A multicolor fluorescence analysis method for pollutants in food based on artificial ribozymes

Abstract

The present invention discloses a multicolor fluorescence analysis method for pollutants in food based on artificial ribozymes, which belongs to the field of molecular biological detection. The present invention uses gold nanoparticles as carriers to synthesize AuNPs-DNA-antibody detection probes, antibodies are used to capture targets, and primer DNA is used to amplify signals. Under optimal conditions, the method of the present invention achieves the detection of CAP, 17β-E 2 and AFM 1 The detection ranges were 0.333ng / mL-3.333μg / mL, 3.333ng / mL-3.333μg / mL, 3.333fg / mL-3.333ng / mL, and the limits of quantification were 0.333ng / mL, 3.333ng / mL, and 3.333fg / mL, respectively. The detection accuracy, sensitivity, and stability of the present invention are high. Compared with the prior art, the method established by the present invention does not require the addition of additional nuclease during the detection process, thereby reducing the reaction cost.

Description

Technical Field

[0001] The invention relates to a multicolor fluorescence analysis method for pollutants in food based on artificial ribozymes, and belongs to the field of molecular biology detection. Background Art

[0002] In recent years, with the improvement of people's quality of life and concept level, milk has gradually become a necessity in the family. Milk is rich in nutrients, but data show that the content of antibiotics, estrogens, and microbial toxins in modern milk has increased. Long-term intake may endanger human health, especially the growth and development of adolescents and the reproductive system. Therefore, monitoring the content of contaminants in food is crucial to ensure food safety. So far, a variety of analytical detection methods have been developed for the joint detection of contaminants in food, including instrumental analysis and enzyme-linked immunosorbent assay (ELISA). However, these methods may have disadvantages such as cumbersome pretreatment steps and strong cross-reactions. Therefore, it is necessary to develop a simple and efficient multicolor fluorescence detection method.

[0003] AuNPs-DNA-antibody is a detection probe that combines nanomaterials and immunoassay methods, in which antibodies are used to compete for immune targets and trigger DNA is used to amplify signals. At present, this probe has been widely used in the field of biosensors. In addition, many isothermal amplification techniques based on DNAzymes are also hot topics in recent years. DNAzyme is a class of DNA molecules with catalytic functions. Like protein and RNA catalytic enzymes, DNAzyme can catalyze various types of biochemical reactions and has been widely used in asymmetric catalysis, biosensors, DNA nanotechnology and clinical diagnosis. In summary, combining AuNPs-DNA-antibody specific detection probes with DNAzyme-mediated nucleic acid amplification (DANA), according to the different fluorescent signals designed by the detection probe, high-sensitivity detection and analysis of different trace pollutants in food can be achieved. Summary of the invention

[0004] [Technical issues]

[0005] The problem of simultaneous detection of multiple pollutants by fluorescent immunoassay is solved, and a convenient method of multi-color fluorescence high-sensitivity detection is provided.

[0006] [Technical solution]

[0007] The first object of the present invention is to provide a method for simultaneously detecting multiple pollutants, the specific steps of the method are:

[0008] (1) Synthesis of detection probes: primer DNA and corresponding specific antibodies of chloramphenicol (CAP), estradiol (17β-E2) or aflatoxin M1 (AFM1) were modified on the surface of AuNPs to obtain three detection probes for CAP, 17β-E2 and AFM1, respectively;

[0009] (2) The corresponding antigens of CAP, 17β-E2, and AFM1 were mixed and added to a black polystyrene microplate and blocked with BSA;

[0010] (3) diluting the detection probes of CAP, 17β-E2, and AFM1 prepared in step (1), respectively, mixing them with the sample to be tested, and adding them to the black polystyrene microplate in step (2), incubating and washing the black polystyrene microplate;

[0011] (4) Signal DNA targeting CAP, 17β-E2, and AFM1 and Mg 2+ Add them to the black polystyrene microplate in step (3) respectively, incubate and detect the fluorescence intensity signal.

[0012] In one embodiment of the present invention, the method for synthesizing the detection probe in step (1) is as follows: taking a certain amount of AuNPs, adding CAP, 17β-E2 or AFM1 monoclonal antibodies, incubating at room temperature to form three AuNPs-antibody dispersions; TCEP-activated primer DNAs of CAP, 17β-E2 or AFM1 modified with thiol groups are added to the corresponding AuNPs-antibody dispersions, respectively, mixed and frozen, and PEG20000 and PBS are added after dissolution to obtain AuNP-DNA-antibody dispersions; BSA is added for incubation, and the supernatant is taken by centrifugation to obtain the detection probe.

