CN114563572A

CN114563572A - Tobacco leaf all-in-one heavy metal rapid quantitative detection card and detection method

Info

Publication number: CN114563572A
Application number: CN202210447569.7A
Authority: CN
Inventors: 吴晓迪; 米军; 罗超; 刘畅; 刘松洁; 黄磊; 潘虹
Original assignee: Chengdu Anpro Biotechnology Co ltd
Current assignee: Chengdu Anpro Biotechnology Co ltd
Priority date: 2022-04-27
Filing date: 2022-04-27
Publication date: 2022-05-31
Anticipated expiration: 2042-04-27
Also published as: CN114563572B

Abstract

The invention belongs to the field of heavy metal detection, and relates to a tobacco leaf all-in-one heavy metal rapid quantitative detection card and a detection method, wherein a gold-labeled antibody formed by combining modified colloidal gold and an antibody is stable, the antagonistic action between the antibodies during the reaction of the gold-labeled antibody and an antigen is reduced, the reaction sensitivity is effectively improved, the antibody dosage is reduced, and the immunoreaction during detection is effectively overcome; the all-in-one heavy metal rapid quantitative detection card using the gold-labeled antibody can simultaneously detect multiple heavy metals, realize rapid detection and quantification of the multiple heavy metals, greatly simplify the operation process and improve the detection efficiency.

Description

Tobacco leaf all-in-one heavy metal rapid quantitative detection card and detection method

Technical Field

The invention belongs to the field of heavy metal detection, and relates to a tobacco leaf all-in-one heavy metal rapid quantitative detection card and a detection method.

Background

Heavy metal is defined as a metal having a specific gravity greater than 5. Heavy metals in terms of environmental pollution mainly refer to heavy elements with significant biological toxicity, such As mercury (Hg), cadmium (Cd), lead (Pb), chromium (Cr), and metalloid arsenic (As). Heavy metals cannot be biodegraded, but can be enriched hundreds of times under the biological amplification of the food chain, and finally enter the human body. Heavy metals can strongly interact with proteins, enzymes and the like in human bodies, so that the heavy metals lose activity and are accumulated in certain organs of the human bodies to cause chronic poisoning. The main toxic effects are anemia, neurological dysfunction, kidney damage, severe bone softening, cunning and cutting, digestive system symptoms and skin lesions, etc. The affected people include children, the old and people with low immunity. Heavy metals are mainly derived from various paints, storage batteries, electroplating, cosmetics, hair dyes, tableware, fire coal, fruits and vegetables, food, traditional Chinese medicinal materials, tap water pipes and the like.

The main characteristics of heavy metal pollution in China are as follows: (1) environmental pollution effects are the most important factors. Researches find that the overproof rate of cultivated land points of Sichuan basins, Yangtze river midstream and Jianghuai regions, Huang-Huai-Hai plain, Song-Nen plain and Sanjiang plain is 43.55%, 30.64%, 12.22%, 9.35% and 1.67%, and the pollution of cultivated land in south is heavier than that in north. The main pollutants are cadmium (Cd), nickel (Ni), copper (Cu), zinc (Zn) and mercury (Hg), and the standard exceeding rates are 17.39%, 8.41%, 4.04%, 2.84% and 2.56%, respectively. At present, the environmental pollution of surface water, atmosphere, soil and the like in China is serious, 1200 ten thousand tons of grains are polluted by heavy metal in soil every year, and agricultural non-point source pollutants exceed 7.5 times of that of the industry; (2) the contamination lasts long. The environmental pollution is persistent, the treatment effect is poor, and a new industrial pollution activity causes the persistence of the corresponding heavy metal food safety problem. Since the 80 s in the 20 th century, the soil heavy metal content of cultivated land in five major food production areas in China is increased, and the overall site overproof rate is increased by 14.91 percent; (3) the complexity is increasing. Problem food has been expanded from the past grain and the like to bean products, aquatic products, fruits, wines, dried goods, dairy products, traditional Chinese medicinal materials, tobaccos and the like, from the original heavy metal cadmium to elements such as lead, arsenic, mercury, chromium and the like, and from food source pollution to processing technology pollution and storage and sale process pollution.

The problem of heavy metal pollution in the tobacco field is also outstanding, and the tobacco heavy metal detection technology is more and more paid attention to by the industry.

Among the current numerous analytical techniques for heavy metal ion detection, atomic absorption spectroscopy (reas), Atomic Fluorescence Spectrometry (AFS), inductively coupled plasma mass spectrometry (ICP-MS) are commonly used. These methods have higher sensitivity, but are more expensive in instrumentation and detection, require larger sample volumes, and are more time consuming. Therefore, the current requirements of mass samples and field detection cannot be met.

The existing main heavy metal rapid detection methods on the market mainly comprise a chemical color development method, an anodic stripping voltammetry method and an X fluorescence method, and due to the limitation of sample matrixes and sensitivity requirements, the methods can only be applied to the detection and analysis of a small part of food (rice), or the detection and analysis of the content of various heavy metals cannot be realized simultaneously due to single detection item, so that the rapid detection of most samples including tobacco is useless.

Instrumental methods generally have higher sensitivity, multiple heavy metal detection capability, but the instrumentation and detection costs are higher, a larger sample volume is required to obtain higher sensitivity, and the time consumption is longer because samples must be analyzed sequentially.

The immunoassay mainly uses a colloidal gold method, is a heavy metal detection method which is started in recent years, and mainly comprises an indirect competitive ELISA method and a colloidal gold rapid detection methodThe immunochromatography requires the development of colloidal gold when the immunochromatography is used for detection. The colloidal gold is prepared from chloroauric acid (HAuCl)₄) Under the action of reducing agent such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, etc., gold particles with a certain size can be polymerized, and become a stable colloidal state due to electrostatic interaction to form a hydrophobic gel solution with negative charge, and become a stable colloidal state due to electrostatic interaction, so the gold colloid is called gold colloid. The immunoassay is an analysis method for detecting various substances (drugs, hormones, proteins, microorganisms, etc.) by developing a color of a color-developing agent labeled with an antibody by utilizing an antigen-antibody specific binding reaction. The colloidal gold has negative charge in weak alkali environment and can be combined with the positive charge group of the antibody, and the combination is electrostatic combination, so the biological characteristics of the antibody are not influenced. Therefore, when the colloidal gold is used for immunoassay, the method has the advantages of convenience, rapidness, no need of special equipment and reagents, visual result judgment and the like, and is widely used.

However, since colloidal gold can be effectively bound to an antibody only in a weakly alkaline environment, once the pH of a detection object is not weakly alkaline, accurate detection cannot be performed, and detection of heavy metals requires strong acid extraction, although detection can be barely performed by increasing the dilution factor, the detection result is inaccurate.

At present, the heavy metal colloidal gold products in the market mainly take single qualitative items as main materials, and have the advantages of rapidness, good specificity, high sensitivity and the like, but the traditional colloidal gold detection card is generally used for detecting single items (single heavy metals), a two-line method mode is adopted, namely a quality control line C and a detection line T, and a gold-labeled antibody is a single-item antibody, so that the detection card can only detect one item, cannot detect multiple items and cannot perform quantitative analysis, and is difficult to adapt to the trend that the current detection of heavy metals is developed from the detection of single heavy metal varieties to the simultaneous detection of multiple heavy metal components; the conventional heavy metal quantitative detection system does not adopt a masking measure, can not effectively mask the interference of a natural chelating agent in a complex sample of an agricultural product such as tobacco and the like on the detection chelating process, and has low detection sensitivity, can not detect various heavy metals simultaneously and lower detection efficiency. The market needs a detection mode that can be fast, simple and convenient, accurate, high-efficient unification quantitative determination's heavy metal, conveniently carry moreover.

