Analytical performance of an ultrasonic particle focusing flow cytometer

Creation of inexpensive small-flow cytometers is important for applications ranging from disease diagnosis in resource-poor areas to use in distributed sensor networks. In conventional-flow cytometers, hydrodynamics focus particles to the center of a flow stream for analysis, which requires sheath fluid that increases consumable use and waste while dramatically reducing instrument portability. Here we have evaluated, using quantitative measurements of fluorescent microspheres and cells, the performance of a flow cytometer that uses acoustic energy to focus particles to the center of a flow stream. This evaluation demonstrated measurement precision for fluorescence and side scatter CVs for alignment microspheres of 2.54% and 7.7%, respectively. Particles bearing 7 x 10(3) fluorophores were well resolved in a background of 50 nM free fluorophore. The lower limit of detection was determined to be about 650 fluorescein molecules. Analysis of Chinese hamster cells on the system demonstrated that acoustic focusing had no effect on cellular viability. These results indicate that the ultrasonic flow cytometer has the necessary performance for most flow cytometry applications. Furthermore, through robust engineering approaches and the combination of acoustic focusing with low-cost light sources, detectors, and data acquisition systems, it will be possible to achieve a low-cost, truly portable flow cytometer.     

High Throughput‐Per‐Footprint Inertial Focusing

Matching the scale of microfluidic flow systems with that of microelectronic chips for realizing monolithically integrated systems still needs to be accomplished. However, this is appealing only if such re‐scaling does not compromise the fluidic throughput. This is related to the fact that the cost of microelectronic circuits primarily depends on the layout footprint, while the performance of many microfluidic systems, like flow cytometers, is measured by the throughput. The simple operation of inertial particle focusing makes it a promising technique for use in such integrated flow cytometer applications, however, microfluidic footprints demonstrated so far preclude monolithic integration. Here, the scaling limits of throughput‐per‐footprint (TPFP) in using inertial focusing are explored by studying the interplay between theory, the effect of channel Reynolds numbers up to 1500 on focusing, the entry length for the laminar flow to develop, and pressure resistance of the microchannels. Inertial particle focusing is demonstrated with a TPFP up to 0.3 L/(min cm²) in high aspect‐ratio rectangular microfluidic channels that are readily fabricated with a post‐CMOS integratable process, suggesting at least a 100‐fold improvement compared to previously demonstrated techniques. Not only can this be an enabling technology for realizing cost‐effective monolithically integrated flow cytometry devices, but the methodology represented here can also open perspectives for miniaturization of many biomedical microfluidic applications requiring monolithic integration with microelectronics without compromising the throughput.     

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.     

High-throughput and high-resolution flow cytometry in molded microfluidic devices

We describe the design, fabrication, and operation of two types of flow cytometers based on microfluidic devices made of a single cast of poly(dimethylsiloxane). The stream of particles or cells injected into the devices is hydrodynamically focused in both transverse and lateral directions, has a uniform velocity, and has adjustable diameter and shape. The cytometry system built around the first microfluidic device has fluorescence detection accuracy comparable with that of a commercial flow cytometer and can analyze as many as 17 000 particles/s. This high-throughput microfluidic device could be used in inexpensive stand-alone cytometers or as a part of integrated microanalysis systems. In the second device, a stream of particles is focused to a flow layer of a submicrometer thickness that allows imaging the particles with a high numerical aperture microscope objective. To take long-exposure, low-light fluorescence images of live cells, the device is placed on a moving stage, which accurately balances the translational motion of particles in the flow. The achieved resolution is comparable to that of still micrographs. This high-resolution device could be used for analysis of morphology and fluorescence distribution in cells in continuous flow.     

Demonstration of submersible high-throughput microfluidic immunosensors for underwater explosives detection

