Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.     

Electrochemical oxidation of chlorophenols at a boron-doped diamond electrode and their determination by high-performance liquid chromatography with amperometric detection

Anodically pretreated diamond electrodes have been used for the detection of chlorophenols (CPs) in environmental water samples after high-performance liquid chromatographic (HPLC) separation. The anodization of as-deposited boron-doped polycrystalline diamond thin-film electrodes has enabled the stable determination of phenols over a wide concentration range. Prior to the HPLC analysis, a comparative study with ordinary glassy carbon, as-deposited diamond, and anodized diamond was made to examine the oxidative behavior of phenols by cyclic voltammety and flow injection analysis with amperometric detection. At anodized diamond electrodes, reproducible, well-defined cyclic voltammograms were obtained even at high CP concentration (5 mM), due to a low proclivity for adsorption of the oxidation products on the surface. In addition, after prolonged use, the partially deactivated diamond could be reactivated on line by applying a highly anodic potential (2.64 Vvs SCE) for 4 min, which enabled the destruction of the electrodeposited polymer deposits. Hydroxyl radicals produced by the high applied potential, in which oxygen evolution occurs, are believed to be responsible for the oxidation of the passivating layer on the surface. When coupled with flow injection analysis (FIA), anodized diamond exhibited excellent stability, with a response variability of 2.3% (n = 100), for the oxidation of a high concentration (5 mM) of chlorophenol. In contrast, glassy carbon exhibited a response variability of 39.1%. After 100 injections, the relative peak intensity, for diamond decreased by 10%, while a drastic decrease of 70% was observed for glassy carbon. The detection limit obtained in the FIA mode for 2,4-dichlorophenol was found to be 20 nM (S/N = 3), with a linear dynamic range up to 100 microM. By coupling with the column-switching technique, which enabled on-line preconcentration (50 times), the detection limit was lowered to 0.4 nM (S/N = 3). By use of this technique, anodized diamond electrodes were demonstrated for the analysis of CPs in drainwater that was condensed from the flue gas of waste incinerators.     

Performance of pyrolyzed photoresist carbon films in a microchip capillary electrophoresis device with sinusoidal voltammetric detection

Pyrolyzed photoresist films (PPF) are introduced as planar carbon electrodes in a PDMS-quartz hybrid microchip device. The utility of PPF in electroanalytical applications is demonstrated by the separation and detection of various neurotransmitters. PPF is found to form a stable, low-capacitance, durable layer on quartz, which can then be used in conjunction with a microchip capillary electrophoretic device. Sinusoidal voltammetric detection at PPF electrodes is shown to be very sensitive, with a detection limit (S/N = 3) of 100 nM for dopamine, corresponding to a mass detection limit (S/N = 3) of 2 amol. The selectivity of analysis in the frequency domain is demonstrated by isolating each individual signal in a pair of analytes that are chromatographically unresolved. Effectively decoupling the electrophoresis and electrochemical systems allows the electrodes to be placed just inside the separation channel, which results in efficient separations (80 000-100 000 plates/m).     

Attomole amino acid determination by capillary zone electrophoresis with thermooptical absorbance detection

Attomole quantities of 4-(dimethylamino)azobenzene-4'-sulfonyl chloride derivatized amino acids are separated by using capillary zone electrophoresis in a mixed acetonitrile/aqueous buffer system. Detection is performed with an on-column thermooptical absorbance detection technique based on a 130-mW argon ion pump laser. Detection limits for the concentration of analyte injected onto the column range from 5 x 10(-8) M for methionine to 5 x 10(-7) M for aspartic acid. Only 37 amol of methionine and 450 amol of aspartic acid are contained within the subnanoliter injection volume. It is interesting to note that these limits are a factor of 4 superior to the best fluorescence detection limit reported for chromatographic separation of amino acids. A subnanoliter sample of derivatized human urine was analyzed with this technique; quantities of amino acids contained within the sample are 3 orders of magnitude greater than the detection limit.     

