基于单螺旋微流控芯片的细菌气溶胶的高效富集

设计了一种单螺旋通道的聚二甲基硅氧烷(Poly(dimethylsiloxane),PDMS)微流控芯片,用于副溶血性弧菌气溶胶的快速有效富集。该芯片的特征在于其通道呈螺旋分布,且通道内部含有均匀分布的鱼骨形结构。结果表明,在不同富集时间段内,采用该芯片方法捕获的细菌总数均远高于传统落板法。对于传统落板法无法有效捕获的低浓度样本(10~4CFU/mL)的缺陷,该方法的优势在于:芯片内部的螺旋通道可增大对气溶胶中微生物的离心力;鱼骨形结构的设计增加了待测样品与芯片内壁间的接触几率。此外,以无鱼骨形的螺旋芯片作为对照,验证了鱼骨形结构对于高效富集的意义。此芯片设计巧妙、易于制备、高效便携、富集效果较好,在气溶胶污染严重的水产加工等场所具有较大的应用前景。     

Simultaneous sample washing and concentration using a "trapping-and-releasing" mechanism of magnetic beads on a microfluidic chip

Simultaneous washing and concentration of functionalized magnetic beads in a complex sample solution were demonstrated by applying a rotational magnetic actuation system to a microfluidic chip under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to the fluidic channel carrying a magnetic bead suspension allows trapping and releasing of the beads along the fluidic channel in a periodical manner. Each trapping and releasing event resembles one washing cycle. A purification efficiency of magnetic beads out of a mixed magnetic and non-magnetic bead sample solution of 83±4% at a flow rate of 0.5 μL min(-1), and a magnetic bead recovery or concentration efficiency of 91±5% were achieved using a flow rate of 0.2 μL min(-1). The detection performance of the device was experimentally evaluated with two different bioassays, using either streptavidin-coated magnetic beads in combination with biotinylated fluorescent isothiocyanate (FITC), or a mouse antigen (Ag)-antibody (Ab) system.     

Integrated SPPS on continuous-flow radial microfluidic chip

A novel integrated continuous-flow microfluidic system was designed and fabricated for solid phase peptide synthesis (SPPS) using conventional reactants. The microfluidic system was composed of a glass-based radial reaction chip, a diffluent chip, amino acid feeding reservoirs and continuous-flow reagent pathways. A tri-row cofferdam-fence structure was designed for solid phase supports trapping. Highly cross-linked, porous and high-loading 4-(hydroxymethyl)phenoxymethyl polystyrene (HMP) beads were prepared for microfluidic SPPS. The transfer losses, hazardous handling and time-consuming processes in traditional peptide cleavage steps were avoided by being replaced with the on-chip cleavage treatment. Six peptides from an antibody affinity peptide library against β-endorphin with different lengths and sequences were obtained simultaneously on the constructed continuous-flow microfluidic system within a short time. This microfluidic system is automatic, integrated, effective, low-cost, recyclable and environment-friendly for not only SPPS but also other solid phase chemical syntheses.     

微流控芯片实验室

以作者所在课题组近年来的研究工作为基础,就芯片实验室平台建设及相应的以系统生物学为最终目标的功能化研究作一说明,对在分子和细胞层面,甚至是单分子、单细胞水平上实现以规模集成为特征的临床诊断和药物筛选的努力予以特别的关注。     

Low‐Voltage Pulsed Electric Field Sterilization on a Microfluidic Chip

A polyimide substrate based microfluidic chip with thousands of comb‐shaped microelectrodes has been designed, fabricated, and tested for sterilization of bacteria by using pulsed electric field. The performance of bacteria sterilization as functions of the electric field strength, pulse number and width, treatment buffer, bacteria growth status, and bacteria enrichment by positive dielectrophoresis has been experimentally investigated on the microfluidic chip. Experimental results show that only 100 V are sufficient to obtain good sterilization of Escherichia coli. Higher electric field strength, bacteria enrichment by positive dielectrophoresis, longer pulse time, buffer with fewer components and nutritions, and suitable bacteria growth status also improve the sterilization of bacteria. In addition, configuration of the microelectrode array affects bacteria sterilization. This microfluidic device allows one to preconcentrate bacteria to a region with high electric field strength by using positive dielectrophoresis, and subsequently kill the enriched bacteria by applying a pulsed electric field through the same microelectrode array.     

