Here you'll see what we were able to measure with our own device, as well as how to build your own, low-priced iRIf device.

Result of the binding of rabbit/anti-rabbit measured in our own device. A: The first picture of the measurement. B: The last picture of the measurement. C: The quotient picture of first and last picture. D: Schematic illustration of the spots on the slide

For testing our device we bound proteins derived from rabbits onto an iRIf slide in distinct spots (Fig 1 D). The proteins used were polyclonal anti-HCV (hepatitis C virus) antibodies, which we then aimed to detect with anti-rabbit antibodies. The reason for using the rabbit/anti-rabbit couple in this series of experiments was to reproduce a successful measurement with this binding couple which we previously performed in our regular measuring device. The binding layer on the iRIf slide consisted of an APTES/PDITC surface. The spots on the slide were made by pipetting 3 µl (500 µg/ml) rabbit-anti-HCV protein and 3 µl (1 mg/ml) BSA in an alternating pattern onto the slide. After incubation, the slide was blocked for 30 min in BSA solution. A Canon 50D camera was used to record the measurement and was set to take one picture every 5 seconds. The exposure time was set so that the pixels in the image were approx. 80% of maximum light saturation before the solution was flown onto the chip. The antibody solution was pipetted into the flow-chamber without the use of any microfluidics device. Instead a syringe was loaded with 500 µl [5 µg/ml] anti-rabbit antibody solution (diluted in PBS) and slowly released into the binding chamber of the device by gently dispensing the solution from the syringe. As can be seen in the figure C, the quotient picture clearly showed binding of anti-rabbit to the rabbit protein spots. The BSA control-spots showed none or negligible unspecific binding.

An illustration showing the exact setup of our device from a top perspective

As can be seen in the illustration below, the basic setup is fairly simple. Light from an LED enters a lense where the light rays are made parallel. To achieve this, the distance from LED to the lense has to be exactly one focal length. The light then hits the iRIf slide, where it gets reflected (reflecting at the same angle it hit the slide) and enters a second lense, whose purpose is to project a sharp image of the slide onto the CCD chip of the camera.

A major problem that we confronted when building the device from scratch was to assure that all components are at the exact distance and angle to each other. This is crucial as a slight misplacement of a component may lead to lower signal strengh, blurred images or in the worst case no signal at all. This can be difficult since our device doesn't rely on straight angles. We overcame this problem by designing a case for the device that ensures the right placement of the components in the device. This was archieved by calculating all the distances

An image of the vector file used to order the parts. The vector file is used to laser out parts of a 5 mm acrylic glass board. Blue lines are cut out by the laser, gray lines/areas are engravings. Refer to the download link to downloaded the SVG/Vector file.

A 3D model of the design of our device, build from the vector files used to order the parts

The 3D model of our device without walls or top part. Parts have been colorized for clarification - Green: The wall where light exits the device and enters into the camera; Pink: a platform holding Cooling-Element+LED in place; Blue: platforms for holding the lenses in place; White: rear end wall where the flow chamber is attached to. The slits in the top and bottom part are where the magnets have to be fixed

A: The LED glued to the cooling element with thermal adhesive; B: The 10 magnets, each 5x5x5 mm as well as a PDMS flowcell (depth of the flowchamber: 30 µm)