Disclosure of Invention

The invention provides a tobacco leaf all-in-one heavy metal rapid quantitative detection card and a detection method, which solve the technical problems, utilize sulfydryl and metal to easily form a strong coordination bond to replace the traditional colloidal gold to be marked by a way of electrostatic adsorption of an antibody, improve the stability of a gold-labeled antibody, and simultaneously, through a hydrophilic polymer polyethylene glycol (PEG) chain coupling antibody, effectively overcome immunoreaction during detection, reduce the antagonism between the antibodies during the reaction of the antibody and an antigen, effectively improve the reaction sensitivity and reduce the dosage of the antibody; the all-in-one heavy metal rapid quantitative detection card is prepared by utilizing a colloidal gold immunity technology, simultaneous detection of various heavy metals can be realized by one-time operation, and rapid quantification of detection is realized by matching with a constant temperature reaction device and a detection card reading instrument, so that the operation flow is greatly simplified, and the detection efficiency is improved; the influence of background compounds in agricultural products such as tobacco and the like on the detection of the target heavy metal is effectively masked by a masking technology, and the detection applicability is effectively improved.

The technical scheme for solving the technical problems is as follows:

the invention provides a preparation method of modified colloidal gold, which comprises the following preparation steps:

taking 100 parts of colloidal gold solution with diameter of 10-60nm, and using 0.1M K₂CO₃Adjusting the pH value of the colloidal gold solution to be between 6 and 9, adding an HS-PEG-COOH solution which accounts for 0.01 to 1 percent of the mass of the colloidal gold solution, stirring and reacting for 0.5 to 2 hours at the speed of 100 plus 400r/min, then centrifuging for 2 to 10 minutes at the speed of 4000 plus 10000r/min, and removing the supernatant, namely removing the redundant unmarked HS-PEG-COOH to obtain the modified colloidal gold. Wherein the colloidal gold solution is prepared by a chloroauric acid-trisodium citrate reduction method.

Further, the concentration of the HS-PEG-COOH solution is 1%, wherein the molecular weight of PEG is 1000-100000.

The invention also provides the modified colloidal gold prepared by the method.

The invention also provides a preparation method of the gold-labeled antibody, which comprises the following preparation steps:

s101, activating-COOH on the surface of the modified colloidal gold by using NHS and EDC to obtain activated modified colloidal gold;

s102, adding the antibody into the activated and modified colloidal gold obtained in the step S1, and activating-COOH on the surface of the modified colloidal gold and-NH on the surface of the antibody₂Forming amido bond, thereby coupling the antibody on the surface of the colloidal gold to form the gold-labeled antibody.

Preferably, in step S102, the antibody is a Cd monoclonal antibody, a Pb monoclonal antibody, a Hg monoclonal antibody, an As monoclonal antibody, a Cr monoclonal antibody, or a Cu monoclonal antibody.

The invention also provides the gold-labeled antibody prepared by the method.

The invention also provides the application of the modified colloidal gold or the gold-labeled antibody in immune colloidal gold detection.

Preferably, the immune colloidal gold detection is immune colloidal gold optical lens staining, immune colloidal gold electron microscope staining, colloidal gold immunochromatography.

The invention relates to an all-in-one heavy metal rapid quantitative detection card for detecting multiple heavy metals, which comprises detection test paper arranged in a card shell, wherein the detection test paper comprises a bottom plate, a sample pad, a binding pad containing gold-labeled antibodies respectively marked by corresponding antibodies of multiple heavy metals to be detected, a chromatographic membrane and absorbent paper are sequentially stuck on the bottom plate from one end to the other end, one end of the sample pad is in press connection with one end of the binding pad, the other end of the binding pad is in press connection with one end of the chromatographic membrane, and one end of the absorbent paper close to the chromatographic membrane is in press connection with one end of the chromatographic membrane; the chromatographic membrane is coated with a plurality of detection lines T and a quality control line C, the number of the detection lines T is consistent with the number of the types of the gold-labeled antibodies on the binding pad, any detection line T is coated with a complete antigen of the heavy metal to be detected, and the quality control line C is coated with an anti-mouse IgG antibody; a sample adding hole is formed in the position, corresponding to the sample pad, of the surface of the card shell, and an observation port is formed in the position, corresponding to the chromatographic membrane, of the surface of the card shell; the gold-labeled antibodies on the bonding pad are prepared according to the preparation method of the gold-labeled antibodies.

Wherein, the chromatographic membrane is a nitrocellulose membrane with a porous sample structure with the aperture of 5-12 microns, the sample pad is a glass cellulose membrane or a polyester membrane, and the sample absorbing paper is absorbent filter paper.

Preferably, the complete antigen of the heavy metal coated on any detection line T is formed by complexing the heavy metal with a complexing agent and then coupling the heavy metal with a carrier protein to form an antigen, wherein the complexing agent is one or more of citric acid, ethylenediamine tetraacetic acid, glycine, 1- (4-isothiocyanatobenzyl) ethylenediamine-N, N, N ', N' -tetraacetic acid, diethylenetriamine pentacarboxylate, glutathione and organic polydentate ligand; the carrier protein is one or more of bovine serum albumin, keyhole limpet hemocyanin, ovalbumin and human serum protein.

Preferably, the heavy metal is selected from Cd, Pb, Hg, Cu, Cr, As.

The invention also provides a preparation method of the all-in-one heavy metal rapid quantitative detection card, which comprises the following preparation steps:

s201, preparing complete antigens of various heavy metals to be detected, and preparing the modified colloidal gold;

s202, scribing and coating on a chromatographic membrane, scribing a detection line T for each heavy metal complete antigen obtained in the step S201, and scribing a quality control line C by using anti-mouse IgG;

s203, preparing the monoclonal antibodies of various heavy metals to be detected and the modified colloidal gold obtained in the step S201 respectively to obtain gold-labeled antibodies according to the preparation method of the gold-labeled antibodies;

s205, mixing the gold-labeled antibodies of the heavy metals to be detected obtained in the step S203 to prepare a gold-labeled antibody solution, and spraying the gold-labeled antibody solution onto the bonding pad in an amount of 1-10 mu L/cm, wherein the gold-labeled antibody solution comprises the following components: PBS 2mM, BSA 0.1-5 wt.%, sucrose 1-20 wt.%, tween-200.01-1 wt.%, and gold-labeled antibodies of several heavy metals to be detected, wherein the gold-labeled antibodies are 0.001-0.1mg/mL respectively;

and S206, a sample pad, the bonding pad obtained in the step S205, the chromatographic membrane coated by the drawn line in the step S202 and absorbent paper are sequentially adhered to a bottom plate, so that the all-in-one heavy metal rapid quantitative detection card is obtained.

In step S202, streaking is performed with a streaking instrument at a rate of 0.5 to 2. mu.L/cm, and the complete antigen concentrations of heavy metals in the coating solution are each 0.01 to 3 mg/ml.

