CN114280049A - Colorimetric-photothermal dual-mode test strip for detecting allergen protein and preparation method thereof - Google Patents
Colorimetric-photothermal dual-mode test strip for detecting allergen protein and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a colorimetric-photothermal dual-mode test strip for detecting allergen protein and a preparation method thereof, wherein the colorimetric-photothermal dual-mode test strip comprises the following steps: (1) preparation of Au-CuFeSe2The surface of the composite nano particle adsorbs an allergen protein antibody to be detected, namely a first primary antibody, so as to obtain a signal probe; (2) assembling test paper strips, and respectively fixing the test paper strips to be detected on the detection area and the quality control areaAllergen protein antibodies, i.e., secondary and secondary antibodies; (3) after mixing the solutions to be detected, inserting the sample pad end of the test strip into the mixed solution for chromatography; (4) detection, (4.1) colorimetric mode: the color depth of the detection area is in direct proportion to the concentration of the sample to be detected, and the detection area is qualitative by naked eyes and quantitative by gray level analysis; (4.2) photothermal mode: irradiating the detection area by laser, collecting temperature by thermal imaging or temperature measuring equipment, and analyzing and quantifying according to the direct proportion of the temperature of the detection area and the concentration of the sample to be measured. The invention has the advantages of flexible detection mode, wide linear range, high sensitivity and good specificity.
Description
Technical Field
The invention relates to a rapid detection technology of allergen protein in food, in particular to a colorimetric-photothermal dual-mode test strip for detecting allergen protein in food and a preparation method thereof, and specifically relates to a test strip based on Au-CuFeSe2The colorimetric-photothermal dual-mode lateral flow immunochromatographic test strip for detecting the allergen protein in food belongs to the technical field of analysis and detection.
Background
With the improvement of living standard of human beings, food structure is more and more abundant, the probability of generating anaphylactic reaction is also improved along with the same, more and more people appear food anaphylactic reaction, in recent years the anaphylactic reaction that arouses by food is also more and more paid attention to, and some allergens in the anaphylactic food can arouse including severe food anaphylactic reaction such as anaphylactic shock and allergic death, but the anaphylactic reaction that most food arouses is relative mild. There is no effective treatment means for food allergy so far, and the only feasible measure is to avoid food which can cause anaphylactic reaction, so that it is necessary to develop a technology capable of quickly, effectively and accurately detecting allergen in food.
The lateral flow immunochromatographic test paper (LFIA) has the characteristics of high analytical performance, good selectivity, low cost, small sample quantity requirement, easy large-scale production and stable long-term storage, and is widely applied to the design of portable analytical detection sensors.
In conventional LFIAs, colloidal gold nanoparticles are commonly used as signaling probes. The qualitative or semi-quantitative detection can be carried out by direct observation with naked eyes. However, the colorimetric LFIA mainly based on colloidal gold has the defects of single detection mode, low sensitivity, narrow linear range, lack of quantitative analysis and the like, for example, the sensitivity of the lateral flow immunochromatographic test strip for detecting peanut allergen protein on the market currently is only about 5 μ g/ml for detecting peanut total protein, the sensitivity of the lateral flow immunochromatographic test strip for detecting allergen protein in cow milk is only about 10 μ g/ml, and the application of the test strips in certain high-precision detection is limited. In order to improve the sensitivity of analytical detection, researchers have developed fluorescent LFIAs by using various fluorescent nanoparticles as probes, however, these fluorescent substances are interfered by autofluorescence, resulting in low signal-to-noise ratio. In addition, signal acquisition is often performed with expensive and complex instruments, which also reduces the applicability of their field testing.
In recent years, the surface plasmon resonance (LSPR) effect of gold nanoparticles is discovered, and is applied to LFIA again in the form of a photothermal signal probe, and compared with a colorimetric mode, the sensitivity is improved by dozens to hundreds of times, but the current photothermal conversion performance of the material is not good enough, and the further improvement of the sensitivity of photothermal mode test paper is still limited, and a new nano material with higher photothermal conversion efficiency is still needed as a signal probe to form a more obvious thermal contrast so as to further improve the performance of photothermal LFIA.
Nanomaterials with LSPR effect were investigatedIn the favor of people, different kinds of materials are successively synthesized, including noble metal nanoparticles, carbon-based materials, metal and non-metal compounds, and various nano-composite materials. With the discovery of unique physical and chemical properties of some ternary semiconductor nanomaterials, such as higher absorption coefficient and higher photothermal conversion efficiency in a near infrared region and low toxicity, the ternary semiconductor nanomaterials have wide application prospects in the fields of biomedicine and the like such as photoacoustic imaging and photothermal therapy. CuFeSe with high light-heat conversion efficiency2Ternary nanomaterials have been successfully used for cancer diagnosis and photothermal therapy. At present, CuFeSe is used2The material is applied to LFIA and takes Au-CuFeSe2The photothermal quantitative immunochromatographic test strip with the probe as the compound is not reported.
