CN115792216B - Lateral chromatography test strip for detecting toxins, preparation method and use method - Google Patents
Lateral chromatography test strip for detecting toxins, preparation method and use method Download PDFInfo
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application provides a lateral chromatography test strip for detecting toxins, a preparation method and a use method, wherein the lateral chromatography test strip comprises the following components: the device comprises a bottom plate, wherein a sample pad, a bonding pad, a chromatographic membrane and an absorption pad are sequentially arranged on the bottom plate along the length direction, and a detection line and a quality control line are arranged on the chromatographic membrane; a toxin marker antigen is attached to the detection line; the quality control line is attached with a secondary antibody; the binding pad is attached with a detection antibody connected with a Raman probe, and the Raman probe comprises a bound Raman beacon molecule and silica-coated gold nanoparticles. The lateral chromatography test strip for detecting toxins, the preparation method and the use method have the advantages of good stability of the Raman probe, high detection intensity of Raman signals and high detection sensitivity of the test strip, can detect various mycotoxins in a complex environment, and provides guarantee for the safety of grain feed.
Description
Technical Field
The application relates to the technical field of toxin detection, in particular to a lateral chromatography test strip for detecting toxin, a preparation method and a use method.
Background
Toxins are often secondary metabolites produced by bacteria during harvesting, transportation and storage of cereal and feed, and the misingestion of the metabolites by humans or animals may cause diseases or death due to mishandling of the metabolites, and therefore are of great importance for the detection of mycotoxins in cereal feeds. The detection methods commonly used at present mainly comprise high performance liquid chromatography, liquid chromatography-mass spectrometry tandem method, gas chromatography-mass spectrometry tandem method and the like, and although the detection methods can accurately detect toxins, pretreatment of detection samples is complex, and the instrument cost is high, so that the methods cannot be used for on-site instant detection.
The lateral flow immunochromatography test strip has the advantages of simple operation, low cost, short detection time, convenient carrying and the like, is one of important methods for on-site instant detection, however, the traditional lateral chromatography test strip has the defects of low detection sensitivity and the like, and the surface enhanced Raman scattering SERS technology has the advantages of high sensitivity, no interference of aqueous solution, no damage to a sample, narrow spectral half-peak width, easy operation and the like, so that the SERS technology and the lateral flow immunochromatography are combined to provide a new method for on-site high-sensitivity multiplex detection of mycotoxins.
However, the existing SERS lateral chromatography test strip based on gold nanoparticles has a small enhancement effect on detection signals, is particularly easy to be influenced by detection environments, and has extremely poor stability, so that development of the SERS lateral chromatography test strip with high stability and capability of enhancing the detection signal intensity is needed.
Disclosure of Invention
In view of the above, the present application aims to provide a lateral chromatography test strip for detecting toxins, a preparation method and a use method thereof.
In view of the above object, a first aspect of the present application provides a lateral chromatography test strip for detecting toxins, comprising: the device comprises a bottom plate, wherein a sample pad, a bonding pad, a chromatographic membrane and an absorption pad are sequentially arranged on the bottom plate along the length direction, and a detection line and a quality control line are arranged on the chromatographic membrane; a toxin marker antigen is attached to the detection line; the quality control line is attached with a secondary antibody; the binding pad is attached with a detection antibody connected with a Raman probe, and the Raman probe comprises a bound Raman beacon molecule and silica-coated gold nanoparticles.
Further, at least two toxin marker antigens are attached to the detection line, at least two detection antibodies corresponding to the toxin marker antigens are attached to the binding pad, and at least two secondary antibodies corresponding to the detection antibodies are attached to the quality control line.
Further, the thickness of the sample pad and the bonding pad is 0.3mm-0.5mm, and the total length of the sample pad and the bonding pad is 15mm-20mm; the length of the chromatographic membrane is 22mm-30mm, and the distance between the detection line and the quality control line is 6mm-8mm; the thickness of the absorption pad is 0.5mm-0.8mm, and the length is 25mm-30mm; the length of the bottom plate is 75mm-90mm, and the thickness of the bottom plate is 3mm-5mm.
