Extracellular vesicles (EVs) have emerged as promising biomarkers for various diseases. Encapsulating biomolecules reflective of their parental cells, EVs are readily accessible in bodily fluids. The prospect for minimally invasive, repeatable molecular testing has stimulated significant research; however, challenges persist in isolating EVs from complex biological matrices and characterizing their limited molecular cargo. Technical advances have been pursued to address these challenges, producing innovative EV-specific platforms. This review highlights recent technological developments, focusing on EV isolation and molecular detection methodologies. Furthermore, it explores the translation of these laboratory innovations to clinical applications through the analysis of patient samples, providing insights into the potential diagnostic and prognostic utility of EV-based technologies.

Extracellular vesicles (EVs) are heterogeneous lipid containers carrying complex molecular cargoes, including proteins, nucleic acids, glycans, etc. These vesicles are closely associated with specific physiological characteristics, which makes them invaluable in the detection and monitoring of various diseases. However, traditional isolation methods are often labour-intensive, inefficient, and time-consuming. In addition, single biomarker analyses are no longer accurate enough to meet diagnostic needs. Routine isolation and molecular analysis of high-purity EVs in clinical applications is even more challenging. In this review, we discuss a promising solution, microfluidic-based techniques, that combine efficient isolation and multiplex detection of EVs, to further demystify EV heterogeneity. These microfluidic-based EV multiplexing platforms will hopefully facilitate development of liquid biopsies and offer promising opportunities for personalised therapy.

A deeper understanding of disease heterogeneity highlights the urgent need for precision medicine. Microfluidics, with its unique advantages, such as high adjustability, diverse material selection, low cost, high processing efficiency, and minimal sample requirements, presents an ideal platform for precision medicine applications. As nanoparticles, both of biological origin and for therapeutic purposes, become increasingly important in precision medicine, microfluidic nanoparticle separation proves particularly advantageous for handling valuable samples in personalized medicine. This technology not only enhances detection, diagnosis, monitoring, and treatment accuracy, but also reduces invasiveness in medical procedures. This review summarizes the fundamentals of microfluidic nanoparticle separation techniques for precision medicine, starting with an examination of nanoparticle properties essential for separation and the core principles that guide various microfluidic methods. It then explores passive, active, and hybrid separation techniques, detailing their principles, structures, and applications. Furthermore, the review highlights their contributions to advancements in liquid biopsy and nanomedicine. Finally, it addresses existing challenges and envisions future development spurred by emerging technologies such as advanced materials science, 3D printing, and artificial intelligence. These interdisciplinary collaborations are anticipated to propel the platformization of microfluidic separation techniques, significantly expanding their potential in precision medicine.

This work reports on experimental investigations into the operational parameters of nanoelectrokinetic purification and preconcentration, especially utilizing on ion concentration polarization (ICP). ICP as a nanoscale electrokinetic phenomenon has demonstrated promising advances in various fields utilizing an ion depletion zone (IDZ) with a steep electric field gradient inside the ICP layer. However, the inevitable electrokinetic instability occurring within the IDZ has posed a challenge in operating the ICP system stably. To address the need for a stable and efficient ICP operation in various devices and applications, we propose an operational strategy along with conducted research to determine optimal operating ranges. In order to investigate the operational parameters, a unit voltage (VTH) is introduced as the threshold for initiating ICP. We examined the applicability of VTH across various operating ranges to ensure its effectiveness and versatility. In ICP purification, we categorize three modes (steady, burst, and unsteady) based on IDZ expansion and stability under varying VTH and flow rate conditions, presenting optimal operational conditions that minimize the voltage margin. In ICP preconcentration, a systematic investigation is conducted to observe the influence of background electrolyte concentration and voltage conditions on preconcentration efficiency, offering insights into the correlation between preconcentration factor, electrical conditions, and preconcentration time. Therefore, this research would contribute to the practical understanding of nanoelectrokinetics, providing insight into experimental designs. These findings are expected to offer valuable guidance to researchers aiming to utilize ICP's potential across a spectrum of applications, from purification to preconcentration, in the realm of micro/nanofluidic systems.

Microfluidic technologies are revolutionizing the synthesis of nanoscale lipid particles and enabling new opportunities for the production of lipid-based nanomedicines. By harnessing the benefits of microfluidics for controlling diffusive and advective transport within microfabricated flow cells, microfluidic platforms enable unique capabilities for lipid nanoparticle synthesis with precise and tunable control over nanoparticle properties. Here we present an assessment of the current state of microfluidic technologies for lipid-based nanoparticle and nanomedicine production. Microfluidic techniques are discussed in the context of conventional production methods, with an emphasis on the capabilities of microfluidic systems for controlling nanoparticle size and size distribution. Challenges and opportunities associated with the scaling of manufacturing throughput are discussed, together with an overview of emerging microfluidic methods for lipid nanomedicine post-processing. The impact of additive manufacturing on current and future microfluidic platforms is also considered.

