Aqueous two-phase extraction for bovine serum albumin (BSA) with co-laminar flow in a simple coaxial capillary microfluidic device

Microfluidic extraction based on a co-laminar flow of aqueous two-phase system is used to separate bovine serum albumin (BSA). Mass transfer between the continuous two-phase flows is demonstrated by the extraction of BSA in a microfluidic device. The protein concentrations of the BSA samples were determined using the Bradford method. Polyethylene glycol 4000 and ammonium sulfate ((NH₄)₂SO₄) served as model aqueous two-phase solutions. The appropriate flow rates of the aqueous two phases were thus determined. We can flexibly control the mass transfer area and time by simply adjusting the flow rate. It takes only 3.6 s for three extraction cycles in a coaxial microfluidic device to achieve a BSA recovery yield of 71.1 %, which is superior to the traditional beaker aqueous two-phase extraction process. In this study, co-laminar flow-based continuous microextraction is demonstrated and its mass transfer is analyzed by solving the diffusion model, based on a large specific interfacial area and surface renewal.     

Automated genomic and proteomic applications on the Biomek NX laboratory automation workstation

This technical paper describes the utilization of a new automated liquid handler from Beckman Coulter, Inc., the Biomek^® NX Laboratory Automation Workstation, for genomic and proteomic applications. For genomic applications, methodology for plasmid DNA purification using Promega Wizard^® SV 96 reagents was developed for the Biomek NX. A single plate of bacterial pellets can be processed to purified plasmid DNA without user interaction after initial setup. DNA quantity and quality were assessed by spectrophotometric analysis, restriction digestion, PCR (The PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman La Roche, Ltd.), and capillary sequencing. Additionally, the plasmid preparation method was used to purify plasmid DNA from bacterial clones isolated in a bacterial two-hybrid screening procedure. In this case, the system quickly and efficiently prepared clones for rapid identification of target sequences. For proteomic applications, His-tag proteins were purified from bacterial cultures in a 96-well plate format. Following purification, a Bradford assay was used to determine the quantitative yields of the His-tag protein products in each of the aliquots from the purified samples. The AD 340 Automated Labware Positioner (ALP), an integrated absorbance reader, was used for absorbance measurements in the Bradford assay. Given the placement of this ALP on the deck of the Biomek NX, the entire process of protein purification and quantitation was performed in a complete walk-away automated format. Results obtained when purifying proteins, from both uninduced and induced bacterial cultures, on the worksurface of the Biomek NX will be described.     

Automated digital microfluidic sample preparation for next-generation DNA sequencing

Next-generation sequencing (NGS) technology is a promising tool for identifying and characterizing unknown pathogens, but its usefulness in time-critical biodefense and public health applications is currently limited by the lack of fast, efficient, and reliable automated DNA sample preparation methods. To address this limitation, we are developing a digital microfluidic (DMF) platform to function as a fluid distribution hub, enabling the integration of multiple subsystem modules into an automated NGS library sample preparation system. A novel capillary interface enables highly repeatable transfer of liquid between the DMF device and the external fluidic modules, allowing both continuous-flow and droplet-based sample manipulations to be performed in one integrated system. Here, we highlight the utility of the DMF hub platform and capillary interface for automating two key operations in the NGS sample preparation workflow. Using an in-line contactless conductivity detector in conjunction with the capillary interface, we demonstrate closed-loop automated fraction collection of target analytes from a continuous-flow sample stream into droplets on the DMF device. Buffer exchange and sample cleanup, the most repeated steps in NGS library preparation, are also demonstrated on the DMF platform using a magnetic bead assay and achieving an average DNA recovery efficiency of 80%±4.8%.     

