A microfabricated capillary electrophoresis chip with multiple buried optical fibers and microfocusing lens for multiwavelength detection

We present a new microfluidic device utilizing multiwavelength detection for high-throughput capillary electrophoresis (CE). In general, different fluorescent dyes are only excited by light sources with appropriate wavelengths. When excited by an appropriate light source, a fluorescent dye emits specific fluorescence signals of a longer wavelength. This study designs and fabricates plastic micro-CE chips capable of performing multiple-wavelength fluorescence detection by means of multimode optic fiber pairs embedded downstream of the separation channel. For detection purposes, the fluorescence signals are enhanced by positioning microfocusing lens structures at the outlets of the excitation fibers and the inlets of the detection fibers, respectively. The proposed device is capable of detecting multiple samples labeled with different kinds of fluorescent dyes in the same channel in a single run. The experimental results demonstrate that various proteins, including bovine serum albumin and beta-casein, can be successfully injected and detected by coupling two light sources of different wavelengths to the two excitation optic fibers. Furthermore, the proposed device also provides the ability to measure the speed of the samples traveling in the microchannel. The developed multiwavelength micro-CE chip could have significant potential for the analysis of DNA and protein samples.     

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.     

Magnification in excess of 100-times of the microscopic photothermal lensing signal from solute molecules by two-color excitation with continuous-wave lasers.

A microscopic photothermal lensing measurement under two-color continuous-wave laser excitation was performed to investigate a signal enhancement owing to the transient absorption by photoexcited solute molecules in liquid solutions. An intensity-modulated 409 nm laser beam and an un-modulated 532 nm laser beam were used for excitation, and a 670 nm probe beam was used for detecting the modulation amplitude of thermal lensing signals generated with a microscopic objective lens focusing laser beams into a capillary flow cell of 0.1 mm optical path length. The amplitude of the modulated signal increased as the power of the un-modulated laser beam increased, and a 143-times magnification was observed for an iso-propanol solution of naphthacene having 4.6 x 10(-4) absorbance at 409 nm and a negligible absorbance at 532 nm. A four-level model explaining the signal enhancement is proposed, and an important role of the transient absorption by photoexcited molecules is discussed.     

Discretely tunable optofluidic compound microlenses

We report a novel method to fabricate high zoom-ratio optofluidic compound microlenses using poly(dimethylsiloxane) with multi-layer architecture. The layered structure of deformable lenses, biconvex and plano-concave, are self-aligned as a group. The refractive index contrast of each lens, which is controlled by filling the chambers with a specific medium, is the key factor for determining the device's numerical aperture. The chip has multiple independent pneumatic valves that can be digitally switched on and off, pushing the liquid into the lens chambers with great accuracy and consistency. This quickly and precisely tunes the focal length of the microlens device from centimetres to sub-millimetre. The system has great potential for applications in portable microscopic imaging, bio-sensing, and laser beam configuration.     

Simultaneous high speed optical and impedance analysis of single particles with a microfluidic cytometer

We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 μm high and 200 μm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer.     

An integrated optics microfluidic device for detecting single DNA molecules

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.     

Two-Color Two-Photon Excitation of Fluorescence

Abstract— We report the observation of two-photon excitation of an organic fluorophore with two different wavelengths, a phenomenon we refer to as two-color two-photon (2C2P) excitation. Ultraviolet emission of p -Merphenyl at 340 nm was observed when the sample was illuminated with both 375 and 750 nm pulses from a picosecond dye laser. The emission of p -terphenyl was about 100-fold and more than 1000-fold less for illumination at only 375 or 750 nm, respectively. Observation of the 2C2P signal required temporal and spatial overlap of the 375 and 750 nm pulses. The amplitude of the signal depended on the polarization of each beam. 2C2P excitation can have applications in fluorescence microscopy and elsewhere when spatially localized excitation is desirable.     

