阅读说明：本技术 胃蛋白酶检测薄膜及其制备方法、应用和胃蛋白酶检测试剂盒 (Pepsin detection film, preparation method and application thereof, and pepsin detection kit ) 是由 张书鹏 段晓东 于 2020-07-24 设计创作，主要内容包括：本发明提供了一种胃蛋白酶检测薄膜及其制备方法、应用和胃蛋白酶检测试剂盒,涉及胃蛋白酶检测技术领域。该胃蛋白酶检测薄膜的制备方法,包括以下步骤：提供聚合物薄膜基材；将所述聚合物薄膜基材浸入染料中,使染料附着于聚合物薄膜基材,得到胃蛋白酶检测薄膜。本发明的制备方法操作简单,成本低,技术门槛低,对检测和操作人员要求低,本发明能够缓解现有技术中胃蛋白酶检测存在的技术难度大、测试复杂、高成本等问题。(The invention provides a pepsin detection film, a preparation method and application thereof, and a pepsin detection kit, and relates to the technical field of pepsin detection. The preparation method of the pepsin detection film comprises the following steps: providing a polymeric film substrate; and immersing the polymer film substrate into a dye to attach the dye to the polymer film substrate to obtain the pepsin detection film. The preparation method disclosed by the invention is simple to operate, low in cost, low in technical threshold and low in requirements on detection and operating personnel, and can be used for relieving the problems of high technical difficulty, complex test, high cost and the like in pepsin detection in the prior art.)

1. A preparation method of a pepsin detection film is used for detecting pepsin and is characterized by comprising the following steps:

providing a polymeric film substrate;

immersing the polymer film substrate into a dye to attach the dye to the polymer film substrate to obtain a pepsin detection film;

wherein the dye is adapted to interact with pepsin, the dye is different in color in different pepsin concentration environments, and the dye comprises at least one of bromophenol blue dye, xylenol orange dye, triarylmethane dye, azo dye, cyanine dye or metal complex dye.

2. The method for preparing the pepsin detection film according to claim 1, wherein the concentration of the dye is 0.5-5 mg/mL, preferably 1-3 mg/mL;

preferably, the solvent of the dye comprises water and/or an alcohol solvent;

preferably, the temperature for immersing the polymer film substrate into the dye is 20-40 ℃, preferably 25-35 ℃, and the time for immersing the polymer film substrate into the dye is 15-30 min, preferably 18-22 min.

3. The method for preparing a pepsin detecting film according to claim 1, wherein the method for preparing the polymer film substrate comprises the following steps:

providing a substrate assembly comprising a substrate and a first lubricant disposed on the substrate;

placing the film-forming solution of the polymer film substrate on the substrate assembly, and covering a cover plate with a second lubricant on the film-forming solution, wherein the film-forming solution is in contact with the first lubricant and the second lubricant respectively;

after the polymerization reaction of the raw materials in the film-forming solution, separating the polymer film substrate from the substrate assembly and the cover plate to obtain the polymer film substrate;

preferably, the substrate assembly further comprises tinfoil, the tinfoil being disposed on the substrate, the first lubricant being disposed on the tinfoil.

4. The method of manufacturing a pepsin detecting film according to claim 3, wherein the first lubricant and the second lubricant are each independently selected from at least one of white petrolatum, silicone oil, paraffin, mineral oil, or grease.

5. The method for preparing a pepsin detecting film according to claim 3, wherein the polymerization reaction is carried out under the irradiation of ultraviolet light; the wavelength of the ultraviolet light is preferably 250-400 nm; and/or the time of ultraviolet irradiation is preferably 10-30 min;

preferably, after the ultraviolet light irradiation, the curing box is placed for 5-10 min.

6. The method of claim 3, wherein the separating the polymer film substrate from the substrate assembly and the cover plate comprises:

removing the substrate assembly, placing the cover plate attached with the polymer film substrate in a standing solution, standing for 10-30 min, and removing the cover plate to obtain the polymer film substrate;

or removing the cover plate, placing the substrate assembly attached with the polymer film substrate in a standing solution, standing for 10-30 min, and removing the substrate assembly to obtain the polymer film substrate;

preferably, removing the substrate, placing the cover plate and the tinfoil attached with the polymer film base material in a standing solution, standing for 10-30 min, and removing the tinfoil to obtain the polymer film base material;

preferably, after separating the polymer film substrate from the substrate assembly and the cover plate, the method further comprises a step of cleaning the polymer film substrate, wherein the cleaning comprises sequentially performing ultrasonic cleaning in clean water, an alcohol solution and clean water.

7. The method for producing a pepsin detection film according to any one of claims 3 to 6, wherein the method for producing the deposition solution comprises:

uniformly mixing an ionic liquid monomer and a base membrane monomer, adding a cross-linking agent and an initiator, and then carrying out second ultrasonic treatment to obtain the film forming solution;

preferably, the time of the second ultrasonic treatment is 10-30 min;

preferably, the method for preparing the film-forming solution further comprises the step of carrying out first ultrasonic treatment on the ionic liquid monomer, wherein the time of the first ultrasonic treatment is 10-30 min;

preferably, the film-forming solution comprises at least one of imidazole ionic liquid, pyridine ionic liquid, quaternary ammonium salt ionic liquid, quaternary phosphine ionic liquid or pyrrolidine ionic liquid;

preferably, the ionic liquid monomers include bromobutane and vinylimidazole; preferably, the molar ratio of bromobutane to vinylimidazole is from 2:1 to 1: 1;

preferably, the base film monomer includes acrylonitrile; preferably, the mass of acrylonitrile is greater than or equal to the sum of the masses of bromobutane and vinylimidazole;

preferably, the crosslinking agent comprises N, N-methylene bisacrylamide; preferably, the mass of the cross-linking agent is 8-12 wt% calculated by the total mass of bromobutane, vinyl imidazole and acrylonitrile;

preferably, the initiator comprises 2,4,6- (trimethylbenzoyl) diphenylphosphine oxide; preferably, the mass of the initiator is 1 wt% to 4 wt% calculated on the total mass of bromobutane, vinylimidazole and acrylonitrile.

8. A pepsin detection film, which is prepared by the preparation method of the pepsin detection film as claimed in any one of claims 1 to 7.

