One-way multiplexed immunoassay strategy for simultaneous determination of multi-analytes by microchip electrophoresis

An integrated microchip electrophoresis (MCE) system with online immunoreaction and laser induced fluorescence (LIF) detection has been developed for simultaneous determination of multi-analytes. In this system, the multiplexed immunoreactions between multiple antibody-immobilized glass beads with analytes and respective fluorescently labeled antigens were performed in a sample reservoir. After online incubation, the immunoreaction solution was injected into a one-way separation channel, and free fluorescently labeled antigens were separated and detected in the separation channel. With the help of glass beads, the immunocomplex can not move into the separation channel, which simplifies the separation of fluorescently labeled antigens. With the use of phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ) and theophylline (Th) as proof-of-principle analytes, the one-way multiplexed immunoassay could be completed within 20 min, resulting in a response curve over the range of 4.0-400 nM for each analyte. Detection limits (S/N = 3) for the drugs tested were in the range of 1.8 × 10(-9) to 2.5 × 10(-9) M. Compared with the conventional immunoassays, this assay is simple, rapid, sensitive and low cost, and provides an accurate procedure for a multiplex immunoassay. The present method has been applied for the simultaneous determination of PB, PHT, CBZ and Th in human serum, which showed a promise of automated clinical application.     

A sensitive and rapid immunoassay for quantification of testosterone by microchip electrophoresis with enhanced chemiluminescence detection

A sensitive and rapid approach to perform testosterone (T) competitive immunoassay by microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed. The assay is based on the competitive immunoreactions between T and N-(4-aminobutyl)-N-ethylisoluminol-labeled T (ABEI-T) with a limited amount of antibody (Ab), and the rapid electrophoretic separation of an equilibrated mixture of ABEI-T-Ab complex and free ABEI-T, followed by CL detection using horseradish peroxidase-catalyzed ABEI-H(2)O(2) system. Free ABEI-T and the ABEI-T-Ab complex are well separated within 30 s under the assay conditions. The developed method could be used to determine T with good precision and a detection limit lower than 1.0 nM. This method was applied for the quantification of T in human serum. The results demonstrated that the current MCE-CL-based competitive immunoassay maybe served as an alternative tool for clinical analysis.     

Hydrophobic labeling of amino acids: transient trapping-capillary/microchip electrophoresis

Transient trapping (tr-trapping) was developed as one of the on-line sample preconcentration techniques to improve a low concentration-sensitivity in microchip electrophoresis (MCE), providing highly effective preconcentration and separation based on the trap-and-release mechanism. However, a poor performance to hydrophilic analytes limited the applicability of tr-trapping. To overcome this drawback, tr-trapping was combined with a sample labeling using a hydrophobic reagent in CE. Three commercially available fluorescent dyes, fluorescein isothiocyanate, succinimidyl esters of Alexa Fluor 488 and BODIPY FL-X, were tested as derivatization reagents to increase the hydrophobicity of amino acids (AAs) that were undetectable due to no fluorescence/UV-absorbance. As a result, it was confirmed that BODIPY labeling allowed various AAs to be analyzed in tr-trapping-micellar electrokinetic chromatography (tr-trapping-MEKC) by the increase in the hydrophobicity. In tr-trapping-MEKC, both the improvement of the resolution and 106-125-fold enhancements of the detectability of labeled AAs were achieved relative to the conventional capillary zone electrophoresis. The limit of detection of labeled phenylalanine was improved from 800 to 5 pM by applying tr-trapping-MEKC. In tr-trapping-microchip MEKC, furthermore, an 80-160-fold enhancement of the peak intensity and a baseline separation was also achieved within 30 s. These results clearly demonstrate that the tr-trapping technique with hydrophobic labeling will make CE/MCE more sensitive for various analytes.     

