A polydimethylsiloxane/glass capillary electrophoresis microchip for the analysis of biogenic amines using indirect fluorescence detection

A polydimethylsiloxane-glass capillary microchip is fabricated for the rapid analysis of a mixture of common biogenic amines using indirect fluorescence detection. Using a running buffer of phosphate and 2-propanol, and Rhodamine 110 as a background fluorophore, both co-ionic and counter-ionic systems are explored. Studies demonstrate the separation and analysis of cations using indirect fluorescence detection for the first time in a chip-based system. Resulting electrophoretic separations are achieved within a few tens of seconds with detection limits of approximately 6 microM. The reduced sample handling and rapid separations afforded by the coupling of indirect fluorescence detection with chip-based capillary electrophoresis provide a highly efficient method for the analysis and detection of molecules not possessing a chromophore or fluorophore. Furthermore, limits of detection are on a par with reported chip-based protocols that incorporate precolumn derivatisation with fluorescence detection. The current device circumvents lengthy sample preparation stages and therefore provides an attractive alternative technique for the analysis biogenic amines.     

Chip electrophoresis of active banana ingredients with label-free detection utilizing deep UV native fluorescence and mass spectrometry

In the present work, we report on a rapid and straightforward approach for the determination of biologically active compounds in bananas applying microchip electrophoresis (MCE). For this purpose, we applied label-free detection utilizing deep UV fluorescence detection with excitation at 266 nm. Using this approach, we could identify dopamine and serotonin, their precursors tryptophan and tyrosine and also the isoquinoline alkaloid salsolinol in less than 1 min. In bananas, after 10 days of ripening, we additionally found the compound levodopa which is a metabolite of the tyrosine pathway. Quantitative analysis of extracts by external calibration revealed concentrations of serotonin, tryptophan, and tyrosine from 2.7 to 7.6 μg/mL with relative standard deviations of less than 3.5%. The corresponding calibration plots showed good linearity with correlation coefficients higher than 0.985. For reliable peak assignment, the compounds were also analyzed by coupling chip electrophoresis with mass spectrometry. This paper demonstrates exemplarily the applicability of MCE with native fluorescence detection for rapid analysis of natural compounds in fruits and reveals the potential of chip-based separation systems for the analysis of complex mixtures.     

Nonaqueous electrophoresis on microchips: A review

The use of organic solvents as electrolytic medium in electrophoresis has become an important alternative for the analysis of compounds that exhibit low or no solubility in water. In recent years, nonaqueous electrophoresis has been extensively explored in conventional capillary systems for different applications. On the other hand, this separation strategy is still not as popular as free solution electrophoresis on chip-based platforms due to the effects of solvent in the background electrolyte on the sample injection, detection performance, and microfluidic platform compatibility. In this way, this review summarizes the main achievements on nonaqueous microchip electrophoresis (NAME). To the best of our knowledge, this is the first review dedicated to discuss exclusively nonaqueous electrophoresis on chip-based systems. For this purpose, some important theoretical aspects involved when separations are performed in organic medium, such as equilibrium, interactions and electrophoretic considerations, are included in the review. In addition, the main challenges, advantages and influences of nonaqueous media on the sample injection, detection as well as the choice of the substrate to fabricate chip-based electrophoresis devices are highlighted. Last, examples showing the feasibility of nonaqueous microchip electrophoresis for applications exploiting different methodologies, operational, and instrumental conditions are summarized and discussed. We hope this review can be useful to spread the huge potential of nonaqueous electrophoresis on microfluidic platforms.     

Electrochemical techniques for lab-on-a-chip applications

During the last few years there has been a rapid increase in the use of electrochemical reactions in lab-on-a-chip devices. This development, which has so far mainly focussed on electrochemical detection in chip-based capillary electrophoresis, can be explained by the fact that electrochemical techniques and devices are particularly well-suited for inclusion in lab-on-a-chip systems. The most important reason for this is that the required electrodes can readily be manufactured and miniaturised without loss of analytical performance using conventional microfabrication methods. In this Research Highlight article, the developments during the last three years concerning electrochemical techniques for lab on-a-chip applications are discussed, with particular focus on emerging electrochemical methods for sample clean-up and preconcentration, electrochemical derivatisation and electrochemical detection in chip-based capillary electrophoresis.     