In a second aspect of the present application, there is provided a method for preparing a lateral chromatography test strip for detecting toxins according to the first aspect, comprising: preparing a Raman probe; ligating the raman probe to a detection antibody; attaching the detection antibody connected with the Raman probe to a binding pad, attaching a toxin marker antigen to a detection line of a chromatographic membrane, and attaching a secondary antibody to a quality control line of the chromatographic membrane; and arranging the sample pad, the combination pad, the chromatographic membrane and the absorption pad on a bottom plate to obtain the lateral chromatography test strip.
Further, the preparing of the raman probe includes: centrifuging colloidal gold combined with Raman beacon molecules to obtain first precipitate, adding water and isopropanol into the first precipitate, and stirring to obtain a first solution; adding ammonia water into the first solution, adjusting the pH value to a preset value, gradually adding a tetraethyl silicate isopropanol solution, and stirring for reaction to obtain a second solution; and refrigerating and storing the second solution for a preset time, performing centrifugal operation to obtain a second precipitate, washing the second precipitate, and then re-suspending the second precipitate in water to obtain a third solution containing the Raman probe.
Further, the raman beacon molecule comprises one or more of nielblue a, p-mercaptobenzoic acid, methylene blue, 5-dithiocarbamic acid, rhodamine 6G, p-aminophenylsulfol, malachite green, 4-mercaptopyridine, p-mercaptoaniline, or p-aminophenylsulfol.
Further, adding ammonia water into the first solution, adjusting the pH value to a preset value, gradually adding a tetraethyl silicate isopropanol solution, and stirring for reaction to obtain a second solution, wherein the method comprises the following steps: ammonia water is added into the first solution, the pH value is regulated to 10, tetraethyl silicate isopropanol solution is gradually added in 5h, and stirring reaction is carried out at normal temperature for 3h to obtain a second solution.
Further, the step of performing a centrifugation operation after the second solution is stored in a refrigerated state for a preset time to obtain a second precipitate includes: and (3) storing the second solution at 3-5 ℃ for at least 12 hours, and then performing centrifugal operation to obtain a second precipitate.
Further, the linking the raman probe with the detection antibody comprises: adding 3- (triethoxysilylpropyl carbamoyl) butanoic acid ethanol solution into the third solution for shaking reaction, and centrifuging to obtain a third precipitate; and (3) washing the third precipitate, then, re-suspending in water to obtain a fourth solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and sulfonated N-hydroxysuccinimide into the fourth solution for activation, adding a detection antibody for incubation reaction, and centrifuging to obtain the detection antibody connected with the Raman probe.
In a third aspect of the present application, there is provided a method for using the lateral chromatography test strip for detecting toxins according to the first aspect, comprising: dripping a detection sample on the sample pad of the lateral chromatography test strip; after the preset reaction time, obtaining a detection result according to the color development conditions of the detection line and the quality control line; and measuring the Raman signal intensity of the detection line, and determining the toxin concentration according to the Raman signal intensity.
From the above, the lateral chromatography test strip for detecting toxins, the preparation method and the using method provided by the application are characterized in that a bottom plate is provided with a sample bearing pad, a binding pad, a chromatographic membrane and an absorption pad; providing a sample pad for absorbing a test sample; the binding pad is attached with a detection antibody, and is used for allowing a detection sample flowing through the sample pad to interact with the detection antibody and flow to the chromatographic membrane; the chromatographic membrane is sequentially provided with a detection line and a quality control line, and a toxin marker antigen is attached to the detection line and used for combining with a detection antibody to develop color so as to form a competition relationship with toxin in a detection sample; the secondary antibody is attached to the quality control line and used for combining with the detection antibody to develop color, and verifying whether the detection is effective; the absorption pad is arranged to absorb the detection liquid through the chromatographic membrane by utilizing capillary action and collect the treated waste liquid, so that a larger amount of detection sample can be used, and the test sensitivity is improved; the detection antibody is combined with the Raman probe, so that the intensity and sensitivity of a detection signal can be improved, the Raman probe comprises a combined Raman beacon molecule and silica-coated gold nanoparticles, the Raman beacon molecule is used for Raman signal detection, the silica-coated gold nanoparticles can enhance the electromagnetic field intensity at a core-shell gap so as to improve the Raman signal intensity, and the silica shell has stable chemical property, has an anti-corrosion function, can protect the gold nanoparticles from being interfered by a detection environment, reduces the possibility of agglomeration, has good biocompatibility, and can further improve the detection sensitivity and reduce the measurement limit value; the lateral chromatography test strip for detecting toxins, the preparation method and the use method have the advantages of good stability of the Raman probe, high detection intensity of Raman signals and high detection sensitivity of the test strip, can detect various mycotoxins in a complex environment, and provides guarantee for the safety of grain feed.
