CN114371298A - SERS immunochromatographic test strip for detecting cow milk allergen alpha-lactalbumin and application thereof - Google Patents

SERS immunochromatographic test strip for detecting cow milk allergen alpha-lactalbumin and application thereof Download PDF

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CN114371298A
CN114371298A CN202210017529.9A CN202210017529A CN114371298A CN 114371298 A CN114371298 A CN 114371298A CN 202210017529 A CN202210017529 A CN 202210017529A CN 114371298 A CN114371298 A CN 114371298A
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lactalbumin
alpha
sers
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魏新林
李靖
朱永智
徐佳
彭斓兰
魏康
王元凤
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Shanghai Jiaotong University
Shanghai Normal University
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Abstract

The invention discloses an SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin and application thereof. The SERS probe contains Raman beacon molecules, is captured by a capture antibody to react to form a double-antibody sandwich structure when reaching a detection line, the detection line is colored, redundant SERS probes are combined with a goat anti-rabbit secondary antibody on the control line along with buffer chromatography, the control line is colored, the detection sensitivity of the milk allergen alpha-lactalbumin can be improved through the combination of a colorimetric method and a Raman method, and the accurate assessment of the allergy risk is facilitated.

Description

SERS immunochromatographic test strip for detecting cow milk allergen alpha-lactalbumin and application thereof
Technical Field
The invention relates to an SERS immunochromatographic test strip, in particular to an SERS immunochromatographic test strip for detecting milk allergen alpha-lactalbumin and application thereof, and belongs to the technical field of food safety detection.
Background
The milk is the source of intake of vitamins (vitamin A, vitamin B6) and minerals (calcium), is a nutrient substance necessary for the growth and development of infants, plays a key role in bones, hairs, skin and teeth of human bodies, and is one of the first foods eaten by infants. However, milk is also the first and most common cause of food allergy in early childhood. Allergic diseases caused by allergens have two remarkable characteristics, namely, the allergic diseases start in the infant stage and are not changed after the food allergy lasts for a long time; secondly, the sensitizing dose has great crowd difference. Thus, some cows' milk allergy sufferers develop an allergic reaction from potentially very small amounts of milk. The allergens in food are mostly proteinaceous substances, and each 100ml of cow's milk contains about 3 g of proteins, and at least 25 different proteins, all of which are likely to be allergens, and alpha-Lactalbumin (alpha-LA) is currently considered to be one of the major allergens in cow's milk.
The methods for detecting cow milk allergens mainly comprise an immunoassay technology, a liquid chromatography technology, a biological mass spectrometry technology, a real-time PCR (polymerase chain reaction), an electrospray ionization mass spectrometry and the like, and although the methods have high sensitivity, the methods need long detection time or expensive instrument equipment.
Surface Enhanced Raman Scattering (SERS) is a metal nanostructure-mediated signal enhancement effect, has higher resolution and sensitivity than ordinary raman spectroscopy, can realize 10-14 times of single-molecule raman signal enhancement, has a narrow band of a characteristic raman spectrum of SERS, and can provide abundant structural information for an analyte under various interferences of a complex system. The lateral chromatography immunity technology is a technology that antigen and antibody generate immunoreaction with high specificity and high affinity in the chromatography process, and sample detection is realized through visual markers such as colloidal gold. The colloidal gold immunochromatography technology has been widely applied to detection of pathogenic bacteria, proteins, pesticide and veterinary drug residues and the like. However, the colloidal gold test strip mainly observes the result through naked eyes, has low detection sensitivity and mainly performs qualitative and semi-quantitative detection.
Therefore, the combination of SERS and immunochromatography can be considered, the immunochromatography has the advantages of instantaneity, rapidness, simplicity and high specificity of detection, the defect that colloidal gold needs to be aggregated in a large quantity to develop color is overcome, the detection sensitivity is improved, the linear relation corresponding to the concentration of a detected object and the Raman characteristic peak intensity is established, and the rapid, efficient and accurate quantitative detection of the target alpha-lactalbumin is realized.
Disclosure of Invention
The invention provides an SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin, aiming at the problems of low detection sensitivity and inaccurate quantification of the traditional colloidal gold test strip, and the SERS immunochromatographic test strip is obtained by combining an SERS technology and an immunochromatographic technology.
