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 device for detecting allergic proteins. The method detects allergenic proteins based on the coffee ring effect, and can quickly detect whether the allergenic protein to be tested is contained without complex instruments and operations. Simple, convenient and rapid detection of allergenic proteins to be tested.

Description

A method and device for detecting allergic proteins 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 detection device for detecting allergic proteins.

Background technique

Fish allergy is due to the abundance of a large number of heterologous proteins in fish, which activate immune cells directly or indirectly, causing the release of chemical mediators, and then producing a series of complex biochemical reactions. Allergic reactions can range from mild local symptoms to severe systemic reactions, which can lead to anaphylactic shock and even life-threatening reactions. Parvalbumin (PV) is the main allergen in fish, and 90% of fish allergy patients will have an allergic reaction to PV. At present, the detection methods of the allergic protein mainly include ELISA (enzyme-linked immunosorbent assay) method and PCR (polymerase chain reaction) method. These methods require expensive equipment and professional operators, and the operation is highly technical and time-consuming. Due to limitations in convenience and detection speed, it is difficult to meet the application needs of the market. A variety of colorimetric sensors based on gold nanoparticles have been reported at this stage, but they are susceptible to interference and have low sensitivity.

The "coffee ring effect" refers to the phenomenon in which the solute will deposit a darker ring-shaped deposit on the edge as the droplet evaporates. This phenomenon generally exists in colloidal particles, nanoparticles and small molecule systems. A large number of studies have shown that the identification and quantitative detection of various targets to be detected can be successfully achieved through the "coffee ring effect" of droplets, which has great application prospects in the fields of diagnostic analysis and biosensing technology.

Promoting and inhibiting the "coffee ring effect" is of great significance for practical technical applications: when DNA sediments are subjected to spectral analysis, the more uniform the sediment is, the more accurate the analysis is, and the more accurate analysis patterns can be obtained by inhibiting the coffee ring effect; the use of coffee ring sieving The function of particles can realize the separation and purification of organic macromolecules; diseases can be quickly detected according to the difference in the pattern of blood after drying, which is suitable for areas with poor medical conditions; microcircuits or transparent circuits can be prepared by using the coffee ring effect. At the same time, the assembly of particles by droplet evaporation does not require additional energy, and is simple and easy to operate. It can be used to separate different types of blood cells to diagnose diseases. Useful place.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a method and device for detecting allergic proteins. The method is based on the coffee ring effect for the detection of allergenic proteins, with good accuracy and high sensitivity, and can be used by non-professionals to quickly detect whether the allergenic protein to be detected is contained without complex instruments and operations.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a method for detecting allergic proteins based on the coffee ring effect, comprising:

Mix the sample to be tested and the detection reagent evenly to obtain a mixed solution;

Put the mixed droplets onto a glass slide, let stand, and wait for the droplets to evaporate to form a "coffee ring" precipitate;

Perform ultraviolet irradiation on the precipitate, and determine whether the sample to be tested contains an allergic protein according to the fluorescent signal;

The detection reagent is gold nanoparticles modified with nucleic acid aptamers;

The nucleic acid aptamer is a nucleic acid aptamer that specifically binds to the allergic protein to be tested.

"Coffee ring effect" means that when the droplet evaporates, due to the uneven distribution of the evaporation flux of the droplet, the evaporation flux at the edge is larger, resulting in uneven concentration inside the droplet, so the inside of the droplet will produce a flow from the inside to the outside. compensating flow, which is called capillary flow. During the evaporation process, due to the action of capillary flow, most of the particles inside the droplet will be brought to the three-phase contact line at the edge of the droplet, and they will accumulate together to form a deposit, pin the contact line, and finally the particles will be deposited on the edge. A phenomenon of darker annular deposits.

The invention detects allergenic proteins based on the coffee ring effect, and takes parvalbumin as an example for illustration. When parvalbumin is present in the sample to be tested, the complex formed by the specific binding of nucleic acid aptamer and parvalbumin has greater resistance and lower mobility than gold particles, so the gold particles can be affected by capillary flow. It accumulates on the edge of the droplet, and the nucleic acid aptamer-protein complex forms a precipitate in the middle part; because gold nanoparticles are a strong energy acceptor, they have the effect of fluorescence resonance energy transfer (FRET), which is a good Fluorescence quencher, after the nucleic acid aptamer carrying a fluorophore is combined with gold nanoparticles, the fluorophore is close to the gold nanoparticles, resulting in fluorescence quenching. When the nano-gold-aptamer probe recognizes parvalbumin, the aptamer specifically binds to parvalbumin to form a complex, which disassembles the aptamer-nano-gold assembly, thereby making the nucleic acid suitable for The fluorophore on the ligand moves away from the gold nanoparticles, and the fluorophore restores fluorescence.

