A visual detection method and detection kit for lipase based on liquid viscosity change

Abstract

The present invention relates to a visual detection method and a detection kit for lipase based on changes in liquid viscosity. The lipase detection method provided by the present invention generates a hydrogel after ions intervene in the reaction system to cause phase separation. Due to different viscosities and water contents in the supernatant, different flow distances are presented on the paper base; the flow distance on the paper base can be measured to achieve quantitative detection of the object to be detected. In addition, the invention relates to a paper-based biosensing method for detecting enzymes due to viscosity changes caused by phase separation, taking lipase as an example. The present invention combines sodium alginate and calcium ion cross-linking gelation, enzymatic hydrolysis reaction, phase separation to cause viscosity changes, and a detection method combined with a paper base to design a convenient, simple and sensitive sensing method. Taking lipase as an example, a two-step reaction of sodium alginate and calcium ions, triglycerides and lipase is combined to design a lipase paper-based detection method with high sensitivity, good specificity and simple operation, which has the potential for commercial detection.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a lipase visualization detection method based on liquid viscosity change and a paper-based biosensor kit applied to the above detection. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] In the field of analytical testing, paper analytical devices (PADs) use porous cellulose to store reagents and transport sample liquids through capillary action. Due to its advantages such as low cost, fast speed, simple operation, easy use and portability, PADs have become attractive analytical method tools in recent years and can be used for detection in the fields of environmental monitoring, medical diagnosis, instant testing and food safety control. Among them, PADs based on detection distance is a new quantitative method, which relies on the relationship between the flow distance of signal molecules along the paper substrate and the concentration of the analyte to establish a quantitative method for detecting the analyte. This new type of distance-specific PADs is simple to operate and low in cost, and is a new biosensing method with great potential and appeal.

[0004] Hydrogels are polymers with three-dimensional network structures that can absorb a large amount of water during their formation. There are two types of gel transformation processes, one is gelation without phase separation, and the other is gelation driven by phase separation. At present, the development of hydrogel biosensors is mainly based on gel-sol transition under external stimuli. In order to quantify the formation of hydrogels or the remaining hydrogels, this method usually requires the transfer of hydrogels, molecular labeling or encapsulation of functionalized particles, and the assistance of professional instruments. In addition, due to the uneven distribution of high-viscosity hydrogels on the sensing interface, these methods have the disadvantages of low sensitivity, slow response, and poor reproducibility. Constructing a biosensor based on viscosity change caused by phase separation to directly monitor the viscosity change of aqueous solutions can effectively overcome the above problems, but there are few related reports. Lipase is a glycerol ester hydrolase that catalyzes the hydrolysis of triglycerides into glycerol and fatty acids. It is abundant in the pancreas and adipose tissue and is an essential enzyme for the digestion, transportation and processing of human fat. At the same time, pancreatic lipase, as an important exocrine enzyme from the pancreas, is an important biomarker for the early diagnosis and progression of acute pancreatitis. Acute pancreatitis is a common and serious gastrointestinal inflammatory disease with a high mortality rate. Therefore, the detection of lipase activity in vivo is of great significance and value for the clinical diagnosis of acute pancreatitis. The existing lipase detection methods mainly include colorimetry, fluorescence, etc. Although these methods are of great significance for the detection of lipase, they usually require complex sample preparation, time-consuming procedures or expensive equipment. Therefore, the development of a simple, convenient, and economical method for rapid and label-free detection of pancreatic lipase is still a great demand.

[0005] The inventors found that the paper-based sensor using the change in viscosity caused by phase separation has not been developed yet, and it can be combined with the existing small molecule reaction to achieve the detection of biomarkers such as enzymes and antibodies. The present invention can be combined with the paper-based sensing method of viscosity change caused by phase separation to quantitatively measure lipase. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a paper-based biosensor detection method based on viscosity change caused by phase separation. The method can generate hydrogels after divalent metal ions are introduced into the reaction system to cause phase separation, and the supernatant shows viscosity differences on the paper base; after the ions to be tested react and combine with the system, the concentration gradient of the ions to be tested is shown on the paper base. The flow distance of the supernatant on the paper base can be measured to achieve quantitative detection of the ions to be tested.