The invention also provides application of the SERS immunochromatographic test strip for detecting the milk allergen alpha-lactalbumin in the fixed detection and high-sensitivity quantitative detection of the milk allergen alpha-lactalbumin within 5-10 min.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention provides an SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin, which sequentially comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane (NC membrane) and a water absorption pad, wherein the NC membrane is provided with a detection line (T line) and a control line (C line), wherein:
the T line is arranged at one end close to the combination pad, and a capture antibody alpha-lactalbumin mouse monoclonal antibody is fixed on the T line;
the C line is arranged at one end close to the adsorption pad, and a goat anti-rabbit secondary antibody is fixed on the C line;
and an SERS probe is sprayed on the combination pad and is obtained by coupling and combining silver-coated gold nanoparticles, Raman beacon molecules and a detection antibody alpha-lactalbumin rabbit polyclonal antibody.
Further, the particle size of the silver-coated gold nanoparticles is 20-40nm, the shape of the silver-coated gold nanoparticles is spherical, and the silver-coated gold nanoparticles are selected from one of silver-coated gold nanoparticles with different silver shell layer thicknesses; and/or the Raman beacon molecule is Raman beacon molecule 4-mercaptobenzoic acid (4-MBA).
Further, the Raman beacon molecule and the silver-coated gold nanoparticles are coupled in a covalent mode and then coupled to the alpha-lactalbumin rabbit polyclonal antibody of the detection antibody to obtain the SERS probe.
The invention also provides a preparation method of the SERS immunochromatographic test strip for detecting the cow milk allergen alpha-lactalbumin, which comprises the following steps:
(1) preparing silver-coated gold nanoparticles;
(2) detecting the combination of the alpha-lactalbumin rabbit polyclonal antibody, the silver-coated gold nanoparticles and the Raman beacon molecules to form an SERS probe, and spraying the SERS probe on a combination pad;
(3) the capture antibody alpha-lactalbumin mouse monoclonal antibody is scratched to be covered on a T line on an NC membrane, and the goat anti-rabbit secondary antibody is scratched to be covered on a C line on the NC membrane;
(4) and the sample pad, the combining pad, the NC membrane and the water absorption pad are sequentially laminated and adhered on the bottom plate, and the sample pad, the combining pad, the NC membrane and the water absorption pad are sliced and assembled to obtain the water-based ink.
Further, the silver-coated gold nanoparticles are prepared by reducing a 10mM silver nitrate solution, wherein the addition amount of the 10mM silver nitrate solution is 1-5 mL, and/or the dropping speed of the 10mM silver nitrate solution is 0.1-0.3 mL/min.
Further, the preparation method of the SERS probe comprises the following steps: uniformly mixing silver-coated gold nanoparticles with 1mM Raman beacon molecule 4-MBA, adjusting the pH value to 7.0-9.0, magnetically stirring in a glass bottle for 8-12 h, centrifuging at 6000-8000 g for 10-20min, removing supernatant, and re-suspending in water to form a Raman probe; then adding a detection antibody alpha-lactalbumin rabbit polyclonal antibody, oscillating at room temperature for 2-4 h, adjusting the pH value to 8.0-9.0, adding BSA, and oscillating at room temperature for 8-12 h to obtain an SERS probe;
wherein the volume ratio of the silver-coated gold nanoparticles to the 4-MBA in the Raman probe is 1000-2000: 1;
and/or the addition amount of the alpha-lactalbumin rabbit polyclonal antibody in the SERS probe is 20-60 mug/mL of Raman probe solution;
and/or the addition amount of BSA in the SERS probe is 50-150 mug/mL of Raman probe solution.
Further, after the Raman probe and the alpha-lactalbumin rabbit polyclonal antibody of the detection antibody are combined to form an SERS probe, centrifuging for 10-20min at 8000g of 6000-; further, adding trehalose into the SERS probe after the PBS buffer solution is resuspended, and spraying the SERS probe on the bonding pad treated by the treatment solution, wherein the addition amount of the trehalose is 20-40%;
and/or the gold spraying amount is 2-8 mu L/cm;
and/or the treatment fluid is PBS buffer solution containing 0.5-2% BSA and 0.5-2% PVP;
and/or drying the combined pad after the treatment of the treatment liquid, and drying at 37 ℃ for 0.5-2 h.
Further, the concentration of the capture antibody alpha-lactalbumin mouse monoclonal antibody is 1-4 mg/mL, and/or the concentration of the goat anti-rabbit secondary antibody is 1-4 mg/mL, and/or the NC membrane after membrane scratching is dried for 2-5 h at 37 ℃.
Further, the sample pad is laminated at a position 1-2 mm from the combination pad, and/or the combination pad is laminated at a position 1-2 mm from the NC membrane, and/or the water absorption pad is laminated at a position 1-2 mm from the NC membrane, and/or the width of the SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin is 3-4 mm.
