WO2023168822A1 - Artificial ribozyme-based multi-color fluorescence analysis method for contaminants in food product - Google Patents

Artificial ribozyme-based multi-color fluorescence analysis method for contaminants in food product Download PDF

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WO2023168822A1
WO2023168822A1 PCT/CN2022/092166 CN2022092166W WO2023168822A1 WO 2023168822 A1 WO2023168822 A1 WO 2023168822A1 CN 2022092166 W CN2022092166 W CN 2022092166W WO 2023168822 A1 WO2023168822 A1 WO 2023168822A1
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dna
chloramphenicol
aflatoxin
aunps
signal
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French (fr)
Chinese (zh)
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郭亚辉
沈维韦
桑潘婷
陆华进
谢云飞
姚卫蓉
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杭州傲敏生物科技有限公司
江南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A20/00—Water conservation; Efficient water supply; Efficient water use
    • Y02A20/20—Controlling water pollution; Waste water treatment

Definitions

  • the invention relates to a multi-color fluorescence analysis method for contaminants in food based on artificial ribozymes, and belongs to the field of molecular biology detection.
  • AuNPs-DNA-antibody is a detection probe that combines nanomaterials and immunoassay methods, where the antibody is used for immune competition targets and trigger DNA is used to amplify the signal.
  • this probe has been widely used in the field of biosensors.
  • DNAzyme-based isothermal amplification technologies have also been a hot research topic in recent years.
  • DNAzyme is a type of DNA molecule with catalytic function. Like protein and RNA catalytic enzymes, DNAzymes can catalyze various types of biochemical reactions and have been widely used in asymmetric catalysis, biosensors, DNA nanotechnology, and clinical diagnosis.
  • the first object of the present invention is to provide a method for detecting multiple pollutants simultaneously.
  • the specific steps of the method are:
  • Synthetic detection probe modify the AuNPs surface with chloramphenicol (CAP), estradiol hormone (17 ⁇ -E 2 ) or aflatoxin M1 (AFM 1 ) primer DNA and corresponding specific antibodies to obtain respectively Three detection probes for CAP, 17 ⁇ -E 2 and AFM 1 ;
  • CAP chloramphenicol
  • AFM 1 aflatoxin M1
  • step (3) Dilute the detection probes of CAP, 17 ⁇ -E 2 and AFM 1 prepared in step (1) respectively and mix them with the sample to be tested, then add them to the black polystyrene microplate in step (2), incubate and Clean black polystyrene microplates;
  • step (3) Add the signal DNA and Mg 2+ for CAP, 17 ⁇ -E 2 , and AFM 1 to the black polystyrene microplate in step (3), incubate and detect the fluorescence intensity signal.
  • the method for synthesizing the detection probe in step (1) is: take a certain amount of AuNPs, add CAP, 17 ⁇ -E 2 or AFM 1 monoclonal antibody, and incubate at room temperature to form three types of AuNPs -Antibody dispersion; after TCEP activation of the primer DNA of thiol-modified CAP, 17 ⁇ -E 2 or AFM 1 , add the corresponding AuNPs-antibody dispersion, mix and freeze, add PEG20000 and PBS after dissolution, and obtain AuNPs-DNA- Antibody dispersion; add BSA for incubation, centrifuge and take the supernatant to obtain the detection probe.
  • the method for synthesizing the detection probe in step (1) is:
  • the sequence of the primer DNA of CAP is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.
  • the sequence of the primer DNA of 17 ⁇ -E 2 is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'.
  • the sequence of the primer DNA of AFM 1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.
  • sequence of the signal DNA of CAP is: 5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’.
  • sequence of the signal DNA of 17 ⁇ -E 2 is: 5'-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3'.
  • the sequence of the signal DNA of AFM 1 is: 5'-Texas red -CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3'.
  • step (2) the corresponding antigens of CAP, 17 ⁇ -E 2 and AFM 1 are mixed and added to a black polystyrene microplate, and incubated at 35-38°C for 1.5-2.5 h, add 220 ⁇ L 2% BSA and block at 35-38°C for 0.5-1.5 h.
  • step (2) the added concentrations of the corresponding antigens of CAP, 17 ⁇ -E 2 and AFM 1 are 0.6 to 1.5 ⁇ g/mL.
  • step (3) the dilution factor of the detection probes for CAP, 17 ⁇ -E 2 and AFM 1 is 1/80 to 1/60.
  • the incubation condition is incubation at 35-38°C for 0.5-1.5 hours.
  • the added concentrations of signal DNA of CAP, 17 ⁇ -E 2 and AFM 1 are 0.01-0.1 ⁇ g/mL, and the concentration of Mg 2+ is 5-15 mM.
  • the incubation condition is incubation at 35-38°C for 0.5-1.5 hours.
  • fluorescence intensity signals are detected at 489/521nm, 532/568nm and 592/622nm respectively.
  • the invention also provides a multi-color fluorescence detection kit.
  • the kit includes detection probes for chloramphenicol, estradiol hormone and aflatoxin M1, and is coated with chloramphenicol, estradiol hormone and aflatoxin.
  • the preparation method of the detection probe is: take a certain amount of AuNPs, add CAP, 17 ⁇ -E 2 or AFM 1 monoclonal antibody, and incubate at room temperature to form three AuNPs-antibody dispersions. liquid; the primer DNA of thiol-modified CAP, 17 ⁇ -E 2 or AFM 1 was activated by TCEP and then added to the corresponding AuNPs-antibody dispersion, mixed and frozen. After dissolving, add PEG20000 and PBS to obtain the AuNPs-DNA-antibody dispersion. ;Add BSA for incubation, centrifuge and take the supernatant to obtain the detection probe.
  • the preparation method of the detection probe is:
  • the sequence of the primer DNA of CAP is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.
  • the sequence of the primer DNA of 17 ⁇ -E 2 is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'.
  • the sequence of the primer DNA of AFM 1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.
  • sequence of the signal DNA of CAP is: 5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’.
  • sequence of the signal DNA of 17 ⁇ -E 2 is: 5'-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3'.
  • the sequence of the signal DNA of AFM 1 is: 5'-Texas red-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3'.
  • the present invention uses gold nanoparticles as carriers to synthesize AuNPs-DNA-antibody detection probes.
  • the antibodies are used to capture target objects, and the primer DNA is used to amplify signals.
  • 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; excess probe and target substances are washed away
  • signal DNA and Mg 2+ are added to complete the signal amplification step with the primer DNA on the detection probe.
  • the present invention establishes a fluorescence immunoassay method for DNAzyme-assisted signal amplification for simultaneous quantitative detection of CAP/17 ⁇ -E 2 /AFM 1. Under optimal conditions, this method achieves the detection of CAP, 17 ⁇ -E 2 and AFM 1 . Simultaneous detection, the detection ranges are 0.333ng/mL-3.333 ⁇ g/mL, 3.333ng/mL-3.333 ⁇ g/mL, 3.333fg/mL-3.333ng/mL, and the quantitation limits are 0.333ng/mL, 3.333ng/mL respectively. ,3.333fg/mL.
  • Figure 1 is a schematic diagram of the principle of the present invention.
  • Figure 2 shows the influence of different fluoresceins on the experimental results; a) The detection effect of fluorescence signals using 6-FAM, Cy3, and Texas red as probes respectively; b) Using 6-FAM, TAMRA, and Cy5 as probe fluorescence respectively Signal detection effect diagram; c) Detection effect diagram under the amplification of 6-FAM, Cy3, Texas red fluorescent probe; d) Detection effect diagram under the amplification of 6-FAM, TAMRA, Cy5 fluorescent probe.
  • Figure 3 shows the feasibility verification of the scheme; a) detecting CAP; b) detecting E 2 ; c) detecting AFM 1 ; d) changes in fluorescence intensity without using CAP concentration; e) changes in fluorescence intensity under different E 2 concentrations; f is different Fluorescence intensity change at AFM 1 concentration).
  • Figure 4 is a diagram showing the optimization of the concentration of coated antigen and the concentration of the detection probe; a) the optimization of the concentration of the CAP coated antigen and the concentration of the detection probe; b) the optimization of the concentration of the 17 ⁇ -E 2 coating antigen and the concentration of the detection probe; c )AFM 1 coating antigen concentration and detection probe concentration optimization.
  • Figure 5 shows the concentration optimization diagram of signal DNA and Mg 2+ a) The concentration optimization of CAP signal DNA and Mg 2+ ; b) The concentration optimization of 17 ⁇ -E 2 signal DNA and Mg 2+ ; c) AFM 1 signal DNA and Mg 2+ concentration optimization.
  • Figure 6 shows the standard curve for detecting linear relationships; a) CAP standard curve; b) 17 ⁇ -E 2 standard curve; c) AFM 1 standard curve.
  • Figure 7 shows the specificity and stability study diagram; a) the detection effect diagram of the method for different antibiotics, hormones and toxins; b) the detection effect diagram of the detection method at different times.
  • CAP standards were purchased from Zhenxiang Technology Co., Ltd. (Beijing, China). Trisodium citrate, Tween 20, bovine albumin (BSA), MgCl and polyethylene glycol 20000 (PEG 20000) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). TCEP was purchased from Biyuntian Co., Ltd. (Shanghai, China). Monoclonal antibodies were purchased from Gene Tex (USA). CAP-BSA was purchased from Oster Technology Co., Ltd. (Nanning, China).
  • 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 Na 2 HPO 4 , 0.02M NaH 2 PO 4 ). All oligonucleotides were synthesized, modified and purified by Sangon (Shanghai, China).
  • AuNPs were prepared by citrate reduction method. Heat 100 mL of 0.01% HAuCl to boiling on a magnetic stirrer. Then 2.5 mL of freshly prepared 1% trisodium citrate was added to the boiling solution with vigorous stirring. Boil the mixture continuously for about 15 minutes, until the color turns orange-red. The solution was then cooled to ambient temperature with slow stirring, replenished to 100 mL of dispersion, and stored at 4°C.
  • step 2) Add the primer DNA activated in step 2) for CAP, 17 ⁇ -E 2 and AFM 1 into the corresponding mixture in step 1).
