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1.
The emerging technologies on mobile-based diagnosis and bioanalytical detection have enabled powerful laboratory assays such as enzyme-linked immunosorbent assay (ELISA) to be conducted in field-use lab-on-a-chip devices. In this paper, we present a low-cost universal serial bus (USB)-interfaced mobile platform to perform microfluidic ELISA operations in detecting the presence and concentrations of BDE-47 (2,2′,4,4′-tetrabromodiphenyl ether), an environmental contaminant found in our food supply with adverse health impact. Our point-of-care diagnostic device utilizes flexible interdigitated carbon black electrodes to convert electric current into a microfluidic pump via gas bubble expansion during electrolytic reaction. The micropump receives power from a mobile phone and transports BDE-47 analytes through the microfluidic device conducting competitive ELISA. Using variable domain of heavy chain antibodies (commonly referred to as single domain antibodies or Nanobodies), the proposed device is sensitive for a BDE-47 concentration range of 10−3–104 μg/l, with a comparable performance to that uses a standard competitive ELISA protocol. It is anticipated that the potential impact in mobile detection of health and environmental contaminants will prove beneficial to our community and low-resource environments.  相似文献   

2.
In the era of the targeted therapy identification of EGFR mutation detection in lung cancer is extremely helpful to predict the treatment efficacy of EGFR tyrosine kinase inhibitors (TKIs). Unfortunately, the inadequacy and quality of the biopsy samples are the major obstacles in molecular testing of EGFR mutation in lung cancer. To address this issue, the present study intended to use liquid biopsy as the non-invasive method for EGFR mutation detection. A total of 31 patients with an advanced stage of lung cancer were enrolled in the study from which cell-free DNA (cfDNA) and FFPE tissue DNA was extracted. Extracted DNA samples were analyzed for further EGFR exon specific mutation analysis by ARMS-PCR. Data were analyzed statistically using SPSS software. In cfDNA samples, the prevalence of wild type EGFR was 48% while the prevalence of TKI resistant and TKI sensitive mutations were 3%. Conversely, in tissue DNA samples, the prevalence of wild type, TKI sensitive and TKI resistant mutations were 48%, 19%, and 3%, respectively. The overall concordance of EGFR mutation between cfDNA and tissue DNA was 83%. McNemar’s test revealed that there was no significant difference between EGFR expression of cfDNA and tissue DNA samples. Additionally, the significant-high incidence of TKI resistant mutations was observed in tobacco habituates, indicating the role of carcinogens present in the tobacco in developing resistant mutations. In conclusion, our data suggest that evaluation of EGFR mutation from cfDNA samples is practicable as a non-invasive tool in patients with advanced-stage of lung cancer.  相似文献   

3.
We employed direct-current electric fields (dcEFs) to modulate the chemotaxis of lung cancer cells in a microfluidic cell culture device that incorporates both stable concentration gradients and dcEFs. We found that the chemotaxis induced by a 0.5 μM/mm concentration gradient of epidermal growth factor can be nearly compensated by a 360 mV/mm dcEF. When the effect of chemical stimulation was balanced by the electrical drive, the cells migrated randomly, and the path lengths were largely reduced. We also demonstrated electrically modulated chemotaxis of two types of lung cancer cells with opposite directions of electrotaxis in this device.  相似文献   