Drawings
In order to more clearly illustrate the technical solutions of the present application or related art, the drawings that are required to be used in the description of the embodiments or related art will be briefly described below, and it is apparent that the drawings in the following description are only embodiments of the present application, and other drawings may be obtained according to the drawings without inventive effort to those of ordinary skill in the art.
FIG. 1 is a schematic diagram of a lateral chromatography test strip for detecting toxins according to an embodiment of the present application;
FIG. 2 is a schematic diagram of an electron microscope structure of a Raman probe prepared after being stored for 12 hours in a refrigerated manner in the embodiment of the application;
FIG. 3 is a schematic diagram of an electron microscope structure of a Raman probe prepared after being stored for 8 hours in a refrigerated manner in the embodiment of the application;
FIG. 4 is a schematic diagram of an electron microscope structure of a Raman probe prepared after 4 hours of cold storage in an embodiment of the application;
FIG. 5 is a schematic diagram of an electron microscope of a Raman probe prepared without cold storage in an embodiment of the application;
FIG. 6 shows aflatoxin B concentration for different concentrations in the examples of the application 1 (AFB 1 ) And a raman spectrum for ochratoxin a (OTA) detection;
FIG. 7 shows the Raman signal strength and AFB in an embodiment of the application 1 A concentration relationship diagram;
fig. 8 is a graph showing the relationship between the raman signal intensity and the OTA concentration in the embodiment of the application.
Reference numerals: 1. a bottom plate; 2. a sample pad; 3. a bonding pad; 4. a chromatographic membrane; 4-1, detecting lines; 4-2, a quality control line; 5. an absorbent pad.
Detailed Description
For the purposes of promoting an understanding of the principles and advantages of the disclosure, reference will now be made in detail to the following specific examples.
It should be noted that unless otherwise defined, technical terms used in the following examples have the same meaning as commonly understood by those skilled in the art to which the present application pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
Toxins are often secondary metabolites produced by bacteria during harvesting, transportation and storage of cereal and feed, and the misingestion of the metabolites by humans or animals may cause diseases or death due to mishandling of the metabolites, and therefore are of great importance for the detection of mycotoxins in cereal feeds. The detection methods commonly used at present mainly comprise high performance liquid chromatography, liquid chromatography-mass spectrometry tandem method, gas chromatography-mass spectrometry tandem method and the like, and although the detection methods can accurately detect toxins, pretreatment of detection samples is complex, and the instrument cost is high, so that the methods cannot be used for on-site instant detection.
The lateral flow immunochromatography test strip has the advantages of simple operation, low cost, short detection time, convenient carrying and the like, is one of important methods for on-site instant detection, however, the traditional lateral chromatography test strip has the defect of low detection sensitivity and the like, and the surface enhanced Raman scattering SERS technology is a signal enhancement effect mediated by a metal nano structure, can realize single-molecule Raman signal enhancement of which the sensitivity is up to 10-14 times, has the advantages of high sensitivity, no interference by aqueous solution, no damage to a sample, narrower spectral half-peak width, easy operation and the like, and can be widely applied to the fields of food safety, medical diagnosis, environmental detection and the like, so that the SERS technology and the lateral flow immunochromatography are combined to provide a new method for on-site high-sensitivity multi-element detection of mycotoxins.
However, the existing SERS lateral chromatography test strip based on gold nanoparticles has a small enhancement effect on detection signals, is particularly easy to be influenced by detection environments, has extremely poor stability, and is relatively sensitive to external interferents due to high surface energy, so that the gold nanoparticles are easy to agglomerate and precipitate, and therefore, the stability of the gold nanoparticles is poor in detection, and the application of the gold nanoparticles in complex environments is limited, so that the SERS lateral chromatography test strip with high stability and capability of enhancing the detection signal intensity is needed to be developed.
