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.
