Preparation and detection device of multicolor fluorescent signal open competitive immunochromatographic test strip

Abstract

A multicolor fluorescent signal open type competitive immunochromatographic test strip preparation and detection device, the test strip uses "red, orange, yellow, green, blue" five-color quantum dot fluorescence as the background signal of the five fungal toxin detection lines, silver nanoparticles are respectively combined with monoclonal antibodies of the five fungal toxins as the inner filter probe of the quantum dot excitation light, and anti-rabbit IgG and rabbit IgG are used as independent control line systems. During the detection, the test strip is inserted into the fluorescence reading box, and the fluorescence signal is analyzed by the smartphone APP, and the ratio of the fluorescence signal intensity of each detection line to the fluorescence signal intensity of the control line is used for accurate quantification. The present invention can be used for the simultaneous rapid quantitative detection of five fungal toxins such as aflatoxin B1, ochratoxin A, zearalenone, deoxynivalenol and fumonisin B1 in grain and oil products.

Description

Technical Field

[0001] The present invention relates to the field of rapid detection technology, and in particular to a multicolor fluorescence "signal-on" competitive immunochromatographic test strip and a detection device thereof. Background Art

[0002] my country is a major grain producer and consumer, and strengthening grain quality control is of great significance. my country's National Food Safety Standard (2017) has set limit requirements for four mycotoxins in grains: aflatoxin B1 (AFB1) 5~20µg / kg, ochratoxin A (OTA) 5µg / kg, zearalenone (ZEN) 60µg / kg and deoxynivalenol (DON) 1000µg / kg. In addition, due to the toxicity of fumonisin and the severity of pollution, the 2019 draft for comments on the limit of mycotoxins in the national food safety standard has added the limit requirements for the total amount of fumonisin B1, B2 and B3: the limits for corn, cornmeal (dregs) and cereal products containing corn raw materials are 4000, 2000 and 1000µg / kg respectively. Establishing a highly sensitive and rapid detection method for the simultaneous detection of multiple mycotoxins is of great significance for maintaining food safety.

[0003] Immunochromatography is a commonly used method for rapid on-site screening because of its advantages such as simple operation, rapid speed, and strong resistance to matrix interference. Mycotoxins are small molecules, and a competitive reaction mode is usually used in immunochromatography. In this method, the number of analytes is inversely proportional to the signal intensity on the detection line, and the "signal disappearance" on the detection line (T line) is usually used as the judgment standard, and the sensitivity is not high. Constructing a competitive immunochromatography technology based on the "signal on" mode, that is, the detection signal increases from zero to one as the concentration of the small molecule analyte increases, can significantly improve the detection sensitivity of the method.

[0004] Some scholars have proposed a "signal-on" immunochromatography method based on fluorescence resonance energy transfer (FRET), which has higher sensitivity than traditional competitive immunochromatography. However, FRET can only act on the light emitted by fluorescent substances. For multi-color fluorescence to construct a "signal-on" mode of competitive immunochromatography, energy acceptors with multiple absorption spectra are required. Nanoparticles with different absorption spectra have large differences in morphology, size, biocompatibility, suitable pH value and buffer salt concentration, and are not suitable for unified use on the same test strip. Therefore, the competitive immunochromatography based on the "signal-on" mode of FRET is not suitable for multiple detections indicated by multi-color fluorescence.

[0005] To address this problem, the present invention proposes a "signal-on" competitive immunochromatographic test strip based on the inner filter effect (IFE). IFE refers to the strong absorption effect of precious metal nanoparticles on the specific excitation light or emission light of fluorescent substances. Since IFE can act on the excitation light and some fluorescent substances such as quantum dots have the characteristics of a wide excitation spectrum, the "signal-on" of multi-color fluorescence can be achieved through IFE of a single excitation light.

[0006] The sensitivity of the IFE system is closely related to the following three factors: first, the effective overlap between the absorption spectrum of the absorber and the excitation or emission spectrum of the fluorescent agent (this is a necessary condition), and the higher the degree of overlap, the more significant the IFE; second, the molar extinction coefficient of the absorber. The higher the molar extinction coefficient, the stronger its absorption of light, and thus the more significant the IFE; third, the performance of the fluorescent agent. Fluorescence is the output signal of the system. The higher the luminescence intensity and the better the stability of the fluorescent agent, the greater the degree of change in the fluorescence signal caused by IFE, that is, the more sensitive the system is.

