An in vitro platform for study of the human gut microbiome under an oxygen gradient.

The complex, dynamic environment of the human lower gastrointestinal tract is colonized by hundreds of bacterial species that impact health and performance. Ex vivo study of the functional interactions between microbial community members in conditions representative of those in the gut is an ongoing challenge. We have developed an in vitro 40-plex platform that provides an oxygen gradient to support simultaneous maintenance of microaerobic and anaerobic microbes from the gut microbiome that can aid in rapid characterization of microbial interactions and direct comparison of individual microbiome samples. In this report, we demonstrate that the platform more closely maintained the microbial diversity and composition of human donor fecal microbiome samples than strict anaerobic conditions. The oxygen gradient established in the platform allowed the stratification and subsequent sampling of diverse microbial subpopulations that colonize microaerobic and anaerobic micro-environments. With the ability to run forty samples in parallel, the platform has the potential to be used as a rapid screening tool to understand how the gut microbiome responds to environmental perturbations such as toxic compound exposure, dietary changes, or pharmaceutical treatments.

PERSIA for Direct Fluorescence Measurements of Transcription, Translation, and Enzyme Activity in Cell-Free Systems.

Quantification of biology's central dogma (transcription and translation) is pursued by a variety of methods. Direct, immediate, and ongoing quantification of these events is difficult to achieve. Common practice is to use fluorescent or luminescent proteins to report indirectly on prior cellular events, such as turning on a gene in a genetic circuit. We present an alternative approach, PURExpress-ReAsH-Spinach In-vitro Analysis (PERSIA). PERSIA provides information on the production of RNA and protein during cell-free reactions by employing short RNA and peptide tags. Upon synthesis, these tags yield quantifiable fluorescent signal without interfering with other biochemical events. We demonstrate the applicability of PERSIA in measuring cell-free transcription, translation, and other enzymatic activity in a variety of applications: from sequence-structure-function studies, to genetic code engineering, to testing antiviral drug resistance.

Emulation of Colonic Oxygen Gradients in a Microdevice.

Gut-on-a-chip in vitro modeling is an emerging field, as the human gut epithelium and gut microbiome have been recently identified as novel drug targets for a wide variety of diseases. Realistic in vitro gut models require a variety of precise environmental cues, such as chemical and gas gradients, in combination with substrates like mucus that support the growth of microbial communities. This technical brief describes a microfluidic architecture capable of developing a physiologically relevant oxygen gradient that emulates the oxygen profile proximal to the epithelial inner lining of the human colon. The device generates stable and repeatable defined oxygen gradients from 0% to 4 % partial pressure O2 over a length scale of hundreds of microns, and was applied to study the effects of oxygenation on the structure of native mucus that lines the colon wall. Using simulation as a design tool for hybrid gas-liquid microfluidic devices enables on-chip creation of defined, physiologically oxygen gradients. These microfluidic architectures have powerful potential applications for gut physiology, including providing optimal oxygenation conditions for the culture of mammalian epithelial cells in the gut lining, as well as creating a realistic mimic of the oxygen gradient found in the intestinal lumen for complex microbiome cultures.

Bioelectronic measurement and feedback control of molecules in living cells.

We describe an electrochemical measurement technique that enables bioelectronic measurements of reporter proteins in living cells as an alternative to traditional optical fluorescence. Using electronically programmable microfluidics, the measurement is in turn used to control the concentration of an inducer input that regulates production of the protein from a genetic promoter. The resulting bioelectronic and microfluidic negative-feedback loop then serves to regulate the concentration of the protein in the cell. We show measurements wherein a user-programmable set-point precisely alters the protein concentration in the cell with feedback-loop parameters affecting the dynamics of the closed-loop response in a predictable fashion. Our work does not require expensive optical fluorescence measurement techniques that are prone to toxicity in chronic settings, sophisticated time-lapse microscopy, or bulky/expensive chemo-stat instrumentation for dynamic measurement and control of biomolecules in cells. Therefore, it may be useful in creating a: cheap, portable, chronic, dynamic, and precise all-electronic alternative for measurement and control of molecules in living cells.

Fluidic microoptics with adjustable focusing and beam steering for single cell optogenetics.

Electrically controlled micron-scale liquid lenses have been designed, fabricated and demonstrated, that provide both adjustable focusing and beam steering, with the goal of applying them to optogenetic in vivo mapping of brain activity with single cell resolution. The liquid lens is formed by the interface between two immiscible liquids which are contained in a conically tapered lens cavity etched into a fused silica substrate. Interdigitated electrodes have been patterned along the sidewall of the taper to control the liquid lens curvature and tilt. Microlenses with apertures ranging in size from 30 to 80 µm were fabricated and tunable focusing ranging from 0.25 to 3 mm and beam steering of ± 1 degree have been demonstrated.

