Method for detecting alkaline phosphatase activities and concentration of phosphatase inhibitors
A technology of phosphatase activity and phosphatase, which is applied in the field of analysis and testing, can solve the problems of long detection time, poor dispersion and instability of Cu-MOFs, and achieve the effect of short detection time
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Embodiment 1
[0030] This embodiment relates to a method for detecting alkaline phosphatase (ALP) activity, specifically comprising the following steps:
[0031] Add 80μL Tris-HCl (pH 7.4), 10μL 7mM PPi and 10μL different concentrations of ALP (0.5, 1, 2, 4, 6, 8, 10, 20, 40mU / mL) into a 1.5mL centrifuge tube, mix well Then place in a 37°C water bath and incubate for 1 hour. Then, add 700 μL acetate buffer solution (pH 4.0), 100 μL 2.5 mg / mL CeO 2 Nanoparticles and 100 μL 5mM TMB were incubated in a water bath at 37°C for 3 minutes and then measured by UV-Vis absorption spectrum, as shown in figure 2 shown. Using ΔA and ALP activity to carry out linear fitting, the linear equations obtained are respectively: ΔA=-0.0031+0.0293c (c is ALP activity, the unit is mU / mL)), and the linear coefficient is 0.995, such as image 3 shown. In this embodiment, the experimental conditions are optimized, including pH of acetic acid buffer solution, CeO 2 Concentrations of nanoparticles and TMB, incub...
Embodiment 2
[0033] The present embodiment relates to a kind of detection sodium vanadate (Na 3 VO 4 ) concentration method, which specifically includes the following steps: mix 70 μL Tris-HCl (pH 7.4), 10 μL 2U / mL ALP and 10 μL different concentrations of Na 3 VO 4 (0, 0.005, 0.01, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.20, 0.25mM) were sequentially added to a 1.5mL centrifuge tube, mixed evenly and incubated in a 37°C water bath for 30min; Then put 10 μL of 7mM PPi into the centrifuge tube and incubate in a 37°C water bath for 1 hour. Then, add 700 μL acetate buffer solution (pH 4.0), 100 μL 2.5 mg / mL CeO 2 Nanoparticles and 100 μL 5mM TMB were incubated in a water bath at 37°C for 3 minutes and then measured by UV-Vis absorption spectrum, as shown in Figure 4 shown. We take the suppression efficiency and Na 3 VO 4 Concentration fitting curve, such as Figure 5 shown, get its IC 50 The value is 71 μM.
[0034] Test results: The best experimental conditions are: pH of a...
Embodiment 3
[0036] This embodiment involves an anti-interference experiment, and the specific steps are:
[0037] Add 80 μL Tris-HCl (pH 7.4), 10 μL 7mM PPi and 10 μL 0.1mg / mL interfering substances (bovine serum albumin, lysozyme, pepsin, trypsin and pancreatin) into a 1.5mL centrifuge tube in turn, mix well Place in a 37°C water bath and incubate for 1 hour. Then, 700 μL of acetate buffer solution (pH 4.0), 100 μL of 2.5 mg / mL CeO 2 Nanoparticles and 100 μL of 5 mM TMB were incubated in a water bath at 37°C for 3 minutes, and then measured by UV-Vis absorption spectrum.
[0038] Add 70μL Tris-HCl (pH 7.4), 10μL 7mM PPi, 10μL 0.1mg / mL alkaline phosphatase and interfering substances (bovine serum albumin, lysozyme, pepsin, trypsin and pancreatin) to a 1.5mL centrifuge tube Mix well and incubate in a 37°C water bath for 1 hour. Then, add 700 μL acetate buffer solution (pH 4.0), 100 μL 2.5 mg / mL CeO 2 Nanoparticles and 100 μL of 5 mM TMB were incubated in a water bath at 37°C for 3 minu...
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