CN111175271B - Lipase activity detection method based on fluorescent probe - Google Patents
Lipase activity detection method based on fluorescent probe Download PDFInfo
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Abstract
The invention discloses a lipase activity detection method based on a fluorescent probe, which takes citric acid as a carbon source, ethylenediamine as a carbon-nitrogen source and chloroauric acid as a gold source, adopts a hydrothermal method to prepare a nitrogen-gold co-doped carbon nanodot as the fluorescent probe, mixes the fluorescent probe with copper ions and a hydrolysis product of lipase, and establishes a standard curve between lipase activity and probe fluorescence intensity by utilizing the difference of the action intensity of the fluorescent probe and the hydrolysis product of lipase on the copper ions, thereby realizing the efficient, sensitive and accurate detection of the lipase activity. Through the mode, the optical performance and the fluorescence quantum yield of the carbon nano-dots can be effectively improved, so that the detection has higher sensitivity; the detection method is simple and easy to operate, has the advantages of high detection speed, wide detection range, low detection limit and the like, can accurately detect the lipase with low activity, has a wide application range, and has a good application prospect.
Description
Technical Field
The invention relates to the technical field of lipase activity detection, in particular to a lipase activity detection method based on a fluorescent probe.
Background
The lipase is an enzyme which is universally existed in organisms and has various catalytic capacities, can catalyze hydrolysis, alcoholysis, esterification, transesterification and reverse synthesis reaction of triacylglycerol and other water-insoluble esters, has important physiological significance and potential value of industrial application, and is widely applied to the fields of food, medicines, leather, daily chemical industry and the like at present. Since the catalytic ability of lipase is closely related to its activity, accurate detection of lipase activity is crucial to the study of lipase catalytic performance and its application.
At present, methods for detecting the activity of lipase mainly comprise a plate method, a turbidity analysis method, a titration method, a colorimetric method, a chromatography method and a fluorescence analysis method. The plate method, the turbidity analysis method, the titration method and the colorimetric method are simple and convenient to operate, but have the defects of long time consumption, large error, low sensitivity and the like; chromatography methods require the use of expensive equipment and are not suitable for the detection of large numbers of samples; the fluorescence analysis method is based on the detection of lipase activity by a fluorescent probe, has the advantages of high detection speed, high sensitivity and the like, and is often used for quantitative detection of lipase activity. When the lipase activity is tested by a fluorescence analysis method, the selection of the fluorescent probe directly affects the precision and accuracy of the test result, so how to select the most suitable fluorescent probe to test the activity of the lipase is the current research focus.
Patent publication No. CN109724953A provides a method for fluorescence detection of lipase activity, and the patent prepares a Tween-curcumin complex micelle as a fluorescence signal probe by mixing a non-ionic surfactant and curcumin, thereby realizing high-throughput detection of lipase activity. However, the curcumin used in the patent is a hydrophobic substance, the water solubility of the curcumin is poor, although the hydrophilicity of the curcumin can be improved to a certain extent by using the curcumin and tween in a compounding way, the effect is limited, and the coating of the tween has certain influence on the release of the fluorescence effect of the curcumin, so that the detection result is not accurate enough; in addition, the curcumin has low fluorescence efficiency, so that the sensitivity is low when the lipase activity is detected, the detection limit is high, and the lipase with low activity is difficult to accurately detect.
In view of the above, there is still a need to research a lipase activity detection method based on a fluorescent probe, so as to improve the sensitivity of lipase activity detection by preparing an optimum fluorescent probe, and to make the detection result more accurate, so as to meet the requirements of practical applications.
Disclosure of Invention
Aiming at the problems, the invention provides a lipase activity detection method based on a fluorescent probe, which is characterized in that a nitrogen-gold co-doped carbon nanodot with high fluorescence efficiency and sensitivity is prepared as the fluorescent probe, and a standard curve between lipase activity and probe fluorescence intensity is established by utilizing the difference of the action intensity of the fluorescent probe and lipase hydrolysate on copper ions, so that the lipase activity is detected efficiently, sensitively and accurately.