Microfluidic distillation chip for methanol concentration detection

An integrated microfluidic distillation system is proposed for separating a mixed ethanol-methanol-water solution into its constituent components. The microfluidic chip is fabricated using a CO₂ laser system and comprises a serpentine channel, a boiling zone, a heating zone, and a cooled collection chamber filled with de-ionized (DI) water. In the proposed device, the ethanol-methanol-water solution is injected into the microfluidic chip and driven through the serpentine channel and into the collection chamber by means of a nitrogen carrier gas. Following the distillation process, the ethanol-methanol vapor flows into the collection chamber and condenses into the DI water. The resulting solution is removed from the collection tank and reacted with a mixed indicator. Finally, the methanol concentration is inversely derived from the absorbance measurements obtained using a spectrophotometer. The experimental results show the proposed microfluidic system achieves an average methanol distillation efficiency of 97%. The practicality of the proposed device is demonstrated by detecting the methanol concentrations of two commercial fruit wines. It is shown that the measured concentration values deviate by no more than 3% from those obtained using a conventional bench top system.     

3D Insulator‐based dielectrophoresis using DC‐biased,AC electric fields for selective bacterial trapping

Insulator‐based dielectrophoresis (iDEP) is a well‐known technique that harnesses electric fields for separating, moving, and trapping biological particle samples. Recent work has shown that utilizing DC‐biased AC electric fields can enhance the performance of iDEP devices. In this study, an iDEP device with 3D varying insulating structures analyzed in combination with DC biased AC fields is presented for the first time. Using our unique reactive ion etch lag, the mold for the 3D microfluidic chip is created with a photolithographic mask. The 3D iDEP devices, whose largest dimensions are 1 cm long, 0.18 cm wide, and 90 μm deep are then rapidly fabricated by curing a PDMS polymer in the glass mold. The 3D nature of the insulating microstructures allows for high trapping efficiency at potentials as low as 200 Vpp. In this work, separation of Escherichia coli from 1 μm beads and selective trapping of live Staphylococcus aureus cells from dead S. aureus cells is demonstrated. This is the first reported use of DC‐biased AC fields to selectively trap bacteria in 3D iDEP microfluidic device and to efficiently separate particles where selectivity of DC iDEP is limited.     

A microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow

Biological cells in vivo typically reside in a dynamic flowing microenvironment with extensive biomechanical and biochemical cues varying in time and space. These dynamic biomechanical and biochemical signals together act to regulate cellular behaviors and functions. Microfluidic technology is an important experimental platform for mimicking extracellular flowing microenvironment in vitro. However, most existing microfluidic chips for generating dynamic shear stress and biochemical signals require expensive, large peripheral pumps and external control systems, unsuitable for being placed inside cell incubators to conduct cell biology experiments. This study has developed a microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow. Further, based on the lumped-parameter and distributed-parameter models of multiscale fluid dynamics, the oscillatory flow field and the concentration field of biochemical factors has been simulated at the cell culture region within the designed microfluidic chip. Using the constructed experimental system, the feasibility of the designed microfluidic chip has been validated by simulating biochemical factors with red dye. The simulation results demonstrate that dynamic shear stress and biochemical signals with adjustable period and amplitude can be generated at the cell culture chamber within the microfluidic chip. The amplitudes of dynamic shear stress and biochemical signals is proportional to the pressure difference and inversely proportional to the flow resistance, while their periods are correlated positively with the flow capacity and the flow resistance. The experimental results reveal the feasibility of the designed microfluidic chip. Conclusively, the proposed microfluidic generator based on autonomously oscillatory flow can generate dynamic shear stress and biochemical signals without peripheral pumps and external control systems. In addition to reducing the experimental cost, due to the tiny volume, it is beneficial to be integrated into cell incubators for cell biology experiments. Thus, the proposed microfluidic chip provides a novel experimental platform for cell biology investigations.     

