WO2021046740A1 - 一种基于表面增强拉曼技术的生物传感器及制备方法 - Google Patents

一种基于表面增强拉曼技术的生物传感器及制备方法 Download PDF

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WO2021046740A1
WO2021046740A1 PCT/CN2019/105287 CN2019105287W WO2021046740A1 WO 2021046740 A1 WO2021046740 A1 WO 2021046740A1 CN 2019105287 W CN2019105287 W CN 2019105287W WO 2021046740 A1 WO2021046740 A1 WO 2021046740A1
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detection
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gold core
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张茂峰
熊良钟
熊清爵
陈敏
阮志燕
王振
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亳州市新健康科技有限公司
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  • the invention relates to the field of Raman scattering, in particular to a sandwich immune detection biosensor based on surface enhanced Raman technology.
  • the commonly used immunoassay method is enzyme-linked immunosorbent assay (ELISA), which specifically binds antibodies to enzyme complexes, and then performs color detection to detect trace substances in body fluids.
  • ELISA enzyme-linked immunosorbent assay
  • the detection limit of this method can reach pg/ml, however, substances with higher sensitivity requirements cannot be detected.
  • SERS surface-enhanced Raman
  • the current selection of SERS detection substrate material is used to bond Manra detection molecules, of which the best effect is the P-ATP molecule, the sensitivity is ng/ml-pg/ml, although the P-ATP is bonded After the molecule, the sensitivity of SERS detection has been improved, but the detection sensitivity and detection range width required by many detected objects are higher.
  • the present invention provides a sandwich immunodetection biosensor based on surface-enhanced Raman technology, including:
  • the top layer is a gold core-silver shell nanorod bonded with vitamin K molecules, a functional modification and a detection antibody; and the substrate is a gold core silver shell nanorod bonded with a capture antibody.
  • the functional modification includes glutaraldehyde and cysteamine.
  • biosensor will form a sandwich structure of "detection antibody-antigen to be tested-capture antibody" during the working process.
  • the present invention provides a sandwich immune detection biosensor based on surface enhanced Raman technology.
  • Both the top layer and the substrate adopt gold core silver shell nanorods as base materials.
  • the gold core silver shell nanorods can significantly improve the surface Raman detection process.
  • the top layer of gold-core-silver-shell nanorods firmly bond the diazotized sulfhydryl vitamin K4 molecules through the sulfhydryl group, so that the signal is further enhanced significantly, and at the same time, the functionalized modified molecule E is bonded Dialdehyde and cysteine make the top gold-core-silver-shell nanorods firmly immobilize the detection antibody, and the gold-core-silver-shell nanorods as the substrate are bonded to the capture antibody, which is used to fix the test antigen through specific binding.
  • the sandwich immune detection biosensor based on surface enhanced Raman technology provided by the present invention can significantly improve the sensitivity of surface enhanced Raman detection.
  • Figure 1 shows the sandwich structure of "detection antibody-antigen to be tested-capture antibody” formed by the biosensor and the antigen to be tested in the present invention
  • Figure 2 shows the Raman spectrum of the SERS immunoassay with IL-6 concentration of 1fg/mL and a blank sample in Example 1;
  • Figure 3 shows the curve of Raman intensity versus KIM1 concentration
  • Figure 4 shows the variation curve of the Raman intensity of the peak at 1145 cm -1 with the concentration of KIM1, and the inset shows its linear dynamic response range.
  • This embodiment provides a sandwich immunoassay biosensor based on surface-enhanced Raman technology.
  • the sensor is used to perform SERS immunoassay on IL-6 solutions and blank samples with a concentration of 1fg/mL.
  • the specific process is as follows:
  • step 6) Repeat step 5) three times, and store the precipitate in CTAC solution to obtain gold nanorod solution
  • the gold core silver shell nanorod solution was functionalized with 25 mM cysteamine solution, and then 2 ⁇ L of 25% wt glutaraldehyde solution was added to functionalize the gold core silver shell nanorod solution, and then the gold core silver shell nanorod solution was functionalized.
  • This embodiment provides a sandwich immune detection biosensor based on surface-enhanced Raman technology, which is used to measure KIM1 in artificial urine, specifically as follows:
  • the top layer is bonded with KIM1 detection antibody, and the substrate is bonded with KIM1 capture antibody, as follows:
  • the gold core silver shell nanorod solution was functionalized with 150 mM cysteamine solution. Then, 12 ⁇ L of 25% wt glutaraldehyde solution was added to functionalize the gold core silver shell nanorod solution. Add 12mL of 20 ⁇ g/mL KIM1 capture antibody solution to the transformed gold core silver shell nanorod solution and store it at 4°C for 12 hours. Next, add 12mL of 1% BSA solution to the stored solution and let it stand for 1 hour. In order to block non-specific binding to the active site, a substrate structure solution is obtained.
  • the substrate solution was equally divided into 6 parts, and 6 kinds of urine to be tested were added to the 6 parts of substrate solution respectively, and incubated at room temperature for 1 hour to obtain the KIM1 urine to be tested.

