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1.
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   

2.
Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels.  相似文献   

3.
Li Y  Fu YQ  Brodie SD  Alghane M  Walton AJ 《Biomicrofluidics》2012,6(1):12812-128129
This paper presents integrated microfluidic lab-on-a-chip technology combining surface acoustic wave (SAW) and electro-wetting on dielectric (EWOD). This combination has been designed to provide enhanced microfluidic functionality and the integrated devices have been fabricated using a single mask lithographic process. The integrated technology uses EWOD to guide and precisely position microdroplets which can then be actuated by SAW devices for particle concentration, acoustic streaming, mixing and ejection, as well as for sensing using a shear-horizontal wave SAW device. A SAW induced force has also been employed to enhance the EWOD droplet splitting function.  相似文献   

4.
We present an integrated microfluidic chip for detection of β-amyloid (Aβ) peptides. Aβ peptides are major biomarkers for the diagnosis of Alzheimer''s disease (AD) in its early stages. This microfluidic device consists of three main parts: (1) An immunocapture microcolumn based on self-assembled magnetic beads coated with antibodies specific to Aβ peptides, (2) a nano-porous membrane made of photopolymerized hydrogel for preconcentration, and (3) a microchip electrophoresis (MCE) channel with fluorescent detection. Sub-milliliter sample volume is either mixed off-chip with antibody coated magnetic beads and injected into the device or is injected into an already self-assembled column of magnetic beads in the microchannel. The captured peptides on the beads are then electrokinetically eluted and re-concentrated onto the nano-membrane in a few nano-liters. By integrating the nano-membrane, total assay time was reduced and also off-chip re-concentration or buffer exchange steps were not needed. Finally, the concentrated peptides in the chip are separated by electrophoresis in a polymer-based matrix. The device was applied to the capture and MCE analysis of differently truncated peptides Aβ (1–37, 1–39, 1–40, and 1–42) and was able to detect as low as 25 ng of synthetic Aβ peptides spiked in undiluted cerebrospinal fluid (CSF). The device was also tested with CSF samples from healthy donors. CSF samples were fluorescently labelled and pre-mixed with the magnetic beads and injected into the device. The results indicated that Aβ1-40, an important biomarker for distinguishing patients with frontotemporal lobe dementia from controls and AD patients, was detectable. Although the sensitivity of this device is not yet enough to detect all Aβ subtypes in CSF, this is the first report on an integrated or semi-integrated device for capturing and analyzing of differently truncated Aβ peptides. The method is less demanding and faster than the conventional Western blotting method currently used for research.  相似文献   

5.
The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today''s diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein.  相似文献   

6.
Digital microfluidics is an elegant technique based on single droplets for the design, composition, and manipulation of microfluidic systems. In digital microfluidics, especially in the electrowetting on dielectric (EWOD) system, each droplet acts as an independent reactor, which enables a wide range of multiple parallel biological and chemical reactions at the microscale. EWOD digital microfluidics reduces reagent and energy consumption, accelerates analysis, enables point-of-care diagnostic, simplifies integration with sensors, etc. Such a digital microfluidic system is especially relevant for droplet digital PCR (ddPCR), thanks to its nanoliter droplets and well-controlled volume distribution. At low DNA concentration, these small volumes allow less than one DNA strand per droplet on average (limited dilution) so that after a fixed number of PCR cycles (endpoint PCR), only the DNA in droplets containing the sequence of interest has been amplified and can be detected by fluorescence to yield an accurate count of the sequences of interest using statistical models. Focusing on ddPCR, this article summarizes the latest development and research on EWOD technology for droplet PCR over the last decade.  相似文献   

