High-precision screening and sorting of double emulsion droplets.

Mounting evidence suggests that cell populations are extremely heterogeneous, with individual cells fulfilling different roles within the population. Flow cytometry (FC) is a high-throughput tool for single-cell analysis that works at high optical resolution. Sub-populations with unique properties can be screened, isolated and sorted through fluorescence-activated cell sorting (FACS), using intracellular fluorescent products or surface-tagged fluorescent products of interest. However, traditional FC and FACS methods cannot identify or isolate cells that secrete extracellular products of interest. Double emulsion (DE) droplets are an innovative approach to retaining these extracellular products so cells producing them can be identified and isolated with FC and FACS. The water-in-oil-in-water structure makes DE droplets compatible with the sheath flow of flow cytometry. Single cells can be encapsulated with other reagents into DEs, which act as pico-reactors. These droplets allow biological activities to take place while allowing for cell cultivation monitoring, rare mutant identification, and cellular events characterization. However, using DEs in FACS presents technical challenges, including rupture of DEs, poor accuracy and low sorting efficiency. This study presents high-performance sorting using fluorescent beads (as simulants for cells). This study aims to guide researchers in the use of DE-based flow cytometry, offering insights into how to resolve the technical difficulties associated with DE-based screening and sorting using FC.

Biomechanics of circulating cellular and subcellular bioparticles: beyond separation.

Biomechanical attributes have emerged as novel markers, providing a reliable means to characterize cellular and subcellular fractions. Numerous studies have identified correlations between these factors and patients' medical status. However, the absence of a thorough overview impedes their applicability in contemporary state-of-the-art therapeutic strategies. In this context, we provide a comprehensive analysis of the dimensions, configuration, rigidity, density, and electrical characteristics of normal and abnormal circulating cells. Subsequently, the discussion broadens to encompass subcellular bioparticles, such as extracellular vesicles (EVs) enriched either from blood cells or other tissues. Notably, cell sizes vary significantly, from 2 µm for platelets to 25 µm for circulating tumor cells (CTCs), enabling the development of size-based separation techniques, such as microfiltration, for specific diagnostic and therapeutic applications. Although cellular density is relatively constant among different circulating bioparticles, it allows for reliable density gradient centrifugation to isolate cells without altering their native state. Additionally, variations in EV surface charges (-6.3 to -45 mV) offer opportunities for electrophoretic and electrostatic separation methods. The distinctive mechanical properties of abnormal cells, compared to their normal counterparts, present an exceptional opportunity for diverse medical and biotechnological approaches. This review also aims to provide a holistic view of the current understanding of popular techniques in this domain that transcend conventional boundaries, focusing on early harvesting of malignant cells from body fluids, designing effective therapeutic options, cell targeting, and resonating with tissue and genetic engineering principles.

Surface Modification of Polystyrene with Boronic Acid for Immunoaffinity-Based Cell Enrichment.

Obtaining an enriched and phenotypically pure cell population from heterogeneous cell mixtures is important for diagnostics and biosensing. Existing techniques such as fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require preincubation with antibodies (Ab) and specialized equipment. Cell immunopanning removes the need for preincubation and can be done with no specialized equipment. The majority of the available antibody-mediated analyte capture techniques require a modification to the Abs for binding. In this work, no antibody modification is used because we take advantage of the carbohydrate chain in the Fc region of Ab. We use boronic acid as a cross-linker to bind the Ab to a modified surface. The process allows for functional orientation and cleavable binding of the Ab. In this study, we created an immunoaffinity matrix on polystyrene (PS), an inexpensive and ubiquitous plastic. We observed a 37% increase in Ab binding compared with that of a passive adsorption approach. The method also displayed a more consistent antibody binding with 17 times less variation in Ab loading among replicates than did the passive adsorption approach. Surface topography analysis revealed that a dextran coating reduced nonspecific antibody binding. Elemental analysis (XPS) was used to characterize the surface at different stages and showed that APBA molecules can bind upside-down on the surface. While upside-down antibodies likely remain functional, their elution behavior might differ from those bound in the desired way. Cell capture experiments show that the new surface has 43% better selectivity and 2.4-fold higher capture efficiency compared to a control surface of passively adsorbed Abs. This specific surface chemistry modification will allow the targeted capture of cells or analytes with the option of chemical detachment for further research and characterization.