Extracellular vesicles (EVs) are cell-derived particles that exhibit diverse sizes, molecular contents, and clinical implications for various diseases depending on their specific subpopulations. However, fractionation of EV subpopulations with high resolution, efficiency, purity, and yield remains an elusive goal due to their diminutive sizes. In this study, we introduce a novel strategy that effectively separates EV subpopulations in a gel-free and label-free manner, using two-dimensional (2D) electrophoresis in a microfluidic artificial sieve. The microfabricated artificial sieve consists of periodically arranged micro-slit-well structures in a 2D array and generates an anisotropic electric field pattern to size fractionate EVs into discrete streams and steer the subpopulations into designated outlets for collection within a minute. Along with fractionating EV subpopulations, contaminants such as free proteins and short nucleic acids can be simultaneously directed to waste outlets, thus accomplishing both size fractionation and purification of EVs with high performance. Our platform offers a simple, rapid, and versatile solution for EV subpopulation isolation, which can potentially facilitate the discovery of biomarkers for specific EV subtypes and the development of EV-based therapeutics.

Small extracellular vesicles (sEVs; <200 nm) that contain lipids, nucleic acids, and proteins are considered promising biomarkers for a wide variety of diseases. Conventional methods for sEV isolation from blood are incompatible with routine clinical workflows, significantly hampering the utilization of blood-derived sEVs in clinical settings. Here, we present a simple, viscoelastic-based microfluidic platform for label-free isolation of sEVs from human blood. The separation performance of the device is assessed by isolating fluorescent sEVs from whole blood, demonstrating purities and recovery rates of over 97 and 87%, respectively. Significantly, our viscoelastic-based microfluidic method also provides for a remarkable increase in sEV yield compared to gold-standard ultracentrifugation, with proteomic profiles of blood-derived sEVs purified by both methods showing similar protein compositions. To demonstrate the clinical utility of the approach, we isolate sEVs from blood samples of 20 patients with cancer and 20 healthy donors, demonstrating that elevated sEV concentrations can be observed in blood derived from patients with cancer.

A nanoelectrokinetic phenomenon called ion concentration polarization (ICP) has been recently applied to microfluidic paper-based devices for the high fold preconcentration of low-abundant analytes. The inherent microstructural characteristics of cellulose papers can sufficiently stabilize the chaotic electroconvection of ICP, which is a significant annoyance for typical engineered microfluidic channels. However, a high electrical voltage to induce ICP in a paper-fluidic channel can increase unavoidable electrophoretic forces over drag forces so that the preconcentrated plug is rapidly receded with severe dispersion. In order to enhance the hydraulic drag force that helps the preconcentration of analytes, here we introduce a multilayered paper structure into paper-fluidic channel. We theoretically and experimentally demonstrate that a hierarchical capillary structure in a multilayered paper-fluidic channel can effectively increase the hydraulic drag force. For the practical utility in the field of diagnostics, the mechanism is verified by a simple example of the immunoassay using biotin-streptavidin complexation.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, characterized by an initial and progressive loss of dopaminergic neurons of the substantia nigra pars compacta via a potentially substantial contribution from protein aggregates, the Lewy bodies, mainly composed of α-Synuclein among other factors. Distinguishing symptoms of PD are bradykinesia, muscular rigidity, unstable posture and gait, hypokinetic movement disorder and resting tremor. Currently, there is no cure for PD, and palliative treatments, such as Levodopa administration, are directed to relieve the motor symptoms but induce severe side effects over time. Therefore, there is an urgency for discovering new drugs in order to design more effective therapeutic approaches. The evidence of epigenetic alterations, such as the dysregulation of different miRNAs that may stimulate many aspects of PD pathogenesis, opened a new scenario in the research for a successful treatment. Along this line, a promising strategy for PD treatment comes from the potential exploitation of modified exosomes, which can be loaded with bioactive molecules, such as therapeutic compounds and RNAs, and can allow their delivery to the appropriate location in the brain, overcoming the blood-brain barrier. In this regard, the transfer of miRNAs within Mesenchymal stem cell (MSC)-derived exosomes has yet to demonstrate successful results both in vitro and in vivo. This review, besides providing a systematic overview of both the genetic and epigenetic basis of the disease, aims to explore the exosomes/miRNAs network and its clinical potential for PD treatment.

Extracellular vesicles (EVs) are cell-derived bioparticles that play significant roles in various biological processes including cell-to-cell communication and intercellular delivery. Additionally, they hold great potential as liquid biopsy biomarkers for pre-diagnostic applications. However, the isolation of EV subpopulations, especially exosomes from a biological fluid remains a challenge due to their submicron range. Here, we demonstrate continuous-flow label-free size fractionation of EVs for the first time through a synergistic combination of electrothermal fluid rolls and dielectrophoresis in a microfluidic device. The device features three dimensional microelectrodes with unique sidewall contours that give rise to effective electrothermal fluid rolls in cooperation with dielectrophoretic forces for the electrokinetic manipulation and size separation of submicron particles. We first validate the device functionality by separating submicron polystyrene particles from binary mixtures with a cut-off size of ∼200 nm and then isolate intact exosomes from cell culture medium or blood serum with a high recovery rate and purity (∼80%). The device operation in a high-conductivity medium renders the method ideal for the purification of target bioparticles directly from physiological fluids, and may offer a robust and versatile platform for EV related diagnostic applications.