Evaluation of band broadening in chemiluminescence detection coupled to pressurized capillary electrochromatography with an off-column coaxial flow interface

A system of off-column coaxial flow chemiluminescence (CL) detection coupled to pressurized CEC (pCEC) was described. The interface utilized a reactor that introduced postcolumn CL reagent into the capillary effluents in a sheathing flow profile. To compare and evaluate band broadening of analytes caused by the detector, the typical CL compounds luminol and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) were separated and detected by pCEC or capillary HPLC (cHPLC) coupled to CL and UV detector, respectively. The results demonstrated that the band broadening caused by off-column detection interface was minimized due to the fast kinetic nature of the CL reaction. With the proposed pCEC-CL system, the detection limits of luminol and ABEI were 1.0x10(-8) and 8.0x10(-8) mol/L, respectively, which were approximately 100-fold more sensitive than those obtained with UV absorption. In addition, separation and detection of the ABEI-labeled L-lysine (L-Lys) and L-arginine (L-Arg) were accomplished by pCEC-CL method based on the principle of ABEI-potassium ferricyanide-alkaline medium CL reaction system. Under the optimum conditions, good results could be achieved compared with pCEC-UV.     

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.     

Chemiluminescent determination of the activity of caspase-3 using a specific peptide substrate and magnetic beads

We report on a new scheme for the determination of the activity of caspase-3 using a specific peptide labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as a chemiluminescent (CL) probe and on the development of magnetic separation technology. Firstly, the ABEI-labeled and biotinylated peptide was prepared and conjugated to streptavidin-coated magnetic beads (MBs) to form f-MBs (functionalized magnetic beads). The f-MBs contain a site (DEVD, Asp-Glu-Val-Asp) that is cleaved by caspase-3. Upon cleavage, the terminal residue attached to ABEI can dissociate from the f-MBs and can be used for CL detection. CL intensity is linearly related to the concentration of caspase-3 in the range 1.0 to 600 ng mL^?1, with a detection limit of 0.3 ng mL^?1. The relative standard deviation of the assay is 3.6 % at a level of 50 ng mL^?1 of caspase-3 (for n?=?11). The CL assay has been applied to the determination of caspase-3 in Jurkat cell extract with recoveries between 96.6 % and 106.1 % (n?=?5).

Micellar electrokinetic chromatography on microchips

This review highlights the methodological and instrumental developments in microchip micellar EKC (MCMEKC) from 1995. The combination of higher separation efficiencies in micellar EKC (MEKC) with high-speed separation in microchip electrophoresis (MCE) should provide high-throughput and high-performance analytical systems. The chip-based separation technique has received considerable attention due to its integration ability without any connector. This advantage allows the development of a multidimensional separation system. Several types of 2-D separation microchips are described in the review. Since complicated channel configurations can easily be fabricated on planar substrates, various sample manipulations can be carried out prior to MCMEKC separations. For example, mixing for on-chip reactions, on-line sample preconcentration, on-chip assay, etc., have been integrated on MEKC microchips. The application of on-line sample preconcentration to MCMEKC can provide not only sensitivity enhancement but also the elucidation of the preconcentration mechanism due to the visualization ability of MCE. The characteristics of these sample manipulations on MEKC microchips are presented in this review. The scope of applications in MCMEKC covers mainly biogenic compounds such as amino acids, peptides, proteins, biogenic amines, DNA, and oestrogens. This review provides a comprehensive table listing the applications in MCMEKC in relation to detection methods.     

Multiplex immunoassays of equine virus based on fluorescent encoded magnetic composite nanoparticles

A new detection format for multiplexed analysis based on fluorescent encoded magnetic composite nanoparticles is presented. Two kinds of virus were analyzed by this new method: equine influenza virus (EIV) and equine infectious anemia virus (EIAV). Firstly, EIV antigen and EIAV antigen were conjugated to two kinds of fluorescent encoded magnetic composite nanoparticles, while the green-emitting CdTe quantum dots (QDs) were attached to the antibody of EIV and EIAV. Then both green-emitting CdTe QD-labeled antibodies and antigens labeled with fluorescent encoded magnetic composite nanoparticles were used to form an immunoassay system for the detection of EIV and EIAV antigens. The method is time-saving and has higher sensitivity (1.3 ng mL⁻¹ for EIV antigens and 1.2 ng mL⁻¹ for EIAV antigens) than the conventional methods. A competitive immunoassay method based on this analysis system was used to detect EIV and EIAV antigens in spiked serum samples with satisfactory results.     

