High intensity light emitting diode array as an alternative exposure source for the fabrication of electrophoretic microfluidic devices

A commercially available array of light emitting diodes (LEDs), namely a UV Shark series LED high flux array, was evaluated as a light source for photolithographic patterning of SU-8 photoresist for the fabrication of templates suitable to make poly(dimethylsiloxane) (PDMS) microchips for electrophoresis. At a distance of 15 cm from the substrate, a relatively even intensity of 0.76+/-0.05 mW/cm(2) was obtained over an area sufficient for patterning a 10 cm (4 in.) silicon wafer. The exposure source was evaluated using a spiral mask design covering a 10 cm wafer. PDMS replicates of this template made in a 25 microm thick layer of SU-8 3025 showed little variation in width over the surface of the substrate, with a variation of 3.2% RSD (n=36) and a maximum range in widths of 7.8% of the mean channel width. The use of the optional metal reflector available with the LED array provided partial collimation of the light allowing near vertical structures to be produced across the entire wafer, something which was not possible without the reflector. SU-8 masters prepared using the LED array were compared to masters made using an alternative cheap lithographic source, namely a gel crosslinker. The SU-8 features were much narrower with the LED array than the crosslinker due to the multiple light sources in the crosslinker. A PDMS microchip made using a SU-8 template created using the Shark UV LED array was used for the electrophoretic separation of three anionic fluorescent dyes, with efficiencies up to 32,000 plates. Given that the LED array can be purchased and assembled for less than US$ 500, the Shark UV LED array is a promising alternative to more expensive lithographic light sources and will have significant appeal to many researchers wishing to undertake research in microfluidics around the world.     

Spectrometer with microreaction chamber and tri-colour light emitting diode as a light source

This paper describes a tri-colour light emitting diode (LED)-photoresistor (PR)-based in situ spectrometer with geometry that differs from the previously presented and allows for small volumes of test solution (350 μl) and effective homogenisation of reagents. The emission maximums of the tri-colour LED (TC-LED) are at 470, 565 and 660 nm. The basic measuring characteristics of the prototype were evaluated. The prototype was tested for determination of calcium in natural waters with the o-cresolphtalein complexon and was proved to be useful for real life applications. The in situ spectrometer with microreaction chamber enables the rapid optimisation of experimental conditions, as was demonstrated on the example of oxidation of alcohols with potassium dichromate. The drop-based experimental approach to the ruggedness test is fast, economic and gives reliable results with the minimum waste production. The prototype, connected to the computer, functions as a kinetic microreactor suitable also for following rapid changes. In spite of the manual addition of the initialising reagent, the data are lost only during the first 3 s.     

Whole column fluorescence imaging on a microchip by using a programmed organic light emitting diode array as a spatial-scanning light source and a single photomultiplier tube as detector

A novel miniaturized, integrated whole-column imaging detection (WCID) system on a microchip is presented. In this system, a program controlled organic light emitting diode (OLED) array was used as a spatial-scanning light source, to achieve imaging by the time sequence of the excited fluorescence. By this mechanism, a photomultiplier tube (PMT) instead of a charge coupled detector (CCD) can be applied to the imaging. Unlike conventional systems, no lenses, fibers or any mechanical components are required either. The novel flat light source provides uniform excitation light without size limitations and outputs a stronger power by pulse driving. The scanning mode greatly reduced the power consumption of the light source, which is valuable for a portable system. Meanwhile, this novel simplified system has a broader linear range, higher sensitivity and higher efficiency in data collection. Isoelectric focusing of R-phycoerythrin (PE) and monitoring of the overall process with WCID were performed on this system. The limit of detection (LOD) was 38 ng mL(-1) or 3.2 pg at 85 nL per column injection of PE. The system provides a technique for WCID capillary isoelectric focusing (cIEF) on chip and can be used for throughput analysis.     

Dual wavelength excitation fluorescence detector for capillary electrophoresis using a pulsed bi-colour light emitting diode

