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1.
A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm in radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to derive quantities for sub-cellular units. Clausius-Mossotti factor of Jurkat cells is extracted from the impedance spectrum. Effects of cellular heterogeneity are discussed and parameterized. Jurkat cells are also tested with a time domain reflectometry system for verification of the microfluidic device. Results indicate good agreement of values obtained with both techniques. The device can be used as a unique cell diagnostic tool to yield information on sub-cellular units.  相似文献   

2.
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   

3.
Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.  相似文献   

4.
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   

5.
This paper presents a vertically positioned microfluidic system made of poly(dimethylsiloxane) (PDMS) and glass, which can be applied as a microbubble column (μBC) for biotechnological screening in suspension. In this μBC, microbubbles are produced in a cultivation chamber through an integrated nozzle structure. Thus, homogeneous suspension of biomass is achieved in the cultivation chamber without requiring additional mixing elements. Moreover, blockage due to produced carbon dioxide by the microorganisms—a problem predominant in common, horizontally positioned microbioreactors (MBRs)—is avoided, as the gas bubbles are released by buoyancy at the upper part of the microsystem. The patterned PDMS layer is based on an optimized two-lithographic process. Since the naturally hydrophobic PDMS causes problems for the sufficient production of microbubbles, a method based on polyelectrolyte multilayers is applied in order to allow continuous hydrophilization of the already bonded PDMS-glass-system. The μBC comprises various microelements, including stabilization of temperature, control of continuous bubble formation, and two optical configurations for measurement of optical density with two different sensitivities. In addition, the simple and robust application and handling of the μBC is achieved via a custom-made modular plug-in adapter. To validate the scalability from laboratory scale to microscale, and thus to demonstrate the successful application of the μBC as a screening instrument, a batch cultivation of Saccharomyces cerevisiae is performed in the μBC and compared to shake flask cultivation. Monitoring of the biomass growth in the μBC with the integrated online analytics resulted in a specific growth rate of 0.32 h−1, which is almost identical to the one achieved in the shake flask cultivation (0.31 h−1). Therefore, the validity of the μBC as an alternative screening tool compared to other conventional laboratory scale systems in bioprocess development is proven. In addition, vertically positioned microbioreactors show high potential in comparison to conventional screening tools, since they allow for high density of integrated online analytics and therefore minimize time and cost for screening and guarantee improved control and analysis of cultivation parameters.  相似文献   

6.
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   

7.
We developed a novel strategy for fabrication of microfluidic paper-based analytical devices (μPADs) by selective wet etching of hydrophobic filter paper using a paper mask having a specific design. The fabrication process consists of two steps. First, the hydrophilic filter paper was patterned hydrophobic by using trimethoxyoctadecylsilane (TMOS) solution as the patterning agent. Next, a paper mask penetrated with NaOH solution (containing 30% glycerol) was aligned onto the hydrophobic filter paper, allowing the etching of the silanized filter paper by the etching reagent. The masked region turned highly hydrophilic whereas the unmasked region remains highly hydrophobic. Thus, hydrophilic channels, reservoirs, and detection zones were generated and delimited by the hydrophobic barriers. The effects of some factors including TMOS concentration, etching temperature, etching time, and NaOH concentration on fabrication of μPAD were studied. Being free of any expensive equipment, metal mask and expensive reagents, this rapid, simple, and cost-effective method could be used to fabricate μPAD by untrained personnel with minimum cost. A flower-shaped μPAD fabricated by this presented method was applied to the glucose assay in artificial urine samples with good performance, indicating its feasibility as a quantitative analysis device. We believe that this method would be very attractive to the development of simple microfluidic devices for point-of-care applications in clinical diagnostics, food safety, and environmental protection.  相似文献   

8.
We propose a blood separation microfluidic device suitable for point-of-care (POC) applications. By utilizing the high gas permeability of polydimethylsiloxane (PDMS) and phaseguide structures, a simple blood separation device is presented. The device consists of two main parts. A separation chamber with the phaseguide structures, where a sample inlet, a tape-sealed outlet, and a dead-end ring channel are connected, and pneumatic chambers, in which manually operating syringes are plugged. The separation chamber and pneumatic chambers are isolated by a thin PDMS wall. By manually pulling out the plunger of the syringe, a negative pressure is instantaneously generated inside the pneumatic chamber. Due to the gas diffusion from the separation chamber to the neighboring pneumatic chamber through the thin permeable PDMS wall, low pressure can be generated, and then the whole blood at the sample inlets starts to be drawn into the separation chamber and separated through the phaseguide structures. Reversely, after removing the tape at the outlet and manually pushing in the plunger of the syringe, a positive pressure will be created which will cause the air to diffuse back into the ring channel, and therefore allow the separated plasma to be recovered at the outlet on demand. In this paper, we focused on the study of the plasma separation and associated design parameters, such as the PDMS wall thickness, the air permeable overlap area between the separation and pneumatic chambers, and the geometry of the phaseguides. The device required only 2 μl of whole blood but yielding approximately 0.38 μl of separated plasma within 12 min. Without any of the requirements of sophisticated equipment or dilution techniques, we can not only separate the plasma from the whole blood for on-chip analysis but also can push out only the separated plasma to the outlet for off-chip analysis.  相似文献   