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Abstract

一种基于表面增强拉曼技术的生物传感器及制备方法，生物传感器为三层夹心结构，包括上层结构：键合拉曼检测分子和检测抗体的金核银壳纳米棒；中层结构：抗原；下层结构：键合捕获抗体的金核银壳纳米棒，基于表面增强拉曼技术的夹心免疫检测生物传感器通过拉曼检测分子与金核银壳纳米棒的结合，大大增强了检测信号，对于超痕量物质及单分子检测的灵敏度显著提高，最低限可达fg/mL。

Description

一种基于表面增强拉曼技术的生物传感器及制备方法 技术领域
本发明涉及拉曼散射领域，特别涉及一种基于表面增强拉曼技术的夹心免疫检测生物传感器。
背景技术
目前，普遍使用的免疫测定方法为酶联免疫吸附测定法(ELISA)，该方法通过抗体与酶复合物特异性结合，然后进行显色检测来检测体液中微量物质，该方法的检测限可达到pg/ml，但是，对于灵敏度要求更高的物质则无法检测出。
因此，为了能够可靠、准确地检测超痕量甚至单分子水平的分析物，表面增强拉曼(SERS)逐渐的发展起来，并广泛应用于各种化学传感、生物分析、生物传感和早期癌症诊断等领域。典型的SERS免疫分析检测方法是基于夹心免疫复合物能够捕获抗体固定在固体底物上。从样品溶液中捕获相应的抗原，并在三明治结构形成后通过SERS标记抗体的特征拉曼光谱进行检测。定量信息从剂量依赖性SERS实验中获得。
Schlücker及其合作者在金/银纳米壳-玻璃切片夹层结构基础上开发了夹心型SERS免疫分析法用于检测白细胞介素-6(IL-6)，其检测限(LOD)为1pg/mL。近年，基于银-金双金属表面-金纳米颗粒夹层结构，在皮克检测灵敏度下实现了对IL-6、IL-8和IL-18的同时多重识别。此外，在金底物-金纳米颗粒夹层平台上已经证实了对来自胰腺癌患者的血清样品中CA19-9，MMP7和MUC4的多重检测，检测限低至2ng/mL。然而，以往的夹层免疫检测设计方法多采用光滑的宏观玻璃、金、银膜或其双金属膜衬底，导致灵敏度有限。
为了提高SERS检测的灵敏度，目前选用在SERS检测的基底材料 上键合曼拉检测分子，其中效果最佳的为P-ATP分子，灵敏度为ng/ml-pg/ml，虽然键合P-ATP分子后，SERS检测的灵敏度有所提升，但是很多被检测物所需要的检测灵敏度以及检测范围宽度要求更高。
发明内容
本发明为了解决现有技术中夹心型SERS免疫分析法灵敏度有限的问题，提供了一种基于表面增强拉曼技术的夹心免疫检测生物传感器，包括：
顶层，为金核银壳纳米棒键合维生素K分子、功能化修饰物以及检测抗体；和衬底，为金核银壳纳米棒键合捕获抗体。
进一步地，所述功能化修饰物包括戊二醛和半胱胺。
进一步地，所述生物传感器在工作过程中会形成“检测抗体-待测抗原-捕获抗体”夹心结构。
本发明提供一种基于表面增强拉曼技术的夹心免疫检测生物传感器，顶层和衬底均采用金核银壳纳米棒作为基底材料，金核银壳纳米棒可以显著的提高表面拉曼检测过程中的电磁信号，在此基础上，顶层的金核银壳纳米棒通过巯基牢固的键合经重氮化巯解的巯基维生素K4分子，使其信号进一步显著增强，同时键合功能化修饰分子戊二醛和半胱氨酸使顶层金核银壳纳米棒可以牢固的固定检测抗体，作为衬底的金核银壳纳米棒键合捕获抗体，用于通过特异性结合固定待测抗原，综合以上结构，本发明提供的一种基于表面增强拉曼技术的夹心免疫检测生物传感器可以显著的提高表面增强拉曼检测的灵敏度。
应当理解，前述大体的描述和后续详尽的描述均为示例性说明和解释，并不应当用作对本发明所要求保护内容的限制。
附图说明
参考随附的附图，本发明更多的目的、功能和优点将通过本发明实 施方式的如下描述得以阐明，其中：
图1示出本发明中生物传感器与待测抗原形成的“检测抗体-待测抗原-捕获抗体”夹心结构；
图2示出实施例1中具有1fg/mL的IL-6浓度和空白样品的SERS免疫测定的拉曼光谱；
