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.

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.

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.

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.

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.

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.

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.

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.

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.

Microfluidic Obstacle Arrays Induce Large Reversible Shape Change in Red Blood Cells.

Red blood cell (RBC) shape change under static and dynamic shear stress has been a source of interest for at least 50 years. High-speed time-lapse microscopy was used to observe the rate of deformation and relaxation when RBCs are subjected to periodic shear stress and deformation forces as they pass through an obstacle. We show that red blood cells are reversibly deformed and take on characteristic shapes not previously seen in physiological buffers when the maximum shear stress was between 2.2 and 25 Pa (strain rate 2200 to 25,000 s-1). We quantify the rates of RBC deformation and recovery using Kaplan-Meier survival analysis. The time to deformation decreased from 320 to 23 milliseconds with increasing flow rates, but the distance traveled before deformation changed little. Shape recovery, a measure of degree of deformation, takes tens of milliseconds at the lowest flow rates and reached saturation at 2.4 s at a shear stress of 11.2 Pa indicating a maximum degree of deformation was reached. The rates and types of deformation have relevance in red blood cell disorders and in blood cell behavior in microfluidic devices.

Deterministic Lateral Displacement: Challenges and Perspectives.

The advent of microfluidics in the 1990s promised a revolution in multiple industries from healthcare to chemical processing. Deterministic lateral displacement (DLD) is a continuous-flow microfluidic particle separation method discovered in 2004 that has been applied successfully and widely to the separation of blood cells, yeast, spores, bacteria, viruses, DNA, droplets, and more. Deterministic lateral displacement is conceptually simple and can deliver consistent performance over a wide range of flow rates and particle concentrations. Despite wide use and in-depth study, DLD has not yet been fully elucidated or optimized, with different approaches to the same problem yielding varying results. We endeavor here to provide up-to-date expert opinion on the state-of-art and current fundamental, practical, and commercial challenges with DLD as well as describe experimental and modeling opportunities. Because these challenges and opportunities arise from constraints on hydrodynamics, fabrication, and operation at the micro- and nanoscale, we expect this Perspective to serve as a guide for the broader micro- and nanofluidic community to identify and to address open questions in the field.

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.

Targeting of externalized αB-crystallin on irradiated endothelial cells with pro-thrombotic vascular targeting agents: Potential applications for brain arteriovenous malformations.

BACKGROUND: Vascular targeting uses molecular markers on the surface of diseased vasculature for ligand-directed drug delivery to induce vessel occlusion or destruction. In the absence of discriminatory markers, such as in brain arteriovenous malformations (AVMs), stereotactic radiosurgery may be used to prime molecular changes on the endothelial surface. This study explored αB-crystallin (CRYAB) as a radiation induced target and pre-tested the specificity and efficacy of a CRYAB-targeting coaguligand for in vitro thrombus induction. METHODS: A parallel-plate flow system was established to circulate human whole blood over a layer of human brain endothelial cells. A conjugate of anti-CRYAB antibody and thrombin was injected into the circuit to compare binding and thrombus formation on cells with or without prior radiation treatment (0-25 Gy). RESULTS: Radiation increased CRYAB expression and surface exposure in human brain endothelial cells. In the parallel-plate flow system, the targeted anti-CRYAB-thrombin conjugate increased thrombus formation on the surface of irradiated cells relative to non-irradiated cells and to a non-targeting IgG-thrombin conjugate. Fibrin deposition and accumulation of fibrinogen degradation products increased significantly at radiation doses at or above 15 Gy with conjugate concentrations of 1.25 and 2.5 µg/mL. CONCLUSIONS: CRYAB exposure can be detected at the surface of human brain endothelial cells in response to irradiation. Pro-thrombotic CRYAB-targeting conjugates can bind under high flow conditions and in the presence of whole blood induce stable thrombus formation with high specificity and efficacy on irradiated surfaces. CRYAB provides a novel radiation marker for potential vascular targeting in irradiated brain AVMs.

Focusing of sub-micrometer particles in microfluidic devices.