Protein analysis is an important approach for disease diagnosis, in which sample pretreatment is an essential step since protein samples are often complex and many protein biomarkers are of low abundance. Here, given the good openness and light transmission of liquid plasticine (LP), which is a liquid entity formed by SiO₂ nanoparticles and encapsulated aqueous solution, we developed a LP-based field-amplified sample stacking (FASS) system for protein enrichment. The system was composed of a LP container, a sample solution and a Tris-HCl solution containing hydroxyethyl cellulose (HEC). The system design, mechanism investigation, optimization of experimental parameters and characterization of LP-FASS performance for protein enrichment were well studied. Under the optimized experimental conditions of 1 % HEC, 100 mm Tris-HCl and 100 V in the LP-FASS system, a 40-80 times enrichment of proteins was obtained in 40 min using bovine hemoglobin (BHb) as the model protein using the constructed LP-FASS system. The simultaneous enrichment of multiple proteins (phycocyanin, BHb and cytochrome C) was also realized using the system. The LP-FASS system can serve as a new platform for protein enrichment which is easy to be combined with online and offline detections.

Manipulation and separation of submicron and nanoparticles are indispensable in many chemical, biological, medical, and environmental applications. Conventional technologies such as ultracentrifugation, ultrafiltration, size exclusion chromatography, precipitation and immunoaffinity capture are limited by high cost, low resolution, low purity or the risk of damage to biological particles. Microfluidics can accurately control fluid flow in channels with dimensions of tens of micrometres. Rapid microfluidics advancement has enabled precise sorting and isolating of nanoparticles with better resolution and efficiency than conventional technologies. This paper comprehensively studies the latest progress in microfluidic technology for submicron and nanoparticle manipulation. We first summarise the principles of the traditional techniques for manipulating nanoparticles. Following the classification of microfluidic techniques as active, passive, and hybrid approaches, we elaborate on the physics, device design, working mechanism and applications of each technique. We also compare the merits and demerits of different microfluidic techniques and benchmark them with conventional technologies. Concurrently, we summarise seven standard post-separation detection techniques for nanoparticles. Finally, we discuss current challenges and future perspectives on microfluidic technology for nanoparticle manipulation and separation.

Extracellular vesicles (EVs) are phospholipidic bi-layer enclosed nanoparticles secreted naturally by all cell types. They are attracting increasing attention in the fields of nanomedicine, nutraceutics and cosmetics as biocompatible carriers for drug delivery, with intrinsic properties beneficial to human health. Scientific work now focuses on developing techniques for isolating EVs that can translate into industrial-scale production and meet rigorous clinical requirements. The science of EVs is ongoing, and many pitfalls must be addressed, such as the requirement for standard, reproducible, inexpensive, and Good Manufacturing Practices (GMP) adherent EV processing techniques. Researchers are exploring the use of alternative sources to EVs derived from mammalian cultures, such as plant EVs, as well as the use of bacteria, algae and milk. Regarding the downstream processing of EVs, many alternative techniques to the ultracentrifugation (UC) protocols most commonly used in the laboratory are emerging. In the context of process scale-up, membrane-based processes for isolation and purification of EVs are the most promising, either as stand-alone processes or in combination with chromatographic techniques. This review discusses current trends on EVs source selection and EVs downstream processing techniques, with a focus on plant-derived EVs and membrane-based techniques for EVs enrichment.

This study uses computational design to explore the performance of a novel electro-membrane microfluidic diode consisting of physically conjugated nanoporous and micro-perforated ion-exchange layers. Previously, such structures have been demonstrated to exhibit asymmetric electroosmosis, but the model was unrealistic in several important respects. This numerical study investigates two quantitative measures of performance (linear velocity of net flow and efficiency) as functions of such principal system parameters as perforation size and spacing, the thickness of the nanoporous layer and the zeta potential of the pore surface. All of these dependencies exhibit pronounced maxima, which is of interest for future practical applications. The calculated linear velocities of net flows are in the range of several tens of liters per square meter per hour at realistically applied voltages. The system performance somewhat declines when the perforation size is increased from 2 µm to 128 µm (with a parallel increase of the inter-perforation spacing) but remains quite decent even for the largest perforation size. Such perforations should be relatively easy to generate using inexpensive equipment.

Microfluidic preconcentration enables the collection or extraction of low-abundance analytes at specific locations. It has attracted considerable attention as an essential technology in bioengineering, particularly for detection and diagnosis. Herein, we investigated the key parameters in the preconcentration of fluorescent dyes based on electrophoresis in a microfluidic electromembrane system. Commercial ion-exchange membrane (IEM)-integrated polydimethylsiloxane microfluidic devices were fabricated, and Alexa Fluor 488 and Rhodamine 6G were used as fluorescent dyes for sample preconcentration. Through experimental studies, the effect of the channel concentration ratio (CCR, concentration ratio of the main and buffer channels) on the performance of the sample preconcentration was studied. The results show that the preconcentration of the target sample occurs more effectively for a high CCR or high salt concentration of the main channel when the CCR is constant. We also demonstrate a phenomenon that the salt concentration in the electrolyte solution increases as the preconcentration progresses. Our results provide consolidated conditions for electrophoresis-based sample preconcentration in electromembrane systems.

