A microfluidic transistor for automatic control of liquids.

Microfluidics have enabled notable advances in molecular biology1,2, synthetic chemistry3,4, diagnostics5,6 and tissue engineering7. However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity and scalability of electronic circuits8-10. Just as the electronic transistor enabled unprecedented advances in the automatic control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the automatic control of reagents, droplets and single cells on a microfluidic chip. Previous works on creating a microfluidic analogue to the electronic transistor11-13 did not replicate the transistor's saturation behaviour, and could not achieve proportional amplification14, which is fundamental to modern circuit design15. Here we exploit the fluidic phenomenon of flow limitation16 to develop a microfluidic element capable of proportional amplification with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. We then use this microfluidic transistor to directly translate fundamental electronic circuits into the fluidic domain, including the amplifier, regulator, level shifter, logic gate and latch. We also combine these building blocks to create more complex fluidic controllers, such as timers and clocks. Finally, we demonstrate a particle dispenser circuit that senses single suspended particles, performs signal processing and accordingly controls the movement of each particle in a deterministic fashion without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic-transistor-based circuits enable fluidic automatic controllers to manipulate liquids and single suspended particles for lab-on-a-chip platforms.

Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells.

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.

Oscillatory inertial focusing in infinite microchannels.

Inertial microfluidics (i.e., migration and focusing of particles in finite Reynolds number microchannel flows) is a passive, precise, and high-throughput method for microparticle manipulation and sorting. Therefore, it has been utilized in numerous biomedical applications including phenotypic cell screening, blood fractionation, and rare-cell isolation. Nonetheless, the applications of this technology have been limited to larger bioparticles such as blood cells, circulating tumor cells, and stem cells, because smaller particles require drastically longer channels for inertial focusing, which increases the pressure requirement and the footprint of the device to the extent that the system becomes unfeasible. Inertial manipulation of smaller bioparticles such as fungi, bacteria, viruses, and other pathogens or blood components such as platelets and exosomes is of significant interest. Here, we show that using oscillatory microfluidics, inertial focusing in practically "infinite channels" can be achieved, allowing for focusing of micron-scale (i.e. hundreds of nanometers) particles. This method enables manipulation of particles at extremely low particle Reynolds number (Rep < 0.005) flows that are otherwise unattainable by steady-flow inertial microfluidics (which has been limited to Rep > ∼10-1). Using this technique, we demonstrated that synthetic particles as small as 500 nm and a submicron bacterium, Staphylococcus aureus, can be inertially focused. Furthermore, we characterized the physics of inertial microfluidics in this newly enabled particle size and Rep range using a Peclet-like dimensionless number (α). We experimentally observed that α >> 1 is required to overcome diffusion and be able to inertially manipulate particles.

Engineering of a silica encapsulation platform for hydrocarbon degradation using Pseudomonas sp. NCIB 9816-4.

Industrial application of encapsulated bacteria for biodegradation of hydrocarbons in water requires mechanically stable materials. A silica gel encapsulation method was optimized for Pseudomonas sp. NCIB 9816-4, a bacterium that degrades more than 100 aromatic hydrocarbons. The design process focused on three aspects: (i) mechanical property enhancement; (ii) gel cytocompatibility; and (iii) reduction of the diffusion barrier in the gel. Mechanical testing indicated that the compressive strength at failure (σf ) and elastic modulus (E) changed linearly with the amount of silicon alkoxide used in the gel composition. Measurement of naphthalene biodegradation by encapsulated cells indicated that the gel maintained cytocompatibility at lower levels of alkoxide. However, significant loss in activity was observed due to methanol formation during hydrolysis at high alkoxide concentrations, as measured by FTIR spectroscopy. The silica gel with the highest amount of alkoxide (without toxicity from methanol) had a biodegradation rate of 285 ± 42 nmol/L-s, σf = 652 ± 88 kPa, and E = 15.8 ± 2.0 MPa. Biodegradation was sustained for 1 month before it dropped below 20% of the initial rate. In order to improve the diffusion through the gel, polyvinyl alcohol (PVA) was used as a porogen and resulted in a 48 ± 19% enhancement in biodegradation, but it impacted the mechanical properties negatively. This is the first report studying how the silica composition affects biodegradation of naphthalene by Pseudomonas sp. NCIB 9816-4 and establishes a foundation for future studies of aromatic hydrocarbon biodegradation for industrial application.

