Rapid Micromolding of Sub-100 µm Microfluidic Channels Using an 8K Stereolithographic Resin 3D Printer.

Engineering microfluidic devices relies on the ability to manufacture sub-100 micrometer fluidic channels. Conventional lithographic methods provide high resolution but require costly exposure tools and outsourcing of masks, which extends the turnaround time to several days. The desire to accelerate design/test cycles has motivated the rapid prototyping of microfluidic channels; however, many of these methods (e.g., laser cutters, craft cutters, fused deposition modeling) have feature sizes of several hundred microns or more. In this paper, we describe a 1-day process for fabricating sub-100 µm channels, leveraging a low-cost (USD 600) 8K digital light projection (DLP) 3D resin printer. The soft lithography process includes mold printing, post-treatment, and casting polydimethylsiloxane (PDMS) elastomer. The process can produce microchannels with 44 µm lateral resolution and 25 µm height, posts as small as 400 µm, aspect ratio up to 7, structures with varying z-height, integrated reservoirs for fluidic connections, and a built-in tray for casting. We discuss strategies to obtain reliable structures, prevent mold warpage, facilitate curing and removal of PDMS during molding, and recycle the solvents used in the process. To our knowledge, this is the first low-cost 3D printer that prints extruded structures that can mold sub-100 µm channels, providing a balance between resolution, turnaround time, and cost (~USD 5 for a 2 × 5 × 0.5 cm3 chip) that will be attractive for many microfluidics labs.

Digital Assays Part II: Digital Protein and Cell Assays.

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

Digital Assays Part I: Partitioning Statistics and Digital PCR.

A digital assay is one in which the sample is partitioned into many small containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, …). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotypes and phenotypes. Part I of this review begins with the benefits and Poisson statistics of partitioning, including sources of error. The remainder focuses on digital PCR (dPCR) for quantification of nucleic acids. We discuss five commercial instruments that partition samples into physically isolated chambers (cdPCR) or droplet emulsions (ddPCR). We compare the strengths of dPCR (absolute quantitation, precision, and ability to detect rare or mutant targets) with those of its predecessor, quantitative real-time PCR (dynamic range, larger sample volumes, and throughput). Lastly, we describe several promising applications of dPCR, including copy number variation, quantitation of circulating tumor DNA and viral load, RNA/miRNA quantitation with reverse transcription dPCR, and library preparation for next-generation sequencing. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows. Part II focuses on digital protein and cell assays.

Programming microbes using pulse width modulation of optical signals.

Cells transmit and receive information via signalling pathways. A number of studies have revealed that information is encoded in the temporal dynamics of these pathways and has highlighted how pathway architecture can influence the propagation of signals in time and space. The functional properties of pathway architecture can also be exploited by synthetic biologists to enable precise control of cellular physiology. Here, we characterised the response of a bacterial light-responsive, two-component system to oscillating signals of varying frequencies. We found that the system acted as a low-pass filter, able to respond to low-frequency oscillations and unable to respond to high-frequency oscillations. We then demonstrate that the low-pass filtering behavior can be exploited to enable precise control of gene expression using a strategy termed pulse width modulation (PWM). PWM is a common strategy used in electronics for information encoding that converts a series of digital input signals to an analog response. We further show how the PWM strategy extends the utility of bacterial optogenetic control, allowing the fine-tuning of expression levels, programming of temporal dynamics, and control of microbial physiology via manipulation of a metabolic enzyme.

Droplet morphometry and velocimetry (DMV): a video processing software for time-resolved, label-free tracking of droplet parameters.

