CN107576702B - Preparation method of electrochemical sensor for detecting concentration of galectin-3 in serum - Google Patents
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Abstract
本发明涉及引发2型糖尿病的胰岛素灵敏度降低早期诊断生物标志物‑galectin‑3(Gal‑3)的血液浓度水平检测的电化学传感器的制备方法及应用,属于电化学检测技术领域。其特征在于:首先分别合成N‑GNRs‑Fe‑MOFs@AuPNs传感器基底修饰纳米复合材料和AuPt‑MB纳米复合信号材料,再将能与Gal‑3特异性结合检测抗体与AuPt‑MB复合材料混合,制得生物信号探针。然后在N‑GNRs‑Fe‑MOFs@AuPNs复合材料修饰的基底平台上接合Gal‑3特异性捕获抗体,以便于特异性捕获目标物Gal‑3。最后通过Gal‑3与其检测抗体和捕获抗体的特异性结合,制备得到了Gal‑3的血液浓度水平检测的电化学传感器,该传感器成功的用于血清中Gal‑3的检测。本发明的优点在于灵敏度高,特异性强,检测迅速,方便。本发明为引发2型糖尿病的胰岛素抵抗提供早期的诊断依据。
The invention relates to a preparation method and application of an electrochemical sensor for detecting the blood concentration level of a biomarker-galectin-3 (Gal-3) for early diagnosis of early diagnosis biomarker-galectin-3 (Gal-3) caused by the decrease in insulin sensitivity of type 2 diabetes, and belongs to the technical field of electrochemical detection. It is characterized by: firstly synthesizing N‑GNRs‑Fe‑MOFs@AuPNs sensor substrate modified nanocomposite material and AuPt‑MB nanocomposite signal material, and then mixing detection antibody that can specifically bind to Gal‑3 and AuPt‑MB composite material , to prepare biosignal probes. Then, the Gal-3-specific capture antibody was attached to the substrate platform modified by the N-GNRs-Fe-MOFs@AuPNs composite material to facilitate the specific capture of the target Gal-3. Finally, through the specific binding of Gal-3 to its detection antibody and capture antibody, an electrochemical sensor for detecting the blood concentration level of Gal-3 was prepared, and the sensor was successfully used for the detection of Gal-3 in serum. The invention has the advantages of high sensitivity, strong specificity, rapid detection and convenience. The invention provides early diagnosis basis for the insulin resistance causing type 2 diabetes mellitus.
Description
技术领域:Technical field:
本发明涉及引发2型糖尿病的胰岛素灵敏度降低早期诊断生物标志物-galectin-3(Gal-3)的血液浓度水平检测的电化学传感器的制备方法及应用,尤其是基于AuPt-MB纳米复合材料作为新型信号探针制备的夹心型免疫生物传感器,用于血清中galectin-3(Gal-3)浓度检测,属于电化学检测领域。The invention relates to a preparation method and application of an electrochemical sensor for detecting the blood concentration level of the early diagnosis biomarker-galectin-3 (Gal-3) caused by the decrease in insulin sensitivity of
背景技术:Background technique:
糖尿病在全球范围内迅速增长,对人类健康已构成严重威胁。特别是由胰岛素灵敏度降低引起的2型糖尿病(T2D)能够导致致死性的心血管并发症,并占据糖尿病患病率的90%。在最近的研究中发现,galectin-3(Gal-3)能直接与胰岛素受体结合抑制下游胰岛素受体(IR)信号传导和链接炎症降低胰岛素敏感性,最终导致胰岛素抵抗和葡萄糖不耐受,引发2型糖尿病。因此,监测血液中Gal-3浓度水平的变化,对预防T2D及其心血管并发症尤为重要。Diabetes is growing rapidly worldwide and has posed a serious threat to human health. In particular,