4.
Cytokines are small proteins secreted by leukocytes in blood in response to infections, thus offering valuable diagnostic information. Given that the same cytokines may be produced by different leukocyte subsets in blood, it is beneficial to connect production of cytokines to specific cell types. In this paper, we describe integration of antibody (Ab) microarrays into a microfluidic device to enable enhanced cytokine detection. The Ab arrays contain spots specific to cell-surface antigens as well as anti-cytokine detection spots. Infusion of blood into a microfluidic device results in the capture of specific leukocytes (CD4 T-cells) and is followed by detection of secreted cytokines on the neighboring Ab spots using sandwich immunoassay. The enhancement of cytokine signal comes from leveraging the concept of reconfigurable microfluidics. A three layer polydimethylsiloxane microfluidic device is fabricated so as to contain six microchambers (1 mm × 1 mm × 30 μm) in the ceiling of the device. Once the T-cell capture is complete, the device is reconfigured by withdrawing liquid from the channel, causing the chambers to collapse onto Ab arrays and enclose cell/anti-cytokine spots within a 30 nl volume. In a set of proof-of-concept experiments, we demonstrate that ∼90% pure CD4 T-cells can be captured inside the device and that signals for three important T-cell secreted cytokines, tissue necrosis factor-alpha, interferon-gamma, and interleukin-2, may be enhanced by 2 to 3 folds through the use of reconfigurable microfluidics.  相似文献   

5.
In this paper, we present an on-chip hand-powered membrane pump using a robust patient-to-chip syringe interface. This approach enables safe sample collection, sample containment, integrated sharps disposal, high sample volume capacity, and controlled downstream flow with no electrical power requirements. Sample is manually injected into the device via a syringe and needle. The membrane pump inflates upon injection and subsequently deflates, delivering fluid to downstream components in a controlled manner. The device is fabricated from poly(methyl methacrylate) (PMMA) and silicone, using CO2 laser micromachining, with a total material cost of ∼0.20 USD/device. We experimentally demonstrate pump performance for both deionized (DI) water and undiluted, anticoagulated mouse whole blood, and characterize the behavior with reference to a resistor-capacitor electrical circuit analogy. Downstream output of the membrane pump is regulated, and scaled, by connecting multiple pumps in parallel. In contrast to existing on-chip pumping mechanisms that typically have low volume capacity (∼5 μL) and sample volume throughput (∼1–10 μl/min), the membrane pump offers high volume capacity (up to 240 μl) and sample volume throughput (up to 125 μl/min).  相似文献   

6.
In this report, we demonstrate a simple and low cost method that can be reproducibly used for fabrication of microfluidic devices in nitrocellulose. The fluidic patterns are created via a laser-based direct-write technique that induces polymerisation of a photo-polymer previously impregnated in the nitrocellulose. The resulting structures form hydrophobic barriers that extend through the thickness of the nitrocellulose and define an interconnected hydrophilic fluidic-flow pattern. Our experimental results show that using this method it is possible to achieve microfluidic channels with lateral dimensions of ∼100 μm using hydrophobic barriers that form the channel walls with dimensions of ∼60 μm; both of these values are considerably smaller than those that can be achieved with other current techniques used in the fabrication of nitrocellulose-based fluidic devices. A simple grid patterned nitrocellulose device was then used for the detection of C-reactive protein via a sandwich enzyme-linked immunosorbent assay, which served as a useful proof-of-principle experiment.  相似文献   

7.
An electro-osmosis (EOS) diode pumping platform capable of culturing cells in fluidic cellular micro-environments particularly at low volume flow rates has been developed. Diode pumps have been shown to be a viable alternative to mechanically driven pumps. Typically electrokinetic micro-pumps were limited to low-concentration solutions (≤10 mM). In our approach, surface mount diodes were embedded along the sidewalls of a microchannel to rectify externally applied alternating current into pulsed direct current power across the diodes in order to generate EOS flows. This approach has for the first time generated flows at ultra-low flow rates (from 2.0 nl/s to 12.3 nl/s) in aqueous solutions with concentrations greater than 100 mM. The range of flow was generated by changing the electric field strength applied to the diodes from 0.5 Vpp/cm to 10 Vpp/cm. Embedding an additional diode on the upper surface of the enclosed microchannel increased flow rates further. We characterized the diode pump-driven fluidics in terms of intensities and frequencies of electric inputs, pH values of solutions, and solution types. As part of this study, we found that the growth of A549 human lung cancer cells was positively affected in the microfluidic diode pumping system. Though the chemical reaction compromised the fluidic control overtime, the system could be maintained fully functional over a long time if the solution was changed every hour. In conclusion, the advantage of miniature size and ability to accurately control fluids at ultra-low volume flow rates can make this diode pumping system attractive to lab-on-a-chip applications and biomedical engineering in vitro studies.  相似文献   

