CN113092750A - Preparation method of multicolor fluorescence immunochromatographic test strip for naked eye observation - Google Patents

Abstract

A preparation method of a multicolor fluorescence immunochromatographic test strip for naked eye observation comprises the steps of taking red, orange, yellow, green and blue five-color quantum dot fluorescence as background signals, coupling the five-color quantum dots with bovine serum albumin respectively, then uniformly mixing the five-color quantum dots with five mycotoxin detection antigens respectively, and spraying the mixture on a test strip detection area; the silver nano particles are respectively combined with monoclonal antibodies of five mycotoxins to be used as an inner filtering probe of quantum dot exciting light. When the content of a certain mycotoxin in a detection sample is greater than or equal to the national limit standard, the corresponding fluorescent signal is turned on, and whether the five mycotoxins meet the national standard or not can be visually judged by naked eyes. The test strip prepared by the invention can be used for simultaneously and rapidly detecting aflatoxin B in grain and oil products₁Ochratoxin A, zearalenone, deoxynivalenol and fumonisin B₁And the five mycotoxins are equal.

Description

Preparation method of multicolor fluorescence immunochromatographic test strip for naked eye observation

Technical Field

The invention relates to the technical field of rapid detection, in particular to a multicolor fluorescence signal opening type competitive immunochromatographic test strip based on naked eye observation, namely, the immunochromatographic test strip is constructed based on the internal filtration of silver nanoparticles to multicolor quantum dot excitation light, and the aflatoxin in grain and oil products is rapidly detectedElement B₁Ochratoxin A, zearalenone, deoxynivalenol and fumonisin B₁And the five mycotoxins are equal.

Background

China is a large country for grain production and consumption, and has great significance for enhancing the quality control of grains. Crops are easily polluted by fungi in the planting, harvesting, processing, transporting and storing processes, and certain fungi toxigenic strains can generate a class of micromolecular secondary metabolites, namely mycotoxin, under the condition of proper temperature and humidity, so that serious health risks are brought to human beings and animals. The implementation of regulatory and regulatory limits on mycotoxins in food products is an important measure to ensure food safety.

National food safety standards (2017) in China set limit requirements for four mycotoxins in grains: aflatoxin B₁（aflatoxin B₁, AFB₁) 5-20 mug/kg, 5 mug/kg of ochratoxin A (OTA), 60 mug/kg of Zearalenone (ZEN) and 1000 mug/kg of Deoxynivalenol (DON). In addition, due to toxicity of Fumonisin (Fumonisin) and severity of pollution, Fumonisin B was added to the national standard mycotoxin limit survey for food safety in 2019₁，B₂And B₃The limit requirement for the total amount: the corn, corn flour (grits) and the cereal product containing corn material limit amounts are 4000, 2000 and 1000 mug/kg, respectively. The establishment of a high-sensitivity rapid detection method for simultaneously detecting the mycotoxins has important practical significance.

The immunochromatography method has the advantages of simple and convenient operation, rapidness, strong matrix interference resistance and the like, and is a common method for on-site rapid screening. The immunochromatography method is generally a competitive reaction mode for the analysis of small molecule analytes such as mycotoxins. That is, the detection antigen fixed on the detection line (T line) and the detected object compete for the antibody marked on the signal substance together, and the detection signal intensity and the content of the detected object are in inverse proportion. In qualitative or semi-quantitative analysis, the "signal disappearance" on the T-line is usually used as the interpretation standard, and when the amount of the analyte is large, the antibody labeled on the signal substance is saturated and not "captured" by the detection antigen on the T-line. Therefore, immunochromatographic methods based on "signal disappearance" patterns are not highly sensitive for qualitative or semi-quantitative analysis. A competitive immunochromatography technology based on a signal opening mode is constructed, namely, a detection signal is increased from nothing to nothing along with the increase of the concentration of a small molecule object to be detected, and the detection sensitivity of the methodology can be obviously improved.

