CN113552122B - Detection method of biological molecules and inhibitor molecules - Google Patents
Detection method of biological molecules and inhibitor molecules Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
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Abstract
The disclosure belongs to the technical field of analysis and detection, and particularly provides a detection method of biomolecules and inhibitor molecules. The detection method of the concentration of the biological molecules comprises the following steps: and mixing the object to be detected with the viscous solution or the hydrogel to form a mixed solution, placing the mixed solution at one end of an indicator test strip, and detecting the object to be detected by different amounts by using the different amounts to cause different hydrolysis degrees of the viscous solution or the hydrogel, so that the content of water released by decomposition of the viscous solution or the hydrogel is different, different distances can be generated on the pH test strip to move, and the object to be detected is detected by the length of the moving distance. The method solves the problems that the common biomolecule detection method in the prior art has complicated operation steps, needs to train professionals to operate related equipment, has long detection time and low efficiency, causes resource waste and cost increase, and has environmental background factors such as matrixes and the like so as to influence detection sensitivity.
Description
Technical Field
The disclosure belongs to the technical field of analysis and detection, and particularly provides a detection method of biomolecules and inhibitor molecules.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Some biomolecules can be objectively measured as markers and used as evaluation indexes to judge normal biological processes, pathological processes or post-healing reactions, and are also important early warning indexes when organisms are damaged or diseased. For this reason, laboratory personnel often need to perform biomolecule detection when exploring pathology or other biological studies.
The detection methods commonly used at present mainly comprise an enzyme-linked immunoassay method, a chemiluminescent immunoassay method, an electrochemical assay method and the like. Generally, the methods have higher selectivity and specificity, but have complicated operation steps, require training professionals to operate related equipment, have long detection time and low efficiency, cause resource waste and cost increase, and have environmental background factors such as matrixes and the like to influence the detection sensitivity.
Disclosure of Invention
Aiming at the problems that the common biomolecule detection method in the prior art has complicated operation steps, needs to train professionals to operate related equipment, has long detection time and low efficiency, causes resource waste and cost increase, and has environmental background factors such as matrixes and the like so as to influence detection sensitivity, in one or some embodiments of the disclosure, the biomolecule detection method is provided, and comprises the following steps: mixing the object to be detected with a viscous solution (such as a polymer solution, hydrogel and the like) to form a mixed solution, placing the mixed solution at one end of an indicator test strip, and hydrolyzing the components of the viscous solution to different degrees by different amounts of target detection objects, so that the contents of water released by decomposition of the viscous solution or the hydrogel are different, different distances of movement can be generated on the pH test strip, and the detection of the target detection objects is realized by the length of the movement distance.
In one or some embodiments of the present disclosure, a method for testing inhibitor molecules is provided, wherein a target molecule, a potential inhibitor molecule and a hydrogel are mixed to form a mixed solution, and the mixed solution is placed at one end of an indicator test strip;
the target molecule causes the hydrogel to hydrolyze, so that the water released by the hydrogel decomposition can move on the pH test strip for a certain distance, and the detection of the target detection object is realized by measuring the length of the moving distance;
the inhibitor molecules inhibit target molecules to play a role, the hydrolysis of hydrogel is reduced, the mixed solution can also move on the pH test strip for a certain distance, and the inhibition capability of the inhibitor molecules is tested by measuring the moving distance.
One or some of the above technical solutions have the following advantages or beneficial effects:
1) The detection principle related to the present disclosure is simple, the target detection object induces the viscous solution or the hydrogel to split the water for discharging, and the target detection object is detected by moving the released water on the indicative test strip by a distance. The method has the advantages of low cost of the used materials, simple and convenient operation, rapid detection, long-time preservation of the result, capability of avoiding interference of background factors, nonspecific absorption and the like.
2) The method has the advantages that the requirement on the detection device is simple, the image data recording and the qualitative analysis can be completed only by one intelligent mobile phone with a photographing function or one measuring ruler, the defects that the traditional detection needs a large laboratory instrument and the like are overcome, the outdoor detection or the home self-detection can be realized, the economic value and the social benefit are good, and the method is worthy of popularization and application.
