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1.
This paper describes a new and facile approach for the formation of pore-spanning bilayer lipid membranes (BLMs) within a poly(dimethylsiloxane) (PDMS) microfluidic device. Commercially, readily available polycarbonate (PC) membranes are employed for the support of BLMs. PC sheets with 5 μm, 2 μm, and 0.4 μm pore diameters, respectively, are thermally bonded into a multilayer-stack, reducing the pore density of 0.4 μm-pore PC by a factor of 200. The BLMs on this support are considerably stable (a mean lifetime: 17 h). This multilayer-stack PC (MSPC) membrane is integrated into the PDMS chip by an epoxy bonding method developed to secure durable bonding under the use of organic solvents. The microchip has a special channel for guiding a micropipette in the proximity of the MSPC support. With this on-site injection technique, tens to hundreds of nanoliters of solutions can be directly dispensed to the support. Incorporating gramicidin ion channels into BLMs on the MSPC support has confirmed the formation of single BLMs, which is based on the observation from current signals of 20 pS conductance that is typical to single channel opening. Based on the bilayer capacitance (1.4 pF), about 15% of through pores across the MSPC membrane are estimated to be covered with BLMs.  相似文献   

2.
For the first time, we report on the preliminary evaluation of gold coated optical fibers (GCOFs) as three-dimensional (3D) electrodes for a membraneless glucose/O2 enzymatic biofuel cell. Two off-the-shelf 125 μm diameter GCOFs were integrated into a 3D microfluidic chip fabricated via rapid prototyping. Using soluble enzymes and a 10 mM glucose solution flowing at an average velocity of 16 mm s−1 along 3 mm long GCOFs, the maximum power density reached 30.0 ± 0.1 μW cm−2 at a current density of 160.6 ± 0.3 μA cm−2. Bundles composed of multiple GCOFs could further enhance these first results while serving as substrates for enzyme immobilization.  相似文献   

3.
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   

4.
Blood analysis plays a major role in medical and science applications and white blood cells (WBCs) are an important target of analysis. We proposed an integrated microfluidic chip for direct and rapid trapping WBCs from whole blood. The microfluidic chip consists of two basic functional units: a winding channel to mix and arrays of two-layer trapping structures to trap WBCs. Red blood cells (RBCs) were eliminated through moving the winding channel and then WBCs were trapped by the arrays of trapping structures. We fabricated the PDMS (polydimethylsiloxane) chip using soft lithography and determined the critical flow velocities of tartrazine and brilliant blue water mixing and whole blood and red blood cell lysis buffer mixing in the winding channel. They are 0.25 μl/min and 0.05 μl/min, respectively. The critical flow velocity of the whole blood and red blood cell lysis buffer is lower due to larger volume of the RBCs and higher kinematic viscosity of the whole blood. The time taken for complete lysis of whole blood was about 85 s under the flow velocity 0.05 μl/min. The RBCs were lysed completely by mixing and the WBCs were trapped by the trapping structures. The chip trapped about 2.0 × 103 from 3.3 × 103 WBCs.  相似文献   

