Name: Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System (Video) | JoVE Journal
Uploaded: 2010-06-13T17:00:00.000+00:00
Duration: 6 min 58 s
Description: Learn rapid homogeneous detection of biological molecules using magnetic modulation biosensing (MMB). This method detects IL-8 via sandwich immunoassay with magnetic beads and fluorescent labeling, eliminating washing steps.

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5 minutes

This work was partly supported by the Ishaya Horowitz Fund.

IL-8 assay:
<ol>
 <li>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. </li>
 <li>A control reaction is prepared the same way without the IL-8 target. </li>
 <li>The reactions are later placed without any separation or washing step in the cuvettes and inspected using the MMB system.</li>
</ol>
MMB system:
<ol>
 <li>Place the cuvette in its position between the two electromagnetic poles.</li>
 <li>Operate the modulation current and wait 30 seconds to allow aggregation and condensation of the beads</li>
 <li>Measure the signal of the lock-in amplifier using an oscilloscope. </li>
</ol>
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).
<img src="/files/ftp_upload/1935/1935fig1.jpg" alt="Spectroscopy analysis; transient absorption spectra illustrating molecular excitation, experiment results." data-altupdatedat="1788235205000" data-alt="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).
<img src="/files/ftp_upload/1935/1935fig2.jpg" alt="Pole modulation clock and PMT signal, lock-in amplifier voltage, graph, time vs voltage analysis." data-altupdatedat="1788235205000" data-alt="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.

<ol><li>Danielli, A., Arie, A., Porat, N., Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+fluorescent-labeled+probes+at+sub-picomolar+concentrations+by+magnetic+modulation%5BTitle%5D)+AND+%22Optics+Express%22%5BJournal%5D)">Detection of fluorescent-labeled probes at sub-picomolar concentrations by magnetic modulation.</a> Optics Express. 16, 19253-19259 (2008).</li>
<li>Danielli, A., Porat, N., Arie, A., Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+homogenous+detection+of+the+Ibaraki+virus+NS3+cDNA+at+pico-molar+concentrations+by+magnetic+modulation%5BTitle%5D)+AND+%22Biosensors+and+Bioelectronics%22%5BJournal%5D)">Rapid homogenous detection of the Ibaraki virus NS3 cDNA at pico-molar concentrations by magnetic modulation.</a> Biosensors and Bioelectronics. 25, 858-863 (2009).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Bio-Rad+%22Human+angiogenesis+assay+panel%22%22">Bio-Rad "Human angiogenesis assay panel"</a>. , Bio-Plex Pro. Forthcoming.</li>
<li>Instruction Manual. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Bio-Plex+Precision+Pro+Cytokine+Assay%22">Bio-Plex Precision Pro Cytokine Assay</a>. , Bio-Rad Laboratories. Hercules, CA. Forthcoming.</li>
</ol>

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.

<table><tbody><tr><td>Bio-Plex Pro Human Cytokine IL-8 set</td><td>Bio-Rad</td><td>171-B5008M</td><td></td></tr><tr><td>Recombinant human IL-8 target</td><td>Thermo Fisher Scientific, Inc.</td><td>R202625</td><td></td></tr><tr><td>Biotinylated monoclonal mouse anti human IL-8 antibody G265-8</td><td>BD Biosciences</td><td>554718</td><td></td></tr><tr><td>Streptavidin coupled fluorescent protein</td><td>Bio-Rad</td><td>Streptavidin PE</td><td></td></tr></tbody></table>

rapid homogeneous detection of biological assays using magnetic modulation biosensing system

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

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.

