CN107247136A - A kind of preparation method of Norfloxacin electrochemical immunosensor - Google Patents
A kind of preparation method of Norfloxacin electrochemical immunosensor Download PDFInfo
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- 229960004050 aminobenzoic acid Drugs 0.000 claims description 4
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- UKJLNMAFNRKWGR-UHFFFAOYSA-N cyclohexatrienamine Chemical group NC1=CC=C=C[CH]1 UKJLNMAFNRKWGR-UHFFFAOYSA-N 0.000 claims description 3
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Abstract
本发明公开了一种诺氟沙星电化学免疫传感器的制备方法，通过差分脉冲方法进行检测，线性范围为1μg L‑1～10mg L‑1。本方法基于抗原抗体特异性结合的原理，通过化学键的作用将抗体固定在电极表面，之后滴加辣根过氧化物酶标记的抗原和目标物的混合物，达到吸附平衡后将电极浸入含苯酚、双氧水的PBS溶液中用差分脉冲法进行检测。本方法将传统的免疫技术与传感器技术相结合，为小分子物质诺氟沙星的痕量检测提供了一种新的快速、便捷、灵敏的检测方法，克服了原有的检测方法中检测时间长、仪器昂贵且操作复杂、灵敏度低、特异性差、检测范围小等技术问题。
The invention discloses a preparation method of norfloxacin electrochemical immunosensor, which is detected by a differential pulse method, and the linear range is 1 μg L ‑1 to 10 mg L ‑1 . This method is based on the principle of antigen-antibody specific binding. The antibody is fixed on the surface of the electrode through the action of chemical bonds, and then the mixture of antigen and target substance labeled with horseradish peroxidase is added dropwise. After the adsorption equilibrium is reached, the electrode is immersed in phenol, Hydrogen peroxide in PBS solution was detected by differential pulse method. This method combines traditional immune technology with sensor technology, provides a new rapid, convenient and sensitive detection method for the trace detection of small molecule substance norfloxacin, overcomes the detection time in the original detection method There are technical problems such as long time, expensive equipment and complicated operation, low sensitivity, poor specificity, and small detection range.
Description
技术领域technical field
本发明属于分子检测技术领域，具体涉及一种诺氟沙星电化学免疫传感器的制备方法。The invention belongs to the technical field of molecular detection, and in particular relates to a preparation method of norfloxacin electrochemical immunosensor.
背景技术Background technique
诺氟沙星为第三代喹诺酮类抗菌药，会阻碍消化道内致病细菌的DNA旋转酶(DNAGyrase)的作用，阻碍细菌DNA复制，对细菌有抑制作用，是治疗肠炎痢疾的常用药。但此药对未成年人骨骼形成有延缓作用，会影响到发育。Norfloxacin is a third-generation quinolone antibacterial drug, which can hinder the action of DNA gyrase (DNAGyrase) of pathogenic bacteria in the digestive tract, hinder bacterial DNA replication, and have an inhibitory effect on bacteria. It is a commonly used drug for the treatment of enteritis and dysentery. However, this drug has a delaying effect on the formation of bones in minors, which will affect development.
电化学免疫传感器将电化学检测技术和免疫检测技术相结合，来监测免疫分析过程。它是以抗原抗体的特异性反应为基础，可以用来进行特异性定量或者半定量的集成器件，抗原或者抗体作为分子的识别性原件和电化学的传感性元件直接接触，然后通过传感性元件把待测的物质的浓度信号转变成响应的电信号。其能特异性识别目标物，增加了检测结果的准确性。The electrochemical immunosensor combines electrochemical detection technology and immunoassay technology to monitor the immunoassay process. It is based on the specific reaction of antigens and antibodies, and can be used for specific quantitative or semi-quantitative integrated devices. Antigens or antibodies are directly in contact with electrochemical sensing elements as molecular recognition elements, and then through sensing The sexual element converts the concentration signal of the substance to be measured into a corresponding electrical signal. It can specifically identify the target object, increasing the accuracy of the detection result.
