共查询到20条相似文献，搜索用时 31 毫秒
1.
Uwe Tangen Gabriel Antonio S. Minero Abhishek Sharma Patrick F. Wagler Rafael Cohen Ofir Raz Tzipy Marx Tuval Ben-Yehezkel John S. McCaskill 《Biomicrofluidics》2015,9(4)
Nanoscale synthetic biology can benefit from programmable nanoliter-scale processing of DNA in microfluidic chips if they are interfaced effectively to biochemical arrays such as microwell plates. Whereas active microvalve chips require complex fabrication and operation, we show here how a passive and readily fabricated microchip can be employed for customizable nanoliter scale pipetting and reaction control involving DNA. This recently developed passive microfluidic device, supporting nanoliter scale combinatorial droplet generation and mixing, is here used to generate a DNA test library with one member per droplet exported to addressed locations on microwell plates. Standard DNA assembly techniques, such as Gibson assembly, compatible with isothermal on-chip operation, are employed and checked using off-chip PCR and assembly PCR. The control of output droplet sequences and mixing performance was verified using dyes and fluorescently labeled DNA solutions, both on-chip and in external capillary channels. Gel electrophoresis of products and DNA sequencing were employed to further verify controlled combination and functional enzymatic assembly. The scalability of the results to larger DNA libraries is also addressed by combinatorial input expansion using sequential injection plugs from a multiwell plate. Hence, the paper establishes a proof of principle of the production of functional combinatorial mixtures at the nanoliter scale for one sequence per well DNA libraries. 相似文献
2.
In this study, droplet formations in microfluidic double T-junctions (MFDTD) are investigated based on a two-dimensional numerical model with volume of fluid method. Parametric ranges for generating alternating droplet formation (ADF) are identified. A physical background responsible for the ADF is suggested by analyzing the dynamical stability of flow system. Since the phase discrepancy between dispersed flows is mainly caused by non-symmetrical breaking of merging droplet, merging regime becomes the alternating regime at appropriate conditions. In addition, the effects of channel geometries on droplet formation are studied in terms of relative channel width. The predicted results show that the ADF region is shifted toward lower capillary numbers when channel width ratio is less than unity. The alternating droplet size increases with the increase of channel width ratio. When this ratio reaches unity, alternating droplets can be formed at very high water fraction (wf = 0.8). The droplet formation in MFDTD depends significantly on the viscosity ratio, and the droplet size in ADF decreases with the increase of the viscosity ratio. The understanding of underlying physics of the ADF phenomenon is useful for many applications, including nanoparticle synthesis with different concentrations, hydrogel bead generation, and cell transplantation in biomedical therapy. 相似文献
3.
A new microfluidic device with liquid-droplet merging and droplet storage functions for the controlled release of drugs from microcapsules is reported. A switching channel is designed and integrated within the microfluidic device, facilitating the generation and capturing of uniform droplets by the storage chambers. The drug model is the MnCO3 microparticle, which is encapsulated by a microcapsule and fabricated using a simple layer-by-layer nanoassembly process. The merging function is used for dynamically adding the control solution into the droplets, which contain drugs within the microcapsules (DWμCs) and water. The storage chambers are used for collecting DWμCs-laden droplets so that the controlled-drug release in specific droplets can be monitored for an extended period of time, which has been experimentally implemented successfully. This technology could offer a promising technical platform for the long-term observation and studies of drug effects on specific cells in a controlled manner, which is especially useful for single cell analysis. 相似文献
4.
Jiseok Lim Ouriel Caen Jérémy Vrignon Manfred Konrad Valérie Taly Jean-Christophe Baret 《Biomicrofluidics》2015,9(3)
Droplet-based microfluidic technologies are powerful tools for applications requiring high-throughput, for example, in biochemistry or material sciences. Several systems have been proposed for the high-throughput production of monodisperse emulsions by parallelizing multiple droplet makers. However, these systems have two main limitations: (1) they allow the use of only a single disperse phase; (2) they are based on multiple layer microfabrication techniques. We present here a pipette-and-play solution offering the possibility of manipulating simultaneously 10 different disperse phases on a single layer device. This system allows high-throughput emulsion production using aqueous flow rates of up to 26 ml/h (>110 000 drops/s) leading to emulsions with user-defined complex chemical composition. We demonstrate the multiplex capabilities of our system by measuring the kinetics of β-galactosidase in droplets using nine different concentrations of a fluorogenic substrate. 相似文献
5.