7.
Oscillating electrowetting on dielectrics (EWOD) with coplanar electrodes is investigated in this paper as a way to provide efficient stirring within a drop with biological content. A supporting model inspired from Ko et al. [Appl. Phys. Lett. 94, 194102 (2009)] is proposed allowing to interpret oscillating EWOD-induced drop internal flow as the result of a current streaming along the drop surface deformed by capillary waves. Current streaming behaves essentially as a surface flow generator and the momentum it sustains within the (viscous) drop is even more significant as the surface to volume ratio is small. With the circular electrode pair considered in this paper, oscillating EWOD sustains toroidal vortical flows when the experiments are conducted with aqueous drops in air as ambient phase. But when oil is used as ambient phase, it is demonstrated that the presence of an electrode gap is responsible for a change in drop shape: a pinch-off at the electrode gap yields a peanut-shaped drop and a symmetry break-up of the EWOD-induced flow pattern. Viscosity of oil is also responsible for promoting an efficient damping of the capillary waves which populate the surface of the actuated drop. As a result, the capillary network switches from one standing wave to two superimposed traveling waves of different mechanical energy, provided that actuation frequency is large enough, for instance, as large as the one commonly used in electrowetting applications (f ∼ 500 Hz and beyond). Special emphasis is put on stirring of biological samples. As a typical application, it is demonstrated how beads or cell clusters can be focused under flow either at mid-height of the drop or near the wetting plane, depending on how the nature of the capillary waves is (standing or traveling), and therefore, depending on the actuation frequency (150 Hz–1 KHz).  相似文献   

8.
Determination of amyloid β (Aβ) isoforms and in particular the proportion of the Aβ 1-42 isoform in cerebrospinal fluid (CSF) of patients suspected of Alzheimer's disease might help in early diagnosis and treatment of that illness. Due to the low concentration of Aβ peptides in biological fluids, a preconcentration step prior to the detection step is often necessary. This study utilized on-chip immunoprecipitation, known as micro-immunoprecipitation (μIP). The technique uses an immunosorbent (IS) consisting of magnetic beads coated with specific anti-Aβ antibodies organized into an affinity microcolumn by a magnetic field. Our goal was to thoroughly describe the critical steps in developing the IS, such as selecting the proper beads and anti-Aβ antibodies, as well as optimizing the immobilization technique and μIP protocol. The latter includes selecting optimal elution conditions. Furthermore, we demonstrate the efficiency of anti-Aβ IS for μIP and specific capture of 5 Aβ peptides under optimized conditions using various subsequent analytical methods, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), capillary electrophoresis, microchip electrophoresis, and immunoblotting. Synthetic Aβ peptides samples prepared in buffer and spiked in human CSF were analyzed. Finally, on-chip immunoprecipitation of Aβ peptides in human CSF sample was performed.  相似文献   

9.
A microfluidic device based on a pair of slant-finger interdigital transducers (SFITs) is developed to achieve a selective and flexible manipulation of microbubbles (MBs) by surface acoustic waves (SAWs). The resonance frequency of SAWs generated by the SFITs depends on the location of its parallel pathway; the particles at different locations of the SAWs'' pathway can be controlled selectively by choosing the frequency of the excitation signal applied on the SFITs. By adjusting the input signal continuously, MBs can be transported along the acoustic aperture precisely. The displacement of MBs has a linear relationship with the frequency shift. The resolution of transportation is 15.19 ± 2.65 μm when the shift of input signal frequency is at a step of 10 kHz. In addition, the MBs can be controlled in a two-dimensional plane by combining variations of the frequency and the relative phase of the excitation signal applied on the SFITs simultaneously. This technology may open up the possibility of selectively and flexibly manipulating MBs using a simple one-dimensional device.  相似文献   

10.
Dielectric breakdown is a common problem in a digital microfluidic system, which limits its application in chemical or biomedical applications. We propose a new fabrication of an electrowetting-on-dielectric (EWOD) device using Si3N4 deposited by low-pressure chemical vapor deposition (LPCVD) as a dielectric layer. This material exhibits a greater relative permittivity, purity, uniformity, and biocompatibility than polymeric films. These properties also increase the breakdown voltage of a dielectric layer and increase the stability of an EWOD system when applied in biomedical research. Medium droplets with mouse embryos were manipulated in this manner. The electrical properties of the Si3N4 dielectric layer—breakdown voltage, refractive index, relative permittivity, and variation of contact angle with input voltage—were investigated and compared with a traditional Si3N4 dielectric layer deposited as a plasma-enhanced chemical vapor deposition to confirm the potential of LPCVD Si3N4 applied as the dielectric layer of an EWOD digital microfluidic system.  相似文献   