9. The detection film prepared by the method for preparing a pepsin detection film according to any one of claims 1 to 7 or the application of the pepsin detection film according to claim 8 in detecting pepsin, wherein the detection method comprises the following steps:

and placing the pepsin detection film in the liquid to be detected, and enabling the dye in the pepsin detection film to contact or interact with the liquid to be detected.

10. The use according to claim 9, wherein the detection method further comprises: observing the color change of the pepsin detection film.

11. The use according to claim 9, wherein the detection method further comprises: and in the environment containing the liquid to be detected with different pepsin concentrations, the colors of the pepsin detection films are different, and the pepsin concentrations are judged according to the different colors displayed by the pepsin detection films.

12. A pepsin detection kit, characterized by comprising the detection film prepared by the preparation method of the pepsin detection film according to any one of claims 1 to 7 or the pepsin detection film according to claim 8.

13. The pepsin detection kit according to claim 12, characterized in that the pepsin detection kit further comprises a pepsin detection standard colorimetric card.

Technical Field

The invention relates to the technical field of pepsin detection, and particularly relates to a pepsin detection film, a preparation method and application thereof, and a pepsin detection kit.

Background

Pepsin is a digestive protease that helps break down and absorb proteins in food, the precursor of which is pepsinogen, which functions to break down food proteins into small peptide fragments. Pepsin in gastric juice has become a biological marker of gastritis and gastric cancer. Pepsin secretion is reduced when intestinal metaplasia, atypical hyperplasia and gastric cancer occur; or, when helicobacter pylori is infected, or gastric ulcer and duodenal ulcer are caused, the pepsin value is increased. At present, a large number of statistical analyses show that the relationship between the content change of the serum pepsinogen and gastric diseases considers that the detection of the serum pepsinogen plays an important role in early diagnosis of gastric cancer.

Currently, the pepsin detection method is commonly used for analysis by collecting serum of a subject. Briefly, the method involves collecting a sample in vivo and testing it in vitro to analyze the specific amount of pepsin. However, in the actual process, the time between the step of collecting the sample and the step of detecting the sample is long, and thus, there is a limitation in monitoring the pepsin content in the gastric juice in real time. In addition, in vitro detection is easily affected by the environment, so that the test result is inaccurate. In addition, the existing pepsin detection method has higher technical requirements on operators and higher cost.

Disclosure of Invention

The invention aims to provide a pepsin detection film, a preparation method thereof, application thereof and a pepsin detection kit, which have the advantages of simple structure, easy operation, low requirement on operators, low cost and the like, and can overcome the problems or at least partially solve the technical problems.

In order to achieve the purpose, the technical scheme adopted by the application is as follows:

according to one aspect of the present application, there is provided a method for preparing a pepsin detection film, comprising the steps of:

providing a polymeric film substrate;

and immersing the polymer film substrate into a dye to attach the dye to the polymer film substrate to obtain the pepsin detection film.

Wherein the dye is adapted to interact with pepsin, the dye differing in color in environments where the concentration of pepsin is different; the dye comprises at least one of bromophenol blue dye, xylenol orange dye, triarylmethane dye, azo dye, cyanine dye or metal complex dye.

In one possible implementation mode, the concentration of the dye is 0.5-5 mg/mL, preferably 1-3 mg/mL;

preferably, the solvent of the dye comprises water and/or an alcohol solvent;

preferably, the temperature for immersing the polymer film substrate into the dye is 20-40 ℃, preferably 25-35 ℃; the polymer film substrate is immersed in the dye for 15-30 min, preferably 18-22 min.

In one possible implementation, the method for preparing the polymer film substrate comprises the following steps:

providing a substrate assembly comprising a substrate and a first lubricant disposed on the substrate;

placing the film-forming solution of the polymer film substrate on the substrate assembly, and covering a cover plate with a second lubricant on the film-forming solution, wherein the film-forming solution is in contact with the first lubricant and the second lubricant respectively;

after the polymerization reaction of the raw materials in the film-forming solution, separating the polymer film substrate from the substrate assembly and the cover plate to obtain the polymer film substrate;

preferably, the substrate assembly further comprises tinfoil, the tinfoil being disposed on the substrate, the first lubricant being disposed on the tinfoil.

In one possible implementation, the first lubricant and the second lubricant are each independently selected from at least one of white petrolatum, silicone oil, paraffin wax, mineral oil, or grease.

In one possible implementation, the polymerization reaction is carried out under irradiation of ultraviolet light; wherein the wavelength of the ultraviolet light is preferably 250-400 nm; and/or the time of ultraviolet irradiation is preferably 10-30 min;

preferably, after the ultraviolet light irradiation, the curing box is placed for 5-10 min.

In one possible implementation, the separating the polymer film substrate from the substrate assembly and the cover plate includes: removing the substrate assembly, placing the cover plate attached with the polymer film substrate in a standing solution, standing for 10-30 min, and removing the cover plate to obtain the polymer film substrate;

or removing the cover plate, placing the substrate assembly attached with the polymer film substrate in a standing solution, standing for 10-30 min, and removing the substrate assembly to obtain the polymer film substrate;

preferably, removing the substrate, placing the cover plate and the tinfoil attached with the polymer film base material in a standing solution, standing for 10-30 min, and removing the tinfoil to obtain the polymer film base material;

preferably, after separating the polymer film substrate from the substrate assembly and the cover plate, the method further comprises a step of cleaning the polymer film substrate, wherein the cleaning comprises sequentially performing ultrasonic cleaning in clean water, an alcohol solution and clean water.

In one possible implementation, the method for preparing the deposition solution comprises:

uniformly mixing an ionic liquid monomer and a base membrane monomer, adding a cross-linking agent and an initiator, and then carrying out second ultrasonic treatment to obtain the film forming solution;

preferably, the time of the second ultrasonic treatment is 10-30 min;

preferably, the method for preparing the film-forming solution further comprises performing first ultrasonic treatment on the ionic liquid monomer, wherein the time of the first ultrasonic treatment is 10-30 min;

preferably, the ionic liquid monomers include bromobutane and vinylimidazole; preferably, the molar ratio of bromobutane to vinylimidazole is from 2:1 to 1: 1;

preferably, the base film monomer includes acrylonitrile; preferably, the mass of acrylonitrile is greater than or equal to the sum of the masses of bromobutane and vinylimidazole;

preferably, the crosslinking agent comprises N, N-methylene bisacrylamide; preferably, the mass of the cross-linking agent is 8-12 wt% calculated by the total mass of bromobutane, vinyl imidazole and acrylonitrile;

preferably, the initiator comprises 2,4,6- (trimethylbenzoyl) diphenylphosphine oxide; preferably, the mass of the initiator is 1 wt% to 4 wt% calculated on the total mass of bromobutane, vinylimidazole and acrylonitrile.