The invention also provides a detection method of the all-in-one heavy metal rapid quantitative detection card, which is used for detecting various heavy metals and comprises the following detection steps:

s301, crushing a sample to be detected, and screening the sample through a 40-mesh screen to obtain a sample to be detected;

s302, taking the sample to be detected after sieving in the step S301, adding an extracting agent, reacting for 2-30min under the condition that the oscillation frequency is 2-60cpm, centrifuging, and taking the supernatant for later use; the extractant is a dilute acid solution of 1-15% nitric acid, hydrochloric acid, sulfuric acid and perchloric acid, and the volume weight ratio (mL: g) of the extractant to the sample to be detected is (2-20): 1;

s303, taking the supernatant obtained in the step S302, adding a diluent with the volume of 2-40 times that of the supernatant, and uniformly mixing to obtain a chromatography liquid; the diluent is a buffer solution containing a surfactant, a heavy metal chelating agent and a masking agent;

s304, simultaneously putting the all-in-one heavy metal rapid quantitative detection card and the chromatography liquid obtained in the step S303 into a constant-temperature incubator, keeping the constant temperature for 2-15min, and controlling the temperature to be 20-45 ℃;

s305, adding the constant-temperature chromatographic solution obtained in the step S304 into a sample adding hole of the all-in-one heavy metal rapid quantitative detection card, and continuing to perform constant-temperature reaction for 5-30 min;

s306, placing the all-in-one heavy metal rapid quantitative detection card reacted in the step S305 in a reading instrument, comparing the all-in-one heavy metal rapid quantitative detection card with a preset standard curve, and reading a detection result.

In step S303, the surfactant is one of tween-20, tween-80, triton X-100, S7 and S9 with the concentration of 0.5-2%, and the heavy metal complexing agent is one or more of citric acid, ethylenediamine tetraacetic acid, aminoacetic acid, 1- (4-isothiocyanatobenzyl) ethylenediamine-N, N, N ', N' -tetraacetic acid, diethylenetriamine pentacarboxylate, glutathione and organic polydentate ligand with the concentration of 0.01-100 mg/L.

In step S306, the reader is a scanning type colloidal gold reader.

Further, in step S303, the masking agent is one of bismuth, antimony and mercury with a concentration of 0.01-100 mg/L.

The invention adopts the reaction of heavy metal ions and a heavy metal chelating agent to prepare the metal ion chelate, effectively overcomes the property of no immunogenicity of the metal ions, prepares the corresponding complete antigen and monoclonal antibody of the heavy metal ion chelate, and prepares the all-in-one heavy metal rapid quantitative detection card by utilizing the colloidal gold immunity technology, thereby realizing the all-in-one detection of the heavy metals. During detection, heavy metal ions in a detection sample are dissociated by dilute acid, then form corresponding chelates with a metal ion chelating agent, and are added into the detection card to react with the gold label marked on the corresponding monoclonal antibody on the detection card, so that the reaction of the corresponding whole antigen coated detection line and the gold labeled monoclonal antibody is inhibited when the detection line passes through the chromatographic membrane area of the detection card, the color of the detection line is weakened, the higher the content of the metal ions is, the lighter the color is, and the content of the heavy metal ions is accurately detected by comparing the color gray scale of the detection line with the color gray scale of a preset standard curve by using a card reader.

The invention has the beneficial effects that:

1. the invention utilizes sulfydryl (HS-) to easily form strong coordination bond with metal, HS-of HS-PEG-COOH and metal ion on the surface of colloidal gold particle form strong coordination bond to connect in preparing modified colloidal gold, and carboxyl (-COOH) after activation on the surface of modified colloidal gold and amino (-NH) on antibody are reacted with antibody₂) The gold-labeled antibody is coupled on the surface of the colloidal gold to form a gold-labeled antibody by condensation reaction, the phenomenon that the traditional colloidal gold labels the gold-labeled antibody by means of electrostatic adsorption of the antibody is changed, the stability of the gold-labeled antibody, particularly the stability to acid and alkali, is greatly improved due to the protective effect of a polymer on the colloidal gold, the phenomena of adsorption and aggregation and the like are difficult to stably occur when the pH value of the traditional colloidal gold-labeled antibody is lower than 6, the gold-labeled antibody marked by the modified colloidal gold can stably exist within the range of pH value of 2-14, and the liquid can stably exist in a 10% NaCl solution;the modified colloidal gold is connected with the colloidal gold and the antibody through the macromolecular hydrophilic coupling arm, namely polyethylene glycol, so that the stability of the gold-labeled antibody is enhanced by utilizing the strong acting force of a coordination bond, and the binding site of the modified colloidal gold and the antibody is far away from the surface of the colloidal gold, so that the distance between the connected antibodies is increased, the mutual antagonism caused by too close distance between the antibodies in the reaction of the gold-labeled antibody and the antigen is effectively reduced, the reaction sensitivity of the gold-labeled antibody is improved, the using amount of the antibody is greatly reduced, the performance of a detection product is effectively improved, the detection cost is reduced, the requirement on a detection object is small, the application range is wide, and the modified colloidal gold can be applied to detection of various immune colloidal gold;

2. the combined pad of the all-in-one heavy metal rapid quantitative detection card contains two or more gold-labeled antibodies respectively labeled by antibodies corresponding to heavy metals to be detected, the chromatographic membrane is coated with two or more detection lines T corresponding to the gold-labeled antibodies on the combined pad, and each detection line T is coated with a complete antigen of the heavy metals to be detected, so that the simultaneous detection of multiple heavy metals is realized, the detection time is greatly shortened, the operation steps are reduced, and the detection efficiency is greatly improved; the invention unifies the manufacturing processes of different detection items through the modified colloidal gold process, and overcomes the problem that in the prior art, a single detection card is required to be prepared for multiple heavy metal detection items due to inconsistent manufacturing process conditions, so that the all-in-one detection card is easier to prepare, and the preparation operation is simple and convenient;

3. according to the invention, a sample pretreatment masking technology is adopted, and a masking agent is added into a diluent, so that the masking agent consumes a natural chelating agent extracted from a sample, the influence of the natural chelating agent in agricultural products such as tobacco on a detection chelating process is masked, the influence of the reaction of the chelating agent and target metal ions during detection is reduced, and the detection accuracy of the heavy metal ions is effectively improved;

4. the all-in-one heavy metal rapid quantitative detection card detection method has the advantages of simple sample pretreatment, sensitive detection and interference resistance, and can complete rapid quantitative detection of various heavy metals on site by matching the detection card with simple reagents and equipment such as a constant temperature incubator, a reading instrument and the like, so that the detection cost is greatly reduced, and the all-in-one heavy metal rapid quantitative detection card detection method has great application prospects.

Drawings

FIG. 1 is a schematic structural diagram of a test paper of the all-in-one heavy metal rapid quantitative detection card of the present invention;

FIG. 2 is a schematic structural diagram of a card shell of the all-in-one heavy metal rapid quantitative detection card of the present invention;

FIG. 3 is a transmission electron micrograph of a modified gold colloid according to the present invention;

FIG. 4 is a transmission electron micrograph of unmodified colloidal gold according to the present invention;

FIG. 5 is a schematic diagram of the rapid quantitative detection card for Cd, Pb and Cu three-in-one heavy metal of the invention.

Reference numerals: 1-bottom plate, 2-sample pad, 3-combined pad, 4-chromatographic membrane, 6-absorbent paper, 7-sample adding hole and 8-observation hole.

Detailed Description

The principles and features of this invention are described below (in conjunction with the following figures), which are set forth by way of illustration only and are not intended to limit the scope of the invention.

Example 1

The embodiment of the invention provides a preparation method of modified colloidal gold, which comprises the following preparation steps:

1.1 heating 100mL of 0.01% chloroauric acid aqueous solution to boiling, accurately adding 1.8mL of 1% trisodium citrate aqueous solution under stirring, changing the golden chloroauric acid aqueous solution into red within 2min, and continuously boiling for 15min to obtain 40nm colloidal gold solution;

1.2 taking 100mL of colloidal gold solution with the diameter of 40nm, adding 400-800 mu L of 0.1M K₂CO₃Adjusting the pH value of the colloidal gold solution to 7.5, adding 0.01-1% of the mass of the colloidal gold solution of HS-PEG20000-COOH solution with the concentration of 1%, stirring and reacting for 1h at 200r/min, centrifuging for 5min at 8000r/min to remove the supernatant, and obtaining the lower-layer centrifugate which is the modified colloidal gold.