Tuneable Cell-Laden Double-Emulsion Droplets for Enhanced Signal Detection.

Water-in-oil-in-water (w/o/w) or double-emulsion (DE) droplets have been widely used for cellular assays at a single-cell level because of their stability and biocompatibility. The oil shell of w/o/w droplets plays the role of a semipermeable membrane that allows substances with low molecular weight (e.g., water) to travel through but restricts those with high molecular weight (e.g., fluorescent biomarkers). Therefore, the core of DEs can be manipulated using osmosis, resulting in the shrinking or swelling of the core. Water leaves the inner aqueous phase to the outer phase via the oil shell when the osmotic pressure of the outer phase is higher than that in the inner phase, causing the shrinkage of DEs and vice versa. These processes can be achieved by transferring the DEs to hypertonic or hypotonic solutions. Manipulation of the core size of DEs can be beneficial to cellular assays. First, due to the selectivity of the oil shell of DEs, the concentration of biomarkers in the core increases when the inner aqueous phase is shrunk, resulting in the enhancement of biosignals. We demonstrate this by encapsulating the Bgl3 enzyme-secreting yeast with a substrate that displays fluorescence after hydrolyzation. In a second application, a single GFP-tagged yeast cell was encapsulated in DEs. After swelling the core of DEs, we observe that the larger core of DEs promotes cell growth compared to those with the smaller cores, leading to more intracellular proteins (green-fluorescent protein) for screening. These osmotic manipulations provide new tools for droplet-based biochemistry.

Microfluidic Separation and Enrichment of Escherichia coli by Size Using Viscoelastic Flows.

Here, we achieve the separation and enrichment of Escherichia coli clusters from its singlets in a viscoelastic microfluidic device. E. coli, an important prokaryotic model organism and a widely used microbial factory, can aggregate in clusters, leading to biofilm development that can be detrimental to human health and industrial processes. The ability to obtain high-purity populations of E. coli clusters is of significance for biological, biomedical, and industrial applications. In this study, polystyrene particles of two different sizes, 1 and 4.8 µm, are used to mimic E. coli singlets and clusters, respectively. Experimental results show that particles migrate toward the channel center in a size-dependent manner, due to the combined effects of inertial and elastic forces; 4.8 and 1 µm particles are found to have lateral equilibrium positions closer to the channel centerline and sidewalls, respectively. The size-dependent separation performance of the microdevice is demonstrated to be affected by three main factors: channel length, the ratio of sheath to sample flow rate, and poly(ethylene oxide) (PEO) concentration. Further, the separation of E. coli singlets and clusters is achieved at the outlets, and the separation efficiency is evaluated in terms of purity and enrichment factor.

Inertial Separation of Particles Assisted by Symmetrical Sheath Flows in a Straight Microchannel.

Over the past two decades, inertial microfluidics, which works at an intermediate range of Reynolds number (∼1 < Re < ∼100), has been widely used for particle separation due to its high-throughput and label-free features. This work proposes a novel method for continuous separation of particles by size using inertial microfluidics, with the assistance of symmetrical sheath flows in a straight microchannel. Here, larger particles (>3 µm) are arranged close to the channel sidewalls, while smaller particles (<2 µm) remain flowing along the channel centerline. This conclusion is supported by experimental data with particles of different sizes ranging from 0.79 to 10.5 µm. Symmetrical Newtonian sheath flows are injected on both sides of particle mixtures into a straight rectangular microchannel with an aspect ratio (AR = height/width) of 2.5. Results show that the separation performance of the developed microfluidic device is affected by three main factors: channel length, total flow rate, and flow rate ratio of sheath to sample. Besides, separation of platelets from whole blood is demonstrated. The developed microfluidic platform owns the advantages of low fabrication cost, simple experiment setup, versatile selections of particle candidates, and stable operations. This systematic study provides a new perspective for particle separation, which is expected to find applications across various fields spanning physics, biology, biomedicine, and industry.

