CN108267603A - Diclofenac quantum dot immune chromatography detection card and detection method based on signal amplifying system - Google Patents

Abstract

The invention relates to a diclofenac quantum dot immune chromatography detection card and a detection method based on a signal amplification system. The detection card includes a liner, a detection antigen release pad, a quantum dot probe release pad, a chromatographic membrane and a water-absorbing pad; the detection antigen release pad contains a detection antigen coupled by ovalbumin and diclofenac; the quantum dot probe The release pad contains quantum dot probes labeled with anti-ovalbumin antibody; the chromatographic membrane is provided with a detection line and a quality control line, the detection line is fixed with anti-diclofenac antibody, and the quality control line is fixed with ovalbumin antigen. The detection antigen dissolves into the detection system from the release pad, forms a ternary system immune complex with the detection antibody and the labeled antibody, and generates a fluorescent detection signal. Under the action of the detection reference of the quality control line, it is judged whether the sample contains diclofenac according to the fluorescent signal on the detection line, which increases the signal strength and stability of the detection, and improves the detection sensitivity and quantitative precision.

Description

Diclofenac Quantum Dot Immunochromatography Detection Card and Detection Based on Signal Amplification System method

technical field

The present invention relates to the field of food and drug safety detection, in particular to a method of applying immunochemical detection technology to the detection of illegally added chemicals in health care products and Chinese patent medicines, especially a diclofenac quantum dot immunochromatographic detection card and detection method based on a signal amplification system .

Background technique

The chemical name of diclofenac is 2-[(2,6-dichloro-3-methylphenyl)amino]benzoic acid, which belongs to non-steroidal anti-inflammatory drugs, has anti-inflammatory, analgesic and antipyretic effects, and is used for rheumatism Arthritis, adhesive spondylitis, non-inflammatory joint pain, arthritis, non-articular rheumatism, pain caused by non-articular inflammation, various neuralgia, cancer pain, post-traumatic pain and fever caused by various inflammations etc., but there are certain side effects, which are clinically prescribed drugs and must be taken under the guidance of a doctor.

Rheumatic diseases are mainly characterized by pain in joints, muscles, soft tissues, and nerves, and the course of the disease is mostly chronic and recurrent. my country has a tradition of regulating chronic diseases through "diet therapy" or traditional Chinese medicine. Against the background of many side effects of chemically synthesized drugs, health food (mainly health wine) and Chinese patent medicines for rheumatic diseases have been widely favored by the market in recent years.

Diclofenac has quick pain-relieving effect and low price, so some unscrupulous manufacturers illegally add diclofenac to health food and Chinese patent medicines to enhance the curative effect and seek illegitimate benefits. Improper use of diclofenac may cause adverse reactions such as nausea, headache or allergic rash, damage the digestive tract, liver and kidneys, and even lead to anaphylactic shock and acute renal failure in severe cases. Long-term consumption of health food containing diclofenac without the knowledge of patients is very harmful, and the supervision and inspection of illegally added diclofenac products must be strengthened. At present, the confirmatory methods for detecting illegally added diclofenac are high performance liquid chromatography and liquid chromatography-mass spectrometry. However, these methods require large investment in equipment, high operating costs, and complicated sample pretreatment, and cannot be tested on-site, making it difficult to screen for illegal additions on a large scale.

The existing rapid detection methods for diclofenac are mainly chemical detection methods and thin-layer chromatography detection methods, and there is a need to improve the detection sensitivity and anti-interference ability. Immunological detection methods are sensitive, specific, rapid and cheap, and have been widely used in the fields of environmental monitoring and food safety. The currently reported immunological methods for the detection of small molecule compounds are all based on the "dual system immunocompetition method" of detection antigen and detection antibody, in which the signal substance is labeled on the detection antibody, and the detection antigen immobilized on the chromatographic membrane is formed. The immune complex produces a detection signal. The "binary system immunocompetition method" has the following disadvantages: 1. It is necessary to label the signal substance for each detection antibody separately, so that the signal substance cannot be prepared uniformly; 2. The detection antibody is in the liquid phase and has insufficient stability; 3. The detection signal intensity and Less sensitive.