3) The disclosure provides a novel method for detecting biomolecules based on hydrolysis degree of viscous solution or hydrogel, namely, different amounts of target detection objects cause different hydrolysis degrees of the hydrolysis of the viscous solution or the hydrogel, at the moment, different distances can be generated on an indicative test strip due to different contents of water released by the decomposition of the viscous solution or the hydrogel, and the detection of the target detection objects is realized through the length of the moving distances. The method is suitable for various target objects to be detected, and specifically, the method can be used for detecting the objects to be detected as long as the viscous solution or the hydrogel can be hydrolyzed.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate and explain the exemplary embodiments of the disclosure and together with the description serve to explain the disclosure, and do not constitute an undue limitation on the disclosure.
Fig. 1 is a schematic diagram of the structure and principle of a detection device according to the present disclosure; wherein: 1. a viscous solution or hydrogel droplet for detection; 2. a hydrophobic substrate board, a ph test strip; 4. a support bar; 5. a water movement distance; 6. a target detection object.
FIG. 2 is a graph showing the results of optimizing the concentration of gelatin used in example 1;
FIG. 3 is a graph showing the results of the serum trypsin assay in example 1;
FIG. 4 is a graph showing the results of aprotinin assay in example 4.
FIG. 5 is a graph showing the results of optimizing the concentration of hyaluronic acid used in example 5.
Detailed Description
The following will clearly and fully describe the technical solutions in the embodiments of the present disclosure, and it is apparent that the described embodiments are only some embodiments of the present disclosure, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of this disclosure without making any inventive effort, are intended to be within the scope of this disclosure.
Aiming at the problems that the common biomolecule detection method in the prior art has complicated operation steps, needs to train professionals to operate related equipment, has long detection time and low efficiency, causes resource waste and cost increase, and has environmental background factors such as matrixes and the like so as to influence detection sensitivity.
In one or some embodiments of the present disclosure, there is provided a method of detecting a biomolecule, comprising the steps of: and mixing the object to be detected with the viscous solution or the hydrogel to form a mixed solution, placing the mixed solution at one end of an indicator test strip, and detecting the object to be detected by different amounts by using the different amounts to cause different hydrolysis degrees of the viscous solution or the hydrogel, so that the content of water released by decomposition of the viscous solution or the hydrogel is different, and the movement of different distances can be generated on the pH test strip, and the detection of the object to be detected is realized by the length of the movement distance.
The detection principle of the detection method of the biological molecule disclosed by the disclosure is as follows: when the detection object exists, the hydrolysis degree of the viscous solution or the hydrogel is positively correlated with the amount of the detection object, and the higher the concentration of the detection object is, the higher the hydrolysis degree of the viscous solution or the hydrogel is, the larger the released water amount is, and the longer the moving distance is on the indicative test strip or the higher the proportion of the water mark is. When no detection object exists, the viscous solution is not hydrolyzed, and a large amount of water movement trace does not appear on the indicator test strip.
The hydrogel is used as an elastic material with a three-dimensional polymer network structure, can absorb and retain a large amount of water, has good biocompatibility, and is widely applied to the fields of drug delivery, biological tissue engineering, flexible sensors, soft driving and the like.
By utilizing the characteristic of high water content of the viscous solution or hydrogel, the novel method for detecting the biomolecules based on the hydrolysis degree of the viscous solution or hydrogel is developed, mainly utilizing a detection object to induce the viscous solution or hydrogel to divide and explain water release, and detecting a target object by moving the released water on an indicative test strip. The method has the advantages of simplicity, easiness in operation, low cost, rapid detection, long-time preservation of results, capability of avoiding interference of background factors, non-specific adsorption and the like.
Preferably, the viscous solution or hydrogel is hyaluronic acid solution, gelatin solution, starch solution, glucose solution, pectin solution, sodium alginate solution, agar solution, xanthan gum solution, locust bean gum solution, methylcellulose hot solution;
preferably, the object to be detected is plant polysaccharide or animal polysaccharide;
preferably, the object to be detected is levan, mannan, rhamnose, galacturonic acid, mucopolysaccharide or chitosan; microbial polysaccharides such as peptidoglycans; algal polysaccharide; a proteinaceous substance;
further preferably, the proteinaceous material is an enzyme, preferably an amylase, a glucose oxidase, a galacturonase, a pectolytic enzyme, a pectinase, an agarase, a xanthan liquefying enzyme, a mannanase, a cellulase;
preferably, the hydrogel and the analyte have the following properties: the hydrogel changes along with the concentration of the object to be detected, so that the decomposing capability is enhanced; it should be understood that any analyte that hydrolyzes and converts a hydrogel to a gel state can be used in the methods described in this disclosure.