A simple and novel two-colour fluorescence detector for capillary electrophoresis was created using a single bi-colour light emitting diode (LED), multi-band pass excitation and emission filters and a single detector. Excitation light from a blue/red (470/635 nm) bi-colour LED was filtered through a 390/482/563/640 nm multi-band bandpass filter, with emitted light filtered through a 446/523/600/677 nm multi-band bandpass filter before being detected using a photon counting detector. Sequential pulsing of the blue/red LED and deconvolution of the collected fluorescence data allowed extracted electropherograms to be obtained corresponding to excitation with the blue and red LEDs. Optimisation of the pulsed LED conditions revealed an optimum LED on-time of 50 ms, off-time of 30 ms with a pulsed current of 40 mA, giving an effective data acquisition rate of 6.25 Hz. The characteristics of this system were validated by the simultaneous separation and determination of six fluorescent dyes: fluorescein, FITC, coumarin 334, dibromo(R)fluorescein (Ex/Em 470/525 nm), and Cy 5 and the Agilent Bioanalyser DNA dye (Ex/Em 635/670 nm). Under optimum conditions, the detection limits for FITC, fluorescein and Cy 5 were 69 nM, 42 nM and 289 nM (S/N = 3), respectively. These were lower than those obtained with continuous operation of the individual wavelengths at a constant current of 20 mA, but were slightly higher than those obtained using dedicated single wavelength filter combinations designed specifically for use with these fluorophores. The intraday repeatability (n = 6) of migration times was less than 1.0% and less than 3.4% for peak areas, while interday (n = 3) migration time and peak area reproducibility were less than 0.9% and 3.6%, respectively. This simple detector is capable of performing quantitative two-wavelength excitation without the need for complex optics and light source configurations.     

Cell-based high content screening using an integrated microfluidic device

High content screening (HCS) has quickly established itself as a core technique in the early stage of drug discovery for secondary compound screening. It allows several independent cellular parameters to be measured in a single cell or populations of cells in a single assay. In this work, we describe high content screening for the multiparametric measurement of cellular responses in human liver carcinoma (HepG2) cells using an integrated microfluidic device. This device consists of multiple drug gradient generators and parallel cell culture chambers, in which the processes of liquid dilution and diffusion, micro-scale cell culture, cell stimulation and cell labeling can be integrated into a single device. The simple assay provides multiparametric measurements of plasma membrane permeability, nuclear size, mitochondrial transmembrane potential and intracellular redox states in anti-cancer drug-induced apoptosis of HepG2 cells. The established platform is able to rapidly extract the maximum of information from tumor cells in response to several drugs varying in concentration, with minimal sample and less time, which is very useful for basic biomedical research and cancer treatment.     

Pulsed multi-wavelength excitation using fiber-in-capillary light emitting diode induced fluorescence detection in capillary electrophoresis

A novel instrument was developed using a multi-wavelength pulsed LED array with in-column optic-fiber induced fluorescence detection by capillary electrophoresis. The light from 2 different wavelength LEDs (450 nm and 480 nm) was pulsed for short intervals at high intensity. The beam from each LED was collimated and reshaped with the gradient index (GRIN) lens group to achieve a highly effective coupling between LED light source and an optical fiber. The optical fiber was placed inside the capillary for in-capillary LED-induced fluorescence detection. The advantages of this system were validated by the simultaneous determination of vitamin B2 and fluorescein. Detection limits for vitamin B2 and fluorescein were estimated to be 5 nM and 0.29 nM (S/N = 3), respectively. The relative standard deviations (RSDs, n = 6) of the both compounds for migration time and peak area were better than 0.83%, 2.20% and 1.21%, 2.75%, respectively. The method was applied to the determination of vitamin B2 in commercial tablets and fluorescein in fluorescein sodium injection and the recoveries obtained were in the range of 96.6-102.0% and 99.9-102.8%, respectively. It was also applied to human serum, where the recoveries were found to be in the range of 94.4-97.0% and 92.6-96.4%, respectively. The system has been successfully applied in separation and determination of the both biological samples with acceptable analytical performance.     

A luminescent cyclometalated platinum(II) complex and its green organic light emitting device with high device performance

A luminescent cyclometalated platinum(II) complex has been demonstrated to show green electrophosphorescence in multilayer organic light-emitting devices (OLEDs) using a dual emissive layer with high current and external quantum efficiencies of 38.9 cd A(-1) and 11.5%, respectively.     

Thin-film polymer light emitting diodes as integrated excitation sources for microscale capillary electrophoresis

We report the use of a thin-film polymer light emitting diode as an integrated excitation source for microfabricated capillary electrophoresis. The polyfluorene-based diode has a peak emission wavelength of 488 nm, an active area of 40 microm x 1000 microm and a thickness of similar 2 mm. The simple layer-by-layer deposition procedures used to fabricate the polymer component allow facile integration with planar chip-based systems. To demonstrate the efficacy of the approach, the polyfluorene diode is used as an excitation source for the detection of fluorescent dyes separated on-chip by electrophoresis. Using a conventional confocal detection system the integrated pLED is successfully used to detect fluorescein and 5-carboxyfluorescein at concentrations as low as 10(-6) M with a mass detection limit of 50 femtomoles. The drive voltages required to generate sufficient emission from the polymer diode device are as low as 3.7 V.     

