CN114280049B - Colorimetric-photothermal dual-mode test strip for detecting allergen proteins and preparation method thereof - Google Patents
Colorimetric-photothermal dual-mode test strip for detecting allergen proteins and preparation method thereof Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses a colorimetric-photothermal dual-mode test strip for detecting allergen proteins and a preparation method thereof, comprising the following steps of: (1) Preparation of Au-CuFeSe 2 The surface of the composite nanoparticle is adsorbed with an allergen protein antibody to be detected, namely a first antibody, so as to obtain a signal probe; (2) Assembling a test strip, and respectively fixing an allergen protein antibody to be tested, namely a second primary antibody and a second antibody, on a detection area and a quality control area; (3) Mixing the liquid to be detected, and inserting the sample pad end of the test strip into the mixed liquid 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 naked eye is qualitative and the gray level analysis is quantitative; (4.2) photo-thermal mode: and irradiating the detection area by laser, collecting the temperature by using thermal imaging or temperature measuring equipment, and analyzing and quantifying according to the proportion of the temperature of the detection area and the concentration of the sample to be detected. The invention has flexible detection mode, wide linear range, high sensitivity and good specificity.
Description
Technical Field
The invention relates to a rapid detection technology of allergen proteins in food, in particular to a colorimetric-photothermal dual-mode test strip for detecting allergen proteins in food and a preparation method thereof, and in particular relates to an Au-CuFeSe-based test strip 2 A colorimetric-photothermal dual-mode lateral flow immunochromatographic test strip of a compound for detecting allergen proteins in food belongs to the technical field of analysis and detection.
Background
With the improvement of human living standard, the food structure is more and more abundant, the probability of anaphylactic reaction is also improved, more and more people are subjected to food anaphylactic reaction, more and more importance is attached to the anaphylactic reaction caused by food in recent years, and serious food anaphylactic reaction including anaphylactic shock, anaphylactic death and the like can be caused by the allergen in some allergic foods, but the anaphylactic reaction caused by most foods is relatively mild. So far, no effective treatment means for food allergy exists, and the only viable measure is to avoid eating food which can cause anaphylactic reaction, so that development of a technology capable of rapidly, effectively and accurately detecting allergen in food is particularly necessary.
The lateral flow immunochromatographic test paper (LFIA) has the characteristics of quick analysis performance, good selectivity, low cost, small sample size requirement, easy mass production and stable long-term storage, and is widely applied to the design of portable analysis and detection sensors.
In conventional LFIA, colloidal gold nanoparticles are typically used as signaling probes. The naked eye can directly observe to perform qualitative or semi-quantitative detection. However, the colorimetric LFIA 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 current commercial lateral flow immunochromatographic test paper for detecting peanut allergen proteins is only about 5 mug/ml for detecting peanut total proteins, the sensitivity of the current lateral flow immunochromatographic test paper for detecting allergen proteins in cow milk is only about 10 mug/ml, and the application of the test paper in certain high-precision detection is limited. To improve the sensitivity of analytical detection, researchers have developed fluorescent LFIA by using various fluorescent nanoparticles as probes, however, these fluorescent substances suffer from interference of autofluorescence, resulting in lower signal-to-noise ratio. In addition, signal acquisition is typically performed with expensive and complex instrumentation, which also reduces its applicability to field detection.
In recent years, the surface plasmon resonance (LSPR) effect of gold nanoparticles is found to be applied to LFIA again in the form of a photo-thermal signal probe, and compared with a colorimetric mode, the sensitivity is improved by tens to hundreds times, but the photo-thermal conversion performance of the current material is not good enough, still limits the further improvement of the photo-thermal mode test paper sensitivity, and a new nano material with higher photo-thermal conversion efficiency is still needed to be used as the signal probe to form more obvious thermal contrast so as to further improve the performance of the photo-thermal LFIA.
Nanomaterials with LSPR effect are favored by researchers and different kinds of materials are synthesized sequentially, including noble metal nanoparticles, carbon-based materials, metallic and non-metallic compounds, and various nanocomposite materials. With the discovery of unique physical and chemical properties of some ternary semiconductor nano materials, for example, the higher absorption coefficient and higher photo-thermal conversion efficiency in the near infrared region and low toxicity, the ternary semiconductor nano materials have wide application prospects in the fields of biomedical science such as photo-acoustic imaging, photo-thermal treatment and the like. CuFeSe with high photo-thermal conversion efficiency 2 Ternary nanomaterials have been successfully used for diagnosis of cancer and photothermal therapy. Currently, cuFeSe is used 2 The material is applied to LFIA and is used as Au-CuFeSe 2 The photothermal quantitative immunochromatographic test strip with the complex as a probe has not been reported yet.
