Method of fluorescence detection for lipase activity
A fluorescence detection, lipase technology, applied in fluorescence/phosphorescence, measurement devices, instruments, etc., can solve the problems of limited practical application, poor stability, poor water solubility of curcumin, etc., and achieves a wide range of applications, low cost, and good detection effect. Effect
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Embodiment 1
[0045] (1) Pipette 1 mL of 300 μmol / L Tween 40 and 10 μL of 25 μmol / L curcumin into 2 mL centrifuge tubes, shake and mix to obtain sample 1; add 5 μL of 100 μmol / L CuCl 2 , Obtain mixed solution 1, wherein, the mol ratio of curcumin and soluble copper salt is 1:2, and the concentration of Tween 40 is 300 μ mol / L;
[0046] (2) Add 250 μL phosphate buffer solution to 750 μL 1.5 mmol / L methyl thioglycolate solution to obtain mixed solution 2 (pH 7);
[0047] (3) Add Novozymes 435 lipase or inactivated Novozymes 435 lipase (final concentration 100U / mL) to the mixed solution 2, wherein the concentration of methyl thioglycolate is 1.125mmol / L; , filter out lipase to obtain mixed solution 3, without adding lipase as a control;
[0048] (4) Take 50 μL of mixed solution 3 and add it to mixed solution 1, and react at room temperature 25° C. for 5 minutes to obtain mixed solution 4 .
[0049] Fluorescence spectroscopy was used to verify the above sensing mechanism, and the fluorescence...
Embodiment 2
[0051] Example 2 Quencher Cu 2+ Effect of Concentration
[0052] (1) Pipette 1 mL of 300 μmol / L Tween 40 and 10 μL of 25 μmol / L curcumin into 2 mL centrifuge tubes, shake and mix to obtain sample 1; add 5 μL of different concentrations of CuCl 2 (40, 60, 80, 100, 120, 140, 160 μmol / L), to obtain mixed solution 1;
[0053] (2) Transfer sample 1 and mixed solution 1 to a 96-well plate for fluorescence detection in a microplate reader. With ΔF (ΔF=F a -F b ) is the ordinate, Cu 2+ The concentration is the abscissa, draw the curve, F a Indicates the fluorescence intensity of the Tween 40-curcumin complex, F b Indicates the addition of Cu 2+ Fluorescence intensity of post-Tween 40-curcumin system.
[0054] see results image 3 A, with Cu 2+ As the concentration increases, the fluorescence difference increases gradually, and the quenching effect also increases accordingly. When Cu 2+ When the concentration reached 100μmol / L, the fluorescence signal difference of Tween 40-...
Embodiment 3
[0055] The influence of embodiment 3 substrate methyl thioglycolate concentration
[0056] (1) Pipette 1 mL of 300 μmol / L Tween 40 and 10 μL of 25 μmol / L curcumin into 2 mL centrifuge tubes, shake and mix to obtain sample 1; add 5 μL of 100 μmol / L CuCl 2 , Obtain mixed solution 1, wherein, the mol ratio of curcumin and soluble copper salt is 1:2, and the concentration of Tween 40 is 300 μ mol / L;
[0057] (2) Add 250 μL of phosphate buffer solution to 750 μL of different concentrations of methyl thioglycolate solutions (0.5, 1.0, 1.5, 2.0, 2.5 mmol / L) to obtain mixed solution 2 (pH 7);
[0058] (3) Add Novozymes 435 lipase (final concentration 100U / mL) to the mixed solution 2, wherein the concentrations of methyl thioglycolate are respectively 0.375, 0.75, 1.125, 1.5mmol / L; after reacting in a 35°C water bath for 15min, filter Remove lipase to obtain mixed solution 3;
[0059] (4) Take 50 μL of mixed solution 3 and add it to mixed solution 1, react at room temperature 25°C fo...
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