[0007] Among traditional fluorescent materials and light-absorbing materials, there are few fluorescent agent-absorber combinations that meet the above three elements, which limits the application of this method in biosensing. With the development of new nanomaterials, more options for fluorescent agent-absorber combinations are provided, which greatly promotes the application of IFE in biodetection. The molar extinction coefficient of silver nanoparticles is greater than 10 10 cm -1 M -1 , 100 times higher than colloidal gold, and it has excellent properties such as adjustable absorption spectrum, stability and good biocompatibility, making it an ideal absorbent for the IFE system. Compared with conventional organic fluorescent dyes, quantum dots have the advantages of high fluorescence yield, good photostability, wide excitation spectrum and narrow and symmetrical emission spectrum, and spectrum with different colors as the particle size changes, making them ideal fluorescent agents. On the immunochromatographic test strip, the absorption spectrum of silver nanoparticles and the excitation spectrum of multicolor quantum dots are effectively overlapped, combined with the strong extinction characteristics of silver nanoparticles, and the number of silver nanoparticles is exponentially converted into the fluorescence signal of multicolor quantum dots through IFE, which can achieve simultaneous high-sensitivity detection of multiple small molecule analytes.

[0008] When immunochromatography detects a single target, the T / C ratio method (i.e., the ratio of the signal intensity of the T line to the control line) is often used for quantification. This method can eliminate the influence of external interference on the immunological reaction to a certain extent, so the quantitative detection result is relatively accurate. However, in multiple detections, the control line (C line, usually sprayed with anti-mouse IgG antibodies) signal is simultaneously affected by the concentrations of multiple analytes in the detection system, and cannot be quantified by the T / C ratio method. Therefore, the immunochromatography multiple quantitative analysis method often uses the T line signal for quantification. However, since the T line signal of the test strip is a dynamic change process, the signal will gradually change with the extension of the reaction time until it tends to be relatively stable, and this dynamic process is affected by many uncertain external factors such as the production process of the test strip, the detection environment, the sample to be tested, and the differences in operating habits, the quantitative deviation is large during actual detection. In response to this problem, the present invention designs an independent C line signal system so that the C line signal is not interfered by multiple analytes, thereby realizing multiple quantitative detection by the T / C ratio method and ensuring the accuracy of multiple detections.

[0009] In recent years, optical detection devices based on smartphones have been widely studied. By combining some terminal components and using smartphones as optical signal receiving and processing tools, researchers have achieved high-sensitivity detection of colorimetric signals, fluorescence signals, Raman scattering signals, etc. For example, Yeo et al. designed a signal detector for fluorescent immunochromatographic test strips based on smartphones; Zheng et al. used a color recognition smartphone APP based on hue-saturation-brightness (HSL) to collect and analyze the degree of colloidal gold aggregation. Summary of the invention

[0010] The purpose of the present invention is to provide a preparation and detection device for a multicolor fluorescent signal open type competitive immunochromatographic test strip.

[0011] The present invention is achieved through the following technical solutions.

[0012] The preparation of a multicolor fluorescent signal open type competitive immunochromatographic test strip of the present invention comprises the following steps.

[0013] (1) Selection of an independent control line system.

[0014] Rabbit IgG and anti-rabbit IgG antibodies that had no cross-reaction with mouse monoclonal antibodies against five fungal toxins were screened and used as an independent C-line system. There was no cross-reaction between the control line system and the detection probe.

[0015] Under the optimal ratio of the control probe quantity and the anti-rabbit IgG antibody dosage, the fluorescence of the control line was reduced by 50%.

[0016] (2) Preparation of silver inner filter probe.

[0017] In a 1000mL system, adjust the concentration of sodium citrate (SC) to 5 mM and tannic acid (TA) to 0.025 mM, heat to boiling under vigorous stirring, add 10 mL of 25 mM silver nitrate, and the solution turns bright yellow. Remove 200 mL of the reaction solution, add 170 mL of water to the reaction system, set the temperature to 90°C, add 5 mL of 25 mM SC, 15 mL of 2.5 mM TA, and 10 mL of 25 mM silver nitrate, and keep at 90°C for 30 minutes. Repeat the above removal and addition steps for a total of 11 times to obtain a silver nanoparticle solution with a maximum absorption peak of 450 nm.

