CN107796938B - Nucleic acid aptamer fluorescent test strip and preparation method and application thereof - Google Patents
Nucleic acid aptamer fluorescent test strip and preparation method and application thereof Download PDFInfo
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- CN107796938B CN107796938B CN201710969466.6A CN201710969466A CN107796938B CN 107796938 B CN107796938 B CN 107796938B CN 201710969466 A CN201710969466 A CN 201710969466A CN 107796938 B CN107796938 B CN 107796938B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
Abstract
The invention provides an aptamer fluorescent test strip and a preparation method and application thereof, and relates to the technical field of biological detection. A nucleic acid aptamer fluorescent test strip comprises a bottom plate, a sample pad, a nano-material pad, a reaction membrane and a water absorption pad, wherein the reaction membrane is provided with a detection line which is arranged at one end close to the nano-material pad; the nano material pad is attached with fluorescein-labeled aptamer and/or fluorescence-labeled aptamer with a connecting sequence and free fluorescein, and the complementary strand of the aptamer or the complementary strand of the aptamer connecting sequence is fixed on the detection line. The test strip has simple structure, sensitive detection and high accuracy. A preparation method of a nucleic acid aptamer fluorescent test strip comprises the following steps: and sequentially adhering the sample pad, the nano material pad, the reaction membrane and the water absorption pad to the same side of the bottom plate. The operation is simple and the flexibility is strong.
Description
Technical Field
The invention relates to the technical field of biological detection, and particularly relates to a nucleic acid aptamer fluorescent test strip and a preparation method and application thereof.
Background
The test strip is widely used in clinical diagnosis, food safety and environmental monitoring as a simple and rapid analysis tool. The existing test strip mainly has two detection modes of colloidal gold and fluorescence, wherein the colloidal gold test strip displays a result through a color reaction and has the advantage of being visible by naked eyes without other instruments; however, the disadvantage is the low sensitivity and the semi-quantitative result can only be achieved by comparing the shade of the color. Compared with a colloidal gold test strip, the fluorescent test strip has high sensitivity, and can accurately quantify a target object in a sample by matching with a portable fluorescent detector, so that the fluorescent test strip becomes a development direction of the test strip in recent years.
However, both colloidal gold test strips and fluorescent test strips are basically test strips based on antigen-antibody reaction at present, and the antigen-antibody as active protein needs to be stored and transported at low temperature, so the shelf life is short, and the application of the technology in remote laggard areas is limited. Moreover, because many small-molecule substances are difficult to prepare haptens and difficult to stimulate high-affinity antigen antibodies, the application of the technology is limited.
The aptamer is a recognition molecule newly discovered in recent years, mainly refers to single-stranded DNA with the length of 10-100 bases, and can be combined with a target molecule with high affinity and high specificity after being folded by conformation. The aptamer is obtained by in vitro screening, so compared with an antibody, the aptamer is not limited in target, the aptamer can be screened from metal ions as small as protein molecules, the screened aptamer can be synthesized in batches after sequencing, the aptamer is easy to modify and good in stability, can be used in various antibody analysis methods instead of an antibody, and is also called a chemical antibody.
The same aptamers have also been used in test strip development instead of antibodies, in a similar principle to conventional test strips. However, the current colloidal gold or fluorescent test strips based on nucleic acid aptamers are not many, and the main reason is that different aptamer sequences are different in length, most of the aptamer sequences are relatively long, so that the affinity of the aptamer sequences to form a double strand with a complementary strand is greater than that of the aptamer sequences to form a complex with a target, and therefore, the aptamer sequences combined with the target are still dissociated at a detection line, and accurate detection of the target cannot be realized. Although the competitive power of the detection line for fixing the antigen is consistent, for many small molecular targets, after the small molecular targets are combined with the coupling protein, the combination with the aptamer is limited, so that the aptamer cannot be combined at the detection line, and sensitive and accurate detection cannot be realized.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the aptamer fluorescent test strip which is simple in structure, sensitive in detection and high in accuracy.
