Showing papers in "Lab on a Chip in 2020"

Electrochemical paper-based devices: sensing approaches and progress toward practical applications

Abstract: Paper-based sensors offer an affordable yet powerful platform for field and point-of-care (POC) testing due to their self-pumping ability and utility for many different analytical measurements. When combined with electrochemical detection using small and portable electronics, sensitivity and selectivity of the paper devices can be improved over naked eye detection without sacrificing portability. Herein, we review how the field of electrochemical paper-based analytical devices (ePADs) has grown since it was introduced a decade ago. We start by reviewing fabrication methods relevant to ePADs with more focus given to the electrode fabrication, which is fundamental for electrochemical sensing. Multiple sensing approaches applicable to ePADs are then discussed and evaluated to present applicability, advantages and challenges associated with each approach. Recent applications of ePADs in the fields of clinical diagnostics, environmental testing, and food analysis are also presented. Finally, we discuss how the current ePAD technologies have progressed to meet the analytical and practical specifications required for field and/or POC applications, as well as challenges and outlook.

Current commercialization status of electrowetting-on-dielectric (EWOD) digital microfluidics

Abstract: The emergence of electrowetting-on-dielectric (EWOD) in the early 2000s made the once-obscure electrowetting phenomenon practical and led to numerous activities over the last two decades. As an eloquent microscale liquid handling technology that gave birth to digital microfluidics, EWOD has served as the basis for many commercial products over two major application areas: optical, such as liquid lenses and reflective displays, and biomedical, such as DNA library preparation and molecular diagnostics. A number of research or start-up companies (e.g., Phillips Research, Varioptic, Liquavista, and Advanced Liquid Logic) led the early commercialization efforts and eventually attracted major companies from various industry sectors (e.g., Corning, Amazon, and Illumina). Although not all of the pioneering products became an instant success, the persistent growth of liquid lenses and the recent FDA approvals of biomedical analyzers proved that EWOD is a powerful tool that deserves a wider recognition and more aggressive exploration. This review presents the history around major EWOD products that hit the market to show their winding paths to commercialization and summarizes the current state of product development to peek into the future. In providing the readers with a big picture of commercializing EWOD and digital microfluidics technology, our goal is to inspire further research exploration and new entrepreneurial adventures.

Computational inertial microfluidics: a review

Abstract: Since the discovery of inertial focusing in 1961, numerous theories have been put forward to explain the migration of particles in inertial flows, but a complete understanding is still lacking. Recently, computational approaches have been utilized to obtain better insights into the underlying physics. In particular, fundamental aspects of particle focusing inside straight and curved microchannels have been explored in detail to determine the dependence of focusing behavior on particle size, channel shape, and flow Reynolds number. In this review, we differentiate between the models developed for inertial particle motion on the basis of whether they are semi-analytical, Navier-Stokes-based, or built on the lattice Boltzmann method. This review provides a blueprint for the consideration of numerical solutions for modeling of inertial particle motion, whether deformable or rigid, spherical or non-spherical, and whether suspended in Newtonian or non-Newtonian fluids. In each section, we provide the general equations used to solve particle motion, followed by a tutorial appendix and specified sections to engage the reader with details of the numerical studies. Finally, we address the challenges ahead in the modeling of inertial particle microfluidics for future investigators.

Smartphone-based multiplex 30-minute nucleic acid test of live virus from nasal swab extract.

Abstract: Rapid, sensitive and specific detection and reporting of infectious pathogens is important for patient management and epidemic surveillance. We demonstrated a point-of-care system integrated with a smartphone for detecting live virus from nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human diseases such as COVID-19. Specific nucleic acid sequences of five pathogens were amplified by loop-mediated isothermal amplification on a microfluidic chip and detected at the end of reactions by the smartphone. Pathogen-spiked horse nasal swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional lab-based test, polymerase chain reaction, with results achieved in ∼30 minutes.

