CN112881701A - Test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicine and preparation method thereof - Google Patents
Test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of drug detection, in particular to a test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicines and a preparation method thereof, wherein the test strip comprises the following components: the test strip comprises a nitrocellulose membrane, a sample pad, a conjugate release pad, a water absorption pad and a bottom plate, wherein the nitrocellulose membrane comprises a detection antigen coated with aristolochic acid and bovine serum albumin coupled and a goat anti-mouse IgG antibody, the conjugate release pad is coated with an aristolochic acid monoclonal antibody-colloidal gold marker, and the detection sensitivity of the test strip reaches 6.0 ng/mL. The test strip has the characteristics of strong specificity, high sensitivity, simplicity and convenience in operation, rapidness and easiness in detection of aristolochic acid in a traditional Chinese medicine sample, and can be popularized and applied in a large range.
Description
Technical Field
The invention relates to the technical field of drug detection, in particular to a test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicines and a preparation method thereof.
Background
Aristolochic Acids (AAs) are also called total Aristolochic acids, phagocytic acids or akebia base, and are a class of nitro phenanthrene carboxylic acids. Aristolochic acids a and B are mainly present in plants of the aristolochiaceae family, such as Aristolochia (Aristolochia) and Asarum (Asarum). In the aristolochic acid compounds, the main toxic components are aristolochic acid I and aristolochic acid II (namely aristolochic acid A and aristolochic acid B), one part of the aristolochic acid is reduced into aristolochia lactam under the catalysis of nitroreductase, and the other part of the aristolochic acid further reacts with DNA in the reduction process to form adducts which have mutagenic and carcinogenic toxicity. At present, in addition to caulis aristolochiae manshuriensis which has been withdrawn from Chinese pharmacopoeia, 6 kinds of medicinal materials which are definitely contained in 2000 edition pharmacopoeia and national medicine standard and contain aristolochia manshuriensis are fourstamen stephania root, dutchmanspipe vine, aristolochia debilis, wooly mollissima and ardisia crenata, and hundreds of Chinese patent medicine varieties which are contained in Chinese pharmacopoeia and national medicine standard and contain aristolochia manshuriensis are contained.
The detection method of aristolochic acid A and aristolochic acid B mainly comprises the following steps: high performance liquid chromatography, thin layer chromatography and the like, but the methods are generally only suitable for laboratory detection, are time-consuming and expensive, require complex instruments and complex sample pretreatment, require professional operation, are difficult to popularize and popularize at the basic level, and are not suitable for rapid screening in fields such as quality inspection departments, consumers and the like.
At present, enzyme-linked immunosorbent assay (ELISA) method for detecting aristolochic acid A and aristolochic acid B is reported. For example, Nantiegui et al, preparation of aristolochic acid A monoclonal antibody as a nephrotoxic component caused by traditional Chinese medicines, and establishment of enzyme-linked immunoassay (analytical chemistry, 2010, 38(8): 1206-1210). However, the detection of the method needs to be equipped with a professional enzyme-labeling instrument, needs to be operated by professional personnel, is only suitable for detection in a laboratory, and is not suitable for rapid field screening.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicines and a preparation method thereof, and solves the problems that the existing detection method for the aristolochic acid A and the aristolochic acid B is only suitable for laboratory detection, needs to be operated by professionals and is not suitable for rapid field screening of quality inspection departments, consumers and the like.
(II) technical scheme
The invention provides a test strip for detecting aristolochic acid A and B in traditional Chinese medicine and a preparation method thereof, which are a colloidal gold immune rapid detection method based on the reaction of an antigen and an antibody on a nitrocellulose membrane, and are high in detection sensitivity and can be used for detecting the total amount of aristolochic acid A and B at the same time.
In order to achieve the purpose, the invention adopts the following specific technical scheme:
a test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicines comprises: a nitrocellulose membrane, a sample pad, a conjugate release pad, a water absorbent pad, and a base plate; wherein the nitrocellulose membrane comprises a detection antigen coated with AAs coupled with BSA and a goat anti-mouse IgG antibody, and the conjugate release pad is coated with an AAs monoclonal antibody-colloidal gold marker.