In order to achieve the purpose, the invention adopts the technical scheme that:
a lipase activity detection method based on a fluorescent probe comprises the following steps:
s1, mixing citric acid, ethylenediamine and chloroauric acid according to a preset molar ratio, dissolving in deionized water, and preparing nitrogen-gold co-doped carbon nanodots by a hydrothermal method, wherein the nitrogen-gold co-doped carbon nanodots are used as fluorescent probes and stored for later use;
s2, uniformly mixing a lipase standard sample with certain activity and a methyl thioglycolate solution according to a preset volume ratio, and performing full hydrolysis reaction to obtain a hydrolysis reaction solution;
s3, uniformly mixing the hydrolysis reaction liquid obtained in the step S2 with a copper salt solution and the nitrogen-gold co-doped carbon nanodots prepared in the step S1 according to a preset volume ratio, and performing fluorescence detection after full reaction at room temperature;
s4, adjusting the activity of the lipase standard sample, repeating the steps S2-S3, and drawing a standard curve of the change of fluorescence intensity along with the lipase activity;
s5, replacing the lipase standard sample with a lipase sample to be detected, detecting the fluorescence intensity of the lipase sample to be detected according to the steps S2-S3, and calculating the activity of the lipase sample to be detected according to the standard curve obtained in the step S4.
Further, in step S1, the predetermined molar ratio of citric acid, ethylenediamine and chloroauric acid is 100 (10-15): 1-2.
Further, in step S1, the hydrothermal method for preparing the nitrogen-gold co-doped carbon nanodots includes the following steps:
s11, mixing citric acid, ethylenediamine and chloroauric acid according to the preset molar ratio, dissolving in deionized water, heating and stirring at 80 ℃ for 10min, transferring the solution into a high-pressure kettle, heating at 160 ℃ for 12h, and cooling to room temperature to obtain a nitrogen-gold co-doped carbon nanodot crude product;
s12, centrifuging the crude nitrogen-gold co-doped carbon nanodot product obtained in the step S11, and filtering the centrifuged supernatant by using a 0.22-micron microporous filter membrane to obtain a filtrate, namely the purified nitrogen-gold co-doped carbon nanodot.
Further, in step S2, the activity of the lipase standard sample is 0-800 mU/mL, and the concentration of the methyl thioglycolate solution is 1-2 mmol/L.
Further, in step S2, the preset volume ratio of the lipase standard sample to the methyl thioglycolate solution is 1: 25.
Further, in step S2, the hydrolysis reaction is performed at a temperature of 35 to 40 ℃ for 15 to 30 min.
Further, in step S3, the preset volume ratio of the hydrolysis reaction solution to the copper salt solution to the nitrogen-gold co-doped carbon nanodots prepared in step S1 is 100 (20-25) to (6-8).
Further, in step S3, the copper salt solution is one or more of a copper chloride solution, a copper sulfate solution and a copper nitrate solution; the concentration of copper ions in the copper salt solution is 100mu mol/L.
Further, in step S3, the reaction time for the sufficient reaction at room temperature is 5 to 10 min.
Further, in step S3, the excitation wavelength of the fluorescence detection process is 340-370 nm, and the detection wavelength is 450 nm.
The principle of lipase activity test of the present invention is as follows:
the nitrogen-gold co-doped carbon nanodot prepared by the method has excellent fluorescence property, can generate a strong fluorescence signal, and the amino group on the surface of the nitrogen-gold co-doped carbon nanodot can be coordinated with copper ions, so that the copper ions are adsorbed to the nitrogen-gold co-doped carbon nanodot, and the copper ions can generate a quenching effect on the nitrogen-gold co-doped carbon nanodot, so that the fluorescence intensity of the nitrogen-gold co-doped carbon nanodot is reduced; meanwhile, the hydrolysis product obtained by hydrolyzing methyl thioglycolate with lipase is thioglycolic acid, and the copper ions can be preferentially combined with the thioglycolic acid because the combination strength of sulfydryl in the thioglycolic acid and the copper ions is greater than the coordination strength between the nitrogen-gold co-doped carbon nano-dots and the copper ions. Therefore, the higher the lipase activity is, the more mercaptoacetic acid is obtained as a product by hydrolysis, more copper ions can be combined with mercaptoacetic acid, so that the content of copper ions adsorbed on the surface of the nitrogen-gold co-doped carbon nanodots is reduced, the fluorescence quenching effect of the copper ions on the nitrogen-gold co-doped carbon nanodots is weakened, the fluorescence intensity of the nitrogen-gold co-doped carbon nanodots is increased, and therefore the linear relation between the lipase activity and the fluorescence intensity of the nitrogen-gold co-doped carbon nanodots can be established, and the activity of the lipase is tested.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the lipase activity detection method based on the fluorescent probe, the nitrogen-gold co-doped carbon nanodots with high fluorescence efficiency and sensitivity are prepared to serve as the fluorescent probe, and the standard curve between the lipase activity and the fluorescence intensity of the probe is established by utilizing the difference of the action intensity of the fluorescent probe and the action intensity of the lipase hydrolysate on copper ions, so that the lipase activity is detected efficiently and sensitively, the detection result is high in accuracy, and the requirement of practical application can be met.