The invention also provides a preparation method of the nucleic acid aptamer fluorescent test strip, which is simple to operate and strong in flexibility.
The invention is realized by the following steps:
the invention provides a nucleic acid aptamer fluorescent test strip which comprises a bottom plate, a sample pad, a nano material pad, a reaction membrane and a water absorption pad, wherein the end parts of the sample pad, the nano material pad, the reaction membrane and the water absorption pad are sequentially connected to form a detection layer;
the nano material pad is attached with fluorescein-labeled aptamer and/or fluorescence-labeled aptamer with a connecting sequence and free fluorescein, a complementary chain of the aptamer or the nucleic acid aptamer connecting sequence is fixed on the detection line, and an anti-fluorescein antibody corresponding to the fluorescein is arranged at the quality control line.
The invention provides a preparation method of a nucleic acid aptamer fluorescent test strip, which comprises the following steps: and sequentially adhering the sample pad, the nano material pad, the reaction membrane and the water absorption pad to the same side of the bottom plate.
The invention provides an application of a nucleic acid aptamer fluorescent test strip in food safety, environmental monitoring and clinical diagnosis.
The beneficial effect of above-mentioned scheme:
according to the aptamer fluorescent test strip provided by the embodiment of the invention, a gold-labeled pad in a conventional test strip is replaced by a nano material pad, a nano material point can tightly adsorb a fluorescein-labeled aptamer and quench the fluorescence of the fluorescein-labeled aptamer, when a target exists, only the aptamer combined with the target dissociates, the fluorescence recovers and advances to a detection line along with the sample, and the detection line is enabled to generate fluorescence and is enhanced along with the increase of the concentration of the target through the combination of the aptamer, a complementary chain or an aptamer connecting sequence and a corresponding complementary chain. The method has universality, can be applied to aptamers of various lengths and also is suitable for targets of various molecular sizes; the method has sensitive, accurate and reliable measurement results, and avoids the inconsistency of the affinity of the aptamer complementary strand and the aptamer target and the inaccurate measurement caused by the change of the affinity due to the coupling of the small molecular target and the antigen. The test strip adsorbs the aptamer marked by the fluorescein to the nano material, and the stability of the fluorescein is improved through the quenching effect of the nano material on the fluorescein.
The test strip adopts fluorescent labels, has higher sensitivity, and can realize accurate quantitative detection of target molecules by measuring the fluorescence intensity through a fluorescence card reader.
The test strip adopts free fluorescein as quality control, and provides guarantee for the effectiveness of the test strip through the fluorescence labeling of a quality control line.
The test strip adopts the ratio of the detection line fluorescence signal to the quality control line fluorescence signal as the basis of the detection result, avoids the difference caused by different test strips and different operating environment conditions, improves the comparability between different operations, and ensures that the result is more accurate and reliable.
The test strip has the advantages of simple structure, convenient detection, sensitive and accurate detection, room-temperature storage and wide application prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic structural diagram of a nucleic acid aptamer fluorescent test strip provided by the invention;
FIG. 2 is a standard curve for detecting sulfadimethoxine in example 2 of the present invention;
FIG. 3 is a standard curve for detecting ochratoxin A in example 3 of the present invention.
Icon: 100-nucleic acid aptamer fluorescent test paper; 110-sample pad; 120-nanomaterial pad; 130-a reaction membrane; 131-a detection line; 133-quality control line; 140-absorbent pad; 150-a detection layer; 160-bottom plate.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following describes a nucleic acid aptamer fluorescent test strip, a preparation method and an application thereof in embodiments of the present invention.
The invention provides a nucleic acid aptamer fluorescent test strip which comprises a bottom plate, a sample pad, a nano material pad, a reaction membrane and a water absorption pad, wherein the end parts of the sample pad, the nano material pad, the reaction membrane and the water absorption pad are sequentially connected to form a detection layer;
the nano material pad is attached with fluorescein-labeled aptamer and/or fluorescence-labeled aptamer with a connecting sequence and free fluorescein, a complementary chain of the aptamer or the nucleic acid aptamer connecting sequence is fixed on the detection line, and an anti-fluorescein antibody corresponding to the fluorescein is arranged at the quality control line.
