Rapid, sensitive, and quantitative detection of pathogenic DNA at the point of care through microfluidic electrochemical quantitative loop-mediated isothermal amplification

Single-step DNA detection: a microfluidic electrochemical loop mediated isothermal amplification platform is reported for rapid, sensitive, and quantitative detection of pathogen genomic DNA at the point of care. DNA amplification was electrochemically monitored in real time within a monolithic microfluidic device, thus enabling the detection of as few as 16 copies of Salmonella genomic DNA through a single-step process in less than an hour.     

Single-molecule emulsion PCR in microfluidic droplets

The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.     

Controlled generation of droplets using an electric field in a flow-focusing paper-based device

Droplet-based microfluidics is a modular platform in high-throughput single-cell and small sample analyses. However, this droplet microfluidic system was widely fabricated using soft lithography or glass capillaries, which is expensive and technically demanding for various applications, limiting use in resource-poor settings. Besides, the variation in droplet size is also restricted due to the limitations on the operating forces that the paper-based platform is able to withstand. Herein, we develop a fully integrated paper-based droplet microfluidic platform for conducting droplet generation and cell encapsulation in independent aqueous droplets dispersed in a carrier oil by incorporating electric fields. Through imposing an electric field, the droplet size would decrease with increasing the electric field and smaller droplets can be produced at high applied voltage. The droplet diameter can be adjusted by the ratio of inner and outer flow velocities as well as the applied electric field. We also demonstrated the proof of concept encapsulation application of our paper device by encapsulating yeast cells under an electric field. Using a simple wax printing method, carbon electrodes can be integrated on the paper. The integrated paper-based microfluidic platform can be fabricated easily and conducted outside of centralized laboratories. This microfluidic system shows great potential in drug and cell investigations by encapsulating cells in resource-limited environments.     

Microfluidic DNA amplification—A review

The application of microfluidic devices for DNA amplification has recently been extensively studied. Here, we review the important development of microfluidic polymerase chain reaction (PCR) devices and discuss the underlying physical principles for the optimal design and operation of the device. In particular, we focus on continuous-flow microfluidic PCR on-chip, which can be readily implemented as an integrated function of a micro-total-analysis system. To overcome sample carryover contamination and surface adsorption associated with microfluidic PCR, microdroplet technology has recently been utilized to perform PCR in droplets, which can eliminate the synthesis of short chimeric products, shorten thermal-cycling time, and offers great potential for single DNA molecule and single-cell amplification. The work on chip-based PCR in droplets is highlighted.     

Sequential microfluidic droplet processing for rapid DNA extraction

This work describes a novel droplet-based microfluidic device, which enables sequential droplet processing for rapid DNA extraction. The microdevice consists of a droplet generation unit, two reagent addition units and three droplet splitting units. The loading/washing/elution steps required for DNA extraction were carried out by sequential microfluidic droplet processing. The movement of superparamagnetic beads, which were used as extraction supports, was controlled with magnetic field. The microdevice could generate about 100 droplets per min, and it took about 1 min for each droplet to perform the whole extraction process. The extraction efficiency was measured to be 46% for λ-DNA, and the extracted DNA could be used in subsequent genetic analysis such as PCR, demonstrating the potential of the device for fast DNA extraction.     

Droplet-based microscale colorimetric biosensor for multiplexed DNA analysis via a graphene nanoprobe

The development of simple and inexpensive DNA detection strategy is very significant for droplet-based microfluidic system. Here, a droplet-based biosensor for multiplexed DNA analysis is developed with a common imaging device by using fluorescence-based colorimetric method and a graphene nanoprobe. With the aid of droplet manipulation technique, droplet size adjustment, droplet fusion and droplet trap are realized accurately and precisely. Due to the high quenching efficiency of graphene oxide (GO), in the absence of target DNAs, the droplet containing two single-stranded DNA probes and GO shows dark color, in which the DNA probes are labeled carboxy fluorescein (FAM) and 6-carboxy-X-rhodamine (ROX), respectively. The droplet changes from dark to bright color when the DNA probes form double helix with the specific target DNAs leading to the dyes far away from GO. This colorimetric droplet biosensor exhibits a quantitative capability for simultaneous detection of two different target DNAs with the detection limits of 9.46 and 9.67 × 10⁻⁸ M, respectively. It is also demonstrated that this biosensor platform can become a promising detection tool in high throughput applications with low consumption of reagents. Moreover, the incorporation of graphene nanoprobe and droplet technique can drive the biosensor field one more step to some extent.     