[0013] In one embodiment of the present invention, the method for synthesizing the detection probe in step (1) is:

[0014] a) 3 mL of the dispersion containing 13 nm AuNPs was taken, the pH was adjusted to 8.5-9, and the mixture was equally divided into three centrifuge tubes. 18 μg of CAP, 17β-E2 or AFM1 monoclonal antibody was added respectively, and the mixture was incubated at room temperature for 1 h to form three dispersions containing AuNPs-antibodies;

[0015] b) The primer DNA of CAP modified with thiol, the primer DNA of 17β-E2 and the primer DNA of AFM1 were activated by TCEP (molar ratio 1:100) and added to the corresponding AuNPs-antibody dispersion respectively, and frozen at -20°C for 30 min. After dissolving, 30% PEG20000 and 0.1M PBS were added, and the mixture was mixed for 5 min. The mixture was salt aged at 4°C for 2 h to form a dispersion containing AuNPs-DNA-antibody.

[0016] c) Add 10% BSA, incubate at room temperature for 40 min, centrifuge at 13000 rpm for 15 min, and finally disperse the synthesized probe in 200 μL 0.01 M PBS (containing 1% PEG20000 and 1% BSA, pH=7.4), store at 4°C in the dark, and use within one week.

[0017] In one embodiment of the present invention, the sequence of the primer DNA of CAP is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.

[0018] In one embodiment of the present invention, the sequence of the primer DNA of 17β-E2 is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'.

[0019] In one embodiment of the present invention, the sequence of the primer DNA of AFM1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.

[0020] In one embodiment of the present invention, the sequence of the signal DNA of CAP is: 5'-FAM-ACGCACTAT / rA / GGAAGAGAT-BHQ1-3'.

[0021] In one embodiment of the present invention, the sequence of the signal DNA of 17β-E2 is: 5'-Cy3-TAGCTTCAT / rA / GGACAATCA-BHQ2-3'.

[0022] In one embodiment of the present invention, the sequence of the signal DNA of AFM1 is: 5'-Texas red-CGTTCTAAT / rA / GGAGTAGCC-BHQ2-3'.

[0023] In one embodiment of the present invention, in step (2), the corresponding antigens of CAP, 17β-E2 and AFM1 are mixed and added to a black polystyrene microplate, incubated at 35-38°C for 1.5-2.5h, and 220 μL of 2% BSA is added and blocked at 35-38°C for 0.5-1.5h.

[0024] In one embodiment of the present invention, in step (2), the added concentration of the corresponding antigens of CAP, 17β-E2, and AFM1 is 0.6 to 1.5 μg / mL.

[0025] In one embodiment of the present invention, in step (3), the dilution factor of the detection probes of CAP, 17β-E2, and AFM1 is 1 / 80-1 / 60.

[0026] In one embodiment of the present invention, in step (3), the incubation condition is 35-38° C. for 0.5-1.5 h.

[0027] In one embodiment of the present invention, in step (4), the concentration of the added signal DNA of CAP, 17β-E2, and AFM1 is 0.01 to 0.1 μg / mL, and the concentration of Mg is 0.01 to 0.1 μg / mL. 2+ The concentration is 5~15mM.

[0028] In one embodiment of the present invention, in step (3), the incubation condition is 35-38° C. for 0.5-1.5 h.

[0029] In one embodiment of the present invention, in step (4), the fluorescence intensity signals are detected at 489 / 521 nm, 532 / 568 nm and 592 / 622 nm, respectively.

[0030] The present invention also provides a multicolor fluorescence detection kit, which includes detection probes for chloramphenicol, estradiol hormone and aflatoxin M1, a black polystyrene microplate coated with chloramphenicol, estradiol hormone and aflatoxin M1 coated antigens, BSA, chloramphenicol standard, estradiol hormone standard, aflatoxin M1 standard, signal DNA and Mg 2+ .

[0031] In one embodiment of the present invention, the preparation method of the detection probe is as follows: taking a certain amount of AuNPs, adding CAP, 17β-E2 or AFM1 monoclonal antibodies, incubating at room temperature to form three AuNPs-antibody dispersions; TCEP-activated primer DNAs of CAP, 17β-E2 or AFM1 modified with thiol groups are added to the corresponding AuNPs-antibody dispersions, mixed and frozen, and PEG20000 and PBS are added after dissolution to obtain AuNP-DNA-antibody dispersions; BSA is added for incubation, and the supernatant is obtained by centrifugation to obtain the detection probe.