9.
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   

10.
We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-μl pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, “amplicon-in-answer-out” operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis.  相似文献   

11.
Acoustic trapping of minute bead amounts against fluid flow allows for easy automation of multiple assay steps, using a convenient aspirate/dispense format. Here, a method based on acoustic trapping that allows sample preparation for immuno-matrix-assisted laser desorption/ionization mass spectrometry using only half a million 2.8 μm antibody covered beads is presented. The acoustic trapping is done in 200 × 2000 μm2 glass capillaries and provides highly efficient binding and washing conditions, as shown by complete removal of detergents and sample processing times of 5-10 min. The versatility of the method is demonstrated using an antibody against Angiotensin I (Ang I), a peptide hormone involved in hypotension. Using this model system, the acoustic trapping was efficient in enriching Angiotensin at 400 pM spiked in plasma samples.  相似文献   

12.
We present an optofluidic microvalve utilizing an embedded, surface plasmon-enhanced fiber optic microheater. The fiber optic microheater is formed by depositing a titanium thin film on the roughened end-face of a silica optical fiber that serves as a waveguide to deliver laser light to the titanium film. The nanoscale roughness at the titanium-silica interface enables strong light absorption enhancement in the titanium film through excitation of localized surface plasmons as well as facilitates bubble nucleation. Our experimental results show that due to the unique design of the fiber optic heater, the threshold laser power required to generate a bubble is greatly reduced and the bubble growth rate is significantly increased. By using the microvalve, stable vapor bubble generation in the microchannel is demonstrated, which does not require complex optical focusing and alignment. The generated vapor bubble is shown to successfully block a liquid flow channel with a size of 125 μm × 125 μm and a flow rate of ∼10 μl/min at ∼120 mW laser power.  相似文献   

13.
Microfluidic paper-based analytical devices (μPADs) are widely used for performing diagnostic assays. However, in many assays, time-delay valves are required to improve the sensitivity and specificity of the results. Accordingly, this study presents a simple, low-cost method for realizing time-delay valves using a color wax printing process. In the proposed approach, the time-delay effect is controlled through a careful selection of both the color and the saturation of the wax content. The validity of the proposed method is demonstrated by performing nitrite and oxalate assays using both a simple two-dimensional μPAD and a three-dimensional μPAD incorporating a colored wax-printed timer. The experimental results confirm that the flow time can be controlled through an appropriate selection of the color and the wax content. In addition, it is shown that nitrite and oxalate assays can be performed simultaneously on a single device. In general, the results presented in this study show that the proposed μPADs provide a feasible low-cost alternative to conventional methods for performing diagnostic assays.  相似文献   

14.
Recent studies have shown that there are systematic differences among total and free prostate-specificantigen (PSA) immunoassays. In this study we analyzedintermethod differences in total PSA (tPSA) and free PSA(fPSA) measurement using ARCHITECT i2000SR (Abbott Diagnostics) and COBAS E601 (Roche Diagnostics). A number of 160 blood samples were tested for tPSA and 50 samples for fPSA (selecting only sampleswith tPSA: 4.1–10.0 μg/L). Passing–Bablok regression analysis was used to compare the two analytical methods fortPSA, fPSA and percentage of fPSA (%fPSA). A strong correlation was noticed between ARCHITECT i2000SR and COBAS E601 for tPSA, fPSA and %fPSA (r between 0.94 and 0.99). Concentrations of tPSA and fPSA measured by COBAS E601 were higher thanthose measured by ARCHITECT i2000SR with a bias of 0.8 μg/L for tPSA and 0.14 μg/L for fPSA. Analyzing therelative difference between methods for fPSA and %fPSA, COBAS E601 exceed a 10% relative difference limit. Our study confirms that there are differences in measured concentrations of tPSA and fPSA byvarious commercial methods. Because clinical judgment on subsequent diagnostic procedures, such as prostatebiopsy, is based on tPSA and fPSA results, tests harmonization should be a priority.  相似文献   

