旋转激光扫描共焦荧光检测式毛细管阵列电泳

研制了一种新型毛细管阵列电泳仪——旋转激光扫描共焦荧光检测式毛细管阵列电泳。采用圆形毛细管阵列,激光扫描共聚焦荧光检测方式,利用了高速直流电机旋转反射镜来进行激光扫描,并且加快了采样速率;采用旋转编码器获得毛细管的绝对位置并对数据采集系统触发来采集信号,并利用研制的8通道毛细管阵列电泳对酪氨酸、赖氨酸、谷氨酸及天冬氨酸的混合物进行了分离分析,获得了良好的分离效果。     

Home-made capillary array electrophoresis for high-throughput amino acid analysis

A capillary array electrophoresis (CAE) with confocal rotary scanner for high-throughput carboxytetramethylrhodamine succinimidyl ester (TAMRA)-labeled amino acid (AA) analysis is presented. Performance of the CAE setup was evaluated with AA samples. Up to 128 capillaries could be detected in parallel. For the first time, the device was applied to separate the enantiomers of isoleucine, cysteine, and glutamic acid with cyclodextrin-modified electrolytes by capillary zone electrophoresis. Baseline separation of seven AAs is also demonstrated using micellar electrokinetic chromatography method.     

532 nm激光诱导荧光毛细管阵列电泳仪的研制

基于共聚焦激光诱导荧光检测技术,研制了一台旋转扫描高效毛细管阵列电泳装置.以波长为532nm的半导体二极管激光器作为激发光源,多根毛细管阵列采用圆形布局,微型高速直流电机带动旋转反射镜进行激光扫描,加快了数据采集的速度;采用旋转编码器实现了毛细管的定位与位置读出,并触发数据采集系统进行荧光信号采集.以罗丹明6G和罗丹明B为分析样品,考察了16通道毛细管阵列电泳仪的基本性能.     

532nm激光诱导荧光毛细管阵列电泳仪的研制

基于共聚焦激光诱导荧光检测技术,研制了一台旋转扫描高效毛细管阵列电泳装置。以波长为532nm的半导体二极管激光器作为激发光源,多根毛细管阵列采用圆形布局,微型高速直流电机带动旋转反射镜进行激光扫描,加快了数据采集的速度;采用旋转编码器实现了毛细管的定位与位置读出,并触发数据采集系统进行荧光信号采集。以罗丹明6G和罗丹明B为分析样品,考察了16通道毛细管阵列电泳仪的基本性能。     

Capillary array electrophoresis with confocal fluorescence rotary scanner

A capillary array electrophoresis system with a rotary confocal fluorescence scanner is reported. A high speed direct current rotary motor, combined with a rotary encoder and a reflection mirror, has been designed to direct the excitation laser beam precisely to a round array of capillaries which are symmetrically distributed around the motor. The rotary encoder is introduced to accurately orientate the position of each capillary and its output signal triggers the data acquisition system to record the fluorescence signal corresponding to each capillary. Separation of enantiomers of glutamic acid, methionine and tryptophan with different additives are demonstrated by this system. The experimental results indicate that this setup can be used to optimize separation methods for capillary electrophoresis as quickly as possible.     

On-line wall-free cell for laser-induced fluorescence detection in capillary electrophoresis

A wall-free detection method based on liquid junction in a capillary gap was proposed for laser-induced fluorescence (LIF) of capillary electrophoresis (CE). The capillary gap of the wall-free cell was fabricated by etching a 10-mm × 50-μm I.D. fused-silica capillary to obtain a polyimide coating sleeve, decoating about 6 mm at one end of both 50 μm I.D. separation and liquid junction capillary, inserting the treated capillary ends into the coating sleeve oppositely, fixing the capillaries with a gap distance of 140 μm by epoxy glue and removing the coating sleeve by burning. The theoretical model, experimental results and wall-free cell images indicated that the gap distance and applied voltage were main influence factors on the wall-free detection. Since the wall-free cell increased the absorption light path and avoided the stray light from the capillary wall, it improved the ratio of signal to noise and limit of detection (LOD) of CE-LIF. Three flavin compounds of riboflavin (RF), flavin mononucleotide sodium (FMN) and flavin adenine dinucleotide disodium (FAD) were used to evaluate the wall-free detection method. Compared with on-column cell, the LODs of the wall-free cell were improved 15-, 6- and 9-fold for RF, FMN and FAD, respectively. The linear calibration concentrations of the flavins ranged from 0.005 to 5.0 μmol/L. The column efficiency was in the range from 1.0 × 10⁵ to 2.5 × 10⁵ plates. The wall-free detection of CE-LIF was applied to the analysis of the flavins in spinach and lettuce leaves.     

