the second chapter, a standard direct binding assay and sandwich assay were carried out to detect CEA using SPR sensor. Antibody coupling conditions were optimized, including pH, flow rate, concentration. pH 4.5, 10 μL/min, 100μg/m L were chosed as the condition of optimization in immobilaizition processes. The immobilization of antibodies on the sensor chip caused a shift of 8475 RU, which corresponds to 8.475 ng mm-2. The the limit of detection(LOD) is 13.78 ng/mL in direc format assay. To improve the sensitivity, sandwich direct format was used to detect CEA. The LOD of this enhanced sandwich assay for CEA detection was reduced to be 3.30 ng/mL. The method of the sandwich assay with mAbCEA-B5 gave about 4.2-fold enhancement in the sensitivity compared to that of the direct detection method. The recovery(102.56%~106.86%) and relative standard deviation(RSD)(between 4.96%~8.99%) were acceptable. Rapid and sensitive detection of CEA was performed.In the third chapter, GNPs enhanced sandwich format using bio-mAbCEA-B5 and SA-GNPs were used to detect CEA in buffer based on SPR sensor. Quantitative detection of SA adsorbed on the GNPs surface and the binding sites of SA were carried out. The amount of SA adsorbed to GNPs was calculated to be 8.50 μg. The results show that 42.5% of SA adsorbed on the GNPs. About 11.92 SA tetramermolecules are bound to one particle and about 33.10 biotin bindingsites are accessible at the surface of a single particle. This corresponds to 10.54 biotin binding sites 100 nm-2 particle surface. The calibration plot showed a good linear relationship between SPR response and CEA concentration in the range of 1~60 ng/m L with a correlation coefficient of 0.9772. The LOD of this enhanced sandwich assay for CEA detection using SPR biosensor was 1.0 ng/mL. Linear equation is: Y=59.8100+6.8540X(ng/mL). The sensitivity of GNPs enhanced sandwich assay format was about 13.8-fold better than that using direct assay format and 3.3-fold better than that using sandwich assay format without GNPs. The experimental results confirmed that the proposed SPR biosensor possessed high sensitivity, good selectivity for CEA detection, which indicated that the developed SPR biosensor provides a possible application for the detection of CEA in clinical diagnostics.In the fourth chapter, qualitative and semiquantitative detection of CEA was conducted based on the combination of IBMs with fluoroimmunoassay. Carboxyl groups on the magnetic beads were first activated with a mixture of EDC and NHS to give reactive succinimide esters. The mAbCEA-C3 was then passed over the surface and esters react spontaneously with amino groups to link mAbCEA-C3 covalently to magnetic beads to form IBMs. In the mixture samples, CEA was captured by mAbCEA-C3 with high specificity and affinity. The conditions, such as washing times and immune reaction time were optimized. The final choice of immune time is 45 min, three times of washing. The results indicated that the fluorescence intensity increased with the increment of CEA concentration in the optimal condition.