In recent years, some research groups have tried a fluorescence quenching competitive immunochromatography mode based on colloidal gold, in which a fluorescent material is immobilized as a background signal on a detection line (T line) or sprayed on the detection area of the entire nitrocellulose membrane (NC membrane). When the sample solution has no object to be detected, the gold-labeled antibody is electrophoresed to the T line and is combined with the detection antigen to cause fluorescence quenching; when a trace amount of the object to be detected exists in the sample solution, the quantity of the gold-labeled antibody captured by the T line is reduced, so that a fluorescence signal of the T line is opened. Compared with the traditional competitive immunochromatography, the method has the advantages that the fluorescence signal is increased from nothing to nothing along with the increase of the concentration of the object to be detected, and the detection sensitivity can be obviously improved. For example, Fu et al establishes a signal open type immunochromatography method based on Fluorescence Resonance Energy Transfer (FRET) between colloidal gold and fluorescein, which is used for detecting heavy metal chromium ions and clenbuterol (clenbuterol), and the detection limit is reduced by more than ten times compared with the traditional signal disappearance mode.

FRET is an energy transfer phenomenon between two fluorescent substances or between a fluorescent substance and an energy acceptor, which are in close proximity (within 10 nm). The production conditions are such that the emission spectrum of the donor fluorescent substance overlaps with the absorption spectrum of the acceptor, and the distance between the two molecules is within 10 nm. If a plurality of targets need to be detected simultaneously and various objects to be detected are distinguished in different colors, the competitive immunochromatography based on the FRET signal opening mode needs to use a plurality of nanoprobes with absorption spectra to correspond to emission spectra of fluorescent substances with different colors, and nanoparticles with different absorption spectra have larger differences in aspects of appearance, size, biocompatibility, proper pH value, buffer solution salt concentration and the like, so that the nanoparticles are difficult to be uniformly used on the same test strip. Furthermore, immunological detection systems based on antigen-antibody binding do not have a high FRET efficiency, considering the size of the antibody itself (8 to 10 nm). Competitive immunochromatography for FRET-based signal-on modes is not suitable for multiplex detection of multicolor fluorescence indications.

The Internal Filter Effect (IFE) refers to a strong absorption effect of noble metal nanoparticles on a specific excitation light or emission light of a fluorescent substance, which is not limited by a distance of 10nm compared to FRET, and can act on the excitation light of the fluorescent substance. The molar extinction coefficient of the silver nano particles is far higher than that of colloidal gold, so that the silver nano particles are an ideal absorbent for an IFE system; the quantum dot has the advantages of high fluorescence yield, good light stability, wide excitation spectrum, narrow and symmetrical emission spectrum, spectrum with different colors along with the change of particle size and the like, and is an ideal fluorescent agent for an IFE system. The immunochromatography test strip is high-sensitivity, rapid and simultaneous detection of a multicolor fluorescence signal opening mode of various micromolecule objects to be detected on the immunochromatography test strip can be realized by effectively overlapping the silver nanoparticle absorption spectrum and the multicolor quantum dot excitation spectrum, combining the strong extinction characteristic of the silver nanoparticles and converting the number of the silver nanoparticles into fluorescence signals of the multicolor quantum dots in an exponential relationship through IFE.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a preparation method of a multicolor fluorescence immunochromatographic test strip for naked eye observation, silver nanoparticles are used as a common internal filter of red, orange, yellow, green and blue multicolor quantum dot exciting light, an IFE-based multicolor fluorescence signal opening type competition immunochromatographic detection method is established, and AFB in grains is realized₁OTA, ZEN, DON and FB₁The five mycotoxins are simultaneously detected by naked eyes with high sensitivity and high speed.

The invention is realized by the following technical scheme.

The invention relates to a preparation and use method of a multicolor fluorescence immunochromatographic test strip for naked eye observation.

(1) Preparing a silver inner filtering probe.

The sodium citrate concentration was adjusted to 5mM and tannic acid 0.025mM in a 1000mL system, heated to boiling with vigorous stirring, and 10mL of 25mM silver nitrate was added and the solution turned bright yellow. Removing 200mL of reaction liquid, adding 170mL of water into the reaction system, setting the temperature at 90 ℃, adding 5mL of 25mM sodium citrate, 15mL of 2.5mM tannic acid and 10mL of 25mM silver nitrate, and keeping the temperature at 90 ℃ for 30 minutes; the removal addition procedure was repeated and the uv absorption spectrum of each removal was monitored until a silver nanoparticle solution with a maximum absorption peak of 450nm was obtained.

