CN114518348A - Method and device for detecting allergic protein based on coffee ring effect - Google Patents

Abstract

The invention relates to the technical field of biological analysis and detection, in particular to a method and a device for detecting allergic protein. The method is used for detecting the allergic protein based on the coffee ring effect, can quickly detect whether the food contains the allergic protein to be detected or not without complex instruments and operations, has good accuracy and high sensitivity, and can realize simple, convenient and quick detection of whether the food contains the allergic protein to be detected or not by non-professional personnel in any place on site.

Description

Method and device for detecting allergic protein based on coffee ring effect

Technical Field

The invention relates to the technical field of medical analysis and detection, in particular to a method and a device for detecting allergic protein.

Background

Fish allergy is caused by the fact that fish is rich in a large number of foreign proteins which directly or indirectly activate immune cells, cause the release of chemical mediators and then generate a series of complex biochemical reactions. The extent of anaphylaxis can range from mild local symptoms to severe systemic reactions, which in severe cases can produce anaphylactic shock and even life-threatening. Parvalbumin (PV) is a major allergen in fish, and 90% of fish allergic patients develop allergic reactions to PV. At present, the detection methods of the allergic protein mainly comprise an ELISA (enzyme-linked immunosorbent assay) method and a PCR (polymerase chain reaction) method. These methods require expensive equipment and specialized operators, are technically demanding to operate, and are time consuming. Due to the limitations in the aspects of convenience, detection speed and the like, the application requirements of the market are difficult to meet. Many nanogold-based colorimetric sensors have been reported at the present stage, but are susceptible to interference and have low sensitivity.

The "coffee ring effect" refers to the phenomenon that when the liquid drops are evaporated, the solute can deposit a ring-shaped deposition stain with darker color on the edge. This phenomenon is prevalent in colloidal particle, nanoparticle and small molecule systems. A large number of researches show that the identification and quantitative detection of various targets to be detected can be successfully realized through the coffee ring effect of liquid drops, and the method has great application prospects in the fields of diagnostic analysis, biosensing technology and the like.

Promoting and inhibiting the "coffee ring effect" is of great significance for practical technical applications: when the DNA sediment is subjected to spectral analysis, the more uniform and more accurate the sediment is, and the more accurate analysis map can be obtained by inhibiting the coffee ring effect; the separation and purification of organic macromolecules can be realized by utilizing the function of screening particles by a coffee ring; the method can quickly detect diseases according to the difference of patterns after blood is evaporated to dryness, and is suitable for areas with poor medical conditions; the coffee ring effect can be used to make microcircuits or transparent circuits. Meanwhile, the particles are assembled through liquid drop evaporation without additional energy supply, the operation is simple and easy, the method can be used for separating different types of blood cell diagnosis diseases, and the method has great application in the fields of biological large particle sorting, sample concentration, uniform spraying, signal enhancement and analysis and the like.

Disclosure of Invention

In view of the above, the present invention provides a method and apparatus for detecting allergenic proteins. The method is used for detecting the allergic protein based on the coffee ring effect, has good accuracy and high sensitivity, and can be used for non-professionals to quickly detect whether the allergic protein to be detected is contained or not without complex instruments and operations.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a method for detecting allergic protein based on coffee ring effect, which comprises the following steps:

uniformly mixing a sample to be detected with a detection reagent to obtain a mixed solution;

dripping the mixed liquid on a glass slide, standing, and forming a 'coffee ring' precipitate after the liquid is evaporated;

carrying out ultraviolet irradiation on the precipitate, and judging whether the sample to be detected contains the allergic protein or not according to the fluorescent signal;

the detection reagent is gold nanoparticles modified with aptamer;

the aptamer is specifically bound with the allergen protein to be detected.

The "coffee ring effect" means that when the liquid drop is evaporated, the evaporation flux at the edge is larger due to the uneven distribution of the evaporation flux of the liquid drop, so that the concentration inside the liquid drop is uneven, and therefore a compensation flow from inside to outside is generated inside the liquid drop, and the flow is called capillary flow. During the evaporation process, most particles in the liquid drop are carried to the three-phase contact line of the edge of the liquid drop due to the action of capillary flow, and are piled together to form deposition, the contact line is pinned, and finally, the particles deposit a ring-shaped deposition phenomenon with darker color at the edge.

