Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> Focusing of particles/cells in the vertical direction inside a micromachined flow cytometer is a critical issue while using an embedded optical detection system aligned with microchannels. Even if the particles/cells have been focused centrally in the horizontal direction using coplanar sheath flows, appreciable errors may still arise if they are randomly distributed in the vertical direction. This work presents a vertical focusing device utilizing dielectrophoretic (DEP) forces and its application on micromachined flow cytometer. A pair of parallel microelectrodes is deposited on the upper and bottom surface of the microfluidic channel to drive particles/cells into the vertical center of the sample flow. This new microfluidic device is capable of three-dimensional (3-D) focusing of microparticles/cells and thus improves the uniformity of the optical detection signals. This 3-D focusing feature of the sample flow is realized utilizing the combination of dielectrophoretic and hydrodynamic forces. Initially, two sheath flows are used to focus the sample flow horizontally by means of hydrodynamic forces, and then two embedded planar electrodes apply negative DEP forces to focus the particles/cells vertically. A new micromachined flow cytometer integrated with an embedded optical detection mechanism is then demonstrated. Numerical simulation is used to analyze the operation conditions and the dimension of the microelectrodes for DEP manipulation. The dynamic trace of the moving particles/cells within a flow stream under the DEP manipulation is calculated numerically. Micro polystyrene beads and diluted human red blood cells (RBC) are used to test the performance of the proposed device. The experimental results confirm the suitability of the proposed device for applications requiring precise counting of particles or cells. Experimental data indicates the proposed method can provide more stable signals over the other types of micromachined flow cytometers that were previously reported. <s> BIB001 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> We introduce a novel on-chip microparticle focusing technique using standing surface acoustic waves (SSAW). Our method is simple, fast, dilution-free, and applicable to virtually any type of microparticle. <s> BIB002 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities—such as automated sample staining—are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers. <s> BIB003 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> This paper presents a fundamental study of particle electrokinetic focusing in a single microchannel constriction. Through both experiments and simulations, we demonstrate that such dielectrophoresis-induced particle focusing can be implemented in a much smaller magnitude of DC-biased AC electric fields (10 kV/m in total) as compared to pure DC electric fields (up to 100 kV/m). This is attributed to the increase in the ratio of cross-stream particle dielectrophoretic velocity to streamwise electrokinetic velocity as only the DC field component contributes to the latter. The effects of the 1 kHz frequency AC to DC electric field ratio on particle trajectories and velocity variations through the microchannel constriction are also examined, which are found to agree with the simulation results. <s> BIB004 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> We describe a novel technique that utilizes simultaneous implementation of dielectrophoresis (DEP) and magnetophoresis (MAP) to focus magnetic particles into streams for optical analysis of biological samples. This technique does not require sheath flow and utilizes a novel interdigitated electrode array chip that yields multiple streams of flowing magnetic particles in single-file columns. The MAP force placed particles in close proximity to the microelectrodes where they were subjected to a strong DEP force that generated the particle focusing effect. Particle focusing efficiency was improved using this combination DEP?MAP technique compared to DEP alone: particle stream widths were reduced ~47% and stream width variability was reduced 80% for focused streams of 8.5 ?m diameter magnetic particles. 3 ?m diameter magnetic particles were strongly focused with DEP?MAP (~4 ?m wide streams with sub-?m variability in stream width) while DEP alone provided minimal focusing. Additional components of a prototype detection system were also demonstrated including an integrated magnetic pelleting component, a hand-held MHz frequency signal generator and a bench-top near-confocal microscope for optical analysis of flowing particles. Preliminary testing of a sandwich assay performed on the surface of magnetic particles showed 50 ppb detection levels of a surrogate biotoxin (ovalbumin) in a raw milk sample. <s> BIB005 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> Three-dimensional (3D) continuous microparticle focusing has been achieved in a single-layer polydimethylsiloxane (PDMS) microfluidic channel using a standing surface