Universally applicable three-dimensional hydrodynamic microfluidic flow focusing

We present a simple technique to generate stable hydrodynamically focused flows by driving the flow with hydrostatic pressure from liquid columns connected to the inlets of a microfluidic device. Importantly, we compare the focused flows generated by hydrostatic pressure and classical syringe pump driven flows, and find that the stability of the hydrostatic pressure driven technique is significantly better than the stability achieved via syringe pumps, providing fluctuation-free focused flows that are suitable for sensitive microfluidic flow cytometry applications. We show that the degree of flow focusing with the hydrostatic method can be accurately controlled by the simple tuning of the liquid column heights. We anticipate that this approach to stable flow focusing will find many applications in microfluidic cytometry technologies. Keywords: Microfluidics, hydrodynamic flow focusing, hydrostatic pressure driven flows, flow cytometry

Author response for "A High-Sensitivity and Clogging-Free Microfluidic Impedance Flow Cytometer based on Three-Dimensional Hydrodynamic Focusing"

The Application of Microfluidics in Preparing Nano Drug Delivery Systems

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.

Multidisciplinary single-cell optical cytometry.

Miniature flow cytometer models enable fast and cost-effective management of diseases in vulnerable and low-end settings. The single-line focusing of cell or particle samples is achieved using hydrodynamic forces in the microfluidic channels. The two common configurations among them are the single-sheath and dual-sheath flows wherein the sample is directed through the main channel, and the surrounding sheath fluids are directed into the main channel through inlets on either side of the main channel. Most models predict the width of the focused sample stream based on hydrodynamic focusing in the low Reynolds number regime (Re << 1), where the viscous forces dominate the inertial forces. In this work, we present comparative analysis of particle focusing by single-sheath and dual-sheath configurations for focusing of micron-sized cells/particles in the range 2 to 20 μm in the higher Re (10 < Re < 80) laminar regime. A quantitative analysis of the relative focused stream width (wf/wch) as a function of flow rate ratio (FRR = Sample flow rate/Sheath flow rate) for the two configurations is presented. The particle tracing results are also compared with the experimental fluorescent microscopy results at various FRR. The deviations of the results from the theoretical predictions of hydrodynamic focusing at Re << 1, are explained analytically. These findings clearly outline the range of flow parameters and relative particle sizes that can be used for cytometry studies for a given channel geometry. This is a highly predictive modeling method as it provides substantial results of particle positions across the microchannel width according to their size and FRR for single-line focusing of particles. Such information is crucial for one to engineer miniaturized flow cytometry for screening of desired cells or particles.

Electrical impedance-based particle detection or Coulter counting, offers a lab-on-chip compatible method for flow cytometry. Developments in this area will produce devices with greater portability, lower cost, and lower power requirements than fluorescence-based flow cytometry. Because conventional Coulter apertures are prone to clogging, hydrodynamic focusing improves the device by creating fluid-walled channels with variable width to increase sensitivity without the associated risk of blocking the channel. We describe a device that focuses the sample in three dimensions, creating a narrow sample stream on the floor of the channel for close interaction with sensing electrodes. The key to this design is a stepped outlet channel fabricated in a single layer with soft lithography. In contrast to previous impedance-based designs, the new design requires minimal alignment with the substrate. Three-dimensional focusing maximizes the sensitivity of the device to cell-size particles within much larger channels. Impedance-based particle sensing experiments within this device show an increase in percentage conductivity change by a factor of 2.5 over devices that only focus the sample in the horizontal direction.

The development of microfluidic chip-based cytometers has become an important area due to their advantages of compact size and low cost. Herein, we demonstrate a sheathless microfluidic cytometer which integrates a standing surface acoustic wave (SSAW)-based microdevice capable of 3D particle/cell focusing with a laser-induced fluorescence (LIF) detection system. Using SSAW, our microfluidic cytometer was able to continuously focus microparticles/cells at the pressure node inside a microchannel. Flow cytometry was successfully demonstrated using this system with a coefficient of variation (CV) of less than 10% at a throughput of ~1000 events s(-1) when calibration beads were used. We also demonstrated that fluorescently labeled human promyelocytic leukemia cells (HL-60) could be effectively focused and detected with our SSAW-based system. This SSAW-based microfluidic cytometer did not require any sheath flows or complex structures, and it allowed for simple operation over a wide range of sample flow rates. Moreover, with the gentle, bio-compatible nature of low-power surface acoustic waves, this technique is expected to be able to preserve the integrity of cells and other bioparticles.