在目前对Gal-3进行检测的主要传统方法有:酶联免疫吸附测定(ELISA),免疫组化和“常规Gal-3”测定(雅培诊断)。然而,这些传统方法相对存在着昂贵,费时,半定量,需要熟练的操作者,灵敏度有限和线性范围窄等缺点的限制。特别是不能满足早期检测的临床需求。因此,探索研究一种用于Gal-3的简便,快速和灵敏的检测方法显得格外重要。与传统的检测方法相比,电化学免疫传生物感器检测方法因其具有灵敏度高,特异性强和快速检测等优点,近年来在临床蛋白生物标志物检测方面越来越受到重视。同时,基于纳米材料的电化学生物传感器由于简便,快速,成本低,灵敏度高等优势而被广泛应用于生物样品的检测。因此,基于纳米复合材料构建一种夹心式电化学免疫生物传感器为实现血清中Gal-3的低浓度高灵敏检测提供了一种新思路。The main traditional methods for the detection of Gal-3 at present are: enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and "conventional Gal-3" assay (Abbott Diagnostics). However, these traditional methods are relatively expensive, time-consuming, semi-quantitative, require skilled operators, limited sensitivity and narrow linear range. In particular, it cannot meet the clinical needs of early detection. Therefore, it is very important to explore a simple, rapid and sensitive detection method for Gal-3. Compared with traditional detection methods, electrochemical immunosensor detection methods have attracted more and more attention in the detection of clinical protein biomarkers in recent years due to their advantages of high sensitivity, strong specificity and rapid detection. Meanwhile, electrochemical biosensors based on nanomaterials are widely used in the detection of biological samples due to their simplicity, rapidity, low cost, and high sensitivity. Therefore, the construction of a sandwich electrochemical immunobiosensor based on nanocomposites provides a new idea for the realization of low-concentration and high-sensitivity detection of Gal-3 in serum.
亚甲基蓝(MB)是一种吩噻嗪染料的衍生物,由于其结构中存在富含电子的硫和氮杂原子作为一种新兴的电化学氧化还原活性物质被广泛用地应用于催化、感测和氧化还原指示等领域。然而,由于其不均匀的形态使其在免疫传感器信号探针方面的应用收到限制。为解决这一问题,本发明创新性地引入了具有比表面积大、导电性强和生物分子兼容性好AuPt双金属纳米材料与MB发生氧化聚合反应,得到了具有均匀棒状形态AuPt-MB纳米复合材料,并且显示出显著的电化学活性。在本发明中将AuPt-MB纳米复合材料与Gal-3特异性检测抗体Ab2结合,并用BSA做封闭剂制备得到新型纳米信号探针。Methylene blue (MB), a derivative of phenothiazine dyes, has been widely used as an emerging electrochemical redox active species due to the presence of electron-rich sulfur and nitrogen heteroatoms in its structure for catalysis, sensing and Redox indication and other fields. However, its application in immunosensor signaling probes is limited due to its heterogeneous morphology. In order to solve this problem, the present invention innovatively introduces AuPt bimetallic nanomaterials with large specific surface area, strong electrical conductivity and good biomolecular compatibility to undergo oxidative polymerization with MB, and obtain AuPt-MB nanocomposite with uniform rod shape. material and showed remarkable electrochemical activity. In the present invention, the AuPt-MB nanocomposite material is combined with the Gal-3 specific detection antibody Ab2, and BSA is used as a blocking agent to prepare a novel nanometer signal probe.
