利用磁调制生物传感系统的生物检测的快速均质检测

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Summary

利用磁调制生物传感系统,迅速,灵敏,简单地检测生物检测的DNA分子和蛋白质,如。

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Danielli, A., Porat, N., Ehrlich, M., Arie, A. Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System. J. Vis. Exp. (40), e1935, doi:10.3791/1935 (2010).

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Abstract

磁调制生物传感系统(MMB)[1,2]迅速,并在没有任何清洗或分离步骤的低浓度均匀检测生物指标。当IL - 8的目标,目前,附有一个“sandwich'为基础的检测IL - 8的捕获抗体的磁珠的IL - 8的目标和生物素标记的IL - 8抗体的链霉通过耦合荧光蛋白。磁珠操纵振荡运动,通过采用两个电磁两极交变磁场梯度。凝结成的荧光蛋白质,这是附着在磁珠检测区域和他们的运动和正交激光束产生一个周期性的使用同步检测荧光信号解调。以前使用的磁调制生物传感系统检测茨城病毒非结构蛋白3(NS3区)互补DNA(cDNA)的[2]的编码序列。外部操纵和颗粒凝结在这项工作中表现出的技术可用于其他应用程序,如提供磁耦合的药物

Protocol

IL - 8含量:

  1. 反应混合物包括以下四个部分,在各自的浓度100μL缓冲液的最终体积。 10μL100珠/μL终浓度,2μL生物素IL - 8抗体nano-gram/μl终浓度为1,1μL和素荧光蛋白20nano-gram/μl终浓度与捕获抗体的磁珠, 0.48pico-gram/μl和1μl的IL - 8的目标。这些组件被添加缓冲液,然后30分钟动摇。
  2. 一个控制反应是准备没有IL - 8的目标相同的方式。
  3. 反应后放置的试管中,没有任何分离或洗涤步骤和检查使用MMB系统。

MMB的系统:

  1. 将在其两者之间的电磁极点地位的比色皿。
  2. 操作的调制电流,并等待30秒,让聚合和凝结珠
  3. 在锁定放大器使用示波器测量信号。

代表性的成果:

聚合珠的目视检查,提出了一个与目标之间的反应和没有目标的反应的明显差异。在所有的反应与目标,珠形成一个单一的,密集的聚集出的激光束在一个团结的方式提出。然而,在没有目标的反应,珠少汇总和它们的运动是不太团结(见图1)。

图1
图1:目视检查三明治“免疫法(一)没有目标的IL - 8(二)随着IL - 8的目标。

极调制时钟(黄色)和光电倍增管的输出信号(洋红色),同时检测0.48微微克的IL - 8的目标是在图1(a)。调制频率为每极2赫兹。然而,因为这是理论上的预期,珠通过激光束时,光电倍增管检测荧光光有一个光电倍增管的输出电压的峰值。因此,解调频率为4赫兹。当光电倍增管的信号和一倍调制时钟(4赫兹)是美联储锁定放大器中,敏感的阶段检测器检测到的同步和高电压(见图2)的结果。

图2
图2:(一)调制时钟(黄色)和光电倍增管的信号(洋红色)当检测0.48微微克的IL - 8的目标。 (b)在导致放大器的电压锁定在两个不同的扫描。

放大器的锁,没有检测到任何信号,当控制样品测试。这个事实,一起聚集在视觉上的差异表明,MMB系统可以清楚地识别存在的IL - 8的目标。

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Discussion

综上所述,我们发现,MMB系统可用于检测低浓度(0.5微微克的生物复杂精密临的细胞因子的检测[3,4]的检测限)的IL - 8的目标存在。该系统的能力并不局限于IL - 8和可用于检测其他蛋白质。 MMB系统的优点是能够迅速地检测到目标,并没有任何分离或洗涤步骤。因此,它有利于在检测过程中,允许系统在现场应用中使用。

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Acknowledgements

部分支持这项工作是由Ishaya霍洛维茨基金。

Materials

Name Company Catalog Number Comments
Bio-Plex Pro Human Cytokine IL-8 set Bio-Rad 171-B5008M
Recombinant human IL-8 target Thermo Fisher Scientific, Inc. R202625
Biotinylated monoclonal mouse anti human IL-8 antibody G265-8 BD Biosciences 554718
Streptavidin coupled fluorescent protein Bio-Rad Streptavidin PE

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References

  1. Danielli, A., Arie, A., Porat, N., Ehrlich, M. Detection of fluorescent-labeled probes at sub-picomolar concentrations by magnetic modulation. Optics Express. 16, 19253-19259 (2008).
  2. Danielli, A., Porat, N., Arie, A., Ehrlich, M. Rapid homogenous detection of the Ibaraki virus NS3 cDNA at pico-molar concentrations by magnetic modulation. Biosensors and Bioelectronics. 25, 858-863 (2009).
  3. Bio-Rad "Human angiogenesis assay panel". Bio-Plex Pro. Forthcoming.
  4. Instruction Manual. Bio-Plex Precision Pro Cytokine Assay. Bio-Rad Laboratories. Hercules, CA. Forthcoming.

Comments

2 Comments

  1. In regards to the IL-8 Assay: [1] Did you have a specific concentration values on hand? [²] Is there a specific pH range that you used? [3] How did you optimize so that the target antigen would bind to both the magnetic bead and the fluorescent protein?

    Reply
    Posted by: Anonymous
    August 19, 2010 - 2:26 PM
  2. Hi Michael,
    Here are the answers to your questions:
    [1] Did you have a specific concentration values on hand?

    We made several concentrations according to the Bio-Rad kit that we used. We chose to present the results with the smallest concentration that we made.

    [²] Is there a specific pH range that you used?

    We used the assay buffer of the kit. I guess that it had the optimal ph for the reaction.

    [3] How did you optimize so that the target antigen would bind to both the magnetic bead and the fluorescent protein?

    This kit is optimized already. You may check the reference list of the JoVE manuscript and see the kit&#x²019;s specifications. For further information on the MMB system as well as on the IL-8 kit, I suggest that you look at additional paper that we published: A. Danielli, N. Porat, M. Ehrlich, and A. Arie, &#x²01C;Magnetic modulation biosensing for rapid and homogeneous detection of biological targets at low concentrations&#x²01D;, Current Pharmaceutical Biotechnology, 11, 1²8-137 (²010)

    Best regards,

    Amos Danielli

    Reply
    Posted by: Amos D.
    November 2, 2010 - 3:44 PM

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