CN109254001B - Preparation and application of starch rapid test card - Google Patents
Preparation and application of starch rapid test card Download PDFInfo
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- CN109254001B CN109254001B CN201811265774.1A CN201811265774A CN109254001B CN 109254001 B CN109254001 B CN 109254001B CN 201811265774 A CN201811265774 A CN 201811265774A CN 109254001 B CN109254001 B CN 109254001B
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Abstract
The invention relates to the fields of nano materials, catalysis and analytical chemistry, and particularly relates to preparation and application of a starch rapid test card. The method utilizes cobaltosic oxide nanospheres with peroxidase activity, glucoamylase, glucose oxidase and a color-developing agent to modify filter paper to obtain a rapid detection card, and realizes simple and rapid detection of starch. The novel colorimetric starch detection method has strong practicability, so the method has wide application prospects in the fields of food detection, analytical chemistry, environmental engineering and agriculture.
Description
Technical Field
The invention relates to the fields of nano materials, catalysis and analytical chemistry, and particularly relates to preparation and application of a starch rapid test card.
Background
Starch (starch) is a polysaccharide of the formula (C)6H10O5) n, is one way for plants to store energy. Starch can be regarded as a high polymer of glucose. Besides eating, starch is industrially used for preparing dextrin, maltose, glucose, alcohol and the like, and is also used for preparing printing paste, textile sizing, paper sizing, medicine tablet pressing and the like. Various methods have been reported for detecting starch, for example, Chattoplahy et al, Co-immobilization of glucoamylase and glucose oxidase for electrochemical sequencing of starch for static biosensior and biobasic cell, electrochemically (Biosensors and Bioelectronics 2014, 51, 158-. Beckles et al, Structural investments and Morphology of Tomato Starch, used a method of gel electrophoresis to detect Starch in Tomato (Journal of Agricultural and Food Chemistry 2009, 57, 282-291). However, these methods have the disadvantages of complicated operation process and high cost, and therefore, it is necessary to develop a sensitive, simple and fast method for detecting starch.
The test strip has been widely used in life as a portable rapid detection method, such as a blood glucose test strip, a urine analysis test strip, and the like. Therefore, many documents have also conducted some research on test strips. Yan et al, in Nanozyme-strip for rapid local diagnosis of Ebola, replaced the colloidal gold in the conventional test strip with magnetic nanoenzyme, detected Ebola virus by color development, and increased the sensitivity of the test strip by 100-fold (Biosensors and Bioelectronics 2015,74, 134-141). Cai et al, in A simple and rapid colloidal gold-based immunochromatogrophicstrin for detection of FMDV derotype A, combined immunoassay with test strips for the detection of foot and mouth disease virus (ACS Applied Materials & Interfaces 2014,6, 21624-. Although the test strip has been applied to a certain extent, the existing method has a complex manufacturing process and high cost, and the action mechanism of the test strip is essentially different from that of starch detection. Therefore, there is a need to develop a new method for simple and rapid preparation and starch detection.
Disclosure of Invention
The invention aims to provide preparation and application of a starch rapid test card.
The preparation of the starch rapid test card is characterized in that: the starch rapid test card is composed of a substrate, a composite catalytic material and a color developing agent, wherein the composite catalytic material is cobaltosic oxide nanospheres (Co nanospheres) modified with saccharifying enzyme and glucose oxidase (GOx)3O4NPs) with a developer, the composite catalytic material being fixed to a substrate.
In order to achieve the purpose, the invention adopts the technical scheme that:
the preparation method of the starch rapid test card is characterized in that the preparation process of the starch rapid test card is as follows:
(1) dropping a Bovine Serum Albumin (BSA) solution with the mass concentration of 1% on a substrate, standing for 1 hour and drying at room temperature;
(2) respectively dripping 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with the mass concentration of 2% on the substrate modified with BSA in the step (1), standing for 5 hours, washing with water and drying at room temperature;
(3) uniformly dripping the solution of the composite catalytic material on the substrate obtained in the step (2), standing overnight at 4 ℃, washing with water and drying;
(4) and (4) dropwise coating the organic color developing agent solution on the substrate modified with the composite catalytic material in the step (3), and drying at 4 ℃ to obtain the rapid test card.
The preparation process of the composite catalytic material is characterized by comprising the following steps:
(1) adding a certain amount of sodium borohydride into a mixed solution containing BSA and divalent cobalt ions, reacting, centrifuging and drying to obtain Co3O4 NPs；
(2) A certain amount of fourCobaltosic oxide nanosphere (Co)3O4NPs) is added into a 0.2mM tert-butyl carbamate (BOC) solution, and the mixture is washed and dried after being kept stand for 2 hours;
(3) the Co obtained in the step (2) is mixed3O4Adding NPs into a mixed solution of EDC and NHS with the mass concentration of 2%, standing for 5 hours, washing with water and drying;
(4) the Co obtained in the step (3) is put into3O4NPs are added into a mixed solution of Glucoamylase (Glucoamylase) and glucose oxidase (GOx), reacted overnight at 4 ℃, washed and dried to prepare the composite catalytic material.