Digital LAMP in a sample self-digitization (SD) chip

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource-limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we accomplished the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings.     

1-Million droplet array with wide-field fluorescence imaging for digital PCR

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.     

Droplet-based microextraction in the aqueous two-phase system

This report is about microfluidic extraction systems based on droplets of aqueous two-phase system. Mass transfer between continuous phase and dispersed droplet is demonstrated by microextraction of ruthenium red in a microfluidic device. Droplets are generated with electrohydrodynamic method in the same device. By comparing brightness in the digital image of a solution with known concentrations of ruthenium red and those of a droplet in the microextraction, ruthenium red concentration was measured along the microextraction channel, resulting in good agreement with a simple diffusion model. The maximum partition coefficient was 9.58 in the experiment with the 70-mm-long-channel microextractor. The method is usable for terminating microextraction by electrohydrodynamic manipulation of droplet movement direction. Droplets of different ruthenium red concentration, 0.12 and 0.24% (w/w) in this experiment, can be moved to desired place of microfluidic system for further reaction through respectively branched outlets. In this study droplet-based microextraction is demonstrated and the mass transport is numerically analyzed by solving the diffusion–dissolution model.     

Spontaneous generation of emulsion droplets by autonomous fluid-pumping using the gas permeability of poly(dimethylsiloxane) (PDMS)

Conventional droplet-based microfluidic systems require expensive, bulky external apparatuses, such as electric power supplies and pressure-driven pumps for fluid transportation. This study demonstrates an alternative way to produce emulsion droplets by autonomous fluid-handling based on the gas permeability of poly(dimethylsiloxane) (PDMS). Furthermore, basic concepts of fluid-handling are expanded to control the direction of the microfluid in the microfluidic device. The alternative pumping energy resulting from the high gas permeability of PDMS is used to generate water-in-oil (W/O) emulsions, which require no additional structures apart from microchannels. We can produce emulsion droplets by simple loading of the oil and aqueous solutions into the inlet reservoirs. During the operation of the microfluidic device, changes in droplet size, volumetric flow rate, and droplet generation frequency were quantitatively analyzed. As a result, we found that changes in the wetting properties of the microchannel greatly influence the volumetric flow rate and droplet generation frequency. This alternative microfluidic approach for preparing emulsion droplets in a simple and efficient manner is designed to improve the availability of emulsion droplets for point of care bioanalytical applications, in situ synthesis of materials, and on-site sample preparation tools.     

Picoliter droplet microfluidic immunosorbent platform for point-of-care diagnostics of tetanus

We have developed a sensitive, specific, rapid and low cost picoliter microsphere-based platform for bioanalyte detection and quantification. In this method, a biological sample, biosensing microspheres, and fluorescently labeled detection (secondary) antibodies are co-encapsulated to capture the analyte (here: human anti-tetanus immunoglobulin G) on the surface of the microsphere in microfluidic pL-sized droplets. The absorption of the analyte and detecting antibodies on the microsphere concentrate the fluorescent signal in correlation with analyte concentration. Using our platform and commercially available antibodies, we were able to quantify anti-tetanus antibodies in human serum. In comparison to standard bulk immunosorbent assays, the microfluidic droplet platform presented here reduces the reagent volume by four orders of magnitude, while fast reagent mixing reduces the detection time from hours to minutes. We consider this platform to be a major leap forward in the miniaturization of immunosorbent assays and to provide a rapid and low cost tool for global point-of-care.