Disclosure of Invention
Technical problems: 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 the Au-CuFeSe with high photo-thermal conversion efficiency 2 Composite nano material and creates a nano material based on Au-CuFeSe 2 The colorimetric-photothermal dual-mode lateral flow immunochromatographic test strip of the complex 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 invention provides 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 adsorbed with first primary antibody of allergen protein to be detected 2 A composite material;
the main structure of the test strip is that a sample pad, a nitrocellulose membrane, namely an 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 on an overlapped lower layer; the NC film is used for realizing separation and detection of 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 film comprises a detection area, namely a T area, and a quality control area, namely a C area, wherein a second primary antibody of an allergen protein to be detected is fixed on the detection area, and a second primary 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, and the allergen proteins are macromolecular allergen proteins which exist in foods and have non-unique antigenic sites, including but not limited to nut allergy proteins, natural milk allergy proteins, aquatic product allergy proteins and egg allergy proteins; the first antibody and the second antibody are independently two antibodies combined with different antigenic sites of allergen proteins;
the secondary antibodies include, but are not limited to, goat anti-mouse secondary antibodies, rabbit anti-mouse secondary antibodies, goat anti-rabbit secondary antibodies, donkey anti-rabbit secondary antibodies.
The signaling probes may be used independently of the dipstick format or may be dried on a conjugate pad and then applied between the sample pad and NC membrane.
The detection object of the application is allergen protein, in particular to macromolecular protein, and common proteins capable of causing allergy are basically only present in foods, including animal foods and plant foods, including but not limited to nut allergy proteins, natural milk allergy proteins, aquatic product allergy proteins, egg allergy proteins and the like.
The invention also aims to provide a preparation method of the colorimetric-photothermal dual-mode test strip for allergen proteins in food, which comprises the following preparation steps:
(1) Preparation of Signal Probe Au-CuFeSe 2 A composite material;
(2)Au-CuFeSe 2 preparation of Au-CuFeSe by composite material and first primary antibody 2 The mAb mixed solution is the signal probe;
(3) Diluting the second primary antibody by using a PBS (phosphate buffer solution) solution to prepare a detection area solution, and diluting the second antibody by using the PBS solution to prepare a quality control area solution;
(4) Constructing a test strip main body: and (3) dropwise adding or spraying a solution in the detection area in the T area to fix the second antibody, dropwise adding or spraying a solution in the quality control area in the C area to fix the second antibody, and drying and then preserving in vacuum for later use.
Further, the specific method of the step (1) is as follows: cuFeSe prepared by wet chemical method in aqueous solution 2 Diluting the nanocrystalline dispersion liquid, and taking diluted CuFeSe 2 Adding the solution into a centrifuge tube, adding trisodium citrate solution, mixing uniformly, and adding HAuCl 4 Adding ultrapure water into the solution, immediately placing the solution on a vortex oscillator, and carrying out oscillation reaction at room temperature to obtain Au-CuFeSe, wherein the color of the solution is changed from light brown to purple 2 The composite material is stored in an environment of 4 ℃ for standby.
Further, the specific method of the step (2) is as follows: taking Au-CuFeSe 2 Adding K into a centrifuge tube 2 CO 3 And regulating the pH value of the solution to 6-8, adding the prepared allergen protein monoclonal antibody to be tested, namely the first antibody solution, after shaking uniformly, incubating at room temperature, and shaking for 1h. After the reaction is finishedAdding 20% BSA aqueous solution and 10% PEG-20000, incubating at room temperature for 30min, and centrifuging at high speed for 15min at 4deg.C. The supernatant was discarded and the pellet was dissolved in 100. Mu.L containing 10mmol/L Na 3 PO 4 In running buffer solution of 1-10% BSA, 0.15-1% Tween-20 and 5-15% sucrose to obtain Au-CuFeSe 2 -mAb cocktail, stored in 4 ℃ environment for later use. Here Na 3 PO 4 Other buffers such as PBS, etc. are also possible.
Further, the specific method of the step (3) is as follows: the primary antibody and the secondary antibody of the antibody to be detected are respectively diluted by 10mM PBS solution to prepare a detection area solution and a quality control area solution, and monoclonal antibody sources for modifying the composite material include, but are not limited to, mice, rats and rabbits. Correspondingly, the secondary antibody fixed in the quality control region is a secondary antibody aiming at the source of the first primary antibody.
The small molecule compound monoclonal antibody modified composite material is stored in a sealed container including a centrifuge tube in a 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 detected, namely the second antibody, is dripped or sprayed in the T region, and 5-20mM PBS solution containing 0.05-10mg/mL of the second antibody is dripped or sprayed in the C region.
It is another object of the present invention to provide a detection device for detecting an allergen protein,
when the colorimetric mode detection is adopted, the detection device comprises the allergen protein colorimetric-photothermal dual-mode test strip, namely a test strip main body and an allergen protein monoclonal antibody modified composite material Au-CuFeSe 2 -mAb, signaling probe;
when the photo-thermal mode detection is adopted, the detection device comprises the allergen protein colorimetric-photo-thermal 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, or other wavelength laser light sources), a thermal imaging or temperature measuring device and an intelligent display end, wherein the detection device takes laser as a light source and Au-CuFeSe as a light source 2 -mAb is a photothermal conversion material.