The preparation and application of the starch rapid test card, and the composite catalytic material can be used for detecting starch.
Preferably, the substrate may be a nitrocellulose membrane, a cellulose acetate membrane, a polyester fiber, a glass fiber, a non-woven fabric, or the like; the organic color developing agent is 2,2 ' -dinitro-bis (3-ethylbenzthiazoline-6-sulfonic acid) diamine salt, 3 ', 5,5 ' -tetramethyl benzidine, dopamine or phenol.
Preferably, the starch rapid test card is characterized by comprising the following steps:
(1) taking two speed measuring cards, adding a certain amount of starch solution on one speed measuring card, not adding starch on the other speed measuring card, and standing for 5 minutes to ensure that the starch and the composite catalytic material fully react;
(2) observing the color change of the rapid-determination card to realize the qualitative detection of the starch, if the color change of the rapid-determination card added with the starch is compared with that of the rapid-determination card not added with the starch, indicating that the rapid-determination card can be used for realizing the detection of the starch;
(3) the intelligent mobile phone is used for photographing, and the photographing can be carried out under the irradiation of the LED lamp during photographing so as to prevent the pictures from being fuzzy. Reading the specific RGB value of the speed measuring card in the photo by using color recognition software of the mobile phone, and obtaining the RGB value by BNCalculating to obtain B/(R + G + B)NSubstituted into BNAnd a concentration working curve equation is used for calculating the concentration of the starch, so that the quantitative detection of the starch is realized.
The invention has the following effects:
1. the invention utilizes the series reaction of cobaltosic oxide nanospheres with the activity of mimic peroxidase, saccharifying enzyme and glucose oxidase to construct a sensor for rapidly detecting starch.
2. The composite catalytic material synthesized by the invention and the prepared test strip realize starch detection by a one-pot method, and avoid the step-by-step reaction.
3. The invention has certain practical value in actual life and can be expanded to quantitative analysis, catalysts and other aspects in the fields of food fermentation, biomedicine, chemical industry, environment, biotechnology and the like.
Drawings
Fig. 1 is a flowchart of a manufacturing process of a speed measuring card according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of a detection of a speed measurement card according to an embodiment of the present invention.
Fig. 3 illustrates the feasibility of the speed measuring card detection provided by the embodiment of the present invention.
Fig. 4 is a one-pot pH optimization of the composite catalytic material provided in the embodiments of the present invention.
Fig. 5 is a photograph of a qualitative test of the speed test card according to the embodiment of the present invention.
FIG. 6 is a standard working curve for starch quantification provided by an embodiment of the present invention.
Detailed Description
In order to further illustrate the contents of the present invention, some examples will be further illustrated below, but the present invention is not limited to the illustrated examples. The specific experimental conditions or methods in the following examples, if not noted, were carried out according to conventional conditions or methods in the art.
Example 1
The preparation process of the composite catalytic material is characterized by comprising the following steps:
(1) adding a certain amount of sodium borohydride into a mixed solution containing BSA and divalent cobalt ions, and obtaining Co through the reduction of the sodium borohydride and the oxidation of oxygen3O4NPs are centrifuged and dried to obtain solid;
(2) a certain amount of Co3O4NPs were added to a 0.2mM BOC solution to protect Co3O4Standing amino groups on the NPs for 2 hours, washing with water, and drying;
(3) the Co obtained in the step (2) is mixed3O4NPs were added to a mixed solution of EDC and NHS at a mass concentration of 2% to activate Co3O4Standing carboxyl on NPs for 5 hours, washing with water and drying;
(4) the Co after the carboxyl activation obtained in the step (3) is treated3O4NPs are added into the mixed solution of saccharifying enzyme and GOx, and amino groups on the saccharifying enzyme and the GOx are mixed with Co3O4 The carboxyl groups on the NPs are subjected to an amide reaction to be fixed to Co3O4And (4) carrying out an overnight reaction on the NPs at the temperature of 4 ℃, washing with water, and drying to obtain the composite catalytic material.
