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3.
In this study, we constructed a novel microfluidic device for continuous-flow ATP amplification, using the SU-8:PDMS method. Sepharose beads immobilized with adenylate kinase and pyruvate kinase was packed into a microfluidic chamber to form lamination layer. Dry film type SU-8 was suitable to form a very thick mold for beads column reactor and its dam structure. A good correlation between amplified luminescence and initial ATP concentration was observed in this system. The gradient of amplification when performing six cycles of continuous-flow ATP amplification was 1.72 N. 相似文献
4.
Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible
healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain
reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified
from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and
cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral
targets (human platelet antigen 1 ( HPA1), fibroblast growth factor receptor 2 ( FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving.
The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR
chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were
present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative
but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating
the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for
inexpensive and faster clinical diagnostics. 相似文献
7.
This work presents the combination and acceleration of PCR and fluorescent labelling within a disposable microfluidic chip.
The utilised geometry consists of a spiral meander with 40 turns, representing a cyclic-flow PCR system. The used reaction
chemistry includes Cy3-conjugated primers leading to a one-step process accelerated by cyclic-flow PCR. DNA of three different
bacterial samples ( Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) was processed and successfully amplified and labelled with detection limits down to 10 2 cells per reaction. The specificity of species identification was comparable to the approach of separate PCR and labelling.
The overall processing time was decreased from 6 to 1.5 h. We showed that a disposable polycarbonate chip, fabricated by injection
moulding is suitable for the significant acceleration of DNA microarray assays. The reaction output led to high-sensitivity
bacterial identification in a short time, which is crucial for an early and targeted therapy against infectious diseases. 相似文献
8.
In this article, we report a novel approach to fabricating a low molecular weight cut-off membrane that could readily be employed
for several microfluidic applications. The reported structure was created by selectively retaining a precursor solution [5%
(w/v) maleic anhydride, 21% (v/v) (37:1) acrylamide/bisacrylamide, and 0.2% (w/v) VA-086 photoinitiator] in a chosen location
of a microfluidic network via capillary forces and then photo-polymerizing the mixture. The pores in the resulting membrane
were subsequently filled with 3-aminopropyltriethoxysilane, heated, and then treated with sodium silicate solution and heated
again, giving a structure having reduced porosity. The composite membrane thus created has been shown to have a molecular
weight cut-off that is at least an order of magnitude smaller than other photo-polymerized microfluidic membranes reported
in the literature. Moreover, this polymer–silicate structure was observed to be capable of blocking electroosmotic flow, thereby
generating a pressure gradient around its interface with an open microchannel upon application of an electric field across
the microchannel-membrane junction. In this study, a fraction of the resulting hydrodynamic flow was successfully guided to
an electric field free analysis channel to implement a pressure-driven assay. With our current design pressure-driven velocities,
up to 1.8 mm/s was generated in the electric field free analysis channel for an applied voltage of 2 kV in the pumping section.
Finally, the functionality of this integrated microfluidic device was demonstrated by implementing a reverse phase chromatographic
separation using the pressure-driven flow generated on-chip. 相似文献
9.
We present a microfluidic biosensor that integrates membrane-based preconcentration with fluorescence detection. The concentration
membrane was fabricated in polyacrylamide by an in situ photopolymerization technique at the junction of glass microchannels.
Liposomes entrapping sulforhodamine B dye molecules were used for signal amplification. The biotin–streptavidin binding system
was a model system for evaluating device performance. Biotinylated liposomes were preconcentrated at the membrane by applying
an electric field across the membrane. The electric field causes the liposomes to migrate toward the membrane where they are
concentrated by a sieving effect. Two orders of magnitude concentration was achieved after applying the electric field for
only 2 min. The concentrated bolus was then eluted toward the detection unit, where the biotinylated liposomes were captured
by immobilized streptavidin. The integrated system with the preconcentration module shows a 14-fold improvement in signal
as opposed to a system that does not include preconcentration. 相似文献
10.
This paper describes the fabrication of a microfluidic device for use in protein-based bioassays that effectively incorporates
poly(ethylene glycol) (PEG) hydrogel microparticles within a defined region. The microfluidic device is composed of a polymerization
chamber and reaction chamber that are serially connected through the microchannel. Various shapes and sizes of hydrogel microparticles
were fabricated in the polymerization chamber by photopatterning and moved to the reaction chamber by pressure-driven flow.
All of the hydrogel microparticles were retained within the reaction chamber due to an in-chamber integrated microfilter with
smaller mesh size than hydrogel microparticles. Hydrogel microparticles were able to encapsulate enzymes without losing their
activity, and different concentrations of glucose were detected by sequential bienzymatic reaction of hydrogel-entrapped glucose
oxidase (GOX) and peroxidase (POD) inside the microfluidic device using fluorescence method. Importantly, there was a linear
correspondence between fluorescence intensity and the glucose concentration over the physiologically important range of 1.00–10.00 mM.
D. Choi and E. Jang contributed equally to this work. 相似文献
11.
