Label-free fluorescence detection in capillary and microchip electrophoresis

Herein, we summarize the current status of native fluorescence detection in microchannel electrophoresis, with a strong focus on chip-based systems. Fluorescence detection is a powerful technique with unsurpassed sensitivity down to the single-molecule level. Accordingly fluorescence detection is attractive in combination with miniaturised separation techniques. A drawback is, however, the need to derivatize most analytes prior to analysis. This can often be circumvented by utilising excitation light in the UV spectral range in order to excite intrinsic fluorescence. As sensitive absorbance detection is challenging in chip-based systems, deep-UV fluorescence detection is currently one of the most general optical detection techniques in microchip electrophoresis, which is especially attractive for the detection of unlabelled proteins. This review gives an overview of research on native fluorescence detection in capillary (CE) and microchip electrophoresis (MCE) between 1998 and 2008. It discusses material aspects of native fluorescence detection and the instrumentation used, with particular focus on the detector design. Newer developments, featured techniques, and their prospects in the future are also included. In the last section, applications in bioanalysis, drug determination, and environmental analysis are reviewed with regard to limits of detection.     

Detection method for microchip separations

The features of analytical systems utilizing microfluidic devices, especially detection methods, are described. Electrochemical detection (EC), laser-induced fluorescence (LIF), mass spectrometry (MS), and chemical luminescence (CL) methods are covered. EC enables detection without labeling and has been used in recent years because of its low cost and sensitivity. LIF is the most generally used detection method in microchip separations. Use of LED as an excitation source for fluorescence measurement was also developed for the purpose of miniaturization of the entire system, including detection and separation. Although MS enables highly sensitive analysis, the interface between MS and micro channels is still under examination. This review with fifty-two references introduces interesting detection methods for microchip separations. Related separation methods using microfluidic devices are also discussed.     

A hybrid microdevice with a thin PDMS membrane on the detection window for UV absorbance detection

We exploited a PDMS-quartz hybrid microchip with a thin PDMS membrane on the concave detection window for UV absorbance detection. The thickness of the PDMS membrane is about 100 mum, with high UV transmittance. As compared to a PDMS-quartz hybrid chip with a common detection window, the proposed one exhibited over an one order of magnitude sensitivity enhancement, and an about two orders of magnitude S/N increase for gastrodin (p-hydroxymethylphenyl-beta-D-glucopyranoside). In addition, the limit of the detection wavelength has been extended from 240 to 210 nm, which is otherwise impossible for a traditional PDMS-quartz hybrid microchip. This kind of microchip has the potential for a large range of applications in an integrated microfluidic system with UV detection.     

Ultraviolet absorbance detection of colchicine and related alkaloids on a capillary electrophoresis microchip

The separation and UV absorbance detection of four toxic alkaloids, colchicine, thiocolchicine, colchicoside, and thiocolchicoside, on a microchip-based capillary electrophoresis device are reported. To increase the sensitivity of UV absorbance detection, optical cells with extended path lengths were integrated into the separation channel during the microfabrication process. The absorbance values realized on the microchip using these optical cells were proportional to the increase in average depths according to the Beer-Lambert Law, resulting in sensitivity enhancements by as much as five times. Linearity of response was observed from 5.0 to 500 mg L⁻¹ of colchicine, with detection limits ranging from 2 to 6 mg L⁻¹ depending upon the specific alkaloid and the dimension of the optical cell. The extraction of colchicine from spiked milk samples was performed and an average recovery rate of 83% with a relative standard deviation of 3.8% was determined using the optimized conditions on the microchip.     

Two‐photon excitation in chip electrophoresis enabling label‐free fluorescence detection in non‐UV transparent full‐body polymer chips

One of the most commonly employed detection methods in microfluidic research is fluorescence detection, due to its ease of integration and excellent sensitivity. Many analytes though do not show luminescence when excited in the visible light spectrum, require suitable dyes. Deep‐ultraviolet (UV) excitation (<300 nm) allows label‐free detection of a broader range of analytes but also mandates the use of expensive fused silica glass, which is transparent to UV light. Herein, we report the first application of label‐free deep UV fluorescence detection in non‐UV transparent full‐body polymer microfluidic devices. This was achieved by means of two‐photon excitation in the visible range (λ_ex = 532 nm). Issues associated with the low optical transmittance of plastics in the UV range were successfully circumvented in this way. The technique was investigated by application to microchip electrophoresis of small aromatic compounds. Various polymers, such as poly(methyl methacrylate), cyclic olefin polymer, and copolymer as well as poly(dimethylsiloxane) were investigated and compared with respect to achievable LOD and ruggedness against photodamage. To demonstrate the applicability of the technique, the method was also applied to the determination of serotonin and tryptamine in fruit samples.     

High-throughput screening and optimization approaches for chiral compounds by means of microfluidic devices

Chiral separations facilitated using microchip devices are reviewed in this paper. The first research paper on this topic was published in 1999. It was seen that analysis times are greatly reduced compared with more conventional techniques such as liquid chromatography and capillary electrophoresis, and that these devices enable the separation of chiral molecules. Method optimization can be conducted in a rather easy manner, reducing the total method development time. Finally, minute amounts of sample and buffer are used during analysis, which makes the systems ultra-economical. Although the number of applications in the chiral separation field on these miniaturized systems is still rather limited, they exhibit much potential towards high-throughput screening. Some efforts are, however, still needed regarding detection modes, because derivatisation of the samples is often needed to enable their detection.     