Microchip capillary electrophoresis with electrochemical detection

A novel microchip capillary electrophoresis system with electrochemical detection, using the replaceable microelectrode, is first reported. This kind of electrode can be fabricated in general laboratories and can be replaced quickly with electrodes of different materials according to the requirements of experiments. The end-column electrochemical detection on microchip CE was achieved by fixing the working electrode (such as carbon fiber, Pt, or Au, etc.) through a guide tube on the end of the separation channel. The experiment results indicate that the alignment of the electrode with the channel outlet can be carried out accurately and reproducibly, and therefore, the detection device has low noise and good reproducibility. The detection limit of dopamine is 2.4 x 10(-7) M, which is the lowest result reported so far. The separation and detection of dopamine, 5-hydroxytryptamine and epinephrine using carbon fiber and Pt microdisk electrodes within 50 s was successfully performed.     

Improving the compatibility of contact conductivity detection with microchip electrophoresis using a bubble cell

A new approach for improving the compatibility between contact conductivity detection and microchip electrophoresis was developed. Contact conductivity has traditionally been limited by the interaction of the separation voltage with the detection electrodes because the applied field creates a voltage difference between the electrodes, leading to unwanted electrochemical reactions. To minimize the voltage drop between the conductivity electrodes and therefore improve compatibility, a novel bubble cell detection zone was designed. The bubble cell permitted higher separation field strengths (600 V/cm) and reduced background noise by minimizing unwanted electrochemical reactions. The impact of the bubble cell on separation efficiency was measured by imaging fluorescein during electrophoresis. A bubble cell four times as wide as the separation channel led to a decrease of only 3% in separation efficiency at the point of detection. Increasing the bubble cell width caused larger decreases in separation efficiency, and a 4-fold expansion provided the best compromise between loss of separation efficiency and maintaining higher field strengths. A commercial chromatography conductivity detector (Dionex CD20) was used to evaluate the performance of contact conductivity detection with the bubble cell. Mass detection limits (S/N = 3) were as low as 89 +/- 9 amol, providing concentration detection limits as low as 71 +/- 7 nM with gated injection. The linear range was measured to be greater than 2 orders of magnitude, from 1.3 to 600 microM for sulfamate. The bubble cell improves the compatibility and applicability of contact conductivity detection in microchip electrophoresis, and similar designs may have broader application in electrochemical detection as the expanded detection zone provides increased electrode surface area and reduced analyte velocity in addition to the reduction of separation field effects.     

Quantum dot biolabeling coupled with immunomagnetic separation for detection of Escherichia coli O157:H7

A sensitive, specific, and rapid method for the detection of E. coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-E. coli O157 antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-E. coli antibodies were added to form sandwich immuno complexes. After magnetic separation, the immuno complexes were labeled with QDs via biotin-streptavidin conjugation. This was followed by a fluorescence measurement using a laptop-controlled portable device, which consisted of a blue LED and a CCD-array spectrometer. The peak intensity of the fluorescence emission was proportional to the initial cell concentration of E. coli O157:H7 in the range of 10(3)-10(7) CFU/mL with a detection limit at least 100 times lower than that of the FITC-based method. The total detection time was less than 2 h. Neither E. coli K12 nor Salmonella typhimurium interfered with the detection of E. coli O157:H7.     

Electrochemical detection of peptides

A general method of wide applicability for the determination of peptides is described. Peptides longer than dipeptides react in the classical biuret reaction with Cu(II) to yield electroactive Cu(II)-peptide complexes that can be oxidized to the corresponding Cu(III) complexes. This allows the sensitive electrochemical detection of peptides following their separation by reversed-phase liquid chromatography. The reaction chemistry, which is reversible, allows for the determination of peptides that lack an electroactive group or a primary amine. Selectivity for a model peptide is 10(3)-10(4) over nonelectroactive amino acids.     