[0007] In a first aspect, the present invention provides a visual detection method for lipase based on liquid viscosity change, the detection method comprising the following steps: adding triglyceride to a lipase sample to be tested to cause a complete enzymatic hydrolysis reaction; preparing a certain amount of calcium ion solution, adding it to the reaction system after enzymatic hydrolysis until no white precipitate is produced, adding the unreacted calcium ion solution to a sodium alginate solution and letting it stand for a period of time until the solution is separated into a supernatant layer and a hydrogel layer; taking the supernatant layer and adding it to one end of a paper-based sensor, and recording the flow of the supernatant on the paper-based sensor.

[0008] In the above detection method, after the enzymatic hydrolysis reaction is completed, the triglyceride in the reaction system is hydrolyzed into oleic acid and glycerol. A pre-prepared calcium ion solution is added to the system after the enzymatic hydrolysis reaction, and oleic acid reacts with calcium ions to generate white calcium oleate precipitates. The remaining calcium ion solution is added to the sodium alginate solution to form a supernatant layer and a hydrogel layer. A portion of the supernatant is added dropwise to one end of the paper base. The viscosity difference can be converted into a flow difference of distance on the paper base. According to the coverage of the supernatant on the paper strip, the concentration of calcium ions can be determined, thereby determining the dosage of the object to be tested.

[0009] Preferably, the flow condition includes the coverage distance or coverage area of ​​the supernatant on the paper strip; in a specific example, the present invention selects the ratio of the coverage area after the liquid flows to the area of ​​the paper strip as the detection signal Cr.

[0010] Preferably, the detection method further comprises preparing a series of concentrations of lipase reference substances, performing detection according to the method described in the first aspect and recording the flow of the supernatant on the paper-based sensor, and establishing a linear regression equation between the lipase concentration C and the detection signal Cr.

[0011] In a specific embodiment of the above preferred technical solution, the concentration of the lipase C = 37.7-6.66Cr; the Cr = P mark / P total × 100%, where the coverage area of ​​the supernatant solution is P mark and the area of ​​the test strip P total Can be read by manual measurement or image recognition software.

[0012] In a second aspect, the present invention provides a paper-based biosensor kit, which includes a sodium alginate solution, a calcium ion-containing solution, triglyceride and a paper-based biosensor, wherein the paper-based biosensor includes an indicator paper strip and a hydrophobic substrate.

[0013] The paper-based biosensor described in the second aspect is applied to the detection of lipase or lipase inhibitors, and can perform visual detection of lipase based on the detection method described in the first aspect. In one embodiment verified by the present invention, the lipase is pancreatic lipase; correspondingly, the inhibitor of pancreatic lipase is, for example, orlistat.

[0014] Preferably, in the sodium alginate solution, the sodium alginate has no limitation on molecular weight and can be a commercially available product. In a specific embodiment of the present invention, the ratio of β-D-mannuronic acid (β-D-mannuronic, M) to α-L-guluronic acid (α-L-guluronic, G) in the sodium alginate is 2:1.

[0015] Preferably, the concentration of the sodium alginate solution is 0.2%-0.7%.

[0016] Preferably, the calcium ion-containing solution refers to a buffer solution containing free calcium ions, and the source of the calcium ions is mainly calcium salts, such as calcium chloride and calcium nitrate.

[0017] Preferably, the hydrophobic substrate material is one including but not limited to a polycarbonate board, a polystyrene board, a polymethyl methacrylate board, and a PVC board.

[0018] Preferably, the indicator paper strip is a cellulose paper strip, including but not limited to pH test paper, filter paper, and microporous cellulose membrane. In a further preferred embodiment of the present invention, the indicator paper strip is a pH test paper, and after the supernatant is dripped onto the pH test paper, the test paper can undergo an obvious color change, which is more convenient for reading the detection signal.

[0019] In the above paper-based biosensor, the indicator paper strip is placed on a hydrophobic substrate, and the paper-based biosensor should be kept dry before use, without any excess liquid interfering with the detection.

[0020] Preferably, the kit further comprises a buffer, and the buffer is a Tris-HCl buffer with a pH of 7.2 to 7.4.

[0021] The beneficial effects of one or more of the above technical solutions are:

[0022] (1) The present invention uses paper as the detection element, which is simple, green and friendly.

[0023] (2) The present invention combines enzymatic hydrolysis reaction, so that the reaction has good specificity and high stability.