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

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Magnetic modulation BIOSENSING or MMB is a novel technique for the detection of biological molecules. Here a proof of principle assay is shown in which interleukin eight is detected when interleukin eight, also called IL eight, is added to a reaction mixture containing antibody coated magnetic beads by at Inated anti IL eight antibody and fluorescently labeled strep din. A molecular sandwich is created effectively linking the fluorescent molecule to the magnetic bead.The reaction is placed into a vete and alternating magnetic field. Gradients are applied through two electromagnetic poles, which maneuver the beads into oscillatory motion. The periodic motion of the bead attached fluorescent proteins in and out of an orthogonal laser beam produces a fluorescent signal that is collected by A PMT and demo modulated.Using a lock-in amplifier, the movement of the beads can be visualized using A CCD camera in place of the PMT synchronous movement is seen in samples where the magnetic beads have been linked to fluorescent proteins via IL eight. Hi, I'm Adia from the Nonlinear Optics and Laser Lab at the Department of Physical Electronics Telaviv University. I'm NoGo from the Department of Biological Sciences in University of Illinois at Chicago, And I'm Marcelo Erlich from the Cell Research and Immunology Department of the Tel Aviv University.Today we will show you a method for rapid homogeneous detection of biological assays using a magnetic modulation biosensing system. We are using this procedure in our laboratory to detect specific proteins and DNA sequences at low concentrations. So let's get started.Begin this procedure by setting up and labeling a tube for each reaction, including a target sample and a non-target control sample. To each tube, add the following components of the bio plex Precision Pro cytokine assay, one by one coupled magnetic beads detection IL eight antibody streptavidin PE and IL eight.Target. Bring the volume up to 100 microliters with assay buffer, then shake for 30 minutes on a compact orbital shaker.After the samples have been shaken for 30 minutes at room temperature, place them directly into cuvettes for analysis. Using the MMB system clear cuvettes with lens cleaning tissue and 70%ethanol. The instrument used for analysis consists of a 488 nanometer frequency doubled laser used as an excitation light source directed using a 562 nanometer dichroic beam splitter onto a microscope objective lens.The microscope objective lens focuses the laser to a 27 micrometer beam waste in a 400 micrometer wide BO silicate glass tube. The emitted light is spectrally filtered using two successive band pass filters detected by a photo multiplier or PMT and demo modulated using a lock-in amplifier. A homemade current modulator is used to deliver a square wave successively to each electromagnet.The amplitude should be 1.4 amps with a frequency of one to six hertz since the beads enter and leave the laser beam twice every cycle. The demodulation frequency at the lockin amplifier is two to 12 hertz. Initiate the system by turning the 488 nanometer laser key to the on position and switching on the power.Then set the laser power to minimum roughly four to six milliwatts. Turn on the oscilloscope, then turn on the PMT. Next place the sample vete between the two electromagnetic poles.Use the rotational stage of the vete to ensure that the laser beam passes through the vete and doesn't hit the vet's walls. Use the XY, Z translation stage of the vete to change its position with respect to the objective lens and electromagnetic poles. To optimize the coupling of fluorescent light into the detector, the vete should be positioned slightly behind the poles line.Now set the oscilloscope to time trace measurement. Set the PMT gain control to 0.5 bolts. Next power on the current modulator, and set the frequency to three hertz.Wait 30 seconds to allow aggregation and condensation of the beads. The magnetic beads will begin to oscillate moving close to the front of the tube. When the laser beam hits the oscillating beads, a flicker of reflected laser light should be seen from the beads.Notice that the beads move faster as the current modulation is increased and slower as it is decreased. The position of the beads in the tube can be adjusted by using the micrometer controlled stages. To move the poles bead movement can be visualized using a CCD camera that is placed instead of the PMT.The oscilloscope detects the periodic signal from the fluorescent coupled beads record the time trace of the oscilloscope. The saved data can be analyzed using MATLAB reaction mixtures with and without IL eight were assayed by MMB. In all the reactions with the target, the beads formed a single dense aggregate that moved in and out of the laser beam in sync.However, without the target, the beads were less aggregated and their motion was less united. Here the pole modulation clock shown in yellow and the PMT output signal shown in magenta depict the modulation frequency for each pole at two hertz. Therefore, the demodulation frequency is at four hertz as 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 when the PMT signal and the double modulation clock are fed to the lock-in amplifier. The sensitive phase detector the synchronization and results with high voltage. The two colors indicate two different scans at the same target concentration.The black lines represent the average of each signal. The lock-in amplifier didn't detect any signal when the control sample was tested. The lock-in amplifier didn't provide any locked signal for this case.This fact, together with the visual difference in aggregation, suggests that the MMB system and the difference in the time trace measurements of the PMT output signal can clearly identify the presence of ILH target. We've just shown you how to rapidly and homogeneously detect biological molecules using magnetic modulation. Biosensing with IL eight as an example.When doing this procedure, it is important to remember to follow laser safety procedures. So that's it. Thanks for watching and good luck with your experiments.

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Learn rapid homogeneous detection of biological molecules using magnetic modulation biosensing (MMB). This method detects IL-8 via sandwich immunoassay with magnetic beads and fluorescent labeling, eliminating washing steps.

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

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