Assays and Devices for Rapid Fully Integrated Nucleic Acid Testing at the Point of Care
Angelika Niemz, Jim Sterling, and external collaborators
Nucleic acid amplification enables sensitive and specific pathogen diagnosis. Point-of-care testing in low resource settings requires rapid, simple to use, and inexpensive assays and related devices for nucleic acid testing that address the entire process from sample in to answer out in an integrated format. We are developing such a platform technology by integrating the isothermal Exponential Amplification Reaction (EXPAR), lateral flow-based detection, and novel sample preparation approaches. Targeted pathogens include Herpes Simplex Virus (HSV), Mycobacterium tuberculosis, and Human Immunodeficiency Virus (HIV).
Isothermal DNA Amplification Through Polymerase and Nicking Enzyme Activities
The isothermal Exponential Amplification Reaction (EXPAR) efficiently amplifies short oligonucleotides through thermostable polymerase and nicking endonuclease activities. We are investigating how EXPAR is influenced by thermodynamic, enzyme kinetic, and sequence related parameters, using experimental and computational methods. We are developing new assay formats and are conducting systematic assay optimization to achieve robust reproducible assay performance, with sensitive and specific target detection, a pre-requisite for application of EXPAR in clinical diagnostics.
Nucleic Acid Sample Preparation: What Happens at the Surface?
Our goal is to further the understanding, on a molecular level, of what determines the reversible interactions of nucleic acids with silica surfaces (including composites and modified surfaces). Based on this understanding, reaction conditions, surface composition and functionalization can be altered to more effectively switch from binding to release, particularly release of nucleic acids from the surface into a small elution volume compatible with subsequent nucleic acid amplification. Such understanding can lead to improved solid phase extraction methods for nucleic acid testing.
Microfluidics and Mucosal Bioengineering
Our research is aimed at the development of miniaturized and automated systems for biomolecular analysis. We have developed automated sample-to-answer systems for infectious-disease diagnostics with focus on compact, rapid, disposable systems that manipulate samples using cartridges or electrowetting-based (aka digital-microfluidic, or DMF) systems. We are also developing methods of studying polysaccharide-rich biohydrogels that are present in the extraceullar matrix and at mucosal surfaces. The mucosal glycocalyx gel controls transport of nutrients, biomolecules, and therapeutics to and across the mucosal surface and also controls interaction with pathogens, commensals, and immune responses. Bioengineering of the glycocalyx represents a fundamentally new opportunity to improve human health.
Engineering of Compact, Automated Systems for Pathogen Detection
Biothreat detection research at KGI is being conducted in several multi-disciplinary, multi-team efforts. With funding from the Department of Homeland Security, National Institutes of Health and the Defense Advanced Research Projects Agency, the focus of the research is on bioanalytical methods and engineering instrument/device development for nucleic acid diagnostics. The projects are focused mainly on: (1) sample preparation methods including electrical and mechanical sample lysis and nucleic acid purification components, (2) thermally-controlled DNA amplification systems integrated with compact lateral-flow or optical fluorescence systems, and (3) chip-based free-solution capillary electrophoresis of short oligonucleotides. The efforts address primarily the ability to detect-to-protect by diagnosing the presence of bacterial and viral pathogens in swab or air-filtered samples.
Identification of Biomarker Panels for Diagnosis of Disease
Jim Osborne, Craig Adams
Biomarkers are measurable entities used to diagnose disease and monitor clinical responses to therapy. Many diseases are difficult to diagnose, and patients with the same diagnosis often respond differently to a given therapy. Most approved diagnostic tests are based on a single biomarker. The Center for Biomarker Research is investigating the use of biomarker panels to better diagnose disease and stratify patient populations for selection of therapy. Flow cytometry is a very powerful technique for measuring multiple markers on the surface and inside of cells. Nucleic acid and protein assays can also be multiplexed in a flow cytometer by designing assays on multicolored beads. We are developing and validating protocols using multiplex flow cytometry to investigate biomarker panels for diseases that have good therapeutic options, but are difficult to diagnose.
Biomarker for Diagnosis and Monitoring of Hereditary Inclusion Body Myopathy
Craig Adams, Jim Osborne, in collaboration with Dr. Daniel Darvish
Hereditary Inclusion Body Myopathies (HIBM) are a diverse group of muscle wasting disorders that share similar histopathology with sporadic Inclusion Body Myositis and senile plaques seen in Alzheimer’s brain disease. Various forms of HIBM are genetically and clinically diverse, with the autosomal recessive form (IBM2) as the most common. It usually affects young adults and often leads to severe disability and confinement to a wheelchair. IBM2 is also known as Quadriceps Sparing Myopathy, Distal Myopathy with Rimmed Vacuoles, or Nonaka’s Myopathy. As in many rare disorders, for IBM2 there is a significant need to develop biomarkers that can be useful in clinical and molecular evaluation of the disease. Such biomarkers will allow us to determine the effectiveness of promising therapeutic intervention currently in early clinical trials, which may translate to significant cost and time savings.