The invention also provides application of the SERS immunochromatographic test strip for detecting the milk allergen alpha-lactalbumin in the fixed detection and high-sensitivity quantitative detection of the milk allergen alpha-lactalbumin within 5-10 min.
Further, the method for detecting the cow milk allergen alpha-lactalbumin comprises the following steps: diluting an alpha-lactalbumin sample to be detected by using a sample buffer solution, dropwise adding the diluted alpha-lactalbumin sample to a sample pad of the SERS immunochromatography test strip for detecting the milk allergen alpha-lactalbumin, combining the chromatography with an SERS probe on a combination pad, sequentially passing through a T line and a C line, and carrying out spectrum scanning on points on the T line by using a scanning test line of a quantitative analyzer and a laser confocal Raman instrument to carry out alpha-lactalbumin detection; wherein:
(1) and (3) qualitative detection: visually observing the color change of the T line and the C line within 5-10 min: when the T line is colored and the C line is colored, the detection result is positive, and the alpha-lactalbumin sample to be detected contains alpha-lactalbumin; when the T line is not developed and the C line is developed, the detection result is negative, and the alpha-lactalbumin sample to be detected does not contain the alpha-lactalbumin;
(2) high-sensitivity quantitative detection: scanning an SERS signal at 785nm on a T line by using a confocal micro-Raman spectrometer, and quantitatively analyzing the alpha-lactalbumin by using the Raman signal intensity at a characteristic peak of 4-MBA of Raman beacon molecules.
Advantageous effects
The invention carries out quantitative analysis on alpha-lactalbumin by SERS technology, the SERS signal is strong, the fingerprint information is rich and stable, the noise is reduced smoothly after 1-3 times of accumulation, the higher sensitivity can be detected, and the prepared SERS immunochromatographic test strip is simple and convenient to operate, high in efficiency and low in price.
Drawings
FIG. 1 is a technical schematic diagram of an SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin in the present invention.
FIG. 2 is a photograph (A), a UV spectrum (B) and a scanning electron microscope (C) of Ag-coated Au nanoparticles prepared by using different amounts of 10mM silver nitrate solution in one example.
FIG. 3 is a Raman spectrum (A) of the enhancement effect of silver-coated gold nanoparticles on Raman beacon molecules 4-MBA at 1078cm prepared with different addition amounts of silver nitrate solution in one example-1And 1587cm-Raman intensity at 1 (B).
FIG. 4 is a diagram of an embodiment of the SERS immunochromatographic strip dropped with different concentrations of the alpha-lactalbumin sample (A), a Raman scanning spectrum (B) at the T line, and a Raman spectrum at 1611cm-1Standard plot of raman intensity (C).
Detailed Description
In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or the conditions recommended by the manufacturer.
alpha-Lactalbumin (alpha-LA) is one of the main allergens of cow milk, alpha-LA is a monomer spherical calcium-binding metalloprotein which is composed of 123 amino acid residues, accounts for 25 percent of whey protein, has a molecular weight of about 14.2kDa, and has four disulfide bonds and a high-affinity calcium binding site in the protein structure, thereby ensuring the stability of a secondary structure. In addition, α -LA has high thermostability and refolding ability, has a dense globular tertiary structure, and its potential sensitization is due to the presence of a large number of epitopes that selectively bind IgE, the main antigenic sites being 42-49, 60-80 and 91-96.
The methods for detecting cow milk allergens mainly comprise an immunoassay technology, a liquid chromatography technology, a biological mass spectrometry technology, a real-time PCR (polymerase chain reaction) technology, an electrospray ionization mass spectrometry and the like, and although the methods have high sensitivity, the methods need long detection time or expensive instrument equipment. The SERS is combined with the immunochromatography technology, so that the immunochromatography detection kit has the advantages of instantaneity, rapidness, simplicity, convenience and high specificity of immunochromatography detection, can develop color within 5-10 min, can be combined with a portable Raman instrument to establish a linear relation corresponding to the concentration of a detected object and the Raman characteristic peak intensity, improves the detection sensitivity, and can realize the rapid, efficient and accurate quantitative detection of the target alpha-lactalbumin.