Five 100mL portions of silver nanoparticle solution (particle number about 10) were taken¹¹seed/mL), centrifuging for 15 minutes at 6500 rpm, and discarding the supernatant; the pellets were resuspended in 100mL of anti-AFB-containing suspension₁anti-OTA, anti-ZEN, anti-DON and anti-FB₁0.1M boric acid buffer solution with pH7.5 of the mouse monoclonal antibody ascites; slowly stirring at room temperature for 2h, centrifuging at 6500 rpm for 15 min, and removing the supernatant; the pellet was resuspended in 100mL of a borate buffer (stock solution) containing 0.1% (w/v) Bovine Serum Albumin (BSA), 5 minutes later, 6500 rpm was centrifuged for 15 minutes, and the pellet was resuspended in 10mL of the stock solution and stored at 4 ℃ until use.

Considering fumonisin B in national standard₁、B₂And B₃The total amount limit, selection and FB of the invention₂、FB₃anti-FB with cross-reactivity rate of more than 70%₁The silver Nephrolet probe prepared by the method can effectively capture FB at the same time₁、FB₂And FB₃So as to meet the limit requirement of national standard on the total amount of the three fumonisins.

(2) CdSe/ZnS quantum dots were coupled to BSA.

Five colors of CdSe/ZnS Quantum Dots (QDs), blue (QDs) respectively₅₀₀) Green (QD)₅₃₀) Yellow (QD)₅₇₀) Orange (QD)₆₀₀) And red (QD)₆₃₀) Surface carboxylation treatment, activation by 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride (EDC) and coupling with BSA. Adjusting the concentration of the five carboxylated quantum dots to be 1 mu M, respectively taking 1mL, adjusting the pH to 5.7, adding 0.1mL of freshly prepared 1mg/mL EDC aqueous solution under low-speed stirring at room temperature, activating for 5 minutes, then adding 0.1mL of 1% BSA solution, and reacting for 1 hour at room temperature; then adding 0.12mL of freshly prepared 1mg/mL EDC solution, adding 0.12mL of 1% BSA solution after 5 minutes, and reacting for 1h at room temperature; the third time adding0.15mL of freshly prepared 1mg/mL EDC solution was added, and after 5 minutes, 0.15mL of 1% BSA solution was added and the reaction was carried out at room temperature for 2 hours. Excess polymer was removed by centrifugation at 1000 rpm for 5 minutes and stored at 4 ℃ until use.

(3) And (5) assembling the immunochromatographic test strip.

The immunochromatographic test strip is formed by overlapping and fixing five parts of filter paper, a sample pad, a combination pad, a Nitrocellulose (NC) membrane, absorbent paper and the like on an adhesive card paper according to the mode of figure 1.

The bottom layer is adhesive card paper, the middle part is adhered with NC membrane, the NC membrane is sprayed with five detection antigens and five BSA-QD mixtures in the detection area in advance, and the quality control position is sprayed with donkey anti-mouse IgG antibody and BSA-QD₆₃₀. The combination pad, the sample pad and the filter paper of five kinds of silver inner filter probes of spraying are pasted on the viscidity card paper on NC membrane left side in proper order, and 1 to 2 mm is range upon range of to combination pad right side edge and NC membrane left side edge, and the sample pad is range upon range of on the combination pad, and 1 to 2 mm is pasted on the card paper in the left side staggers, and the filter paper is range upon range of on the sample pad, and 1 to 2 mm is pasted on the card paper in the same left side staggers. And (3) adhering absorbent paper to the right side of the NC membrane, laminating the left side edge of the absorbent paper and the right side edge of the NC membrane by 1-2 mm, and allowing a sample solution to be detected to enter the NC membrane under the capillary action of the absorbent paper through filter paper, a sample pad and a combination pad during detection to flow from left to right.