Development of a low flow‐resistive charged nanoporous membrane in a microchip for fast electropreconcentration

A nanoporous poly‐(styrene sulfonate) (poly‐SS) membrane was developed for fast and selective ion transport in a microfluidic chip. The poly‐SS membrane can be photopolymerized in‐situ at arbitrary location of a microchannel, enabling integrated fluidics design in the microfluidic chip. The membrane is characterized by a low hydraulic resistance and a high surface charge to maximize the electroosmotic flow and charge selectivity. The membrane characteristics were investigated by charge‐selective electropreconcentration method. Experimental results show membranes with various percentages of poly‐SS are able to concentrate anions (fluorescein and TRITC‐labeled BSA). The anion‐selective electropreconcentration process is stable and 26‐times faster than previously reported poly‐AMPS (2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid) based system. The electropreconcentration was also demonstrated to depend on the sample valency and buffer concentration.     

Collagen microsphere production on a chip

We have developed an integrated microfluidic material processing chip and demonstrated the rapid production of collagen microspheres encapsulating cells with high uniformity and cell viability. The chip integrated three material processing steps. Monodisperse microdroplets were generated at a microfluidic T junction between aqueous and mineral oil flows. The flow was heated immediately to 37 °C to initiate collagen fiber assembly within a gelation channel. Gelled microspheres were extracted from the mineral oil phase into cell culture media within an extraction chamber. Collagen gelation immediately after microdroplet generation significantly reduced coalescence among microdroplets that led to non-uniform microsphere production. The microfluidic extraction approach led to higher microsphere recovery and cell viability than when a conventional centrifugation extraction approach was employed. These results indicate that chip-based material processing is a promising approach for cell-ECM microenvironment generation for applications such as tissue engineering and stem cell delivery.     

On-chip sample pretreatment using a porous polymer monolithic column for solid-phase microextraction and chemiluminescence determination of catechins in green tea

A porous polymer monolithic column for solid-phase microextraction and chemiluminescence detection was integrated into a simple microfluidic chip for the extraction and determination of catechins in green tea. The porous polymer was prepared by poly(glycidyl methacrylate-co-ethylene dimethacrylate) and modified with ethylenediamine. Catechins can be concentrated in the porous polymer monolithic column and react with potassium permanganate to give chemiluminescence. The microfluidic chip is reusable with high sensitivity and very low reagent consumption. The on-line preconcentration and detection can be realized without an elution step. The enrichment factor was calculated to be about 20 for catechins. The relative chemiluminescence intensity increased linearly with concentration of catechin from 5.0 × 10(-9) to 1.0 × 10(-6) M and the limit of detection was 1.0 × 10(-9) M. The proposed method was applied to determine catechin in green tea. The recoveries are from 90% to 110% which benefits the actual application for green tea samples.     

Polyacrylamide gel plugs enabling 2-D microfluidic protein separations via isoelectric focusing and multiplexed sodium dodecyl sulfate gel electrophoresis

In situ photopolymerized polyacrylamide (PAAm) gel plugs are used as hydrodynamic flow control elements in a multidimensional microfluidic system combining IEF and parallel SDS gel electrophoresis for protein separations. The PAAm gel plugs offer a simple method to reduce undesirable bulk flow and limit reagent/sample crosstalk without placing unwanted constraints on the selection of separation media, and without hindering electrokinetic ion migration in the complex microchannel network. In addition to improving separation reproducibility, the discrete gel plugs integrated into critical regions of the chip enable the use of a simple pressure-driven sample injection method which avoids electrokinetic injection bias. The gel plugs also serve to greatly simplify operation of the spatially multiplexed system by eliminating the need for complex external fluidic interfaces. Using an FITC-labeled Escherichia coli cell lysate as a model system, the use of gel plugs is shown to significantly enhance separation reproducibility in a chip containing five parallel CGE channels, with an average variance in peak elution time of only 4.1%.     