Characterization of optofluidic devices for the sorting of sub-micrometer particles.

In this work, we investigate methods of fabricating a device for the optical actuation of nanoparticles. To create the microfluidic channel, we pursued three fabrication methods: SU-8 to molded polydimethylsiloxane soft lithography, laser etching of glass, and deep reactive ion etching of fused silica. We measured the surface roughness of the etched sidewalls, and the laser power transmission through each device. We then measured the radiation pressure on 0.5-µm particles in the best-performing fabricated device (etched fused silica) and in a square glass capillary.

A mobility shift assay for DNA detection using nanochannel gradient electrophoresis.

Conventional detection of pathogenic or other biological contamination relies on amplification of DNA using sequence-specific primers. Recent work in nanofluidics has shown very high concentration enhancement of biomolecules with some degree of simultaneous separation. This work demonstrates the combination of these two approaches by selectively concentrating a mobility-shifted hybridization product, potentially enabling rapid detection of rare DNA fragments such as highly specific 16S ribosomal DNA. We have performed conductivity gradient electrofocusing within nanofluidic channels and have shown concentration of hybridized peptide nucleic acids and DNA oligomers. We also show selectivity to single base-pair mismatch on 18-mer oligos. This approach may enable sensitive optical detection of small amounts of DNA.

Characterization of the interaction between heterodimeric αvß6 integrin and urokinase plasminogen activator receptor (uPAR) using functional proteomics.

Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvß6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvß6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvß6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo ß6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvß6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, ß1, ß3, and ß6 alone). Our data suggest that interaction with uPAR requires expression of the complete αß heterodimer (e.g., αvß6), not individual subunits (i.e., αv, ß1, ß3, or ß6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvß6 and uPAR are located in uPAR domains II and III.

Isoelectric focusing in a silica nanofluidic channel: effects of electromigration and electroosmosis.

Isoelectric focusing of proteins in a silica nanofluidic channel filled with citric acid and disodium phosphate buffers is investigated via numerical simulation. Ions in the channel migrate in response to (i) the electric field acting on their charge and (ii) the bulk electroosmotic flow (which is directed toward the cathode). Proteins are focused near the low pH (anode) end when the electromigration effect is more significant and closer to the high pH (cathode) end when the electroosmotic effect dominates. We simulate the focusing behavior of Dylight labeled streptavidin (Dyl-Strep) proteins in the channel, using a relationship between the protein's charge and pH measured in a previous experiment. Protein focusing results compare well to previous experimental measurements. The effect of some key parameters, such as applied voltage, isoelectric point (pI), bulk pH, and bulk conductivity, on the protein trapping behavior in a nanofluidic channel is examined.

Stationary chemical gradients for concentration gradient-based separation and focusing in nanofluidic channels.

Previous work has demonstrated the simultaneous concentration and separation of proteins via a stable ion concentration gradient established within a nanochannel (Inglis Angew. Chem., Int. Ed. 2001, 50, 7546-7550). To gain a better understanding of how this novel technique works, we here examine experimentally and numerically how the underlying electric potential controlled ion concentration gradients can be formed and controlled. Four nanochannel geometries are considered. Measured fluorescence profiles, a direct indicator of ion concentrations within the Tris-fluorescein buffer solution, closely match depth-averaged fluorescence profiles calculated from the simulations. The simulations include multiple reacting species within the fluid bulk and surface wall charge regulation whereby the deprotonation of silica-bound silanol groups is governed by the local pH. The three-dimensional system is simulated in two dimensions by averaging the governing equations across the (varying) nanochannel width, allowing accurate numerical results to be generated for the computationally challenging high aspect ratio nanochannel geometries. An electrokinetic circuit analysis is incorporated to directly relate the potential drop across the (simulated) nanochannel to that applied across the experimental chip device (which includes serially connected microchannels). The merit of the thick double layer, potential-controlled concentration gradient as a particle focusing and separation tool is discussed, linking this work to the previously presented protein trapping experiments. We explain why stable traps are formed when the flow is in the opposite direction to the concentration gradient, allowing particle separation near the low concentration end of the nanochannel. We predict that tapered, rather than straight nanochannels are better at separating particles of different electrophoretic mobilities.