Ovarian cancer is a malignant tumor of the female reproductive system, with a very poor prognosis and high mortality rates. Chemotherapy and radiotherapy are the most common treatments for ovarian cancer, with unsatisfactory results. Exosomes are a subpopulation of extracellular vesicles, which have a diameter of approximately 30-100 nm and are secreted by many different types of cells in various body fluids. Exosomes are highly stable and are effective carriers of immunotherapeutic drugs. Recent studies have shown that exosomes are involved in various cellular responses in the tumor microenvironment, influencing the development and therapeutic efficacy of ovarian cancer, and exhibiting dual roles in inhibiting and promoting tumor development. Exosomes also contain a variety of genes related to ovarian cancer immunotherapy that could be potential biomarkers for ovarian cancer diagnosis and prognosis. Undoubtedly, exosomes have great therapeutic potential in the field of ovarian cancer immunotherapy. However, translation of this idea to the clinic has not occurred. Therefore, it is important to understand how exosomes could be used in ovarian cancer immunotherapy to regulate tumor progression. In this review, we summarize the biomarkers of exosomes in different body fluids related to immunotherapy in ovarian cancer and the potential mechanisms by which exosomes influence immunotherapeutic response. We also discuss the prospects for clinical application of exosome-based immunotherapy in ovarian cancer.

Ribonucleoproteins (RNPs), particularly microRNA-induced silencing complex (miRISC), have been associated with cancer-related gene regulation. Specific RNA-protein associations in miRISC complexes or those found in let-7 lin28A complexes can downregulate tumor-suppressing genes and can be directly linked to cancer. The high protein-RNA electrostatic binding affinity is a particular challenge for the quantification of the associated microRNAs (miRNAs). We report here the first microfluidic point-of-care assay that allows direct quantification of RNP-associated RNAs, which has the potential to greatly advance RNP profiling for liquid biopsy. Key to the technology is an integrated cation-anion exchange membrane (CEM/AEM) platform for rapid and irreversible dissociation (k = 0.0025 s-1) of the RNP (Cas9-miR-21) complex and quantification of its associated miR-21 in 40 minutes. The CEM-induced depletion front is used to concentrate the RNP at the depletion front such that the high electric field (>100 V cm-1) within the concentration boundary layer induces irreversible dissociation of the low KD (∼0.5 nM) complex, with ∼100% dissociation even though the association rate (kon = 6.1 s-1) is 1000 times higher. The high field also electrophoretically drives the dissociated RNA out of the concentrated zone without reassociation. A detection limit of 1.1 nM is achieved for Cy3 labelled miR-21.

There is a great need for techniques which enable reproducible separation of extracellular vesicles (EVs) from biofluids with high recovery, purity and throughput. The development of new techniques for isolation of EVs from minute sample volumes is instrumental in enabling EV-based biomarker profiling in large biobank cohorts and paves the way to improved diagnostic profiles in precision medicine. Recent advances in microfluidics-based devices offer a toolbox for separating EVs from small sample volumes. Microfluidic devices that have been used in EV isolation utilise different fundamental principles and rely largely on benefits of scaling laws as the biofluid processing is miniaturised to chip level. Here, we review the progress in the practicality and performance of both passive devices (such as mechanical filtering and hydrodynamic focusing) and active devices (using magnetic, electric or acoustic fields). As it stands, many microfluidic devices isolate intact EV populations at higher purities than centrifugation, precipitation or size-exclusion chromatography. However, this comes at a cost. We address challenges (in particular low throughput, clogging risks and ability to process biofluids) and highlight the need for more improvements in microfluidic devices. Finally, we conclude that there is a need to refine and standardise these lab-on-a-chip techniques to meet the growing interest in the diagnostic and therapeutic value of purified EVs.

Extracellular vesicles (EVs) are double-layered lipid membrane vesicles released by cells. Currently, EVs are attracting a lot of attention in the biological and medical fields due to their role as natural carriers of proteins, lipids, and nucleic acids. Thus, they can transport useful genomic information from their parental cell through body fluids, promoting cell-to-cell communication even between different organs. Due to their functionality as cargo carriers and their protein expression, they can play an important role as possible diagnostic and prognostic biomarkers in various types of diseases, e.g., cancers, neurodegenerative, and autoimmune diseases. Today, given the invaluable importance of EVs, there are some pivotal challenges to overcome in terms of their isolation. Conventional methods have some limitations: they are influenced by the starting sample, might present low throughput and low purity, and sometimes a lack of reproducibility, being operator dependent. During the past few years, several microfluidic approaches have been proposed to address these issues. In this review, we summarize the most important microfluidic-based devices for EV isolation, highlighting their advantages and disadvantages compared to existing technology, as well as the current state of the art from the perspective of the use of these devices in clinical applications.