The design principle of the invention is that an alpha-lactalbumin sample to be detected is dripped on a sample pad and is combined with an antibody on an SERS probe on a combination pad, the SERS probe contains a Raman beacon molecule and is captured by a captured antibody to react to form a double-antibody sandwich structure when reaching a T line, the T line develops color, the redundant SERS probe can be combined with a goat anti-rabbit secondary antibody on a C line along with the chromatography of a buffer solution, the C line develops color, and the result is obtained by a colorimetric method and a Raman method. The colorimetric detection is to scan the color development degree of the test line and the C line by using a quantitative instrument. The Raman method detection is to utilize a laser confocal Raman instrument to carry out Raman spectrum scanning on a T line, and establish a linear relation corresponding to the concentration of a detected object and the Raman characteristic peak intensity by analyzing the peak height of a Raman beacon molecule 4-MBA characteristic peak, so that an actual sample can be analyzed.
The technical solution, its implementation and principles will be further explained as follows.
The following embodiment provides a preparation method of an SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin, which comprises the following steps:
(1) preparing silver-coated gold nanoparticles capable of enhancing Raman signals;
(2) detecting the combination of the alpha-lactalbumin rabbit polyclonal antibody, the silver-coated gold nanoparticles and the Raman beacon molecules to form an SERS probe, and spraying the SERS probe on a combination pad;
(3) the capture antibody alpha-lactalbumin mouse monoclonal antibody is scratched to be covered on a T line on an NC membrane, and the goat anti-rabbit secondary antibody is scratched to be covered on a C line on the NC membrane;
(4) and (3) sequentially laminating the sample pad, the combining pad, the NC film and the water absorption pad on the bottom plate, and slicing and assembling to obtain the water-absorbing material.
In some embodiments, silver-coated gold nanoparticles are prepared using a reduction process.
Further, the amount of the added 10mM silver nitrate solution was 1-5 mL.
Further, the dropping speed of the 10mM silver nitrate solution is 0.1-0.3 mL/min.
In some embodiments, the method of making comprises: uniformly mixing silver-coated gold nanoparticles with 1mM 4-MBA, magnetically stirring for 8-12 h in a glass bottle, centrifuging for 10-20min at 8000g of 6000-plus protein, removing supernatant, suspending in water to form a Raman probe, adding a detection antibody alpha-lactalbumin rabbit polyclonal antibody, carrying out oscillation reaction for 2-4 h at room temperature, adding BSA (bovine serum albumin), and oscillating for 8-12 h at room temperature to obtain the SERS probe.
Further, the volume ratio of the silver-coated gold nanoparticles to the 4-MBA in the Raman probe is 1000-2000: 1.
Furthermore, the addition amount of the alpha-lactalbumin rabbit polyclonal antibody detected in the SERS probe is 20-60 mug/mL of Raman probe solution.
Furthermore, the adding amount of BSA in the SERS probe is 50-150 mug/mL of Raman probe solution.
In some embodiments, the method of making comprises: after the Raman probe is combined with the rabbit polyclonal antibody, the system is centrifuged at 6000-.
In a preferred embodiment, the preparation method of the SERS probe specifically comprises: mixing the nano particles and Raman beacon molecules, centrifugally suspending, adjusting the pH value to 7.0-9.0, combining the Raman probe and rabbit polyclonal antibody, adjusting the pH value to 8.0-9.0, centrifuging the SERS probe for 10-20min, discarding the supernatant, and then re-suspending with PBS (phosphate buffer solution) containing BSA (bovine serum albumin).
In some embodiments, the method of making comprises: and adding trehalose into the resuspended SERS probe, and spraying the SERS probe onto the bonding pad treated by the treatment solution.
Further, the addition amount of trehalose is 20% -40%.
Further, the amount of sprayed gold is 2-8. mu.L/cm.
Further, the conjugate pad treatment solution was PBS buffer containing 0.5-2% BSA and 0.5-2% PVP.
Further, after the bonding pad is treated with the treating fluid, it is dried by spin-drying and dried at 37 ℃ for 0.5 to 2 hours.
In some embodiments, the method of making comprises: and scratching a T line of an NC membrane by a capture antibody alpha-lactalbumin mouse monoclonal antibody, scratching a C line of an NC membrane by a goat anti-rabbit secondary antibody, wherein the concentration of the mouse monoclonal antibody is 1-4 mg/mL, and the concentration of the goat anti-rabbit secondary antibody is 1-4 mg/mL.
In a preferred embodiment, the treatment method after scribing specifically comprises: and drying the scratched NC film at 37 ℃ for 2-5 h.