Disclosure of Invention
The technical problem is as follows: in order to overcome the technical defects of low colorimetric mode sensitivity and narrow linear range of the traditional colloidal gold test paper, the invention develops Au-CuFeSe with high photothermal conversion efficiency2A composite nano material and creates a nano material based on Au-CuFeSe2The colorimetric-photothermal dual-mode lateral flow immunochromatographic test strip of the compound is used for quantitative detection of allergen proteins in food.
The technical scheme is as follows: the technical scheme of the invention is as follows:
the first purpose of the invention is to provide a colorimetric-photothermal dual-mode test strip for detecting allergen proteins in food, which comprises a test strip main body and a signal probe.
The signal probe is Au-CuFeSe absorbed with the first primary antibody2A composite material;
the test strip main body structure is that a sample pad, a nitrocellulose membrane (namely NC membrane) and an absorption pad are sequentially overlapped and stuck on a PVC bottom plate along the horizontal direction, and two ends of the NC membrane are positioned at the overlapped lower layer; the NC membrane is used for separating and detecting analytes and other substances in a sample, the sample pad is used for loading the sample, the absorption pad is used for absorbing excessive liquid, and the PVC bottom plate provides physical support for the test paper;
the NC membrane comprises a detection area, namely a T area, and a quality control area, namely a C area, wherein a second primary antibody is fixed on the detection area, and a second antibody, namely an anti-primary antibody, is fixed on the quality control area;
the first primary antibody and the second primary antibody are both primary antibodies, the primary antibodies are monoclonal antibodies of allergen proteins to be detected, the allergen proteins are macromolecular allergen proteins which exist in food and have non-unique antigenic sites, and the allergen proteins include but are not limited to nut allergen proteins, natural milk allergen proteins, aquatic product allergen proteins and egg allergen proteins; the first primary antibody and the second primary antibody are two antibodies which are combined with different antigenic sites of the allergen protein independently;
the secondary antibody includes but is not limited to goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, goat anti-rabbit secondary antibody and donkey anti-rabbit secondary antibody.
The signaling probes can be used independently of the strip format or can be dried onto the conjugate pad and then laminated between the sample pad and the NC membrane.
The detection object is allergen protein, in particular to macromolecular protein, and common protein capable of causing allergy is only basically present in food, including animal food and plant food, including but not limited to nut allergy protein, natural milk allergy protein, aquatic product allergy protein, egg allergy protein and the like.
Another objective of the present invention is to provide a method for preparing a colorimetric-photothermal dual-mode test strip for allergen protein in food, which comprises the following steps:
(1) preparation of Signal Probe Au-CuFeSe2A composite material;
(2)Au-CuFeSe2the composite material and the first primary antibody are used for preparing Au-CuFeSe2-a mAb cocktail, i.e. a signaling probe;
(3) diluting the second primary antibody with a PBS (phosphate buffer solution) solution to prepare a detection area solution, and diluting the second antibody with the PBS solution to prepare a quality control area solution;
(4) constructing a test strip main body: and dripping or spraying a detection area solution in the area T to fix the secondary antibody, dripping or spraying a quality control area solution in the area C to fix the secondary antibody, drying and then preserving in vacuum for later use.
Further, the specific method in the step (1) comprises the following steps: wet chemical methodCuFeSe prepared in aqueous solution by the method2Diluting the nanocrystalline dispersion, and taking diluted CuFeSe2Adding the solution into a centrifuge tube, adding trisodium citrate solution, mixing uniformly, and adding HAuCl4Adding ultrapure water into the solution, immediately placing the solution on a vortex oscillator, oscillating the solution at room temperature for reaction, and changing the color of the solution from light brown to purple to obtain Au-CuFeSe2The composite material is stored for standby at the temperature of 4 ℃.
Further, the specific method in the step (2) is as follows: taking Au-CuFeSe2Adding K into a centrifuge tube2CO3Adjusting the pH value of the solution adjusting system to 6-8, adding the prepared allergen protein monoclonal antibody to be detected, namely the first primary antibody solution after uniform oscillation, and incubating and oscillating for 1h at room temperature. After the reaction, 20% BSA aqueous solution and 10% PEG-20000 were added, incubated at room temperature for 30min, and centrifuged at high speed at 4 ℃ for 15 min. The supernatant was discarded and the pellet was dissolved in 100. mu.L of a solution containing 10mmol/LNa3P041-10% BSA, 0.15-1% Tween-20 and 5-15% sucrose to obtain Au-CuFeSe2mAb mixture, stored at 4 ℃ for use. Here Na3PO4Other buffers such as PBS and the like are also possible.
Further, the specific method in the step (3) is as follows: respectively diluting an antibody of a substance to be detected, namely a first primary antibody and a second antibody, with 10mM PBS solution to prepare a detection area solution and a quality control area solution, wherein the sources of the monoclonal antibody for modifying the composite material include but are not limited to mice, rats and rabbits. Correspondingly, the secondary antibody fixed on the quality control area is a secondary antibody aiming at the primary antibody source.
The composite material modified by the small molecule compound monoclonal antibody is stored in a closed container comprising a centrifuge tube in the state of solution or freeze-dried powder.