为了进一步增加本夹心型免疫传感器的灵敏度和稳定性。引入了一种新型的纳米复合材料氮参杂石墨烯纳米带(N-GNRs)-铁金属有机框架(Fe-MOFs)-金纳米粒(AuNPs)-(N-GNRs-Fe-MOFs@AuNPs)作为传感器基底修饰材料。金属-有机骨架(MOFs)是一种类沸石状结晶多孔材料,它们具有较大的表面积,灵活的孔隙率,易于定制组合成分和活性位点多等优点,作为新型多功能材料,它们已被广泛地用于储气/分离,催化和传感等领域。本发明中引入的Fe-MOFs具有稳定性好、比表面积大和低毒性等特点。因其具有很大的比表面积可以通过Au-N化学键在其表面结合大量的AuNPs纳米粒增加其导电性,同时有助于下步固载结合Gal-3特异性捕获抗体Ab1。为了进一步增加导电性,将N-GNRs与Fe-MOFs@AuNPs混合反应得到N-GNRs-Fe-MOFs@AuNPs纳米复合材料。N-GNRs由广泛用于提高改性电极表面的导电性能的N掺杂多壁碳纳米管(N-MWCNTs)合成。与碳纳米管相比,N-GNR具有反应性边界和固有的梯田和阶梯结构,可以增强化学活性和吸附能力。令人很意外的事,最后N-GNRs-Fe-MOFs@AuNPs纳米复合材料不仅拥有了N-GNRs、Fe-MOFs和AuNPs的优点,还在导电性方面显示出显著的综合效应。因此,该新型纳米复合材料应用于本电化学免疫传感器的基底修饰以进一步增强传感器的灵敏度和稳定性。In order to further increase the sensitivity and stability of the sandwich immunosensor. Introduced a novel nanocomposite of nitrogen-doped graphene nanoribbons (N-GNRs)-iron metal organic frameworks (Fe-MOFs)-gold nanoparticles (AuNPs)-(N-GNRs-Fe-MOFs@AuNPs) As a sensor substrate modification material. Metal-organic frameworks (MOFs) are a kind of zeolite-like crystalline porous materials, they have the advantages of large surface area, flexible porosity, easy to customize composition composition, and many active sites, etc. As new multifunctional materials, they have been widely used. It is widely used in the fields of gas storage/separation, catalysis and sensing. The Fe-MOFs introduced in the present invention have the characteristics of good stability, large specific surface area and low toxicity. Because of its large specific surface area, a large number of AuNPs nanoparticles can be bound on its surface through Au-N chemical bonds to increase its conductivity, and at the same time, it is helpful to immobilize and bind Gal-3 specific capture antibody Ab 1 in the next step. To further increase the electrical conductivity, N-GNRs-Fe-MOFs@AuNPs nanocomposites were obtained by mixing N-GNRs with Fe-MOFs@AuNPs. N-GNRs are synthesized from N-doped multi-walled carbon nanotubes (N-MWCNTs), which are widely used to improve the electrical conductivity of modified electrode surfaces. Compared with carbon nanotubes, N-GNRs possess reactive boundaries and inherent terraced and stepped structures, which can enhance chemical activity and adsorption capacity. Surprisingly, the final N-GNRs-Fe-MOFs@AuNPs nanocomposite not only possesses the advantages of N-GNRs, Fe-MOFs, and AuNPs, but also shows a significant combined effect in electrical conductivity. Therefore, the novel nanocomposite material is applied in the substrate modification of the electrochemical immunosensor to further enhance the sensitivity and stability of the sensor.
本发明基于AuPt-MB纳米复合材料构建新型纳米信号探针和N-GNRs-Fe-MOFs@AuNPs纳米复合材料作为免疫传感器的基底修饰材料,建立了一种用于血清中galectin-3(Gal-3)浓度检测的电化学传感器制备方法与应用,为引发2型糖尿病的胰岛素抵抗提供早期的诊断依据。In the present invention, a novel nano-signal probe is constructed based on AuPt-MB nanocomposite materials and N-GNRs-Fe-MOFs@AuNPs nanocomposite materials are used as substrate modification materials for immunosensors, and a new type of nano-signal probe for galectin-3 (Gal- 3) The preparation method and application of an electrochemical sensor for concentration detection provide an early diagnosis basis for insulin resistance of
发明内容:Invention content:
本发明的目的是提供一种用于血清中galectin-3(Gal-3)浓度检测的电化学传感器制备方法及应用,其特征在于包括以下步骤:The object of the present invention is to provide a kind of electrochemical sensor preparation method and application for the detection of galectin-3 (Gal-3) concentration in serum, it is characterized in that comprising the following steps:
(1)氮参杂石墨烯纳米带(N-GNRs)-铁金属有机框架(Fe-MOFs)-金纳米粒(AuNPs)传感器基底修饰纳米复合材料的制备。(1) Preparation of nitrogen-doped graphene nanoribbons (N-GNRs)-iron metal organic frameworks (Fe-MOFs)-gold nanoparticles (AuNPs) sensor substrate modified nanocomposites.
(2)金铂双金属(AuPt)-亚甲蓝(MB)-Gal-3特异性检测抗体(Ab2)纳米信号探针的制备。(2) Preparation of gold-platinum bimetallic (AuPt)-methylene blue (MB)-Gal-3 specific detection antibody (Ab 2 ) nano-signal probe.