Two phase micromixing and analysis using electrohydrodynamic instabilities

Organic–aqueous liquid (phenol) extraction is one of many standard techniques to efficiently purify DNA directly from cells. Effective mixing of the two fluid phases increases the surface area over which biological component partitioning may occur. In this work, two phase mixing has been demonstrated in a three inlet microfluidic device geometry. Mixing between the two phases has been achieved by producing an electrohydrodynamic instability at the liquid–liquid interface between the two phases. The initial instability is modeled by considering the small signal linearized analysis for interfacial stresses from both fluid and electrical stress tensors for both inviscid and viscous models. These models predict the onset of instability and the stability criteria over a range of unstable wavenumbers of the mixing process. These models may be applied to relevant microscale geometries, where the unstable wavenumbers and fastest growth wavenumber are determined. At an applied electric field of ∼8.0×10⁵ V/m an instability is experimentally observed by labeling the organic phase with a fluorescent dye and visualizing interfacial perturbations by microscopy. Increasing the electric field increases the instability growth rate and results in an increase of the level of mixing. These results show an increase in conductive fluid entrainment into the nonconducting fluid core measured as a percentage of area of entrainment into the fluorescently labeled organic phase. The entrainment area is seen to increase from 1.9 to 28.6% as the applied field is increased from 8.0×10⁵ to 9.0×10⁵ V/m. The mixing images are converted into a power spectrum using a fast Hartley transform and the band of unstable wavenumbers of the mixing process are determined. From these results, the theoretical field strengths required to produce these unstable wavenumbers are calculated using the theoretical model, determining the maximum field strength required to excite the largest measured unstable wavenumber. At lower field strengths tested, the theoretically predicted maximum electric field and fastest growth wavenumber compare favorably with the initially applied field and measured fastest growth wavenumber whereas at higher field strengths the theoretical field is much larger than the initially applied field. This is attributed to the larger level of mixing and the ability of the instability to grow beyond the linear range and the field increases as the mixing process occurs due to entrainment of highly conductive fluid decreasing the effective dielectric spacing so that the linearized models underpredict the instability growth rates and interfacial perturbations.     

A two-stage electrophoretic microfluidic device for nucleic acid collection and enrichment

Sample purification and enrichment is an important and usually time-consuming step for on-chip nucleic acid detection and analysis. This paper presents an electrophoretic DNA focusing method in microfluidic devices to enrich nucleic acid concentration by around 2700-fold. The electrical waveforms applied to five individual electrodes are such designed that DNAs move successively to the collection electrodes at high speed, while the interferences from bubbles due to electrohydrolysis are minimized. In a spiral channel with a total length of 48 cm, 1 ml DNA sample is purified and enriched by 57 times at a flow rate of 30 μl/min at first. The captured DNAs are then released and transported to the second microfluidic chamber where DNAs are collected and concentrated by 49 times. Thus, in about 40 min, the two-stage device can extract DNAs from 1 ml sample volume and enrich its concentration by 2790-fold. A trade-off exists between the process throughput and the DNA collection efficiency. A DNA capture efficiency of 99.7 % is reached when the flow rate is 1 μl/min, and the maximum DNA capture throughput is achieved at a flow rate of 30 μl/min. As a platform technology, the device can be integrated into bio-sensing and genetic analysis assays for DNA extraction and pre-concentration.     

A magnetic bead-based DNA extraction and purification microfluidic device

This article introduces a novel magnetic bead-based DNA extraction and purification device using active magnetic mixing approach. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in cell lysis buffer in a circular chamber that is sandwiched between two external magnetic coils. Non-uniform nature of magnetic field causes temporal and spatial distribution of beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom magnetic coil. DNA molecules are extracted from magnetic beads by washing and re-suspension processes. In this study, a circular PMMA microchamber, 25 μL in volume, 500 μm in depth and 8 mm in diameter was fabricated to purify DNA from spiked bacterial cell cultures into the whole blood sample using Promega Magazorb DNA extraction kit. The lysis efficiency was evaluated using a panel of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial cells cultures into the blood sample to achieve approximately 100,000 copy levels inside the chip. Manufacturer’s standard extraction protocol was modified to a more simplified process suitable for chip-based extraction. The lysis step was performed using 5 min incubation at 56 °C followed by 5 min incubation at room temperature for binding process. Temperature rise was generated and maintained by the same external magnetic coils used for active mixing. The yield/purity and recovery levels of the extracted DNA were evaluated using quantitative UV spectrophotometer and real-time PCR assay, respectively. Real-time PCR results indicated efficient chip-based bacterial DNA extraction using modified extraction protocol comparable to the standard bench-top extraction process.     

工业有机污染物治理过程的多目标优化控制EI北大核心CSCD

工业有机污染物具有多相态共存、治理工艺复杂、治理成本高等特点.因此针对有机污染物的临氧裂解/催化氧化治理工艺,研究了一种基于改进的NSGA–Ⅱ的优化控制方法.首先,根据治理过程的工艺要求和设备操作参数,使用Aspen Plus建立了稳态模拟系统,并对关键控制参数进行了灵敏度分析.然后以最低的总能耗和最大的废水治理量为优化目标,使用改进的NSGA–Ⅱ优化处理过程的控制参数,获得一组帕累托最优解.最后,基于能量平衡设计了厂级有机污染物综合处理的能量自平衡控制方案,并进行动态模拟和实验装置测试.模拟和实验结果表明,该系统具有良好的动态响应性能,所设计的实验装置稳定运行状态下能耗低,净化效率高.     