Analysis of inorganic and small organic ions with the capillary electrophoresis microchip

Capillary electrophoresis microchip devices are receiving considerable attention due to their versatility, portability, and sample handling capabilities. This article is a comprehensive review of the analysis of inorganic and small, charged organic species on microchip platforms. The application of conductivity, amperometry, laser-induced fluorescence, absorbance, and chemiluminescence detection methods are discussed. The potential utilization of these devices for miniaturized analytical systems is described.     

A far-red fluorescent probe for selective G-quadruplex DNA targeting

The development of rapid and simple approaches for detection of G-quadruplex DNA structures has attracted significant attention to disclose their diverse physiological and pathological functions. Thiazole orange (TO) is a common fluorescence probe used for the detection of G-quadruplexes. However, it still suffers from some common problems like non-selective for G-quadruplex and emission in the lower wavelength region of spectrum, thus hampering its further applications. Probes with turn-on fluorescence in the far-red region are highly sought-after due to minimal auto-fluorescence and cellular damage. In this paper, we described a far-red fluorescent probe (L-1) by introducing an amine group into styrylquinolinium scaffold. The experimental results indicated that L-1 exhibited significant fluorescence enhancement when treated with G-quadruplexes but retained weak fluorescence in the presence of duplex DNAs. In addition, this probe also displayed higher binding affinity for parallel G-quadruplexes. The characteristics of L-1 were further investigated with UV–vis spectrophotometry, fluorescence, circular dichroism, KI quenching, FID assay and molecular docking to validate optical photophysical properties, as well as the selectivity, sensitivity and detailed binding mode toward G-quadruplex DNAs.     

Fluorometric determination of sulfite and nitrite in aqueous samples using a novel detection unit of a microfluidic device.

On-chip fluorescence determination of sulfite and nitrite with N-(9-acridinyl)maleimide (NAM) and 2,3-diaminonaphthalene (DAN) has been developed using a novel fluorescence detection unit for microchip analysis. Usually, these fluorescence reagents are derivatized and detected separately in microchip analysis because different fluorescence wavelengths are emitted. The proposed fluorescence detection unit has optical fibers with no optical filter, and plural wavelengths of fluorescence were detected sensitively, even in the microchip. In this study, the simultaneous determination of sulfite and nitrite in environmental samples was performed with a polymer microchip analysis system. The calibration curves of sulfite and nitrite showed linear relations (R2 = 0.998 (sulfite) and R2 = 0.990 (nitrite)), and the relative standard deviations (RSD) for 4 runs were 2.1% (20 microM sulfite) and 1.3% (20 microM nitrite), respectively. The proposed method was applied to the recovery test of sulfite and nitrite in environmental samples.     

Performance of an in-plane detection cell with integrated waveguides for UV/Vis absorbance measurements on microfluidic separation devices

A microfluidic device with integrated waveguides and a long path length detection cell for UV/Vis absorbance detection is presented. The 750 microm U-cell detection geometry was evaluated in terms of its optical performance as well as its influence on efficiency for electrophoretic separations in the microdevice. Stray light was found to have a strong effect on both, the sensitivity of the detection and the available linear range. The long path length U-cell showed a 9 times higher sensitivity when compared to a conventional capillary electrophoresis (CE) system with a 75 microm inner diameter (ID) capillary, and a 22 times higher sensitivity than with a 50 microm ID capillary. The linear range was comparable to that achieved in a 75 microm ID capillary and more than twice as large as in a 50 microm ID capillary. The use of the 750 microm U-cell did not contribute significantly to band broadening; however, a clear quantification was made difficult by the convolution of several other band broadening sources.     

Label-free analysis in chip electrophoresis applying deep UV fluorescence lifetime detection

Herein we introduce deep UV fluorescence lifetime detection in microfluidics applied for label-free detection and identification of various aromatic analytes in chip electrophoresis. For this purpose, a frequency quadrupled Nd:YAG (neodymium-doped yttrium aluminum garnet) picosecond laser at 266 nm was incorporated into an inverse fluorescence microscope setup with time-correlated single photon counting detection. This allowed recording of photon timing with sub-nanosecond precision. Thereby fluorescence decay curves are gathered on-the-fly and average lifetimes can be determined for each substance in the electropherogram. The aromatic compounds serotonin, propranolol, 3-phenoxy-1,2-propanediol and tryptophan were electrophoretically separated using a fused-silica microchip. Average lifetimes were independently determined for each compound via bi-exponential tail fitting. Time-correlated single photon counting also allows the discrimination of background fluorescence in the time domain. This results in improved signal-to-noise-ratios as demonstrated for the above model analytes. Microchip electrophoretic separations with fluorescence lifetime detection were also performed with a protein mixture containing lysozyme, trypsinogen and chymotrypsinogen emphasizing the potential for biopolymer analysis.     