The sample pad adsorbs a sample to be measured, and the sample flows from the sample pad to the nanomaterial pad through chromatography, preferably, the sample pad is made of a cellulose membrane. The absorbent pad is used for absorbing the sample and can be absorbent filter paper.
Preferably, the nanomaterial pad may be a porous nanomaterial membrane or a glass cellulose membrane modified by a nanomaterial. The nanomaterial includes, but is not limited to, at least one of graphene oxide, reduced graphene, nanogold, molybdenum disulfide, and carbon nanotubes. The cellulose film includes, but is not limited to, at least one of glass fiber, cellulose filter paper, and polyester film.
The key point of the invention is that the gold-labeled pad of the conventional test strip is replaced by the nano-material pad, and the test strip can be suitable for aptamers with various lengths and various targets. The conventional test strip fixes the aptamer marked by colloidal gold or fluorescein on the gold-marked pad, and then reacts with the antigen or the complementary strand on the detection line to develop color or emit fluorescence. This approach is often limited: firstly, if a complementary strand is fixed on a detection line, for an aptamer with a longer sequence, the affinity of the aptamer and the complementary strand is greater than that of the aptamer and a target, so that a competitive reaction cannot be realized; secondly, if the target is fixed on the detection line, for the small molecular target, the small molecular target needs to be fixed after being coupled with the carrier protein, and the coupling limits the full combination of the aptamer and the target molecule, so that the affinity is reduced, and even the small molecular target cannot be combined finally, so that the detection cannot be finally performed.
The test paper strip disclosed by the invention has the following detection principle:
when the aptamer fluorescent test strip is used for detecting a sample, the sample to be detected is dripped on the sample pad, the sample can be chromatographed upwards under the capillary action, when the sample enters the nano material pad, a target in the sample can be combined with the fluorescence-labeled aptamer to form a compound, the fluorescein-labeled aptamer is dissociated from the surface of the nano material, and simultaneously, free fluorescein in the nano material pad is chromatographed on the nitrocellulose membrane together with the target-aptamer compound under the drive of the sample.
When the target-aptamer complex reaches the detection line, the target-aptamer complex is combined with a complementary strand of the biotin-modified aptamer or a complementary strand of the aptamer connecting sequence fixed on the detection line, and fluorescence appears in the detection line. The more the target amount is, the more dissociated target-aptamer complexes are, the more fluorescently-labeled aptamers bound to the detection line are, and the stronger the fluorescence signal of the detection line is; conversely, the fewer the targets in the sample, the lower the fluorescence signal at the detection line, so that the target in the sample can be quantified by giving the signal strength through fluorescence.
If the sample does not contain the target to be detected, the fluorescein-labeled aptamer is tightly bound to the nanomaterial without being dissociated, so that no fluorescence is present at the detection line. Whether the sample has the target to be detected or not, the free fluorescein on the nano material pad is combined by the anti-fluorescein antibody at the quality control line when the free fluorescein ascends to the quality control line, and the quality control line generates fluorescence. If no fluorescence appears at the quality control line, the test strip is invalid, and the test strip is replaced for retesting. The quality control line can timely know whether the test strip is invalid or not, and the detection accuracy is improved.
A preparation method of the aptamer fluorescent test strip comprises the following steps:
the preparation method of the nano material pad modified by the nano material comprises the following steps: selecting a cellulose membrane as a raw material for preparing the nano material pad, spraying the nano material suspension solution on the cellulose membrane, drying for 7-9 hours at the temperature of 110-130 ℃, and removing water on the glass cellulose membrane to obtain the cellulose membrane modified by the nano material. And cutting according to the requirement to obtain the nano material pad.