Preferably, the hydrogel is gelatin hydrogel system, hyaluronic acid hydrogel system, DNA
A hydrogel-like system;
preferably, the hydrogel is a gelatin hydrogel system, and the object to be detected is trypsin in serum;
preferably, the hydrogel is a hyaluronic acid hydrogel system, and the object to be detected is hyaluronidase;
preferably, the hydrogel is a DNA hydrogel system, and the object to be detected is DNA shearing enzyme or ochratoxin A (OTA);
after the combination is verified by a test, the change of the combination along with the concentration on the test paper to be tested is obvious, the distinguishing degree is high, and the method is suitable for the concentration detection of the method.
Further preferably, the hydrogel is gelatin after phosphoric acid activation, and the object to be detected is trypsin in serum;
further preferably, the proportion of phosphoric acid in the gelatin is 1 to 5wt%, further preferably 5wt%.
Preferably, the viscous solution or hydrogel is hyaluronic acid, and the concentration of hyaluronic acid is 0.2-1wt%, more preferably 1wt%.
Experiments show that the 5wt% phosphate buffer solution can enable the liquid drop to become gel after 5 minutes, has higher sensitivity and can meet the requirement of rapid detection.
Preferably, the indicator test strip is one of a pH test strip, a polyester film test strip, a carbon cellulose film test strip, a glass fiber test strip and the like, and is preferably a pH test strip;
further preferably, the pH test strip is a common wide-range test strip, and the pH range is 1-14.
Preferably, the detection method of the concentration of the biological molecules is carried out on a hydrophobic substrate, the mixed solution is firstly dripped on the hydrophobic substrate, and one end of the indicator test strip is arranged on the upper surface of the liquid drop after a period of time;
preferably, the hydrophobic substrate material comprises one of a polycarbonate plate, a polystyrene plate, a polymethyl methacrylate plate, a PVC plate, and the like;
preferably, the period of time is 1 to 30 minutes, more preferably 5 minutes.
Preferably, the method also comprises an optimization process of the concentration of the viscous solution or the hydrogel, and the method comprises the following steps of: preparing viscous solutions with different mass concentrations by using phosphate buffer solution, respectively dripping the viscous solutions onto a hydrophobic substrate plate, observing whether the liquid drops can become gel after a period of time, and selecting the corresponding concentration of the viscous solution or hydrogel when the liquid drops become gel state as the optimal concentration for the next detection;
preferably, the period of time is 1 to 30 minutes, more preferably 5 minutes;
or, the method also comprises a hydrophobic substrate plate cleaning process, which comprises the following steps: ultrasonically cleaning for a plurality of times by using a mixed solution of ethanol and deionized water;
preferably, the volume ratio of ethanol to deionized water is 1:1.5-2.5, more preferably 1:2;
preferably, the number of times of ultrasound is 3 to 5 times, further preferably 3 times;
preferably, each time the ultrasound is performed for 4-6 minutes, more preferably 5 minutes;
or, the method further comprises the incubation process of the sample to be detected, comprising the steps of mixing the sample to be detected with the viscous solution, and incubating the mixed solution in a proper temperature environment for a period of time;
preferably, the volume ratio of the detection sample to the viscous solution is 1:1.2, preferably 1:1;
preferably, the mixed solution is incubated at 37℃for 15 minutes.
Preferably, the method further comprises an indicating test strip scale acquisition process, which comprises the following steps: mixing the to-be-detected objects with different standard concentrations with the respectively viscous solutions or the hydrogels, respectively placing the to-be-detected objects at one end of the indicator test strip, and recording scales corresponding to the concentrations for subsequent comparison of the target detection concentrations.
In one or some embodiments of the present disclosure, a method for testing inhibitor molecules is provided, wherein a target molecule, an inhibitor molecule and a hydrogel are mixed to form a mixed solution, and the mixed solution is placed at one end of an indicator test strip;
the target molecule causes the hydrogel to hydrolyze, so that the water released by the hydrogel decomposition can move on the pH test strip for a certain distance, and the detection of the target detection object is realized by measuring the length of the moving distance;
the inhibitor molecules inhibit target molecules to play a role, the hydrolysis of hydrogel is reduced, the mixed solution can also move on the pH test strip for a certain distance, and the inhibition capability of the inhibitor molecules is tested by measuring the moving distance.