Emerging assays in droplet microfluidics require the measurement of parameters such as drop size, velocity, trajectory, shape deformation, fluorescence intensity, and others. While micro particle image velocimetry (µPIV) and related techniques are suitable for measuring flow using tracer particles, no tool exists for tracking droplets at the granularity of a single entity. This paper presents droplet morphometry and velocimetry (DMV), a digital video processing software for time-resolved droplet analysis. Droplets are identified through a series of image processing steps which operate on transparent, translucent, fluorescent, or opaque droplets. The steps include background image generation, background subtraction, edge detection, small object removal, morphological close and fill, and shape discrimination. A frame correlation step then links droplets spanning multiple frames via a nearest neighbor search with user-defined matching criteria. Each step can be individually tuned for maximum compatibility. For each droplet found, DMV provides a time-history of 20 different parameters, including trajectory, velocity, area, dimensions, shape deformation, orientation, nearest neighbour spacing, and pixel statistics. The data can be reported via scatter plots, histograms, and tables at the granularity of individual droplets or by statistics accrued over the population. We present several case studies from industry and academic labs, including the measurement of 1) size distributions and flow perturbations in a drop generator, 2) size distributions and mixing rates in drop splitting/merging devices, 3) efficiency of single cell encapsulation devices, 4) position tracking in electrowetting operations, 5) chemical concentrations in a serial drop dilutor, 6) drop sorting efficiency of a tensiophoresis device, 7) plug length and orientation of nonspherical plugs in a serpentine channel, and 8) high throughput tracking of >250 drops in a reinjection system. Performance metrics show that highest accuracy and precision is obtained when the video resolution is >300 pixels per drop. Analysis time increases proportionally with video resolution. The current version of the software provides throughputs of 2-30 fps, suggesting the potential for real time analysis.

Optical microplates for high-throughput screening of photosynthesis in lipid-producing algae.

It is well known that biological systems respond to chemical signals as well as physical stimuli. The workhorses of high throughput screening, microplates and pipetting robots, are well suited for screening chemical stimuli; however, there are fewer options for screening physical stimuli, particularly those which involve temporal patterns. This paper presents an optical microplate for photonic high-throughput screening. The system provides addressable intensity and temporal control of LED light emission in each well, and operates on standard black-wall clear-bottom 96-well microplates, which prevent light spillover. Light intensity can be controlled to 7-bit resolution (128 levels), with a maximum intensity of 120 mE cm(-2). The temporal resolution, useful for studying dynamics of light-driven bioprocesses, can be as low as 10 µs. The microplate is used for high-throughput studies of light-dependent growth rates and photosynthetic efficiency in the model organism Dunaliella tertiolecta, a lipid-producing algae of interest in 2(nd) generation biofuels. By conducting 96 experiments in parallel, photoirradiance studies, which would require 2 years using conventional tools, can be completed in <2 weeks. In a 12 day culture, algal growth rates increase with total photon flux, as expected. Interestingly, the lipid production efficiency, defined as lipid production per unit photon flux per capita, increases nearly 5 fold at low light intensity (constant light) and at low duty cycle (pulsed light). High throughput protocols enabled by this system are conducive to systematic studies and discovery in the fields of photobiology and photochemistry.

Field-free particle focusing in microfluidic plugs.

Particle concentration is a key unit operation in biochemical assays. Although there are many techniques for particle concentration in continuous-phase microfluidics, relatively few are available in multiphase (plug-based) microfluidics. Existing approaches generally require external electric or magnetic fields together with charged or magnetized particles. This paper reports a passive technique for particle concentration in water-in-oil plugs which relies on the interaction between particle sedimentation and the recirculating vortices inherent to plug flow in a cylindrical capillary. This interaction can be quantified using the Shields parameter ([Formula: see text]), a dimensionless ratio of a particle's drag force to its gravitational force, which scales with plug velocity. Three regimes of particle behavior are identified. When [Formula: see text] is less than the movement threshold (region I), particles sediment to the bottom of the plug where the internal vortices subsequently concentrate the particles at the rear of the plug. We demonstrate highly efficient concentration (∼100%) of 38 µm glass beads in 500 µm diameter plugs traveling at velocities up to 5 mm/s. As [Formula: see text] is increased beyond the movement threshold (region II), particles are suspended in well-defined circulation zones which begin at the rear of the plug. The length of the zone scales linearly with plug velocity, and at sufficiently large [Formula: see text], it spans the length of the plug (region III). A second effect, attributed to the co-rotating vortices at the rear cap, causes particle aggregation in the cap, regardless of flow velocity. Region I is useful for concentrating/collecting particles, while the latter two are useful for mixing the beads with the solution. Therefore, the two key steps of a bead-based assay, concentration and resuspension, can be achieved simply by changing the plug velocity. By exploiting an interaction of sedimentation and recirculation unique to multiphase flow, this simple technique achieves particle concentration without on-chip components, and could therefore be applied to a range of heterogeneous screening assays in discrete nl plugs.