The invention takes the coffee ring effect as the theoretical basis to detect the allergic protein, and takes the parvalbumin as an example for explanation. When parvalbumin exists in a sample to be detected, a complex formed after the specific binding of the aptamer and the parvalbumin has larger resistance and lower mobility compared with the gold particles, so the gold particles are accumulated at the edge of the liquid drop under the action of capillary flow, and the aptamer-protein complex forms a precipitate in the middle part; because the nano-gold is a stronger energy receptor, has Fluorescence Resonance Energy Transfer (FRET) effect and is a good fluorescence quencher, after the nucleic acid aptamer carrying the fluorescent group is combined with the nano-gold particles, the fluorescent group leads to fluorescence quenching because of being close to the gold nano-particles. And after the nano gold-aptamer probe is identified with the parvalbumin, the aptamer is specifically combined with the parvalbumin to form a compound, so that the aptamer-nano gold assembly is disassembled and assembled, a fluorescent group on the aptamer is far away from the nano gold particles, and the fluorescent group recovers fluorescence.

In the invention, the sample to be detected is a food sample, the food source is not limited at all, and in some common embodiments in the field, the food form is not limited at all, and the food sample is prepared into a solution for detection. In some embodiments, the sample to be tested comprises a food sample such as raw fish meat, cooked fish, fish-containing snacks, and condiments.

In some embodiments, the allergenic protein includes at least one of parvalbumin, bovine serum albumin, ovalbumin, and like common allergenic proteins or combinations thereof. In some embodiments, the allergenic protein is parvalbumin.

In some embodiments, the aptamer of the allergenic protein to be detected is linked to a fluorophore. The type of the fluorescent group is not particularly limited, and any type known in the art can be used for the fluorescent reaction after being irradiated by an ultraviolet lamp.

Before the method provided by the invention is adopted for detecting the allergic protein, after sampling, the sample is prepared into a solution to obtain the sample to be detected. In some embodiments, the sample to be tested and the detection reagent are mixed homogeneously.

In some embodiments, the time of standing is 20 to 40 min. Upon standing, the droplets evaporate and then form a "coffee ring" precipitate.

In some embodiments, the method of determining is:

if the fluorescence signal is detected, judging that the sample to be detected contains the allergic protein; and if the fluorescence signal is not detected, judging that the sample to be detected does not contain the allergic protein.

The invention also provides a device for detecting the allergic protein based on the coffee ring effect, which comprises a reagent tube, a glass slide, an ultraviolet lamp and a detection reagent; the detection reagent is gold nanoparticles modified with the aptamer of the allergic protein to be detected.

According to the method and the device for detecting the allergic protein based on the coffee ring effect, when the allergic protein (such as parvalbumin) exists, the gold particles and the aptamer-protein complex carrying the fluorescent group are effectively separated under the action of the coffee ring effect, so that the fluorescent reaction is more obvious after illumination, and the result identification is facilitated. In addition, the operation flow is simple, complex tools are not needed, the detection can be carried out on site by non-professional personnel, the sensitivity is high, the result judgment is not influenced by subjective factors, and the existence of the anaphylactic protein-parvalbumin can be qualitatively determined.

Drawings

FIG. 1 is a schematic diagram showing the operation of the present invention for detecting allergenic proteins;

FIG. 2 shows a schematic diagram of capillary flow as a droplet evaporates;

FIG. 3 shows a schematic diagram of solute particle flow within a droplet in the presence of parvalbumin;

FIG. 4 is a schematic diagram showing the mechanism of binding of the nanogold-aptamer probe to the target protein;

FIG. 5 shows a schematic view of a fluorescent "OFF-ON" probe;

FIG. 6 shows a reaction scheme after ultraviolet irradiation of the annular precipitate;

FIG. 7 is a schematic diagram showing the result of discrimination based on the fluorescence reaction.

Detailed Description

The invention provides a method and a device for detecting allergic protein. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Unless otherwise specified, the test materials used in the present invention are all common commercial products and are all available on the market.

It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content. In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following description is provided with reference to the accompanying drawings to clearly and completely describe the technical solutions in the embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and therefore should not be considered as limiting the scope of protection. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The invention is further illustrated by the following examples:

example 1

The detection device comprises a reagent tube, a glass slide, an ultraviolet lamp and a detection reagent; the detection reagent is a gold nanoparticle carrying a fluorescent group and modified with a parvalbumin aptamer.

