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What is iRIf?

Imaging Reflectometric interference (iRIf) is an optical detection technology that can detect and visualize binding processes between biomolecules in real-time. For example the binding of antibodies to their corresponding antigens can be analyzed. The detection method is based on interference of light that is reflected at biolayers. A biolayer in this scenario would for example be antibodies that bind to immobilized antigens which cover the surface of a transparent material (see Figure 1). Adding such an additional biolayer to a surface changes the optical properties (optical thickness), resulting in an increased intensity of the reflected light. The intensity of the reflected light is detected by the CCD sensor of a camera and can be visualized with appropriate software.

Why use iRIf?

Labelfree: Many analyzing approaches to detect binding between molecules are dependent on labelling with a fluorophore or enzyme (ELISA, WB). Since iRIf is a labelfree optical method no expensive labeling reagents are required. Fast and simultenous: Within minutes it is possible to screen a complex sample for binding partners on a microarray. This for example enables us to screen a blood sample within 20 minutes for potentially hundreds of different pathogenic antigens or their corresponding antibodies, respectively. Real-time: With iRIf it is possible to observe binding events during your measurement in real-time! Small samples amounts: The iRIf detection method is very sensitive and can detect even tiny target molecule concentrations. Therefore, usually only small sample volumes (in µl range) are necessary to get reliable results. Hence, the iRIf detection is mostly coupled with a microfluidic device.

Applications

As large as the number of specific binding processes is the number of applications for labelfree reflectometric intereference techniques like iRIf. Applications include amongst others: drug discovery (Birkert & Gauglitz, 2001), kinetic interaction studies (Daaboul et al. 2011), food analysis (Rau et al., 2014), biomarker research (Ewald et al., 2015) and (serologic) diagnostics (Nagel et al., 2007). Usually, basis of each application is some kind of biomolecule microarray (often proteins) which are arranged in distinct spots on a transparent glass side. Protein microarrays facilitate high-throughput screening with a small quantity of sample (Lin et al., 2009).

Our focuses

(Serological) diagnostic of infectious diseases Concerning serological diagnostics iRIf can be used to simultaneously detect potential antibodies for hundreds of diseases from a few drops of blood. For that, one needs to arrange an antigen peptide array (or antibody array) and flood it with the blood of a patient (just like our Dia-Chip works). Different studies show successful detection of pathogenic antigens or the corresponding antibodies within the blood with this technique, e.g. for Tuberculosis (Nagel et al., 2007), Hughes-Syndrom (Bleher et al., 2014), Influenza (Schwarz et al., 2010) or Pox (Proll et al., 2014). Our Dia-chip shows promising results for the detection of Salmonella (Link zum Ergebnis) and Tetanus (Link zum Ergebnis) antibodies within a complex mixture like blood. Determining status of vaccinaton Another application is to examine wheter the vaccination of a patient is still up to date. Since iRIf measurements allow to quantify the amount of antibodies binding to the antigens, this provides a way of adequately quantifying the current vaccination statuses. Too little antibodies would indicate that a vaccination has to be refreshed (Link vergleich Vaccination before(after measurement).

What does an iRIf measurement look like?

[Video] The video shows an exemplary iRIf measurement. Three different proteins are arranged in 3 distinct spots on the glass slide. One after another, three different antibodies, which bind to the corresponding antigen spot, are flushed over the slide. Upon binding, the optical properties of the spots are changed, resulting in an increased intensity of the reflected light at this location of binding. Using a look-up-table(LUT) the amount of bound protein is visualized by different colors.

How it works

To understand the basic physics of iRIf only three propositions of ideal wave optics have to be known:

(P1)	Everytime light is confronting a (non-absorbing) different medium than the one it is propagating through it splits into two parts: one part is going to be reflected and one part is refracted to the next medium.
(P2)	If two waves of light are in the same place they interfere with each other: They are seen as a new wave resulting from summing up the original two.
(P3)	The light intensity is proportional to the square of its amplitude. This also holds up for waves due to interference.