  • the amount of primer DNA added is 1OD and let stand at -20°C for 0.5h.
  • 30% PEG 20000 final concentration: 1%) and 0.1M PBS (final concentration: 0.01M) to the mixture, mix for 5 minutes, and perform salt aging at 4°C for 2 hours.
  • the primer DNA1 of CAP is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.
  • Primer DNA2 of 17 ⁇ -E 2 is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'.
  • Primer DNA 3 of AFM 1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.
  • the fluorometer test results show that no matter what kind of fluorophore is used, after Example 1
  • the detection signals are much amplified compared to the original fluorescence signal, that is, the signal when only signal DNA exists, and the peak positions are clear and there is no interference between each other.
  • Group1 represents the combination of signal DNA1 and primer DNA1 with 6-FAM(1) as the fluorescein, and so on for group2 (Table 1). From the figure, it can be It can be seen that the amplification effect of 6-FAM(4), TAMRA(5), and Cy5(6) is not as good as that of the detection probes using 6-FAM(1), Cy3(2), and Texas red(3) as fluorescein respectively. Needle.
  • the signal DNA1 of CAP is: 5’-fluorescein-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’.
  • the signal DNA2 of 17 ⁇ -E 2 is: 5'-fluorescein-TAGCTTCAT/rA/GGACAATCA-BHQ2-3'.
  • the signal DNA 3 of AFM 1 is: 5'-fluorescein-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3'.
  • 10 ⁇ L of the primer DNA (0.3 ⁇ M) for CAP, 17 ⁇ -E 2 or AFM 1 in Example 1, and 10 ⁇ L of the signal DNA for CAP, 17 ⁇ -E 2 or AFM 1 ( 0.6 ⁇ M) and 20 ⁇ L MgCl 2 (50mM) were incubated in 0.1M Tris-HCl (containing 0.75M NaCl, pH 8.3) for 1 hour at 37°C, and the fluorescence was measured at 489/521nm, 532/568nm and 592/622nm respectively.
  • Figure 3d-f show the detection effect of this method on CAP, E2 and AFM1 respectively. As the concentration of the three targets increases, the fluorescence intensity gradually decreases, indicating that this solution is feasible.
  • the checkerboard method was used to optimize different antigen coating concentrations (0.6, 0.9, 1.2, 1.5 ⁇ g/mL) and AuNPs-antibody-DNA concentrations (1/80, 1/60) were optimized, and the inhibition rate was used as the evaluation index.
  • the concentrations of signal DNA and Mg 2+ are critical conditions for DNAzyme-mediated isothermal nucleic acid amplification.
  • set the signal DNA concentration S1: 0.005, 0.01, 0.02, 0.03, 0.04 ⁇ M; S2: 0.025, 0.05 , 0.1, 0.15, 0.2 ⁇ M; S3: 0.05, 0.1, 0.2, 0.3, 0.4 ⁇ M
  • Mg 2+ 3, 5, 8, 10, 13, 15, 18mM
  • 1/60 dilution of AuNPs-antibody-DNA, 0.9 ⁇ g/mL antigen, 10mM Mg 2+ and 0.01 ⁇ M signal DNA was selected for CAP detection, and 1/60 dilution of AuNPs-antibody-DNA, 0.9 ⁇ g/mL antigen, 10mM Mg 2+ and 0.025 ⁇ M signal DNA were used for E 2 detection, 1/60 diluted AuNPs-antibody-DNA, 1.5 ⁇ g/mL antigen, 10 mM Mg 2+ and 0.1 ⁇ M signal DNA were used for AFM 1 detection.
  • Example 1 to synthesize the detection probe, mix the CAP, E 2 and AFM 1 standard solutions with a concentration of 1.5 ⁇ g/mL according to the volume ratio of 1:1:1 to prepare a mixed sample solution, and select 1/60 to dilute the AuNPs-antibody-DNA , 0.9 ⁇ g/mL CAP antigen, 10mM Mg 2+ and 0.01 ⁇ M signal DNA1 are used for CAP detection, 1/60 diluted AuNPs-antibody-DNA, 0.9 ⁇ g/mL E 2 antigen, 10mM Mg 2+ and 0.025 ⁇ M signal DNA2 are used For E 2 detection, 1/60 dilution of AuNPs-antibody-DNA, 1.5 ⁇ g/mL AFM 1 antigen, 10 mM Mg 2+ and 0.01 ⁇ M signal DNA 3 was used for AFM 1 detection. Change the concentration of the mixed sample, measure at least 20 blank samples, record the values, find the mean (x) and standard deviation (SD), find x+3SD or x-3SD and bring
  • the limits of quantification were 0.333ng/mL, 3.333ng/mL, and 3.333fg/mL respectively.
  • KANA kanamycin
  • SM streptomycin
  • E1 estrone
  • E3 estriol
  • ZEN zearalenone
  • KANA+E1+OTA KANA+E1+OTA will be used as a group
  • SM+E3+ZEN SM+E3+ZEN will be used as a group to prepare their respective standard solutions to form a mixed sample solution.
  • Each sample will have 3 Repeat, apply the established method for testing, and perform statistics, analysis and evaluation of the results. The results are shown in Figure 7a, with lower cross-reactivity measured.
  • the stability of the probe was studied over a period of one month.
  • the probes were stored at 4°C and tested on 1, 15 and 30 days respectively.
  • the inhibition rate was used as the evaluation index (CAP, 1-F 10 /F 0.001 ; E 2 , 1-F 10 /F 0.01 ; AFM 1 , 1-F 0.01 /F 0.00001 ).
  • the probe still showed good detection results after being stored for 1 month.
  • the recovery test was performed by spiking standard solutions of the three target compounds into milk samples in triplicate at different concentration levels (0.005 and 0.01 ⁇ g/mL for CAP, 0.05 and 0.1 ⁇ g/mL for E 2 , and 0.05 and 0.1 ⁇ g/mL for AFM 1 ). 0.00005 and 0.005 ⁇ g/mL), the results are shown in Table 2. Average recoveries range from 93% to 115%, with RSDs ranging from 5% to 10%. The results show that the detection method provided by the invention has good accuracy.

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Abstract

An artificial ribozyme-based multi-color fluorescence analysis method for contaminants in a food product. Gold nanoparticles are used as a carrier, an AuNPs-DNA-antibody detection probe is synthesized, an antibody is used for capturing a target object, and a primer DNA is used for amplifying a signal; simultaneous detection of CAP, 17β-E2, and AFM1 is implemented, detection ranges are respectively 0.333 ng/mL to 3.333 μg/mL, 3.333 ng/mL to 3.333 μg/mL, and 3.333 fg/mL to 3.333 ng/mL, and the limits of quantitation are respectively 0.333 ng/mL, 3.333 ng/mL, and 3.333 fg/mL. Detection accuracy, sensitivity, and stability are relatively high, in contrast to the prior art, an extra exonuclease does not need to be added during detection, and reaction costs are reduced.

Description

一种基于人工核酶的食品中污染物多色荧光分析方法A multicolor fluorescence analysis method for contaminants in food based on artificial ribozymes 技术领域Technical field
本发明涉及一种基于人工核酶的食品中污染物多色荧光分析方法，属于分子生物学检测领域。The invention relates to a multi-color fluorescence analysis method for contaminants in food based on artificial ribozymes, and belongs to the field of molecular biology detection.
背景技术Background technique
近年来随着人们生活质量与观念水平的提高，牛奶已经逐渐成为家庭中的必需品，牛奶营养丰富，但有资料显示现代牛奶中抗生素、雌激素、微生物毒素含量增加，长时间摄入可能危害着人类健康尤其是对青少年生长发育和生殖系统有不利影响。因此，监测食品中污染物的含量对于确保食品安全至关重要。迄今为止，已经开发了多种分析检测方法用于联合检测食品中的污染物，包括仪器分析法和酶联免疫法(ELISA)。然而，这些方法可能预处理步骤繁琐、交叉反应较强等缺点。因此，有必要开发一种简单高效的多色荧光检测方法。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. However, data shows that the content of antibiotics, estrogen, and microbial toxins in modern milk has increased. Long-term intake may be harmful to the body. It has adverse effects on human health, especially on adolescent growth and development and reproductive system. Therefore, monitoring the levels of contaminants in food is critical to ensuring food safety. To date, a variety of analytical detection methods have been developed for the joint detection of contaminants in food, including instrumental analysis and enzyme-linked immunoassays (ELISA). However, these methods may have shortcomings such as cumbersome pretreatment steps and strong cross-reactivity. Therefore, it is necessary to develop a simple and efficient multicolor fluorescence detection method.
AuNPs-DNA-抗体是一种将纳米材料和免疫分析方法相结合的检测探针，其中抗体用于免疫竞争靶标，trigger DNA用于扩增信号。目前，该探针已被广泛应用到生物传感器领域。此外，很多基于DNAzyme的等温扩增技术也是近年来研究的热点。DNAzyme是一类具有催化功能的DNA分子。同蛋白质和RNA催化酶一样，DNAzyme能够催化多种类型的生化反应，并在不对称催化、生物传感器、DNA纳米技术以及临床诊断方面得到了广泛的应用。综上，结合AuNPs-DNA-抗体特异性检测探针与DNAzyme介导的核酸扩增(DANA)，根据检测探针设计的不同荧光素信号，实现食品中不同痕量污染物的高灵敏度检测分析。AuNPs-DNA-antibody is a detection probe that combines nanomaterials and immunoassay methods, where the antibody is used for immune competition targets and trigger DNA is used to amplify the signal. At present, this probe has been widely used in the field of biosensors. In addition, many DNAzyme-based isothermal amplification technologies have also been a hot research topic in recent years. DNAzyme is a type of DNA molecule with catalytic function. Like protein and RNA catalytic enzymes, DNAzymes can catalyze various types of biochemical reactions and have been widely used in asymmetric catalysis, biosensors, DNA nanotechnology, and clinical diagnosis. In summary, by combining the AuNPs-DNA-antibody specific detection probe and DNAzyme-mediated nucleic acid amplification (DANA), based on the different fluorescein signals designed by the detection probe, highly sensitive detection and analysis of different trace contaminants in food can be achieved. .