Efficient and stable non-doped deep-blue organic light emitting diode based on an anthracene derivative

A new anthracene derivative 9,10-bis[3,5-di(4-tert-butylphenyl)phenyl]anthracene (BPPA) was synthesized via Suzuki coupling reaction and characterized by 1H NMR spectrum,mass spectrum,and elemental analysis.BPPA exhibits deep-blue emission both in solution and in solid thin film.This compound has a non-planar structure that results in high thermal stability and the phenomenon of polymorphism.The non-doped device based on this material shows stable deep-blue emission with the 1931 Commission international de...     

Flexible planar microfluidic chip employing a light emitting diode and a PIN-photodiode for portable flow cytometers

Detection of fluorescence particles is a key method of flow cytometry. We evaluate the performance of a design for a microfluidic fluorescence particle detection device. Due to the planar design with low layer thicknesses, we avoid optical components such as lenses or dichroic mirrors and substitute them with a shadow mask and colored film filters. A commercially available LED is used as the light source and a PIN-photodiode as detector. This design approach reduces component cost and power consumption and enables supplying the device with power from a standard USB port. From evaluation of this design, we obtain a maximum particle detection frequency of up to 600 particles per second at a sensitivity of better than 4.7 × 10(5) MESF (molecules of equivalent soluble fluorochrome) measured with particles for FITC sensitivity calibration. Lowering the flow rate increases the instrument sensitivity by an order of magnitude enabling the detection of particles with 4.5 × 10(4) MESF.     

New low-molecular-weight electroluminescent materials for green organic light emitting diodes

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Characterizing doxorubicin-induced apoptosis in HepG2 cells using an integrated microfluidic device

Apoptosis has now established its importance in numerous areas of biology and is recently receiving great attention as an important topic related to the development of diseases. In this work, an integrated microfluidic device was developed to characterize doxorubicin-induced apoptosis in human hepatocellular carcinoma (HepG2) cells. A continuous concentration gradient of stimulator (doxorubicin) was generated in the upstream network and used to perfuse downstream cultured HepG2 cells. The appropriate fluorescent dyes were introduced into cells from the inlets connected to the cell culture chambers, allowing one to distinguish apoptotic cells from nonapoptotic or necrotic cells. The resultant fluorescence of cellular population was monitored and quantified with single-cell resolution to infer the apoptosis process being studied. The feasibility of studying apoptosis was demonstrated by measuring several apoptotic events, including morphological alterations, plasma membrane phosphatidylserine externalization, and mitochondrial membrane potential collapse. This microfluidic device, integrating the cell culture, stimulation, staining, and washing steps into a single device, can simultaneously generate a number of experimental conditions and investigate multiple parameters relating stimulation to apoptosis. It offers a unique platform to characterize various cellular responses in a high-throughput fashion, which is otherwise impossible with conventional methods.     

Fibre coupled micro-light emitting diode array light source with integrated band-pass filter for fluorescence detection in miniaturised analytical systems

In this work, a new type of miniaturized fibre-coupled solid-state light source is demonstrated as an excitation source for fluorescence detection in capillary electrophoresis. It is based on a parabolically shaped micro-light emitting diode (μ-LED) array with a custom band-pass optical interference filter (IF) deposited at the back of the LED substrate. The GaN μ-LED array consisted of 270 individual μ-LED elements with a peak emission at 470 nm, each about 14 μm in diameter and operated as a single unit. Light was extracted through the transparent substrate material, and coupled to an optical fibre (OF, 400 μm in diameter, numerical aperture NA = 0.37), to form an integrated μ-LED-IF-OF light source component. This packaged μ-LED-IF-OF light source emitted approximately 225 μW of optical power at a bias current of 20 mA. The bandpass IF filter was designed to reduce undesirable LED light emissions in the wavelength range above 490 nm. Devices with and without IF were compared in terms of the optical power output, spectral characteristics as well as LOD values. While the IF consisted of only 7.5 pairs (15 layers) of SiO₂/HfO₂ layers, it resulted in an improvement of the baseline noise as well as the detection limit measured using fluorescein as test analyte, both by approximately one order of magnitude, with a LOD of 1 × 10⁻⁸ mol L⁻¹ obtained under optimised conditions. The μ-LED-IF-OF light source was then demonstrated for use in capillary electrophoresis with fluorimetric detection. The limits of detection obtained by this device were compared to those obtained with a commercial fibre coupled LED device.     