Electrochemical oxidation of histamine and serotonin at highly boron-doped diamond electrodes

The electrochemistry of histamine and serotonin in neutral aqueous media (pH 7.2) was investigated using polycrystalline, boron-doped diamond thin-film electrodes. Cyclic voltammetry, hydrodynamic voltammetry, and flow injection analysis (FIA) with amperometric detection were used to study the oxidation reactions. Comparison experiments were carried out using polished glassy carbon (GC) electrodes. At diamond electrodes, highly reproducible and well-defined cyclic voltammograms were obtained for histamine with a peak potential at 1.40 V vs SCE. The voltammetric signal-to-background ratios obtained at diamond were 1 order of magnitude higher than those obtained for GC electrodes at and above 100 microM analyte concentrations. A linear dynamic range of 3-4 orders of magnitude and a detection limit of 1 microM were observed in the voltammetric measurements. Well-defined sweep rate-dependent voltammograms were also obtained for 5-hydroxytryptamine (5-HT). The characteristics of the voltammogram indicated lack of adsorption of its oxidation products on the surface. No fouling or deactivation of the electrode was observed within the experimental time of several hours. A detection limit of 0.5 microM (signal-to-noise ratio 13.8) for histamine was obtained by use of the FIA technique with a diamond electrode. A remarkably low detection limit (10 nM) was obtained for 5-HT on diamond by the same method. Diamond electrodes exhibited a linear dynamic range from 10 nM to 100 microM for 5-HT determination and a range of 0.5-100 microM for histamine determination. The FIA response was very reproducible from film to film, and the response variability was below 7% at the actual detection limits.     

Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection

An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification. The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation. Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors. In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions. After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation. An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence. Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences. The limit of detection is about six copies of target DNA.     

Simple and sensitive electrode design for microchip electrophoresis/electrochemistry

A simple and sensitive electrode design for microchip capillary electrophoresis/electrochemistry (CE-EC) is presented. The system employs metal microwires as the working electrodes for electrochemical detection. Two general approaches for integration of electrodes in microchip CE-EC are commonly used, end-channel and microfabrication. The end-channel approach allows electrode cleaning and the use of chemically modified electrodes; however, the designs generally lack portability and the ability to incorporate multiple electrodes. Microfabrication allows the incorporation of multiple electrodes on-chip and is easily made portable; however, it requires the use of expensive metallization and clean room facilities, and integration of more than one electrode material is challenging. The reported approach aligns a solid metal microwire through the separation channel allowing integration of multiple electrodes and the use of different electrode materials without sacrificing the portability. A detection limit of 100 nM for dopamine was achieved without the use of a decoupler as a result of a higher collection efficiency with the new design.     

Insulin oxidation and determination at carbon electrodes

The redox chemistry of insulin was investigated at glassy carbon (GC) electrodes that were coated with films of chitosan (CHIT) and multiwalled carbon nanotubes (CNT). While bare electrodes deactivated quickly during insulin oxidation, the GC electrodes coated with CHIT and CHIT-CNT films generated stable insulin currents. The GC/CHIT-CNT electrodes were used for investigating the electrooxidation process of insulin and amperometric determination of insulin. The mass spectrometric, electron paramagnetic resonance, and separation studies of electrolyzed insulin solutions suggested that the loss of 4 mass units upon insulin oxidation at CNT could be accounted for by the formation of two dityrosine cross-links intramolecularly. At a potential of 0.700 V and physiological pH 7.40, the GC/CHIT-CNT electrodes displayed a detection limit of approximately 30 nM insulin (S/N = 3), sensitivity of 135 mA M(-1) cm(-2), linear dynamic range from 0.10 to 3.0 microM (R2 = 0.995), and superior operational and long-term stability. The CNT-based electrodes are promising new insulin detectors for diabetes-related studies such as fast chromatographic analysis of therapeutic insulin formulations or evaluation of quality of pancreatic islets prior to their transplantation.     