Channel innovations for inertial microfluidics

Abstract: Inertial microfluidics has gained significant attention since first being proposed in 2007 owing to the advantages of simplicity, high throughput, precise manipulation, and freedom from an external field. Superior performance in particle focusing, filtering, concentrating, and separating has been demonstrated. As a passive technology, inertial microfluidics technology relies on the unconventional use of fluid inertia in an intermediate Reynolds number range to induce inertial migration and secondary flow, which depend directly on the channel structure, leading to particle migration to the lateral equilibrium position or trapping in a specific cavity. With the advances in micromachining technology, many channel structures have been designed and fabricated in the past decade to explore the fundamentals and applications of inertial microfluidics. However, the channel innovations for inertial microfluidics have not been discussed comprehensively. In this review, the inertial particle manipulations and underlying physics in conventional channels, including straight, spiral, sinusoidal, and expansion-contraction channels, are briefly described. Then, recent innovations in channel structure for inertial microfluidics, especially channel pattern modification and unconventional cross-sectional shape, are reviewed. Finally, the prospects for future channel innovations in inertial microfluidic chips are also discussed. The purpose of this review is to provide guidance for the continued study of innovative channel designs to improve further the accuracy and throughput of inertial microfluidics.

Exosome-mediated microRNA-497 delivery for anti-cancer therapy in a microfluidic 3D lung cancer model

Abstract: Non-small cell lung cancer (NSCLC) is one of the leading causes of death from cancer worldwide. The delivery and controlled regulation of miRNAs via exosomes is known as a potential therapeutic approach in the treatment of cancer. In this study, human cell-derived exosomes were used as delivery vehicles for miRNAs, and we investigated their anti-tumor and anti-angiogenic effects on NSCLCs that were cultured in 2D and 3D microfluidic devices. We demonstrated that exosomes that contained miRNA-497 (miR-497) effectively suppressed tumor growth and the expression of their associated genes, i.e., yes-associated protein 1 (YAP1), hepatoma-derived growth factor (HDGF), cyclin E1 (CCNE1), and vascular endothelial growth factor-A (VEGF-A), in A549 cells. Also, the level of VEGF-A-mediated angiogenic sprouting was decreased drastically in human umbilical vein endothelial cells (HUVECs) cultured in a microfluidic device. To mimic the in vivo-like tumor microenvironment of NSCLC, A549 cells were co-cultured with HUVECs in a single device, and miR-497-loaded exosomes were delivered to both types of cells. As a result, both the tube formation of endothelial cells and the migration of tumor decreased dramatically compared to the control. This indicated that miR-497 has synergistic inhibitory effects that target tumor growth and angiogenesis, so exosome-mediated miRNA therapeutics combined with the microfluidic technology could be a predictive, cost-efficient translational tool for the development of targeted cancer therapy.

High-throughput screening by droplet microfluidics: perspective into key challenges and future prospects.

Abstract: In two decades of development, impressive strides have been made for automating basic laboratory operations in droplet-based microfluidics, allowing the emergence of a new form of high-throughput screening and experimentation in nanoliter to femtoliter volumes. Despite advancements in droplet storage, manipulation, and analysis, the field has not yet been widely adapted for many high-throughput screening (HTS) applications. Broad adoption and commercial development of these techniques require robust implementation of strategies for the stable storage, chemical containment, generation of libraries, sample tracking, and chemical analysis of these small samples. We discuss these challenges for implementing droplet HTS and highlight key strategies that have begun to address these concerns. Recent advances in the field leave us optimistic about the future prospects of this rapidly developing technology.

Towards practical sample preparation in point-of-care testing: user-friendly microfluidic devices.

Abstract: Microfluidic technologies offer a number of advantages for sample preparation in point-of-care testing (POCT), but the requirement for complicated external pumping systems limits their wide use. To facilitate sample preparation in POCT, various methods have been developed to operate microfluidic devices without complicated external pumping systems. In this review, we introduce an overview of user-friendly microfluidic devices for practical sample preparation in POCT, including self- and hand-operated microfluidic devices. Self-operated microfluidic devices exploit capillary force, vacuum-driven pressure, or gas-generating chemical reactions to apply pressure into microchannels, and hand-operated microfluidic devices utilize human power sources using simple equipment, including a syringe, pipette, or simply by using finger actuation. Furthermore, this review provides future perspectives to realize user-friendly integrated microfluidic circuits for wider applications with the integration of simple microfluidic valves.

A flexible 3-dimensional microelectrode array for in vitro brain models.