In the preparation method provided in this example, the isoelectric point of HS-PEG-COOH is within 6-9, colloidal gold is easily aggregated when the pH of the colloidal gold solution is lower than 6, and is not easily labeled when the pH of the colloidal gold solution is higher than 9, so K is used₂CO₃Adjusting colloidal goldThe pH value of the solution is 7.5, so that the solution is easier to mark, and the influence of colloidal gold aggregation on the modification effect is avoided;

the molecular weight of PEG in HS-PEG-COOH is selected to be 20000, so that a hydrophilic arm with a certain length is formed, the binding site of the modified colloidal gold and the antibody is far away from the surface of the colloidal gold, and the antagonistic action between the coupled antibodies is weakened;

the addition amount of HS-PEG-COOH is 0.01-1% of the mass of the colloidal gold solution, so as to ensure the full modification; the adverse effect of too low addition of HS-PEG-COOH is insufficient modification, empty uncombined positions exist on the surface of the colloidal gold, the charge is not uniform, the stability is poor, and aggregation is easy; when the addition amount of HS-PEG-COOH is too large, more free HS-PEG-COOH exists, and the free HS-PEG-COOH consumes additional antibody when combined with the antibody, thereby increasing the cost.

Example 2

The embodiment of the invention provides a preparation method of a gold-labeled antibody, which comprises the following preparation steps:

2.1 activation

1) Adding 100mL of the modified colloidal gold solution obtained in the example 1 into a triangular flask, slowly adding 40mL of MES buffer solution at a constant speed, uniformly mixing for 5min in a spiral mixer, centrifuging for 5min under the condition of 10000r/min, removing supernatant, and observing redissolution; adding 100mL MES, carrying out ultrasonic resuspension for 1-3min, carrying out ultrasonic power 50%, and observing the redissolution condition;

2) respectively adding 5mg of EDC [1- (3-dimethylaminopropyl) -3-ethylcarbodiimide ] and 5mg of NHS (N-hydroxysuccinimide) into a centrifuge tube, respectively adding 1mL of MES buffer solution, and fully and uniformly mixing to obtain EDC solution and NHS solution, wherein the EDC solution is prepared for use on site, and is preferably protected from light;

3) slowly adding 0.2mL of the NHS solution obtained in the step 2) into the modified colloidal gold solution subjected to ultrasonic resuspension in the step 1), uniformly mixing, slowly adding 0.05mL of the EDC solution obtained in the step 2), uniformly mixing, and reacting for 30min in a dark place; centrifuging at 10000r/min for 5min after reaction, removing supernatant, ultrasonically resuspending and washing the lower-layer centrifugate by using 50mL of HEPES buffer solution, centrifuging to remove supernatant, adding 100mL of HEPES buffer solution, providing an alkaline reaction environment with pH of 8, preparing an environment for coupling an antibody, and obtaining an activated modified colloidal gold solution;

2.2 coupling

1) Adding 10-500 mu g of antibody diluted in 1mL of HEPES buffer solution into 100mL of the obtained activated modified colloidal gold solution, wherein the antibody needs to be added in an adherent manner when the antibody is added, and the addition is finished for 30s, so that the aggregation of the colloidal gold caused by overhigh local concentration is prevented; the antibody is one of a Cd monoclonal antibody, a Pb monoclonal antibody, an Hg monoclonal antibody, an As monoclonal antibody, a Cr monoclonal antibody or a Cu monoclonal antibody, and can be selected according to actual detection requirements;

2) placing the activated modified colloidal gold solution system added with the antibody in the step 1) in a dark room, and slightly vibrating for 60min at room temperature; centrifuging at 10000r/min for 5min, and then washing for 2 times by using 100mL HEPES buffer solution to obtain a coupling gold label;

2.3 sealing

Adding 10ml of 10% BSA HEPES buffer solution into the coupling gold label, and reacting for 30min by soft mixing; centrifuging at 10000r/min for 5min after reaction, removing supernatant, adding 100mL HEPES buffer solution, and resuspending; centrifuging at 10000r/min for 5min, pouring the supernatant, adding 100mL HEPES buffer again, resuspending, centrifuging, pouring the supernatant, and diluting the centrifuged precipitate to 5mL by using 0.1M PBS solution containing 0.1% glycine to obtain the gold-labeled antibody.

In the preparation method provided by the embodiment, the MES buffer solution added in the step 2.1, activation 1) has the function of adjusting the pH value of the reaction environment to about 5-6, so as to create conditions for the next activation; the reason why the light-shielding reaction is carried out for 30min in the step 2.1 activation 3) is that the activation is easily weakened by light to influence the coupling, and the light-shielding reaction avoids the activation from being weakened by light to influence the subsequent coupling.

Example 3

The embodiment of the invention provides an all-in-one heavy metal rapid quantitative detection card, the structure of the detection card refers to fig. 1 and fig. 2, the detection card comprises detection test paper arranged in a card shell, the detection test paper comprises a bottom plate 1, the bottom plate 1 is a plastic bottom plate, a sample pad 2, a combination pad 3 containing gold-labeled antibodies respectively labeled by corresponding antibodies of various heavy metals to be detected, a chromatographic membrane 4 and absorbent paper 6 are sequentially adhered to the bottom plate 1 from one end to the other end, one end of the sample pad 2 is in press-connection with one end of the combination pad 3, the other end of the combination pad 3 is in press-connection with one end of the chromatographic membrane, one end, close to the chromatographic membrane 4, of the absorbent paper 6 is in press-connection with one end of the chromatographic membrane 4, and the absorbent paper 6 is absorbent paper; the chromatographic membrane 4 is coated with a plurality of detection lines T (T1, T2, T3 and the like) and a quality control line C, the distance between the detection lines T is controlled to be 1-2.5mm, the detection lines T are uniformly distributed and do not intersect, the number of the detection lines T is consistent with the number of the types of heavy metals to be detected, each detection line T is coated with complete antigens of the heavy metals to be detected, and the quality control line C is coated with an anti-mouse IgG antibody; the position of card shell surface correspondence sample pad 2 is equipped with application of sample hole 7, the card shell surface correspondence the position of chromatographic carrier 4 is equipped with viewing aperture 8.

Two or more heavy metals of Cd, Pb, Hg, Cu, Cr and As can be selected As heavy metals to be detected according to actual detection requirements; three heavy metals of Cd, Pb and Cu are selected as heavy metals to be detected, and the preparation of the Cd, Pb and Cu all-in-one heavy metal rapid quantitative detection card is taken as an example:

preparation of the conjugate pad 3: mixing three gold-labeled antibodies (Cd, Pb and Cu gold-labeled antibodies) with equal amount, which are obtained according to the preparation method of the embodiment 2, spraying the mixture on a 2cm wide glass cellulose membrane or polyester membrane according to 4 mu L/cm, and drying the mixture at 37 ℃ for 18-24 h to prepare a binding pad 3;

the complete antigen of the heavy metal is formed by complexing Cd, Pb and Cu with complexing agents respectively and then coupling with carrier protein respectively, and in the process, the complexing agents are one or more of citric acid, ethylenediamine tetraacetic acid, aminoacetic acid, 1- (4-isothiocyanatobenzyl) ethylenediamine-N, N, N ', N' -tetraacetic acid, diethylenetriamine pentacarboxylate, glutathione and organic polydentate ligand; the carrier protein is one or more of bovine serum albumin, keyhole limpet hemocyanin, ovalbumin and human serum protein;

the coating process of the chromatographic membrane 4 is as follows: respectively preparing complete antigens of Cd, Pb and Cu into solutions with the concentration of 0.01-3 mg/mL by using 0.01M phosphate buffer solution with the pH value of 7.4, preparing goat anti-mouse IgG into a solution with the concentration of 0.01-3 mg/mL, respectively scribing and coating a quality control line C (goat anti-mouse IgG), a detection line T1 (Cd), a detection line T2 (Pb) and a detection line T3 (Pb) at the speed of 1 mu l/cm by using a scribing instrument, and then placing in an oven for drying at 37 ℃ for 5-16 hours.

