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The growing interest in regenerative medicine has opened new avenues for novel cell therapies using stem cells. Bone marrow aspirate (BMA) is an important source of stromal mesenchymal stem cells (MSCs). Conventional MSC harvesting from BMA relies on archaic centrifugation methods, often leading to poor yield due to osmotic stress, high centrifugation force, convoluted workflow, and long experimental time (∼2–3 hours). To address these issues, we have developed a scalable microfluidic technology based on deterministic lateral displacement (DLD) for MSC isolation. This passive, label-free cell sorting method capitalizes on the morphological differences between MSCs and blood cells (platelets and RBCs) for effective separation using an inverted L-shaped pillar array. To improve throughput, we developed a novel multi-chip DLD system that can process 2.5 mL of raw BMA in 20 ± 5 minutes, achieving a 2-fold increase in MSC recovery compared to centrifugation methods. Taken together, we envision that the developed DLD platform will enable fast and efficient isolation of MSCs from BMA for effective downstream cell therapy in clinical settings., National Research Foundation Singapore

Significance Bioelectronic medicine relies on electrical stimulation for most applications in the peripheral nervous system. It faces persistent challenges in selectively activating bundled nerve fibers. Here, we investigated ion-concentration modulation with ion-selective membranes and whether this modality may enhance the functional selectivity of peripheral nerve stimulation. We designed a multimodal stimulator that could control Ca 2+ concentrations within a focused volume. Acutely implanting it on the sciatic nerve of a rat, we demonstrated that Ca 2+ depletion could increase the sensitivity of the nerve to electrical stimulation in vivo. We provided evidence that it selectively influenced individual fascicles of the nerve, allowing selective activation by electrical current. Improved functional selectivity may improve outcomes for important therapeutic modalities.

Confined plunging jets are investigated as potential outfalls for the discharge of desalination brine. Compared to offshore submerged outfalls that rely on momentum to induce mixing, plunging jets released above the water surface utilize both momentum and negative buoyancy. Plunging jets also introduce air into the water column, which can reduce the possibility of hypoxic zones. In contrast to unconfined plunging jets, confined plunging jets include a confining tube, or downcomer, around the jet, which increases the penetration depth of the bubbles and can provide better aeration. However, the presence of this downcomer can hinder mixing with surrounding water. Therefore, laboratory measurements of dilution are reported here and compared to the dilution of unconfined plunging jets. In addition, qualitative observations of bubble penetration depth are also used to discuss aeration potential. For designs that increase the bubble penetration depth as compared to unconfined plunging jets, results show that dilution decreases as the depth of the downcomer is increased. However, it is shown that confined plunging jets can be designed with a short downcomer to provide higher dilution than unconfined jets. The effect of the diameter of downcomer on dilution is also investigated and a non-monotonic effect is observed.

The differential count of white blood cells (WBCs) is one widely used approach to assess the status of a patient’s immune system. Currently, the main methods of differential WBC counting are manual counting and automatic instrument analysis with labeling preprocessing. But these two methods are complicated to operate and may interfere with the physiological states of cells. Therefore, we propose a deep learning-based method to perform label-free classification of three types of WBCs based on their morphologies to judge the activated or inactivated neutrophils. Over 90% accuracy was finally achieved by a pre-trained fine-tuning Resnet-50 network. This deep learning-based method for label-free WBC classification can tackle the problem of complex instrumental operation and interference of fluorescent labeling to the physiological states of the cells, which is promising for future point-of-care applications.

A deformation-free and mass-producible plastic spiral inertial microfluidic device was developed, which provides continuous, clogging-free, and industry-level-throughput cell manipulation.

Objective The zonal properties of articular cartilage critically contribute to the mechanical support and lubrication of the tissue. Current treatments for articular cartilage have yet to regenerate this zonal architecture, thus compromising the functional efficacy of the repaired tissue and leading to tissue degeneration in the long term. In this study, the efficacy of zonal cartilage regeneration through bilayered implantation of expanded autologous zonal chondrocytes was investigated in a porcine chondral defect model. Design Autologous chondrocytes extracted from articular cartilage in the non-weight bearing trochlea region of the knee were subjected to an expansion-sorting strategy, integrating dynamic microcarrier (dMC) culture, and spiral microchannel size-based zonal chondrocyte separation. Zonal chondrocytes were then implanted as bilayered fibrin hydrogel construct in a porcine knee chondral defect model. Repair efficacy was compared with implantation with cell-free fibrin hydrogel and full thickness (FT) cartilage-derived heterogenous chondrocytes. Cartilage repair was evaluated 6 months after implantation. Results Sufficient numbers of zonal chondrocytes for implantation were generated from the non-weight bearing cartilage. Six-month repair outcomes showed that bilayered implantation of dMC-expanded zonal chondrocytes resulted in substantial recapitulation of zonal architecture, including chondrocyte arrangement, specific Proteoglycan 4 distribution, and collagen alignment, that was accompanied by healthier underlying subchondral bone. Conclusion These results demonstrate that with appropriate expansion and isolation of zonal chondrocytes, the strategy of stratified zonal chondrocyte implantation represents a significant advan

