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Cellphone Based Molecular iDiagnostics
by TIRF Labs
iDiagnostics and TIRF-EC Microarrays - Ultimate Biodetection Technologies
for Rapid and Accurate Molecular Diagnostics
Alexander N. Asanov, Ph.D.
President and Scientific Director, TIRF Labs, Inc.
106 Grendon Place, Cary, NC 27519-6945
Advances in biology and medicine, along with recent cost constraints, are driving the demand for rapid and accurate, yet affordable Molecular Diagnostics (MDx) technologies. After 9-11 followed by anthrax letters attack, the U.S. government spent over $70 billion on biodefense, including an estimated $40 billion on MDx. There is no analytical technique which has not been tried for MDx. However, a few highly efficient technologies have been developed; iDiagnostics and iTIRF-EC microarrays are among the most promising technologies. Nevertheless, inefficient approaches, including so-called label-free methods, such as SPR, QCM, and field-effect transistors block the way for advanced MDx, distract significant financial resources that otherwise could be used for the development of useful methods. The problem is that the U.S. government, including NIH, NSF, DARPA, DTRA, and other DOD agencies, continue to solicit the development of MDx based on approaches that have proven to be too inaccurate, too slow, and/or too inefficient. Among these inefficient approaches are so-called “label-free” methods (see the note “Misconception of Label-Free Biodetection”). The purpose of this synopsis is to inform the MDx community that rapid and accurate, yet affordable MDx technologies have been developed. They are termed iDiagnostics and TIRF-EC Microarrays.
The MDx market is large and rapidly growing: was $37 billion in 2011, projected to grow to $92B by 2016. The market demands accurate and rapid MDx, because false negative and false positive responses and delays in obtaining the results cause enormous damages. iDiagnostics and TIRF-EC Microarrays are such technologies that are exceptionally well-suited for advanced MDx. iDiagnostics is well-suited for field and point-of-care applications, while TIRF-EC - for laboratory use. Both iDiagnostics and TIRF-EC microarray technologies are super sensitive multiplexed platforms with rapid and accurate responses.
iDiagnostics is highly cost-effective. Both iDiagnostics and TIRF-EC are based on a synergistic combination of fluorescence spectroscopy, microfluidics, nanoengineered bioassays, and electronics. Both iDiagnostics and TIRF-EC yield a single-molecule limit of detection, high levels of simultaneous multiplexing across multiple classes of molecular markers - including DNA, RNA, proteins, metabolites, and chemical agents. The parallel detection of multiple nucleic acid, protein, and metabolite markers is of paramount importance for accurate MDx. Recent advances in biomedical sciences have proven that the detection of just one or two markers is not sufficient - the rate of false positive responses is too high. Unlike traditional methods, iDiagnostics and TIRF-EC can detect multiple markers, which ensures high accuracy. iDiagnostics and TIRF-EC require no or minimal sample preparation, resulting in rapid response - from several seconds to a few minutes. Additionally, the iDiagnostics platform can be miniaturized to yield affordable and versatile handheld devices with disposable cartridges. Affordable handheld sensors are very necessary for point-of-care MDx, biodefense, food safety, and combating pandemics.
Both iDiagnostics and TIRF-EC microarrays employ Total Internal Reflection Fluorescence (TIRF) as a biodetection method. TIRF-EC is additionally coupled with ElectroChemistry and Electric field Control (EC). Disposable iDiagnostics and TIRF-EC cartridges are adaptable for numerous tasks: the measurement of multiple medically significant analytes, including the early detection of cancer, monitoring epidemics and pandemics, food and water safety, agricultural, environmental, forensic, military, and biodefense applications. TIRF Labs is currently marketing benchtop and portable TIRF instruments (Fig. 1), as well as TIRF accessories for spectroscopy and microscopy. Our customers have proven that our existing TIRF products are powerful tools for the analysis of biomolecular interactions, cell biology studies, and other applications in fundamental sciences (see TIRF Application Notes). We also have developed and prototyped an iDiagnostics sensor (Fig. 1), which is reprogrammable, via smartphone equipped with Wi-Fi, Bluetooth, and a GPS system. Future versions of iDiagnostics will incorporate modules for the detection of chemical, nuclear, and radiological agents, the functionalities necessary for first responders. Another version of iDiagnostics will incorporate cartridges for the detection of metabolites in blood, saliva, urine, and other biological fluids, the functionalities necessary for clinical analyses. In other words, the iDiagnostics sensor can be a single device that supports first responders, clinicians, patients, and can be used in numerous other civilian and military applications.
