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Carnosine is an endogenous dipeptide composed of β-alanine and L-histidine, possessing a multimodal pharmacodynamic profile that includes anti-inflammatory and anti-oxidant activities. Carnosine has also shown its ability to modulate cell proliferation, cell cycle arrest, apoptosis, and even glycolytic energy metabolism, all processes playing a key role in the context of cancer. Cancer is one of the most dreaded diseases of the 20th and 21st centuries. Among the different types of cancer, breast cancer represents the most common non-skin cancer among women, accounting for an estimated 15% of all cancer-related deaths in women. The main aim of the present review was to provide an overview of studies on the anti-cancer activity of carnosine, and in particular its activity against breast cancer. We also highlighted the possible advantages and limitations involved in the use of this dipeptide. The first part of the review entailed a brief description of carnosine's biological activities and the pathophysiology of cancer, with a focus on breast cancer. The second part of the review described the anti-tumoral activity of carnosine, for which numerous studies have been carried out, especially at the preclinical level, showing promising results. However, only a few studies have investigated the therapeutic potential of this dipeptide for breast cancer prevention or treatment. In this context, carnosine has shown to be able to decrease the size of cancer cells and their viability. It also reduces the levels of vascular endothelial growth factor (VEGF), cyclin D1, NAD+, and ATP, as well as cytochrome c oxidase activity in vitro. When tested in mice with induced breast cancer, carnosine proved to be non-toxic to healthy cells and exhibited chemopreventive activity by reducing tumor growth. Some evidence has also been reported at the clinical level. A randomized phase III prospective placebo-controlled trial showed the ability of Zn–carnosine to prevent dysphagia in breast cancer patients undergoing adjuvant radiotherapy. Despite this evidence, more preclinical and clinical studies are needed to better understand carnosine's anti-tumoral activity, especially in the context of breast cancer. [ABSTRACT FROM AUTHOR]

A method for the determination of selected aromatic amino acid biomarkers of oxidative stress using microchip electrophoresis with electrochemical detection is described. The separation of the major reaction products of phenylalanine and tyrosine with reactive nitrogen and oxygen species was accomplished using ligand exchange micellar electrokinetic chromatography with a PDMS/glass hybrid chip. Electrochemical detection was achieved using a pyrolyzed photoresist film working electrode. The system was evaluated for the analysis of the products of the Fenton reaction with tyrosine and phenylalanine, and the reaction of peroxynitrite with tyrosine. [ABSTRACT FROM AUTHOR]

Peroxynitrite ([ONOO.sup.-]) is a highly reactive species implicated in the pathology of several cardiovascular and neurodegenerative diseases. It is generated in vivo by the diffusion-limited reaction of nitric oxide ([NO.sup.*]) and superoxide anion ([sup.*][O.sub.2.sup.-]) and is known to be produced during periods of inflammation. Detection of ONOO is made difficult by its short half-life under physiological conditions (~1 s). Here we report a method for the separation and detection of [ONOO.sup.-] from other electroactive species utilizing a microchip electrophoresis device incorporating an amperometric detection scheme. Microchip electrophoresis permits shorter separation times (~25 s for [ONOO.sup.-]) and higher temporal resolution than conventional capillary electrophoresis (several minutes). This faster analysis allows ONOO to be detected before substantial degradation occurs, and the increased temporal resolution permits more accurate tracking of dynamic changes in chemical systems. 10.1021/ac902821v