[0032] In one embodiment of the present invention, the preparation method of the detection probe is:

[0033] a) 3 mL of the dispersion containing 13 nm AuNPs was taken, the pH was adjusted to 8.5-9, and the mixture was equally divided into three centrifuge tubes. 18 μg of CAP, 17β-E2 or AFM1 monoclonal antibody was added respectively, and the mixture was incubated at room temperature for 1 h to form three dispersions containing AuNPs-antibodies;

[0034] b) The primer DNA of CAP modified with thiol, the primer DNA of 17β-E2 and the primer DNA of AFM1 were activated by TCEP (molar ratio 1:100) and added to the corresponding AuNPs-antibody dispersion respectively, and frozen at -20°C for 30 min. After dissolving, 30% PEG20000 and 0.1M PBS were added, and the mixture was mixed for 5 min. The mixture was salt aged at 4°C for 2 h to form a dispersion containing AuNPs-DNA-antibody.

[0035] c) Add 10% BSA, incubate at room temperature for 40 min, centrifuge at 13000 rpm for 15 min, and finally disperse the synthesized probe in 200 μL 0.01 M PBS (containing 1% PEG20000 and 1% BSA, pH=7.4), store at 4°C in the dark, and use within one week.

[0036] In one embodiment of the present invention, the sequence of the primer DNA of CAP is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.

[0037] In one embodiment of the present invention, the sequence of the primer DNA of 17β-E2 is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'.

[0038] In one embodiment of the present invention, the sequence of the primer DNA of AFM1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.

[0039] In one embodiment of the present invention, the sequence of the signal DNA of CAP is: 5'-FAM-ACGCACTAT / rA / GGAAGAGAT-BHQ1-3'.

[0040] In one embodiment of the present invention, the sequence of the signal DNA of 17β-E2 is: 5'-Cy3-TAGCTTCAT / rA / GGACAATCA-BHQ2-3'.

[0041] In one embodiment of the present invention, the sequence of the signal DNA of AFM1 is: 5'-Texas red-CGTTCTAAT / rA / GGAGTAGCC-BHQ2-3'.

[0042] Beneficial effects:

[0043] 1. The present invention uses gold nanoparticles as carriers to synthesize AuNPs-DNA-antibody detection probes. Antibodies are used to capture the target, and primer DNA is used to amplify the signal. After the black 96-well microplate is coated with antigens, blocked, and washed, the AuNPs-DNA-antibody detection probe is added, and the coated antigen competes with the sample to be tested for binding to the antibody on the probe; the excess probe and the probe bound to the target are washed away, and the signal DNA and Mg are added. 2+ , and the primer DNA on the detection probe completes the signal amplification step.

[0044] 2. The present invention establishes a fluorescent immunoassay method for DNAzyme-assisted signal amplification for simultaneous quantitative detection of CAP / 17β-E2 / AFM1. Under optimal conditions, the method achieves simultaneous detection of CAP, 17β-E2 and AFM1, with detection ranges of 0.333 ng / mL-3.333 μg / mL, 3.333 ng / mL-3.333 μg / mL, and 3.333 fg / mL-3.333 ng / mL, respectively, and quantitative limits of 0.333 ng / mL, 3.333 ng / mL, and 3.333 fg / mL, respectively.

[0045] 3. The cross-reaction rate with antibiotics (KANA and SM), two hormones (E1 and E3) and two toxins (OTA and ZEN) is low, and it has high selectivity for chloramphenicol, 17β-E2 and AFM1. The detection accuracy, sensitivity and stability of the present invention are high. Compared with the prior art, the method established by the present invention does not need to add additional nuclease during the detection process, thereby reducing the reaction cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the principle of the present invention.

[0047] Figure 2 The diagram shows the influence of different fluorescent substances on the experimental results; a) The detection effect diagram of the fluorescence signal using 6-FAM, Cy3, and Texas red as probes respectively; b) The detection effect diagram of the fluorescence signal using 6-FAM, TAMRA, and Cy5 as probes respectively; c) The detection effect diagram under the amplification of 6-FAM, Cy3, and Texas red fluorescence probes; d) The detection effect diagram under the amplification of 6-FAM, TAMRA, and Cy5 fluorescence probes.

[0048] Figure 3 To verify the feasibility of the scheme; a) CAP detection; b) E2 detection; c) AFM1 detection; d) Fluorescence intensity changes under different CAP concentrations; e) Fluorescence intensity changes under different E2 concentrations; f) Fluorescence intensity changes under different AFM1 concentrations).

