WO2023123806A1 - 一种基于DNAzyme辅助信号放大的荧光免疫分析方法 - Google Patents

一种基于DNAzyme辅助信号放大的荧光免疫分析方法 Download PDF

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WO2023123806A1
WO2023123806A1 PCT/CN2022/092153 CN2022092153W WO2023123806A1 WO 2023123806 A1 WO2023123806 A1 WO 2023123806A1 CN 2022092153 W CN2022092153 W CN 2022092153W WO 2023123806 A1 WO2023123806 A1 WO 2023123806A1
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dna
chloramphenicol
antibody
aunp
add
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郭亚辉
桑潘婷
沈维韦
陆华进
于航
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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6804—Nucleic acid analysis using immunogens
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

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  • the invention relates to a fluorescence immunoassay method based on DNAzyme auxiliary signal amplification, which belongs to the technical field of molecular biology detection.
  • Antibiotics are a class of small molecules that can interfere with the developmental functions of other cells. Antibiotics have been widely used in livestock farming in recent years to speed up growth, prevent disease and fight inflammation. However, due to the abuse of antibiotics, the current problem of bacterial resistance to antibiotics has become very serious and has become a threat to global health. Antibiotics may cause harm to the human body while killing pathogenic bacteria. Penicillin, for example, can cause allergic reactions and even have adverse effects on the nervous system. Therefore, monitoring the presence of antibiotic residues in food is essential to ensure food safety. To date, a variety of analytical assays have been developed for the detection of antibiotics, including instrumental assays and enzyme-linked immunoassays (ELISA). However, these methods may have disadvantages such as cumbersome pretreatment steps and expensive experimental equipment. Therefore, it is necessary to develop a simple and efficient method for antibiotic detection.
  • ELISA enzyme-linked immunoassays
  • AuNP-DNA-antibody As a highly sensitive detection probe, AuNP-DNA-antibody has been used in various detection fields. Among them, antibodies are used for immune competition targets, and Trigger DNA is used for nucleic acid amplification. As a large number of DNA sequences can be modified on a single AuNP, the amplification efficiency is significantly improved. At present, the probe has been used to construct various composite technologies, such as immune-PCR, immune-LAMP and so on. In addition, with the development of isothermal nucleic acid technology, various enzyme-free amplification techniques are gradually applied to the detection field. For example, in the presence of specific metal ions, such as Mg 2+ and Mn 2+ , DNAzymes can rapidly and efficiently catalyze and continuously cleave single RNA bonds in complementary DNA sequences.
  • specific metal ions such as Mg 2+ and Mn 2+
  • DNAzyme has excellent characteristics such as high catalytic efficiency and good specificity, it has been applied in various biomedicine and biosensors. Based on this, the indirect immunocompetitive method combined with DNAzyme-mediated nucleic acid amplification (DANA) combined with AuNP-DNA-antibody detection probes can provide an advanced strategy with more effective signal amplification for trace antibiotic detection.
  • DANA DNAzyme-mediated nucleic acid amplification
  • the first object of the present invention is to provide a probe for detecting chloramphenicol, the probe comprises sulfhydryl-modified Trigger DNA and chloramphenicol antibody, and the Trigger DNA and chloramphenicol antibody are linked to gold nanoparticles superior.
  • the sequence of the Trigger DNA is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.
  • a method for preparing a probe for detecting chloramphenicol comprising the following processes:
  • HAuCl 4 is dispersed in water to prepare a 0.01V% HAuCl 4 solution.
  • trisodium citrate is added as a 1 wt% aqueous solution.
  • the volume ratio of 1wt% trisodium citrate solution to 0.01V% HAuCl4 solution is 2.5mL: 100mL.
  • the mixing reaction time is 15-20min.
  • the obtained gold nanoparticles have a particle diameter of 13 nm, and are stored at 4° C. in the dark.
  • the pH in the step (2), can be adjusted to 8.5.
  • the incubation time is 1 h.
  • the sequence of the thiol-modified Trigger DNA is: 5'-HS-(T) 28 TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3'.
  • the activation refers to activating the sulfhydryl-modified DNA by using TCEP.
  • the molar ratio of TCEP to sulfhydryl-modified DNA is 100:1.
  • 20 N ⁇ g of chloramphenicol monoclonal antibody is added to each N mL of gold nanoparticle dispersion, prepared into an AuNP-antibody dispersion, and then N OD Trigger DNA is added.
  • the freezing reaction includes: mixing the activated Trigger DNA and the AuNP-antibody, and then standing at -20°C for 1 hour.
