INTERMODULATION PEAK DETECTION OF BRANCHED DNA FOR COMPACT-APPARATUS VIRAL LOAD M
Daniel James Laser, President And Ceo
Wave 80 Biosciences, Inc., 2325 3rd St, Ste 215, San Francisco, Ca 94107
Grant 1R43AI080454-01A1 from National Institute Of Allergy And Infectious Diseases
Abstract: HIV viral load measurement in resource-limited settings is an area of persistent need. Branched DNA (bDNA), a non-PCR nucleic acid method based on multitiered solution-phase probe structures, is a promising approach to meeting the need for HIV viral load measurement in resource-limited settings. On reference lab instruments, the branched DNA chemistry supports detection of as few as 75 copies of viral RNA in a 1 mL sample without target amplification. Branched DNA is inherently resistant to sample contamination, requires no thermal cycling or extreme temperatures, and is compatible with enclosed-cartridge-format assay instruments. This Phase I project focuses on a specific technical barrier to the development of cartridge-format bDNA systems suitable for use in resource-limited settings detecting bound, bDNA probe-hybridized viral RNA to determine the assay outcome. While standard bDNA chemiluminescence detection yields excellent results in traditional clinical laboratories, this method presents stability concerns for bDNA systems intended for use outside of tightly controlled environments. To overcome this challenge, a novel method for detecting bound bDNA probe-hybridized viral RNA is proposed intermodulation peak detection of nonlinear superparamagnetic effects in materials subjected to oscillating fields. Intermodulation peak detection is a recently developed magnetic detection method which shares the robustness of more well established giant magnetoresistance methods, but affords advantages in terms of detecting spatially distributed probes. This method has recently been demonstrated for immunoassays, but the work proposed here is (to our knowledge) the first use of intermodulation peak detection as a tool for solution-phase nucleic acid assays. Preliminary studies indicate that intermodulation peak detection methods can support bDNA assay sensitivities of as few as 10 bDNA-complexed viral RNA molecules within a bDNA-optimized porous structure and an assay dynamic range of at least four orders of magnitude. The project work encompasses bench-level prototype detector construction and extensive experimentation, modeling, and analysis. Reaching these milestones will demonstrate feasibility intermodulation peak detection as a component of the ultimate goal, a compact, low-maintenance, battery-powered, FDA-approved system which analyzes fingerstick samples with a two-hour turnaround time. The proposed research is pioneering, exploring a novel combination of bDNA signal amplification with intermodulation peak detection; it is rigorous, with exhaustive calibration protocols and careful mapping of relevant parameter spaces; and it is of high value, addressing a prominent technical barrier to the deployment of diagnostic devices with the potential to facilitate significant improvement in the level of care afforded HIV patient populations in resource-limited settings across the globe. The central hypothesis of this project is that intermodulation peak detection of bDNA complexes bound within a porous structure supports viral RNA measurement with a limit of detection of 5,000 copies per mL or better, upper bound of dynamic range at least 500,000 copies per mL, and total assay duration less than 120 minutes. Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project explores new ways of making viral load measurements less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities
Keywords: 1H-Thieno(3, 4-d)imidazole-4-pentanoic acid, hexahydro-2-oxo-, (3aS-(3aalpha, 4beta, 6aalpha))-; AIDS; AIDS Vaccines; AIDS Virus; AIDS, International; AIDS/HIV; AIDS/HIV problem; ATGN; ATP; ATP8A2; ATP8A2 gene; ATPIB; Academy; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immune Deficiency Syndrome Virus; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Acquired Immunodeficiency Syndrome Virus; Address; Amplifiers; Antigens; Apparatus and Instruments; Area; Assay; Autoimmune Diseases; Binding; Binding (Molecular Function); Binding Sites; Bioassay; Biochemical; Biologic Assays; Biological Assay; Biotin; Blood Plasma; Blood Tests; Body Fluids; Branched DNA Assay; Branched DNA Signal Amplification Assay; Calibration; Cancers; Caring; Cell Communication and Signaling; Cell Signaling; Chemistry; Clinic; Clinical; Clinical Research; Clinical Study; Clinical Trials; Clinical Trials, Unspecified; Combining Site; Communicable Diseases; Complex; Computer Systems Development; Consult; Controlled Environment; Coupling; DKFZP434B1913; DNA; DNA Hybridization Probes; DNA Probes; Data; Deoxyribonucleic Acid; Detection; Development; Development and Research; Development, Computer Systems; Devices; Diagnostic; Diamond; Disease Management; Disorder Management; Electromagnetic, Laser; Engineering; Engineerings; Environment, Controlled; Environmental Engineering technology; Equilibrium; Equipment and Supplies; FDA approved; FLR; Failure (biologic function); Family; Formosa; Foundations; Frequencies (time pattern); Frequency; Funding; Gene Products, RNA; Generations; Goals; HIV; HIV/AIDS; HIV/AIDS problem; HTLV-III; Head; Health; Hematologic Tests; Hematological Tests; Hematology Testing; Hour; Human; Human Figure; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human body; Human, General; Immunoassay; Immunologic Deficiency Syndrome, Acquired; In Vitro; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Instrumentation, Other; International AIDS; Intracellular Communication and Signaling; Investigators; Knowledge; LAV-HTLV-III; Label; Laboratories; Lasers; Licensing; Life; Lymphadenopathy-Associated Virus; ML-1; Magnetism; Maintenance; Maintenances; Malaria; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Manufacturer; Manufacturer Name; Maps; Math Models; Measurement; Mechanics; Medical; Methods; Modeling; Molecular Interaction; Natural Sciences; Noise; Nucleic Acids; Optics; Outcome; PROV; Paludism; Patients; Performance; Phase; Plasma; Plasmodium Infections; Preparation; Principal Investigator; Process; Production; Programs (PT); Programs [Publication Type]; Proteins; Protocol; Protocols documentation; Province; R & D; R&D; RNA; RNA Binding; RNA, Non-Polyadenylated; RNA, Viral; Radiation, Laser; Reactive Site; Reading; Reagent; Reporter; Republic of China; Research; Research Personnel; Research Resources; Researchers; Resistance; Resources; Reticuloendothelial System, Serum, Plasma; Ribonucleic Acid; Running; SBIR; SBIRS (R43/44); Sampling; Science; Science of Chemistry; Serum, Plasma; Signal Transduction; Signal Transduction Systems; Signaling; Small Business Innovation Research; Small Business Innovation Research Grant; Solid; Solutions; Strepavidin; Streptavidin; Structure; Surface; System; System, LOINC Axis 4; Systems Development; Taiwan; Technology; Temperature; Testing; Therapeutic; Time; V (voltage); Viral Burden; Viral Load; Viral Load result; Viral load measurement; Virus-HIV; Vitamin H; Washington; Work; artificial environment; autoimmune disorder; bDNA Assay; bDNA Signal Amplification Assay; balance; balance function; base; biological signal transduction; biomarker; clinical investigation; coenzyme R; design; designing; detector; environmental engineering; experience; experimental analysis; extreme temperature; failure; gene product; immunogen; information processing; instrument; instrumentation; magnetic; malignancy; mathematical model; mathematical modeling; meetings; member; neoplasm/cancer; novel; patient population; product development; programs; prototype; public health relevance; research and development; resistant; stability testing; superparamagnetic beads; tool; viral RNA; virus RNA; virus load; voltage
Relevance: Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project explores new ways of making viral load measurements less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities
Project start date: 2009-01-01
Project end date: 2010-06-30
Budget start date: 1-JAN-2009
Budget end date: 30-JUN-2010
PFA/PA: PA-06-134
1R43AI080454-01A1 (2009): $299852
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Daniel James Laser
Flow-Chamber Hybridization Rate Enhancement For Distributed Viral Load Measuremen
Daniel James Laser, President
Wave 80 Biosciences, Inc.
