CN103076326B - Nano-gold colorimetric method for detecting lipase activity - Google Patents
Nano-gold colorimetric method for detecting lipase activity Download PDFInfo
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- 235000019626 lipase activity Nutrition 0.000 title claims abstract description 26
- 238000004737 colorimetric analysis Methods 0.000 title claims abstract description 22
- 239000010931 gold Substances 0.000 title claims description 121
- 229910052737 gold Inorganic materials 0.000 title claims description 62
- 229920000136 polysorbate Polymers 0.000 claims abstract description 55
- 102000004882 Lipase Human genes 0.000 claims abstract description 44
- 108090001060 Lipase Proteins 0.000 claims abstract description 44
- 239000004367 Lipase Substances 0.000 claims abstract description 44
- 235000019421 lipase Nutrition 0.000 claims abstract description 44
- 239000000758 substrate Substances 0.000 claims abstract description 8
- 239000003381 stabilizer Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 111
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 60
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 46
- 239000011780 sodium chloride Substances 0.000 claims description 23
- 230000004048 modification Effects 0.000 claims description 20
- 238000012986 modification Methods 0.000 claims description 20
- 238000001514 detection method Methods 0.000 claims description 17
- 238000002371 ultraviolet--visible spectrum Methods 0.000 claims description 14
- 230000007062 hydrolysis Effects 0.000 claims description 12
- 238000006460 hydrolysis reaction Methods 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 12
- 239000008363 phosphate buffer Substances 0.000 claims description 11
- 239000001164 aluminium sulphate Substances 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 9
- 238000002835 absorbance Methods 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 230000009467 reduction Effects 0.000 claims description 7
- 239000004220 glutamic acid Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
- NMWONDKHBZEDMY-UHFFFAOYSA-H [Au](Cl)(Cl)Cl.C(CC(O)(C(=O)[O-])CC(=O)[O-])(=O)[O-].[Na+].[Na+].[Na+] Chemical compound [Au](Cl)(Cl)Cl.C(CC(O)(C(=O)[O-])CC(=O)[O-])(=O)[O-].[Na+].[Na+].[Na+] NMWONDKHBZEDMY-UHFFFAOYSA-H 0.000 claims description 4
- 238000013016 damping Methods 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- CSVGEMRSDNSWRF-UHFFFAOYSA-L disodium;dihydrogen phosphate Chemical compound [Na+].[Na+].OP(O)([O-])=O.OP(O)([O-])=O CSVGEMRSDNSWRF-UHFFFAOYSA-L 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 10
- -1 salt ions Chemical class 0.000 abstract description 5
- 238000004945 emulsification Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 239000007864 aqueous solution Substances 0.000 abstract 1
- 230000031700 light absorption Effects 0.000 abstract 1
- 238000010521 absorption reaction Methods 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 5
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- 238000002360 preparation method Methods 0.000 description 5
- 230000005540 biological transmission Effects 0.000 description 4
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- 239000004246 zinc acetate Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
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- 230000035484 reaction time Effects 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OAGSFHDUINSAMQ-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;sodium;hydrate Chemical compound O.[Na].OC(=O)CC(O)(C(O)=O)CC(O)=O OAGSFHDUINSAMQ-UHFFFAOYSA-N 0.000 description 1
- 238000006136 alcoholysis reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
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- NICDRCVJGXLKSF-UHFFFAOYSA-N nitric acid;trihydrochloride Chemical compound Cl.Cl.Cl.O[N+]([O-])=O NICDRCVJGXLKSF-UHFFFAOYSA-N 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
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- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- PHYFQTYBJUILEZ-IUPFWZBJSA-N triolein Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC PHYFQTYBJUILEZ-IUPFWZBJSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a colorimetric analysis method for quickly and conveniently detecting lipase activity in an aqueous solution. The invention utilizes the characteristic that nanogold has high light absorption coefficient, takes the nanogold as a color development signal element, and simultaneously uses Tween as a substrate of lipase and a nanogold stabilizer to resist high-concentration salt ions. Develops a colorimetric analysis method with the characteristics of low cost, simple operation, convenience and quickness. The Tween adopted by the invention has amphipathy, an emulsification process is not needed, and the stable nanogold is very stable and has very strong capability of resisting high-concentration salt ions, so that the Tween is suitable for popularization and application.