  • PEG20000 is added as a 30% aqueous solution; the final concentration of PEG20000 in the solution is 1%.
  • step (3) in the step (3), add 0.1M PBS; add until the concentration of PBS in the solution is 0.01M.
  • the reaction includes: first mixing and reacting for 5 minutes, and then aging with salt at 4° C. for 2 hours.
  • the concentration of BSA is 10wt%; it is added until the final concentration of BSA in the solution is 1%.
  • the incubation temperature is room temperature and the incubation time is 40 minutes.
  • the solid-liquid separation is by centrifugation at 13000 rpm for 10-20 min.
  • the preparation method specifically includes the following steps:
  • Trigger DNA was activated by TCEP (molar ratio 1:100), it was frozen at -20°C for 1 hour, dissolved and then added with 30% PEG20000, 0.1M PBS, and mixed for 5 minutes. After aging for 2 hours, a dispersion liquid containing AuNP-DNA-antibody was formed.
  • the present invention also provides a kind of method of detecting chloramphenicol content, described method comprises following process:
  • the 96-well black microplate is coated with chloramphenicol-coated antigen (CAP-BSA), and after BSA is blocked, the 96-well black microplate with chloramphenicol-coated antigen (CAP-BSA) is obtained;
  • the needle is diluted with a buffer to configure the probe diluent; then the sample to be tested and the probe diluent are mixed and added to the 96 wells of the CAP-BSA coated plate, and incubated for competition reaction (at this time, the CAP sample and the coated antigen Competing with the probe binding), after the end, wash the 96-well black microwell plate, after washing away the unbound probe and the probe bound to the sample, the probe bound to the coated antigen on the remaining microplate plate; then Add signal DNA and Mg 2+ , mix and react to form a detection sample solution; detect the fluorescence intensity signal at 489/517nm.
  • the sequence of the signal DNA is: 5'-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ 1 -3'.
  • the probe diluent refers to resuspending the probe prepared by using the above-mentioned AuNP dispersion solution per 1 mL in 200 ⁇ L of buffer.
  • the dilution is performed with 60-fold buffer solution, and then used for detection.
  • the buffer is 0.01M PBS, 1% PEG 20000, 1% BSA.
  • the concentration of the signal DNA in the detection sample solution is 0.2 ⁇ M.
  • the concentration of Mg 2+ in the detection sample liquid is 75 mM.
  • the chloramphenicol-coated antigen is added to the antibody on the probe, and the concentration in the detection sample solution is 1.8 ⁇ g/mL.
  • the detection method specifically includes the following process:
  • the present invention also provides a test kit for detecting chloramphenicol, which includes the above-mentioned probe, 96 black microwell plates coated with chloramphenicol-coated antigens, BSA, chloramphenicol standard items, signal DNA and Mg 2+ .
  • the sequence of the signal DNA is 5'-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ 1 -3'.
  • the present invention uses gold nanoparticles as a carrier to synthesize AuNP-DNA-antibody detection probes, the antibody is used to capture the target, and the Trigger DNA is used to amplify the signal. After the black 96 microwell plate is coated with antigen, blocked, and washed, add AuNP-DNA-antibody detection probe, and the coated antigen competes with the sample to be tested for the antibody on the probe; wash away excess probe and target substance Add signal DNA and Mg 2+ to the bound probe, and complete the signal amplification step with the Trigger DNA on the detection probe (Fig. 1).
  • the present invention establishes an immunosorbent analysis method for DNAzyme-assisted signal amplification for the quantitative detection of chloramphenicol.
  • the detection limit of chloramphenicol is 0.1ng/L, and the linear range is 0.0000001-1 ⁇ g/mL, which is compatible with streptomycin, Gentamicin, kanamycin, etc. have low cross-reactivity rates and are highly selective for chloramphenicol.
  • the invention has high sensitivity and strong operability, and can be well applied in the field of antibiotic detection. Compared with the prior art, the method established by the present invention does not need to add additional exonuclease during the detection process, thereby reducing the reaction cost.
  • Fig. 1 schematic diagram of the principle of the present invention.
  • Figure 2 (a) TEM image of synthesized 13nm AuNP; (b) UV image of AuNP and AuNP-DNA-antibody.
  • Figure 3 is the feasibility verification of the scheme, (a) DANA effect verification; (b) DA-NAFIA fluorescence spectrum verification; (c) DA-NAFIA gel electrophoresis verification.
  • Figure 4 shows the condition optimization, (a) signal DNA concentration optimization; (b) Mg 2+ concentration optimization.