Example 2
Preparation process of the speed measuring card (fig. 1):
(1) dropping a BSA solution with the mass concentration of 1% on a cellulose membrane, standing for 1 hour and drying at room temperature;
(2) respectively dripping EDC and NHS with the mass concentration of 2% on the cellulose membrane (selecting the cellulose nitrate membrane with good water absorbability) modified with BSA in the step (1) to activate the carboxyl of the BSA, standing for 5 hours, washing with water and drying at room temperature;
(3) uniformly dripping the solution of the composite catalytic material on the cellulose membrane obtained in the step (2), enabling amino on the composite catalytic material to react with carboxyl on BSA (bovine serum albumin) to enable the composite catalytic material to be fixed on the cellulose membrane, standing overnight at 4 ℃, washing with water and drying;
(4) and (4) dropwise coating the organic color developing agent solution on the cellulose membrane modified with the composite catalytic material in the step (3), and drying at 4 ℃ to obtain the rapid test card.
Detection principle of the tach card (fig. 2):
the starch rapid test card is composed of a substrate, an enzyme and a color developing agent, wherein the substrate adopts nitrocellulose filter paper, and Co connected with saccharifying enzyme and GOx3O4NPs and a color developing agent are fixed on a substrate, and the detection basic principle is as follows: when adding starch, the starch is first addedGenerating glucose under the catalytic action of saccharifying enzyme, and continuously reacting the generated glucose with GOx to generate H2O2Then Co with peroxidase-mimetic activity3O4NPs catalyze H2O2The ABTS is changed in color, and the speed measuring card is changed from colorless to green; the color of the rapid test card without adding starch is changed slightly or not, and the quantitative analysis of the starch can be realized according to the color change condition of the rapid test card.
Example 3
Feasibility of rapid test card detection:
catalytic reaction system (a) the colorimetric card was cut into pieces and placed in a starch (1 mmol/L) solution (pH = 5.0), and after 5 minutes of reaction, the reaction solution was scanned at full wavelength. For another set of control experiments (b) the cards were cut into pieces and placed in buffer (pH = 5.0) and after 5 minutes of reaction, the reaction solution was scanned at full wavelength.
As shown in FIG. 3, the catalytic reaction system (a) has a distinct UV absorption peak, and the catalytic reaction system (b) has no UV absorption peak, which proves that the catalytic reaction can be carried out only in the presence of starch.
Example 4
One-pot pH optimization of the composite catalytic material:
the catalytic reaction system comprises a composite catalytic material (30 mu g/mL), starch (1 mmol/L) and buffers with different pH values (pH 1.0-2.0, glycine-hydrochloric acid buffer solution, pH 3.0-6.0, acetic acid-sodium acetate buffer solution, pH 7.0-8.0, phosphate buffer solution, pH 9.0, Tris-hydrochloric acid buffer solution). After 5 minutes of reaction at room temperature (25 ℃), the absorbance of the reaction solution at 650 nm was measured. As shown in FIG. 4 (a), the optimum pH was about 4.0.
Another set of control experiments was performed, in which the catalytic reaction system included Co3O4NPs (30. mu.g/mL), glucoamylase (0.3 mg/mL), GOx (0.1 mg/mL), starch (1 mmol/L) and buffers of different pH (pH 1.0-2.0, glycine-hydrochloric acid buffer; pH 3.0-6.0, acetic acid-sodium acetate buffer; pH 7.0-8.0, phosphate buffer; pH 9.0-10.0, Tris-hydrochloric acid buffer). After 5 minutes at room temperature (25 ℃), the reaction solution was measuredAbsorbance at 650 nm. As shown in FIG. 4 (b), the activity was higher at a pH of about 3.0 to 4.5, but the activity was lower than that of 4 (a).
Example 5
Qualitative detection of the speed test card:
(1) taking three speed measuring cards, wherein starch is not added in the speed measuring card (a), 1mM starch is added in the speed measuring card (b), 100 mM starch is added in the speed measuring card (c), and the three speed measuring cards are placed for 5 minutes to ensure that the starch and the composite catalytic material fully act;
(2) the intelligent mobile phone is used for photographing, and the photographing can be carried out under the irradiation of the LED lamp during photographing so as to prevent the pictures from being fuzzy. As shown in fig. 5, the quick test card (a) without starch does not change color, the quick test card (b) with starch of smaller concentration becomes darker than the quick test card (a), and the quick test card (c) with starch of larger concentration becomes darker than the quick test card (b), which indicates that the starch concentration dropped on the quick test card (c) is the largest, the quick test card (b) is the second time, and the quick test card (a) does not add starch, which again proves the feasibility of the starch quick test card in detecting starch, so that the starch quick test card can detect starch.