Microfluidic-SERS Technologies for CTC: A Perspective on Clinical Translation.

Enumeration and phenotypic profiling of circulating tumor cells (CTCs) provide critical information for clinical diagnosis and treatment monitoring in cancer. To achieve this goal, an integrated system is needed to efficiently isolate CTCs from patient samples and sensitively evaluate their phenotypes. Such integration would comprise a high-throughput single-cell processing unit for the isolation and manipulation of CTCs and a sensitive and multiplexed quantitation unit to detect clinically relevant signals from these cells. Surface-enhanced Raman scattering (SERS) has been used as an analytical method for molecular profiling and in vitro cancer diagnosis. More recently, its multiplexing capability and power to create distinct molecular signatures against their targets have garnered attention. Here, we share our insights into the combined power of microfluidics and SERS in realizing CTC isolation, enumeration, and detection from a clinical translation perspective. We highlight the key operational factors in CTC microfluidic processing and SERS detection from patient samples. We further discuss microfluidic-SERS integration and its clinical utility as a paradigm shift in clinical CTC-based cancer diagnosis and prognostication. Finally, we summarize the challenges and attempt to look forward to what lies ahead of us in potentially translating the technique into real clinical applications.

Nanochannel pH gradient electrofocusing of proteins.

We demonstrate matrix-free pH gradient electrofocusing of proteins within an 85 nm deep nanochannel. In contrast to conventional isoelectric focusing where the fluid does not move, this pH gradient method traps protein molecules flowing through a channel by balancing electric forces due to pH-dependent protein charge and viscous drag forces caused by electro-osmosis. The nanoscale depth of the device and the low voltage used limit convection relative to diffusion, thus producing a stable focused band of protein. R-Phycoerythrin (RPE) and Dylight labeled streptavidin (Dyl-Strep) were focused within a nanochannel using applied voltages between 0.4 and 1.6 V. Concentration enhancement factors of over 380 have been achieved within 5 min. Varying the buffer pH (between 2.7 and 7.2) at the boundaries of the nanochannel affected the shape of the focused bands. For RPE, a pH span of 4.5 (pH 2.7 to 7.2) yielded the narrowest peak while a span of 2.4 (pH 2.7 to 5.1) produced a significantly wider peak. Such matrix-free nanofluidic devices with pH gradient electrofocusing may enable on-chip integration of orthogonal separation techniques with mass spectrometry offering labor savings and enhanced performance.

Emerging Microfluidic Devices for Sample Preparation of Undiluted Whole Blood to Enable the Detection of Biomarkers.

Blood testing allows for diagnosis and monitoring of numerous conditions and illnesses; it forms an essential pillar of the health industry that continues to grow in market value. Due to the complex physical and biological nature of blood, samples must be carefully collected and prepared to obtain accurate and reliable analysis results with minimal background signal. Examples of common sample preparation steps include dilutions, plasma separation, cell lysis, and nucleic acid extraction and isolation, which are time-consuming and can introduce risks of sample cross-contamination or pathogen exposure to laboratory staff. Moreover, the reagents and equipment needed can be costly and difficult to obtain in point-of-care or resource-limited settings. Microfluidic devices can perform sample preparation steps in a simpler, faster, and more affordable manner. Devices can be carried to areas that are difficult to access or that do not have the resources necessary. Although many microfluidic devices have been developed in the last 5 years, few were designed for the use of undiluted whole blood as a starting point, which eliminates the need for blood dilution and minimizes blood sample preparation. This review will first provide a short summary on blood properties and blood samples typically used for analysis, before delving into innovative advances in microfluidic devices over the last 5 years that address the hurdles of blood sample preparation. The devices will be categorized by application and the type of blood sample used. The final section focuses on devices for the detection of intracellular nucleic acids, because these require more extensive sample preparation steps, and the challenges involved in adapting this technology and potential improvements are discussed.