  • Figure 5 is the detection effect verification, (a) standard curve diagram, the concentration is set to 0, 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 ⁇ g/mL; (b) scheme selectivity verification, with streptomycin, Kanamycin and thiamphenicol were used as reference objects.
  • CAP standard was purchased from Zhenxiang Technology Co., Ltd. (Beijing, China). Trisodium citrate, Tween 20, bovine albumin (BSA), MgCl 2 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 5X 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).
  • Embodiment 1 synthetic detection probe
  • AuNPs were prepared by the citrate reduction method. Heat 100 mL of 0.01% HAuCl to boiling on a magnetic stirrer. 2.5 mL of freshly prepared 1% trisodium citrate was then added to the boiling solution with vigorous stirring. Boil the mixture continuously for about 15 minutes until it turns orange in color. The solution was then cooled to ambient temperature with slow stirring, replenished to 100 mL of dispersion, and stored at 4°C.
  • step 3 Add the Trigger DNA from step 2) to the mixture in step 1), the amount of Trigger DNA added is 1OD, and stand at -20°C for 1h. After dissolving, 30% PEG 20000 (final concentration: 1%) and 0.1M PBS (final concentration: 0.01M) were added to the mixture, mixed for 5 minutes, and aged at 4°C for 2 hours. Finally, 10% BSA was added to block the remaining active sites on the AuNP surface. To remove excess oligonucleotides, the solution was centrifuged at 13000 rpm and 4°C for 15 minutes to obtain AuNP-DNA-antibody detection probes. Probes were then resuspended in 200 ⁇ L of storage buffer (0.01M PBS, 1% PEG 20000, 1% BSA) and stored at 4°C.
  • the synthesized AuNP particle size is about 13nm, well dispersed, and no aggregation, the ultraviolet peak appears at 518nm, after connecting Trigger DNA and antibody, the ultraviolet light at 518nm The peak red shifted to 523nm, and the DNA peak appeared at 260nm, indicating the successful synthesis of AuNP-DNA-antibody detection probe.
  • the signal DNA, AuNP-DNA-antibody detection probe, 5 ⁇ reaction buffer and 75mM MgCl 2 were mixed and reacted for 1h to obtain the amplification product.
  • 10 ⁇ L of the amplification product was mixed with 2 ⁇ L of 6 ⁇ loading buffer (instant view TM red fluorescent DNA loading buffer, 6 ⁇ bromophenol blue), and added to the wells of 2% agarose gel.
  • 6 ⁇ loading buffer instant view TM red fluorescent DNA loading buffer, 6 ⁇ bromophenol blue
  • 1 ⁇ TBE was used as the electrophoresis buffer, and after electrophoresis at 90V for 2 hours, the bands were analyzed using a gel imaging system.
  • Example 3 (1) the concentration of the coating antigen CAP-BSA and AuNP-DNA-antibody detection probe was changed, as shown in Table 1, and the fluorescent signal was detected while other things were kept unchanged.
  • the concentration of the signal DNA was changed to 0.2, 0.4, 0.6, 0.8, 1.0 ⁇ M respectively, and the others were kept unchanged for detection.
  • the calculation formula of inhibition rate is: (fluorescence signal when chloramphenicol solution is 0 ⁇ g/mL-fluorescence signal when chloramphenicol solution is 1 ⁇ g/mL)/fluorescence signal when chloramphenicol solution is 0 ⁇ g/mL.
  • the concentration of Mg 2+ was changed to 25, 50, 75, 100, and 125 mM, respectively, and the others were kept unchanged for detection.
  • the concentration of coating antigen CAP-BSA is set to be 1.8 ⁇ g/mL
  • the dilution factor of AuNP-DNA-antibody detection probe is 60 times
  • the concentration of signal DNA is 0.2 ⁇ M
  • the concentration of Mg 2+ is 75mM.
  • a series of diluted CAP standard sample concentrations (0, 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 ⁇ g/mL) were detected. Fluorescence (FAM/BHQ1) was obtained at 489nm and 517nm.
  • Antibiotics that may appear in milk including streptomycin, kanamycin, and thiamphenicol, were selected as test samples to evaluate the specificity of the method.
  • the coating antigen CAP-BSA concentration was 1.8 ⁇ g/mL, and incubated at 37°C for 2h. After washing three times with 0.05% PBST, 250 ⁇ L of 2% BSA was added to block at 37° C. for 1 h.