[0024] (3) The reagents and materials used in the present invention, such as sodium alginate, divalent metal ions, and pH test strips, are all commercial reagents on the market, and are stable in nature, low in cost, and highly practical, making them easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 Schematic diagram of the detection principle of paper-based biosensor;

[0027] Wherein, a is the flow of sodium alginate and calcium ion solutions of different concentrations (0-10 mM) on the paper substrate; b is the flow of the paper strip corresponding to a (expressed by Cr).

[0028] Figure 2 The results of the correlation study of the lipase detection method described in Example 1;

[0029] a is the enzymatic reaction formula of lipase and the process of binding with calcium ions;

[0030] b is to detect the flow effect of different concentrations of lipase on the paper substrate;

[0031] c is the flow condition of the paper strip corresponding to b (expressed by Cr) and the linear relationship between the horizontal axis function and the lipase concentration after processing.

[0032] Figure 3 This is a schematic diagram of the paper-based biosensing method for detecting lipase using viscosity change caused by phase separation in Example 1. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] Terminology Note:

[0036] Cr value: Cr = P mark / P total ×100%

[0037] P mark : Coverage area of ​​supernatant

[0038] P total : Area of ​​pH test strip

[0039] G:α-L-guluronic acid

[0040] M:β-D-mannuronic acid

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0042] Example 1

[0043] In this embodiment, a method for detecting pancreatic lipase is provided, and the detection method comprises the following steps:

[0044] Ⅰ. Test strip processing: The indicative test strip used in this embodiment is a pH test strip, and the test strip is cut into 5.75 mm × 60 mm;

[0045] Ⅱ. Place the test strip on the polycarbonate plate for use;

[0046] III. Dissolve sodium alginate in 100 mM Tris-HCl solution at pH 7.2 to 7.4 to obtain a 0.2% sodium alginate aqueous solution;

[0047] IV. Dissolve calcium chloride in the same buffer solution and mix well to obtain a 16 mM calcium chloride solution;

[0048] V. Add 5 μL of triglyceride to the pancreatic lipase sample to be tested, vortex for 30s-60s to mix evenly, and maintain the temperature of the reaction system at 37°C to allow enzymatic hydrolysis to generate oleic acid and glycerol;

[0049] VI. Add the solution prepared in IV to the reaction system in V and shake for 3.5 hours to allow oleic acid to react with calcium ions to form calcium oleate precipitate;

[0050] VII. Take 180 μL of the unreacted calcium ion solution in the calcium oleate precipitation system in system VI and mix it with the same volume of sodium alginate aqueous solution in system III, let it stand for 2 minutes, and the solution will have a hydrogel layer in the lower part and a solution layer in the upper part;

[0051] VIII. Take 30 μL of the supernatant of VII and drop it on the test strip of Ⅱ, and observe the flow distance;

[0052] IX. Based on Figure 2 The data were processed using the linear function given in c to obtain the lipase concentration.

[0053] Figure 2a is the enzymatic reaction formula of lipase and the process of binding with calcium ions; b is the flow effect on the paper base after reaction of different concentrations of lipase; c is the flow length of the paper strip corresponding to b (expressed by Cr) and the linear relationship between the horizontal axis function and the lipase concentration after processing.

[0054] Example 2

[0055] In this embodiment, a method for detecting orlistat, a lipase inhibitor, is provided, and the detection method comprises the following steps:

[0056] Ⅰ. Test strip processing: The indicative test strip used in this embodiment is a microporous cellulose membrane, and the test strip is cut into 5.75 mm×60 mm;

[0057] Ⅱ. Place the test strip on the polystyrene plate for use;

[0058] III. Dissolve sodium alginate in 100 mM Tris-HCl solution at pH 7.2 to 7.4 to obtain a 0.2% sodium alginate aqueous solution;

[0059] IV. Dissolve calcium ions in the same buffer solution as in III and mix;

[0060] V. Dissolve orlistat with DMSO, then dilute with 100 mM Tris-HCl solution with pH 7.2-7.4, and react the orlistat solution with lipase at 25-37° C. for 30 min;

[0061] VI. The triglyceride reacts with the solution of V at 37°C;

[0062] VII. Add an appropriate solution of IV to the system after the reaction of VI, and shake for 3.5 hours to allow oleic acid to react with calcium ions to form calcium oleate precipitate;

[0063] VIII. Take 180 μL of the unreacted calcium ion solution in system VII and mix it with the same volume of sodium alginate aqueous solution in system III, let it stand for 2 minutes, and the solution will have a hydrogel layer in the lower part and a solution layer in the upper part;

[0064] IX. Take 30 μL of the supernatant from VIII and drop it onto the test strip from II, and observe the flow distance;

[0065] Ⅹ. Data processing to obtain the orlistat concentration to be tested.