This work describes the fabrication and evaluation of a poly(dimethyl)siloxane (PDMS)-based device that enables the discrete injection of a sample plug from a continuous-flow stream into a microchannel for subsequent analysis by electrophoresis. Devices were fabricated by aligning valving and flow channel layers followed by plasma sealing the combined layers onto a glass plate that contained fittings for the introduction of liquid sample and nitrogen gas. The design incorporates a reduced-volume pneumatic valve that actuates (on the order of hundreds of milliseconds) to allow analyte from a continuously flowing sampling channel to be injected into a separation channel for electrophoresis. The injector design was optimized to include a pushback channel to flush away stagnant sample associated with the injector dead volume. The effect of the valve actuation time, the pushback voltage, and the sampling stream flow rate on the performance of the device was characterized. Using the optimized design and an injection frequency of 0.64 Hz showed that the injection process is reproducible (RSD of 1.77%, n = 15). Concentration change experiments using fluorescein as the analyte showed that the device could achieve a lag time as small as 14 s. Finally, to demonstrate the potential uses of this device, the microchip was coupled to a microdialysis probe to monitor a concentration change and sample a fluorescein dye mixture.

Microdialysis sampling is a technique that has been used for in vivo and in vitro monitoring of compounds of pharmaceutical, biomedical, and environmental interest. The coupling of a commercially available microdialysis probe to a microchip-based capillary electrophoresis (CE) system is described. A continuously flowing dialysate stream from a microdialysis probe was introduced into the microchip, and discrete injections were achieved using a valveless gating approach. The effect of the applied voltage and microdialysis flow rate on device performance was investigated. It was found that the peak area varied linearly with the applied voltage. Higher voltages led to lower peak response but faster separations. Perfusion flow rates of 0.8 and 1.0 [micro]L/min were found to provide optimal performance. The on-line microdialysis/microchip CE system was used to monitor the hydrolysis of fluorescein mono-[beta]-D-galactopyranoside (FMG) by [beta]-D-galactosidase. A decrease of the FMG substrate with an increase in the fluorescein product was observed. The temporal resolution of the device, which is dependent on the CE separation time, was 30 s. To the best of our knowledge, this is the first reported coupling of a microdialysis sampling probe to a microchip capillary electrophoresis device.

The fabrication and evaluation of a palladium decoupler and working electrode for microchip capillary electrophoresis (CE) with electrochemical detection is described. The use of the Pd decoupler allows the working electrode to be placed directly in the separation channel and eliminates the band-broadening characteristic of the end-channel configuration. The method used for fabrication of the decoupler and working electrode was based on thin-layer deposition of titanium followed by palladium onto a glass substrate. When employed as the cathode in CE, palladium absorbs the hydrogen gas that is generated by the hydrolysis of water. The effect of the decoupler size on the ability to remove hydrogen was evaluated with regard to reproducibility and longevity. Using boric acid and TES buffer systems, 500 [micro]m was determined to be the optimum decoupler size, with effective voltage isolation lasting for ~6 h at a constant field strength of 600 V/cm. The effect of distance between the decoupler and working electrode on noise and resolution for the separation of dopamine and epinephrine was also investigated. It was found that 250 [micro]m was the optimum spacing between the decoupler and working electrode. At this spacing, laser-induced fluorescence detection at various points around the decoupler established that the band broadening due to pressure-induced flow that occurs after the decoupler did not significantly affect the separation efficiency of fluorescein. Limits of detection, sensitivity, and linearity for dopamine (500 nM, 3.5 pA/[micro]M, [r.sup.2] = 0.9996) and epinephrine (2.1 [micro]M, 2.6 pA/[micro]M, [r.sup.2] = 0.9996) were obtained using the palladium decoupler in combination with a Pd working electrode.

An indirect method for monitoring nitric oxide (NO) by determining nitrate and nitrite using microchip capillary electrophoresis (CE) with electrochemical (EC) detection has been developed. This method combines determination of nitrite by direct amperometric detection following a microchip-based CE separation and conversion of nitrate to nitrite by chemical reduction using Cu-coated Cd granules. The amount of nitrate is quantified by calculating the difference in the amount of nitrite in the sample before and after the reduction of nitrate. Optimization of the separation, injection, detection, and reduction reaction conditions, as well as studies involving integration of the reduction reaction onto the microchip, are described. It was found that nitrite can be separated and detected in ~45 s by microchip CEEC. The reduction reaction was successfully integrated on-chip and carried out in ~1 min following activation of the Cd granules. The usefulness of this device was demonstrated by monitoring the amount of nitrate and nitrite produced from 3-morpholinosydnonimine, a NO-releasing compound.

