The analytical power of flow cytometry makes it invaluable for numerous biomedical applications that require the enumeration of cell populations and the analysis of multicellular model systems or organisms. However, sample analysis flow rates of typical flow cytometers are limited to less than 250 uL/min, analytical rates are limited o 70,000 cells/s, and particle diameters must be less than 70 um. These limitations are driven by a number of factors that include pressure induced by high linear velocity fluid flows, turbulence in wide channels, and the single point analysis of stochastically arriving particles. Therefore, flow cytometry requires significant additional sample preparation steps to be effective in the analysis of very rare cell populations, uses offline particle concentration to analyze particles in large volume samples, and requires special purpose large flow channel cytometers using low linear velocity hydrodynamic focusing in wide channels to analyze particles that are >70 um in diameter at low analysis rates (200 s-1). Such limitations severely reduce its effectiveness in many critical applications including the detection of rare blood cell populations, the detection of pathogens in liquid samples, and the high throughput analysis model systems (e.g. multicellular model organisms, cellular spheroids, and one-bead-one-compound chemical libraries) that use large particles. To provide the analytical power of flow cytometry to these critical applications, we must dramatically increase the analytical rate, volumetric sample delivery, and the useable particle size of flow cytometers. To this end, we have developed acoustic flow cells that generate up to 300 focused parallel streams of particles using both acoustically resonant micro fabricated channels and multi-node acoustic standing waves. These flow cells focus particles up to 200 um in diameter at volumetric delivery rates as high as 25 mL/min. In this proposal, we will optimize the fluidics and optical properties of our flow cells and couple them with new approaches for highly parallel optical detection to create an affordable parallel acoustic flow cytometer (APAfc) platform. To address the broad set of unmet application needs, the APAfc platform will analyze cells or particles, ranging from 1 to 1000 um in diameter, at flow rates up t 50 mL/min, and at rates up to 1 x 106 particles/s. Importantly, the APAfc will achieve these specifications while retaining the analytical properties of flow cytometry (sensitivity, resolutionof free vs. bound probes, correlated multipara meter analysis) that make it the technology of choice for cell and particle analysis. Furthermore, the APAfc platform will be designed using affordable technologies to ensure that when translated into a commercial product, it will cost about what a low- end flow cytometer does today (~$50 to $100K). We will demonstrate the effectiveness of the APAfc platform using relevant models of clinical and research assays that are directly limited by analytical rates, volumetric throughput, or particle size. Development of the APAfc will have significant impact on both biomedical research and clinical diagnostics. It will provide a prototype instrument that provides highly sensitive and precise multipara meter optical analysis at analytical and volumetric delivery rates sufficient to provide a cost effective solution to routine detection of rare cells in blood or environmental samples, dramatically increase sample processing rates for HTS applications, and dramatically speed the analysis of multicellular particles and model organisms. We anticipate that if we are successful, our approaches to large volume high throughput flow cytometry will bring powerful analytical techniques to bear on a new spectrum of clinical and research problems.

Public Health Relevance

This project will develop an affordable parallel acoustic flow cytometer (APAfc) platform that has particle analysis rates as high as 1x106/s, sampling rates up to 50 mL/min, can handle cells/particles 1 to 1000 um in diameter, and analyze up to 100 samples or flow streams in parallel. This will create an entirely new generation of flow cytometers that combine parallel flow cytometry data acquisition, excitation, and detectors with multi-node acoustic focusing to provide an economical solution for many biomedical applications that include rare cell detection in blood, analysis of model organisms, and high throughput screening. Therefore, we feel that this proposal is ideally suited to this call by developing a new instrument that is of broad value to biomedical research and clinical applications.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZGM1-BBCB-A (BT))
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Friedman, Fred K
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University of New Mexico
Biomedical Engineering
Schools of Engineering
United States
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Kalb, Daniel M; Olson, Robert J; Sosik, Heidi M et al. (2018) Resonance control of acoustic focusing systems through an environmental reference table and impedance spectroscopy. PLoS One 13:e0207532
Gautam, Gayatri P; Burger, Tobias; Wilcox, Andrew et al. (2018) Simple and inexpensive micromachined aluminum microfluidic devices for acoustic focusing of particles and cells. Anal Bioanal Chem 410:3385-3394
Kalb, Daniel M; Fencl, Frank A; Woods, Travis A et al. (2017) Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry. Anal Chem 89:9967-9975
Piyasena, Menake E; Graves, Steven W (2014) The intersection of flow cytometry with microfluidics and microfabrication. Lab Chip 14:1044-59