[0066] Figure 2 a is the enzymatic reaction formula of lipase and the process of binding with calcium ions; b is the flow effect on the paper base after reaction of different concentrations of lipase; c is the flow length of the paper strip corresponding to b (expressed by Cr) and the linear relationship between the horizontal axis function and the lipase concentration after processing.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A visual detection method for lipase based on changes in liquid viscosity, characterized in that: The detection method comprises the following steps: adding triglyceride oil to a lipase sample to be tested to make it undergo a complete enzymatic hydrolysis reaction; preparing a certain amount of calcium ion solution, adding it to the reaction system after enzymatic hydrolysis until no white precipitate is generated, adding the unreacted calcium ion solution in the reaction system to a sodium alginate solution and letting it stand for a period of time until the solution is separated into a supernatant layer and a hydrogel layer; taking the supernatant layer and adding it to one end of a paper-based sensor, and recording the flow of the supernatant on the paper-based sensor.

2. The method for visual detection of lipase based on liquid viscosity change according to claim 1, characterized in that: The flow condition includes the coverage distance or coverage area of ​​the supernatant on the paper strip.

3. The method for visual detection of lipase based on liquid viscosity change according to claim 2, characterized in that: The ratio of the area covered by the liquid after flowing to the area of ​​the paper strip is used as a detection signal C r.

4. The method for visual detection of lipase based on liquid viscosity change according to claim 3, characterized in that: The detection method further comprises: preparing a series of concentrations of lipase reference substances for detection according to the method of claim 1 and recording the flow of the supernatant on the paper-based sensor to establish a relationship between the lipase concentration C and the detection signal C The linear regression equation between r.

5. The method for visual detection of lipase based on liquid viscosity change according to claim 4, characterized in that: Said C r = P mark / P total × 100%, where the coverage area of ​​the supernatant is P mark and the area of ​​the test strip P total Can be read by manual measurement or image recognition software.

6. A paper-based biosensor kit, characterized in that: The kit comprises a sodium alginate solution, a calcium ion solution, triglyceride and a paper-based biosensor, and the paper-based biosensor comprises an indicator paper strip and a hydrophobic substrate.

7. The paper-based biosensor kit according to claim 6, characterized in that: The paper-based biosensor is used for detecting lipase or lipase inhibitor, the lipase is pancreatic lipase, and the pancreatic lipase inhibitor is orlistat.

8. The paper-based biosensor kit according to claim 6, characterized in that: In the sodium alginate solution, the ratio of β-D-mannuronic acid to α-L-guluronic acid in the sodium alginate is 2:1; Alternatively, the concentration of the sodium alginate solution is 0.2wt%-0.7wt%.

9. The paper-based biosensor kit according to claim 6, characterized in that: The calcium ion-containing solution refers to a buffer solution containing free calcium ions, and the calcium ions are derived from calcium salts.

10. The paper-based biosensor kit according to claim 9, characterized in that: The calcium ions are derived from one of calcium chloride and calcium nitrate.

11. The paper-based biosensor kit according to claim 6, characterized in that: The hydrophobic substrate material is one of a polycarbonate board, a polystyrene board, a polymethyl methacrylate board and a PVC board.

12. The paper-based biosensor kit according to claim 6, characterized in that: The indicative paper strip is a cellulose paper strip.

13. The paper-based biosensor kit according to claim 12, characterized in that: The indicator paper strip is one of pH test paper, filter paper, and microporous cellulose membrane.

14. The paper-based biosensor kit according to claim 13, characterized in that: The indicative paper strip is selected from pH test paper.

15. The paper-based biosensor kit according to claim 6, characterized in that: The kit also includes a buffer solution, which is a Tris-HCl buffer solution with a pH of 7.2 to 7.4.

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