Bacterial Cyanuric Acid Hydrolase for Water Treatment.

Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 µM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 µM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation.

Manufacturing of bioreactive nanofibers for bioremediation.

Recombinant Escherichia coli (E. coli) cells were successfully encapsulated in reactive membranes comprised of electrospun nanofibers that have biocompatible polyvinyl alcohol (PVA)-based cores entrapping the E. coli and silica-based, mechanically sturdy porous shells. The reactive membranes were produced in a continuous fashion using a coaxial electrospinning system coupled to a microfluidic timer that mixed and regulated the reaction time of the silica precursor and the PVA solution streams. A factorial design method was employed to investigate the effects of the three critical design parameters of the system (the flow rate of the core solution, protrusion of the core needle, and the viscosity of the core solution) and to optimize these parameters for reproducibly and continuously producing high-quality core/shell nanofibers. The feasibility of using the reactive membranes manufactured in this fashion for bioremediation of atrazine, a herbicide, was also investigated. The atrazine degradation rate (0.24 µmol/g of E. coli/min) of the encapsulated E. coli cells expressing the atrazine-dechlorinating enzyme AtzA was measured to be relatively close to that measured with the free cells in solution (0.64 µmol/g of E. coli/min). We show here that the low cost, high flexibility, water insolubility, and high degradation efficiency of the bioreactive membranes manufactured with electrospinning makes it feasible for their wide-spread use in industrial scale bioremediation of contaminated waters.

A Microfluidic Transistor for Liquid Signal Processing.

Microfluidics have enabled significant advances in molecular biology 1-3 , synthetic chemistry 4,5 , diagnostics 6,7 , and tissue engineering 8 . However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity, and scalability of electronic circuits 9-11 . Just as the electronic transistor enabled unprecedented advances in the control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the complex, scalable control of reagents, droplets, and single cells on an autonomous microfluidic chip. Prior works on creating a microfluidic analogue to the electronic transistor 12-14 could not replicate the transistor's saturation behavior, which is crucial to perform analog amplification 15 and is fundamental to modern circuit design 16 . Here we exploit the fluidic phenomenon of flow-limitation 17 to develop a microfluidic element with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. As this microfluidic transistor successfully replicates all of the key operating regimes of the electronic transistor (linear, cut-off and saturation), we are able to directly translate a variety of fundamental electronic circuit designs into the fluidic domain, including the amplifier, regulator, level shifter, logic gate, and latch. Finally, we demonstrate a "smart" particle dispenser that senses single suspended particles, performs liquid signal processing, and accordingly controls the movement of said particles in a purely fluidic system without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic transistor-based circuits are easy to integrate at scale, eliminate the need for external flow control, and enable uniquely complex liquid signal processing and single-particle manipulation for the next generation of chemical, biological, and clinical platforms.

In-flow measurement of cell-cell adhesion using oscillatory inertial microfluidics.

