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Yim S.W.,Nesher Technologies | Kim T.,Nesher Technologies | Laurence T.A.,Nesher Technologies | Partono S.,Nesher Technologies | And 6 more authors.
Clinical Chemistry | Year: 2012

BACKGROUND: Single-molecule detection (SMD) technologies are well suited for clinical diagnostic applications by offering the prospect of minimizing precious patient sample requirements while maximizing clinical information content. Not yet available, however, is a universal SMD-based platform technology that permits multiplexed detection of both nucleic acid and protein targets and that is suitable for automation and integration into the clinical laboratory work flow. METHODS: We have used a sensitive, specific, quantitative, and cost-effective homogeneous SMD method that has high single-well multiplexing potential and uses alternating-laser excitation (ALEX) fluorescenceaided molecule sorting extended to 4 colors (4c-ALEX). Recognition molecules are tagged with different-color fluorescence dyes, and coincident confocal detection of ≥2 colors constitutes a positive target-detection event. The virtual exclusion of the majority of sources of background noise eliminates washing steps. Sorting molecules with multidimensional probe stoichiometries (S) and single-molecule fluorescence resonance energy transfer efficiencies (E) allows differentiation of numerous targets simultaneously. RESULTS: We show detection, differentiation, and quantification-in a single well-of (a) 25 different fluorescently labeled DNAs; (b) 8 bacterial genetic markers, including 3 antibiotic drug-resistance determinants found in 11 septicemia-causing Staphylococcus and Enterococcus strains; and (c) 6 tumor markers present in blood. CONCLUSIONS: The results demonstrate assay utility for clinical molecular diagnostic applications by means of multiplexed detection of nucleic acids and proteins and suggest potential uses for early diagnosis of cancer and infectious and other diseases, as well as for personalized medicine. Future integration of additional technology components to minimize preanalytical sample manipulation while maximizing throughput should allow development of a user-friendly ("sample in, answer out") point-of-care platform for next-generation medical diagnostic tests that offer considerable savings in costs and patient sample. © 2011 American Association for Clinical Chemistry.


Rozenberg O.,Nesher Technologies | Lerner A.,Pediatric Gastroenterology and Nutrition Unit | Lerner A.,Technion - Israel Institute of Technology | Pacht A.,Pediatric Gastroenterology and Nutrition Unit | And 4 more authors.
Clinical Reviews in Allergy and Immunology | Year: 2012

There is an urgent clinical need for a better laboratory celiac disease diagnosis with both less false positive results and minimal underdetection. The aim of the present study was to evaluate the performance and diagnostic accuracy of different assays in an outpatient population setting for the diagnosis for celiac disease (CD) in order to design an optimal algorithm. We used 15 different ELISA assays to assess 47 blood samples of newly diagnosed children (positive biopsy results) and 52 samples from age- and sex-matched children with negative biopsy results for CD. Scoring criteria were established for grading the assays performance and characteristics. The combined gliadin and tTG assays exhibited the best sensitivity (100%). The addition of other assays to the CeliCheck neo-epitopes assay improved specificity so that the final algorithm had 100% sensitivity, 96.2% specificity, and 98.1% accuracy. The clinical demand for both maximal sensitivity and maximal specificity cannot be achieved with a single test. Using a combination of a sensitive assay together with specific assays improved celiac disease detection rates, with an acceptable number of false positive results. This model, however, needs to be confirmed prospectively in both children and adults. © Springer Science+Business Media, LLC 2011.


Kim T.,Nesher Technologies | Reitmair A.,Nesher Technologies
International Journal of Molecular Sciences | Year: 2013

Noncoding RNAs (ncRNAs) have been found to have roles in a large variety of biological processes. Recent studies indicate that ncRNAs are far more abundant and important than initially imagined, holding great promise for use in diagnostic, prognostic, and therapeutic applications. Within ncRNAs, microRNAs (miRNAs) are the most widely studied and characterized. They have been implicated in initiation and progression of a variety of human malignancies, including major pathologies such as cancers, arthritis, neurodegenerative disorders, and cardiovascular diseases. Their surprising stability in serum and other bodily fluids led to their rapid ascent as a novel class of biomarkers. For example, several properties of stable miRNAs, and perhaps other classes of ncRNAs, make them good candidate biomarkers for early cancer detection and for determining which preneoplastic lesions are likely to progress to cancer. Of particular interest is the identification of biomarker signatures, which may include traditional protein-based biomarkers, to improve risk assessment, detection, and prognosis. Here, we offer a comprehensive review of the ncRNA biomarker literature and discuss state-of-the-art technologies for their detection. Furthermore, we address the challenges present in miRNA detection and quantification, and outline future perspectives for development of next-generation biodetection assays employing multicolor alternating-laser excitation (ALEX) fluorescence spectroscopy. © 2013 by the authors; licensee MDPI, Basel, Switzerland.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2015