Recent developments in optical detection methods for microchip separations

This paper summarizes the features and performances of optical detection systems currently applied in order to monitor separations on microchip devices. Fluorescence detection, which delivers very high sensitivity and selectivity, is still the most widely applied method of detection. Instruments utilizing laser-induced fluorescence (LIF) and lamp-based fluorescence along with recent applications of light-emitting diodes (LED) as excitation sources are also covered in this paper. Since chemiluminescence detection can be achieved using extremely simple devices which no longer require light sources and optical components for focusing and collimation, interesting approaches based on this technique are presented, too. Although UV/vis absorbance is a detection method that is commonly used in standard desktop electrophoresis and liquid chromatography instruments, it has not yet reached the same level of popularity for microchip applications. Current applications of UV/vis absorbance detection to microchip separations and innovative approaches that increase sensitivity are described. This article, which contains 85 references, focuses on developments and applications published within the last three years, points out exciting new approaches, and provides future perspectives on this field.     

Capillary electrophoresis and microchip capillary electrophoresis with electrochemical and electrochemiluminescence detection

Recent advances and key strategies in capillary electrophoresis and microchip CE with electrochemical detection (ECD) and electrochemiluminescence (ECL) detection are reviewed. This article consists of four main parts: CE-ECD; microchip CE-ECD; CE-ECL; and microchip CE-ECL. It is expected that ECD and ECL will become powerful tools for CE microchip systems and will lead to the creation of truly disposable devices. The focus is on papers published in the last two years (from 2005 to 2006).     

Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA

Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) using polymer solutions are two of the most powerful techniques for the analysis of DNA. Problems, such as the difficulty of filling polymer solution to small separation channels, recovering DNA, and narrow separation size ranges, have put a pressure on developing new techniques for DNA analysis. In this review, we deal with DNA separation using chip-based nanostructures and nanomaterials in CE and MCE. On the basis of the dependence of the mobility of DNA molecules on the size and shape of nanostructures, several unique chip-based devices have been developed for the separation of DNA, particularly for long DNA molecules. Unlike conventional CE and MCE methods, sieving matrices are not required when using nanostructures. Filling extremely low-viscosity nanomaterials in the presence and absence of polymer solutions to small separation channels is an alternative for the separations of DNA from several base pairs (bp) to tens kbp. The advantages and shortages of the use of nanostructured devices and nanomaterials for DNA separation are carefully addressed with respect to speed, resolution, reproducibility, costs, and operation.     

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.     

Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis

Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.     

Field amplified sample stacking coupled with chip-based capillary electrophoresis using negative pressure sample injection technique

A multi-T microchip for integrated field amplified sample stacking (FASS) with CE separation to increase the chip-based capillary electrophoresis (chip-based CE) sensitivity was developed. Volumetrically defined large sample plug was formed in one step within 5s by the negative pressure in headspace of the two sealed sample waste reservoirs produced using a syringe pump equipped with a 3-way valve. Stacking and separation can proceed only by switching the 3-way valve to release the vacuum in headspace of the two sample waste reservoirs. This approach considerably simplified the operations and the equipments for FASS in chip-based CE systems. Migration time precisions of 3.3% and 1.3% RSD for rhodamine123 (Rh123) and fluorescien sodium salt (Flu) in the separation of a mixture of Flu and Rh123 were obtained for nine consecutive determinations with peak height precisions of 4.8% and 3.4% RSD, respectively. Compared with the chip-based CE on the cross microchip, the sensitivity for analysis of FlTC, FITC-labeled valine (Val) and Alanine (Ala) increased 55-, 41- and 43-fold, respectively.     