Description
Technical field
The invention belongs to analytical chemistry field, be specifically related to a kind of nm of gold that uses as signal element, the tween not only substrate as lipase but also the chemical colorimetry of the detection lipase activity as nm of gold stabilizing agent in aqueous.
Background technology
Lipase (lipase E. C. 3. 1. 1. 3) is the enzyme that a class has multiple catalysis, the hydrolysis of major catalytic triglyceride and some other insoluble ester class, alcoholysis, esterification, reverse reaction that is transesterification and ester class react, and also have the ability that hydrolysis of racemic potpourri and ester and peptide are bonded to. and lipase has been widely used in the numerous areas such as food, chemical industry, medicine synthesis.Along with the application of lipase day by day widen, market scale progressively expands, lipase detects and also becomes more and more important.
The assay method of lipase hydrolysis activity mainly contains p-NPP method and constant potential automatic titering process.The substrate used mainly contains olein, tributyrin and olive oil.But these substrates exist preparation trouble, need to add emulsifier, and the emulsifying agent added can affect the mensuration that enzyme is lived, and has the restriction of pH scope, although p-NPP method is simple to operate, p-NPP reagent is expensive, and under-sensitive shortcoming.
Nanogold particle refers to the gold grain of particle diameter between 1 ~ 100 nanometer.Often be dispersed in water formed aurosol with gold grain to apply, therefore also known as collaurum or aurosol.In recent years, nanogold particle, as a kind of novel material, by means of the physicochemical characteristics of its uniqueness, as high-specific surface area, high surface reaction activity, strong adsorbability etc., all obtains in fields such as material science, clinical medicine, life sciences and applies widely.The absorptivity of the gold grain of 13 nanometer diameters is 2.7 × 108 M
-1cm
-1, larger than traditional organic chromophores 3 orders of magnitude.In addition, dispersion nm of gold is in the solution after assembling mutually, and color can become blue from redness.Therefore, in colorimetric analysis, nm of gold is a kind of desirable color signal element.
The technical scheme that the unexposed activity using nm of gold as color signal element to lipase of prior art detects.
Summary of the invention
The object of this invention is to provide the effective colorimetric analysis of a kind of cheap and simple for detecting lipase.Nanogold colorimetric method is combined with lipase natural substrate by the method first, lives for detecting its enzyme, and the concrete tween that uses is as the specific substrate of lipase, and nm of gold is as signal element.
Object of the present invention can be achieved through the following technical solutions:
Detect a nanogold colorimetric method for lipase activity, use nm of gold as signal element, to hold concurrently nm of gold stabilizing agent using tween as the substrate of lipase simultaneously.
More properly, method of the present invention specifically comprises the steps:
(1) reduction of sodium citrate gold chloride is adopted to obtain claret nano-Au solution,
(2) add tween solution in the nano-Au solution obtained in step (1) to modify nm of gold;
(3) in the nano-Au solution after modification, add sodium chloride solution, make to be in salt solusion through the nm of gold that tween is stable;
(4) nano-Au solution obtained in step (3) adds the adjustment that buffer solution carries out pH; After add lipase solution to be measured, mixing, regulate temperature of reaction, make the tween on nm of gold surface by lipase hydrolysis, nm of gold is exposed in salt solusion again assembles, and detects the uv-vis spectra of solution, obtain the uv-vis spectra that different time is corresponding, then with absorbance ratio E
620/ E
520as ordinate, the time is horizontal ordinate, draws the kinetic curve of lipase activity.
Method of the present invention, in described step (1), the concentration of the nano-Au solution that reduction of sodium citrate legal system is standby is 1.8-2.5 nmol/L, and the particle diameter of nm of gold is 13 ± 2 nm.The concentration of preferred nano-Au solution is 2 nmol/L, and the particle diameter of nm of gold is 13 nm.