Abstract: Three-dimensional (3D) in vitro models have become increasingly popular as systems to study cell-cell and cell-ECM interactions dependent on the spatial, mechanical, and chemical cues within the environment of the tissue, which is limited in traditional two-dimensional (2D) models. Although electrophysiological recordings of neuronal action potentials through 2D microelectrode arrays (MEAs) are a common and trusted method of evaluating neuronal function, network communication, and response to chemicals and biologicals, there are currently limited options for measuring electrophysiological activity from many locations simultaneously throughout a 3D network of neurons in vitro. Here, we have developed a thin-film, 3D flexible microelectrode array (3DMEA) that non-invasively interrogates a 3D culture of neurons and can accommodate 256 channels of recording or stimulation. Importantly, the 3DMEA is straightforward to fabricate and integrates with standard commercially available electrophysiology hardware. Polyimide probe arrays were microfabricated on glass substrates and mechanically actuated to collectively lift the arrays into a vertical position, relying solely on plastic deformation of their base hinge regions to maintain vertical alignment. Human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes were entrapped in a collagen-based hydrogel and seeded onto the 3DMEA, enabling growth of suspended cells in the matrix and the formation and maturation of a neural network around the 3DMEA probes. The 3DMEA supported the growth of functional neurons in 3D with action potential spike and burst activity recorded over 45 days in vitro. This platform is an important step in facilitating noninvasive electrophysiological characterization of 3D networks of electroactive cells in vitro.

Intelligent image-activated cell sorting 2.0

Abstract: The advent of intelligent image-activated cell sorting (iIACS) has enabled high-throughput intelligent image-based sorting of single live cells from heterogeneous populations. iIACS is an on-chip microfluidic technology that builds on a seamless integration of a high-throughput fluorescence microscope, cell focuser, cell sorter, and deep neural network on a hybrid software-hardware data management architecture, thereby providing the combined merits of optical microscopy, fluorescence-activated cell sorting (FACS), and deep learning. Here we report an iIACS machine that far surpasses the state-of-the-art iIACS machine in system performance in order to expand the range of applications and discoveries enabled by the technology. Specifically, it provides a high throughput of ∼2000 events per second and a high sensitivity of ∼50 molecules of equivalent soluble fluorophores (MESFs), both of which are 20 times superior to those achieved in previous reports. This is made possible by employing (i) an image-sensor-based optomechanical flow imaging method known as virtual-freezing fluorescence imaging and (ii) a real-time intelligent image processor on an 8-PC server equipped with 8 multi-core CPUs and GPUs for intelligent decision-making, in order to significantly boost the imaging performance and computational power of the iIACS machine. We characterize the iIACS machine with fluorescent particles and various cell types and show that the performance of the iIACS machine is close to its achievable design specification. Equipped with the improved capabilities, this new generation of the iIACS technology holds promise for diverse applications in immunology, microbiology, stem cell biology, cancer biology, pathology, and synthetic biology.

A point-of-care selenium nanoparticle-based test for the combined detection of anti-SARS-CoV-2 IgM and IgG in human serum and blood.

Abstract: COVID-19 is a widespread and highly contagious disease in the human population. COVID-19 is caused by SARS-CoV-2 infection. There is still a great demand for point-of-care tests for detection, epidemic prevention and epidemiological investigation, both now and after the epidemic. We present a lateral flow immunoassay kit based on a selenium nanoparticle-modified SARS-CoV-2 nucleoprotein, which detects anti-SARS-CoV-2 IgM and anti-SARS-CoV-2 IgG in human serum, and the results can be read by the naked eye in 10 minutes. We expressed and purified the SARS-CoV-2 nucleoprotein in HEK293 cells, with a purity of 98.14% and a concentration of 5 mg mL-1. Selenium nanoparticles were synthesized by l-ascorbic acid reduction of seleninic acid at room temperature. After conjugation with the nucleoprotein, a lateral flow kit was successfully prepared. The IgM and IgG detection limits of the lateral flow kit reached 20 ng mL-1 and 5 ng mL-1, respectively, in human serum. A clinical study sample comprising 90 COVID-19-diagnosed patients and 263 non-infected controls was used to demonstrate a sensitivity and specificity of 93.33% and 97.34%, respectively, based on RT-PCR and clinical results. No cross-reactions with rheumatoid factor and positive serum for anti-nuclear antibodies, influenza A, and influenza B were observed. Moreover, the lateral flow kit remained stable after storage for 30 days at 37 °C. Our results demonstrate that the selenium nanoparticle lateral flow kit can conveniently, rapidly, and sensitively detect anti-SARS-CoV-2 IgM and IgG in human serum and blood; it can also be suitable for the epidemiological investigation of COVID-19.