In some embodiments, the method of making comprises: the method comprises the following steps of sequentially connecting a sample pad, a combination pad, an NC membrane and a water absorption pad on a bottom plate to finish assembly of the SERS immunochromatography test strip, wherein the sample pad is overlapped at a position 1-2 mm away from the combination pad, the combination pad is overlapped at a position 1-2 mm away from the NC membrane, the water absorption pad is overlapped at a position 1-2 mm away from the NC membrane, assembling slices, and the test strip is 3-4 mm wide, so that detection of an alpha-lactalbumin standard and a sample by a colorimetric method and a Raman method is finished within 5-10 min.
In a preferred embodiment, the specific steps for preparing the SERS immunochromatographic strip for detecting bovine milk allergen α -lactalbumin are as follows:
1) preparation of silver-coated gold nanoparticles
Adding 100mL of 0.01% chloroauric acid solution into a 250mL triangular flask, constantly stirring and heating until boiling, and controlling the reaction temperature at 100-; boiling the solution for 5min, quickly adding 2mL of 1% trisodium citrate solution, continuously boiling for 10min, stopping reaction when the solution is relatively transparent, cooling to room temperature, and supplementing to 100mL with ultrapure water; adding 100mL of ultrapure water into the triangular flask again, heating to boil, adding 3mL of 1% sodium citrate solution, and adding 0, 1, 2, 3, 4, 5mL of 10mM AgNO dropwise at 0.2mL/min speed by using a syringe pump3Heating the solution, stirring for 60min, stopping heating, cooling to room temperature, diluting to 200mL with ultrapure water, centrifuging the obtained solution at 8000rpm for 15min, removing supernatant, redissolving with ultrapure water, and storing at 4 deg.C.
2) Preparation of SERS probes
Uniformly mixing 1mL of silver-coated gold nanoparticles with 10 mu L of Raman beacon molecule 4-MBA with the concentration of 1mM, adjusting the pH value to 7.0-9.0, magnetically stirring in a glass bottle for 12h, centrifuging at 6900g for 10min, removing the supernatant, suspending in water to form a Raman probe, adding 20 mu g of alpha-lactalbumin rabbit polyclonal antibody, oscillating at room temperature for 2h, adjusting the pH value to 8.0-9.0, adding BSA, oscillating at room temperature for 12h to obtain an SERS probe, centrifuging at 8000g, removing the supernatant, and suspending with PBS buffer containing 1% BSA.
3) Spraying gold
Soaking the bonding pad in PBS buffer solution containing 0.5% BSA and 0.5% PVP as bonding pad treating solution, spin-drying, oven-drying, adding 20% -40% trehalose into the resuspended SERS probe, spraying the solution onto the treated bonding pad, spraying gold at 4 μ L/cm, and drying at 37 deg.C for 30 min.
4) Scribing film
Diluting a capture antibody alpha-lactalbumin mouse monoclonal antibody to the concentration of 2mg/mL by using a T-line diluent, scratching a membrane at the T line of an NC membrane with the scratching amount of 1 mu L/cm, wherein the T-line diluent is a PBS (phosphate buffer solution) containing 3% trehalose, diluting a goat anti-rabbit secondary antibody to the concentration of 2mg/mL by using a C-line diluent, scratching a membrane at the C line on the NC membrane, wherein the C-line diluent is a PBS (phosphate buffer solution) containing 3% trehalose, and drying the NC membrane after scratching at 37 ℃ for 2 h.
5) Assembly
Sequentially superposing a sample pad, a combination pad, an NC membrane and a water absorption pad on a PVC base plate, superposing the sample pad on the combination pad at a position of 1-2 mm, superposing the combination pad on the NC membrane at a position of 1-2 mm, superposing the water absorption pad on the NC membrane at a position of 1-2 mm, cutting the combined sample pad into test strips with the width of 3mm by a slicer, and assembling the test strips by a card shell to finish the preparation of the SERS immunochromatographic test strip.
Another aspect of embodiments of the present invention also provides silver-coated gold nanoparticles prepared by the foregoing method.
Furthermore, the particle size of the silver-coated gold nanoparticles is between 20 and 40nm, and the enhancement effect on Raman signals is different.
The embodiment of the invention also provides an application of the alpha-lactalbumin SERS test strip prepared by the method in detection of alpha-lactalbumin by a standard sample and an actual sample.
In conclusion, the Raman enhancement effect of the silver-coated gold nanoparticles prepared by different silver nitrate solution addition amounts is evaluated, and the nanoparticles are characterized by a transmission electron microscope and ultraviolet spectrum scanning. The invention evaluates the detection limit of the alpha-lactalbumin SERS test strip under three detection methods of naked eyes, a colorimetric method and a Raman method and the cross reaction condition with beta-lactoglobulin, casein, BSA, ovalbumin and soybean protein, and the prepared alpha-lactalbumin SERS test strip has good practicability in practical samples such as pure milk, coconut juice, jelly, desensitized milk powder and the like.