Dielectrophoretic separation of microalgae cells in ballast water in a microfluidic chip

The composition of the ship's ballast water is complex and contains a large number of microalgae cells, bacteria, microplastics, and other microparticles. To increase the accuracy and efficiency of detection of the microalgae cells in ballast water, a new microfluidic chip for continuous separation of microalgae cells based on alternating current dielectrophoresis was proposed. In this microfluidic chip, one piece of 3‐dimensional electrode is embedded on one side and eight discrete electrodes are arranged on the other side of the microchannel. An insulated triangular structure between electrodes is designed for increasing the inhomogeneity of the electric field distribution and enhancing the dielectrophoresis (DEP) force. A sheath flow is designed to focus the microparticles near the electrode, so as to increase the suffered DEP force and improve separation efficiency. To demonstrate the performance of the microfluidic separation chip, we developed two species of microalgae cells (Platymonas and Closterium) and a kind of microplastics to be used as test samples. Analyses of the related parameters and separation experiments by our designed microfluidic chip were then conducted. The results show that the presented method can separate the microalgae cells from the mixture efficiently, and this is the first time to separate two or more species of microalgae cells in a microfluidic chip by using negative and positive DEP force simultaneously, and moreover it has some advantages including simple operation, high efficiency, low cost, and small size and has great potential in on‐site pretreatment of ballast water.     

Evaporation Induced Spontaneous Micro-Vortexes through Engineering of the Marangoni Flow

Vortex flow fields are widely used to manipulate objects at the microscale in microfluidics. Previous approaches to produce the vortex flow field mainly focused on inertia flows. It remains a challenge to create vortexes in Stokes flow regime. Here we reported an evaporation induced spontaneous vortex flow system in Stokes flow regime by engineering Marangoni flow in a micro-structured microfluidic chip. The Marangoni flow is created by nonuniform evaporation of surfactant solution. Various vortexes are constructed by folding the air–water interface via microstructures. Patterns of vortexes are programmable by designing the geometry of the microstructures and are predictable using numerical simulations. Moreover, rotation of micro-objects and enrichment of micro-particles using vortex flow is demonstrated. This approach to create vortexes will provide a promising platform for various microfluidic applications such as biological analysis, chemical synthesis, and nanomaterial assembly.     

Microfluidic device for concentration and SERS-based detection of bacteria in drinking water

There is a constant need for the development of easy-to-operate systems for the rapid and unambiguous identification of bacterial pathogens in drinking water without the requirement for time-consuming culture processes. In this study, we present a disposable and low-cost lab-on-a-chip device utilizing a nanoporous membrane, which connects two stacked perpendicular microfluidic channels. Whereas one of the channels supplies the sample, the second one attracts it by potential-driven forces. Surface-enhanced Raman spectrometry (SERS) is employed as a reliable detection method for bacteria identification. To gain the effect of surface enhancement, silver nanoparticles were added to the sample. The pores of the membrane act as a filter trapping the bodies of microorganisms as well as clusters of nanoparticles creating suitable conditions for sensitive SERS detection. Therein, we focused on the construction and characterization of the device performance. To demonstrate the functionality of the microfluidic chip, we analyzed common pathogens (Escherichia coli DH5α and Pseudomonas taiwanensis VLB120) from spiked tap water using the optimized experimental parameters. The obtained results confirmed our system to be promising for the construction of a disposable optical platform for reliable and rapid pathogen detection which couples their electrokinetic concentration on the integrated nanoporous membrane with SERS detection.     