DESCRIPTION provided by applicant 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 Diabetes T D 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 color to 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 andquot single molecule optofluidicsandquot and ii enhanced throughput using a multifoci excitation detection geometry NTIandapos 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 T D testing Our Specific Aims are Initial reagent development for a multiplex autoantibody and miRNA based next generation test for T D Separate as well as multiplexed biomarker detection and quantification using spiked samples ALEX based analysis of 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 andquot sample in answer outandquot diagnostic system offering significant cost and patient sample savings PUBLIC HEALTH RELEVANCE Type 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 T D 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 T D 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 T D 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


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2015

DESCRIPTION provided by applicant Systemic lupus erythematosus SLE is a systemic autoimmune disease with worldwide distribution Despite medical treatment morbidity and mortality from renal disease are still common in lupus patients However early diagnosis and prompt treatment can significantly improve long term prognosis Anti double stranded ds DNA autoantibodies are a serologic hallmark of patients with SLE In addition circulating microRNAs miRNAs have been shown recently to be systematically altered unveiling 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 both protein and nucleic acid targets 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 color to 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 andquot single molecule optofluidicsandquot and ii enhanced throughput using a multi foci excitation detection geometry NTIandapos 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 SLE testing Our Specific Aims are Initial reagent development for a multiplex miRNA andamp autoantibody based next generation test for SLE Separate as well as multiplexed biomarker detection and quantification using spiked samples ALEX based analysis of 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 andquot sample in answer outandquot diagnostic system offering significant cost and patient sample savings PUBLIC HEALTH RELEVANCE Systemic lupus erythematosus SLE an autoimmune disease with an unpredictable course involving flares and remissions adversely affecting organ functions remains associated with an appreciably shortened life span As current single marker tests for SLE 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 SLE refine prognosis guide management target treatment and finally improve patient survival 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 SLE monitoring a panel of microRNA and autoantibody 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


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 119.53K | Year: 2012

DESCRIPTION (provided by applicant): Botulinum Neurotoxins (BoNTs) are the most toxic substances known and classified as Category A biothreat agents. BoNTs are zinc metalloproteases that cleave and inactivate proteins involved in synaptic vesicle fusion and acetylcholine release. Therapeutic applications of botulinum toxins have increased steadily over the years requiring new and reliable assays to assess the potency and stability of BoNT products. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for a revolutionary quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimalsample requirements, and extremely simplified handling procedures (no separation/washing steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby two (or more) target recognition molecules are tagged with different color fluorescence dyes. Coincident confocal detection of two (or more) colors constitutes a positive target detection event, allowing identification of biomolecules in solution and detection of numerous targets simultaneously. NTI recently achieved expansion from 2-color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power. Furthermore, recent work by our consultants Profs. Stephen Quake and Shimon Weiss demonstrates successful merging of microfluidics-based sample handling with 2c-ALEX spectroscopy for quantification of enzymatic activity, a new breakthrough approach for assay miniaturization and increased throughput termed single molecule optofluidics . NTI's long-term goal is to develop rapid, highly multiplexed (with a capacity of gt100 analytes per sample), ultrasensitive and -specific, quantitative, cost-effective, and fully automated, protein- and nucleic acid-based syndrome-driven tests that require minimal patient samples. In this Phase I SBIR application, we propose to adapt ALEX-based single molecule optofluidics for development of an innovative, completely solution-based in vitro endopeptidase assay, overcoming limitations of animal- and cell-based assays, to determine BoNT/A potency and stability based on quantifying its protease activity by measuring fluorophore-labeled substrate cleavage. Specific Aims are: 1. Substrate peptide synthesis and fluorophore/quencher conjugation. 2. BoNT/A detection and quantification using ALEX-based single molecule optofluidics. 3. Determination of specificity, sensitivity (d100 fM), linear range of quantification (e4 log orders), and time (1 hr). Phase II will be dedicated to assay optimization, automation, prototype development, and, if considered desirable, expansion to include other BoNT substrates for rapid multiplexed (single-well) discrimination and quantification. Software will be developed suitable for user-friendly operation of a fully integrated ALEX-based testing system for routine commercial release testing of BoNTproducts. It may serve as basis for future development of an FDA-approved diagnostic system for BoNT and other infectious diseases agents testing. PUBLIC HEALTH RELEVANCE: Nesher Technologies, Inc. intends to develop an innovative alternative approach, based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, to the currently used mouse intraperitoneal injection assay which measures activity in Mouse Units (MU), with 1 MU of activity defined as the LD50 dose (death within 72 to 96 hours in 50% of the mice), in order to allow for standardization of potency units for Botulinum Neurotoxin products made by different manufacturers. This will greatly reduce use of animals and will also complement Nesher Technologies' federally-funded efforts of instrument and reagent development for tests for bioterror agents, infectious and genetic diseases, and early cancer detection, thereby translating cutting-edge innovations in nanobiotechnology into benefits for the society at large by saving human lives, monitoring the population for bioterror attacks and disease outbreaks, and reducing healthcare costs.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 300.00K | Year: 2012