TIRF Labs and its predecessors received $4.2M in U.S. federal grants. Using these funds we developed the advanced TIRF-EC Microarray platform and a family of TIRF products for spectroscopy and microscopy (www.tirf-labs.com). Using our own resources generated by the sales of our TIRF products, we further progressed to the iDiagnostics platform. Currently, the iDiagnostics platform uses existing consumer applications of smartphones to acquire the response of iDiagnostics arrays. The acquired data is transmitted to a computer with TIRF Labs’ data analysis software. In the future, all data acquisition, analysis and reporting will be performed by a smartphone application downloadable from the Apple Store or a similar Internet store.
We are seeking additional government funding to finalize the development of our iDiagnostics sensor for first responder applications and the early detection of cancer. Additionally, we are willing to enter into a partnership with a large corporation with existing engineering and manufacturing infrastructures and established marketing network to increase sales of our existing TIRF products, co-develop a family of clinical, food safety, agricultural, and first-responder applications, pass FDA approvals, and roll out new TIRF products, including iDiagnostics to the MDx market. We believe that iDiagnostics and TIRF-EC array technologies are uniquely well-positioned among competing MDx methods. Sensitivity, selectivity, rate of response, multiplicity, minimal sample preparation, and efficient costs of MDx analysis are the parameters of paramount importance for MDx. iDiagnostics and TIRF-EC microarray technologies meet these challenging requirements for several orders of magnitude better than competing technologies. Rapid, accurate, and inexpensive handheld devices will eventually dominate the MDx market. iDiagnostics sensor is well-suited for this purpose. iDiagnostics is not incrementally, it is several orders of magnitude more accurate than competition, yet affordable. We believe that the iDiagnostics platform and devices have excellent chances to succeed in the point-of-care MDx and other molecular sensing markets due to their unique set of advantages, providing capabilities several orders of magnitude better than competing technologies, yet at affordable costs.
Principles of iDiagnostics and TIRF-EC microarray technologies. iDiagnostics and TIRF-EC real-time microarrays employ a microfluidics-enabled microarray format, which can be used to detect unlabeled, unmodified target molecules using reagentless bioassays [1,2,4,9] (see also TIRF Technologies web pages). The assays contain embedded fluorescence reporters that change their fluorescence upon binding to unlabeled target molecules. iDiagnostics and TIRF-EC microarrays employ Total Internal Reflection Fluorescence to detect nucleic acid, protein and metabolite markers. In the case of nucleic acids the arrays use DNA molecular beacons. Protein and metabolite markers are detected using aptamer- or antibody-based assays similar to molecular beacons that change their luminescence upon binding to respective unlabeled, unmodified targets. Alternative technologies that use “label-free” detection (SPR, QCM, FET, SiNW) have much poorer sensitivity due to large fluctuating background and inability to suppress interferences from nonspecific interactions. Total Internal Reflection Fluorescence (TIRF) efficiently rejects the background (unwanted scatter of light and fluorescence from irrelevant and unbound molecules) greatly boosting signal-to-noise ratio and providing the ultimate sensitivity - down to single molecules. Due to the fact of ultimate sensitivity, TIRF has become a method of choice for single molecule detection studies. Over two hundred articles have been published that used TIRF for single molecule detection. Only a few other technologies are capable of detecting single molecules. However, none of alternative technologies is downsizeable to an affordable handheld device. Additionally, a series of unique advantages is enabled in TIRF-EC microarray by the use of electric field. In particular, electric field can be used to perform sample preparation and acceleration of the transport of target molecules. Electric field control also facilitates highly selective detection. Distinctive features that differentiate iDiagnostics and TIRF-EC from existing and emerging technologies include:
Ultimate sensitivity. iDiagnostics and TIRF-EC are sensitive down to single molecules [1, 2]. Most alternative technologies are at least 3 to 4 orders of magnitude less sensitive. Solution-based detection systems, bead-based methods, and traditional microarray technologies display the disadvantage of high background and low signal-to-noise ratio.