Further, in the step (4), 5-20mM PBS solution containing 0.5-10mg/mL of the allergen protein antibody to be tested, i.e., the secondary antibody, is dripped or sprayed on the T area, and 5-20mM PBS solution containing 0.05-10mg/mL of the secondary antibody is dripped or sprayed on the C area.
Another object of the present invention is to provide a detection apparatus for detecting an allergen protein,
when the colorimetric mode is adopted for detection, the detection device comprises the allergen protein colorimetric-photothermal dual-mode test strip, namely a test strip main body and the allergen protein monoclonal antibody modified composite material Au-CuFeSe2-mAb, a signaling probe;
when the photothermal detection mode is adopted, the detection device comprises the allergen protein colorimetric-photothermal dual-mode test strip (namely a test strip main body and a signal probe), a laser light source (such as a 808nm laser light source, and also can be laser light sources with other wavelengths), thermal imaging or temperature measurement equipment and an intelligent display end, the detection device takes laser as a light source, and Au-CuFeSe is taken as an Au-CuFeSe light source2-mAb is a photothermal conversion material.
Furthermore, the thermal imaging or temperature measuring device comprises a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, a handheld infrared thermal imaging analyzer or an infrared thermal imaging temperature measuring gun, the intelligent display end comprises but is not limited to a computer or a smart phone, and the thermal imaging or temperature measuring device collects photo-thermal imaging photos and outputs and displays the photo-thermal imaging photos through the intelligent display end connected with the thermal imaging or temperature measuring device. The common thermal imaging or temperature measuring equipment comprises various models of mobile phone infrared thermal imaging analysis accessories, an infrared thermal imager, a handheld infrared thermal imaging analyzer or an infrared thermal imaging temperature measuring gun, and common product models can be selected, such as a Gao De Zhi sense FLIR E50 handheld thermal imager. When the test paper strip is used, corresponding application software is downloaded on the smart phone, then the thermal imaging or temperature measuring equipment such as the handheld infrared thermal imaging analyzer is connected with the smart phone (wireless or wired according to actual conditions), the probe of the handheld infrared thermal imaging analyzer is aligned to the test paper strip main body, and the corresponding temperature can be displayed on the application software of the smart phone. The specific method of the infrared thermal image analyzer depends on its own method of use, and is not particularly limited in the present invention.
Another object of the present invention is to provide a method for using the test strip, which comprises the following steps: dissolving a sample to be detected in an operation buffer solution, putting a signal probe and a test strip main body into the operation buffer solution, detecting by adopting a colorimetric method and/or a photothermal method,
when the colorimetric method is adopted for detection, after 15-30min, the qualitative judgment can be carried out preliminarily through naked eyes according to the color development condition of the T area, or the colorimetric result of the test strip can be read by analyzing the gray value of the T area by using Image analysis software such as Image J software, and the quantitative judgment of the content of the target object can be carried out;
when the optothermal method is adopted for detection, the test strip is dried and then is excited under a laser light source with the wavelength of 808nm, and the optothermal imaging is obtained by using thermal imaging or temperature measuring equipment and an intelligent display terminal, so that the temperature result is read.
Further, the running buffer comprises 10mM Na3PO45-15% of sucrose, 1-10% of bovine serum albumin solution (BSA), 0.15-1% of Tween-20, and a pH solution of 6.5-8.0, wherein the buffer solution can also be a borate buffer solution, a carbonate buffer solution and the like.
Further, the signal probe is used independently from the strip, or is dried on a bonding pad and then is superposed between a sample pad and an NC membrane of the strip body.
When the signal probe is used independently of the test strip, the specific method is as follows: placing a sample to be detected and the signal probe in an operation buffer solution, mixing for 3-10min, placing the mixture into the test strip main body, and detecting by adopting a colorimetric method or a photothermal method;
when the signal probe is superposed between the sample pad and the NC membrane of the test strip main body after being dried on the combination pad, the specific method comprises the following steps: and dropwise adding the signal probe on the bonding pad, drying, assembling and superposing the bonding pad between a sample pad and an NC membrane of the test strip main body, mixing a sample to be detected with a running buffer solution (the same running buffer solution as the running buffer solution), putting the mixed sample into the test strip main body containing the bonding pad, and detecting by adopting a colorimetric method or a photothermal method.
Furthermore, the area C is used as a reference for verifying the validity of the test paper result and is always purple; the excitation temperature intensity of the T area is positively correlated with the content of the allergen protein to be detected in the sample, and the color development intensity is positively correlated with the content of the allergen protein in the sample, specifically comprising the following steps:
when the sample does not contain allergen protein, the T area does not develop color, the photothermal effect is weak, and the temperature is low;
when the sample contains allergen protein, the T area is purple, the photothermal effect is strong, and the temperature is high.
When the sample does not contain the allergen protein to be detected, the signal probe cannot be combined with the target object, so that the signal probe cannot be combined with the second primary antibody of the T area, and the T area does not contain the signal probe, so that the T area does not develop color, does not have a photothermal effect and is low in temperature;
when the sample contains the allergen protein to be detected, the signal probe is combined with the target substance and can be combined with the second primary antibody of the T area, and the T area contains the signal probe, so that the T area is colored, has photothermal effect and is increased in temperature.