Plug-and-play, single-chip photoplethysmography.

Remote patient monitoring (RPM) relies on low-cost, low-power, wearable sensors for continuous physiological assessment. Photoplethysmographic (PPG) sensors generally require >10 components, occupy an area >300 mm(2), consume >10 mW power, and cost >$20 USD. Although the principle of PPG sensing is straightforward, in practice, a robust implementation requires a careful design including optical alignment, analog circuits, ambient light cancellation, and power management. This paper reports the first use of digital optical proximity sensors (OPS) for "plug-and-play" PPG. OPS have traditionally been used for distance sensing in smartphones and factory automation. Here we show that a digital OPS can perform PPG functions in a single 4×4 mm package which also provides a direct digital interface to a microcontroller. By exploiting its key features, a digital OPS can provide substantial performance advantages over existing state-of-the-art PPGs, including: i) 10X lower power consumption (200 µW) due to pulse operation; ii) high signal to noise ratio (>90), as a result of built-in optical barriers, filters, and ambient light cancellation; iii) 10X lower cost ($2 USD); and iv) 12X smaller area. We show single wavelength PPG measurements in multiple anatomical locations, including fingertips and earlobes. The results suggest that a digital OPS can provide an elegant solution for battery-powered, wearable physiological monitors. To the authors' knowledge, this is the smallest and lowest power PPG sensor reported to date.

Capillary fractionation of HPLC substrates by a microfluidic droplet generator for high throughput analysis.

Biochemical samples are complex mixtures containing 1000's of components which often must be fractionated prior to analysis. Conventional fraction collectors, which can only accommodate 10's of fractions, are not well suited for high throughput analysis. This paper describes microfractionation in droplets (µFD), a scalable microfluidic technique for generating thousands of fractions. A drop generator, placed downstream from a high performance liquid chromatography (HPLC) column, encapsulates the separated components into a serial array of monodisperse droplets. The droplets can be stored in a capillary or immediately used in subsequent assays. Using µFD, a mixture of 3 dyes separated in a C18 column was fractionated into 2,160 droplets in <6 min. The volume and frequency of the droplet fractions are governed by the capillary number (Ca), which depends on the viscosity of the carrier fluid, flow rate, and interfacial tension. With HPLC-compatible flow rates of 0.38-0.7 mL/min, in a 1.5 mm Teflon capillary, fractions contain volumes of 1-6 µL and are generated at 2-10 drops/s. Droplet fractions can be mixed with a subsequent reagent using a downstream tee junction. In theory, µFD can be coupled to a wide variety of separation processes, enabling high throughput fractionation and screening of complex mixtures in µL to sub-nL volumes.

Optical microplates for photonic high throughput screening of algal photosynthesis and biofuel production.

Biological systems respond not only to chemical stimuli (drugs, proteins) but also to physical stimuli (light, heat, stress). Though there are many high throughput tools for screening chemical stimuli, no such tool exists for screening of physical stimuli. This paper presents a novel instrument for photonic high throughput screening of photosynthesis, a light-driven bioprocess. The optical microplate has a footprint identical to a standard 96 well plate, and it provides temporal and intensity control of light in each individual well. Intensity control provides 128 dimming levels (7-bit resolution), with maximum intensity 120 mE/cm(2). Temporal modulation, used for studying dynamics and regulation of photosynthesis, can be as low as 10 µs. We used photonic screening for high throughput studies of algal growth rates and photosynthetic efficiency, using the model organism Dunaliella tertiolecta, a lipid producing algae of interest in biofuel production. Due to the ability to conduct 96 studies in parallel, experiments that would require 2 years using conventional tools can be completed in 1 week. This instrument opens up novel high throughput protocols for photobiology and the growing field of phenomics.

Shape dependent Laplace vortices in deformed liquid-liquid slug flow.