The technical solution of the present invention will be described in further detail below with reference to specific examples.
Example 1
(1) Preparation of silver-coated gold nanoparticles
Adding 100mL of 0.01% chloroauric acid solution into a 250mL triangular flask, constantly stirring and heating until boiling, and controlling the reaction temperature at 100-; boiling for 5min, quickly adding 2mL of 1% trisodium citrate solution, boiling for 10min, stopping reaction when the solution is relatively transparent, cooling to room temperature, adding ultrapure water to 100mL, adding 100mL of ultrapure water into a triangular flask, heating to boil, adding 3mL of 1% sodium citrate solution, and adding 2mL of 10mM AgNO dropwise at 0.2mL/min speed by using a syringe pump3Heating the solution, stirring for 60min, stopping heating, cooling to room temperature, diluting to 200mL with ultrapure water, centrifuging the obtained solution at 8000rpm for 15min, removing supernatant, redissolving with ultrapure water, and storing at 4 deg.C.
(2) Preparation of SERS probes
Uniformly mixing 1mL of silver-coated gold nanoparticles with 10 mu L of Raman beacon molecule 4-MBA with the concentration of 1mM, adjusting the pH value to 8.0, magnetically stirring for 12h in a glass bottle, centrifuging for 20min at 8000g, removing the supernatant, suspending in water to form a Raman probe, adding 40 mu g of alpha-lactalbumin rabbit polyclonal antibody, adjusting the pH value to 9.0, oscillating for 4h at room temperature, adding BSA, oscillating for 8h at room temperature to obtain an SERS probe, centrifuging at 6000g, removing the supernatant, and suspending with PBS buffer containing 3% BSA.
(3) Spraying gold
Soaking the bonding pad in PBS buffer solution containing 1% BSA and 1% PVP as bonding pad treating solution, drying, adding 25% trehalose into the resuspended SERS probe, spraying the solution onto the treated bonding pad, spraying gold at 4 μ L/cm, and drying at 37 deg.C for 2 hr.
(4) Scribing film
Diluting a capture antibody alpha-lactalbumin mouse monoclonal antibody to the concentration of 2.5mg/mL by using a T-line diluent, scratching a membrane at the T line of an NC membrane with the scratching amount of 1 mu L/cm, wherein the T-line diluent is a PBS (phosphate buffer solution) containing 2% trehalose, diluting a goat anti-rabbit secondary antibody to the concentration of 2mg/mL by using a C-line diluent, scratching the membrane at the C line on the NC membrane, wherein the C-line diluent is a PBS buffer solution containing 2% trehalose, and drying the NC membrane after scratching at 37 ℃ for 3 h.
(5) Assembly
Sequentially superposing a sample pad, a combination pad, an NC membrane and a water absorption pad on a PVC base plate, superposing the sample pad on the combination pad at a position of 1-2 mm, superposing the combination pad on the NC membrane at a position of 1-2 mm, superposing the water absorption pad on the NC membrane at a position of 1-2 mm, cutting the combined sample pad into test strips with the width of 3mm by a slicer, and assembling the test strips by a card shell to finish the preparation of the alpha-lactalbumin SERS immunochromatography test strip.
Example 2
The difference between this embodiment and example 1 is that 2mL of 10mM AgNO was added dropwise at a rate of 0.2mL/min using a syringe pump during the preparation of silver-coated gold nanoparticles3A solution, a preparation method for preparing the SERS probe in example 2, was performed according to the following steps:
uniformly mixing 1mL of silver-coated gold nanoparticles with 15 mu L of Raman beacon molecule 4-MBA with the concentration of 1mM, adjusting the pH value to 7.5, magnetically stirring for 12h in a glass bottle, centrifuging for 15min at 6900g, removing the supernatant, suspending in water to form a Raman probe, adding 20 mu g of alpha-lactalbumin rabbit polyclonal antibody, adjusting the pH value to 9.0, oscillating for 4h at room temperature, adding BSA (bovine serum albumin) for sealing, oscillating for 8h at room temperature to obtain an SERS (surface enhanced Raman scattering) probe, centrifuging at 8000g, removing the supernatant, and suspending with 1% BSA-containing PBS (phosphate buffer solution).