[0018] Take six 100 mL portions of silver nanoparticle solution (the number of particles is about 10 11 / mL), centrifuged at 6500 rpm for 15 minutes, and discarded the supernatant; the precipitates were resuspended in 100 mL of 0.1 M pH 7.5 boric acid buffer containing rabbit IgG and five kinds of mycotoxin mouse monoclonal antibody ascites; slowly stirred at room temperature for 2 hours, centrifuged at 6500 rpm for 15 minutes, and discarded the supernatant; the precipitates were resuspended in boric acid buffer (storage solution) containing 0.1% (w / v) bovine serum albumin (BSA), after 5 minutes, centrifuged at 6500 rpm for 15 minutes, and the precipitates were resuspended in 10 mL of storage solution and stored at 4°C for later use.

[0019] Considering the total amount limit of fumonisins B1, B2 and B3 in the national standard, the present invention selects anti-FB1 mouse monoclonal antibody with a cross-reaction rate with FB2 and FB3 greater than 70%, and the silver probe prepared thereby can reflect the total amount of fumonisins B1, B2 and B3.

[0020] (3) Coupling of CdSe / ZnS quantum dots (QD) with BSA.

[0021] Five colors of CdSe / ZnS QD, blue (QD 500 )、Green(QD 530 )、Yellow(QD 570 )、Orange(QD 600 ) and red (QD 630), surface carboxylation treatment, activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and coupled with BSA. The concentration of five carboxylated quantum dots was adjusted to 1μM, and 1mL was taken respectively, and the pH was adjusted to 5.7. Under low-speed stirring at room temperature, 0.1mL of freshly prepared 1mg / mL EDC aqueous solution was added for activation for 5 minutes, followed by 0.1mL of 1% BSA solution, reaction at room temperature for 1h, and then 0.12mL of freshly prepared 1mg / mL EDC solution was added. After 5 minutes, 0.12mL of 1% BSA solution was added, and reaction at room temperature for 1h. 0.15mL of freshly prepared 1mg / mL EDC solution was added for the third time, and 0.15mL of 1% BSA solution was added after 5 minutes, and reaction at room temperature for 2h. Centrifuge at 1000 rpm for 5 minutes to remove excess polymer and store at 4℃ for use.

[0022] (4) Assembly of immunochromatographic test strips.

[0023] The test strip consists of filter paper, sample pad, conjugate pad, nitrocellulose membrane (NC membrane) and absorbent paper. Figure 1 The cards are fixed on the sticky card paper in an overlapping manner.

[0024] The bottom layer is sticky cardboard, and the middle part of it is pasted with NC membrane, which is pre-sprayed with rabbit IgG, five detection antigens and five-color BSA-QD mixture. The sticky cardboard on the left side of the NC membrane is pasted with the conjugate pad, sample pad and filter paper sprayed with six silver inner filter probes in sequence. The right edge of the conjugate pad overlaps with the left edge of the NC membrane by 1 to 2 mm, the sample pad is overlapped on the conjugate pad, and the left side is offset by 1 to 2 mm and pasted on the cardboard. The filter paper is overlapped on the sample pad, and the left side is also offset by 1 to 2 mm and pasted on the cardboard. The absorbent paper is pasted on the right side of the NC membrane, and the left edge of the absorbent paper overlaps with the right edge of the NC membrane by 1 to 2 mm. During the test, the test liquid passes through the filter paper, sample pad, and conjugate pad, and enters the NC membrane under the capillary action of the absorbent paper, flowing from left to right.

[0025] The sample pad was pre-wetted with 50 mM pH 7.5 phosphate buffered saline (containing 1% BSA, 0.5% Tween 20 and 0.05% sodium azide) and vacuum dried.

[0026] The conjugate pad carried five detection probes and an independent control line probe, which was soaked and dried with 10 mM pH 7.5 phosphate buffered saline (containing 0.2% Tween 20 and 2% sucrose). Then, a mixture of six silver inner filter probes was sprayed on the conjugate pad at 25 μL / cm and dried in a vacuum drying oven at 37 °C for 6 h.