发明内容Contents of the invention
[技术问题][technical problem]
解决荧光免疫法同时检测多种污染物的问题，提供一种多色荧光高灵敏检测的便捷方法。It solves the problem of simultaneous detection of multiple pollutants by fluorescence immunoassay and provides a convenient method for highly sensitive detection of multi-color fluorescence.
[技术方案][Technical solutions]
本发明的第一个目的是提供一种同时检测多种污染物的方法，所述方法的具体步骤为：The first object of the present invention is to provide a method for detecting multiple pollutants simultaneously. The specific steps of the method are:
(1)合成检测探针：在AuNPs表面修饰氯霉素(CAP)、雌二醇激素(17β-E 2)或黄曲霉毒素M1(AFM 1)的引物DNA和相应的特异性抗体，分别得到针对CAP、17β-E 2、AFM 1的三种检测探针； (1) Synthetic detection probe: modify the AuNPs surface with chloramphenicol (CAP), estradiol hormone (17β-E 2 ) or aflatoxin M1 (AFM 1 ) primer DNA and corresponding specific antibodies to obtain respectively Three detection probes for CAP, 17β-E 2 and AFM 1 ;
(2)将CAP、17β-E 2、AFM 1相应抗原进行混合并添加至黑色聚苯乙烯微孔板，BSA封闭； (2) Mix the corresponding antigens of CAP, 17β-E 2 and AFM 1 and add them to a black polystyrene microplate, and seal with BSA;
(3)将步骤(1)制备的CAP、17β-E 2、AFM 1的检测探针分别稀释后与待测样品混合后加入至步骤(2)中的黑色聚苯乙烯微孔板，孵育并清洗黑色聚苯乙烯微孔板； (3) Dilute the detection probes of CAP, 17β-E 2 and AFM 1 prepared in step (1) respectively and mix them with the sample to be tested, then add them to the black polystyrene microplate in step (2), incubate and Clean black polystyrene microplates;
(4)将针对CAP、17β-E 2、AFM 1的信号DNA和Mg 2+分别加入至步骤(3)中的黑色聚苯乙烯微孔板，孵育并检测荧光强度信号。 (4) Add the signal DNA and Mg 2+ for CAP, 17β-E 2 , and AFM 1 to the black polystyrene microplate in step (3), incubate and detect the fluorescence intensity signal.
在本发明的一种实施方式中，步骤(1)中合成检测探针的方法为：取一定量的AuNPs，加入CAP、17β-E 2或AFM 1单克隆抗体，室温孵育，形成三种AuNPs-抗体分散液；巯基修饰的CAP、17β-E 2或AFM 1的引物DNA经TCEP活化后分别加入对应AuNPs-抗体分散液，混匀并冷冻，溶解后加入PEG20000和PBS，获得AuNPs-DNA-抗体分散液；加入BSA进行孵育，离心取上清获得检测探针。 In one embodiment of the present invention, the method for synthesizing the detection probe in step (1) is: take a certain amount of AuNPs, add CAP, 17β-E 2 or AFM 1 monoclonal antibody, and incubate at room temperature to form three types of AuNPs -Antibody dispersion; after TCEP activation of the primer DNA of thiol-modified CAP, 17β-E 2 or AFM 1 , add the corresponding AuNPs-antibody dispersion, mix and freeze, add PEG20000 and PBS after dissolution, and obtain AuNPs-DNA- Antibody dispersion; add BSA for incubation, centrifuge and take the supernatant to obtain the detection probe.
在本发明的一种实施方式中，步骤(1)中合成检测探针的方法为：In one embodiment of the present invention, the method for synthesizing the detection probe in step (1) is:
a)取3mL含有13nm AuNPs的分散液，调节pH至8.5-9，等分至3个离心管中，分别加入18μg CAP、17β-E 2或AFM 1单克隆抗体，室温孵育1h，形成三种含AuNPs-抗体的分散液； a) Take 3 mL of the dispersion containing 13nm AuNPs, adjust the pH to 8.5-9, divide it equally into 3 centrifuge tubes, add 18 μg of CAP, 17β-E 2 or AFM 1 monoclonal antibody respectively, and incubate at room temperature for 1 hour to form three Dispersions containing AuNPs-antibodies;
b)巯基修饰的CAP的引物DNA、17β-E 2的引物DNA和AFM 1的引物DNA经TCEP活化后(摩尔比1：100)分别加入对应AuNPs-抗体分散液，置于-20℃冷冻30min，溶解后加入30％PEG20000、0.1M PBS，持续混匀5min后，4℃条件下盐老化2h，形成含有AuNPs-DNA-抗体的分散液； b) After thiol-modified CAP primer DNA, 17β-E 2 primer DNA and AFM 1 primer DNA are activated by TCEP (molar ratio 1:100), add the corresponding AuNPs-antibody dispersion and freeze at -20°C for 30 minutes. , after dissolving, add 30% PEG20000 and 0.1M PBS, continue mixing for 5 minutes, and then salt-age for 2 hours at 4°C to form a dispersion containing AuNPs-DNA-antibody;
c)加入10％BSA，室温孵育40min，13000rpm离心15min，最终合成的探针分散于200μL 0.01M PBS中(含1％PEG20000和1％BSA，pH＝7.4)，4℃避光保存，一周内使用完。c) Add 10% BSA, incubate at room temperature for 40 minutes, and centrifuge at 13000 rpm for 15 minutes. The final synthesized probe is dispersed in 200 μL 0.01M PBS (containing 1% PEG20000 and 1% BSA, pH=7.4), and stored in the dark at 4°C within one week. finish using.
在本发明的一种实施方式中，CAP的引物DNA的序列为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’。 In one embodiment of the present invention, the sequence of the primer DNA of CAP is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.
在本发明的一种实施方式中，17β-E 2的引物DNA的序列为：5’-HS-(T) 28GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3’。 In one embodiment of the present invention, the sequence of the primer DNA of 17β-E 2 is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'.
在本发明的一种实施方式中，AFM 1的引物DNA的序列为：5’-HS-(T) 28GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3’。 In one embodiment of the invention, the sequence of the primer DNA of AFM 1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.
在本发明的一种实施方式中，CAP的信号DNA的序列为：5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’。In one embodiment of the invention, the sequence of the signal DNA of CAP is: 5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’.
在本发明的一种实施方式中，17β-E 2的信号DNA的序列为：5’-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3’。 In one embodiment of the invention, the sequence of the signal DNA of 17β-E 2 is: 5'-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3'.
在本发明的一种实施方式中，AFM 1的信号DNA的序列为：5’-Texas red -CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3’。 In one embodiment of the invention, the sequence of the signal DNA of AFM 1 is: 5'-Texas red -CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3'.
在本发明的一种实施方式中，步骤(2)中，将CAP、17β-E 2、AFM 1相应抗原进行混合并添加至黑色聚苯乙烯微孔板，35～38℃下孵育1.5～2.5h，加入220μL 2％BSA在35～38℃下封闭0.5～1.5h。 In one embodiment of the invention, in step (2), the corresponding antigens of CAP, 17β-E 2 and AFM 1 are mixed and added to a black polystyrene microplate, and incubated at 35-38°C for 1.5-2.5 h, add 220 μL 2% BSA and block at 35-38°C for 0.5-1.5 h.
在本发明的一种实施方式中，步骤(2)中，CAP、17β-E 2、AFM 1相应抗原的添加浓度为0.6～1.5μg/mL。 In one embodiment of the present invention, in step (2), the added concentrations of the corresponding antigens of CAP, 17β-E 2 and AFM 1 are 0.6 to 1.5 μg/mL.
在本发明的一种实施方式中，步骤(3)中，CAP、17β-E 2、AFM 1的检测探针的稀释倍数为1/80～1/60。 In one embodiment of the present invention, in step (3), the dilution factor of the detection probes for CAP, 17β-E 2 and AFM 1 is 1/80 to 1/60.
在本发明的一种实施方式中，步骤(3)中，孵育的条件为在35～38℃下孵育0.5～1.5h。In one embodiment of the present invention, in step (3), the incubation condition is incubation at 35-38°C for 0.5-1.5 hours.
在本发明的一种实施方式中，步骤(4)中，CAP、17β-E 2、AFM 1的信号DNA的添加浓度为0.01～0.1μg/mL，Mg 2+的浓度为5～15mM。 In one embodiment of the present invention, in step (4), the added concentrations of signal DNA of CAP, 17β-E 2 and AFM 1 are 0.01-0.1 μg/mL, and the concentration of Mg 2+ is 5-15 mM.
在本发明的一种实施方式中，步骤(3)中，孵育的条件为在35～38℃下孵育0.5～1.5h。In one embodiment of the present invention, in step (3), the incubation condition is incubation at 35-38°C for 0.5-1.5 hours.
在本发明的一种实施方式中，步骤(4)中，分别于489/521nm、532/568nm和592/622nm处检测荧光强度信号。In one embodiment of the present invention, in step (4), fluorescence intensity signals are detected at 489/521nm, 532/568nm and 592/622nm respectively.
本发明还提供一种多色荧光检测试剂盒，试剂盒包括针对氯霉素、雌二醇激素和黄曲霉毒素M1的检测探针、包被有氯霉素、雌二醇激素和黄曲霉毒素M1包被抗原的黑色聚苯乙烯微孔板、BSA、氯霉素标准品、雌二醇激素标准品、黄曲霉毒素M1标准品、信号DNA和Mg 2+。 The invention also provides a multi-color fluorescence detection kit. The kit includes detection probes for chloramphenicol, estradiol hormone and aflatoxin M1, and is coated with chloramphenicol, estradiol hormone and aflatoxin. Black polystyrene microplate coated with M1 antigen, BSA, chloramphenicol standard, estradiol hormone standard, aflatoxin M1 standard, signal DNA and Mg 2+ .