According to another aspect of the present application, there is provided a pepsin detection film, which is prepared by the above-mentioned method for preparing a pepsin detection film.

According to another aspect of the present application, there is provided a detection film prepared by the method for preparing a pepsin detection film or an application of the pepsin detection film in detecting pepsin, wherein the detection method comprises:

and placing the pepsin detection film in the liquid to be detected, and enabling the dye in the pepsin detection film to contact or interact with the liquid to be detected.

In one possible implementation manner, the detection method further includes: observing the color change of the pepsin detection film.

In one possible implementation manner, the detection method further includes: and in the environment containing the liquid to be detected with different pepsin concentrations, the colors of the pepsin detection films are different, and the pepsin concentrations are judged according to the different colors displayed by the pepsin detection films.

According to another aspect of the present application, the present application further provides a pepsin detection kit, comprising the detection film prepared by the above pepsin detection film preparation method or the above pepsin detection film.

In one possible implementation mode, the pepsin detection kit further comprises a pepsin detection standard colorimetric card.

Compared with the prior art, the technical scheme provided by the invention can achieve the following beneficial effects:

the preparation method of the pepsin detection film provided by the invention has the characteristics of simple synthesis, easy operation, low technical threshold and the like, and is easy to realize large-scale production. Moreover, the pepsin detection film prepared by the method can be applied to pepsin detection, can relieve the conditions of high technical difficulty, high cost and single function of the existing pepsin detection mode, and meets the current requirements.

The pepsin detection kit of the invention has all the characteristics and advantages of the pepsin detection film, and the details are not repeated.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below.

FIG. 1 is a graph showing the relationship between pepsin concentration and blue B value provided by one embodiment of the present invention;

FIG. 2 is a table showing the relationship between pepsin concentration and film color according to one embodiment of the present invention.

Detailed Description

In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be clearly and completely described below with reference to the embodiments of the present application, and it should be apparent that the described embodiments are some but not all of the embodiments of the present application. All other embodiments obtained by those skilled in the art without any creative effort based on the technical solutions and the given embodiments provided in the present application belong to the protection scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

It should be noted that the term "and/or"/"used herein is only one kind of association relationship describing associated objects, and means that there may be three relationships, for example, a and/or B, and may mean: a exists alone, A and B exist simultaneously, and B exists alone. As used in the examples of this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For numerical ranges, one or more new numerical ranges may be obtained by combining the individual values, or by combining the individual values.

All the technical features mentioned herein, as well as preferred features, may be combined with each other to form new solutions, if not mentioned specifically. Unless defined or indicated otherwise, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art.

As the technical background, the skilled in the art understands that the existing pepsin detection method or the used instrument and equipment have the defects of great technical difficulty, complex structure or equipment, complex test, high cost and the like, cannot meet the current requirements, and still needs to be improved. For example, the prior art discloses a pepsin detection capsule, a detection system and a detection method, the pepsin detection capsule comprises a capsule body, the capsule body comprises a shell and an upper light-transmitting sheet and a lower light-transmitting sheet which are arranged in the shell, a reaction cavity is arranged between the upper light-transmitting sheet and the lower light-transmitting sheet, the upper light-transmitting sheet and the lower light-transmitting sheet are the cavity walls of the reaction cavity, the shell is provided with a through hole for communicating the reaction cavity and the shell, and a biological semipermeable membrane is arranged on the through hole; the shell is internally provided with a light source generator and a detection emitter which are opposite to each other, the light source generator and the detection emitter are respectively arranged on the outer sides of the upper layer light-transmitting sheet and the lower layer light-transmitting sheet and are opposite to each other one by one, a monochromator is arranged between the light source generator and the upper layer light-transmitting sheet, the output end of the detection emitter is connected with the input end of the signal receiving unit, and the detection emitter detects absorbance and sends the absorbance to the signal receiving unit. Although this system and method can detect pepsin, the apparatus is complicated, involves many parts, and is costly and cumbersome to operate. For another example, the prior art also discloses a pepsin chemiluminescence immunoassay kit and a preparation method thereof, and the pepsin chemiluminescence immunoassay kit comprises a pepsin antigen calibrator, a sample collecting solution, a sample diluent, a pepsin antibody coated microporous plate, a pepsin antibody marker, chemiluminescence substrate solution and concentrated washing solution. The kit can detect the content of pepsin in gastric juice, esophageal contents and laryngeal secretions of pharynx, judge whether gastroesophageal reflux exists according to whether the pepsin can be detected, and judge the treatment effect and the disease change of stomach lesions according to the content of the pepsin. Although the kit and the method can be used for detecting the pepsin, the process is complex, the requirements on operators and detection equipment are high, and the cost is high.

In addition, the prior art also discloses a plurality of detection kits or detection methods for pepsinogen, and exemplarily discloses a test strip/kit for quantitatively detecting serum pepsinogen by using a colloidal gold immunochromatography technology, a preparation method and an application thereof, wherein the kit comprises test strips for pepsinogen I and pepsinogen II respectively. Or, the prior art discloses a pepsinogen I and pepsinogen II detection method and a kit thereof. The kit and the method can detect pepsinogen, but the pepsinogen is detected, and the real-time feedback of the pepsin in the body cannot be realized.

Therefore, in order to overcome the defects of the prior art and further meet the use requirements at present, the technical scheme of the embodiment of the invention provides a pepsin detection film, a preparation method and application thereof, and a pepsin detection kit, and the preparation of the pepsin detection film is carried out by a simple and efficient preparation method. The application realizes the effects of simple and convenient synthesis, short preparation period, low technical threshold, reusability of the prepared pepsin detection film and the like.