[0049] Figure 4The diagrams are for optimizing the concentration of coated antigen and detection probe; a) CAP coating antigen concentration and detection probe concentration optimization; b) 17β-E2 coating antigen concentration and detection probe concentration optimization; c) AFM1 coating antigen concentration and detection probe concentration optimization.

[0050] Figure 5 For signal DNA and Mg 2+ Concentration optimization diagram of a) CAP signal DNA and Mg 2+ Optimization of concentrations of 17β-E2 signal DNA and Mg 2+ Optimization of concentrations of AFM1 signal DNA and Mg 2+ Optimization of concentration.

[0051] Figure 6 Standard curves for detecting linear relationships; a) CAP standard curve; b) 17β-E2 standard curve; c) AFM1 standard curve.

[0052] Figure 7 Figure 1 shows the specificity and stability study; a) The effect of the method on the detection of different antibiotics, hormones and toxins; b) The effect of the detection method at different times. DETAILED DESCRIPTION

[0053] Reagents and materials: CAP standards were purchased from Zhenxiang Technology Co., Ltd. (Beijing, China). Trisodium citrate, Tween 20, bovine albumin (BSA), MgCl2, and polyethylene glycol 20000 (PEG 20000) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). TCEP was purchased from Bio-Sky Co., Ltd. (Shanghai, China). Monoclonal antibodies were purchased from Gene Tex (USA). CAP-BSA was purchased from Oster Technology Co., Ltd. (Nanning, China).

[0054] Coating buffer (0.06M CBS, pH 9.6) and 5× reaction buffer (0.1M Tris-HCl, 0.75M NaCl, pH 8.3). 0.1M PBS (1.35M NaCl, 0.047M KCl, 0.1M Na2HPO4, 0.02M NaH2PO4). All oligonucleotides were synthesized, modified and purified by Sangon (Shanghai, China).

[0055] Instruments: Tecnai G220 transmission electron microscope (TEM) (USA); H1M1 enzyme-linked immunosorbent analyzer (Guangzhou Darui Biotechnology Co., Ltd.); T9-UV-vis spectrophotometer (Beijing Puxi General Instrument Co., Ltd.); shaking incubator (USA, Thermo Fisher).

[0056] Example 1 Synthesis of detection probe

[0057] (1) Preparation of AuNPs:

[0058] AuNPs were prepared by citrate reduction method. 100 mL of 0.01% HAuCl4 was heated to boiling on a magnetic stirrer. 2.5 mL of freshly prepared 1% trisodium citrate was then added to the boiling solution under vigorous stirring. The mixture was boiled continuously for about 15 minutes until the color turned orange-red. The solution was then cooled to ambient temperature under slow stirring, made up to 100 mL of dispersion, and stored at 4°C.

[0059] (2) Synthesis of AuNPs-DNA-antibody detection probe:

[0060] 1) 1 mL of AuNPs dispersion (pH 8.5-9.0) was mixed with 18 μg of CAP / 17β-E2 / AFM1 monoclonal antibody and incubated for 1 h at room temperature with gentle stirring to obtain mixed solutions of AuNPs-CAP, AuNPs-17β-E2 and AuNPs-AFM1.

[0061] 2) TCEP was used to activate the thiol-modified primer DNAs for CAP, 17β-E2, and AFM1, respectively.

[0062] 3) Add the primer DNA for CAP, 17β-E2, and AFM1 activated in step 2) to the corresponding mixed solution in step 1), the amount of primer DNA added is 1OD, and stand at -20°C for 0.5h. After dissolution, add 30% PEG20000 (final concentration is 1%) and 0.1M PBS (final concentration is 0.01M) to the mixed solution, mix for 5 minutes, and perform salt aging at 4°C for 2h. Finally, add 10% BSA, incubate at room temperature for 40min, centrifuge at 13000rpm for 15min, and the synthesized probe is finally dispersed in 200μL 0.01M PBS (containing 1% PEG20000 and 1% BSA, pH=7.4).

[0063] Primer DNA1 for CAP is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.

[0064] Primer DNA2 for 17β-E2 is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'.

[0065] Primer DNA3 of AFM1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.

[0066] Example 2 Selection of fluorescent marker for signal DNA

[0067] (1) Fluorescence signal detection

[0068] The 1.5 μg / mL CAP, E2, and AFM1 standard solutions were mixed at a volume ratio of 1:1:1 to prepare a mixed sample solution, and the 0.3 μM CAP, 17β-E2, and AFM1 AuNPs-DNA-antibody prepared in Example 1 were also mixed at a volume ratio of 1:1:1 to prepare an AuNPs-antibody-DNA probe mixed solution.