Significant security threats posed by highly energetic nitroaromatic compounds in aquatic environments and the demilitarization and pending cleanup of areas previously used for munitions manufacture and storage represent a challenge for less expensive, faster, and more sensitive systems capable of analyzing groundwater and seawater samples for trace levels of explosive materials. Presented here is an inexpensive high throughput microfluidic immunosensor (HTMI) platform intended for the rapid, highly selective quantitation of nitroaromatic compounds in the field. Immunoaffinity and fluorescence detection schemes were implemented in tandem on a novel microfluidic device containing 39 parallel microchannels that were 500 μm tall, 250 μm wide, and 2.54 cm long with covalently tethered antibodies that was engineered for high-throughput high-volume sample processing. The devices were produced via a combination of high precision micromilling and hot embossing. Mass transfer limitations were found in conventional microsystems and were minimized due to higher surface area to volume ratios that exceeded those possessed by conventional microdevices and capillaries. Until now, these assays were limited to maximum total volume flow rates of ~1 mL/min due in part to kinetics and high head pressures of single microchannels. In the design demonstrated here, highly parallelized microchannels afforded up to a 100-fold increase in total volume flow rate while maintaining favorable kinetic constraints for efficient antigen-antibody interaction. The assay employed total volume throughput of up to 6 mL/min while yielding signal-to-noise ratios of >15 in all cases. In addition to samples being processed up to 60 times faster than in conventional displacement-based immunoassays, the current system was capable of quantitating 0.01 ng/mL TNT samples without implementing offline preconcentration, thereby, demonstrating the ability to improve sensitivity by as much as 2 orders of magnitude while decreasing total analysis times up to 60-fold.     

Microchip flow cytometry using electrokinetic focusing

Flow cytometry of fluorescently labeled and unlabeled latex particles is demonstrated on a microfabricated device. The latex particles were detected and counted using laser light scattering and fluorescence coincidence measurements. Sample confinement was accomplished using electrokinetic focusing at a cross intersection, and detection occurred 50 μm downstream from the intersection. Particles with diameters of 1 and 2 μm were analyzed and distinguished from each other based on their light scattering intensity and fluorescence. A maximum sample throughput of 34 particles/s was achieved. Sample mixtures with varying proportions of fluorescently labeled and unlabeled particles were also analyzed and found to be within experimental error of the expected ratios.     

Smartphone-based cytometric biosensors for point-of-care cellular diagnostics

Analysis on a single-cell basis is both fundamental and meaningful in biomedical research and clinical practice.Flow cytometry is one of the most popular approaches in this field with broad applications in cell sorting,counting, and identification of rare cells. However, the complicated design and bulky size of conventional flow cytometry have restricted their applications mainly in centralized laboratories. With the recent development of smartphone devices, smartphone-based cytometry has been explored and tested for single-cell analysis. Compared with traditional cytometers, smartphone-based cytometric biosensors are more suitable for point-of-care(POC) uses, such as on-site disease diagnosis and personal health monitoring. In this review article, the history of traditional flow cytometry is introduced, and advances of smartphone-enabled cytometry are summarized in detail based on different working principles. Representative POC applications of smartphone cytometers are also discussed. The achievements demonstrated so far illustrate the potential of smartphone-based cytometric devices to transform single-cell measurement in general, with a significant impact in POC diagnostics, preventive medicine, and cell biology.     

Standing Surface Acoustic Wave (SSAW)‐Based Fluorescence‐Activated Cell Sorter

Microfluidic fluorescence‐activated cell sorters (μFACS) have attracted considerable interest because of their ability to identify and separate cells in inexpensive and biosafe ways. Here a high‐performance μFACS is presented by integrating a standing surface acoustic wave (SSAW)‐based, 3D cell‐focusing unit, an in‐plane fluorescent detection unit, and an SSAW‐based cell‐deflection unit on a single chip. Without using sheath flow or precise flow rate control, the SSAW‐based cell‐focusing technique can focus cells into a single file at a designated position. The tight focusing of cells enables an in‐plane‐integrated optical detection system to accurately distinguish individual cells of interest. In the acoustic‐based cell‐deflection unit, a focused interdigital transducer design is utilized to deflect cells from the focused stream within a minimized area, resulting in a high‐throughput sorting ability. Each unit is experimentally characterized, respectively, and the integrated SSAW‐based FACS is used to sort mammalian cells (HeLa) at different throughputs. A sorting purity of greater than 90% is achieved at a throughput of 2500 events s^?1. The SSAW‐based FACS is efficient, fast, biosafe, biocompatible and has a small footprint, making it a competitive alternative to more expensive, bulkier traditional FACS.     

High‐Throughput Continuous Flow Production of Nanoscale Liposomes by Microfluidic Vertical Flow Focusing

Liposomes represent a leading class of nanoparticles for drug delivery. While a variety of techniques for liposome synthesis have been reported that take advantage of microfluidic flow elements to achieve precise control over the size and polydispersity of nanoscale liposomes, with important implications for nanomedicine applications, these methods suffer from extremely limited throughput, making them impractical for large‐scale nanoparticle synthesis. High aspect ratio microfluidic vertical flow focusing is investigated here as a new approach to overcoming the throughput limits of established microfluidic nanoparticle synthesis techniques. Here the vertical flow focusing technique is utilized to generate populations of small, unilamellar, and nearly monodisperse liposomal nanoparticles with exceptionally high production rates and remarkable sample homogeneity. By leveraging this platform, liposomes with modal diameters ranging from 80 to 200 nm are prepared at production rates as high as 1.6 mg min⁻¹ in a simple flow‐through process.     