The following describes the technical solution of the present application in detail by specific embodiments in conjunction with fig. 1 to 8.
In some embodiments of the present application, there is provided a lateral chromatography test strip for detecting toxins, as shown in fig. 1, comprising: the device comprises a bottom plate 1, wherein a sample pad 2, a bonding pad 3, a chromatographic membrane 4 and an absorption pad 5 are sequentially arranged on the bottom plate 1 along the length direction, and a detection line 4-1 and a quality control line 4-2 are arranged on the chromatographic membrane 4; a toxin marker antigen is attached to the detection line 4-1; the secondary antibody is attached to the quality control line 4-2; the binding pad 3 is attached with a detection antibody connected with a Raman probe, and the Raman probe comprises a bound Raman beacon molecule and silica-coated gold nanoparticles.
By providing the base plate 1 to carry the sample pad 2, the conjugate pad 3, the chromatographic carrier 4 and the absorbent pad 5, the material of the base plate 1 may be polyvinyl chloride resin, polymethyl methacrylate, polyethylene terephthalate, polyethylene or the like, and is not particularly limited.
The sample pad 2 is arranged for absorbing the detection sample, can separate and filter impurities in the detection sample, pre-treat the sample, balance the pH value of the detection sample, adjust the salt ion intensity and the like, so that the detection sample can keep certain uniformity and controllability in the chromatography process, and the material of the sample pad 2 can be glass fiber paper without limitation.
The conjugate pad 3 has a detection antibody attached thereto, and the conjugate pad 3 is provided for interaction of a detection sample flowing through the sample pad 2 with the detection antibody, for example, AFB, and flows to the chromatographic carrier 4 1 The material of the conjugate pad 3 for binding to the toxin such as a detection antibody may be glass fiber paper, and is not particularly limited.
The chromatographic membrane 4 can be made of nitrocellulose, the pore diameter of the chromatographic membrane 4 can be 2-8 mu m, the chromatographic membrane 4 is not limited in particular, a detection line 4-1 and a quality control line 4-2 are sequentially arranged on the chromatographic membrane 4, and a toxin marker antigen is attached to the detection line 4-1 and used for combining with a detection antibody to develop color so as to form a competition relationship with toxin in a detection sample; the quality control line 4-2 is attached with a secondary antibody for combining with a detection antibody for color development, and verifying whether the detection is effective; toxin marker antigens are, for example, haptens to which the toxin marker binds to bovine serum albumin, and secondary antibodies are, for example, capable of binding to AFB 1 The antibody to which the detection antibody binds is not particularly limited.
The material of the absorbent pad 5 may be absorbent paper, and is not particularly limited, and the absorbent pad 5 is provided to absorb the detection liquid through the chromatographic carrier 4 by capillary action and collect the treated waste liquid, so that a larger amount of the detection sample can be used, thereby improving the test sensitivity.
The detection antibody is combined with a Raman probe, so that the intensity and sensitivity of detection signals can be improved, the Raman probe comprises combined Raman beacon molecules and silica coated gold nanoparticles, the Raman beacon molecules are used for Raman signal detection, a basis is provided for quantitative analysis of toxin concentration, and the silica coated gold nanoparticles are Au@SiO 2 The core-shell particles can enhance the electromagnetic field intensity at the gap of the core-shell, further improve the Raman signal intensity, and the silicon dioxide shell has stable chemical property, has an anti-corrosion function, can protect gold nanoparticles from being interfered by detection environment, reduces the possibility of agglomeration, has good biocompatibility, and can further improve the detection sensitivity and reduce the measurement limit value.
The lateral chromatography test strip for detecting toxins has the advantages that the stability of the Raman probe is good, the detection intensity of Raman signals is high, the detection sensitivity of the test strip is high, various mycotoxins can be detected under a complex environment, the safety of cereal feeds is guaranteed, the concentration of toxins can be quantitatively analyzed through the Raman signal intensity of the lateral chromatography test strip after detection and test, and a basis is provided for accurate judgment.