Combining five parts of filter paper, a sample pad, a combination pad, an NC membrane, absorbent paper and the like, cutting into strips with the width of 4mm, putting the strips into a test paper strip card shell, and then putting the test paper strip card shell and a drying agent into a light-proof bag for sealing and storing. A test strip fixing clamping groove is formed in the test strip clamping shell, a sample adding hole is formed in the position of filter paper of the test strip, detection observation ports are formed in the five detection line areas of the NC film, and quality control observation ports are formed in the position of the quality control line.

The sample pad was treated by soaking in 50 mM phosphate buffered saline pH7.5 (containing 1% BSA, 0.5% Tween 20 and 0.05% sodium azide) and dried in vacuo.

The conjugate pad carries five detection probes, and after infiltration with 10mM phosphate buffered saline pH7.5 (containing 0.2% Tween 20 and 2% sucrose) and drying treatment, a mixture of five silver filtered probes was sprayed onto the conjugate pad at 20 μ L/cm and dried in a vacuum oven at 37 ℃ for 6 h.

FB is sprayed on the NC film in sequence₁Detection of antigen with BSA-QD₅₀₀Mixture, DON detection antigen and BSA-QD₅₃₀Mixture, ZEN detection antigen and BSA-QD₅₇₀Mixture, OTA detection antigen and BSA-QD₆₀₀Mixture, AFB₁Detection of antigen with BSA-QD₆₃₀Mixture and donkey anti-murine Secondary antibody with BSA-QD₆₃₀Mixing to form five T lines and one quality control line; the detection antigen is conjugate of each mycotoxin and BSA, the molar coupling ratio is 15:1, and the spraying concentration is 1.5mg/mL calculated by the mass of the BSA; the BSA-QD spraying amount of each color takes the condition that an obvious strip is observed by naked eyes under an LED lamp with the wavelength of 450nm as an adjustment basis, and when the content of mycotoxin in a sample is equal to the national standard requirement, a fluorescence signal on a corresponding detection line is opened until the observation of the sample by the naked eyes is visible; and (3) drying the sprayed NC membrane in a vacuum drying oven at 37 ℃ for 6 h.

Commercial filter paper and absorbent paper were used without special treatment.

(4) The test paper strip is used.

Weighing 5g of crushed grains, placing the crushed grains in a 50mL centrifuge tube, adding 25mL of methanol-water solution (60: 40, v/v), after vigorous shaking extraction for 25min, centrifuging for L0min at 12000 r, diluting supernatant by 15 times, adding 80 mu L of the crushed grains into a test strip sample adding hole, and after 15 min, changing the quality control observation hole into brown, which indicates that the test strip state is normal. Irradiating by using a 450nm LED light source, if the content of mycotoxin in a grain sample is more than or equal to the national limit standard, enabling the detection line to have naked eye visible fluorescence, wherein the blue color is fumonisin, the green color is deoxynivalenol, the yellow color is zearalenone, the orange color is ochratoxin A, and the red color is aflatoxin B₁。

The invention provides an IFE-based signal open type competitive immunochromatography test strip. Compared with FRET, the IFE is not limited by the distance of 10nm, and can act on the exciting light of a fluorescent substance, and the IFE acts on the exciting light of quantum dots with different colors through the silver nano particles with high molar extinction performance, so that a multicolor fluorescence signal is opened simultaneously; the detection sensitivity of the competitive immunochromatographic test strip is improved through a signal opening mode, so that five fungaltoxins which are not less than the national limit standard can be observed by naked eyes at the same time.

Drawings

FIG. 1 is a schematic diagram of a test strip according to the method of the present invention.

Detailed Description

In order that the invention may be more clearly understood, reference will now be made in detail to the following examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The five mycotoxin monoclonal antibodies ascites and donkey anti-mouse IgG antibodies referred to in the examples were provided by Wuxi Zhongdeberber Biotechnology, Inc.; the detection antigens of the five mycotoxins are synthesized in a laboratory; five carboxyl modified quantum dots were used as provided by Ocean-nano, usa; the related mycotoxin standard substances, EDC, BSA, sodium citrate, tannic acid and AgNO₃Etc. were purchased from Sigma company; the chemicals involved were purchased from Aladdin corporation; the 450nm LED light source was customized to Shanghai women scientific instruments Inc.