(1) The detection principle is as follows: due to the uneven distribution of the evaporation flux of the droplets, the evaporation flux at the edges is large, resulting in uneven concentration inside the droplets, and thus inside-out compensation flow inside the droplets is generated, which is called capillary flow. During evaporation, most of the particles inside the droplet are carried to the three-phase contact line at the edge of the droplet due to capillary flow and are piled together to form a deposit, pinning the contact line, as shown in fig. 2. The final particles will deposit a ring-like deposit with darker color on the edges. In the presence of the allergenic protein, the aptamer-allergenic protein specifically binds to the allergenic protein to form a complex with greater resistance and lower mobility than gold particles, so that gold particles accumulate at the edges of the droplets under capillary flow, while the aptamer-allergenic protein complex forms a precipitate in the middle, as shown in FIG. 3; because the nano-gold is a stronger energy receptor, fluorescent molecules can be quenched through a Fluorescence Resonance Energy Transfer (FRET) effect, and after the nucleic acid aptamer carrying the fluorescent group is combined with the nano-gold particles, the fluorescent group is quenched due to the fact that the fluorescent group is close to the gold nano-particles. After the nanogold-aptamer probe is identified with the allergenic protein, the aptamer is specifically combined with the allergenic protein to form a complex, so that the aptamer-nanogold assembly is disassembled, as shown in fig. 4. Thereby leading the fluorescent group on the nucleic acid aptamer to be far away from the gold nanoparticles and leading the fluorescent group to recover fluorescence. As shown in fig. 5.

(2) The operation process is as follows: taking a food sample to prepare a protein solution to be detected, uniformly mixing the protein solution with a specific reagent (a nanogold-nucleic acid probe carrying a fluorescent group) prepared in advance, carrying out mutual recognition and interaction between the nanogold-nucleic acid aptamer probe and the protein to be detected, dripping the mixed solution onto a glass sheet, standing and evaporating, and waiting for subsequent detection. As in fig. 1. After evaporation of the droplets, the annular precipitate was subjected to ultraviolet irradiation, as shown in FIG. 6. Whether the precipitate shows a fluorescent reaction is judged to contain the allergenic protein. And (4) analyzing results: the coffee ring effect can separate gold particles from the aptamer-protein complex after the liquid drops are evaporated, most of the gold particles are on the ring, most of the aptamer-protein complex can be uniformly distributed in the middle part by aggregation and precipitation, and the fluorescent group in the aptamer-protein complex has a fluorescent reaction after illumination, namely contains the allergic protein (positive); if the allergen protein is not contained, the nanogold-nucleic acid aptamer is positioned on the ring, and the aptamer is not far away from the gold particle, and the fluorescent group carried on the aptamer does not have a fluorescent reaction (negative) after illumination because of the fluorescence quenching capacity of the nanogold particle, as shown in fig. 7.

(3) Analysis of reaction conditions: the specific binding of the aptamer and the protein, the disassembly and assembly of the aptamer-nanogold assembly and the generation of annular precipitates by droplet evaporation are all natural phenomena, and external force and energy do not need to be provided additionally. And the whole process does not need high-end equipment and a severe environment, and can directly take objects on the spot for rapid detection.

Example 2 detection of parvalbumin in Fish food

Sampling fish food to prepare a parvalbumin solution. The method comprises the following specific steps:

(1) adding the parvalbumin solution and the specific reagent into a reagent tube, and uniformly mixing for 1.5h, wherein the concentration of the parvalbumin solution is 100 mug/mL; parvalbumin aptamer concentration of 0.1 mM; the concentration of the nano gold solution is 20 nM.

(2) The reacted mixture was extracted 5. mu.L and dropped on a glass slide to be horizontally left to evaporate for 36min to obtain a precipitate. The precipitates are edge annular precipitates and middle precipitates.

(3) The precipitate was irradiated with an ultraviolet lamp and the middle precipitate was observed to exhibit a fluorescent reaction.

(4) The positive result in this example indicates the presence of parvalbumin in the prepared solution.

Example 3 specific detection

Preparing bovine serum albumin solution. The method comprises the following specific steps:

(1) adding a bovine serum albumin solution and a specific reagent into a reagent tube, and uniformly mixing for 1.5h, wherein the concentration of the bovine serum albumin solution is 100 mug/mL; the rest of the specific reagents are the same as in example 2.

(2) The reacted mixture was extracted 5. mu.L and dropped on a glass slide to be horizontally left to evaporate for 38min to obtain a precipitate. The precipitate was an edge ring precipitate.

(3) The precipitate was irradiated with an ultraviolet lamp and no fluorescence reaction occurred.