In the present invention, the sample to be tested is a food sample, and there is no restriction on the source of the food, which is common in the art. In some embodiments, there is no restriction on the form of the food, and the food sample is prepared into a solution for detection. In some embodiments, the samples to be tested include food samples such as raw fish food, cooked fish food, fish-containing snacks and condiments.

In some embodiments, the allergenic protein includes at least one of common allergenic proteins such as parvalbumin, bovine serum albumin, ovalbumin, or the like. In some specific embodiments, the allergenic protein is parvalbumin.

In some embodiments, the nucleic acid aptamer of the allergenic protein to be tested is linked with a fluorescent group. The types of fluorescent groups are not particularly limited, and those well-known in the art can be irradiated with an ultraviolet lamp and have a fluorescent reaction.

Before using the method provided by the present invention to detect allergic proteins, and after sampling, the sample is made into a solution to obtain the sample to be tested. In some embodiments, the sample to be tested and the detection reagent are uniformly mixed.

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

In some embodiments, the method of judging is:

If a fluorescent signal is detected, it is determined that the sample to be tested contains an allergic protein; if no fluorescent signal is detected, it is determined that the sample to be tested does not contain an allergic protein.

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

The detection method and device for allergenic proteins based on the coffee ring effect provided by the present invention When the allergenic proteins (such as parvalbumin) are present, the gold particles and the nucleic acid aptamer-protein complex carrying a fluorescent group are under the effect of the coffee ring effect. Effective separation makes the fluorescence reaction more obvious after illumination, which is convenient for the identification of results. In addition, the operation process is simple and does not require complex tools, which can be detected by non-professionals on site, and the sensitivity is high. The result judgment is not affected by subjective factors, and the presence or absence of the allergenic protein - parvalbumin can be qualitatively determined.

Description of drawings

Fig. 1 shows the operation flow schematic diagram that the present invention detects allergic protein;

Figure 2 shows a schematic diagram of capillary flow when droplets evaporate;

Figure 3 shows a schematic diagram of the flow of solute particles in a droplet in the presence of parvalbumin;

Figure 4 shows a schematic diagram of the binding mechanism between the nano-gold-aptamer probe and the target protein;

Figure 5 shows a schematic diagram of a fluorescent "OFF-ON" probe;

Fig. 6 shows the reaction schematic diagram after ring-shaped precipitation carries out ultraviolet irradiation;

FIG. 7 is a schematic diagram showing the identification results according to the fluorescence reaction.

Detailed ways

The present invention provides a method and device for detecting allergic proteins. Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.

Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

It should be understood that the one or more method steps mentioned in the present invention do not exclude that there are other method steps before and after the combination step or other method steps may be inserted between these explicitly mentioned steps; it should also be understood that these The examples are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, rather than limiting the arrangement order of each method step or limiting the scope of the present invention. In the case where the technical content is not substantially changed, it should also be regarded as the scope in which the present invention can be implemented. In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following will describe the technical solutions in the embodiments of the present invention clearly and completely with reference to the accompanying drawings. It should be understood that the described embodiments are only the present invention. Some of the embodiments, but not all of the embodiments, should not be construed as limiting the scope of protection. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1

The detection device of the present invention includes a reagent tube, a glass slide, an ultraviolet lamp and a detection reagent; the detection reagent is a nano-gold particle carrying a fluorescent group and modified with a parvalbumin nucleic acid aptamer.