[0069] 60 μL of the coated antigens CAP-BSA, E2-BSA and AFM1-BSA at a concentration of 0.9 μg / mL were mixed together, coated in a black microplate, and incubated at 37°C for 2 hours. After washing three times with 0.05% PBST, 250 μL of 2% BSA was added and blocked at 37°C for 1 hour. Then 90 μL of the mixed sample solution and 90 μL of the AuNPs-antibody-DNA probe mixed solution were added and incubated at 37°C for 1 hour for immune competition reaction. After washing, 36 μL of Tris-HCl, 18 μL of signal DNA and 36 μL of MgCl2 were added, and the reaction system was supplemented to 180 μL with water and incubated at 37°C for 1 hour. The fluorescence spectrum curve was measured with a fluorescence spectrophotometer, and the fluorescence signals at 489 / 521nm, 532 / 568nm and 592 / 622nm were measured with a multifunctional microplate reader ( Figure 1 ).

[0070] (2) Selection of fluorescent markers

[0071] In order to better distinguish the detection signals of the three target pollutants CAP, 17β-E2, and AFM1, different fluorescent substances were connected to the 3' end of the signal DNA for CAP, 17β-E2, and AFM1, and the effects of different fluorescent substances on the detection sensitivity were further tested. Therefore, two combinations of 6-FAM (1), Cy3 (2), Texas red (3) and 6-FAM (4), TAMRA (5), and Cy5 (6) were selected to test the effects of fluorescent substances on the experimental results. Figure 2 As shown in ab, the letter S represents signal DNA, I represents primer DNA, and the number represents the fluorescein serial number connected to the signal DNA (Table 1). The fluorescence instrument test results show that no matter which fluorescein is used, the detection signal after Example 1 is much amplified than the original fluorescence signal, that is, the signal when only signal DNA exists, and the peak position is clear without interference with each other.

[0072] The actual amplification effect was compared using an ELISA reader, such as Figure 2cd, group 1 represents the combination of signal DNA 1 and primer DNA 1 with 6-FAM (1) as the fluorescent element, and group 2 is similar (Table 1). As can be seen from the figure, the amplification effect of 6-FAM (4), TAMRA (5), and Cy5 (6) is not as good as that of the detection probes with 6-FAM (1), Cy3 (2), and Texas red (3) as the fluorescent element. The signal of the detection probe with TAMRA (5) as the fluorescent element was only amplified by 14 times ((FS+I) / FS), and the signal of the detection probe with Cy5 (6) as the fluorescent element was only amplified by 20 times, which is obviously not as good as the detection probe with Cy3 (38 times) or Texas red (37 times) as the fluorescent signal. Therefore, 6-FAM (1), Cy3 (2), and Texas red (3) were selected as the three labeled fluorescent elements.

[0073] The signal DNA1 of CAP is: 5'-fluorescein-ACGCACTAT / rA / GGAAGAGAT-BHQ1-3'.

[0074] The signal DNA2 of 17β-E2 is: 5'-fluorescein-TAGCTTCAT / rA / GGACAATCA-BHQ2-3'.

[0075] The signal DNA3 of AFM1 is: 5'-fluorescein-CGTTCTAAT / rA / GGAGTAGCC-BHQ2-3'.

[0076] Table 1

[0077] Group Representative sequence S1 SignalDNA1-6-FAM(1) S2 Signal DNA2-Cy3(2) S3 Signal DNA3-Texas red (3) S4 SignalDNA1-6-FAM(4) S5 Signal DNA2-TAMRA (5) S6 Signal DNA3-Cy5(6) Group1 <![CDATA[Signal DNA 1-6-FAM(1) + Primer DNA 1 + Mg 2+ > Group2 <![CDATA[Signal DNA2-Cy3(2) + Primer DNA2 + Mg 2+ > Group3 <![CDATA[Signal DNA 3 - Texas red(3) + Primer DNA 3 + Mg 2+ > Group4 <![CDATA[Signal DNA 1-6-FAM(4) + Primer DNA 1 + Mg 2+ > Group5 <![CDATA[Signal DNA2-TAMRA(5) + Primer DNA2 + Mg 2+ > Group6 <![CDATA[Signal DNA 3 - Texas red(3) + Primer DNA 3 + Mg 2+ >

[0078] Example 3 Fluorescence Detection Feasibility Analysis

[0079] To verify the amplification effect of the three groups of sequences, 10 μL of primer DNA (0.3 μM) for CAP, 17β-E2 or AFM1 in Example 1, 10 μL of signal DNA (0.6 μM) for CAP, 17β-E2 or AFM1 and 20 μL of MgCl2 (50 mM) were incubated in 0.1 M Tris-HCl (containing 0.75 M NaCl, pH 8.3) at 37°C for 1 h, and the fluorescence was measured at 489 / 521 nm, 532 / 568 nm and 592 / 622 nm, respectively.