15.
A novel microstirring strategy is applied to accelerate the digestion rate of the substrate Nα-benzoyl-L-arginine-4-nitroanilide (L-BAPA) catalyzed by sol-gel encapsulated trypsin. We use an ac nonlinear electrokinetic vortex flow to stir the solution in a microfluidic reaction chamber to reduce the diffusion length between the immobilized enzyme and substrate in the solution. High-intensity nonlinear electroosmotic microvortices, with angular speeds in excess of 1 cm∕s, are generated around a small (∼1.2 mm) conductive ion exchange granule when ac electric fields (133 V∕cm) are applied across a miniature chamber smaller than 10 μl. Coupling between these microvortices and the on-and-off electrophoretic motion of the granule in low frequency (0.1 Hz) ac fields produces chaotic stream lines to stir substrate molecules sufficiently. We demonstrate that, within a 5-min digestion period, the catalytic reaction rate of immobilized trypsin increases almost 30-fold with adequate reproducibility (15%) due to sufficient stirring action through the introduction of the nonlinear electrokinetic vortices. In contrast, low-frequency ac electroosmotic flow without the granule, provides limited stirring action and increases the reaction rate approximately ninefold with barely acceptable reproducibility (30%). Dye molecules are used to characterize the increases in solute diffusivity in the reaction reservoir in which sol-gel particles are placed, with and without the presence of granule, and compared with the static case. The solute diffusivity enhancement data show respective increases of ∼30 and ∼8 times, with and without the presence of granule. These numbers are consistent with the ratios of the enhanced reaction rate.  相似文献   

16.
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   

17.
A novel microflow cytometer is proposed in which the particles are focused in the horizontal and vertical directions by means of the Saffman shear lift force generated within a micro-weir microchannel. The proposed device is fabricated on stress-relieved glass substrates and is characterized both numerically and experimentally using fluorescent particles with diameters of 5 μm and 10 μm, respectively. The numerical results show that the micro-weir structures confine the particle stream to the center of the microchannel without the need for a shear flow. Moreover, the experimental results show that the particles emerging from the micro-weir microchannel pass through the detection region in a one-by-one fashion. The focusing effect of the micro-weir microchannel is quantified by computing the normalized variance of the optical detection signal intensity. It is shown that the focusing performance of the micro-weir structure is equal to 99.76% and 99.57% for the 5-μm and 10-μm beads, respectively. Overall, the results presented in this study confirm that the proposed microcytometer enables the reliable sorting and counting of particles with different diameters.  相似文献   

18.
A wide range of diseases and conditions are monitored or diagnosed from blood plasma, but the ability to analyze a whole blood sample with the requirements for a point-of-care device, such as robustness, user-friendliness, and simple handling, remains unmet. Microfluidics technology offers the possibility not only to work fresh thumb-pricked whole blood but also to maximize the amount of the obtained plasma from the initial sample and therefore the possibility to implement multiple tests in a single cartridge. The microfluidic design presented in this paper is a combination of cross-flow filtration with a reversible electroosmotic flow that prevents clogging at the filter entrance and maximizes the amount of separated plasma. The main advantage of this design is its efficiency, since from a small amount of sample (a single droplet  ～ 10 μl) almost 10% of this (approx 1 μl) is extracted and collected with high purity (more than 99%) in a reasonable time (5–8 min). To validate the quality and quantity of the separated plasma and to show its potential as a clinical tool, the microfluidic chip has been combined with lateral flow immunochromatography technology to perform a qualitative detection of the thyroid-stimulating hormone and a blood panel for measuring cardiac Troponin and Creatine Kinase MB. The results from the microfluidic system are comparable to previous commercial lateral flow assays that required more sample for implementing fewer tests.  相似文献   

19.
Microfluidic paper-based analytical devices (μPADs) are a promising solution for a wide range of point-of-care applications. The feasibility of inducing ion concentration polarization (ICP) on μPADs has thus far attracted little attention. Accordingly, this study commences by demonstrating the ICP phenomenon in a μPAD with a Nafion ion-selective membrane. We are the first to measure the current-voltage curve on a Nafion-coated μPAD in order to indicate that the ion depletion occurs and the ICP is triggered when the current reaches the limiting current. The ICP effect is then exploited to preconcentrate fluorescein on μPADs incorporating straight and convergent channels. By an optimal geometric design, it is shown that the convergent channel results in a greater preconcentration effect than the straight channel. Specifically, a 20-fold enhancement in the sample concentration is achieved after 130 s given an initial concentration of 10?5 M and an external potential of 50 V. By contrast, the straight channel yields only a 10-fold improvement in the concentration after 180 s. Further, the practical feasibility of the proposed convergent-channel μPAD is demonstrated using fluorescein isothiocyanate labeled bovine serum albumin. The experimental results show that a 15-fold enhancement of the initial sample concentration (10?5 M) is obtained after 120 s given an external potential of 50 V.  相似文献   

20.
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   

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