JoVE Science Education
Biochemistry
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JoVE Science Education Biochemistry
Surface Plasmon Resonance (SPR)
  • 00:00Overview
  • 01:06Principles of SPR
  • 03:49SPR Sample Preparation and Experimental Protocol
  • 04:58Applications
  • 06:28Summary

表面等离子体共振 (SPR)

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Overview

表面等离子体共振 (SPR) 是无标签生物传感器背后的基本光学现象, 用来评估生物分子的亲和性、动力学、特异性和浓度。在 SPR 中, 生物分子之间的相互作用发生在一个由薄薄的金属层在棱镜上。生物分子的实时相互作用可以通过测量反射在金属下侧的光的变化来监测.

该视频描述了 SPR 的基本概念, 以及它是如何用于分析和可视化生物分子相互作用的。接下来是一个样品制备和实验协议, 用于调查结合率利用 SPR。在应用部分, 对 spr 成像、局部 spr 和量子点增强 spr 进行了探讨.

表面等离子体共振 (或 SPR) 是某些无标签生物传感器背后的基本现象, 用于评估生物分子的结合和吸附作用。需要标记的结合化验, 例如 ELISA, 可能是一个费时的过程, 并且也许改变分析物的功能。在 SPR 中, 生物分子之间的相互作用发生在一个特殊的传感器, 由薄薄的金属层在一个棱镜的表面。通过监测金属底部反射光的变化, SPR 仪器可以在不使用标签的情况下 real-time 这些相互作用的可视化。本视频将介绍 spr 的基本原理, spr 成像的一般程序, 以及在生物化学中的一些应用.

SPR 传感器通常由在棱镜表面上的一层贵金属组成。为了从传感器中读取读数, 光线从棱镜金属界面反射到光电探测器中。反射光除了在一定角度外, 还具有高强度, 与金属表面的电子特性有关, 称为 #34; 表面等离子体共振角和 #34;.

当分子与表面结合时, 金属的电子性质会发生变化, 进而调整角度。随着新的蛋白质附着, 形成复合物, 角度将进一步转移。通过测量 SPR 角度的相对变化, 可以在 real-time 中监视类似的相互作用.

另一种称为本地化或 #34 的技术; L 和 #34; SPR, 使用金属纳米粒子作为传感器表面。影响 SPR 角度的特性高度地定位于每个纳米粒子, 从而提高灵敏度和信号分辨率.

当研究与标准 SPR 的绑定交互时, 传感器通常安装在一个平台上, 该工作台将成为仪器中流动单元的地板。感兴趣的生物分子通过缓冲液通过流动细胞。传感器表面通常先涂上具有高亲和力的基体。这就确保了大量的配体, 反过来对感兴趣的分析物, 将固定在传感器上, 并减少了在过程中配体将离解的可能性.

一旦配体固定在传感器上, 分析物就会流过传感器的缓冲液中。当分析物与配体结合时, 通过监测 SPR 角度随着时间的变化, 可以计算出束缚率和其他动力学信息.

反射率数据也可用于 SPR 成像或 SPRi, 方法是将反射光定向到 CCD 探测器。这将产生整个传感器表面的高对比度图像。利用 SPR 和相关技术, 可以回答有关分子亲和性、动力学、特异度和浓度等问题.

现在, 您了解在 SPR 实验中测量的是什么, 让 & #39; 我们来看一个调查绑定利率的程序.

在开始该过程之前, 必须准备运行和示例缓冲区。运行缓冲区用于将配体沉积到传感器上, 并使用样本缓冲区来存放分析物。传感器芯片被仔细地清洗并且被装载入鞘。然后, 该设备被放置到仪器, 它成为流细胞的底部。为实验和后续分析建立了仪器软件。如有必要, 传感器表面上配有衬底以捕获配体。所述配体流经传感器表面, 在所述运行缓冲器上, 在传感器表面上由所述基板捕获.

然后, 样本缓冲区中的分析物通过流动单元运行, 在这里它有选择地绑定到固定的配体。对反射率的变化进行了绘制和比较对照, 以确定速率常数和其他反应动力学数据的调查反应.

现在您了解了 spr 实验是如何执行的, 请 #39; 我们来看看在生物化学中 spr 的其他一些应用.

在这里, SPR 成像被用来评估蛋白质与阵列的十一受体在传感器上. 根据每种蛋白质的反射率数据, 制备了3D 反射率与时间和受体浓度的关系图。这些和 #34;p rofiles 和 #34; 是每个蛋白质的特征, 因此可以随后用于蛋白质鉴定.

在本实验中, 使用 custom-made LSPR 传感器对细胞分泌物进行了研究。该传感器还兼容 SPRi 和荧光显微镜。在传感器上沉积细胞后, 可以用高空间分辨率测量细胞分泌物与纳米粒子阵列的相互作用.

在这里, 量子点的使用, 纳米半导体, 作为 SPR 信号增强剂混合与分析物进行了研究。这种增强和 #34; 纳米 SPRi 和 #34; 方法与标准 SPRi 和 ELISA 法进行了比较。纳米 SPRi 法大大提高了检测的灵敏度和限制, 同时也比 ELISA 方法的耗时少.