发明内容Contents of the invention
本发明在于树状大分子包裹的纳米金复合材料在增强电信号的同时利用表面大量的氨基来固定更多的抗体，从而增加检测方法的灵敏度及扩大检测范围。通过辣根过氧化物酶标记抗原与目标物竞争抗体来实现电信号的变化，从而达到对目标物的定量检测。The invention lies in that the nano-gold composite material wrapped by the dendritic macromolecule uses a large amount of amino groups on the surface to immobilize more antibodies while enhancing the electrical signal, thereby increasing the sensitivity of the detection method and expanding the detection range. The change of the electrical signal is realized by the horseradish peroxidase-labeled antigen competing with the target antibody, so as to achieve the quantitative detection of the target.
原理：在固定了辣根过氧化物酶标记诺氟沙星的抗原的浓度、体积以及目标物的体积之后，通过改变目标物的浓度来形成抗原与酶标抗原之间的竞争关系，实现对目标物的定量检测。随着目标物浓度的增大，与抗体结合的酶标抗原的量会相应减少，从而催化双氧水的能力减弱，产生的电信号会有所降低。Principle: After fixing the concentration, volume and target volume of the horseradish peroxidase-labeled norfloxacin antigen, the competition relationship between the antigen and the enzyme-labeled antigen is formed by changing the concentration of the target to realize the Quantitative detection of target objects. As the concentration of the target substance increases, the amount of enzyme-labeled antigen bound to the antibody will decrease accordingly, so the ability to catalyze hydrogen peroxide will weaken, and the generated electrical signal will decrease.
这种电化学免疫传感器的制备方法具体步骤为：The specific steps of the preparation method of this electrochemical immunosensor are as follows:
(1)树状大分子包裹的纳米金的制备(1) Preparation of gold nanoparticles wrapped by dendrimers
将2mL浓度为1mmol L-1的HAuCl4加入到2mL 4代氨基端PAMAM溶液和2mL甲酸的混合液中，剧烈搅拌2min，溶液由淡黄色变为酒红色，在紫外分光光度计下测得吸收峰在520nm左右，透射电镜可观察到纳米金粒径在5nm以内。Add 2mL of HAuCl4 with a concentration of 1mmol L -1 into the mixture of 2mL of 4th -generation amino-terminal PAMAM solution and 2mL of formic acid, stir vigorously for 2min, the solution changes from light yellow to wine red, and the absorption is measured under an ultraviolet spectrophotometer. The peak is around 520nm, and the nano-gold particle size can be observed within 5nm by transmission electron microscope.
树状大分子分为半代和整代，0.5、1、1.5、2、2.5、3、3.5、4代等，代数越大表面带有的基团越多。还分为氨基端和羧基端的，本方法中为了更好的与抗体的羧基端相连，选取了4代氨基端的树状大分子。4代大分子为球形结构，内部有空腔，内径在5nm左右，氯金酸进入其空腔后在还原剂的作用下形成小于5nm的树状大分子包裹的纳米金。Dendrimers are divided into half-generation and full-generation, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 generations, etc. The larger the generation number, the more groups on the surface. It is also divided into amino-terminal and carboxyl-terminal. In this method, in order to better connect with the carboxyl-terminal of the antibody, 4th generation of dendrimers with amino-terminal are selected. The 4th generation macromolecule has a spherical structure with a cavity inside, and the inner diameter is about 5nm. After the chloroauric acid enters the cavity, under the action of a reducing agent, a gold nanometer wrapped in a dendrimer macromolecule smaller than 5nm is formed.