DESCRIPTION (provided by applicant): Recently, microRNAs (miRNAs) have been implicated in initiation and progression of human malignancies including cancer. Identified expression patterns in body fluids and biopsies underscore their potential as biomarkers. As patient samples are limited and miRNA concentrations often extremely low, serial analysis is not practical. Thus only new, extremely sensitive methodologies with high multiplexing power will be able to significantly boost diagnostic value and advanceour understanding of the biological roles of miRNAs. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for a revolutionary quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimal sample requirements, and extremely 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). Based on confocal microscopy, it allows ultrasensitive detection of biomolecules in solution, differentiation of numerous targets simultaneously, and direct quantification via molecule counting. NTI recently achieved expansion from 2-color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power, and demonstrated diagnosticutility for ultrasensitive miRNA quantification at clinically relevant concentrations without amplification. Furthermore, recent work by our consultants Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, a new breakthrough approach for assay miniaturization 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 (with a capacity of gt100 analytes per sample), ultrasensitive and -specific, quantitative, cost- effective, and fully automated, nucleic acid- and protein-based diagnostic tests that require minimal sample sizes. In this Phase I SBIR application, we propose to adapt4c-ALEX for fast ultrasensitive multiplexed detection and quantification of microRNAs. Proof-of-principle will be established via amplification-free characterization of cancer-specific miRNAs signatures in serum samples. Subsequent technology commercialization will target basic research, pharmaceuticals, and diagnostics markets. Our Specific Aims are: 1. Separate detection and quantification of seven lung cancer-related miRNAs in spiked samples by 4c-ALEX single molecule fluorescence spectroscopy and method comparison to qPCR. 2. Multiplexed (single-well) discrimination and quantification of these miRNAs. 3. 4c-ALEX-based analysis of 100 archived clinical serum samples (60 cancer patients and 40 controls). SBIR Phase II will be dedicated to assay expansion, miniaturization, and instrument prototype development. PUBLIC HEALTH RELEVANCE: Dysregulated expression of microRNAs in various tissues has recently been associated with a variety of diseases and it has been shown that miRNA signatures can beused as novel biomarkers, potentially offering more sensitive and specific tests than those currently available for early diagnosis of cancer and other diseases. Nesher Technologies, Inc. intends to develop an innovative test for microRNA signatures basedon patent-protected alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, offering the prospect to detect cancers at an early stage with high sensitivity and specificity which would significantly improve survival rates of this deadly disease. This will complement Nesher Technologies' federally-funded efforts of instrument and reagent development for tests for detection of early cancer, infectious and genetic diseases, bioterror agents, and others, thereby translating cutting-edge innovations in nanobiotechnology into benefits for the society at large by saving human lives and reducing healthcare costs.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 462.69K | Year: 2013