5.
Accurate measurement of blood viscoelasticity including viscosity and elasticity is essential in estimating blood flows in arteries, arterials, and capillaries and in investigating sub-lethal damage of RBCs. Furthermore, the blood viscoelasticity could be clinically used as key indices in monitoring patients with cardiovascular diseases. In this study, we propose a new method to simultaneously measure the viscosity and elasticity of blood by simply controlling the steady and transient blood flows in a microfluidic analogue of Wheastone-bridge channel, without fully integrated sensors and labelling operations. The microfluidic device is designed to have two inlets and outlets, two side channels, and one bridge channel connecting the two side channels. Blood and PBS solution are simultaneously delivered into the microfluidic device as test fluid and reference fluid, respectively. Using a fluidic-circuit model for the microfluidic device, the analytical formula is derived by applying the linear viscoelasticity model for rheological representation of blood. First, in the steady blood flow, the relationship between the viscosity of blood and that of PBS solution (μBlood/μPBS) is obtained by monitoring the reverse flows in the bridge channel at a specific flow-rate rate (QPBSSS/QBloodL). Next, in the transient blood flow, a sudden increase in the blood flow-rate induces the transient behaviors of the blood flow in the bridge channel. Here, the elasticity (or characteristic time) of blood can be quantitatively measured by analyzing the dynamic movement of blood in the bridge channel. The regression formula (ABlood (t) = Aα + Aβ exp [−(t − t0)/λBlood]) is selected based on the pressure difference (ΔP = PA − PB) at each junction (A, B) of both side channels. The characteristic time of blood (λBlood) is measured by analyzing the area (ABlood) filled with blood in the bridge channel by selecting an appropriate detection window in the microscopic images captured by a high-speed camera (frame rate = 200 Hz, total measurement time = 7 s). The elasticity of blood (GBlood) is identified using the relationship between the characteristic time and the viscosity of blood. For practical demonstrations, the proposed method is successfully applied to evaluate the variations in viscosity and elasticity of various blood samples: (a) various hematocrits form 20% to 50%, (b) thermal-induced treatment (50 °C for 30 min), (c) flow-induced shear stress (53 ± 0.5 mL/h for 120 min), and (d) normal rat versus spontaneously hypertensive rat. Based on these experimental demonstrations, the proposed method can be effectively used to monitor variations in viscosity and elasticity of bloods, even with the absence of fully integrated sensors, tedious labeling and calibrations.  相似文献   

6.
Deterministic lateral displacement (DLD) is a microfluidic size-based particle separation or filter technology with applications in cell separation and enrichment. Currently, there are no cost-effective manufacturing methods for this promising microfluidic technology. In this fabrication paper, however, we develop a simple, yet robust protocol for thermoplastic DLD devices using regulatory-approved materials and biocompatible methods. The final standalone device allowed for volumetric flow rates of 660 μl min−1 while reducing the manufacturing time to <1 h. Optical profilometry and image analysis were employed to assess manufacturing accuracy and precision; the average replicated post height was 0.48% less than the average post height on the master mold and the average replicated array pitch was 1.1% less than the original design with replicated posts heights of 62.1 ± 5.1 μm (mean ± 6 standard deviations) and replicated array pitches of 35.6 ± 0.31 μm.  相似文献   

7.
Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml−1), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.  相似文献   

8.
To sequentially handle fluids is of great significance in quantitative biology, analytical chemistry, and bioassays. However, the technological options are limited when building such microfluidic sequential processing systems, and one of the encountered challenges is the need for reliable, efficient, and mass-production available microfluidic pumping methods. Herein, we present a bubble-free and pumping-control unified liquid handling method that is compatible with large-scale manufacture, termed multilayer microfluidic sample isolated pumping (mμSIP). The core part of the mμSIP is the selective permeable membrane that isolates the fluidic layer from the pneumatic layer. The air diffusion from the fluidic channel network into the degassing pneumatic channel network leads to fluidic channel pressure variation, which further results in consistent bubble-free liquid pumping into the channels and the dead-end chambers. We characterize the mμSIP by comparing the fluidic actuation processes with different parameters and a flow rate range of 0.013 μl/s to 0.097 μl/s is observed in the experiments. As the proof of concept, we demonstrate an automatic sequential fluid handling system aiming at digital assays and immunoassays, which further proves the unified pumping-control and suggests that the mμSIP is suitable for functional microfluidic assays with minimal operations. We believe that the mμSIP technology and demonstrated automatic sequential fluid handling system would enrich the microfluidic toolbox and benefit further inventions.  相似文献   