(2)对氨基苯基酸修饰电极的自组装(2) Self-assembly of p-aminophenyl acid modified electrodes
将浓盐酸稀释成0.01mol L-1的稀盐酸，准确称取13.7mg对氨基苯甲酸，加入到50mL稀盐酸溶液中，超声加速溶解，氮气除氧2min，即得到2mmol L-1的对氨基苯甲酸聚合液，该溶液现配先用。将预先处理干净的玻碳电极插入上述电聚合溶液中，0-1v施加电压，扫速为40mv/s，扫描5圈，即得到表面修饰有一层羧基膜的电极。Dilute concentrated hydrochloric acid into 0.01mol L -1 dilute hydrochloric acid, accurately weigh 13.7mg p-aminobenzoic acid, add it to 50mL dilute hydrochloric acid solution, accelerate dissolution by ultrasonic, deoxygenate with nitrogen for 2min, and obtain 2mmol L -1 p-aminobenzoic acid Benzoic acid polymerization solution, the solution is prepared before use. Insert the pre-treated glassy carbon electrode into the above electropolymerization solution, apply a voltage of 0-1v, scan at a speed of 40mv/s, and scan 5 times to obtain an electrode modified with a carboxyl film on the surface.
(3)树状大分子包裹的纳米金复合材料的自组装(3) Self-assembly of gold nanocomposites wrapped in dendrimers
将10μL合成的树状大分子纳米复合材料均匀滴涂在修饰有羧基的电极表面，待其自然晾干，用双蒸水冲洗除去未结合的材料。利用纳米金复合材料表面丰富的氨基与电极表面大量的羧基结合，使纳米材料牢固的结合在电极表面。10 μL of the synthesized dendrimer nanocomposite material was evenly drop-coated on the surface of the electrode modified with carboxyl groups, and allowed to dry naturally, then rinsed with double distilled water to remove unbound materials. The combination of abundant amino groups on the surface of the nano-gold composite material and a large number of carboxyl groups on the electrode surface enables the nano-materials to be firmly combined on the electrode surface.
(4)抗体的自组装(4) Self-assembly of antibodies
将连有树状大分子纳米金复合材料的电极表面滴涂上10μL纯化好的浓度为50μgmL-1的诺氟沙星的抗体，纳米复合材料表面丰富的氨基可以连接更多的抗体。连接好抗体之后，用10μL浓度为1％的BSA封闭电极表面未结合位点30min，冲掉多余的溶液，将修饰好的电极备用。10 μL of purified norfloxacin antibody with a concentration of 50 μg mL -1 was drip-coated on the surface of the electrode connected with the dendrimer nano-gold composite material, and more antibodies could be connected to the abundant amino groups on the surface of the nanocomposite material. After the antibody was connected, 10 μL of 1% BSA was used to block the unbound sites on the surface of the electrode for 30 min, the excess solution was washed away, and the modified electrode was used for later use.
(5)检测方法：(5) Detection method:
将稀释1000倍的辣根过氧化物酶标记的诺氟沙星抗原分别与不同浓度的目标物诺氟沙星标准液等体积混合，取10μL混合液，均匀滴涂在制备好的电极表面，待电极表面的抗体与抗原吸附50min，达到吸附平衡状态，此时，抗原与抗体的结合量达到最大值，用PBS冲洗未结合的溶液。之后，将修饰有辣根过氧化物酶标记的诺氟沙星抗原与目标物的电极放在3mL含双氧水和苯酚的pH为7.4的PBS溶液中进行差分脉冲检测，电位范围为-0.5～0.3v。Mix the horseradish peroxidase-labeled norfloxacin antigen diluted 1000 times with the target substance norfloxacin standard solution of different concentrations in equal volumes, take 10 μL of the mixed solution, and evenly drop-coat it on the surface of the prepared electrode. Wait until the antibody and antigen on the surface of the electrode are adsorbed for 50 minutes to reach an adsorption equilibrium state. At this time, the binding amount of antigen and antibody reaches the maximum value, and the unbound solution is washed with PBS. After that, place the electrode modified with horseradish peroxidase-labeled norfloxacin antigen and the target object in 3 mL of PBS solution containing hydrogen peroxide and phenol at a pH of 7.4 for differential pulse detection, and the potential range is -0.5 to 0.3 v.