The colorimetric-photothermal dual-mode test strip for detecting the allergen protein in the food has the following detection principle: the area C is used as a reference for verifying the validity of the test paper result and is always purple; sample solution and Au-CuFeSe2The mAb after premixing moves towards the absorbent paper by capillary action,
when the sample contains allergen protein, Au-CuFeSe2The mAb binds to the allergen protein, is captured by the secondary antibody in the T-zone, is purple in the visible T-zone, and is excited by laser light to form Au-CuFeSe2An LSPR effect is produced and the temperature rises. As the concentration of allergen protein in the sample increases, Au-CuFeSe trapped in the T region2Increasing mAb, darker T-zone and increasing temperature, proportional to analyte concentration; excess Au-CuFeSe2The mAb will be captured by a secondary antibody in the C-domain so that the C-domain is also purple;
Au-CuFeSe when there is no allergen protein in the sample2The mAb is unable to bind to the secondary antibody of the T-region, thus rendering the T-region colorless and unable to bind Au-CuFeSe2mAb, to abolish photothermal effect and decrease temperature. Excess Au-CuFeSe2The mAb will be captured by the secondary antibody in the C-domain so that the C-domain will appear purple.
Has the advantages that:
the colorimetric-photothermal dual-mode test strip for detecting the allergen protein in food is based on the plasma resonance effect, and has high photothermal conversion efficiency and small-sized Au-CuFeSe2The composite material is fixed in the T areaThe signal probe and the near-infrared laser light source are excited and combined with the intelligent display end to realize the acquisition of photo-thermal signals, and the substrate color of the sample liquid and the fluorescent background interference of the test paper are effectively removed, so that the signal-to-noise ratio is improved. The method has lower detection limit, wider detection range and better specificity, has higher sensitivity than other photo-thermal detection test paper, and is suitable for quickly detecting the allergen protein in food.
Drawings
FIG. 1 is a schematic diagram of a test strip assembly;
FIG. 2 is a graph showing the positive case of zone C temperature distribution in photothermal mode;
FIG. 3 shows the positive T-zone temperature profile in photothermal mode;
FIG. 4 is a graph showing the temperature distribution of zone C in the negative case in photothermal mode;
FIG. 5 is a graph showing the temperature distribution of the negative T-zone in photothermal mode;
FIG. 6 is a schematic diagram showing the positive gray scale detection in the colorimetric mode;
FIG. 7 is a schematic diagram showing the gray scale detection of negative case in colorimetric mode;
FIG. 8 shows CuFeSe2An electron microscope representation of (1);
fig. 9 shows potential characterization of the materials and composites.
Detailed Description
The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.
The invention discloses Au-CuFeSe for detecting allergen protein2The compound colorimetric-photothermal dual-mode test strip and the preparation method thereof comprise the following steps:
(1) preparation of Au-CuFeSe2The surface of the composite nano particle adsorbs an antibody of an object to be detected, namely a first primary antibody to obtain a signal probe;
(2) assembling a test strip, and respectively fixing another antibody, namely a second primary antibody and a secondary antibody, namely an anti-primary antibody, of the substance to be detected on the detection area and the quality control area;
(3) inserting the test strip sample pad end into the sample solution to be detected for chromatography;
(4) detection of
(4.1) colorimetric mode: the color depth of the detection area is in direct proportion to the concentration of the sample to be detected, and the detection area is qualitative by naked eyes and quantitative by gray level analysis;
(4.2) photothermal mode: and (3) irradiating the detection area by using 808nm laser, collecting the temperature by using thermal imaging or temperature measuring equipment, and carrying out quantitative analysis according to the linear relation between the temperature of the detection area and the concentration of the sample to be detected.
The invention has the advantages of flexible detection mode, wide linear range, high sensitivity and good specificity.
The following examples illustrate the preparation of the test strip and the use of the test device in full detail, using the peanut allergen Ara H1 protein and bovine beta-lactoglobulin as the allergen proteins in food.
Example 1: Au-CuFeSe for peanut allergen Ara H1 protein2Composite photothermal quantitative dual-mode test strip detection
1. Preparation of test paper material
1.1 preparation of CuFeSe2Nanocrystal
Preparation of CuFeSe in aqueous solution by wet chemical method2And (4) nanocrystals. 39.48mg of Se powder was dispersed in 100mL of ultrapure water, and 50mg of NaBH was then added4And the reduction is carried out under the environmental condition protected by nitrogen flow. Preparation of CuCl separately2·2H2O(42.62mg)、FeSO4·7H25ml of a mixture of O (69.75mg) and PTMP-PMAA (400 mg). And immediately adding the mixture into the Se precursor solution after the Se powder is completely reduced to form a black solution. The resulting solution was ultrafiltered at 3500rpm through a membrane with a molecular weight cut-off (MWCO) of 100 kDa. The supernatant was dialyzed against ultrapure water (MWCO of 8-14kDa) for 48 hours to remove impurities. Purified CuFeSe2The NC solution was concentrated by a similar ultrafiltration method and stored at 4 ℃ for further use.