图3示出拉曼强度随KIM1浓度变化曲线；
图4示1145cm -1峰值的拉曼强度随KIM1浓度的变化曲线，插图显示了其线性动态响应范围。
具体实施方式
通过参考示范性实施例，本发明的目的和功能以及用于实现这些目的和功能的方法将得以阐明。然而，本发明并不受限于以下所公开的示范性实施例；可以通过不同形式来对其加以实现。说明书的实质仅仅是帮助相关领域技术人员综合理解本发明的具体细节。
在下文中，将参考附图描述本发明的实施例。在附图中，相同的附图标记代表相同或类似的部件，或者相同或类似的步骤。
实施例1
本实施例提供一种基于表面增强拉曼技术的夹心免疫检测生物传感器，使用该传感器对具有1fg/mL浓度的IL-6溶液和空白样品进行SERS免疫测定，具体过程如下：
1、金纳米棒的制备
1)在20mL的玻璃瓶中将0.1mL浓度为25mM HAuCl 4溶液用去离子水稀释至5mL，向稀释液中加入5mL 0.2M的CTAB溶液，得到溶液一；
2)将0.6mL0.01M的NaBH 4溶液快速注入溶液一中，NaBH 4溶液现用现配，对混合溶液进行磁力搅拌，速度为1200rpm，搅拌2min，最 后获得的种子溶液在30℃静置30min待用；
3)将7.0g CTAB和1.234g油酸钠溶解于水中250mL 50℃水中，自然冷却到30℃，然后加入18ml,4.0mM的硝酸银溶液，保温1min，得到溶液二；
4)一边磁力搅拌一边向溶液二中注射250ml 1.0mM的HAuCl 4溶液，在700rpm速度下搅拌90min，改变第二次磁力搅拌速度为400rpm，一边搅拌一边加入2.1ml 37wt％的HCl溶液，搅拌15min，最后加入1.25ml 0.064M的抗坏血酸溶液，进行第三次搅拌，搅拌速度为1200rpm，搅拌30s，得到生长液；
5)将0.4mL种子溶液注入生长液中1500rpm速度下搅拌30s，最后，将混合液在30℃条件下静置10h，将静置后的混合液在8000r/min的速度速度下离心10min，收集沉淀物，将沉淀物分散于80mM的CTAC溶液中；
6)将步骤5)重复三次，将所述得沉淀物储存在CTAC溶液中，得到金纳米棒溶液
2、金核银壳纳米棒的制备
将0.5mL金纳米棒溶液用水稀释至4ml，向稀释液中加入2.5ml 10mM的硝酸银溶液，在1000Hz频率下超声波处理2min，后加入2.5ml 0.1M的抗坏血酸溶液，经65℃水浴保存4h、8000r/min离心10min，收集沉淀物分散到1ml去离子水中，得到金核银壳纳米棒悬浮液。
3、拉曼检测分子的键合
将2μL,5mM的维生素K4乙醇溶液经重氮化巯解引入巯基，再添加到1.0ml金核银壳纳米棒悬浮液中得到混合物，温和摇动混合物2h，得到维生素K4标记的金核银壳纳米棒溶液。
4、带标记检测抗体的顶层结构制备
将2μL，5mM的生素K4乙醇溶液经重氮化巯解引入巯基，再添加到1.0ml金核银壳纳米棒溶液中，然后将混合物温和摇动2h得到维生素K4标记的金核银壳纳米棒溶液，然后在7000rpm下离心10分钟并且分散到1.0mL去离子水中。接下来，将2μL，25％wt戊二醛溶液添加到1.0ml维生素K4标记的金核银壳纳米棒溶液中，轻度摇动1.5h，产物在6000rpm下离心10min，然后分散到1mL，9μg/mL的IL-6的检测抗体溶液中，将该混合溶液在4℃条件下保存12h，然后用1.0mL 1×PBS缓冲液离心纯化，在4℃条件下保存待用。
5、带捕获抗体的衬底结构制备
首先用25mM的半胱胺溶液对金核银壳纳米棒溶液进行氨基功能化处理，然后，加入2μL，25％wt戊二醛溶液，使金核银壳纳米棒溶液功能化，之后在功能化后的金核银壳纳米棒溶液中加入2mL，20μg/mL IL-6的捕获抗体溶液，于4℃温度下保存12h，接下来，向保存后的溶液加入2mL，1％BSA溶液，静置1h以阻断非特异性结合活性位点。
6、IL-6抗原的固定
