High-sensitivity, disposable lab-on-a-chip with thin-film organic electronics for fluorescence detection

We report a high-sensitivity, disposable lab-on-a-chip with a thin-film organic light-emitting diode (OLED) excitation source and an organic photodiode (OPD) detector for on-chip fluorescence analysis. A NPB/Alq3 thin-film green OLED with an active area of 0.1 cm(2) was used as the excitation source, while a CuPC/C(60) thin-film OPD with 0.6 cm(2) active area was used as a photodetector. A novel cost-effective, cross-polarization scheme was used to filter out excitation light from a fluorescent dye emission spectrum. The excitation light from the OLED was linearly polarized and used to illuminate a microfluidic device containing a 1 microL volume of dye dissolved in ethanol. The detector was shielded by a second polarizer, oriented orthogonally to the excitation light, thus reducing the photocurrent due to excitation light leakage on the detector by approximately 25 dB. The fluorescence emission light, which is randomly polarized, is only attenuated by approximately 3 dB. Fluorescence signals from Rhodamine 6G (peak emission wavelength of 570 nm) and fluorescein (peak emission wavelength of 494 nm) dyes were measured in a dilution series in the microfluidic device with emission signals detected by the OPD. A limit-of-detection of 100 nM was demonstrated for Rhodamine 6G, and 10 microM for fluorescein. This suggests that an integrated microfluidic device, with an organic photodiode and LED excitation source and integrated polarizers, can be fabricated to realize a compact and economical lab-on-a-chip for point-of-care fluorescence assays.     

A microfluidic device using a green organic light emitting diode as an integrated excitation source

A simply fabricated microfluidic device using a green organic light emitting diode (OLED) and thin film interference filter as integrated excitation source is presented and applied to fluorescence detection of proteins. A layer-by-layer compact system consisting of glass/PDMS microchip, pinhole, excitation filter and OLED is designed and equipped with a coaxial optical fiber and for fluorescence detection a 300 microm thick excitation filter is employed for eliminating nearly 80% of the unwanted light emitted by OLEDs which has overlaped with the fluorescence spectrum of the dyes. The distance between OLED illuminant and microchannels is limited to approximately 1 mm for sensitive detection. The achieved fluorescence signal of 300 microM Rhodamine 6G is about 13 times as high as that without the excitation filter and 3.5 times the result of a perpendicular detection structure. This system has been used for fluorescence detection of Rhodamine 6G, Alexa 532 and BSA conjugates in 4% linear polyacrymide (LPA) buffer (in 1 x TBE, pH 8.3) and 1.4 fmol and 35 fmol mass detection limits at 0.7 nl injection volume for Alexa and Rhodamine dye have been obtained, respectively.     

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.     

Algal fluorescence sensor integrated into a microfluidic chip for water pollutant detection

We report the first miniaturized fluorescent sensor based on algae, with an organic light emitting diode (OLED) and an organic photodetector (OPD) integrated into a microfluidic chip. The blue emission OLED was used as the excitation source, while a blend of PTB3/PC(61)BM was used for the fabrication of the organic photodetector. Excitation and emission color filters based on acid/base dyes and a metal complex were developed and assembled with the organic optoelectronic components in order to complete the fluorescent detection system. The detection system was then integrated in a microfluidic chip made from (poly)dimethylsiloxane (PDMS). The complete sensor is designed to detect algal fluorescence in the microfluidic chamber. Algal chlorophyll fluorescence enables evaluation of the toxicity of pollutants like herbicides and metals-ions from agricultural run-offs. The entirely organic bioassay here presented allowed detection of the toxic effects of the herbicide Diuron on Chlamydomonas reinhardtii green algae that gave 50% inhibition of the algae photochemistry (EC(50)) with a concentration as low as 11 nM.     

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.     

A simple,low-cost,remote fiber-optic micro volume fluorescence flowcell for capillary flow-injection analysis

A small volume flowcell for fluorescence detection in capillary flow injection (CFI) analysis has been created by using a low cost, commercially available fluidic device. Fluorescence detection is achieved using an optical fiber to deliver excitation light to the sample flowing through the device and another optical fiber to collect fluorescence emission. The flowcell is a standard fluidic cross with a swept volume of 721 nL. Optical fibers were oriented at right angles using standard sleeves and ferrules to set their position near the cross intersection. Multiple excitation sources were used including a low power UV laser and blue and UV light emitting diodes (LED). The full emission spectrum detection limits, using the laser, for fluorescein and bovine serum albumin (BSA) were 0.30 ppb and 2.1 x 10(-4)% (w/w), respectively. Two fluidic crosses were used in series for multi-wavelength fluorescence excitation using fiber-optically coupled LED.     