The assembly of the detection card is carried out in a room with the temperature of 20-30 ℃ and the humidity of less than 40%, a plastic base plate 1 is taken, a coated chromatographic membrane 4 is pasted and placed in the middle of the plastic base plate, a combination pad 3 is placed on one side of a T1 detection line of the chromatographic membrane 4, the combination pad 3 is placed on an NC membrane for pasting by 1mm, a sample pad 2 is lapped and pasted on the other side of the combination pad 3, the sample pad 2 is aligned with the lower edge of detection test paper, absorbent paper 6 is lapped and pasted on one side of the chromatographic membrane 4 and is placed on the chromatographic membrane 4 by 1mm, finally, a strip cutting machine is used for cutting the pasted plastic base plate into test strips with the width of 3-5 mm, and the test strips are then loaded into a plastic card shell to be assembled into the detection card.

Example 4

The embodiment of the invention provides a detection method of an all-in-one heavy metal rapid quantitative detection card, which is used for heavy metal detection by using the all-in-one heavy metal rapid quantitative detection card in the embodiment 3 and comprises the following detection steps:

s301, crushing a required detection sample by using a crusher, and sieving the sample by using a 40-mesh sieve to obtain a sample to be detected;

s302, adding an extracting agent into the sample to be detected after sieving in the step S301, reacting for 5min under the condition that the oscillation frequency is 2-60cpm, centrifuging, taking the supernatant for later use, and dissociating Cd, Pb, Hg, Cu, Cr, As and other ions in the sample; the extractant is a dilute acid solution of 5% nitric acid, hydrochloric acid, sulfuric acid and perchloric acid, and the volume-to-weight ratio (mL: g) of the extractant to the sample to be detected is 4: 1;

s303, taking the supernatant obtained in the step S302, adding 20 times of diluent, and uniformly mixing to obtain a chromatography liquid; the diluent is added with 1 percent tween20, 5mg/L bismuth ions and 1 multiplied by 10^-5ITCBE (benzylethylenediaminetetraacetic acid isothiocyanate) g/mL in 0.2M PBS;

s304, simultaneously putting the all-in-one heavy metal rapid quantitative detection card and the chromatographic solution obtained in the step S303 into a constant-temperature incubator, keeping the constant temperature for 5min, and controlling the temperature to be 37 ℃;

s305, adding the constant-temperature chromatographic solution obtained in the step S304 into a sample adding hole of the all-in-one heavy metal rapid quantitative detection card, and continuing to perform constant-temperature reaction for 8 min;

In the detection method provided by this embodiment, in step S301, the function of sieving with a 40-mesh sieve after crushing the detection sample is to ensure the recovery rate of metal ion extraction, so as to ensure the accuracy of the quantitative detection of heavy metals, and incomplete extraction of metal ions caused by too large particle size of the detection sample; in step S303, a diluent is added to provide a stable environment for the reaction of the chelating agent with the metal ions, and to provide a stable acid-base salt environment for the detection of the immune reaction; in step S305, the isothermal reaction is continued for 8min to ensure that the chromatography process is in an isothermal environment.

Wherein the constant-temperature incubator is purchased from Shenzhen Tunengda science and technology Limited; the reading instrument is a portable optical instrument, is smaller than 0.001m3 in volume, is simple and convenient to operate, can detect a plurality of items, has a GPS positioning function, can store and print detection data, and is used for quantitatively judging a detection result.

The traditional colloidal gold judges whether the detection line on the colloidal gold detection card has negative or positive content of the target detection object by naked eyes, and can only judge for a limited value, the colloidal gold quantitative technology is that gray level scanning is carried out on the color of the detection line through a scanning technology on the traditional colloidal gold technology, and the color depth of the detection line is related to the content of the target detection object, the concentration of the target detection object of the detection object can be obtained by comparing the color depth of the scanning detection line with a preset standard curve, the whole detection range data can be judged, and the detection accuracy and the convenience are greatly improved.

The advantageous effects of the present invention are described below by way of experimental examples.

Experimental example 1

The modified colloidal gold and the unmodified 40nm colloidal gold obtained in example 1 were characterized by using a JEM-100CX type Transmission Electron Microscope (TEM), and the results are shown in FIGS. 3 and 4. JEM-100CX type transmission electron microscope is available from JEOL (JEOL).

FIG. 3 is a transmission electron microscope image of modified colloidal gold, FIG. 4 is a transmission electron microscope image of unmodified colloidal gold, and it can be seen from FIGS. 3 and 4 that the surface of modified colloidal gold has a film layer, but the surface of unmodified colloidal gold does not have a film layer, which proves that the invention successfully connects PEG to colloidal gold particles to form a PEG film layer, and the thickness of the PEG film is 5-10 nm.

Therefore, in the combination of the traditional colloidal gold and the antibody, the antibody is directly and electrostatically adsorbed on the colloidal gold, and the closer distance between the antibody and the antibody leads the antagonistic action to occur when the traditional colloidal gold labeled antibody reacts with the antigen, thereby reducing the reaction sensitivity₂The condensation reaction is carried out to generate amido bonds, so that the antibody is coupled on the surface of the colloidal gold to form the gold-labeled antibody, and the binding sites of the modified colloidal gold and the antibody are far away from the surface of the colloidal gold, so that the distance between the antibodies is increased, the antagonistic action between the coupled antibodies is effectively weakened, and the reaction sensitivity of the gold-labeled antibody is improved.

Experimental example 2 stability test

The gold-labeled antibody is dark red in color when being stable, the maximum absorption peak is 530-535 nm, the change fluctuation of the OD value is small, when the stability of the gold-labeled antibody is damaged due to external reasons, colloidal gold is gathered, the color of the gold-labeled antibody is changed into purple firstly, then is changed into black, and finally is colorless; the maximum absorption peak is gradually larger than the normal peak, and the OD value is gradually reduced to 0.

Respectively using the modified colloidal gold obtained in the example 1 to perform labeling treatment on the heavy metal cadmium monoclonal antibody, the lead monoclonal antibody and the copper monoclonal antibody according to the preparation method of the example 2 to obtain a cadmium novel gold label, a lead novel gold label and a copper novel gold label; and simultaneously, labeling the cadmium monoclonal antibody, the lead monoclonal antibody and the copper monoclonal antibody by using 40nm colloidal gold according to a traditional method to obtain a traditional cadmium gold label, a traditional lead gold label and a traditional copper gold label. The following tests were performed on the above labeled gold markers:

1) the gold specimen was stored in a refrigerator at 4 ℃ and observed to increase with time, and the change in the absorption peak and OD value of the gold specimen was measured using an ultraviolet spectrophotometer, and the results are shown in Table 1:

the results are given in table 1 below:

TABLE 1 table of results of storage stability test of novel gold label and traditional gold label

As can be seen from Table 1, the maximum absorption peaks of the cadmium novel gold label, the lead novel gold label and the copper novel gold label are between 530 nm and 535nm after being stored for 7 days and 14 days, and the OD value change fluctuation is small, which proves that the antibody marked by the modified colloidal gold is always in a stable state along with the increase of the storage time; after the cadmium traditional gold mark, the lead traditional gold mark and the copper traditional gold mark are stored for 7 days, the maximum absorption peaks of the lead traditional gold mark and the copper traditional gold mark exceed 535nm, the OD value changes greatly, after the cadmium traditional gold mark, the lead traditional gold mark and the copper traditional gold mark are stored for 14 days, the maximum absorption peaks of the cadmium traditional gold mark, the lead traditional gold mark and the copper traditional gold mark exceed 535nm, and the OD value changes greatly, which indicates that the storage stability of the antibody marked by the traditional colloidal gold is poor, and the traditional gold mark gradually changes to be purple and aggregates along with the increase of time; therefore, the modified colloidal gold-labeled antibody of the present invention effectively improves the storage stability of the gold-labeled antibody, and the gold-labeled antibody of the present invention has good storage stability.