In a first aspect, in some embodiments, there is provided a method of making a pepsin detection membrane, comprising the steps of:

providing a polymeric film substrate;

immersing the polymer film substrate into a dye to attach the dye to the polymer film substrate to obtain a pepsin detection film;

wherein the dye is adapted to interact with pepsin, the dye having a different color in environments with different concentrations of pepsin; the dye includes, but is not limited to, at least one of bromophenol blue dye, xylenol orange dye, triarylmethane-based dye, azo-based dye, cyanine-based dye, or metal complex-based dye.

The preparation method of the pepsin detection film is a simple, convenient and efficient preparation method, and has the advantages of simple preparation, easy operation, low technical threshold, low cost, low requirements on detection and operation personnel, easy large-scale production and use and the like. The pepsin detection film prepared by the method can be applied to pepsin detection, has the advantages of simplicity and convenience in operation, low cost, safety and the like, and can judge the concentration of pepsin according to color, so that the pepsin detection film has great application potential.

In the pepsin detection film, the polymer film base material can be a polyion film, namely a polyion liquid type film, has the excellent performances of both the ionic liquid and the polymer, can overcome the fluidity of the ionic liquid, has unique physicochemical properties, and can be well applied to the field of medical detection.

In the pepsin detection film, the dye is a compound which has certain color and can make other substances obtain bright or more obvious color. In the examples of the present application, the dye detects that pepsin can change color, and the dye and the polymer film substrate can form good stable coordination. Specifically, the polymer film substrate can adsorb the dye, or the dye can be well attached to the polymer film substrate, and the dye can be well kept on the polymer film substrate due to the strong ionic interaction between the two.

Compared with the prior art, the embodiment of the application utilizes the combination of the dye and the pepsin or the reaction (interaction) to develop the color, and has the advantages of simple operation, low technical threshold, obvious color development change, low cost, quick detection and the like.

Specifically, the specific shape of the pepsin detection film can be various types, for example, the specific shape can be any shape such as a circle, a square, a polygon or other irregular shapes, or can be a sheet, a strip or the like, and the specific shape of the pepsin detection film is not limited in the embodiments of the present application.

Specifically, the size of the pepsin detection film may be adjusted according to actual conditions, and the specific size of the pepsin detection film is not limited in the embodiments of the present application.

In particular, the dye is adapted to interact with pepsin, the color of the dye being different in environments where the concentration of pepsin is different. That is, the dye has different colors when the pepsin concentration is different. Thus, the interaction between the dye and pepsin is used for developing color, and the color change of the pepsin detection film is observed, so that the detection result can be obtained. In addition, different color development effects can be achieved by dye mixing or changing the concentration of the dye.

Specifically, the dye includes, but is not limited to, at least one of bromophenol blue dye, xylenol orange dye, triarylmethane-based dye, azo-based dye, cyanine-based dye, or metal complex-based dye.

It should be noted that, the dye may be bromophenol blue dye, xylenol orange dye, triarylmethane dye, azo dye, etc., but is not limited thereto. More generally, the dye may be any dye that satisfies the following three conditions: (1) can be stably combined with a polymer film substrate; (2) capable of binding to or interacting with pepsin; (3) meets the requirement of biological safety.

The bromophenol blue dye is safe and nontoxic, can be combined with pepsin, and after combination, phenol in the bromophenol blue dye undergoes deprotonation reaction, so that the color of the bromophenol blue dye changes, and the pepsin detection film displays different colors before and after detection. In addition, in the detection process, the bromophenol blue dye of the pepsin detection film shows different colors for the concentrations of pepsin with different concentrations, and the pepsin concentration can be quantitatively detected through color change.

In particular, in the pepsin detection film, in one aspect, the polymeric film substrate may be a carrier or platform for the dye. Because the polymer film substrate and the dye, especially bromophenol blue dye, xylenol orange dye and the like have stronger ionic interaction, the dye can be well kept or attached on the polymer film substrate, namely the dye can be stably combined with the polymer film substrate. On the other hand, dyes, particularly bromophenol blue dye, xylenol orange dye and the like, are safe and nontoxic, can be combined with pepsin to change the color of the pepsin detection film, for example, the pepsin detection film can be changed from blue to green, and the shade (tone) of the color change is different according to the concentration of the pepsin, so that the pepsin condition can be quantitatively detected through the color change. Of course, in other embodiments of the present application, the dye may also change color due to other types of physical or chemical reactions between pepsin and the dye, and will not be described in detail herein.

It can be seen from the above that, in the embodiment of the present application, dyes such as bromophenol blue dye, xylenol orange dye, etc. are directly utilized to combine or react with pepsin, so as to achieve the purpose of dye color change, thereby achieving the detection effect. Moreover, the concentration of the pepsin in the liquid environment can be judged by detecting the color change degrees before and after the detection, for example, the concentration of the pepsin is judged by the color shade (tone) change, thereby assisting the examination and the corresponding treatment. The pepsin detection film is applied to detection of pepsin, has the characteristics of simple operation, obvious color change, quick detection, high efficiency and the like, and has lower requirements on detection personnel.

In some embodiments, the polymeric film substrate comprises a polyionic liquid film (polyionic film) comprising an ionic liquid.

Preferably, the ionic liquid includes, but is not limited to, at least one of imidazole ionic liquid, pyridine ionic liquid, quaternary ammonium salt ionic liquid, quaternary phosphine ionic liquid or pyrrolidine ionic liquid. It is understood that the ionic liquid used to prepare the polymer film substrate may be a functionalized ionic liquid commonly used in the art, depending on the function that the ionic liquid performs. Illustratively, the ionic liquid may be imidazole ionic liquid, pyridine ionic liquid, quaternary ammonium salt ionic liquid, pyrrolidine ionic liquid, or the like. The specific type of ionic liquid in the examples of the present application is not limited, and several of the ionic liquids listed above may be used, and other types of ionic liquids known in the art may also be used.

Preferably, the ionic liquid monomers forming the polyionic liquid membrane include bromobutane and vinylimidazole. It is understood that bromobutane and vinylimidazole can react and form ionic liquid monomers.

Preferably, the base film monomer forming the polyionic liquid film includes, but is not limited to, acrylonitrile. The base film monomer can be acrylonitrile, or a mixture of acrylonitrile and styrene, or other base film monomers with similar functions or functions commonly used in the field.