[0080] The results are as follows Figure 3 As shown, in the primer DNA, signal DNA and Mg 2+In the presence of both primer DNA and auxiliary factors, enhanced fluorescence was obtained at 489 / 521nm, 532 / 568nm and 592 / 622nm, respectively, indicating that the amplification reaction can only proceed in this case. In the absence of primer DNA or auxiliary factors, the amplification reaction will not occur and the fluorescence signal is weak. Figure 3 As can be seen from ac, the primer DNA cannot act on the mismatched signal DNA, and the fluorescent signal cannot be obtained. In the reaction between one of the primer DNAs and all the signal DNAs, the fluorescent signal is hardly disturbed.

[0081] Furthermore, the feasibility of the scheme was verified using AuNPs-antibody-DNA against CAP, 17β-E2 or AFM1 in Example 1.

[0082] The 1.5 μg / mL CAP, E2, and AFM1 standard solutions were mixed at a volume ratio of 1:1:1 to prepare a mixed sample solution, and the 0.3 μM CAP, 17β-E2, and AFM1 AuNPs-DNA-antibody prepared in Example 1 were also mixed at a volume ratio of 1:1:1 to prepare an AuNPs-antibody-DNA probe mixed solution.

[0083] 60 μL of 0.9 μg / mL coated antigens CAP-BSA, E2-BSA and AFM1-BSA were mixed together, coated in a black microplate, and incubated at 37°C for 2 h. After washing three times with 0.05% PBST, 250 μL of 2% BSA was added and blocked at 37°C for 1 h. Then, 90 μL of mixed sample solutions with concentrations of 0 μg / mL, 0.005 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, and 5 μg / mL and 90 μL of AuNPs-antibody-DNA probe mixed solution were added and incubated at 37°C for 1 h for immune competition reaction. After washing, 36 μL Tris-HCl, 18 μL signal DNA and 36 μL MgCl2 were added, and the reaction system was supplemented to 180 μL with water and incubated at 37°C for 1 h. The fluorescence spectrum curve was measured with a fluorescence spectrophotometer, and the fluorescence signals at 489 / 521 nm, 532 / 568 nm and 592 / 622 nm were measured with a multifunctional microplate reader.

[0084] like Figure 3 df respectively represent the detection effect of this method on CAP, E2 and AFM1. As the concentrations of the three targets increase, the fluorescence intensity gradually decreases, indicating that the scheme is feasible.

[0085] Example 4 Condition Optimization

[0086] (1) Explore the concentration of coated antigen and detection probe:

[0087] Referring to the method of simultaneously detecting the contents of three target pollutants, CAP, 17β-E2 and AFM1, in Example 2 (1), the checkerboard method was used to optimize different antigen coating concentrations (0.6, 0.9, 1.2, 1.5 μg / mL) and AuNPs-antibody-DNA concentrations (1 / 80, 1 / 60), and the inhibition rate was used as the evaluation index. Figure 4 As shown, when the probe dilution was 1 / 60, 1 / 60, 1 / 60 and the antigen coating concentration was 0.9, 0.9 and 1.5 μg / mL, the inhibition rates (1-F1 / F0, 1-F1 / F0, 1-F0.05 / F0) reached the highest, which were 82.439%, 62.052% and 68.222%, respectively.