In this paper, we propose a continuous flow droplet-based microfluidic platform for magnetic particle-based assays by employing in-droplet washing. The droplet-based washing was implemented by traversing functionalized magnetic particles across a laterally merged droplet from one side (containing sample and reagent) to the other (containing buffer) by an external magnetic field. Consequently, the magnetic particles were extracted to a parallel-synchronized train of washing buffer droplets, and unbound reagents were left in an original train of sample droplets. To realize the droplet-based washing function, the following four procedures were sequentially carried in a droplet-based microfluidic device: parallel synchronization of two trains of droplets by using a ladder-like channel network; lateral electrocoalescence by an electric field; magnetic particle manipulation by a magnetic field; and asymmetrical splitting of merged droplets. For the stable droplet synchronization and electrocoalescence, we optimized droplet generation conditions by varying the flow rate ratio (or droplet size). Image analysis was carried out to determine the fluorescent intensity of reagents before and after the washing step. As a result, the unbound reagents in sample droplets were significantly removed by more than a factor of 25 in the single washing step, while the magnetic particles were successfully extracted into washing buffer droplets. As a proof-of-principle, we demonstrate a magnetic particle-based immunoassay with streptavidin-coated magnetic particles and fluorescently labelled biotin in the proposed continuous flow droplet-based microfluidic platform. 相似文献
6.
Zhichao Guan Yuan Zou Mingxia Zhang Jiangquan Lv Huali Shen Pengyuan Yang Huimin Zhang Zhi Zhu Chaoyong James Yang 《Biomicrofluidics》2014,8(1)
Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins. 相似文献
7.
Droplet-based microfluidic systems enable a variety of biomedical applications from point-of-care diagnostics with third world implications, to targeted therapeutics alongside medical ultrasound, to molecular screening and genetic testing. Though these systems maintain the key advantage of precise control of the size and composition of the droplet as compared to conventional methods of production, the low rates at which droplets are produced limits translation beyond the laboratory setting. As well, previous attempts to scale up shear-based microfluidic systems focused on increasing the volumetric throughput and formed large droplets, negating many practical applications of emulsions such as site-specific therapeutics. We present the operation of a parallel module with eight flow-focusing orifices in the dripping regime of droplet formation for the generation of uniform fine droplets at rates in the hundreds of kilohertz. Elevating the capillary number to access dripping, generation of monodisperse droplets of liquid perfluoropentane in the parallel module exceeded 3.69 × 105 droplets per second, or 1.33 × 109 droplets per hour, at a mean diameter of 9.8 μm. Our microfluidic method offers a novel means to amass uniform fine droplets in practical amounts, for instance, to satisfy clinical needs, with the potential for modification to form massive amounts of more complex droplets. 相似文献
8.
This paper presents a continuous flow microfluidic device for the separation of DNA from blood using magnetophoresis for biological applications and analysis. This microfluidic bio-separation device has several benefits, including decreased sample handling, smaller sample and reagent volumes, faster isolation time, and decreased cost to perform DNA isolation. One of the key features of this device is the use of short-range magnetic field gradients, generated by a micro-patterned nickel array on the bottom surface of the separation channel. In addition, the device utilizes an array of oppositely oriented, external permanent magnets to produce strong long-range field gradients at the interfaces between magnets, further increasing the effectiveness of the device. A comprehensive simulation is performed using COMSOL Multiphysics to study the effect of various parameters on the magnetic flux within the separation channel. Additionally, a microfluidic device is designed, fabricated, and tested to isolate DNA from blood. The results show that the device has the capability of separating DNA from a blood sample with a purity of 1.8 or higher, a yield of up to 33 μg of polymerase chain reaction ready DNA per milliliter of blood, and a volumetric throughput of up to 50 ml/h. 相似文献
9.
We report the design and implementation of capacitive detection and control of microfluidic droplets in microfluidic devices. Integrated microfluidic chip(s) with detection∕control circuit enables us to monitor in situ the individual volume of droplets, ranging from nanoliter to picoliter, velocity and even composition, with an operation frequency of several kilohertz. Through electronic feedback, we are able to easily count, sort, and direct the microfluidic droplets. Potential applications of this approach can be employed in the areas of biomicrofluidic processing, microchemical reactions as well as digital microfluidics. 相似文献
10.