Taking aprotinin and trypsin as examples, the principle of the method for testing the inhibition capacity of the inhibitor molecules of the present disclosure is as follows: when aprotinin and trypsin are simultaneously present, trypsin activity is inhibited, gelatin cannot be hydrolyzed or the degree of hydrolysis is low (no water is released or the amount of released water is low at the moment), and no trace or short moving distance of water is generated on the test paper; when trypsin is present and aprotinin is absent, the gelatin water is interpreted to release water, and a trace of water movement is observed on the indicator strip.
Preferably, the target molecule is an enzyme and the inhibitor molecule is an enzyme inhibitor;
preferably, the target molecule is aprotinin and the inhibitor molecule is trypsin.
Preferably, the method further comprises a concentration inhibition test process, comprising the following steps: the target molecules are target molecules with different concentrations, and the inhibitor is inhibitor with different concentrations.
Preferably, the method for testing the inhibition capacity of the inhibitor molecules is carried out on a hydrophobic substrate, the mixed solution is firstly dripped on the hydrophobic substrate, and one end of an indicator test strip is arranged on the upper surface of the liquid drop after a period of time;
preferably, the hydrophobic substrate material comprises one of a polycarbonate plate, a polystyrene plate, a polymethyl methacrylate plate and a PVC plate;
preferably, the period of time is 1 to 30 minutes, more preferably 5 minutes.
Example 1:
the embodiment provides a method for detecting the concentration of trypsin in serum, which uses gelatin as a hydrogel matrix and successfully realizes the detection of trypsin based on the detection device and principle shown in fig. 1. The specific operation steps are as follows:
step one, optimizing and selecting the using concentration of gelatin:
gelatin solutions (e.g., 1wt%,2wt%,3wt%,4wt%,5wt%, etc.) of different mass concentrations were prepared using phosphate buffer solution (10 mmol PBS), after being sufficiently dissolved at 37 ℃, 30 μl was respectively dropped onto the hydrophobic base plate, and at room temperature (20 ℃) it was observed whether the droplets could become gels after 5 minutes, the corresponding hydrogel concentration when the droplets became gel state was selected as the optimum concentration, where the hydrogel optimum use concentration was determined to be 5wt%, and as shown in fig. 2, the hydrogel detection concentration used in the subsequent series of detection was 5wt%.
Step two, treating a hydrophobic substrate plate and a pH test strip:
the indicator test strip used here is a pH test strip, and as an example, the pH test strip is cut to 50mm by 70mm;
cleaning a hydrophobic substrate plate: a mixed solution of ethanol and deionized water (volume ratio of 1:2) was used for ultrasonic cleaning 3 times for 5 minutes each.
Step three, sample testing:
(1) Preparing trypsin solutions with different concentrations by using PBS and normal human serum mixed solution (wherein the volume ratio of PBS to serum is 3:2), wherein the concentrations are respectively 2 multiplied by 10 -1 mg/mL,2×10 -2 mg/mL,2×10 -3 mg/mL,2×10 -4 mg/mL,2×10 -5 mg/mL,2×10 -6 mg/mL; as a control group, trypsin was not added
(2) A 10wt% gelatin solution was prepared using PBS;
(3) Mixing the sample prepared in the step (1) with a gelatin solution according to a volume ratio of 1:1, and incubating the mixed solution for 15 minutes at 37 ℃;
(4) Dripping 30uL of the reacted solution on a hydrophobic PS substrate board, putting one end of a pH test strip on the upper surface of the liquid drop after 5 minutes, recording and observing the moving distance of water released after gelatin hydrolysis on the pH test strip by using a photographing tool such as a smart phone, so as to realize the detection of a target object;
(6) The ratio of the moving distance to the whole pH test strip can be counted by using software tools such as Photoshop, origin and the like to carry out quantitative calculation;
the present example is based on the principle that: the degree of gelatin hydrolysis is positively correlated with the amount of trypsin, and the higher the concentration of trypsin, the greater the degree of gelatin hydrolysis and the greater the amount of water released, the longer the distance of movement on the pH test strip or the higher the proportion of the place where the watermark appears. When trypsin is not present, gelatin is not hydrolyzed and converted into a gel state, and no trace of water movement appears on the pH test strip.