Carbon nanotube and diamond as electrochemical detectors in microchip and conventional capillary electrophoresis

As two important polymorphs of carbon, carbon nanotube (CNT) and diamond have been widely employed as electrode materials for electrochemical sensing. This review focuses on recent advances and the key strategies in the fabrication and application of electrochemical detectors in microchip and conventional capillary electrophoresis (CE) using CNT and boron-doped diamond. The subjects covered include CNT-based electrochemical detectors in microchip CE, CNT-based electrochemical detectors in conventional CE, boron-doped diamond electrochemical detectors in microchip CE, and boron-doped diamond electrochemical detectors in conventional CE. The attractive properties of CNT and boron-doped diamond make them very promising materials for the electrochemical detection in microchip and conventional CE systems and other microfluidic analysis systems.     

Conductivity detection for conventional and miniaturised capillary electrophoresis systems

Since the introduction of capillary electrophoresis (CE), conductivity detection has been an attractive means of detection. No additional chemical properties are required for detection, and no loss in sensitivity is expected when miniaturising the detector to scale with narrow-bore capillaries or even to the microchip format. Integration of conductivity and CE, however, involves a challenging combination of engineering issues. In conductivity detection the resistance of the solution is most frequently measured in an alternating current (AC) circuit. The influence of capacitors both in series and in parallel with the solution resistance should be minimised during conductivity measurements. For contact conductivity measurements, the positioning and alignment of the detection electrodes is crucial. A contact conductivity detector for CE has been commercially available, but was withdrawn from the market. Microfabrication technology enables integration and precise alignment of electrodes, resulting in the popularity of conductivity detection in microfluidic devices. In contactless conductivity detection, the alignment of the electrodes with respect to the capillary is less crucial. Contactless conductivity detection (CCD) was introduced in capillary CE, and similar electronics have been applied for CCD using planar electrodes in microfluidic devices. A contactless conductivity detector for capillaries has been commercialised recently. In this review, different approaches towards conductivity detection in capillaries and chip-based CE are discussed. In contrast to previous reviews, the focus of the present review is on the technological developments and challenges in conductivity detection in CE.     

Recent progress of on-line sample preconcentration techniques in microchip electrophoresis

This review highlights recent developments and applications of on-line sample preconcentration techniques to enhance the detection sensitivity in microchip electrophoresis (MCE); references are mainly from 2008 and later. Among various developed techniques, we focus on the sample preconcentration based on the changes in the migration velocity of analytes in two or three discontinuous solutions system, since they can provide the sensitivity enhancement with relatively easy experimental procedures and short analysis times. The characteristic features of the on-line sample preconcentration applied to microchip electrophoresis (MCE) are presented, categorized on the basis of "field strength-" or "chemically" induced changes in the migration velocity. The preconcentration techniques utilizing field strength-induced changes in the velocity include field-amplified sample stacking, isotachophoresis and transient-isotachophoresis, whereas those based on chemically induced changes in the velocity are sweeping, transient-trapping and dynamic pH junction.     

Recent developments in electrochemical detection for microchip capillary electrophoresis

Significant progress in the development of miniaturized microfluidic systems has occurred since their inception over a decade ago. This is primarily due to the numerous advantages of microchip analysis, including the ability to analyze minute samples, speed of analysis, reduced cost and waste, and portability. This review focuses on recent developments in integrating electrochemical (EC) detection with microchip capillary electrophoresis (CE). These detection modes include amperometry, conductimetry, and potentiometry. EC detection is ideal for use with microchip CE systems because it can be easily miniaturized with no diminution in analytical performance. Advances in microchip format, electrode material and design, decoupling of the detector from the separation field, and integration of sample preparation, separation, and detection on-chip are discussed. Microchip CEEC applications for enzyme/immunoassays, clinical and environmental assays, as well as the detection of neurotransmitters are also described.     