Open-source, community-driven microfluidics with Metafluidics.

Microfluidic devices have the potential to automate and miniaturize biological experiments, but open-source sharing of device designs has lagged behind sharing of other resources such as software. Synthetic biologists have used microfluidics for DNA assembly, cell-free expression, and cell culture, but a combination of expense, device complexity, and reliance on custom set-ups hampers their widespread adoption. We present Metafluidics, an open-source, community-driven repository that hosts digital design files, assembly specifications, and open-source software to enable users to build, configure, and operate a microfluidic device. We use Metafluidics to share designs and fabrication instructions for both a microfluidic ring-mixer device and a 32-channel tabletop microfluidic controller. This device and controller are applied to build genetic circuits using standard DNA assembly methods including ligation, Gateway, Gibson, and Golden Gate. Metafluidics is intended to enable a broad community of engineers, DIY enthusiasts, and other nontraditional participants with limited fabrication skills to contribute to microfluidic research.

High-throughput dental biofilm growth analysis for multiparametric microenvironmental biochemical conditions using microfluidics.

Dental biofilm formation is not only a precursor to tooth decay, but also induces more serious systematic health problems such as cardiovascular disease and diabetes. Understanding the conditions promoting colonization and subsequent biofilm development involving complex bacteria coaggregation is particularly important. In this paper, we report a high-throughput microfluidic 'artificial teeth' device offering controls of multiple microenvironmental factors (e.g. nutrients, growth factors, dissolved gases, and seeded cell populations) for quantitative characteristics of long-term dental bacteria growth and biofilm development. This 'artificial teeth' device contains multiple (up to 128) incubation chambers to perform parallel cultivation and analyses (e.g. biofilm thickness, viable-dead cell ratio, and spatial distribution of multiple bacterial species) of bacteria samples under a matrix of different combinations of microenvironmental factors, further revealing possible developmental mechanisms of dental biofilms. Specifically, we applied the 'artificial teeth' to investigate the growth of two key dental bacteria, Streptococci species and Fusobacterium nucleatum, in the biofilm under different dissolved gas conditions and sucrose concentrations. Together, this high-throughput microfluidic platform can provide extended applications for general biofilm research, including screening of the biofilm properties developing under combinations of specified growth parameters such as seeding bacteria populations, growth medium compositions, medium flow rates and dissolved gas levels.

In situ microfluidic SERS assay for monitoring enzymatic breakdown of organophosphates.

In this paper, we report on a method to probe the breakdown of the organophosphate (OP) simulants o,s-diethyl methyl phosphonothioate (OSDMP) and demeton S by the enzyme organophosphorous hydrolase (OPH) in a microfluidic device by surface enhanced Raman spectroscopy (SERS). SERS hotspots were formed on-demand inside the microfluidic device by laser-induced aggregation of injected Ag NPs suspensions. The Ag NP clusters, covering micron-sized areas, were formed within minutes using a conventional confocal Raman laser microscope. These Ag NP clusters were used to enhance the Raman spectra of the thiol products of OP breakdown in the microfluidic device: ethanethiol (EtSH) and (ethylsulfanyl) ethane-1-thiol (2-EET). When the OPH enzyme and its substrates OSDMP and demeton S were introduced, the thiolated breakdown products were generated, resulting in changes in the SERS spectra. With the ability to analyze reaction volumes as low as 20 nL, our approach demonstrates great potential for miniaturization of SERS analytical protocols.

Realization and efficiency evaluation of a micro-photocatalytic cell prototype for real-time blood oxygenation.

A novel, miniaturized, high-efficiency photocatalytic cell, able to work in dynamic conditions, has been designed and validated in this study. Microfluidic channels were molded out of polydimethylsiloxane (PDMS) by means of standard soft lithography techniques, so as to work as photocatalytic cells, where the coupling of anatase titanium dioxide thin films and platinum electrodes, allows an electrically assisted photocatalytic reaction to produce dissolved oxygen gas from the water content of flowing fluid (e.g. blood). The thin films were deposited onto quartz glass substrates at room temperature (300 K) using reactive radio-frequency sputtering with a titanium metal target. The photocatalytic activity was evaluated through reduction rate of methylene blue solution. The results of the current study, as a proof of concept, have shown that the device can generate oxygen at a rate of 4.06 µM O(2)/(cm(2)min), thus extending its possible application range to the full oxygenation of flowing venous blood.

Liquid waveguide-based evanescent wave sensor that uses two light sources with different wavelengths.