The preparation of concrete nano-Au solution can adopt preparation method disclosed in prior art, and disclosed in China's application 201110052259.7, the present invention is not particularly limited this.
Wherein, in described step (2), the concentration of tween solution is 3-6 mmol/L, and the volume ratio of tween solution and nano-Au solution is 1:100-1:50, preferred 1:100.In step (2), the modification time is 10-60 min, preferred 30min.
Wherein, in the nano-Au solution after described step (3) modification, the ultimate density of sodium chloride is 0.02-0.1mol/L, preferably 0.1 mol/L.
The nanogold colorimetric method of detection lipase activity of the present invention, damping fluid described in step (4) is sodium hydrogen phosphate-sodium dihydrogen phosphate buffer, concentration and the concrete consumption of this damping fluid are understood by those skilled in the art and are grasped, specifically be adjusted to 6-8 with nano-Au solution pH value to be the most at last as the criterion, preferably 6.5.
The nanogold colorimetric method of detection lipase activity of the present invention, in described step (4), temperature of reaction is 30-60 DEG C, preferably 50 DEG C; The addition of lipase solution to be measured is the 1:20-1:5 of nano-Au solution volume, preferred 1:20.
More specifically, the nanogold colorimetric method of detection lipase activity of the present invention, comprises the steps:
(1) reduction of sodium citrate gold chloride is adopted to obtain the claret nano-Au solution that concentration is 1.8-2.5 nmol/L,
(2) add tween solution in the nano-Au solution obtained in step (1) to modify nm of gold; The concentration of tween solution is 3-6 mmol/L, and the volume ratio of tween solution and nano-Au solution is 1:100-1:50; The modification time is 10-60 min;
(3) in the nano-Au solution after modification, add sodium chloride solution, make to be in salt solusion through the nm of gold that tween is stable; Wherein, the ultimate density of sodium chloride is 0.02-0.1mol/L;
(4) nano-Au solution obtained in step (3) adds the adjustment that buffer solution carries out pH, and pH value is adjusted to 6-8; After add lipase solution to be measured, mixing, regulate temperature of reaction to 30-60 DEG C, make the tween on nm of gold surface by lipase hydrolysis, nm of gold is exposed in salt solusion again, and nm of gold is assembled, the uv-vis spectra of rear detection solution per minute, obtain the uv-vis spectra that different time is corresponding, then with absorbance ratio E
620/ E
520as ordinate, the time is horizontal ordinate, draws the kinetic curve of lipase activity.
The present invention adopts and is joined in nano-Au solution by certain density tween solution.After certain hour, add sodium chloride solution and the PB damping fluid of certain solubility.Because the tween solution added can make nm of gold resist salt ion, nm of gold color does not change.After add certain density lipase in this solution, tween starts by lipase hydrolysis, and under nm of gold is exposed to salt ion again, salt ion makes nm of gold assemble, and causes solution colour to change.The change of color is lived relevant with the enzyme of lipase, therefore, it is possible to live for the enzyme detecting lipase.
The substrate tween dissolubility that technical solutions according to the invention adopt is good, without the need to the emulsification link needed for conventional art, simple and convenient, and cheap.In addition, the present invention is based on nanometer colourimetry sensing, therefore highly sensitive, be visually applicable to high-throughout monitoring, simultaneously tween can under high ionic strength condition stabilized nanoscale gold, can be used for the detection of lipase in practical systems.Therefore, be conducive to applying.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture of (a) 13nm nm of gold; Transmission electron microscope picture after the gathering of (b) 13nm nm of gold.
Fig. 2 is (a) nano-Au solution; B () is containing 0.1 mol L
-1sodium chloride and 0.01 mol L
-1the nano-Au solution of phosphate buffer; C () is containing 0.1 mol L
-1sodium chloride and 0.01 mol L
-1the nano-Au solution that the tween (3 mmol/L) of phosphate buffer is stable; D () adds inactivation lipase after, containing 0.1 mol L
-1sodium chloride and 0.01 mol L
-1the nano-Au solution that the tween of phosphate buffer is stable; E () adds lipase after, containing 0.1 mol L
-1the uv-vis spectra of the nano-Au solution that the tween of sodium chloride and 0.01 mol L-1 phosphate buffer is stable.