Example 2:
the present example provides a method for detecting hyaluronidase, which is similar to that of example 1 in that the detection object is changed to hyaluronidase and the hydrogel is changed to hyaluronic acid hydrogel, based on the same device and principle, so that the detection of hyaluronidase can be realized.
Example 3:
the present example provides a method for detecting DNA shearing enzyme, which is similar to that of example 1 in that the detection object is replaced with DNA shearing enzyme, the hydrogel is replaced with DNA hydrogel, and the detection of DNA shearing enzyme can be realized.
Example 4:
provided is a method for detecting the inhibitory effect of an enzyme inhibitor.
The present embodiment provides a method for testing aprotinin inhibitory activity, wherein when aprotinin and trypsin are present at the same time, trypsin activity is inhibited, gelatin cannot be hydrolyzed or the degree of hydrolysis is low (no water is released or the amount of released water is low at this time), and no trace of water movement or short movement distance is found on a pH test strip; when trypsin is present and aprotinin is absent, gelatin hydrolyzes to release water, and water movement marks are observed on the pH test strip.
Comparative example 1
This example provides a method for measuring the concentration of trypsin in serum, which is similar to example 1 in that a solution containing no trypsin or other enzyme solution is added, and the gelatin solution cannot be used at pH
The test strip moves up.
Comparative example 2
This example provides a method for detecting the concentration of trypsin in serum, which is based on the same device and principle as in example 1, and replaces gelatin with agar, and it is found that agar is difficult to hydrolyze and cannot move on a pH paper.
From the comparison of comparative examples 1 and 2 with example 1, it is apparent that the matching of the gelatin solution activated by the phosphate buffer solution and the pH test paper can be well matched with the hydrolysis of the hydrogel and the moving speed of the pH test paper, and the observation effect is good.
The inventors tried to conduct the test without forming hydrogel, using only hyaluronic acid solution, and specifically conducted the following test:
example 5:
in the embodiment, hyaluronic acid is used as a substrate, and the detection of the hyaluronidase is successfully realized based on the detection device and the principle shown in fig. 1. The specific operation steps are as follows:
step one, optimizing and selecting the use concentration of hyaluronic acid:
hyaluronic acid solutions (for example, 0.2wt%,0.4wt%,0.6wt%,0.8wt%,1wt%, etc.) with different mass concentrations are prepared by using a 0.1M Phosphate Buffer Solution (PBS), 30uL of each solution is dropped onto a pH test strip after being heated and fully swelled, the flowing condition of the droplets on the pH test strip is observed at room temperature (25 ℃) and the concentration of the droplet with the shortest flowing distance is selected as the optimal concentration, wherein the optimal use concentration of hyaluronic acid is determined to be 1wt%, and the detection concentration of hyaluronic acid used in the subsequent series of detection is about 1wt%, as shown in fig. 2.
Step two, treating a hydrophobic substrate plate and a pH test strip:
the indicator test strip used here is a pH test strip, and as an example, the pH test strip is cut to a size of 5mm by 70mm;
cleaning a hydrophobic substrate plate: the mixture of ethanol and deionized water (volume ratio of 1:2) was used for ultrasonic cleaning 3 times, each for 5min.
Step three, hyaluronidase detection:
(1) Different concentrations of hyaluronidase solutions, 200U/ml,20U/ml,2U/ml, 2X 10-1U/ml, 2X 10-2U/ml, 2X 10-3U/ml, 2X 10-4U/ml, were prepared using PBS and hyaluronidase solutions, respectively, as control groups, no hyaluronidase was added;
(2) A 1.6wt% hyaluronic acid solution was prepared using PBS;
(3) Mixing the sample prepared in the step (1) with the hyaluronic acid solution according to the volume ratio of 1:2, and incubating the mixed solution for 45 minutes at 37 ℃;
(4) Dripping 45ul of the reacted solution onto a pH test strip, and recording and observing the moving distance of the low-viscosity solution after the decomposition of the hyaluronic acid on the pH test strip by using a photographing tool such as a smart phone, so as to realize the detection of a target object;
(6) The ratio of the moving distance to the whole pH test strip can be counted by using software tools such as Photoshop, origin and the like to carry out quantitative calculation;
the present example is based on the principle that: the degree of decomposition of the hyaluronic acid is positively correlated with the amount of the hyaluronidase, and the higher the concentration of the hyaluronidase is, the higher the degree of decomposition of the hyaluronic acid is, the more the solution viscosity is reduced, and the longer the moving distance on the pH test strip or the higher the proportion of the watermark is. When the hyaluronidase is not present, the hyaluronic acid is not decomposed, the solution viscosity is not changed, and at the moment, the trace of the solution movement cannot appear on the pH test strip.
