Stable microfluidic flow focusing using hydrostatics

We have demonstrated a microfluidic device that can not only achieve three-dimensional flow focusing but also confine particles to the center stream along the channel. The device has a sample channel of smaller height and two sheath flow channels of greater height, merged into the downstream main channel where 3D focusing effects occur. We have demonstrated that both beads and cells in our device display significantly lower CVs in velocity and position distributions as well as reduced probability of coincidental events than they do in conventional 2D-confined microfluidic channels. The improved particle confinement in the microfluidic channel is highly desirable for microfluidic flow cytometers and in fluorescence-activated cell sorting (FACS). We have also reported a novel method to measure the velocity of each individual particle in the microfluidic channel. The method is compatible with the flow cytometer setup and requires no sophisticated visualization equipment. The principles and methods of device design and characterization can be applicable to many types of microfluidic systems.

The Application of Microfluidics in Preparing Nano Drug Delivery Systems

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.

Microfluidic gradient systems offer a very precise means to probe the response of cells to graded biomolecular signals in vitro, for example to model how morphogen proteins affect cell fate during developmental processes. However, existing gradient makers are designed for non-physiological plastic or glass cell culture substrates that are often limited in maintaining the phenotype and function of difficult-to-culture mammalian cell types, such as stem cells. To address this bottleneck, we combine hydrogel engineering and microfluidics to generate tethered protein gradients on the surface of biomimetic poly(ethylene glycol) (PEG) hydrogels. Here we used software-assisted hydrodynamic flow focusing for exposing and rapidly capturing tagged proteins to gels in a step-wise fashion, resulting in immobilized gradients of virtually any desired shape and composition. To render our strategy amenable for high-throughput screening of multifactorial artificial cellular microenvironments, a dedicated microfluidic chip was devised for parallelization and multiplexing, yielding arrays of orthogonally overlapping gradients of up to 4 × 4 proteins. To illustrate the power of the platform for stem cell biology, we assessed how gradients of tethered leukemia inhibitory factor (LIF) influence embryonic stem cell (ESC) behavior. ESC responded to LIF gradients in a binary manner, maintaining the pluripotency marker Rex1/Zfp42 and forming self-renewing colonies above a threshold concentration of 85 ng cm(-2). Our concept should be broadly applicable to probe how complex signaling microenvironments influence stem cell fate in culture.

Single-cell analysis commonly requires the confinement of cell suspensions in an analysis chamber or the precise positioning of single cells in small channels. Hydrodynamic flow focusing has been broadly utilized to achieve stream confinement in microchannels for such applications. As imaging flow cytometry gains popularity, the need for imaging-compatible microfluidic devices that allow for precise confinement of single cells in small volumes becomes increasingly important. At the same time, high-throughput single-cell imaging of cell populations produces vast amounts of complex data, which gives rise to the need for versatile algorithms for image analysis. In this work, we present a microfluidics-based platform for single-cell imaging in-flow and subsequent image analysis using variational autoencoders for unsupervised characterization of cellular mixtures. We use simple and robust Y-shaped microfluidic devices and demonstrate precise 3D particle confinement towards the microscope slide for high-resolution imaging. To demonstrate applicability, we use these devices to confine heterogeneous mixtures of yeast species, brightfield-image them in-flow and demonstrate fully unsupervised, as well as few-shot classification of single-cell images with 88% accuracy.

A central challenge in the development of drug-encapsulated polymeric nanoparticles is the inability to control the nanoparticle physicochemical properties that affect their biodistribution, drug release, and efficacy. Nanoparticles may be developed by mixing and nanoprecipitation of polymers and drugs dissolved in organic solvents with non-solvents. Inadequate control over this mixing process is a source of variability in the synthesis of these nanoparticles by nanoprecipitation. We demonstrate that rapid and tunable mixing through hydrodynamic flow focusing in a microfluidic device can be used to control nanoprecipitation of poly(lactic-co-glycolic acid)-bpoly(ethylene glycol) (PLGA-PEG) diblock copolymers as a model polymeric biomaterial for drug delivery.

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.

The benefits of LST are many. This directly translates to applications, making it attractive, and can be touted as the tool for next-generation technologies. High-throughput interrogation systems used in medical and biological sciences are set to benefit directly from sheet-based technology. Existing technologies are limited by field of interrogation, low throughput, lack of volume imaging capability, specificity, and complex instrumentation. LST overcomes most of these limitations and has additional features such as single-shot sectional imaging, diffraction-limited resolution, and lab-on-a-chip solution. Expressly, flow-based imaging/cytometry is set to benefit directly from sheet illumination. The fact that point illumination requires additional complex instrumentation (additional sheath fluid, hydrodynamic flow focusing, and precise matching of system PSF with flowing samples) makes it somewhat limited. In this respect, a light sheet provides a suitable replacement. Overall, the emerging LST has the potential to advance a range of fields from biomedical sciences to medicine and applied physics to engineering sciences.

