Time-resolved microfluidic flow cytometer for decoding luminescence lifetimes in the microsecond region.

Time-resolved luminescence detection using long-lived probes with lifetimes in the microsecond region have shown great potential in ultrasensitive and multiplexed bioanalysis. In flow cytometry, however, the long lifetime poses a significant challenge to measure wherein the detection window is often too short to determine the decay characteristics. Here we report a time-resolved microfluidic flow cytometer (tr-mFCM) incorporating an acoustic-focusing chip, which allows slowing down of the flow while providing the same detection conditions for every target, achieving accurate lifetime measurement free of autofluorescence interference. Through configuration of the flow velocity and detection aperture with respect to the time-gating sequence, a multi-cycle luminescence decay profile is captured for every event under maximum excitation and detection efficiency. A custom fitting algorithm is then developed to resolve europium-stained polymer microspheres as well as leukemia cells against abundant fluorescent particles, achieving counting efficiency approaching 100% and lifetime CVs (coefficient of variation) around 2-6%. We further demonstrate lifetime-multiplexed detection of prostate and bladder cancer cells stained with different europium probes. Our acoustic-focusing tr-mFCM offers a practical technique for rapid screening of biofluidic samples containing multiple cell types, especially in resource-limited environments such as regional and/or underdeveloped areas as well as for point-of-care applications.

Resonance control of acoustic focusing systems through an environmental reference table and impedance spectroscopy.

Acoustic standing waves can precisely focus flowing particles or cells into tightly positioned streams for interrogation or downstream separations. The efficiency of an acoustic standing wave device is dependent upon operating at a resonance frequency. Small changes in a system's temperature and sample salinity can shift the device's resonance condition, leading to poor focusing. Practical implementation of an acoustic standing wave system requires an automated resonance control system to adjust the standing wave frequency in response to environmental changes. Here we have developed a rigorous approach for quantifying the optimal acoustic focusing frequency at any given environmental condition. We have demonstrated our approach across a wide range of temperature and salinity conditions to provide a robust characterization of how the optimal acoustic focusing resonance frequency shifts across these conditions. To generalize these results, two microfluidic bulk acoustic standing wave systems (a steel capillary and an etched silicon wafer) were examined. Models of these temperature and salinity effects suggest that it is the speed of sound within the liquid sample that dominates the resonance frequency shift. Using these results, a simple reference table can be generated to predict the optimal resonance condition as a function of temperature and salinity. Additionally, we show that there is a local impedance minimum associated with the optimal system resonance. The integration of the environmental results for coarse frequency tuning followed by a local impedance characterization for fine frequency adjustments, yields a highly accurate method of resonance control. Such an approach works across a wide range of environmental conditions, is easy to automate, and could have a significant impact across a wide range of microfluidic acoustic standing wave systems.

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry.

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 µL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.

Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coli.

Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx 1 , stx 2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.

Inertial manipulation and transfer of microparticles across laminar fluid streams.

A general strategy for controlling particle movement across streams would enable new capabilities in single-cell analysis, solid-phase reaction control, and biophysics research. Transferring cells across streams is difficult to achieve in a well-controlled manner, since it requires precise control of fluid flow along with external force fields or precisely manufactured mechanical structures. Herein a strategy is introduced for particle transfer based on passive inertial lift forces and shifts in the distribution of these forces for channels with shifting aspect ratios. Uniquely, use of the dominant wall-effect lift parallel to the particle rotation direction is explored and utilized to achieve controllable cross-stream motion. In this way, particles are positioned to migrate across laminar streams and enter a new solution without significant disturbance of the interface at rates exceeding 1000 particles per second and sub-millisecond transfer times. The capabilities of rapid inertial solution exchange (RInSE) for preparation of hematological samples and other cellular assays are demonstrated. Lastly, improvements to inline flow cytometry after RInSE of excess fluorescent dye and focusing for downstream analysis are characterized. The described approach is simply applied to manipulating cells and particles and quickly exposing them to or removing them from a reacting solution, with broader applications in control and analysis of low affinity interactions on cells or particles.