2. According to the invention, citric acid is used as a carbon source, ethylenediamine is used as a carbon nitrogen source, chloroauric acid is used as a gold source, and a hydrothermal method is adopted to prepare the nitrogen-gold co-doped carbon nanodot. Compared with single carbon nanodots, the Fermi level of the carbon nanodots can be changed by co-doping of nitrogen and gold, the optical performance and the fluorescence quantum yield of the carbon nanodots are effectively improved, and the carbon nanodots have higher fluorescence efficiency, so that the detection sensitivity is improved; meanwhile, the nitrogen-gold co-doping can also reduce the influence of the pH value on the fluorescence performance of the carbon nanodots, so that the prepared nitrogen-gold co-doped carbon nanodots can be suitable for different pH conditions, and the application range is wider. In addition, the ethylene diamine used in the invention can be used as a carbon nitrogen source to provide carbon and nitrogen, and can also be used as a passivator to further improve the surface performance of the carbon nanodots and promote the uniform dispersion of the carbon nanodots, so that the fluorescence intensity is enhanced; the raw materials used by the method can provide functional groups such as amino, carboxyl, hydroxyl and the like for the carbon nanodots, and the functional groups can coordinate with copper ions to ensure that the detection process is carried out smoothly, and can ensure that the carbon nanodots have excellent hydrophilicity to meet the requirements of practical application.
3. The lipase activity detection method based on the fluorescent probe is simple and easy to operate, and meets the requirements of practical application; the method has the advantages of high detection speed, wide detection range, low detection limit, capability of accurately detecting the lipase with low activity, wide application range and good application prospect.
Drawings
FIG. 1 is a graph of the fluorescence spectra of solutions of different compositions of example 1;
FIG. 2 is a standard curve of lipase activity versus fluorescence intensity;
FIG. 3 is a graph showing fluorescence spectra of examples 2 to 3 and comparative examples 1 to 2;
FIG. 4 is a graph showing the change of fluorescence intensity with pH in example 3 and comparative examples 1 to 2;
FIG. 5 is a fluorescence spectrum of nitrogen-gold co-doped carbon nanodots at different excitation wavelengths.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without any inventive step, are within the scope of the present invention.
The invention provides a lipase activity detection method based on a fluorescent probe, which comprises the following steps:
s1, mixing citric acid, ethylenediamine and chloroauric acid according to a preset molar ratio, dissolving in deionized water, and preparing nitrogen-gold co-doped carbon nanodots by a hydrothermal method, wherein the nitrogen-gold co-doped carbon nanodots are used as fluorescent probes and stored for later use;
s2, uniformly mixing a lipase standard sample with certain activity and a methyl thioglycolate solution according to a preset volume ratio, and performing full hydrolysis reaction to obtain a hydrolysis reaction solution;
s3, uniformly mixing the hydrolysis reaction liquid obtained in the step S2 with a copper salt solution and the nitrogen-gold co-doped carbon nanodots prepared in the step S1 according to a preset volume ratio, and performing fluorescence detection after full reaction at room temperature;
s4, adjusting the activity of the lipase standard sample, repeating the steps S2-S3, and drawing a standard curve of the change of fluorescence intensity along with the lipase activity;
s5, replacing the lipase standard sample with a lipase sample to be detected, detecting the fluorescence intensity of the lipase sample to be detected according to the steps S2-S3, and calculating the activity of the lipase sample to be detected according to the standard curve obtained in the step S4.