在本发明的一种实施方式中，所述检测探针的制备方法为：取一定量的AuNPs，加入CAP、17β-E 2或AFM 1单克隆抗体，室温孵育，形成三种AuNPs-抗体分散液；巯基修饰的CAP、17β-E 2或AFM 1的引物DNA经TCEP活化后分别加入对应AuNPs-抗体分散液，混匀并冷冻，溶解后加入PEG20000和PBS，获得AuNPs-DNA-抗体分散液；加入BSA进行孵育，离心取上清获得检测探针。 In one embodiment of the invention, the preparation method of the detection probe is: take a certain amount of AuNPs, add CAP, 17β-E 2 or AFM 1 monoclonal antibody, and incubate at room temperature to form three AuNPs-antibody dispersions. liquid; the primer DNA of thiol-modified CAP, 17β-E 2 or AFM 1 was activated by TCEP and then added to the corresponding AuNPs-antibody dispersion, mixed and frozen. After dissolving, add PEG20000 and PBS to obtain the AuNPs-DNA-antibody dispersion. ;Add BSA for incubation, centrifuge and take the supernatant to obtain the detection probe.
在本发明的一种实施方式中，所述检测探针的制备方法为：In one embodiment of the invention, the preparation method of the detection probe is:
a)取3mL含有13nm AuNPs的分散液，调节pH至8.5-9，等分至3个离心管中，分别加入18μg CAP、17β-E 2或AFM 1单克隆抗体，室温孵育1h，形成三种含AuNPs-抗体的分散液； a) Take 3 mL of the dispersion containing 13nm AuNPs, adjust the pH to 8.5-9, divide it equally into 3 centrifuge tubes, add 18 μg of CAP, 17β-E 2 or AFM 1 monoclonal antibody respectively, and incubate at room temperature for 1 hour to form three Dispersions containing AuNPs-antibodies;
b)巯基修饰的CAP的引物DNA、17β-E 2的引物DNA和AFM 1的引物DNA经TCEP活化后(摩尔比1：100)分别加入对应AuNPs-抗体分散液，置于-20℃冷冻30min，溶解后加入30％PEG20000、0.1M PBS，持续混匀5min后，4℃条件下盐老化2h，形成含有 AuNPs-DNA-抗体的分散液； b) After thiol-modified CAP primer DNA, 17β-E 2 primer DNA and AFM 1 primer DNA are activated by TCEP (molar ratio 1:100), add the corresponding AuNPs-antibody dispersion and freeze at -20°C for 30 minutes. , after dissolving, add 30% PEG20000 and 0.1M PBS, continue mixing for 5 minutes, and then salt-age for 2 hours at 4°C to form a dispersion containing AuNPs-DNA-antibody;
c)加入10％BSA，室温孵育40min，13000rpm离心15min，最终合成的探针分散于200μL 0.01M PBS中(含1％PEG20000和1％BSA，pH＝7.4)，4℃避光保存，一周内使用完。c) Add 10% BSA, incubate at room temperature for 40 minutes, and centrifuge at 13000 rpm for 15 minutes. The final synthesized probe is dispersed in 200 μL 0.01M PBS (containing 1% PEG20000 and 1% BSA, pH=7.4), and stored in the dark at 4°C within one week. finish using.
在本发明的一种实施方式中，CAP的引物DNA的序列为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’。 In one embodiment of the present invention, the sequence of the primer DNA of CAP is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.
在本发明的一种实施方式中，17β-E 2的引物DNA的序列为：5’-HS-(T) 28GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3’。 In one embodiment of the present invention, the sequence of the primer DNA of 17β-E 2 is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'.
在本发明的一种实施方式中，AFM 1的引物DNA的序列为：5’-HS-(T) 28GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3’。 In one embodiment of the invention, the sequence of the primer DNA of AFM 1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.
在本发明的一种实施方式中，CAP的信号DNA的序列为：5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’。In one embodiment of the invention, the sequence of the signal DNA of CAP is: 5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’.
在本发明的一种实施方式中，17β-E 2的信号DNA的序列为：5’-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3’。 In one embodiment of the invention, the sequence of the signal DNA of 17β-E 2 is: 5'-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3'.
在本发明的一种实施方式中，AFM 1的信号DNA的序列为：5’-Texas red-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3’。 In one embodiment of the invention, the sequence of the signal DNA of AFM 1 is: 5'-Texas red-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3'.
有益效果：Beneficial effects:
1、本发明以金纳米颗粒为载体，合成AuNPs-DNA-抗体检测探针，抗体用于捕获目标物，引物DNA用于放大信号。在黑色96微孔板经抗原包板、封闭、洗涤后，加入AuNPs-DNA-抗体检测探针，包被抗原与待测样品竞争结合探针上的抗体；洗去多余探针及与目标物结合的探针，加入信号DNA及Mg 2+，与检测探针上的引物DNA完成信号放大步骤。 1. The present invention uses gold nanoparticles as carriers to synthesize AuNPs-DNA-antibody detection probes. The antibodies are used to capture target objects, and the primer DNA is used to amplify signals. After the black 96 microwell plate is coated with the antigen, 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; excess probe and target substances are washed away To the bound probe, signal DNA and Mg 2+ are added to complete the signal amplification step with the primer DNA on the detection probe.
2、本发明建立用于CAP/17β-E 2/AFM 1同步定量检测的DNAzyme辅助信号放大的荧光免疫分析方法，在最佳条件下，该方法实现了CAP、17β-E 2和AFM 1的同时检测，检测范围分别为0.333ng/mL-3.333μg/mL、3.333ng/mL-3.333μg/mL、3.333fg/mL-3.333ng/mL，定量限分别为0.333ng/mL、3.333ng/mL、3.333fg/mL。 2. The present invention establishes a fluorescence immunoassay method for DNAzyme-assisted signal amplification for simultaneous quantitative detection of CAP/17β-E 2 /AFM 1. Under optimal conditions, this method achieves the detection of CAP, 17β-E 2 and AFM 1 . Simultaneous detection, the detection ranges are 0.333ng/mL-3.333μg/mL, 3.333ng/mL-3.333μg/mL, 3.333fg/mL-3.333ng/mL, and the quantitation limits are 0.333ng/mL, 3.333ng/mL respectively. ,3.333fg/mL.
3、与抗生素(KANA和SM)、两种激素(E 1和E 3)和两种毒素(OTA和ZEN)交叉反应率低，对氯霉素、17β-E 2和AFM 1具有高选择性。本发明检测准确度、灵敏度和稳定性较高，相比现有技术，本发明建立的方法在检测过程中，无需添加额外的核酸外切酶，降低反应成本。 3. Low cross-reaction rate with antibiotics (KANA and SM), two hormones (E 1 and E 3 ) and two toxins (OTA and ZEN), and high selectivity for chloramphenicol, 17β-E 2 and AFM 1 . The detection accuracy, sensitivity and stability of the present invention are higher. Compared with the existing technology, the method established by the present invention does not need to add additional exonuclease during the detection process, thus reducing the reaction cost.
附图说明Description of the drawings
图1本发明原理示意图。Figure 1 is a schematic diagram of the principle of the present invention.
图2为不同荧光素对实验结果的影响图；a)分别以6-FAM、Cy3、Texas red为探针荧光信号的检测效果图；b)分别以6-FAM、TAMRA、Cy5为探针荧光信号的检测效果图；c)在6-FAM、Cy3、Texas red荧光探针扩增下的检测效果图；d)在6-FAM、TAMRA、Cy5荧光探针扩增下的检测效果图。Figure 2 shows the influence of different fluoresceins on the experimental results; a) The detection effect of fluorescence signals using 6-FAM, Cy3, and Texas red as probes respectively; b) Using 6-FAM, TAMRA, and Cy5 as probe fluorescence respectively Signal detection effect diagram; c) Detection effect diagram under the amplification of 6-FAM, Cy3, Texas red fluorescent probe; d) Detection effect diagram under the amplification of 6-FAM, TAMRA, Cy5 fluorescent probe.
图3为方案可行性验证；a)检测CAP；b)检测E 2；c)检测AFM 1；d)不用CAP浓度下的荧光强度变化；e)不同E 2浓度下的荧光强度变化；f不同AFM 1浓度下的荧光强度变化)。 Figure 3 shows the feasibility verification of the scheme; a) detecting CAP; b) detecting E 2 ; c) detecting AFM 1 ; d) changes in fluorescence intensity without using CAP concentration; e) changes in fluorescence intensity under different E 2 concentrations; f is different Fluorescence intensity change at AFM 1 concentration).
图4为包被抗原浓度和检测探针的浓度优化图；a)CAP包被抗原浓度和检测探针的浓度优化；b)17β-E 2包被抗原浓度和检测探针的浓度优化；c)AFM 1包被抗原浓度和检测探针的浓度优化。 Figure 4 is a diagram showing the optimization of the concentration of coated antigen and the concentration of the detection probe; a) the optimization of the concentration of the CAP coated antigen and the concentration of the detection probe; b) the optimization of the concentration of the 17β-E 2 coating antigen and the concentration of the detection probe; c )AFM 1 coating antigen concentration and detection probe concentration optimization.
图5为信号DNA和Mg 2+的浓度优化图a)CAP信号DNA和Mg 2+的浓度优化；b)17β-E 2信号DNA和Mg 2+的浓度优化；c)AFM 1信号DNA和Mg 2+的浓度优化。 Figure 5 shows the concentration optimization diagram of signal DNA and Mg 2+ a) The concentration optimization of CAP signal DNA and Mg 2+ ; b) The concentration optimization of 17β-E 2 signal DNA and Mg 2+ ; c) AFM 1 signal DNA and Mg 2+ concentration optimization.
图6为检测线性关系标准曲线图；a)CAP标准曲线图；b)17β-E 2标准曲线图；c)AFM 1标准曲线图。 Figure 6 shows the standard curve for detecting linear relationships; a) CAP standard curve; b) 17β-E 2 standard curve; c) AFM 1 standard curve.
