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.
デジタルアッセイとはサンプルを多数の微細なコンテナに分配し、各パーティションに個々の数（0、1、2、3…）の生物学的実体が含まれるようにする方法である。生物学者用ツールキットの有力な手段であるデジタルアッセイは、核酸の定量、種々のタンパク質とそれらの酵素活性の測定、および単細胞由来の遺伝子型と表現型の探索の精度を新たなレベルに引き上げる。本レビューのパート1では始めに、パーティショニングのベネフィットおよびエラーの発生源を含めたPoisson分布の統計処理について述べる。次に核酸を定量するデジタルPCR（dPCR）に注目する。サンプルを物理的に隔離されたチャンバー（cdPCR）またはエマルジョン化したドロップレット（ddPCR）内に分配する5種類の市販ツールについて述べる。dPCRの長所（絶対定量、高精度、希少ターゲットまたは変異ターゲットの検出力）を、従来のPCR、すなわちリアルタイム定量PCRの長所（ダイナミックレンジ、多サンプル量、およびハイスループット）と比較する。最後に、将来有望ないくつかのdPCRの応用について述べる、例えば、コピー数多型の解析、循環腫瘍DNAおよびウイルス負荷の定量、逆転写反応dPCRによるRNA/miRNAの定量、および次世代シーケンシングのためのライブラリの作成などである。本レビューの目的は、デジタルアッセイを自身のワークフローに採用することに関心のある科学者に、幅広い展望を示すことである。パート2ではタンパク質と細胞のデジタルアッセイに注目する。
数字测定表示将样本分别装入众多小容器中，以便每个分区包含不同数量的生物实体（0、1、2、3……）。作为生物学家工具包中一项功能出色的技术，数字测定可以全面提升核酸定量、蛋白质及其酶活性测量以及单细胞基因型和显性检测的精度。本文第一部分将介绍分区的优势和泊松统计，包括误差源。第二部分将关注用于定量核酸的数字PCR（dPCR）。我们将讨论5种用于将样本分割为物理隔离室（cdPCR）或滴乳液（ddPCR）的仪器，并就dPCR（绝对定量、精度、能够探测罕见或突变目标）与之前采用的实时定量PCR（动态范围、更大的采样量和更高的效率）的优劣进行比较。最后，我们将介绍dPCR的数种潜在应用，包括拷贝数变异、定量循环肿瘤DNA和病毒量、应用逆转录dPCR的RNA/miRNA定量以及制备库以用于新一代排序。本文旨在为有意引入数字测定的科学家提供全面的介绍。第二部分关注数字蛋白质和细胞测定。
数字测定表示将样本分别装入众多小容器中，以便每个分区包含不同数量的生物实体（0、1、2、3……）。作为生物学家工具包中一项功能出色的技术，数字测定可以全面提升核酸定量、蛋白质及其酶活性测量以及单细胞基因型和显性检测的精度。本文第一部分将介绍分区的优势和泊松统计，包括误差源。第二部分将关注用于定量核酸的数字PCR（dPCR）。我们将讨论5种用于将样本分割为物理隔离室（cdPCR）或滴乳液（ddPCR）的仪器，并就dPCR（绝对定量、精度、能够探测罕见或突变目标）与之前采用的实时定量PCR（动态范围、更大的采样量和更高的效率）的优劣进行比较。最后，我们将介绍dPCR的数种潜在应用，包括拷贝数变异、定量循环肿瘤DNA和病毒量、应用逆转录dPCR的RNA/miRNA定量以及制备库以用于新一代排序。本文旨在为有意引入数字测定的科学家提供全面的介绍。第二部分关注数字蛋白质和细胞测定。
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.
Sensitive, inline detection of proteins is required for post-chromatographic analyses in proteomics, cell-based assays, and drug discovery workflows. Among the common inline methods, post-column ...derivatization requires chemical labels, while label-free methods are either expensive (mass spectrometry) or have limited sensitivity at small length scales (UV–Vis). This paper presents a label-free detection technique based on the concept that dissolved proteins can function as surfactants and decrease the dynamic interfacial tension (IFT) of an immiscible (water–oil) interface. Existing methods for measuring IFT, such as axisymmetric drop shape analysis (ADSA), operate in batch mode and are not suitable for continuous detection. Here we show that a microfluidic flow-focusing droplet generator operating at a frequency of > 100 Hz can track IFT changes continuously, with high temporal resolution and small detection volumes. Variations in protein concentration alter the size and shape of the drops/plugs formed, and these changes can be quantified in time using a high-speed camera and in-house image processing software. Moreover, the continuously refreshing interface alleviates issues related to surface aging. Two applications are demonstrated: (1) direct injection of a single protein into a microfluidic chip. (2) post-column detection of protein mixtures separated by high performance size exclusion chromatography (SEC HPLC). Of interest, the dynamic range of protein (bovine serum albumin, BSA) was 50 -10
4
μg/ml without using HPLC unit. The lowest limit of detection without HPLC unit was ~ 1 μg/ml of thyroglobulin protein in a 1 nl droplet, which equates to 1 fg of total protein. When used as a detector, the aforementioned detection method offered a sensitivity of six orders of magnitude higher than conventional UV–VIS detectors.
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.
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•Precise control of gene expression is essential to synthetic biology.•We find that a light-responsive, two-component system acts as a low-pass filter.•PWM can be used to transform digital signals to analog gene expression outputs.•PWM enables precise programming of expression dynamics.•This strategy is further validated by controlling an essential metabolic pathway.
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.
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.
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.
A common issue in biomicrofluidic systems is that fluidic channels may become contaminated when ampiphilic molecules adsorb to the hydrophobic channel walls. Desorption rates are often measured using ...optical methods, many of which require a chromophore or a fluorophore label. This paper describes label-free desorption measurements using a drop frequency sensor (DFS), a microfluidic sensor reported recently by our group. The DFS is based on a surfactant retardation effect which measures the drag of surface-active agents on microdroplets generated in a tee junction. We measure desorption curves of Tween, a small molecule surfactant, from the walls of a polydimethylsiloxane channels. Typical desorption times increase with Tween concentration, ranging from 45 to 324 seconds at Tween concentrations between 10 and 1000 ppm. The limit of detection is 10 ppm.
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 (
θ
), 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
θ
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
θ
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
θ
, 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.
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