In step S1, the predetermined molar ratio of citric acid, ethylenediamine and chloroauric acid is 100 (10-15): 1-2.
In step S1, the hydrothermal method for preparing nitrogen-gold co-doped carbon nanodots includes the following steps:
s11, mixing citric acid, ethylenediamine and chloroauric acid according to the preset molar ratio, dissolving in deionized water, heating and stirring at 80 ℃ for 10min, transferring the solution into a high-pressure kettle, heating at 160 ℃ for 12h, and cooling to room temperature to obtain a nitrogen-gold co-doped carbon nanodot crude product;
s12, centrifuging the crude nitrogen-gold co-doped carbon nanodot product obtained in the step S11, and filtering the centrifuged supernatant by using a 0.22-micron microporous filter membrane to obtain a filtrate, namely the purified nitrogen-gold co-doped carbon nanodot.
In step S2, the activity of the lipase standard sample is 0-800 mU/mL, and the concentration of the methyl thioglycolate solution is 1-2 mmol/L.
In step S2, the preset volume ratio of the lipase standard sample to the methyl thioglycolate solution is 1: 25.
In step S2, the hydrolysis reaction is performed at a temperature of 35 to 40 ℃ for 15 to 30 min.
In step S3, the preset volume ratio of the hydrolysis reaction liquid to the copper salt solution to the nitrogen-gold co-doped carbon nanodots prepared in step S1 is 100 (20-25) to (6-8).
In step S3, the copper salt solution is one or more of a copper chloride solution, a copper sulfate solution and a copper nitrate solution; the concentration of copper ions in the copper salt solution is 100mu mol/L.
In step S3, the reaction time for the sufficient reaction at room temperature is 5 to 10 min.
In step S3, the excitation wavelength of the fluorescence detection process is 340-370 nm, and the detection wavelength is 450 nm.
The lipase activity test method based on the fluorescent probe according to the present invention will be described with reference to the following examples and accompanying drawings.
Example 1
The embodiment provides a lipase activity test method based on a fluorescent probe, which comprises the following steps:
s1, mixing 1.92g of citric acid, 0.72g of ethylenediamine and 0.61g of chloroauric acid, and dissolving the mixture in 30mL of deionized water to obtain a mixed solution, wherein the molar ratio of the citric acid to the ethylenediamine to the chloroauric acid is 100:12: 1.5; heating and stirring the mixed solution at 80 ℃ for 10min, transferring the mixed solution into an autoclave with a polytetrafluoroethylene lining, heating the mixed solution at 160 ℃ for 12h, and naturally cooling the heated mixed solution to room temperature to obtain a nitrogen-gold co-doped carbon nanodot crude product; centrifuging the crude product of the nitrogen-gold co-doped carbon nanodots at the rotating speed of 10000r/min for 10min, removing larger particles, and filtering the centrifuged supernatant by using a 0.22 mu m microporous filter membrane to obtain filtrate, namely the purified nitrogen-gold co-doped carbon nanodots.
S2, mixing 2mL of lipase standard samples with the activity of 0mU/mL, 0.1mU/mL, 0.5mU/mL, 2mU/mL, 5mU/mL, 10mU/mL, 20mU/mL, 40mU/mL, 60mU/mL, 80mU/mL, 100mU/mL, 200mU/mL, 400mU/mL, 600mU/mL and 800mU/mL respectively with 50mL of methyl thioglycolate solution with the concentration of 1.5mmol/L uniformly, wherein the volume ratio of the lipase standard sample to the methyl thioglycolate solution is 1: 25; reacting the obtained mixed solution at 37 ℃ for 20min to obtain hydrolysis reaction solution of lipase with different activities.
S3, respectively taking 500mu L of hydrolysis reaction liquid of the lipase with different activities obtained in the step S2, respectively and uniformly mixing the hydrolysis reaction liquid with 125 mu L of copper chloride solution with the concentration of 100mu mol/L and 35 mu L of nitrogen-gold co-doped carbon nano-dots prepared in the step S1, respectively metering the volume to 1mL by deionized water, reacting for 5min at room temperature, and then transferring the mixture into a 96-hole enzyme label plate for fluorescence detection; at the moment, the volume ratio of the hydrolysis reaction liquid to the copper salt solution and the nitrogen-gold co-doped carbon nanodots is 100:25:7, the excitation wavelength in the fluorescence detection process is 360nm, and the detection wavelength is 450 nm.