图7为特异性和稳定性研究图；a)方法对不同抗生素、激素及毒素的检测效果图b)不同时间下检测方法的效果图。Figure 7 shows the specificity and stability study diagram; a) the detection effect diagram of the method for different antibiotics, hormones and toxins; b) the detection effect diagram of the detection method at different times.
具体实施方式Detailed ways
试剂和材料：CAP的标准品购自振翔科技有限公司(中国北京)。柠檬酸三钠，吐温20，牛白蛋白(BSA)、MgCl 2和聚乙二醇20000(PEG 20000)购自国药集团化学试剂有限公司(中国上海)。TCEP购自碧云天有限公司(中国上海)。单克隆抗体购自Gene Tex(美国)。CAP-BSA购自奥斯特科技有限责任公司(中国南宁)。 Reagents and materials: CAP standards were purchased from Zhenxiang Technology Co., Ltd. (Beijing, China). Trisodium citrate, Tween 20, bovine albumin (BSA), MgCl and polyethylene glycol 20000 (PEG 20000) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). TCEP was purchased from Biyuntian Co., Ltd. (Shanghai, China). Monoclonal antibodies were purchased from Gene Tex (USA). CAP-BSA was purchased from Oster Technology Co., Ltd. (Nanning, China).
包被缓冲液(0.06M CBS，pH 9.6)和5×反应缓冲液(0.1M Tris-HCl,0.75M NaCl,pH 8.3)。0.1M PBS(1.35M NaCl,0.047M KCl,0.1M Na 2HPO 4,0.02M NaH 2PO 4)。所有寡核苷酸均由生工(中国上海)合成，修饰和纯化。 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 Na 2 HPO 4 , 0.02M NaH 2 PO 4 ). All oligonucleotides were synthesized, modified and purified by Sangon (Shanghai, China).
仪器：Tecnai G2 20透射电子显微镜(TEM)(美国)；H1M1酶联免疫分析仪(广州达瑞生物技术有限公司)；T9-UV-vis分光光度计(北京普析通用仪器有限公司)。震荡孵育机(美国，赛默飞)。Instrument: Tecnai G2 20 transmission electron microscope (TEM) (USA); H1M1 enzyme-linked immunoassay analyzer (Guangzhou Darui Biotechnology Co., Ltd.); T9-UV-vis spectrophotometer (Beijing Puxi General Instrument Co., Ltd.). Shaking incubator (Thermo Fisher, USA).
实施例1合成检测探针Example 1 Synthesis of detection probes
(1)AuNP的制备：(1) Preparation of AuNPs:
通过柠檬酸盐还原法制备AuNP。在磁力搅拌器上将100mL的0.01％HAuCl 4加热至沸 腾。随后在剧烈搅拌下将2.5mL新鲜制备的1％柠檬酸三钠加入沸腾溶液中。将混合物连续煮沸约15分钟，直到颜色变成橘红色。然后在缓慢搅拌下将溶液冷却至环境温度，补充至100mL分散液后，在4℃下保存。 AuNPs were prepared by citrate reduction method. Heat 100 mL of 0.01% HAuCl to boiling on a magnetic stirrer. Then 2.5 mL of freshly prepared 1% trisodium citrate was added to the boiling solution with vigorous stirring. Boil the mixture continuously for about 15 minutes, until the color turns orange-red. The solution was then cooled to ambient temperature with slow stirring, replenished to 100 mL of dispersion, and stored at 4°C.
(2)合成AuNPs-DNA-抗体检测探针：(2) Synthesis of AuNPs-DNA-antibody detection probe:
1)取1mL AuNPs分散液(pH 8.5-9.0)分别与18μg CAP/17β-E 2/AFM 1单克隆抗体在室温下温和搅拌并孵育1h，获得混合液AuNPs-CAP、AuNPs-17β-E 2和AuNPs-AFM 1。 1) Take 1 mL of AuNPs dispersion (pH 8.5-9.0) and 18 μg of CAP/17β-E 2 /AFM 1 monoclonal antibody, stir gently at room temperature and incubate for 1 hour to obtain a mixture of AuNPs-CAP and AuNPs-17β-E 2 and AuNPs-AFM 1 .
2)利用TCEP分别活化巯基修饰的针对CAP、17β-E 2、AFM 1的引物DNA。 2) Use TCEP to activate the thiol-modified primer DNA targeting CAP, 17β-E 2 , and AFM 1 respectively.
3)将步骤2)中活化的针对CAP、17β-E 2、AFM 1的引物DNA加入至步骤1)中对应的混合液中，引物DNA的添加量为1OD，-20℃静置0.5h。待溶解后，再向混合液中加入30％PEG 20000(终浓度为1％)和0.1M PBS(终浓度为0.01M)混合5分钟，并在4℃下进行盐老化2h。最后，加入10％BSA，室温孵育40min，13000rpm离心15min，最终合成的探针分散于200μL 0.01M PBS中(含1％PEG20000和1％BSA，pH＝7.4)。 3) Add the primer DNA activated in step 2) for CAP, 17β-E 2 and AFM 1 into the corresponding mixture in step 1). The amount of primer DNA added is 1OD and let stand at -20°C for 0.5h. After dissolving, add 30% PEG 20000 (final concentration: 1%) and 0.1M PBS (final concentration: 0.01M) to the mixture, mix for 5 minutes, and perform salt aging at 4°C for 2 hours. Finally, 10% BSA was added, incubated at room temperature for 40 min, centrifuged at 13000 rpm for 15 min, and the finally synthesized probe was dispersed in 200 μL of 0.01M PBS (containing 1% PEG20000 and 1% BSA, pH=7.4).
CAP的引物DNA1为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’。 The primer DNA1 of CAP is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.
17β-E 2的引物DNA2为：5’-HS-(T) 28GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3’。 Primer DNA2 of 17β-E 2 is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'.
AFM 1的引物DNA3为：5’-HS-(T) 28GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3’。 Primer DNA 3 of AFM 1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.
实施例2信号DNA的标记荧光素的选择Example 2 Selection of labeled fluorescein for signal DNA
(1)荧光信号检测(1) Fluorescence signal detection
将1.5μg/mL浓度的CAP、E 2、AFM 1标准液按照1:1:1的体积比混合制备混合样品溶液，将实施例1中制备的0.3μM浓度的CAP、17β-E 2、AFM 1的AuNPs-DNA-抗体也按照1:1:1的体积比混合制备AuNPs-抗体-DNA探针混合溶液。 Mix the standard solutions of CAP, E 2 and AFM 1 at a concentration of 1.5 μg/mL according to the volume ratio of 1:1:1 to prepare a mixed sample solution. Mix the CAP, 17β-E 2 and AFM at a concentration of 0.3 μM prepared in Example 1. The AuNPs-DNA-antibody of 1 was also mixed according to the volume ratio of 1:1:1 to prepare the AuNPs-antibody-DNA probe mixed solution.
将60μL浓度为0.9μg/mL的包被抗原CAP-BSA、E 2-BSA和AFM 1-BSA混合在一起，包被在黑色微孔板中，37℃孵育2h。用0.05％PBST洗涤3次后，加入250μL 2％BSA在37℃下封闭1h。然后加入90μL混合样品溶液和90μL AuNPs-抗体-DNA探针混合溶液在37℃下孵育1h进行免疫竞争反应。洗涤后，加入36μL Tris-HCl、18μL信号DNA和36μL MgCl 2，用水补足反应体系至180μL，并于37℃孵育1h。用荧光分光光度计测量荧光光谱曲线，同时用多功能酶标仪测量489/521nm、532/568nm和592/622nm处的荧光信号(图1)。 Mix 60 μL of coating antigens CAP-BSA, E 2 -BSA and AFM 1 -BSA with a concentration of 0.9 μg/mL together, coat them in a black microplate, and incubate at 37°C for 2 hours. After washing three times with 0.05% PBST, 250 μL of 2% BSA was added and blocked for 1 h at 37°C. Then add 90 μL mixed sample solution and 90 μL AuNPs-antibody-DNA probe mixed solution and incubate at 37°C for 1 h to perform immune competition reaction. After washing, add 36 μL Tris-HCl, 18 μL signal DNA and 36 μL MgCl 2 , make up the reaction system to 180 μL with water, and incubate at 37°C for 1 hour. Use a fluorescence spectrophotometer to measure the fluorescence spectrum curve, and use a multifunctional microplate reader to measure the fluorescence signals at 489/521nm, 532/568nm and 592/622nm (Figure 1).
(2)荧光标记素的选择(2) Selection of fluorescent markers
为了更好的区分CAP、17β-E 2、AFM 1三种目标污染物的检测信号，在针对CAP、17β-E 2、AFM 1的信号DNA的3’端连接不同的荧光素，并进一步检测不同的荧光素对于检测灵敏度的影响，因此选用6-FAM(1)、Cy3(2)、Texas red(3)和6-FAM(4)、TAMRA(5)、Cy5 (6)两种组合测试荧光素对实验结果的影响。如图2a-b所示，字母S代表信号DNA，I代表引物DNA，数字代表信号DNA上连接的荧光素序号(表1)，荧光仪测试结果显示无论使用何种荧光素，实施例1后的检测信号均比原始荧光信号即仅存在信号DNA时的信号放大了许多，且出峰位置清晰，相互之间无干扰。 In order to better distinguish the detection signals of the three target pollutants CAP, 17β-E 2 and AFM 1 , different fluorophores were connected to the 3' end of the signal DNA for CAP, 17β-E 2 and AFM 1 and further detected. The impact of different fluoresceins on detection sensitivity, so two combinations of 6-FAM(1), Cy3(2), Texas red(3) and 6-FAM(4), TAMRA(5), Cy5(6) were selected for testing Effect of fluorescein on experimental results. As shown in Figure 2a-b, the letter S represents signal DNA, I represents primer DNA, and the numbers represent the fluorophore serial number connected to the signal DNA (Table 1). The fluorometer test results show that no matter what kind of fluorophore is used, after Example 1 The detection signals are much amplified compared to the original fluorescence signal, that is, the signal when only signal DNA exists, and the peak positions are clear and there is no interference between each other.