Very low optical absorptions and analyte concentrations in water measured by Optimized Thermal Lens Spectrometry

Thermal Lens Spectrometry has traditionally been carried out in the single-beam and the mode-mismatched dual-beam configurations. Recently, a much more sensitive dual-beam TL setup was developed, where the probe beam is expanded and collimated. This feature optimizes Thermal Lens (TL) signal and allows the use of thicker samples, further improving the sensitivity. In this paper, we have made comparisons between the conventional and optimized TL configurations, and presented applications such as measurements of very low absorptions and concentrations in water and Cr(III) aqueous solution in the UV-vis range. For pure water we found linear absorption coefficients as low as the Raman scattering one due to the stretching vibrational modes of OH group. The detection limit was estimated 1 × 10⁻⁶ cm⁻¹ with a 180-mW excitation power using a 100-mm cell length. This sensitivity is very high, considering that water has a photothermal enhancement factor ∼33 times smaller than CCl₄, for example. For Cr(III) species in aqueous solution, the limit of detection (LOD) was estimated in ∼40 ng mL⁻¹ at 514 nm, or ∼10 ng mL⁻¹ at 405 nm, which is ∼30 times smaller than the LOD achieved with conventional transmission techniques. The more recent TL configuration is very attractive to obtain absorption spectra, since the result does not depend critically on the beam parameters, unlike the other configurations. The main drawbacks of this optimized TL configuration are the longer acquisition time and the need for larger samples.     

Capillary scale light emitting diode based multi-reflection absorbance detector

We describe a light emitting diode (LED) based multi-reflection capillary scale absorbance detector based on both square and round capillaries and compare their performance with a conventional single-pass on-tube detector. The optical path length is extended by silver coating, the external surface of the capillary. The reflective geometry has been reported to be less prone to artifacts induced by refractive index changes; we do find this to be true. Although the detection volume/illuminated volume is increased some, a multi-reflection cell based on a 180 μm bore capillary with a ∼2-cm long illuminated volume shows over a 50-fold gain in signal-to-noise (S/N) compared to a single-pass on-tube configuration with the same capillary. The limit of detection (LOD) is 4.4 fmol (2.6 pg, 1 μL of 22.0 nM injected dye) BTB under pulseless (pneumatic) flow conditions. The cells behave as multipath devices where the effective path lengths are greater at low absorbance values. In our experiments, where non-coherent light is launched through optical fibers that are large compared to capillary bore dimensions, increase in the effective path length of the cell do not occur in a predictable fashion with the angle of incidence of the light beam. Although the effective path length almost linearly increases with increasing distance between the light entry and exit windows, the absolute values of the effective path lengths are always lower than this physical distance, suggesting that after some passage through the solution, light largely travels through or along the glass wall. Square capillaries have better light transmission and offer some performance advantages. Multi-reflection cells can indeed be of value for sensitive detection in microflow systems.     

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.     

A simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence detection for the analysis of chelerythrine and sanguinarine in Chinese herbal medicines

Laser-induced fluorescence (LIF) is a highly sensitive detection method for capillary electrophoresis (CE). However, it usually requires analyte to be derivatized, unless the wavelength of native fluorescence of analyte matches the laser's. That limits its application in drug analysis. In this work, we introduced a rapid, simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence (NACE-LIF) detection for the analysis of chelerythrine and sanguinarine for the first time. As these two alkaloids have some native fluorescence, they were directly detected using a commercially available Ar⁺ laser without troublesome fluorescent derivatization. The fluorescence was enhanced by nonaqueous media. Compared with previously reported UV detection method, lower limit of detection (LOD) is achieved thanks to the high sensitivity of LIF detection (2.0 ng/mL for chelerythrine and 6.3 ng/mL for sanguinarine). Moreover, with NACE, the baseline separation of these alkaloids is finished within 3.5 min. This method is successfully applied to determine the contents of chelerythrine and sanguinarine in Macleaya cordata (Willd.) R. Br. and Chelidonium majus L.     