Further, the thermal imaging or temperature measuring equipment comprises a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imaging instrument, 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 equipment collects photo-thermal imaging photos and can output and display the photo-thermal imaging photos through the intelligent display end connected with the photo-thermal imaging or temperature measuring equipment. Common thermal imaging or temperature measuring equipment comprises various types of mobile phone infrared thermal imaging analysis accessories, infrared thermal imagers, handheld infrared thermal imaging analyzers or infrared thermal imaging temperature measuring guns, and common product types, such as Gao Dezhi induction FLIR E50 handheld thermal imagers, can be selected. When the intelligent mobile phone is used, corresponding application software is downloaded on the intelligent mobile phone, then a thermal imaging or temperature measuring device such as a handheld infrared thermal imaging analyzer is connected with the intelligent mobile phone (wireless or wired according to actual conditions), a probe of the handheld infrared thermal imaging analyzer is aligned to the main body of the test paper strip, and the corresponding temperature can be displayed on the application software of the intelligent mobile phone. The specific method of the different infrared thermal imaging analyzers depends on the own use method, 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, comprising the following steps: dissolving a sample to be detected in an operation buffer solution, putting a signal probe and a test strip main body, detecting by adopting a colorimetric method and/or a photothermal method,
when the colorimetric method is adopted for detection, the qualitative judgment can be preliminarily carried out through naked eyes according to the color development condition of the T region after 15-30min, or the gray value of the T region can be analyzed by Image analysis software such as Image J software to further read the colorimetric result of the test strip, so that the quantitative judgment of the content of the target object can be carried out;
when the photothermal method is adopted for detection, the test strip is dried and then is excited under a 808nm laser light source, a thermal imaging or temperature measuring device and an intelligent display end are used for obtaining the photothermal imaging, and a temperature result is read.
Further, the running buffer is a buffer containing 10mM Na 3 PO 4 5-15% sucrose, 1-10% Bovine Serum Albumin (BSA), 0.15-1% Tween-20, pH 6.5-8.0, and optionally boric acidSalt buffers, carbonate buffers, and the like.
Further, the signaling probe is used independently of the test strip or is used after being dried on a binding pad and then being superposed between a sample pad and an NC film of the test 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 into an operation buffer solution, mixing for 3-10min, placing the sample into the test strip main body, and detecting by adopting a colorimetric method or a photothermal method;
when the signal probe is dried on the binding pad and then is overlapped between the sample pad and the NC film of the test strip main body, the specific method is as follows: and (3) dropwise adding the signal probe on the bonding pad, drying, assembling and superposing the bonding pad between the sample pad and the NC film of the test strip main body, mixing a sample to be tested and an operation buffer solution (the same operation buffer solution as the above), putting the mixture into the test strip main body containing the bonding pad, and detecting by adopting a colorimetric method or a photothermal method.
Further, the C area is used as a reference for verifying the validity of the test paper result and always turns purple; the excitation temperature intensity of the T region is positively correlated with the content of allergen protein to be detected in the sample, and the color development intensity is positively correlated with the content of allergen protein in the sample, specifically:
when the sample does not contain allergen protein, the T region does not develop color, the photo-thermal effect is weak, and the temperature is low;
when the sample contains allergen protein, the T region is purple, the photo-thermal effect is strong, and the temperature is high.
When the sample does not contain allergen protein to be detected, the signaling probe cannot be combined with the target object, so that the signaling probe cannot be combined with the second antibody of the T region, and the T region does not contain the signaling probe, so that the T region does not develop color, does not have a photo-thermal effect and has low temperature;
when the sample contains allergen protein to be detected, the signal probe is combined with the target object and can be combined with the second antibody of the T region, and the T region contains the signal probe, so that the T region develops color, has a photo-thermal effect and has a temperature rise.
The invention relates to a colorimetric-photothermal dual-mode test strip detection principle for detecting allergen proteins in foodThe explanation is as follows: the C area is used as a reference for verifying the validity of the test paper result and always shows purple; sample solution and Au-CuFeSe 2 The mAb moves towards the absorbent paper under capillary action after premixing,
when the sample contains allergen protein, au-CuFeSe 2 The mAb binds to the allergen protein, is captured by the second antibody in the T region, and the T region is visible as purple, while under laser excitation the Au-CuFeSe 2 The LSPR effect is produced and the temperature rises. Au-CuFeSe trapped in the T region as the concentration of allergen protein in the sample increases 2 More and more mAb, darker T-region and progressively higher temperature, proportional to analyte concentration; redundant Au-CuFeSe 2 The mAb will be captured by the secondary antibody of the C region and thus the C region is also purple;
Au-CuFeSe when no allergen protein is present in the sample 2 The mAb cannot bind to the T region secondary antibody, thus rendering the T region non-chromogenic and unable to bind to Au-CuFeSe 2 mAb, causing the photothermal effect to disappear, temperature to drop. Redundant Au-CuFeSe 2 The mAb will be captured by the secondary antibody of the C region and the C region will appear purple.