利用酶标仪比较实际的扩增效果，如图2c-d，group1代表信号DNA1和引物DNA1以6-FAM(1)为荧光素的组合，group2以此类推(表1)，由图中可以看到，以6-FAM(4)、TAMRA(5)、Cy5(6)的扩增效果不如分别以6-FAM(1)、Cy3(2)、Texas red(3)为荧光素的检测探针。以TAMRA(5)为荧光素的检测探针信号只扩增了14倍((FS+I)/FS)，以Cy5(6)为荧光素的检测探针信号只扩增了20倍，明显不如以Cy3(38倍)或Texas red(37倍)为荧光信号的检测探针。因此选择6-FAM(1)、Cy3(2)、Texas red(3)作为三种标记荧光素。Use a microplate reader to compare the actual amplification effects, as shown in Figure 2c-d. Group1 represents the combination of signal DNA1 and primer DNA1 with 6-FAM(1) as the fluorescein, and so on for group2 (Table 1). From the figure, it can be It can be seen that the amplification effect of 6-FAM(4), TAMRA(5), and Cy5(6) is not as good as that of the detection probes using 6-FAM(1), Cy3(2), and Texas red(3) as fluorescein respectively. Needle. The signal of the detection probe using TAMRA(5) as the fluorescein only amplified 14 times ((FS+I)/FS), and the signal of the detection probe using Cy5(6) as the fluorescein only amplified 20 times, obviously It is better to use Cy3 (38 times) or Texas red (37 times) as the fluorescence signal detection probe. Therefore, 6-FAM(1), Cy3(2), and Texas red(3) were selected as the three labeled fluoresceins.
CAP的信号DNA1为：5’-荧光素-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’。The signal DNA1 of CAP is: 5’-fluorescein-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’.
17β-E 2的信号DNA2为：5’-荧光素-TAGCTTCAT/rA/GGACAATCA-BHQ2-3’。 The signal DNA2 of 17β-E 2 is: 5'-fluorescein-TAGCTTCAT/rA/GGACAATCA-BHQ2-3'.
AFM 1的信号DNA3为：5’-荧光素-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3’。 The signal DNA 3 of AFM 1 is: 5'-fluorescein-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3'.
表1Table 1
组别Group 代表序列representative sequence
S1S1 信号DNA1-6-FAM(1)SignalDNA1-6-FAM(1)
S2S2 信号DNA2-Cy3(2)SignalDNA2-Cy3(2)
S3S3 信号DNA3-Texasred(3)SignalDNA3-Texasred(3)
S4S4 信号DNA1-6-FAM(4)SignalDNA1-6-FAM(4)
S5S5 信号DNA2-TAMRA(5)Signal DNA2-TAMRA(5)
S6S6 信号DNA3-Cy5(6)SignalDNA3-Cy5(6)
Group1Group1 信号DNA1-6-FAM(1)+引物DNA1+Mg 2+ Signal DNA1-6-FAM(1)+Primer DNA1+Mg 2+
Group2Group2 信号DNA2-Cy3(2)+引物DNA2+Mg 2+ Signal DNA2-Cy3(2)+Primer DNA2+Mg 2+
Group3Group3 信号DNA3-Texasred(3)+引物DNA3+Mg 2+ Signal DNA3-Texasred(3)+Primer DNA3+Mg 2+
Group4Group4 信号DNA1-6-FAM(4)+引物DNA1+Mg 2+ Signal DNA1-6-FAM(4)+Primer DNA1+Mg 2+
Group5Group5 信号DNA2-TAMRA(5)+引物DNA2+Mg 2+ Signal DNA2-TAMRA(5)+Primer DNA2+Mg 2+
Group6Group6 信号DNA3-Texasred(3)+引物DNA3+Mg 2+ Signal DNA3-Texasred(3)+Primer DNA3+Mg 2+
实施例3荧光检测可行性分析Example 3 Fluorescence Detection Feasibility Analysis
为验证三组序列的扩增效果，将10μL实施例1中的针对CAP、17β-E 2或AFM 1的引物DNA(0.3μM)、10μL针对CAP、17β-E 2或AFM 1的信号DNA(0.6μM)和20μLMgCl 2(50mM)在0.1M Tris-HCl(含0.75M NaCl，pH 8.3)中于37℃孵育1h，于489/521nm、532/568nm和592/622nm处分别测定荧光。 In order to verify the amplification effect of the three sets of sequences, 10 μL of the primer DNA (0.3 μM) for CAP, 17β-E 2 or AFM 1 in Example 1, and 10 μL of the signal DNA for CAP, 17β-E 2 or AFM 1 ( 0.6μM) and 20μL MgCl 2 (50mM) were incubated in 0.1M Tris-HCl (containing 0.75M NaCl, pH 8.3) for 1 hour at 37°C, and the fluorescence was measured at 489/521nm, 532/568nm and 592/622nm respectively.
结果如图3所示，在引物DNA、信号DNA和Mg 2+的同时存在的情况下，分别在489/521nm、532/568nm和592/622nm处获得了增强荧光，表明扩增反应仅在这种情况下可以进行。当缺少引物DNA或者辅助因子时，扩增反应不会发生，荧光信号较弱。另外，由图3a-c可知，引物DNA无法作用于错配的信号DNA，荧光信号无法获得。在其中一种引物DNA与所有信号DNA的反应中，荧光信号几乎不会受到干扰。 The results are shown in Figure 3. In the presence of primer DNA, signal DNA and Mg 2+ at the same time, enhanced fluorescence was obtained at 489/521nm, 532/568nm and 592/622nm respectively, indicating that the amplification reaction only occurs here. This can be done in this case. When there is a lack of primer DNA or cofactors, the amplification reaction will not occur and the fluorescence signal will be weak. In addition, it can be seen from Figure 3a-c that the primer DNA cannot act on the mismatched signal DNA, and the fluorescence signal cannot be obtained. In the reaction of one of the primer DNAs with all signal DNAs, there is little interference in the fluorescence signal.
进一步的，利用实施例1中的针对CAP、17β-E 2或AFM 1的AuNPs-抗体-DNA验证了方案的可行性。 Furthermore, the feasibility of the scheme was verified using the AuNPs-antibody-DNA directed against CAP, 17β-E 2 or AFM 1 in Example 1.
将1.5μg/mL浓度的CAP、E 2、AFM 1标准液按照1:1:1的体积比混合制备混合样品溶液，将实施例1中制备的0.3μM浓度的CAP、17β-E 2、AFM 1的AuNPs-DNA-抗体也按照1:1:1的体积比混合制备AuNPs-抗体-DNA探针混合溶液。 Mix the standard solutions of CAP, E 2 and AFM 1 at a concentration of 1.5 μg/mL according to the volume ratio of 1:1:1 to prepare a mixed sample solution. Mix the CAP, 17β-E 2 and AFM at a concentration of 0.3 μM prepared in Example 1. The AuNPs-DNA-antibody of 1 was also mixed according to the volume ratio of 1:1:1 to prepare the AuNPs-antibody-DNA probe mixed solution.
将60μL浓度为0.9μg/mL的包被抗原CAP-BSA、E 2-BSA和AFM 1-BSA混合在一起，包被在黑色微孔板中，37℃孵育2h。用0.05％PBST洗涤3次后，加入250μL 2％BSA在37℃下封闭1h。然后分别加入90μL浓度为0μg/mL、0.005μg/mL、0.05μg/mL、0.1μg/mL、0.5μg/mL、5μg/mL的混合样品溶液和90μL AuNPs-抗体-DNA探针混合溶液在37℃下孵育1h进行免疫竞争反应。洗涤后，加入36μL Tris-HCl、18μL信号DNA和36μL MgCl 2，用水补足反应体系至180μL，并于37℃孵育1h。用荧光分光光度计测量荧光光谱曲线，同时用多功能酶标仪测量489/521nm、532/568nm和592/622nm处的荧光信号。 Mix 60 μL of coating antigens CAP-BSA, E 2 -BSA and AFM 1 -BSA with a concentration of 0.9 μg/mL together, coat them in a black microplate, and incubate at 37°C for 2 hours. After washing three times with 0.05% PBST, 250 μL of 2% BSA was added and blocked for 1 h at 37°C. 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 solutions were added at 37 Incubate for 1 hour at ℃ for immune competition reaction. After washing, add 36 μL Tris-HCl, 18 μL signal DNA and 36 μL MgCl 2 , make up the reaction system to 180 μL with water, and incubate at 37°C for 1 hour. Use a fluorescence spectrophotometer to measure the fluorescence spectrum curve, and use a multifunctional microplate reader to measure the fluorescence signals at 489/521nm, 532/568nm and 592/622nm.
如图3d-f分别表示该方法对CAP、E2和AFM1的检测效果，随着三个靶标浓度的增加，荧光强度逐渐降低，表明该方案可行。Figure 3d-f show the detection effect of this method on CAP, E2 and AFM1 respectively. As the concentration of the three targets increases, the fluorescence intensity gradually decreases, indicating that this solution is feasible.
实施例4条件优化Example 4 Condition Optimization
(1)探究包被抗原浓度和检测探针的浓度：(1) Explore the concentration of coated antigen and detection probe:
参照实施例2(1)同时检测CAP、17β-E 2和AFM 1三种目标污染物含量的方法，用棋盘法优化对不同的抗原包被浓度(0.6、0.9、1.2、1.5μg/mL)和AuNPs-抗体-DNA的浓度(1/80、1/60)进行优化，并以抑制率作为评价指标。如图4所示，当探针稀释度为1/60、1/60、1/60，抗原包被浓度为0.9、0.9和1.5μg/mL时，抑制率(1-F1/F0,1-F1/F0,1-F0.05/F0)达到最高， 分别为82.439％、62.052％和68.222％。 Referring to the method of simultaneously detecting the contents of three target contaminants of CAP, 17β-E 2 and AFM 1 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) were optimized, and the inhibition rate was used as the evaluation index. As shown in Figure 4, when the probe dilutions are 1/60, 1/60, and 1/60, and the antigen coating concentrations are 0.9, 0.9, and 1.5 μg/mL, the inhibition rate (1-F1/F0,1- F1/F0, 1-F0.05/F0) reached the highest, which were 82.439%, 62.052% and 68.222% respectively.
