Cu<2+> colorimetric detection method based on MarR protein regulation
A detection method and protein technology, applied in the fields of analytical chemistry and food safety, can solve the problems of long detection time and inability to use real-time detection, and achieve the effects of high sensitivity, short detection time and strong specificity
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Embodiment 1
[0017] The preparation of embodiment 1MarR protein
[0018] The wild-type MarR gene was cloned into the pET30a expression vector, and introduced into the host strain BL21(DE3). The transformed cells were placed in LB medium containing 30 μg / ml kana antibiotics, and cultured in a shaker at 37°C. When the OD 600 When = 0.6, IPTG was added to make the final concentration 1 mM, the temperature was adjusted to 18° C., and induced for 12 hours. Cells were collected by centrifugation and protein extraction buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA with 1 mM phenylmethylsulfonyl fluoride) was used. When the solution was sonicated until the solution became clear, the cell debris was removed by centrifugation at 12,000rpm for 10 minutes. The supernatant was passed through a 1mL Histrap prepacked column (GE), and the column was eluted with different concentrations of imidazole, and the solution of the target protein was selected for ultrafiltration. Proteins were collected ...
Embodiment 2
[0019] The preparation of embodiment 2 colorimetric probes
[0020] (1) Preparation of 20nm colloidal gold particles
[0021] Take 100 mL of 0.01% HAuCl 4 The solution was added to a conical beaker with a volume of 250mL, heated and stirred to boil. Then add 1.8mL 1% trisodium citrate solution and keep stirring. When the color of the solution turns wine red for 45s, boil for 5min and stop heating. The final prepared colloidal gold solution is stirred for 10min and then put into a glass bottle and stored in the dark at 4°C. All glassware was acid washed and rinsed with distilled water.
[0022] (2) MarR protein labeled colloidal gold
[0023] Utilize 0.2mol L -1 K 2 CO 3 Adjust the pH of the 20nm colloidal gold solution to 8.2. Take 1.4mL concentration to be 137.5mg L -1 The MarR protein solution was added to 6mL colloidal gold solution, stirred rapidly and then allowed to stand at room temperature for 4h. Centrifuge the colloidal gold solution at 10,000×g at 4°C for 2...
Embodiment 3
[0024] Embodiment 3 colorimetric probe is to Cu 2+ Feasibility determination of detection
[0025] Prepare separate gold nanoparticles at the same concentration, MarR protein-labeled gold nanoparticles, 20 μM Cu 2+ Mixed gold nanoparticles, 20 μM Cu 2+ Mixed MarR protein-labeled gold nano-solutions, recorded the colors of the solutions under four conditions and the corresponding UV-Vis absorption spectra. figure 2 showed that only MarR protein-labeled gold nanoparticles in 20 μM Cu 2+ In the case of Cu 2+ Induces the aggregation of gold nanoprobes labeled with MarR protein, resulting in a change in the color of the solution. At the same time, observe its corresponding ultraviolet-visible light spectrum, only the gold nanoparticles labeled with MarR protein 2+ In the case of , the UV-Vis spectrum of the solution will be red-shifted. The above results demonstrate that the colorimetric probe of MarR protein-labeled gold nanoparticles is sensitive to Cu 2+ detection feasib...
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