Example 6:
based on the same device and principle, the concentration of hyaluronic acid in the solution and all solutions with viscosity change can be directly detected, and plant polysaccharide such as glycan, mannan, rhamnose and galacturonic acid; animal polysaccharides such as glycosaminoglycans and chitosan; microbial polysaccharides such as peptidoglycans; algal polysaccharide; proteins, and the like.
Example 7:
based on the same device and principle, the detection object is replaced by trypsin, and the hyaluronic acid solution is replaced by gelatin solution, so that the trypsin can be detected.
Example 8:
based on the same device and principle, the detection object is replaced by amylase and the like, and the hyaluronic acid solution is replaced by starch solution, so that the amylase can be detected.
Example 9:
based on the same device and principle, the detection object is replaced by glucose oxidase and the like, and the hyaluronic acid solution is replaced by glucose solution, so that the detection of the glucose oxidase can be realized.
Example 10:
based on the same device and principle, the detection object is replaced by galacturonase, pectolytic enzyme and the like, and the hyaluronic acid solution is replaced by pectin solution, so that the detection of the galacturonase, pectolytic enzyme and pectolytic enzyme can be realized.
Example 11:
based on the same device and principle, the detection object is replaced by sodium alginate lyase and the like, and the hyaluronic acid solution is replaced by sodium alginate solution, so that the detection of the sodium alginate lyase can be realized.
Example 12:
based on the same device and principle, the detection object is replaced by agarase and the like, and the hyaluronic acid solution is replaced by the agar solution, so that the detection of the agarase can be realized.
Example 13:
based on the same device and principle, the detection object is replaced by xanthan gum liquefying enzyme and the like, and the hyaluronic acid solution is replaced by xanthan gum solution, so that the detection of the xanthan gum liquefying enzyme can be realized.
Example 14:
based on the same device and principle, the detection object is replaced by mannanase and the like, and the hyaluronic acid solution is replaced by locust bean gum solution, so that the detection of the mannanase can be realized.
Example 15:
based on the same device and principle, the detection object is replaced by cellulase and the like, and the hyaluronic acid solution is replaced by methyl cellulose hot solution, so that the detection of the cellulase can be realized.
Example 16:
based on the same device and principle, the detection of humic acid with different concentrations can be realized.
Example 17:
based on the same device and principle, the gelation behavior of hydrogen peroxide can be rapidly detected based on the temperature sensitivity and shear thixotropic properties of the responsive hydrogen peroxide gel.
Example 18:
based on the same device and principle, a DNA hydrogel system can be studied, and the object to be detected is DNA shearing enzyme and ochratoxin A.
Example 19:
in this example, some enzyme inhibitors were added to viscous solutions or hydrogels to effect detection of enzyme inhibitors. For example, a hyaluronidase inhibitor, when the hyaluronidase inhibitor and the hyaluronidase are simultaneously present, the hyaluronidase activity is inhibited, the hyaluronic acid cannot be decomposed or the decomposition degree is low (at the moment, the solution viscosity is unchanged or the viscosity change is small), and no trace or short moving distance of the solution exists on the pH test strip; when hyaluronidase is present and the inhibitor is not present, the solution viscosity becomes low due to decomposition of hyaluronic acid, and water movement trace can be observed on the pH test strip.
Example 20:
in enzyme-regulated repairable polymer hydrogel systems, the amount of enzyme added is proportional to the gel stability and viscosity and inversely proportional to the change in the distance traveled by the solution dropped onto the test strip.