2325 3rd St, Ste 215
san Francisco, Ca 94107
Grant 1R43AI073221-01A2 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: Viral load measurement in resource-limited settings is a persistent area of need. Recent advances in compact, easy-to-use instrumentation with low per-test costs is promising for the development of highly portable assayers (HPAs) for HIV RNA quantitation. Lacking thermal cycling and minimally susceptible to contamination, the branched DNA (bDNA) HIV RNA sandwich nucleic acid hybridization/signal amplification assay is a good candidate for viral load measurement in resource-limited settings. However, successful development of HPA-bDNA methods and instruments requires speeding hybridization of HIV RNA to solid- phase capture probes. In the current bDNA protocol, hybridization takes place overnight; such a lengthy hybridization step is impractical for HPA viral load testing in resource-limited settings. The central hypothesis of this feasibility study is that microscale fluid mechanical processes and nonlinear diffusion effects can be leveraged to significantly accelerate flow-chamber HIV RNA hybridization-to the extent that a sufficiently large fraction of total viral RNA in a sample binds to the solid phase in less than twenty minutes. A novel method referred to as sequential delamination, which takes advantage of the microscale fluid flow laminarity and nonlinear scaling of diffusive effects, will be studied. Preliminary modeling indicates that sequential delamination-enhanced flow chambers can achieve analyte capture efficiencies approaching those of lateral flow membranes (hybridization of over 30% of HIV RNA in a 100 microliter sample with a primary hybridization step only ten minutes in duration) while maintaining the operational parameter control of fully active fluid transport. To determine feasibility and lay the groundwork for HPA-bDNA system development, prototype flow chambers will be fabricated and tested using bDNA probes and reference analytes, finite element modeling will be used for design optimization and performance parameter estimation, and system requirements will be established. Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project explores new ways of making viral load measurements less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities
Keywords: RNA, measurement, model, performance AIDS, DNA, antigen, back, blood test, chemical, computer, copying, diffusion, element, environment, fluid, fluid flow, gait, membrane, motivation, nucleic acid hybridization, plasma, quality of life, sectioning, solution, stroke
Project start date: 2007-08-01
Project end date: 2008-07-31
1R43AI073221-01A2 (2007): $299999
CARTRIDGE-FORMAT BRANCHED DNA SYSTEM FOR HIV VIRAL LOAD MEASUREMENT IN LOW-RESOUR
Daniel James Laser, President And Ceo
Wave 80 Biosciences, Inc., 2325 3rd St, Ste 215, San Francisco, Ca 94107
Grant 5R44AI073221-03 from National Institute Of Allergy And Infectious Diseases
Abstract: HIV viral load measurement in low-resource settings is an area of persistent need. A combination of two technologies holds great promise for improving access to HIV viral load measurement among underserved patient populations. The first is branched DNA (bDNA), a gold-standard nucleic acid chemistry which labels each strand of viral RNA with as many as 10,000 signaling molecules to enable detection of as few as 75 copies of viral RNA in a 1 mL plasma sample with low risk of test failure from sample contamination. The second is SCA microfluidic technology for high-performance on-cartridge sample/reagent processing with a static interface between cartridge and instrument. The starting point for this Phase II project is the successful Phase I work to shorten the bDNA assay´s duration while simultaneously adapting the assay to an enclosed-cartridge format. The newly developed cartridge- format bDNA assay, the EO-NAT HIV Assay, includes re-engineered capture probes and signaling chemistry while retaining the original assay´s multi-tiered probe configuration and high-specificity synthetic bases. Running in enclosed cartridges with cartridge-integrated SCA micropumps, the primary hybridization step in the bDNA assay has been speeded up eight-fold whereas the primary hybridization step takes 16 hours running on conventional reference laboratory instruments, in the EO-NAT HIV cartridge-format assay this step requires only 120 minutes. Total test turnaround time has been reduced to less than four hours, a duration which facilitates treatment of HIV/AIDS patients with limited access to care, for whom achieving favorable clinical outcomes often depends on carrying out viral load testing and developing a management plan in the same visit. The Phase II project work incorporates two additional technological elements into the EO-NAT HIV assay. Detection of hybridized bDNA complexes is by a specialized form of electrochemiluminescence with sub- nanomolar concentrations of signaling molecules and multiplexing. Sample preparation, including lysing and preconcentration, is by cartridge-integrated photopolymerized monoliths. During the second year of Phase II, cartridges integrating all four technological elements-cartridge-optimized bDNA chemistry, SCA microfluidic technology, mECL detection, and extraction monoliths-are produced. These cartridges and prototype EO- NAT instruments are tested on clinical specimens to validate the clinical sensitivity and specificity of the system. In the final portion of Phase II, pilot ISO 13485-compliant manufacturing is ramped up in preparation for filings with regulatory agencies. The central hypothesis of this project is that the EO-NAT HIV Assay running on EO-NAT instruments detects as few as 50 copies of HIV RNA in 20 microliter samples with 98% sensitivity (n=100) and exhibits specificity greater than 90% in testing of negative samples (n=100). Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project develops a new system which makes viral load measurement less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities
Keywords: AIDS Virus; AIDS test; AIDS/HIV; AIDS/HIV problem; AIDS/HIV test; ATGN; Access to Care; Access to Health Care; Access to Healthcare; Accessibility of health care; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Adherence; Adherence (attribute); Anonymous Testing; Antigens; Apoplexy; Architecture; Area; Assay; Au element; Availability of Health Services; Bioassay; Biocompatible Materials; Biologic Assays; Biologic Sciences; Biological Assay; Biological Sciences; Biomaterials; Blood Plasma; Blood Tests; Boston; Branched DNA Assay; Branched DNA Signal Amplification Assay; Businesses; Calibration; Carcinoma of the Liver Cells; Care, Health; Caring; Cell Communication and Signaling; Cell Function; Cell Process; Cell Signaling; Cell physiology; Cellular Function; Cellular Physiology; Cellular Process; Cerebral Stroke; Cerebrovascular Apoplexy; Cerebrovascular Stroke; Cerebrovascular accident; Chemistry; Chemistry, Physical; City of Boston; Clinical; Clinical Evaluation; Clinical Sensitivity; Clinical Testing; Communicable Diseases; Complex; Coupling; Cytolysis; DNA; Deoxyribonucleic Acid; Detection; Development; Development and Research; Diagnosis; Diagnostic; Disease; Disorder; Dose; Electromagnetic, Laser; Elements; Employee; Engineering; Engineering / Architecture; Engineerings; Exhibits; FLR; Failure (biologic function); Favorable Clinical Outcome; Gene Products, RNA; Gold; HCC; HIV; HIV test; HIV/AIDS; HIV/AIDS problem; HTLV-III; Health; Health Services Accessibility; Healthcare; Hematologic Tests; Hematological Tests; Hematology Testing; Hepatocellular Carcinoma; Hepatocellular cancer; Hepatoma; Hour; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human immunodeficiency virus test; Improve Access; Infectious Disease Pathway; Infectious Diseases; Infectious Diseases and Manifestations; Infectious Disorder; Instrumentation, Other; Intracellular Communication and Signaling; Investigation; Investigators; LAV-HTLV-III; Label; Laboratories; Lasers; Lead; Leadership; Life; Life Sciences; Lymphadenopathy-Associated Virus; Lysis; Marketing; Measurement; Measures; Mechanics; Mediating; Medical; Medical Device; Methods; Microfluidic; Microfluidics; Monitor; NAT; Nucleic Acid Amplification Tests; Nucleic Acid Testing; Nucleic Acids; Patients; Pb element; Performance; Phase; Physical Chemistry; Plasma; Plastics; Position; Positioning Attribute; Preparation; Primary carcinoma of the liver cells; Process; Production; Programs (PT); Programs [Publication Type]; R & D; R&D; RNA; RNA, Non-Polyadenylated; RNA, Viral; Radiation, Laser; Ramp; Reagent; Regulation; Reporter; Research; Research Personnel; Research Resources; Research Specimen; Researchers; Resources; Reticuloendothelial System, Serum, Plasma; Ribonucleic Acid; Risk; Role; Run-On Assays; Running; Sample Size; Sampling; Scheme; Science; Science of Chemistry; Sensitivity and Specificity; Serum, Plasma; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Molecule; Solid; Specificity; Specimen; Speed; Speed (motion); Staging; Stratification; Stroke; Structure; Subcellular Process; Surface; System; System, LOINC Axis 4; Systems Integration; Technology; Testing; Time; Universities; V (voltage); Validation; Variant; Variation; Vascular Accident, Brain; Viral; Viral Burden; Viral Load; Viral Load result; Viral load measurement; Virus-HIV; Visit; Work; access to services; access to treatment; assay development; availability of services; bDNA Assay; bDNA Signal Amplification Assay; base; biological signal transduction; biomarker; brain attack; cerebral vascular accident; clinical test; commercialization; cost; density; design; designing; detector; disease/disorder; failure; health care availability; health care service access; health care service availability; health services availability; healthcare access availability; healthcare service access; healthcare service availability; heavy metal Pb; heavy metal lead; immunogen; innovate; innovation; innovative; instrument; instrumentation; novel; patient population; pre-clinical; preclinical; product development; programs; prototype; research and development; research clinical testing; response; social role; stroke; viral RNA; virus RNA; virus load; voltage
Relevance: Plasma viral load, determined by complex blood tests, is an indication of how sick an HIV/AIDS patient is and how well he or she is responding to treatment. Regular viral load measurement is important in caring for HIV/AIDS patients, but these tests are currently not available for everyone. This project develops a new system which makes viral load measurement less expensive and more readily available to doctors and patients who live far from sophisticated medical facilities
Project start date: 2006-10-01
Project end date: 2012-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-06-134
5R44AI073221-03 (2010): $699223