(4) The negative result in this example indicates that parvalbumin is not present in the prepared solution. The parvalbumin aptamer cannot be specifically combined with bovine serum albumin, so that the kit disclosed by the invention has good specificity.

Example 4 sensitivity detection

Preparing parvalbumin solutions with different concentrations. The method comprises the following specific steps:

(1) respectively adding the parvalbumin solution with different concentrations and the specific reagent into different reagent tubes, and uniformly mixing for 1.5h, wherein the concentration of the parvalbumin solution is respectively 1, 2.5, 5, 10, 20, 50 and 100 mu g/mL; the rest of the specific reagents are the same as in example 2.

(2) And extracting 5 mu L of the mixed solution after reaction respectively, dripping the mixed solution on a glass sheet, horizontally standing and evaporating to obtain a precipitate. The precipitates are edge ring precipitates and middle precipitates when the concentration of the parvalbumin solution is more than 10 mu g/mL.

(3) The precipitate was irradiated with an ultraviolet lamp, and the fluorescent reaction was observed in the middle precipitate at a parvalbumin solution concentration of 10. mu.g/mL or more. The fluorescence reaction was not evident at a concentration of 5. mu.g/mL or less.

(4) The test result of this example is positive when the concentration of the parvalbumin solution is 10. mu.g/mL or more, which indicates that the test method of the present invention can effectively detect the result when the concentration of the sample is 10. mu.g/mL or more.

Example 5 detection of accuracy

Respectively preparing a solution to be detected: parvalbumin solution and bovine serum albumin solution. The method comprises the following specific steps:

(1) simultaneously adding the parvalbumin solution, the bovine serum albumin solution and the specific reagent into a reagent tube, and uniformly mixing for 1.5h, wherein the concentration of the parvalbumin solution is 100 mu g/mL; the concentration of the bovine serum albumin solution is 100 mug/mL; the rest of the specific reagents are the same as in example 2.

(2) The reacted mixture was extracted 5. mu.L and dropped on a glass slide to be horizontally left to evaporate for 35min to obtain a precipitate. The precipitates are edge annular precipitates and middle precipitates.

(3) The precipitate was irradiated with an ultraviolet lamp and the middle precipitate was observed to exhibit a fluorescent reaction.

(4) The result of the test in this example is positive, indicating that parvalbumin is present in the test solution. When the target protein and other proteins exist simultaneously, the invention can still effectively detect the existence of the target protein.

In conclusion, the method detects the existence of the parvalbumin in the sample based on the coffee ring effect, has positive detection result on the parvalbumin solution, has negative result on other allergic proteins which are not the parvalbumin, and has specificity. The kit can detect positive results for parvalbumin with the concentration of more than 10 mug/mL, and has better sensitivity. The test can still show positive results after the parvalbumin is mixed with other proteins, and has better accuracy. And the method is simple to operate, short in detection time and capable of detecting without complex tools and professionals.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the terminology used in the description presented above should not be understood as necessarily referring to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (7)

1. A method for detecting an allergenic protein based on the coffee ring effect, comprising:

uniformly mixing a sample to be detected with a detection reagent to obtain a mixed solution;

Dripping the mixed liquid onto a glass slide, standing and evaporating, and forming a 'coffee ring' precipitate after the liquid is evaporated;

carrying out ultraviolet irradiation on the precipitate, and judging whether the sample to be detected contains the allergic protein or not according to the fluorescent signal;

the detection reagent is gold nanoparticles modified with aptamer;

the aptamer is specifically bound with the allergen protein to be detected.

2. The method of claim 1, wherein the sample to be tested is a food sample comprising one or more of raw fish, cooked fish, fish-containing snack, or fish-containing seasoning.

3. The method according to claim 1, wherein the allergenic protein comprises at least one of common allergenic proteins such as parvalbumin, bovine serum albumin, ovalbumin, and the like.

4. The method according to claim 1, wherein the aptamer of the test allergenic protein is linked to a fluorophore.

5. The method according to claim 1, wherein the main operation for the reaction droplets is static evaporation for 20-40 min to generate coffee ring effect.

6. The method according to claim 1, wherein the judging method is as follows:

If the fluorescence signal is detected, judging that the sample to be detected contains the allergic protein; and if the fluorescence signal is not detected, judging that the sample to be detected does not contain the allergic protein.

7. The device for detecting the allergic protein based on the coffee ring effect is characterized by comprising a reagent tube, a glass slide, an ultraviolet lamp and a detection reagent; the detection reagent is gold nanoparticles modified with the aptamer of the allergic protein to be detected.

Images