Ion selective membrane (ISM) is widely used in electrochemical engineering and micro-fluidic processes, yet accurate modeling of the ISM is still challenging due to many scientific issues. So far, assumptions on the "ideal ISM"have been used in most simulation studies involving ion transport and electrokinetic flow in ion concentration polarization systems, but the validity or accuracy of those assumptions has never been investigated. In this paper, using a two-dimensional nanochannel system with practical significance, we verify the validity of the ideal ISM model by making comparisons between the idealized ISM system and a more realistic permselective nanochannel system in terms of simplifications over the electrical potential, counter-ion concentration, and zero co-ion flux. Our results show that the simplifications of fixed voltage and fixed counter-ion concentration in the ideal ISM model are largely accurate in most situations, especially under high applied voltage and/or with high charge density inside the ISM. However, zero co-ion flux simplification is not exactly accurate in most occasions. Significant errors may be incurred by the zero co-ion flux assumption when steady state solutions are sought using the ISM model. Some discussions over the influences of structures of the nanochannel system are also added. The obtained results will help in obtaining detailed understanding of the transport features inside the nanoporous ISM, especially when the comparison between simulation and experimental data is necessary.

© 2021, The Author(s). Chinese hamster ovary (CHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins, such as monoclonal antibodies. Enhancement of productivity of these CHO cells is one of the top priorities in the biopharmaceutical industry to reduce manufacturing cost. Although there are many different methods (e.g. temperature, pH, feed) to improve protein production in CHO cells, the role of physiologically relevant hydrostatic pressure in CHO cell culture has not been reported yet. In this study, four different hydrostatic pressures (0, 30, 60, and 90 mmHg) were applied to batch CHO cells, and their cell growth/metabolism and IgG1 production were examined. Our results indicate that hydrostatic pressure can increase the maximum cell concentration by up to 50%. Moreover, overall IgG1 concentration on Day 5 showed that 30 mmHg pressure can increase IgG1 production by 26%. The percentage of non-disulphide-linked antibody aggregates had no significant change under pressure. Besides, no significant difference was observed between 30 mmHg and no pressure conditions in terms of cell clumping formation. All these findings are important for the optimization of fed-batch or perfusion culture for directing cell growth and improving antibody production.

Comet assay is a standard approach for studying DNA damage in malaria, but high-throughput options are not available. The CometChip was previously developed using mammalian cells as a high-throughput version of the comet assay. It is based on the same principle as the comet assay but provides greater efficacy, automated data processing, and improved consistency between experiments. In this protocol, we present MalariaCometChip to quantitatively assess drug-induced DNA damage in Plasmodium falciparum. For complete details on the use and execution of this protocol, please refer to Xiong et al. (2020).

© 2017 The Royal Society of Chemistry. With various promising applications demonstrated, nanofluidics has been of broad research interest in the past decade. As nanofluidics matures from a proof of concept towards practical applications, it faces two major barriers: expensive nanofabrication and ultra-low throughput. To date, the only material that enables nanofabrication-free, high-throughput, yet precisely controllable nanofluidic systems is the close-packed nanoparticle array, i.e. nanofluidic crystals. Recently, significant progress in nanofluidics has been made using nanofluidic crystals, including high-current ionic diodes, high-power energy harvesters, efficient biomolecular separation, and facile biosensors. Nanofluidic crystals are seen as a key to applying nanofluidic concepts to real-world applications. In this review, we introduce the key concepts and models in nanofluidic crystals, summarize the fabrication methods, and discuss the various applications of nanofluidic crystals in depth, highlighting their advantages in terms of simple fabrication, low cost, flexibility, and high throughput. Finally, we provide our perspectives on the future of nanofluidic crystals and their potential impacts.

Sepsis is a critical illness characterized by dysregulated inflammatory responses lacking counter-regulation. Specialized proresolving mediators are agonists for antiinflammation and for promoting resolution, and they are protective in preclinical sepsis models. Here, in human sepsis, we mapped resolution circuits for the specialized proresolving mediators resolvin D1 and resolvin D2 in peripheral blood neutrophils and monocytes, their regulation of leukocyte activation and function ex vivo, and their relationships to measures of clinical severity. Neutrophils and monocytes were isolated from healthy subjects and patients with sepsis by inertial microfluidics and resolvin D1 and resolvin D2 receptor expression determined by flow cytometry. The impact of these resolvins on leukocyte activation was determined by isodielectric separation and leukocyte function by stimulated phagolysosome formation. Leukocyte proresolving receptor expression was significantly higher in sepsis. In nanomolar concentrations, resolvin D1 and resolvin D2 partially reversed sepsis-induced changes in leukocyte activation and function. Principal component analyses of leukocyte resolvin receptor expression and responses differentiated sepsis from health and were associated with measures of sepsis severity. These findings indicate that resolvin D1 and resolvin D2 signaling for antiinflammation and resolution are uncoupled from leukocyte activation in early sepsis and suggest that indicators of diminished resolution signaling correlate with clinical disease severity.