Preferably, the crosslinking agent for forming the polyion liquid film includes, but is not limited to, N-methylenebisacrylamide (N, N' -methanediylbisprop-2-enamide, abbreviated as MBA). The MBA is used as a cross-linking agent, plays a role in bridging among molecular monomers, and molecules are mutually bonded and cross-linked to form a net structure to promote the inter-molecular chain bonding of the polymer. In addition, the crosslinking agent may be other crosslinking agents having similar properties or functions commonly used in the art.

Preferably, the initiator for forming the polyion liquid film includes, but is not limited to, 2,4,6- (trimethylbenzoyl) diphenylphosphine oxide (Diphenyl (2,4,6-trimethylbenzoyl) phosphinoxide, abbreviated as TPO). TPO belongs to a photoinitiator, is a light yellow solid, mainly plays a role in photocuring, is a high-efficiency universal ultraviolet photoinitiator, and can be used for initiating the UV polymerization reaction of an unsaturated prepolymerization system. In addition, the initiator may be other initiators commonly used in the art having similar properties or functional effects. For example, the initiator may be an initiator commonly used in the art, such as photoinitiator 907, photoinitiator 184, azobisisobutyronitrile, benzoin and derivatives thereof, and the like. One skilled in the art can select an appropriate initiator depending on the specific type of ionic liquid monomer, base film monomer, etc.

It should be noted that, in the embodiments of the present application, the specific type of the above-mentioned ionic liquid, the specific type of the base film monomer, the specific type of the cross-linking agent, the specific type of the initiator, and the like are not limited, and various types commonly used in the art may be adopted as long as the purpose and the application scenario of the present invention are not limited. For convenience of description, in the examples of the present application, MBA is mainly used as a cross-linking agent, TPO is mainly used as an initiator, acrylonitrile is used as a basement membrane monomer, and an ionic liquid monomer formed by bromobutane and vinyl imidazole is used as an example to specifically describe the pepsin detection membrane and the preparation method thereof. However, those skilled in the art will understand that the principles of the present disclosure may be implemented in any suitable polymer film substrate stock. Further, descriptions of well-known functions or actions may be omitted for clarity and conciseness.

Preferably, the concentration of the dye is 0.5-5 mg/mL, preferably 1-3 mg/mL; typically, but not by way of limitation, it can be, for example, 0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, and the like. The dye with a proper concentration range is adopted, so that the final display effect is better, and the method is more beneficial to accurately detecting the concentration of the pepsin.

Preferably, the solvent of the dye includes water and/or an alcohol solvent, and may be, for example, a lower alcohol, and further may be an alcohol solvent such as ethanol. That is, bromophenol blue or xylenol orange or the like may be dissolved in water or in an alcohol solvent to form the dye.

Preferably, the solvent of the dye is a water-ethanol mixed solution, and the mass ratio of the water to the ethanol is preferably 1: 4.

Specifically, in some embodiments, the polymer film substrate is immersed in the dye at a temperature of 20 to 40 ℃, preferably 25 to 35 ℃; the immersion temperature may be room temperature, and may be, for example, 20 ℃, 25 ℃, 30 ℃, 40 ℃ or the like.

Preferably, the polymer film substrate is immersed in the dye for 15 to 30min, preferably 18 to 22min, typically but not limited to, for example, 15min, 20min, 22min, 25min, 30min, and the like. Therefore, under the appropriate time and temperature range, the polymer film substrate and the dye can be better combined, the property of the dye can not be changed, and the obtained pepsin detection film has good performance and high efficiency.

In some embodiments, a method of making a polymeric film substrate comprises the steps of:

providing a substrate assembly, wherein the substrate assembly comprises a substrate and a first lubricant arranged on the substrate;

placing the film-forming solution of the polymer film substrate on the substrate assembly, and covering the cover plate with a second lubricant on the film-forming solution, wherein the film-forming solution is respectively contacted with the first lubricant and the second lubricant;

and after the polymerization reaction of the raw materials in the film forming solution, separating the polymer film substrate, the substrate assembly and the cover plate to obtain the polymer film substrate.

It will be appreciated that after the polymerization reaction has taken place and before the separation, a composite structure comprising the polymeric film substrate, the cover plate and the substrate assembly is obtained, and then, after the separation step, the polymeric film substrate is separated from the cover plate and substrate assembly to obtain the polymeric film substrate.

In some embodiments, the substrate assembly further comprises a tinfoil, the tinfoil disposed on the substrate, and the first lubricant disposed on the tinfoil. It is to be understood that the substrate assembly may include the substrate and the first lubricant, or may also include the substrate, the tinfoil, and the first lubricant. When the substrate assembly comprises tinfoil, the tinfoil needs to be flatly laid on the substrate or the tinfoil is flatly wrapped on the substrate, and then the first lubricant can be arranged on the tinfoil, which is more beneficial to the preparation of the polymer film substrate.

The preparation method of the polymer film substrate provided by the embodiment of the invention has the characteristics of high product rate, high reaction rate and energy consumption saving. Meanwhile, the whole process is simple and convenient to operate, low in requirement on experimental equipment and high in processing safety. And the prepared polymer film base material has stable structure, easy stripping and low irritation.

According to the preparation method of the polymer film base material, the tin foil and the lubricant are used, so that direct contact between a film forming solution and a base plate or a cover plate before film forming is avoided, the integrity of the polymer film base material and the thickness uniformity of the polymer film base material are ensured, and the time required for separating the polymer film base material can be greatly shortened. In addition, the method is easy to operate, short in time consumption, easy to separate products, and uniform and controllable in film thickness.

Specifically, the substrate may be a glass plate, a stainless steel plate, a hard plastic plate that is not easily penetrated by ultraviolet light, or the like. Among these substrates, the substrate is preferably a glass plate. The glass plate has the advantages of easily available raw materials and low cost, and has good heat resistance, and is easy to cool after polymerization reaction under ultraviolet irradiation, thereby shortening the operation time.

Specifically, the cover plate may be a glass plate, a rigid plastic plate, or the like. Preferably, the cover plate can be a transparent glass plate or a transparent rigid plastic plate. The transparent glass plate or the transparent hard plastic plate can enable the polymerization reaction to be carried out under the irradiation of ultraviolet, so that the reaction speed is increased, and an alternative scheme is provided for the polymerization reaction.

In particular, the lubricant used, such as the first lubricant or the second lubricant, needs to be inert to and non-interfering with ultraviolet light. The first lubricant and the second lubricant may be of the same type or of different types.