Design of an optimized ECCA microchannel for particle manipulation utilizing dean flow coupled elasto-inertial method

In this paper, an Eulerian-Lagrangian simulation was conducted to achieve optimal Expanded-Contracted Cavity Arrays microchannel. First, a new code was developed to solve the viscoelastic flow field, and then the particles were solved by adding appropriate forces to the OpenFOAM Lagrangian solver. This code was then validated for both Eulerian and Lagrangian models. Subsequently, the effect of different parameters such as flow rate, distance from the inlet, cavity depth and distance, and particle size were also studied to obtain the proper geometry for particle focusing. Finally, the selected channel was integrated with a straight channel to separate 4.8 and 13 μm particles. The results of current research can be used to find a proper design of an Expanded-Contracted Cavity Arrays channel to achieve precise focusing and efficient, continuous, and sheathless particle/cell separation, which is much worthy for applications such as high-speed cytometry, cell counting, sorting, and many biological applications.     

导叶式旋风管内短路流颗粒夹带的研究

采用数值模拟方法,结合试验与理论分析,研究Shell型导叶式旋风管内短路流颗粒夹带问题。结果表明:Shell型旋风管直筒芯管下口存在短路流现象,计算得知短路流量占进口总流量的39.3%。理论分析发现,短路流主要夹带粒径小于9μm的颗粒,短路流夹带颗粒临界粒径为9μm。另外,数值模拟跟踪颗粒逃逸的轨迹证明,Shell型旋风管能将粒径大于9μm的颗粒全部除尽;粒径小于9μm的颗粒既有经排尘口返混逃逸,又有短路流夹带逃逸,其中短路流夹带逃逸占主要部分,且随着粒径的增加,经芯管下口短路夹带逃逸的数目减小。     

High-Resolution Elemental Bioimaging of Ca, Mn, Fe, Co, Cu, and Zn Employing LA-ICP-MS and Hydrogen Reaction Gas

Imaging of trace metal distribution in tissue sections by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is typically performed using spatial resolutions of 30 μm(2) and above. Higher resolution imaging is desirable for many biological applications in order to approach the dimensions of a single cell. The limiting factor for increasing resolution is sensitivity, where signal-to-noise ratios are poor due to inherent background spectral interferences and reduced sample volume with decreasing laser beam diameter. Several prominent spectral interferences are present for a number of biologically relevant isotopes, including the (40)Ar(16)O(+) spectral interference on (56)Fe(+). We examined if H(2) as a reaction gas could improve the analytical performance of imaging experiments for a range of masses with spectral interferences. At low (<1 mL min(-1)) H(2) flow rates, greater spectral interference due to H(+) adducts was observed for (55)Mn, (57)Fe, and (59)Co. At higher flow rates of up to 3 mL H(2) per minute, the spectral interferences were reduced leading to improvement in limits of analysis for masses with O- and N-based polyatomic interferences. Enhanced sensitivity with the reaction cell allowed construction of high resolution (6 μm(2)) imaging of (56)Fe in the mouse brain that approached the dimensions of single cells.     

Axial particle trajectory measurements in high-gradient magnetic separation

A technique has been developed for studying the trajectories of paramagnetic particles in a high-gradient magnetic separator. Human red blood cells are used as the paramagnetic particle and their trajectories in relation to a single magnetized stainless-steel wire are imaged. The flow velocity is parallel to the wire and has a value of approximately 50 μm . s^-1. The magnetic field of 1.4 T is applied perpendicular to the wire. A phase modulation technique is used to image the red blood cells with a resolution of less than 0.5 μm. The measured particle trajectories confirm the main features of current theoretical models.     

Nanoflow low pressure high peak capacity single dimension LC-MS/MS platform for high-throughput, in-depth analysis of mammalian proteomes

The use of narrow bore LC capillaries operated at ultralow flow rates coupled with mass spectrometry provides a desirable convergence of figures of merit to support high-performance LC-MS/MS analysis. This configuration provides a viable means to achieve in-depth protein sequence coverage while maintaining a high rate of data production. Here we explore potential performance improvements afforded by use of 25 μm × 100 cm columns fabricated with 5 μm diameter reversed phase particles and integrated electrospray emitter tips. These columns achieve a separation peak capacity of ≈750 in a 600-min gradient, with average chromatographic peak widths of less than 1 min. At room temperature, a pressure drop of only ≈1500 psi is sufficient to maintain an effluent flow rate of ≤10 nL/min. Using mouse embryonic stem cells as a model for complex mammalian proteomes, we reproducibly identify over 4000 proteins across duplicate 600 min LC-MS/MS analyses.     