[0027] Anti-rabbit IgG and BSA-QD were sprayed on the NC membrane in sequence. 630 (Red) Mixture, FB1 detection antigen and BSA–QD500 (Blue) Mixture, DON detection antigen and BSA–QD 530 (Green) Mixture, ZEN detection antigen and BSA–QD 570 (Yellow) Mixture, OTA detection antigen and BSA–QD 600 (Orange) Mixture and AFB1 detection antigen and BSA–QD 630 (red) mixture, forming a control line and five T lines; the detection antigen was the conjugate of each fungal toxin and BSA, the conjugation molar ratio was 15:1, and the spraying concentration was 1.5 mg / mL calculated based on the mass of BSA; the spraying amount of each color BSA-QD was adjusted based on the obvious bands observed under 450nm LED light; the NC membrane after spraying was placed in a vacuum drying oven at 37 °C for 6 h.

[0028] Commercial filter paper and absorbent paper were used directly without special treatment.

[0029] After the filter paper, sample pad, conjugate pad, NC membrane and absorbent paper are combined, they are cut into strips with a width of 4 mm each, placed in the test strip card case, and then sealed in a light-proof bag together with a desiccant for storage. The test strip card case has a test strip fixing slot, a sample addition hole is set at the filter paper position of the test strip, and a detection observation port is set at the NC membrane control line and the five detection line areas.

[0030] The invention discloses a detection device for a multicolor fluorescent signal open-type competitive immunochromatographic test strip, comprising a fluorescent reading box of the test strip and a smart phone.

[0031] The test strip fluorescence reading box is a dark-colored plastic card box with a dark field of view. A test strip insertion hole is provided at one end of the bottom of the card box. A set of 450nm LED light sources are placed on both sides of the inner wall of the card box. A switch is provided outside the card box to control the LED light source. A filter and an observation window are provided on the top of the card box. The fluorescence signal can be qualitatively interpreted by naked eyes and quantitatively interpreted by a smart phone through the observation window.

[0032] Install the "HSL Image Analysis APP" on your smartphone, pre-set the program and input the corresponding equations of the fluorescence signals of each test line. During quantitative testing, place the mobile phone camera flat on the observation window of the test strip fluorescence reading box, turn on the 450nm LED light source in the reading box, and the smartphone will automatically take pictures and perform HSL analysis and calculation of the image, directly giving the test results of five fungal toxins in the sample.

[0033] Test strips use.

[0034] Weigh 5g of crushed grains, place them in a 50mL centrifuge tube, add 25mL of methanol-water solution (60:40, v / v), and extract vigorously for 25min. Centrifuge at 12,000 rpm for 10min, dilute the supernatant 15 times, take 80μL and add it to the test strip sample well. After 15 minutes, insert the test strip into the fluorescence reading box, turn on the LED light source, and the smartphone automatically takes a photo and performs HSL analysis and calculation of the image, directly giving the detection results of five fungal toxins in the sample.

[0035] The present invention adopts silver nanoparticles as the common inner filter of "red, orange, yellow, green and blue" multicolor quantum dot excitation light, establishes a multicolor fluorescence "signal-on" competitive immunochromatographic detection method based on IFE, and realizes simultaneous, highly sensitive and rapid quantitative detection of five fungal toxins, including aflatoxin B1, ochratoxin A, zearalenone, deoxynivalenol and fumonisin B1 (AFB1, OTA, ZEN, DON and FB1), in grain and oil products by designing an independent C-line signal system and quantifying with T / C ratio, combined with smartphone HSL-APP analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the test strip structure of the method of the present invention.

[0037] Figure 2 The method of the present invention is a test strip detection device. DETAILED DESCRIPTION

[0038] In order to make the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0039] The five fungal toxin monoclonal antibody ascites, rabbit IgG (NC05) and anti-rabbit IgG antibody (NC05-G11) involved in the examples were provided by Wuxi Zhongdeber Biotechnology Co., Ltd.; the five carboxyl-modified quantum dots used were provided by Ocean-nano, USA; the fungal toxin standard substances, EDC, BSA, SC, TA, and AgNO3 involved were purchased from Sigma; the chemical reagents involved were purchased from Aladdin; the fluorescence reading box was custom assembled for this laboratory; the smartphone image analysis and processing software was provided by the research group of Professor Lin Jianhan of China Agricultural University.