S4, drawing a standard curve of fluorescence intensity changing with lipase activity according to the fluorescence intensity corresponding to different active lipases measured in the step S3, wherein the activity of the lipase is used as an abscissa, and the difference between the fluorescence intensity corresponding to the lipase and the fluorescence intensity corresponding to the lipase with the activity of 0 is used as an ordinate.
S5, replacing the lipase standard sample with a lipase sample to be detected, detecting the fluorescence intensity of the lipase sample to be detected according to the steps S2-S3, and calculating the activity of the lipase sample to be detected according to the standard curve obtained in the step S4.
In order to verify the inspection mechanism of the method, the solution containing only nitrogen-gold co-doped carbon nanodots, the solution containing nitrogen-gold co-doped carbon nanodots and copper salt, and the solution containing nitrogen-gold co-doped carbon nanodots, copper salt and lipase hydrolysate were subjected to fluorescence detection, and the results are shown in fig. 1. In FIG. 1, a represents a solution consisting of 35. mu.L of the nitrogen-containing gold co-doped carbon nano-dots obtained in step S1 and 625. mu.L of deionized water; b represents a solution consisting of 35 mu L of the nitrogen-containing gold co-doped carbon nanodots, 125 mu L of copper chloride solution with the concentration of 100mu mol/L and 500mu L of deionized water; c represents a solution consisting of 35. mu.L of the nitrogen-containing gold co-doped carbon nanodots, 125. mu.L of a copper chloride solution having a concentration of 100. mu. mol/L, and 500. mu.L of a hydrolysis reaction solution of lipase.
As can be seen from fig. 1, the solution a containing only nitrogen-gold co-doped carbon nanodots has the highest fluorescence intensity, the fluorescence intensity of the solution b obtained after adding copper ions is significantly reduced, and the fluorescence intensity is partially recovered after continuously adding the lipase hydrolysis solution. Mainly because the nitrogen and gold co-doped carbon nanodots have excellent fluorescence performance and can generate strong fluorescence signals; after the copper ions are mixed with the copper ions, amino groups on the surface of the nitrogen-gold co-doped carbon nano dots can be coordinated with the copper ions, so that the copper ions are adsorbed to the nitrogen-gold co-doped carbon nano dots, and the copper ions can generate a quenching effect on the nitrogen-gold co-doped carbon nano dots, so that the fluorescence intensity of the nitrogen-gold co-doped carbon nano dots is reduced; if hydrolysis product thioglycollic acid obtained by hydrolyzing methyl thioglycollate by adding lipase is added, because the bonding strength of sulfydryl and copper ions in the thioglycollic acid is greater than the coordination strength between the nitrogen-gold co-doped carbon nano dots and the copper ions, the copper ions can be preferentially bonded with the thioglycollic acid, so that the content of the copper ions on the surface of the nitrogen-gold co-doped carbon nano dots is reduced, the fluorescence quenching effect of the copper ions on the nitrogen-gold co-doped carbon nano dots is weakened, and the fluorescence intensity is partially recovered.
Therefore, the method provided by the present invention can be used for testing the activity of lipase, and the standard curve established according to step S4 is shown in FIG. 2. As can be seen from FIG. 2, the fluorescence intensity is dependent on the lipaseThe activity is in a linear relation within the range of 0-800 mU/mL, and the linear equation is y =2.25698x +14.88168, and the standard deviation R2=0.99857, three times according to the standard deviation rule (LOD = 3R)2/k，R2Standard deviation, k is slope) was calculated to have a limit of detection (LOD) of 1.327 mU/mL.
The fluorescence intensity of the lipase test sample was measured according to the above method, the fluorescence intensity corresponding to the lipase having an activity of 0 was subtracted from the actual measured value of the fluorescence intensity of the lipase test sample to obtain a fluorescence intensity of 1152.7, and the fluorescence intensity value was substituted into the above linear equation to calculate that the activity of the lipase test sample was 504.1 mU/mL, which was 0.82% different from the actual value of 500 mU/mL.