We demonstrate a prototypic optofluidic evanescent wave sensor made of poly(dimethylsiloxane) (PDMS) elastomer in which two light sources with different wavelengths are coupled into an optofluidic liquid-core/liquid-cladding (L(2)) waveguide. The exponentially decaying evanescent wave interacts with analyte molecules dissolved in the cladding fluids or products formed by in situ reactions at the core-cladding interface. The analyte molecules exhibit distinctly different light absorbance at the two wavelengths during the light-analyte interaction. Therefore, by using the normalized absorbance calculated from the intensity ratio of the two wavelengths instead of the absolute magnitude of either signal, unwanted effects from omnipresent external noise sources can be reduced. In addition, the differential absorption of the two beams by the analyte solutions can be used to enhance the resolution of sample analysis. The evanescent wave sensor based on a liquid waveguide can also be used for real-time monitoring of chemical reactions, because the core and cladding fluids in the L(2) waveguide are slightly miscible at the core-cladding interface due to the diffusional mixing.

Chemical control of Vorticella bioactuator using microfluidics.

In this report, we demonstrate a microfluidic platform to control the stalk contraction and extension of Vorticella convallaria by changing concentration of Ca2+ with pneumatically-actuated elastomeric microvalves. Habitation, extraction and control of V. convallaria were carried out in a PDMS-based microfluidic device. By treating the cells with the permeant saponin, external actuation of cell-anchoring stalk between an extended and contracted state was achieved by cyclic exposure of the cells to a Ca2+ buffer (10(-6) M) and a rinse buffer containing EGTA as a chelation agent. When solutions were switched, the stalk contracted and extended responding to the ambient Ca2+ concentration change. The length of the stalk changed between 20 and 60 microm, resulting in a working distance of about 40 microm.

Ultrafast high-pressure AC electro-osmotic pumps for portable biomedical microfluidics.

This paper details the development of an integrated AC electro-osmotic (ACEO) microfluidic pump for dilute electrolytes consisting of a long serpentine microchannel lined with three dimensional (3D) stepped electrode arrays. Using low AC voltage (1 V rms, 1 kHz), power (5 mW) and current (4.5 mA) in water, the pump is capable of generating a 1.3 kPa head pressure, a 100-fold increase over prior ACEO pumps, and a 1.3 mm/s effective slip velocity over the electrodes without flow reversal. The integrated ACEO pump can utilize low ionic strength solutions such as distilled water as the working solution to pump physiological strength (100 mM) biological solutions in separate microfluidic devices, with potential applications in portable or implantable biomedical microfluidic devices. As a proof-of-concept experiment, the use of the ACEO pumps for DNA hybridization in a microfluidic microarray is demonstrated.

Dissolution of a liquid microdroplet in a nonideal liquid-liquid mixture far from thermodynamic equilibrium.

A droplet placed in a liquid-liquid solution is expected to grow, or shrink, in time as approximately t;{1/2}. In this Letter, we report experimental evidence that when the composition in the interface is far from thermodynamic equilibrium due to the nonideality of the mixture, a droplet shrinks as approximately t. This scaling is due to the coupling between mass and momentum transfer known as Korteweg forces as a result of which the droplet self-propels around. The consequent hydrodynamic convection greatly enhances the mass transfer between the droplet and the bulk phase. Thus, the combined effect of nonideality and nonequilibrium modifies the dynamical behavior of the dissolving droplet.

Microvalve thickness and topography measurements in microfluidic devices by white-light confocal microscopy.

A microvalve is a key part in a multilayer microfluidic device to control the fluid flow, and its thickness directly determines its performance. In this paper, a three-dimensional measurement technology using a white-light confocal microscope is developed for measuring both the topography and thickness of microvalves. The impact of system parameters and sample parameters on measurement accuracy is discussed in detail, particularly for measurement with a dry objective. With this technique, the microvalve thicknesses before and after bonding were characterized with submicrometer measurement sensitivity and about 1 microm measurement accuracy.

Microfluidic electrospinning of biphasic nanofibers with Janus morphology.

In this paper a method of electrospinning conducting and nonconducting biphasic Janus nanofibers using microfluidic polydimethylsiloxane (PDMS)-based manifolds is described. Key benefits of using microfluidic devices for nanofiber synthesis include rapid prototyping, ease of fabrication, and the ability to spin multiple Janus fibers in parallel through arrays of individual microchannels. Biphasic Janus nanofibers of polyvinylpyrrolidone (PVP)+polypyrrole (PPy)PVP nanofibers with an average diameter of 250 nm were successfully fabricated using elastomeric microfluidic devices. Fiber characterization and confirmation of the Janus morphology was subsequently carried out using a combination of scanning electron microscopy, energy dispersion spectroscopy, and transmission electron microscopy.

Culturing aerobic and anaerobic bacteria and mammalian cells with a microfluidic differential oxygenator.