The beneficial effects are that:
the colorimetric-photothermal dual-mode test strip for detecting allergen proteins in food is based on a plasma resonance effect, and has Gao Guangre conversion efficiency and micro-size of Au-CuFeSe 2 The composite material is a T-zone fixed signal probe, and the near infrared laser light source is excited to be combined with the intelligent display end to collect photo-thermal signals, so that the background color of the sample liquid and the fluorescent background interference of the test paper are effectively removed, and the signal to noise ratio is further improved. The method has lower detection limit, wider detection range and better specificity, has higher sensitivity than other photo-thermal detection test papers, and is suitable for rapid detection of allergen proteins in food.
Drawings
FIG. 1 is a schematic diagram of a test strip assembly;
FIG. 2 shows a positive C-zone temperature distribution diagram in a photo-thermal mode;
FIG. 3 shows a positive case T zone temperature distribution diagram in a photo-thermal mode;
FIG. 4 shows a graph of the temperature distribution of the C region in the negative case of the photo-thermal mode;
FIG. 5 shows a temperature distribution diagram of a negative T region in a photo-thermal mode;
FIG. 6 is a schematic diagram showing gray scale detection of a positive condition in a colorimetric mode;
FIG. 7 is a schematic diagram showing the gray scale detection of a negative case in a colorimetric mode;
FIG. 8 shows CuFeSe 2 Is characterized by an electron microscope;
fig. 9 shows potential characterization of materials and composites.
Detailed Description
The invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are illustrative of the present invention and should not be construed as limiting the invention described in detail in the claims.
The invention discloses an Au-CuFeSe for detecting allergen proteins 2 The compound colorimetric-photothermal dual-mode test strip and the preparation method thereof comprise the following steps:
(1) Preparation of Au-CuFeSe 2 The surface of the composite nano-particle is adsorbed with an antibody of an object to be detected, namely a first antibody, so as to obtain a signal probe;
(2) Assembling a test strip, and respectively fixing a second antibody, namely a first antibody, of an object to be tested and a second antibody, namely a first antibody, on a detection area and a quality control area;
(3) The sample pad end of the test paper strip is inserted into the sample liquid to be tested 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 naked eye is qualitative and the gray level analysis is quantitative;
(4.2) photo-thermal mode: and (3) irradiating the detection area by 808nm laser, collecting the temperature by using thermal imaging or temperature measuring equipment, and quantitatively analyzing according to the linear relation between the temperature of the detection area and the concentration of the sample to be detected.
The invention has flexible detection mode, wide linear range, high sensitivity and good specificity.
The following examples illustrate the preparation of the entire test strip and the use of the test device using the peanut allergen Ara H1 protein and bovine beta-lactoglobulin as examples of allergen proteins in food.
Example 1: au-CuFeSe for peanut allergen Ara H1 protein 2 Compound photo-thermal quantitative dual-mode test strip detection
1. Preparation of test paper material
1.1 preparation of CuFeSe 2 Nanocrystalline crystal
Preparation of CuFeSe in aqueous solution by wet chemical method 2 And (3) nanocrystalline. 39.48mg Se powder was dispersed in 100mL of ultrapure water, followed by addition of 50mg of NaBH 4 The reduction is carried out under ambient conditions protected by a stream of nitrogen. Preparation of CuCl separately 2 ·2H 2 O(42.62mg)、FeSO 4 ·7H 2 5ml of a mixture of O (69.75 mg) and PTMP-PMAA (400 mg). Immediately after complete reduction of the Se powder, the above mixture was added to the Se precursor solution to form a black solution. The resulting solution was subjected to ultrafiltration through a membrane having a molecular weight cut-off (MWCO) of 100kDa at a speed of 3500 rpm. The supernatant was dialyzed against ultrapure water (MWCO 8-14 kDa) for 48 hours to remove impurities. Purified CuFeSe 2 NC solution is concentrated by adopting a similar ultrafiltration method, and is preserved at 4 ℃ for standby.
1.2 preparation of Au-CuFeSe 2 Composite material
CuFeSe 2 Diluting the dispersion to volume fraction of 10%, and collecting 320 μl of diluted CuFeSe 2 Adding 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 HAuCl 4 Adding 3.59mL of ultrapure water into the solution (m/v=1%), immediately placing the solution on a vortex oscillator, and performing oscillation reaction at room temperature for 10min to obtain Au and CuFeSe after the solution changes from light brown to purple 2 Is of composite nano material Au-CuFeSe 2 . FIG. 8 is a view of CuFeSe 2 The electron microscope characterization of (C) shows that the prepared Au-CuFeSe 2 The particle size of the composite material is below 20nm, and the particle size is uniformly distributed; FIG. 9 is a potential characterization of the composite material, showing the prepared Au-CuFeSe 2 The composite material has better stability.