Optical fiber light-emitting diode-induced fluorescence detection for capillary electrophoresis

A highly sensitive optical fiber light-emitting diode (LED)-induced fluorescence detector for CE has been constructed and evaluated. In this detector, a violet or blue LED was used as the excitation source and an optical fiber with 40 microm OD was used to transmit the excitation light. The upper end of the fiber was inserted into the separation capillary and was situated right at the detection window. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a cutoff filter before reaching the photomultiplier tube. Output signals were recorded and processed with a computer using in-house written software. The present CE/fluorescence detector deploys a simple and inexpensive optical system that requires only an LED as the light source. Its utility was successfully demonstrated by the separation and determination of amino acids (AAs) labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and FITC. Low detection limits were obtained ranging from 17 to 23 nM for NDA-tagged AAs and 8 to 12 nM for FITC-labeled AAs (S/N=3). By virtue of such valuable features as low cost, convenience, and miniaturization, the presented detection scheme was proven to be attractive for sensitive fluorescence detection in CE.     

Fabrication of an integrated PDMS microchip incorporating an LED-induced fluorescence device

A microfluidic device with an integrated fluorescence detection system has been developed in order to miniaturize the entire analytical system. A blue or green light-emitting diode (LED) and an optical fiber were mounted in a polydimethylsiloxane-based microchip. The performance of this device was evaluated by microchip electrophoresis. When a green LED was used as the light source, the calibration curve of Sulforhodamine-101 was linear over the range 1–100 M. The detection limit was found to be 600 nM (240 amol) for a S/N ratio of 3. When using a blue LED, the calibration curve of Fluorescein was linear over the range 0.2–100 M. The detection limit was estimated to be 120 nM (50 amol) (S/N=3). The detection sensitivity per unit power was comparable to that of LIF. The RSD values for the migration time, peak height and peak area were 0.74, 7.18 and 9.45%, respectively. The integrated microfluidic device was successfully used to determine amino acid derivatives.     

Compact fluorescence detection using in-fiber microchannels-its potential for lab-on-a-chip applications

This paper reports a compact and practical fluorescence sensor using an in-fiber microchannel. A blue LED, a multimode PMMA or silica fiber and a mini-PMT were used as an excitation source, a light guide and a fluorescence detector, respectively. Microfluidic channels of 100 microm width and 210 microm depth were fabricated in the optical fibers using a direct-write CO(2) laser system. The experimental results show that the sensor has high sensitivity, able to detect 0.005 microg L(-1) of fluorescein in the PBS solution, and the results are reproducible. The results also show that the silica fiber sensor has better sensitivity than that of the PMMA fiber sensor. This could be due to the fouling effect of the frosty layer formed at the microchannel made within the PMMA fiber. It is believed that this fiber sensor has the potential to be integrated into microfluidic chips for lab-on-a-chip applications.     

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.     

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 light emitting diode-induced fluorescence detector for capillary electrophoresis

An integrated light emitting diode (LED)-induced fluorescence detector was described and evaluated. The LED and its related components including lens and interference filter, the optical fiber used to collect fluorescence, and the capillary column are integrated into a substrate block, which eliminates the need of align procedure of the fiber and the capillary. Forty-fold enhancement of sensitivity was obtained compared with our previous work and the detection limit for fluorescein was 5 nM. Application of the detector for the analysis of FITC-labeled Ephedrine extract was demonstrated.     

发光二极管诱导荧光用于毛细管电泳检测

利用发光二极管作为激发光源，组装了用于毛细管电泳的荧光检测器。光纤用于传输荧光信号；光纤端面修饰成球形使耦合效率比平面端光纤提高了50．8％；光阑、光纤及毛细管检测池之间的光学校准简单、便捷。荧光素染料用于评价该体系性能，得到了fmol的质量检出限。     

A low-cost light-emitting diode induced fluorescence detector for capillary electrophoresis based on an orthogonal optical arrangement

In this work, a simple and low-cost miniaturized light-emitting diode induced fluorescence (LED-IF) detector based on an orthogonal optical arrangement for capillary electrophoresis (CE) was developed, using a blue concave light-emitting diode (LED) as excitation source and a photodiode as photodetector. A lens obtained from a waste DVD-ROM was used to focus the LED light beam into an ∼80 μm spot. Fluorescence was collected with an ocular obtained from a pen microscope at 45° angle, and passed through a band-pass filter to a photodiode detector. The performance of the LED-IF detector was demonstrated in CE separations using sodium fluorescein and fluorescein isothiocyanate (FITC)-labeled amino acids as model samples. The limit of detection for sodium fluorescein was 0.92 μM with a signal-to-noise ratio (S/N) of 3. The total cost of the LED-IF detector was less than $ 50.     

A lamp light-emitting diode-induced fluorescence detector for capillary electrophoresis

A light-emitting diode-induced fluorescence detector (LED-FD) for capillary electrophoresis was constructed and evaluated. A lamp LED with an enhanced emission spectrum and a band pass filter was used as the excitation light source. Refractive index matching fluid (RIMF) was used in the detection cell to reduce scattering light and the noise level. The limit of detection (LOD) for fluorescein was 1.5 nM (SNR=3). The system exhibited linear responses in the range of 1 x 10(-8) to 5 x 10(-6)M (R=0.999). Application of the lamp LED-FD for the analysis of FITC-labeled ephedra herb extract by capillary electrophoresis was demonstrated.     

