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1.
This work describes the development of a prototypic microfluidic platform for the generation of stepwise concentration gradients of drugs. A sensitive apoptotic analysis method is integrated into this microfluidic system for studying apoptosis of HeLa cells under the influence of anticancer drug, etoposide, with various concentrations in parallel; it measures the yellow fluorescent protein∕cyan fluorescent protein fluorescence resonance energy transfer (FRET) signal that responds to the activation of caspase-3, an indicator of cell apoptosis. Sets of microfluidic valves on the chip generate stepwise concentration gradient of etoposide in various cell-culture microchambers. The FRET signals from multiple chambers are simultaneously monitored under a fluorescent microscope for long-time observation and the on-chip results are compared with those from 96-well plate study and the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The microfluidic platform shows several advantages including high-throughput capacity, low drug consumption, and high sensitivity.  相似文献   

2.
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   

3.
分子生物传感器是由生物大分子通过基因重组或DNA合成所构成的传感器,能够实时、可视化探测活细胞及活体内关键分子事件。目前研究热度高、应用广的分子生物传感器包括分子信标(MB)、共振能量转移系统(荧光共振能量转移和生物发光共振能量转移)和分子荧光互补系统(如双分子荧光互补、三分子荧光互补等)。文章介绍了这几类分子生物传感器的原理和特点,重点强调了它们在活细胞分子影像学中的运用,如:研究细胞内蛋白之间的相互作用,探索生物大分子在细胞中的定位、运动和动力学等。此外,还讨论了分子生物传感器的局限性和面临的挑战,并展望了未来发展方向。"眼见为实",分子生物传感器在这方面发挥独特的作用,它使我们前所未有地深入到细胞内部去观察生物分子事件乃至生物学过程,从而解答更多的生物学难题。  相似文献   

4.
Kantak C  Zhu Q  Beyer S  Bansal T  Trau D 《Biomicrofluidics》2012,6(2):22006-220069
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 F?rster 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.
Flow-through gold film perforated with periodically arrayed sub-wavelength nano-holes can cause extraordinary optical transmission (EOT), which has recently emerged as a label-free surface plasmon resonance sensor in biochemical detection by measuring the transmission spectral shift. This paper describes a systematic study of the effect of microfluidic field on the spectrum of EOT associated with the porous gold film. To detect biochemical molecules, the sub-micron-thick film is free-standing in a microfluidic field and thus subject to hydrodynamic deformation. The film deformation alone may cause spectral shift as measurement error, which is coupled with the spectral shift as real signal associated with the molecules. However, this microfluid-induced measurement error has long been overlooked in the field and needs to be identified in order to improve the measurement accuracy. Therefore, we have conducted simulation and analytic analysis to investigate how the microfluidic flow rate affects the EOT spectrum and verified the effect through experiment with a sandwiched device combining Au/Cr/Si3N4 nano-hole film and polydimethylsiloxane microchannels. We found significant spectral blue shift associated with even small flow rates, for example, 12.60 nm for 4.2 μl/min. This measurement error corresponds to 90 times the optical resolution of the current state-of-the-art commercially available spectrometer or 8400 times the limit of detection. This really severe measurement error suggests that we should pay attention to the microfluidic parameter setting for EOT-based flow-through nano-hole sensors and adopt right scheme to improve the measurement accuracy.  相似文献   

6.
We describe the integration of an actively controlled programmable microfluidic sample processor with on-chip optical fluorescence detection to create a single, hybrid sensor system. An array of lifting gate microvalves (automaton) is fabricated with soft lithography, which is reconfigurably joined to a liquid-core, anti-resonant reflecting optical waveguide (ARROW) silicon chip fabricated with conventional microfabrication. In the automaton, various sample handling steps such as mixing, transporting, splitting, isolating, and storing are achieved rapidly and precisely to detect viral nucleic acid targets, while the optofluidic chip provides single particle detection sensitivity using integrated optics. Specifically, an assay for detection of viral nucleic acid targets is implemented. Labeled target nucleic acids are first captured and isolated on magnetic microbeads in the automaton, followed by optical detection of single beads on the ARROW chip. The combination of automated microfluidic sample preparation and highly sensitive optical detection opens possibilities for portable instruments for point-of-use analysis of minute, low concentration biological samples.  相似文献   