[0088] (2) Exploring signal DNA and Mg 2+ Concentration:

[0089] Signal DNA and Mg 2+ The concentration of Mg is a key condition for DNAzyme-mediated isothermal nucleic acid amplification. Referring to the method for simultaneously detecting the contents of three target pollutants, CAP, 17β-E2 and AFM1, the signal DNA concentration (S1: 0.005, 0.01, 0.02, 0.03, 0.04 μM; S2: 0.025, 0.05, 0.1, 0.15, 0.2 μM; S3: 0.05, 0.1, 0.2, 0.3, 0.4 μM) and Mg are set. 2+ (3, 5, 8, 10, 13, 15, 18 mM) for analysis, e.g. Figure 5 As shown. When the signal DNA and Mg 2+ When the concentrations were 0.01μM, 0.025μM, 0.1μM and 10mM, 10mM, 10mM, the inhibition rates were the highest, which were 49.600%, 65.419% and 46.472%, respectively. Finally, the 1 / 60 dilution of AuNPs-antibody-DNA, 0.9μg / mL antigen, 10mM Mg 2+ and 0.01 μM signal DNA for CAP detection, 1 / 60 dilution of AuNPs-antibody-DNA, 0.9 μg / mL antigen, 10 mM Mg 2+ and 0.025 μM signal DNA for E2 detection, 1 / 60 dilution of AuNPs-antibody-DNA, 1.5 μg / mL antigen, 10 mM Mg 2+ and 0.1 μM signal DNA for AFM1 detection.

[0090] Example 5 Methodology Verification

[0091] To ensure the practicability of this multi-target strategy, the linearity and limit of detection (LOD) of this method were verified.

[0092] The detection probe was synthesized by referring to Example 1. The 1.5 μg / mL concentration of CAP, E2, and AFM1 standard solutions were mixed in a volume ratio of 1:1:1 to prepare a mixed sample solution. The 1 / 60 dilution of AuNPs-antibody-DNA, 0.9 μg / mL CAP antigen, and 10 mM Mg 2+ and 0.01 μM signal DNA1 for CAP detection, 1 / 60 dilution of AuNPs-antibody-DNA, 0.9 μg / mL E2 antigen, 10 mM Mg 2+ and 0.025 μM signal DNA2 for E2 detection, 1 / 60 diluted AuNPs-antibody-DNA, 1.5 μg / mL AFM1 antigen, 10 mM Mg 2+ and 0.01 μM signal DNA3 for AFM1 detection. The concentration of the mixed sample was changed, at least 20 blank samples were measured, the values ​​were recorded, the mean value (x) and standard deviation (SD) were calculated, x+3SD or x-3SD was calculated and substituted into the curve equation to obtain the detection limit.

[0093] The results are as follows Figure 6 As shown, as the concentration of the target increases, the corresponding fluorescence intensity decreases. This trend can be explained by the fact that when the target and the detection probe are added to the well at the same time, the target and the antigen will compete with the antibody on the AuNPs-antibody-DNA. As the concentration of the target molecule increases, the number of probes bound to the antigen decreases and the fluorescence intensity decreases. The detection ranges of CAP, E2, and AFM1 are 0.333ng / mL-3.333μg / mL, 3.333ng / mL-3.333μg / mL, and 3.333fg / mL-3.333ng / mL, respectively. The standard curve shows good linearity (CAP, y=-436.96x+657.13, R 2 =0.96; E2, y=-33.27x+109.23, R 2 =0.94;AFM1,y=-41.57x-27.82,R 2 =0.94). The limits of quantification were 0.333 ng / mL, 3.333 ng / mL, and 3.333 fg / mL, respectively.

[0094] Example 6 Specificity Study

[0095] Two antibiotics, kanamycin (KANA) and streptomycin (SM), two hormones, estrone (E1) and estriol (E3), and two toxins, zearalenone (ZEN) and ochratoxin A (OTA), were selected for specificity study. KANA+E1+OTA and SM+E3+ZEN were used as a group to prepare mixed sample solutions. Each sample was repeated three times and tested using the established method. The results were statistically analyzed and evaluated. Figure 7 As shown in a, the cross-reactivity measured was low.

[0096] Example 7 Stability Study

[0097] The stability of the probe was studied over a period of one month. The probe was stored at 4°C and tested at 1, 15, and 30 days. The inhibition rate was used as the evaluation index (CAP, 1-F 10 / F 0.001 ; E2, 1-F 10 / F 0.01 ;AFM1,1-F 0.01 / F 0.00001 ).like Figure 7 As shown in b, the probe still showed good detection effect after being stored for 1 month.

[0098] Example 8 Determination of actual samples

[0099] The recovery test was performed by spiking the standard solutions of the three target substances into the milk samples at different concentration levels in triplicate (CAP was 0.005 and 0.01 μg / mL, E2 was 0.05 and 0.1 μg / mL, and AFM1 was 0.00005 and 0.005 μg / mL), and the results are shown in Table 2. The average recovery ranged from 93% to 115%, and the RSD ranged from 5% to 10%. The results show that the detection method provided by the present invention has good accuracy.