We present dual-mode, on-demand droplet routing in a multiple-outlet microfluidic device using an oil-based magnetic fluid. Magnetite (Fe3O4) nanoparticle-contained oleic acid (MNOA) was used as a carrier phase for droplet generation and manipulation. The water-in-MNOA droplets were selectively distributed in a curved microchannel with three branches by utilizing both a hydrodynamic laminar flow pattern and an external magnetic field. Without the applied magnetic field, the droplets travelled along a hydrodynamic centerline that was displaced at each bifurcating junction. However, in the presence of a permanent magnet, they were repelled from the centerline and diverted into the desired channel when the repelled distance exceeded the minimum offset allocated to the channel. The repelled distance, which is proportional to the magnetic field gradient, was manipulated by controlling the magnet''s distance from the device. To evaluate routing performance, three different sizes of droplets with diameters of 63, 88, and 102 μm were directed into designated outlets with the magnet positioned at varying distances. The result demonstrated that the 102-μm droplets were sorted with an accuracy of ∼93%. Our technique enables on-demand droplet routing in multiple outlet channels by simply manipulating magnet positions (active mode) as well as size-based droplet separation with a fixed magnet position (passive mode). 相似文献
11.
This article describes a fabrication process for the generation of a leak proof paper based microfluidic device and a new design strategy for convenient incorporation of externally prepared test zones. Briefly, a negative photolithographic method was used to prepare the device with a partial photoresist layer on the rear of the device to block the leakage of sample. Microscopy and Field Emission Scanning Electron Microscopy data validated the formation of the photoresist layer. The partial layer of photoresist on the device channel limits sample volume to 7 ± 0.2 μl as compared to devices without the partial photoresist layer which requires a larger sample volume of 10 ± 0.1 μl. The design prototype with a customized external test zone exploits the channel protrusions on the UV exposed photoresist treated paper to bridge the externally applied test zone to the sample and absorbent zones. The partially laminated device with an external test zone has a comparatively low wicking speed of 1.8 ± 0.9 mm/min compared to the completely laminated device with an inbuilt test zone (3.3 ± 1.2 mm/min) which extends the reaction time between the analyte and reagents. The efficacy of the prepared device was studied with colorimetric assays for the non-specific detection of protein by tetrabromophenol blue, acid/base with phenolphthalein indicator, and specific detection of proteins using the HRP-DAB chemistry. The prepared device has the potential for leak proof detection of analyte, requires low sample volume, involves reduced cost of production (∼$0.03, excluding reagent and lamination cost), and enables the integration of customized test zones. 相似文献
12.
We present a novel use for channel structures in microfluidic devices, whereby two two-phase emulsions, one created on-chip, the other off-chip, are rapidly mixed with each other in order to allow for the coalescence of one emulsion with the other. This approach has been motivated by the difficulty in introducing aqueous cross linking agents into droplets by utilising conventional approaches. These conventional approaches include continuous introduction of the different aqueous reagents before droplet formation or alternatively formation of individual droplets of each reagent and subsequent droplet merging later in the microfluidic device. We show that our approach can decrease the mixing time for these fluidic systems by a factor greater than 10 times when compared to a standard microfluidic channel without structures, thereby also allowing for additional reaction time within the microfluidic device. This method shows an application for microfluidic channel structures not before demonstrated, also demonstrating an alternative method for introducing reagents such as cross linkers which link polymer chains to form particles, and provides an example where enzymes are immobilized in monodisperse particles. 相似文献
13.