8.
This paper presents a continuous flow microfluidic device for the separation of DNA from blood using magnetophoresis for biological applications and analysis. This microfluidic bio-separation device has several benefits, including decreased sample handling, smaller sample and reagent volumes, faster isolation time, and decreased cost to perform DNA isolation. One of the key features of this device is the use of short-range magnetic field gradients, generated by a micro-patterned nickel array on the bottom surface of the separation channel. In addition, the device utilizes an array of oppositely oriented, external permanent magnets to produce strong long-range field gradients at the interfaces between magnets, further increasing the effectiveness of the device. A comprehensive simulation is performed using COMSOL Multiphysics to study the effect of various parameters on the magnetic flux within the separation channel. Additionally, a microfluidic device is designed, fabricated, and tested to isolate DNA from blood. The results show that the device has the capability of separating DNA from a blood sample with a purity of 1.8 or higher, a yield of up to 33 μg of polymerase chain reaction ready DNA per milliliter of blood, and a volumetric throughput of up to 50 ml/h.  相似文献   

9.
In this paper, we present a low cost and equipment-free blood filtration device capable of producing plasma from blood samples with mL-scale capacity and demonstrate its clinical application for hepatitis B diagnosis. We report the results of in-field testing of the device with 0.8–1 ml of undiluted, anticoagulated human whole blood samples from patients at the National Hospital for Tropical Diseases in Hanoi, Vietnam. Blood cell counts demonstrate that the device is capable of filtering out 99.9% of red and 96.9% of white blood cells, and the plasma collected from the device contains lower red blood cell counts than plasma obtained from a centrifuge. Biochemistry and immunology testing establish the suitability of the device as a sample preparation unit for testing alanine transaminase (ALT), aspartate transaminase (AST), urea, hepatitis B “e” antigen (HBeAg), hepatitis B “e” antibody (HBe Ab), and hepatitis B surface antibody (HBs Ab). The device provides a simple and practical front-end sample processing method for point-of-care microfluidic diagnostics, enabling sufficient volumes for multiplexed downstream tests.  相似文献   

10.
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   

11.
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   

12.
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   

13.
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   

14.
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   

15.
This article describes a fabrication process for the generation of a leak proof paper based microfluidic device and a new design strategy for convenient incorporation of externally prepared test zones. Briefly, a negative photolithographic method was used to prepare the device with a partial photoresist layer on the rear of the device to block the leakage of sample. Microscopy and Field Emission Scanning Electron Microscopy data validated the formation of the photoresist layer. The partial layer of photoresist on the device channel limits sample volume to 7 ± 0.2 μl as compared to devices without the partial photoresist layer which requires a larger sample volume of 10 ± 0.1 μl. The design prototype with a customized external test zone exploits the channel protrusions on the UV exposed photoresist treated paper to bridge the externally applied test zone to the sample and absorbent zones. The partially laminated device with an external test zone has a comparatively low wicking speed of 1.8 ± 0.9 mm/min compared to the completely laminated device with an inbuilt test zone (3.3 ± 1.2 mm/min) which extends the reaction time between the analyte and reagents. The efficacy of the prepared device was studied with colorimetric assays for the non-specific detection of protein by tetrabromophenol blue, acid/base with phenolphthalein indicator, and specific detection of proteins using the HRP-DAB chemistry. The prepared device has the potential for leak proof detection of analyte, requires low sample volume, involves reduced cost of production (∼$0.03, excluding reagent and lamination cost), and enables the integration of customized test zones.  相似文献   