High through-put aflatoxin determination in plant material by automated solid-phase extraction on-line coupled to laser-induced fluorescence screening and determination by liquid chromatography–triple quadrupole mass spectrometry

A screening test based on laser-induced fluorescence (LIF) and a method for individual identification – quantitation of aflatoxins (AFs) in olive leaves and drupes, based on chromatographic separation and triple-quad mass-spectrometry detection with electrospray ionization in positive mode, is here reported. The sensitivity and selectivity of both methods are enhanced by a preconcentration–cleanup step developed by a Prospekt station. The analysis frequency is at least 3.5 samples/h. The screening test makes able to detect the target analytes at concentrations of 0.7 μg/kg without “false negatives”. The LC–MS/MS method provides limits of detection (LOD) and quantification (LOQ) ranging between 0.01–0.03 and 0.03–0.11 μg/kg, respectively. The linear dynamic range is between LOQ–50 μg/kg. The between-day precision, expressed as relative standard deviation, ranges between 0.97–2.86% and the within laboratory reproducibility, also expressed as RSD, between 1.63% and 4.84%.     

Determination of mono- and dichloroacetic acids in betaine media by liquid chromatography

A simple and sensitive method has been developed for the analysis of residue amounts of chloroacetic acids in betaine samples based on derivatization by 1-naphthylamine (NA). The derivatized compounds are analyzed by reverse phase high performance liquid chromatography using methanol and water as mobile phase in the ratio of 32/68 (v/v) and phenyl column and PDA detection at 222 nm. The detection limits (LOD) of monochloroacetic acid (MCA) and dichloroacetic acid (DCA) are 0.1 and 0.15 μg mL⁻¹, respectively. The limits of quantification (LOQ) and the linear dynamic ranges (LDR) of MCA are found to be 1 and 1-400 μg mL⁻¹, respectively, and for DCA are found to be 3 and 3-400 μg mL⁻¹, respectively. The precision at the 5 ppm level for MCA and DCA are about 3% and 2%, (n = 5), respectively. The average recovery for MCA and DCA spiked to betaine samples are 98% and 97%, respectively.     

Simultaneous detection of ultraviolet B-induced DNA damage using capillary electrophoresis with laser-induced fluorescence

An immunoassay based on CE–LIF was developed for the simultaneous detection of cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) in genomic DNA irradiated with UVB or natural sunlight. Human cells were first exposed to varying amounts of UVB or natural sunlight to induce DNA damage. Genomic DNA was extracted and incubated with anti-CPD and anti-6-4PP primary antibodies attached to secondary antibodies with a fluorescent quantum dot (QD) reporter that emitted either red or yellow fluorescence. CE was used to separate the unbound antibodies from those bound to the photoproducts, and LIF with appropriate optical filters was used to separate the fluorescence signals from each QD to individual photomultiplier tubes for simultaneous photoproduct detection. Using this strategy, photoproducts were detected from ∼6 ng (200 ng μL⁻¹) of DNA under a low UVB fluence of 65 J m⁻² for CPDs or 195 J m⁻² for 6-4PPs. This assay was also the first to demonstrate the detection of CPDs in human cells after only 15 min of irradiation under natural sunlight.     

Fabrication and characterisation of high performance polypyrrole modified microarray sensor for ascorbic acid determination

In this study, gold microelectrode array (Au/MEA) with electrode of 12 μm diameter was fabricated by photolithography technique. Subsequently, polypyrrole (Ppy) modified gold microarrays sensor (Ppy/Au/MEA) was prepared by cyclic voltammetry technique. The deposition potential range and number of cycles were optimised in order to get optimum thickness of Ppy film. Scanning Electron Microscope and Atomic Force Microscope investigations reveal that Ppy coating formed at 3 cycles is porous with thickness of 1.5 μm which exhibiting high catalytic current for ascorbic acid (AA) in square wave technique (SWV). In contrast to earlier sensors designs, these Ppy/Au/MEA sensors exhibits lower detection limit (LOD) of 10 nm towards AA at physiological conditions. It also exhibits enhanced sensitivity (2.5 mA cm⁻² mM⁻¹) and long range of linear detection limit from 10 nm to 2.8 mM. In the same way, polypyrrole modified macro Au (Ppy/Au/MA) biosensor was also fabricated and its electro catalytic property towards AA was compared with that of Ppy/Au/MEA. The Ppy/Au/MA exhibits sensitivity of only 0.27 mA cm⁻² mM⁻¹, LOD of 5 μM and linear range of 10 μM to 2.2 mM. Hence, our investigations indicate that the Ppy/Au/MEA could serve as highly sensitive sensor for AA than any of the earlier designs. So, the Ppy/Au/MEA electrode was utilised for determination AA in a wide variety of real samples.     

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.     

Development of a ligase detection reaction/CGE method using a LIF dual‐channel detection system for direct identification of allelic composition of mutated DNA in a mixed population of excess wild‐type DNA

We developed an inexpensive LIF dual‐channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low‐abundance point mutations in a large excess of wild‐type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K‐ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K‐ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild‐type sequence‐dominated population, and to disclose their allelic composition, was thus demonstrated successfully.     