Sepsis is a potentially lethal condition that might benefit from early monitoring of circulating activated leukocytes for faster stratification of severity of illness and improved administration of targeted treatment. Characterization of the intrinsic electrical properties of leukocytes is lable-free and can provide a quick way to quantify the number of activated cells as sepsis progresses. Isodielectric separation (IDS) uses dielectrophoresis (DEP) to characterize the electrical signatures of cells. Here we use IDS to show that activated and non-activated leukocytes have different electrical properties. We then present a double-sided version of the IDS platform to increase throughput to characterize thousands of cells. This new platform is less prone to cell fouling and allows faster characterization. Using peripheral blood samples from a cecal-ligation and puncture (CLP) model of polymicrobial sepsis in mice, we estimate the number of activated leukocytes by looking into differences in the electrical properties of cells. We show for the first time using animal models that electrical cell profiling correlates with flow cytometry (FC) results and that IDS is therefore a good candidate to provide rapid monitoring of sepsis by quantifying the number of circulating activated leukocytes., National Institutes of Health (U.S.) (U24AI118656), Space and Naval Warfare Systems Center San Diego (U.S.) (N66001-11-1-4182)

© Copyright © 2021 Flavin, Paul, Lim, Abdulhamed, Lissandrello, Ajemian, Lin and Han. For many peripheral neuro-modulation applications, the cuff electrode has become a preferred technology for delivering electrical current into targeted volumes of tissue. While basic cuffs with low spatial selectivity, having longitudinally arranged contacts, can be produced from relatively straightforward processes, the fabrication of more complex electrode configurations typically requires iterative design and clean-room fabrication with skilled technicians. Although facile methods for fabricating cuff electrodes exist, their inconsistent products have limited their adoption for rapid manufacturing. In this article, we report a fast, low-cost fabrication process for patterning of electrode contacts in an implantable peripheral nerve cuff. Using a laser cutter as we have prescribed, the designer can render precise contact geometries that are consistent between batches. This method is enabled by the use of silicone/carbon black (CB) composite electrodes, which integrate with the patterned surface of its substrate—tubular silicone insulation. The size and features of its products can be adapted to fit a wide range of nerve diameters and applications. In this study, we specifically documented the manufacturing and evaluation of circumpolar cuffs with radial arrays of three contacts for acute implantation on the rat sciatic nerve. As part of this method, we also detail protocols for verification—electrochemical characterization—and validation—electrophysiological evaluation—of implantable cuff electrodes. Applied to our circumpolar cuff electrode, we report favorable electrical characteristics. In addition, we report that it reproduces expected electrophysiological behaviors described in prior literature. No specialized equipment or fabrication experience was required in our production, and we encountered negligible costs relative to commercially available solutions. Since, as we demo

Confined plunging jets are investigated as potential outfalls for the discharge of desalination brine. Compared to offshore submerged outfalls that rely on momentum to induce mixing, plunging jets released above the water surface utilize both momentum and negative buoyancy. Plunging jets also introduce air into the water column, which can reduce the possibility of hypoxic zones. In contrast to unconfined plunging jets, confined plunging jets include a confining tube, or downcomer, around the jet, which increases the penetration depth of the bubbles and can provide better aeration. However, the presence of this downcomer can hinder mixing with surrounding water. Therefore, laboratory measurements of dilution are reported here and compared to the dilution of unconfined plunging jets. In addition, qualitative observations of bubble penetration depth are also used to discuss aeration potential. For designs that increase the bubble penetration depth as compared to unconfined plunging jets, results show that dilution decreases as the depth of the downcomer is increased. However, it is shown that confined plunging jets can be designed with a short downcomer to provide higher dilution than unconfined jets. The effect of the diameter of downcomer on dilution is also investigated and a non-monotonic effect is observed.

© 2017 Author(s). Electrokinetic concentration devices based on the ion concentration polarization (ICP) phenomenon have drawn much attention due to their simple setup, high enrichment factor, and easy integration with many subsequent processes, such as separation, reaction, and extraction etc. Despite significant progress in the experimental research, fundamental understanding and detailed modeling of the preconcentration systems is still lacking. The mechanism of the electrokinetic trapping of charged particles is currently limited to the force balance analysis between the electric force and fluid drag force in an over-simplified one-dimensional (1D) model, which misses many signatures of the actual system. This letter studies the particle trapping phenomena that are not explainable in the 1D model through the calculation of the two-dimensional (2D) force fields. The trapping of charged particles is shown to significantly distort the electric field and fluid flow pattern, which in turn leads to the different trapping behaviors of particles of different sizes. The mechanisms behind the protrusions and instability of the focused band, which are important factors determining overall preconcentration efficiency, are revealed through analyzing the rotating fluxes of particles in the vicinity of the ion-selective membrane. The differences in the enrichment factors of differently sized particles are understood through the interplay between the electric force and convective fluid flow. These results provide insights into the electrokinetic concentration effect, which could facilitate the design and optimization of ICP-based preconcentration systems.