[0040] Example

[0041] Preparation of multicolor fluorescent signal open-type competitive immunochromatographic test strips and their detection device for quantitative detection of five fungal toxins in corn.

[0042] (1) Preparation of silver inner filter probe.

[0043] Silver nanoparticles with a particle size of 70nm were synthesized by seed growth method, and their SPR characteristic absorption peak was 450nm. The specific scheme is as follows:

[0044] (a) Synthesis of seed silver: In a 100 mL system, adjust the SC concentration to 5 mM and the TA concentration to 0.025 mM. Heat to boiling with vigorous stirring. Add 1 mL of AgNO3 (25 mM). The solution immediately turns bright yellow. At this time, the seed size is about 15 nm.

[0045] (b) Gradual growth of silver: After the synthesis of the seed silver, 19.5 mL of the reaction solution was removed (stored at 4 °C for later use), 16.5 mL of water was added to the reaction system, the temperature was set to 90 °C, 0.5 mL of SC (25 mM), 1.5 mL of TA (2.5 mM), and 1 mL of AgNO3 were added, and this process was repeated every 30 min. The absorption spectrum of the silver nanoparticles taken out each time was monitored by UV scanning until silver nanoparticles with an SPR absorption peak of 450 nm were obtained. Under the conditions of this embodiment, the SPR absorption peak of 450 nm was reached after 11 rounds of growth of the silver seed.

[0046] The silver-antibody fluorescent inner filter probe was prepared by free adsorption method. The specific scheme is as follows: take six portions of 1 mL silver nanoparticle solution (the number of particles is about 10 11 / mL), centrifuged at 7000 rpm for 10 min, and discarded the supernatant; the precipitates were resuspended in 1 mL of 0.1 M boric acid solution (pH value adjusted to 7.5 by NaOH) containing ascites of mouse monoclonal antibodies against rabbit IgG, anti-AFB1, anti-OTA, anti-ZEN, anti-DON and anti-FB1; stirred at low speed at room temperature for 2 h, centrifuged at 7000 rpm for 10 min, and discarded the supernatant; the precipitates were resuspended in boric acid solution (storage solution) containing 0.1% (w / v) BSA, and after 5 min, centrifuged at 7000 rpm for 10 min, and the precipitates were resuspended in 0.1 mL of storage solution and stored at 4°C for later use.

[0047] (2) Coupling of quantum dots with BSA.

[0048] Surface carboxyl-modified QDs 500 , QD 530 , QD 570 , QD 600 and QD 630, the concentration was 2μM, 0.05mL and 0.05mL phosphate buffer (8mM, pH 5.7) were taken in a small beaker and mixed, stirred at low speed on a magnetic stirrer, 0.1mL freshly prepared 1mg / mL EDC solution was added, activated for 5min, then 0.1mL 1% BSA solution was added, reacted at room temperature for 1h, then 0.12mL freshly prepared 1mg / mL EDC solution was added, 0.12mL 1% BSA solution was added 5 minutes later, reacted at room temperature for 1h, 0.15mL freshly prepared 1mg / mL EDC solution was added for the third time, 0.15mL 1% BSA solution was added 5 minutes later, reacted at room temperature for 2h. Centrifuged at 1000 rpm for 5 minutes, and the supernatant was stored at 4℃ for later use.

[0049] (3) Assembly of immunochromatographic test strips.

[0050] The test strip consists of five parts: filter paper, sample pad, conjugate pad, NC membrane and absorbent paper.

[0051] The sample pad was infiltrated with 50 mM pH 7.5 phosphate buffered saline (containing 1% BSA, 0.5% Tween 20 and 0.05% sodium azide) and dried under vacuum.

[0052] The conjugate pad was soaked with 10 mM pH 7.5 phosphate buffered saline (containing 0.2% Tween 20 and 2% sucrose) and dried. Then, the six silver inner filter probe mixtures were sprayed on the conjugate pad at 25 μL / cm and dried in a vacuum drying oven at 37 °C for 6 h.