1.3Au-CuFeSe 2 Adsorption with first primary antibody
Taking Au-CuFeSe 2 Adding 0-5 mu L of 27.6mg/mL K into a centrifuge tube 2 CO 3 And regulating the pH value of the solution to 6-8, adding 3-5 mu L of 0.65mg/mL of the first antibody of the allergen protein to be detected, which is prepared in advance, after shaking uniformly, incubating and shaking for 1h at room temperature. After the reaction was completed, 10. Mu.L of 20% aqueous BSA solution and 5. Mu.L of 10% PEG-20000 were added, and incubated at room temperature for 30min and centrifuged at 12000rpm at 4℃for 15min. The supernatant was discarded, and the pellet was dissolved in 80. Mu.L working buffer (20 mmol/L Na 3 PO 4 5% BSA, 0.25% Tween-20, 10% sucrose) to give Au-CuFeSe 2 -mAb cocktail, stored in 4 ℃ environment for later 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 give a T-block solution. The second primary antibody herein differs from the binding site of the first primary antibody in 1.3.
1.5 preparation of solutions in quality control zone (zone C)
The goat anti-mouse secondary antibody was diluted to 0.4mg/mL with 10mM PBS to give a solution in zone C.
2. Preparation of test strip main body
According to the conventional lateral flow immunochromatography test strip combination mode, as shown in fig. 1, an NC film is stuck in 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 film, so that the NC film is covered and pressed by about 2mm, and the built large card is cut into strips with the width of 3mm, so that a blank test strip is obtained. A solution of 0.5 mu L T area and a solution of 0.5 mu L C area were added dropwise to the T area and the C area, respectively. The test strips after spotting were placed in an oven, dried at 37 ℃ for 60min, and stored in a vacuum bag for further use.
3. Dual mode detection of peanut allergen Ara H1 protein
3.1 colorimetric Pattern detection
10mL of 1mg/mL peanut allergen protein standard solution is prepared by purified water, and diluted with PBS solution to a 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, 500ng/mL are used as the test solution. 80 mu L of the sample to be testedLiquid and 10. Mu.L of Au-CuFeSe 2 mAb and 10. Mu.L running buffer (10 mM PBS containing 5% sucrose, 1% BSA,1% Tween-20, pH 7.4) were mixed in the centrifuge tube for 10min, and the test strip was inserted into the tube for 20min before reading the results.
And then, analyzing the gray value of the T area by using Image J software so as to read the colorimetric result of the test strip, and analyzing the colorimetric result to obtain a working curve so as to quantitatively judge the content of the target object.
3.2 photo-thermal mode detection
10mL of 1mg/mL peanut allergen protein standard solution is prepared by purified water, diluted with PBS solution to a 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, 500ng/mL are used as the test solution. 80. Mu.L of the test solution and 10. Mu.L of Au-CuFeSe are mixed 2 mAb and 10. Mu.L running buffer (10 mM PBS containing 5% sucrose, 1% BSA,1% Tween-20, pH 7.4) were mixed in a centrifuge tube for 10min, the strip was inserted into the tube and after drying the strip was applied with a 808nm laser (power 1.36W/cm) 2 ) And irradiating for 3min, using a smart phone and a high intelligent FLIR E50 handheld thermal imager, monitoring the temperature of the T region of the test strip by the handheld thermal imager, recording the temperature by means of the infrared thermal imaging software of the smart phone, monitoring the temperature change, and observing the temperature values of the T region and the C region to obtain an experimental result in a photo-thermal mode.
Drawing a standard curve graph with one-to-one correspondence between temperature values and concentrations of different peanut allergen proteins Ara H1, setting the horizontal axis coordinate as the concentration of a target object, and marking the target object as a letter x; the vertical axis coordinate 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, which is marked as y=f (x); the standard equation reflects the linear relation between the temperature of the detection area and the concentration of the sample to be detected, and 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
In colorimetric mode, au-CuFeSe is used when peanut allergen protein Ara H1 is not contained in the sample 2 The detection chain cannot be captured by the T-region capture chain, and no apparent color change is visible in the macroscopic T-region, as shown in fig. 7;
when the peanut allergen protein Ara H1 is contained in the sample, the color of the T region is positively related to the concentration range of the peanut allergen protein Ara H1, and the more the peanut allergen protein Ara H1 is contained in the sample, the darker the color of the T region is, as shown in FIG. 6; when the concentration of peanut allergen protein Ara H1 is increased to 5ng/mL, the T region has macroscopic purple, and the concentration is taken as a colorimetric mode detection limit;
in the photo-thermal mode, when peanut allergen protein Ara H1 is not contained in the sample, au-CuFeSe 2 The detection chain cannot be captured by the T region capturing chain, so that the T region does not generate LSPR effect under the excitation of laser, the temperature is low, and the temperature distribution diagram of the T region is a negative condition under the photo-thermal mode shown in figure 5;
when the sample contains peanut allergen protein Ara H1, the peanut allergen protein Ara H1 and part of Au-CuFeSe 2 The detection strand is complementary to the capture strand portion of the T region to form a sandwich structure, thus the T region Au-CuFeSe 2 The LSPR effect of (a) increases and the temperature increases as shown in fig. 3; the temperature of the positive T region is highest, and when the concentration of peanut allergen protein Ara H1 is 2ng/mL, the temperature of the T region is higher than that of the C region, and the concentration is taken as the detection limit of a photo-thermal mode.