© 2015 Huang et al. Infections by malaria parasites can lead to very different clinical outcomes, ranging from mild symptoms to death. Differences in the ability of the spleen to deal with the infected red blood cells (iRBCs) are linked to differences in virulence. Using virulent and avirulent strains of the rodent malaria parasite Plasmodium yoelii, we investigated how parasite virulence modulates overall spleen function. Following parasite invasion, a difference in parasite virulence was observed in association with different levels of spleen morphology and iRBC rigidity, both of which contributed to enhanced parasite clearance. Moreover, iRBC rigidity as modulated by the spleen was demonstrated to correlate with disease outcome and thus can be used as a robust indicator of virulence. The data indicate that alterations in the biomechanical properties of iRBCs are the result of the complex interaction between host and parasite. Furthermore, we confirmed that early spleen responses are a key factor in directing the clinical outcome of an infection.

© 2017 World Scientific Publishing Europe Ltd. This paper studies the mechanism of preconcentration of charged particles in a straight microchannel embedded with permselective membranes by numerically solving the coupled transport equations of ions, charged particles and solvent fluid without any simplifying assumptions. It is demonstrated that trapping and preconcentration of charged particles are determined by the interplay between drag force from the electroosmotic fluid flow and the electrophoretic force applied through the electric field. Several insightful characteristics are revealed, including the diverse dynamics of co-ions and counter ions, replacement of co-ions by focused particles, lowered ion concentrations in particle-enriched zone, and enhanced electroosmotic pumping effect, etc. Conditions for particles that can be concentrated are identified in terms of charges, sizes and electrophoretic mobilities of particles and co-ions. Dependences of enrichment factor on cross-membrane voltage, initial particle concentration and buffer ion concentrations are analyzed and the underlying reasons are elaborated. Finally, post priori condition for the validity of decoupled simulation model is given based on the charges carried by focused particles and buffer co-ions. These results provide an important guidance in the design and optimization of nanofluidic preconcentration and other related devices.

© 2017 The Royal Society of Chemistry. With various promising applications demonstrated, nanofluidics has been of broad research interest in the past decade. As nanofluidics matures from a proof of concept towards practical applications, it faces two major barriers: expensive nanofabrication and ultra-low throughput. To date, the only material that enables nanofabrication-free, high-throughput, yet precisely controllable nanofluidic systems is the close-packed nanoparticle array, i.e. nanofluidic crystals. Recently, significant progress in nanofluidics has been made using nanofluidic crystals, including high-current ionic diodes, high-power energy harvesters, efficient biomolecular separation, and facile biosensors. Nanofluidic crystals are seen as a key to applying nanofluidic concepts to real-world applications. In this review, we introduce the key concepts and models in nanofluidic crystals, summarize the fabrication methods, and discuss the various applications of nanofluidic crystals in depth, highlighting their advantages in terms of simple fabrication, low cost, flexibility, and high throughput. Finally, we provide our perspectives on the future of nanofluidic crystals and their potential impacts.

© 2019, The Author(s). Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy at the species level and 100% accuracy at the family level, enabling all critical clinical distinctions. In pilot studies on primary clinical specimens, including sputum, blood cultures, and pus, bacteria from 5 different phyla were identified.

© 2019 The Authors. Published by Elsevier Ltd on behalf of The Chinese Society of Theoretical and Applied Mechanics U-shaped micro-nanochannels can generate significant flow disturbance as well as locally amplified electric field, which gives itself potential to be microfluidic mixers, electrokinetic pumps, and even cell lysis process. Numerical simulation is utilized in this work to study the hidden characteristics of the U-shaped micro-nanochannel system, and the effects of key controlling parameters (the external voltage and pressure) on the device output metrics (current, maximum values of electric field, shear stress and flow velocity) were evaluated. A large portion of current flowing through the whole system goes through the nanochannels, rather than the middle part of the microchannel, with its value increasing linearly with the increase of voltage. Due to the local ion depletion near micro-nanofluidic junction, significantly enhanced electric field (as much as 15 fold at V=1 V and P 0 =0) as well as strong shear stress (leading to electrokinetic flow) is generated. With increasing external pressure, both electric field and shear stress can be increased initially (due to shortening of depletion region length), but are suppressed eventually at higher pressure due to the destruction of ion depletion layer. Insights gained from this study could be useful for designing nonlinear electrokinetic pumps and other systems.

We demonstrate a new micro/nanofluidic system for continuous and automatic monitoring of protein product size and quantity directly from the culture supernatant during a high-cell-concentration CHO cell perfusion culture. A microfluidic device enables clog-free cell retention for a bench-scale (350 mL) perfusion bioreactor that continuously produces the culture supernatant containing monoclonal antibodies (IgG1). A nanofluidic device directly monitors the protein size and quantity in the culture supernatant. The continuous-flow and fully automated operation of this nanofluidic protein analytics reduces design complexity and offers more detailed information on protein products than offline and batch-mode conventional analytics. Moreover, chemical and mechanical robustness of the nanofluidic device enables continuous monitoring for several days to a week. This continuous and online protein quality monitoring could be deployed at different steps and scales of biomanufacturing to improve product quality and manufacturing efficiency., SMART Innovation Centre (Grant ING137075-BIO), SMART Innovation Centre (Grant ING1510101-BIO), U.S. Food and Drug Administration (Award 1-U01-FD006751-01)