Extensive range of detected molecular classes. It is advantageous for an MDx technology to be able to simultaneously detect molecular markers of different classes: DNA, RNA, proteins, and metabolites. This is necessary for accurate diagnostics. Detecting of just one class of markers is not sufficient. For example, detecting only nucleic acids frequently results in high rate of false responses. iDiagnostics and TIRF-EC microarrays are capable of parallel detection of molecular markers of different classes [1-3], a feature necessary for accurate MDx. Alternative technologies detect just one class of molecular markers at a time.
High selectivity. Unlike many alternative technologies that struggle to distinguish highly similar analytes, iDiagnostics and TIRF-EC efficiently discriminate between perfectly matched targets, including DNA, RNA, protein, and metabolite markers, and their close homologs [1-4]. Monitoring the kinetics of interactions and measuring the dependence of kinetics on EC polarization allow for superior selectivity.
Multiplicity. iDiagnostics and TIRF-EC microarrays are capable of simultaneous detecting up to 10,000 DNA, RNA, protein, and metabolite markers [1-4]. The iDiagnostics and TIRF-EC sensor cartridges can be configured for performing detection from one to 10,000 molecular markers, depending on the application. In most cases, detection of 100 or fewer biomarkers may suffice.
Rate of response. iDiagnostics and TIRF-EC microarrays responds in several seconds to a few minutes [1,2].
Real-time detection. iDiagnostics and TIRF-EC microarrays measure the real-time kinetics of interactions between target and sensor molecules [1-4]. The kinetics provides additional information on specificity of molecular binding. Traditional microarrays and bead-based methods measure only end-point results.
Minimal sample preparation. iDiagnostics and TIRF-EC require minimal or no sample preparation. Whole blood and other complex biofluids can be rapidly analyzed without laborious sample preparation.
Accelerated mass transfer. Electric field confers unique advantages to iDiagnostics, including accelerated mass transfer of target biomarker molecules from bulk solution to their detection sites [1,2].
EC programs for lysis and driving fluids. Certain programs of EC polarization induce disintegration of cell membranes (lysis) to provide access to intracellular biomarkers [1,2]. EC polarization is also capable of driving solutions through microfluidic channels.
Broad dynamic range. Unlike traditional methods that measure only end-point results, real-time iDiagnostics and TIRF-EC microarrays measure the entire course of the kinetics of interactions between biomarker and the bioassay, which provides broader dynamic range for analysis. Additionally, iDiagnostics and TIRF-EC microarrays can be equipped with assays with different affinities to target molecules to increase the dynamic range of their detection to 9-10 orders of magnitude [1,2].
Internal positive and negative controls and reference standards for normalization and calibration can be included in iDiagnostics arrays to improve accuracy and reliability of the analysis [1,2].
Regenereable cartridges. EC polarization can be used to regenerate iDiagnostics sensors for re-use.
Detecting rapidly dissociating analytes. iDiagnostics microarrays detect the entire range of bioanalytes, including reversibly bound, rapidly dissociating markers. Traditional microarrays and bead-based methods involve washing steps, measure only end-point results, and do not detect rapidly dissociating bioanalytes. In certain cases, rapidly dissociating bioanalytes represent important class of molecular markers that are necessary for accurate MDx.
Adaptable to new biomarkers. iDiagnostics microarrays are adaptable to new biomarkers of different classes, including DNA, RNA, proteins, metabolites, and chemical agents. Chemical agents can be detected in liquid and even in gas phase.
Robust portable and handheld networkable devices. iDiagnostics devices are available in simple to operate portable and handheld form factors. iDiagnostics sensors can be configured to include remotely controlled analysis. Portable and handheld sensors are equipped with Wi-Fi, Bluetooth, and cell phone connectivity, and a GPS system.
No equivalent technology exists with this superior combination of features, yet affordable cost. Consequently, we believe that iDiagnostics sensors will successfully compete for significant presence in the MDx market.
TIRF Labs has received $4.2M in U.S. federal grants to develop advanced Molecular Diagnostics (MDx) technology. We have developed such platform technology called the TIRF-EC Microarray and are currently marketing benchtop and portable TIRF-EC biosensors. We also developed a cell phone based sensor, termed iDiagnostics, which targets the multibillion market of point-of-care MDx. Unlike benchtop and portable TIRF-EC biosensors that use scientific grade expensive EM CCD cameras, iDiagnostics employs enhanced TIRF arrays and can use the CCD cameras of smartphones for detection. Smartphones are also capable of performing TIRF array image analysis and processing using future applications dedicated to iDiagnostics arrays. At the present time (April 2016) cell phone records the response of iDiagnostics array and sends it to a computer, which performs data analysis and processing.