Therefore, the lipase activity detection method provided by the embodiment has the advantages of wide detection range, low detection limit, high accuracy and the like, and can meet the requirements of practical application.
Examples 2 to 3 and comparative examples 1 to 2
Examples 2 to 3 and comparative examples 1 to 2 each provide a method for preparing a carbon nanodot, which is different from step S1 of example 1 in that the amount of ethylenediamine as a nitrogen source and chloroauric acid as a gold source are changed. The molar ratios of citric acid, ethylenediamine and chloroauric acid in examples 2 to 3 and comparative examples 1 to 2 are shown in table 1.
TABLE 1 molar ratios of citric acid, ethylenediamine and chloroauric acid for examples 2-3 and comparative examples 1-2
| Examples/comparative examples | Molar ratio of citric acid, ethylenediamine and chloroauric acid |
| Example 2 | 100:10:1 |
| Example 3 | 100:15:2 |
| Comparative example 1 | 100:12:0 |
| Comparative example 2 | 100:0:0 |
Fluorescence detection was performed on the carbon nanodots prepared in examples 2 to 3 and comparative examples 1 to 2, and when the excitation wavelength was 360nm, the fluorescence spectra of the carbon nanodots prepared in each example are shown in fig. 3. Fig. 3 shows that a, b, c, and d respectively represent the carbon nanodots prepared in example 2, example 3, comparative example 1, and comparative example 2, and it can be seen from fig. 3 that the fluorescence intensity of the prepared carbon nanodots increases with the increase of the doping amount of the nitrogen source and the gold source, indicating that nitrogen-gold co-doping can effectively improve the optical performance and the fluorescence quantum yield of the carbon nanodots, so that the fluorescence probe has higher sensitivity.
In addition, solutions having pH values of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 were prepared using a hydrochloric acid solution and a sodium hydroxide solution, and the carbon nanodots prepared in example 3 and comparative examples 1 to 2 were placed in the solutions having the respective pH values, respectively, and the change of fluorescence intensity at 450nm with the pH value at an excitation wavelength of 360nm was tested, and the results are shown in fig. 4. In FIG. 4, a, b, and c show the change curves of fluorescence intensity with pH for example 3, comparative example 1, and comparative example 2, respectively. As can be seen from fig. 4, compared with a single carbon nanodot and a carbon nanodot doped with nitrogen only, the carbon nanodot doped with nitrogen provided by the present invention can be stable at different pH values, can be suitable for measurement at different pH values, and has a wider application range.
Examples 4 to 6
Examples 4 to 6 each provide a method for detecting lipase activity based on a fluorescent probe, which is different from example 1 in that the excitation wavelength of the fluorescence detection process in step S3 is changed, and the excitation wavelengths corresponding to examples 4 to 6 are 340nm, 350nm, and 370nm, respectively.
Fluorescence spectrograms of the nitrogen-gold co-doped carbon nanodots prepared in the embodiments 4 to 6 and 1 under different excitation wavelengths are shown in fig. 5. In FIG. 5, the excitation wavelengths for a, b, c, d are 340nm (example 4), 350nm (example 5), 360nm (example 1) and 370nm (example 6), respectively. As can be seen from fig. 5, the maximum fluorescence intensity value of the obtained fluorescence spectrum increases and then decreases as the excitation wavelength increases. Therefore, in order to enable the nitrogen-gold co-doped carbon nanodots to have high fluorescence intensity, the excitation wavelength of the fluorescence detection process is preferably 340-370 nm, wherein when the excitation wavelength is 370nm, the obtained fluorescence spectrogram has the maximum fluorescence intensity at 450nm, and the lipase activity can be accurately and sensitively detected.
In summary, according to the lipase activity testing method based on the fluorescent probe provided by the invention, citric acid is used as a carbon source, ethylenediamine is used as a carbon-nitrogen source, chloroauric acid is used as a gold source, a hydrothermal method is adopted to prepare the nitrogen-gold co-doped carbon nanodots as the fluorescent probe, the fluorescent probe is mixed with copper ions and a hydrolysis product of lipase, and a standard curve between the lipase activity and the fluorescence intensity of the probe is established by utilizing the difference of the action intensity of the fluorescent probe and the hydrolysis product of lipase on the copper ions, so that the efficient, sensitive and accurate testing of the lipase activity is realized. Through the mode, the optical performance and the fluorescence quantum yield of the carbon nano-dots can be effectively improved, so that the detection has higher sensitivity; the detection method is simple and easy to operate, has the advantages of high detection speed, wide detection range, low detection limit and the like, can accurately detect the lipase with low activity, has a wide application range, and has a good application prospect.
