i-Diagnostics Technology

How Does i-Diagnostics Work?

The underlying technology of i-Diagnostics uses the principles of  real-time TIRF microarrays to simultaneously detect 4 classes of molecular markers in bodily fluids: DNA, RNA, proteins, and metabolites. “Real-time” means that the entire kinetic curve of the microarray response is recorded and analyzed in the real-time mode, rather than a single end-point is measured as in the case of “off-line” microarrays. TIRF microarrays measure not only the concentration of molecular markers that are signatures of diseases, but also the entire course of the dynamics of molecular marker interactions with bioassay molecules. The dynamics of interactions contains useful information about molecular markers. These markers appear in bodily fluids several months before a cancer tumor develops, and at least several weeks before a heart attack strikes, giving us enough time to take preventive measures against the disease.

i-Diagnostics is capable of detecting multiple molecular markers in several droplets of saliva, urine, sweat, blood, or other bodily fluid. It is designed to be minimally invasive and highly precise.  Since many important biomarkers are not stable and do not endure shipping and certain sample preparation procedures, it is important to perform the measurements in situ or shortly after taking the sample.  i-Diagnostics offers such an opportunity – it requires no or minimal sample preparation to perform the test.

Fig. 1 shows a photo (top) and the schematics of i-Diagnostics prototype (bottom). The detection of biomarkers occurs in the middle of the cartridge, where an array of fluorescent bioassays is printed at the surface of the TIRF slide. If a specific biomarker is present in the bodily fluid, it binds to a specific spot in the microarray, and the binding event results in the increase of fluorescence of this spot, which is recorded by the camera. The cradle and cartridge integrate optics, microfluidics, electronics, and nanoengineered bioassays into one autonomous handheld device, which implements TIRF microarray technology [1, 2]. A cellphone camera can be used as a photodetector (as shown in Figs, 1, 2). Alternatively, the i-Diagnostics reader can be equipped with a dedicated Bluetooth camera.

Fig. 2 shows that the excitation light enters the slide at its end, reflects from the top and the bottom of the slide, generates the evanescent wave at the slide surface, and excites fluorescence of multiple bioassay spots (green spots) that are arrayed at the bottom of the slide. Because TIRF is a surface-selective technique, which probes only a sub-micron layer of the fluid, bioassays that are immobilized at the surface are excited and fluoresce, while molecules in the bulk of solution are not excited, and respectively, do not fluoresce. This provides exceptional sensitivity – down to single molecules, and allows for the testing of complex biological fluids, such as whole blood, with no or minimal sample preparation. The combination of these features drastically reduces the turn-around time between the moments of “sample-in” and  “result-out.” Classical 2D TIRF microarrays operate with small, sub-monolayer amounts of antibodies or DNA probes immobilized on the surface. Their fluorescence signal is weak, which translates into the necessity of using a low light photodetector (e.g. EMCCD camera which is bulky and expensive). In i-Diagnostics the signal of TIRF arrays is enhanced by 3D encapsulation by silk fibroin protein, which captures the excitation light and becomes an integral part of the lightguide [http://tirf-labs.com/lightguidetirf]. 3D encapsulation by silk fibroin allows for the usage of larger amounts of antibodies or DNA probes.  The fluorescence signal from such arrays is a thousand-fold greater than that in classical arrays so CCD cameras of cellphones are sensitive enough to detect it. In summary, i-Diagnostics is an ideal platform for interfacing antibody-based bioassays for detecting proteins and metabolites, and molecular beacon assays for measuring nucleic acids.

For the detection of DNA and RNA markers, i-Diagnostics uses bioassays, termed “molecular beacons.” The principle of their operation is shown in the left panel of Fig. 3. In the absence of target DNA or RNA, the fluorescence of a molecular beacon is quenched. Upon binding to target DNA or RNA the molecular beacon opens and its fluorescence is dequenched. Irrelevant, non-target DNA or RNA do not open the molecular beacon; its fluorescence remains quenched.

For the detection of protein and metabolite markers, i-Diagnostics employs immunoassays based on antibodies, as shown in the right panel of Fig. 3. Capture antibody, which is immobilized at the surface, selectively binds protein or metabolite molecular marker. The binding of the detection antibody labeled by a fluorescent tag results in increase of fluorescence.  For more information about the principles of 3D-enhanced TIRF microarrays contact us by email Info@tirf-labs.com

LITERATURE CITED


1. Asanov A, Zepeda A, Vaca L. A platform for combined DNA and protein microarrays based on total internal reflection fluorescence. Sensors (Basel). 2012;12(2):1800-15. doi: 10.3390/s120201800. Epub 2012 Feb 9.


2.  Asanov A. “TIRF-EC Biosensors Massively parallel DNA and Protein Microarrays for Accurate and Rapid Detection of Pathogens.”  Biodetection Technologies 2007. Knowledge Press, Brookline, MA. 4th Edition,  2007, Chapter 17, p. 373-396, isbn # 1-59430-126-3.