Investigation of the optimum optical design for pulsed-laser crossed-beam thermal lens spectrometry in infinite and finite samples

Optimization of the optical design for pulsed-laser crossed-beam thermal lens (PLCBTL) spectrometry has been investigated. Experiments have been carried out with large samples as well as for very small samples in a microchannel and using different lens combinations to focus the probe and excitation beams. The results have been interpreted in terms of the influence of the excitation beam size as well as the degree of mode-mismatching of the excitation and probe beams on the optimum sample position and on the amplitude and decay of the photothermal signal. A semi-empirical formula that describes the influence of the sample position with respect to the probe beam waist has been established. We have shown that the optimum signal is inversely proportional to the waist of the excitation beam and is independent of the sample size as long as the size of the excitation beam is smaller than the microchannel. Time-resolved experiments have also shown that when the excitation beam is smaller than the sample, the signal decay depends not only on the size of the excitation beam but also on the mode-mismatching factor. Otherwise, the temporal characteristics are closely related to the size of the microchannel.     

Sensitivity enhancement of fluorescence detection in CE by coupling and conducting excitation light with tapered optical fiber

This paper reports the enhancement of sensitivity of detection for in‐column fiber optic‐induced fluorescence detection system in CE by tapered optical fiber (TOF). Two types of optical fiber, TOF and conventional cylindrical optical fiber (COF), were employed to construct the CE (TOF‐CE and COF‐CE) and were compared for sensitivity to riboflavin (RF). The fluorescence intensities from a RF sample with excitation light sources and fibers at various coupling angles were investigated. The fluorescence signal from TOF‐CE was ca. ten times that of COF‐CE. In addition, the detection performance of four excitation light source‐fiber configurations including Laser‐TOF, Laser‐COF, LED‐TOF, and LED‐COF were compared. The LODs for RF were 0.21, 0.82, 0.80, and 7.5 nM, respectively, for the four excitation light source–fiber configurations. The results demonstrate that the sensitivity obtained by LED‐TOF is close to that of Laser‐COF. Both Laser‐TOF and LED‐TOF can greatly improve the sensitivity of detection in CE. TOF has the major attribute of collecting and focusing the excitation light intensity. Thus, the sensitivity obtained by LED‐TOF without focusing lens is just same as that of LED‐COF with a focusing lens. This demonstrates that the CE system can be further simplified by eliminating the focusing lens for excitation light. LED‐TOF‐CE and LED‐COF‐CE system were applied to the separation and determination of RF in real sample (green tea), respectively. The tapered fiber optic‐induced fluorescence detection system in CE is an ideal tool for trace analysis.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

PDMS 2D optical lens integrated with microfluidic channels: principle and characterization

In this paper, the fabrication and characterization of PDMS 2D-optical lenses are reported. These lenses are designed in order to improve the performance of fluorescent spectroscopy detection performed on a portable chip using optical fibers. The fabrication process of the PDMS layer is first detailed, and the patterns are then checked with a SEM. By comparing various interfacial structures, it is shown that the beam properties of the light coming out from the fiber can be modified depending on the lens curvature radius. As a consequence, for a constant dye concentration, the use of such lenses can increase the intensity of fluorescent response close to the fiber or far from the fiber, compared to the same design with a flat interface. This excitation improvement corresponding to a stronger response from the dye then consequently leads to around three times higher sensitivity of the on-chip detection method for fluorescent spectroscopy.     

强脉冲激光激发的热透镜光度分析法

用自装的热透镜测量装置,观察了强脉冲激光作用下的热透镜行为。测量丙酮/水混合液中的Cocl₂,检测限为3×10^-7M,相当于6×10^-6的吸光度。将差分放大技术用于热透镜测量,降低了He-Ne激光振幅噪声的影响,改善了信噪比。     

Fast DNA hybridization on a microfluidic mixing device based on pneumatic driving