  • Add 50 ⁇ L of CAP standard solutions with concentrations of 0 ⁇ g/mL, 0.001 ⁇ g/mL, 0.01 ⁇ g/mL, 0.1 ⁇ g/mL, and 1 ⁇ g/mL for the standard curve, and the samples to be tested add 50 ⁇ L of AuNP with a dilution factor of 60 times -The DNA-antibody detection probe was mixed with the CAP standard solution and the sample to be tested respectively, and incubated at 37° C. for 1 h to carry out the competition reaction. After washing, 0.2 ⁇ M signal DNA, 75 mM Mg 2+ and reaction buffer were added for reaction.
  • Embodiment 7 Accuracy
  • the coating antigen CAP-BSA concentration was 1.8 ⁇ g/mL, and incubated at 37°C for 2h. After washing three times with 0.05% PBST, 250 ⁇ L of 2% BSA was added to block at 37° C. for 1 h.
  • Add 50 ⁇ L of CAP standard solutions with concentrations of 0 ⁇ g/mL, 0.001 ⁇ g/mL, 0.01 ⁇ g/mL, 0.1 ⁇ g/mL, and 1 ⁇ g/mL for the standard curve, and the samples to be tested add 50 ⁇ L of AuNP with a dilution factor of 60 times -The DNA-antibody detection probe was mixed with the CAP standard solution and the sample to be tested respectively, and incubated at 37° C. for 1 h to carry out the competition reaction. After washing, 0.2 ⁇ M signal DNA, 75 mM Mg 2+ and reaction buffer were added for reaction.

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Abstract

本发明公开了一种基于DNAzyme辅助信号放大的荧光免疫分析方法，属于分子生物学检测技术领域。本发明以金纳米颗粒为载体，合成AuNP-DNA-抗体检测探针，抗体用于捕获目标物，Trigger DNA用于放大信号。本方法对氯霉素的检测限为0.1ng/L。

Description

一种基于DNAzyme辅助信号放大的荧光免疫分析方法 技术领域
本发明涉及一种基于DNAzyme辅助信号放大的荧光免疫分析方法，属于分子生物学检测技术领域。
背景技术
抗生素是一类可以干扰其它细胞发育功能的小分子物质。近年来，抗生素在畜牧业中被广泛使用，以加快生长速度、预防疾病和对抗炎症。然而,由于抗生素的滥用,当前细菌对抗生素产生耐药性的问题变得十分严重,已对全球健康构成威胁。抗生素在杀死病原菌的同时，可能会对人体造成伤害。例如，青霉素会引起过敏反应，甚至对神经系统产生不良影响。因此，监测食品中抗生素残留的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检测抗生素，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、需要昂贵的实验设备等缺点。因此，有必要开发一种简单高效的抗生素检测方法。
AuNP-DNA-抗体作为一种高灵敏的检测探针，已被用于各种检测领域。其中抗体用于免疫竞争靶标，Trigger DNA用于核酸扩增。由于可以在单个AuNP上修饰大量DNA序列，因此显着提高了扩增效率。目前，该探针已被用于构建各种复合技术，如免疫-PCR、免疫-LAMP等。此外，随着等温核酸技术的发展，各种无酶扩增技术也逐渐应用于检测领域。例如，在特定金属离子，如Mg 2+和Mn 2+的存在下，DNAzyme可以快速有效地催化并连续切割互补DNA序列中的单个RNA键。由于DNAzyme具有催化效率高、特异性好等优良特性，已被应用于各种生物医学和生物传感器中。基于此，结合AuNP-DNA-抗体检测探针将间接免疫竞争法与DNAzyme介导的核酸扩增(DANA)相结合，可以为痕量抗生素检测提供一种具有更有效信号放大效果的先进策略。