7.
We propose biofunctionalized nanofluidic slits (nanoslits) as an effective platform for real-time fluorescence-based biosensing in a reaction-limited regime with optimized target capture efficiency. This is achieved by the drastic reduction of the diffusion length, thereby a boosted collision frequency between the target analytes and the sensor, and the size reduction of the sensing element down to the channel height comparable to the depletion layer caused by the reaction. Hybridization experiments conducted in DNA-functionalized nanoslits demonstrate the analyte depletion and the wash-free detection ∼10 times faster compared to the best microfluidic sensing platforms. The signal to background fluorescence ratio is drastically increased at lower target concentrations, in favor of low-copy number analyte analysis. Experimental and simulation results further show that biofunctionalized nanoslits provide a simple means to study reaction kinetics at the single-pixel level using conventional fluorescence microscopy with reduced optical depth.  相似文献   

8.
The recent development of microfluidic "lab on a chip" devices requiring sample sizes <100 μL has given rise to the need to concentrate dilute samples and trap analytes, especially for surface-based detection techniques. We demonstrate a particle collection device capable of concentrating micron-sized particles in a predetermined area by combining AC electroosmosis (ACEO) and dielectrophoresis (DEP). The planar asymmetric electrode pattern uses ACEO pumping to induce equal, quadrilateral flow directed towards a stagnant region in the center of the device. A number of system parameters affecting particle collection efficiency were investigated including electrode and gap width, chamber height, applied potential and frequency, and number of repeating electrode pairs and electrode geometry. The robustness of the on-chip collection design was evaluated against varying electrolyte concentrations, particle types, and particle sizes. These devices are amenable to integration with a variety of detection techniques such as optical evanescent waveguide sensing.  相似文献   

9.
We developed an automated laser induced fluorescence system utilizing microfluidic chips for detection and quantification of immunoglobulins. Microchips were fabricated from polydimethysiloxane (PDMS) using the so-called "prepolymerization technique." The microchip structure helped minimize the effects of PDMS autofluorescence and light scattering. Furthermore, a thin and uniform PDMS layer forming the top of the microchip enabled proper focusing and collection of the excitation beam and the emitted fluorescence, respectively. The developed system was tested for the detection of mouse immunoglobulins. The capturing antibodies were immobilized on internal microchannel walls in the form of a polyelectrolyte. We clearly show that this immobilization technique, if correctly realized, gives results with high reproducibility. After sample incubation and washing, secondary antibodies labeled by fluorescein isothiocyanate were introduced into microchannels to build a detectable complex. We show that mouse antibodies can be quantified in a wide concentration range, 0.01-100 μg ml(-1). The lower detection limit was below 0.001 μg ml(-1) (6.7 pM). The developed laser induced fluorescence (LIF) apparatus is relatively cheap and easy to construct. The total cost of the developed LIF detector is lower than a typical price of plate readers. If compared to classical ELISA (enzyme linked immunosorbent assay) plate systems, the detection of immunoglobulins or other proteins in the developed PDMS microfluidic device brings other important benefits such as reduced time demands (10 min incubation) and low reagent consumption (less than 1 μl). The cost of the developed PDMS chips is comparable with the price of commercial ELISA plates. The main troubleshooting related to the apparatus development is also discussed in order to help potential constructors.  相似文献   

10.
For the diagnosis of biochemical reactions, the investigation of microflow behavior, and the confirmation of simulation results in microfluidics, experimentally quantitative measurements are indispensable. To characterize the mixing and reaction of fluids in microchannel devices, we propose a mixing quality index (Mqi) to quantify the cross-sectional patterns (also called mixing patterns) of fluids, captured with a confocal-fluorescence microscope (CFM). The operating parameters of the CFM for quantification were carefully tested. We analyzed mixing patterns, flow advection, and mass exchange of fluids in the devices with overlapping channels of two kinds. The mixing length of the two devices derived from the analysis of Mqi is demonstrated to be more precise than that estimated with a commonly applied method of blending dye liquors. By means of fluorescence resonance-energy transfer (FRET), we monitored the hybridization of two complementary oligonucleotides (a FRET pair) in the devices. The captured patterns reveal that hybridization is a progressive process along the downstream channel. The FRET reaction and the hybridization period were characterized through quantification of the reaction patterns. This analytical approach is a promising diagnostic tool that is applicable to the real-time analysis of biochemical and chemical reactions such as polymerase chain reaction (PCR), catalytic, or synthetic processes in microfluidic devices.  相似文献   