Intercellular cross talk between cancer cells and stromal and immune cells is essential for tumor progression and metastasis. Extracellular vesicles and particles (EVPs) are a heterogeneous class of secreted messengers that carry bioactive molecules and that have been shown to be crucial for this cell-cell communication. Here, we highlight the multifaceted roles of EVPs in cancer. Functionally, transfer of EVP cargo between cells influences tumor cell growth and invasion, alters immune cell composition and function, and contributes to stromal cell activation. These EVP-mediated changes impact local tumor progression, foster cultivation of pre-metastatic niches at distant organ-specific sites, and mediate systemic effects of cancer. Furthermore, we discuss how exploiting the highly selective enrichment of molecules within EVPs has profound implications for advancing diagnostic and prognostic biomarker development and for improving therapy delivery in cancer patients. Altogether, these investigations into the role of EVPs in cancer have led to discoveries that hold great promise for improving cancer patient care and outcome.

Organ-on-a-chip (OOC) is an emerging technology that has been proposed as a new powerful cell-based tool to imitate the pathophysiological environment of human organs. For most OOC systems, a pivotal step is to culture cells in microfluidic devices. In active cell capturing techniques, external actuators, such as electrokinetic, magnetic, acoustic, and optical forces, or a combination of these forces, can be applied to trap cells after ejecting cell suspension into the microchannel inlet. This review paper distinguishes the characteristics of biomaterials and evaluates microfluidic technology. Besides, various types of OOC and their fabrication techniques are reported and various active cell capture microstructures are analyzed. Furthermore, their constraints, challenges, and future perspectives are provided.

Exosomes are small extracellular vesicles with sizes ranging from 30-150 nanometers that contain proteins, lipids, mRNAs, microRNAs, and double-stranded DNA derived from the cells of origin. Exosomes can be taken up by target cells, acting as a means of cell-to-cell communication. The discovery of these vesicles in body fluids and their participation in cell communication has led to major breakthroughs in diagnosis, prognosis, and treatment of several conditions (e.g., cancer). However, conventional isolation and evaluation of exosomes and their microRNA content suffers from high cost, lengthy processes, difficult standardization, low purity, and poor yield. The emergence of microfluidics devices with increased efficiency in sieving, trapping, and immunological separation of small volumes could provide improved detection and monitoring of exosomes involved in cancer. Microfluidics techniques hold promise for advances in development of diagnostic and prognostic devices. This review covers ongoing research on microfluidics devices for detection of microRNAs and exosomes as biomarkers and their translation to point-of-care and clinical applications.

A circular nonuniform electric field strategy coupled with gel electrophoresis was proposed to control the precise separation and efficient concentration of nano- and microparticles. The circular nonuniform electric field has the feature of exponential increase in the electric field intensity along the radius, working with three functional zones of migration, acceleration, and concentration. The distribution form of electric field lines is regulated in functional zones to control the migration behaviors of particles for separation and concentration by altering the relative position of the ring electrode (outside) and rodlike electrode (inner). The circular nonuniform electric field promotes the target-type and high-precision separation of nanoparticles based on the difference in charge-to-size ratio. The concentration multiple of nanoparticles is also controlled randomly with the alternation of radius, taking advantage of vertical extrusion and concentric converging of the migration path. This work provides a brand new insight into the simultaneous separation and concentration of particles and is promising for developing a versatile tool for the separation and preparation of various samples instead of conventional methods.

Herein, we demonstrate digital microfluidics-like manipulations of preconcentrated biomolecule plugs within a continuous flow that is different from the commonly known digital microfluidics involving discrete (i.e. droplets) media. This is realized using one- and two-dimensional arrays of individually addressable ion-permselective membranes with interconnecting microfluidic channels. The location of powered electrodes, dictates which of the membranes are active and generates either enrichment/depletion diffusion layers, which, in turn, control the location of the preconcentrated plug. An array of such powered membranes enables formation of multiple preconcentrated plugs of the same biosample as well as of preconcentrated plugs of multiple biosample types introduced via different inlets in a selective manner. Moreover, digital-microfluidics operations such as up-down and left-right translation, merging, and splitting, can be realized, but on preconcentrated biomolecule plugs instead of on discrete droplets. This technology, based on nanoscale electrokinetics of ion transport through permselective medium, opens future opportunities for smart and programmable digital-like manipulations of preconcentrated biological particle plugs for various on-chip biological applications.

Ion concentration polarization (ICP) accomplishes preconcentration for bioanalysis by localized depletion of electrolyte ions, thereby generating a gradient in electric field strength that facilitates electrokinetic focusing of charged analytes by their electromigration against opposing fluid flow. Such ICP focusing has been shown to accomplish up to a million-fold enrichment of nucleic acids and proteins in single-stage preconcentrators. However, the rate at which the sample volume is swept is limited, requiring several hours to achieve these high enrichment factors. This limitation is caused by two factors. First, an ion depleted zone (IDZ) formed at a planar membrane or electrode may not extend across the full channel cross section under the flow rate employed for focusing, thereby allowing the analyte to "leak" past the IDZ. Second, within the IDZ, large fluid vortices lead to mixing, which decreases the efficiency of analyte enrichment and worsens with increased channel dimensions. Here, we address these challenges with faradaic ICP (fICP) at a three-dimensional (3D) electrode comprising metallic microbeads. This 3D-electrode distributes the IDZ, and therefore, the electric field gradient utilized for counter-flow focusing across the full height of the fluidic channel, and its large area, microstructured surface supports smaller vortices. An additional bed of insulating microbeads restricts flow patterns and supplies a large area for surface conduction of ions through the IDZ. Finally, the resistance of this secondary bed enhances focusing by locally strengthening sequestering forces. This easy-to-build platform lays a foundation for the integration of enrichment with user-defined packed bed and electrode materials.