发明内容
[技术问题]
解决传统免疫法的低灵敏度问题，提供一种氯霉素高灵敏检测的便捷方法。
[技术方案]
本发明的第一个目的是提供一种检测氯霉素的探针，所述探针包含经巯基修饰的Trigger DNA和氯霉素抗体，所述Trigger DNA和氯霉素抗体连接在金纳米颗粒上。
在本发明的一种实施方式中，所述Trigger DNA的序列为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’。
一种制备检测氯霉素的探针的方法，所述方法包括如下过程：
(1)合成金纳米颗粒AuNP：将HAuCl 4溶液加热至沸腾，加入柠檬酸三钠，保持沸腾直至得到橘红色溶液体系，冷却至室温，即为金纳米颗粒分散液；
(2)调节金纳米颗粒分散液的pH至8.5-9.0，加入氯霉素单克隆抗体孵育，获得AuNP-抗体分散液；
(3)将活化后的巯基修饰的Trigger DNA加入到AuNP-抗体分散液中，混匀并冷冻，将溶液溶解后，加入PEG20000、PBS进行反应，获得AuNP-DNA-抗体分散液；
(4)在所得AuNP-DNA-抗体分散液中加入BSA进行孵育，孵育结束后，固液分离，得到探针。
在本发明的一种实施方式中，所述步骤(1)中，HAuCl 4分散在水中配制成0.01V％HAuCl 4溶液。
在本发明的一种实施方式中，所述步骤(1)中，柠檬酸三钠配制成1wt％的水溶液进行添加。
在本发明的一种实施方式中，所述步骤(1)中，1wt％的柠檬酸三钠溶液与0.01V％HAuCl 4溶液的体积比为2.5mL：100mL。
在本发明的一种实施方式中，所述步骤(1)中，混匀反应的时间为15-20min。
在本发明的一种实施方式中，所述步骤(1)中，所得金纳米颗粒的粒径为13nm，置于4℃避光保存。
在本发明的一种实施方式中，所述步骤(2)中，pH具体可调至8.5。
在本发明的一种实施方式中，所述步骤(2)中，孵育的时间为1h。
在本发明的一种实施方式中，所述有巯基修饰的Trigger DNA的序列为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’。
在本发明的一种实施方式中，所述步骤(3)中，活化是指利用TCEP活化有巯基修饰的DNA。其中，TCEP与巯基修饰的DNA的摩尔比为100：1。
在本发明的一种实施方式中，每N mL金纳米颗粒分散液中加入20Nμg氯霉素单克隆抗体，制备成AuNP-抗体分散液后再添加N OD的Trigger DNA。
在本发明的一种实施方式中，所述步骤(3)中，冷冻反应包括：将活化的Trigger DNA与AuNP-抗体混匀后，-20℃静置1h。
在本发明的一种实施方式中，所述步骤(3)中，PEG20000制成30％的水溶液进行添加；添加至使溶液中PEG 20000的终浓度为1％。
在本发明的一种实施方式中，所述步骤(3)中，加入0.1M的PBS；添加至使溶液中的PBS浓度为0.01M。
在本发明的一种实施方式中，所述步骤(3)中，反应包括：先混匀反应5min然后4℃盐老化2h。
在本发明的一种实施方式中，所述步骤(4)中，BSA的浓度为10wt％；添加至使溶液中BSA的终浓度为1％。
在本发明的一种实施方式中，所述步骤(4)中，孵育的温度为室温，时间为40min。
在本发明的一种实施方式中，所述步骤(4)中，固液分离是通过在13000rpm离心10-20min。
在本发明的一种实施方式中，将步骤(4)所得的探针分散于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。
在本发明的一种实施方式中，所述制备方法具体包括如下步骤：
(1)首先合成13nm的金纳米颗粒(AuNP)：取100mL 0.01％HAuCl 4加入250mL烧杯中，加热至沸腾；搅拌下快速加入2.5mL 1％的柠檬酸三钠；继续搅拌15-20min，直至颜色变为橘红色；温度调0，转速调低，冷却后，获得含有AuNP的分散液，4℃避光保存。
(2)取1mL含有13nm AuNP的分散液，用K 2CO 3调节pH至8.5，加入20μg氯霉素单克隆抗体，室温孵育1h，形成含AuNP-抗体的分散液。
(3)巯基修饰的Trigger DNA经TCEP活化后(摩尔比1：100)，置于-20℃冷冻1h，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AuNP-DNA-抗体的分散液。
(4)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。
本发明还提供了一种检测氯霉素含量的方法，所述方法包括如下过程：