Contents of the invention

Based on this, the object of the present invention is to provide a diclofenac quantum dot immunochromatography detection card and detection method based on a signal amplification system, which has the advantages of increasing the strength and stability of the detection signal and improving the detection sensitivity.

The purpose of the present invention is achieved through the following technical solutions:

Diclofenac quantum dot immunochromatography detection card based on signal amplification system, including liner, detection antigen release pad, quantum dot probe release pad, and chromatographic membrane that are sequentially adhered to the liner and partially overlapped between adjacent ones and a water-absorbing pad; the detection antigen release pad comprises a detection antigen coupled by ovalbumin and diclofenac; the quantum dot probe release pad comprises a quantum dot probe marked with an anti-ovalbumin antibody; the layer The membrane analysis is provided with a detection line and a quality control line, the detection line is fixed with anti-diclofenac antibody, and the quality control line is fixed with ovalbumin antigen.

Compared with the traditional "dual system immunocompetition method", the detection card of the present invention utilizes the principle of "ternary system immunocompetition method". In addition to the two immune detection elements of "detection antigen" and "detection antibody", Added "labeled antibody" for detection of antigen carrier protein. The present invention covalently connects the detection substance (diclofenac) to the carrier protein (ovalbumin) to synthesize the detection antigen, marks the labeled antibody (anti-ovalbumin antibody) on the quantum dots, and immobilizes the detection antibody (anti-diclofenac antibody) on the layer The detection antibody captures the detection substance coupled to the detection antigen, and the labeled antibody binds to the carrier protein coupled to the detection antigen to form a double-antibody sandwich immune complex to generate a fluorescent detection signal. When the detection antibody is bound by the free detection substance, the fluorescence intensity will be suppressed, resulting in an inhibitory detection signal.

Specifically, during detection, the sample solution is dropped onto the detection antigen release pad. During the movement of the sample solution, the detection antigen and quantum dot probes are dissolved and carried to the detection line and quality control line. Anti-ovalbumin (OVA) antibody, detection antigen and anti-diclofenac antibody on the detection line form a double-antibody sandwich immunoconjugate, which makes the detection line generate fluorescent signals; the carried quantum dot probe-labeled anti-OVA antibody and the quality control line The OVA antigen immobilized on the surface is bound to accumulate on the quality control line to form a fluorescent signal. When the free diclofenac in the sample reaches a certain concentration, the immune reaction on the detection line will be blocked by competition, and the fluorescent signal will be suppressed; while the fluorescent signal on the quality control line will not be affected by the concentration of diclofenac.

At present, there are few relevant reports on the detection of diclofenac by fluorescent quantum dot immunochromatography, and the present invention provides a new tool for food and drug safety detection. Quantum dots are nano-semiconductor crystals with 200-10000 atoms gathered in three spherical spaces. The diameter of the spherical crystal nucleus is 2-8nm. In order to increase the water solubility and biocompatibility of quantum dots, the outer layer of the spherical crystal nucleus is usually covered with organic molecules. Coating introduces functional groups, and the diameter will increase to 15-30nm. It has good colloidal properties and dynamic properties, and is suitable as a nano-marker material for biological detection. Compared with nanospheres filled with traditional organic fluorescent dyes, quantum dots have a wide and continuous distribution of excitation spectrum, narrow and symmetrical emission peaks; strong stability, strong resistance to photobleaching; high light energy conversion efficiency, brightness is Dozens of times or even more than organic dyes.