In this manuscript, we report on the culture of anaerobic and aerobic species within a disposable multilayer polydimethylsiloxane (PDMS) microfluidic device with an integrated differential oxygenator. A gas-filled microchannel network functioning as an oxygen-nitrogen mixer generates differential oxygen concentration. By controlling the relative flow rate of the oxygen and nitrogen input gases, the dissolved oxygen (DO) concentration in proximal microchannels filled with culture media are precisely regulated by molecular diffusion. Sensors consisting of an oxygen-sensitive dye embedded in the fluid channels permit dynamic fluorescence-based monitoring of the DO concentration using low-cost light-emitting diodes. To demonstrate the general utility of the platform for both aerobic and anaerobic culture, three bacteria with differential oxygen requirements (E. coli, A. viscosus, and F. nucleatum), as well as a model mammalian cell line (murine embryonic fibroblast cells (3T3)), were cultured. Growth characteristics of the selected species were analyzed as a function of eight discrete DO concentrations, ranging from 0 ppm (anaerobic) to 42 ppm (fully saturated).

A self-contained microfluidic cell culture system.

Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.

Noninvasive metabolic profiling using microfluidics for analysis of single preimplantation embryos.

Noninvasive analysis of metabolism at the single cell level will have many applications in evaluating cellular physiology. One clinically relevant application would be to determine the metabolic activities of embryos produced through assisted reproduction. There is increasing evidence that embryos with greater developmental capacity have distinct metabolic profiles. One of the standard techniques for evaluating embryonic metabolism has been to evaluate consumption and production of several key energetic substrates (glucose, pyruvate, and lactate) using microfluorometric enzymatic assays. These assays are performed manually using constriction pipets, which greatly limits the utility of this system. Through multilayer soft-lithography, we have designed a microfluidic device that can perform these assays in an automated fashion. Following manual loading of samples and enzyme cocktail reagents, this system performs sample and enzyme cocktail aliquotting, mixing of reagents, data acquisition, and data analysis without operator intervention. Optimization of design and operating regimens has resulted in the ability to perform serial measurements of glucose, pyruvate, and lactate in triplicate with submicroliter sample volumes within 5 min. The current architecture allows for automated analysis of 10 samples and intermittent calibration over a 3 h period. Standard curves generated for each metabolite have correlation coefficients that routinely exceed 0.99. With the use of a standard epifluorescent microscope and CCD camera, linearity is obtained with metabolite concentrations in the low micromolar range (low femtomoles of total analyte). This system is inherently flexible, being easily adapted for any NAD(P)H-based assay and scaled up in terms of sample ports. Open source JAVA-based software allows for simple alterations in routine algorithms. Furthermore, this device can be used as a standalone device in which media samples are loaded or be integrated into microfluidic culture systems for in line, real time metabolic evaluation. With the improved throughput and flexibility of this system, many barriers to evaluating metabolism of embryos and single cells are eliminated. As a proof of principle, metabolic activities of single murine embryos were evaluated using this device.

High throughput microfluidic cell array for cytotoxicity screening.

A microfluidic array with micro cell sieves was designed to fabricate multiple cells array for high-throughput cell cytotoxicity screening. Micro channels in both directions (column and row) were individually addressable by using a set of elastomeric monolithic array valves, enabling parallel loading of various cell lines in one direction and introducing of different toxins in the other direction. Several micro cell sieves were designed in each chamber. Cell number and distribution in chambers can be conveniently controlled by the adjustment of cell sieve number, distribution and size. Fluid transport property and cell loading in chamber were simulated and verified experimentally. The capability of the microfluidic array was demonstrated for seeding multiple mammalian cell lines in chambers and observing the cell viabilities after cells exposed to different toxins with a fluorescence microscope.

Computational and functional evaluation of a microfluidic blood flow device.

The development of microfluidic devices supporting physiological blood flow has the potential to yield biomedical technologies emulating human organ function. However, advances in this area have been constrained by the fact that artificial microchannels constructed for such devices need to achieve maximum chemical diffusion as well as hemocompatibility. To address this issue, we designed an elastomeric microfluidic flow device composed of poly (dimethylsiloxane) to emulate the geometry and flow properties of the pulmonary microcirculation. Our chip design is characterized by high aspect ratio (width > height) channels in an orthogonally interconnected configuration. Finite element simulations of blood flow through the network design chip demonstrated that the apparent pressure drop varied in a linear manner with flow rate. For simulated flow rates <250 mul min, the simulated pressure drop was <2000 Pa, the flow was laminar, and hemolysis was minimal. Hemolysis rate, assayed in terms of [total plasma hemoglobin (TPH) (sample - control)/(TPH control)] during 6 and 12 hour perfusions at 250 mul/min, was <5.0% through the entire period of device perfusion. There was no evidence of microscopic thrombus at any channel segment or junction under these perfusion conditions. We conclude that a microfluidic blood flow device possessing asymmetric and interconnected microchannels exhibits uniform flow properties and preliminary hemocompatibility. Such technology should foster the development of miniature oxygenators and similar biomedical devices requiring both a microscale reaction volume and physiological blood flow.