Optofluidic fluorescent imaging cytometry on a cell phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings.     

Introducing Enantioselective Ultrahigh-Pressure Liquid Chromatography (eUHPLC): Theoretical Inspections and Ultrafast Separations on a New Sub-2-μm Whelk-O1 Stationary Phase

A new chiral stationary phase for ultrahigh-pressure liquid chromatography (UHPLC) applications was prepared by covalent attachment of the Whelk-O1 selector to spherical, high-surface-area 1.7-μm porous silica particles. Columns of varying dimensions (lengths of 50, 75, 100, and 150 mm and internal diameters of 3.0 or 4.6 mm) were packed and characterized in terms of permeability, efficiency, retention, and enantioselectivity, using both organic and water-rich mobile phases. A conventional HPLC Whelk-O1 column based on 5.0-μm porous silica particles and packed in a 250 mm × 4.6 mm column was used as a reference. Van Deemter curves, generated with low-molecular-weight solutes on a 100 mm × 4.6 mm column packed with the 1.7-μm particles, showed H(min) (μm) and μ(opt) (mm/s) values of 4.10 and 5.22 under normal-phase and 3.74 and 4.34 under reversed-phase elution conditions. The flat C term of the van Deemter curves observed with the 1.7-μm particles allowed the use of higher-than-optimal flow rates without significant efficiency loss. Kinetic plots constructed from van Deemter data confirmed the ability of the column packed with the 1.7-μm particles to afford subminute separations with good efficiency and its superior performances in the high-speed regime, compared to the column packed with 5.0-μm particles. Resolutions in the time scale of seconds were obtained using a 50-mm-long column in the normal phase or polar organic mode. The intrinsic kinetic performances of 1.7-μm silica particles are retained in the Whelk-O1 chiral stationary phase, clearly demonstrating the potentials of enantioselective UHPLC in terms of high speed, throughput, and resolution.     

Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells

Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts; however, the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW to 179 kW/cm(2)). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: S(beam3)/S(beam1)) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching.     

Microfluidic sorting with a moving array of optical traps

Optical sorting was demonstrated by selective trapping of a set of microspheres (having specific size or composition) from a flowing mixture and guiding these in the desired direction by a moving array of optical traps. The approach exploits the fact that whereas the fluid drag force varies linearly with particle size, the optical gradient force has a more complex dependence on the particle size and also on its optical properties. Therefore, the ratio of these two forces is unique for different types of flowing particles. Selective trapping of a particular type of particles can thus be achieved by ensuring that the ratio between fluid drag and optical gradient force on these is below unity whereas for others it exceeds unity. Thereafter, the trapped particles can be sorted using a motion of the trapping sites towards the output. Because in this method the trapping force seen by the selected fraction of particles can be suitably higher than the fluid drag force, the particles can be captured and sorted from a fast fluid flow (about 150 μm/s). Therefore, even when using a dilute particle suspension, where the colloidal trafficking issues are naturally minimized, due to high flow rate a good throughput (about 30 particles/s) can be obtained. Experiments were performed to demonstrate sorting between silica spheres of different sizes (2, 3, and 5 μm) and between 3 μm size silica and polystyrene spheres.     

Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high‐force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi‐analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti‐epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof‐of‐concept, EpCAM‐labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument.     

Analytical electric field and sensitivity analysis for two microfluidic impedance cytometer designs

Microfabricated impedance cytometers have been developed to measure the electrical impedance of single biological particles at high speed. A general approach to analytically solve the electric field distributions for two different designs of cytometers: parallel facing electrodes and coplanar electrodes, using the Schwarz-Christoffel Mapping method is presented. Compared to previous analytical solutions, our derivations are more systematic and solutions are more straightforward. The solutions have been validated by comparison with numerical simulations performed using the finite element method. The influences on the electric field distribution due to the variations in the geometry of the devices have been discussed. A simple method is used to determine the impedance sensitivity of the system and to compare the two electrode designs. For identical geometrical parameters, we conclude that the parallel electrodes design is more sensitive than the coplanar electrodes.