Therefore, the detection limit of the constructed dual-mode test paper can reach 2ng/mL.
In summary, the colorimetric-photothermal dual-mode test strip detection principle for detecting allergen proteins in food is explained by taking peanut allergen Ara H1 protein in the embodiment as an example as follows: the C area is used as a reference for verifying the validity of the test paper result and always shows purple; sample solution and Au-CuFeSe 2 After premixing, the mAb, the signaling probe, moves in the direction of the absorbent paper by capillary action, and then the following two conditions exist:
when the sample contains Ara H1 protein, au-CuFeSe 2 The mAb binds to the Ara H1 protein in the sample, is captured by the T-region second antibody, and the T-region appears purple to the naked eyeUnder the excitation of laser, au-CuFeSe 2 Generating LSPR effect, and increasing the temperature; as the concentration of Ara H1 protein in the sample increases, au-CuFeSe trapped in the T region 2 More and more mAb, darker T-region and progressively higher temperature, proportional to analyte concentration; redundant Au-CuFeSe 2 The mAb will be captured by the secondary antibody of the C region and thus the C region will also appear purple, as shown in figure 2;
Au-CuFeSe when Ara H1 protein is not present in the sample 2 The mAb is unable to bind to the second antibody of the T region, so that the T region is not developed and is unable to bind to Au-CuFeSe 2 mAb, such that the photothermal effect disappears and the temperature drops; redundant Au-CuFeSe 2 mAb will be captured by the secondary antibody of region C and thus the region C will appear purple, as shown in the negative case of the photothermal pattern of the region C temperature profile shown in fig. 4.
Example 2: au-CuFeSe for bovine beta-lactoglobulin 2 The specificity verification of the compound photo-thermal quantitative dual-mode test strip comprises the following steps:
1. preparation of test paper material
As in example 1, the allergen protein was changed to bovine beta-lactoglobulin, and the corresponding test strip body and signaling probe were changed accordingly.
2. Preparation of test paper strip
As in example 1.
3. Working curve drawing
As in example 1.
4. Sample pretreatment
10mL of 2mg/mL of bovine beta-lactoglobulin, almond protein, ovotransferrin, mung bean protein, oat protein and sesame protein standard solution are prepared by purified water, and the solution is diluted to a concentration of 200ng/mL by PBS solution to be used as a liquid to be tested.
5. Sample detection
80. Mu.L of the test solution and 10. Mu.L of Au-CuFeSe are mixed 2 mAb and 10. Mu.L running buffer (10 mM PBS containing 5% sucrose, 1% BSA,1% Tween-20, pH 7.4) were mixed in a centrifuge tube for 10min and then assayed.
5.1 colorimetric mode:
the test strip main body is inserted into a centrifuge tube, and the colorimetric result is read after 25 min.
5.2 photo-thermal mode:
after the test paper has dried, it is irradiated with a 808nm laser (power 1.36W/cm 2 ) The temperature change is monitored by using a mobile phone and a handheld infrared thermal imaging analyzer after 3min of irradiation, colorimetric and photo-thermal mode results show that only bovine beta-lactoglobulin groups have positive color development and temperature distribution results, and the T area results of test paper of almond protein, egg transferrin, mung bean protein, oat protein and sesame protein are light color development, low in temperature and good in specific recognition result. Thus, the dual-mode test strip of the embodiment of the present application is only responsive to bovine beta-lactoglobulin and not responsive to other allergen proteins, so it is considered that the test strip of the embodiment of the present application has detection specificity to allergen proteins.
Example 3: au-CuFeSe for Ara H1 protein 2 Drying the compound photo-thermal quantitative dual-mode test strip-signal probe on a bonding pad
The difference from example 1 is that the signal probes were dried on the conjugate pad and then stacked between the sample pad and NC membrane in the steps 2, 3.