(2)探究信号DNA和Mg 2+的浓度： (2) Explore the concentrations of signal DNA and Mg 2+ :
信号DNA和Mg 2+的浓度是DNAzyme介导的等温核酸扩增的关键条件。参照实施例2(1)同时检测CAP、17β-E 2和AFM 1三种目标污染物含量的方法，设置信号DNA浓度(S1：0.005、0.01、0.02、0.03、0.04μM；S2：0.025、0.05、0.1、0.15、0.2μM；S3：0.05、0.1、0.2、0.3、0.4μM)和Mg 2+(3、5、8、10、13、15、18mM)进行分析，如图5所示。当信号DNA和Mg 2+的浓度分别为0.01μM、0.025μM、0.1μM和10mM、10mM、10mM时，抑制率最高，分别为49.600％、65.419％和46.472％。最终，选择1/60稀释AuNPs-抗体-DNA、0.9μg/mL抗原、10mM Mg 2+和0.01μM信号DNA用于CAP检测，1/60稀释AuNPs-antibody-DNA、0.9μg/mL抗原、10mM Mg 2+和0.025μM信号DNA用于E 2检测、1/60稀释AuNPs-抗体-DNA、1.5μg/mL抗原、10mM Mg 2+和0.1μM信号DNA用于AFM 1检测。 The concentrations of signal DNA and Mg 2+ are critical conditions for DNAzyme-mediated isothermal nucleic acid amplification. Referring to the method of simultaneously detecting the contents of three target contaminants of CAP, 17β-E 2 and AFM 1 in Example 2 (1), set 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, 0.2μM; S3: 0.05, 0.1, 0.2, 0.3, 0.4μM) and Mg 2+ (3, 5, 8, 10, 13, 15, 18mM) were analyzed, as shown in Figure 5. When the concentrations of signal DNA and Mg 2+ were 0.01μM, 0.025μM, 0.1μM and 10mM, 10mM and 10mM respectively, the inhibition rates were the highest, which were 49.600%, 65.419% and 46.472% respectively. Finally, 1/60 dilution of AuNPs-antibody-DNA, 0.9μg/mL antigen, 10mM Mg 2+ and 0.01μM signal DNA was selected for CAP detection, and 1/60 dilution of AuNPs-antibody-DNA, 0.9μg/mL antigen, 10mM Mg 2+ and 0.025 μM signal DNA were used for E 2 detection, 1/60 diluted AuNPs-antibody-DNA, 1.5 μg/mL antigen, 10 mM Mg 2+ and 0.1 μM signal DNA were used for AFM 1 detection.
实施例5方法学验证Example 5 Methodology Verification
为保证该多靶标策略的实用性，对该方法的线性、检出限(LOD)进行了验证。To ensure the practicability of this multi-target strategy, the linearity and limit of detection (LOD) of the method were verified.
参照实施例1合成检测探针，将1.5μg/mL浓度的CAP、E 2、AFM 1标准液按照1:1:1的体积比混合制备混合样品溶液，选择1/60稀释AuNPs-抗体-DNA、0.9μg/mL CAP抗原、10mM Mg 2+和0.01μM信号DNA1用于CAP检测，1/60稀释AuNPs-antibody-DNA、0.9μg/mL E 2抗原、10mM Mg 2+和0.025μM信号DNA2用于E 2检测、1/60稀释AuNPs-抗体-DNA、1.5μg/mL AFM 1抗原、10mM Mg 2+和0.01μM信号DNA3用于AFM 1检测。改变混合样品的浓度，测定至少20个空白样品，记录数值，求出平均值(x)和标准差(SD)，求出x+3SD或x-3SD将其带入曲线方程，得到检出限。 Refer to Example 1 to synthesize the detection probe, mix the CAP, E 2 and AFM 1 standard solutions with a concentration of 1.5 μg/mL according to the volume ratio of 1:1:1 to prepare a mixed sample solution, and select 1/60 to dilute the AuNPs-antibody-DNA , 0.9μg/mL CAP antigen, 10mM Mg 2+ and 0.01μM signal DNA1 are used for CAP detection, 1/60 diluted AuNPs-antibody-DNA, 0.9μg/mL E 2 antigen, 10mM Mg 2+ and 0.025μM signal DNA2 are used For E 2 detection, 1/60 dilution of AuNPs-antibody-DNA, 1.5 μg/mL AFM 1 antigen, 10 mM Mg 2+ and 0.01 μM signal DNA 3 was used for AFM 1 detection. Change the concentration of the mixed sample, measure at least 20 blank samples, record the values, find the mean (x) and standard deviation (SD), find x+3SD or x-3SD and bring it into the curve equation to get the detection limit .
结果如图6所示，随着目标物浓度的增加，对应的荧光强度下降。这一趋势可以解释为：当靶标和检测探针同时加入孔中时，靶标和抗原将与AuNPs-抗体-DNA上的抗体竞争。随着靶分子浓度的增加，与抗原结合的探针减少，荧光强度下降。CAP、E 2、AFM 1的检测范围分别在0.333ng/mL-3.333μg/mL、3.333ng/mL-3.333μg/mL、3.333fg/mL-3.333ng/mL。标准曲线显示出良好的线性(CAP，y＝-436.96x+657.13，R 2＝0.96；E 2，y＝-33.27x+109.23，R 2＝0.94；AFM 1，y＝-41.57x-27.82，R 2＝0.94)。定量限分别为0.333ng/mL、3.333ng/mL、3.333fg/mL。 The results are shown in Figure 6. As the target concentration increases, the corresponding fluorescence intensity decreases. This trend can be explained by the fact that when the target and detection probe are added to the well simultaneously, the target and antigen will compete with the antibodies 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, E 2 and AFM 1 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; E 2 , y=-33.27x+109.23, R 2 =0.94; AFM 1 , y=-41.57x-27.82, R 2 =0.94). The limits of quantification were 0.333ng/mL, 3.333ng/mL, and 3.333fg/mL respectively.
实施例6特异性研究Example 6 Specificity Study
分别选择两种抗生素：卡那霉素(KANA)和链霉素(SM)、两种激素：雌酮(E1)和雌三醇(E3)和两种毒素：玉米赤霉烯酮(ZEN)和赭曲霉毒素A(OTA)进行特异性研究， 将以KANA+E1+OTA为一组、SM+E3+ZEN为一组将其各自的标准溶液配成混合样品溶液，每个样品设3个重复，应用建立的方法进行检测，对结果进行统计、分析和评价。结果如图7a所示，测得的交叉反应性较低。Choose two antibiotics: kanamycin (KANA) and streptomycin (SM), two hormones: estrone (E1) and estriol (E3), and two toxins: zearalenone (ZEN). To conduct specificity research with Ochratoxin A (OTA), KANA+E1+OTA will be used as a group, and SM+E3+ZEN will be used as a group to prepare their respective standard solutions to form a mixed sample solution. Each sample will have 3 Repeat, apply the established method for testing, and perform statistics, analysis and evaluation of the results. The results are shown in Figure 7a, with lower cross-reactivity measured.
实施例7稳定性研究Example 7 Stability Study
研究了一个月的时间内探针的稳定性。将探针置于4℃保存，分别于1、15和30天进行检测。以抑制率作为评价指标(CAP，1-F 10/F 0.001；E 2，1-F 10/F 0.01；AFM 1，1-F 0.01/F 0.00001)。如图7b所示，探针存放1个月后仍显示出良好的检测效果。 The stability of the probe was studied over a period of one month. The probes were stored at 4°C and tested on 1, 15 and 30 days respectively. The inhibition rate was used as the evaluation index (CAP, 1-F 10 /F 0.001 ; E 2 , 1-F 10 /F 0.01 ; AFM 1 , 1-F 0.01 /F 0.00001 ). As shown in Figure 7b, the probe still showed good detection results after being stored for 1 month.
实施例8实际样品的测定Example 8 Measurement of actual samples
回收率测试是将三种目标物的标准溶液以不同浓度水平一式三份加标到牛奶样品中进行的(CAP为0.005和0.01μg/mL，E 2为0.05和0.1μg/mL，AFM 1为0.00005和0.005μg/mL)，结果如表2所示。平均回收率范围为93％至115％，RSD范围为5％至10％。结果表明，本发明提供的检测方法具有良好的准确性。 The recovery test was performed by spiking standard solutions of the three target compounds into milk samples in triplicate at different concentration levels (0.005 and 0.01 μg/mL for CAP, 0.05 and 0.1 μg/mL for E 2 , and 0.05 and 0.1 μg/mL for AFM 1 ). 0.00005 and 0.005μg/mL), the results are shown in Table 2. Average recoveries range from 93% to 115%, with RSDs ranging from 5% to 10%. The results show that the detection method provided by the invention has good accuracy.
表2回收率验证结果Table 2 Recovery rate verification results
Figure PCTCN2022092166-appb-000001
Figure PCTCN2022092166-appb-000001
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed above in terms of preferred embodiments, they are 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 protection scope of the present invention should be defined by the claims.