1.2 preparation of Au-CuFeSe2Composite material
Mixing CuFeSe2Diluting the dispersion liquid to 10% by volume, and taking 320 mu L of diluted CuFeSe2Adding 20 mu L of trisodium citrate solution with the concentration of 194mmol/L into a centrifuge tube, uniformly mixing, and adding 50 mu L of LHAuCl4Adding 3.59mL of ultrapure water into the solution (m/v is 1 percent), then immediately placing the solution on a vortex oscillator, oscillating and reacting for 10min at room temperature, and changing the color of the solution from light brown to purple to obtain Au and CuFeSe2The composite nano material Au-CuFeSe2. FIG. 8 is CuFeSe2The electron microscope characterization shows that the prepared Au-CuFeSe2The particle size of the composite material is below 20nm, and the particle sizes are uniformly distributed; FIG. 9 is a potential characterization of the composite material, showing the Au-CuFeSe prepared2The composite material has better stability.
1.3 Au-CuFeSe2Adsorption to the first antibody
Taking Au-CuFeSe2Adding 0-5 mu L27.6mg/mL K into a centrifuge tube2CO3Adjusting the pH value of the system to 6-8 by using the solution, uniformly shaking, adding 3-5 mu L of 0.65mg/mL allergen protein primary antibody to be detected, which is prepared in advance, incubating and shaking at room temperature for 1 h. After the reaction, 10. mu.L of 20% BSA aqueous solution and 5. mu.L of 10% PEG-20000 were added, incubated at room temperature for 30min, and centrifuged at 12000rpm at 4 ℃ for 15 min. The supernatant was discarded and the pellet was dissolved in 80. mu.L of working buffer (20 mmol/LNa)3 PO 45% BSA, 0.25% Tween-20, 10% sucrose) to obtain Au-CuFeSe2mAb mixture, stored at 4 ℃ for use.
1.4 preparation of detection zone (T zone) solution
The Ara H1 protein secondary antibody was diluted to 0.5mg/mL with 10mM PBS to obtain a T region solution. The second antibody here differs from the binding site of the first antibody in 1.3.
1.5 preparation of solution in the quality control zone (zone C)
The goat anti-mouse secondary antibody was diluted to 0.4mg/mL with 10mM PBS to obtain a solution in the C region.
2. Preparation of test strip main body
According to a conventional combination mode of the lateral flow immunochromatographic test strip, as shown in fig. 1, an NC membrane is adhered to the middle of a PVC bottom plate, a sample pad and a water absorption pad are respectively lapped at the left end and the right end of the NC membrane to enable the NC membrane to be covered and pressed by about 2mm, and the built large card is cut into a paper strip with the width of 3mm, so that a blank test strip is obtained. The solution in zone 0.5. mu. L T and the solution in zone 0.5. mu. L C were added dropwise to zone T and zone C, respectively. The test strips after spotting are placed in an oven, dried for 60min at 37 μ, and stored in a vacuum bag for further use.
3. Dual-mode detection of peanut allergen Ara H1 protein
3.1 colorimetric mode detection
Preparing 10mL of 1mg/mL peanut allergen protein standard solution by using purified water, and diluting the peanut allergen protein standard solution to the concentration of 0ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL by using a PBS solution to serve as a liquid to be detected for later use. Mixing 80 μ L of the solution to be tested with 10 μ LAu-CuFeSe2mAb and 10 μ L running buffer (10mM PBS solution, 5% sucrose, 1% BSA, 1% tween-20, pH 7.4) were mixed in the centrifuge tube for 10min, then the strip was inserted into the centrifuge tube and the results read after 20 min.
And subsequently, analyzing the gray value of the T area by using Image J software, reading the colorimetric result of the test strip, analyzing the colorimetric result, thus preparing a working curve and carrying out quantitative judgment on the content of the target object.
3.2 photothermal mode detection
Preparing 10mL of 1mg/mL peanut allergen protein standard solution by using purified water, and diluting the solution to the concentration of 0ng/mL, 0.01ng/mL, 0.02ng/mL, 0.05ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL by using a PBS solution to serve as a test solution for standby. Mixing 80 μ L of the solution to be tested with 10 μ LAu-CuFeSe2mAb and 10. mu.L running buffer (10mM PBS containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4) were mixed in a centrifuge tube for 10min, and then the strip was inserted into the centrifuge tube and dried using a 808nm laser (power 1.36W/cm)2) Irradiating for 3min, monitoring test strip T zone temperature by using a smart phone and a high German intelligence FLIR E50 handheld thermal imager, recording temperature and monitoring temperature change by using smart phone infrared thermal imaging software, and observing T zone and C zoneThe temperature value of the temperature sensor is the experimental result in the photo-thermal mode.
