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1.
Nam J  Lim H  Kim C  Yoon Kang J  Shin S 《Biomicrofluidics》2012,6(2):24120-2412010
This study presents a method for density-based separation of monodisperse encapsulated cells using a standing surface acoustic wave (SSAW) in a microchannel. Even though monodisperse polymer beads can be generated by the state-of-the-art technology in microfluidics, the quantity of encapsulated cells cannot be controlled precisely. In the present study, mono-disperse alginate beads in a laminar flow can be separated based on their density using acoustophoresis. A mixture of beads of equal sizes but dissimilar densities was hydrodynamically focused at the entrance and then actively driven toward the sidewalls by a SSAW. The lateral displacement of a bead is proportional to the density of the bead, i.e., the number of encapsulated cells in an alginate bead. Under optimized conditions, the recovery rate of a target bead group (large-cell-quantity alginate beads) reached up to 97% at a rate of 2300 beads per minute. A cell viability test also confirmed that the encapsulated cells were hardly damaged by the acoustic force. Moreover, cell-encapsulating beads that were cultured for 1 day were separated in a similar manner. In conclusion, this study demonstrated that a SSAW can successfully separate monodisperse particles by their density. With the present technique for separating cell-encapsulating beads, the current cell engineering technology can be significantly advanced.  相似文献   

2.
Understanding the mechanical properties of optically transparent polydimethylsiloxane (PDMS) microchannels was essential to the design of polymer-based microdevices. In this experiment, PDMS microchannels were filled with a 100 μM solution of rhodamine 6G dye at very low Reynolds numbers (∼10−3). The deformation of PDMS microchannels created by pressure-driven flow was investigated by fluorescence microscopy and quantified the deformation by the linear relationship between dye layer thickness and intensity. A line scan across the channel determined the microchannel deformation at several channel positions. Scaling analysis widely used to justify PDMS bulging approximation was allowed when the applied flow rate was as high as 2.0 μl/min. The three physical parameters (i.e., flow rate, PDMS wall thickness, and mixing ratio) and the design parameter (i.e., channel aspect ratio = channel height/channel width) were considered as critical parameters and provided the different features of pressure distributions within polymer-based microchannel devices. The investigations of the four parameters performed on flexible materials were carried out by comparison of experiment and finite element method (FEM) results. The measured Young''s modulus from PDMS tensile test specimens at various circumstances provided reliable results for the finite element method. A thin channel wall, less cross-linker, high flow rate, and low aspect ratio microchannel were inclined to have a significant PDMS bulging. Among them, various mixing ratios related to material property and aspect ratios were one of the significant factors to determine PDMS bulging properties. The measured deformations were larger than the numerical simulation but were within corresponding values predicted by the finite element method in most cases.  相似文献   

3.
The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10–100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning.  相似文献   

4.
Here, we utilize microfluidic droplet technology to generate photopolymerizeable polyethylene glycol (PEG) hydrogel microbeads incorporating a fluorescence-based glucose bioassay. A microfluidic T-junction and multiphase flow of fluorescein isothiocyanate dextran, tetramethyl rhodamine isothiocyanate concanavalin A, and PEG in water were used to generate microdroplets in a continuous stream of hexadecane. The microdroplets were photopolymerized mid-stream with ultraviolet light exposure to form PEG microbeads and were collected at the outlet for further analysis. Devices were prototyped in PDMS and generated highly monodisperse 72 ± 2 μm sized microbeads (measured after transfer into aqueous phase) at a continuous flow rate between 0.04 ml/h—0.06 ml/h. Scanning electron microscopy analysis was conducted to analyze and confirm microbead integrity and surface morphology. Glucose sensing was carried out using a Förster resonance energy transfer (FRET) based assay. A proportional fluorescence intensity increase was measured within a 1–10 mM glucose concentration range. Microfluidically synthesized microbeads encapsulating sensing biomolecules offer a quick and low cost method to generate monodisperse biosensors for a variety of applications including cell cultures systems, tissue engineering, etc.  相似文献   