Novel fused-LEDs devices as optical sensors for colorimetric analysis

The development of a novel, low power optical sensing platform based on light emitting diodes (LEDs) is described. The sensor is constructed from a pair of LEDs fused together at an angle where one LED functions as the light source and the other LED is reverse biased to function as a light detector. Sensor function is based on the level of light received by the detector diode, which varies with the reflectance of the interface between the device and its environment, or the chemochromic membrane that covers the device. A simple microprocessor circuit is used to measure the time taken for the photon-induced current to discharge the detector LED from an initial 5 V (logic 1) to 1.7 V (logic zero). This sensing device has been successfully used for colour and colour-based pH measurements and offers extremely high sensitivity, enabling detection down to the sub micro molar level of dyes.     

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.     

Small-angle optical deflection from collinear configuration for sensitive detection in microfluidic systems

This paper describes a novel detection system based on small-angle optical deflection from the collinear configuration of a microfluidic chip. In this system, the incident light beam was focused on the microchannel through the edge of a lens, resulting in a small deflection angle that deviated 20° from the collinear configuration. The emitted fluorescence was collected through the center of the same lens and delivered to a photomultiplier tube in the vertical direction; the reflection light of the chip plate was kept away from the detector. In contrast to traditional confocal and nonconfocal laser-induced fluorescence detection systems, background levels resulting from scattered excitation light, reflection and refraction from the microchip was significantly eliminated. Significant enhancement of the signal-to-noise ratio was obtained by shaping a laser beam that combined an attenuator with a spectral filter to optimize laser power and the dimensions of the laser beam. FITC and FITC-labeled amino acid were used as model analytes to demonstrate the performance sensitivity, separation efficiency, and reproducibility of this detection system by using a hybrid polydimethylsiloxane/glass microfluidic device. The limit of detection of FITC was estimated to be 2 pM (0.55 zmol) (S/N = 3). Furthermore, the single cell analysis for the determination of intracellular glutathione in a single 3T3 mouse fibroblast cell was demonstrated. The results suggest that the proposed optical arrangements will be promising for development of sensitive, low-cost microfluidic systems.     

Micro fluorescent analysis system integrating GaN-light-emitting-diode on a silicon platform

A micro fluorescent analysis system is proposed using silicon micromachining. GaN blue light-emitting diode (LED) monolithically integrated on a silicon substrate is used as a light source for the fluorescent analysis system. The blue light suits the excitation of several dyes used commonly in fluorescent analysis. Silicon photodiode (Si-PD) that matches the visible and near infrared fluorescent wavelengths of dyes is integrated on a silicon substrate. Polydimethylsiloxane (PDMS) micro-channels are also stacked for flowing dye-sensitized liquid. Therefore, the proposed system is an integrated system that can be composed on a silicon platform, i.e. a bottom layer of Si-PD, a middle layer of GaN-LED on silicon substrate and a top layer of micro PDMS channel. An aperture is opened into the GaN-LED layer by deep reactive ion etching to create a ring-shaped GaN-LED and a through-hole for detection. The light from the ring-shaped GaN-LED in the middle layer excites the dye-sensitized liquid in the top micro-channel layer. The fluorescence emitted from dye is detected by the Si-PD on the bottom layer at an angle larger than 90 degrees from the direction of excitation. Therefore, the detection optics consist basically of a dark-field illumination optical system. In order to evaluate the performance of the integrated system, fluorescence of fluorescein isothiocyanate (FITC) solution flowing in the micro channel is measured. From the measurement, the noise, sensitivity and limit of detection in the fabricated system are evaluated for FITC dye to be 0.57 pA, 1.21 pA μM(-1) and 469 nM, respectively. From these results, a compact fluorescence analysis system is demonstrated.     

A glycerol assisted light-emitting diode-induced fluorescence detector for capillary flow systems

A glycerol assisted light-emitting diode (LED)-induced fluorescence detector (IF) for capillary flow systems was constructed and evaluated. A blue LED was used as the excitation source, and optical fibers (OF) were used to transmit the excitation light and collect the fluorescence. A commercial available 5-port manifold was used as detection cell, where the capillary tube and the OF were fixed into the manifold. The precision of the holes on the manifold ensured a self-alignment of optical path. A refractive index matching fluid (RIMF)-glycerol was used to eliminate the interfaces between the OF and the LED, as well as between the fused silica capillary and the transmitting/collecting fiber. The enhancement of excitation light led to 2.8-folds improvement on the signal-to-noise ratio. The use of RIMF also eliminates focusing effect of the capillary wall and reduces both the excitation light directed to the detection cell and background signal, resulting in reduction in the fluorescence intensity and noise level. The intensity was reduced to 47-63% for laser and 60-77% for LED, respectively, for capillaries with i.d. from 50 to 250 microm; while the noise level was reduced to 1/3 when RIMF was used for both laser and LED on the tested capillaries. About 5.6-fold enhancement in signal-to-noise ratio was obtained in total. The detection limit of the LED-IF for fluorescein isothiocyanate (FITC) was 4 nM. Application of the LED-IF for the analysis of FITC-labeled amino acids by electrophoresis was demonstrated.