Compared with the prior art, the detection card of the present invention has the following advantages: 1. Quantum dots labeled with anti-carrier protein antibodies are uniformly used to generate detection signals, and only one kind of fluorescent probe needs to be prepared in a concentrated manner, which is conducive to the scale and efficiency of detection work. Standardization; 2. The detection antibody is immobilized on the chromatographic membrane. By sealing and drying the membrane, it is more beneficial to the activity protection of the antibody and increases the stability of the antibody; 3. The steric hindrance of the immune combination is smaller and the detection signal is improved. Intensity and sensitivity; 4. Strong specificity, short detection time (5-10 minutes), can be operated on site, and the fluorescent signal can be excited with the help of a portable 360nm light source, and the results can be interpreted by naked eyes. The detection cost is low and the operation is simple, suitable for grassroots detection personnel operate.

Further, the detection antigen release pad is prepared by absorbing a PBS solution containing detection antigen, surfactant, mannitol and sucrose through a glass cellulose membrane. Preferably, a glass cellulose membrane with a thickness of 0.85 mm is used to fully absorb the PBS solution containing 10 μg/mL detection antigen, 50 μg/mL surfactant, 30 mg/mL mannitol, and 50 mg/mL sucrose. Among them, mannitol is used as a freeze-dried scaffold to ensure the rapid dissolution of the detection antigen during the detection process; sucrose is used to adjust the viscosity of the detection solution to control the development speed of chromatography; surfactants are used to eliminate non-specific adsorption during the detection process, preferably polyethylene glycol Octylphenyl ether (Triton X-100); the detection antigen is synthesized by covalent coupling of OVA and diclofenac, which can be bound by both the detection antibody on the membrane and the labeled antibody on the quantum dot.

Further, the quantum dot probe release pad is prepared by absorbing a PBS solution containing quantum dot fluorescent probes, polyethylene glycol, mannitol, sucrose, and glycine through an artificial cellulose membrane. Preferably, an artificial cellulose film with a thickness of 0.34 mm is used to absorb the 20 μg/mL quantum dot fluorescent probe, 60 μg/mL polyethylene glycol (PEG-500), 10 mg/mL mannitol, 60 mg/mL sucrose, 10 mg/mL mL glycine in PBS. Among them, mannitol is used as a freeze-dried scaffold to ensure the rapid dissolution of quantum dot probes during the detection process; polyethylene glycol and sucrose are used to adjust the viscosity of the detection solution to control the development speed of chromatography; glycine is used to eliminate non-specific adsorption during the detection process; Quantum dot fluorescent probes are composed of quantum dot surface-labeled anti-OVA antibodies, which can bind to the detection antigen captured on the detection line and the OVA antigen immobilized on the quality control line to generate fluorescent detection signals respectively.

Further, the chromatographic membrane adopts a nitrocellulose membrane, and the detection line is prepared by spraying a PBS solution containing anti-diclofenac antibody and sucrose on the nitrocellulose membrane. Preferably, a 0.05M PBS solution (pH 7.4) containing 0.3 mg/mL anti-diclofenac antibody and 10 mg/mL sucrose is sprayed on the nitrocellulose membrane at an amount of 1.0-4.0 μg/cm. The basis for the formation of the fluorescent signal of the detection line is the immune reaction between the antibody and the diclofenac coupled to the detection antigen, which will be competitively inhibited by the free diclofenac in the test product.

Further, the quality control line is prepared by spraying a PBS solution containing ovalbumin and sucrose on a nitrocellulose membrane. Preferably, a 0.05M PBS solution (pH 7.4) containing 0.2 mg/mL OVA and 10 mg/mL sucrose is sprayed on the nitrocellulose membrane at an amount of 1.0-3.0 μg/cm. The basis for the formation of the fluorescent signal of the quality control line is the immune reaction between the anti-OVA antibody of the quantum dot and the quality control OVA antigen, which has nothing to do with the diclofenac test product and will not be competitively inhibited by the free diclofenac in the test product.