The preparation method of the nano material pad adsorbed with the fluorescence labeling aptamer comprises the following steps: and (2) dropwise adding the fluorescence-labeled aptamer and/or the fluorescence-labeled aptamer with a connecting sequence and free fluorescein to the nano material, incubating for 15-25 min, and drying at 35-40 ℃ to remove the moisture of the nano material. Preferably, fluorescein can be Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), anthocyanin 5(Cy5), carboxyfluorescein (FAM), quantum dots, upper transfer, fluorescent microspheres, and the like, which are not limited in the present invention. The aptamer selects specific aptamers according to different targets and designs corresponding connecting sequences. Preferably, the aptamer connection sequence comprises one of polyTn, polyAn, polyCn and polyGn, wherein n is an integer of 6-25.
The reaction membrane is a portion where a fluorescent color reaction occurs. In the embodiment of the present invention, a nitrocellulose membrane is used as the reaction membrane, and in other embodiments of the present invention, other materials may be used for the reaction membrane. The reaction membrane is provided with a detection line for detecting the target.
The preparation method of the reaction membrane detection line comprises the following steps: and incubating the complementary strand of the aptamer modified by biotin or the complementary strand of the aptamer connecting sequence with avidin for 25-35 min, and directly spraying the incubated complementary strand to a detection line. Preferably, the complementary strand of the aptamer linker corresponding to the aptamer linker comprises one of polyAn, polyTn, polyGn and polyCn, wherein n is an integer of 6 to 25.
One end of the reaction membrane close to the water absorption pad is also provided with a quality control line for verifying whether the test strip is effective. The preparation method of the reaction film quality control line comprises the following steps: and spraying the anti-fluorescein antibody corresponding to the fluorescein for marking the aptamer to the position of the quality control line, and drying.
After the nano material pad and the reaction film are prepared, the sample pad, the nano material pad, the reaction film and the water absorption pad are sequentially adhered to the same side of the bottom plate. The fluorescent test strip has the advantages of simple structure, sensitive detection and high accuracy.
The invention also provides a detection application of the nucleic acid aptamer fluorescent test strip in food safety, environmental monitoring and clinical diagnosis, which comprises the following steps:
s1, detecting a target standard substance by using a test strip to obtain a fluorescence signal intensity ratio of the standard substance in a detection line and a quality control line of the test strip, making a linear regression curve of the concentration of the target standard substance corresponding to the signal intensity ratio, and calculating a regression equation;
s2, detecting the sample to be detected by using the test strip to obtain the fluorescence signal intensity ratio of the sample to be detected in the detection line and the quality control line of the test strip, and obtaining the content of the target object in the sample to be detected according to the regression equation obtained in S1.
The method for detecting the fluorescence signal intensity ratio of the detection line and the quality control line comprises the following steps: dripping a target standard substance or a sample to be detected on a sample absorption pad of the test paper, after 8-12 min, putting the test paper into a test paper detection instrument ESEQuant-LR3 for detection, and respectively detecting the fluorescence signal intensity of a detection line and a quality control line; and comparing the fluorescence signal intensity (T) of the detection line with the fluorescence signal intensity (C) of the quality control line to obtain a T/C value, namely the fluorescence signal ratio of the detection line and the quality control line.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Referring to fig. 1, the present invention provides a nucleic acid aptamer fluorescent test strip 100, which includes a bottom plate 160, a sample pad 110, a nano-material pad 120, a reaction membrane 130, and a water absorption pad 140, wherein the ends of the sample pad 110, the nano-material pad 120, the reaction membrane 130, and the water absorption pad 140 are sequentially connected to form a detection layer 150, and the detection layer 150 is connected to the bottom plate 160.
In the embodiment of the present invention, the aptamer fluorescent test strip 100 may be a conventional strip, and the sample pad 110, the nanomaterial pad 120, the reaction membrane 130 and the absorbent pad 140 are all corresponding strips, in other embodiments, the aptamer fluorescent test strip 100 may be a circle or other shapes, and the shapes of the sample pad 110, the nanomaterial pad 120, the reaction membrane 130 and the absorbent pad 140 are also changed accordingly, which is not limited by the present invention.