Background: Emergence of drug-resistant cancer phenotypes is a challenge for anti-cancer therapy. Cancer stem cells are identified as one of the ways by which chemoresistance develops. Method: We investigated the anti-inflammatory combinatorial treatment (DA) of doxorubicin and aspirin using a preclinical microfluidic model on cancer cell lines and patient-derived circulating tumour cell clusters. The model had been previously demonstrated to predict patient overall prognosis. Results: We demonstrated that low-dose aspirin with a sub-optimal dose of doxorubicin for 72 h could generate higher killing efficacy and enhanced apoptosis. Seven days of DA treatment significantly reduced the proportion of cancer stem cells and colony-forming ability. DA treatment delayed the inhibition of interleukin-6 secretion, which is mediated by both COX-dependent and independent pathways. The response of patients varied due to clinical heterogeneity, with 62.5% and 64.7% of samples demonstrating higher killing efficacy or reduction in cancer stem cell (CSC) proportions after DA treatment, respectively. These results highlight the importance of using patient-derived models for drug discovery. Conclusions: This preclinical proof of concept seeks to reduce the onset of CSCs generated post treatment by stressful stimuli. Our study will promote a better understanding of anti-inflammatory treatments for cancer and reduce the risk of relapse in patients., Singapore. National Medical Research Council (Grant NMRC)

Diabetes mellitus is one of the fastest-growing health burdens globally. Oxidative stress, which has been implicated in the pathogenesis of diabetes complication (e.g., cardiovascular event), remains poorly understood. We report a new approach to rapidly manipulate and evaluate the redox states of blood using a point-of-care NMR system. Various redox states of the hemoglobin were mapped out using the newly proposed (pseudo) two-dimensional map known as T1–T2 magnetic state diagram. We exploit the fact that oxidative stress changes the subtle molecular motion of water proton in the blood, and thus inducing a measurable shift in magnetic resonance relaxation properties. We demonstrated the clinical utilities of this technique to rapidly stratify diabetes subjects based on their oxidative status in conjunction to the traditional glycemic level to improve the patient stratification and thus the overall outcome of clinical diabetes care and management.

Long-term management for leukemia is challenging due to the painful and invasive procedure of bone marrow (BM) biopsy. At present, non-invasive liquid (blood) biopsy is not utilized for leukemia, due to lower counts of leukemia blast cells in the blood. Here, we described a robust system for the simultaneous detection and enrichment of rare blast cells. Enrichment of blast cells was achieved from blood with a one-step microfluidic blast cell biochip (BCB) sorting system, without specific targeting of proteins by antibodies. Non-target cells encountered a differential net force as compared to stiffer blast cells and were removed. The efficiency of the BCB promotes high detection sensitivity (1 in 106 cells) even from patients with minimal residual disease. The procedure was validated using actual blast cells from patients with various types of leukemia. Outcomes were compared to current evaluation standards, such as flow cytometry, using BM aspirates. Blast cell detection efficiency was higher in 55.6% of the patients using the BCB as compared to flow cytometry, despite the lower concentrations of blast cells in liquid biopsy. These studies promote early-stage detection and routine monitoring for minimal residual disease in patients.

The production of endogenous hydrogen sulfide (H₂S) has been shown to confer antibiotic tolerance in all bacteria studied to date. Therefore, this mediator has been speculated to be a universal defense mechanism against antibiotics in bacteria. This is assuming that all bacteria produce endogenous H₂S. In this study, we established that the pathogenic bacteria Acinetobacter baumannii does not produce endogenous H₂S, giving us the opportunity to test the effect of exogenous H₂S on antibiotic tolerance in a bacterium that does not produce it. By using a H₂S-releasing compound to modulate the sulfide content in A. baumannii, we demonstrated that instead of conferring antibiotic tolerance, exogenous H₂S sensitized A. baumannii to multiple antibiotic classes, and was able to revert acquired resistance to gentamicin. Exogenous H₂S triggered a perturbation of redox and energy homeostasis that translated into hypersensitivity to antibiotic killing. We propose that H₂S could be used as an antibiotic-potentiator and resistance-reversion agent in bacteria that do not produce it.

Developments in Dean flow fractionation (DFF) and deterministic lateral displacement (DLD) for label-free purification of cultured RBCs from human hematopoietic stem cells. An advancement in sorting and closed-loop manufacturing of viable human RBCs.

Southeast Asia (SEA) has been the breeding ground for malaria drug resistance, including resistance to artemisinin, the first-line anti-malarial drug. In this study, Xiong et al. find a relationship between DNA damage repair and artemisinin resistance in SEA, leading to insights on the establishment and spread of current artemisinin resistance.

Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis., National Institutes of Health (U.S.) (Grant U19AI10975), National Institutes of Health (U.S.) (Grant R01AI117043)

Upon Plasmodium falciparum infection of the red blood cells (RBCs), the parasite replicates and consumes haemoglobin resulting in the release of free heme which is rapidly converted to hemozoin crystallites. The bulk magnetic susceptibility of infected RBCs (iRBCs) is changed due to ferric (Fe 3+ ) paramagnetic state in hemozoin crystallites which induce a measurable change in spin-spin relaxation (transverse relaxation) rate in proton nuclear magnetic resonance (NMR) of iRBCs. Earlier, our group reported that this transverse relaxation rate (R 2 ) can be measured by an inexpensive, portable 0.5 Tesla bench top magnetic resonance relaxometry (MRR) system with minimum sample preparation and is able to detect very low levels of parasitemia in both blood cultures as well as animal models. However, it was challenging to diagnose malaria in human blood using MRR, mainly due to the inherent variation of R 2 values of clinical blood samples, caused by many physiological and genotypic differences not related to the parasite infection. To resolve the problem of baseline R 2 rates, we have developed an improved lysis protocol for removing confounding molecular and cellular background for MRR detection. With this new protocol and by processing larger volume of blood (>1 ml), we are able to reliably detect very low level of parasitemia (representing early stage of infection, ~0.0001%) with a stable baseline and improved sensitivity using the current MRR system. ©2019, The Author(s)., National Research Foundation Singapore, Biosystems and Micromechanics (BioSYM) IRG research program, SMART Postdoctoral Research Fellows Programme, SMART Graduate Fellowship

A techno-economic analysis is used to evaluate the economic feasibility of ion concentration polarization (ICP) desalination for seawater desalination and brine management. An empirical optimization model based on a limited set of experimental data, which was obtained from a lab-scale ICP desalination prototype, was established to calculate the required energy and membrane area for a given set of operating parameters. By calculating operating and capital expenses in various feed and product cases, the optimal levelized cost of water is determined over a range of feed salinities, mostly above seawater salinity (35 g/kg). Through these analyses, we study the economic feasibility of three applications: 1) partial desalination of brine discharge by ICP (feed varied from 35 to 75 g/kg) to common seawater RO feed level (35 g/kg) in a hybrid ICP-RO system; 2) the concentration of seawater desalination brine for salt production, and 3) partial desalination of oilfield wastewater. The economic feasibility of ICP desalination processes has been evaluated and the rough cost of treatment has been generated for several relevant applications. The approach taken in this work could be employed for other new and existing desalination processes, where a priori process modeling and optimization is scientifically and/or numerically challenging. Keywords: Electromembrane desalination; High salinity desalination; Brine management; Economic analysis; Ion concentration polarization

A techno-economic analysis is used to evaluate the economic feasibility of ion concentration polarization (ICP) desalination for seawater desalination and brine management. An empirical optimization model based on a limited set of experimental data, which was obtained from a lab-scale ICP desalination prototype, was established to calculate the required energy and membrane area for a given set of operating parameters. By calculating operating and capital expenses in various feed and product cases, the optimal levelized cost of water is determined over a range of feed salinities, mostly above seawater salinity (35 g/kg). Through these analyses, we study the economic feasibility of three applications: 1) partial desalination of brine discharge by ICP (feed varied from 35 to 75 g/kg) to common seawater RO feed level (35 g/kg) in a hybrid ICP-RO system; 2) the concentration of seawater desalination brine for salt production, and 3) partial desalination of oilfield wastewater. The economic feasibility of ICP desalination processes has been evaluated and the rough cost of treatment has been generated for several relevant applications. The approach taken in this work could be employed for other new and existing desalination processes, where a priori process modeling and optimization is scientifically and/or numerically challenging. Keywords: Electromembrane desalination; High salinity desalination; Brine management; Economic analysis; Ion concentration polarization

Recent research has shown that interactions between acoustic waves and microfluidic channels can generate microscale interference patterns with the application of a traveling surface acoustic wave (SAW), effectively creating standing wave patterns with a traveling wave. Forces arising from this interference can be utilized for precise manipulation of micron-sized particles and biological cells. The patterns that have been produced with this method, however, have been limited to straight lines and grids from flat channel walls, and where the spacing resulting from this interference has not previously been comprehensively explored. In this work we examine the interaction between both straight and curved channel interfaces with a SAW to derive geometrically deduced analytical models. These models predict the acoustic force-field periodicity near a channel interface as a function of its orientation to an underlying SAW, and are validated with experimental and simulation results. Notably, the spacing is larger for flat walls than for curved ones and is dependent on the ratio of sound speeds in the substrate and fluid. Generating these force-field gradients with only travelling waves has wide applications in acoustofluidic systems, where channel interfaces can potentially support a range of patterning, concentration, focusing and separation activities by creating locally defined acoustic forces.

Microfluidic tumor model has the unique advantage of recapitulating tumor microenvironment in a comparatively easier and representative fashion. In this review, we aim to focus more on the possibility of generating clinically actionable information from these microfluidic systems, not just scientific insight.