Blood viscosity has been considered as one of important biophysical parameters for effectively monitoring variations in physiological and pathological conditions of circulatory disorders. Standard previous methods make it difficult to evaluate variations of blood viscosity under cardiopulmonary bypass procedures or hemodialysis. In this study, we proposed a unique microfluidic device for simultaneously measuring viscosity and flow rate of whole blood circulating in a complex fluidic network including a rat, a reservoir, a pinch valve, and a peristaltic pump. To demonstrate the proposed method, a twin-shaped microfluidic device, which is composed of two half-circular chambers, two side channels with multiple indicating channels, and one bridge channel, was carefully designed. Based on the microfluidic device, three sequential flow controls were applied to identify viscosity and flow rate of blood, with label-free and sensorless detection. The half-circular chamber was employed to achieve mechanical membrane compliance for flow stabilization in the microfluidic device. To quantify the effect of flow stabilization on flow fluctuations, a formula of pulsation index (PI) was analytically derived using a discrete fluidic circuit model. Using the PI formula, the time constant contributed by the half-circular chamber is estimated to be 8 s. Furthermore, flow fluctuations resulting from the peristaltic pumps are completely removed, especially under periodic flow conditions within short periods (T < 10 s). For performance demonstrations, the proposed method was applied to evaluate blood viscosity with respect to varying flow rate conditions [(a) known blood flow rate via a syringe pump, (b) unknown blood flow rate via a peristaltic pump]. As a result, the flow rate and viscosity of blood can be simultaneously measured with satisfactory accuracy. In addition, the proposed method was successfully applied to identify the viscosity of rat blood, which circulates in a complex fluidic network. These observations confirm that the proposed method can be used for simultaneous measurement of viscosity and flow rate of whole blood circulating in the complex fluid network, with sensorless and label-free detection. Furthermore, the proposed method will be used in evaluating variations in the viscosity of human blood during cardiopulmonary bypass procedures or hemodialysis. 相似文献
14.
We report the successful fabrication and testing of 3D printed microfluidic devices with integrated membrane-based valves. Fabrication is performed with a low-cost commercially available stereolithographic 3D printer. Horizontal microfluidic channels with designed rectangular cross sectional dimensions as small as 350 μm wide and 250 μm tall are printed with 100% yield, as are cylindrical vertical microfluidic channels with 350 μm designed (210 μm actual) diameters. Based on our previous work [Rogers et al., Anal. Chem. 83, 6418 (2011)], we use a custom resin formulation tailored for low non-specific protein adsorption. Valves are fabricated with a membrane consisting of a single build layer. The fluid pressure required to open a closed valve is the same as the control pressure holding the valve closed. 3D printed valves are successfully demonstrated for up to 800 actuations. 相似文献
15.
Dilution of microfluidic droplets where the concentration of a reagent is incrementally varied is a key operation in drop-based biological analysis. Here, we present an electrocoalescence based dilution scheme for droplets based on merging between moving and parked drops. We study the effects of fluidic and electrical parameters on the dilution process. Highly consistent coalescence and fine resolution in dilution factor are achieved with an AC signal as low as 10 V even though the electrodes are separated from the fluidic channel by insulator. We find that the amount of material exchange between the droplets per coalescence event is high for low capillary number. We also observe different types of coalescence depending on the flow and electrical parameters and discuss their influence on the rate of dilution. Overall, we find the key parameter governing the rate of dilution is the duration of coalescence between the moving and parked drop. The proposed design is simple incorporating the channel electrodes in the same layer as that of the fluidic channels. Our approach allows on-demand and controlled dilution of droplets and is simple enough to be useful for assays that require serial dilutions. The approach can also be useful for applications where there is a need to replace or wash fluid from stored drops. 相似文献
16.
Zhuoqi Li Weiran Liu Tian Qiu Lan Xie Weixing Chen Ran Liu Ying Lu Keith Mitchelson Jundong Wang Jie Qiao Jing Cheng 《Biomicrofluidics》2014,8(2)
Thermotaxis has been demonstrated to be an important criterion for sperm evaluation, yet clinical assessment of thermotaxis capacity is currently lacking. In this article, the on-chip thermotaxis evaluation of human sperm is presented for the first time using an interfacial valve-facilitated microfluidic device. The temperature gradient was established and accurately controlled by an external temperature gradient control system. The temperature gradient responsive sperm population was enriched into one of the branch channels with higher temperature setting and the non-responsive ones were evenly distributed into the two branch channels. We employed air-liquid interfacial valves to ensure stable isolation of the two branches, facilitating convenient manipulation of the entrapped sperm. With this device, thermotactic responses were observed in 5.7%-10.6% of the motile sperm moving through four temperature ranges (34.0-35.3 °C, 35.0-36.3 °C, 36.0-37.3 °C, and 37.0-38.3 °C, respectively). In conclusion, we have developed a new method for high throughput clinical evaluation of sperm thermotaxis and this method may allow other researchers to derive better IVF procedure. 相似文献
17.