[0100] Table 2 Recovery verification results

[0101]

[0102] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for simultaneously detecting multiple pollutants, characterized in that: The specific steps of the method are: (1) Synthetic detection probes: Primer DNA of chloramphenicol, estradiol or aflatoxin M1 and corresponding specific antibodies were modified on the surface of AuNPs to obtain three detection probes for chloramphenicol, estradiol and aflatoxin M1 respectively; the sequence of primer DNA of chloramphenicol is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'; the sequence of the primer DNA for estradiol hormone is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'; the sequence of the primer DNA for aflatoxin M1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'; (2) Chloramphenicol, estradiol, and aflatoxin M1 corresponding antigens were mixed and added to a black polystyrene microplate and blocked with BSA; (3) diluting the detection probes of chloramphenicol, estradiol hormone, and aflatoxin M1 prepared in step (1) respectively, mixing them with the sample to be tested, and adding them to the black polystyrene microplate in step (2), incubating and washing the black polystyrene microplate; (4) Signal DNA and Mg targeting chloramphenicol, estradiol, aflatoxin M1 2+ Add them to the black polystyrene microplate in step (3) respectively, incubate and detect the fluorescence intensity signal; the sequence of the signal DNA of chloramphenicol is: 5'-FAM-ACGCACTAT / rA / GGAAGAGAT-BHQ1-3', the sequence of the signal DNA of estradiol hormone is: 5'-Cy3-TAGCTTCAT / rA / GGACAATCA-BHQ2-3', and the sequence of the signal DNA of aflatoxin M1 is: 5'-Texas red -CGTTCTAAT / rA / GGAGTAGCC-BHQ2-3'.

2. The method according to claim 1, characterized in that In step (2), chloramphenicol, estradiol hormone, and aflatoxin M1 corresponding antigen were mixed and added to a black polystyrene microplate, incubated at 35-38°C for 1.5-2.5 h, and blocked with 220 μL 2% BSA at 35-38°C for 0.5-1.5 h.

3. The method according to claim 1 or 2, characterized in that: In step (2), the added concentration of chloramphenicol, estradiol hormone, and aflatoxin M1 corresponding antigen is 0.6~1.5 μg / mL.

4. The method according to claim 1, characterized in that In step (3), the dilution ratio of the detection probes for chloramphenicol, estradiol, and aflatoxin M1 is 1 / 80-1 / 60.

5. The method according to claim 1, characterized in that In step (4), the concentration of signal DNA of chloramphenicol, estradiol, and aflatoxin M1 is 0.01-0.1 μg / mL, and Mg 2+ The concentration is 5~15 mM.

6. A multicolor fluorescence detection kit, characterized in that: The kit comprises detection probes for chloramphenicol, estradiol hormone and aflatoxin M1, a black polystyrene microplate coated with chloramphenicol, estradiol hormone and aflatoxin M1 coated antigens, BSA, chloramphenicol standard, estradiol hormone standard, aflatoxin M1 standard, signal DNA and Mg 2+ ; The primer DNA of chloramphenicol, estradiol or aflatoxin M1 and the corresponding specific antibodies were modified on the surface of AuNPs to obtain three detection probes for chloramphenicol, estradiol and aflatoxin M1 respectively; the sequence of the primer DNA of chloramphenicol is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'; the sequence of the primer DNA for estradiol hormone is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'; the sequence of the primer DNA for aflatoxin M1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'; The sequence of the signal DNA of chloramphenicol is: 5'-FAM-ACGCACTAT / rA / GGAAGAGAT-BHQ1-3', the sequence of the signal DNA of estradiol hormone is: 5'-Cy3-TAGCTTCAT / rA / GGACAATCA-BHQ2-3', and the sequence of the signal DNA of aflatoxin M1 is: 5'-Texas red -CGTTCTAAT / rA / GGAGTAGCC-BHQ2-3'.

7. The multicolor fluorescence detection kit according to claim 6, characterized in that: The preparation method of the detection probe is as follows: taking a certain amount of AuNPs, adding chloramphenicol, estradiol hormone or aflatoxin M1 monoclonal antibody, incubating at room temperature to form three AuNPs-antibody dispersions; TCEP-activated primer DNAs of chloramphenicol, estradiol hormone or aflatoxin M1 modified by thiol are added to the corresponding AuNPs-antibody dispersions, respectively, mixed and frozen, and PEG20000 and PBS are added after dissolution to obtain AuNP-DNA-antibody dispersions; BSA is added for incubation, and the supernatant is taken by centrifugation to obtain the detection probe.

Images