Exosomes, powerful extracellular nanovesicles released from almost all types of living cells, are considered the communication engines (messengers) that control and reprogram physiological pathways inside target cells within a community or between different communities. The cell-like structure of these extracellular vesicles provides a protective environment for their proteins and DNA/RNA cargos, which serve as biomarkers for many malicious diseases, including infectious diseases and cancers. Cancer-derived exosomes control cancer metastasis, prognosis, and development. In addition to the unique structure of exosomes, their nanometer size and tendency of interacting with cells makes them a viable novel drug delivery solution. In recent years, numerous research efforts have been made to quantify and characterize disease-derived exosomes for diagnosis, monitoring, and therapeutic purposes. This review aims to (1) relate exosome biomarkers to their origins, (2) focus on current isolation and detection methods, (3) discuss and evaluate the proposed technologies deriving from exosome research for cancer treatment, and (4) form a conclusion about the prospects of the current exosome research.

Nano structured ion-selective membranes (ISMs) are very attractive materials for a wide range of sensing and ion separation applications. The present review focuses on the design principles of various ISMs; nanostructured and ionophore/ion acceptor doped ISMs, and their use in biomedical engineering. Applications of ISMs in the biomedical field have been well-known for more than half a century in potentiometric analysis of biological fluids and pharmaceutical products. However, the emergence of nanotechnology and sophisticated sensing methods assisted in miniaturising ion-selective electrodes to needle-like sensors that can be designed in the form of implantable or wearable devices (smartwatch, tattoo, sweatband, fabric patch) for health monitoring. This article provides a critical review of recent advances in miniaturization, sensing and construction of new devices over last decade (2011-2021). The designing of tunable ISM with biomimetic artificial ion channels offered intensive opportunities and innovative clinical analysis applications, including precise biosensing, controlled drug delivery and early disease diagnosis. This paper will also address the future perspective on potential applications and challenges in the widespread use of ISM for clinical use. Finally, this review details some recommendations and future directions to improve the accuracy and robustness of ISMs for biomedical applications.

Exosomes are cell-derived structures packaged with lipids, proteins, and nucleic acids. They exist in diverse bodily fluids and are involved in physiological and pathological processes. Although their potential for clinical application as diagnostic and therapeutic tools has been revealed, a huge bottleneck impeding the development of applications in the rapidly burgeoning field of exosome research is an inability to efficiently isolate pure exosomes from other unwanted components present in bodily fluids. To date, several approaches have been proposed and investigated for exosome separation, with the leading candidate being microfluidic technology due to its relative simplicity, cost-effectiveness, precise and fast processing at the microscale, and amenability to automation. Notably, avoiding the need for exosome labeling represents a significant advance in terms of process simplicity, time, and cost as well as protecting the biological activities of exosomes. Despite the exciting progress in microfluidic strategies for exosome isolation and the countless benefits of label-free approaches for clinical applications, current microfluidic platforms for isolation of exosomes are still facing a series of problems and challenges that prevent their use for clinical sample processing. This review focuses on the recent microfluidic platforms developed for label-free isolation of exosomes including those based on sieving, deterministic lateral displacement, field flow, and pinched flow fractionation as well as viscoelastic, acoustic, inertial, electrical, and centrifugal forces. Further, we discuss advantages and disadvantages of these strategies with highlights of current challenges and outlook of label-free microfluidics toward the clinical utility of exosomes.

Exosomes are small extracellular vesicles that can be obtained from several body fluids such as blood and urine. Since these vesicles can carry biomarkers and other cargo, they have application in healthcare diagnostics and therapeutics, such as liquid biopsies and drug delivery. Yet, their identification and separation from a sample remain challenging due to their high degree of heterogeneity and their co-existence with other bioparticles. In this contribution, we review the state-of-the-art on electrical techniques and methods to displace, selectively trap/isolate, and detect/characterize exosomes in microfluidic devices. Although there are many reviews focused on exosome separation using benchtop equipment, such as ultracentrifugation, there are limited reviews focusing on the use of electrical phenomena in microfluidic devices for exosome manipulation and detection. Here, we highlight contributions published during the past decade and present perspectives for this research field for the near future, outlining challenges to address in years to come.

Point-of-care testing (POCT) is a specific format of diagnostic testing that is conducted without accompanying infrastructure or sophisticated instrumentation. Traditionally, such rapid sample-to-answer assays provide inferior analytical performances to their laboratory counterparts when measuring cardiac biomarkers. Hence, their potentially broad applicability is somewhat bound by their inability to detect clinically relevant concentrations of cardiac troponin (cTn) in the early stages of myocardial injury. However, the continuous refinement of biorecognition elements, the optimization of detection techniques, and the fabrication of tailored fluid handling systems to manage the sensing process has stimulated the production of commercial assays that can support accelerated diagnostic pathways. This review will present the latest commercial POC assays and examine their impact on clinical decision-making. The individual elements that constitute POC assays will be explored, with an emphasis on aspects that contribute to economically feasible and highly sensitive assays. Furthermore, the prospect of POCT imparting a greater influence on early interventions for medium to high-risk individuals and the potential to re-shape the paradigm of cardiovascular risk assessments will be discussed.