We are seeking additional government funding and/or institutional or private investments to develop applications for iDiagnostics sensor for first-responder, food and water safety applications, management of pandemics, and early detection and accurate prognosis of cancer. We are also willing to enter into a partnership with large corporation with existing engineering and manufacturing infrastructures and established a marketing network to increase the sales of our existing TIRF products, co-develop a family of clinical, food safety, agricultural, and first-responder applications, pass FDA approvals, and roll out new TIRF products, including the iDiagnostics sensor in the MDx market.
We believe that TIRF-EC and iDiagnostics technologies are uniquely well-positioned among competing MDx methods. Sensitivity, selectivity, rate of response, multiplicity, minimal sample preparation, and efficient costs of MDx analysis - are the parameters of paramount importance for MDx. iDiagnostics and TIRF-EC Microarrays meet these challenging requirements and for several orders of magnitude exceed competing technologies. Rapid, accurate, and inexpensive handheld devices will eventually dominate the MDx market. The iDiagnostics sensor is a well-suited device for this purpose. We believe that the iDiagnostics platform has excellent chances to succeed in the MDx market due to its unique set of advantages, providing capabilities several orders of magnitude better than competing technologies, yet at affordable costs.
Figure. 1. Benchtop, portable TIRF-EC, and iTIRF microarray chem-biosensors. The sensors simultaneously detect from a few to as many as several thousands of DNA, RNA, proteins, toxins, metabolite markers, and chemical agents. TIRF sensors provide accurate detection and respond in several seconds to a few minutes. Highly multiplexed and supersensitive analysis minimizes the probability of false positive and false negative responses. Portable and iTIRF sensors operate on batteries up to 8 hours. Portable TIRF sensor is equipped with disposable or reusable cartridges, Wi-Fi, Bluetooth, cellular, and USB connectivity, and a GPS. iTIRF renders similar functionality using smartphones.
Literature (Selected publications of our customers and our own data generated by our TIRF and TIRF-EC products)
1. 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.
2. Asanov A. “TIRF-EC Biosensors with Reagentless Bioassays for Rapid and Accurate Detection of Pathogens.” Biodetection Technologies 2008. Knowledge Press, Brookline, MA. 5th Edition, 2008, Chapter 8, p. 155-190, isbn # 1-59430-137-9.
3. Asanov A., Wilson W., and Oldham P. “Regenerable biosensor using total internal reflection fluorescence with electrochemical control”. US Patent 6,511,854. Publication Date: 2003-01-28. (Active patent.)
4. Asanov A., Zepeda A., and Vaca L. “A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence.” Sensors, 2012, 12, 1800.
5. Patents pending 2012-2013.
6. Willoughby D., Everett K., Halls M., Pacheco J., Skroblin P., Vaca L., Klussmann E., and Cooper D. “Direct Binding Between Orai1 and AC8 Mediates Dynamic Interplay Between Ca2+ and cAMP Signaling.” Science Signaling, 2012, 5 (219), 29.
7. Sampieri A., Zepeda A., Asanov A., and Vaca L. “Visualizing the store operated channel complex assembly in real time: Identification of SERCA2 as a new member.” Cell Calcium, 2009, 45, 439.
8. Asanov A., Zepeda A., and Vaca L. “A novel form of Total Internal Reflection Fluorescence Microscopy (LG-TIRFM) reveals different and independent lipid raft domains in living cells.“ Biochim. Biophys. Acta, 2010, 1801, 147.
9. Asanov A., Sherry R., Sampieri A., and Vaca L. “A relay mechanism between EB1 and APC facilitate STIM1 puncta assembly at endoplasmic reticulum.plasma membrane junctions”. Cell Calcium, 2013, 54, 246– 256
10.Luz-Madrigal A., Asanov A., Camacho-Zarco A.R., Sampieri A., and Vaca L. “A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells”. Journal of Virology, 2013, 87 (2), 11894–11907.
11. Additional publications of our customers with data generated by our TIRF and TIRF-EC products are available at the URL: www.tirf-labs.com