acoustic wave (SSAW). The SSAW was generated by the interference of two identical surface acoustic waves (SAWs) created by two parallel interdigital transducers (IDTs) on a piezoelectric substrate with a microchannel precisely bonded between them. To understand the working principle of the SSAW-based 3D focusing and investigate the position of the focal point, we computed longitudinal waves, generated by the SAWs and radiated into the fluid media from opposite sides of the microchannel, and the resultant pressure and velocity fields due to the interference and reflection of the longitudinal waves. Simulation results predict the existence of a focusing point which is in good agreement with our experimental observations. Compared with other 3D focusing techniques, this method is non-invasive, robust, energy-efficient, easy to implement, and applicable to nearly all types of microparticles. <s> BIB006 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> Focusing particles and cells into a tight stream is often required in order for continuous flow detection, counting and sorting. So far a variety of particle focusing methods have been developed in microfluidic devices, among which magnetic focusing is still relatively new. We?develop in this work an approach to embedding symmetrically two repulsive permanent magnets about a straight rectangular microchannel in a PDMS-based microfluidic device. The closest distance between the magnets is limited only by the sizes of the embedded and holder magnets involved in the fabrication process. The developed device is used to implement and investigate the three-dimensional magnetic focusing of polystyrene particles in ferrofluid microflow with both the top- and side-view visualizations. The effects of flow speed and particle size on the particle focusing effectiveness are studied. The developed device is also applied to magnetically focus yeast cells in ferrofluid, which proves to be biocompatible as verified by a cell viability test. In addition, an analytical model is developed and found to be able to predict the experimentally observed particle and cell focusing behaviors with reasonable agreement. <s> BIB007 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> We describe a versatile microfluidic fluorescence-activated cell sorter that uses acoustic actuation to sort cells or drops at ultra-high rates. Our acoustic sorter combines the advantages of traditional fluorescence-activated cell (FACS) and droplet sorting (FADS) and is applicable for a multitude of objects. We sort aqueous droplets, at rates as high as several kHz, into two or even more outlet channels. We can also sort cells directly from the medium without prior encapsulation into drops; we demonstrate this by sorting fluorescently labeled mouse melanoma cells in a single phase fluid. Our acoustic microfluidic FACS is compatible with standard cell sorting cytometers, yet, at the same time, enables a rich variety of more sophisticated applications. <s> BIB008 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> In this paper, we propose a novel hybrid microfluidic device, for the first time, which integrates and fully couples both dielectrophoresis (DEP) and inertial focusing. A DEP force is coupled with inertial lift force to adjust particle's equilibrium position in the vertical direction in real time. The focusing pattern and position of the particles along the horizontal plane can be concurrently adjusted by adjusting their vertical position with DEP forces. The magnitude of secondary flow drag dramatically increases when particles are levitated towards the centre of the channel in the vertical direction. The paper analytically and experimentally investigates the mechanism of this tuneable DEP-inertial microfluidic device. The proposed hybrid device possesses the advantages of both DEP and inertial microfluidic devices, working in a high-throughput manner, as well as having precise controllability in real-time. This DEP-inertial microfluidic device is potentially a versatile and robust platform for a feedback-controlled manipulation and separation of particles and cells. <s> BIB009 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> Flow cytometers have been optimized for use in portable platforms, where cell separation, identification and counting can be achieved in a compact and modular format. This feature can be combined with magnetic detection, where magnetoresistive sensors can be integrated within microfluidic channels to detect magnetically labelled cells. This work describes a platform for in-flow detection of magnetically labelled cells with a magneto-resistive based cell cytometer. In particular, we present an example for the validation of the platform as a magnetic counter that identifies and quantifies Streptococcus agalactiae in milk. <s> BIB010 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> Increasing research efforts have been recently devoted to the coupling of microfluidic chip-integrated ionization sources to mass spectrometry (MS). Considering the limitations