Portable flow-injection analyzer with liquid-core waveguide based fluorescence, luminescence, and long path length absorbance detector

We describe a multifunctional flow analysis instrument that is portable ( cm, 2.3 kg) for facile field deployment. Using a 50 cm long Teflon^® AF tubing as final reaction and optical measurement conduit, we combine a liquid-core waveguide (LCW) based fluorescence detector that is transversely illuminated by an addressable light emitting diode array, a chemiluminescence (CL) detector and an absorbance detector with a solid-state broadband (400-700 nm) source. Several illustrative experiments have been carried out to test the performance of the instrument in different detection modes. A S/N=3 limit of detection (LOD) of 0.25 μg l⁻¹ for chromium(VI) was established using the diphenylcarbazide chemistry, and an LOD of 5 μg l⁻¹ was similarly established for Al(III), using Pyrocatechol Violet (PCV) as the chelating chromogenic dye, in both cases using long path absorption detection. The LOD for aqueous hydrogen peroxide was 16 nM using a fluorescence method based on the formation of thiochrome from thiamine and 4 nM by a luminol chemiluminescence method. With a Nafion membrane diffusion scrubber (DS), the LOD was 8.0 pptv for gaseous hydrogen peroxide by the fluorescence method.     

An economic approach for construction of a multiplexed capillary electrophoresis system

We describe in this report an economic approach to construct a multiplexed capillary electrophoresis (CE) system with optic-fiber UV detection. To demonstrate the feasibility of this approach, a seven-lane CE setup is built. The system has a background noise of ∼10⁻⁵ absorbance unit, a limit of detection of 3-4 μM for uracil at 254 nm, and a linear dynamic range of 2.5 orders of magnitude. The apparatus consists of three major components: a UV light source, a photodiode array (PDA) and optical module assembly, and a computer. Compared to the recently reported 96-lane capillary electrophoresis system with UV-vis absorbance detection, the cost of goods is reduced by more than 10-fold, and the noise level is improved by 2 to 3 folds. Parallel CE separations of a protein mixture have been performed on this apparatus, and reproducible and high resolution separation results have been obtained. Detailed construction and characterization of the system have also been discussed.     

Lamp-based native fluorescence detection of proteins in capillary electrophoresis

The potential of a recently developed lamp-based fluorescence detector for the analysis of underivatised proteins by capillary electrophoresis (CE) was investigated. Fluorescence detection (Flu) was achieved using optical light guides to deliver excitation light from a Xenon–Mercury lamp to the capillary detection window and to collect fluorescence emission and lead it to a photomultiplier. The performance of the detector was evaluated by monitoring the native fluorescence of the amino acid tryptophan and the proteins α-chymotrypsinogen A, carbonic anhydrase II, lysozyme and trypsinogen upon excitation at 280 nm. The test compounds were analysed using background electrolytes (BGEs) of sodium phosphate at pH 3.0 and 11.3. The results were compared to experiments of CE with UV absorbance detection. For tryptophan, a linear fluorescence response was obtained with a dynamic range of over 4 orders of magnitude, and a limit of detection (LOD) of 6.7 nM. This LOD was a factor of 200 more favourable than UV detection at 280 nm, and a factor of 20 better than detection at low-UV wavelengths. All tested proteins showed linear fluorescence responses up to 250 μg/mL. LODs were typically in the 10–20 nM range. These LODs were a factor of 25 lower than for UV detection at 280 nm, and comparable to UV detection at low-UV wavelengths. Overall, Flu yields much more stable baselines, especially with a BGE of high pH. The applicability of CE–Flu is demonstrated by the analysis of a degraded protein mixture, and of an expired formulation of the protein drug human growth hormone, indicating that protein degradation products can be selectively detected.     

Study of the voltammetric behaviour of maleic hydrazide and its determination at a hanging mercury drop electrode

Voltammetric behaviour of maleic hydrazide pesticide dissolved in a Britton-Robertson buffer was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). It was found that the process at the Hg electrode was diffusion controlled; the reaction was irreversible and involved a change of one proton and a transfer of one electron. A quantitative differential pulse voltammetric method for determination of maleic hydrazide was developed on the basis of these studies involving the reduction of the compound at a hanging mercury drop electrode. A linear calibration was obtained in the range of 0.5-5.5 mg l⁻¹, and the developed DPV methodology was then applied for the determination of maleic hydrazide in spiked vegetable samples by the standard addition method. Satisfactory percentage R.S.D. (∼2%), percentage recovery values (∼85%) and LOD (0.215 mg l⁻¹) were obtained. These compared well with the results from the alternative spectrophotometric method.