Pumpless deterministic lateral displacement separation using a paper capillary wick.

Deterministic lateral displacement (DLD) is a passive separation method that separates particles by hydrodynamic size. This label-free method is a promising technique for cell separation because of its high size resolution and insensitivity to flow rate. Development of capillary-driven microfluidic technologies allows microfluidic devices to be operated without any external power for fluid pumping, lowering their total cost and complexity. Herein, we develop and test a DLD-based particle and cell sorting method that is driven entirely by capillary pressure. We show microchip self-filling, flow focusing, flow stability, and capture of separated particles. We achieve separation efficiency of 92% for particle-particle separation and more than 99% efficiency for cell-particle separation. The high performance of driven flow and separation along with simplicity of the operation and setup make it a valuable candidate for point-of-care devices.

Visible 532 nm laser irradiation of human adipose tissue-derived stem cells: effect on proliferation rates, mitochondria membrane potential and autofluorescence.

BACKGROUND AND OBJECTIVE: The photobiological effect of laser light on cells and tissues originates from light absorption by endogenous chromophores and hence it depends on the wavelength of light source and cell type. Earlier studies regarding the biostimulation effects of green laser light investigated a wide variety of cells but not adipose tissue-derived stem cells (ADSCS). In this study we reported the in vitro effect of 532-nm Nd:YAG laser on proliferation, mitochondrial activity of these mesenchymal stem cells (MSCs) on the autofluorescence emission at wavelengths associated with nicotinamide adenine dinucleotide (NADH) and flavoproteins. MATERIALS AND METHODS: ADSCS were exposed to 532 nm second harmonic generation laser light at moderate power density (0.153 W/cm(2)) for periods of 30, 45, 60, 180, and 300 seconds. Mitochondrial membrane potential was measured using JC1 stain and confocal laser scanning microscopy, cell proliferation rates, and cellular autofluorescence emission at 450 and 540 nm wavelengths were measured using micro plate spectrofluorometer 48 hours after irradiation. RESULTS: Shorter (30-60 seconds) exposure times led to significantly increased proliferation, attributed to increased mitochondrial activity (P < 0.05). At longer exposures we observed a significant decrease in proliferation and autofluorescence (P < 0.05). Strong correlation was observed between proliferation rates of cells and autofluorescence intensity. CONCLUSION: Our results show that autofluorescence of the respiratory chain components and key autofluorescent metabolites offers a non-invasive method to quantify cellular response to laser irradiation.

Mild Positive Pressure Improves the Efficacy of Benzalkonium Chloride against Staphylococcus aureus Biofilm.

Current protocols using liquid disinfectants to disinfect heat-sensitive hospital items frequently fail, as evidenced by the continued isolation of bacteria following decontamination. The contamination is, in part, due to biofilm formation. We hypothesize that mild positive pressure (PP) will disrupt this biofilm structure and improve liquid disinfectant/detergent penetration to biofilm bacteria for improved killing. Staphylococcus aureus biofilm, grown on polycarbonate coupons in the biofilm reactor under shear at 35 °C for 3 days, was treated for 10 min and 60 min with various dilutions of benzalkonium chloride without PP at 1 atmosphere (atm), and with PP at 3, 5, 7, and 10 atm. The effect on biofilm and residual bacterial viability was determined by standard plate counts, confocal laser scanning microscopy, and scanning electron microscopy. Combined use of benzalkonium chloride and PP up to 10 atm significantly increased biofilm killing up to 4.27 logs, as compared to the treatment using disinfectant alone. Microscopy results were consistent with the viability plate count results. PP improved disinfectant efficacy against bacterial biofilm. The use of mild PP is possible in many flow situations or if equipment/contaminated surfaces can be placed in a pressure chamber.

