| Recently, with the occurrence of widespread heavy cyanobacterial blooms, several serious water and food safety episodes involving microcystins is attracting global attention. Microcystin-LR (MC-LR) is the most common and toxic in all cyanophycean toxin.Existing analytical methods for MC-LR have many disadventages such as high cost, tedious sample preparation and low efficiency. Therefore, the development of a highly sensitive and rapid detection method for MC-LR is of significance. In this paper, through the preparation of immune antigen and specific antibodies to MC-LR, an immunosensor which based on immunoassay technique and self-assembled nano-material was developed for the determination of MC-LR. The main points of this dissertation are summarized as follows:The immuno and detection antigen were prepared using the methods of"active ester"and"EDC catalyze". High selectivity polyclonal antibody against MC-LR was obtained from the New Zealand rabbit immunized with the immuno-antigen. The antiserum was tested by indirected ELISA. The titer of this antibody is higher than 5.2×104 after purification; The calibration curve by indirected ELISA shows that the range of quantitative detection was 0.25～4μg/L with an IC50 value of 0.68μg/L.The cross reactivity with MC-RR was 21.9%,and were all lower than 1% with MC-LW and MC-LF.A label-free immunosensor for detection of MC-LR has been developed by immobilizing anti-MCLR on Nano-Au/L-cysteine coated electrode. Using the developed method, MC-LR could be determined with a detection limit of 1.82×10-2 ng/mL(S/N=3) and linearity between 0.05 and 300.0 ng/mL. The whole detecting time was only 15 min. The average recovery rate was in the range of 96.8～112.5%. The immunosensor exhibited long-term stability and good reproducibility of the signal could be obtained up to 42 times by CV, The coefficient of variation was 3.58% ; Furthermore, good stability in 30 days at 4℃.Immobilization of anti-MCLR has been based on self-assemble technique of 1,6-hexanedithiol and Nano-Au. The resulting immunosensor displayed a high sensitivity and a wide linear range for the detection of MC-LR, and responded to the MC-LR in the range of 0.25～95.0 ng/mL with a low detection limit of 8.5×10-2 ng/mL(S/N=3). Moreover, the relative standard deviation for eight parallel determinations is 7.48% and good stability in 30 days at 4℃. The average recovering was in the range of 92.6%～118.3% . The detetion time was 15 min.Immobilizing anti-MCLR on multi-walled carbon nanotubes (MWNTs)/ room temperature ionic liquids (RTILs) coated electrode, an immunosensor for detection of MC-LR was developed.MC-LR could be determined with a lower detection limit of 1.7×10-3 ng/mL(S/N=3)and linearity between 5×10-3 ng/mL and 1×102 ng/mL. The average recovery was in the range of 80.95%～127.23%. Some common ions in water have no interference with MC-LR.In view of storage conditions of immunosensors, a solution which can protect electrodes is first put forward. The modified electrodes were stored in a pH 7.4 phosphate buffer solution containing a series of room temperature ionic liquid at 4℃in 60 days. The solution maintains activity of antibody effectively was selected as the optimum one. Circular dichromism spectra was empolyed to study on the mechanism of interaction between room temperature ionic liquid and activity of antibody. The experiment showed the change of protein conformation in the room temperature ionic liquid.In conclusion, the simple, highly sensitive and label-free impedimetric immunosensors for detection of MC-LR in water have been developed.They have good practical value and provide important experimental basis for the further study on implementation of direct, real-time, in-situ , on-line ,daily examination. |
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