11.
Yazdi SH  White IM 《Biomicrofluidics》2012,6(1):14105-141059
We report the demonstration of an optofluidic surface enhanced Raman spectroscopy (SERS) device that leverages a nanoporous microfluidic matrix to improve the SERS detection performance by more than two orders of magnitude as compared to a typical open microfluidic channel. Although it is a growing trend to integrate optical biosensors into microfluidic channels, this basic combination has been detrimental to the sensing performance when applied to SERS. Recently, however, synergistic combinations between microfluidic functions and photonics (i.e., optofluidics) have been implemented that improve the detection performance of SERS. Conceptually, the simplest optofluidic SERS techniques reported to date utilize a single nanofluidic channel to trap nanoparticle-analyte conjugates as a method of preconcentration before detection. In this work, we leverage this paradigm while improving upon the simplicity by forming a 3D nanofluidic network with packed nanoporous silica microspheres in a microfluidic channel; this creates a concentration matrix that traps silver nanoclusters and adsorbed analytes into the SERS detection volume. With this approach, we are able to achieve a detection limit of 400 attomoles of Rhodamine 6G after only 2 min of sample loading with high chip-to-chip repeatability. Due to the high number of fluidic paths in the nanoporous channel, this approach is less prone to clogging than single nanofluidic inlets, and the loading time is decreased compared to previous reports. In addition, fabrication of this microsystem is quite simple, as nanoscale fabrication is not necessary. Finally, integrated multimode fiber optic cables eliminate the need for optical alignment, and thus the device is relevant for portable and automated applications in the field, including point-of-sample and point-of-care detection. To illustrate a relevant field-based application, we demonstrate the detection of 12 ppb of the organophosphate malathion in water using the nanofluidic SERS microsystem.  相似文献   

12.
Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.  相似文献   

13.
In this work, we demonstrate an integrated, single-layer, miniature flow cytometry device that is capable of multi-parametric particle analysis. The device integrates both particle focusing and detection components on-chip, including a “microfluidic drifting” based three-dimensional (3D) hydrodynamic focusing component and a series of optical fibers integrated into the microfluidic architecture to facilitate on-chip detection. With this design, multiple optical signals (i.e., forward scatter, side scatter, and fluorescence) from individual particles can be simultaneously detected. Experimental results indicate that the performance of our flow cytometry chip is comparable to its bulky, expensive desktop counterpart. The integration of on-chip 3D particle focusing with on-chip multi-parametric optical detection in a single-layer, mass-producible microfluidic device presents a major step towards low-cost flow cytometry chips for point-of-care clinical diagnostics.  相似文献   

14.
Optical chromatography involves the elegant combination of opposing optical and fluid drag forces on colloidal samples within microfluidic environments to both measure analytical differences and fractionate injected samples. Particles that encounter the focused laser beam are trapped axially along the beam and are pushed upstream from the laser focal point to rest at a point where the optical and fluid forces on the particle balance. In our recent devices particles are pushed into a region of lower microfluidic flow, where they can be retained and fractionated. Because optical and fluid forces on a particle are sensitive to differences in the physical and chemical properties of a sample, separations are possible. An optical chromatography beam focused to completely fill a fluid channel is operated as an optically tunable filter for the separation of inorganic, polymeric, and biological particle samples. We demonstrate this technique coupled with an advanced microfluidic platform and show how it can be used as an effective method to fractionate particles from an injected multicomponent sample. Our advanced three-stage microfluidic design accommodates three lasers simultaneously to effectively create a sequential cascade optical chromatographic separation system.  相似文献   

15.
Optical chromatography relies on the balance between the opposing optical and fluid drag forces acting on a particle. A typical configuration involves a loosely focused laser directly counter to the flow of particle-laden fluid passing through a microfluidic device. This equilibrium depends on the intrinsic properties of the particle, including size, shape, and refractive index. As such, uniquely fine separations are possible using this technique. Here, we demonstrate how matching the diameter of a microfluidic flow channel to that of the focusing laser in concert with a unique microfluidic platform can be used as a method to fractionate closely related particles in a mixed sample. This microfluidic network allows for a monodisperse sample of both polystyrene and poly(methyl methacrylate) spheres to be injected, hydrodynamically focused, and completely separated. To test the limit of separation, a mixed polystyrene sample containing two particles varying in diameter by less than 0.5 μm was run in the system. The analysis of the resulting separation sets the framework for continued work to perform ultra-fine separations.  相似文献   