The diclofenac quantum dot immunochromatographic detection method based on the signal amplification system comprises the following steps:

S1: Preparation of detection antigen, quantum dot probe, detection line and quality control line; the detection antigen is formed by coupling ovalbumin and diclofenac, the quantum dot probe is labeled with anti-ovalbumin antibody, and the detection The anti-diclofenac antibody is fixed on the line, and the ovalbumin antigen is fixed on the quality control line;

S2: Mix the sample solution, detection antigen and quantum dot probe, and send them to the detection line and quality control line together, and judge whether the sample contains diclofenac according to the fluorescent signals on the detection line and quality control line.

Diclofenac is a small molecule compound. The principle of immunoassay for routine detection of small molecule compounds is the "dual system immunocompetition method" formed by "detection antigen" and "detection antibody". , on the basis of "detection antigen" and "detection antibody", the present invention adds "labeled antibody" to form a "ternary system immune competition method", and also adjusts the combination of each immune reaction element in the chromatography system, Fix the detection antibody on the detection line, label the anti-OVA antibody on the quantum dot to form a fluorescent probe, "bridge" with the detection antigen (OVA-diclofenac) to form a "double antibody sandwich" immune complex, and the fluorescent probe accumulates to form a detection signal , increase the signal strength and stability of the detection, and improve the detection sensitivity. When the detection antibody is bound by the free detection substance, the fluorescence intensity will be suppressed, thereby generating an inhibitory detection signal. Utilizing the detection method of the present invention, the minimum detection limit of diclofenac in the sample solution is 2.0ng/mL, and the minimum detection limit of diclofenac and its analogues illegally added in health care products and Chinese patent medicines is 2.0 μg/kg, and can be used in The rapid test is completed within 5 minutes.

Further, in step S2, the following method is used to judge: the detection line and the quality control line both display fluorescence signals, and the result is determined to be negative, and the sample does not contain diclofenac; the detection line does not display fluorescence signals, and the quality control line displays fluorescence signals, it is concluded that The result was positive and the sample contained diclofenac. Among them, the quality control line is set to check whether the method is effective or not. The quality control line shows a fluorescent signal, indicating that the method is effective, and the quality control line does not show a fluorescent signal, indicating that the method itself is invalid; while the detection line forms a double-antibody sandwich immune complex. Fluorescent signal, when there is free diclofenac in the sample, the immune reaction on the detection line will be blocked by competition, and the fluorescent signal will disappear.

Further, the quantum dot probe generates 630nm emission light when excited by a 365nm light source.

For better understanding and implementation, the present invention will be described in detail below in conjunction with the accompanying drawings.

Description of drawings

FIG. 1 is a schematic structural view of the diclofenac quantum dot immunochromatography detection card based on the signal amplification system of the embodiment.

Fig. 2a is a schematic diagram of an immune reaction occurring on the detection line and a fluorescent signal generated.

Fig. 2b is a schematic diagram showing that the immune reaction on the detection line is blocked by competition and the fluorescent signal disappears.

Fig. 3 is a schematic diagram of the immunoreaction on the quality control line and the generation of fluorescent signals.

Fig. 4 shows the principle of generating fluorescent immunoassay results and the judgment principle of the test results.

Detailed ways

Please refer to Figure 1, which is the diclofenac quantum dot immunochromatographic detection card based on the signal amplification system of this embodiment, including a PVC liner, and detection antigens that are sequentially adhered to the PVC liner and partially overlapped between adjacent ones Release pad, quantum dot probe release pad, nitrocellulose membrane (NC membrane) and water-absorbing pad; The detection antigen release pad comprises the detection antigen formed by OVA and diclofenac coupling; The quantum dot probe release pad comprises A quantum dot probe labeled with an anti-OVA antibody; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is fixed with an anti-diclofenac antibody, and the quality control line is fixed with an OVA antigen.

Specifically, the preparation method of the detection antigen release pad is as follows: a glass cellulose membrane with a thickness of 0.85 mm is used to fully absorb the reagent containing 10 μg/mL detection antigen, 50 μg/mL Triton X-100, 30 mg/mL mannitol, and 50 mg/mL sucrose. PBS solution, freeze-dried for later use.