The reaction membrane 130 is provided with a detection line 131 and a quality control line 133, the detection line 131 is disposed at one end close to the nanomaterial pad 120, and the quality control line 133 is disposed at one end close to the water absorption pad 140. A certain distance is kept between the detection line 131 and the quality control line 133 to distinguish the color development reason of fluorescence and distinguish a target from free fluorescein.
Example 2
The embodiment provides a sulfadimethoxine aptamer test strip, a preparation method, detection and application thereof.
The preparation method of the test strip for the sulfadimethoxine aptamer comprises the following steps:
selecting a glass cellulose membrane as a raw material for preparing the nano material pad, spraying the graphene oxide suspension solution to the glass cellulose membrane, drying for 8 hours at 120 ℃, and removing water on the glass cellulose membrane to obtain the graphene oxide modified glass fiber. Cutting to obtain the nanometer material.
The Cy5 fluorescein-labeled sulfadimethoxine aptamer (Cy5-GAGGG-CAACGAGTGTTTATAGATTTTTTTTTTTTTTT) containing the linked sequence of polyT15 was added dropwise to the nanomaterial pad, incubated for 20 minutes, dried at 37 ℃ and the nanomaterial was dehydrated.
Selecting a nitrocellulose membrane as a reaction membrane. Incubating a complementary sequence polyA15(Btiotin-AAAAAAAAAAAAAAA) of the biotin-modified aptamer connecting chain with avidin for 30min, and spraying the avidin to a detection line; antibody against Cy5 was sprayed at the mass control line.
And sequentially adhering the sample pad, the nano material pad, the reaction membrane and the water absorption pad to the same side of the bottom plate.
Secondly, the detection principle of the test strip is as follows:
as shown in fig. 1, when the sample containing sulfadimethoxine was added to the sample pad, the sample flowed to the nanomaterial pad by capillary action, and the sulfadimethoxine in the sample bound to the aptamer on the nanomaterial pad, causing it to dissociate from the nanomaterial pad and continue to flow forward with the free fluorescein. When flowing to the detection line, the connecting sequence on the aptamer combined with the target can be combined with the complementary sequence of the connecting sequence on the detection line, so that fluorescence appears at the detection line, and the more sulfadimethoxine in the sample, the stronger the fluorescence intensity on the detection line; conversely, the less the sulfadimethoxine in the sample, the lower the fluorescence intensity on the detection line, so that the sulfadimethoxine in the sample can be quantitatively detected according to the fluorescence intensity. Meanwhile, free fluorescein in the liquid continuously ascends to the position of the quality control line and is captured by the antibody at the position, and a fluorescence signal appears. Fluorescence on the detection line and the quality control line can be detected by a test strip detection instrument ESEQuant-LR3 to detect the intensity of a fluorescence signal.
As shown in fig. 1, when the sample does not contain sulfadimethoxine, the aptamer cannot be dissociated from the nanomaterial pad, so that no aptamer is bound at the detection line, and no fluorescence signal is generated. And the free fluorescein on the nano material pad can still move upwards to the quality control line of the reaction membrane under the drive of the sample liquid and is captured by the anti-fluorescein antibody, and the quality control line generates a fluorescence signal.
And thirdly, a detection method of the test strip of the sulfadimethoxine aptamer.
Dripping a sulfadimethoxine standard substance with a series of concentrations on a sample absorption pad of the test paper, after 10 minutes, putting the test paper into a test paper detection instrument ESEQuant-LR3 for detection, and respectively determining the fluorescence signal intensity of a detection line and a quality control line; comparing the fluorescence signal intensity (T) of the detection line with the fluorescence signal intensity (C) of the quality control line to obtain a T/C value, making a linear regression curve of the concentration logarithm of the sulfadimethoxine standard substance corresponding to the fluorescence signal T/C ratio, and calculating a regression equation;
replacing the amine dimethoxy pyrimidine standard substance with a sample to be detected, detecting the sample to be detected by using the test paper, recording the ratio of fluorescence signals of a detection line and a quality control line of the test paper, and obtaining the concentration of the aflatoxin in the sample to be detected according to the regression equation in the step (1).