A novel fluid mixing strategy was developed which significantly enhanced the efficiency of DNA hybridization. A pneumatic micro-mixing device consisting of two pneumatic chambers and an underneath DNA microarray chamber was built up. The fluid in the array chamber was pneumatically pumped alternately by the two pneumatic chambers. The chaotic oscillatory flow caused by the pumping greatly intensified the fluidic mixing. A homogeneous distribution of the tracer dye solution in the microarray chamber was observed after 2 s mixing with a pumping frequency of 24 Hz. Microarray DNA hybridization was substantially accelerated using this device, and the fluorescence intensity showed a plateau after oscillating 30 s at room temperature. The corresponding signal level of the dynamic hybridization was 12.5-fold higher than that of the static hybridization performed at 42 °C. A signal-to-noise ratio of 117 was achieved and the nonspecific adsorption of the targets to the sample array was minimized, which might be attributed to the strong shearing force generated during the pneumatic mixing process.     

Selective parent ion axialization for improved efficiency of collision-induced dissociation in laser desorption-ionization fourier transform ion cyclotron resonance mass spectrometry

We have systematically established the excitation frequency, amplitude, duration, and buffer gas pressure for optimal axialization efficiency and mass selectivity of quadrupolar excitation-collisional cooling for isolation of parent ions for collision-induced dissociation in Fourier transform ion cyclotron resonance mass spectrometry. For example, at high quadrupolar excitation amplitude, ion axialization efficiency and selectivity are optimal when the applied quadrupolar excitation frequency is lower than the unperturbed ion cyclotron frequency by up to several hundred hertz. Moreover, at high buffer gas pressure (10^?6 Torr), quadrupolar excitation duration can be quite short because of efficient collisional cooling of the cyclotron motion produced by magnetron-to-cyclotron conversion. Efficiency, detected signal magnitude, and mass resolving power for collision-induced dissociation (CID) product ions are significantly enhanced by prior parent ion axialization. With this method, we use argon CID to show that C ₉₄ ⁺ (m/z 1128) formed by Nd:YAG laser desorption-ionization behaves as a closed-cage structure.     

Signal optimisation in cw-laser crossed-beam photothermal spectrometry: influence of the chopping frequency, sample size and flow rate

Optimisation of the optical design for cw-laser crossed-beam thermal lens spectrometry in infinite and finite samples has been investigated using different excitation beam waists and various lens combinations. The characteristics of the photothermal signal depending on the position of the sample with respect to the probe beam waist, the chopping frequency, the sample size and the flow rate have been considered. Depending on the irradiation duration, the size of the thermal element at the measurement time can be much greater than the waist of the excitation beam. As a result, the optimum sample position is closely related to the probe beam to thermal element size ratio and therefore depends on the chopping frequency and of the sample size. At low frequencies, the size of the thermal element is almost independent of the degree of focusing of the excitation beam because a smaller beam waist induces a faster thermal expansion. As a result, the amplitude of the optimum signal does not depend on the waist of the excitation beam. In contrast, at high frequency, the size of the thermal element remains closer to the size of the excitation beam and the signal is inversely proportional to the waist of the excitation beam as previously demonstrated under pulsed-laser excitation. Moreover, at moderate flow velocities, the signal is significantly enhanced because the negative effect produced by the displacement of the thermal element across the probe beam axis is more than compensated by a decrease of the effective thermal time constant due to radial mixing.     