Exosomes (EXOs) are nano-sized vesicles secreted by most cell types. They are abundant in bio-fluids and harbor specific molecular constituents from their parental cells. Due to these characteristics, EXOs have a great potential in cancer diagnostics for liquid biopsy and personalized medicine. Despite this unique potential, EXOs are not yet widely applied in clinical settings, with two main factors hindering their translational process in diagnostics. Firstly, conventional extraction methods are time-consuming, require large sample volumes and expensive equipment, and often do not provide high-purity samples. Secondly, characterization methods have some limitations, because they are often qualitative, need extensive labeling or complex sampling procedures that can induce artifacts. In this context, novel label-free approaches are rapidly emerging, and are holding potential to revolutionize EXO diagnostics. These methods include the use of nanodevices for EXO purification, and vibrational spectroscopies, scattering, and nanoindentation for characterization. In this progress report, we summarize recent key advances in label-free techniques for EXO purification and characterization. We point out that these methods contribute to reducing costs and processing times, provide complementary information compared to the conventional characterization techniques, and enhance flexibility, thus favoring the discovery of novel and unexplored EXO-based biomarkers. In this process, the impact of nanotechnology is systematically highlighted, showing how the effectiveness of these techniques can be enhanced using nanomaterials, such as plasmonic nanoparticles and nanostructured surfaces, which enable the exploitation of advanced physical phenomena occurring at the nanoscale level.

One of the most cited limitations of biochemical detection is its poor sensitivity, owing to the relatively high complexity of micro-samples. Moreover, some samples cannot be easily self-replicated and their abundance cannot be increased through traditional technologies. Therefore, the preconcentration of low-abundance samples is a key requirement for microfluidic biological analysis. In recent years, the ion-concentration polarization phenomenon has aroused widespread interest in the application of microfluidic technology. In addition, paper-based materials are readily available, easy to modify, and exhibit good hydrophilicity. The study of the ion-concentration polarization preconcentration of micro-samples in paper-based microfluidic chips is of considerable significance. In this review, we discuss the development and applications of ion-concentration polarization paper-based preconcentrator in the past 5 years, with emphasis on key progresses in chip fabrication and performance optimization under different conditions. The current needs and development prospects in this field have also been discussed.

Exosomes are a class of cell-secreted, nano-sized extracellular vesicles with a bilayer membrane structure of 30-150 nm in diameter. Their discovery and application have brought breakthroughs in numerous areas, such as liquid biopsies, cancer biology, drug delivery, immunotherapy, tissue repair, and cardiovascular diseases. Isolation of exosomes is the first step in exosome-related research and its applications. Standard benchtop exosome separation and sensing techniques are tedious and challenging, as they require large sample volumes, multi-step operations that are complex and time-consuming, requiring cumbersome and expensive instruments. In contrast, microfluidic platforms have the potential to overcome some of these limitations, owing to their high-precision processing, ability to handle liquids at a microscale, and integrability with various functional units, such as mixers, actuators, reactors, separators, and sensors. These platforms can optimize the detection process on a single device, representing a robust and versatile technique for exosome separation and sensing to attain high purity and high recovery rates with a short processing time. Herein, we overview microfluidic strategies for exosome isolation based on their hydrodynamic properties, size filtration, acoustic fields, immunoaffinity, and dielectrophoretic properties. We focus especially on advances in label-free isolation of exosomes with active biological properties and intact morphological structures. Further, we introduce microfluidic techniques for the detection of exosomal proteins and RNAs with high sensitivity, high specificity, and low detection limits. We summarize the biomedical applications of exosome-mediated therapeutic delivery targeting cancer cells. To highlight the advantages of microfluidic platforms, conventional techniques are included for comparison. Future challenges and prospects of microfluidics towards exosome isolation applications are also discussed. Although the use of exosomes in clinical applications still faces biological, technical, regulatory, and market challenges, in the foreseeable future, recent developments in microfluidic technologies are expected to pave the way for tailoring exosome-related applications in precision medicine.

Liquid biopsy offers non-invasive and real-time molecular profiling of individual patients, and is thus considered a revolutionary technology in precision medicine. Exosomes have been acknowledged as significant biomarkers in liquid biopsy, as they play a central role in cell-cell communication and are closely related to the pathogenesis of most human malignancies. Nevertheless, in biofluids exosomes always co-exist with other particles, and the cargo components of exosomes are highly heterogeneous. Thus, the isolation and molecular characterization of exosomes are still technically challenging. Microfluidics technology effectively addresses this challenge by virtue of its inherent advantages, such as precise manipulation of fluids, low consumption of samples and reagents, and a high level of integration. Recent advances in microfluidics allow in situ exosome capture and molecular detection with unprecedented selectivity and sensitivity. In this review, the state-of-the-art developments in microfluidics-based exosome research, including exosome isolation approaches and molecular detection strategies, with highlights of the characterization of exosomal biomarkers in cancer liquid biopsy is summarized. The major challenges are also discussed and some perspectives for the future directions of exosome-based liquid biopsy in microfluidic systems are presented.