Example 21:
based on the same device and principle, a microorganism system can be studied, and detection of microorganisms is achieved by detecting enzymes produced by the microorganisms.
The foregoing disclosure is merely illustrative of the presently preferred embodiments of the disclosure and is, of course, not to be construed as limiting the scope of the disclosure, for the purpose of describing and claiming equivalent variations thereto, which fall within the scope of the disclosure.
Claims (44)
1. A method for detecting a biomolecule, comprising the steps of: mixing an object to be detected with a viscous solution or hydrogel to form a mixed solution, placing the mixed solution at one end of an indicator test strip, and detecting the object by different amounts according to the object to be detected, wherein the different amounts of the object to be detected cause different hydrolysis degrees of the viscous solution or hydrogel, so that the content of water released by decomposition of the viscous solution or hydrogel is different, and the object to be detected can move on the pH test strip at different distances according to the length of the moving distance;
the viscous solution or hydrogel is hyaluronic acid, gelatin solution, starch solution, glucose solution, pectin solution, sodium alginate solution, agar solution, xanthan gum solution, locust bean gum solution, and methylcellulose hot solution;
the object to be detected is plant polysaccharide or animal polysaccharide.
2. The method for detecting a biological molecule according to claim 1, wherein the analyte is levan, mannan, rhamnose, galacturonic acid, glycosaminoglycan, chitosan; microbial polysaccharides such as peptidoglycans; algal polysaccharide; a proteinaceous substance.
3. The method for detecting a biomolecule according to claim 2, wherein the proteinaceous substance is an enzyme.
4. The method for detecting a biological molecule according to claim 2, wherein the protein is amylase, glucose oxidase, galacturonase, pectolytic enzyme, pectinase, agarase, xanthan liquefying enzyme, mannanase, or cellulase.
5. The method for detecting a biological molecule according to claim 1, wherein the viscous solution or the hydrogel is a hydrogel, and the hydrogel and the analyte have the following properties: the hydrogel changes along with the concentration of the object to be detected, and the decomposing capacity is enhanced.
6. The method for detecting a biomolecule according to claim 1, wherein the hydrogel is a gelatin-based hydrogel system, a hyaluronic acid-based hydrogel system, or a DNA-based hydrogel system.
7. The method for detecting biological molecules according to claim 1, wherein the hydrogel is a gelatin-based hydrogel system, and the analyte is trypsin in serum.
8. The method for detecting a biological molecule according to claim 1, wherein the hydrogel is a hyaluronic acid hydrogel system, and the analyte is hyaluronidase.
9. The method for detecting biological molecules according to claim 1, wherein the hydrogel is a DNA-based hydrogel system, and the analyte is DNA-cleaving enzyme, ochratoxin a (OTA).
10. The method for detecting a biological molecule according to claim 1, wherein the hydrogel is gelatin after activation with phosphoric acid, and the analyte is trypsin in serum.
11. The method for detecting a biological molecule according to claim 10, wherein the proportion of said phosphoric acid in gelatin is 1 to 5wt%.
12. The method for detecting a biological molecule according to claim 10, wherein the proportion of said phosphoric acid in gelatin is 5wt%.
13. The method for detecting a biological molecule according to claim 1, wherein the viscous solution or the hydrogel is hyaluronic acid, and the concentration of the hyaluronic acid is 0.2-1 wt%.
14. The method of claim 1, wherein the viscous solution or hydrogel is hyaluronic acid and the concentration of hyaluronic acid is 1wt%.
15. The method according to claim 1, wherein the indicator strip is one of a pH strip, a polyester film strip, a carbon cellulose film strip, and a glass fiber strip.
16. The method of claim 1, wherein the indicator strip is a pH strip.
17. The method for detecting biological molecules according to claim 1, wherein the pH test strip is a common broad-range test strip, and the pH range is 1-14.
18. The method for detecting biomolecules according to claim 1, wherein the method for detecting the concentration of biomolecules is carried out on a hydrophobic substrate, wherein the mixed solution is first dropped on the hydrophobic substrate, and then one end of the indicator strip is placed on the upper surface of the drop after a period of time.
19. The method of claim 18, wherein the hydrophobic substrate material comprises one of a polycarbonate plate, a polystyrene plate, a polymethyl methacrylate plate, and a PVC plate.