Example 1. Preparation and use of a multicolor fluorescence signal open type immunochromatography test strip for detecting five mycotoxins in corn flour (residues).

(1) Preparing a silver inner filtering probe.

Silver nano particles with the ultraviolet absorption peak of 450nm are synthesized by a seed growth method, and the average particle size of the silver nano particles is about 70 nm. The specific scheme is as follows: (a) synthesizing seed silver: adjusting sodium citrate concentration to 5mM and tannin 0.025mM in 100mL system, heating to boil under vigorous stirring, adding 1mL AgNO₃(25 mM), the solution immediately turned bright yellow, at which point the seed size was about 15 nm. (b) Gradual growth of silver: after the seed silver was synthesized, 19.5mL of the reaction solution was removed (stored at 4 ℃ C. for further use), 16.5mL of water was added to the reaction system, the temperature was set to 90 ℃ C., and 0.5mL of sodium citrate (25 mM), 1.5mL of tannic acid (2.5 mM), 1mL of AgNO were added₃This process was repeated every 30 min. Ultraviolet scanning is carried out to monitor the absorption spectrum of the silver nano particles taken out each time until the silver nano particles with the ultraviolet absorption peak of 450nm are obtained, under the condition of the embodiment, the silver seeds grow for 11 rounds to achieve ultraviolet absorptionThe peak is 450 nm.

Five 1mL portions of silver nanoparticle solution (particle number about 10) were taken¹¹individual/mL), 7000 turns and centrifugates for 10min, abandons the supernatant; the pellets were resuspended in 1mL of anti-AFB-containing suspension₁anti-OTA, anti-ZEN, anti-DON and anti-FB₁0.1M boric acid solution (pH value adjusted by NaOH 7.5) of the mouse monoclonal antibody ascites; slowly stirring at room temperature for 2h, centrifuging at 7000 rpm for 10min, and removing the supernatant; the pellet was resuspended in a boric acid solution (stock solution) containing 0.1% (w/v) BSA and after 5min, 7000-rpm centrifugation was carried out for 10min, and the pellet was resuspended in 0.25mL of stock solution and stored at 4 ℃ for further use.

(2) The quantum dots were coupled to BSA.

QDs surface carboxyl modified₅₀₀(blue), QD₅₃₀(Green), QD₅₇₀(yellow), QD₆₀₀(orange) and QD₆₃₀(Red), the concentration is 2 μ M, 0.05mL and 0.05mL of phosphate buffer (8 mM, pH 5.7) are respectively taken and mixed in a small beaker, stirred at low speed on a magnetic stirrer, 0.1mL of freshly prepared 1mg/mL EDC solution is added, activation is carried out for 5min, then 0.1mL of 1% BSA solution is added, reaction is carried out for 1h at room temperature, 0.12mL of freshly prepared 1mg/mL EDC solution is added, 0.12mL of 1% BSA solution is added after 5min, reaction is carried out for 1h at room temperature, 0.15mL of freshly prepared 1mg/mL EDC solution is added for the third time, 0.15mL of 1% BSA solution is added after 5min, and reaction is carried out for 2h at room temperature. Centrifuging for 5 minutes at 1000 rpm, and storing the supernatant at 4 ℃ for later use.

Respectively diluting the five prepared BSA-QDs to 1-30 nM, spraying an NC membrane at 0.45 mu L/cm, and observing under a 450nM LED lamp. The concentration of BSA-QD with a fluorescent strip clearly visible to the naked eye was selected as the background fluorescence concentration of the test strip.

(3) And (5) assembling the immunochromatographic test strip.

The test paper strip consists of five parts, namely filter paper, a sample pad, a combination pad, an NC membrane, absorbent paper and the like.