9.
Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency.  相似文献   

10.
Cryptosporidiosis has been reported to be associated with HIV/acquired immune deficiency syndrome, which greatly reduces the quality of life and shortens the life expectancy of HIV-infected patients. In order to properly treat the infected patients, accurate and automatic diagnostic tools need to be developed. In this study, a novel microfluidic immunochip system was presented for the surveillance and the rapid detection of Cryptosporidium infection in 190 HIV-infected patients from Guangxi, China, using the P23 antigen of Cryptosporidium. The procedure of detection can be completed within 10 min with 2 μl sample consumption. The system also was evaluated using the standard ELISA method. Among 190 HIV-infected individuals, the rate of P23 positivity was 13.7%. Seropositivity in HIV-infected individuals was higher in female patients. The seropositivity to P23 was higher in HIV-infected individuals with high viral load, although the difference was statistically insignificant. Significantly higher Cryptosporidium seropositivity was observed in HIV-infected individuals with a CD4+ T-cell count of <200 cells/μl than in those with ≥200 cells/μl. Our results also demonstrate that a lower CD4+ T-cell count may reflect an increased accumulated risk for cryptosporidiosis. The detection system was further validated using the standard ELISA method and good correlation between the two methods was found (r = 0.80). Under the same sensitivity, this new microfluidic chip device had a specificity of 98.2%. This developed system may provide a powerful platform for the fast screening of Cryptospordium infection in HIV-infected patients.  相似文献   

11.
We have performed microfluidic experiments with erythrocytes passing through a network of microchannels of 20–25 μm width and 5 μm of height. Red blood cells (RBCs) were flowing in countercurrent directions through microchannels connected by μm pores. Thereby, we have observed interesting flow dynamics. All pores were blocked by erythrocytes. Some erythrocytes have passed through pores, depending on the channel size and cell elasticity. Many RBCs split into two or more smaller parts. Two types of splits were observed. In one type, the lipid bilayer and spectrin network were cut at the same time. In the second type, the lipid bilayer reconnected, but the part of spectrin network stayed outside the cell forming a rope like structure, which could eventually break. The microporous membrane results in multiple breakups of the cells, which can have various clinical implications, e.g., glomerulus hematuria and anemia of patients undergoing dialysis. The cell breakup procedure is similar to the one observed in the droplet breakage of viscoelastic liquids in confinement.  相似文献   

12.
We developed a microfluidic device to culture cellular spheroids of controlled sizes and suitable for live cell imaging by selective plane illumination microscopy (SPIM). We cocultured human umbilical vein endothelial cells (HUVECs) within the spheroids formed by hepatocellular carcinoma cells, and studied the distributions of the HUVECs over time. We observed that the migration of HUVECs depended on the size of spheroids. In the spheroids of ∼200 μm diameters, HUVECs migrated outwards to the edges within 48 h; while in the spheroids of ∼250 μm diameters, there was no outward migration of the HUVECs up to 72 h. In addition, we studied the effects of pro-angiogenic factors, namely, vascular endothelial growth factor (VEGF) and fibroblast growth factor (β-FGF), on the migration of HUVECs in the carcinoma cell spheroid. The outward migration of HUVECs in 200 μm spheroids was hindered by the treatment with VEGF and β-FGF. Moreover, some of the HUVECs formed hollow lumen within 72 h under VEGF and β-FGF treatment. The combination of SPIM and microfluidic devices gives high resolution in both spatial and temporal domains. The observation of HUVECs in spheroids provides us insight on tumor vascularization, an ideal disease model for drug screening and fundamental studies.  相似文献   

13.
We present a method to perform sample concentration within a lab-on-a-chip using a microfluidic structure which controls the liquid-gas interface through a micropillar array fabricated in polydimethylsiloxane between microfluidic channels. The microstructure confines the liquid flow and a thermal gradient is used to drive evaporation at the liquid-gas-interface. The evaporation occurs in-plane to the microfluidic device, allowing for precise control of the ambient environment. This method is demonstrated with a sample containing 1 μm, 100 nm fluorescent beads and SYTO-9 labelled Escherichia coli bacteria. Over 100 s, the fluorescent beads and bacteria are concentrated by a factor of 10.  相似文献   