In some embodiments, at least two kinds of the toxin-marker antigens are attached to the detection line 4-1, at least two kinds of the detection antibodies corresponding to the toxin-marker antigens are attached to the binding pad 3, and at least two kinds of the secondary antibodies corresponding to the detection antibodies are attached to the quality control line 4-2.
By arranging at least two toxin marker antigens on the detection line 4-1, arranging at least two secondary antibodies on the quality control line 4-2 and arranging at least two detection antibodies on the binding pad 3, multiple toxins can be detected on one lateral chromatographic test strip at the same time, and each set of toxin marker antigen, secondary antibody and detection antibody correspondingly detects one toxin, so that the on-site detection efficiency can be improved, and the resource utilization rate is also improved; different raman beacon molecules can be bound for each detection antibody, providing a basis for quantitative analysis of different toxin concentrations.
In some embodiments, the sample pad 2 and the conjugate pad 3 each have a thickness of 0.3mm to 0.5mm, and the total length of the sample pad 2 and the conjugate pad 3 is 15mm to 20mm; the length of the chromatographic membrane 4 is 22mm-30mm, and the distance between the detection line 4-1 and the quality control line 4-2 is 6mm-8mm; the thickness of the absorption pad 5 is 0.5mm-0.8mm, and the length is 25mm-30mm; the length of the bottom plate 1 is 75mm-90mm, and the thickness is 3mm-5mm; the overlapping portion between the sample pad 2, the conjugate pad 3, the chromatographic carrier 4 and the absorbent pad 5 may be 2mm to 4mm, and is not particularly limited.
In some embodiments, a method of preparing a lateral chromatography test strip for detecting a toxin is provided, comprising the steps of:
s1, preparing a Raman probe.
S2, connecting the Raman probe with a detection antibody.
S3, attaching the detection antibody connected with the Raman probe to a binding pad 3, attaching a toxin marker antigen to a detection line 4-1 of a chromatographic membrane 4, and attaching a secondary antibody to a quality control line 4-2 of the chromatographic membrane 4.
And S4, arranging the sample pad 2, the combination pad 3, the chromatographic membrane 4 and the absorption pad 5 on the bottom plate 1 to obtain the lateral chromatography test strip.
In some embodiments, the preparing a raman probe comprises:
s101, carrying out centrifugal operation on colloidal gold combined with a Raman beacon molecule to obtain a first precipitate, adding water and isopropanol into the first precipitate, and stirring to obtain a first solution.
The raman beacon molecule may comprise one or more of nielan a, p-mercaptobenzoic acid, methylene blue, 5-dithiocarbamic acid, rhodamine 6G, p-aminophenylthiophenol, malachite green, 4-mercaptopyridine, p-mercaptoaniline, or p-aminophenylthiophenol; colloidal gold is a conjugate widely used in lateral chromatography test strips because it can produce intense color, is easy to couple, and has stable quality.
The colloidal gold combined with the Raman beacon molecules can react for 20min-30min by dripping the Raman beacon molecules into the colloidal gold, and the Raman beacon molecules are connected to the surfaces of the metal nano particles through physical adsorption or covalent bonding.
Step S101 can take 20mL of colloidal gold with the size of 45-60 nm and combined with the Raman beacon molecules, centrifuge the colloidal gold at 4000 rpm to obtain first precipitate of the colloidal gold combined with the Raman beacon molecules, collect the first precipitate in a centrifuge tube to remove floating excess Raman beacon molecules, then add 20mL of deionized water and 100mL of isopropanol solution into the centrifuge tube to stir and mix, and manufacture an organic system, so that subsequent crust formation is facilitated.
S102, adding ammonia water into the first solution, adjusting the pH value to a preset value, gradually adding a tetraethyl silicate isopropanol solution, and stirring for reaction to obtain a second solution.
The preset value is, for example, 9.5 to 10.5, and is not particularly limited.
Step S102 may be to add 2.5mL ammonia water with a mass concentration of 30% into the first solution, adjust the pH value to 10, then gradually add 2mL tetraethyl silicate isopropyl alcohol solution with a concentration of 10mM in 5h, for example add 0.4mL tetraethyl silicate isopropyl alcohol solution every 1h, hydrolyze tetraethyl silicate by ammonia water, stir at normal temperature for 3h, make the silica coated nano-particle gold shell, and obtain the second solution, where the second solution contains the Raman probe, that is, the mutually combined silica coated gold core-shell structure nano-particle and Raman beacon molecule, where the Raman beacon molecule is physically adsorbed or covalently connected on the surface of the nano-gold particle or in the gap of the core-shell structure nano-particle.