本发明分别在10、25、50、100μg mL-1的抗体浓度与酶标抗原稀释500、1000、1500、2000倍的条件下进行4×4试验，最后得到在抗体浓度为50μg mL-1，酶标抗原稀释1000倍时结果最佳。In the present invention, the antibody concentration of 10, 25, 50, 100 μg mL-1 and enzyme-labeled antigen were diluted 500, 1000, 1500 and 2000 times respectively, and the 4×4 test was carried out, and finally the antibody concentration was 50 μg mL-1, The best results were achieved when the enzyme-labeled antigen was diluted 1000 times.
(6)诺氟沙星含量的测定(6) Determination of Norfloxacin Content
根据辣根过氧化物酶标记的抗原与目标物对抗体的竞争作用，来实现目标物的定量检测。随着目标物浓度的增加，与抗体结合的酶标抗原就会减少，连接在电极表面的辣根过氧化物酶就会减少，其对苯酚-双氧水体系的催化能力会降低，从而呈现出来的电信号有所降低。所以，随着目标物浓度的增加，响应的电流值会降低。差分脉冲的响应值与诺氟沙星的浓度在1μg L-1～10mg L-1之间呈良好的线性关系：y＝-3.33442x+47.84746，斜率为-3.33442，相关系数R2为0.99237，最低检出限为0.3837μg L-1。Quantitative detection of the target is achieved based on the competition between the horseradish peroxidase-labeled antigen and the target on the antibody. As the concentration of the target increases, the enzyme-labeled antigen bound to the antibody will decrease, and the horseradish peroxidase attached to the surface of the electrode will decrease, and its catalytic ability to the phenol-hydrogen peroxide system will decrease, thus presenting the The electrical signal has decreased. Therefore, as the target concentration increases, the corresponding current value will decrease. There is a good linear relationship between the response value of the differential pulse and the concentration of norfloxacin between 1 μg L -1 and 10 mg L -1 : y=-3.33442x+47.84746, the slope is -3.33442, and the correlation coefficient R 2 is 0.99237, The lowest detection limit was 0.3837μg L -1 .
本发明优点是：本发明采用的树状大分子包裹的纳米金复合材料不但能增强电信号还能利用表面丰富的氨基连接更多的诺氟沙星抗体，采用苯酚-双氧水体系，通过诺氟沙星酶标抗原与目标物对抗体的竞争反应，来产生不同的电信号，从而更准确的对小分子目标物的定量检测。该方法克服了其他原有方法检测时间长、仪器昂贵且操作复杂、灵敏度低、特异性差、检测范围小等技术问题。The advantages of the present invention are: the nano-gold composite material wrapped by dendrimers used in the present invention can not only enhance the electrical signal, but also use the abundant amino groups on the surface to connect more norfloxacin antibodies. The competition reaction between the sandacin enzyme-labeled antigen and the target object to the antibody generates different electrical signals, so that the quantitative detection of the small molecule target object is more accurate. This method overcomes the technical problems of other original methods such as long detection time, expensive instruments, complicated operation, low sensitivity, poor specificity, and small detection range.
附图说明Description of drawings
图1本发明的实施例诺氟沙星浓度与相应差分脉冲电流值的关系图。Fig. 1 is a graph showing the relationship between the norfloxacin concentration and the corresponding differential pulse current value in an embodiment of the present invention.
具体实施方式detailed description
实施例1Example 1
(1)树状大分子包裹的纳米金的制备(1) Preparation of gold nanoparticles wrapped by dendrimers
将2mL浓度为1mmol L-1的HAuCl4加入到2mL 4代氨基端PAMAM溶液和2mL甲酸的混合液中，剧烈搅拌2min，溶液由淡黄色变为酒红色，在紫外分光光度计下测得吸收峰在520nm左右，透射电镜可观察到纳米金粒径在5nm以内。Add 2mL of HAuCl4 with a concentration of 1mmol L -1 into the mixture of 2mL of 4th -generation amino-terminal PAMAM solution and 2mL of formic acid, stir vigorously for 2min, the solution changes from light yellow to wine red, and the absorption is measured under an ultraviolet spectrophotometer. The peak is around 520nm, and the nano-gold particle size can be observed within 5nm by transmission electron microscope.