Claims (13)

  1. 一种同时检测多种污染物的方法，其特征在于，所述方法的具体步骤为：A method for detecting multiple pollutants simultaneously, characterized in that the specific steps of the method are:
    (1)合成检测探针：在AuNPs表面修饰氯霉素、雌二醇激素或黄曲霉毒素M1的引物DNA和相应的特异性抗体，分别得到针对氯霉素、雌二醇激素、黄曲霉毒素M1的三种检测探针；(1) Synthetic detection probe: Modify the primer DNA of chloramphenicol, estradiol hormone or aflatoxin M1 on the surface of AuNPs and the corresponding specific antibodies to obtain chloramphenicol, estradiol hormone and aflatoxin respectively. M1’s three detection probes;
    (2)将氯霉素、雌二醇激素、黄曲霉毒素M1相应抗原进行混合并添加至黑色聚苯乙烯微孔板，BSA封闭；(2) Mix chloramphenicol, estradiol hormone, and aflatoxin M1 corresponding antigen and add them to a black polystyrene microplate, and seal with BSA;
    (3)将步骤(1)制备的氯霉素、雌二醇激素、黄曲霉毒素M1的检测探针分别稀释后与待测样品混合后加入至步骤(2)中的黑色聚苯乙烯微孔板，孵育并清洗黑色聚苯乙烯微孔板；(3) Dilute the detection probes for chloramphenicol, estradiol hormone, and aflatoxin M1 prepared in step (1) respectively, mix them with the sample to be tested, and then add them to the black polystyrene micropores in step (2). plate, incubate and wash black polystyrene microplates;
    (4)将针对氯霉素、雌二醇激素、黄曲霉毒素M1的信号DNA和Mg 2+分别加入至步骤(3)中的黑色聚苯乙烯微孔板，孵育并检测荧光强度信号。 (4) Add the signal DNA and Mg 2+ for chloramphenicol, estradiol hormone, and aflatoxin M1 respectively to the black polystyrene microplate in step (3), incubate and detect the fluorescence intensity signal.
  2. 根据权利要求1所述的方法，其特征在于，氯霉素的引物DNA的序列为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’；雌二醇激素的引物DNA的序列为：5’-HS-(T) 28GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3’；黄曲霉毒素M1的引物DNA的序列为：5’-HS-(T) 28GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3’。 The method according to claim 1, characterized in that the sequence of the primer DNA of chloramphenicol is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'; the sequence of the primer DNA of estradiol hormone is: 5' -HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'; the sequence of the primer DNA of aflatoxin M1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.
  3. 根据权利要求1所述的方法，其特征在于，氯霉素的信号DNA的序列为：5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’，雌二醇激素的信号DNA的序列为：5’-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3’，黄曲霉毒素M1的信号DNA的序列为：5’-Texas red-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3’。The method according to claim 1, wherein the sequence of the signal DNA of chloramphenicol is: 5'-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3', and the sequence of the signal DNA of the estradiol hormone is: 5'-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3', the signal DNA sequence of aflatoxin M1 is: 5'-Texas red-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3'.
  4. 根据权利要求1所述的方法，其特征在于，步骤(2)中，将氯霉素、雌二醇激素、黄曲霉毒素M1相应抗原进行混合并添加至黑色聚苯乙烯微孔板，35～38℃下孵育1.5～2.5h，加入220μL 2％BSA在35～38℃下封闭0.5～1.5h。The method according to claim 1, characterized in that, in step (2), chloramphenicol, estradiol hormone, and aflatoxin M1 corresponding antigen are mixed and added to a black polystyrene microplate, 35 to Incubate at 38℃ for 1.5~2.5h, add 220μL 2% BSA and block at 35~38℃ for 0.5~1.5h.
  5. 根据权利要求1或4所述的方法，其特征在于，步骤(2)中，氯霉素、雌二醇激素、黄曲霉毒素M1相应抗原的添加浓度为0.6～1.5μg/mL。The method according to claim 1 or 4, characterized in that in step (2), the added concentration of chloramphenicol, estradiol hormone, and aflatoxin M1 corresponding antigen is 0.6 to 1.5 μg/mL.
  6. 根据权利要求1所述的方法，其特征在于，步骤(3)中，氯霉素、雌二醇激素、黄曲霉毒素M1的检测探针的稀释倍数为1/80-1/60。The method according to claim 1, characterized in that in step (3), the dilution factor of the detection probes for chloramphenicol, estradiol hormone, and aflatoxin M1 is 1/80-1/60.
  7. 根据权利要求1所述的方法，其特征在于，步骤(3)中，孵育的条件为在35～38℃下孵育0.5～1.5h。The method according to claim 1, characterized in that in step (3), the incubation condition is incubation at 35-38°C for 0.5-1.5 hours.
  8. 根据权利要求1所述的方法，其特征在于，步骤(4)中，氯霉素、雌二醇激素、黄曲霉毒素M1的信号DNA的添加浓度为0.01～0.1μg/mL，Mg 2+的浓度为5～15mM。 The method according to claim 1, characterized in that in step (4), the added concentration of signal DNA of chloramphenicol, estradiol hormone, and aflatoxin M1 is 0.01 to 0.1 μg/mL, and the concentration of Mg 2+ The concentration is 5~15mM.
  9. 一种多色荧光检测试剂盒，其特征在于，所述试剂盒包括针对氯霉素、雌二醇激素和黄曲霉毒素M1的检测探针、包被有氯霉素、雌二醇激素和黄曲霉毒素M1包被抗原的黑色聚苯乙烯微孔板、BSA、氯霉素标准品、雌二醇激素标准品、黄曲霉毒素M1标准品、信号DNA和Mg 2+。 A multi-color fluorescence detection kit, characterized in that the kit includes detection probes for chloramphenicol, estradiol hormone and aflatoxin M1, coated with chloramphenicol, estradiol hormone and aflatoxin M1. Black polystyrene microplate coated with aspergillus toxin M1 antigen, BSA, chloramphenicol standard, estradiol hormone standard, aflatoxin M1 standard, signal DNA and Mg 2+ .
  10. 根据权利要求9所述的多色荧光检测试剂盒，其特征在于，所述检测探针的制备方法为：取一定量的AuNPs，加入氯霉素、雌二醇激素或黄曲霉毒素M1单克隆抗体，室温孵育，形成三种AuNPs-抗体分散液；巯基修饰的氯霉素、雌二醇激素或黄曲霉毒素M1的引物DNA经TCEP活化后分别加入对应AuNPs-抗体分散液，混匀并冷冻，溶解后加入PEG20000和PBS，获得AuNPs-DNA-抗体分散液；加入BSA进行孵育，离心取上清获得检测探针。The multi-color fluorescence detection kit according to claim 9, characterized in that the preparation method of the detection probe is: taking a certain amount of AuNPs, adding chloramphenicol, estradiol hormone or aflatoxin M1 monoclonal Antibodies were incubated at room temperature to form three AuNPs-antibody dispersions; the primer DNA of thiol-modified chloramphenicol, estradiol hormone or aflatoxin M1 was activated by TCEP and added to the corresponding AuNPs-antibody dispersions respectively, mixed and frozen , after dissolving, add PEG20000 and PBS to obtain the AuNPs-DNA-antibody dispersion; add BSA for incubation, and centrifuge to take the supernatant to obtain the detection probe.
  11. 根据权利要求10所述的多色荧光检测试剂盒，其特征在于，所述检测探针的制备方法为：The multi-color fluorescence detection kit according to claim 10, wherein the preparation method of the detection probe is:
    a)取3mL含有13nm AuNPs的分散液，调节pH至8.5-9，等分至3个离心管中，分别加入18μg氯霉素、雌二醇激素和黄曲霉毒素M1单克隆抗体，室温孵育1h，形成三种含AuNPs-抗体的分散液；a) Take 3 mL of the dispersion containing 13 nm AuNPs, adjust the pH to 8.5-9, divide it equally into 3 centrifuge tubes, add 18 μg of chloramphenicol, estradiol hormone and aflatoxin M1 monoclonal antibody respectively, and incubate at room temperature for 1 hour , forming three dispersions containing AuNPs-antibodies;
    b)巯基修饰的氯霉素的引物DNA、雌二醇激素的引物DNA和黄曲霉毒素M1的引物DNA经TCEP活化后(摩尔比1：100)分别加入对应AuNPs-抗体分散液，置于-20℃冷冻30min，溶解后加入30％PEG20000、0.1M PBS，持续混匀5min后，4℃条件下盐老化2h，形成含有AuNPs-DNA-抗体的分散液；b) After thiol-modified chloramphenicol primer DNA, estradiol hormone primer DNA and aflatoxin M1 primer DNA were activated by TCEP (molar ratio 1:100), they were added to the corresponding AuNPs-antibody dispersion and placed - Freeze at 20°C for 30 minutes, add 30% PEG20000 and 0.1M PBS after dissolution, continue mixing for 5 minutes, and then salt-age for 2 hours at 4°C to form a dispersion containing AuNPs-DNA-antibody;
    c)加入10％BSA，室温孵育40min，13000rpm离心15min，最终合成的探针分散于200μL 0.01M PBS中(含1％PEG20000和1％BSA，pH＝7.4)，4℃避光保存，一周内使用完。c) Add 10% BSA, incubate at room temperature for 40 minutes, and centrifuge at 13000 rpm for 15 minutes. The final synthesized probe is dispersed in 200 μL 0.01M PBS (containing 1% PEG20000 and 1% BSA, pH=7.4), and stored in the dark at 4°C within one week. finish using.
  12. 根据权利要求9所述的多色荧光检测试剂盒，其特征在于，氯霉素的信号DNA的序列为：5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3’，雌二醇激素的信号DNA的序列为：5’-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3’，黄曲霉毒素M1的信号DNA的序列为：5’-Texas red-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3’。The multi-color fluorescence detection kit according to claim 9, characterized in that the sequence of the signal DNA of chloramphenicol is: 5'-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ1-3', and the signal of the estradiol hormone The sequence of DNA is: 5'-Cy3-TAGCTTCAT/rA/GGACAATCA-BHQ2-3', and the sequence of signal DNA of aflatoxin M1 is: 5'-Texas red-CGTTCTAAT/rA/GGAGTAGCC-BHQ2-3'.
  13. 根据权利要求10或11所述的多色荧光检测试剂盒，其特征在于，氯霉素的引物DNA的序列为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’；雌二醇激素的引物DNA的序列为：5’-HS-(T) 28GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3’；黄曲霉毒素M1的引物DNA的序列为：5’-HS-(T) 28GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3’。 The multi-color fluorescence detection kit according to claim 10 or 11, characterized in that the sequence of the chloramphenicol primer DNA is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'; the primer of the estradiol hormone The DNA sequence is: 5'-HS-(T) 28 GATTGTCTCCGAGCCGGTCGAAATGAAGCTA-3'; the primer DNA sequence of aflatoxin M1 is: 5'-HS-(T) 28 GCTACTCTCCGAGCCGGTCGAAATTAGAACG-3'.
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