[0053] Anti-rabbit IgG and BSA-QD were sprayed on the NC membrane in sequence. 630 (Red) Mixture, FB1 detection antigen and BSA–QD 500 (Blue) Mixture, DON detection antigen and BSA–QD 530 (Green) Mixture, ZEN detection antigen and BSA–QD 570 (Yellow) Mixture, OTA detection antigen and BSA–QD 600 (Orange) Mixture and AFB1 detection antigen and BSA–QD 630 (red) mixture, forming a control line and five test lines. The test antigens were conjugates of each mycotoxin and BSA, and the molar conjugation ratio of mycotoxin to BSA was 15:1. The concentration of anti-rabbit IgG in the spray solution was 0.75 mg / mL, the concentration of each test antigen was 1.5 mg / mL calculated based on the mass of BSA, and the amount of each BSA–QD was calculated based on the QD concentration: BSA–QD 630 3nM, BSA–QD 500 30nM, BSA-QD 530 10nM, BSA-QD570 10nM, BSA-QD 600 4.5nM and BSA–QD 630 3nM.

[0054] Commercial filter paper and absorbent paper were used directly without special treatment.

[0055] The filter paper, sample pad, conjugate pad, NC membrane and absorbent paper were divided into five parts according to Figure 1 The test strips are fixed on the sticky cardboard in an overlapping manner, with an overlap of about 1.5 mm. Cut into strips with a width of 4 mm each, put them into the test strip card holder, and store them in a dry place away from light.

[0056] Figure 1 This is a schematic diagram of the test strip structure of this embodiment. The bottom layer is sticky cardboard, and the middle part of it is pasted with NC membrane. The NC membrane is pre-sprayed with rabbit IgG and five detection antigens and a five-color BSA-QD mixture. The sticky cardboard on the left side of the NC membrane is pasted with a conjugation pad, a sample pad and a filter paper sprayed with six silver inner filter probes in sequence. The right edge of the conjugation pad is overlapped with the left edge of the NC membrane by 1 to 2 mm. The sample pad is overlapped on the conjugation pad and pasted on the cardboard with a 1 to 2 mm offset on the left side. The filter paper is overlapped on the sample pad and also pasted on the cardboard with a 1 to 2 mm offset on the left side. The absorbent paper is pasted on the right side of the NC membrane, and the left edge of the absorbent paper is overlapped with the right edge of the NC membrane by 1 to 2 mm. During the test, the test liquid passes through the filter paper, the sample pad, and the conjugation pad, and enters the NC membrane under the capillary action of the absorbent paper, flowing from left to right.

[0057] (4) Determination of quantitative standard curve.

[0058] Ten mixed standard solutions of five mycotoxins were prepared, with the concentration series of AFB1, OTA and ZEN being 0, 0.01, 0.02, 0.04, 0.078, 0.156, 0.3125, 0.625, 1.25 and 2.5 µg / L, respectively; the concentration series of DON and FB1 being 0, 0.4, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 and 100 µg / L, respectively. Take 80µL of each mixed standard solution and add it to the test strip (make three parallel samples). After 15 minutes, insert the test strip into the fluorescence reading box, open the smartphone image analysis and processing software, place the mobile phone camera flat on the observation window of the test strip fluorescence reading box, turn on the 450nm LED light source in the reading box, and the smartphone automatically takes pictures and processes the data. The HSL intensity ratio of each T line and the control line is used as the vertical coordinate, and the logarithm of the concentration of five fungal toxins is used as the horizontal coordinate to draw a standard curve to obtain the standard equation for quantitative detection of each fungal toxin. Since the corn sample is extracted with 5 times the mass volume of solvent and then diluted 15 times to remove matrix interference, the fungal toxins in the actual sample are diluted 75 times to correspond to the standard curve. In order to directly obtain the fungal toxin content in the actual sample detection, the horizontal coordinates of each standard equation are enlarged 75 times and entered into the image processing software program.