5.
Droplet microfluidics is a powerful method used to characterize chemical reactions at high throughput. Often detection is performed via in-line optical readout, which puts high demands on the detection system or makes detection of low concentration substrates challenging. Here, we have developed a droplet acoustofluidic chip for time-controlled reactions that can be combined with off-line optical readout. The principle of the platform is demonstrated by the enzymatic conversion of fluorescein diphosphate to fluorescein by alkaline phosphatase. The novelty of this work is that the time of the enzymatic reaction is controlled by physically removing the enzymes from the droplets instead of using chemical inhibitors. This is advantageous as inhibitors could potentially interact with the readout. Droplets containing substrate were generated on the chip, and enzyme-coupled microbeads were added into the droplets via pico-injection. The reaction starts as soon as the enzyme/bead complexes are added, and the reaction is stopped when the microbeads are removed from the droplets at a channel bifurcation. The encapsulated microbeads were focused in the droplets by acoustophoresis during the split, leaving the product in the side daughter droplet to be collected for the analysis (without beads). The time of the reaction was controlled by using different outlets, positioned at different lengths from the pico-injector. The enzymatic conversion could be measured with fluorescence readout in a separate PDMS based assay chip. We show the ability to perform time-controlled enzymatic assays in droplet microfluidics coupled to an off-line optical readout, without the need of enzyme inhibitors.  相似文献   

6.
This paper reports the use of a recent composite material, noted hereafter i-PDMS, made of carbonyl iron microparticles mixed in a PolyDiMethylSiloxane (PDMS) matrix, for magnetophoretic functions such as capture and separation of magnetic species. We demonstrated that this composite which combine the advantages of both components, can locally generate high gradients of magnetic field when placed between two permanent magnets. After evaluating the magnetic susceptibility of the material as a function of the doping ratio, we investigated the molding resolution offered by i-PDMS to obtain microstructures of various sizes and shapes. Then, we implemented 500 μm i-PDMS microstructures in a microfluidic channel and studied the influence of flow rate on the deviation and trapping of superparamagnetic beads flowing at the neighborhood of the composite material. We characterized the attraction of the magnetic composite by measuring the distance from the i-PDMS microstructure, at which the beads are either deviated or captured. Finally, we demonstrated the interest of i-PDMS to perform magnetophoretic functions in microsystems for biological applications by performing capture of magnetically labeled cells.  相似文献   

7.
We present a 91 MHz surface acoustic wave resonator with integrated microfluidics that includes a flow focus, an expansion region, and a binning region in order to manipulate particle trajectories. We demonstrate the ability to change the position of the acoustic nodes by varying the electronic phase of one of the transducers relative to the other in a pseudo-static manner. The measurements were performed at room temperature with 3 μm diameter latex beads dispersed in a water-based solution. We demonstrate the dependence of nodal position on pseudo-static phase and show simultaneous control of 9 bead streams with spatial control of −0.058 μm/deg ± 0.001 μm/deg. As a consequence of changing the position of bead streams perpendicular to their flow direction, we also show that the integrated acoustic-microfluidic device can be used to change the trajectory of a bead stream towards a selected bin with an angular control of 0.008 deg/deg ± 0.000(2) deg/deg.  相似文献   

8.
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   

9.
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   

10.
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   

11.
Label-free isolation of single cells is essential for the growing field of single-cell analysis. Here, we present a device which prints single living cells encapsulated in free-flying picoliter droplets. It combines inkjet printing and impedance flow cytometry. Droplet volume can be controlled in the range of 500 pl–800 pl by piezo actuator displacement. Two sets of parallel facing electrodes in a 50 μm × 55 μm channel are applied to measure the presence and velocity of a single cell in real-time. Polystyrene beads with <5% variation in diameter generated signal variations of 12%–17% coefficients of variation. Single bead efficiency (i.e., printing events with single beads vs. total number of printing events) was 73% ± 11% at a throughput of approximately 9 events/min. Viability of printed HeLa cells and human primary fibroblasts was demonstrated by culturing cells for at least eight days.  相似文献   