Identification and analysis of phytoplankton is important for understanding the environmental parameters that are influenced by the oceans, including pollution and climate change. Phytoplanktons are studied at the single cell level using conventional light-field and fluorescence microscopy, but the technique is labour intensive. Flow cytometry enables rapid and quantitative measurements of single cells and is now used as an analytical tool in phytoplankton analysis. However, it has a number of drawbacks, including high cost and portability. We describe the fabrication of a microfluidic (lab-on-a-chip) device for high-speed analysis of single phytoplankton. The device measures fluorescence (at three wavelength ranges) and the electrical impedance of single particles. The system was tested using a mixture of three algae (Isochrysis Galbana, Rhodosorus m., Synechococcus sp.) and the results compared with predictions from theory and measurements using a commercial flow cytometer (BD FACSAria). It is shown that the microfluidic flow cytometer is able to distinguish and characterise these different taxa and that impedance spectroscopy enables measurement of phytoplankton biophysical properties.

Fluorescence-based detection schemes provide for multiparameter analysis in a broad range of applications in the chemical and biological sciences. Toward the realization of fully portable analysis systems, microfluidic devices integrating diverse functional components have been implemented in a range of out-of-lab environments. That said, there still exits an unmet and recognized need for miniaturized, low-cost, and sensitive optical detection systems, which provide not only for efficient molecular excitation, but also enhanced photon collection capabilities. To this end, an optofluidic platform that is adept at enhancing fluorescence light collection from microfluidic channels is presented. The central component of the detection module is a monolithic parabolic mirror located directly above the microfluidic channel, which acts to enhance the number of emitted photons reflected toward the detector. In addition, two-photon polymerization is used to print a microscale-lens below the microfluidic flow channel and directly opposite the mirror, to enhance the delivery of excitation radiation into the channel. Using such an approach, it is demonstrated that fluorescence signals can be enhanced by over two orders of magnitude, with component parallelization enabling the detection of pL-volume droplets at rates up to 40 000 droplets per second.

This paper presents numerical and experimental analysis of the hydrodynamic flow focusing in a rectangular microchannel. Aim of the study is to improve performance of the Particle Image Velocimetry (PIV) technique applied to micro-scale flow analysis. The symmetric flow focusing system of two channels crossed at right angle is investigated. The numerical model is used to analyse the effect of Reynolds number on the flow focusing mechanism. In the experiment, the flow focusing is applied to concentrate seeding tracers into a thin sheet at the channel axis. Such a modification removes the out of focus images of the seeding particles, effectively improving PIV evaluation of vector fields in microchannel. Based on the experimental and numerical results we have found that expected improvement is possible for the flow at Reynolds number less than 10 only.

This paper develops a microfluidic chip and a binary optical element (BOE) for flow cytometry. The chip is capable of achieving three-dimensional (3D) hydrodynamic focusing, where the sample flow is focused down to 13–23 μm in a velocity range of 0.9–10 m/s. The BOE is used to shape and homogenize laser beam, providing a flat-top 10 μm × 50 μm rectangular spot for illumination. The two devices were combined to build a micro flow cytometer. It was able to estimate microbead sizes and count at 1.3–20k eps with high signal quality, which was competitive to commercial instruments.

A central challenge in the development of drug-encapsulated polymeric nanoparticles is the inability to control the mixing processes required for their synthesis resulting in variable nanoparticle physicochemical properties. Nanoparticles may be developed by mixing and nanoprecipitation of polymers and drugs dissolved in organic solvents with nonsolvents. We used rapid and tunable mixing through hydrodynamic flow focusing in microfluidic channels to control nanoprecipitation of poly(lactic- co-glycolic acid)- b-poly(ethylene glycol) diblock copolymers as a model polymeric biomaterial for drug delivery. We demonstrate that by varying (1) flow rates, (2) polymer composition, and (3) polymer concentration we can optimize the size, improve polydispersity, and control drug loading and release of the resulting nanoparticles. This work suggests that microfluidics may find applications for the development and optimization of polymeric nanoparticles in the newly emerging field of nanomedicine.

In this work, a series of experiments and numerical simulations performed using a volume-of-fluid approach were carried out to investigate the flow of miscible viscous fluid systems through microfluidic flow focusing devices with one central inlet stream (with “fluid 1”) and two lateral inlet streams (with “fluid 2”). The combined effect of the fluid viscosity ratio and the inlet velocity ratio on the characteristics of the central focused outlet stream was assessed in microfluidic channels with different aspect ratios. An analytical expression for the two-dimensional case, relating the width of the central focused stream in the outlet channel with the velocity ratio and the viscosity ratio, was also derived from first principles. The analytical results are in excellent agreement with the two-dimensional numerical results, and the expression is also able to represent well the experimental findings for the configuration with an aspect ratio of 0.84. The width of the central focused outlet stream at the center plane is seen to decrease with both the velocity ratio and the viscosity ratio. The results of the three-dimensional numerical simulations and experimental measurements are in good agreement, producing further insight into the curved interface known to exist when high viscosity contrasts are present in parallel flow systems. It was observed that the interface curvature across the depth of the channel cross section is strongly dependent on the ratio of inlet viscosities and microchannel aspect ratio, highlighting the three-dimensional nature of the flow, in which confinement plays a significant role.

A portable microfluidic flow cytometer based on simultaneous detection of impedance and fluorescence