Specifically, the preparation method of the quantum dot probe release pad is as follows: first, use the active ester method to label the carboxylated quantum dots, take 1.0 mL of carboxylated quantum dots and dilute them in 20 mL of ultrapure water, add 0.05 mL of freshly prepared EDC solution (10mg/mL), stirred at room temperature for 20 minutes to activate the carboxyl group; added 0.45mg anti-OVA antibody to the pre-activated microspheres, stirred at room temperature for 30 minutes to connect the activated carboxyl group to the amino group of the antibody; the solution was centrifuged at 16000rpm for 30 minutes at low temperature, discarded The supernatant was removed to separate unbound antibodies; the quantum dot probes were resuspended in PBS solution containing 10% Triton X-100, and stored in the dark at 4°C for later use. Then, a Whatman 85 man-made cellulose membrane with a thickness of 0.34 mm was used to absorb the PBS solution containing 20 μg/mL quantum dot fluorescent probe, 60 μg/mL PEG-500, 10 mg/mL mannitol, 60 mg/mL sucrose, and 10 mg/mL glycine , and freeze-dried for later use.

Specifically, the preparation method of the detection line is as follows: 0.05M PBS solution (pH 7.4) containing 0.3 mg/mL anti-diclofenac specific antibody and 10 mg/mL sucrose is sprayed on the nitrocellulose membrane in an amount of 1.00 μg/cm , forming a detection line.

Specifically, the preparation method of the quality control line is as follows: 0.05MPBS solution (pH 7.4) containing 0.2mg/mL OVA and 10mg/mL sucrose is sprayed on the nitrocellulose membrane in an amount of 1.00μg/cm to form a quality control line. Wire.

The method for inducing and producing specific antibodies of diclofenac and its analogues, the method for inducing and producing anti-OVA antibodies, the methods for labeling anti-OVA antibodies on quantum dots, and preparing quantum dot fluorescent probes all adopt the prior art.

The diclofenac quantum dot immunochromatographic detection method based on the signal amplification system of the present embodiment is as follows:

Take 0.2g (or 0.2mL) sample, dissolve it in 2.0mL absolute ethanol to fully extract the target sample, let the extract stand for 10 minutes to fully precipitate impurities, take 0.2mL supernatant and add it to 20mL sample buffer, fully Shake well to become a sample solution; drop 3-4 drops of sample solution on the detection antigen release pad with a dropper, and the detection antigen and quantum dot probes are released during the movement of the sample solution to the nitrocellulose membrane, and then cross the detection line and Quality control line, according to the fluorescent signal on the detection line and the quality control line to determine whether the sample contains diclofenac.

The anti-diclofenac antibody on the nitrocellulose membrane detection line and the anti-OVA antibody labeled on the quantum dot probe are respectively combined with the detection antigen to form a double-antibody sandwich immunoconjugate, so that the detection line generates a fluorescent detection signal, as shown in Figure 2a Show. When the free diclofenac in the sample reaches a certain concentration, the immune reaction on the detection line will be blocked by competition, and the fluorescent signal will disappear, as shown in Figure 2b. The quantum dot probe binds to the OVA antigen immobilized on the quality control line of the nitrocellulose membrane through the labeled anti-OVA antibody, and accumulates on the quality control line to form a fluorescent signal, as shown in Figure 3. The quality control line is set to test whether the method itself is valid or not. The color development is valid, and the non-color development indicates that the method itself is invalid.

As shown in Figure 4, the quantum dot probe produces 630nm emission light under the excitation of a 365nm light source. The judgment principle of the detection result is: both the detection line and the quality control line show fluorescent signals, and the result is determined to be negative (A). Does not contain diclofenac; the detection line does not show a fluorescent signal, and the quality control line shows a fluorescent signal, and the results are determined to be positive (B and C), and the sample contains diclofenac. Among them, the concentration of diclofenac in the positive result B is 1.0ng/mL, and the positive result C The concentration of diclofenac in the drug was higher than 2.0ng/mL.