This work presents a polydimethylsiloxane (PDMS) microfluidic device for packaging CMOS MEMS impedance sensors. The wrinkle
electrodes are fabricated on PDMS substrates to ensure a connection between the pads of the sensor and the impedance instrument.
The PDMS device can tolerate an injection speed of 27.12 ml/h supplied by a pump. The corresponding pressure is 643.35 Pa.
The bonding strength of the device is 32.44 g/mm 2. In order to demonstrate the feasibility of the device, the short circuit test and impedance measurements for air, de-ionized
water, phosphate buffered saline (PBS) at four concentrations (1, 2 × 10 −4, 1 × 10 −4, and 6.7 × 10 −5 M) were performed. The experimental results show that the developed device integrated with a sensor can differentiate various
samples. 相似文献
12.
This article introduces an on-demand microfluidic hydrogen generator that can be integrated with a micro-proton exchange membrane
(PEM) fuel cell. The catalytic reaction, reactant circulation, gas/liquid separation, and autonomous control functionalities
are all integrated into a single microfluidic device. It generates hydrated hydrogen gas from an aqueous ammonia borane solution
which is circulated and exchanged between the microfluidic reactor and a rechargeable fuel reservoir without any parasitic
power consumption. Ammonia borane is chosen instead of sodium borohydride because of its faster hydrogen generation rate,
higher hydrogen storage capability, stability, and better catalyst durability. The self-circulation of the ammonia borane
solution was achieved using directional growth and selective venting of hydrogen bubbles in micro-channels, which leads to
agitation and addition of fresh solution without consumption of electrical power. The self-regulation mechanism ensures that
hydrogen can be supplied to a fuel cell according to the exact demand of the current output of the fuel cell. The circulation
flow rate of ammonia borane solution is also automatically regulated by the venting rate of hydrogen at the gas outlet. Design,
fabrication, and testing results of a prototype system are described. The hydrogen generator is capable of generating hydrogen
gas at a maximum rate of 0.6 ml/min (2.1 ml/min cm 2) and circulating aqueous ammonia borane at a maximum flow rate of ~15.7 μl/min. The device has also been connected with a
micro-PEM fuel cell to demonstrate the feasibility of its practical applications in a high-impedance system. 相似文献
13.
Propidium monoazide (PMA) is a membrane impermeable molecule that covalently bonds to double stranded DNA when exposed to light and inhibits the polymerase activity, thus enabling DNA amplification detection protocols that discriminate between viable and non-viable entities. Here, we present a microfluidic device for inexpensive, fast, and simple PMA labeling for viable qPCR and qLAMP assays. The three labeling stages of mixing, incubation, and cross-linking are completed within a microfluidic device that is designed with Tesla structures for passive microfluidic mixing, bubble trappers to improve flow uniformity, and a blue LED to cross-link the molecules. Our results show that the on-chip PMA labeling is equivalent to the standard manual protocols and prevents the replication of DNA from non-viable cells in amplification assays. However, the on-chip process is faster and simpler (30 min of hands-off work), has a reduced likelihood of false negatives, and it is less expensive because it only uses 1/20th of the reagents normally consumed in standard bench protocols. We used our microfluidic device to perform viable qPCR and qLAMP for the detection of S. typhi and E. coli O157. With this device, we are able to specifically detect viable bacteria, with a limit of detection of 7.6 × 10 3 and 1.1 × 10 3 CFU/mL for S. typhi and E. coli O157, respectively, while eliminating amplification from non-viable cells. Furthermore, we studied the effects of greater flow rates to expedite the labeling process and identified a maximum flow rate of 0.7 μL/min for complete labeling with the current design. 相似文献
14.
Evaporation is of great importance when dealing with microfluidic devices with open air/liquid interfaces due to the large
surface-to-volume ratio. For devices utilizing a thermal reaction (TR) reservoir to perform a series of biological and chemical
reactions, excessive heat-induced microfluidic evaporation can quickly lead to reaction reservoir dry out and failure of the
overall device. In this study, we present a simple, novel method to decrease heat-induced fluid evaporation within microfluidic
systems, which is termed as heat-mediated diffusion-limited (HMDL) method. This method does not need complicated thermal isolation
to reduce the interfacial temperature, or external pure water to be added continuously to the TR chamber to compensate for
evaporation loss. The principle of the HMDL method is to make use of the evaporated reaction content to increase the vapor
concentration in the diffusion channel. The experimental results have shown that the relative evaporation loss ( V
loss/ V
ini) based on the HMDL method is not only dependent on the HMDL and TR region’s temperatures ( T
HMDL and T
TR), but also on the HMDL and TR’s channel geometries. Using the U-shaped uniform channel with a diameter of 200 μm, the V
loss/ V
ini within 60 min is low to 5% ( T
HMDL = 105°C, T
TR = 95°C). The HMDL method can be used to design open microfluidic systems for nucleic acid amplification and analysis such
as isothermal amplification and PCR thermocycling amplification, and a PCR process has been demonstrated by amplifying a 135-bp
fragment from Listeria monocytogenes genomic DNA. 相似文献
15.
