Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 3D Hydrodynamic Flow Focusing <s> Abstract This paper presents a novel microfabrication technology of three-dimensional (3D) microstructures with inclined/rotated UV lithography using negative thick photoresist, SU-8. In exposure process, a photomask and a SU-8 coated substrate are fixed together, and tilted or tilted and rotated to a UV source. The reflected UV at the interface between the resist and the substrate is also exploited as well as the incident UV. With the 3D microfabrication technology, various 3D microstructures are easily fabricated such as oblique cylinders, embedded channels, bridges, V-grooves, truncated cones, and so on. The angles between fabricated structures and normal lines are 19.5°, when the incident angle of UV in air is 32°. Thus, the refractive index of SU-8 and the maximum refractive angle at the interface between SU-8 and air are expected about 1.6 and 39°, respectively. <s> BIB001 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 3D Hydrodynamic Flow Focusing <s> This paper presents a generalization of the hydrodynamic focusing technique to three-dimensions. Three-dimensional (3-D) hydrodynamic focusing offers the advantages of precision positioning of molecules in both vertical and lateral dimensions and minimizing the interaction of the sample fluid with the surfaces of the channel walls. In an ideal approach, 3-D hydrodynamic focusing could be achieved by completely surrounding the sample flow by a cylindrical sheath flow that constrains the sample flow to the center of the channel in both the lateral and the vertical dimensions. We present here design and simulation, 3-D fabrication, and experimental results from a piecewise approximation to such a cylindrical flow. Two-dimensional (2-D) and 3-D hydrodynamic focusing chips were fabricated using micromolding methods with polydimethylsiloxane (PDMS). Three-dimensional hydrodynamic focusing chips were fabricated using the "membrane sandwich" method. Laser scanning confocal microscopy was used to study the hydrodynamic focusing experiments performed in the 2-D and 3-D chips with Rhodamine 6G solution as the sample fluid and water as the sheath fluid. <s> BIB002 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 3D Hydrodynamic Flow Focusing <s> This paper presents the inclined UV lithography in water medium and 3D mixing microchannel application. The theoretical limit of the inclination angle of the structure is determined by the difference of the refractive index between air and SU-8. In order to reduce the mismatch of the refractive index between air and photoresist, DI water was used as exposure medium instead of air. As a result, the maximum angle of the inclined structure was improved from 38.7° (in air) to 56.2° (in water). Fine and high aspect ratio SU-8 structures were obtained using UV exposure in water medium. As a microfluidic application, a microchannel having 3D slanted grooves was designed and fabricated. 45° slanted grooves were formed successfully on the sidewalls of the microchannel by using inclined UV exposure in water medium. Mixing efficiency of this microchannel was evaluated by CFD simulation and cross-sectional fluorescence image detection using confocal microscope. <s> BIB003 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 3D Hydrodynamic Flow Focusing <s> Abstract In this work we describe the design and fabrication of a novel three-dimensional flow focusing device with channel dimensions of 50 μm × 50 μm at the focusing region. The device is suitable for one-by-one cell or particle detection near the channel bottom, in for example a Coulter counter or for optical near field detection. The device comprises a silicon-glass sandwich with SU-8 resist in between. The sample flow is hydrodynamically focused between three sheath flows. At the fourth side the sample flow is moving along the channel bottom. We investigated the focusing limitation of the sample flow and found that the minimum width amounts to 2.5 μm at a channel width of 50 μm. Confocal laser scanning measurements were constituted to obtain the sample flow profile. We show the experimental and numerical images of the focused stream shape from a cross-section perspective. Furthermore, we analyzed the measured fluorescent color intensity of the sample stream and from that, calculated the sample flow height. These results were compared with the corresponding confocal images. Additionally, the influence of the hydrodynamic focusing on the sample profile and its uniformity over a certain distance was investigated. <s> BIB004 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 3D Hydrodynamic Flow Focusing <s> In this paper, we have designed, fabricated and characterized an efficient 3-dimensional hydrodynamic focusing microfluidic device (3D-HFMD) in numerical and experimental ways. The 3D-HFMD achieved vertical focusing of core sample flow effectively in the pressure driven flow with the help of locally increased aspect ratio of thickness to width without any horizontal separation wall, thereby enabling us to realize 3D focusing after simple horizontal focusing. Extensive numerical analysis shows that the 3D-HFMD enhances the performance of vertical focusing compared with the previous designs over a wide range of the Reynolds numbers, even in the case of a the global aspect ratio less than 0.5. Experimental results from the fabricated 3D-HFMD confirmed that 3D hydrodynamic focusing could be successfully achieved, even when the dimension of the channel was kept consistent with that of general microfluidic channels. The 3D-HFMD suggested in this study could be applied to various integrated biological or chemical microfluidic devices. <s> BIB005 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 3D Hydrodynamic Flow Focusing <s> A microflow cytometer was developed that ensheathed the sample (core) fluid on all sides and interrogated each particle in the sample stream at four different wavelengths. Sheathing was achieved by first sandwiching the core fluid with the sheath fluid laterally via fluid focusing. Chevron-shaped groove features fabricated in the top and bottom of the channel directed sheath fluid from the sides to the top and bottom of the channel, completely surrounding the sample stream. Optical fibers inserted into guide channels provided excitation light from diode lasers at 532 and 635 nm and collected the emission wavelengths. Two emission collection fibers were connected