of microfluidic chips coupled with MS such as liquid spreading, dead volume, and manufacturing troubles, this paper proposed a new three-dimensional (3D) flow focusing (FF)-based microfluidic ionizing source. This source was fabricated by using the two-layer soft lithography method with the nozzle placed inside the chip. The proposed FF microfluidic chip can realize two-phase FF with liquid in air regardless of the viscosity ratio of the continuous and dispersed phases. MS results indicated that the proposed FF microfluidic chip can work as a typical electrical ionization source when supplied with high voltage and can serve as a sonic ionization source without high voltage. The electro-sonic FF ionization microfluidic chip is expected to have various applications, particularly in the integrated and portable applications of ionization sources coupling with portable MS in the future. <s> BIB011 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Other Methods <s> Trapping and sorting of micro-sized objects is one important application of lab on a chip devices, with the use of acoustic bubbles emerging as an effective, non-contact method. Acoustically actuated bubbles are known to exert a secondary radiation force (FSR) on micro-particles and stabilize them on the bubble surface, when this radiation force exceeds the external hydrodynamic forces that act to keep the particles in motion. While the theoretical expression of FSR has been derived by Nyborg decades ago, no direct experimental validation of this force has been performed, and the relationship between FSR and the bubble's ability to trap particles in a given lab on a chip device remains largely empirical. In order to quantify the connection between the bubble oscillation and the resultant FSR, we experimentally measure the amplitude of bubble oscillations that give rise to FSR and observe the trapping and release of a single microsphere in the presence of the mean flow at the corresponding acoustic parameters using an acoustofluidic device. By combining well-developed theories that connect bubble oscillations to the acoustic actuation, we derive the expression for the critical input voltage that leads to particle release into the flow, in good agreement with the experiments. <s> BIB012
Besides hydrodynamic focusing, acoustics BIB002 BIB006 BIB012 BIB008 BIB011 , dielectrophoresis (DEP) force BIB004  BIB009  , electrokinetics and magnetophoresis (MAP) BIB005 BIB007 BIB010 can be used alternatively to focus particles and cells in a microflow cytometer. Acoustic-based focusing methods do not need sheath fluids for 3D focusing. The standing surface acoustic waves (SSAW) field generated by two parallel interdigital transducers (IDTs) applies lateral and vertical acoustic radiation force to the particles or cells, as shown in Figure 3a . Particles or cells are focused in the center of the microchannels where the pressure node is located. Recently, Chen et al. created an SSAW-based 3D focusing microflow cytometer BIB012 , as shown in Figure 3b . Dielectrophoresis (DEP) is another approach to achieve 3D focusing in microchannels without sheath fluids. In DEP, a non-uniform oscillating electric field creates a dipole on the cells or particles that will experience a negative or positive force depending on the dipole's phase with the applied AC field and the strength of the electric field at each end of the dipole. By changing the frequency of the field, it is possible to tune the strength of the force on the particles or even to switch the direction of the DEP force. The DEP force can adjust particle's or cell's equilibrium position-normally at the center of the channel in the vertical position by utilizing a pair of parallel microelectrodes on the top and bottom surface of the microchannel. Usually, particles or cells experience a negative force as a positive DEP force pulls the particles or cells towards the surface of the electrode where the greatest field gradient occurs that could destroy the cells BIB003 . The DEP force depends on the size and electrical properties of the particles or cells, the electrical properties of the sample fluids and the electric field. Large particles or cell move slower than smaller particles or cells, and the focusing pattern of each cell or particle is different. Many microchip-based flow cytometers now use more than one technique to confine sample flow. Lin et al. BIB001 combined the 2D hydrodynamic focusing and DEP method to obtain 3D focusing: two electrodes exerted a DEP force from the vertical direction on the particles and cells, which had already been focused laterally hydrodynamically by two sheath fluids. More recently, Zhang et al. presented a novel DEP-inertial microflow cytometer, which combined the DEP force and inertial force to achieve vertical-focusing BIB009 . The MAP theory is similar to DEP force, except that the electric field is replaced by the magnetic field. In addition, particles or cells need to be attached to a magnetic bead so that they can move to the interrogation point exactly. 