An integrated enzyme-linked immunosorbent assay system with an organic light-emitting diode and a charge-coupled device for fluorescence detection

A fluorescence detection system for a microfluidic device using an organic light-emitting diode (OLED) as the excitation light source and a charge-coupled device (CCD) as the photo detector was developed. The OLED was fabricated on a glass plate by photolithography and a vacuum deposition technique. The OLED produced a green luminescence with a peak emission at 512 nm and a half bandwidth of 55 nm. The maximum external quantum efficiency of the OLED was 7.2%. The emission intensity of the OLED at 10 mA/cm(2) was 13 μW (1.7 mW/cm(2)). The fluorescence detection system consisted of the OLED device, two band-pass filters, a five microchannel poly(dimethylsiloxane) (PDMS) microfluidic device and a linear CCD. The fluorescence detection system was successfully used in a flow-based enzyme-linked immunosorbent assay on a PDMS microfluidic device for the rapid determination of immunoglobulin A (IgA), a marker for human stress. The detection limit (S/N=3) for IgA was 16.5 ng/mL, and the sensitivity was sufficient for evaluating stress. Compared with the conventional 96-well microtiter plate assay, the analysis time and the amounts of reagent and sample solutions could all be reduced.     

Synthesis and characterization of carbazole-based dendrimers as bipolar host materials for green phosphorescent organic light emitting diodes

A group of novel, carbazole-based dendrimers comprised of the electron-accepting dibenzothiophene core and the electron-donating oligo-carbazole dendrons, namely G1 SF and G2 SF, are synthesized utilizing the Ullmann C–N coupling reaction. The dendrimers are designed in such a way to show good solubility in common organic solvents, excellent thermochemical stability with decomposition temperatures(Td) up to430 8C, and high HOMO levels in a range from 5.45 e V to 5.37 e V. Results of density functional theory calculations(DFT) indicate G2 SF has an almost complete separation of HOMO and LUMO levels at the holeand electron-transporting moieties; while G1 SF exhibits only partial separation of the HOMO and LUMO levels possibly due to intramolecular charge transfer. Green phosphorescent OLEDs were fabricated by the spin coating method with the dendrimers as hosts and traditional green iridium phosphor as doped emitter. Under ambient conditions, a maximum luminance efficiency(hL) of 19.83 cd A~-(1)and a maximum external quantum efficiency of 5.85% are achieved for G1 SF, and 15.50 cd A (-1)and 4.57% for G2 SF.     

A single-bead analysis on a disk-shaped microfluidic device using an antigen-immobilized bead.

A disk-shaped microfluidic device (lab-on-a-Disk) was developed to allow the evaluation of mental stress. As a standard sample, secretory immunoglobulin A (sIgA), which is a candidate marker of mental stress, was measured by a heterogeneous enzyme immunoassay (EIA) on the lab-on-a-Disk. Centrifugal force provided a microfluidic control on the lab-on-a-Disk. We examined the relationship between the rotational speed, the channel profile, and the position of the microfluidic chambers from the center of rotation to manipulate sample solutions into each reaction reservoir through microchannels sequentially, i.e., retain in a reservoir or flow into a subsequent reservoir. A single glass bead with immobilized sIgA on its surface was injected into a reservoir for a competitive antigen-antibody reaction, and applied to a specific surface in a heterogeneous assay. It is expected that the lab-on-a-Disk would be suitable for miniaturization and automation of the processes in EIA compared with a conventional EIA using a titer plate.     

Dynamic trapping and high-throughput patterning of cells using pneumatic microstructures in an integrated microfluidic device

Microfluidic trapping methods create significant opportunities to establish highly controlled cell positioning and arrangement for the microscale study of numerous cellular physiological and pathological activities. However, a simple, straightforward, dynamic, and high-throughput method for cell trapping is not yet well established. In the present paper, we report a direct active trapping method using an integrated microfluidic device with pneumatic microstructures (PμSs) for both operationally and quantitatively dynamic localization of cells, as well as for high-throughput cell patterning. We designed and fabricated U-shape PμS arrays to replace the conventional fixed microstructures for reversible trapping. Multidimensional dynamics and spatial consistency of the PμSs were optically characterized and quantitatively demonstrated. Furthermore, we performed a systematic trapping investigation of the PμSs actuated at a pressure range of 0 psi to 20 psi using three types of popularly applied mammalian cells, namely, human lung adenocarcinoma A549 cells, human hepatocellular liver carcinoma HepG2 cells, and human breast adenocarcinoma MCF-7 cells. The cells were quantitatively trapped and controlled by the U-shape PμSs in a programmatic and parallel manner, and could be opportunely released. The trapped cells with high viability were hydrodynamically protected by the real-time actuation of specifically designed umbrella-like PμSs. We demonstrate that PμSs can be applied as an active microfluidic component for large-scale cell patterning and manipulation, which could be useful in many cell-based tissue organization, immunosensor, and high-throughput imaging and screening.     