3) Adding NaCl solutions with different high concentrations into the gold standard, measuring the change of an absorption peak and an OD value after 30min by using ultraviolet, wherein the test results are shown in Table 2:

table 2 table of salt tolerance stability test results of new and traditional gold labels

As can be seen from table 2, the OD value of the cadmium new type gold label, the lead new type gold label, and the copper new type gold label in the concentrations of 0% NaCl, 5% NaCl, and 10% NaCl respectively has small fluctuation, while the OD value of the cadmium conventional gold label, the lead conventional gold label, and the copper conventional gold label has large fluctuation, thereby proving that the modified colloidal gold-labeled antibody of the present invention effectively improves the stability of the gold-labeled antibody to salt, and the gold-labeled antibody of the present invention has good salt tolerance stability.

4) Different amounts of 0.1M hydrochloric acid were added to the gold standard, and the changes in the absorption peak and OD value were measured after 30min by UV, and the test results are shown in Table 3:

table 3 acid-resistant stability test result table for novel gold mark and traditional gold mark

As can be seen from table 3, the modified colloidal gold-labeled antibody of the present invention effectively improves the stability of the gold label against acid, compared to the conventional gold label.

5) Different amounts of 1M sodium hydroxide were added to the gold standard, and the change in the absorption peak and OD value was measured after 30min using UV, the test results are shown in Table 4:

table 4 alkali-resistant stability test result table for novel gold mark and traditional gold mark

As can be seen from Table 4, the modified colloidal gold-labeled antibody of the present invention effectively improved the stability of the gold label to alkali.

As can be seen from tables 1-4, the modified colloidal gold of the present invention has very stable binding with the antibody, and the gold labeled antibody has good stability, is not affected by acid-base environment and ionic strength, and is acid-resistant, alkali-resistant and salt-resistant.

As can be seen from the above, in the preparation of the modified colloidal gold, the HS-of the HS-PEG-COOH forms a strong coordination bond with metal ions on the surface of the colloidal gold particles for connection, and in the reaction process of the modified colloidal gold and the antibody, the activated-COOH on the surface of the modified colloidal gold and-NH on the antibody₂Generates an amido bond by condensation reaction, thereby coupling the antibody on the surface of the colloidal gold to form a stable gold-labeled antibody, and changing the condition that the traditional colloidal gold depends on electrostatic absorptionThe gold-labeled antibody is labeled in an antibody attaching mode, so that the stability of the gold-labeled antibody is greatly improved, and compared with the traditional colloidal gold-labeled antibody, the gold-labeled antibody disclosed by the invention is not influenced by acid-base environment and ionic strength, is acid-resistant, alkali-resistant and salt-resistant, and has very strong stability.

Experimental example 3 antibody dose comparison experiment

Different amounts of Cd, Pb and Cu monoclonal antibodies were labeled with 100mL of the modified colloidal gold obtained in example 1 according to the preparation method of example 2, while different amounts of Cd, Pb and Cu monoclonal antibodies were labeled with 100mL of 40nm unmodified colloidal gold according to the conventional method, and the optimal labeling amounts were compared, and the experimental results are shown in Table 5:

TABLE 5 comparison of optimal labeling amounts of antibodies

As can be seen from Table 5, the amount of the antibody labeled with the modified colloidal gold is relatively stable, the unmodified colloidal gold is greatly influenced by the properties of the antibody, and the difference of the labeled amount is large; marking the same heavy metal antibody, coupling 100mL of modified colloidal gold with 60 microgrammes of Cd monoclonal antibody, and coupling 100mL of unmodified colloidal gold with 600 microgrammes of Cd monoclonal antibody, wherein compared with the modified colloidal gold, the dosage of the antibody correspondingly used by the unmodified colloidal gold in the preparation of the gold-labeled antibody is 10 times higher; from the above, compared with the modified colloidal gold, the dosage of the antibody corresponding to the unmodified colloidal gold is 10 to 70 times higher, so that the modified colloidal gold disclosed by the invention can greatly reduce the dosage of the antibody in the preparation of the gold-labeled antibody and enhance the process stability of the labeled antibody.

Experimental example 4 Rapid quantitative detection card for Cd, Pb and Cu three-in-one heavy metal

(1) Principle of principle

The colloidal gold immunochromatography method is used for detecting heavy metals and is based on the specific recognition and reaction of antigens and antibodies. Because heavy metal ions have no immunogenicity, the heavy metal ions can be combined with protein to form complete antigen only after being coupled with certain functional groups to form hapten, and then immunoassay can be carried out.

The detection principle is that a nitrocellulose membrane is used as a carrier and is coated with heavy metal holoantigen (T line), after a sample to be detected is added, a heavy metal ion chelate is combined with a colloidal gold-labeled monoclonal antibody, the unbound colloidal gold-labeled monoclonal antibody is specifically combined with a complete detection antigen (T line) and is intercepted, and a displayed red strip is formed. And the color depth of the T line is measured by an instrument and is compared with a standard curve preset in the instrument, so that the content of the metal ions is accurately judged.

(2) Principal materials and instruments

1) Cadmium (Cd), lead (Pb), copper (Cu) standard samples: a product of a national analysis and test center for nonferrous metals and electronic materials; cd-specific monoclonal antibody, Pb-specific monoclonal antibody, Cu-specific monoclonal antibody, Cd complete antigen, Pb complete antigen, Cu complete antigen, goat anti-mouse IgG: product of Chengdu's Anpuno Biotech limited; gold chloride acid: a product of Sigma Co; a nitrocellulose membrane: MILLIPORE (usa), M135; bovine Serum Albumin (BSA): sigma product; HS-PEG20000-COOH, NHS, EDC: shanghai Tankou Technique, Inc.; glass cellulose membrane, absorbent pad, bottom plate: shanghai jie a biological product; other common reagents are analytically pure reagents;

2) tobacco leaf samples containing heavy metals of Cd, Pb and Cu are obtained on the market or by a detection mechanism, the content of the tobacco leaf samples is determined by using a first method of national standard GB/T5009.15-2003, wherein the distribution interval of the Cd content is 200-5000 mug/kg, the distribution interval of the Pb content is 20-1000 mug/kg, and the distribution interval of the Cu content is 500-10000 mug/kg.

3) Constant temperature incubator, readout appearance, purchase from Shenzhen Can reach science and technology Limited.