您和 #39; 我刚刚看了朱庇特和 #39 的表面等离子体共振的视频。这种现象被用来监测和图像生物分子的相互作用, 而不使用标签。该视频介绍了 spr 的原理、spr 实验的典型协议以及 spr 在生物化学中的一些应用.

感谢收看!

Procedure

Surface plasmon resonance (SPR) is the underlying optical phenomenon behind label-free biosensors to evaluate the molecular affinity, kinetics, specificity, and concentration of biomolecules. In SPR, biomolecular interactions occur on a biosensor made of a thin layer of metal on a prism. Real-time interactions of biomolecules can be monitored by measuring the changes of light reflected off the underside of the metal. This video describes the basic concepts of SPR and how it is used to analyze and visualize biomolecular interactions. T…

Disclosures

No conflicts of interest declared.

Transcript

Surface plasmon resonance, or SPR, is the underlying phenomenon behind certain label-free biosensors for evaluating binding and adsorption interactions of biomolecules. Binding assays that require labeling, such as ELISA, can be a time-consuming process, and may alter the functionality of the analyte. In SPR, biomolecular interactions occur on a special sensor made of a thin layer of metal on one face of a prism. By monitoring the changes in light reflected off of the underside of the metal, SPR instruments visualize these interactions in real-time without the use of labels. This video will introduce the principles of SPR, a general procedure for SPR imaging, and some applications of in biochemistry.

An SPR sensor is usually made of a thin layer of a noble metal atop the face of a prism. To take readings from the sensor, light is reflected off of the prism-metal interface into a photodetector. The reflected light will have a high intensity except at a certain angle, related to the electronic properties of the metal surface, known as the “surface plasmon resonance angle”.

As molecules bind to the surface, the electronic properties of the metal change, which in turn adjusts the angle. As new proteins attach, forming complexes, the angle will shift further. By measuring relative changes in the SPR angle, interactions like these can be monitored in real-time.

Another technique called localized, or “L”SPR, uses metal nanoparticles as the sensor surface. The properties that affect the SPR angle are highly localized to each nanoparticle, which improves sensitivity and signal resolution.

When investigating binding interactions with standard SPR, the sensor is generally mounted in a platform that becomes the floor of a flow cell in the instrument. The biomolecules of interest are carried through the flow cell by buffer solution. The sensor surface is often first coated with a substrate that has a high affinity for the metal. This ensures that a significant amount of ligand, which in turn binds to the analyte of interest, will be immobilized onto the sensor and reduces the likelihood that the ligand will dissociate during the procedure.

Once the ligand is immobilized on the sensor, the analyte is flowed over the sensor in buffer. By monitoring the change in the SPR angle over time as the analyte binds to the ligand, the binding rate and other kinetic information can be calculated.

The reflectance data can also be used for SPR imaging, or SPRi, by directing the reflected light to a CCD detector. This produces a high-contrast, high-resolution image of the entire sensor surface. Using SPR and the related techniques, questions can be answered about molecular affinity, kinetics, specificity, and concentration.

Now that you understand what is being measured in an SPR experiment, let’s look at a procedure for investigating binding rates.

Before beginning the procedure, the running and sample buffers must be prepared. The running buffer is used to deposit the ligand onto the sensor, and the sample buffer is used to deposit the analyte. The sensor chip is carefully cleaned and loaded into a sheath. Then, the device is placed into the instrument, where it becomes the bottom of the flow cell. The instrument software is set up for the experiment and subsequent analysis. If necessary, the sensor surface is primed with a substrate to capture the ligand. The ligand is flowed over the sensor surface in the running buffer, where it is captured by the substrate on the sensor surface.

Then, the analyte in the sample buffer is run through the flow cell, where it selectively binds to the immobilized ligand. The change in reflectance is plotted and compared against controls to determine rate constants and other reaction kinetics data for the investigated reaction.

Now that you understand how an SPR experiment is performed, let’s look at a few other applications of SPR in biochemistry.

Here, SPR imaging was used to evaluate proteins with an array of eleven receptors on a sensor. 3D graphs of reflectivity versus time and receptor concentration were prepared from the reflectivity data for each protein. These “profiles” are characteristic to each protein, and thus could subsequently be used for protein identification.

In this experiment, cell secretions were studied using a custom-made LSPR sensor. The sensor was also compatible with SPRi and fluorescence microscopy. Upon depositing the cell on the sensor, the interaction of cell secretions with the nanoparticle array could be measured with high spatial resolution.

Here, the use of quantum dots, nanoscale semiconductors, as an SPR signal enhancement agent mixed with the analyte was investigated. This enhanced “nano-SPRi” method was compared to assays by standard SPRi and the ELISA method. The nano-SPRi method significantly improved the sensitivity and limit of detection while still being less time-consuming than the ELISA method.

You’ve just watched JoVE’s video on surface plasmon resonance. This phenomenon is used to monitor and image biomolecular interactions without the use of labels. This video introduced the principles of SPR, a typical protocol for performing an SPR experiment, and a few applications of SPR in biochemistry.

Thanks for watching!

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JoVE Science Education Database. JoVE Science Education. Surface Plasmon Resonance (SPR). JoVE, Cambridge, MA, (2023).

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