Preferably, the amount of coated AAs monoclonal antibody-colloidal gold label on the conjugate release pad is 2-5. mu.L/cm.
Preferably, the nitrocellulose membrane comprises a test area coated with AAs-BSA conjugate and a quality control area coated with goat anti-mouse IgG antibody.
Preferably, the nitrocellulose membrane comprises a test area coated with AAs-BSA conjugate and a quality control area coated with goat anti-mouse IgG antibody.
Preferably, the amount of test area AAs-BSA conjugate on the nitrocellulose membrane is 0.5-1.0. mu.L/cm.
Preferably, the amount of the goat anti-mouse IgG antibody in the quality control region on the nitrocellulose membrane is 0.5-1.0. mu.L/cm.
Preferably, the use method of the test strip is as follows:
(1) weighing the crushed sample in a conical flask, adding 10-20 times of methanol as an extracting solution, fully shaking for 3-10min, filtering with qualitative filter paper, collecting filtrate, taking 1mL of the filtrate, diluting 4-7 times by adopting PBST buffer solution, and fully and uniformly mixing to obtain a solution called as a loading solution;
(2) and (3) dripping the sample loading liquid obtained in the step (2) on a test strip to be detected, and observing a detection result after 10 min.
Preferably, the PBST buffer solution in the step (1) is 0.05-0.1mol/L PBS solution containing 0.5% -5% Tween-20 and fully dissolved after stirring.
In order to achieve the second object of the present invention, the present invention further provides a preparation method of the test strip for detecting aristolochic acid a and aristolochic acid B in traditional Chinese medicine, comprising the following steps:
(1) AAs monoclonal antibody-colloidal gold marker
Adding K to the colloidal gold solution2CO3Adjusting the pH of the solution to 6.0-7.0; adding 1.0-2.0 mu g of AAs antibody into every 1mL of colloidal gold, and reacting for 10-65min by rapid stirring; adding BSA to a final concentration of 0.5-1.5%, and reacting for 10-40 min; adding PEG-20000 to a final concentration of 0.1-0.5%, and reacting for 10-40 min; centrifuging the solution at 8000-12000rpm for 20-60min, discarding supernatant, and re-dissolving precipitate with PBS solution containing 5.0-15.0% sucrose, 0.1-2% BSA, 0.1-0.5% PEG-20000, and 0.1-3.5% trehalose to obtain AAs monoclonal antibody-colloidal gold marker.
(2) Coating of conjugate release pads
And (2) putting the conjugate release pad into 0.01-0.1mol/L PBS solution containing 5.0% -15.0% of sucrose, 0.1% -5% of BSA, 0.1% -0.5% of PEG-20000 and 0.1% -3.5% of trehalose to soak for 5-40min, drying at 37 ℃ in vacuum, uniformly spraying the AAs monoclonal antibody-colloidal gold marker obtained in the step (1) onto the conjugate release pad, spraying 2.0-5.0 mu LAAs monoclonal antibody-colloidal gold marker onto each 1cm of the conjugate release pad, and drying at 37 ℃ in vacuum.
(3) Coating of nitrocellulose membranes
Spraying detection antigen with concentration of 0.1-2.0mg/mL BSA coupled AAs on nitrocellulose membrane as detection line, spraying amount of 0.5-1.0 μ L/cm, spraying goat anti-mouse IgG with concentration of 0.1-1.0mg/mL at a distance of 5-10mm as quality control line, spraying amount of 0.5-1.0 μ L/cm, vacuum drying at 37 deg.C, and sealing.
(4) Production of test paper
Sticking the nitrocellulose membrane sprayed with the AAs detection antigen and the goat anti-mouse IgG obtained in the step (3) on a PVP bottom plate; one end of the PVP bottom plate is a sample pad, the other end of the PVP bottom plate is an adsorption pad, the two ends of the nitrocellulose membrane are respectively connected with the adsorption pad and the binder release pad in an overlapped mode, the connecting portion is 1-2mm, the sample pad is pressed on the binder release pad, and the test strip for rapid detection is obtained after the test strip is cut into strips with the width of 3-4 mm.