AI on a chip

Abstract: Artificial intelligence (AI) has dramatically changed the landscape of science, industry, defence, and medicine in the last several years. Supported by considerably enhanced computational power and cloud storage, the field of AI has shifted from mostly theoretical studies in the discipline of computer science to diverse real-life applications such as drug design, material discovery, speech recognition, self-driving cars, advertising, finance, medical imaging, and astronomical observation, where AI-produced outcomes have been proven to be comparable or even superior to the performance of human experts. In these applications, what is essentially important for the development of AI is the data needed for machine learning. Despite its prominent importance, the very first process of the AI development, namely data collection and data preparation, is typically the most laborious task and is often a limiting factor of constructing functional AI algorithms. Lab-on-a-chip technology, in particular microfluidics, is a powerful platform for both the construction and implementation of AI in a large-scale, cost-effective, high-throughput, automated, and multiplexed manner, thereby overcoming the above bottleneck. On this platform, high-throughput imaging is a critical tool as it can generate high-content information (e.g., size, shape, structure, composition, interaction) of objects on a large scale. High-throughput imaging can also be paired with sorting and DNA/RNA sequencing to conduct a massive survey of phenotype-genotype relations whose data is too complex to analyze with traditional computational tools, but is analyzable with the power of AI. In addition to its function as a data provider, lab-on-a-chip technology can also be employed to implement the developed AI for accurate identification, characterization, classification, and prediction of objects in mixed, heterogeneous, or unknown samples. In this review article, motivated by the excellent synergy between AI and lab-on-a-chip technology, we outline fundamental elements, recent advances, future challenges, and emerging opportunities of AI with lab-on-a-chip technology or "AI on a chip" for short.

Liver microphysiological systems development guidelines for safety risk assessment in the pharmaceutical industry

Abstract: The liver is critical to consider during drug development because of its central role in the handling of xenobiotics, a process which often leads to localized and/or downstream tissue injury. Our ability to predict human clinical safety outcomes with animal testing is limited due to species differences in drug metabolism and disposition, while traditional human in vitro liver models often lack the necessary in vivo physiological fidelity. To address this, increasing numbers of liver microphysiological systems (MPS) are being developed, however the inconsistency in their optimization and characterization often leads to models that do not possess critical levels of baseline performance that is required for many pharmaceutical industry applications. Herein we provide a guidance on best approaches to benchmark liver MPS based on 3 stages of characterization that includes key performance metrics and a 20 compound safety test set. Additionally, we give an overview of frequently used liver injury safety assays, describe the ideal MPS model, and provide a perspective on currently best suited MPS contexts of use. This pharmaceutical industry guidance has been written to help MPS developers and end users identify what could be the most valuable models for safety risk assessment.

Clinical decision support tool and rapid point-of-care platform for determining disease severity in patients with COVID-19.

Abstract: SARS-CoV-2 is the virus that causes coronavirus disease (COVID-19) which has reached pandemic levels resulting in significant morbidity and mortality affecting every inhabited continent. The large number of patients requiring intensive care threatens to overwhelm healthcare systems globally. Likewise, there is a compelling need for a COVID-19 disease severity test to prioritize care and resources for patients at elevated risk of mortality. Here, an integrated point-of-care COVID-19 Severity Score and clinical decision support system is presented using biomarker measurements of C-reactive protein (CRP), N-terminus pro B type natriuretic peptide (NT-proBNP), myoglobin (MYO), D-dimer, procalcitonin (PCT), creatine kinase-myocardial band (CK-MB), and cardiac troponin I (cTnI). The COVID-19 Severity Score combines multiplex biomarker measurements and risk factors in a statistical learning algorithm to predict mortality. The COVID-19 Severity Score was trained and evaluated using data from 160 hospitalized COVID-19 patients from Wuhan, China. Our analysis finds that COVID-19 Severity Scores were significantly higher for the group that died versus the group that was discharged with median (interquartile range) scores of 59 (40-83) and 9 (6-17), respectively, and area under the curve of 0.94 (95% CI 0.89-0.99). Although this analysis represents patients with cardiac comorbidities (hypertension), the inclusion of biomarkers from other pathophysiologies implicated in COVID-19 (e.g., D-dimer for thrombotic events, CRP for infection or inflammation, and PCT for bacterial co-infection and sepsis) may improve future predictions for a more general population. These promising initial models pave the way for a point-of-care COVID-19 Severity Score system to impact patient care after further validation with externally collected clinical data. Clinical decision support tools for COVID-19 have strong potential to empower healthcare providers to save lives by prioritizing critical care in patients at high risk for adverse outcomes.