14.
Cui S  Liu Y  Wang W  Sun Y  Fan Y 《Biomicrofluidics》2011,5(3):32003-320038
This paper examined the feasibility of a microfluidics chip for cell capturing and pairing with a high efficiency. The chip was fabricated by the polydimethylsiloxane-based soft-lithography technique and contained two suction duct arrays set in parallel on both sides of a main microchannel. Cells were captured and paired by activating two sets of suction ducts one by one with the help of syringe pumps along with switching the cell suspensions inside the main microchannel correspondingly. The effects of suction flow rate and the dimensions of suction channels on the cell capturing and pairing efficiency were characterized. The present chip was capable of creating 1024 pairs of two different cell populations in parallel. The preliminary experimental results showed that the cell capturing efficiency was 100% and the pairing one was 88% with an optimal suction rate of 5 μl/min in the chip in the 2 μm-sized suction duct chip. The cell viability after capture inside the microfluidic device was 90.0 ± 5.3%. With this cell capturing and pairing chip, interaction between cells in a single pair mode can be studied. The ability to create cell pairs has a number of biological applications for cell fusion, cell-cell interaction studies, and cell toxicity screening.  相似文献   

15.
In this study, a 3D passivated-electrode, insulator-based dielectrophoresis microchip (3D πDEP) is presented. This technology combines the benefits of electrode-based DEP, insulator-based DEP, and three dimensional insulating features with the goal of improving trapping efficiency of biological species at low applied signals and fostering wide frequency range operation of the microfluidic device. The 3D πDEP chips were fabricated by making 3D structures in silicon using reactive ion etching. The reusable electrodes are deposited on second glass substrate and then aligned to the microfluidic channel to capacitively couple the electric signal through a 100 μm glass slide. The 3D insulating structures generate high electric field gradients, which ultimately increases the DEP force. To demonstrate the capabilities of 3D πDEP, Staphylococcus aureus was trapped from water samples under varied electrical environments. Trapping efficiencies of 100% were obtained at flow rates as high as 350 μl/h and 70% at flow rates as high as 750 μl/h. Additionally, for live bacteria samples, 100% trapping was demonstrated over a wide frequency range from 50 to 400 kHz with an amplitude applied signal of 200 Vpp. 20% trapping of bacteria was observed at applied voltages as low as 50 Vpp. We demonstrate selective trapping of live and dead bacteria at frequencies ranging from 30 to 60 kHz at 400 Vpp with over 90% of the live bacteria trapped while most of the dead bacteria escape.  相似文献   

16.
Separation and sorting of biological entities (viruses, bacteria, and cells) is a critical step in any microfluidic lab-on-a-chip device. Acoustofluidics platforms have demonstrated their ability to use physical characteristics of cells to perform label-free separation. Bandpass-type sorting methods of medium-sized entities from a mixture have been presented using acoustic techniques; however, they require multiple transducers, lack support for various target populations, can be sensitive to flow variations, or have not been verified for continuous flow sorting of biological cells. To our knowledge, this paper presents the first acoustic bandpass method that overcomes all these limitations and presents an inherently reconfigurable technique with a single transducer pair for stable continuous flow sorting of blood cells. The sorting method is first demonstrated for polystyrene particles of sizes 6, 10, and 14.5 μm in diameter with measured purity and efficiency coefficients above 75 ± 6% and 85 ± 9%, respectively. The sorting strategy was further validated in the separation of red blood cells from white blood cells and 1 μm polystyrene particles with 78 ± 8% efficiency and 74 ± 6% purity, respectively, at a flow rate of at least 1 μl/min, enabling to process finger prick blood samples within minutes.  相似文献   