分别将200μL，1fg/mL的IL-6抗原和空白样品滴在带捕获抗体的衬底上，并在室温下温育1h。
7、“检测抗体-待测抗原-捕获抗体”夹心结构组装
将200μL维生素K4标记的，键合IL-6检测抗体的金核银壳纳米棒溶液滴在已固定好待测抗原的衬底溶液中，经特异性结合后，形成了检测抗体-待测抗原-捕获抗体“夹心”结构。
实施例2
本实施例提供一种基于表面增强拉曼技术的夹心免疫检测生物传感器，使用该传感器对人工尿液中的KIM1进行测定，具体如下：
使用实施例1中的基于表面增强拉曼技术的夹心免疫检测生物传感 器的结构，顶层键合KIM1检测抗体，衬底键合KIM1捕获抗体，具体如下：
首先，取6.0ml带维生素K4标记的金核银壳纳米棒溶液，在7000rpm下离心10分钟并且分散到6.0mL去离子水中。接下来，将12μL，25％wt戊二醛溶液添加到6.0ml维生素K4标记的金核银壳纳米棒溶液中，轻度摇动1.5h，产物在6000rpm下离心10min，将沉淀物分散到6ml，9μg/mL的KIM1检测抗体溶液中，得到顶层结构溶液，将该混合溶液在4℃条件下保存12h，然后用1.0mL 1×PBS缓冲液离心纯化，在4℃条件下保存待用。
然后，用150mM的半胱胺溶液对金核银壳纳米棒溶液进行氨基功能化处理，然后，加入12μL，25％wt戊二醛溶液，使金核银壳纳米棒溶液功能化，之后在功能化后的金核银壳纳米棒溶液中加入12mL，20μg/mL KIM1的捕获抗体溶液，于4℃温度下保存12h，接下来，向保存后的溶液加入12mL，1％BSA溶液，静置1h以阻断非特异性结合活性位点，得到衬底结构溶液。
取6份人工尿液溶液各200μL，分别在6份溶液中加入浓度为1ng/mL、10pg/mL、100fg/mL、1fg/mL、0.1fg/mL和0fg/mL的KIM1，混合均匀后得到待测尿液，待用。
将衬底溶液平均分为6份，分别在6份衬底溶液中加入6种待测尿液，在室温下温育1h，得到待测KIM1尿液。
最后，在每份待测KIM1尿液中加入200μL顶层结构溶液，经特异性结合后形成待测的检测抗体-待测抗原-捕获抗体“夹心”结构溶液。
试验例1
采用Renishaw inVia共焦拉曼光谱仪，以20倍物镜和785nm激光为激发源，在徕卡显微镜上采集实施例1中样品拉曼光谱。光谱在 800-1800cm -1范围内，曝光时间为10s，测的结果如图2所示，结果表明，本发明提供的生物传感器对IL-6的检测限度可达到fg/ml。
试验例2
采用Renishaw inVia共焦拉曼光谱仪，以20倍物镜和785nm激光为激发源，在徕卡显微镜上采集实施例2中样品拉曼光谱。光谱在800-1800cm -1范围内，曝光时间为10s，测的结果如图3-4所示，从图5可以看出拉曼强度随KIM1浓度的增加呈单调下降趋势，从图4中可以看出1145cm -1峰值的拉曼强度随KIM1浓度的变化，图4中的插图显示了其较宽的线性动态响应范围，从1ng/mL到0.1fg/mL。
结合这里披露的本发明的说明和实践，本发明的其他实施例对于本领域技术人员都是易于想到和理解的。说明和实施例仅被认为是示例性的，本发明的真正范围和主旨均由权利要求所限定。

Claims (4)

  1. 一种基于表面增强拉曼技术的夹心免疫检测生物传感器，其特征在于，所述生物传感器包括：
    顶层，为金核银壳纳米棒键合拉曼检测分子、功能化修饰物以及检测抗体；和衬底，为金核银壳纳米棒键合捕获抗体。
  2. 根据权利要求1所述的生物传感器，其特征在于，所述拉曼检测分子为维生素K4，所述维生素K4分子经重氮化巯解引入巯基。
  3. 根据权利要求1所述的生物传感器，其特征在于，所述功能化修饰物包括戊二醛和半胱胺。
  4. 根据权利要求1所述的生物传感器，其特征在于，所述生物传感器在工作过程中会形成“检测抗体-待测抗原-捕获抗体”夹心结构。
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