We recently reported the successful use of the loop-mediated isothermal amplification (LAMP) reaction for hepatitis B virus (HBV) DNA amplification and its optimal primer design method. In this study, we report the development of an integrated isothermal device for both amplification and detection of targeted HBV DNA. It has two major components, a disposable polymethyl methacrylate (PMMA) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of DNA amplification by-product, magnesium pyrophosphate. We have established a correlation curve ( R2 = 0.99) between the concentration of pyrophosphate ions and the level of turbidity by using a simulated chemical reaction to evaluate the characteristics of our device. For the applications of rapid pathogens detection, we also have established a standard curve ( R2 = 0.96) by using LAMP reaction with a standard template in our device. Moreover, we also have successfully used the device on seven clinical serum specimens where HBV DNA levels have been confirmed by real-time PCR. The result indicates that different amounts of HBV DNA can be successfully detected by using this device within 1 h. 相似文献
16.
We report a new technique for conducting immuno-diagnostics on a microfluidic platform. Rather than handling fluid reagents
against a stationary solid phase, the platform manipulates analyte-coated magnetic beads through stationary plugs of fluid
reagents to detect an antigenic analyte. These isolated but accessible plugs are pre-encapsulated in a microchannel by capillary
force. We call this platform microfluidic inverse phase enzyme-linked immunosorbent assay (μIPELISA). μIPELISA has distinctive
advantages in the family of microfluidic immunoassay. In particular, it avoids pumping and valving fluid reagents during assaying,
thus leading to a lab-on-a-chip format that is free of instrumentation for fluid actuation and control. We use μIPELISA to
detect digoxigenin-labeled DNA segments amplified from E. coli O157:H7 by polymerase chain reaction (PCR), and compare its detection capability with that of microplate ELISA. For 0.259 ng μl −1 of digoxigenin-labeled amplicon, μIPELISA is as responsive as the microplate ELISA. Also, we simultaneously conduct μIPELISA
in two parallel microchannels. 相似文献
18.
This paper presents design and analysis of microelectromechanical system (MEMS) based displacement amplification mechanism actuated using thermal actuators with enhanced performance. The proposed model consists of chevron shaped thermal actuators, an amplification mechanism capable of amplifying displacement 20 times and an electrostatic comb drives for sensing displacements. When voltage is applied to thermal chevrons, displacement is produced which is then amplified 20 times. Steady state static thermal electrical analysis is performed under variable resistivity and voltage bias of 2 V. In-plane reaction forces of magnitude 194.2 and 150.91 µN along X and Y-axis, respectively, thus producing displacement of 0.11 and 2.22 µm along X and Y-axis, respectively. Time domain simulations of device are carried with constant electrical resistivity, variable voltage and convective boundary conditions. Modal analysis of the mechanism is carried out to predict the natural frequencies and associated mode shapes of mechanism during free vibrations. The desired mode is at frequency of 286.160 kHz. Dynamic simulations including direct integration-transient, transient modal and steady state modal analysis are performed on the device for time span of 0.0006 s, under application of 25 g and frequency range of 200–300 kHz. Simulation results prove the viability of the mechanism as an amplification device with enhanced voltage–stroke ratio. 相似文献
19.
An on-chip polymerase chain reaction (PCR) device employing a magnetic beads-droplet-handling system was developed. Actuation with a magnet offers a simple system for droplet manipulation that allows separation and fusion of droplets containing magnetic beads by handling with a magnet. The device consists of a reaction chamber channel and two magnet-handling channels for the manipulation of micro-droplets containing magnetic beads. Micro-droplets were placed inside a reaction chamber filled with oil and manipulated with a magnet. When a droplet containing NaOH and magnetic beads was manipulated towards a droplet containing phenol red, a color change was observed after fusion. Sample preparation was performed by fusion of droplets containing a forward primer, reverse primer, template DNA and PCR mixture, using a droplet containing magnetic beads. PCR amplification or RT-PCR was also successfully performed, with efficiency comparable to manual methods that use this device by placing it on a thermal cycler for amplification. With a magnetic beads-manipulation step, purification of amplified DNA was also accomplished by using magnetic beads as the carrier. The amplified DNA was captured on streptavidin conjugated magnetic beads using a biotinylated primer, purified by washing and digested for separation of the target DNA. 相似文献
20.
In this work, we demonstrated an all-dry, top-down, and one-step rapid process to fabricate paper-based microfluidic devices using fluorocarbon plasma polymerization. This process is able to create fluorocarbon-coated hydrophobic patterns on filter paper substrates while maintaining the trench and detection regions intact and free of contamination after the fabrication process, as confirmed by attenuated total reflectance–Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. We have shown that the processing time is one critical factor that influences the device performance. For the device fabricated with a sufficiently long processing time (180 s), the sample fluid flow can be well confined in the patterned trenches. By testing the device with an 800 μm channel width, a sample solution amount as small as 4.5 μL is sufficient to perform the test. NO 2 ? assay is also performed and shows that such a device is capable for biochemical analysis. 相似文献
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