Silicon–glass instrumented solid-phase extraction–zone electrophoresis microchip with thin amorphous silicon film electrodes: performance in immunoaffinity analysis

Silicon–glass microchips were designed and fabricated for on-chip solid phase extraction (SPE) and zone electrophoresis studies. The solvent channels for extraction and the separation channels for analyses were fabricated sequentially on the silicon device. Electrical contacts were integrated in a fused silica glass lid. Amorphous silicon thin film electrodes were fabricated for high voltage and conductivity detection. A chip installation rack with electrical and fluidic contacts was constructed to facilitate the experiments. Simulation was used to elucidate both the liquid flow and the electric field distribution. The operational performance of the microchips was demonstrated by using a fluorescein isothiocyanate (FITC)-labelled testosterone derivative as the model analyte and fluorescein as both the negative control and the calibration compounds. In SPE an immunosorbent, based on recombinant anti-testosterone Fab-fragments, was immobilized to activated Sepharose gel. Simultaneous monitoring of the movement of FITC-testosterone from SPE cavity through the channel to the detection point was performed with a laser-induced fluorescence detector. The observed limit of detection for FITC-testosterone was 2 μM.     

Sample preparation of chemical warfare agent simulants on a digital microfluidic (DMF) device using magnetic bead-based solid-phase extraction

In the present study, the purification and extraction of five chemical warfare agent (CWA) simulants, dimethyl methyl phosphonate, di(propylene glycol) methyl ether, methyl salicylate, triethyl phosphate, and diethyl phthalate, on a digital microfluidic (DMF) device were demonstrated. The DMF on-chip purification and extraction was performed using a magnetic bead (MB)-based diol solid-phase extraction procedure. The extracted CWA simulants were detected using both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and gas chromatography-mass spectrometry (GC-MS). The method detection limits of the DMF-MS approach using diol-MBs were also determined. In particular, for the DMF-GC-MS, the quantitative analysis ability was validated by determining accuracy, precision, calibration curve performance, and recovery efficiency. This study clearly shows that the CWA analyses can be automated on the DMF-MS platform, thereby minimizing human involvement.     

Precision moulding of biomimetic disposable chips for droplet-based applications

In this study, we present a biomimetic approach to improve the stability and reproducibility of droplet generation processes and to reduce the adhesion of aqueous droplets to channel surfaces of microfluidic polymer chips. The hierarchical structure of the lotus leaf was used as a template for a partial laser structuring of the moulds that were used for casting the polymer chips. The hydrophobic wax layer of the lotus leaf was technologically replicated by coating the polymer chips using a plasma deposition process. The resulting microfluidic polymer chip surfaces reveal a topography and a surface free energy similar to those of the lotus leaf. Subsequent droplet-based microfluidic experiments were performed using a 2D flow focussing set-up. Droplets from both, serum-supplemented cell culture medium and anticoagulated human whole blood, could be generated stably and reproducibly using a fluorocarbon as continuous phase. The presented results illustrate the application potential of the lotus-leaf-like polymer chips in life sciences, e.g. in the field of personalised medicine.     

On-chip polymerase chain reaction microdevice employing a magnetic droplet-manipulation system

An on-chip polymerase chain reaction (PCR) device employing a magnetic beads-droplet-handling system was developed. Actuation with a magnet offers a simple system for droplet manipulation that allows separation and fusion of droplets containing magnetic beads by handling with a magnet. The device consists of a reaction chamber channel and two magnet-handling channels for the manipulation of micro-droplets containing magnetic beads. Micro-droplets were placed inside a reaction chamber filled with oil and manipulated with a magnet. When a droplet containing NaOH and magnetic beads was manipulated towards a droplet containing phenol red, a color change was observed after fusion. Sample preparation was performed by fusion of droplets containing a forward primer, reverse primer, template DNA and PCR mixture, using a droplet containing magnetic beads. PCR amplification or RT-PCR was also successfully performed, with efficiency comparable to manual methods that use this device by placing it on a thermal cycler for amplification. With a magnetic beads-manipulation step, purification of amplified DNA was also accomplished by using magnetic beads as the carrier. The amplified DNA was captured on streptavidin conjugated magnetic beads using a biotinylated primer, purified by washing and digested for separation of the target DNA.     