16.
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   

17.
A variety of methods have been used to introduce chemicals into a stream or to mix two or more streams of different compositions using microfluidic devices. In the following paper, the introduction of cryoprotective agents (CPAs) used during cryopreservation of cells in order to protect them from freezing injuries and increase viability post thaw is described. Dimethylsulphoxide (DMSO) is the most commonly used CPA. We aim to optimize the operating conditions of a two-stream microfluidic device to introduce a 10% vol/vol solution of DMSO into a cell suspension. Transport behavior of DMSO between two streams in the device has been experimentally characterized for a spectrum of flow conditions (0.7 < Re < 10), varying initial donor stream concentrations, (1% vol/vol < Co < 15% vol/vol) and different flow rate fractions (0.23 < fq < 0.77). The outlet cell stream concentration is analyzed for two different flow configurations: one with the cell stream flowing on top of the DMSO-rich donor stream, and the other with the cell stream flowing beneath the heavy DMSO-laden stream. We establish a transition from a diffusive mode of mass transfer to gravity-influenced convective currents for Atwood numbers (At) in the range of (1.7 × 10−3 < At < 3.1 × 10−3) for the latter configuration. Flow visualization with cells further our understanding of the effect of At on the nature of mass transport. Cell motion studies performed with Jurkat cells confirm a high cell recovery from the device while underscoring the need to collect both the streams at the outlet of the device and suggesting flow conditions that will help us achieve the target DMSO outlet concentration for clinical scale flow rates of the cell suspension.  相似文献   

18.
A novel microfluidic device which consists of two stages for particle focusing and separation using a viscoelastic fluid has been developed. A circular capillary tube was used for three-dimensional particle pre-alignment before the separation process, which was inserted in a polydimethylsiloxane microchannel. Particles with diameters of 5 and 10 μm were focused at the centerline in the capillary tube, and the location of particles was initialized at the first bifurcation. Then, 5 and 10 μm particles were successfully separated in the expansion region based on size-dependent lateral migration, with ∼99% separation efficiency. The proposed device was further applied to separation of MCF-7 cells from leukocytes. Based on the cell size distribution, an approximate size cutoff for separation was determined to be 16 μm. At 200 μl/min, 94% of MCF-7 cells were separated with the purity of ∼97%. According to the trypan blue exclusion assay, high viability (∼90%) could be achieved for the separated MCF-7 cells. The use of a commercially available capillary tube enables the device to be highly versatile in dealing with particles in a wide size range by using capillary tubes with different inner diameters.  相似文献   

19.
Monocytes represent a class of immune cells that play a key role in the innate and adaptive immune response against infections. One mechanism employed by monocytes for sensing foreign antigens is via toll-like receptors (TLRs)—transmembrane proteins that distinguish classes of foreign pathogens, for example, bacteria (TLR4, 5, and 9) vs. fungi (TLR2) vs. viruses (TLR3, 7, and 8). Binding of antigens activates a signaling cascade through TLR receptors that culminate in secretion of inflammatory cytokines. Detection of these cytokines can provide valuable clinical data for drug developers and disease investigations, but this usually requires a large sample volume and can be technically inefficient with traditional techniques such as flow cytometry, enzyme-linked immunosorbent assay, or luminex. This paper describes an approach whereby antibody arrays for capturing cells and secreted cytokines are encapsulated within a microfluidic device that can be reconfigured to operate in serial or parallel mode. In serial mode, the device represents one long channel that may be perfused with a small volume of minimally processed blood. Once monocytes are captured onto antibody spots imprinted into the floor of the device, the straight channel is reconfigured to form nine individually perfusable chambers. To prove this concept, the microfluidic platform was used to capture monocytes from minimally processed human blood in serial mode and then to stimulate monocytes with different TLR agonists in parallel mode. Three cytokines, tumor necrosis factor-α, interleukin (IL)-6, and IL-10, were detected using anti-cytokine antibody arrays integrated into each of the six chambers. We foresee further use of this device in applications such as pediatric immunology or drug/vaccine testing where it is important to balance small sample volume with the need for high information content.  相似文献   

20.
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   

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