16.
This paper describes an integrated microfluidic chip that is capable of rapidly and quantitatively measuring the concentration of a bladder cancer biomarker, apolipoprotein A1, in urine samples. All of the microfluidic components, including the fluid transport system, the micro-valve, and the micro-mixer, were driven by negative pressure, which simplifies the use of the chip and facilitates commercialization. Magnetic beads were used as a solid support for the primary antibody, which captured apolipoprotein A1 in patients'' urine. Because of the three-dimensional structure of the magnetic beads, the concentration range of the target that could be detected was as high as 2000 ng ml−1. Because this concentration is 100 times higher than that quantifiable using a 96-well plate with the same enzyme-linked immunosorbent assay (ELISA) kit, the dilution of the patient''s urine can be avoided or greatly reduced. The limit of detection was determined to be approximately 10 ng ml−1, which is lower than the cutoff value for diagnosing bladder cancer (11.16 ng ml−1). When the values measured using the microfluidic chip were compared with those measured using conventional ELISA using a 96-well plate for five patients, the deviations were 0.9%, 6.8%, 9.4%, 1.8%, and 5.8%. The entire measurement time is 6-fold faster than that of conventional ELISA. This microfluidic device shows significant potential for point-of-care applications.  相似文献   

17.
Optical based analysis in microfluidic and lab-on-a-chip systems are currently considered the gold standard methodology for the determination of end point reactions for various chemical and biological reaction processes. Typically, assays are performed using bulky ancillary apparatus such as microscopes and complex optical excitation and detection systems. Such instrumentation negates many of the advantages offered by device miniaturisation, particularly with respect to overall portability. In this article, we present a CO2 laser ablation technique for rapidly prototyping on-chip planar lenses, in conjunction with capillary action based autonomous microfluidics, to create a miniaturised and fully integrated optical biosensing platform. The presented self-aligned on-chip optical components offer an efficient means to direct excitation light within microfluidics and to directly couple light from a LED source. The device has been used in conjunction with a miniaturised and bespoke fluorescence detection platform to create a complete, palm sized system (≈60 × 80 × 60 mm) capable of performing fluoro-immunoassays. The system has been applied to the detection of cardiac Troponin I, one of the gold standard biomarkers for the diagnosis of acute myocardial infarction, achieving a lower detection limit of 0.08 ng/ml, which is at the threshold of clinically applicable concentrations. The portable nature of the complete system and the biomarker detection capabilities demonstrate the potential of the devised instrumentation for use as a medical diagnostics device at the point of care.  相似文献   

18.
In this work, we demonstrate an integrated, single-layer, miniature flow cytometry device that is capable of multi-parametric particle analysis. The device integrates both particle focusing and detection components on-chip, including a "microfluidic drifting" based three-dimensional (3D) hydrodynamic focusing component and a series of optical fibers integrated into the microfluidic architecture to facilitate on-chip detection. With this design, multiple optical signals (i.e., forward scatter, side scatter, and fluorescence) from individual particles can be simultaneously detected. Experimental results indicate that the performance of our flow cytometry chip is comparable to its bulky, expensive desktop counterpart. The integration of on-chip 3D particle focusing with on-chip multi-parametric optical detection in a single-layer, mass-producible microfluidic device presents a major step towards low-cost flow cytometry chips for point-of-care clinical diagnostics.  相似文献   

19.
Optofluidics may hold the key to greater success of photocatalytic water treatment. This is evidenced by our findings in this paper that the planar microfluidic reactor can overcome the limitations of mass transfer and photon transfer in the previous photocatalytic reactors and improve the photoreaction efficiency by more than 100 times. The microreactor has a planar chamber (5 cm×1.8 cm×100 μm) enclosed by two TiO(2)-coated glass slides as the top cover and bottom substrate and a microstructured UV-cured NOA81 layer as the sealant and flow input∕output. In experiment, the microreactor achieves 30% degradation of 3 ml 3×10(-5)M methylene blue within 5 min and shows a reaction rate constant two orders higher than the bulk reactor. Under optimized conditions, a reaction rate of 8% s(-1) is achieved under solar irradiation. The average apparent quantum efficiency is found to be only 0.25%, but the effective apparent quantum efficiency reaches as high as 25%. Optofluidic reactors inherit the merits of microfluidics, such as large surface∕volume ratio, easy flow control, and rapid fabrication and offer a promising prospect for large-volume photocatalytic water treatment.  相似文献   

20.
Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml−1), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.  相似文献   

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