(3)建立电化学免疫生物传感器,检测血清中Gal-3,绘制标准曲线。(3) Establish an electrochemical immunobiosensor, detect Gal-3 in serum, and draw a standard curve.
本发明所述N-GNRs-Fe-MOFs@AuNPs复合物的制备过程具体包括以下步骤,其特征在于包括以下步骤:The preparation process of the N-GNRs-Fe-MOFs@AuNPs composite of the present invention specifically includes the following steps, and is characterized in that it includes the following steps:
(1)N-GNRs材料的制备:(1) Preparation of N-GNRs materials:
称取0.1g氮参杂多壁碳纳米管(N-MWCNTs)加入至10mL的H2SO4和H3PO4混合溶液中(H2SO4∶H3PO4=9∶1)混匀。将上述混合溶液置于高压反应釜中在磁力搅拌条件下加热至140℃持续10min,再将反应混合溶液转移至65℃油浴中在搅拌条件下加入0.25g KMnO4持续反应8min。反应结束后控制溶液降温至室温,所得溶液经10000r/min,离心5min,用超纯水清洗3次。将所得沉淀置于真空干燥24h后,4℃保存备用。Weigh 0.1 g of nitrogen-doped multi-walled carbon nanotubes (N-MWCNTs) into 10 mL of a mixed solution of H 2 SO 4 and H 3 PO 4 (H 2 SO 4 : H 3 PO 4 =9:1) and mix well . The above mixed solution was placed in an autoclave and heated to 140 °C for 10 min under magnetic stirring, and then the reaction mixture was transferred to an oil bath at 65 °C, and 0.25 g of KMnO was added under stirring to continue the reaction for 8 min. After the reaction, the solution was controlled to cool down to room temperature, and the obtained solution was centrifuged at 10,000 r/min for 5 min, and washed with ultrapure water for 3 times. The obtained precipitate was dried in vacuum for 24 h and stored at 4°C for later use.
(2)Fe-MOFs材料的制备(2) Preparation of Fe-MOFs
分别称取0.187g FeCl3·6H2O和0.126g 2-氨基对苯二甲酸加入15mL的二甲基甲酰胺(DMF)溶液中,混匀。将上述混合溶液置于120℃硅油中加热4h,在开始加热后15min时,向混合溶液中加入200μL的冰醋酸。加热结束后控制溶液降温至室温,所得溶液经10000r/min,离心5min,分别用DMF,无水乙醇,超纯水各清洗3次。将所得沉淀置于真空干燥24h后,4℃保存备用。0.187g FeCl 3 ·6H 2 O and 0.126g 2-aminoterephthalic acid were respectively weighed and added to 15 mL of dimethylformamide (DMF) solution, and mixed well. The above mixed solution was heated in silicone oil at 120° C. for 4 hours, and 200 μL of glacial acetic acid was added to the mixed
(3)Fe-MOFs@AuNPs纳米复合材料的制备(3) Preparation of Fe-MOFs@AuNPs nanocomposites
取2mL HAuCl4·4H2O(1%)溶液加入到制备好的2mLFe-MOFs(1mg mL-1)溶液中,剧烈超声30分钟后,向上述溶液中逐滴缓慢加入4mL NaBH4(0.1M)同时放在磁力搅拌器上进行搅拌反应30min,所得溶液经10000r/min,离心5min,用超纯水清洗3次。将所得沉淀再分散至4mL的去离子水中,4℃保存备用。Take 2 mL of HAuCl 4 ·4H 2 O (1%) solution and add it to the prepared 2 mL Fe-MOFs (1 mg mL -1 ) solution. After vigorously sonicating for 30 minutes, slowly add 4 mL of NaBH 4 (0.1 M) to the above solution dropwise. ) at the same time on a magnetic stirrer to carry out stirring reaction for 30min, the obtained solution was centrifuged at 10000r/min for 5min, and washed 3 times with ultrapure water. The obtained precipitate was redispersed in 4 mL of deionized water and stored at 4°C for later use.