Drawing a standard curve graph by corresponding temperature values and concentrations of different peanut allergen protein Ara H1 concentrations one by one, setting a horizontal axis coordinate as a target object concentration, and marking as a letter x; the coordinate of the vertical axis is set as a temperature change value and is marked as a letter y; performing linear fitting by using computer software (such as Excel) to obtain a standard curve equation, and recording as y ═ f (x); the standard equation embodies the linear relation between the detection area temperature and the concentration of the sample to be detected, and can be used for obtaining the concentration of peanut allergen Ara H1 through the temperature change value under the actual detection condition, namely, the standard curve equation can be used for further quantitative analysis.
4. Analysis of results
In colorimetric mode, when peanut allergen protein Ara H1 is not in the sample, Au-CuFeSe2The detection strand cannot be captured by the capture strand in the T-zone, and no significant color change in the T-zone is visible to the naked eye, as shown in fig. 7;
when the sample contains peanut allergen protein Ara H1, the color of the T region is in positive correlation with the concentration range of peanut allergen protein Ara H1, and the color of the T region is darker as the peanut allergen protein Ara H1 is more, as shown in FIG. 6; when the concentration of peanut allergen protein Ara H1 is increased to 5ng/mL, the color of the T area appears to be purple, and the concentration is taken as the detection limit of the colorimetric mode;
in photothermal mode, when the peanut allergen protein Ara H1 is absent in the sample, Au-CuFeSe2The detection chain cannot be captured by the T-zone capture chain, so that the T-zone does not generate LSPR effect under the excitation of the laser, and the temperature is low, as shown in the T-zone temperature distribution diagram of the negative case in the photothermal mode in FIG. 5;
when the sample contains the peanut allergen protein Ara H1, the peanut allergen protein Ara H1 and part of Au-CuFeSe2Complementary pairing of the detection strands and partial complementarity to the capture strand of the T region to form a sandwich structure, the T region thus being Au-CuFeSe2The LSPR effect of (a) increases and the temperature increases, as shown in fig. 3; the temperature of the positive T area is the highest, when the concentration of peanut allergen protein Ara H1 is 2ng/mL, the temperature of the T area is higher than that of the C area, and the concentration is taken as the detection limit of a photothermal mode.
Therefore, the detection limit of the constructed dual-mode test paper can reach 2 ng/mL.
In summary, the principle of the colorimetric-photothermal dual-mode test strip for detecting allergen protein in food according to the present invention is explained by taking the peanut allergen Ara H1 protein in this embodiment as an example as follows: the area C is used as a reference for verifying the validity of the test paper result and is always purple; sample solution and Au-CuFeSe2The mAb, the signaling probe, was pre-mixed and then wicked towards the absorbent paper, then two cases were:
when the sample contains Ara H1 protein, Au-CuFeSe2The mAb binds to Ara H1 protein in the sample, is captured by the secondary antibody in the T region, the T region is purple visible to the naked eye, and the Au-CuFeSe is excited by laser2The LSPR effect is generated, and the temperature is increased; as the concentration of Ara H1 protein in the sample increases, the Au-CuFeSe trapped in the T region2Increasing mAb, darker T-zone and increasing temperature, proportional to analyte concentration; excess Au-CuFeSe2The mAb will be captured by the secondary antibody of the C-domain so that the C-domain is also purple, as shown in FIG. 2;
Au-CuFeSe when Ara H1 protein is not present in the sample2The mAb is unable to bind to the secondary antibody of the T-region, thus rendering the T-region colorless and unable to bind Au-CuFeSe2mAb, to allow the photothermal effect to disappear, the temperature to drop; excess Au-CuFeSe2mAb was captured by the secondary antibody in C-zone and C-zone appeared purple, as shown in the negative C-zone temperature profile in photothermal mode in FIG. 4.
Example 2: Au-CuFeSe for bovine beta-lactoglobulin2The specificity verification of the compound photo-thermal quantitative dual-mode test strip comprises the following steps:
1. preparation of test paper material
In the same manner as in example 1, the allergen protein was changed to bovine beta-lactoglobulin, and the corresponding test strip body and signal probe were changed accordingly.
2. Preparation of test paper strip
The same as in example 1.
3. Drawing working curve
The same as in example 1.
4. Sample pretreatment
Preparing 10mL of 2mg/mL bovine beta-lactoglobulin, almond protein, ovotransferrin, mung bean protein, oat protein and sesame protein standard solution by using purified water, and diluting the standard solution to a concentration of 200ng/mL by using a PBS solution to serve as a to-be-detected solution for later use.
5. Sample detection
Mixing 80 μ L of the solution to be tested with 10 μ LAu-CuFeSe2mAb and 10. mu.L running buffer (10mM PBS solution containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4) were mixed in a centrifuge tube for 10min before detection.
5.1 colorimetric mode:
and (4) inserting the test strip main body into a centrifuge tube, and reading a colorimetric result after 25 min.