In some embodiments, the first lubricant and the second lubricant are each independently selected from at least one of white petrolatum, silicone oil, paraffin wax, mineral oil, or grease. Illustratively, the first lubricant may be white petrolatum, may be silicone oil, may be mineral oil, etc., and the second lubricant may be white petrolatum, may be silicone oil, may be grease, etc.

In the field of medical devices, the above lubricants employed need to be non-toxic, non-corrosive, non-residue and transparent in the coating. Especially for the fields with high safety requirements, such as sensors, drug controlled release and the like, the lubricant has higher requirements, and common medical grade lubricants can be adopted.

Specifically, in the method for producing the polymer film substrate, a deposition solution may be prepared in advance. And then spreading the tinfoil on the substrate flatly, or wrapping the substrate flatly by the tinfoil, or wiping the tinfoil by a dust-free cloth until the tinfoil has no wrinkles, wherein the smooth surface of the tinfoil faces upwards, then coating a first lubricant such as white vaseline on the tinfoil, and continuously wiping the tinfoil by the dust-free cloth until the surface is smooth. Then covering the prepared film-forming liquid on the tinfoil with the first lubricant, uniformly coating, and covering the cover plate which is coated with the second lubricant in advance on the tinfoil with the film-forming liquid; it is understood that the upper and lower surfaces of the deposition solution may be contacted with the second lubricant and the first lubricant, respectively.

Optionally, the substrate is wetted with a wetting solution prior to laying the tinfoil on the substrate. The purpose of wetting the substrate is to exclude air between the substrate and the tinfoil. And, through the laminating nature of wetting solution and tin foil increase the cohesion of base plate and tin foil, smooth the tin foil more easily to improve the roughness on tin foil surface. The wetting solution for wetting the substrate may be various, but is preferably water, ethanol or a mixed solution thereof from the viewpoint of source, cost and environmental protection.

Additionally, in other embodiments, the substrate assembly may include only the substrate. Specifically, in the method for preparing the polymer film substrate, a deposition solution may be prepared in advance, and then a first lubricant such as white vaseline is applied on the substrate, and the first lubricant is wiped with a dust-free cloth until the surface is smooth. Then, the prepared deposition solution was coated on the substrate with the first lubricant, and uniformly coated, and then the cover plate previously coated with the second lubricant was coated on the substrate carrying the deposition solution. It is understood that the upper and lower surfaces of the deposition solution may be contacted with the second lubricant and the first lubricant, respectively.

Specifically, in some embodiments, a method of preparing a deposition solution comprises:

uniformly mixing the ionic liquid monomer and the base membrane monomer, adding a cross-linking agent and an initiator, and then carrying out second ultrasonic treatment to obtain a film forming solution;

preferably, the time of the second ultrasonic treatment is 10-30 min; typical but non-limiting examples are 10min, 15min, 20min, 25min, 30min, etc.

Preferably, the method for preparing the film-forming solution further comprises performing first ultrasonic treatment on the ionic liquid monomer, wherein the time of the first ultrasonic treatment is 10-30 min; typical but non-limiting examples are 10min, 15min, 20min, 25min, 30min, etc.

Compared with the existing 'two-step method' for preparing ionic liquid, the preparation method provided by the embodiment of the application can obviously improve the preparation efficiency of the deposition solution by using an ultrasonic method, greatly shortens the preparation time, and is easy to operate and good in controllability. Therefore, the preparation efficiency of the polymer film substrate and the pepsin detection film can be further improved by adopting the preparation method of the film-forming solution.

According to the embodiment of the invention, different deposition solutions can be selected according to different requirements. The ionic liquid in the film-forming solution may be one or more of imidazole ionic liquid, pyridine ionic liquid, quaternary ammonium salt ionic liquid, quaternary phosphine ionic liquid or pyrrolidine ionic liquid, or may be other types of ionic liquids known in the art.

Preferably, the ionic liquid monomers include bromobutane and vinylimidazole.

Preferably, the base film monomer comprises acrylonitrile.

Preferably, the crosslinking agent comprises N, N-Methylenebisacrylamide (MBA).

Preferably, the initiator comprises 2,4,6- (trimethylbenzoyl) diphenylphosphine oxide (TPO).

Specifically, in the preparation of the deposition solution, the molar ratio of bromobutane to vinylimidazole may be from 2:1 to 1:1, for example, 1: 1. To ensure complete reaction, the mass of acrylonitrile needs to be greater than or equal to the sum of the masses of bromobutane and vinylimidazole. Considering the reaction ratio, conversion rate, dosage and subsequent cleaning treatment process of the three, the mass of the acrylonitrile is preferably the sum of the mass of the bromobutane and the mass of the vinylimidazole. The mass of the crosslinking agent is 8 to 12 wt%, for example, 8, 9, 10, 12 wt%, etc., based on the total mass of bromobutane, vinylimidazole and acrylonitrile. The mass of the initiator is 1 to 4 weight percent calculated by the total mass of bromobutane, vinyl imidazole and acrylonitrile; for example, it may be 1 wt%, 1.5 wt%, 2 wt%, 3 wt%, 4 wt%, etc. It should be understood that the ratio of the above raw materials is not limited thereto, and may be adjusted appropriately according to the actual process conditions.

Specifically, the preparation of the deposition solution may comprise the steps of: mixing bromobutane and vinyl imidazole in equal molar ratio, and carrying out ultrasonic treatment on the obtained mixed solution for 15min until the bromobutane and the vinyl imidazole are fully mixed. After removing impurities, acrylonitrile which is equal to the sum of the mass of bromobutane and vinyl imidazole is added. Then adding MBA accounting for 8 wt% of the total mass of the bromobutane, the vinyl imidazole and the acrylonitrile and TPO accounting for 1 wt% of the total mass of the bromobutane, the vinyl imidazole and the acrylonitrile, and carrying out ultrasonic treatment for 15min after the addition to obtain a film-forming solution, wherein the obtained film-forming solution is clear and transparent liquid. In the preparation step, ultrasonic treatment can add an energy field to the mixed liquid of the bromobutane and the vinyl imidazole, thereby accelerating the reaction.

Further, in the method for preparing the pepsin detection thin film, each raw material in the deposition solution is subjected to polymerization reaction, and in some embodiments, the polymerization reaction is performed under irradiation of ultraviolet light.