(4)N-GNRs-Fe-MOFs@AuNPs纳米复合材料的制备(4) Preparation of N-GNRs-Fe-MOFs@AuNPs nanocomposites
称取0.2mg N-GNRs超声溶解于2mL的去离子水中,将其与4mL Fe-MOFs@AuNPs复合物溶液超声混匀30min,在磁力搅拌500r/min条件下,搅拌过夜。所得溶液经8000r/min,离心5min,用超纯水清洗3次。将所得沉淀再分散至4mL的去离子水中,4℃保存备用。0.2 mg of N-GNRs were weighed and dissolved in 2 mL of deionized water by ultrasonic, which was mixed with 4 mL of Fe-MOFs@AuNPs complex solution by ultrasonic for 30 min, and stirred overnight under the condition of magnetic stirring at 500 r/min. The resulting solution was centrifuged at 8000 r/min for 5 min, and washed three times with ultrapure water. The obtained precipitate was redispersed in 4 mL of deionized water and stored at 4°C for later use.
本发明所述AuPt-MB-Ab2纳米信号探针的制备过程具体包括以下步骤,其特征在于包括以下步骤:The preparation process of the AuPt-MB-Ab 2 nano-signal probe of the present invention specifically includes the following steps, and is characterized in that it includes the following steps:
(1)AuPt-MB纳米复合材料的制备:(1) Preparation of AuPt-MB nanocomposites:
分别取600μL HCL(0.1M)和6mL十二烷基三甲基溴化铵(DTAB 1.14mM)加入至1mL的MB(9.8mM)溶液中,剧烈搅拌10min。再同时加入100μL HAuCl4(4wt%,)和100μL H2PtCl6(4wt%,),持续室温搅拌6h。所得溶液经8000r/min,离心10min,用超纯水清洗3次。将所得沉淀再分散至2mLPBS缓冲溶液(0.1M pH 7.0)中,4℃保存备用。600 μL of HCL (0.1 M) and 6 mL of dodecyltrimethylammonium bromide (DTAB 1.14 mM) were added to 1 mL of MB (9.8 mM) solution and stirred vigorously for 10 min. 100 μL of HAuCl 4 (4 wt %,) and 100 μL of H 2 PtCl 6 (4 wt %, ) were added simultaneously, and stirring was continued at room temperature for 6 h. The obtained solution was centrifuged at 8000 r/min for 10 min, and washed three times with ultrapure water. The obtained precipitate was redispersed into 2 mL of PBS buffer solution (0.1 M pH 7.0), and stored at 4°C for later use.
(2)AuPt-MB-Ab2纳米信号探针的制备:(2) Preparation of AuPt-MB-Ab 2 nano-signal probe:
取100μL Gal-3特异性检测抗体Ab2加入至2mL AuPt-MB纳米复合材料溶液中,在4℃条件下温和搅拌,持续12h。加入40μL牛血清白蛋白BSA(1wt%)在4℃条件下持续温和搅拌6h。所得溶液经8000r/min,离心10min,将所得沉淀再分散至2mLPBS缓冲溶液(0.1M pH7.0)中,4℃保存备用。Add 100 μL of Gal-3-specific detection antibody Ab 2 to 2 mL of AuPt-MB nanocomposite solution, and stir gently at 4°C for 12 h. 40 μL of bovine serum albumin BSA (1 wt%) was added and gentle stirring was continued for 6 h at 4°C. The obtained solution was centrifuged at 8000 r/min for 10 min, and the obtained precipitate was redispersed into 2 mL of PBS buffer solution (0.1 M pH 7.0), and stored at 4° C. for later use.
本发明所述的建立电化学免疫生物传感器,检测血清中Gal-3,绘制标准曲线,其特征在于包括以下步骤:The establishment of an electrochemical immune biosensor according to the present invention, detecting Gal-3 in serum, and drawing a standard curve, is characterized in that the following steps are included:
(1)分别用0.3和0.05μm的Al2O3粉末将电极抛光成镜面,然后分别按超纯水、无水乙醇、超纯水的顺序超声电极各5min,室温干燥备用;(1) Polish the electrode into a mirror surface with Al 2 O 3 powder of 0.3 and 0.05 μm, respectively, then ultrasonically sonicate the electrode in the order of ultrapure water, absolute ethanol, and ultrapure water for 5 minutes each, and dry at room temperature for use;
(2)将6μL所制备的N-GNRs-Fe-MOFs@AuNPs纳米复合材料溶液滴加在电极表面,室温干燥。(2) 6 μL of the prepared N-GNRs-Fe-MOFs@AuNPs nanocomposite solution was dropped on the electrode surface and dried at room temperature.