Effect of process parameters on separation efficiency in a deterministic lateral displacement device.

Deterministic lateral displacement (DLD) is a hydrodynamic method known for its high-resolution sorting of particles. It achieves this through a periodic array of obstacles and laminar flow that passively directs particles along in two different directions depending on the particles' diameter. Many prior publications have been dedicated to the structural and geometrical development of DLD arrays to improve separation performance; however, a successful separation requires much more than a well-designed array. This paper shows how separation performance is affected by process parameters. For this purpose, the design and fabrication of a DLD device are described. Then three experiments show how process parameters affect the performance of the device. The first experiment uses dye solutions to visualize the formation of a hydrodynamically focused sample stream. The second experiment shows that the particle separation performance (of 7- & 15-µm particles) is affected by the way output fluids are collected. Finally, the third experiment looks at the particle separation efficiency as the input flow rates and the ratio of buffer to sample are changed. The results show that the proper range for buffer and sample flow rate in this device is 1-10 and 0.1-1 (µl/min), respectively. The buffer to sample flow rate ratio of 10 gives the highest separation efficiency, but at a lower sample throughput. The optimized values are specific for our device but demonstrate processes that we believe are universal for DLD separations.

Shape-based separation of drug-treated Escherichia coli using viscoelastic microfluidics.

Here, we achieve shape-based separation of drug-treated Escherichia coli (E. coli) by viscoelastic microfluidics. Since shape is critical for modulating biological functions of E. coli, the ability to prepare homogeneous E. coli populations adopting uniform shape or sort bacterial sub-population based on their shape has significant implications for a broad range of biological, biomedical and environmental applications. A proportion of E. coli treated with 1 µg mL-1 of the antibiotic mecillinam were found to exhibit changes in shape from rod to sphere, and the heterogeneous E. coli populations after drug treatment with various aspect ratios (ARs) ranging from 1.0 to 5.5 were used for experiment. We demonstrate that E. coli with a lower AR, i.e., spherical E. coli (AR ≤ 1.5), are directed toward the middle outlet, while rod-shaped E. coli with a higher AR (AR > 1.5) are driven to the side outlets. Further, we demonstrate that the separation performance of the viscoelastic microfluidic device is influenced by two main factors: sheath-to-sample flow rate ratio and the concentration of poly-ethylene-oxide (PEO). To the best of our knowledge, this is the first report on shape-based separation of a single species of cells smaller than 4 µm by microfluidics.

Hydrodynamic metamaterials: microfabricated arrays to steer, refract, and focus streams of biomaterials.

We show that it is possible to direct particles entrained in a fluid along trajectories much like rays of light in classical optics. A microstructured, asymmetric post array forms the core hydrodynamic element and is used as a building block to construct microfluidic metamaterials and to demonstrate refractive, focusing, and dispersive pathways for flowing beads and cells. The core element is based on the concept of deterministic lateral displacement where particles choose different paths through the asymmetric array based on their size: Particles larger than a critical size are displaced laterally at each row by a post and move along the asymmetric axis at an angle to the flow, while smaller particles move along streamline paths. We create compound elements with complex particle handling modes by tiling this core element using multiple transformation operations; we show that particle trajectories can be bent at an interface between two elements and that particles can be focused into hydrodynamic jets by using a single inlet port. Although particles propagate through these elements in a way that strongly resembles light rays propagating through optical elements, there are unique differences in the paths of our particles as compared with photons. The unusual aspects of these modular, microfluidic metamaterials form a rich design toolkit for mixing, separating, and analyzing cells and functional beads on-chip.