Example 6
Standard working curve for starch quantification:
(1) taking 12 rapid measurement cards, dripping starch (0-10 mM) with different concentrations on the rapid measurement cards, and standing for 5 minutes to ensure that the starch and the composite catalytic material fully act;
(2) the intelligent mobile phone is used for photographing, and the photographing can be carried out under the irradiation of the LED lamp during photographing so as to prevent the pictures from being fuzzy. Reading the specific RGB value of the speed measuring card in the photo by using color recognition software of the mobile phone, and obtaining the RGB value by BNCalculating to obtain B/(R + G + B)NObtaining a standard curve, wherein the specific working curve is shown in FIG. 6, the linear range is 0-0.3 mM, and the equation is y = 0.6062x +0.0998 (R)2=0.9918）。
Claims (4)
1. A preparation method of a starch rapid test card is characterized by comprising the following steps: the starch rapid test card is composed of a substrate, a composite catalytic material and a color developing agent, wherein the composite catalytic material is a tetraoxide modified with saccharifying enzyme and glucose oxidase (GOx)Cobaltosic nanosphere (Co)3O4NPs), the composite catalytic material and the color developing agent are fixed on the substrate; the preparation method of the starch rapid test card comprises the following steps:
(1) dropping a Bovine Serum Albumin (BSA) solution with the mass concentration of 1% on a substrate, standing for 1 hour and drying at room temperature;
(2) respectively dripping 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with the mass concentration of 2% on the substrate modified with BSA in the step (1), standing for 5 hours, washing with water and drying at room temperature;
(3) uniformly dripping the solution of the composite catalytic material on the substrate obtained in the step (2), standing overnight at 4 ℃, drying and washing with water;
(4) and (4) dropwise coating the organic color developing agent solution on the substrate modified with the composite catalytic material in the step (3), and drying at 4 ℃ to obtain the rapid test card.
2. The method for preparing the starch tach according to claim 1, wherein the preparation steps of the composite catalytic material are as follows:
(1) adding sodium borohydride into a mixed solution containing BSA and divalent cobalt ions, reacting, centrifuging and drying to obtain Co3O4NPs；
(2) Mixing Co3O4NPs are added into a 0.2mM tert-butyl carbamate (BOC) solution, kept stand for 2 hours, washed with water and dried;
(3) the Co obtained in the step (2) is mixed3O4Adding NPs into a mixed solution of EDC and NHS with the mass concentration of 2%, standing for 5 hours, washing with water and drying;
(4) the Co obtained in the step (3) is put into3O4And (3) adding the NPs into the mixed solution of the saccharifying enzyme and the GOx, reacting overnight at 4 ℃, washing with water, and drying to obtain the composite catalytic material.
3. The method for preparing a starch speed measuring card according to claim 1, wherein the method comprises the following steps: the substrate is a nitrocellulose membrane, a cellulose acetate membrane, polyester fiber, glass fiber, non-woven fabric and the like; the organic color developing agent is 2,2 ' -dinitro-bis (3-ethylbenzthiazoline-6-sulfonic acid) diamine salt, 3 ', 5,5 ' -tetramethyl benzidine, dopamine or phenol.
4. The method for preparing a starch tach according to claim 1, wherein the application steps for detecting a starch tach are as follows:
(1) taking two rapid measuring cards, adding starch solution on one rapid measuring card, not adding starch on the other rapid measuring card, and standing for 5 minutes to ensure that the starch and the composite catalytic material fully react;
(2) observing the color change of the rapid-determination card to realize the qualitative detection of the starch, if the color change of the rapid-determination card added with the starch is compared with that of the rapid-determination card not added with the starch, indicating that the rapid-determination card can be used for realizing the detection of the starch;
(3) taking a picture by using a smart phone, taking the picture under the irradiation of an LED lamp during the picture taking to prevent the picture from blurring, reading the specific RGB value of the speed measuring card in the picture by using color recognition software of the smart phone, and taking the picture by BNB is calculated as B/(R + G + B)NSubstituted into BNAnd a concentration working curve equation is used for calculating the concentration of the starch, so that the quantitative detection of the starch is realized.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105388150A (en) * | 2015-10-29 | 2016-03-09 | 大连理工大学 | Oxytetracycline test paper based on chromatic aberration comparison, using method and making method |
| CN105606604A (en) * | 2016-01-08 | 2016-05-25 | 昆明泊银科技有限公司 | Rapid hydrogen peroxide test paper and preparation method thereof |
| CN107857307A (en) * | 2017-11-20 | 2018-03-30 | 青岛农业大学 | A kind of new strategy for realizing one kettle way grape grape color developing detection |
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| US9121831B2 (en) * | 2011-08-24 | 2015-09-01 | Multisorb Technologies, Inc. | Copper, starch and iodide moisture indicator |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105388150A (en) * | 2015-10-29 | 2016-03-09 | 大连理工大学 | Oxytetracycline test paper based on chromatic aberration comparison, using method and making method |
| CN105606604A (en) * | 2016-01-08 | 2016-05-25 | 昆明泊银科技有限公司 | Rapid hydrogen peroxide test paper and preparation method thereof |
| CN107857307A (en) * | 2017-11-20 | 2018-03-30 | 青岛农业大学 | A kind of new strategy for realizing one kettle way grape grape color developing detection |
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