(3)滴加6μL Gal-3特异性捕获抗体Ab1溶液至被修饰的电极表面,4℃孵育12h。(3) Add 6 μL of Gal-3 specific capture antibody Ab 1 solution dropwise to the modified electrode surface, and incubate at 4° C. for 12 h.
(4)用双蒸水将孵育后的电极冲洗干净,在电极表面滴加6μL BSA(1wt%)封闭剂,室温封闭30min。(4) Rinse the incubated electrode with double distilled water, drop 6 μL of BSA (1 wt %) blocking agent on the surface of the electrode, and block at room temperature for 30 min.
(5)将上述BSA封闭后的电极用清洗缓冲液(10mM Na2HPO4,2mM KH2PO4,37mMNaCl,2.7mM KCl,pH 7.4)冲洗干净并在氮气中干燥。(5) The above-mentioned BSA-blocked electrode was rinsed with washing buffer (10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 37 mM NaCl, 2.7 mM KCl, pH 7.4) and dried in nitrogen.
(6)将6μL不同浓度的目标Gal-3滴加至电极表面,37℃孵育1h用清洗缓冲液冲洗干净并在氮气中干燥。(6) 6 μL of different concentrations of target Gal-3 were added dropwise to the electrode surface, incubated at 37° C. for 1 h, rinsed with washing buffer, and dried in nitrogen.
(7)在干燥后电极表面滴加10μL所制备的AuPt-MB-Ab2纳米信号探针溶液,37℃孵育1h用清洗缓冲液冲洗干净并在氮气中干燥。(7) Drop 10 μL of the prepared AuPt-MB-Ab 2 nano-signal probe solution on the electrode surface after drying, incubate at 37°C for 1 h, rinse with washing buffer and dry in nitrogen.
(8)将电极置于5mL,0.1M PBS(PH 7.0)中进行表征,测量其电流变化电流响应峰值。(8) The electrode was placed in 5mL, 0.1M PBS (PH 7.0) for characterization, and the peak value of its current change current response was measured.
(9)根据所得电流变化电流响应峰值与Gal-3浓度呈线性关系,绘制工作曲线。(9) According to the obtained current change, the current response peak value and the Gal-3 concentration have a linear relationship, and the working curve is drawn.
与现有技术相比,本发明的一种用于血清中galectin-3(Gal-3)浓度检测的电化学传感器制备方法及应用,其突出的特点是:Compared with the prior art, a preparation method and application of an electrochemical sensor for the detection of galectin-3 (Gal-3) concentration in serum of the present invention, its outstanding features are:
(1)将N-GNRs-Fe-MOFs@AuNPs纳米复合材料用于电化学免疫生物传感器基底修饰,不仅提供了大量的活性位点固载捕获抗体,而且促进了电子传导速率,进而提高了电化学免疫生物传感器的灵敏度和生物相容性;(1) The N-GNRs-Fe-MOFs@AuNPs nanocomposite was used for electrochemical immunobiosensor substrate modification, which not only provided a large number of active sites to immobilize the capture antibody, but also promoted the electron conduction rate, thereby improving the electrical conductivity. Sensitivity and biocompatibility of chemoimmunobiosensors;
(2)将基于AuPt-MB的纳米复合材料构建信号探针引入到电化学免疫生物传感器的制备中,其中AuPt双金属纳米材料不仅具有放大其电化学信号功能,而且增加了其对检测抗体的生物相容性。(2) The AuPt-MB-based nanocomposite construction signal probe was introduced into the preparation of electrochemical immunobiosensors, in which the AuPt bimetallic nanomaterial not only has the function of amplifying its electrochemical signal, but also increased its ability to detect antibodies. Biocompatibility.