The treatment solution of the binding pad is 0.5% BSA and 0.5% PVP PBS buffer solution, the amount of trehalose added into the resuspended SERS probe is 30%, the drying time after gold spraying is 1h, the concentration of the capture antibody alpha-lactalbumin mouse monoclonal antibody coated on the T line is 2mg/mL, and the NC membrane after membrane scratching is dried for 2h at 37 ℃.
Example 3
The difference between this embodiment and example 2 is that 10mM AGNO is added dropwise during the preparation of silver-coated gold nanoparticles3The amount of the solution is 4mL, and the preparation method for preparing the SERS probe in the example 3 is carried out according to the following steps:
uniformly mixing 1mL of silver-coated gold nanoparticles with 20 mu L of Raman beacon molecule 4-MBA with the concentration of 1mM, adjusting the pH value to 9.0, magnetically stirring for 8h in a glass bottle, centrifuging for 20min at 8000g, removing the supernatant, suspending in water to form a Raman probe, adding 60 mu g of alpha-lactalbumin rabbit polyclonal antibody, adjusting the pH value to 8.5, oscillating for 4h at room temperature, adding BSA confining liquid, oscillating for 10h at room temperature to obtain an SERS probe, centrifuging at 8000g, removing the supernatant, and suspending with PBS buffer containing 2% BSA.
The treatment solution of the binding pad is 1% BSA and 1% PVP PBS buffer solution, the trehalose is added into the resuspended SERS probe in an amount of 35%, the gold spraying amount is 5 muL/cm, the concentration of the detection antibody goat anti-rabbit secondary antibody coated on the C line is 4mg/mL, and the NC membrane after membrane scratching is dried at 37 ℃ for 4 hours.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims (11)

1. The utility model provides a detect SERS immunochromatographic test paper strip of cow milk allergen alpha-lactalbumin which characterized in that includes bottom plate, sample pad, combination pad, nitrocellulose membrane (NC membrane) and the pad that absorbs water in proper order, be equipped with detection line (T line) and control line (C line) on the NC membrane, wherein:
the T line is arranged at one end close to the combination pad, and a capture antibody alpha-lactalbumin mouse monoclonal antibody is fixed on the T line;
the C line is arranged at one end close to the adsorption pad, and a goat anti-rabbit secondary antibody is fixed on the C line;
and an SERS probe is sprayed on the combination pad and is obtained by coupling and combining silver-coated gold nanoparticles, Raman beacon molecules and a detection antibody alpha-lactalbumin rabbit polyclonal antibody.
2. The SERS immunochromatographic strip for detecting bovine milk allergen alpha-lactalbumin as claimed in claim 1, wherein the particle size of the silver-coated gold nanoparticles is 20-40nm, the shape is spherical, the particles are selected from one of silver-coated gold nanoparticles with different silver shell thicknesses, and/or the Raman beacon molecule is Raman beacon molecule 4-mercaptobenzoic acid (4-MBA), and/or the width of the SERS immunochromatographic strip for detecting bovine milk allergen alpha-lactalbumin is 3-4 mm.
3. The SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin of claim 1, wherein the Raman beacon molecule and the silver-coated gold nanoparticles are coupled in a covalent manner and then coupled to the rabbit polyclonal antibody for detecting antibody alpha-lactalbumin to obtain an SERS probe.
4. The method for preparing the SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin of any one of claims 1 to 3, which comprises the following steps:
(1) preparing silver-coated gold nanoparticles;
(2) detecting the combination of the alpha-lactalbumin rabbit polyclonal antibody, the silver-coated gold nanoparticles and the Raman beacon molecules to form an SERS probe, and spraying the SERS probe on a combination pad;
(3) the capture antibody alpha-lactalbumin mouse monoclonal antibody is scratched to be covered on a T line on an NC membrane, and the goat anti-rabbit secondary antibody is scratched to be covered on a C line on the NC membrane;
(4) and the sample pad, the combining pad, the NC membrane and the water absorption pad are sequentially laminated and adhered on the bottom plate, and the sample pad, the combining pad, the NC membrane and the water absorption pad are sliced and assembled to obtain the water-based ink.
5. The method for preparing an SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin as claimed in claim 4, wherein the silver-coated gold nanoparticles are prepared by reduction of 10mM silver nitrate solution, wherein the addition amount of the 10mM silver nitrate solution is 1-5 mL, and/or the dropping speed of the 10mM silver nitrate solution is 0.1-0.3 mL/min.