One-dimensional acoustic standing waves in rectangular channels for flow cytometry.

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry.

Multinode acoustic focusing for parallel flow cytometry.

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 µm, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multinode acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 µm in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of a CD4+ cellular immunophenotyping assay. This approach will have significant impact toward the creation of high throughput flow cytometers for rare cell detection applications (e.g., circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry.

Development of small and inexpensive digital data acquisition systems using a microcontroller-based approach.

Fully digital data acquisition systems for use in flow cytometry provide excellent flexibility and precision. Here, we demonstrate the development of a low cost, small, and low power digital flow cytometry data acquisition system using a single microcontroller chip with an integrated analog to digital converter (ADC). Our demonstration system uses a commercially available evaluation board making the system simple to integrate into a flow cytometer. We have evaluated this system using calibration microspheres analyzed on commercial, slow-flow, and CCD-based flow cytometers. In our evaluations, our demonstration data system clearly resolves all eight peaks of a Rainbow microsphere set on both a slow-flow flow cytometer and a retrofitted BD FACScalibur, which indicates it has the sensitivity and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers.

Evaluation of a green laser pointer for flow cytometry.

Flow cytometers typically incorporate expensive lasers with high-quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom-built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise < or = 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow-flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and < or =1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 micros (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of approximately 75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times.

Microsphere-based protease assays and screening application for lethal factor and factor Xa.

BACKGROUND: Proteases regulate many biological pathways in humans and are components of several bacterial toxins. Protease studies and development of protease inhibitors do not follow a single established methodology and are mostly protease specific. METHODS: We have created recombinant fusion proteins consisting of a biotinylated attachment sequence linked to a GFP via a protease cleavage site to develop a multiplexable microsphere-based protease assay system. Using the proteases lethal factor and factor Xa, we performed kinetic experiments to determine optimal conditions for inhibitor screens and detect known inhibitors using the HyperCyt flow cytometry system. RESULTS: We have demonstrated specific cleavage of lethal factor and factor Xa substrates, optimized screening conditions for these substrates, shown specific inhibition of the proteases, and demonstrated high throughput detection of these inhibitors. CONCLUSIONS: The assay developed here is adaptable to any site-specific protease, compatible with high throughput flow cytometry systems, and multiplexable. Coupled with flow cytometry, which provides continuous time resolution and intrinsic resolution of free vs. bound fluorophores, this assay will be useful for high throughput screening of protease inhibitors in general and could simplify assays designed to determine protease mechanism.

Direct fluorescent staining and analysis of proteins on microspheres using CBQCA.

BACKGROUND: General methods for accurate determination of microsphere surface protein loading are needed for applications from protein arrays to molecular assembly studies. Current methods include bulk absorption measurements of stained microspheres or use of known fluorescently tagged binding partners, which limit sensitivity and general applicability, respectively. METHODS: Microspheres bearing covalently coupled proteins were stained with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) using different incubation times and dye concentrations to determine optimal staining conditions. The CBQCA fluorescence of microspheres (measured by flow cytometry) bearing known amounts of protein were used to generate standard curves of CBQCA fluorescence response versus the amount of microsphere surface protein. CBQCA was also used to stain noncovalent protein interactions. RESULTS: Maximal labeling was attained within 1 h with 1 mM CBQCA. Linear fluorescence response occurred between 8 x 10(4) and 1 x 10(6) proteins/microsphere. CBQCA staining did not disturb noncovalent protein interactions. CONCLUSIONS: We have developed methods using CBQCA and flow cytometry to quickly and simply quantify the amount of protein on the surface of a microsphere. Importantly, this approach could be extended to other formats (e.g., chips). Further, because it does not disturb noncovalent protein-protein interactions, it may be possible to use this approach to detect protein interactions without the use of purified prestained probes.

Microsphere surface protein determination using flow cytometry.

This unit describes an extrinsic staining protocol using the amine-reactive CBQCA dye to measure the amount of protein on the surface of a microsphere. This approach is novel in that it allows microspheres bearing proteins without known binding partners to be accurately quantified on a flow cytometer.