Fig. 3 to add after lipase (a) 0 min for being respectively; (b) 1 min; (c) 2 min; (d) 3 min; (e) 4 min; (f) 5 min; (g) 6 min; (h) 7 min; (i) 8 min; (j) 9 min; (k) 10 after min containing 0.1 mol L
-1sodium chloride and 0.01 mol L
-1the uv-vis spectra of the nano-Au solution that the tween of phosphate buffer is stable.
Fig. 4 is the kinetic curve of lipase activity.
Embodiment
embodiment 1
(1) preparation of nano-Au solution
Nm of gold adopts reduction of sodium citrate gold chloride method to obtain, and (its transmission electron microscope picture is shown in Fig. 1 a, and its ultraviolet-visible spectrogram is shown in Fig. 2 a).Concrete preparation process is:
All glass apparatus are all needed use chloroazotic acid to soak to remove reducing substances residual in glass container.Accurately take HAuCl
4.4H
2o 0.0123 g, in 250 mL there-necked flasks, then adds 100 mL water in there-necked flask.Vigorous stirring, ebuillition of heated refluxes.Accurately take 2 citric acid monohydrate sodium 0.2849 g in 25 mL volumetric flasks.Get a certain amount of sodium citrate solution, heating water bath adds flask fast after 50 DEG C.After 15 minutes, solution is finally claret to purple again from colourless to light blue, continues heating and stops heating after 10 minutes, continuation stirring cool to room temperature after 10 minutes.The size of nm of gold is relevant with the amount of the sodium citrate added.
(2) modification of nano-Au solution; Add tween solution in the nano-Au solution obtained in step (1) to modify nm of gold; The concentration of tween solution is 4.5 mmol/L, and the volume ratio of tween solution and nano-Au solution is 1:100; The modification time is 30 min;
(3) in the nano-Au solution after modification, add sodium chloride solution, make to be in salt solusion through the nm of gold that tween is stable; Wherein, the ultimate density of sodium chloride is that its ultraviolet-visible spectrogram of 0.05mol/L(is shown in Fig. 2 b);
Fig. 2 a is the ultraviolet-visible spectrogram of 1ml nano-Au solution, and only occur absorption peak at 520nm place, illustrate that nm of gold is disperse state, solution is claret.
Fig. 2 b adds 0.1 mol L in 1ml nano-Au solution
-1sodium chloride and 0.01 mol L
-1, there is new absorption peak at 650nm place in the ultraviolet-visible spectrogram of phosphate buffer, and 520nm place absorption peak reduces, and illustrates that nm of gold is state of aggregation, solution au bleu.
Fig. 2 c adds 0.1 mol L in the stable nano-Au solution of 1ml tween (3 mmol/L)
-1sodium chloride and 0.01 mol L
-1the ultraviolet-visible spectrogram of phosphate buffer, occur, and 520nm place absorption peak remains unchanged substantially at 650nm place without new absorption peak, illustrates that tween protection nano-Au solution disperses to exist in high level salt solution.
Fig. 2 d adds 0.1 mol L in the stable nano-Au solution of 1ml tween (3 mmol/L)
-1sodium chloride, 0.01 mol L
-1the ultraviolet-visible spectrogram of the lipase of phosphate buffer and 0.2 mg/ml inactivation, occurs without new absorption peak at 650nm place, illustrates that the lipase of inactivation cannot be hydrolyzed tween, thus nm of gold cannot be caused to assemble.
Fig. 2 e adds 0.1 mol L in the stable nano-Au solution of 1ml tween (3 mmol/L)
-1sodium chloride, 0.01 mol L
-1, there is new absorption peak at 650nm place in the ultraviolet-visible spectrogram of phosphate buffer and 0.2 mg/ml lipase, and 520nm place absorption peak reduces, and illustrate that tween is by lipase hydrolysis, nm of gold is directly exposed in high level salt solution, assembles.