In blood samples from patients with viral infection, it is often important to separate viral particles from human cells, for example, to minimize background in performing viral whole genome sequencing. Here, we present a microfluidic device that uses spiral inertial microfluidics with continuous circulation to separate host cells from viral particles and free nucleic acid. We demonstrate that this device effectively reduces white blood cells, red blood cells, and platelets from both whole blood and plasma samples with excellent recovery of viral nucleic acid. Furthermore, microfluidic separation leads to greater viral genome coverage and depth, highlighting an important application of this device in processing clinical samples for viral genome sequencing., National Institutes of Health (U.S.) (R01AI117043), National Institutes of Health (U.S.) (U24-AI118656), Broad Institute

Acoustic fields have been widely used for manipulation of particles and cells within microfluidic systems. In this Letter, we explore a novel acoustofluidic phenomenon for particle patterning and focusing, where a periodic acoustic pressure field is produced parallel to internal channel boundaries with the imposition of either a traveling or standing surface acoustic wave (SAW). This effect results from the propagation and intersection of edge waves from the channel walls according to the Huygens-Fresnel principle and classical wave fronts from the substrate-fluid interface. We demonstrate versatile control over this effect to produce both one- and two-dimensional acoustic patterning from one-dimensional SAW fields and its utility for continuous particle focusing. Uniquely, this channel-guided acoustic focusing permits the generation of robust acoustic fields without channel resonance conditions and particle focusing positions that are difficult or impossible to produce otherwise.

Mammalian tissues rely on a variety of nutrients to support their physiological functions. It is known that altered metabolism is involved in the pathogenesis of cancer, but which nutrients support the inappropriate growth of intact malignant tumors is incompletely understood. Amino acids are essential nutrients for many cancer cells that can be obtained through the scavenging and catabolism of extracellular protein via macropinocytosis. In particular, macropinocytosis can be a nutrient source for pancreatic cancer cells, but it is not fully understood how the tumor environment influences metabolic phenotypes and whether macropinocytosis supports the maintenance of amino acid levels within pancreatic tumors. Here we utilize miniaturized plasma exchange to deliver labeled albumin to tissues in live mice, and we demonstrate that breakdown of albumin contributes to the supply of free amino acids in pancreatic tumors. We also deliver albumin directly into tumors using an implantable microdevice, which was adapted and modified from ref. 9. Following implantation, we directly observe protein catabolism and macropinocytosis in situ by pancreatic cancer cells, but not by adjacent, non-cancerous pancreatic tissue. In addition, we find that intratumoral inhibition of macropinocytosis decreases amino acid levels. Taken together, these data suggest that pancreatic cancer cells consume extracellular protein, including albumin, and that this consumption serves as an important source of amino acids for pancreatic cancer cells in vivo., National Science Foundation (U.S.) (Grant T32GM007287), National Cancer Institute (U.S.) (Grant F30CA183474), National Institute of General Medical Sciences (U.S.) (Award T32GM007753), National Institutes of Health (U.S.) (Grant P30CA1405141), National Institutes of Health (U.S.) (Grant R01CA168653)

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient's immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils. Keywords: Immunology and Infection, Issue 136, Inertial microfluidics, airway secretion, label-free cell sorting, heterogeneous biofluid, neutrophil enrichment, patient sample preparation, National Institute of Allergy and Infectious Diseases (U.S.) (R21AI119042), National Institutes of Health (U.S.) (U24-AI118656)

Ion exchange membranes (IEMs) have been adopted in various environmental, chemical, and energy applications. However, the formation of ion-depletion regions, caused by concentration polarization near IEMs, often leads to significant energy and efficiency loss. While much research has been devoted to solving this challenge, complete removal of ion-depletion regions is still difficult, especially when the membrane systems are operating under near- or over-limiting conditions. This paper proposes a novel multiscale-pore (MP) IEM to reduce the effect of the ion-depletion region, by allowing a fluid flow through the MP-IEM, thereby limiting the size (and the resulting resistance) of the ion-depletion region. The electrical resistance and energy consumption in MP and conventional IEM-embedded electrochemical systems were investigated, and their performance during water desalination processes were compared. The current-voltage response suggests a secondary ohmic regime attributed to an internal flow rate through the MP-IEM. Moreover, the electrochemical desalination of seawater with MP-IEMs demonstrated up to 75% reduction of energy consumption, compared with conventional IEMs under comparable operating conditions., United States. Defense Advanced Research Projects Agency (Grant DE-AR0000294)

The electrokinetic molecular concentration (EMC) effect at the micro-nanofluidic interface, which enables million-fold preconcentration of biomolecules, is one of the most compelling yet least understood nanofluidic phenomena. Despite the tremendous interests in EMC and the substantial efforts devoted, the detailed mechanism of EMC remains an enigma so far owing to its high complexity, which gives rise to the significant scientific controversies outstanding for over a decade and leaves the precise engineering of EMC devices infeasible. We report a series of experimental and theoretical new findings that decipher the mechanism of EMC. We demonstrate the first elucidation of two separate operating regimes of EMC, and establish the first theoretical model that analytically yet concisely describes the system. We further unveil the dramatically different scaling behaviors of EMC in the two regimes, thereby clarifying the long-lasting controversies. We believe this work represents important progress towards the scientific understanding of EMC and related nano-electrokinetic systems, and would enable the rational design and optimization of EMC devices for a variety of applications., National Institutes of Health (U.S.) (Grant No. U19AI109755), National Science Council (China) (Grant No. 11372229), National Science Council (China) (Grant No. 21576130), National Science Council (China) (Grant No. 21490584)