1. Preparation of test paper material
Same as in example 1
2. Preparation of test strip main body
According to the conventional combination mode of the lateral flow immunochromatography test strip, an NC film is stuck in 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 film, so that the NC film is covered and pressed by about 2mm, and the built large card is cut into strips with the width of 3mm, so that a blank test strip is obtained. And respectively dripping 0.5 mu L T area solution and 0.5 mu L C area solution into the T area and the C area, dispensing 0.5 mu L of signal probe solution on a bonding pad, placing the test strip after sample application in an oven, drying at 37 ℃ for 60min, and storing in a vacuum bag for standby.
3. Dual mode detection of peanut allergen Ara H1 protein
3.1 colorimetric Pattern detection
10mL of 1mg/mL peanut allergen protein standard solution is prepared by purified water, and diluted with PBS solution to a 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, 500ng/mL are used as the test solution. mu.L of the test solution and 20. Mu.L of running buffer (10 mM PBS solution, 5% sucrose, 1% BSA,1% Tween-20, pH 7.4) were mixed in a centrifuge tube, and then a test strip was inserted into the centrifuge tube, and the result was read after 20 min.
And then, analyzing the gray value of the T area by using Image J software so as to read the colorimetric result of the test strip, and analyzing the colorimetric result to obtain a working curve so as to quantitatively judge the content of the target object.
3.2 photo-thermal mode detection
10mL of 1mg/mL peanut allergen protein standard solution is prepared by purified water, and diluted with PBS solution to a 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, 500ng/mL are used as the test solution. mu.L of the test solution and 20. Mu.L of running buffer (10 mM PBS solution, 5% sucrose, 1% BSA,1% Tween-20, pH 7.4) were mixed in a centrifuge tube, and then a test strip was inserted into the centrifuge tube, and after the test strip was dried, a 808nm laser (power 1.36W/cm) was used 2 ) And irradiating for 3min, using a smart phone and a high intelligent FLIR E50 handheld thermal imager, monitoring the temperature of the T region of the test strip by the handheld thermal imager, recording the temperature by means of the infrared thermal imaging software of the smart phone, monitoring the temperature change, and observing the temperature values of the T region and the C region to obtain an experimental result in a photo-thermal mode.
Drawing a standard curve graph with one-to-one correspondence between temperature values and concentrations of different peanut allergen proteins Ara H1, setting the horizontal axis coordinate as the concentration of a target object, and marking the target object as a letter x; the vertical axis coordinate 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, which is marked as y=f (x); the standard equation can be used for solving 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 results of this example were the same as the T-zone color development results of the positive sample in the colorimetric mode shown in FIG. 6 and the T-zone color development results of the negative sample in the colorimetric mode shown in FIG. 7 of example 1.
The photo-thermal detection result of this example is the same as the T-zone temperature distribution result of the positive sample in the photo-thermal mode shown in fig. 3 and the C-zone temperature distribution result of the positive sample in the photo-thermal mode shown in fig. 2 of example 1.
The foregoing is only a preferred embodiment of the invention, it being 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 present invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (17)
1. A colorimetric-photothermal dual-mode test strip for detecting allergen proteins, which is characterized by comprising a test strip main body and a signal probe;
the signal probe is Au-CuFeSe adsorbed with first primary antibody of allergen protein to be detected 2 A composite material;
the test strip main body comprises a bottom plate, a sample pad, an NC film and an absorption pad are sequentially overlapped and stuck on the bottom plate along the horizontal direction, the NC film comprises a detection area, namely a T area, a quality control area, namely a C area, a second primary antibody of allergen proteins to be detected is fixed on the detection area, and a second 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, and the allergen proteins are macromolecular allergen proteins which exist in foods and have non-unique antigenic sites, including but not limited to nut allergy proteins, natural milk allergy proteins, aquatic product allergy proteins and egg allergy proteins; the first antibody and the second antibody are independently two antibodies combined with different antigenic sites of allergen proteins;
the secondary antibodies are primary antibodies, including but not limited to goat anti-mouse secondary antibodies, rabbit anti-mouse secondary antibodies, goat anti-rabbit secondary antibodies and donkey anti-rabbit secondary antibodies.
2. The test strip of claim 1, wherein the region C is always purple as a reference for verifying the validity of the test strip results; the excitation temperature intensity of the T region 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.
3. The test strip of claim 2, wherein the color development intensity of the T-zone is specifically:
when the sample does not contain allergen protein to be detected, the T region does not develop color, the photo-thermal effect is weak, and the temperature is low;
when the sample contains allergen protein to be detected, the T region develops color, the photo-thermal effect is strong, and the temperature is increased.
4. A method of preparing a test strip according to any one of claims 1 to 3, comprising the steps of:
(1) Preparation of Au-CuFeSe 2 A composite material;
(2)Au-CuFeSe 2 preparation of Au-CuFeSe by composite material and first primary antibody 2 The mAb mixed solution is the signal probe;
(3) Diluting the second primary antibody by using a PBS (phosphate buffer solution) solution to prepare a detection area solution, and diluting the second antibody by using the PBS solution to prepare a quality control area solution;
(4) Constructing a test strip main body: and (3) dropwise adding or spraying a solution in the detection area in the T area to fix the second antibody, dropwise adding or spraying a solution in the quality control area in the C area to fix the second antibody, and drying and then preserving in vacuum for later use.