And fourthly, researching the performance of the test paper strip of the sulfadimethoxine.
1. Sensitivity and linear range for detecting sulfadimethoxine
Prepare sulfadimethoxine aqueous solutions with different concentrations, and respectively use the test paper prepared in example 2 to perform determination. The test strip detection instrument ESEQuant-LR3 is used for respectively detecting the fluorescence at the detection line and the quality control lineLight intensity. The ratio of the fluorescence intensity of the detection line to the fluorescence intensity of the quality control line (table 1) is utilized to draw a standard curve for detecting the sulfadimethoxine as shown in figure 2, and the result shows that the sensitivity of the test strip is 20 ng/mL; the linear range is 20 ng/mL-500 ng/mL. The regression equation is that y is 1.4064x-1.8343, R2＝0.9693。
TABLE 1 test results for sulfadimethoxine standard
2. Study of specificity
The test strip is used for detecting sulfadimethoxine solution, chloramphenicol solution, tetracycline solution, enrofloxacin solution and amantadine solution with the concentration of 100ng/mL respectively, and the result (table 2) shows that only sulfadimethoxine has obvious fluorescent signals at the T line and almost no fluorescent signals at other drug detection lines, so that the test strip has good specificity.
TABLE 2 test paper test specificity of sulfadimethoxine
Application of test strip for detecting sulfadimethoxine in milk
1. Drawing of standard curve
In the same way as the standard curve method in the sensitivity and linear range research of detecting sulfadimethoxine in the first part of this example, the regression equation obtained is: 1.4064x-1.8343, R20.9693, which is a standard curve univariate regression equation of sulfadimethoxine.
2. Sample extraction
Selecting 5 milk samples which are determined to be free of sulfadimidine by a national standard method (LC-MSMS), respectively adding sulfadimidine with different concentrations, and then extracting the samples, wherein the extraction method comprises the following steps: putting 2mL of milk into a PVC tube, adding 2mL of deionized water, shaking and shaking for 10min in a full-temperature shaking incubator (27 ℃, 180r), adding 7mL of ethyl acetate, shaking and shaking for 15min under the same condition, centrifuging for 15min at 5000r/4 ℃, taking supernatant, adding 7mL of ethyl acetate, shaking and centrifuging under the same condition, taking supernatant after the second centrifugation is finished, blowing nitrogen to precipitate, and finally dissolving with 2mL of deionized water.
3. Sample assay
Dropwise adding 100 mu L of the prepared sample to be detected on a sample absorption pad of the test strip, and observing the flow of sample chromatography along the nitrocellulose membrane until the sample chromatography is absorbed by the water absorption pad on the nitrocellulose membrane; after 10 minutes, putting the test strip into a test strip detection instrument ESEQuant-LR3 for detection, and respectively detecting the fluorescence signal intensity of a detection line and a quality control line; and (3) comparing the fluorescence signal intensity (T) of the detection line with the fluorescence signal intensity (C) of the quality control line, substituting the obtained calculated value into the regression equation in the step 1, and calculating to obtain the concentration of the sulfadimethoxine in the sample to be detected.
4. And (3) detection results:
the results of the 5 milk samples are shown in Table 3. The results show that the recovery rates of 5 samples are between 89% and 105%, and the recovery rates are good, which indicates that the test strip has high accuracy. In addition, the standard deviation of the method is less than 10%, which shows that the method has good stability. Therefore, the method can be used for detecting the sulfadimethoxine in actual samples.
TABLE 3 result of the test strip of the present invention for detecting sulfadimethoxine in milk
Example 3
The embodiment provides preparation and application of an ochratoxin A test strip.
A preparation method of the ochratoxin A test strip comprises the following steps:
selecting a glass cellulose membrane as a raw material for preparing the nano material pad, spraying the graphene oxide suspension solution to the glass cellulose membrane, drying for 7 hours at the temperature of 110 ℃, and removing water on the glass cellulose membrane to obtain the graphene oxide modified glass fiber. Cutting to obtain the nanometer material.