A “sample-in-multiplex-digital-answer-out” chip for fast detection of pathogens

Abstract: Point-of-care (POC) testing offers rapid diagnostic results. However, the quantification of current methods is performed using standard curves and external references, and not direct and absolute quantification. This paper describes an integrated multiplex digital recombinase polymerase amplification (ImdRPA) microfluidic chip which combines DNA extraction, multiplex digital RPA and fluorescence detection together in one chip, creating a “sample-in-multiplex-digital-answer-out” system. Multi-layer soft lithography technology was used, with polydimethylsiloxane (PDMS) as the chip material and a glass slide as the substrate. This microfluidic chip has a six-layer structure and screw microvalve control function. The sample preparation for the chip involved magnetic bead-based nucleic acid extraction, which was completed within 15 min without any instrument dependence. The dRPA region was divided into 4 regions (3 positive detection areas and 1 negative control area) and included a total of 12 800 chambers, with each chamber being able to contain a volume of 2.7 nL. The screw valve allowed for the reaction components of each specific goal to be pre-embedded in different regions of the chambers. The reagents were passively driven into the dRPA region using vacuum-based self-priming introduction. Furthermore, we successfully demonstrated that the chip can simultaneously detect three species of pathogenic bacteria within 45 min and give digital quantitative results without the need to establish a standard curve in contaminated milk. Moreover, the detection limit of this ImdRPA microfluidic chip was found to be 10 bacterial cells for each kind of pathogen. These characteristics enhance its applicability for rapid detection of foodborne bacteria at the point-of-care (POC). We envision that the further development of this integrated chip will lead to rapid, multiplex and accurate detection of foodborne bacteria in a feasible manner.

Focusing of sub-micrometer particles in microfluidic devices.

Abstract: 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.

Microfluidic Raman biochip detection of exosomes: a promising tool for prostate cancer diagnosis

Abstract: Tumor-derived exosomes, which contain RNA, DNA, and proteins, are a potentially rich non-invasive source of biomarkers. However, no efficient isolation or detection methods are yet available. Here, we developed a microfluidic Raman biochip designed to isolate and analyze exosomes in situ. Anti-CD63 magnetic nanoparticles were used to enrich exosomes through mixing channels of a staggered triangular pillar array. EpCAM-functionalized Raman-active polymeric nanomaterials (Raman beads) allow rapid analysis of exosome samples within 1 h, with a quantitative signal at 2230 cm-1. The limit of detection of this biochip approaches 1.6 × 102 particles per mL with 20 μL samples. The newly developed biochip assay was successfully applied in the determination of exosomes from clinical serum samples. Thus, this novel device may have potential as a clinical exosome analysis tool for prostate cancer.

Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR.

Abstract: Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (blaCTX-M-1 and blaCTX-M-2 families) and CREs (blaOXA-48 and blaKPC), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.

Soft, skin-interfaced microfluidic systems with integrated enzymatic assays for measuring the concentration of ammonia and ethanol in sweat

Abstract: Eccrine sweat is a rich and largely unexplored biofluid that contains a range of important biomarkers, from electrolytes, metabolites, micronutrients and hormones to exogenous agents, each of which can change in concentration with diet, stress level, hydration status and physiologic or metabolic state. Traditionally, clinicians and researchers have used absorbent pads and benchtop analyzers to collect and analyze the biochemical constituents of sweat in controlled, laboratory settings. Recently reported wearable microfluidic and electrochemical sensing devices represent significant advances in this context, with capabilities for rapid, in situ evaluations, in many cases with improved repeatability and accuracy. A limitation is that assays performed in these platforms offer limited control of reaction kinetics and mixing of different reagents and samples. Here, we present a multi-layered microfluidic device platform with designs that eliminate these constraints, to enable integrated enzymatic assays with demonstrations of in situ analysis of the concentrations of ammonia and ethanol in microliter volumes of sweat. Careful characterization of the reaction kinetics and their optimization using statistical techniques yield robust analysis protocols. Human subject studies with sweat initiated by warm-water bathing highlight the operational features of these systems.