Multicellular clusters in circulation can exhibit a substantially different function and biomarker significance compared to individual cells. Notably, clusters of circulating tumor cells (CTCs) are much more effective initiators of metastasis than single CTCs, and correlate with worse patient prognoses. Measuring the cell-cell adhesion strength of CTC clusters is a critical step towards understanding their subsistence in the circulation and mechanism of elevated tumorigenicity. However, measuring cell-cell adhesion forces in flow is elusive using existing methods. Here, we report an oscillatory inertial microfluidics system which exerts a repeating fluidic force profile on suspended cell doublets to determine their cell-cell adhesion strength (Fs), without any biophysical modifications to the cell surface and physiological morphology. Using our system, we analyzed a large number (N > 500) of doublets from a patient-derived breast cancer CTC line. We discovered that the cell-cell adhesion strength of CTC doublets varied almost 20-fold between the weakly adhered (Fs < 28 nN) and strongly bound subpopulations (Fs > 542 nN). Our system can be used with other cancer or noncancer cells without restrictions, and may be used for rapid screening of drugs aiming to disrupt the highly-metastatic CTC clusters in circulation.

Microfluidic concentration and separation of circulating tumor cell clusters from large blood volumes.

Circulating tumor cells (CTCs) are extremely rare in the blood, yet they account for metastasis. Notably, it was reported that CTC clusters (CTCCs) can be 50-100 times more metastatic than single CTCs, making them particularly salient as a liquid biopsy target. Yet they can split apart and are even rarer, complicating their recovery. Isolation by filtration risks loss when clusters squeeze through filter pores over time, and release of captured clusters can be difficult. Deterministic lateral displacement is continuous but requires channels not much larger than clusters, leading to clogging. Spiral inertial focusing requires large blood dilution factors (or lysis). Here, we report a microfluidic chip that continuously isolates untouched CTC clusters from large volumes of minimally (or undiluted) whole blood. An array of 100 µm-wide channels first concentrates clusters in the blood, and then a similar array transfers them into a small volume of buffer. The microscope-slide-sized PDMS device isolates individually-spiked CTC clusters from >30 mL per hour of whole blood with 80% efficiency into enumeration (fluorescence imaging), and on-chip yield approaches 100% (high speed video). Median blood cell removal (in base-10 logs) is 4.2 for leukocytes, 5.5 for red blood cells, and 4.9 for platelets, leaving less than 0.01% of leukocytes alongside CTC clusters in the product. We also demonstrate that cluster configurations are preserved. Gentle, high throughput concentration and separation of circulating tumor cell clusters from large blood volumes will enable cluster-specific diagnostics and speed the generation of patient-specific CTC cluster lines.

Microfluidic arenas for war games between neutrophils and microbes.

Measurements of neutrophil activities such as cell migration and phagocytosis are generally performed using low-content bulk assays, which provide little detail activity at the single cell level, or flow cytometry methods, which have the single cell resolution but lack perspective on the kinetics of the process. Here, we present a microfluidic assay for measuring the essential functions that contribute to the antimicrobial activity of neutrophils: migration towards the target, and killing of microbes. The assay interrogates the interactions between isolated human neutrophils and populations of live, proliferating microbes. The outcome is measured in a binary mode that is reflective of in vivo infections, which are either cleared or endure the host response. The outcome of the interactions is also characterized at single cell resolution for both the neutrophils and the microbes. We applied the assay to test the response of neutrophils from intensive care patients to live Staphylococcus aureus, and observed alterations of antimicrobial neutrophil activity in patients, including those with sepsis. By directly measuring neutrophil activity against live targets at high spatial and temporal resolution, this assay provides unique insights into the life-or-death contest shaping the outcome of interactions between populations of neutrophils and microbes.

Anti-thrombotic strategies for microfluidic blood processing.

The redundant mechanisms involved in blood coagulation are crucial for rapid hemostasis. Yet they also create challenges in blood processing in medical devices and lab-on-a-chip systems. In this work, we investigate the effects of both shear stress and hypothermic blood storage on thrombus formation in microfluidic processing. For fresh blood, thrombosis occurs only at high shear, and the glycoprotein IIb/IIIa inhibitor tirofiban is highly effective in preventing thrombus formation. Blood storage generally activates platelets and primes them towards thrombosis via multiple mechanisms. Thrombus formation of stored blood at low shear can be adequately inhibited by glycoprotein IIb/IIIa inhibitors. At high shear, von Willebrand factor-mediated thrombosis contributes significantly and requires additional treatments with thiol-containing antioxidants-such as N acetylcysteine and reduced glutathione-that interfere with von Willebrand factor polymerization. We further demonstrate the effectiveness of these anti-thrombotic strategies in microfluidic devices made of cyclic olefin copolymer, a popular material used in the healthcare industry. This work identifies effective anti-thrombotic strategies that are applicable in a wide range of blood- and organ-on-a-chip applications.