Microfluidic aptameric affinity sensing of vasopressin for clinical diagnostic and therapeutic applications

We present a microfluidic aptameric biosensor, or aptasensor, for selective detection of clinically relevant analytes with integrated analyte enrichment, isocratic elution and label-free detection by mass spectrometry. Using a microfluidic platform that is coupled to matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), we demonstrate specific purification, enrichment, and label-free detection of trace amounts of arginine vasopressin (AVP), a peptide hormone that is responsible for arterial vasoconstriction. During extreme physical trauma, in particular immunological shock or congestive heart failure, AVP is excreted abnormally and is hence a biomarker for such conditions. The device uses an aptamer, i.e., an oligonucleotide that binds specifically to an analyte via affinity interactions, to achieve highly selective analyte capture and enrichment. In addition, via thermally induced reversible disruption of the aptamer-analyte binding, the device can be easily regenerated for reuse and allows isocratic analyte elution, i.e., release and collection of analytes using a single aqueous solution. Furthermore, the device is coupled to MALDI-MS using a microfluidic flow gate, which directs the eluted analyte onto a MALDI sample plate for mass spectrometry. We first perform systematic characterization of kinetic and thermal release properties, as well as the overall timescale of the assay, using fluorescently labeled AVP. We then demonstrate MALDI-MS detection of unlabeled AVP at clinically relevant concentrations approaching 1 pM.     

Automated droplet measurement (ADM): an enhanced video processing software for rapid droplet measurements

This paper identifies and addresses the bottlenecks that hamper the currently available software to perform in situ measurement on droplet-based microfluidic. The new and more universal object-based background extraction operation and automated binary threshold value selection make the processing step of our video processing software (ADM) fully automated. The ADM software, which is based on OpenCV image processing library, is made to perform measurements with high processing speed using efficient code. As the processing speed is higher than the data transfer speed from the video camera to permanent storage of computer, we integrate the camera software development kit (SDK) with ADM. The integration allows simultaneous operations of the video transfer/streaming and the video processing. As a result, the total time for droplet measurement using the new process flow with the integrated program is shortened significantly. ADM is also validated by comparing with both manual analysis and DMV software. ADM will be publicly released as a free tool. The software can also be used on a video file or files without the integration with the camera SDK.     

Droplet-based synthetic method using microflow focusing and droplet fusion

Droplet generation and droplet-based microreactor are realized by a facile microfluidic device. The relative concentrations of reactants could be well controlled by adjusting the flow rates without bringing reagents into prior contact; this new method shows considerable advantages for the control and rapid mixing of reagents with no dispersion.     

Comparative study of liquid–liquid extraction in miniaturized channels over other conventional extraction methods

We demonstrate the complexion of extracting an aromatic organic compound in microchannel system over other conventional methods like batch extraction, microwave assisted extraction and ultrasonic assisted extraction. Extraction studies were carried out for phenol, an aromatic organic compound from dodecane into distilled water. The extraction process is studied for a varying extraction time, microwave energy, percent ultrasonic power and micro channel diameter. Batch extraction is carried out at 25, 50, 100 and 200 rpm, microwave assisted extraction is carried out at 119, 231, 385, 539 and 700 W, ultrasonic assisted extraction is conducted in a 130 W ultrasonicator at a frequency of 20 kHz and amplitude of 10, 20, 30, 40 and 50 % for a varying extraction time of 1, 2, 4, 8, 16 and 32 min. While, the microsystem based extraction was carried out in a circular T junction microchannel. Among the various flow types in a microchannel, slug flow is intensified for its better hydrodynamic and mass transfer properties. Microchannel having diameter 600, 800, 1,000, 1,200 µm is used and compared for better extraction percentage. Experimental results manifested that 600 µm showed better extraction percentage. When comparing the extraction percentage of phenol acquired in microchannel system with other convention methods we observed the following ladder: slug based extraction > ultrasonic assisted extraction > microwave assisted extraction > batch extraction. Also, the microsystem based extraction needed just half of the operation time required by other conventional methods for achieving maximum extraction percentage. This subsequently causes effective usage of chemicals, thereby reducing chemical wastage. And, high efficient extraction can be obtained at very less time.     