12.
Water-in-oil microdroplets offer microreactors for compartmentalized biochemical reactions with high throughput. Recently, the combination with a sol-gel switch ability, using agarose-in-oil microdroplets, has increased the range of possible applications, allowing for example the capture of amplicons in the gel phase for the preservation of monoclonality during a PCR reaction. Here, we report a new method for generating such agarose-in-oil microdroplets on a microfluidic device, with minimized inlet dead volume, on-chip cooling, and in situ monitoring of biochemical reactions within the gelified microbeads. We used a flow-focusing microchannel network and successfully generated agarose microdroplets at room temperature using the “push-pull” method. This method consists in pushing the oil continuous phase only, while suction is applied to the device outlet. The agarose phase present at the inlet is thus aspirated in the device, and segmented in microdroplets. The cooling system consists of two copper wires embedded in the microfluidic device. The transition from agarose microdroplets to microbeads provides additional stability and facilitated manipulation. We demonstrate the potential of this method by performing on-chip a temperature-triggered DNA isothermal amplification in agarose microbeads. Our device thus provides a new way to generate microbeads with high throughput and no dead volume for biochemical applications.  相似文献   

13.
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   

14.
We present a simple microchip device consisting of an overlaid pattern of micromagnets and microwells capable of capturing magnetically labeled cells into well-defined compartments (with accuracies >95%). Its flexible design permits the programmable deposition of single cells for their direct enumeration and pairs of cells for the detailed analysis of cell-cell interactions. This cell arraying device requires no external power and can be operated solely with permanent magnets. Large scale image analysis of cells captured in this array can yield valuable information (e.g., regarding various immune parameters such as the CD4:CD8 ratio) in a miniaturized and portable platform.The emergent need for point-of-care devices has spurred development of simplified platforms to organize cells across well-defined templates.1 These devices employ passive microwells, immunospecific adhesive islands, and electric, optical, and acoustic traps to manipulate cells.2–6 In contrast, magnetic templating can control the spatial organization of cells through its ability to readily program ferromagnetic memory states.7 While it has been applied to control the deposition of magnetic beads,8–13 it has not been used to direct the deposition of heterogeneous cell pairs, which may help provide critical insight into the function of single cells.14,15 As such, we developed a simple magnetographic device capable of arraying single cells and pairs of cells with high fidelity. We show this magnetic templating tool can use immunospecific magnetic labels for both the isolation of cells from blood and their organization into spatially defined wells.We used standard photolithographic techniques to fabricate the microchips (see supplementary material16). Briefly, an array of 10 × 30 μm cobalt micromagnets were patterned by a photolithographic liftoff process and overlaid with a pattern of dumbbell-shaped microwells formed in SU-8 photoresist (Fig. 1(a)). The micromagnets were designed to produce a predominantly vertical field in the microwells by aligning the ends of the micromagnet at the center of each well of the dumbbell. These features were deposited across an area of ≈400 mm2 (>50 000 well pairs per microchip) (Fig. 1(b)). Depending on the programmed magnetization state with respect to the external field, magnetic beads or cells were attracted to one pole and repelled by the other pole of each micromagnet, leading to a biased deposition (Fig. 1(c)).12Open in a separate windowFIG. 1.Magnetographic array for single cell analysis. (a) SEM image of the dumbbell-shaped well pairs for capturing magnetically labelled cells. (b) Photograph of the finished device. (c) An array of well pairs displaying a pitch of 60 × 120 μm before (top) and 10 min after the deposition of magnetic beads (bottom).To demonstrate the capability of the array to capture cells into a format amenable for rapid image processing, we organized CD3+ lymphocytes using only hand-held permanent magnets. We isolated CD3+ lymphocytes from blood via positive selection using anti-CD3 magnetic nanoparticles (EasySep™, STEMCELL Technologies) with purities confirmed by flow cytometry (97.8%; see supplementary material16). We then stained 1 × 106 CD3+ cells with anti-CD8 Alexa-488 and anti-CD4 Alexa-647 (5 μl of each antibody in 100 μl for 20 min; BD Bioscience) to determine the CD4:CD8 ratio, a prognostic ratio for assessing the immune system.17,18Variably spaced neodymium magnets (0.5 in. × 0.5 in. × 1 in.; K&J Magnetics, Inc.) were fixed on either side of the microchip to generate a tunable magnetic field (0–400 G; Fig. 2(a)). Using this setup, fluorescently labeled cells were deposited, and the populations of CD4+ and CD8+ cells were indiscriminately arrayed, imaged, and enumerated using ImageJ. The resulting CD4:CD8 ratio of 1.84 ± 0.18 (Fig. 2(b)) was confirmed by flow cytometry with a high correlation (5.4% difference; Fig. 2(c)), indicating the magnetographic microarray can pattern cells for the rapid and accurate assessment of critical phenotypical parameters without complex equipment (e.g., function generators or flow cytometers).Open in a separate windowFIG. 