基于微流控双水相液滴流酶促反应的尿素检测

以简单、快速的微流控酶促反应方法实现了尿素浓度的可视化检测。 在微流控双水相液滴流动中,利用脲酶水解尿素生成碳酸铵使液滴内的中性红指示剂变色,并对液滴颜色强度进行分析来确定待测样品中尿素的浓度,检测范围可达到0~50 mg/mL。 双水相体系克服了传统油水分析检测平台生物相容性低的缺陷。 液滴流以较少的试剂消耗、极大的比表面积、微米级的扩散距离大大提高了反应速率,导致了较快的分析检测速度,将检测时间缩短为20 s左右。 为应用化学领域的尿素快速分析检测提供借鉴和参考。     

A microfluidic device for self-synchronised production of droplets

The primary requirement for a mixing operation in droplet-based microfluidic devices is an accurate pairing of droplets of reaction fluids over an extended period of time. In this paper, a novel device for self-synchronous production of droplets has been demonstrated. The device uses a change in impedance across a pair of electrodes introduced due to the passage of a pre-formed droplet to generate a second droplet at a second pair of electrodes. The device was characterised using image analysis. Droplets with a volume of ~23.5 ± 3.1 nl (i.e.~93% of the volume of pre-formed droplets) were produced on applying a voltage of 500 V. The synchronisation efficiency of the device was 83%. As the device enables self-synchronised production of droplets, it has a potential to increase the reliability and robustness of mixing operations in droplet-based microfluidic devices.     

Microfabricated devices for biomolecule encapsulation

Biomolecule encapsulation in droplets is important for miniaturizing biological assays to reduce reagent consumption, cost and time of analysis, and can be most effectively achieved by using microfabricated devices. Microfabricated fluidic devices can generate emulsified drops of uniform size with controlled dimensions and contents. Biological and chemical components such as cells, microgels, beads, hydrogel precursors, polymer initiators, and other droplets can be encapsulated within these drops. Encapsulated emulsions are appealing for a variety of applications since drops can be used as tiny reaction vessels to perform high-throughput reactions at fast rates, consuming minimal sample and solvent amounts due to the small size (micron diameters) of the emulsion drops. Facile mixing and droplet coalescence allow for a diversity of assays to be performed on-chip with tunable parameters. The simplicity of operation and speed of analysis with microencapsulated drops lends itself well to an array of quantitative biomolecular studies such as directed evolution, single-molecule DNA amplification, single-cell encapsulation, high-throughput sequencing, enzyme kinetics, and microfluidic cell culture. This review highlights recent advances in the field of microfabricated encapsulating devices, emphasizing the development of emulsifying encapsulations, device design, and current assays that are performed using encapsulating droplets.     

A homogeneous assay for protein analysis in droplets by fluorescence polarization

We present a novel homogeneous (“mix‐incubate‐read”) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP‐based assay we detect streptavidin concentrations as low as 500 nM and demonstrate that an FP assay allows us to distinguish droplets containing 5 μM rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.     

Integration of field effect transistor-based biosensors with a digital microfluidic device for a lab-on-a-chip application

A new platform for lab-on-a-chip system is suggested that utilizes a biosensor array embedded in a digital microfluidic device. With field effect transistor (FET)-based biosensors embedded in the middle of droplet-driving electrodes, the proposed digital microfluidic device can electrically detect avian influenza antibody (anti-AI) in real time by tracing the drain current of the FET-based biosensor without a labeling process. Digitized transport of a target droplet enclosing anti-AI from an inlet to the embedded sensor is enabled by the actuation of electrowetting-on-dielectrics (EWOD). A reduction of the drain current is observed when the target droplet is merged with a pre-existing droplet on the embedded sensor. This reduction of the drain current is attributed to the specific binding of the antigen and the antibody of the AI. The proposed hybrid device consisting of the FET-based sensor and an EWOD device, built on a coplanar substrate by monolithic integration, is fully compatible with current fabrication technology for control and read-out circuitry. Such a completely electrical manner of inducing the transport of bio-molecules, the detection of bio-molecules, the recording of signals, signal processing, and the data transmission process does not require a pump, a fluidic channel, or a bulky transducer. Thus, the proposed platform can contribute to the construction of an all-in-one chip.     