Microfluidic chip accomplishing self-fluid replacement using only capillary force and its bioanalytical application

A polymer microfluidic chip accomplishing automated sample flow and replacement without external controls and an application of the chip for bioanalytical reaction were described. All the fluidic operations in the chip were achieved by only natural capillary flow in a time-planned sequence. For the control of the capillary flow, the geometry of the channels and chambers in the chip was designed based on theoretical considerations and numerical simulations. The microfluidic chip was made by using polymer replication techniques, which were suitable for fast and cheap fabrication. The test for a biochemical analysis, employing an enzyme (HRP)-catalyzed precipitation reaction, exhibited a good performance using the developed chip. The presented microfluidic method would be applicable to biochemical lab-on-a-chips with integrated fluid replacement steps, such as affinity elution and solution exchange during biosensor signaling.     

Fluorescence affinity sensing by using a self-contained fluid manoeuvring microfluidic chip

An application of a novel polymer microfluidic chip for sample exchange via natural capillary forces for immuno-analysis is described. The microfluidic device was designed to achieve sample replacement by capillary force only, which would therefore be suitable for point-of-care-testing. Complete and automatic replacement of the sample in the reaction chamber with another one makes the chip able to mimic affinity chromatography and immunoassay processes. The microfluidic chip was made using polymer replication techniques, which were suitable for fast and cheap fabrication. Micrometre-sized polystyrene beads were used for the functionalization of biomolecules. Dinitrophenyl (DNP) and anti-DNP antibody coordination was employed on the chip for fluorescence analysis. DNP was immobilized on the polymer beads via a pre-adsorbed dendrimer layer and the beads were placed in the reaction chamber. Fluorescein tagged anti-DNP was successfully observed by a fluorescence microscope after the completion of the entire flow sequence. A calibration curve was registered based on the anti-DNP concentration. A multiplex sensing was accomplished by adding biotin/streptavidin coordination to the system. DNP and biotin conjugated beads were placed in the reaction chamber in an ordered fashion and biospecific bindings of anti-DNP antibody and streptavidin were observed at their expected sites. A ratiometric analysis was carried out with different concentration ratios of anti-DNP/streptavidin. The microfluidic chip described in this work could be applied to various biological and chemical analyses using integrated washing steps or fluid replacement steps with minimum sample handling.     

Disposable on‐chip microfluidic system for buccal cell lysis,DNA purification,and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off‐chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.     

Measuring reaction kinetics in a lab-on-a-chip by microcoil NMR

A microfluidic chip with an integrated planar microcoil was developed for Nuclear Magnetic Resonance (NMR) spectroscopy on samples with volumes of less than a microliter. Real-time monitoring of imine formation from benzaldehyde and aniline in the microreactor chip by NMR was demonstrated. The reaction times in the chip can be set from 30 min down to ca. 2 s, the latter being the mixing time in the microfluidic chip. Design rules will be described to optimize the microreactor and detection coil in order to deal with the inherent sensitivity of NMR and to minimize magnetic field inhomogeneities and obtain sufficient spectral resolution.     

A novel DNA selection and direct extraction process and its application in DNA recombination

In the conventional bench-top approach, the DNA recombination process is time- and effort-consuming due to laborious procedures lasting from several hours to a day. A novel DNA selection and direct extraction process has been proposed, integrated and tested on chip. The integrative microfluidic chip can perform the whole procedure of DNA recombination, including DNA digestion, gel electrophoresis, DNA extraction and insert-vector ligation within 1?h. In this high-throughput design, the manual gel cutting was replaced by an automatic processing system that performed high-quality and high-recovery efficiency in DNA extraction process. With no need of gel-dissolving reagents and manipulation, the application of selection and direct extraction process could significantly eliminate the risks from UV and EtBr and also facilitate DNA recombination. Reliable output with high success rate of cloning has been achieved with a significant reduction in operational hazards, required materials, efforts and time.