An aptamer-based microfluidic device for thermally controlled affinity extraction

We present a microfluidic device for specific extraction and thermally activated release of analytes using nucleic acid aptamers. The device primarily consists of a microchamber that is packed with aptamer-functionalized microbeads as a stationary phase, and integrated with a micro heater and temperature sensor. We demonstrate the device operation by performing the extraction of a metabolic analyte, adenosine monophosphate coupled with thiazole orange (TO-AMP), with high selectivity to an RNA aptamer. Controlled release of TO-AMP from the aptamer surface is then conducted at low temperatures using on-chip thermal activation. This allows isocratic analyte elution, which eliminates the use of potentially harsh reagents, and enables efficient regeneration of the aptamer surfaces when device reusability is desired.     

Numerical and experimental study of a droplet-based PCR chip

A two-temperature continuous-flow polymerase chain reaction (PCR) polymer chip has been constructed that takes advantage of droplet technology to avoid sample contamination and adsorption at the surface. Samples contained in aqueous droplets are continuously moved by an oil carrier-fluid through various temperature zones, introducing the possibility of real-time quantitative PCR. In the present paper, we investigate many of the factors affecting droplet-based PCR chip design, including thermal mass, flow rate, and thermal resistance. The study focuses particularly on the fluid and substrate temperature distribution within the PCR chip and the droplet residence times in critical temperature zones. The simulations demonstrate that the flow rate strongly affects the temperature field within the carrier-fluid. Above a critical flow rate, the carrier-fluid fails to achieve the required temperatures for DNA amplification. In addition, the thermal resistances of the different layers in the chip are shown to have a major impact on the temperature profile in the channel.     

Phase transfer catalyzed esterification: modeling and experimental studies in a microreactor under parallel flow conditions

A liquid–liquid phase transfer catalyzed (PTC) esterification reaction of 4-t-butylphenol in aqueous phase (1 M sodium hydroxide solution) and 4-methoxybenzoyl chloride in organic phase (dichloromethane) in a microchannel under parallel laminar flow conditions was studied in this work. Tetrabutylammonium bromide was used as the PTC. Stable liquid–liquid hydrodynamic flow and a defined specific interfacial area in a microreactor offer considerable benefits over conventional batch reactors and are crucial to study interactions between kinetics and mass transfer effects. Mentioned features were used to develop a 3D mathematical model considering convection in the flow direction, diffusion in all spatial directions, and reactions in organic and aqueous phases. Results have shown a much higher mass transfer rate of the PTC between both phases as the one predicted by the 3D mathematical model. It may be assumed that the instability of parallel flow, along with the mass transfer of catalyst between both phases, causes rippling and erratic pulsation at the interface which then leads to interfacial convection and increased mass transfer rates. With a proposed correlation for mass transfer enhancement due to interfacial convection, all the experimental data were successfully predicted by the model.     

Continuous-flow in-droplet magnetic particle separation in a droplet-based microfluidic platform

This paper presents a continuous-flow in-droplet magnetic particle separation in a droplet-based microfluidic device for magnetic bead-based bioassays. Two functions, electrocoalescence and magnetic particle manipulation, are performed in this device. A pair of charging metallic needles is inserted into two aqueous channels of the device. By electrostatic force, two different solutions can be merged to be mixed at a junction of droplet generation. The manipulation of magnetic particles is achieved using an externally applied magnetic field. The magnetic particles are separated by the magnetic field to one side of the droplet and extracted by splitting the droplet into two daughter droplets: one contains the majority of the magnetic particles and the other is almost devoid of magnetic particles. The applicability of the continuous-flow in-droplet magnetic particle separation is demonstrated by performing a proof-of-concept immunoassay between streptavidin-coated magnetic beads and biotin labelled with fluorescence. This approach will be useful for various biological and chemical analyses and compartmentalization of small samples.     

便携式血液净化装置参数校准仪的研制

研制出一种可以实现多路参数同时检测的血液净化装置参数校准仪。仪器设计了与血液净化装置连接的一个回路装置,并将传感器连接在回路中,使透析液流经这个回路时,传感器就完成了各个参数的数据采集。为了方便检测、携带,电路部分在基于MSP430低功耗单片机的基础上采用集成化、模块化设计,采用高性能、小封装元件,实现了模块化、高精度、多功能、易便携的特点。仪器的电导率的不确定度为0.04 mS/cm,温度的不确定度为±0.1℃,压力的不确定度为±0.05 kPa,流量的不确定度为±20 mL/min,pH值的不确定度为±0.02。