(3) Preparation of test cards

1) Colloidal gold labeling of Cd, Pb and Cu monoclonal antibodies

Optimum addition amount of HS-PEG20000-COOH

The preparation of HS-PEG20000-COOH modified colloidal gold according to the preparation method of example 1: heating 100mL of 0.01% chloroauric acid aqueous solution to boil, accurately adding 1.8mL of 1% trisodium citrate aqueous solution under stirring, allowing the golden chloroauric acid aqueous solution to turn red within 2 minutes, and continuously boiling for 15 minutes to obtain 40nm colloidal gold solution;

taking 5 parts of 40 nm-diameter colloidal gold solution, adding 0.1M K in each part of 100mL each part₂CO₃Adjusting the colloidal gold solution, measuring the pH value to be 7.5 by using a pH meter, respectively adding 10, 20, 40, 60 and 80 mu L of 1% HS-PEG20000-COOH solution, stirring and reacting for 1h at 200r/min, respectively adding 10mL of 10% NaCl solution into each part after the reaction is finished, uniformly mixing, and reacting for 30min at room temperature; observing the color change of the solution, keeping the lowest deep red HS-PEG20000-COOH amount as the optimal labeling amount, and finding out that 40 mu L of 1% HS-PEG20000-COOH solution is added as the optimal labeling amount;

② antibody coupling

The labeled antibody is taken to prepare modified colloidal gold according to the preparation method of the embodiment 1, then the Cd, Pb and Cu monoclonal antibodies are respectively coupled according to the preparation method of the embodiment 2, when the antibody amount is more than 60 mug (100 mL of modified colloidal gold), the test C line strength and the test T line strength are not affected, namely the amount of the coupled labeled antibody corresponding to 100mL of modified colloidal gold is 60 mug (the molecular weight of the monoclonal antibody is about 150 k), and the Cd gold-labeled antibody, the Pb gold-labeled antibody and the Cu gold-labeled antibody are respectively obtained.

2) Test card component preparation

Preparation of combined pad

Uniformly mixing the obtained Cd gold-labeled antibody, Pb gold-labeled antibody and Cu gold-labeled antibody, centrifuging for 5min at 10000r/min, discarding supernatant, redissolving the volume of the mixed gold-labeled antibody by using redissolution (2 mM PBS solution containing 0.1-5 wt.% of BSA, 1-20 wt.% of sucrose and 1-1 wt.% of tween-200.01-1 wt.%) to obtain a mixed gold-labeled antibody original volume, spraying the mixed gold-labeled antibody original volume on a 2 cm-wide glass cellulose membrane according to 4 mul/cm, and drying at 37 ℃ for 18-24 hours to obtain a binding pad;

② chromatographic film coating

Cd, Pb and Cu complete antigens were prepared into 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL solutions with 0.01M phosphate buffer solution with pH7.4, anti-mouse IgG was prepared into 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL solutions, and the solutions were subjected to streaking with a streaking machine at a rate of 1. mu.L/cm to coat lines C, T1, T2 and T3, respectively, and the resulting product was dried in a 37 ℃ oven for 12 hours.

3) Assembly of test cards

Assembling the detection card in a room with the temperature of 20-30 ℃ and the humidity of less than 40%, taking a plastic bottom plate, placing a coated chromatographic membrane (NC membrane) in the middle of the plastic floor for pasting, putting a combination pad on one side of a T1 line of the NC membrane, putting the combination pad on the NC membrane for pasting by 1mm, putting the other side of the combination pad for pasting a sample pad, aligning the sample pad with the lower edge of a test paper, putting a piece of absorbent paper on one side of the NC membrane for lapping by 1mm, finally cutting the pasted plastic plate into a test paper strip with the width of 5mm by using a slitter, and then putting the test paper strip into a plastic card shell to form the detection card, thereby obtaining the three-in-one rapid quantitative detection card for heavy metal Cd, Pb and Cu.

4) Test strip technological parameter debugging

Combining and pairing reagents with different coating amounts to prepare a small sample, testing the reagents by using Cd, Pb and Cu standard samples, and finding out the optimal combination according to the T value, the C value, the T/C value and the test gradient result.

Determining the best combination as: the coating amount of Cd complete antigen is 0.5mg/mL, the coating amount of Pb complete antigen is 1mg/mL, and the coating amount of Cu complete antigen is 0.5 mg/mL.

5) Reagent preparation

The extractant is 5% dilute nitric acid solution.

Preparing a diluent: using 0.2M PBS buffer, tween-20, 1X 10, was added to a final concentration of 1%^-5ITCBE g/mL and 5mg/L bismuth ion were prepared as dilutions.

6) Preparing, converting and recording standard curve

After the technological parameters of the detection card are determined, the detection card is respectively measured by mixed standard samples with Cd and Pb concentrations of 0, 20, 50, 100, 200, 500, 1000, 2000, 5000 mug/kg and Cu concentrations of 0, 100, 200, 500, 1000, 2000, 5000, 10000 mug/kg, standard samples with different concentrations display different gray value intensity detection lines T, the intensity of the detection lines T is measured by a colloidal gold detector, a standard curve is made according to the intensity of the scanning gray value of the concentration and the detection line T, and corresponding parameters are led into the detector, so that the parameter setting of instrument curves is completed.

7) Detection method

Crushing a sample to be detected by using a crusher, and sieving the sample by using a 40-mesh sieve;

accurately weighing 1g of sample to be detected by using a small scale, adding 4mL of extractant into an extraction bottle, and reacting for 5min at room temperature under the condition that the oscillation frequency is 2-60 cpm;

filtering with centrifuge or filter, taking supernatant, transferring 50 μ L of supernatant to 950 μ L of diluent by pipette, and mixing;

fourthly, the detection card and the mixed solution are taken out and put into a constant temperature incubator for 5min, and the temperature is set to be 37 ℃;

taking 100 mu L of the mixed solution by using a pipettor, adding the mixed solution into a sample adding hole of the detection card, and continuously reacting for 8min at the constant temperature of 37 ℃;

sixthly, the concentration of Cd, Pb and Cu in the sample can be displayed by putting the reacted detection card into a reading instrument.

8) Accuracy detection

Tobacco leaf samples containing heavy metals Cd, Pb and Cu are obtained in the market or a detection mechanism, 6 parts in total, and the content of the tobacco leaf samples is determined by using a first method of national standard GB/T5009.15-2003, wherein the distribution interval of the Cd content is 200-5000 mug/kg, the distribution interval of the Pb content is 20-1000 mug/kg, and the distribution interval of the Cu content is 500-10000 mug/kg; the 6 parts of tobacco samples containing heavy metals Cd, Pb and Cu are detected by using a Cd, Pb and Cu three-in-one heavy metal rapid quantitative detection card according to the detection method, and the accuracy of the detection card is compared with the result of an atomic absorption spectrometry to analyze, wherein the detection results are shown in Table 6:

table 6 table of accuracy test results

And (3) performing a matched sample T test method on the group of data, wherein T values of Cd, Pb and Cu are respectively 0.482, 0.253 and 0.600, T double-tail critical value (0.05, 5) =2.571, and T < T double-tail critical value (0.05, 5), which indicates that the detection result of the Cd, Pb and Cu three-in-one heavy metal rapid quantitative detection card has no significant result difference from the detection result of the atomic absorption spectrometry.

9) Detection of precision

Preparing standard sample solutions of Cd, Pb and Cu with the concentrations of 200ppb, 500ppb and 2000ppb respectively, detecting by using a Cd, Pb and Cu three-in-one heavy metal rapid quantitative detection card, analyzing the precision of each concentration detection for 6 times, and obtaining the detection results shown in tables 7-9:

TABLE 7 Cd test result table

TABLE 8 Pb test results Table

TABLE 9 Cu test results Table

As can be seen from tables 7-9, the coefficient of variation of the repeated detection results of the rapid quantitative detection card for Cd, Pb and Cu triple heavy metals is between 1.8 and 11.2%.

