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. 2018 Mar;6(1):1-23.
doi: 10.1142/S2339547818300019. Epub 2018 Mar 16.

Microfluidic flow cytometry: The role of microfabrication methodologies, performance and functional specification

Anil B Shrirao  1 , Zachary Fritz  1 , Eric M Novik  2 , Gabriel M Yarmush  1 , Rene S Schloss  1 , Jeffrey D Zahn  1 , Martin L Yarmush  1
Affiliations

Microfluidic flow cytometry: The role of microfabrication methodologies, performance and functional specification

Anil B Shrirao et al. Technology (Singap World Sci). 2018 Mar.

Abstract

Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers.

Keywords: Electrochemical Detection; Florescence-Based Detection; Flow Cytometry; Flow Focusing; Impedance Spectroscopy; Microfluidics; Micropump; Microvalves; Optical Detection.

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Conflict of interest statement

CONFLICTS OF INTEREST There are no conflicts of interest to declare.

Figures

Figure 1
Figure 1. Schematic of process flow in flow cytometers
Interdependence between performance, fabrication, selection of detection and particle manipulation techniques.
Figure 2
Figure 2. Schematic diagram of the micro-cytometer chip with optical and electrical components
(a) Integrated electrodes and waveguide coupled to an optical fiber for light delivery. (b) Close up of the measurement region, with the lens, waveguide and impedance detection electrodes.
Figure 3
Figure 3. Microfluidic flow cytometer with integrated optical fiber and components for detection
Beam splitters and band pass filters separated light at the termini of the detection optical fibers (yellow arrows). The 635 nm excitation fiber (red arrow) was single mode, all other optical fibers were multimode.
Figure 4
Figure 4. Particle manipulation schemes
(a) Schematic of device with integrated magnetic micro coils to perform spatial manipulations of magnetic particles and eventual sorting. (b) Schematic of the flow configuration for the acoustophoresis cell separation.
Figure 5
Figure 5. Lateral hydrodynamic focusing in microchannels
Fluid 1 is the sheath fluid, Fluid 2 is the sample fluid containing the particles to be focused, and Q1 and Q2 are their respective flow rates.
Figure 6
Figure 6. Clog-proof constriction channel
(I)–(III) shows the normal process of a cell passing through the constriction channel. (IV)–(VI) shows the opening of the pneumatic valve to widen the constriction channel to accommodate clogging material.
Figure 7
Figure 7. Stepped inertial focusing used in focusing a suspension of polystyrene microbeads and Jurkat (human leukemia) cells
(1) The progression (i–iv) of particle focusing with flow through the stepped microchannel, shown in lateral cross section in (2).
Figure 8
Figure 8. Standing surface acoustic waves particle focusing
A pair of interdigitated transducers (IDTs) positioned on either side of the microchannel generate acoustic waves which manipulate dispersed particles (I) into a single file line (II). These focused particles are then analyzed using a standard LIF flow cytometry set up.
Figure 9
Figure 9. Typical optical setup for a LIF microfluidic flow cyto meter
Note the two photomultiplier tubes (PMTs) allowing for sample multiplexing.
Figure 10
Figure 10. Imaging-based flow cytometry detection
(a) Schematic diagram of light sheet based optical system used for image based detection in microfluidic flow cytometers. (b) Microfluidic flow cytometer for leukocyte classification. The inset graph shows the synchronization of the laser pulses and camera exposure times.
Figure 11
Figure 11. Impedance-based flow cytometry detection
(a) The layout of the device. (b) Impedance amplitude data. Peaks correspond to a cell passing through the constriction channel, and peak widths represent cell transit times. (c) Inverted microscope image of a cell passing through the constriction channel, used to calculate the cell’s elongation length.
Figure 12
Figure 12. Schematic diagram of the micro impedance cytometer system utilizing the confocal-optical detection and dual frequency impedance measurement
(a) Simultaneous measurement of fluorescence properties using optical detection and impedance using electrodes to compare the electrical and optical properties on a cell-by-cell basis. (b) A simplified schematic of the impedance detection system with differential measurement. (c) Typical frequency-dependent impedance magnitude signal for a polymer bead and a cell of similar size.
Figure 13
Figure 13. GMR-based flow cytometry
(a) The process involves introducing the SPION-labeled cells (1), which then pass over magnetic Ni chevrons that serve the dual purpose of filtering out unbound SPIONs (2) and focusing the cells (3). The focused cells then pass over the Wheatstone bridges and are detected (4). (b) Data for the magnetic focusing efficiency calculations, determined via microscopy observation of the cells flowing over a GMR band. (c) Photo of the device, with the insets showing an arrangement of the Wheatstone bridges in the GMR and typical time of flight (TOF) data patterns.
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