And S103, refrigerating and storing the second solution for a preset time, performing centrifugal operation to obtain a second precipitate, washing the second precipitate, and then re-suspending the second precipitate in water to obtain a third solution containing the Raman probe.
The refrigerating temperature is, for example, 3 ℃, 4 ℃ or 5 ℃, and the preset time is, for example, 12 hours, 16 hours, 20 hours or 24 hours, etc., and the refrigerating temperature is not particularly limited.
Step S103 can store the second solution at 3-5 ℃ for at least 12h and then carry out centrifugal operation to obtain a second precipitate, so that the structure of the Raman probe is more stable, the second precipitate is washed by ethanol or distilled water to remove impurities, and the second precipitate is resuspended in 20mL of deionized water for storage, so that a third solution containing the Raman probe which is more pure, uniformly dispersed and stable in structure is obtained.
In some embodiments, the linking the raman probe to the detection antibody comprises:
and S201, adding the ethanol solution of the 3- (triethoxysilylpropyl carbamoyl) butyric acid into the third solution for shaking reaction, and centrifuging to obtain a third precipitate.
Step S201 may be to add 4.5mL of 3- (triethoxysilylpropyl carbamoyl) butanoic acid ethanol solution with a concentration of 4.5mM to 8mL of the third solution to hydrolyze the acid anhydride to form carboxyl groups, then add 32mL of ethanol solution, shake and react at room temperature for 2 hours to connect the carboxyl groups with the Raman probe, and centrifuge to obtain a third precipitate, wherein the third precipitate includes the carboxyl-modified Raman probe.
S202, washing the third precipitate, then re-suspending in water to obtain a fourth solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and sulfonated N-hydroxysuccinimide into the fourth solution for activation, adding a detection antibody for incubation reaction, and centrifuging to obtain the detection antibody connected with the Raman probe.
Step S202 can be performed by washing the third precipitate with an ethanol solution for 4 times to remove impurities, then re-suspending the third precipitate in 4mL of deionized water to obtain a fourth solution, adding 5mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 890 mu L of 0.1M phosphate buffer and 10 mu L of 0.5mM sulfonated N-hydroxysuccinimide aqueous solution into 100 mu L of the fourth solution, uniformly mixing the mixture at room temperature for 2 hours to activate carboxyl groups, washing the activated Raman probe three times with the phosphate buffer to remove impurities, then re-suspending the activated Raman probe into 200 mu L of the phosphate buffer, adding a detection antibody and the Raman probe for incubation reaction, and centrifuging the mixture to obtain the detection antibody connected with the Raman probe.
In some embodiments, the application provides a method of using a lateral chromatography test strip for detecting toxins, comprising:
a. the detection sample is dripped on the sample pad 2 of the lateral chromatography test strip.
The detection sample can also be dripped into the sample pad 2 after reacting with the detection antibody in the container, so that the toxin in the detection sample can be conveniently and fully acted with the detection antibody, and the detection result is more accurate.
b. After the preset reaction time, a detection result is obtained according to the color development conditions of the detection line 4-1 and the quality control line 4-2.
The preset reaction time is, for example, 1min, and is not particularly limited.
The lateral chromatography test strip belongs to a competition method test strip, and when the detection line 4-1 and the quality control line 4-2 are both colored and the degree of the color development is the same, the detection result is negative; when the color development degree of the detection line 4-1 is weaker than that of the quality control line 4-2, the detection result is positive, and the lighter the color of the detection line 4-1 is, the higher the concentration of toxin is; and when the quality control line 4-2 does not develop color, the detection result is invalid.
c. And measuring the Raman signal intensity of the detection line 4-1, and determining the toxin concentration according to the Raman signal intensity.