Sub-micrometer particles (0.10-1.0 µm) are of great significance to study, e.g., microvesicles and protein aggregates are targets for therapeutic intervention, and sub-micrometer fluorescent polystyrene (PS) particles are used as probes for diagnostic imaging. Focusing of sub-micrometer particles - precisely control over the position of sub-micrometer particles in a tightly focused stream - has a wide range of applications in the field of biology, chemistry and environment, by acting as a prerequisite step for downstream detection, manipulation and quantification. Microfluidic devices have been attracting great attention as desirable tools for sub-micrometer particle focusing, due to their small size, low reagent consumption, fast analysis and low cost. Recent advancements in fundamental knowledge and fabrication technologies have enabled microfluidic focusing of particles at sub-micrometer scale in a continuous, label-free and high-throughput manner. Microfluidic methods for the focusing of sub-micrometer particles can be classified into two main groups depending on whether an external field is applied: 1) passive methods, which utilize intrinsic fluidic properties without the need of external actuation, such as inertial, deterministic lateral displacement (DLD), viscoelastic and hydrophoretic focusing; and 2) active methods, where external fields are used, such as dielectrophoretic, thermophoretic, acoustophoretic and optical focusing. This article mainly reviews the studies on the focusing of sub-micrometer particles in microfluidic devices over the past 10 years. It aims to bridge the gap between the focusing of micrometer and nanometer scale (1.0-100 nm) particles and to improve the understanding of development progress, current advances and future prospects in microfluidic focusing techniques.

A Nanoparticle-Based Affinity Sensor that Identifies and Selects Highly Cytokine-Secreting Cells.

We developed a universal method termed OnCELISA to detect cytokine secretion from individual cells by applying a capture technology on the cell membrane. OnCELISA uses fluorescent magnetic nanoparticles as assay reporters that enable detection on a single-cell level in microscopy and flow cytometry and fluorimetry in cell ensembles. This system is flexible and can be modified to detect different cytokines from a broad range of cytokine-secreting cells. Using OnCELISA we have been able to select and sort highly cytokine-secreting cells and identify cytokine-secreting expression profiles of different cell populations in vitro and ex vivo. We show that this system can be used for ultrasensitive monitoring of cytokines in the complex biological environment of atherosclerosis that contains multiple cell types. The ability to identify and select cell populations based on their cytokine expression characteristics is valuable in a host of applications that require the monitoring of disease progression.

Droplets for Sampling and Transport of Chemical Signals in Biosensing: A Review.

The chemical, temporal, and spatial resolution of chemical signals that are sampled and transported with continuous flow is limited because of Taylor dispersion. Droplets have been used to solve this problem by digitizing chemical signals into discrete segments that can be transported for a long distance or a long time without loss of chemical, temporal or spatial precision. In this review, we describe Taylor dispersion, sampling theory, and Laplace pressure, and give examples of sampling probes that have used droplets to sample or/and transport fluid from a continuous medium, such as cell culture or nerve tissue, for external analysis. The examples are categorized, as follows: (1) Aqueous-phase sampling with downstream droplet formation; (2) preformed droplets for sampling; and (3) droplets formed near the analyte source. Finally, strategies for downstream sample recovery for conventional analysis are described.

Turn-On Fluorescence Aptasensor on Magnetic Nanobeads for Aflatoxin M1 Detection Based on an Exonuclease III-Assisted Signal Amplification Strategy.

In order to satisfy the need for sensitive detection of Aflatoxin M1 (AFM1), we constructed a simple and signal-on fluorescence aptasensor based on an autocatalytic Exonuclease III (Exo III)-assisted signal amplification strategy. In this sensor, the DNA hybridization on magnetic nanobeads could be triggered by the target AFM1, resulting in the release of a single-stranded DNA to induce an Exo III-assisted signal amplification, in which numerous G-quadruplex structures would be produced and then associated with the fluorescent dye to generate significantly amplified fluorescence signals resulting in the increased sensitivity. Under the optimized conditions, this aptasensor was able to detect AFM1 with a practical detection limit of 9.73 ng kg-1 in milk samples. Furthermore, the prepared sensor was successfully used for detection of AFM1 in the commercially available milk samples with the recovery percentages ranging from 80.13% to 108.67%. Also, the sensor performance was evaluated by the commercial immunoassay kit with satisfactory results.