11.
Determination of amyloid β (Aβ) isoforms and in particular the proportion of the Aβ 1-42 isoform in cerebrospinal fluid (CSF) of patients suspected of Alzheimer’s disease might help in early diagnosis and treatment of that illness. Due to the low concentration of Aβ peptides in biological fluids, a preconcentration step prior to the detection step is often necessary. This study utilized on-chip immunoprecipitation, known as micro-immunoprecipitation (μIP). The technique uses an immunosorbent (IS) consisting of magnetic beads coated with specific anti-Aβ antibodies organized into an affinity microcolumn by a magnetic field. Our goal was to thoroughly describe the critical steps in developing the IS, such as selecting the proper beads and anti-Aβ antibodies, as well as optimizing the immobilization technique and μIP protocol. The latter includes selecting optimal elution conditions. Furthermore, we demonstrate the efficiency of anti-Aβ IS for μIP and specific capture of 5 Aβ peptides under optimized conditions using various subsequent analytical methods, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), capillary electrophoresis, microchip electrophoresis, and immunoblotting. Synthetic Aβ peptides samples prepared in buffer and spiked in human CSF were analyzed. Finally, on-chip immunoprecipitation of Aβ peptides in human CSF sample was performed.  相似文献   

12.
Homogeneous assays possess important advantages that no washing or physical separation is required, contributing to robust protocols and easy implementation which ensures potential point-of-care applications. Optimizing the detection strategy to reduce the number of reagents used and simplify the detection device is desirable. A method of homogeneous bead-agglutination assay based on micro-chip sheathless flow cytometry has been developed. The detection processes include mixing the capture-probe conjugated beads with an analyte containing sample, followed by flowing the reaction mixtures through the micro-chip sheathless flow cytometric device. The analyte concentrations were detected by counting the proportion of monomers in the reaction mixtures. Streptavidin-coated magnetic beads and biotinylated bovine serum albumin (bBSA) were used as a model system to verify the method, and detection limits of 0.15 pM and 1.5 pM for bBSA were achieved, using commercial Calibur and the developed micro-chip sheathless flow cytometric device, respectively. The setup of the micro-chip sheathless flow cytometric device is significantly simple; meanwhile, the system maintains relatively high sensitivity, which mainly benefits from the application of forward scattering to distinguish aggregates from monomers. The micro-chip sheathless flow cytometric device for bead agglutination detection provides us with a promising method for versatile immunoassays on microfluidic platforms.  相似文献   

13.
This study reports an integrated microfluidic system capable of isolation, counting, and sorting of hematopoietic stem cells (HSCs) from cord blood in an automatic format by utilizing a magnetic-bead-based immunoassay. Three functional modules, including cell isolation, cell counting, and cell sorting modules are integrated on a single chip by using microfluidic technology. The cell isolation module is comprised of a four-membrane-type micromixer for binding of target stem cells and magnetic beads, two pneumatic micropumps for sample transport, and an S-shaped channel for isolation of HSCs using a permanent magnet underneath. The counting and sorting of HSCs are performed by utilizing the cell counting and sorting modules. Experimental results show that a separation efficiency as high as 88% for HSCs from cord blood is achieved within 40 min for a sample volume of 100 μl. Therefore, the development of this integrated microfluidic system may be promising for various applications such as stem cell research and cell therapy.  相似文献   

14.
An automated, disk-based, enzyme-linked immunosorbent assay (ELISA) system is presented in this work. Magnetic beads were used as the antibody carriers to improve the assay sensitivity and shorten the reaction time. The magnetic module integrated on the system is capable of controlling the magnetic beads to either move in the incubation stage or immobilize at a specific location during washing stage. This controlling mechanism utilizes a passive controlling approach so that it can be performed through disk spinning without the need of active control from external devices. The movement of the magnetic beads was investigated and the optimal rotational speed was found to be related to the ratio of the processing time to the cycle time of the magnetic beads. Comparing to ELISA conducted on microtiter plates, similar test results could be achieved by the disk-based ELISA but the entire protocol can be finished automatically within 45 min with much less reagent consumption.  相似文献   

15.
In this paper, we demonstrate the possibility to trap and sort labeled cells under flow conditions using a microfluidic device with an integrated flat micro-patterned hard magnetic film. The proposed technique is illustrated using a cell suspension containing a mixture of Jurkat cells and HEK (Human Embryonic Kidney) 293 cells. Prior to sorting experiments, the Jurkat cells were specifically labeled with immunomagnetic nanoparticles, while the HEK 293 cells were unlabeled. Droplet-based experiments demonstrated that the Jurkat cells were attracted to regions of maximum stray field flux density while the HEK 293 cells settled in random positions. When the mixture was passed through a polydimethylsiloxane (PDMS) microfluidic channel containing integrated micromagnets, the labeled Jurkat cells were selectively trapped under fluid flow, while the HEK cells were eluted towards the device outlet. Increasing the flow rate produced a second eluate much enriched in Jurkat cells, as revealed by flow cytometry. The separation efficiency of this biocompatible, compact micro-fluidic separation chamber was compared with that obtained using two commercial magnetic cell separation kits.  相似文献   