For the lateral chromatography test strip with positive detection result, the Raman signal intensity of the detection line 4-1 can be further measured, the actually detected toxin concentration can be determined according to the pre-acquired Raman signal intensity and concentration relation curve, quantitative analysis of toxin is realized, and the guarantee is provided for cereal feed safety.
For example, a plurality of aflatoxin B can be detected simultaneously 1 (AFB 1 ) And ochratoxin A (OTA), wherein AFB is attached to the detection line 4-1 of each lateral chromatography test strip 1 Toxin marker antigen and OTA toxin marker antigen, AFB is attached to the conjugate pad 3 1 Detection antibody and OTA detection antibody, and AFB is attached to quality control line 4-2 1 Secondary antibodies and OTA secondary antibodies.
Testing AFB of different concentrations using the lateral chromatography test strip 1 And an OTA solution sample and measuring the corresponding Raman signal intensity, as shown in the Raman spectrum of FIG. 6, the Raman spectrum having AFB 1 And two characteristic peaks of OTA, because the lateral chromatography test strip belongs to a test strip for detecting by a competition method, the higher the toxin concentration content is, the correspondingThe lower the Raman intensity of the sample, the corresponding relation can be obtained according to different toxin concentrations and corresponding Raman intensities, and the Raman signal intensity-AFB is shown in FIG. 7 1 The concentration relation curve graph is shown in fig. 8 as a raman signal intensity-OTA concentration relation curve graph, and the two relation curves can be used as reference bases, and after the raman signal intensity of the actual sample is detected, the toxin concentration of the actual sample can be determined according to the relation curve graph, so that quantitative analysis is realized.
Example 1
And (3) obtaining a second solution through the step S101 and the step S102, storing the second solution at the temperature of 4 ℃ for 12 hours, performing centrifugal operation to obtain a second precipitate, washing the second precipitate with distilled water, and then re-suspending the second precipitate in water to obtain a third solution.
Example 2
And (3) obtaining a second solution through the step S101 and the step S102, storing the second solution at the temperature of 4 ℃ for 8 hours, performing centrifugal operation to obtain a second precipitate, washing the second precipitate with distilled water, and then re-suspending the second precipitate in water to obtain a third solution.
Example 3
And (3) obtaining a second solution through the step S101 and the step S102, storing the second solution at the temperature of 4 ℃ for 4 hours, performing centrifugal operation to obtain a second precipitate, washing the second precipitate with distilled water, and then re-suspending the second precipitate in water to obtain a third solution.
Example 4
And (3) obtaining a second solution through the step S101 and the step S102, directly centrifuging the second solution to obtain a second precipitate, washing the second precipitate with distilled water, and then, re-suspending the second precipitate in water to obtain a third solution.
Example 1-example 4 different third solutions were prepared by varying the time of storage of the raman probe in cold storage, and scanning electron microscopy was performed on each of the third solutions of example 1 to obtain a corresponding electron micrograph, and the electron micrograph of the third solution of example 1 is shown in fig. 2, i.e., the electron micrograph of the raman probe prepared by storage for 12 hours, it can be seen that the raman probe was uniformly dispersed, siO 2 The thickness of the shell is about 6nm, the detachment phenomenon is avoided, and the structural stability is good; an electron micrograph of the third solution of example 2 is shown in FIG. 3, which shows a pull prepared by refrigerated storage for 8hThe electron microscope image of the Raman probe shows that the Raman probe is dispersed with partial agglomeration phenomenon, siO 2 A small amount of detachment of the shell also occurs; the electron micrograph of the third solution of example 3 is shown in FIG. 4, which shows that the Raman probe prepared by cold storage for 4 hours has relatively poor dispersion, more agglomeration and SiO 2 The shell is separated more, and the stability is poor; the electron micrograph of the third solution of example 4 is shown in FIG. 5, which shows that the aggregation of the Raman probe is serious and a large amount of SiO is observed 2 The shell is separated; the structural stability of the silicon dioxide coated gold nano particles is gradually enhanced along with the extension of the refrigerating and storing time of the Raman probe, and the Raman probe stored for more than 12 hours in the refrigerating and storing process is uniformly dispersed and has stronger anti-interference capability.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined under the idea of the present disclosure, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in details for the sake of brevity.