3D Origami-based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device

A novel 3D microfluidic paper-based immunodevice, integrated with blood plasma separation from whole blood samples, automation of rinse steps, and multiplexed CL detections, was developed for the first time based on the principle of origami (denoted as origami-based device). This 3D origami-based device, comprised of one test pad surrounded by four folding tabs, could be patterned and fabricated by wax-printing on paper in bulk. In this work, a sandwich-type chemiluminescence (CL) immunoassay was introduced into this 3D origami-based immunodevice, which could separate the operational procedures into several steps including (i) folding pads above/below and (ii) addition of reagent/buffer under a specific sequence. The CL behavior, blood plasma separation, washing protocol, and incubation time were investigated in this work. The developed 3D origami-based CL immunodevice, combined with a typical luminuol-H(2)O(2) CL system and catalyzed by Ag nanoparticles, showed excellent analytical performance for the simultaneous detection of four tumor markers. The whole blood samples were assayed and the results obtained were in agreement with the reference values from the parallel single-analyte test. This paper-based microfluidic origami CL detection system provides a new strategy for a low-cost, sensitive, simultaneous multiplex immunoassay and point-of-care diagnostics.     

Rapid separation of microorganisms by quartz microchip capillary electrophoresis.

We developed and optimized a system coupling microchip capillary electrophoresis (MCE) and laser-induced fluorescence (LIF) detection for the analysis of microorganisms. The MCE-LIF system successfully separated pure cultures of lactic acid bacteria and Saccharomyces cerevisiae within 200 s. The results indicate that the MCE system can be conveniently used for the rapid and highly sensitive detection of microorganisms. Thus, MCE can provide a cheap and simple method for the on-line detection of microbial contamination.     

Microchip electrophoresis with chemiluminescence detection for assaying ascorbic acid and amino acids in single cells

A method based on microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed for the determination of ascorbic acid (AA) and amino acids including tryptophan (Trp), glycine (Gly) and alanine (Ala) present in single cells. Cell injection, loading, lysing, electrophoretic separation and CL detection were integrated onto a simple cross microfluidic chip. A single cell was loaded in the cross intersection by electrophoretic means through applying a set of potentials at the reservoirs. The docked cell was lysed rapidly under a direct electric field. The intracellular contents were MCE separated within 130 s. CL detection was based on the enhancing effects of AA and amino acids on the CL reaction of luminol with K₃[Fe(CN)₆]. Rat hepatocytes were prepared and analyzed as the test cellular model. The average intracellular contents of AA, Trp, Gly and Ala in single rat hepatocytes were found to be 38.3, 5.15, 3.78 and 3.84 fmol (n = 12), respectively.     

Quantification of biogenic amines by microchip electrophoresis with chemiluminescence detection

A highly sensitive microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of biogenic amines including agmatine (Agm), epinephrine (E), dopamine (DA), tyramine, and histamine in human urine samples. To achieve a high assay sensitivity, the targeted analytes were pre-column labeled by a CL tagging reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). ABEI-tagged biogenic amines after MCE separation reacted with hydrogen peroxide in the presence of horseradish peroxidase (HRP), producing CL emission. Since no CL reagent was added to the running buffer, the background of the CL detection was extremely low, resulting in a significant improvement in detection sensitivity. Detection limits (S/N = 3) were in the range from 5.9 × 10⁻⁸ to 7.7 × 10⁻⁸ M for the biogenic amines tested, which were at least 10 times lower than those of the MCE–CL methods previously reported. Separation of a urine sample on a 7 cm glass/poly(dimethylsiloxane) (PDMS) microchip channel was completed within 3 min. Analysis of human urine samples found that the levels of Agm, E and DA were in the ranges of 2.61 × 10⁻⁷ to 4.30 × 10⁻⁷ M, 0.81 × 10⁻⁷ to 1.12 × 10⁻⁷ M, and 8.76 × 10⁻⁷ to 11.21 × 10⁻⁷ M (n = 4), respectively.     