Extracellular vesicles (EVs) are heterogenous membrane-bound vesicles released from various origins. EVs play a crucial role in cellular communication and mediate several physiological and pathological processes, highlighting their potential therapeutic and diagnostic applications. Due to the rapid increase in interests and needs to elucidate EV properties and functions, numerous isolation and separation approaches for EVs have been developed to overcome limitations of conventional techniques, such as ultracentrifugation. This review focuses on recently emerging and modern EV isolation and separation techniques, including size-, charge-, and affinity-based techniques while excluding ultracentrifugation and precipitation-based techniques due to their multiple limitations. The advantages and drawbacks of each technique are discussed together with insights into their applications. Emerging approaches all share similar features in terms of being time-effective, easy-to-operate, and capable of providing EVs with suitable and desirable purity and integrity for applications of interest. Combination and hyphenation of techniques have been used for EV isolation and separation to yield EVs with the best quality. The most recent development using an automated on-line system including selective affinity-based trapping unit and asymmetrical flow field-flow fractionation allows reliable isolation and fractionation of EV subpopulations from human plasma.

In this study, an ion depleted zone created by an ion-selective membrane was used to impose a high and uniform constant extracellular potential over an entire ∼1000 cell rat cardiomyocyte (rCM) colony on-a-chip to trigger synchronized voltage-gated ion channel activities while preserving cell viability, thus extending single-cell voltage-clamp ion channel studies to an entire normalized colony. Image analysis indicated that rCM beating was strengthened and accelerated (by a factor of ∼2) within minutes of ion depletion and the duration of contraction and relaxation phases was significantly reduced. After the initial synchronization, the entire colony responds collectively to external potential changes such that beating over the entire colony can be activated or deactivated within 0.1 s. These newly observed collective dynamic responses, due to simultaneous ion channel activation/deactivation by a uniform constant-potential extracellular environment, suggest that perm-selective membrane modules on cell culture chips can facilitate studies of extracellular cardiac cell electrical communication and how ion-channel related pathologies affect cardiac cell synchronization. The future applications of this new technology can lead to better drug screening platforms for cardiotoxicity as well as platforms that can facilitate synchronized maturation of pluripotent stem cells into colonies with high electrical connectivity.

Liquid biopsy, screening cancer non-invasively and frequently by detecting and quantifying molecular markers in physiological fluids, would significantly improve cancer survival rate but it remains a distant goal. The key obstacles presented by the highly heterogeneous samples are rapid/high-yield purification and precise/selective marker capture by their antibody and oligo probes. As irregular expressions of these molecular biomarkers are the key signals, quantifying only those from the cancer cells would greatly enhance the performance of the screening tests. The recent discovery that the biomarkers are carried by nanocarriers, such as exosomes, with cell-specific membrane proteins suggests that such selection may be possible, although a new suite of fractionation and quantification technologies would need to be developed. Although under-appreciated, membrane microfluidics has made considerable contributions to resolving these issues. We review the progress made so far, based on ion-selective, track-etched, and gel membranes and advanced electrophoretic and nano-filtration designs, in this perspective and suggest future directions.

Ion concentration polarization focusing (ICPF) is an electrokinetic technique, in which analytes are enriched and separated along a localized electric field gradient in the presence of a counter flow. This field gradient is generated by depletion of ions of the background electrolyte at an ion permselective junction. In this tutorial review, we summarize the fundamental principles and experimental parameters that govern selective ion transport and the stability of the enriched analyte plug. We also examine faradaic ICP (fICP), in which local ion concentration is modulated via electrochemical reactions as an attractive alternative to ICP that achieves similar performance with a decrease in both power consumption and Joule heating. The tutorial covers important challenges to the broad application of ICPF including undesired pH gradients, low volumetric throughput, samples that induce biofouling or are highly conductive, and limited approaches to on- or off-chip analysis. Recent developments in the field that seek to address these challenges are reviewed along with new approaches to maximize enrichment, focus uncharged analytes, and achieve enrichment and separation in water-in-oil droplets. For new practitioners, we discuss practical aspects of ICPF, such as strategies for device design and fabrication and the relative advantages of several types of ion selective junctions and electrodes. Lastly, we summarize tips and tricks for tackling common experimental challenges in ICPF.

Microfluidic capillary electrophoresis (MCE) is the novel technique resulted from the CE mininaturization as planar separation and analysis device. This review presents and discusses various application fields of this advanced technology published in the period 2017 till mid-2019 in eight different sections including clinical, biological, single cell analysis, environmental, pharmaceuticals, food analysis, forensic and ion analysis. The need for miniaturization of CE and the consequence advantages achieved are also discussed including high-throughput, miniaturized detection, effective separation, portability and the need for micro- or even nano-volume of samples. Comprehensive tables for the MCE applications in the different studied fields are provided. Also, figure comparing the number of the published papers applying MCE in the eight discussed fields within the studied period is included. The future investigation should put into consideration the possibility of replacing conventional CE with the MCE after proper validation. Suitable validation parameters with their suitable accepted ranges should be tailored for analysis methods utilizing such unique technique (MCE).