5.2 photothermal mode:
after the test paper is dried, a 808nm laser (power 1.36W/cm) is used2) Irradiating for 3min, monitoring temperature change by using a mobile phone and a handheld infrared thermal imaging analyzer, and displaying results of colorimetric and photothermal modes, wherein only bovine B-lactoglobulin group has results of positive color development and temperature distribution, and T-zone results of test paper of almond protein, ovotransferrin, mung bean protein, oat protein and sesame protein have the advantages of light color development, low temperature and good specificity identification result. Therefore, the dual-mode test strip of the embodiment of the present application has a response only to bovine β -lactoglobulin and no response to other allergen proteins, and thus the test strip of the embodiment of the present application is considered to have detection specificity for allergen proteins.
Example 3: Au-CuFeSe for Ara H1 protein2Compound photo-thermal quantitative dual-mode test strip-signal probe drying on combination pad
The difference from example 1 is that the signal probes dried on the conjugate pad and then laminated between the sample pad and the NC membrane are used in step 2 and 3.
1. Preparation of test paper material
Same as example 1
2. Preparation of test strip main body
According to the conventional combination mode of the lateral flow immunochromatographic test strip, the NC membrane is adhered to the middle of the PVC bottom plate, the sample pad and the water absorption pad are respectively lapped at the left end and the right end of the NC membrane to enable the NC membrane to be covered and pressed by about 2mm, and the built large card is cut into the paper strips with the width of 3mm, so that the blank test strip is obtained. 0.5 mu LT area solution and 0.5 mu LC area solution are respectively dripped on the T area and the C area, 0.5 mu L of signal probe solution is spotted on the bonding pad, as shown in figure 1, the test strip after spotting is put in an oven, dried for 60min at 37 ℃, and stored in a vacuum bag for standby.
3. Dual-mode detection of peanut allergen Ara H1 protein
3.1 colorimetric mode detection
Preparing 10mL of 1mg/mL peanut allergen protein standard solution by using purified water, and diluting the peanut allergen protein standard solution to the concentration of 0ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL by using a PBS solution to serve as a liquid to be detected for later use. mu.L of the test solution and 20. mu.L of the running buffer (10mM PBS solution, containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4) were mixed in a centrifuge tube, and the test strip was inserted into the centrifuge tube and the results were read after 20 min.
And subsequently, analyzing the gray value of the T area by using Image J software, reading the colorimetric result of the test strip, analyzing the colorimetric result, thus preparing a working curve and carrying out quantitative judgment on the content of the target object.
3.2 photothermal mode detection
Preparing 10mL of 1mg/mL peanut allergen protein standard solution by using purified water, and diluting the solution to the concentration of 0ng/mL, 0.01ng/mL, 0.02ng/mL, 0.05ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL by using a PBS solution to serve as a test solution for standby. mu.L of the test solution and 20. mu.L of the running buffer (10mM PBS solution containing 5% sucrose, 1% BSA, 1% Tween-20, pH 7.4) were mixed in a centrifuge tube, and then the strip was inserted into the centrifuge tube, after drying the strip, using a 808nm laser (power 1.36W/cm)2) Irradiating for 3min, monitoring test strip T zone temperature by using a smart phone and a high German intelligence FLIR E50 handheld thermal imager, and performing infrared thermal imaging by using the smart phoneAnd (3) recording the temperature by software, monitoring the temperature change, and observing the temperature values of the T area and the C area to obtain an experimental result in the photo-thermal mode.
Drawing a standard curve graph by corresponding temperature values and concentrations of different peanut allergen protein Ara H1 concentrations one by one, setting a horizontal axis coordinate as a target object concentration, and marking as a letter x; the coordinate of the vertical axis is set as a temperature change value and is marked as a letter y; performing linear fitting by using computer software (such as Excel) to obtain a standard curve equation, and recording as y ═ f (x); the standard equation can be used for obtaining the concentration of the peanut allergen protein Ara H1 through the temperature change value under the actual detection condition, namely, the standard curve equation can be used for further quantitative analysis.
4. Analysis of results
The colorimetric detection result of this example was the same as the T-zone color development result of the positive sample in the colorimetric mode shown in fig. 6 and the T-zone color development result of the negative sample in the colorimetric mode shown in fig. 7 of example 1.
The photothermal detection results of this example were the same as the results of the T-zone temperature distribution of the positive sample in photothermal mode shown in fig. 3 and the results of the C-zone temperature distribution of the positive sample in photothermal mode shown in fig. 2 of example 1.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (10)
1. The colorimetric-photothermal dual-mode test strip for detecting the allergen protein is characterized by comprising a test strip main body and a signal probe;
the signal probe is Au-CuFeSe of a first primary antibody adsorbed with allergen protein to be detected2A composite material;
the test strip main body comprises a bottom plate, a sample pad, an NC membrane and an absorption pad are sequentially overlapped and stuck on the bottom plate along the horizontal direction, the NC membrane comprises a detection area, namely a T area, and a quality control area, namely a C area, a secondary antibody of the allergen protein to be detected is fixed on the detection area, and a secondary antibody is fixed on the quality control area;
the first primary antibody and the second primary antibody are both primary antibodies, the primary antibodies are monoclonal or polyclonal antibodies of allergen proteins to be detected, the allergen proteins are macromolecular allergen proteins which exist in food and have non-unique antigenic sites, and include but are not limited to nut allergen proteins, natural milk allergen proteins, aquatic product allergen proteins and egg allergen proteins; the first primary antibody and the second primary antibody are two antibodies which are combined with different antigenic sites of the allergen protein independently;
the secondary antibody is an anti-primary antibody, and includes but is not limited to a goat anti-mouse secondary antibody, a rabbit anti-mouse secondary antibody, a goat anti-rabbit secondary antibody and a donkey anti-rabbit secondary antibody.