Determination of intracellular sulphydryl compounds by microchip electrophoresis with selective chemiluminescence detection

An analytical method based on microchip electrophoresis (MCE) and chemiluminescence detection (CL) was developed for the determination of intracellular sulphydryl compounds. Cell injection/loading, cytolysis, electrophoretic separation, and CL detection were integrated onto a simple cross-microfluidic chip. Selective CL detection of sulphydryl compounds was achieved by deploying the luminol–Na₂S₂O₈ reaction. Under the CL conditions selected, many endogenous compounds in biological systems such as amino acids, biogenic amines, peptides and proteins did not produce any CL signal, which further ensured a high selectivity of the proposed MCE–CL assays. Sulphydryl compounds including cysteine (Cys), glutathione (GSH), and hemoglobin (Hb) were selected as the test compounds. The MCE separation was completed within 120 s. The detection limits were estimated to be 7 amol for Cys, 32 amol for GSH and 69 amol for Hb, respectively. The present method was applied to analyze individual red blood cells collected from both healthy subjects and cancer patients. It was found that the average intracellular contents of Cys, GSH and Hb were in the ranges of 26–43 amol/cell, 128–323 amol/cell and 522–667 amol/cell, respectively for cancer patients, compared to 579–609 amol Hb/cell and not detectable Cys and GSH for healthy subjects.     

Signal denoising and baseline correction by discrete wavelet transform for microchip capillary electrophoresis

Signal denoising and baseline correction using discrete wavelet transform (DWT) are described for microchip capillary electrophoresis (MCE). DWT was performed on an electropherogram describing a separation of nine tetramethylrohodamine-5-isothiocyanate labeled amino acids, following MCE with laser-induced fluorescence detection, using Daubechies 5 wavelet at a decomposition level of 6. The denoising efficiency was compared with, and proved to be superior to, other commonly used denoising techniques such as Fourier transform, Savitzky-Golay smoothing and moving average, in terms of noise removal and peak preservation by directly visual inspection. Novel strategies for baseline correction were proposed, with a special interest in baseline drift that frequently occurred in chromatographic and electrophoretic separations.     

A highly efficient and versatile microchip capillary electrophoresis method for DNA separation using gold nanoparticle as a tag

A highly efficient and versatile method for DNA separation using Au nanoparticles (Au NPs) as a tag based on microchip capillary electrophoresis (MCE) was developed. The thiol-modified DNA-binding Au NPs were utilized as a tag. Target DNA was sandwiched between Au NPs and probe DNA labeled with horseradish peroxidase (HRP). In electrophoresis separation, the difference in electrophoretic mobility between free probe and probe-target complex was magnified by Au NPs, which enabled the resulting mixture to be separated with high efficiency by microchip capillary electrophoresis. Horseradish peroxidase was used as a catalytic label to achieve sensitive electrochemical DNA detection via fast catalytic reactions. With this protocol, 27-mer DNA fragments with different sequences were separated with high speed and high resolution. The proposed method was critical to achieve improved DNA separations in hybridization analyses.     

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser‐induced fluorescence detection

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK_a determination of small molecule analytes is demonstrated with the multilane microchip.     

