Resonant waveguide grating surface structure and method for detecting intermolecular interactions using the structure
A resonant waveguide grating and surface structure technology, which is applied in the direction of material analysis, measurement devices, and analysis materials through optical means, can solve the problems that molecular interactions are not suitable for micro-culture, and affect the use of high-throughput screening.
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Embodiment 1
[0079] Embodiment 1: Construction of the surface structure of the resonant waveguide grating according to the present invention
[0080]The surface structure of the resonant waveguide grating in the present invention includes from top to bottom: side chain azo polymer layer, sinusoidal TiO 2 Waveguide grating (commercially available), SU8 coating (commercially available) and ITO glass substrate (commercially available). Wherein, the side chain azo polymer is an azo polymer, specifically polyglycidyl methacrylate-p-nitroazoaniline, and its chemical structural formula is:
[0081]
[0082] The preparation method of described polyglycidyl methacrylate-p-nitroazoaniline is:
[0083] Dissolve 3g of p-nitroaniline in 50ml of distilled water, add 5ml of commercially available 37% concentrated hydrochloric acid, stir and dissolve at room temperature, and slowly add 1.8g of sodium nitrite aqueous solution dropwise at 0°C after the nitroaniline is completely dissolved (Water consum...
Embodiment 2
[0085] Embodiment 2: Preparation of HEV monoclonal antibody
[0086] (1) Preparation of HEV ORF2 used as antigen
[0087] Using the whole HEV gene of human hepatitis E virus (GenBank serial number: D11092) as template, ORF2FP: 5'-atgcgccctcggcca-3' as forward primer, ORF2RP: 5'-aaataaactataactcccga-3' as reverse primer, in PCR thermal cycle The instrument (Biometra T3) performed PCR reaction according to the following conditions: 94°C for 5 minutes; followed by 25 cycles of 94°C for 50 seconds, 57°C for 50 seconds, 72°C for 2 minutes and 30 seconds, and finally 72°C for 10 minutes. A specific HEV ORF2 DNA fragment with a size of about 2kb and used as a template for preparing the polypeptide of the present invention was obtained. The PCR product obtained above was ligated with a commercially available pMD 18-T vector (manufactured by TAKARA), and identified by BamHI / HindIII digestion to obtain a positive clone inserted into the ORF2 gene. In Shanghai Boya Bioengineering Co., ...
Embodiment 3
[0098] Example 3: RGW method to detect the affinity of interaction between ORF2 protein and monoclonal antibody
[0099] Add HEV ORF2 protein to each well of the micro-culture plate according to the present invention, fix overnight at 4°C; after sealing and washing the plate, use a reader to read the baseline value of each well of the micro-plate, and then add PBS buffer to the micro-plate The monoclonal antibody to be tested was serially diluted with liquid, and the irrelevant mouse IgG antibody was used as a negative control, and incubated at 37°C for 2 hours; after the incubation, continue to read the end value with the instrument, and use Biaevaluation software according to the above baseline value and end value Carry out kinetic analysis to obtain the dissociation rate constant Kd, the binding number rate constant Ka and the equilibrium dissociation constant KD of the antibody to be tested, where KD=Kd / Ka, and the monoclonal antibody binding reaction value pm=KD value / conc...
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