(III) advantageous effects
The invention provides a test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicines and a preparation method thereof, and the test strip has the following beneficial effects:
(1) the test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicines, prepared by the invention, has the characteristics of strong specificity, high sensitivity, visual and simple and rapid operation, visual detection result, no need of professional operation, detection result output within 10min, simple operation, suitability for field detection, rapidness, no need of complex instruments, only eye observation, good stability and low detection cost.
(2) The test strip for detecting aristolochic acid A and aristolochic acid B in the traditional Chinese medicine is easy to popularize and apply in a large range, can be widely applied to the requirements of household field detection, traditional Chinese medicine safety detection and the like, and has wide market prospect and great economic and social benefits.
Drawings
FIG. 1 shows the test strip of the present invention with sensitivity, 1-6 respectively showing aristolochic acid concentration: 0, 3, 6, 10, 20 and 30 ng/mL.
Detailed Description
For further understanding of the present invention, the following examples are provided to illustrate a test strip for detecting aristolochic acids a and B in a traditional Chinese medicine and a preparation method thereof, and the scope of the present invention is not limited by the following examples.
Example 1:
the preparation of the test strip for detecting aristolochic acid A and aristolochic acid B in the traditional Chinese medicine comprises the following steps:
(1) preparation of AAs monoclonal antibody-colloidal gold marker
Preparing colloidal gold: diluting 1% chloroauric acid to 0.01% with ultrapure water, placing in an oil bath, heating and boiling under magnetic stirring, adding 10mL of 1% trisodium citrate into every 500mL of 0.01% chloroauric acid, continuing boiling until the liquid is red, stopping heating, cooling to room temperature, and supplementing evaporated water to obtain colloidal gold solution.
② preparation of AAs monoclonal antibody-colloidal gold marker
Adding K into the colloidal gold solution under magnetic stirring2CO3The pH of the solution was adjusted to 6.0, and 1.75. mu.g of anti-AAs monoclonal antibody was added to the colloidal gold solution, and the reaction was continued with stirring for 15 min. 10% BSA was added to a final concentration of 1.0% and stirring was continued for 15 min. Adding 5% PEG-20000 to a final concentration of 0.1%, and stirring for 15 min. Centrifuged at 6000rmp and 4 ℃ for 30 min. Discarding the supernatant, and re-dissolving the precipitate with 5% sucrose solution, 0.1% BSA, 0.3% PEG-20000 and 0.5% trehalose in PBS to obtain the AAs monoclonal antibody-colloidal gold marker.
(1) Coating of conjugate release pads
The conjugate release pad was soaked in PBS containing 0.1% BSA and 5% sucrose for 10min and dried at 37 deg.C. Using a Biodot membrane analyzer to uniformly spray the AAs monoclonal antibody-colloidal gold marker onto the conjugate release pad every 1cm2Spraying 3 μ L AAs monoclonal antibody-colloidal gold marker on the binding pad, vacuum drying, and standing at 4 deg.C.
(3) Coating of nitrocellulose membranes
A Biodot membrane analyzer is adopted to spray a detection antigen with the concentration of 0.2mg/mL BSA coupled AAs on a nitrocellulose membrane to serve as a detection line, the spraying amount is 0.74 mu L/cm, goat anti-mouse IgG with the concentration of 0.1mg/mL is sprayed at the position with the distance of 5mm to serve as a quality control line, and the spraying amount is 0.74 mu L/cm. Vacuum drying, and sealing.
(4) Assembly of colloidal gold test strip
The assembly of the test strip includes: the end head of one end of the bottom plate is a water absorption pad, the end head of the other end of the bottom plate is a sample pad, the two ends of the nitrocellulose membrane are respectively connected with the water absorption pad and the binder release pad in an overlapped mode, and the sample pad is pressed on the binder release pad.