96孔黑色微孔板经氯霉素包被抗原(CAP-BSA)包板，BSA封闭后，获得氯霉素包被抗原(CAP-BSA)包板的96孔黑色微孔板；将上述探针利用缓冲液进行稀释，配置成探针稀释液；然后将待测样本和探针稀释液混合加入到CAP-BSA包板的96孔中，孵育进行竞争反应(此时CAP样品与包被抗原竞争与探针结合)，结束后，清洗96孔黑色微孔板，洗去未结合的探针以及与样品结合的探针后，剩余酶标板上的与包被抗原结合的探针；然后加入信号DNA和Mg 2+，混匀反应，形成检测样液；检测于489/517nm处的荧光强度信号。
在本发明的一种实施方式中，所述信号DNA的序列为：5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ 1-3’。
在本发明的一种实施方式中，所述探针稀释液是指将利用每1mL上述AuNP的分散液制得的探针重悬于200μL缓冲液中。
本发明的一种实施方式中，所述稀释时利用60倍的缓冲液进行稀释，然后用于检测。
在本发明的一种实施方式中，缓冲液为0.01M PBS，1％PEG 20000、1％BSA。
在本发明的一种实施方式中，信号DNA在检测样液的浓度为0.2μM。
在本发明的一种实施方式中，Mg 2+在检测样液中的浓度为75mM。
在本发明的一种实施方式中，氯霉素包被抗原是相对探针上的抗体进行添加，在检测样液的浓度为1.8μg/mL。
在本发明的一种实施方式中，所述检测方法具体包括如下过程：
在96黑色微孔板中加入100μL 1.8μg/mL的氯霉素包被抗原(CAP-BSA)，37℃孵育2h，PBST洗涤三次；加入2％BSA，37℃封闭1h，PBST洗涤3次；加入50μL CAP标准品和50μL稀释60倍的AuNP-DNA-抗体检测探针，37℃竞争1h，PBST洗涤3次；加入0.2μM信号DNA，75mM Mg 2+，37℃反应1h，于489/517nm检测荧光强度信号。
本发明还提供一种检测氯霉素的试剂盒，所述试剂盒包括上述探针、包被有氯霉素包被抗原的96黑色微孔板、BSA、氯霉素标准品、信号DNA和Mg 2+。
在本发明的一种实施方式中，所述信号DNA的序列为5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ 1-3’。
有益效果：
1、本发明以金纳米颗粒为载体，合成AuNP-DNA-抗体检测探针，抗体用于捕获目标物，Trigger 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AuNP-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Trigger DNA完成信号放大步骤(图1)。
2、本发明建立用于氯霉素定量检测的DNAzyme辅助信号放大的免疫吸附分析方法，对氯霉素的检测限为0.1ng/L，线性范围为0.0000001-1μg/mL，与链霉素、庆大霉素、卡那霉素等交叉反应率低，对氯霉素具有高选择性。本发明灵敏度高，可操作性强，能够很好的应用在抗生素检测领域。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。
附图说明
图1本发明原理示意图。
图2(a)合成的13nm AuNP的TEM图；(b)AuNP和AuNP-DNA-抗体紫外图。
图3为方案可行性验证，(a)DANA效果验证；(b)DA-NAFIA的荧光光谱验证；(c)DA-NAFIA的凝胶电泳验证。
图4为条件优化，(a)信号DNA浓度优化；(b)Mg 2+浓度优化。
图5为检测效果验证，(a)标准曲线图，浓度设置为0、0.0000001、0.000001、0.00001、0.0001、0.001、0.01、0.1、1μg/mL；(b)方案选择性验证，以链霉素、卡那霉素和甲砜霉素为参照对象。
具体实施方式
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)。
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。
仪器：Tecnai G220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)。
实施例1合成检测探针
(1)AuNP的制备：
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。
(2)合成AuNP-DNA-抗体检测探针：
1)将1mLAuNP分散液(pH 8.5-9.0)与20μg氯霉素单克隆抗体在室温下温和搅拌并孵育1h，获得混合液。
2)利用10mM TCEP活化巯基修饰的Trigger DNA(序列为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’)。
3)将步骤2)的Trigger DNA加入至步骤1)中的混合液中，Trigger DNA的添加量为1OD，-20℃静置1h。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，添加10％BSA以封闭AuNP表面上剩余的活性位点。为了去除多余的寡核苷酸，将溶液在13000rpm和4℃下离心15分钟，得到AuNP-DNA-抗体检测探针。然后将探针重悬于200μL储存缓冲液(0.01M PBS，1％PEG 20000、1％BSA)中，并保存在4℃下。