Understanding the hydrodynamics of liquid-liquid slug flow is important in the emerging field of plug-based microfluidics; however, the subtle aspects of the vortex geometry are still not comprehensively understood. This paper discusses the hydrodynamics of deformation dependent vortices that develop inside a water-in-oil slug as it flows through a channel. In contrast to prior studies, our simulations and experiments on slug flow reveal multiple vortices inside the moving slug, caused by the deformation of the hemispherical caps by Laplace pressures. These vortices appear in the front and rear of the plug at capillary number between 10(-4) and 10(-2). A theoretical and simulation model shows the cause of asymmetry in slug deformation and the resulting vortices. Understanding the relevant parameters helps in optimizing slug flow for mixing and particle manipulation, which is important for plug-based microreactors and bead based assays.

High speed low noise multiplexed three color absorbance photometry.

Multispectral photometry is often required to distinguish samples in flow injection analysis and flow cytometry; however, the cost of multiple light detectors, filters, and optical paths contribute to the high cost of multicolor and spectral detection systems. This paper describes frequency division multiplexing (FDM), a simple approach for performing multi-wavelength absorbance photometry with a single light detector and a single interrogation window. In previous efforts, modulation frequencies were <10 KHz, resulting in a detector bandwidth of <20 Hz. This paper presents a high frequency FDM circuit which can increase the oscillation frequencies to several 100 KHz, improving the detection bandwidth by a factor of 10 while still maintaining low cost. Light from 3 different LED sources are encoded into unique frequency channels, passed through the detection cell, and later demodulated using phase-sensitive electronics. Electronic multiplexing couples all light sources into a single optical train without spectral filters. Theory and high frequency considerations are demonstrated. Simultaneous three color absorbance detection is demonstrated in solutions and in flowing droplet microreactors. This technique can potentially reduce the cost of multicolor photometry by replacing expensive optical components with low-cost electronics.

A modular approach for the generation, storage, mixing, and detection of droplet libraries for high throughput screening.

The desire to make microfluidic technology more accessible to the biological research community has led to the notion of "modular microfluidics", where users can build a fluidic system using a toolkit of building blocks. This paper applies a modular approach for performing droplet-based screening, including the four integral steps of library generation, storage, mixing, and optical interrogation. Commercially available cross-junctions are used for drop generation, flexible capillary tubing for storage, and tee-junctions for serial mixing. Optical interrogation of the drops is achieved using fiber-optic detection modules which can be incorporated inline at one or more points in the system. Modularity enables the user to hand-assemble systems for functional assays or applications. Three examples are shown: (1) a "mix and read" assay commonly used in high throughput screening (HTS); (2) generation of chemically distinct droplets using microfractionation in droplets (microFD); and (3) in situ encapsulation and culture of eukaryotes. Using components with IDs ranging from 150 microm to 1.5 mm, this approach can accommodate drop assays with volumes ranging from 2 nL to 2 microL, and storage densities ranging from 300 to 3000 drops per metre tubing. Generation rates are up to 200 drops per second and merging rates are up to 10 drops per second. The impact of length scale, carrier fluid viscosity, and flow rates on system performance is considered theoretically and illustratively using 2D CFD simulations. Due to its flexibility, the widespread availability of components, and some favorable material properties compared to PDMS, this approach can be a useful part of a researcher's toolkit for prototyping droplet-based assays.

Microfluidic encapsulation of cells in alginate capsules for high throughput screening.

Microdroplet systems can drastically reduce costs and increase throughput in high throughput screening (HTS) assays. While droplets are well suited for biomolecular screening, cell-based screens are more problematic because eukaryotes typically require attachment to solid supports to maintain viability and function. This paper describes an economical, off-the-shelf microfluidic system which encapsulates eukaryotic cells in gelatinous alginate capsules for the purpose of HTS. The flow-through system consists of i) a cross junction, which forms monodisperse droplets of alginate and cell suspension in an immiscible carrier fluid, followed by ii) a T junction which delivers BaCl(2) to crosslink and solidify each droplet. With an appropriate carrier fluid, the system is self-synchronized and can produce cell-alginate-BaCl(2) capsules with virtually 100% reliability. Droplet volumes and frequency are determined by flow rates and the diameter of the cross junction. The present implementation, which utilizes 1.5 mm Teflon tubing and plastic junctions, can generate 0.4-1.4 microL droplets at frequencies >10 droplets/sec. Cell viability is >80% at 4 hours post-encapsulation. With low recurring cost (