In conclusion, the rapid quantitative detection card for Cd, Pb and Cu three-in-one heavy metal has good performance, can rapidly and accurately detect the content of Cd, Pb and Cu in a sample to be detected, is suitable for rapid quantitative detection in laboratories and sampling sites, and meets the requirements of customers on rapid quantitative detection of Cd, Pb and Cu in food.

EXPERIMENTAL EXAMPLE 5 comparative experiment without addition of masking agent

The method comprises the following steps of (1) detecting 6 tobacco samples in the experimental example 4 by using a Cd, Pb and Cu three-in-one heavy metal rapid quantitative detection card in the experimental example 4, wherein a used diluent is a diluent without a masking agent bismuth (the diluent is prepared by adding tween-20 with the final concentration of 1% and 1 x 10-5 ITCBE g/mL into 0.2M PBS buffer solution), and the rest reagents, detection methods and the like are consistent with those in the experimental example 2; the results of testing 6 tobacco samples using masking agent were compared with those without masking agent, and the difference ratio, i.e., (result with masking agent-result without masking agent)/result with masking agent × 100%, was calculated, and the results of the tests and comparisons are shown in table 10:

TABLE 10 table of examination and comparison results

As can be seen from Table 10, the test results without masking diluent are much smaller than those with masking agent, specifically, the test result of Cd is 28.9-55.2% smaller, and the average is 45.9% smaller; the detection result of Pb is 25.8-47.8% smaller, and the average is 39.4% smaller; the detection result of Cu is 13.5-41.7% less, the average is 33.3% less, therefore, the masking agent has great influence on the detection result, the influence degree on different samples is inconsistent, the masking agent can be eliminated by adding, and the detection result is consistent with the result of an atomic absorption spectrometry.

Experimental example 6 sensitivity comparison

The detection card for the Cd, Pb and Cu three-in-one heavy metal unmodified colloidal gold is manufactured by unmodified colloidal gold according to a traditional colloidal gold labeling method, the Cd, Pb and Cu three-in-one heavy metal unmodified colloidal gold detection card in the experimental example 4 and the Cd, Pb and Cu three-in-one heavy metal detection card in the experimental example 4 are respectively detected by Cd, Pb and Cu metal ion standard solutions with different concentrations, the concentration of the metal ion standard solution used when the strength of a detection line T is half of that of a blank sample detection line T is recorded, and the result is shown in Table 11:

TABLE 11 sensitivity comparison table

As can be seen from Table 11, under the same test conditions, the sensitivity of the Cd, Pb and Cu three-in-one heavy metal test card made of modified colloidal gold was improved by 1-5 times in comparison with the test card made of unmodified colloidal gold, thereby proving that the modified colloidal gold of the present invention improves the reaction sensitivity of the gold-labeled antibody.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. The preparation method of the modified colloidal gold is characterized by comprising the following preparation steps:

taking 100 parts of colloidal gold solution with diameter of 10-60nm, and using 0.1M K₂CO₃Adjusting the pH value of the colloidal gold solution to be between 6 and 9, adding an HS-PEG-COOH solution accounting for 0.01 to 1 percent of the mass of the colloidal gold solution, stirring and reacting for 0.5 to 2 hours under the condition of 100 plus materials and 400r/min, then centrifuging for 2 to 10 minutes under the condition of 4000 plus materials and 10000r/min, and removing the supernatant to obtain the modified colloidal gold.

2. The method as claimed in claim 1, wherein the HS-PEG-COOH solution has a concentration of 1%, and the PEG molecular weight is 1000-100000.

3. A preparation method of a gold-labeled antibody is characterized by comprising the following preparation steps:

s101, activating the modified colloidal gold obtained by the preparation method of any one of claims 1-2 with NHS and EDC to obtain activated modified colloidal gold;

s102, adding an antibody into the activated modified colloidal gold obtained in the step S101 to obtain a gold-labeled antibody.

4. The method of claim 3, wherein in step S102, the antibody is a Cd monoclonal antibody, a Pb monoclonal antibody, a Hg monoclonal antibody, an As monoclonal antibody, a Cr monoclonal antibody or a Cu monoclonal antibody.

5. The all-in-one heavy metal rapid quantitative detection card is characterized by comprising detection test paper arranged in a card shell, wherein the detection test paper comprises a bottom plate (1), a sample pad (2), a combination pad (3) containing various gold-labeled antibodies respectively marked by corresponding antibodies to be detected for heavy metals, a chromatographic membrane (4) and absorbent paper (6) are sequentially pasted on the bottom plate (1) from one end to the other end, one end of the sample pad (2) is in press connection with one end of the combination pad (3), the other end of the combination pad (3) is in press connection with one end of the chromatographic membrane (4), and one end, close to the chromatographic membrane (4), of the absorbent paper (6) is in press connection with one end of the chromatographic membrane (4); the chromatographic membrane (4) is coated with a plurality of detection lines T and a quality control line C, the number of the detection lines T is consistent with the number of the types of the gold-labeled antibodies on the binding pad, any detection line T is coated with a complete antigen of the heavy metal to be detected, and the quality control line C is coated with an anti-mouse IgG antibody; a sample adding hole (7) is formed in the position, corresponding to the sample pad (2), of the surface of the card shell, and an observation port (8) is formed in the position, corresponding to the chromatographic membrane (4), of the surface of the card shell;

the gold-labeled antibody on the conjugate pad (3) is prepared by the method according to any one of claims 3 to 4.

6. The all-in-one heavy metal rapid quantitative detection card according to claim 5, wherein the complete antigen of the heavy metal to be detected coated on the detection line T is formed by complexing the heavy metal with a complexing agent and then coupling with a carrier protein.

7. The all-in-one heavy metal rapid quantitative detection card according to claim 5, wherein the heavy metal is selected from Cd, Pb, Hg, Cu, Cr, As.

8. The preparation method of the all-in-one heavy metal rapid quantitative detection card according to any one of claims 5 to 7, comprising the following preparation steps:

s201, preparing complete antigens of various heavy metals to be detected, and preparing modified colloidal gold according to the preparation method of any one of claims 1-2;

s203, preparing the monoclonal antibodies of various heavy metals to be detected and the modified colloidal gold obtained in the step S201 respectively to obtain gold-labeled antibodies according to the preparation method of any one of claims 3-4;

s204, mixing the gold-labeled antibodies of the heavy metals to be detected obtained in the step S203 to prepare a gold-labeled antibody solution, and spraying the gold-labeled antibody solution onto the bonding pad in an amount of 1-10 mu L/cm, wherein the gold-labeled antibody solution comprises the following components: 2mM PBS contains 0.1-5 wt.% of BSA, 1-20 wt.% of sucrose, 1-1 wt.% of tween-200.01 and 0.001-0.1mg/mL of gold-labeled antibodies of several heavy metals to be detected respectively;

and S205, a sample pad, the bonding pad obtained in the step S204, the chromatographic membrane coated by the drawn line in the step S202 and absorbent paper are sequentially adhered to a bottom plate, so that the all-in-one heavy metal rapid quantitative detection card is obtained.

9. The method for detecting the all-in-one heavy metal rapid quantitative detection card is characterized in that the all-in-one heavy metal rapid quantitative detection card of any one of claims 5 to 7 is used for detecting a plurality of heavy metals, and comprises the following detection steps:

s305, adding the chromatography liquid with the constant temperature in the step S304 into a sample adding hole of the all-in-one heavy metal rapid quantitative detection card, and continuing to perform constant temperature reaction for 5-30 min;

10. The method for detecting the all-in-one heavy metal rapid quantitative detection card according to claim 9, wherein in step S303, the masking agent is one of bismuth, antimony and mercury with a concentration of 0.01-100 mg/L.