16.
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   

17.
Manipulation of magnetic beads plays an increasingly important role in molecular diagnostics. Magnetophoresis is a promising technique for selective transportation of magnetic beads in lab-on-a-chip systems. We investigate periodic arrays of exchange-biased permalloy microstripes fabricated using a single lithography step. Magnetic beads can be continuously moved across such arrays by combining the spatially periodic magnetic field from microstripes with a rotating external magnetic field. By measuring and modeling the magnetophoresis properties of thirteen different stripe designs, we study the effect of the stripe geometry on the magnetophoretic transport properties of the magnetic microbeads between the stripes. We show that a symmetric geometry with equal width of and spacing between the microstripes facilitates faster transportation and that the optimal period of the periodic stripe array is approximately three times the height of the bead center over the microstripes.  相似文献   

18.
We present a method capable of rapidly (∼20 s) determining the density and mass of a single leukemic cell using an optically induced electrokinetics (OEK) platform. Our team had reported recently on a technique that combines sedimentation theory, computer vision, and micro particle manipulation techniques on an OEK microfluidic platform to determine the mass and density of micron-scale entities in a fluidic medium; the mass and density of yeast cells were accurately determined in that prior work. In the work reported in this paper, we further refined the technique by performing significantly more experiments to determine a universal correction factor to Stokes'' equation in expressing the drag force on a microparticle as it falls towards an infinite plane. Specifically, a theoretical model for micron-sized spheres settling towards an infinite plane in a microfluidic environment is presented, and which was validated experimentally using five different sizes of micro polystyrene beads. The same sedimentation process was applied to two kinds of leukemic cancer cells with similar sizes in an OEK platform, and their density and mass were determined accordingly. Our tests on mouse lymphocytic leukemia cells (L1210) and human leukemic cells (HL-60) have verified the practical viability of this method. Potentially, this new method provides a new way of measuring the volume, density, and mass of a single cell in an accurate, selective, and repeatable manner.  相似文献   

19.
Definable surface chemistry is essential for many applications of microfluidic polymer systems. However, small cross-section channels with a high surface to volume ratio enhance passive adsorption of molecules that depletes active molecules in solution and contaminates the channel surface. Here, we present a one-step photochemical process to coat the inner surfaces of closed microfluidic channels with a nanometer thick layer of poly(ethylene glycol) (PEG), well known to strongly reduce non-specific adsorption, using only commercially available reagents in an aqueous environment. The coating consists of PEG diacrylate (PEGDA) covalently grafted to polymer surfaces via UV light activation of the water soluble photoinitiator benzoyl benzylamine, a benzophenone derivative. The PEGDA coating was shown to efficiently limit the adsorption of antibodies and other proteins to <5% of the adsorbed amount on uncoated polymer surfaces. The coating could also efficiently suppress the adhesion of mammalian cells as demonstrated using the HT-29 cancer cell line. In a subsequent equivalent process step, protein in aqueous solution could be anchored onto the PEGDA coating in spatially defined patterns with a resolution of <15 μm using an inverted microscope as a projection lithography system. Surface patterns of the cell binding protein fibronectin were photochemically defined inside a closed microfluidic device that was initially homogeneously coated by PEGDA. The resulting fibronectin patterns were shown to greatly improve cell adhesion compared to unexposed areas. This method opens for easy surface modification of closed microfluidic systems through combining a low protein binding PEG-based coating with spatially defined protein patterns of interest.  相似文献   

20.
An acoustophoresis-based microfluidic flow-chip is presented as a novel platform to facilitate analysis of proteins and peptides loosely bound to the surface of beads or cells. The chip allows for direct removal of the background surrounding the beads or cells, followed by sequential treatment and collection of a sequence of up to five different buffer conditions. During this treatment, the beads/cells are retained in a single flow by acoustic radiation force. Eluted peptides are collected from the outlets and subsequently purified by miniaturized solid-phase extraction and analyzed with matrix assisted laser desorption mass spectrometry. Fundamental parameters such as the system fluidics and dispersion are presented. The device was successfully applied for wash and sequential elution of peptides bound to the surface of microbeads and human spermatozoa, respectively.  相似文献   

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