Individually addressable electrode array for multianalyte electrochemiluminescent immunoassay based on a sequential triggering strategy

Multianalyte immunoassay in a single run is often necessary to monitor or quantitate several components in a complex sample matrix for various purposes. In this paper we present a novel, individually addressable electrode array for sequential electrochemiluminescent (ECL) immunoassay using a non-array detector. An immunosensor array was fabricated by site-selectively immobilizing multiple antigens on different electrodes. With a competitive immunoassay format, the amounts of the bound Ru(bpy)(3)(2+) derivative labeled antibodies decreased with the increase of the antigens in the sample, and the ECL signals from different immunosensors were collected in turn by a photomultiplier with the aid of a home-made single-pore-three-throw switch. Using human IgG and rat IgG as model analytes, this multianalyte immunoassay showed detection limits down to 8.9 and 7.2 ng mL(-1) for them, respectively. The results for real sample analysis demonstrated that this strategy can provide a simple, sensitive, low-cost and high-throughput ECL immunosensor array for clinical diagnosis.     

Fabrication and performance of a three-dimensionally adjustable device for the amperometric detection of microchip capillary electrophoresis

A microchip CE-amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an AD cell containing a one-dimensionally adjustable disk detection electrode in a Plexiglas holder. It facilitates the precise 3-D alignment between the channel outlet and the detection electrode without a complicated 3-D manipulator. The performance of this unique system was demonstrated by separating five aromatic amines (1,4-phenyldiamine, aniline, 2-methylaniline, 4-chloroaniline, and 1-naphthylamine) of environmental concern. Factors influencing their separation and detection processes were examined and optimized. The five analytes have been well separated within 140 s in a 74 cm long separation channel at a separation voltage of +2500 V using a 10 mM phosphate buffer (pH 3.5). Highly linear response is obtained for the five analytes over the range 20-200 microM with the detection limits ranging from 0.46 to 1.44 microM, respectively. The present system demonstrated long-term stability and reproducibility with RSDs of less than 5% for the peak current (n = 9). The new approach for the microchannel-electrode alignment should find a wide range of applications in CE, flowing injection analysis, and other microfluidic analysis systems.     

Impact of reservoir potentials on the analyte behavior in microchip electrophoresis: computer simulation and experimental validation for DNA fragments

Fundamental understanding of the impact of reservoir potentials on the analyte behavior on the microfluidic chips is an important issue in microchip electrophoresis (MCE) for suitable injection and separation of analytes, since the applied potentials may significantly affect the shape of sample plug, sample leakage from the injection channel to the separation channel, injected sample amount, and separation efficiency. This study addressed this issue for the case of a conventional cross-geometry microchip with four reservoirs using computer simulations, the results of which were verified by the analysis of DNA fragments. For the microchip with a definite structure and migration distance, the injected sample amount was shown to be the vital parameter for improving the limit of detection and resolution. During injection, the shape of the sample plug could be adjusted by varying the reservoir potentials. It was demonstrated that a "magnified injection" (applying high voltage on the three reservoirs to the sample reservoir) is useful to enhance the detection sensitivity depending on the analyte composition, although such injection was previously avoided because of introducing too large amounts of the analyte in comparison with two established modes, floating and pinched injection. Optimal magnified injection was proved to improve the sensitivity for about 4 times over that of pinched injection for the analysis of DNA step ladders using microchip gel electrophoresis (MCGE). Sample leakage of DNA fragments could be suppressed by applying a high positive voltage on injection channel during separation, but the voltage degraded the injected amount and resolution.     

Recent advances in microchip enantioseparation and analysis

This review summarizes recent developments (over the past decade) in the field of microfluidics-based solutions for enantiomeric separation and detection. The progress in various formats of microchip electrodriven separations, such as MCE, microchip electrochromatography, and multidimensional separation techniques, is discussed. Innovations covering chiral stationary phases, surface coatings, and modification strategies to improve resolution, as well as integration with detection systems, are reported. Finally, combinations with other microfluidic functional units are also presented and highlighted.