Synthetic polymer nanoparticles (NPs) with biomimetic properties are ideally suited for different biomedical applications such as drug delivery and direct therapy. However, bulk synthetic approaches can suffer from poor reproducibility and scalability when precise size control or multi-step procedures are required. Herein, we report an integrated microfluidic chip for the synthesis of polymer NPs. The chip could sequentially perform homopolymer synthesis and subsequent crosslinking into NPs without intermediate purification. This was made possible by fabrication of the chip with a fluorinated elastomer and incorporation of two microfluidic mixers. The first was a long channel with passive mixing features for the aqueous RAFT synthesis of stimuli-responsive polymers in ambient conditions. The polymers were then directly fed into a hydrodynamic flow focusing (HFF) junction that rapidly mixed them with a crosslinker solution to produce NPs. Compared to microfluidic systems made of PDMS or glass, our chip had better compatibility and facile fabrication. The polymers were synthesized with high monomer conversion and the NP size was found to be influenced by the flow rate ratio between the crosslinker solution and polymer solution. This allowed for the size to be predictably controlled by careful adjustment of the fluid flow rates. The size of the NPs and their stimuli-responses were studied using DLS and SEM imaging. This microfluidic chip design can potentially streamline and provide some automation for the bottom-up synthesis of polymer NPs while offering on-demand size control.

Chapter 7 - Microfluidic mixing and devices for preparing nanoparticulate drug delivery systems

Ionizable lipid nanoparticles (iLNPs) have revolutionized Ribonucleic acid (RNA) therapeutics by enabling precise and efficient delivery of nucleic acids. However, their clinical translation remains challenged by batch-to-batch variability, complex lipid - RNA interactions, and stringent regulatory requirements. This review highlights how advanced microfluidic technologies address these issues by providing precise control over iLNP fabrication through engineered mixer geometries, optimized flow dynamics, and pH-dependent self-assembly. Comparative analyses of hydrodynamic flow focusing (HFF), and staggered herringbone mixers (SHM) demonstrate their distinct influence on particle size, polydispersity index (PDI), and encapsulation efficiency. Furthermore, the integration of design-of-experiments (DoE) methodologies, computational fluid dynamics (CFD) modeling, and machine learning (ML)-assisted optimization enables predictive formulation design and adaptive process control, enhancing reproducibility and scalability. Collectively, this review underscores microfluidics and ML as synergistic technologies that bridge laboratory innovation with Good Manufacturing Practice (GMP)-compliant, large-scale production paving the way for the next generation of intelligent, personalized RNA nanomedicines.

Flow cytometry is a potentially powerful tool to analyze the kinetics of ligand binding, cell response and molecular assembly. The difficulty in adding reactant to cells, achieving adequate mixing, delivering those cells to the laser focal point and establishing stable flow, has historically limited flow cytometry to systems with reactions times longer than 5 s. With the advent of automated syringes and flow injection methods, sample injection times shorter than 1 s have become routine. However, an inherent problem in acquiring time courses starting under 1 s is that rapid sample introduction through the flow tip to the detection point perturbs laminar flow. The purpose of this work was to determine if stable flow could be reestablished more quickly if the sheath flow was reduced during sample introduction, returning to normal sheath and sample rates afterward. We used programmable syringes and valves to control sample mixing as well as sheath and sample delivery through the flow tip to the detection point for stream-in-air detection. Stable flow was monitored by mean particle fluorescence during sample introduction. With no sheath reduction, stable flow recovered after more than 1 s. By reducing sheath flow during the short period (300 msec) of sample mixing and delivery, stable laminar flow recovered within 200 msec. This use of automated syringes to control both sheath and sample flow provides a potential for robust sample handling applicable to kinetic as well as high throughput flow cytometric analysis.

This paper reports a micro flow cytometer fabricated in Polymethylmethacrylate (PMMA) in which a 3d hydrodynamic flow focusing is employed in order to align the particles in a single line along the focused stream. The device has been fabricated by direct micro milling of two parts of PMMA that were finally bonded together. With a suitable choice of the fluidic channel geometry, a circular sample stream located in the center of the channel is obtained. Numerical simulations have been performed in order to investigate the flow characteristic of the structure and find the desiderated geometry. Three dimensional hydrofocusing of the sample fluid was analysed and demonstrated by cross sectional fluorescence imaging in good agreement with numerical simulations. Flow cytometry measurements have been performed by using 10μm particles. From the analysis of the fluorescence signals collected at each transit event we can confirm that the device was capable of creating a single-file particle stream. The results show that the device was capable of discriminating single microparticles with a good signal-to-noise ratio and a high throughput.

Multidisciplinary single-cell optical cytometry.

The leakage problem, which occurs due to the high hydrostatic pressure values of the fluid inside the pipes, is a serious issue in most of the pipeline systems. Therefore, the leak tightness test is required in different standards for verification of almost every kind of pipes used in both academic researches and industrial applications. The behavior of the hydrostatic pressure parameter in the test pipe has to be as smooth as possible during the leak tightness test. The conventional control loops of the servo mechanisms used for pumping the test fluid into the test pipe are weak to provide the minimum overshoot, the rise time and the steady-state error. In this situation, hydrostatic pressure tests are not capable of giving meaningful results. In this study, a leak tightness test machine working with a servo control loop to provide the optimum hydrostatic pressure pattern is designed. The efficacy of the designed leak tightness test machine and proposed optimized servo control loop have been shown with both simulation and experimental results.