Electrophoresis on a microfluidic chip for analysis of fluorescence-labeled human rhinovirus

We report the analysis of human rhinovirus serotype 2 (HRV2) on a commercially available lab-on-a-chip instrument. Due to lack of sufficient native fluorescence, the proteinaceous capsid of HRV2 was labeled with Cy5 for detection by the red laser (lambda ex 630 nm) implemented in the instrument. On the microdevice, electrophoresis of the labeled virus was possible in a BGE without stabilizing detergents, which is in contrast to conventional CE; moreover, analysis times were drastically shortened to the few 10 s range. Resolution of the sample constituents (virions, a contaminant present in all virus preparations, and excess dye) was improved upon adaptation of the separation conditions, mainly by adjusting the SDS concentration of the BGE. Purity of fractions from size-exclusion chromatography after labeling of virus was assessed, and affinity complex formation of the labeled virus with various recombinant very-low-density lipoprotein receptor derivatives differing in the number of concatenated V3 ligand binding repeats was monitored. Virus analysis on microchip devices is of particular interest for experiments with infectious material because of easy containment and disposal of samples. Thus, the employment of microchip devices in routine analysis of viruses appears to be exceptionally attractive.     

Near-infrared laser-induced fluorescence detection in capillary electrophoresis

As capillary electrophoresis continues to focus on miniaturization, either through reducing column dimensions or situating entire electrophoresis systems on planar chips, advances in detection become necessary to meet the challenges posed by these electrophoresis platforms. The challenges result from the fact that miniaturization requires smaller load volumes, demanding highly sensitive detection. In addition, many times multiple targets must be analyzed simultaneously (multiplexed applications), further complicating detection. Near-infrared (NIR) fluorescence offers an attractive alternative to visible fluorescence for critical applications in capillary electrophoresis due to the impressive limits of detection that can be generated, in part resulting from the low background levels that are observed in the NIR. Advances in instrumentation and fluorogenic labels appropriate for NIR monitoring have led to a growing number of examples of the use of NIR fluorescence in capillary electrophoresis. In this review, we will cover instrumental components used to construct ultrasensitive NIR fluorescence detectors, including light sources and photon transducers. In addition, we will discuss various types of labeling dyes appropriate for NIR fluorescence and finally, we will present several applications that have used NIR fluorescence in capillary electrophoresis, especially for DNA sequencing and fragment analysis.     

Sample introduction techniques for microchip electrophoresis: A review

The number of applications of microfluidic analysis systems continues to increase, along with the variety of substrate materials and complexity of the devices themselves. One of the most common features of these devices that has remained relatively unchanged, however, is the introduction of a sample mixture into a separation channel so that individual components can be separated by electrophoresis. Whether a relatively simple mixture of amino acids or a more complex sample of DNA fragments extracted and amplified on-chip, the ability to reliably and reproducibly inject a representative sample is arguably the most significant requirement for an electrophoretic micro total analysis system (μTAS). This review will focus on the different methods reported for sample introduction in microchip electrophoresis, highlighting both pressure-driven and electrokinetic techniques, with an emphasis on the methods employed in μTAS applications.     

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 compactly integrated laser-induced fluorescence detector for microchip electrophoresis

A simple and easy-to-use integrated laser-induced fluorescence detector for microchip electrophoresis was constructed and evaluated. The fluid channels and optical fiber channels in the glass microchip were fabricated using standard photolithographic techniques and wet chemical etching. A 473 nm diode-pumped laser was used as the excitation source, and the collimation and collection optics and mirrors were discarded by using a multimode optical fiber to couple the excitation light straight into the microchannel and placing the microchip directly on the top of the photomultiplier tube. A combination of filter systems was incorporated into a poly(dimethylsiloxane) layer, which was reversibly sealed to the bottom of the microchip to eliminate the scattering excitation light reaching to the photomultiplier tube. Fluorescein/calcein samples were taken as model analytes to evaluate the performance with respect to design factors. The detection limits were 0.05 microM for fluorescein and 0.18 microM for calcein, respectively. The suitability of this simple detector for fluorescence detection was demonstrated by baseline separation of fluorescein isothiocyanate (FITC)-labeled arginine, phenylalanine, and glycine and FITC within 30 s at separation length of 3.8 cm and electrical field strength of 600 V/cm.