2. The method for preparing the test strip of claim 1, comprising the steps of:
(1) preparation of Au-CuFeSe2A composite material;
(2)Au-CuFeSe2the composite material and the first primary antibody are used for preparing Au-CuFeSe2-a mAb cocktail, i.e. a signaling probe;
(3) diluting the second primary antibody with a PBS (phosphate buffer solution) solution to prepare a detection area solution, and diluting the second antibody with the PBS solution to prepare a quality control area solution;
(4) constructing a test strip main body: and dripping or spraying a detection area solution in the area T to fix the secondary antibody, dripping or spraying a quality control area solution in the area C to fix the secondary antibody, drying and then preserving in vacuum for later use.
3. The method according to claim 2, wherein the specific method of step (1) is as follows: CuFeSe to be prepared in aqueous solution by wet chemical method2Diluting the nanocrystalline dispersion, and taking diluted CuFeSe2Adding trisodium citrate solution, mixing, adding HAuCl4Adding ultrapure water into the solution, immediately placing the solution on a vortex oscillator, oscillating the solution at room temperature for reaction, and changing the color of the solution from light brown to purple to obtain Au-CuFeSe2The composite material is stored at 4 ℃ for later use.
4. The method according to claim 2, wherein the specific method of step (2) is as follows: taking Au-CuFeSe2Adding alkalescent buffer solution to adjust the pH value of the system to 6-8, oscillating and mixing uniformly, adding the first primary antibody, oscillating and reacting uniformly, adding BSA (bovine serum albumin) for sealing after the reaction is finished, centrifuging, removing supernatant, and re-dissolving in 100 mu L of Na containing 20mmol/L of Na3PO41-10% BSA, 0.15-1% Tween-20 and 5-15% sucrose to obtain Au-CuFeSe2mAb mixture, stored at 4 ℃ until use.
5. The method of claim 2, wherein in step (4), the 5-20mM PBS solution containing 0.5-10mg/mL of the secondary antibody is dripped or sprayed on the T zone, and the 5-20mM PBS solution containing 0.05-10mg/mL of the secondary antibody is dripped or sprayed on the C zone.
6. A colorimetric-photothermal dual-mode detection device for detecting allergen proteins,
when the colorimetric assay is used, the assay device comprises the strip of claim 1;
when the photothermal detection mode is adopted, the detection device comprises the test strip, the laser light source, the thermal imaging or temperature measuring equipment and the intelligent display end, wherein the laser is used as the light source of the detection device, and the signal probe in the test strip is used as the photothermal conversion material.
7. The detection apparatus according to claim 6, wherein the thermal imaging or temperature measuring device includes but is not limited to a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, a handheld infrared thermal imaging analyzer or an infrared thermal imaging temperature measuring gun, the smart display terminal includes but is not limited to a computer and a smart phone, and the thermal imaging or temperature measuring device collects photo-thermal imaging photos and outputs and displays the photo-thermal imaging photos through the smart display terminal connected with the thermal imaging or temperature measuring device.
8. Method for using a detection device according to claim 6, characterized in that it comprises the following steps: dissolving a sample to be detected in an operation buffer solution, putting a signal probe and a test strip main body, and detecting by adopting a colorimetric method or a photothermal method;
when a colorimetric method is adopted for detection, direct visual observation is carried out after 15-30min, the colorimetric result of the test strip is read, naked eye qualitative judgment is carried out according to the color development condition of the T area, or image analysis software is utilized to analyze the gray value of the T area so as to read the quantitative result of the test strip;
when the photothermal method is adopted for detection, the test strip is dried and then is placed under a laser light source with the wavelength of 808nm for excitation, a thermal imaging photo or a temperature measuring device is used for obtaining a photothermal imaging photo, and a temperature result is read.
9. The use method of claim 6 or 8, wherein the signal probe is used independently from the test strip, or the signal probe is used after being dried on a bonding pad and then is superposed between a sample pad and an NC membrane of the test strip.
10. The strip of claim 1, or the device of claim 6, or the method of use of claim 8, wherein the zone C is always purple when used as a reference for verifying the validity of the strip result; the excitation temperature intensity of the T area is positively correlated with the content of the allergen protein to be detected in the sample, and the color development intensity is positively correlated with the content of the allergen protein to be detected in the sample, specifically comprising the following steps:
when the sample does not contain the allergen protein to be detected, the T area does not develop color, the photothermal effect is weak, and the temperature is low;
when the sample contains the allergen protein to be detected, the T area is developed, the photothermal effect is strong, and the temperature is increased.
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| CN113063938A (en) * | 2021-03-13 | 2021-07-02 | 河南省农业科学院 | High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method |
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