Example 2:
detection of total AAs in traditional Chinese medicine samples:
(1) sample detection: weighing pulverized radix Aristolochiae Fangchi, adding 10-20 times of methanol as extractive solution, shaking for 3-10min, filtering with qualitative filter paper, and collecting filtrate. Taking 1mL of filtrate, diluting by 4-7 times by using 0.05-0.1mol/L PBS solution containing 0.5% -5% Tween-20 after stirring, taking the filtrate as a loading solution after fully mixing, taking 100 mu L of the loading solution to be dropped on a sample pad of the test strip prepared in the example 1 for detection, and observing a detection result after 10 min.
(2) And (4) analyzing results: when the detection line and the quality control line are both red, the sample is negative, namely the content of aristolochic acid A and B in the sample is less than 6.0 ng/mL; when only the detection line shows red, the sample is positive, namely the content of aristolochic acid A and B in the sample is more than 6.0 ng/mL; when only the detection line is colored or the quality control line and the quality control line are not colored, the test strip is invalid.
Example 3:
and (3) detecting each index of the test strip:
(1) test strip effectiveness detection
Weighing aristolochic acid A and aristolochic acid B into a conical flask, adding methanol to dissolve the Arstolochic acid A and the Arstolochic acid B into a solution of 6.0ng/mL, adding a proper amount of PBST buffer solution which is 0.05-0.1mol/L PBS solution containing 0.5% -5% Tween-20 and fully dissolved after stirring, taking 100 mu L for detection, and when the detection line and the quality control line are both red, indicating that the sample is negative, namely the content of the aristolochic acid A and the B in the sample is less than 6.0 ng/mL; when only the detection line shows red, the sample is positive, namely the content of aristolochic acid A and B in the sample is more than 6.0 ng/mL; when only the detection line is colored or the quality control line and the quality control line are not colored, the test strip is invalid.
(2) Sensitivity test
Respectively detecting 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0ng/mL AAs standard substances by using a test strip by adopting the method in the step (1), and repeating for 10 times, wherein two red lines appear at 0, 0.5, 1.0, 2.0 and 4.0ng/mL, and the result is negative; positive results were obtained when only one red line appeared at 6.0, 8.0 and 10.0 ng/mL. Therefore, the sensitivity of the test strip is 6.0 ng/mL.
(2) Homogeneity test
And (2) respectively carrying out 10 parallel detections on 0ng/mL AAs and 6.0ng/mL AAs by adopting the method in the step (1), observing the result after reacting for 10min, wherein two red lines appear at 0ng/mL, only one red line appears at 6.0ng/mL, and the detection color development depth is uniform and the reaction results are consistent.
(3) Stability test
And (3) placing the test strip packaged in vacuum into a 37 ℃ oven for destructive experiments for 20 days, wherein each index accords with the 1-2 indexes, namely the sensitivity reaches 6.0ng/mL, the detection color development depth is uniform, and the reaction results are consistent.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicine is characterized in that: the test strip comprises: a nitrocellulose membrane, a sample pad, a conjugate release pad, a water absorbent pad, and a base plate; wherein the nitrocellulose membrane comprises a detection antigen coated with AAs coupled with BSA and a goat anti-mouse IgG antibody, and the conjugate release pad is coated with an AAs monoclonal antibody-colloidal gold marker.
2. The test strip of claim 1, wherein: the amount of the coated AAs monoclonal antibody-colloidal gold label on the conjugate release pad was 2 to 5 μ L/cm.
3. The test strip of claim 1, wherein: the nitrocellulose membrane comprises a test area coated with AAs-BSA conjugate and a quality control area coated with goat anti-mouse IgG antibody.
4. The test strip of claim 3, wherein: the amount of the test zone AAs-BSA conjugate on the nitrocellulose membrane is 0.5-1.0. mu.L/cm.