to PMTs through a multimode fiber splitter and optical filters for detection at 635 nm (scatter), 665 nm and 700 nm (microsphere identification) and 565 nm (phycoerythrin tracer). The cytometer was capable of discriminating microspheres with different amounts of the fluorophores used for coding and detecting the presence of a phycoerythrin antibody complex on the surface of the microspheres. Assays for Escherichia coli were compared with a commercial Luminex flow cytometer. <s> BIB006 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 3D Hydrodynamic Flow Focusing <s> A microfabricated flow cytometer was used to demonstrate multiplexed detection of bacteria and toxins using fluorescent coded microspheres. Antibody-coated microspheres bound biothreat targets in a sandwich immunoassay format. The microfluidic cytometer focused the microspheres in three dimensions within the laser interrogation region using passive groove structures to surround the sample stream with sheath fluid. Optical analysis at four different wavelengths identified the coded microspheres and quantified target bound by the presence of phycoerythrin tracer. The multiplexed assays in the microflow cytometer had performance approaching that of a commercial benchtop flow cytometer. The respective limits of detection for bacteria (Escherichia coli, Listeria, and Salmonella) were found to be 10(3), 10(5), and 10(4) cfu/mL for the microflow cytometer and 10(3), 10(6), and 10(5) cfu/mL for the commercial system. Limits of detection for the toxins (cholera toxin, staphylococcal enterotoxin B, and ricin) were 1.6, 0.064, and 1.6 ng/mL for the microflow cytometer and 1.6, 0.064, and 8.0 ng/mL for the commercial system. <s> BIB007 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 3D Hydrodynamic Flow Focusing <s> In this article, we demonstrate single-layered, “microfluidic drifting” based three-dimensional (3D) hydrodynamic focusing devices with particle/cell focal positioning approaching submicron precision along both lateral and vertical directions. By systematically optimizing channel geometries and sample/sheath flow rates, a series of “microfluidic drifting” based 3D hydrodynamic focusing devices with different curvature angles are designed and fabricated. Their performances are then evaluated using confocal microscopy, fast camera imaging, and side-view imaging techniques. Using a device with a curvature angle of 180°, we have achieved a standard deviation of ±0.45 μm in particle focal position and a coefficient of variation (CV) of 2.37% in flow cytometric measurements. To the best of our knowledge, this is the best CV that has been achieved using a microfluidic flow cytometry device. Moreover, the device showed the capability to distinguish 8 peaks when subjected to a stringent 8-peak rainbow calibration test, signifying the ability to perform sensitive, accurate tests similar to commercial flow cytometers. We have further tested and validated our device by detection of HEK-293 cells. With its advantages in simple fabrication (i.e., single-layered device), precise 3D hydrodynamic focusing (i.e., submicrometer precision along both lateral and vertical directions), and high detection resolution (i.e., low CV), our method could serve as an important basis for high-performance, mass-producible microfluidic flow cytometry. <s> BIB008 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 3D Hydrodynamic Flow Focusing <s> A micro flow cytometer with a single step 3D hydrodynamic flow focusing has been developed. The proposed design is capable to create a single-file particle stream that is self-aligned with an integrated optical fiber-based detection system, regardless of the flow rate ratio between the focusing and core liquids. The design approach provides the ability to adjust the stream size while keeping the position of the focused stream centered with respect to the focusing channel. The device has been fabricated by direct micro milling of PMMA sheets. Experimental validation of the hydrodynamic sheath focusing effect has been presented and sample stream with tuneable size from about 18 to 50 μm was measured. Flow cytometry measurements have been performed by using 10-23 μm fluorescent particles. From the analysis of the signals collected at each transit event we can confirm that the device was capable to align and measure microparticles with a good coefficient of variance. <s> BIB009
3D hydrodynamic focusing confines the sample fluid to the center of the microchannel in both vertical and horizontal directions, an improvement on the inability of 2D hydrodynamic focusing to focus fluid in the channel's vertical dimension. The straightforward way to achieve 3D hydrodynamic focusing is to use deeper orthogonal sheath fluids, as shown in Figure 2b BIB009 . Two lateral fluids get in above and below the sample fluid in addition to lateral directions and push the sample fluid from both vertical and horizontal directions in a microchannel with a larger dimension than that of the sample channel. Since deeper channels are difficult to fabricate, requiring three fabrication and two alignment steps, 2D hydrodynamic focusing and its one fabrication step is preferred. However, a strategy of using 2D hydrodynamic focusing twice has been applied to achieve 3D hydrodynamic focusing. By using a planar structure, two sheath fluids A and B can be used to focus the sample fluid vertically, and the sample fluid was focused horizontally by another sheath fluid, C BIB005 . Experimental results and numerical simulation results show that the sample stream is focused to a small region in the center of the microchannel. With 3D microfabrication technology, more complex structures are fabricated to focus the particles in two dimensions, such as oblique cylinders or grooves BIB001 . Sundararajan et al. used the "membrane sandwich" method, which contained two sheath fluids from lateral directions and another two on top and at the bottom stacked on the inlet point to create a 3D hydrodynamic focusing microchip BIB002 . Hairer et al. focused the sample fluid by using three sheath fluids in a non-coaxial sheath flow device BIB004 . V-shaped or chevron-shaped grooves were fabricated in microchannels to focus the sample fluid in both lateral and vertical directions BIB006 BIB007 . Similar V-shaped slants were also applied in 3D mixing BIB003 . More recently, Nawaz et al. achieved 3D hydrodynamic focusing by using microfluidic drifting with different curvature angles BIB008 .