DESCRIPTION: To maximize clinical information content obtainable from a single small patient sample (e.g. cerebrospinal fluid-CSF), single molecule detection (SMD) technologies are the most promising for development of next- generation medical diagnostics. Recently, microRNAs (miRNAs) have been implicated in human malignancies including neurodegenerative disorders, offering the exciting prospect to combine traditional protein markers with novel miRNA candidate markers in panels. But as patient samples arelimited and marker concentrations often very low, serial analysis is not practical. Thus, only very sensitive methodologies with high multiplexing power will be able to maximize diagnostic value and be suitable for improved tests for early disease detection. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for a revolutionary, quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimal sample requirements, and extremely simplified workflows (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), allowing, on the same platform, ultrasensitive detection of both proteins and nucleic acids (including miRNAs) in body fluids. NTI recently achieved extension from 2-color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power (particularly when involving FRET), and demonstrated diagnostic utility for ultrasensitive protein as well as miRNA quantification at clinically relevantconcentrations without amplification. Furthermore, recent work by our consultants Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics- based sample handling with ALEX spectroscopy, a new breakthrough approach for assay miniaturizationtermed single molecule opt fluidics , and ii) enhanced throughput using a multifocal excitation/detection geometry. NTI's long-term goal is to develop rapid, highly multiplexed (with a capacity of gt100 analytes per sample), ultrasensitive and -specific,quantitative, cost-effective, and fully automated, protein- and nucleic acid- based diagnostic tests that require minimal sample sizes. Here we propose assay development and clinical validation for improved early-stage Alzheimer's disease (AD) diagnosis,implementing a panel of candidate protein and miRNA biomarkers. Our Specific Aims are: 1. Reagent development for a multiplex protein and microRNA biomarker-based next-generation AD test 2. Separate as well as multiplexed biomarker detection and quantification using spiked samples 3. ALEX-based analysis of archived clinical samples from 108 patients (PRoBE study design implementation) SBIR Phase II will propose assay expansion, miniaturization, and development of a versatile, user-friendly, diagnostic system as useful tool for early detection of AD and other neurodegenerative disorders. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Early detection of neurodegenerative diseases including Alzheimer's disease is crucial for early intervention and to ensure optimal care and implementation of patient management strategies that may support improved quality of life. As disease-modifying treatments are being developed, there will be a great need for versatile next-generation platform technologies todetect and quantify panels of diagnostic and prognostic biomarkers as they become available to decide who should enter particular clinical trials, for determining who should or should not receive a particular therapy, for determining the likelihood of disease progression, and for tracking disease progression. 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 early detection of Alzheimer's disease, monitoring a panel of protein and microRNA biomarkers present in very small patient samples, thereby translating cutting-edge innovations in Nano biotechnology into benefits for the society at large by saving human lives and reducing healthcare costs.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2013

DESCRIPTION (provided by applicant): Arthritis is a heterogeneous disease of the 'whole joint', whereby different combinations of proteolytic enzymes are active in different patient subpopulations leading to progressive joint degeneration. Upfront identification and stratification is critical for appropriate intervention with specific inhibitors, several of which are being evaluated in clinical trials. Nesher Technologies, Inc. (NTI) proposes a next-generation multiplex test for protease activity profilingin minute synovial fluid samples from arthritic joints, using alternating laser excitation (ALEX) single molecule fluorescence spectroscopy. This test should prove highly useful for facilitating treatment (as well as clinical trials) of arthritis with suitale protease inhibitors, or combinations thereof, personalized according to an individual patient's profile. NTI has exclusively licensed the intellectual property for ALEX, a revolutionary quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimal sample requirements, and extremely simplified handling procedures (no separation/washing and amplification steps). Target recognition molecules are tagged with different color fluorescent dyes (and quenchers). Based on confocal microscopy, it allows ultrasensitive detection of biomolecules in solution, differentiation of numerous targets simultaneously, and direct, amplification-free quantification via molecule counting. NTI recently achieved expansion from 2- color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power, and demonstrated diagnostic utility for detection of proteins and nucleicacids. Furthermore, recent work by our consultants Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, a new breakthrough approach for assay miniaturization 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 (with a capacity of gt100 analytes per sample), ultrasensitive and -specific, quantitative, cost-effective, and fully automated diagnostic tests requiring minimal sample sizes. For this SBIR Phase I grant application we propose multiplexed detection and quantification of protease activities for MMP- 13 and ADAMTS-4 as proof-of-principle, to be expanded in Phase II to a panel of up to 10 enzymes. Our Specific Aims are: 1. MMP-13 and ADAMTS-4 substrate peptide synthesis and fluorophore/quencher conjugation; 2. Separate as well as multiplexed protease activity detection and quantification using spiked samples; 3. ALEX-based analysis of 42 archived clinical synovial fluid samples (PRoBE study design implementation). Phase II will be dedicated to assay expansion, miniaturization, and instrument prototype development. Sub- sequent technology commercialization will target pharmaceuticals, diagnostics, and basic research markets. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Despite decades of research and clinical trials, arthritis is still an incurable, progressively degenerative disease involving a multitude of molecular factors, which, through various combinations and pathways, cause irreversible joint destruction. Failure of pharmacological interventions is, in large part, likely due to the inability to stratify patientsub-populations according to molecular criteria and identification of probable responders via use of companion diagnostics. In response, Nesher Technologies, Inc. (NTI) intends to develop an innovative multiplex diagnostic test, based on patent-protected alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, for protease activity profiling in very small sample sizes of precious synovial fluid from arthritic joints, in order to facilitate development o and treatment with specific inhibitors.


Provided herein are methods and compositions for detection and quantification of one or multiple target miRNA(s) in biological fluids and/or tissue samples using alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, and employing such methods and compositions for diagnostic, prognostic, therapeutic, and/or research applications.

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