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Filament Based Point-of-Care Platform
Vanderbilt engineers have developed a robust platform for point-of-care (POC) diagnostics applicable to infectious diseases, biowarfare agents, and environmental and agricultural testing. This technology uses capture antibodies on a polyester filament or a DNA on a gold wire that act as "molecular hooks" to troll for viral or bacterial protein antigens, RNA or capture DNA present in a small biological sample solution. It is envisioned that once the analytical POC device is purchased, a different single-use filament would be sold for each pathogen test of interest.
This robust, simple design uses either capture antibodies on a polyester filament or DNA probes on a gold wire which act as molecular hooks to troll for pathogens present in a test solution via filament motion. Three critical components of the technology are 1) the molecular hook, 2) the processing cassette and 3) the reporter. For antibody detection, the pull-through ELISA processing is followed by fluorescence reporting. In the early prototype device pictured below, capillary reaction chambers are arranged sequentially on a horizontal stage, and a filament with antibody segments immobilized at known locations is threaded through the chambers. One end is attached to the shaft of a precision rotary stage, and a small weight is attached to the other. The rotary stage controls the movement of the antibody segments through each reaction chamber by winding and unwinding the fi lament. In a final device, the chambers will all be part of one removable unit and the filament will attach to hooks at both ends. Initial reports employing this technology are described in the following references:
This technology is being further developed to utilize a DNA based approach without reliance upon microarray technology. In this pencil-sized device a protective cap is removed and the gold wire is exposed to sample viral or bacterial RNA. As the wire is pulled upwards through the device by a small machine the gold tip passes through three reaction chambers. From bottom to top, these chambers wash the gold tip to remove nonspecifically bound virus RNA, expose bound target to binding of a secondary reporter agent, and then wash the tip a second time to remove unbound reporter. The tip is then positioned within the top reporting chamber. In this design, maximum target-hook distance is only 10um. Fundamental design decisions are being driven by the desire to achieve this device's strengths of simplicity, stability and sensitivity.
It is envisioned that this platform could eventually be used in an over the counter capacity for tests with a high enough sensitivity. For example, a strep, flu, HIV or other STD test sold over the counter would offer the quick information consumers seek with the privacy of a home test. For such a test, a person would manually pull the filament through the chambers instead of utilizing a machine that could be used in the office setting. In this case the detection chamber would require a simple colorimetric change in the final step. For example, horseradish peroxidase or alkaline phosphatase can be employed as an optical indicator for ELISA style reactions. Alternative designs to be evaluated include the generation of a gas in the final liquid filled reporter chamber. The gas would produce a large change in the refractive properties and be readily observable. This design is being explored by developing a secondary DNA reagent with complementarity to a region not overlapping with the capture probe and which is also conjugated to an appropriate enzyme (e.g. horseradish peroxidase, catalase). Substrate for the enzyme will be stored in the final reporter chamber and we will quantify the changes in the optical properties produced by the presence of target. While not all the tests developed using this technology will be able to be developed for over the counter use, those that are will tap into a large market hungry for such a product.
Recent efforts in development of diagnostic tests have emphasized nucleic acid amplification (PCR or RT-PCR based methods), which offers greater sensitivity. These methods are, however, subject to a high rate of contamination (false-positives) in clinical settings. In addition, nucleic acids from microorganisms, especially DNA, may persist in the host secretions for prolonged periods beyond the infection, complicating interpretation of a positive test. Currently available molecular diagnostic tests based on nucleic acid detection require large investments in equipment and highly trained technical support, and the turnaround time does not meet the clinical standard of "rapid" when properly performed with confirmation of the specificity of amplified PCR products using PCR-ELISA, Southern Blot, or nucleotide sequence analysis.
This POC platform relies on technology (either ELISA or nucleic acid binding events) that is readily understood by and familiar to the majority of its targeted users. It does not require micro or nanofluidic technology, yet the device is small enough to be easily portable, about the size of a loaf of bread. The fact that it does not depend on traditional microarray technology means it also does not require the accompanying complex machinery. Pulling a filament through a series of chambers and using a fixed detector is a much simpler approach than processing requiring multiple fluid handling steps and it requires only a small sample size. The benefits of even minimal levels of automation will include lower costs and less reliance on lab technician expertise to achieve reliable and repeatable data.
Potential Market Size
U.S. POCT: $1.3B
U.S. Infections Disease POCT: $152M
Worldwide POCT: $6B; growing by an estimated $8 annually.
U.S. POCT: $2.4B
U.S. Infectious Disease POCT: $415M
Current Competitive Products
The infectious disease POC diagnostic market is highly fragmented with a number of companies offering relatively insensitive antibody-based tests. Inverness Medical and Quidel are the major players with their Clearview and QuickVue tests respectively. While test on both platforms can provide results in less than an hour, their sensitivity and specifi city is less than ideal. Inverness is developing a microarray based platform through its acquisition of Clondiag called ArrayStrip. Cepheid, Inc. and GeneOhm (a BD company) currently market real-time PCR POC products for MRSA, Enteroviral meningitis, Strep B and others. HandyLab, Inc. is developing a microfluidic platform for POC diagnostics that uses real-time PCR. The real-time PCR based assays require a considerable amount of staff time and require relatively expensive equipment.
Intellectual Property Status
U.S. Patent 7,521,261, was issued on April 21, 2009.
Inventors:Frederick HaseltonMark McQuain