5. The test strip of claim 3, wherein: the quantity of the goat anti-mouse IgG antibody in the quality control region on the nitrocellulose membrane is 0.5-1.0 mu L/cm.
6. The test strip of claim 3, wherein: the distance between the test area and the quality control area on the nitrocellulose membrane is 5-10 mm.
7. A preparation method of a test strip for detecting aristolochic acid A and aristolochic acid B in traditional Chinese medicines is characterized in that: the method comprises the following steps:
(1) AAs monoclonal antibody-colloidal gold marker
Adding K to the colloidal gold solution2CO3Adjusting the pH of the solution to 6.0-7.0; adding 1.0-2.0 mu g of AAs antibody into every 1mL of colloidal gold, and reacting for 10-65min by rapid stirring; adding BSA to a final concentration of 0.5-1.5%, and reacting for 10-40 min; adding PEG-20000 to a final concentration of 0.1-0.5%, and reacting for 10-40 min; centrifuging the solution at 8000-12000rpm for 20-60min, discarding supernatant, and re-dissolving precipitate with PBS solution containing 5.0-15.0% sucrose, 0.1-2% BSA, 0.1-0.5% PEG-20000, and 0.1-3.5% trehalose to obtain AAs monoclonal antibody-colloidal gold marker.
(2) Coating of conjugate release pads
And (2) putting the conjugate release pad into 0.01-0.1mol/L PBS solution containing 5.0% -15.0% of sucrose, 0.1% -5% of BSA, 0.1% -0.5% of PEG-20000 and 0.1% -3.5% of trehalose, soaking for 5-40min, drying at 37 ℃ in vacuum, uniformly spraying the AAs monoclonal antibody-colloidal gold marker obtained in the step (1) onto the conjugate release pad, spraying 2.0-5.0 mu L of the AAs monoclonal antibody-colloidal gold marker onto each 1cm of the conjugate release pad, and drying at 37 ℃ in vacuum.
(3) Coating of nitrocellulose membranes
Spraying detection antigen with concentration of 0.1-2.0mg/mL BSA coupled AAs on nitrocellulose membrane as detection line, spraying amount of 0.5-1.0 μ L/cm, spraying goat anti-mouse IgG with concentration of 0.1-1.0mg/mL at a distance of 5-10mm as quality control line, spraying amount of 0.5-1.0 μ L/cm, vacuum drying at 37 deg.C, and sealing.
(4) Production of test paper
Sticking the nitrocellulose membrane sprayed with the AAs detection antigen and the goat anti-mouse IgG obtained in the step (3) on a PVP bottom plate; one end of the PVP bottom plate is a sample pad, the other end of the PVP bottom plate is an adsorption pad, the two ends of the nitrocellulose membrane are respectively connected with the adsorption pad and the binder release pad in an overlapped mode, the connecting portion is 1-2mm, the sample pad is pressed on the binder release pad, and the test strip for rapid detection is obtained after the test strip is cut into strips with the width of 3-4 mm.
8. The test strip of any one of claims 1-6, wherein: the use method of the test strip comprises the following steps:
(1) weighing the crushed sample in a conical flask, adding 10-20 times of methanol as an extracting solution, fully shaking for 3-10min, filtering with qualitative filter paper, collecting filtrate, taking 1mL of the filtrate, diluting 4-7 times by adopting PBST buffer solution, and fully and uniformly mixing to obtain a solution called as a loading solution;
(2) and (3) dripping the sample loading liquid obtained in the step (2) on a test strip to be detected, and observing a detection result after 10 min.
9. The method of using the test strip of claim 8, wherein: the PBST buffer solution in the step (1) is 0.05-0.1mol/L PBS solution containing 0.5% -5% Tween-20 and fully dissolved after stirring.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113447463A (en) * | 2021-06-12 | 2021-09-28 | 广西医科大学 | Method for detecting aristolochic acid I by using gold nanoclusters as fluorescent probes |
| CN113504368A (en) * | 2021-07-14 | 2021-10-15 | 广东工业大学 | Test strip for detecting salmonella and preparation method thereof |
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