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 JoVE Biology

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

1,2, 3, 4, 1

1Department of Physical Electronics, Faculty of Engineering, Tel Aviv University, 2Department of Biomedical Engineering, Washington University in St. Louis, 3Department of Biological Sciences, University of Illinois, 4Department of Cell Research and Immunology, Tel Aviv University

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    Summary

    Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

    Date Published: 6/13/2010, Issue 40; doi: 10.3791/1935

    Cite this Article

    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).

    Abstract

    A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

    Protocol

    IL-8 assay:

    1. The reaction mixture included the following four components in 100 μl final volume of assay buffer in their respective concentrations. 10 μl of magnetic beads with capture antibody at 100 beads/μl final concentrations, 2 μl of biotinylated IL-8 antibody at 1 nano-gram/μl final concentration, 1 μl of streptavidin fluorescent protein at 20 nano-gram/μl final concentration, and 1 μl of IL-8 target at 0.48 pico-gram/μl. The components are added one by one to the assay buffer and are then shaken for 30 minutes.
    2. A control reaction is prepared the same way without the IL-8 target.
    3. The reactions are later placed without any separation or washing step in the cuvettes and inspected using the MMB system.

    MMB system:

    1. Place the cuvette in its position between the two electromagnetic poles.
    2. Operate the modulation current and wait 30 seconds to allow aggregation and condensation of the beads
    3. Measure the signal of the lock-in amplifier using an oscilloscope.

    Representative Results:

    Visual inspection of the aggregated beads presented a distinct difference between the reaction with the target and the reaction without the target. In all the reactions with the target, the beads formed a single, dense aggregate that moved in and out the laser beam in a unite manner. However, in all the reaction without the target, the beads were less aggregated and their motion was less unite (see Figure 1).

    Figure 1
    Figure 1: visual inspection of the sandwich' immunoassay (a) without the target IL-8 (b) With the target IL-8.

    The pole modulation clock (yellow) and the PMT output signal (magenta) while detecting 0.48 pico-gram IL-8 target are presented in Figure 1(a). The modulation frequency for each pole is at 2 Hz. However, as it was theoretically expected, when the beads pass the laser beam, the PMT detects the fluorescent light and there is a peak in the PMT output voltage. Therefore, the demodulation frequency is at 4 Hz. When the PMT signal and the doubled-modulation clock (at 4 Hz) are fed to the lock in amplifier, the sensitive phase detector detects the synchronization and results with high voltage (see Figure 2).

    Figure 2
    Figure 2: (a) the modulation clock (yellow) and the PMT signal (magenta) when detection 0.48 pico-gram IL-8 target. (b) The resulted lock in amplifier voltage at two different scans.

    The lock in amplifier didn't detect any signal when the control sample was tested. This fact, together with the visual difference in aggregation suggests that the MMB system can clearly identify the presence of IL-8 target.

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    Discussion

    In summary, we showed that the MMB system can be used to detect the presence of IL-8 target at low concentrations (0.5 pico-gram is the detection limit of the Bio-Plex Precision Pro cytokine assays [3,4]). The ability of the system is not limited to IL-8 and can be used to detect other proteins. The advantages of the MMB system are the ability to detect the target rapidly and without any separation or washing step. Thus, it facilitates the detection process and allows the system to be used in field applications.

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    Disclosures

    Acknowledgements

    This work was partly supported by the Ishaya Horowitz Fund.

    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

    References

    1. Danielli A., Arie A., Porat N., and 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., and 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-Plex Pro, Bio-Rad "Human angiogenesis assay panel", http://www.cancer.dartmouth.edu/shared/Documents/HumanAngiogenesisKit.pdf.
    4. Bio-Plex Precision Pro Cytokine Assay, Instruction Manual, Bio-Rad Laboratories, Hercules, CA.

    Comments

    2 Comments

    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: AnonymousAugust 19, 2010, 2:26 PM

    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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