Structurally integrated organic light emitting device-based sensors for gas phase and dissolved oxygen

A compact photoluminescence (PL)-based O2 sensor utilizing an organic light emitting device (OLED) as the light source is described. The sensor device is structurally integrated. That is, the sensing element and the light source, both typically thin films that are fabricated on separate glass substrates, are attached back-to-back. The sensing elements are based on the oxygen-sensitive dyes Pt- or Pd-octaethylporphyrin (PtOEP or PdOEP, respectively), which are embedded in a polystyrene (PS) matrix, or dissolved in solution. Their performance is compared to that of a sensing element based on tris(4,7-diphenyl-l,10-phenanthroline) Ru II (Ru(dpp)) embedded in a sol-gel film. A green OLED light source, based on tris(8-hydroxy quinoline Al (Alq3), was used to excite the porphyrin dyes; a blue OLED, based on 4,4'-bis(2,2'-diphenylviny1)-1,1'-biphenyl, was used to excite the Ru(dpp)-based sensing element. The O2 level was monitored in the gas phase and in water, ethanol, and toluene solutions by measuring changes in the PL lifetime tau of the O2-sensitive dyes. The sensor performance was evaluated in terms of the detection sensitivity, dynamic range, gas flow rate, and temperature effect, including the temperature dependence of tau in pure Ar and O2 atmospheres. The dependence of the sensitivity on the preparation procedure of the sensing film and on the PS and dye concentrations in the sensing element, whether a solid matrix or solution, were also evaluated. Typical values of the detection sensitivity in the gas phase, S(g) identical with tau(0% O2)/tau(100% O2), at 23 degrees C, were approximately 35 to approximately 50 for the [Alq3 OLED[/[PtOEP dye] pair; S(g) exceeded 200 for the Alq3/PdOEP sensor. For dissolved oxygen (DO) in water and ethanol, S(DO) (defined as the ratio of tau in de-oxygenated and oxygen-saturated solutions) was approximately 9.5 and approximately 11, respectively, using the PtOEP-based film sensor. The oxygen level in toluene was measured with PtOEP dissolved directly in the solution. That sensor exhibited a high sensitivity, but a limited dynamic range. Effects of aggregation of dye molecules, sensing film porosity, and the use of the OLED-based sensor arrays for O2 and multianalyte detection are also discussed.     

Enhancement of signal-to-noise level by synchronized dual wavelength modulation for light emitting diode fluorimetry in a liquid-core-waveguide microfluidic capillary electrophoresis system

The signal-to-noise level of light emitting diode (LED) fluorimetry using a liquid-core-waveguide (LCW)-based microfluidic capillary electrophoresis system was significantly enhanced using a synchronized dual wavelength modulation (SDWM) approach. A blue LED was used as excitation source and a red LED as reference source for background-noise compensation in a microfluidic capillary electrophoresis (CE) system. A Teflon AF-coated silica capillary served as both the separation channel and LCW for light transfer, and blue and red LEDs were used as excitation and reference sources, respectively, both radially illuminating the detection point of the separation channel. The two LEDs were synchronously modulated at the same frequency, but with 180°-phase shift, alternatingly driven by a same constant current source. The LCW transferred the fluorescence emission, as well as the excitation and reference lights that strayed through the optical system to a photomultiplier tube; a lock-in amplifier demodulated the combined signal, significantly reducing its noise level. To test the system, fluorescein isothiocyanate (FITC)-labeled amino acids were separated by capillary electrophoresis and detected by SDWM and single wavelength modulation, respectively. Five-fold improvement in S/N ratio was achieved by dual wavelength modulation, compared with single wavelength modulation; and over 100-fold improvement in S/N ratio was achieved compared with a similar LCW-CE system reported previously using non-modulated LED excitation. A detection limit (S/N = 3) of 10 nM FITC-labeled arginine was obtained in this work. The effects of modulation frequency on S/N level and on the rejection of noise caused by LED-driver current and detector were also studied.