What happens at the device is the following: A green ray of light (520 nm wavelength) produced by an LED is directed onto the iRIf slide. What makes this slide special is an additional layer made out of TaO5 very close (nanometer scale) to the spotted side (why this is necessary can be looked up in the physics section). The ray of light will be reflected everytime the medium changes (P1): On the surface of the slide, at the Ta2O5-layer and on the spotted backside of the slide. Finally the remaining ray is mostly absorbed by the flowcell. Neglecting the first reflection (coherence length, -> physics) we have two reflected waves which are interfering (P2).
(BILD?)
Now what happens if antibodies are bound to a spot? Basically the same happens as if the SiO2 layer would get thicker (optical thickness -> physics) which results in a "delayed" reflection of the ray. This delay especially changes the resulting interference waves' intensity.
Data aquirement at iRIf is done by constantly taking images of the reflected light. Consequently, if this is done while the slide stays in contact with a blood serum (or any solution containing antibodies) possible intensity changes (P3) due to antigen-antibody bindings at the spotted slide will be gathered too. The change is not visible by the naked eye (beyond 1% in relative intensity) but can be seen when comparing later pictures with initial ones using picture division.

(Bild -> Quotientenbild)

Even better, when observing the progression of relative intensity change (relative due to picture division) "binding" curves" can be aquired making quantifications possible.

(Bindekurven-Graph?)

Basic iRIf Setup

Homogenous illumination of the glass slide with monochromatic light source is necessary to archieve good iRIf results. This is best accomplished with a powerful LED light source shining into a lense which is positioned at the distance of one focal length, therefore parallelising the light rays. Since the idea of an iRIf measurement lies in the observation of the minute changes of light intensity of light reflected at the slide, using a sensitive camera with a color depth of 12 bit is advisable. Images in the camera have to be stored lossless, since compression methods such as JPEG or MPEG remove subtle changes in the picture, resulting in the removal of the binding signal. Although not mandatory, microfluidic systems are very well suited for use in conjunction with iRIf, since the iRIf measurement device and the microfluidic chamber can be positioned on opposite sites of the glass slide.

Building your own device

Since the physics behind iRIf is well characterized and the parts necessary for building such a device are easily obtainable, we took it on ourselves to build our very own, affordable iRIf device. The results of this endevour, including a detailed manual on how to build your own iRIf device can be found [here].

Physics behind iRIf

Legal notice

Johannes Landgraf (Karlsruhe, DE) Günther Proll (Denkendorf, DE) and Florian Pröll (Mannheim, DE) from BIAMETRICS (Link Biametrics) own a patent (US 20120058569 A1) for the iRIf detection method (“Method and device for determining reflection coefficients on filter arrangements having thin layers”).

References

Birkert & Gaulitz, 2001. Development of an assay for label-free high-throughput screening of thrombin inhibitors by use of reflectometric interference spectroscopy. Anal Bioanal Chem Vol. 372, doi: 10.1007/s00216-001-1196-4 Bleher et al., 2014. Development of a new parallelized, optical biosensor platform for label-free detection of autoimmunity-related antibodies. Anal Bioanal Chem Vol. 406, doi: 10.1007/s00216-013-7504-y Daaboul et al., 2011. LED-based Interferometric Reflectance Imaging Sensor for quantitative dynamic monitoring of biomolecular interactions. Biosensors and Bioelectronics Vol. 26, doi: 10.1016/j.bios.2010.09.038 Ewald et al., 2015. A multi-analyte biosensor for the simultaneous label-free detection of pathogens and biomarkers in point-of-need animal testing. Anal Bioanal Chem Vol. 407, doi: 10.1007/s00216-015-8562-0 Lin et al., 2009. Development of a novel peptide microarray for large-scale epitope mapping of food allergens. Journal of Allergy and Clinical Immunology Vol. 124, doi: 10.1016/j.jaci.2009.05.024 Nagel et al., 2007. Direct detection of tuberculosis infection in blood serum using three optical label-free approaches. Sensors and Actuators B Vol. 129, doi: 10.1016/j.snb.2007.10.009 Proll et al., 2014. Optical biosensor system for the quick and reliable detection of virus infections – VIROSENS. SPIE Proceedings Vol. 9253, doi: 10.1117/12.2073841 Rau et al., 2014. Label-free optical biosensor for detection and quantification of the non-steroidal anti-inflammatory drug diclofenac in milk without any sample pretreatment. Anal Bioanal Chem Vol. 406, doi: 10.1007/s00216-014-7755-2 Schwarz et al., 2010. Label-free detection of H1N1 virus for point of care testing. Procedia Engineering Vol. 5, doi: 10.1016/j.proeng.2010.09.256