Microchip micellar electrokinetic chromatography separation of alkaloids with UV-absorbance spectral detection

A microchip device is demonstrated for the electrophoretic separation and UV-absorbance spectral detection of four toxic alkaloids: colchicine, aconitine, strychnine, and nicotine. A fused-silica (quartz) microchip containing a simple cross geometry is utilized to perform the separations, and a miniature, fiber-optic CCD spectrometer is coupled to the microchip for detection. Sensitive UV-absorbance detection is achieved via the application of online preconcentration techniques in combination with the quartz microchip substrate which contains an etched bubble-cell for increased pathlength. The miniature CCD spectrometer is configured to detect light between 190 and 645 nm and LabView programming written in-house enables absorbance spectra as well as separations to be monitored from 210 to 400 nm. Consequently, the configuration of this microchip device facilitates qualitative and quantitative separations via simultaneous spatial and spectral resolution of solutes. UV-absorbance limits of quantification for colchicine, 20 microM (8 mg/L); strychnine, 50 microM (17 mg/L); aconitine, 50 microM (32 mg/L); and nicotine, 100 microM (16 mg/L) are demonstrated on the microchip. With the exception of aconitine, these concentrations are > or =20-times more sensitive than lethal dose monitoring requirements. Finally, this device is demonstrated to successfully detect each toxin in water, skim milk, and apple juice samples spiked at sublethal dose concentrations after a simple, SPE procedure.     

Fabrication of calcium fluoride capillary electrophoresis microdevices for on-chip infrared detection

In this paper, we demonstrate microfluidic capillary electrophoresis (CE) devices made in CaF2 , for optical detection in a broad spectral range. We have designed methods for micromachining and enclosing capillaries in CaF2. The utility of these microdevices has been shown through CE analysis of fluorescently labeled amino acids. We have also performed infrared spectroscopy for analyte identification in microfluidic CaF2 channels. These CaF2 microdevices open the door to microchip separations with optical detection in the ultraviolet, visible, and infrared spectral regions.     

Detection of Copper Ion with Laser-Induced Fluorescence in a Capillary Electrophoresis Microchip

A capillary electrophoresis microchip coupled with a confocal laser-induced fluorescence (LIF) detector was successfully constructed for the analysis of trace amounts of heavy metals in environmental sources. A new fluorescence dye, RBPhOH, synthesized from rhodamine B, was utilized in a glass microchip to selectively determine copper with high sensitivity. A series of factors including running buffer concentration, detection voltage, and sample loading time were optimized for maximum LIF detector response and, hence, method sensitivity.     

Capillary gel electrophoresis of single-stranded DNA fragments with UV detection

Capillary gel electrophoresis has proven to be a powerful tool in biomedical research. We report our investigation of some of the critical parameters affecting separations of single-stranded DNA fragments as monitored by ultraviolet (UV) absorbance detection. Although not as sensitive as laser-induced fluorescence (LIF), UV absorbance detection allows one to calculate quite accurately, and inexpensively, the molarity of each separated DNA fragment and, moreover, the signal “fading” effect normally observed with LIF detection can be, in many cases, substituted for fluorescence to detect the many different single-stranded DNAs, as well as for detection of sequencing reactions.     

High-speed separation of proteins by microchip electrophoresis using a polyethylene glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution

In this paper, we describe a method for size-based electrophoretic separation of sodium dodecyl sulfate (SDS)-protein complexes on a polymethyl methacrylate (PMMA) microchip, using a separation buffer solution containing SDS and linear polyacrylamide as a sieving matrix. We developed optimum conditions under which protein separations can be performed, using polyethylene glycol (PEG)-coated polymer microchips and electrokinetic sample injection. We studied the performance of protein separations on the PEG-coated PMMA microchip. The electrophoretic separation of proteins (21.5-116.0 kDa) was completed with separation lengths of 3 mm, achieved within 8 s on the PEG-coated microchip. This high-speed method may be applied to protein separations over a large range of molecular weight, making the PEG-coated microchip approach applicable to high-speed proteome analysis systems.     

Embossed polymer leaky waveguide devices for spectroscopic analysis

A combination of UV embossing, hot embossing and laminating techniques have been exploited during the fabrication of grating-coupled waveguiding devices in which the flow channel materials and geometry are designed for the effective support of light condenser modes through a sample solution. Used in conjunction with LED sources, these devices have been employed as optical platforms to obtain fluorescence emission and molecular absorption spectra in aqueous media for microTAS (micro total analytical system) applications.     

Harnessing dielectric forces for separations of cells, fine particles and macromolecules

A review of conventional dielectrophoresis on a microchip platform is presented. The benefits of miniaturization, some device geometries used to accomplish on-chip separations, and applications of these devices are discussed.     

CE detector based on light-emitting diodes

CE detectors based on light-emitting diodes (LEDs) as light sources are receiving considerable attention due to their exceptionally high stability, high intensity, low cost, and a variety of wavelengths in the UV and visible spectrum. This article is a comprehensive review on CE methods using LED-based detectors with absorbance and fluorescence detection, and several applications on the determination of riboflavin, bacteria, drug, and amino acids in biological samples are described.