通过TEM和UV-可见光谱表征，如图2所示，合成的AuNP粒径约为13nm，分散良好，且无聚集，紫外峰出现在518nm处，连接上Trigger DNA和抗体后，518nm处的紫外峰红移至523nm处，且在260nm出现DNA的峰，表明AuNP-DNA-抗体检测探针的成功合成。
实施例2 DANA单独验证
将10μL 0.2μM Trigger DNA、10μL 0.6μM信号DNA、20μL 5×反应缓冲液、20μL 50mM MgCl 2和40μL H 2O混合并在37℃下反应1小时。在488nm/520nm处获得荧光信号。
结果如图3a所示，相比只含有Trigger DNA和信号DNA的反应体系，添加Mg 2+的反应体系，目标信号增强了40倍。
实施例3 DA-NAFIA验证
(1)荧光光谱
将100μL浓度为1.80μg/mL的包被抗原CAP-BSA加至96孔板中，并在37℃下孵育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50μL浓度分别为0μg/mL、0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL的CAP标准液和50μL稀释60倍的AuNP-DNA-抗体检测探针，并在37℃下孵育1h以进行竞争反应。洗涤后，加入0.2μM信号DNA，75mM Mg 2+和反应缓冲液进行反应。在489nm激发光下扫描荧光光谱。
结果如图3b所示，随着氯霉素标准溶液浓度的升高，荧光强度逐渐降低。
(2)凝胶电泳
首先，信号DNA、AuNP-DNA-抗体检测探针、5×反应缓冲液和75mM MgCl 2混合并反应1h，获得扩增产物。然后，将10μL扩增产物与2μL 6×上样缓冲液(instant view TM红色荧光DNA上样缓冲液，6×溴酚蓝)混合，加入到2％琼脂糖凝胶的孔中。1×TBE用作电泳缓冲液，在90V下电泳2小时后，使用凝胶成像系统分析条带。
结果如图3c所示，加入信号DNA与AuNP-DNA-抗体检测探针反应后，分子量变大，迁移速度变慢(泳道3)。加入Mg 2+后，将信号DNA切成小块片段(泳道4)。
实施例4探究优化
(1)探究包被抗原和检测探针的浓度：
参照实施例3(1)，改变包被抗原CAP-BSA和AuNP-DNA-抗体检测探针的浓度，如表1所示，其他不变，检测荧光信号。
表1棋盘法优化包被抗原和检测探针的浓度
Figure PCTCN2022092153-appb-000001
以抑制率作为评价指标，选择AuNP-DNA-抗体检测探针的60倍稀释和1.8μg/mL包被抗原作为最佳条件。
(2)探究优化信号DNA的浓度条件：
参照实施例3(1)，改变信号DNA的浓度，分别为0.2、0.4、0.6、0.8、1.0μM，其他不变，进行检测。
结果如图4a所示，信号DNA的浓度为0.2μM和0.4μM时具有较高的抑制率，达到84％、83％，检测更加灵敏。
其中，抑制率计算公式：(氯霉素溶液0μg/mL时的荧光信号-氯霉素溶液1μg/mL时的荧光信号)/氯霉素溶液0μg/mL时的荧光信号。
(3)探究Mg 2+的浓度条件：
参照实施例3(1)，改变Mg 2+的浓度，分别为25、50、75、100、125mM，其他不变，进行检测。
结果如图4b所示。Mg 2+的浓度为50mM和75mM时具有较高的抑制率，达到75％、76％，检测更加灵敏。
实施例5线性范围的验证
在实施例3的基础上，设置包被抗原CAP-BSA浓度为1.8μg/mL，AuNP-DNA-抗体检测探针稀释倍数为60倍，信号DNA的浓度为0.2μM，Mg 2+的浓度为75mM。在此条件下，检测一系列稀释的CAP标准样品浓度(0、0.0000001、0.000001、0.00001、0.0001、0.001、0.01、0.1、1μg/mL)。在489nm和517nm处获得荧光(FAM/BHQ1)。
如图5a所示，获得线性范围为0.0000001至1μg/mL的标准曲线，线性方程为y＝-92.35x+887.70(R 2＝0.97)，检出限(LOD)为0.1ng/L。
实施例6特异性
选取牛奶中可能出现的抗生素包括链霉素，卡那霉素，甲砜霉素作为待测样品，评估方法的特异性。
包被抗原CAP-BSA浓度为1.8μg/mL，并在37℃下孵育2h。用0.05％PBST洗涤3次后，加入250μL 2％BSA在37℃下封闭1h。分别加入50μL浓度分别为0μg/mL、0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL的CAP标准液作标准曲线，以及待测样品，加入50μL稀释倍数为60倍的AuNP-DNA-抗体检测探针分别与CAP标准液、待测样品混合，并在37℃下孵育1h以进行竞争反应。洗涤后，加入0.2μM信号DNA，75mM Mg 2+和反应缓冲液进行反应。