Separation high voltage field driven on-chip amperometric detection in capillary electrophoresis

A new potentiostatless detection scheme for amperometric detection in capillary electrophoresis is presented based on the use of microband array electrodes positioned in the capillary electrophoresis electric field. In the present study, the spatial potential difference in the CE separation high-voltage field was measured using two gold microband electrodes positioned in the proximity of the capillary outlet. The induced potential difference between the two electrodes was recorded as a function of the applied separation high voltage and the dependence of the electrochemically generated current on the high-voltage field, and the concentration of a redox couple (Fe(CN)6(4-)/Fe(CN)6(3-)) was investigated. The results show that plots of the generated current versus the CE separation voltage have the same shape as cyclic voltammograms obtained with the same electrodes in a traditional potentiostatic setup and that the current is proportional to the concentration of the redox couple. As a decoupling device is not needed, the described potentiostatless approach significantly simplifies the instrumental setup for amperometric detection. This approach consequently holds great promise for application in inexpensive portable chip-based CE devices.     

Passive conductivity detection for capillary electrophoresis

A passive electrochemical detection principle that can be applied to capillary electrophoresis is presented. The separation electrical field is used to generate a potential difference between two electrodes located along the channel. For constant-current electrophoresis, the generated signal is proportional to the resistance of the solution passing between the two electrodes. Contrary to conductivity detectors that are ac driven and need to be decoupled from the separation field, the passive detection directly takes advantage of the separation field. The signal is simply measured by a high-impedance voltmeter. The detection concept has been validated by numerical simulations showing how the magnitude of the signal is related to the ratio between the electrode distance and the length of the sample plug. As a proof of the principle, this detection concept has been demonstrated by the electrophoretic separation of three alkali ions on a polymer microchip. Based on preliminary results, a detection limit of 20 microM and a dynamic range of up to 3 orders of magnitude have been achieved.     

侧壁四电极电容耦合非接触电导检测器(英文)

研制了一种集成于硅基电泳芯片分离沟道末端侧壁的新型四电极电容耦合非接触电导检测器.研究了该电导检测器的等效模型,对等效电路模型中的参数进行了公式推导,并讨论了影响电导检测响应灵敏度的相关因素.采用深刻蚀及离子注入加工技术制得了用于电导检测的立体电极.制作了基于锁相放大原理的信号处理电路,对该电导检测的频率响应及灵敏度进行了测试分析.实验结果表明,当激励信号频率为300 kHz时,该电导检测器具有最佳线性度;不同浓度Na+溶液响应电压差值为5 mV;检测限达到10-8mol/L;且成功实现了Na+和Li+混合无机阳离子的电泳分离在线检测.     

Enhancement of immunocomplex detection and application to assays for DNA adduct of benzo[a]pyrene

The stability of antibody and formation of immunocomplexes are essential to high-sensitivity capillary electrophoresis immunoassays (CEIA). However, little attention has been paid to enhancing or maintaining immunocomplex formation and antibody stability to improve the performance of CEIA. We report here the use of nonspecific proteins, such as bovine serum albumin (BSA) and rabbit immunoglobulin (rIgG), to enhance immunocomplex formation and to stabilize antibodies and immunocomplexes for immunoassays. Complexes between DNA adducts of benzo[a]pyrenediol epoxide (BPDE) and their antibodies were examined using capillary electrophoresis with laser-induced fluorescence detection (CE-HF). A tetramethylrhodamine (TMR)-labeled single-stranded oligonucleotide (16-mer) containing a single BPDE adduct was used as a fluorescent probe to study its immunocomplexes with a monoclonal antibody (8E11). To examine the formation of larger complexes, a TMR-labeled secondary antibody (anti-mouse), a primary antibody (mouse monoclonal antibody 5D11), and BPDE adducts in cellular DNA were used. We demonstrate that the use of nonspecific proteins stabilized the antibody and greatly enhanced the formation and stability of the immunocomplexes, resulting in substantial improvements in the detection limit (10-fold) and the reproducibility of the analysis. Another advantageous consequence of the stabilization was a 150-fold reduction of the concentration of the antibody needed for the immunoassay, resulting in reduced background and cost. We successfully applied this technique to the determination of DNA adducts of BPDE using a competitive immunoassay. The results from both small complexes (between a primary antibody and an oligonucleotide) and larger complexes (among a secondary antibody, a primary antibody, and cellular DNA) indicate that the technique can be extended to other immunoassays. We suggest that nonspecific proteins may assist the formation and stabilization of antibody-antigen complexes by maintaining the correct conformation of the antibody and antigen for optimum binding.     