(3)本方法制备的电化学免疫生物传感器可为导致胰岛素灵敏度降低的Gal-3血液浓度水平检测提供新思路,为2型糖尿病的预防提供早期诊断依据。(3) The electrochemical immunobiosensor prepared by this method can provide a new idea for the detection of Gal-3 blood concentration level which reduces insulin sensitivity, and provide an early diagnosis basis for the prevention of
(4)使用完全相同的纳米材料和修饰方法,利用捕获抗体,信号探针与目标蛋白的特异性识别,只需通过改变探针的检测抗体和固载在基底的捕获抗体即可实现多种蛋白生物标志的特异性,高灵敏检测,另外,此方法简便,快速,便于实现商品化,从而推进转化医学的发展。(4) Using the exact same nanomaterials and modification methods, using the capture antibody, the specific recognition of the signal probe and the target protein, it is only necessary to change the detection antibody of the probe and the capture antibody immobilized on the substrate to achieve a variety of Specific and highly sensitive detection of protein biomarkers, in addition, this method is simple, fast, and easy to realize commercialization, thereby promoting the development of translational medicine.
附图说明:Description of drawings:
图1为本发明的电化学免疫生物传感器的构建示意图。FIG. 1 is a schematic diagram of the construction of the electrochemical immunobiosensor of the present invention.
图2为本发明的N-GNRs、Fe-MOFs、Fe-MOFs@AuNPs和N-GNRs-Fe-MOFs@AuNPs的扫描电镜图,AuPt-MB的透射电图镜和EDS图。Figure 2 is the scanning electron microscope image of N-GNRs, Fe-MOFs, Fe-MOFs@AuNPs and N-GNRs-Fe-MOFs@AuNPs of the present invention, and the transmission electron microscope and EDS images of AuPt-MB.
图3为Fe-MOFs@AuNPs EDS图Figure 3 shows the EDS image of Fe-MOFs@AuNPs
图4为本发明的电化学免疫生物传感器在检测Gal-3时得到的差示脉冲伏安法的电流响应峰值与浓度对数的线性关系。FIG. 4 is the linear relationship between the current response peak value of differential pulse voltammetry and the logarithm of concentration obtained by the electrochemical immunobiosensor of the present invention when Gal-3 is detected.
图5为传感器的重复性、特异性和稳定性。Figure 5 shows the repeatability, specificity and stability of the sensor.
具体实施方式:Detailed ways:
下面结合具体实施例对本发明进行进一步阐述,应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。The present invention will be further described below with reference to specific embodiments, and it should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.
实施例1Example 1
步骤1.分别称取0.187g FeCl3·6H2O和0.126g 2-氨基对苯二甲酸加入15mL的二甲基甲酰胺(DMF)溶液中,混匀。将上述混合溶液置于120℃硅油中加热4h,在开始加热后15min时,向混合溶液中加入200μL的冰醋酸。加热结束后控制溶液降温至室温,所得溶液经10000r/min,离心5min,分别用DMF,无水乙醇,超纯水各清洗3次。将所得沉淀置于真空干燥24h后,得到Fe-MOFs,4℃保存备用。
步骤2.取2mL HAuCl4·4H2O(1%)溶液加入到制备好的2mL Fe-MOFs(1mg mL-1)溶液中,剧烈超声30分钟后,向上述溶液中逐滴缓慢加入4mL NaBH4(0.1M)同时放在磁力搅拌器上进行搅拌反应30min,所得溶液经10000r/min,离心5min,用超纯水清洗3次。得到Fe-MOFs@AuNPs,将所得Fe-MOFs@AuNPs沉淀物再分散至4mL的去离子水中,4℃保存备用。
步骤3.称取0.2mg N-GNRs超声溶解于2mL的去离子水中,将其与4mL Fe-MOFs@AuNPs复合物溶液超声混匀30min,在磁力搅拌500r/min条件下,搅拌过夜。所得溶液经8000r/min,离心5min,用超纯水清洗3次。得到N-GNRs-Fe-MOFs@AuNPs,将所得N-GNRs-Fe-MOFs@AuNPs沉淀物再分散至4mL的去离子水中,4℃保存备用。
步骤4.分别取600μL HCL(0.1M)和6mL十二烷基三甲基溴化铵(DTAB 1.14mM)加入至1mL的MB(9.8mM)溶液中,剧烈搅拌10min。再同时加入100μL HAuCl4(4wt%,)和100μLH2PtCl6(4wt%,),持续室温搅拌6h。所得溶液经8000r/min,离心10min,用超纯水清洗3次。将所得AuPt-MB沉淀物再分散至2mL PBS缓冲溶液(0.1M pH 7.0)中,4℃保存备用。
步骤5.取100μL Gal-3特异性检测抗体Ab2加入至2mLAuPt-MB纳米复合材料溶液中,在4℃条件下温和搅拌,持续12h。加入40μL牛血清白蛋白BSA(1wt%)在4℃条件下持续温和搅拌6h。所得溶液经8000r/min,离心10min,得到AuPt-MB-Ab2信号探针,将所得信号探针沉淀物再分散至2mL PBS缓冲溶液(0.1M pH 7.0)中,4℃保存备用。
步骤6.分别用0.3和0.05μm的Al2O3粉末将电极抛光成镜面,然后分别按超纯水、无水乙醇、超纯水的顺序超声电极各5min,室温干燥备用;
步骤7.将6μL所制备的N-GNRs-Fe-MOFs@AuNPs纳米复合材料溶液滴加在电极表面,室温干燥。Step 7.