It should be noted that, in step S2, the concentration of the methyl thioglycolate solution may be adjusted within a range of 1 to 2mmol/L, the reaction temperature of the hydrolysis reaction may be adjusted within a range of 35 to 40 ℃, and the reaction time may be 15 to 30min, so that the lipase hydrolysis reaction may be sufficiently performed. Meanwhile, in step S3, the reaction time for the full reaction at room temperature may be 5-10 min, so that the reaction is fully completed; the copper salt solution can be one or more of copper chloride solution, copper sulfate solution and copper nitrate solution, and all fall into the protection scope of the invention.
The above description is only for the purpose of illustrating the technical solutions of the present invention and is not intended to limit the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; all the equivalent structures or equivalent processes performed by using the contents of the specification and the drawings of the invention, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (8)
1. A lipase activity detection method based on a fluorescent probe is characterized by comprising the following steps:
s1, mixing citric acid, ethylenediamine and chloroauric acid in a molar ratio of 100 (10-15) (1-2), dissolving in deionized water, heating and stirring at 80 ℃ for 10min, transferring the solution into an autoclave, heating at 160 ℃ for 12h, and cooling to room temperature to obtain a nitrogen-gold co-doped carbon nanodot crude product; centrifuging the crude product of the nitrogen-gold co-doped carbon nanodots, and filtering the centrifuged supernatant by using a 0.22-micron microporous filter membrane to obtain filtrate, namely purified nitrogen-gold co-doped carbon nanodots serving as a fluorescent probe for storage and later use;
s2, uniformly mixing a lipase standard sample with certain activity and a methyl thioglycolate solution according to a preset volume ratio, and performing full hydrolysis reaction to obtain a hydrolysis reaction solution;
s3, uniformly mixing the hydrolysis reaction liquid obtained in the step S2 with a copper salt solution and the nitrogen-gold co-doped carbon nanodots prepared in the step S1 according to a preset volume ratio, and performing fluorescence detection after full reaction at room temperature;
s4, adjusting the activity of the lipase standard sample, repeating the steps S2-S3, and drawing a standard curve of the change of the fluorescence intensity along with the lipase activity;
s5, replacing the lipase standard sample with the lipase sample to be detected, detecting the fluorescence intensity of the lipase sample to be detected according to the steps S2-S3, and calculating the activity of the lipase sample to be detected according to the standard curve obtained in the step S4.
2. The method of claim 1, wherein the method comprises the steps of: in step S2, the activity of the lipase standard sample is 0-800 mU/mL, and the concentration of the methyl thioglycolate solution is 1-2 mmol/L.
3. The method of claim 1, wherein the method comprises the steps of: in step S2, the preset volume ratio of the lipase standard sample to the methyl thioglycolate solution is 1: 25.
4. The method of claim 1, wherein the method comprises the steps of: in step S2, the hydrolysis reaction is performed at a temperature of 35 to 40 ℃ for 15 to 30 min.
5. The method of claim 1, wherein the method comprises the steps of: in step S3, the preset volume ratio of the hydrolysis reaction liquid to the copper salt solution to the nitrogen-gold co-doped carbon nanodots prepared in step S1 is 100 (20-25) to (6-8).
6. The method of claim 1, wherein the method comprises the steps of: in step S3, the copper salt solution is one or more of a copper chloride solution, a copper sulfate solution and a copper nitrate solution; the concentration of copper ions in the copper salt solution is 100mu mol/L.
7. The method of claim 1, wherein the method comprises the steps of: in step S3, the reaction time for the sufficient reaction at room temperature is 5 to 10 min.
8. The method of claim 1, wherein the method comprises the steps of: in step S3, the excitation wavelength of the fluorescence detection process is 340-370 nm, and the detection wavelength is 450 nm.
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