6. The method for preparing an SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin according to claim 4, wherein the method for preparing the SERS probe comprises the following steps: uniformly mixing silver-coated gold nanoparticles with 1mM Raman beacon molecule 4-MBA, adjusting the pH value to 7.0-9.0, magnetically stirring in a glass bottle for 8-12 h, centrifuging at 6000-8000 g for 10-20min, removing supernatant, and re-suspending in water to form a Raman probe; then adding a detection antibody alpha-lactalbumin rabbit polyclonal antibody, oscillating at room temperature for 2-4 h, adjusting the pH value to 8.0-9.0, adding Bovine Serum Albumin (BSA), and oscillating at room temperature for 8-12 h to obtain an SERS probe; wherein:
the volume ratio of the silver-coated gold nanoparticles to the 4-MBA in the Raman probe is 1000-2000: 1;
and/or the addition amount of the alpha-lactalbumin rabbit polyclonal antibody in the SERS probe is 20-60 mug/mL of Raman probe solution;
and/or the adding amount of BSA in the SERS probe is 50-150 mug/mL of Raman probe solution;
and/or after the Raman probe and the alpha-lactalbumin rabbit polyclonal antibody are combined to form an SERS probe, the SERS probe is centrifuged for 10 to 20min at 8000g of 6000-.
7. The method for preparing an SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin as claimed in claim 6, wherein trehalose is added into an SERS probe resuspended in PBS buffer solution and sprayed on a bonding pad treated by treatment solution, wherein the addition amount of trehalose is 20-40%;
and/or the gold spraying amount is 2-8 mu L/cm;
and/or the treatment fluid is PBS buffer solution containing 0.5-2% BSA and 0.5-2% PVP;
and/or drying the combined pad after the treatment of the treatment liquid, and drying at 37 ℃ for 0.5-2 h.
8. The preparation method of the SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin as claimed in claim 4, wherein the concentration of the capture antibody alpha-lactalbumin murine monoclonal antibody is 1-4 mg/mL, and/or the concentration of the goat anti-rabbit secondary antibody is 1-4 mg/mL, and/or the NC membrane after membrane scratching is dried at 37 ℃ for 2-5 h.
9. The method for preparing an SERS immunochromatographic test strip for detecting bovine milk allergen alpha-lactalbumin according to claim 4, wherein the sample pad is laminated at a position 1-2 mm away from the combination pad, and/or the combination pad is laminated at a position 1-2 mm away from the NC membrane, and/or the absorbent pad is laminated at a position 1-2 mm away from the NC membrane.
10. The use of the SERS immunochromatographic strip for detecting bovine milk allergen α -lactalbumin as claimed in any one of claims 1 to 3 for the qualitative detection and high-sensitivity quantitative detection of bovine milk allergen α -lactalbumin within 5-10 min.
11. The use according to claim 10, wherein the method for detecting the bovine milk allergen α -lactalbumin comprises: diluting an alpha-lactalbumin sample to be detected by using a sample buffer solution, dropwise adding the diluted alpha-lactalbumin sample to a sample pad of the SERS immunochromatography test strip for detecting the milk allergen alpha-lactalbumin, combining the chromatography with an SERS probe on a combination pad, sequentially passing through a T line and a C line, and carrying out spectrum scanning on points on the T line by using a scanning test line of a quantitative analyzer and a laser confocal Raman instrument to carry out alpha-lactalbumin detection; wherein:
(1) and (3) qualitative detection: visually observing the color change of the T line and the C line within 5-10 min: when the T line is colored and the C line is colored, the detection result is positive, and the alpha-lactalbumin sample to be detected contains alpha-lactalbumin; when the T line is not developed and the C line is developed, the detection result is negative, which indicates that the alpha-lactalbumin sample to be detected does not contain the alpha-lactalbumin;
(2) high-sensitivity quantitative detection: scanning an SERS signal at 785nm on a T line by using a confocal micro-Raman spectrometer, and quantitatively analyzing the alpha-lactalbumin by using the Raman signal intensity at a characteristic peak of 4-MBA of Raman beacon molecules.
CN202210017529.9A 2022-01-07 2022-01-07 SERS immunochromatographic test strip for detecting cow milk allergen alpha-lactalbumin and application thereof Pending CN114371298A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115792216A (en) * 2022-11-25 2023-03-14 中国农业科学院北京畜牧兽医研究所 Lateral chromatography test strip for detecting toxin, preparation method and use method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115792216A (en) * 2022-11-25 2023-03-14 中国农业科学院北京畜牧兽医研究所 Lateral chromatography test strip for detecting toxin, preparation method and use method
CN115792216B (en) * 2022-11-25 2023-11-03 中国农业科学院北京畜牧兽医研究所 Lateral chromatography test strip for detecting toxins, preparation method and use method

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