如图5b所示，三种非特异性抗生素均显示较低的交叉反应性。
实施例7准确性
选取市面上常见的全脂和脱脂牛奶为实际样品，添加不同浓度的CAP标准品(0.0000001、0.0001、0.001、0.1ng/L)作为待测样品，评估准确性。
包被抗原CAP-BSA浓度为1.8μg/mL，并在37℃下孵育2h。用0.05％PBST洗涤3次后，加入250μL 2％BSA在37℃下封闭1h。分别加入50μL浓度分别为0μg/mL、0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL的CAP标准液作标准曲线，以及待测样品，加入50μL稀释倍数为60倍的AuNP-DNA-抗体检测探针分别与CAP标准液、待测样品混合，并在37℃下孵育1h以进行竞争反应。洗涤后，加入0.2μM信号DNA，75mM Mg 2+和反应缓冲液进行反应。
如表2所示，得到的回收率的范围为86％至104％，RSD的范围为2％至10％。结果表明，本发明提供的检测氯霉素的方法具有良好的准确性。
表2回收率验证结果
Figure PCTCN2022092153-appb-000002
Figure PCTCN2022092153-appb-000003
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。

Claims (14)

  1. 一种检测氯霉素的探针，其特征在于，所述探针包含经巯基修饰的Trigger DNA和氯霉素抗体，所述Trigger DNA和氯霉素抗体连接在金纳米颗粒上；所述Trigger DNA的序列为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’。
  2. 一种制备检测氯霉素的探针的方法，其特征在于，所述方法包括如下过程：
    (1)合成金纳米颗粒AuNP：将HAuCl 4溶液加热至沸腾，加入柠檬酸三钠，保持沸腾直至得到橘红色溶液体系，冷却至室温，即为金纳米颗粒分散液；
    (2)调节金纳米颗粒分散液的pH至8.5-9.0，加入氯霉素单克隆抗体孵育，获得AuNP-抗体分散液；
    (3)将活化后的巯基修饰的Trigger DNA加入到AuNP-抗体分散液中，混匀并冷冻，将溶液溶解后，加入PEG20000、PBS进行反应，获得AuNP-DNA-抗体分散液；
    (4)在所得AuNP-DNA-抗体分散液中加入BSA进行孵育，孵育结束后，固液分离，得到探针；
    所述有巯基修饰的Trigger DNA的序列为：5’-HS-(T) 28TCTCTTCTCCGAGCCGGTCGAAATAGTGCGT-3’。
  3. 根据权利要求2所述的方法，其特征在于，每N mL纳米金颗粒分散液中加入20Nμg氯霉素单克隆抗体制备成AuNP-抗体分散液，再添加N OD的Trigger DNA。
  4. 根据权利要求2所述的方法，其特征在于，所述步骤(3)中，活化是指利用TCEP活化有巯基修饰的DNA；TCEP与巯基修饰的DNA的摩尔比为100：1。
  5. 根据权利要求2所述的方法，其特征在于，所述步骤(3)中，冷冻反应包括：将活化的Trigger DNA与AuNP-抗体混匀后，-20℃静置1h。
  6. 根据权利要求2所述的方法，其特征在于，所述步骤(3)中，PEG20000制成30％的溶液进行添加；添加至使溶液中PEG20000的终浓度为1％。
  7. 根据权利要求2所述的方法，其特征在于，所述步骤(3)中，加入0.1M的PBS；添加至使溶液中的PBS浓度为0.01M。
  8. 根据权利要求2所述的方法，其特征在于，所述步骤(3)中，反应包括：先混匀反应5min然后4℃盐老化2h。
  9. 根据权利要求2所述的方法，其特征在于，所述步骤(4)中，BSA的浓度为10wt％；添加至使溶液中BSA的终浓度为1％。
  10. 一种检测氯霉素的试剂盒，其特征在于，所述试剂盒包括权利要求1所述的探针、包被有氯霉素包被抗原的96黑色微孔板、BSA、氯霉素标准品、信号DNA和Mg 2+；所述信号DNA的序列为5’-FAM-ACGCACTAT/rA/GGAAGAGAT-BHQ 1-3’。
  11. 一种检测氯霉素含量的方法，其特征在于，所述方法包括如下过程：96孔黑色微孔板经氯霉素包被抗原CAP-BSA包板，BSA封闭，将待测样本和稀释过的权利要求1所述的探针加入到96孔黑色微孔板中，35-40℃孵育50～70min后洗涤并拍干，加入信号DNA和Mg 2+，混匀反应，荧光检测。
  12. 根据权利要求11所述的方法，其特征在于，信号DNA在检测样液的浓度为0.2～0.4μM。
  13. 根据权利要求11所述的方法，其特征在于，Mg 2+在检测样液中的浓度为50～75mM。
  14. 根据权利要求11所述的方法，其特征在于，氯霉素包被抗原在检测样液中的浓度为1.8μg/mL，探针稀释倍数为60倍。
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