(4) nano-Au solution obtained in step (3) adds the adjustment that buffer solution carries out pH, and pH value is adjusted to 6.5; After add lipase solution to be measured, mixing, regulate temperature of reaction to 50 DEG C, make the tween on nm of gold surface by lipase hydrolysis, nm of gold is exposed in salt solusion again, nm of gold is assembled (its transmission electron microscope picture is shown in Fig. 1 b, and its ultraviolet-visible spectrogram is shown in Fig. 2 c), and color becomes purple from claret.The uv-vis spectra of rear detection solution per minute, obtains uv-vis spectra corresponding to different time (see Fig. 3), then uses absorbance ratio E
620/ E
520as ordinate, the time is horizontal ordinate, draws the kinetic curve (see figure 4) of lipase activity.
Fig. 3 is followed successively by (a) 0 min the ultraviolet-visible spectrogram time of 4mg/ml lipase under different time adding 50 μ L in the nano-Au solution of 1 ml; (b) 1 min; (c) 2 min; (d) 3 min; (e) 4 min; (f) 5 min; (g) 6 min; (h) 7 min; (i) 8 min; (j) 9 min; (k) 10 min; Along with the increase of time, the absorption peak near 650nm is increasing, and declines gradually at the peak at 520nm place, and therefore the present invention selects E
650/ E
520ratio be ordinate mapping, ratio is larger represents that the degree of assembling is larger, i.e. more by lipase hydrolysis of tween.Therefore, ratio change speed be exactly sooner lipase activity greatly.
Fig. 4 is after following the uv-vis spectra that records according to (1-10 min) under different time after add lipase in nano-Au solution, with absorbance ratio E
650/ E
520as ordinate, the enzymatic hydrolysis reaction time as horizontal ordinate, the zymetology dynamic criteria curve of drafting.Can see, along with the increase in reaction time, E
650/ E
520ratio is larger, and represent that tween is hydrolyzed gradually, the aggregation extent of nm of gold is larger.Therefore, the present invention can according to E
650/ E
520ratio to lipase activity carry out analysis detect.
embodiment 2
Compared with embodiment 1, distinctive points is only that the operating parameter of step 1-4 is different, and the present embodiment is specially:
(1) concentration of nano-Au solution is 1.8 nmol/L, and the particle diameter of nm of gold is 13nm;
(2) modification of nano-Au solution; Add tween solution in the nano-Au solution obtained in step (1) to modify nm of gold; The concentration of tween solution is 3 mmol/L, and the volume ratio of tween solution and nano-Au solution is 1:50; The modification time is 10 min;
(3) in the nano-Au solution after modification, add sodium chloride solution, make to be in salt solusion through the nm of gold that tween is stable; Wherein, the ultimate density of sodium chloride is 0.02mol/L;
(4) nano-Au solution obtained in step (3) adds the adjustment that buffer solution carries out pH, and pH value is adjusted to 6; After add lipase solution to be measured, mixing, regulate temperature of reaction to 30 DEG C, make the tween on nm of gold surface by lipase hydrolysis, nm of gold is exposed in salt solusion again, and nm of gold is assembled, the uv-vis spectra of rear detection solution per minute, obtain the uv-vis spectra that different time is corresponding, then use absorbance ratio E
620/ E
520as ordinate, the time is horizontal ordinate, draws the kinetic curve of lipase activity.