Integrated all-diamond ultramicroelectrode arrays: optimization of faradaic and capacitive currents

Integrated all-diamond ultramicroelectrode arrays (UMEAs) were fabricated using standard photolithography processes. The array consisted of typically 45 ultramicroelectrodes with a diameter of 10 μm and with a center-to-center spacing of 60 μm. The quasi-reference and counter electrodes were made from conductive diamond and were integrated on a 5 × 5 mm(2) chip. On the UMEA, a high ratio of faradaic current to capacitive current was achieved on heavily boron-doped and hydrogen-terminated diamond surfaces at slow scan rates and in high concentration of supporting electrolyte. A sensitive and reproducible detection of dopamine was achieved on hydrogen-terminated diamond UMEA at slow scan rates. The detection limit of dopamine in the presence of ascorbic acid was 1.0 nM, which is 50-100 times lower than that obtained on the macrosized boron-doped diamond electrodes. This array is promising for sensitive and reproducible detection of analytes in solutions with low detection limits.     

Microchip capillary electrophoresis with an integrated indium tin oxide electrode-based electrochemiluminescence detector

This paper describes an indium tin oxide (ITO) electrode-based Ru(bpy)3(2+) electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 microM (S/N = 3) and a linear range from 5 to 600 microM.     

Potentiometric detection of carboxylic acids, phosphate esters, and nucleotides in liquid chromatography using anion-selective coated-wire electrodes

An all-solid-state ion-selective membrane electrode incorporating a lipophilic anion exchanger was used in a flow-through potentiometric detector for the LC determination of organic anions of biological interest. Different metabolic intermediates (mono-, di-. and tricarboxylic acids, sugar phosphates, and nucleotides) were detected sensitively after separation on a pellicular anion-exchange chromatographic column. The electrode was coated by directly casting the electroactive mixture on a glassy carbon support of 3 mm diameter and used in a wall-jet-type flow cell. The analysis conditions were optimized to obtain both efficient separation and sensitive detection. Calibration curves showed a logarithmic dependence on the injected concentration for concentrations higher than 5.0 x 10(-5) M and a linear dependence for injected concentrations below this value. Under isocratic conditions, detection limits of 5.0 x 10(-7) M (25 pmol) were attained when a sodium hydroxide solution was used as an eluent. No suppressor system was needed in this case. The relative standard deviation for consecutive injections was 0.3% (n = 15), and the electrode lifetime was at least 2 months. The utility of potentiometric detection is further demonstrated in a gradient elution separation for single-run analysis of a synthetic mixture of biochemical compounds containing carboxylic acids, phosphate esters, and nucleotides.     

Selective voltammetric and amperometric detection of uric acid with oxidized diamond film electrodes

Electrochemically anodized diamond film electrodes were used for selective detection of uric acid (UA) in the presence of high concentrations of ascorbic acid (AA) by differential pulse voltammetry and chronoamperometry. Because the oxidation peak potential for AA is approximately 450 mV more positive than that for UA at anodized diamond electrodes, UA can be determined with very good selectivity. By use of chronoamperometry, linear calibration curves were obtained for UA over the concentration range up to 1 x 10(-6) M in 0.1 M HClO4 solution, with the lowest experimental value measured being 5 x 10(-8) M. This is consistent with the fact that a statistical analysis of the calibration curve yielded a detection limit of 1.5 x 10(-8) M (S/N = 3). AA in less than 20-fold excess does not interfere. The practical analytical utility of the method is demonstrated by the measurement of UA in human urine and serum without any preliminary treatment.