In this paper, we demonstrate biphasic microfluidic droplets with broadly tunable internal structures, from simple near-equilibrium drop-in-drop morphologies to complex yet uniform non-equilibrium steady-state structures. The droplets contain an aqueous mixture of poly(ethylene glycol) (PEG) and dextran and are dispensed into an immiscible oil in a microfluidic T-junction device. Above a certain well-defined threshold droplet speed, the inner dextran-rich phase is "stirred" within the outer PEG-rich phase. The stirred polymer mixture is observed to exhibit a near continuum of speed and composition-dependent phase morphologies. There is increasing interest in the use of such aqueous two-phase systems in microfluidic devices for biomolecular applications in a variety of contexts. Our work presents a method to go beyond equilibrium phase morphologies in generating microfluidic "multiple" emulsions and at the same time raises the possibility of biochemical experimentation in benign yet complex biomimetic milieus. 相似文献
18.
Precise temporal control of microfluidic droplets such as synchronization and combinatorial pairing of droplets is required to achieve a variety range of chemical and biochemical reactions inside microfluidic networks. Here, we present a facile and robust microfluidic platform enabling uniform interval control of flowing droplets for the precise temporal synchronization and pairing of picoliter droplets with a reagent. By incorporating microbridge structures interconnecting the droplet-carrying channel and the flow control channel, a fluidic pressure drop was derived between the two fluidic channels via the microbridge structures, reordering flowing droplets with a defined uniform interval. Through the adjustment of the control oil flow rate, the droplet intervals were flexibly and precisely adjustable. With this mechanism of droplet spacing, the gelation of the alginate droplets as well as control of the droplet interval was simultaneously achieved by additional control oil flow including calcified oleic acid. In addition, by parallel linking identical microfluidic modules with distinct sample inlet, controlled synchronization and pairing of two distinct droplets were demonstrated. This method is applicable to facilitate and develop many droplet-based microfluidic applications, including biological assay, combinatorial synthesis, and high-throughput screening. 相似文献
19.
In this study, a microfluidic process is proposed for preparing monodisperse micrometer-sized hydrogel beads. This process utilizes non-equilibrium aqueous droplets formed in a polar organic solvent. The water-in-oil droplets of the hydrogel precursor rapidly shrunk owing to the dissolution of water molecules into the continuous phase. The shrunken and condensed droplets were then gelled, resulting in the formation of hydrogel microbeads with sizes significantly smaller than the initial droplet size. This study employed methyl acetate as the polar organic solvent, which can dissolve water at 8%. Two types of monodisperse hydrogel beads—Ca-alginate and chitosan—with sizes of 6–10 μm (coefficient of variation < 6%) were successfully produced. In addition, we obtained hydrogel beads with non-spherical morphologies by controlling the degree of droplet shrinkage at the time of gelation and by adjusting the concentration of the gelation agent. Furthermore, the encapsulation and concentration of DNA molecules within the hydrogel beads were demonstrated. The process presented in this study has great potential to produce small and highly concentrated hydrogel beads that are difficult to obtain by using conventional microfluidic processes. 相似文献
20.
The specific membrane capacitance (SMC) is an electrical parameter that correlates with both the electrical activity and morphology of the plasma membrane, which are physiological markers for cellular phenotype and health. We have developed a microfluidic device that enables impedance spectroscopy measurements of the SMC of single biological cells. Impedance spectra induced by single cells aspirated into the device are captured over a moderate frequency range (5 kHz–1 MHz). Maximum impedance sensitivity is achieved using a tapered microfluidic channel, which effectively routes electric fields across the cell membranes. The SMC is extracted by curve-fitting impedance spectra to an equivalent circuit model. From our measurement, acute myeloid leukemia (AML) cells are found to exhibit larger SMC values in hypertonic solutions as compared with those in isotonic solutions. In addition, AML cell phenotypes (AML2 and NB4) exhibiting varying metastatic potential yield distinct SMC values (AML2: 16.9 ± 1.9 mF/m2 (n = 23); NB4: 22.5 ± 4.7 mF/m2 (n = 23)). Three-dimensional finite element simulations of the microfluidic device confirm the feasibility of this approach. 相似文献