(2)对氨基苯基酸修饰电极的自组装(2) Self-assembly of p-aminophenyl acid modified electrodes
将浓盐酸稀释成0.01mol L-1的稀盐酸，准确称取13.7mg对氨基苯甲酸，加入到50mL稀盐酸溶液中，超声加速溶解，氮气除氧2min，即得到2mmol L-1的对氨基苯甲酸聚合液，该溶液现配先用。将预先处理干净的玻碳电极插入上述电聚合溶液中，0-1v施加电压，扫速为40mv/s，扫描5圈，即得到表面修饰有一层羧基膜的电极。Dilute concentrated hydrochloric acid into 0.01mol L -1 dilute hydrochloric acid, accurately weigh 13.7mg p-aminobenzoic acid, add it to 50mL dilute hydrochloric acid solution, accelerate dissolution by ultrasonic, deoxygenate with nitrogen for 2min, and obtain 2mmol L -1 p-aminobenzoic acid Benzoic acid polymerization solution, the solution is prepared before use. Insert the pre-treated glassy carbon electrode into the above electropolymerization solution, apply a voltage of 0-1v, scan at a speed of 40mv/s, and scan 5 times to obtain an electrode modified with a carboxyl film on the surface.
(3)树状大分子包裹的纳米金复合材料的自组装(3) Self-assembly of gold nanocomposites wrapped in dendrimers
将10μL合成的树状大分子纳米复合材料均匀滴涂在修饰有羧基的电极表面，待其自然晾干，用双蒸水冲洗除去未结合的材料。利用纳米金复合材料表面丰富的氨基与电极表面大量的羧基结合，使纳米材料牢固的结合在电极表面。10 μL of the synthesized dendrimer nanocomposite material was evenly drop-coated on the surface of the electrode modified with carboxyl groups, and allowed to dry naturally, then rinsed with double distilled water to remove unbound materials. The combination of abundant amino groups on the surface of the nano-gold composite material and a large number of carboxyl groups on the electrode surface enables the nano-materials to be firmly combined on the electrode surface.
(4)抗体的自组装(4) Self-assembly of antibodies
将连有树状大分子纳米金复合材料的电极表面滴涂上10μL纯化好的浓度为50μgmL-1的诺氟沙星的抗体，纳米复合材料表面丰富的氨基可以连接更多的抗体。连接好抗体之后，用10μL浓度为1％的BSA封闭电极表面未结合位点30min，冲掉多余的溶液，将修饰好的电极备用。10 μL of purified norfloxacin antibody with a concentration of 50 μg mL -1 was drip-coated on the surface of the electrode connected with the dendrimer nano-gold composite material, and more antibodies could be connected to the abundant amino groups on the surface of the nanocomposite material. After the antibody was connected, 10 μL of 1% BSA was used to block the unbound sites on the surface of the electrode for 30 min, the excess solution was washed away, and the modified electrode was used for later use.
(5)检测方法：(5) Detection method:
将稀释1000倍的辣根过氧化物酶标记的诺氟沙星抗原分别与不同浓度的目标物诺氟沙星标准液等体积混合，取10μL混合液，均匀滴涂在制备好的电极表面，待电极表面的抗体与抗原吸附50min，达到吸附平衡状态，此时，抗原与抗体的结合量达到最大值，用PBS冲洗未结合的溶液。之后，将修饰有辣根过氧化物酶标记的诺氟沙星抗原与目标物的电极放在3mL含双氧水和苯酚的pH为7.4的PBS溶液中进行差分脉冲检测，电位范围为-0.5～0.3v。Mix the horseradish peroxidase-labeled norfloxacin antigen diluted 1000 times with the target substance norfloxacin standard solution of different concentrations in equal volumes, take 10 μL of the mixed solution, and evenly drop-coat it on the surface of the prepared electrode. Wait until the antibody and antigen on the surface of the electrode are adsorbed for 50 minutes to reach an adsorption equilibrium state. At this time, the binding amount of antigen and antibody reaches the maximum value, and the unbound solution is washed with PBS. After that, place the electrode modified with horseradish peroxidase-labeled norfloxacin antigen and the target object in 3 mL of PBS solution containing hydrogen peroxide and phenol at a pH of 7.4 for differential pulse detection, and the potential range is -0.5 to 0.3 v.