A disposable acoustofluidic chip for nano/microparticle separation using unidirectional acoustic transducers.

Abstract: Separation of nano/microparticles based on surface acoustic waves (SAWs) has shown great promise for biological, chemical, and medical applications ranging from sample purification to cancer diagnosis. However, the permanent bonding of a microchannel onto relatively expensive piezoelectric substrates and excitation transducers renders the SAW separation devices non-disposable. This limitation not only requires cumbersome cleaning and increased labor and material costs, but also leads to cross-contamination, preventing their implementation in many biological, chemical, and medical applications. Here, we demonstrate a high-performance, disposable acoustofluidic platform for nano/microparticle separation. Leveraging unidirectional interdigital transducers (IDTs), a hybrid channel design with hard/soft materials, and tilted-angle standing SAWs (taSSAWs), our disposable acoustofluidic devices achieve acoustic radiation forces comparable to those generated by existing permanently bonded, non-disposable devices. Our disposable devices can separate not only microparticles but also nanoparticles. Moreover, they can differentiate bacteria from human red blood cells (RBCs) with a purity of up to 96%. Altogether, we developed a unidirectional IDT-based, disposable acoustofluidic platform for micro/nanoparticle separation that can achieve high separation efficiency, versatility, and biocompatibility.

Recent advances in microfluidic technologies for circulating tumor cells: enrichment, single-cell analysis, and liquid biopsy for clinical applications.

Abstract: Circulating tumor cells (CTCs) detach from primary or metastatic lesions and circulate in the peripheral blood, which is considered to be the cause of distant metastases. CTC analysis in the form of liquid biopsy, enumeration and molecular analysis provide significant clinical information for cancer diagnosis, prognosis and therapeutic strategies. Despite the great clinical value, CTC analysis has not yet entered routine clinical practice due to lack of efficient technologies to perform CTC isolation and single-cell analysis. Taking the rarity and inherent heterogeneity of CTCs into account, reliable methods for CTC isolation and detection are in urgent demand for obtaining valuable information on cancer metastasis and progression from CTCs. Microfluidic technology, featuring microfabricated structures, can precisely control fluids and cells at the micrometer scale, thus making itself a particularly suitable method for rare CTC manipulation. Besides the enrichment function, microfluidic chips can also realize the analysis function by integrating multiple detection technologies. In this review, we have summarized the recent progress in CTC isolation and detection using microfluidic technologies, with special attention to emerging direct enrichment and enumeration in vivo. Further, few insights into single CTC molecular analysis are also demonstrated. We have provided a review of potential clinical applications of CTCs, ranging from early screening and diagnosis, tumor progression and prognosis, treatment and resistance monitoring, to therapeutic evaluation. Through this review, we conclude that the clinical utility of CTCs will be expanded as the isolation and analysis techniques are constantly improving.

Tumor-on-a-chip for integrating a 3D tumor microenvironment: chemical and mechanical factors

Abstract: Tumor progression, including metastasis, is significantly influenced by factors in the tumor microenvironment (TME) such as mechanical force, shear stress, chemotaxis, and hypoxia. At present, most cancer studies investigate tumor metastasis by conventional cell culture methods and animal models, which are limited in data interpretation. Although patient tissue analysis, such as human patient-derived xenografts (PDX), can provide important clinical relevant information, they may not be feasible for functional studies as they are costly and time-consuming. Thus, in vitro three-dimensional (3D) models are rapidly being developed that mimic TME and allow functional investigations of metastatic mechanisms and drug responses. One of those new 3D models is tumor-on-a-chip technology that provides a powerful in vitro platform for cancer research, with the ability to mimic the complex physiological architecture and precise spatiotemporal control. Tumor-on-a-chip technology can provide integrated features including 3D scaffolding, multicellular culture, and a vasculature system to simulate dynamic flow in vivo. Here, we review a select set of recent achievements in tumor-on-a-chip approaches and present potential directions for tumor-on-a-chip systems in the future for areas including mechanical and chemical mimetic systems. We also discuss challenges and perspectives in both biological factors and engineering methods for tumor-on-a-chip progress. These approaches will allow in the future for the tumor-on-a-chip systems to test therapeutic approaches for individuals through using their cancerous cells gathered through approaches like biopsies, which then will contribute toward personalized medicine treatments for improving their outcomes.

Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery

Abstract: Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate individual variants-of-interest at high-throughput remains challenging. Here, we present sdDE-FACS (ingle roplet ouble mulsion-FACS), a new method that uses a standard flow cytometer to phenotype, select, and isolate individual double emulsion droplets of interest. Using a 130 μm nozzle at high sort frequency (12–14 kHz), we demonstrate detection of droplet fluorescence signals with a dynamic range spanning 5 orders of magnitude and robust post-sort recovery of intact double emulsion (DE) droplets using 2 commercially-available FACS instruments. We report the first demonstration of single double emulsion droplet isolation with post-sort recovery efficiencies >70%, equivalent to the capabilities of single-cell FACS. Finally, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets via qPCR with little to no cross-contamination. sdDE-FACS marries the full power of droplet microfluidics with flow cytometry to enable a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis.

Microfabricated Electrochemical Sensing Devices

Abstract: Electrochemistry provides possibilities to realize smart microdevices of the next generation with high functionalities. Electrodes, which constitute major components of electrochemical devices, can be formed by various microfabrication techniques, and integration of the same (or different) components for that purpose is not difficult. Merging this technique with microfluidics can further expand the areas of application of the resultant devices. To augment the development of next generation devices, it will be beneficial to review recent technological trends in this field and clarify the directions required for moving forward. Even when limiting the discussion to electrochemical microdevices, a variety of useful techniques should be considered. Therefore, in this review, we attempted to provide an overview of all relevant techniques in this context in the hope that it can provide useful comprehensive information.

Integrated human organ-on-a-chip model for predictive studies of anti-tumor drug efficacy and cardiac safety

Abstract: Traditional drug screening models are often unable to faithfully recapitulate human physiology in health and disease, motivating the development of microfluidic organs-on-a-chip (OOC) platforms that can mimic many aspects of human physiology and in the process alleviate many of the discrepancies between preclinical studies and clinical trials outcomes. Linsitinib, a novel anti-cancer drug, showed promising results in pre-clinical models of Ewing Sarcoma (ES), where it suppressed tumor growth. However, a Phase II clinical trial in several European centers with patients showed relapsed and/or refractory ES. We report an integrated, open setting, imaging and sampling accessible, polysulfone-based platform, featuring minimal hydrophobic compound binding. Two bioengineered human tissues – bone ES tumor and heart muscle – were cultured either in isolation or in the integrated platform and subjected to a clinically used linsitinib dosage. The measured anti-tumor efficacy and cardiotoxicity were compared with the results observed in the clinical trial. Only the engineered tumor tissues, and not monolayers, recapitulated the bone microenvironment pathways targeted by linsitinib, and the clinically-relevant differences in drug responses between non-metastatic and metastatic ES tumors. The responses of non-metastatic ES tumor tissues and heart muscle to linsitinib were much closer to those observed in the clinical trial for tissues cultured in an integrated setting than for tissues cultured in isolation. Drug treatment of isolated tissues resulted in significant decreases in tumor viability and cardiac function. Meanwhile, drug treatment in an integrated setting showed poor tumor response and less cardiotoxicity, which matched the results of the clinical trial. Overall, the integration of engineered human tumor and cardiac tissues in the integrated platform improved the predictive accuracy for both the direct and off-target effects of linsitinib. The proposed approach could be readily extended to other drugs and tissue systems.

The emerging role of microfluidics in multi-material 3D bioprinting.

Abstract: To assist the transition of 3D bioprinting technology from simple lab-based tissue fabrication, to fully functional and implantable organs, the technology must not only provide shape control, but also functional control. This can be accomplished by replicating the cellular composition of the native tissue at the microscale, such that cell types interact to provide the desired function. There is therefore a need for precise, controllable, multi-material printing that could allow for high, possibly even single cell, resolution. This paper aims to draw attention to technological advancements made in 3D bioprinting that target the lack of multi-material, and/or multi cell-type, printing capabilities of most current devices. Unlike other reviews in the field, which largely focus on variations in single-material 3D bioprinting involving the standard methods of extrusion-based, droplet-based, laser-based, or stereolithographic methods; this review concentrates on sophisticated multi-material 3D bioprinting using multi-cartridge printheads, co-axial nozzles and microfluidic-enhanced printing nozzles.