(1) Detection principle: Due to the uneven distribution of the evaporation flux of the droplet, the evaporation flux at the edge is large, resulting in uneven concentration inside the droplet, so the inside of the droplet will generate compensation flow from the inside to the outside. The flow is called capillary flow. During the evaporation process, due to the action of capillary flow, most of the particles inside the droplet will be brought to the three-phase contact line at the edge of the droplet, and accumulate together to form a deposit, pinning the contact line, as shown in Figure 2. Eventually the particles will deposit a darker ring around the edges. In the presence of allergen, the complex formed by the specific binding of nucleic acid aptamer and allergen has higher resistance and lower mobility than gold particles, so gold particles accumulate on the edge of the droplet under the action of capillary flow. The nucleic acid aptamer-protein complex forms a precipitate in the middle part, as shown in Figure 3; since gold nanoparticles are a strong energy acceptor, they can quench fluorescent molecules through the fluorescence resonance energy transfer (FRET) effect, and carry After the nucleic acid aptamer of the fluorophore is combined with the gold nanoparticles, the fluorescence quenches due to the proximity of the fluorophore to the gold nanoparticles. When the nano-gold-aptamer probe recognizes the allergy protein, the nucleic acid aptamer specifically binds to the allergy protein to form a complex, and the nucleic acid aptamer-nano-gold assembly is disassembled, as shown in Figure 4. Thus, the fluorophore on the nucleic acid aptamer is kept away from the gold nanoparticles, and the fluorophore restores fluorescence. Figure 5.

(2) Operation process: sample the protein solution to be tested from the food, and mix it with the specific reagent (gold nanoparticles-nucleic acid probes carrying fluorescent groups) prepared in advance. The gold nanoparticles-nucleic acid aptamer probes are mixed with The proteins to be detected recognize and interact with each other, and the mixture is dropped onto the glass slide and left to evaporate for subsequent detection. Figure 1. After the droplets evaporated, their ring-shaped precipitates were subjected to UV irradiation, as shown in Figure 6. The presence of allergenic proteins was judged by whether the precipitate exhibited a fluorescent reaction. Analysis of results: The coffee ring effect can separate gold particles from aptamer-protein complexes after droplet evaporation. Most of the gold particles are on the ring, while the aptamer-protein complexes will aggregate and precipitate and most of them are evenly distributed in the middle part. The fluorescent group in it contains allergenic protein (positive) because of the fluorescent reaction after light exposure; if there is no allergic protein, the nano-gold-nucleic acid aptamer is located on the ring, because the aptamer is not far from the gold particles, the aptamer Due to the fluorescence quenching ability of gold nanoparticles, the fluorophore carried on it will not show a fluorescence reaction (negative) after illumination, as shown in Figure 7.

(3) Analysis of reaction conditions: the specific binding of nucleic acid aptamers to proteins, the disassembly of nucleic acid aptamer-gold nanoparticles, and the formation of ring-shaped precipitates by droplet evaporation are all natural phenomena, and do not require additional external force or energy. And the whole process does not require high-end equipment and harsh environments, and can be directly taken on-site for rapid testing.

Embodiment 2 Detection of parvalbumin in fish food

The parvalbumin solution was prepared by sampling from fish food. Specific steps are as follows:

(1) Add the parvalbumin solution and the specific reagents into the reagent tube and mix them evenly for 1.5 hours, wherein the concentration of the parvalbumin solution is 100 μg/mL; the concentration of the parvalbumin aptamer is 0.1 mM; the concentration of the nanogold solution is 20nM.

(2) 5 μL of the reacted mixture was extracted and dropped on a glass slide and left to stand horizontally for 36 minutes to obtain a precipitate. The precipitation is an edge ring precipitation and a central precipitation.

(3) The precipitate was irradiated with an ultraviolet lamp, and it was observed that the precipitate in the middle showed a fluorescent reaction.

(4) The detection result in this example is positive, indicating that parvalbumin is present in the prepared solution.

Example 3 Specific detection

Prepare bovine serum albumin solution. Specific steps are as follows:

(1) The bovine serum albumin solution and the specific reagent were added to the reagent tube and mixed evenly for 1.5 hours, wherein the concentration of the bovine serum albumin solution was 100 μg/mL; the other specific reagents were the same as those in Example 2.

(2) 5 μL of the reacted mixed solution was extracted and dropped on a glass slide and left to stand horizontally for 38 minutes to obtain a precipitate. The precipitate was an edge-ring precipitate.

(3) The precipitation was irradiated with an ultraviolet lamp, and there was no fluorescent reaction.

(4) The test result in this example is negative, indicating that parvalbumin does not exist in the prepared solution. The parvalbumin nucleic acid aptamer does not specifically bind to bovine serum albumin, indicating that the kit of the present invention has good specificity.