17.
We present a novel 3D hybrid assembly of a polymer microfluidic chip with polycarbonate track-etched membrane (PCTEM) enabling membrane-supported cell culture. Two chip designs have been developed to establish either diffusive or convective reagent delivery using the integrated PCTEM. While it is well suited to a range of cell-based assays, we specifically employ this platform for the screening of a common antitumor chemotoxic agent (mitomycin C – MMC) on the HL60 myeloid leukemia cell line. The toxic activity of MMC is based on the generation of severe DNA damage in the cells. Using either mode of operation, the HL60 cells were cultured on-chip before, during, and after exposure to MMC at concentrations ranging from 0 to 50 μM. Cell viability was analysed off-chip by the trypan blue dye exclusion assay. The results of the on-chip viability assay were found to be consistent with those obtained off-chip and indicated ca. 40% cell survival at MMC concentration of 50 μM. The catalogue of capabilities of the here described cell assay platform comprises of (i) the culturing of cells either under shear-free conditions or under induced through-membrane flows, (ii) the tight time control of the reagent exposure, (iii) the straightforward assembly of devices, (iv) the flexibility on the choice of the membrane, and, prospectively, (v) the amenability for large-scale parallelization.  相似文献   

18.
Despite the myriad of soft lithography based micropatterning methods available to researchers, it is still challenging to define small features (10–100 μm) that are spaced far apart (1–10 mm). In this report, we describe a combined microfluidic-microstencil patterning method that can produce multifunctional substrates of small features, O(10 μm), with a large pitch, O(1 mm). In that, we fabricate microstencils using an UV curable polyurethane (Norland Optical Adhesive 81) with dense arrays of 10–100 μm holes. Overlaying arrays of microfluidic channels over these microstencils allow for the control of the spacing between features and the ability to pattern multiple substrates. We show that this method is capable of patterning soluble proteins, fibrillar insoluble collagen, liposomes, cells, and nanoparticles. We demonstrate the utility of the method by measuring platelet adhesion under flow to three adhesive proteins (insoluble fibrillar collagen, laminin, and reconstituted acid solubilized collagen fibers) in a single assay.  相似文献   

19.
Recent years have witnessed a strong trend towards analysis of single-cells. To access and handle single-cells, many new tools are needed and have partly been developed. Here, we present an improved version of a single-cell printer which is able to deliver individual single cells and beads encapsulated in free-flying picoliter droplets at a single-bead efficiency of 96% and with a throughput of more than 10 beads per minute. By integration of acoustophoretic focusing, the cells could be focused in x and y direction. This way, the cells were lined-up in front of a 40 μm nozzle, where they were analyzed individually by an optical system prior to printing. In agreement with acoustic simulations, the focusing of 10 μm beads and Raji cells has been achieved with an efficiency of 99% (beads) and 86% (Raji cells) to a 40 μm wide center region in the 1 mm wide microfluidic channel. This enabled improved optical analysis and reduced bead losses. The loss of beads that ended up in the waste (because printing them as single beads arrangements could not be ensured) was reduced from 52% ± 6% to 28% ± 1%. The piezoelectric transducer employed for cell focusing could be positioned on an outer part of the device, which proves the acoustophoretic focusing to be versatile and adaptable.  相似文献   

20.
The majority of available cardiomyocyte markers are intercellular proteins, limiting our ability to enrich live cardiomyocytes from heterogeneous cell preparations in the absence of genetic labeling. Here, we describe enrichment of live cardiomyocytes from the hearts of adult mice in a label-free microfluidic approach. The separation device consisted of a vertical column (15 mm long, 700 μm diameter), placed between permanent magnets resulting in a field strength of 1.23 T. To concentrate the field at the column wall, the column was wrapped with 69 μm diameter nickel wire. Before passing the cells through the column, the cardiomyocytes in the cell suspension had been rendered paramagnetic by treatment of the adult mouse heart cell preparation with sodium nitrite (2.5 mM) for 20 min on ice. The cell suspension was loaded into the vertical column from the top and upon settling, the non-myocytes were removed by the upward flow from the column. The cardiomyocytes were then collected from the column by applying a higher flow rate (144 μl/min). We found that by applying a separation flow rate of 4.2 μl/min in the first step, we can enrich live adult cardiomyocytes to 93% ± 2% in a label-free manner. The cardiomyocytes maintained viability immediately after separation and upon 24 h in culture.  相似文献   

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