Positional dependence of particles and cells in microfluidic electrical impedance flow cytometry: origin, challenges and opportunities

Abstract: Microfluidic electrical impedance flow cytometry is now a well-known and established method for single-cell analysis. Given the richness of the information provided by impedance measurements, this non-invasive and label-free approach can be used in a wide field of applications ranging from simple cell counting to disease diagnostics. One of its major limitations is the variation of the impedance signal with the position of the cell in the sensing area. Indeed, identical particles traveling along different trajectories do not result in the same data. The positional dependence can be considered as a challenge for the accuracy of microfluidic impedance cytometers. On the other hand, it has recently been regarded by several groups as an opportunity to estimate the position of particles in the microchannel and thus take a further step in the logic of integrating sensors in so-called “Lab-on-a-chip” devices. This review provides a comprehensive overview of the physical grounds of the positional dependence of impedance measurements. Then, both the developed strategies to reduce position influence in impedance-based assays and the recent reported technologies exploiting that dependence for the integration of position detection in microfluidic devices are reviewed.

Hollow silicon microneedle fabrication using advanced plasma etch technologies for applications in transdermal drug delivery

Abstract: A novel production process flow is presented here for the manufacture of hollow silicon microneedles using deep reactive-ion etching (DRIE) technology The patent-pending three-step process flow has been developed to produce multiple arrays of sharp-tipped, hollow microneedles, which facilitate easy insertion and controlled fluid injection into excised skin samples A bevelled tip and vertical sidewalls for the microneedle have been achieved with good uniformity, despite >45% open etch area Processing steps and etch challenges are discussed, and preliminary skin testing results are presented, showing effective needle insertion and delivery of fluorescent dye into ex vivo skin from human breast tissue

Microfluidic device for high-throughput affinity-based isolation of extracellular vesicles.

Abstract: Immunoaffinity based EV isolation technologies use antibodies targeting surface markers on EVs to provide higher isolation specificity and purity compared to existing approaches. One standing challenge for researchers is how to release captured EVs from the substrate to increase downstream and biological studies. The strong binding between the antibody and antigen or the antibody and substrate is commonly unbreakable without operating at conditions outside of the critical physiological range, making the release of EVs problematic. Additionally, immuno-affinity approaches are usually low-throughput due to their low flow velocity to ensure adequate time for antibody-antigen binding. To overcome these limitations, we modified the OncoBean chip, a previously reported circulating tumor cell isolation microfluidic device. The OncoBean chip is a radial flow microfluidic device with bean-shape microposts functionalized with biotin-conjugated EPCAM antibody through biotin-avidin link chemistry. It was demonstrated that the high surface area and varying shear rate provided by the bean-shaped posts and the radial flow design in the chip, enabled efficient capture of CTCs at high flow rate. We replace the anti-EPCAM with antibodies that recognize common EV surface markers to achieve high-throughput EV isolation. Moreover, by incorporating desthiobiotin-conjugated antibodies, EVs can be released from the device after capture, which offers a significant improvement over the existing isolation. The released EVs were found to be functional by confirming their uptake by cells using flow cytometry and fluorescent microscopy. We believe the proposed technology can facilitate both the study of EVs as cell-to-cell communicators and the further identification of EV markers.

Ultrafast star-shaped acoustic micromixer for high throughput nanoparticle synthesis.

Abstract: We present an acoustically actuated microfluidic mixer, which can operate at flowrates reaching 8 ml min-1, providing a 50-fold improvement in throughput compared to previously demonstrated acoustofluidic approaches. The device consists of a robust silicon based micro-mechanical oscillator, sandwiched between two polymeric channels which guide the fluids in and out of the system. The chip is actuated by application of an oscillatory electrical signal onto a piezoelectric disk coupled to the substrate by adhesive. At the optimal frequency, this acoustofluidic system can homogenise two fluids with a relative mixing efficiency of 91%, within 4.1 ms from first contact. The micromixer has been used to synthesize two different systems: Budesonide nanodrugs with an average diameter of 80 ± 22 nm, and DNA nanoparticles with an average diameter of 63.3 ± 24.7 nm.