[0059] Figure 2 The test strip detection device of this embodiment includes a fluorescence reading box of a multicolor fluorescent signal open type competitive immunochromatographic test strip and a smart phone installed with image processing software. The reading box is a dark-colored plastic card box with a dark field, and a test strip insertion hole is provided at one end of the bottom of the card box. A group of 450nm LED light sources are placed on both sides of the inner wall of the card box, and a switch is provided outside the card box to control the LED light source. A filter and an observation window are provided on the top of the card box, and the fluorescent signal can be qualitatively interpreted by the naked eye through the observation window and quantitatively interpreted by a smart phone. The smart phone is installed with the "HSL Image Analysis APP", the program is pre-set and the corresponding equations of each detection signal are input. When quantitative detection is performed, the mobile phone camera is placed flat on the observation window of the test strip fluorescence reading box, and the 450nm LED light source in the reading box is turned on. The smart phone automatically focuses on taking pictures and performs image analysis, and directly gives the detection results of five fungal toxins in the sample.

[0060] (5) Corn sample testing.

[0061] After corn sampling, it was fully crushed in a grinder. 5g of crushed material was accurately weighed and placed in a 50mL centrifuge tube. 25mL of methanol-water solution (60:40, v / v) was added. After vigorous oscillation extraction for 25min, centrifuged at 12,000 rpm for 10min, 1mL of supernatant was aspirated, 14mL of water was added (diluted 15 times), 80μL was added to the test strip sample well, and the test strip was inserted into the fluorescence reading box after 15 minutes. The smartphone pre-set the program and input the corresponding equations of each detection signal, opened the image processing APP, placed the camera in the reading box observation window, turned on the fluorescence reading box LED light source, the phone automatically focused on the photo and performed image analysis, and calculated according to the standard equations corresponding to each color signal. The blue signal corresponds to FB1, the green signal corresponds to DON, the yellow signal corresponds to ZEN, the orange signal corresponds to OTA, and the red signal corresponds to AFB1, and the detection results of the five fungal toxins in the sample are directly given.