Dripping an ochratoxin A aptamer (FAM-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACATTTTTTTTTTTTTTT) which is marked by FAM fluorescein and contains a connecting sequence polyT15 on the nano material pad, incubating for 15 minutes, drying at 35 ℃, and removing water from the nano material.
Selecting a nitrocellulose membrane as a reaction membrane. And spraying a complementary sequence polyA15(Btiotin-AAAAAAAAAAAAAAA) of the biotin-modified aptamer connecting chain to a detection line, and spraying an antibody resisting Cy5 to a quality control line.
And sequentially adhering the sample pad, the nano material pad, the reaction membrane and the water absorption pad to the same side of the bottom plate.
Second, detection principle of ochratoxin A test strip
The detection principle is the same as that of the test strip for sulfadimethoxine in the embodiment 2.
Detection method of ochratoxin A test strip
The detection method is the same as the detection method of the test strip of sulfadimethoxine in the embodiment 2.
Four, performance research of ochratoxin A test strip
1. Determination of sensitivity for detecting ochratoxin A and preparation of standard curve
Solutions of ochratoxin A with different concentrations were prepared and tested with the test strips prepared in example 3, respectively. And (3) respectively measuring the fluorescence intensity at the detection line and the quality control line by using a test strip detection instrument ESEQuant-LR 3. The ratio of the fluorescence intensity of the detection line to the fluorescence intensity of the quality control line (table 4) is utilized to draw a standard curve for detecting ochratoxin A as shown in figure 3, and the result shows that the sensitivity of the test strip is 2 ng/mL; the linear range is 2 ng/mL-50 ng/mL. Regression equation y 1.2593x-0.1874, R2＝0.941。
TABLE 4 detection results of ochratoxin A standards
2. Study of specificity
The test paper strip is used for detecting ochratoxin A, aflatoxin B1, aflatoxin M1, vomitoxin, fumonisin and zearalanol solutions with the concentrations of 10ng/mL respectively, and results (shown in table 5) show that only ochratoxin A has a remarkable fluorescent signal at a T line, and other toxin detection lines almost have no fluorescent signals, so that the test paper strip has good specificity.
TABLE 5 test paper strip specificity test results for ochratoxin A
Application of ochratoxin A test strip in detection of ochratoxin A in feed
1. Drawing of standard curve
Similar to the standard curve method in the sensitivity and linear range study for detecting ochratoxin A in the first part of this example, the regression equation obtained is that y is 1.2593x-0.1874, R20.941, which is a unitary regression equation of ochratoxin a standard curve.
2. Sample extraction
5 parts of feed sample containing ochratoxin A determined by a national standard method (LC-MSMS) is selected for sample extraction, and the extraction method comprises the following steps: the feed or grain samples were first crushed and passed through a 20 mesh screen. 0.5g of the treated sample was taken and put into a15 ml centrifuge tube. Accurately adding 2mL of purified water and ethyl acetate into a centrifuge tube, tightly sealing a bottle stopper, forcibly oscillating for 5 minutes, and centrifuging for 1 minute at 4000 rpm; 0.6mL of the supernatant was taken with a pipette into a small glass, the filtrate was blown dry, and then the bottom solid was reconstituted with 0.3mL of diluent. The solution is the detection solution.
3. Sample assay
Dropwise adding 100 mu L of the prepared sample to be detected on a sample absorption pad of the test strip, and observing the flow of sample chromatography along the nitrocellulose membrane until the sample chromatography is absorbed by the water absorption pad on the nitrocellulose membrane; after 10 minutes, putting the test strip into a test strip detection instrument ESEQuant-LR3 for detection, and respectively detecting the fluorescence signal intensity of a detection line and a quality control line; and (3) comparing the fluorescence signal intensity (T) of the detection line with the fluorescence signal intensity (C) of the quality control line, substituting the obtained calculated value into the regression equation in the step 1, and calculating to obtain the concentration of ochratoxin A in the sample to be detected.