Wherein the wavelength of the ultraviolet light is preferably 250-400 nm; typically, but not limited to, for example, 250nm, 325nm, 400nm, etc.

The time of ultraviolet irradiation is preferably 10-30 min; typically but not limitatively, for example, 10min, 20min, 30min, etc. may be mentioned.

Alternatively, the polymerization reaction may be carried out under heating while being carried out under irradiation of ultraviolet light. The heating temperature may be 20 to 60 ℃, and typically, but not limited to, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ or the like.

The polymerization reaction is carried out under the condition, which is beneficial to obtaining a polymer film base material with excellent performance and has higher efficiency.

The polymerization reaction may be initiated by an initiator. The initiator may be any initiator commonly used in the art, such as photoinitiator 907, photoinitiator 184, azobisisobutyronitrile, benzoin and derivatives thereof, etc., and those skilled in the art may select an appropriate initiator according to the specific components of the film-forming solution.

Further, in the method for producing a pepsin detection film, the raw materials in the deposition solution are polymerized and then separated (for example, the polymer film base is separated from the cover plate and/or the substrate assembly). In some embodiments, the separating comprises: removing the substrate assembly, placing the cover plate attached with the polymer film substrate in a standing solution, standing for 10-30 min to enable the polymer film substrate and the cover plate to be automatically separated in the standing solution, and removing the cover plate to obtain the polymer film substrate;

or removing the cover plate, placing the substrate assembly attached with the polymer film substrate in a standing solution, standing for 10-30 min to separate the polymer film substrate from the substrate assembly in the standing solution, and removing the substrate assembly to obtain the polymer film substrate.

Further, in the embodiment where the substrate assembly includes the substrate and the tinfoil, when the polymer film base material and the substrate assembly are separated, since the tinfoil and the first lubricant are present between the polymer film base material and the substrate as barriers, the substrate and the tinfoil attached with the polymer film base material can be easily separated. Compared with the separation substrate and the polymer film base material, the thickness of the tin foil is very thin, and the first lubricant is arranged between the tin foil and the polymer film base material, so that the tin foil and the polymer film base material are easier to separate. Specifically, in the process of separating the polymer film substrate from the tinfoil, the tinfoil and the polymer film substrate can be placed in a solution, and the two are naturally separated through the solution, or can be separated through a mechanical separation mode (such as manual stripping of the tinfoil) so as to obtain a complete polymer film substrate.

Alternatively, the standing solution may be water or other solution.

The cover plate or the base plate assembly attached with the polymer film base material is put into a standing solution such as water, and then the polymer film base material is separated from the cover plate or the base plate or the tin foil in a standing mode, namely, the polymer film base material is automatically peeled off from the cover plate or the base plate or the tin foil. By the method, the polymer film substrate can be automatically separated from the cover plate or the base plate or the tinfoil, the operation is simple, the yield is improved, and the production efficiency is improved.

Optionally, the polymer film substrate obtained by separation is cleaned, for example, ultrasonic cleaning can be sequentially performed in clean water, absolute ethyl alcohol and clean water.

As can be seen from the above, according to the preparation method of the embodiment of the present invention, through the use of the tinfoil, the first lubricant and the second lubricant, not only the integrity of the polymer film substrate and the uniform stability of the film thickness can be ensured, but also the polymer film substrate can be more easily separated. Specifically, the preparation method of the polymer film substrate realizes automatic stripping of the polymer film substrate, and greatly shortens the time required by separation. Meanwhile, the preparation of the film-forming solution by adopting an ultrasonic method also greatly shortens the time for preparing the film-forming solution, and has simple and efficient operation. Therefore, compared with the existing preparation method, the preparation method of the polymer film substrate has the characteristics of simplicity, high efficiency, short time and the like.

In a second aspect, in some embodiments, there is provided a pepsin detection film, which is prepared by the above method for preparing a pepsin detection film.

The pepsin detection film comprises:

a polymeric film substrate; and

a dye attached to a polymeric film substrate.

In some embodiments, the dye is adapted to interact with pepsin, the color of the dye being different in environments where the concentration of pepsin is different.

In some embodiments, the dye includes, but is not limited to, at least one of bromophenol blue dye, xylenol orange dye, triarylmethane-based dye, azo-based dye, cyanine-based dye, or metal complex-based dye.

In some embodiments, the polymeric film substrate comprises a polyionic liquid film comprising an ionic liquid;

preferably, the ionic liquid includes but is not limited to at least one of imidazole ionic liquid, pyridine ionic liquid, quaternary ammonium salt ionic liquid, quaternary phosphine ionic liquid or pyrrolidine ionic liquid;

preferably, the ionic liquid monomers forming the polyionic liquid film include, but are not limited to, bromobutane and vinylimidazole;

preferably, the base film monomers forming the polyionic liquid film include, but are not limited to, acrylonitrile;

preferably, the polyionic liquid film-forming crosslinking agent includes, but is not limited to, N-methylenebisacrylamide;

preferably, the initiator for forming the polyionic liquid film includes, but is not limited to, 2,4,6- (trimethylbenzoyl) diphenylphosphine oxide.

It should be understood that the same or similar parts of the pepsin detection film of the second aspect and the pepsin detection film of the first aspect can be referred to the above description of the preparation method of the protease detection film of the first aspect, and the description thereof is omitted.

In a third aspect, in some embodiments, there is provided a use of the pepsin detection film prepared by the above method for preparing a pepsin detection film for detecting pepsin. The detection method comprises the following steps:

and (3) placing the pepsin detection film in the liquid to be detected, and enabling the dye in the pepsin detection film to contact or interact with the liquid to be detected.

In some embodiments, the detection method further comprises: the color change of the pepsin detection film is observed.

In some embodiments, the detection method further comprises: in the environment containing the liquid to be detected with different pepsin concentrations, the colors of the pepsin detection films are different, and the pepsin concentrations are judged according to the different colors displayed by the pepsin detection films.