Claims

1. A preparation of a multicolor fluorescent signal open type competitive immunochromatographic test strip, characterized in that The following steps are involved: (1) Selection of independent control line system: Rabbit IgG and anti-rabbit IgG antibodies that had no cross-reaction with mouse monoclonal antibodies against five fungal toxins were screened as an independent C-line system. There was no cross-reaction between the control line system and the detection probe. Under the optimal ratio of the number of control probes and the amount of anti-rabbit IgG antibodies, the fluorescence of the control line was reduced by 50%. (2) Preparation of silver inner filter probe: Silver nanoparticles with a particle size of 70 nm were synthesized by seed growth method, and their SPR characteristic absorption peak was 450 nm; the specific scheme is as follows: In a 1000mL system, the concentration of sodium citrate was adjusted to 5mM, and the concentration of tannic acid was 0.025mM. The mixture was heated to boiling under vigorous stirring, and 10mL of 25mM silver nitrate was added, and the solution turned bright yellow. 200mL of the reaction solution was removed, and 170mL of water was added to the reaction system. The temperature was set to 90°C, and 5mL of 25mM sodium citrate, 15mL of 2.5mM tannic acid, and 10mL of 25mM silver nitrate were added. The mixture was kept at 90°C for 30 minutes. The removal and addition operation steps were repeated for a total of 11 times to obtain a silver nanoparticle solution with a maximum absorption peak of 450nm. Take six 100mL portions and the number of particles is 10 11 / mL silver nanoparticle solution, centrifuge at 6500 rpm for 15 minutes, and discard the supernatant; resuspend the precipitate in 100mL of 0.1M pH7.5 boric acid buffer containing rabbit IgG and five kinds of mycotoxin mouse monoclonal antibody ascites; slowly stir at room temperature for 2h, centrifuge at 6500 rpm for 15 minutes, and discard the supernatant; resuspend the precipitate in boric acid buffer containing 0.1% (w / v) bovine serum albumin, centrifuge at 6500 rpm for 15 minutes after 5 minutes, resuspend the precipitate in 10mL storage solution, and store at 4℃ for later use; (3) Coupling of quantum dots with bovine serum albumin: Five colors of CdSe / ZnS quantum dots, blue QD 500 , Green QD 530 , Yellow QD 570 , Orange QD 600 and red QD 630 , surface carboxyl treatment, coupled with bovine serum albumin after EDC activation; the concentration of five carboxylation quantum dots was adjusted to 1μM, 1mL was taken respectively, the pH was adjusted to 5.7, and 0.1mL of freshly prepared 1mg / mL EDC aqueous solution was added under low-speed stirring at room temperature for 5 minutes, and then 0.1mL of 1% BSA solution was added to react at room temperature for 1h, and then 0.12mL of freshly prepared 1mg / mL EDC solution was added, and 0.12mL of 1% BSA solution was added after 5 minutes, and reacted at room temperature for 1h, and 0.15mL of freshly prepared 1mg / mLEDC solution was added for the third time, and 0.15mL of 1% BSA solution was added after 5 minutes, and reacted at room temperature for 2h; centrifuged at 1000 rpm for 5 minutes to remove excess polymer, and stored at 4℃ for use; (4) Assembly of immunochromatographic test strips: The test strip consists of filter paper, sample pad, conjugate pad, NC membrane and absorbent paper overlapped and fixed on sticky cardboard; The bottom layer is sticky cardboard, and the middle part of it is pasted with NC membrane, which is pre-sprayed with rabbit IgG, five detection antigens and five-color BSA-QD mixture; the sticky cardboard on the left side of the NC membrane is pasted with a conjugation pad, a sample pad and a filter paper sprayed with six silver inner filter probes in sequence, and the right edge of the conjugation pad is overlapped with the left edge of the NC membrane by 1 to 2 mm, the sample pad is overlapped on the conjugation pad, and the left side is offset by 1 to 2 mm and pasted on the cardboard, and the filter paper is overlapped on the sample pad, and the left side is also offset by 1 to 2 mm and pasted on the cardboard; absorbent paper is pasted on the right side of the NC membrane, and the left edge of the absorbent paper is overlapped with the right edge of the NC membrane by 1 to 2 mm. During the detection, the test liquid passes through the filter paper, the sample pad and the conjugation pad, and enters the NC membrane under the capillary action of the absorbent paper, and flows from left to right; The sample pad was pre-infiltrated with 50 mM pH 7.5 phosphate buffered saline containing 1% BSA, 0.5% Tween 20, and 0.05% sodium azide and then vacuum dried; The conjugate pad carried five detection probes and an independent control line probe, and after being soaked and dried with 10 mM pH 7.5 phosphate buffered saline containing 0.2% Tween 20 and 2% sucrose, a mixture of six silver inner filter probes was sprayed on the conjugate pad at 25 μL / cm and dried in a vacuum drying oven at 37°C for 6 h; Anti-rabbit IgG and BSA-QD were sprayed on the NC membrane in sequence. 630 Mixture, FB1 detection antigen and BSA-QD 500 Mixture, DON detection antigen and BSA-QD 530 Mixture, ZEN detection antigen and BSA-QD 570 Mixture, OTA detection antigen and BSA-QD 600 Mixture and AFB1 detection antigen and BSA-QD 630 mixture to form a control line and five T lines; the detection antigen was a conjugate of each fungal toxin and BSA, the conjugation molar ratio was 15:1, and the spraying concentration was 1.5 mg / mL calculated based on the mass of BSA; the spraying amount of each color BSA-QD was adjusted based on the observation of obvious bands under a 450nm LED light; the sprayed NC membrane was placed in a vacuum drying oven at 37°C and dried for 6 hours.

2. The detection device of a multicolor fluorescent signal opening type competitive immunochromatographic test strip according to claim 1, characterized in that Includes test strip fluorescence reading box and smartphone; The test strip fluorescence reading box is a dark-colored plastic card box with a dark field of view. A test strip insertion hole is provided at one end of the bottom of the card box. A set of 450nm LED light sources are provided on both sides of the inner wall of the card box. A switch is provided outside the card box to control the LED light source. A filter and an observation window are provided on the top of the card box. The fluorescence signal can be qualitatively interpreted by naked eyes and quantitatively interpreted by a smart phone through the observation window. Install the HSL image analysis APP on the smartphone, pre-set the program and input the corresponding equations of the fluorescence signals of each test line. During quantitative testing, place the mobile phone camera flat on the observation window of the test strip fluorescence reading box, turn on the 450nm LED light source in the reading box, and the smartphone will automatically take pictures and perform HSL analysis and calculation of the image, directly giving the test results of five fungal toxins in the sample.

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