Polymer waveguide backplanes for optical sensor interfaces in microfluidics

A polymer optical backplane capable of generic luminescence detection within microfluidic chips is demonstrated using large core polymer waveguides and vertical couplers. The waveguides are fabricated through a new process combining mechanical machining and vapor polishing with elastomer microtransfer molding. A backplane approach enables general optical integration with planar array microfluidics since optical backplanes can be independently designed but still integrated with planar fluidic circuits. Fabricated large core waveguides exhibit a loss of 0.1 dB cm(-1) at 626 nm, a measured numerical aperture of 0.50, and a collection efficiency of 2.86% in an n = 1.459 medium, comparable to a 0.50 NA microscope objective. In addition to vertical couplers for out-of-plane collection and excitation, polymer waveguides are doped with organic dyes to provide wavelength selective filtering within waveguides, further improving optical device integration. With large core low loss waveguides, luminescence collection is improved and measurements can be performed with simple LEDs and photodetectors. Fluorescein detection via fluorescence intensity with a limit of detection (3sigma) of 200 nM in a 1 microL volume is demonstrated. Phosphorescence lifetime based oxygen detection in water in an oxygen controllable microbial cell culture chip with a limit of detection (3sigma) of 0.08% or 35 ppb is also demonstrated utilizing the waveguide backplane. Single waveguide luminescence collection performance is equivalent to a back collection geometry fiber bundle consisting of nine 500 microm diameter collection fibers.     

应用于助航系统的赋形曲面光学系统研究

非对称式光度系统技术可以实现光能量的收集与再分配，已经广泛应用于照明领域。基于非成像光学理论的自由曲面照明光学设计具有体积小、设计自由度高、出光光形准确可控等优点。通过采用自由曲面对LED进行合理的二次配光，可以实现绿色环保的照明，给未来照明行业的发展指明了新的方向。本文通过对关于LED自由曲面的光学设计进行研究，提出了一种满足侧边发光照明方式的自由曲面透镜设计方法。将得到的自由曲面透镜实体模型作为立式滑行道边灯透镜设计的基本结构。仿真与实验结果表明，仅用一颗LED光源即可实现联邦航空局（FAA）对立式滑行道边灯的光强分布要求。     

A thin cover glass chip for contactless conductivity detection in microchip capillary electrophoresis

A microfabricated thin glass chip for contactless conductivity detection in chip capillary electrophoresis is presented in this contribution. Injection and separation channels were photolithographed and chemically etched on the surface of substrate glass, which was bonded with a thin cover glass (100 μm) to construct a new microchip. The chip was placed over an independent contactless electrode plate. Owing to the thinness between channel and electrodes, comparatively low excitation voltage (20–110 V in V_p–p) and frequency (40–65 kHz) were suitable, and favorable signal could be obtained. This microchip capillary electrophoresis device was used in separation and detection of inorganic ions, amino acids and alkaloids in amoorcorn tree bark and golden thread in different buffer solutions. The detection limit of potassium ion was down to 10 μmol/L. The advantages of this microchip system exist in the relative independence between the microchip and the detection electrodes. It is convenient to the replacement of chip and other operations. Detection in different position of the channel would also be available.     

Microchip-based immunoassay system with branching multichannels for simultaneous determination of interferon-gamma

A bead-bed immunoassay system suitable for simultaneous assay of multiple samples was constructed on a microchip. The chip had branching multichannels and four reaction and detection regions; the constructed system could process four samples at a time with only one pump unit. Interferon gamma was assayed by a 3-step sandwich immunoassay with the system coupled to a thermal lens microscope as a detector. The biases of the signal intensities obtained from each channel were within 10%, and coefficients of variation were almost the same level as the single straight channel assay. The assay time for four samples was 50 min instead of 35 min for one sample in the single-channel assay; hence higher throughput was realized with the branching structure chip.     

Radiative decay of nonstationary system

When a finite quantum system, say a fluorescent molecule is attached to a bulk surface and excited by a short laser pulse, the decay dynamics of the system is modulated by the surface and the signal is enhanced due to the bulk surface. We have considered the decay dynamics of a model of displaced distorted molecule whose excited potential surface is coupled to a continuum and then this first continuum is in turn coupled to a second continuum. In the short time scale there is a coherent exchange of energy between the system molecule and the first continuum states. In the long time scale the energy of the whole system plus first continuum drains out to the final continuum states. A dendrimer nanocomposite with the gold surface shows an enhanced light emission. This can be qualitatively understood from the model we proposed here. We have numerically studied the various potential parameters of the molecule which can affect the signal. When the potential surfaces are flat, the band structure of the first continuum states along with its initial excitation has some nontrivial effect on the profile of the radiative decay.