It should be noted that the color of the pepsin detection film is also displayed according to the immersion time or immersion temperature. Specifically, for example, in the case where the time for immersing the polymer film substrate in the dye is the same, and in the case where the temperature for immersing the polymer film substrate in the dye is the same, the color change of the pepsin detection film before and after the detection is affected by the pepsin concentration. In the case where the polymer film substrate is immersed in the dye for a different time or the polymer film substrate is immersed in the dye for a different temperature, the color change of the pepsin detection film before and after the detection may be influenced by the combination of the pepsin concentration and the immersion time or immersion temperature. Therefore, it is necessary to keep the time and temperature for immersing the polymer film substrate in the dye within appropriate ranges, or to keep the time and temperature for immersing the polymer film substrate in the dye the same in order to minimize the influence of the immersion time and immersion temperature on the color change of the pepsin detection film when detecting a series of pepsin concentrations.

The pepsin detection film provided by the embodiment of the invention is applied to pepsin detection, has the advantages of simple and convenient operation, obvious change before and after detection, quick detection, high efficiency and the like, and can relieve the defects of complex equipment, high technical requirement, complex detection, high cost and the like of the existing pepsin detection method.

It should be understood that, in the application of the pepsin detecting film of the third aspect, the same or similar parts as those in the pepsin detecting film of the first aspect and the pepsin detecting film of the second aspect and the preparation method can be referred to the above description of the pepsin detecting film and the preparation method, and the description thereof is omitted.

In a fourth aspect, in some embodiments, there is provided a pepsin detection kit comprising the above pepsin detection membrane.

As can be seen from the above, the pepsin detection kit according to the embodiment of the present invention includes the pepsin detection film, so that the pepsin detection film has at least the same advantages as the pepsin detection film, and the preparation method and the application thereof, and further description thereof is omitted. The pepsin detection kit has the function principle that the dye in the pepsin detection film is combined or reacted with the pepsin for color development, and the pepsin detection film has different colors when the concentration of the pepsin is different, so that the concentration of the pepsin can be judged according to the change degree of the colors of the pepsin detection film before and after detection.

In some embodiments, the pepsin detection kit further comprises a pepsin detection standard colorimetric card, or a pepsin detection standard color scale.

Further, the pepsin detection kit can also comprise a shell and an instruction for use.

The pepsin detection kit can be provided with a shell, a pepsin detection film and a standard colorimetric card can be placed in the shell, and an operation instruction and the like can also be placed in the shell. The number of the pepsin detecting films is not limited, and for example, 1 to 5 pepsin detecting films or more pepsin detecting films can be placed.

According to the embodiment of the invention, the kit for detecting the pepsin comprises the following components:

after the pepsin detection film is prepared, the color of the pepsin detection film before detection can be recorded in a picture shooting mode, or the pepsin detection film is compared with the standard color on the kit for recording;

and then immersing the pepsin detection film in the liquid to be detected, wherein the color of the pepsin detection film can be slowly observed to change after several seconds, for example, the color can be changed from blue to green, after the color is stable, the pepsin detection film is taken out, the color of the pepsin detection film after detection is recorded, and the pepsin detection film before detection is compared with the color of the pepsin detection film before detection for analysis and judgment, or the pepsin detection film can be compared with a color card number for verification, or the pepsin detection film can be compared with a standard color rank or a standard color card in a kit, wherein the color depth change corresponds to different concentrations of the pepsin.

The standard color scale or standard colorimetric card for detecting the pepsin can be prepared by the following method:

preparing a series of pepsin solutions with a concentration, for example, a series of pepsin solutions with a concentration of 0ug/mL, 0.6ug/mL, 3.3ug/mL, 10ug/mL, 25ug/mL, 100 ug/mL;

respectively placing the pepsin solutions with the concentrations in colorimetric containers, respectively adding the same pepsin detection films, standing for a certain time, taking out the pepsin detection films after the color is stable, recording the detected pepsin detection film color development result by using image acquisition equipment such as a camera and the like, and after collecting pictures, forming a pepsin standard color gradation by using pictures with color gradients or printing (through computer processing) to prepare a pepsin detection standard colorimetric card.

In addition, if the film is not sensitive to color, the color of the pepsin detection film can be analyzed by using color analysis software, for example, MATLAB software can be used, the color of the pepsin detection film can be quantified by using color space knowledge, and the relationship between the concentration of pepsin and the color of the film is shown in FIG. 2. It should be noted that the values in the table of fig. 2 may fluctuate by a certain amount due to environmental factors, and the fluctuation range is ± 5.

As described above, in analyzing and processing the relationship between the pepsin concentration and the film color of the pepsin and pepsin detection film, it is possible to use various existing color models, such as an HSI (Hue-preservation-Intensity, HSI) color model, i.e., a color characteristic described by using H, S, I three parameters, or an RGB color model. Wherein h (hue) defines the wavelength of the color, called hue, which represents the human perception of different colors; s (saturation) indicates the shade of a color and the purity of the color, and the higher the saturation is, the more vivid the color looks; i (intensity) represents the intensity or brightness, corresponding to the imaged brightness and image gray scale, and is the brightness level of the color. The HSI model reflects basic attributes of human perceived colors, corresponding to the results of human perceived colors one-to-one, and thus, the HIS model is widely applied to image representation and processing systems perceived by the human visual system. In addition, RGB can convert the HIS model, i.e. RGB color image and HIS model are converted into each other as required, and detailed description is omitted. As can be seen from the data in fig. 2, the blue (B) value and the pepsin concentration have a better linear relationship, specifically, as shown in fig. 1, the abscissa of the blue (B) value is the above-mentioned series of pepsin concentrations, and the ordinate of the blue (B) value is the blue B value, the above-mentioned series of pepsin concentration values and the blue B value are linearly fitted to obtain a better linear relationship, so that the blue B values corresponding to different pepsin concentrations, i.e. different color displays, can be determined according to the linear relationship. Because different colors have respective color value combinations, a standard colorimetric card or a substitute colorimetric card can be prepared according to the color values, so that the corresponding pepsin concentration and the film color can be obtained in the mode.

According to the embodiment of the application, the most intuitive comparison mode can be adopted, namely whether the pepsin is contained or not is judged by detecting whether the color of the film is changed or not by detecting the pepsin before and after the film is detected. And the concentration of pepsin can be judged by a relational expression between the blue (B) value and the concentration of pepsin. Of course, other calculations or other comparisons (other than B) may be used to determine pepsin concentration.

In order to facilitate an understanding of the invention, the invention will now be further described with reference to the following specific examples. In the following specific examples, the raw materials used are all commercially available unless otherwise specified.