There is an increasing need for the desalination of high concentration brine (>TDS 35,000 ppm) efficiently and economically, either for the treatment of produced water from shale gas/oil development, or minimizing the environmental impact of brine from existing desalination plants. Yet, reverse osmosis (RO), which is the most widely used for desalination currently, is not practical for brine desalination. This paper demonstrates technical and economic feasibility of ICP (Ion Concentration Polarization) electrical desalination for the high saline water treatment, by adopting multi-stage operation with better energy efficiency. Optimized multi-staging configurations, dependent on the brine salinity values, can be designed based on experimental and numerical analysis. Such an optimization aims at achieving not just the energy efficiency but also (membrane) area efficiency, lowering the true cost of brine treatment. ICP electrical desalination is shown here to treat brine salinity up to 100,000 ppm of Total Dissolved Solids (TDS) with flexible salt rejection rate up to 70% which is promising in a various application treating brine waste. We also demonstrate that ICP desalination has advantage of removing both salts and diverse suspended solids simultaneously, and less susceptibility to membrane fouling/scaling, which is a significant challenge in the membrane processes.

As key components of autocrine signaling, pericellular proteases, a disintegrin and metalloproteinases (ADAMs) in particular, are known to impact the microenvironment of individual cells and have significant implications in various pathological situations including cancer, inflammatory and vascular diseases. There is great incentive to develop a high-throughput platform for single-cell measurement of pericellular protease activity, as it is essential for studying the heterogeneity of protease response and the corresponding cell behavioral consequences. In this work, we developed a microfluidic platform to simultaneously monitor protease activity of many single cells in a time-dependent manner. This platform isolates individual microwells rapidly on demand and thus allows single-cell activity measurement of both cell-surface and secreted proteases by confining individual cells with diffusive FRET-based substrates. With this platform, we observed dose-dependent heterogeneous protease activation of HepG2 cells treated with phorbol 12-myristate 13-acetate (PMA). To study the temporal behavior of PMA-induced protease response, we monitored the pericellular protease activity of the same single cells during three different time periods and revealed the diversity in the dynamic patterns of single-cell protease activity profile upon PMA stimulation. The unique temporal information of single-cell protease response can help unveil the complicated functional role of pericellular proteases., National Institutes of Health (U.S.) (Grant R01-CA096504), Singapore-MIT Alliance for Research and Technology (SMART)

Chloride ion, the majority salt in nature, is ∼52% faster than sodium ion (D[subscript Na+] = 1.33, D[subscript Cl−] = 2.03[10[superscript −9]m[superscript 2]s[superscript −1]]). Yet, current electrochemical desalination technologies (e.g. electrodialysis) rely on bipolar ion conduction, removing one pair of the cation and the anion simultaneously. Here, we demonstrate that novel ion concentration polarization desalination can enhance salt removal under a given current by implementing unipolar ion conduction: conducting only cations (or anions) with the unipolar ion exchange membrane stack. Combining theoretical analysis, experiment, and numerical modeling, we elucidate that this enhanced salt removal can shift current utilization (ratio between desalted ions and ions conducted through electrodes) and corresponding energy efficiency by the factor ∼(D[subscript −] − D[subscript +])/(D[subscript −] + D[subscript +]). Specifically for desalting NaCl, this enhancement of unipolar cation conduction saves power consumption by ∼50% in overlimiting regime, compared with conventional electrodialysis. Recognizing and utilizing differences between unipolar and bipolar ion conductions have significant implications not only on electromembrane desalination, but also energy harvesting applications (e.g. reverse electrodialysis)., Singapore-MIT Alliance (grant CE programme), United States. Advanced Research Projects Agency-Energy (ARPA-E Award DE-AR0000294)

Detection of bacteria in bloodstream infections and their antibiotic susceptibility patterns is critical to guide therapeutic decision-making for optimal patient care. Current culture-based assays are too slow (>48 h), leading to excessive up-front use of broad-spectrum antibiotics and/or incorrect antibiotic choices due to resistant bacteria, each with deleterious consequences for patient care and public health. To approach this problem, we describe a method to rapidly isolate bacteria from whole blood using inertial microfluidics and directly determine pathogen identity and antibiotic susceptibility with hybridization-based RNA detection. Using the principle of Dean flow fractionation, bacteria are separated from host blood cells in a label-free separation method with efficient recovery of even low abundance bacteria. Ribosomal RNA detection can then be applied for direct identification of low abundance pathogens (~100 per mL) from blood without culturing or enzymatic amplification. Messenger RNA detection of antibiotic-responsive transcripts after brief drug exposure permits rapid susceptibility determination from bacteria with minimal culturing (~105 per mL). This unique coupling of microfluidic cell separation with RNA-based molecular detection techniques represents significant progress towards faster diagnostics (~8 hours) to guide antibiotic therapy., United States. Defense Advanced Research Projects Agency (N66001-11-1-4182)