High-throughput rheology in a microfluidic device

High-throughput rheological measurements in a microfluidic device are demonstrated. A series of microrheology samples are generated as droplets in an immiscible spacer fluid using a microfluidic T-junction. The compositions of the sample droplets are continuously varied over a wide range. Rheology measurements are made in each droplet using multiple particle tracking microrheology. We review critical design and operating parameters, including the droplet size, flow rates and rapid fabrication methods. Validation experiments are performed by measuring the solution viscosity of glycerine and the biopolymer heparin as a function of concentration. Overall, the combination of microrheology with microfluidics maximizes the number of rheological measurements while simultaneously minimizing the sample preparation time and amount of material, and should be particularly suited to the characterization of scarce or expensive materials.     

Fabrication of silver-coated silica microspheres through mussel-inspired surface functionalization

This paper reports a droplet-based microfluidic device composed of patterned co-planar electrodes in an all-in-a-single-plate arrangement and coated with dielectric layers for electrowetting-on-dielectric (EWOD) actuation of discrete droplets. The co-planar arrangement is preferred over conventional two-plate electrowetting devices because it provides simpler manufacturing process, reduced viscous drag, and easier liquid-handling procedures. These advantages lead to more versatile and efficient microfluidic devices capable of generating higher droplet speed and can incorporate various other droplet manipulation functions into the system for biological, sensing, and other microfluidic applications. We have designed, fabricated, and tested the devices using an insulating layer with materials having relatively high dielectric constant (SiO(2)) and compared the results with polymer coatings (Cytop) with low dielectric constant. Results show that the device with high dielectric layer generates more reproducible droplet transfer over a longer distance with a 25% reduction in the actuation voltage with respect to the polymer coatings, leading to more energy efficient microfluidic applications. We can generate droplet speeds as high as 26 cm/s using materials with high dielectric constant such as SiO(2).     

Pneumatic handling of droplets on-demand on a microfluidic device for seamless processing of reaction and electrophoretic separation

Sequential operations of pre-separation reaction process by picoliter droplets and following electrophoretic separation process were realized in a single microfluidic device with pneumatic handling of liquid. The developed device consists of a fluidic chip made of PDMS, an electrode substrate, and a temperature control substrate on which thin film heater/sensor structures are fabricated. Liquid handling, including introduction of liquid samples, droplet generation, and merging of droplets, was implemented by pneumatic manipulation through microcapillary vent structures, allowing air to pass and stop liquid flow. Since the pneumatic manipulations are conducted in a fully automated manner by using a programmable air pressure control system, the user simply has to load liquid samples on each liquid port of the device. Droplets of 420 pL were generated with an accuracy of ± 2 pL by applying droplet generation pressure in the range of 40-100 kPa. As a demonstration, a binding reaction of a 15 mer ssDNA with a peptide nucleic acid oligomer used as an oligoprobe followed by denaturing electrophoresis to discriminate a single-base substitution was performed within 1.5 min. By exploiting the droplet-on-demand capability of the device, the influence of various factors, such as reaction time, mixing ratio and droplet configurations on the ssDNA-peptide nucleic acid binding reaction in the droplet-based process, was studied toward realization of a rapid detection method to discriminate rapid single-base substitution.     

Microfluidic platform for on-demand generation of spatially indexed combinatorial droplets

We propose a highly versatile and programmable nanolitre droplet-based platform that accepts an unlimited number of sample plugs from a multi-well plate, performs digitization of these sample plugs into smaller daughter droplets and subsequent synchronization-free, robust injection of multiple reagents into the sample daughter droplets on-demand. This platform combines excellent control of valve-based microfluidics with the high-throughput capability of droplet microfluidics. We demonstrate the functioning of a proof-of-concept device which generates combinatorial mixture droplets from a linear array of sample plugs and four different reagents, using food dyes to mimic samples and reagents. Generation of a one dimensional array of the combinatorial mixture droplets on the device leads to automatic spatial indexing of these droplets, precluding the need to include a barcode in each droplet to identify its contents. We expect this platform to further expand the range of applications of droplet microfluidics to include applications requiring a high degree of multiplexing as well as high throughput analysis of multiple samples.