5. The method according to claim 4, wherein the specific method of step (1) is as follows: cuFeSe prepared in aqueous solution by wet chemical method 2 Diluting the nanocrystalline dispersion liquid, and taking diluted CuFeSe 2 Adding trisodium citrate solution into the solution, mixing uniformly, and adding HAuCl 4 Adding ultrapure water into the solution, immediately placing the solution on a vortex oscillator, performing oscillation reaction at room temperature, and obtaining the solutionThe color changes from light brown to purple to obtain Au-CuFeSe 2 The composite material is preserved at 4 ℃ for standby.
6. The method according to claim 4, wherein the specific method of step (2) is as follows: taking Au-CuFeSe 2 Adding a weak alkaline buffer solution to adjust the pH value of the system to 6-8, shaking and mixing uniformly, adding the first antibody, shaking and reacting after mixing uniformly, adding BSA for blocking after the reaction is finished, centrifuging to remove the supernatant, and redissolving in 100 mu L of a solution containing 20mmol/L Na 3 PO 4 In an operation buffer solution of 1-10% BSA, 0.15-1% Tween-20 and 5-15% sucrose to obtain Au-CuFeSe 2 -mAb cocktail, stored at 4 ℃ for later use.
7. The method according to claim 4, wherein the specific method in step (3) is as follows: and diluting the first antibody and the second antibody which are the antibodies to be detected by using 10mM PBS solution to prepare a detection area and a quality control area solution.
8. The method according to claim 4, wherein in the step (4), the 5-20mM PBS solution containing 0.5-10mg/mL of the second antibody is dropped or sprayed into the T region, and the 5-20mM PBS solution containing 0.05-10mg/mL of the second antibody is dropped or sprayed into the C region.
9. A colorimetric-photothermal dual-mode detection device for detecting allergen proteins is characterized in that,
when detecting in a colorimetric mode, the detection device comprises the test strip according to any one of claims 1 to 3;
when the photo-thermal mode detection is adopted, the detection device comprises the test strip, a laser light source, thermal imaging or temperature measuring equipment and an intelligent display end according to any one of claims 1-3, wherein the detection device uses laser as a light source and uses a signal probe in the test strip as a photo-thermal conversion material.
10. The detection apparatus according to claim 9, wherein the thermal imaging or temperature measuring device comprises, but is not limited to, a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imaging device, a handheld infrared thermal imaging analyzer or an infrared thermal imaging temperature measuring gun, and the intelligent display terminal comprises, but is not limited to, a computer and a smart mobile phone, and the thermal imaging or temperature measuring device acquires a photo-thermal imaging photo and outputs and displays the photo-thermal imaging photo through the intelligent display terminal connected with the photo-thermal imaging or temperature measuring device.
11. The detection device according to claim 9, wherein the region C is always purple as a reference for verifying the validity of the test paper result; the excitation temperature intensity of the T region 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.
12. The detection device according to claim 11, wherein the color development intensity of the T region is specifically:
when the sample does not contain allergen protein to be detected, the T region does not develop color, the photo-thermal effect is weak, and the temperature is low;
when the sample contains allergen protein to be detected, the T region develops color, the photo-thermal effect is strong, and the temperature is increased.
13. A method of using a detection apparatus according to any one of claims 9 to 12, comprising the steps of: the sample to be tested is dissolved in the running buffer solution, the signal probe and the test strip main body are put in, and the colorimetric method or the photothermal method is adopted for detection.
14. The method according to claim 13, wherein the colorimetric or photothermal detection comprises:
when the colorimetric method is adopted for detection, the colorimetric result of the test strip is directly observed by naked eyes after 15-30min, and the colorimetric result of the test strip is read, and the test strip is qualitatively judged according to the naked eye of the color development condition of the T region, or the gray value of the T region is analyzed by utilizing image analysis software, so that the quantitative result of the test strip is read;
when the photothermal method is adopted for detection, the test strip is dried and then is excited under a 808nm laser light source, a thermal imaging or temperature measuring device is used for obtaining a photothermal imaging photo, and a temperature result is read.
15. The method of claim 13, wherein the signaling probe is used independently of the test strip or is applied between the sample pad and NC membrane of the test strip after the signaling probe is dried on the binding pad.
16. The method of claim 13, wherein the region C is always purple as a reference for verifying the validity of the test paper results; the excitation temperature intensity of the T region 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.
17. The use according to claim 16, wherein the color development intensity of the T-zone is specifically:
when the sample does not contain allergen protein to be detected, the T region does not develop color, the photo-thermal effect is weak, and the temperature is low;
when the sample contains allergen protein to be detected, the T region develops color, the photo-thermal effect is strong, and the temperature is increased.
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