2.CD8 analysis of CD3+ lymphocytes. (a) Photograph of the magnetographic device activated by permanent magnets (covered with green tape). The CD4:CD8 ratio determined by the (b) magnetographic microarray and (c) and (d) flow cytometry was 1.84 and 1.74, respectively.More complex operations, such as the programmed deposition of cell pairs, can be achieved by leveraging the switchable, bistable magnetization of the micromagnets for the detailed studies of cell-cell interactions (Figs. 3(a)–3(d)).12 For these studies, a 200 G horizontal field generated from an electromagnetic coil was used to magnetize the micromagnets.19 We then captured different concentrations of magnetic beads as surrogates for cells (8.4 μm polystyrene, Spherotech, Inc.) and found that higher bead concentrations did not affect the capture accuracy (>95%; see supplementary material16).Open in a separate windowFIG. 3.Programmed pairing of magnetic beads and CD3+ lymphocytes. (a) Schematic of the magnetographic cell pair isolations. (b) Polarized micromagnets isolate cells of one type to one side in a vertical magnetic field and then cells of a second type to the other side when the field is reversed. (c) Fluorescent image of magnetically trapped green stained (top) and red stained (bottom) cell pairs. (d) SEM image of magnetically labeled cells in the microwells. (e) Capture accuracy of magnetic bead pairs. (Each color (and shape) represents the field strength of the reversed field.) (f) Change in the capture accuracy (loss) of initially captured beads after reversing the magnetic field. The capture accuracy of (g) magnetically labeled cell pairs and (h) the second magnetically labeled cell (for (e)–(h): n = 5; time starts from the deposition of the second set of cells or beads).The opposite side of each micromagnet was then populated with the second (yellow fluorescent) bead by reversing the direction of the applied magnetic field. We tested several field strengths (i.e., 10, 25, 40, or 55 G) to optimize the conditions for isolating the desired bead in the opposite well without ejecting the first bead. If the field strength was too large, the previously deposited beads could be ejected from their wells due to the repulsive magnetic force overcoming gravity.12 As shown in Figure 3(e), increasing the field strength from 10 to 25 G significantly increased the capture accuracy at 60 min from the deposition of the second bead (p < 0.01), but increases from 25 to 55 G did not affect the capture accuracy (p > 0.10). As shown in Figure 3(f), higher field strengths (i.e., 40 and 55 G) resulted in lower capture accuracies compared to lower field strengths (i.e., 10 and 25 G) (p < 0.01), which was primarily due to ejection of the initially captured beads when the micromagnets reversed their polarity.We then arranged pairs of membrane dyed (calcein AM, Invitrogen; PKH26, Sigma) magnetically labeled CD3+ lymphocytes. First, red stained cells (150 μl of 2 × 104 cells/ml) were deposited on the microchip in the presence of 250 G vertical magnetic field. After 20 min, the field was reversed (i.e., to 40, 55, and 70 G) and green stained cells (150 μl of 2 × 104 cells/ml) were deposited on the microchip with images taken in 10 min intervals. Fluorescence images were overlaid (Fig. 3(c)) and the capture accuracy of cell pairs was determined (ImageJ).As seen in Figure 3(g), the capture accuracy of pairs of CD3+ lymphocytes was lower than that of magnetic beads (Fig. 3(e)). However, as shown in Figure 3(h), the second set of cells (green fluorescent) exhibited an average capture accuracy of 91.8% ± 1.9%. This indicates that the lower capture accuracy of cell pairs was either due to the ejection of initially captured (red fluorescent) cells or the migration of initially captured cells through the connecting channel, resulting from their relatively high deformability compared to magnetic beads.In summary, we developed a simple device capable of organizing magnetic particles, cells, and pairs of cells into well-defined compartments. A major advantage of this system is the use of specific magnetic labels to both isolate cells and program their deposition. While the design of this device does not enable dynamic control of the spacing between captured cell pairs as does some dielectrophoresis-based devices,20 it can easily capture cells with high fidelity using only permanent magnets and has clinical relevance in the assessment of immune parameters. These demonstrations potentiate a relatively simple and robust device where highly organized spatial arrangement of cells facilitates rapid and accurate analyses towards a functional and low-cost point-of-care device.  相似文献   