步骤8.滴加6μL Gal-3特异性捕获抗体Ab1溶液至被修饰的电极表面,4℃孵育12h。
步骤9.用双蒸水将孵育后的电极冲洗干净,在电极表面滴加6μLBSA(1wt%)封闭剂,室温封闭30min。Step 9. Rinse the incubated electrode with double distilled water, drop 6 μL BSA (1 wt %) blocking agent on the surface of the electrode, and block at room temperature for 30 min.
步骤10.将上述BSA封闭后的电极用清洗缓冲液(10mM Na2HPO4,2mM KH2PO4,37mMNaCl,2.7mM KCl,pH 7.4)冲洗干净并在氮气中干燥。
步骤11.将6μL不同浓度的目标Gal-3滴加至电极表面,37℃孵育1h用清洗缓冲液冲洗干净并在氮气中干燥。Step 11.
步骤12.在干燥后电极表面滴加10μL所制备的AuPt-MB-Ab2纳米信号探针溶液,37℃孵育1h用清洗缓冲液冲洗干净并在氮气中干燥。Step 12. Drop 10 μL of the prepared AuPt-MB-Ab 2 nano-signal probe solution on the electrode surface after drying, incubate at 37° C. for 1 h, rinse with washing buffer and dry in nitrogen.
步骤13.将电极置于5mL,0.1M PBS(PH 7.0)中进行表征,测量其电流变化电流响应峰值。Step 13. The electrode was placed in 5mL, 0.1M PBS (PH 7.0) for characterization, and its current change peak value of current response was measured.
步骤14.根据所得电流变化电流响应峰值与Gal-3浓度呈线性关系,绘制工作曲线。测定结果表明Gal-3浓度在100fg mL-1-50ng mL-1范围内成线性关系,线性相关系数为0.99502,检测限为33.33fg mL-1(S/N=3)。Step 14. According to the obtained current change, the current response peak value and the Gal-3 concentration have a linear relationship, and draw a working curve. The results showed that the concentration of Gal-3 had a linear relationship in the range of 100fg mL -1 -50ng mL -1 , the linear correlation coefficient was 0.99502, and the detection limit was 33.33fg mL -1 (S/N=3).
步骤15.将本发明上述传感器于4℃保存,间断用差示脉冲伏安法检测传感器电流响应,储存3周后电流响应仍为初始电流的95.17%,表面传感器具有良好的稳定性;
步骤16.本发明取同一批次制备的免疫生物传感器5支,在相同条件下对1ng mL-1的Gal-3分别进行测定,每一支电极测定3次,结果电流响应值的相对标准偏差为2.75%,说明传感器的重复性良好。Step 16. The present invention takes 5 immunobiosensors prepared in the same batch, and
步骤17.将本发明上述传感器用于检测100ng mL-1不同干扰物与1ng mL-1Gal-3的混合物,其检测结果的相对标准偏差为1.55%,说明干扰物对传感器检测结果干扰作用很小,传感器的特异性好。Step 17. The above-mentioned sensor of the present invention is used to detect the mixture of 100ng mL -1 of different interfering substances and 1 ng mL -1 Gal-3, and the relative standard deviation of the detection result is 1.55%, indicating that the interfering substances have a great interference effect on the detection result of the sensor. Small, the specificity of the sensor is good.
以上所述仅是本发明的优选实施方式,应当指出的是,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提条件下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and retouching should also be regarded as the protection scope of the present invention.
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