The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Accordingly, any omissions, modifications, equivalents, improvements, and the like, which are within the spirit and principles of the embodiments of the disclosure, are intended to be included within the scope of the disclosure.
Claims (4)
1. A lateral chromatography test strip for detecting toxins, comprising: the device comprises a bottom plate, wherein a sample pad, a bonding pad, a chromatographic membrane and an absorption pad are sequentially arranged on the bottom plate along the length direction, and a detection line and a quality control line are arranged on the chromatographic membrane;
a toxin marker antigen is attached to the detection line;
the quality control line is attached with a secondary antibody;
the binding pad is attached with a detection antibody connected with a Raman probe, and the Raman probe comprises a combined Raman beacon molecule and silica-coated gold nanoparticles;
at least two toxin marker antigens are attached to the detection line, at least two detection antibodies corresponding to the toxin marker antigens are attached to the binding pad, and at least two secondary antibodies corresponding to the detection antibodies are attached to the quality control line;
the thickness of the sample pad and the bonding pad is 0.3mm-0.5mm, and the total length of the sample pad and the bonding pad is 15mm-20mm; the length of the chromatographic membrane is 22mm-30mm, and the distance between the detection line and the quality control line is 6mm-8mm; the thickness of the absorption pad is 0.5mm-0.8mm, and the length is 25mm-30mm; the length of the bottom plate is 75mm-90mm, and the thickness of the bottom plate is 3mm-5mm;
the Raman beacon molecule comprises one or more of Nepal blue A, p-mercaptobenzoic acid, methylene blue, 5-dithiocarbamic acid, rhodamine 6G, p-aminophenylthiophenol, malachite green, 4-mercaptopyridine, p-mercaptoaniline or p-aminophenylthiophenol;
the preparation method of the Raman probe comprises the following steps:
centrifuging colloidal gold combined with Raman beacon molecules to obtain first precipitate, adding water and isopropanol into the first precipitate, and stirring to obtain a first solution;
adding ammonia water into the first solution, adjusting the pH value to a preset value, gradually adding a tetraethyl silicate isopropanol solution, and stirring for reaction to obtain a second solution, wherein the method comprises the following steps of: adding ammonia water into the first solution, adjusting the pH value to 10, gradually adding a tetraethyl silicate isopropanol solution within 5 hours, and stirring at normal temperature for reaction for 3 hours to obtain a second solution;
refrigerating and storing the second solution for a preset time, performing centrifugal operation to obtain a second precipitate, washing the second precipitate, and then re-suspending the second precipitate in water to obtain a third solution containing the Raman probe; and (3) carrying out centrifugal operation after refrigerating and storing the second solution for a preset time to obtain a second precipitate, wherein the method comprises the following steps of: and (3) storing the second solution at 3-5 ℃ for at least 12 hours, and then performing centrifugal operation to obtain a second precipitate.
2. A method of preparing a lateral flow chromatographic test strip for detecting toxins of claim 1, comprising:
preparing a Raman probe;
ligating the raman probe to a detection antibody;
attaching the detection antibody connected with the Raman probe to a binding pad, attaching a toxin marker antigen to a detection line of a chromatographic membrane, and attaching a secondary antibody to a quality control line of the chromatographic membrane;
and arranging the sample pad, the combination pad, the chromatographic membrane and the absorption pad on a bottom plate to obtain the lateral chromatography test strip.
3. The method of claim 2, wherein the step of attaching the raman probe to the detection antibody comprises:
adding 3- (triethoxysilylpropyl carbamoyl) butanoic acid ethanol solution into the third solution for shaking reaction, and centrifuging to obtain a third precipitate;
and (3) washing the third precipitate, then, re-suspending in water to obtain a fourth solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and sulfonated N-hydroxysuccinimide into the fourth solution for activation, adding a detection antibody for incubation reaction, and centrifuging to obtain the detection antibody connected with the Raman probe.
4. A method of using the lateral flow assay test strip of claim 1 for detecting toxins comprising:
dripping a detection sample on the sample pad of the lateral chromatography test strip;
after the preset reaction time, obtaining a detection result according to the color development conditions of the detection line and the quality control line;
and measuring the Raman signal intensity of the detection line, and determining the toxin concentration according to the Raman signal intensity.
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