Compared with the prior art, the "ternary system immunocompetition method" adopted in the present invention has the following advantages: 1. Quantum dots labeled with anti-carrier protein antibodies are uniformly used to generate detection signals, and only one of them needs to be prepared centrally as a fluorescent probe, which is useful. It is conducive to the scale and standardization of detection work; 2. The detection antibody is fixed on the chromatographic membrane. By sealing and drying the membrane, it is more beneficial to the activity protection of the antibody and increases the stability of the antibody; 3. The spatial position of the immune combination The resistance is smaller, and the detection signal strength and sensitivity are improved. Utilizing the detection method of the present invention, the minimum detection limit for diclofenac in the sample solution is 2.0 ng/mL, and the minimum detection limit for illegally adding diclofenac and its analogues in health products and Chinese patent medicines is 2.0 μg/kg, and can Complete rapid test within 5 minutes.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims (8)

1. the Diclofenac quantum dot immune chromatography detection card based on signal amplifying system, it is characterised in that：Including liner plate and Be adhered to successively on liner plate and the detection antigen release pad of overlapping portions between adjacent, quantum dot probe release pad, chromatographic film and Water absorption pad；The detection antigen release pad is included by oralbumin and the diclofenac coupled detection antigen formed；The amount Sub- point probe release pad includes the quantum dot probe for being marked with anti-oralbumin antibody；The chromatographic film is equipped with detection line and matter Line is controlled, the detection line fixes anti-Diclofenac antibody, and the nature controlling line fixes oralbumin antigen.

2. the Diclofenac quantum dot immune chromatography detection card according to claim 1 based on signal amplifying system, special Sign is：The detection antigen release pad using glass fibre element film absorb containing detection antigen, surfactant, mannitol, The PBS solution of sucrose is made.

3. the Diclofenac quantum dot immune chromatography detection card according to claim 1 based on signal amplifying system, special Sign is：The quantum dot probe release pad is absorbed using artificial cellulose's film contains quantum dot fluorescence probe, polyethylene glycol, sweet Dew alcohol, sucrose, glycine PBS solution be made.

4. the Diclofenac quantum dot immune chromatography detection card according to claim 1 based on signal amplifying system, special Sign is：The chromatographic film uses nitrocellulose filter, and the detection line is molten by the PBS for containing anti-Diclofenac antibody and sucrose Liquid is sprayed on nitrocellulose filter and is made.

5. the Diclofenac quantum dot immune chromatography detection card according to claim 4 based on signal amplifying system, special Sign is：The nature controlling line is sprayed on nitrocellulose filter by the PBS solution containing oralbumin and sucrose and is made.

6. the Diclofenac quantum dot immune chromatography detection method based on signal amplifying system, it is characterised in that：Including following step Suddenly：

S1：Prepare detection antigen, quantum dot probe, detection line and nature controlling line；The detection antigen is by oralbumin and double chlorine Fragrant acid is coupled, and the quantum dot probe is marked with anti-oralbumin antibody, and the detection line is fixed anti-Diclofenac and resisted Body, the nature controlling line fix oralbumin antigen；

S2：Sample solution, detection antigen and quantum dot probe are mixed, detection line and nature controlling line are delivered to together, according to detection Fluorescence signal on line and nature controlling line comes in judgement sample whether contain Diclofenac.

7. the Diclofenac quantum dot immune chromatography detection method according to claim 6 based on signal amplifying system, It is characterized in that：In step S2, judged by the following method：Detection line and nature controlling line all show fluorescence signal, conclude result For feminine gender, Diclofenac is free of in sample；Detection line does not show fluorescence signal, and nature controlling line shows fluorescence signal, concludes that result is The positive contains Diclofenac in sample.

8. the Diclofenac quantum dot immune chromatography detection method according to claim 6 based on signal amplifying system, It is characterized in that：The quantum dot probe generates the transmitting light of 630nm under 365nm light source activations.