Example 4 Sensitivity detection

Prepare different concentrations of parvalbumin solutions. Specific steps are as follows:

(1) Add the parvalbumin solutions and specific reagents of different concentrations into different reagent tubes and mix them evenly for 1.5 hours, wherein the concentrations of the parvalbumin solutions are 1, 2.5, 5, 10, 20, 50, and 100 μg/mL, respectively. ; Other specific reagents are the same as in Example 2.

(2) 5 μL of the reacted mixed solution was extracted and dropped on a glass slide and left to stand horizontally to evaporate to obtain a precipitate. When the concentration of parvalbumin solution was above 10 μg/mL, the precipitates were both edge-ring and central.

(3) The precipitate was irradiated with ultraviolet light, and the central precipitate showed fluorescent reaction when the concentration of the parvalbumin solution was 10 μg/mL and above. The fluorescence response was not obvious when the concentration was below 5μg/mL.

(4) The detection result of this embodiment is positive when the concentration of the parvalbumin solution is 10 μg/mL and above, indicating that the detection method of the present invention can effectively detect the result when the sample concentration is 10 μg/mL and above.

Embodiment 5 Accuracy detection

Prepare the solutions to be tested separately: parvalbumin solution and bovine serum albumin solution. Specific steps are as follows:

(1) Add the parvalbumin solution, the bovine serum albumin solution and the specific reagent to the reagent tube at the same time and mix them evenly for 1.5 hours, wherein the concentration of the parvalbumin solution is 100 μg/mL; the concentration of the bovine serum albumin solution is 100 μg/mL; the rest The specific reagents are the same as those in Example 2.

(2) 5 μL of the reacted mixture was extracted and dropped on a glass slide and left to stand horizontally for 35 minutes to obtain a precipitate. The precipitation is an edge ring precipitation and a central precipitation.

(3) The precipitate was irradiated with an ultraviolet lamp, and it was observed that the precipitate in the middle showed a fluorescent reaction.

(4) The detection result of this embodiment is positive, indicating that parvalbumin exists in the solution to be tested. When the target protein and other proteins exist at the same time, the present invention can still effectively detect the existence of the target protein.

In conclusion, the present invention detects the presence of parvalbumin in the sample based on the coffee ring effect, and the detection result is positive for the parvalbumin solution, and negative for other allergic proteins other than parvalbumin, which is specific. Positive results can be detected for parvalbumin with a concentration of more than 10μg/mL, with good sensitivity. When the parvalbumin is mixed with other proteins, the detection can still show a positive result with good accuracy. And the operation is simple, the detection time is short, and the detection can be carried out without complicated tools and professionals.

In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be construed 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, those skilled in the art may combine and combine the different embodiments or examples described in this specification.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

Claims (7)

1. a method for detecting allergic protein based on coffee ring effect, is characterized in that, comprises: Mix the sample to be tested and the detection reagent evenly to obtain a mixed solution; Putting the mixed droplets onto a glass slide, standing to evaporate, and waiting for the droplets to evaporate to form a "coffee ring" precipitation; Perform ultraviolet irradiation on the precipitate, and determine whether the sample to be tested contains an allergic protein according to the fluorescent signal; The detection reagent is gold nanoparticles modified with nucleic acid aptamers; The nucleic acid aptamer is a nucleic acid aptamer that specifically binds to the allergic protein to be tested. 2. The method according to claim 1, wherein the sample to be tested is a food sample, including one of raw fish food, cooked fish food, fish-containing snacks or fish-containing condiments or variety. 3 . The method according to claim 1 , wherein the allergic proteins comprise at least one of common allergic proteins such as parvalbumin, bovine serum albumin, and ovalbumin. 4 . 4 . The method according to claim 1 , wherein the nucleic acid aptamer of the allergenic protein to be tested is linked with a fluorescent group. 5 . 5 . The method according to claim 1 , wherein the main operation for the reaction droplets is to generate a coffee ring effect by standing and evaporating, and the time for standing and evaporating is 20-40 minutes. 6 . 6. The method according to claim 1, wherein the method for judging is: If a fluorescent signal is detected, it is determined that the sample to be tested contains an allergic protein; if no fluorescent signal is detected, it is determined that the sample to be tested does not contain an allergic protein. 7. A device for detecting allergic proteins based on coffee ring effect, characterized in that it comprises reagent tubes, glass slides, ultraviolet lamps and detection reagents; the detection reagents are gold nanoparticles modified with nucleic acid aptamers of the allergic proteins to be detected particles.

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