4. And (3) detection results:
the results of the tests on the 6 feed samples are shown in Table 6. The results show that the recovery rates of 6 samples are all between 87% and 105%, and the test strip has good consistency with the LC-MS/MS method of the national standard method, which indicates that the test strip has higher accuracy. In addition, the standard deviation of the method is less than 10%, which shows that the method has good stability. Therefore, the method can be used for detecting ochratoxin A in feed samples.
TABLE 6 results of test paper strips of the present invention for detecting ochratoxin A in feed
In summary, the nucleic acid aptamer fluorescent test strip provided by the embodiment of the invention replaces a gold-labeled pad in a conventional test strip with a nano-material pad, overcomes the defect that most of the existing nucleic acid aptamers cannot be used for test strip development, and provides a new analysis method for many analytes. Moreover, the embodiment of the invention shows that the method has good consistency with national standard methods such as LC-MS/MS and the like, but the method is simple to operate, has low cost and is more convenient for basic level and field detection application.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (7)
1. A nucleic acid aptamer fluorescent test strip is characterized by comprising a bottom plate, a sample pad, a graphene oxide modified glass fiber membrane, a reaction membrane and a water absorption pad, wherein the ends of the sample pad, the graphene oxide modified glass fiber membrane, the reaction membrane and the water absorption pad are sequentially connected to form a detection layer, the detection layer is connected with the bottom plate, the reaction membrane is provided with a detection line and a quality control line, the detection line is arranged at one end close to the graphene oxide modified glass fiber membrane, and the quality control line is arranged at one end close to the water absorption pad;
the graphene oxide modified glass fiber membrane is attached with a fluorescein-labeled aptamer and/or a fluorescein-labeled aptamer with a connecting sequence and free fluorescein, the detection line is fixed with a complementary chain of the aptamer or a complementary chain of the aptamer connecting sequence, and an anti-fluorescein antibody corresponding to the fluorescein is arranged at the quality control line;
the aptamer connecting sequence comprises one of polyTn, polyAn, polyCn and polyGn, the complementary strand of the corresponding aptamer connecting sequence comprises one of polyAn, polyTn, polyGn and polyCn, and n is an integer of 6-25.
2. The aptamer fluorescent strip of claim 1, wherein the fluorescein is selected from the group consisting of anthocyanidin 5(Cy5) and carboxyfluorescein (FAM).
3. The method for preparing the aptamer fluorescent test strip of claim 1 or 2, which comprises the following steps: and sequentially adhering the sample pad, the glass fiber membrane modified by the graphene oxide, the reaction membrane and the water absorption pad to the same side of the bottom plate.
4. The method for preparing the aptamer fluorescent test strip according to claim 3, wherein the method for preparing the graphene oxide-modified glass fiber membrane comprises the following steps: spraying the graphene oxide suspension solution on a glass cellulose membrane, drying for 7-9 h at 110-130 ℃ to obtain a nano material, dropwise adding the fluorescein-labeled aptamer and free fluorescein to the nano material, reacting for 15-25 min, and drying at 35-40 ℃.
5. The method for preparing the aptamer fluorescent test strip according to claim 4, wherein the method for preparing the reaction membrane comprises the following steps: and incubating the complementary strand of the aptamer modified by biotin or the complementary strand of the aptamer connecting sequence with avidin for 25-35 min, and spraying the incubated product to the detection line.
6. The aptamer fluorescent test strip of claim 1 or 2 for detection applications in food safety, environmental monitoring, and clinical diagnostics.
7. The use of claim 6, wherein the aptamer fluorescent test strip is used for detecting sulfadimethoxine.
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| CN113552360A (en) * | 2021-06-01 | 2021-10-26 | 中国农业科学院农业质量标准与检测技术研究所 | Dual-purpose test strip for detecting enrofloxacin and amantadine as well as preparation method and detection method thereof |
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