embodiment 3
Compared with embodiment 1, distinctive points is only that the operating parameter of step 1-4 is different, and the present embodiment is specially:
(1) concentration of nano-Au solution is 2.5 nmol/L, and the particle diameter of nm of gold is 20 nm;
(2) modification of nano-Au solution; Add tween solution in the nano-Au solution obtained in step (1) to modify nm of gold; The concentration of tween solution is 6 mmol/L, and the volume ratio of tween solution and nano-Au solution is 1:80; The modification time is 60 min;
(3) in the nano-Au solution after modification, add sodium chloride solution, make to be in salt solusion through the nm of gold that tween is stable; Wherein, the ultimate density of sodium chloride is 0.1mol/L;
(4) nano-Au solution obtained in step (3) adds the adjustment that buffer solution carries out pH, and pH value is adjusted to 8; After add lipase solution to be measured, mixing, regulate temperature of reaction to 60 DEG C, make the tween on nm of gold surface by lipase hydrolysis, nm of gold is exposed in salt solusion again, and nm of gold is assembled, the uv-vis spectra of rear detection solution per minute, obtain the uv-vis spectra that different time is corresponding, then use absorbance ratio E
620/ E
520as ordinate, the time is horizontal ordinate, draws the kinetic curve of lipase activity.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (9)
1. detect a nanogold colorimetric method for lipase activity, it is characterized in that, use nm of gold as signal element, to hold concurrently nm of gold stabilizing agent using tween as the substrate of lipase simultaneously;
Specifically comprise the steps:
(1) reduction of sodium citrate gold chloride is adopted to obtain claret nano-Au solution,
(2) add tween solution in the nano-Au solution obtained in step (1) to modify nm of gold; The concentration of tween solution is 3-6 mmol/L, and the volume ratio of tween solution and nano-Au solution is 1:100-1:50, and the modification time is 10-60 min;
(3) in the nano-Au solution after modification, add sodium chloride solution, make to be in salt solusion through the nm of gold that tween is stable, the ultimate density of sodium chloride is 0.02-0.1mol/L;
(4) what the nano-Au solution obtained in step (3) added that buffer solution carries out pH is adjusted to pH value 6-8; After add lipase solution to be measured, the ultimate density of the lipase solution to be measured added is 0.15-0.25 mg/ml, mixing, and adjustment temperature of reaction is 30-60 DEG C, make the tween on nm of gold surface by lipase hydrolysis, nm of gold is exposed in salt solusion again assembles; Solution is detected, obtains the uv-vis spectra that different time is corresponding, then with absorbance ratio E
620/ E
520as ordinate, the time is horizontal ordinate, draws the kinetic curve of lipase activity.
2. the nanogold colorimetric method of detection lipase activity according to claim 1, is characterized in that, in described step (1), the concentration of the nano-Au solution that reduction of sodium citrate legal system is standby is 1.8-2.5 nmol/L, and the particle diameter of nm of gold is 13 ± 2 nm.
3. the nanogold colorimetric method of detection lipase activity according to claim 2, is characterized in that, in described step (1), the concentration of the nano-Au solution that reduction of sodium citrate legal system is standby is 2 nmol/L, and the particle diameter of nm of gold is 13 nm.
4. the nanogold colorimetric method of detection lipase activity according to claim 1, is characterized in that, in described step (2), the volume ratio of tween solution and nano-Au solution is 1:100.
5. the nanogold colorimetric method of detection lipase activity according to claim 1, is characterized in that, in described step (2), the modification time is 30min.
6. the nanogold colorimetric method of detection lipase activity according to claim 1, is characterized in that, in the nano-Au solution after described step (3) modification, the ultimate density of sodium chloride is 0.1 mol/L.
7. the nanogold colorimetric method of detection lipase activity according to claim 1, is characterized in that, in described step (4), described damping fluid is sodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
8. the nanogold colorimetric method of detection lipase activity according to claim 1, is characterized in that, adds buffer solution the pH value of nano-Au solution is adjusted to 6.5 in described step (4).
9. the nanogold colorimetric method of detection lipase activity according to claim 1, is characterized in that, in described step (4), temperature of reaction is 50 DEG C; The ultimate density of the lipase solution to be measured added is 0.2 mg/ml.
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| CN104597044B (en) * | 2014-07-24 | 2019-02-12 | 南京工业大学 | Method for detecting lipase enzyme activity by in-situ synthesized nano-gold colorimetric method |
| CN104597006B (en) * | 2014-07-24 | 2018-02-06 | 南京工业大学 | Fluorescence method for detecting lipase activity |
| CN108593580A (en) * | 2018-04-25 | 2018-09-28 | 南京工业大学 | Method for detecting lipase activity based on gold nanorod etching by colorimetric method |
| CN109374605A (en) * | 2018-09-30 | 2019-02-22 | 东北农业大学 | A kind of method that nanogold colorimetric method detects lipase active in rice bran |
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