SBIR-STTR Award

?Novel single cell assay for quantitative analysis of cell heterogeneity by noninvasive probing of molecular composition of specific organelles in individual cells Project Summary This current SBIR application in response to PA-13-140, is submitted by Advanced Cytometry Instrumentation Systems (ACIS), a company founded in 2001 to commercialize cutting-edge technologies via R&D in the fields of nanotechnology, photonics, and bio photonics. The current proposal is specifically focused on development and commercialization of next generation single cell assay for quantitative analysis of heterogeneity of eukaryotic cells by noninvasive probing of molecular composition in individual cells and/or specific organelles. This technology will enable real-time monitoring of the concentrations of major classes of macromolecules (proteins, lipids, DNA, RNA) as well secondary structures of proteins in regions of interests in live cells. The technology proposed here integrates (i) highly sensitive, high 3D resolution, low phototoxic, miniaturized and robust confocal Raman micro- spectrometry tool, designed to work in both academic and clinical laboratory and (ii) novel concept of user-friendly Biomolecular Component Analysis (BCA) of measured data sets to generate time-sequenced biomolecular composition profiles in individual cells. Based on the above goals the following specific aims are proposed for this Phase I study: Aim 1: Optimization of confocal Raman micro-spectrometer design for multi-cycle long- term measurements of individual eukaryotic cells. Aim 2: Software and database development for semi-automatic Biomolecular Component Analysis. Aim 3: Validation of the proposed micro-Raman-BCA technology for single cell analysis and quantitative characterization of cellular heterogeneity.

Public Health Relevance Statement:


Public Health Relevance:
We propose identification of cellular heterogeneity by time-sequential profiling of subcellular sites of interests (e.g. organelles, macromolecular clusters) fr their macromolecular content. Our approach integrates (i) confocal Raman micro- spectrometry setup modified to enable noninvasive, low phototoxic, real time probing of individual live cells and (ii) novel concept of semi-automated Biomolecular Component Analysis of acquired data to generate time-sequenced biomolecular profiles in individual cells.

Project Terms:
Algorithms; Architecture; base; Biological Assay; Calibration; cell behavior; Cell Nucleolus; Cell physiology; Cells; Classification; Clinical; commercialization; Computer software; Concentration measurement; cost; Cytometry; Data; Data Set; Databases; design; Detection; Development; DNA; Equilibrium; Eukaryotic Cell; Fluorescence; Gene Expression; Goals; Heterogeneity; Individual; instrumentation; interest; Label; Laboratories; Lasers; Life; Lipids; macromolecule; Measurement; Measures; Metabolic; metabolomics; Methods; Microscope; miniaturize; Mitochondria; Modification; Molecular; molecular marker; Molecular Probes; Molecular Profiling; Monitor; Nanotechnology; next generation; novel; novel marker; Organelles; phase 1 study; photonics; Physiological; Power Sources; Proteins; Proteomics; Protocols documentation; public health relevance; Raman Spectrum Analysis; Regulatory Pathway; research and development; Resolution; response; RNA; Secondary Protein Structure; Signal Transduction; single cell analysis; Site; Small Business Innovation Research Grant; Solutions; Spectrometry; System; Techniques; Technology; Time; tool; transcriptomics; user-friendly; Validation; Variant; Work

ACIS, LLC proposes a conceptually novel cellular heterogeneity assay, based on confocal Raman spectrometry and Biomolecular Component Analysis (BCA). The goal of Phase II is to implement a BCA toolbox in a commercial confocal micro- Raman instrument for quantitative assessment of cellular heterogeneity and potential quantitative classification of cellular states based on macromolecular compositions in specific organelles, a single cell assay developed during the Phase I, Phase II focuses on bringing this new device to the market. This project is motivated by the fact, that there is no currently any commercial tool, which provides direct probing of local biomolecular concentration in live cells. At the same time, this tool is extremely valuable for solving a number of problems, which require determination of quantitative markers for different cellular states (i) in diseased cell population (cancer-non-cancer, different cancer stages etc), (ii) during cell-drug interaction, and (iii) in the intracellular processes (apoptosis, proliferation, differentiation, etc). The Phase I research and development of this assay have met stated technical milestones. In particular, it is found that the BCA potential for characterization of cellular heterogeneity significantly exceeds that of the standard math tool of Principal Component Analysis routinely used for analysis of Raman spectra,. The specific aims of this proposal are: (1) Adaptation of a Thermo-Fisher micro-Raman system DXR2 model to meet requirements for BCA applications; (2) Development of Biomolecular Component Analysis Software package in C++ and/or Java and incorporation to DXR2 model of Thermo-Fisher micro-Raman system; (3) Validation of the BCA toolbox integrated with Thermo-Fisher micro-Raman system DXR2 model for ongoing biomedical research (a) Comparative analysis of heterogeneity between IDH- mutated and WT-IDH glioma cells and assessment of cell-drug interaction in IDH-mutant cells in collaboration with NCI, (Bethesda, MD) (b) Molecular profiling of cellular organelles for categorization of Prostate Cancer with RPCI (Buffalo, NY); (4) Pre-market evaluation of the Thermo-Fisher DXR2 micro-Raman system, equipped by the BCA toolbox. The outcome of this project will be a commercial confocal micro-Raman system with an implemented BCA toolbox, along with customized software for quantitative assessment of cellular heterogeneity, which can be used for a broad range of biomedical applications in many sectors such as clinical labs, Biomedical cellular Research labs, Pharmaceutical industries, National Cancer Institutes , etc.

Public Health Relevance Statement:
ACIS, LLC proposes to implement a conceptually novel cellular heterogeneity assay, based on confocal Raman spectrometry and Biomolecular Component Analysis (BCA), in a commercial confocal micro-Raman system. This assay will be implemented as BCA toolbox for quantitative assessment of macromolecular concentrations in specific cellular organelles.

Project Terms:
Analysis of Variance; Apoptosis; assay development; base; Biological Assay; Biomedical Research; Buffaloes; Calibration; Cells; Classification; Clinical; Collaborations; commercialization; comparative; Computer software; cost; curve fitting; Custom; Data; Development; Devices; Disease; Drug Industry; Drug Interactions; Evaluation; Glioma; Goals; graphical user interface; Heterogeneity; Individual; instrument; Java; Least-Squares Analysis; Malignant neoplasm of prostate; Malignant Neoplasms; Manufacturer Name; Mathematics; Modeling; Molecular Probes; Molecular Profiling; mutant; Mutate; National Cancer Institute; neuro-oncology; novel; Optics; Organelles; Outcome; Pharmacology; Phase; Population; Principal Component Analysis; Procedures; Process; Raman Spectrum Analysis; Research; research and development; Site; System; Techniques; Therapeutic; Time; tool; user-friendly; Validation