The bottom layer is adhesive card paper, the middle part is adhered with NC membrane, the NC membrane is sprayed with five detection antigens and five BSA-QD mixtures in the detection area in advance, and the quality control position is sprayed with donkey anti-mouse IgG antibody and BSA-QD₆₃₀. The knot sprayed with five silver inner filtering probes is sequentially stuck on the adhesive card paper on the left side of the NC filmThe paper card comprises a combination pad, a sample pad and filter paper, wherein the right side edge of the combination pad and the left side edge of an NC film are laminated by 1-2 mm, the sample pad is laminated on the combination pad, the left side of the combination pad is staggered by 1-2 mm and is adhered to a card paper, the filter paper is laminated on the sample pad, and the left side of the combination pad is staggered by 1-2 mm and is adhered to the card paper. And (3) adhering absorbent paper to the right side of the NC membrane, laminating the left side edge of the absorbent paper and the right side edge of the NC membrane by 1-2 mm, and allowing a sample solution to be detected to enter the NC membrane under the capillary action of the absorbent paper through filter paper, a sample pad and a combination pad during detection to flow from left to right.

The sample pad was soaked with 50 mM phosphate buffered saline pH7.5 (containing 1% BSA, 0.5% Tween 20 and 0.05% sodium azide) and dried under vacuum.

After the conjugate pad was soaked with 10mM phosphate buffered saline (containing 0.2% Tween 20 and 2% sucrose) at pH7.5 and dried, the five silver filtered probe mixtures were sprayed onto the conjugate pad at 20 μ L/cm and dried in a vacuum oven at 37 ℃ for 6 h.

FB is sprayed on the NC film in sequence with the spraying amount of 0.45 mu L/cm₁Detection of antigen with BSA-QD₅₀₀(blue) mixture, DON detection antigen and BSA-QD₅₃₀(Green) mixture, ZEN detection antigen and BSA-QD₅₇₀(yellow) mixture, OTA detection antigen and BSA-QD₆₀₀(orange) mixture and AFB₁Detection of antigen with BSA-QD₆₃₀(Red) mixture, spray coating donkey anti-murine Secondary antibody with BSA-QD in a spray amount of 0.75. mu.L/cm₆₃₀(red) mixture, forming five T lines and one quality control line. The detection antigen is a conjugate of each mycotoxin and BSA, and the molar coupling ratio of the mycotoxin to the BSA is 15: 1. The concentration of each detected antigen in the spraying liquid is 1.5mg/mL calculated by the mass of BSA, and the concentrations of quantum dots are respectively as follows: BSA-QD₅₀₀ 30nM、BSA–QD₅₃₀ 10nM、BSA–QD₅₇₀ 10nM、BSA–QD₆₀₀ 4.5nM and BSA-QD₆₃₀3 nM. The concentration of the quality control line donkey anti-mouse secondary antibody is 2mg/mL, BSA-QD₆₃₀ 3nM。

Commercial filter paper and absorbent paper were used without special treatment.

Five parts of filter paper, a sample pad, a combination pad, an NC membrane and absorbent paper are overlapped and fixed on the adhesive paperboard according to the mode of figure 1, and the overlapped part is about 1.5 mm. Cutting into strips with the width of 4mm, placing into a test strip card shell, placing into a light-proof bag together with a drying agent, and sealing for storage.

(4) And (3) detecting a corn flour (residue) sample.

After sampling the corn flour (residues), uniformly mixing, accurately weighing 5g, placing the 5g into a 50mL centrifuge tube, adding 25mL of methanol-water solution (60: 40, v/v), after carrying out violent oscillation extraction for 25min, rotating the centrifuge for L0min at 12000, sucking 1mL of supernatant, adding 14mL (diluted by 15 times) of water, adding 80 mu L of supernatant into a test strip sample adding hole, and after 15 min, changing the quality control observation hole into brown, which indicates that the test strip is normal in state. Irradiating by using a 450nm LED light source, if a fluorescent strip appears, indicating that mycotoxin exceeds the standard, when a blue strip appears, the fumonisin content is greater than or equal to 2000 microgram/kg, the green color is that the deoxynivalenol content is greater than or equal to 1000 microgram/kg, the yellow color is that the zearalenone content is greater than or equal to 60 microgram/kg, the orange color is that the ochratoxin A content is greater than or equal to 5 microgram/kg, and the red color is aflatoxin B₁The content is more than or equal to 20 mug/kg.