(6)诺氟沙星含量的测定(6) Determination of Norfloxacin Content
根据辣根过氧化物酶标记的抗原与目标物对抗体的竞争作用，来实现目标物的定量检测。随着目标物浓度的增加，与抗体结合的酶标抗原就会减少，连接在电极表面的辣根过氧化物酶就会减少，其对苯酚-双氧水体系的催化能力会降低，从而呈现出来的电信号有所降低。所以，随着目标物浓度的增加，响应的电流值会降低。差分脉冲的响应值与诺氟沙星的浓度在1μg L-1～10mg L-1之间呈良好的线性关系：y＝-3.33442x+47.84746，斜率为-3.33442，相关系数R2为0.99237，最低检出限为0.3837μg L-1。Quantitative detection of the target is achieved based on the competition between the horseradish peroxidase-labeled antigen and the target on the antibody. As the concentration of the target increases, the enzyme-labeled antigen bound to the antibody will decrease, and the horseradish peroxidase attached to the electrode surface will decrease, and its catalytic ability to the phenol-hydrogen peroxide system will decrease, thus presenting the The electrical signal has decreased. Therefore, as the target concentration increases, the corresponding current value will decrease. There is a good linear relationship between the response value of the differential pulse and the concentration of norfloxacin between 1 μg L -1 and 10 mg L -1 : y=-3.33442x+47.84746, the slope is -3.33442, and the correlation coefficient R 2 is 0.99237, The lowest detection limit was 0.3837μg L -1 .
实施例2Example 2
实际样品中诺氟沙星含量的测定：Determination of norfloxacin content in the actual sample:
利用本发明的纳米金复合材料修饰的工作电极对实际样品(牛奶、鸡蛋、猪肉)中的诺氟沙星进行了分析测定，检出一定含量的诺氟沙星，采用标准加入法进行加标回收实验。选用LK2006型电化学工作站系统中的循环伏安法和差分脉冲法进行检测，循环伏安法扫描电压为0.6～-0.2v，差分脉冲法的扫描范围为0.3～-0.5v。读取电流响应值，带入y＝-3.33442x+47.84746，计算出Cnor。平行测定三次，得到回收率为91.6～106.1％，说明利用本发明的制备方法得到的树状大分子包裹的纳米金复合材料所构建的电化学免疫传感器具有较高的准确度。Norfloxacin in actual samples (milk, eggs, pork) was analyzed and measured by using the working electrode modified by the nano-gold composite material of the present invention, and a certain amount of norfloxacin was detected, and the standard addition method was used to add the standard Recovery experiment. The cyclic voltammetry and differential pulse method in the LK2006 electrochemical workstation system were selected for detection. The scanning voltage of cyclic voltammetry was 0.6~-0.2v, and the scanning range of differential pulse method was 0.3~-0.5v. Read the current response value, enter y=-3.33442x+47.84746, and calculate C nor . The recovery rate is 91.6-106.1% after parallel measurement for three times, which shows that the electrochemical immunosensor constructed by the dendrimer-wrapped nano-gold composite material obtained by the preparation method of the present invention has high accuracy.
以上所述仅为本发明创造的较佳实施例而已，并不用以限制本发明创造，凡在本发明创造的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明创造的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the Within the protection scope of the present invention.
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