Diabetes is a major cause of morbidity and mortality in the U.S. Given that the diabetes epidemic continues to grow worldwide, there is a clear need for improvements in the management of the disease and its complications. Early diagnosis and intervention can significantly improve long-term prognosis. Autoantibodies against islet antigens are a serologic hallmark of patients with Type 1 Diabetes (T1D). In addition, circulating microRNAs (miRNAs) have been shown recently to be systematically altered, indicative of miRNA signatures with diagnostic utility. Growing evidence suggests that a multi-marker strategy, containing a combination of biomarkers with high clinical sensitivity and specificity, may enhance diagnostic and prognostic accuracy in the future compared to single marker tests. To support efforts of identifying the most informative biomarker panels, reliable next- generation platform technologies are needed that permit multiplexed detection of markers in small samples and are suitable for automation and integration into the clinical laboratory work flow. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for an ultrasensitive and - specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with high single-well multiplexing potential, minimal sample requirements, and simplified handling procedures (no separation/washing and amplification steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby target recognition molecules are tagged with different color fluorescent dyes (and quenchers). NTI recently achieved extension from 2-color to 4-color ALEX, substantially expanding its multiplexing power, and demonstrated diagnostic utility for direct protein as well as miRNA quantification. Furthermore, recent work by Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, termed "single molecule optofluidics", and ii) enhanced throughput using a multifoci excitation/detection geometry. NTI's long-term goal is to develop rapid, highly multiplexed, ultrasensitive and -specific, as well as fully automated, nucleic acid- and protein-based diagnostic tests that require minimal sample sizes. Here, we propose assay development and clinical validation of a next-generation test with significantly improved diagnostic, prognostic, and treatment- guiding properties, implementing a panel of autoantibody and miRNA biomarkers, and overcoming limitations of current T1D testing. Our Specific Aims are: 1. Initial reagent development for a multiplex autoantibody- and miRNA-based next-generation test for T1D 2. Separate as well as multiplexed biomarker detection and quantification using spiked samples 3. ALEX-based analysis of 200 archived clinical samples and cross-validation to ELISA and qPCR methods SBIR Phase II will propose assay expansion to include more markers, miniaturization, and development of a user-friendly, "sample in - answer out" diagnostic system offering significant cost and patient sample savings.
Public Health Relevance Statement: Public Health Relevance: Type 1 Diabetes, characterized by a prolonged and variable latent period that culminates in the destruction of pancreatic beta-cells and the development of hyperglycemia, is a debilitating autoimmune disease, and without proper management, patients develop serious complications that reduce their quality of life and life expectancy. As current single-marker tests for T1D are inadequate, there will be a great need for a versatile next-generation platform technology to detect and quantify panels of diagnostic and prognostic protein and nucleic acid biomarkers as they become available, in order to better assist in establishing early diagnosis of T1D, refine prognosis, guide management, target treatment, and finally improve patient outcome. Based on patent-protected alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, Nesher Technologies, Inc. intends to make its single molecule detection (SMD) platform technology robust and easy to use for diagnostic labs as well as the broad research community, and proposes to develop a next-generation test for T1D, monitoring a panel of autoantibody and microRNA biomarkers present in very small patient samples, thereby translating cutting-edge innovations in nanobiotechnology into benefits for the society at large by saving human lives and reducing healthcare costs.
NIH Spending Category: Autoimmune Disease; Bioengineering; Biotechnology; Diabetes; Pediatric; Prevention
Project Terms: adiponectin; Affinity; Antibodies; Antigens; Archives; Area; Autoantibodies; Autoimmune Diseases; Automation; base; Beta Cell; Binding (Molecular Function); Biological Assay; Biological Markers; Biophysics; Blinded; Body Fluids; circulating microRNA; Clinical; clinical assay development; Clinical Sensitivity; Color; Communities; Complex; Conduct Clinical Trials; cost; cytokine; Detection; Development; Diabetes Mellitus; Diagnosis; Diagnostic; diagnostic panel; Diagnostic tests; Disease Management; DNA; Dyes; Early Diagnosis; Early Intervention; endocrine pancreas development; Energy Transfer; Enzyme-Linked Immunosorbent Assay; Epidemic; Evaluation; Evaluation Studies; Exclusion; Fluorescence; Fluorescence Spectroscopy; Fluorescent Dyes; fluorophore; Future; Geometry; Goals; Head; Health Care Costs; Human; Hyperglycemia; improved; Individual; innovation; Insulin; insulin dependent diabetes mellitus onset; Insulin-Dependent Diabetes Mellitus; Intellectual Property; interest; Interferon Type II; islet; Kinetics; Label; Laboratories; Lasers; Legal patent; Licensing; Life Expectancy; Measures; Medical; Methodology; Methods; Microfluidics; MicroRNAs; Miniaturization; Monitor; Morbidity - disease rate; Mortality Vital Statistics; nanobiotechnology; nanolitre; next generation; Noise; Nucleic Acids; Outcome; outcome forecast; Pancreas; Pathogenesis; Patient Monitoring; Patients; Phase; Plasma; Procedures; prognostic; Property; prospective; Proteins; prototype; public health relevance; Quality of life; Reagent; Research; research clinical testing; Research Design; Risk; sample collection; Sample Size; Sampling; Savings; Sensitivity and Specificity; Serological; single molecule; Site; Small Business Innovation Research Grant; Societies; Solutions; Sorting - Cell Movement; Spectrum Analysis; Stratification; Structure of beta Cell of islet; System; Technical Expertise; Technology; Testing; Therapeutic; Time; tool; Translating; Tumor Markers; Tumor Necrosis Factor-alpha; user-friendly; Validation; virtual; Work