Adsorption and Biodegradation of Aromatic Chemicals by Bacteria Encapsulated in a Hydrophobic Silica Gel.

An adsorbent silica biogel material was developed via silica gel encapsulation of Pseudomonas sp. NCIB 9816-4, a bacterium that degrades a broad spectrum of aromatic pollutants. The adsorbent matrix was synthesized using silica precursors methyltrimethoxysilane and tetramethoxysilane to maximize the adsorption capacity of the matrix while maintaining a highly networked and porous microstructure. The encapsulated bacteria enhanced the removal rate and capacity of the matrix for an aromatic chemical mixture. Repeated use of the material over four cycles was conducted to demonstrate that the removal capacity could be maintained with combined adsorption and biodegradation. The silica biogel can thus be used extensively without the need for disposal, as a result of continuous biodegradation by the encapsulated bacteria. However, an inverse trend was observed with the ratio of biodegradation to adsorption as a function of log Kow, suggesting increasing mass-transport limitation for the most hydrophobic chemicals used (log Kow > 4).

Non-equilibrium Inertial Separation Array for High-throughput, Large-volume Blood Fractionation.

Microfluidic blood processing is used in a range of applications from cancer therapeutics to infectious disease diagnostics. As these applications are being translated to clinical use, processing larger volumes of blood in shorter timescales with high-reliability and robustness is becoming a pressing need. In this work, we report a scaled, label-free cell separation mechanism called non-equilibrium inertial separation array (NISA). The NISA mechanism consists of an array of islands that exert a passive inertial lift force on proximate cells, thus enabling gentler manipulation of the cells without the need of physical contact. As the cells follow their size-based, deterministic path to their equilibrium positions, a preset fraction of the flow is siphoned to separate the smaller cells from the main flow. The NISA device was used to fractionate 400 mL of whole blood in less than 3 hours, and produce an ultrapure buffy coat (96.6% white blood cell yield, 0.0059% red blood cell carryover) by processing whole blood at 3 mL/min, or ∼300 million cells/second. This device presents a feasible alternative for fractionating blood for transfusion, cellular therapy and blood-based diagnostics, and could significantly improve the sensitivity of rare cell isolation devices by increasing the processed whole blood volume.

Silica ecosystem for synergistic biotransformation.

Synergistical bacterial species can perform more varied and complex transformations of chemical substances than either species alone, but this is rarely used commercially because of technical difficulties in maintaining mixed cultures. Typical problems with mixed cultures on scale are unrestrained growth of one bacterium, which leads to suboptimal population ratios, and lack of control over bacterial spatial distribution, which leads to inefficient substrate transport. To address these issues, we designed and produced a synthetic ecosystem by co-encapsulation in a silica gel matrix, which enabled precise control of the microbial populations and their microenvironment. As a case study, two greatly different microorganisms: Pseudomonas sp. NCIB 9816 and Synechococcus elongatus PCC 7942 were encapsulated. NCIB 9816 can aerobically biotransform over 100 aromatic hydrocarbons, a feat useful for synthesis of higher value commodity chemicals or environmental remediation. In our system, NCIB 9816 was used for biotransformation of naphthalene (a model substrate) into CO2 and the cyanobacterium PCC 7942 was used to provide the necessary oxygen for the biotransformation reactions via photosynthesis. A mathematical model was constructed to determine the critical cell density parameter to maximize oxygen production, and was then used to maximize the biotransformation rate of the system.