Example 2. Preparation and use of a multicolor fluorescence signal open type immunochromatography test strip for detecting five mycotoxins in corn (raw grain).

(1) The silver inner filter probe was prepared as in example 1.

(2) Quantum dots were coupled to BSA as in example 1.

(3) Assembling immunochromatography test paper strip, and FB on NC membrane₁And adjusting the concentration of the quantum dots in the DON detection line to BSA-QD₅₀₀16nM、BSA–QD₅₃₀ 5nM, other conditions the same as in example 1.

(4) And (4) detecting a corn (raw grain) sample.

After the corn is sampled, the corn is fully crushed in a crusher, 5g of the corn is accurately weighed and placed in a 50mL centrifuge tube, 25mL of methanol-water solution (60: 40, v/v) is added, after the corn is vigorously shaken and extracted for 25min, 12000 is rotated to centrifuge for L0min, 1mL of supernatant is sucked, 14mL (diluted by 15 times) of water is added, 80 mu L of the supernatant is added into a test strip sample adding hole, and after 15 min, the quality control observation hole is brown, so that the test strip state is normal. Irradiation with 450nm LED light sourceIf a naked eye visible fluorescence strip appears, the fact that the mycotoxin exceeds the standard is shown, the fumonisin content is larger than or equal to 4000 microgram/kg when a blue strip appears, the deoxynivalenol content is larger than or equal to 2000 microgram/kg when a green strip appears, the zearalenone content is larger than or equal to 60 microgram/kg when a yellow strip appears, the ochratoxin A content is larger than or equal to 5 microgram/kg when an orange strip appears, and the aflatoxin B when a red strip appears₁The content is more than or equal to 20 mug/kg.

Claims (1)

1. A preparation method of a multicolor fluorescence immunochromatographic test strip for naked eye observation is characterized by comprising the following steps:

(1) preparing a silver inner filtering probe:

adjusting the concentration of sodium citrate to 5mM and tannin to 0.025mM in a 1000mL system, heating to boil under vigorous stirring, adding 10mL of 25mM silver nitrate, and turning the solution to bright yellow; removing 200mL of reaction liquid, adding 170mL of water into the reaction system, setting the temperature at 90 ℃, adding 5mL of 25mM sodium citrate, 15mL of 2.5mM tannic acid and 10mL of 25mM silver nitrate, and keeping the temperature at 90 ℃ for 30 minutes; repeating the operation steps of removing and adding, and monitoring the ultraviolet absorption spectrum of the liquid removed each time until the silver nanoparticle solution with the maximum absorption peak of 450nm is obtained;

five 100mL portions were taken with a particle number of about 10¹¹Centrifuging the silver nanoparticle solution per mL for 15 minutes at 6500 rpm, and discarding the supernatant; the pellets were resuspended in 100mL of anti-AFB-containing suspension₁anti-OTA, anti-ZEN, anti-DON and anti-FB₁0.1M boric acid buffer solution with pH7.5 of the mouse monoclonal antibody ascites; slowly stirring at room temperature for 2h, centrifuging at 6500 rpm for 15 min, and removing the supernatant; resuspending the pellet in 100mL boric acid buffer containing 0.1% (w/v) bovine serum albumin, centrifuging for 15 minutes at 6500 rpm after 5 minutes, resuspending the pellet in 10mL storage solution, and storing at 4 ℃ for later use;

coupling CdSe/ZnS quantum dots with BSA:

five colors of CdSe/ZnS Quantum Dots (QDs), blue QDs respectively₅₀₀Green QD₅₃₀Yellow QD₅₇₀Orange QD₆₀₀And red QD₆₃₀Surface carboxylation treatment, coupling with BSA after EDC activation; five kinds of carboxylAdjusting the concentration of the basic quantum dots to be 1 mu M, respectively taking 1mL, adjusting the pH to 5.7, adding 0.1mL of freshly prepared 1mg/mL EDC aqueous solution under low-speed stirring at room temperature, activating for 5 minutes, then adding 0.1mL of 1% BSA solution, and reacting for 1h at room temperature; then adding 0.12mL of freshly prepared 1mg/mL EDC solution, adding 0.12mL of 1% BSA solution after 5 minutes, and reacting for 1h at room temperature; adding 0.15mL of freshly prepared 1mg/mL EDC solution for the third time, adding 0.15mL of 1% BSA solution after 5 minutes, and reacting at room temperature for 2 hours; centrifuging for 5 minutes at 1000 rpm to remove excessive polymer, and storing at 4 ℃ for later use;

(3) assembling the immunochromatography test strip:

the immunochromatographic test strip is formed by overlapping and fixing five parts of filter paper, a sample pad, a combination pad, an NC membrane and absorbent paper on an adhesive card paper;

the bottom layer is adhesive card paper, the middle part is adhered with NC membrane, the NC membrane is sprayed with five detection antigens and five BSA-QD mixtures in the detection area in advance, and the quality control position is sprayed with donkey anti-mouse IgG antibody and BSA-QD₆₃₀(ii) a Sequentially adhering a combination pad, a sample pad and filter paper sprayed with five silver inner filter probes on an adhesive card paper on the left side of an NC film, laminating the right edge of the combination pad and the left edge of the NC film by 1-2 mm, laminating the sample pad on the combination pad, staggering the left side by 1-2 mm, adhering the left side of the combination pad on the card paper, laminating the filter paper on the sample pad, and staggering the left side by 1-2 mm and adhering the left side of the filter paper on the card paper; the right side of the NC membrane is adhered with absorbent paper, the left side edge of the absorbent paper and the right side edge of the NC membrane are laminated by 1-2 mm, and a sample solution to be detected enters the NC membrane through filter paper, a sample pad and a combination pad under the capillary action of the absorbent paper during detection and flows from left to right;

combining five parts of filter paper, a sample pad, a combination pad, an NC membrane and absorbent paper, cutting into strips with the width of 4mm, putting the strips into a test strip card shell, and then putting the test strip card shell and a drying agent into a light-resistant bag for sealing and storing; a test strip fixing clamping groove is formed in the test strip clamping shell, a sample adding hole is formed in the position of filter paper of the test strip, detection observation ports are formed in the five detection line areas of the NC membrane, and quality control observation ports are formed in the position of the quality control line;

the sample pad was soaked with 50 mM pH7.5 phosphate buffered saline containing 1% BSA, 0.5% tween 20, and 0.05% sodium azide and dried under vacuum;

the combination pad bears five detection probes, after the combination pad is soaked by 10mM phosphate buffered saline with pH7.5 containing 0.2% Tween 20 and 2% sucrose and dried, a mixture of the five silver inner filtering probes is sprayed on the combination pad at 20 muL/cm, and the combination pad is placed in a vacuum drying oven at 37 ℃ for drying for 6 hours;

FB is sprayed on the NC film in sequence₁Detection of antigen with BSA-QD₅₀₀Mixture, DON detection antigen and BSA-QD₅₃₀Mixture, ZEN detection antigen and BSA-QD₅₇₀Mixture, OTA detection antigen and BSA-QD₆₀₀Mixture, AFB₁Detection of antigen with BSA-QD₆₃₀Mixture and donkey anti-murine Secondary antibody with BSA-QD₆₃₀Mixing to form five T lines and one quality control line; the detection antigen is conjugate of each mycotoxin and BSA, the molar coupling ratio is 15:1, and the spraying concentration is 1.5mg/mL calculated by the mass of the BSA; the BSA-QD spraying amount of each color takes the condition that an obvious strip is observed by naked eyes under an LED lamp with the wavelength of 450nm as an adjustment basis, and when the content of mycotoxin in a sample is equal to the national standard requirement, a fluorescence signal on a corresponding detection line is opened until the observation of the sample by the naked eyes is visible; and (3) drying the sprayed NC membrane in a vacuum drying oven at 37 ℃ for 6 h.

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