20. The method of claim 18, wherein the period of time is 3 to 7 minutes.
21. The method of claim 18, wherein the period of time is 5 minutes.
22. The method for detecting biomolecules as claimed in claim 1, further comprising an optimization process of the concentration of the viscous solution or the hydrogel, comprising the steps of: preparing viscous solutions or hydrogel solutions with different mass concentrations by using phosphate buffer solution, respectively dripping the viscous solutions or hydrogel solutions onto a hydrophobic substrate plate, observing whether the liquid drops can become gel after a period of time, and selecting the corresponding viscous solution or hydrogel concentration when the liquid drops become gel state as the optimal concentration for the next detection.
23. The method of claim 22, wherein the period of time is 1 to 30 minutes.
24. The method of claim 22, wherein the period of time is 5 minutes.
25. The method for detecting a biological molecule according to claim 1, further comprising a hydrophobic substrate plate washing process comprising the steps of: ultrasonic cleaning was performed multiple times using a mixed solution of ethanol and deionized water.
26. The method of claim 25, wherein the volume ratio of ethanol to deionized water is 1:1.5-2.5.
27. The method of claim 25, wherein the volume ratio of ethanol to deionized water is 1:2.
28. The method for detecting a biological molecule according to claim 25, wherein the number of times of ultrasound is 3 to 5.
29. The method for detecting a biological molecule according to claim 25, wherein the number of times of ultrasound is 3.
30. The method of claim 25, wherein each time the ultrasound is performed for 4 to 6 minutes.
31. The method of claim 25, wherein each sonication is carried out for 5 minutes.
32. The method for detecting biomolecules according to claim 1, further comprising a step of incubating the sample to be detected, wherein the sample to be detected is mixed with the hydrogel solution, and the mixed solution is incubated in a suitable temperature environment for a period of time.
33. The method of claim 32, wherein the volume ratio of the test sample to the hydrogel solution is 1:1.2.
34. The method of claim 32, wherein the volume ratio of the test sample to the hydrogel solution is 1:1.
35. The method for detecting a biomolecule according to claim 32, wherein the mixed solution is incubated at 37 ℃ for 15 minutes.
36. The method for detecting biological molecules according to claim 1, further comprising an indicator strip scale acquisition process, comprising the steps of: mixing the to-be-detected objects with different standard concentrations with the respective hydrogels, respectively placing the to-be-detected objects at one end of the indicative test strip, and recording scales corresponding to the concentrations for subsequent comparison of the target detection concentration.
37. A method for testing inhibitor molecules is characterized in that target molecules, inhibitor molecules and hydrogel are mixed to form a mixed solution, and the mixed solution is placed at one end of an indicator test strip;
the target molecule causes the hydrogel to hydrolyze, so that the water released by the hydrogel decomposition can move on the pH test strip for a certain distance, and the detection of the target detection object is realized by measuring the length of the moving distance;
the inhibitor molecules inhibit target molecules to play a role, the hydrolysis of hydrogel is reduced, the mixed solution can also move on the pH test strip for a certain distance, and the inhibition capability of the inhibitor molecules is tested by measuring the moving distance.
38. The method of testing an inhibitor molecule according to claim 37, wherein the target molecule is an enzyme and the inhibitor molecule is an enzyme inhibitor.
39. The method of testing an inhibitor molecule according to claim 37, wherein the target molecule is aprotinin and the inhibitor molecule is trypsin.
40. The method of testing an inhibitor molecule according to claim 37, further comprising the step of testing the concentration of the inhibitor comprising: the target molecules are target molecules with different concentrations, and the inhibitor is inhibitor with different concentrations.
41. The method of claim 37, wherein the method of testing the inhibitory capacity of the inhibitor is performed on a hydrophobic substrate, wherein the mixed solution is first dropped on the hydrophobic substrate for a period of time, and then one end of the indicator strip is placed on the upper surface of the drop.
42. The method of testing the inhibitor molecule of claim 41, wherein the hydrophobic substrate material comprises one of a polycarbonate plate, a polystyrene plate, a polymethyl methacrylate plate, and a PVC plate.
43. The method of testing an inhibitor molecule according to claim 41, wherein the period of time is 1 to 30 minutes.
44. The method of testing an inhibitor molecule according to claim 41, wherein the period of time is 5 minutes.
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