15.
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   

16.
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   

17.
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   

18.
The advent and dissemination of next-generation sequencing (NGS) technologies such as Illumina''s sequencing platforms has brought forth vast reductions in the cost, time, and technical difficulties associated with DNA and RNA sequencing. Despite this trend, the workflow required to generate nucleic acid libraries for sequencing remains time-consuming and laborious. The following research proposes a method for simplifying and streamlining this process by replacing the manual washing steps of the common magnetic bead-based cleanup with a novel microfluidic method by integrating magnetic separation and electrokinetic purification (MSEP). Requiring no pumps, pipette mixing, vortexing, or centrifugation, MSEP relies on selective adsorption of target DNA onto the magnetic beads with subsequent transport of beads through a microchannel undergoing an antiparallel electroosmotic flow. The synergetic flow conditions were optimized using a simple electrohydrodynamic flow model. This work demonstrates that MSEP is as effective in eliminating adapter-dimers from the post-ligation library mix as the manual method while also greatly reducing the hands-on time and amount of pipetting required. Although MSEP has been applied specifically toward NGS library preparation at this time, it has the potential to be adapted and employed for any bead-based separation scheme, namely, solid phase extraction, sequence-specific hybridization, and immunoprecipitation on a microscale.  相似文献   

19.
This paper presents the design, fabrication, and testing of a magnetophoretic bioseparation chip for the rapid isolation and concentration of CD4 + T cells from the peripheral blood. In a departure from conventional magnetic separation techniques, this microfluidic-based bioseperation device has several unique features, including locally engineered magnetic field gradients and a continuous flow with a buffer switching scheme to improve the performance of the separation process. Additionally, the chip is capable of processing significantly smaller sample volumes than conventional methods and sample losses are eliminated due to decreased handling. Furthermore, the possibility of sample-to-sample contamination is reduced with the disposable format. The overall dimensions of the device were 22 mm by 60 mm by 1 mm, approximately the size of a standard microscope slide. The results indicate a cell purity of greater than 95% at a sample flow rate of 50 ml/h and a cell recovery of 81% at a sample flow rate of 10 ml/h. The cell purity was found to increase with increasing the sample flow rate. However, the cell recovery decreases with an increase in the flow rate. A parametric study was also performed to investigate the effects of channel height, substrate thickness, magnetic bead size, and number of beads per cell on the cell separation performance.  相似文献   

20.
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   

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