iDiagnostics - the handheld future of precision medicine; Single Molecule Detection, Single Cell Analysis, Cell Membrane Studies, TIRF microscopy, TIRF accessories for fluorometers, TIRF biosensor instruments, Real-time TIRF microarrays, and more>>> visit www.tirf-labs.com
Molecular iDiagnostics for Home Use
by TIRF Labs
Fig. 1. Photo and schematics of iDiagnostics.
Fig. 2. 3D-enhanced TIRF microarray in comparison with classical 2D microarray.
Technology: How Does iDiagnostics Work? Patent pending
Molecular markers are signatures of diseases. They 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 prevent the disease. iDiagnostics is a patent pending technology, which is capable of detecting multiple molecular markers in a droplet of saliva, urine, sweat, blood, or other bodily fluid. It is designed to be minimally invasive and maximally precise. Since many important biomarkers are not stable and do not endure shipping and certain sample preparation procedures, as mentioned above, it is important to perform the measurements shortly after taking the sample.
Fig. 1 shows a photo and the schematics of iDiagnostics. 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 sample, it binds to a specific spot in the microarray, and the event of binding results in the increase of fluorescence intensity of this specific spot, which is recorded by the cellphone. The cradle and cartridge integrate optics, microfluidics, electronics, and nanoengineered bioassays into one handheld device, which implements TIRF microarray technology. Fig. 2 shows that the excitation light enters the slide at its end, is reflected from the top and the bottom of the slide, and excites fluorescence of multiple bioassay spots that are arrayed at the bottom of the slide, where the bodily fluid contacts the array. Because TIRF implements 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 and other particles in the bulk of solution are not excited, and respectively, do not fluoresce. This provides exceptional sensitivity and allows for the testing of complex biological fluids, such as whole blood, with no or minimal sample preparation, and therefore drastically reduces the turn-around time between the moments of "sample-in" and "result-out."
Classical TIRF microarrays operate with small, sub-monolayer amounts of antibodies and DNA probes immobilized on the surface; the fluorescence signal is small, and a low light photodetector (e.g. EMCCD camera) is necessary. In iDiagnostics the signal of TIRF arrays is enhanced by 3D encapsulation, which captures the excitation light and becomes an integral part of the lightguide. 3D encapsulation allows for the usage of larger amounts of antibodies per unit area of bioassay spot. The signal from such arrays is a thousand-fold greater than that in classical arrays. The CCD cameras of cellphones are sensitive enough to detect the signal. In summary, iDiagnostics appears to be the ideal platform for interfacing antibody-based bioassays for detecting proteins and metabolites, and molecular beacon assays for measuring nucleic acids.
Fig. 4 shows main components of the iDiagnostics ADK and uTIRF Biodetection Station and Fig. 5 - main states of microarray printing, assaying and data acquisition and analysis. The set of ADK and uTIRF station will be used by researchers, who are interested in interfacing their existing assays with iDiagnostics platform and who will be developing new assays and panels of assays. Before testing the panels of assays with iDiagnostics cartridges and cradles, the stages of assay development, validation, calibration and other development tasks will be implemented using uTIRF station. For these purposes uTIRF is equipped with open perfusion and closed flow chambers. There are several formats of open perfusion and closed chambers for different tasks. Open perfusion chambers are suitable for easy access to each individual spot of the panel. For more information about iDiagnostics ADK and uTIRF station refer to ADK and uTIRF pdf brochures and contact TIRF Labs via email: firstname.lastname@example.org.
Although the uTIRF Biodetection Station is a brand new product by May 2018, several research groups worldwide have requested this new product along with iDiagnostics ADK for their studies. Distributing uTIRF-ADK and other iDiagnostics development tools, we will keep in mind that the ultimate goal of the iDiagnostics project is to create a decentralized precision diagnostics affordable for all. Fig. 1 shows an early prototype of iDiagnostics and disposable cartridges. In Phase 1, we plan to transform the first prototype into a robust iDiagnostics product well-suited for home use by minimally trained individuals, similar to pregnancy test strip. In Phase 1 we will explore the possibility of including a cellphone camera and a processor into the cradle in order to make iDiagnostics simpler and more user-friendly. By the middle of Phase 2 we will have a well-refined iDiagnostics product ready for manufacturing.
Despite its small size and low cost, iDiagnostics will be an analytical device as powerful as a bench-top laboratory instrument; it also will be used by medical doctors, first responders, clinical researchers, and other specialists. We anticipate that several months after we receive the necessary funds, a number of tests for laboratory use - including food safety, allergens in food, prostate cancer, and breast cancer - will be developed, validated, and used for research purposes in academic, government, and industrial research groups.
Fig. 3. Molecular beacon assay for detection of DNA/RNA and immunoassay for detection of protein markers.
For the detection of DNA and RNA markers, iDiagnostics uses bioassays, termed molecular beacons. The principle of their operation is shown in 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, iDiagnostics employs immunoassays based on antibodies, as shown in 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: email@example.com.
Fig. 5: Printing iDiagnostics microarrays and using uTIRF biodetection station for real-time microarray data acquisition and analysis.