A new electrode configuration for microchip capillary electrophoresis (CE) with electrochemical (EC) detection is described. This approach makes it possible to place the working electrode directly in the separation channel. The 'in-channel' EC detection was accomplished without the use of a decoupler through the utilization of a specially designed, electrically isolated potentiostat. The effect of the working electrode position on the separation performance (in terms of plate height and peak skew) of poly(dimethylsiloxane)-based microchip CEEC devices was evaluated by comparing the more commonly used end-channel configuration with this new in-channel approach. Using catechol as the test analyte, it was found that in-channel EC detection decreased the total plate height by a factor of 4.6 and lowered the peak skew by a factor of 1.3. A similar trend was observed for the small, inorganic ion nitrite. Furthermore, a fluorescent and electrochemically active amino acid derivative was used to directly compare the separation performance of in-channel EC detection to that of a widely used laser-induced fluorescence (LIF) detection scheme. In this case, it was found that the plate height and peak skew for both detection schemes were essentially equal, and the separation performance of in-channel EC detection is comparable to LIF detection.

The development of a poly(dimethylsiloxane)-based (PDMS-based) microchip electrophoresis system employing dual-electrode electrochemical detection is described. This is the first report of dual-electrode electrochemical detection in a microchip format and of electrochemical detection on chips fabricated from PDMS. The device described in this paper consists of a top layer of PDMS containing the separation and injection channels and a bottom glass layer onto which gold detection electrodes have been deposited. The two layers form a tight reversible seal, eliminating the need for high-temperature bonding, which can be detrimental to electrode stability. The channels can also be temporarily removed for cleaning, significantly extending the lifetime of the chip. The performance of the chip was evaluated using catechol as a test compound. The response was linear from 10 to 500 (mu)M with an LOD (S/N = 3) of 4 (mu)M and a sensitivity of 45.9 pA/(mu)M. Collection efficiencies for catechol ranged from 28.7 to 25.9% at field strengths between 200 and 400 V/cm. Dual-electrode detection in the series configuration was shown to be useful for the selective monitoring of species undergoing chemically reversible redox reactions and for peak identification in the electropherogram of an unresolved mixture.

This is the first report of postcolumn amperometric reaction detection for capillary electrophoresis and dual-electrode detection. Bromide present in the run buffer is oxidized to bromine at the first electrode and subsequently detected at a second electrode downstream. Analytes that react with bromine cause a decrease in signal at the downstream electrode that is proportional to analyte concentration. Bromine is known to react with a variety of compounds, including thiols, thioethers, disulfides, amines, and unsaturated organic compounds. In this paper, the development of a new wire-wire on-capillary dual electrode that is well suited to bromine-based post-column reaction detection is described. System performance was evaluated using glutathione, cysteine, and methionine as test analytes. The final optimized system could be operated continuously for 24 h and was stable for day-to-day use for at least two weeks. The response for cysteine was linear from 0.5 to 20 [[micro]molar] with a limit of detection of [approximately]80 nM.

A new dual-electrode detector for capillary electrophoresis is described. The detector consists of an integrated gold tubular electrode as the generator electrode and a gold wire electrode for detection. The detector configuration, including electrode size and position, has been optimized in terms of detection sensitivity and separation efficiency. After amalgamation of the dual electrode with mercury, the capillary electrophoresis/electrochemistry system was employed for simultaneous detection of thiols and disulfides. The response of cystine was found to be linear from 1 [[micro]molar] to 1 mM with a LOD of 0.5 [[micro]molar] (S/N = 3) and sensitivity of 60 pA/[[micro]molar]. The detection limits represent 200-fold improvement over previously reported dual-electrode designs for the detection of disulfides. The use of this detector for identification of thiol- and disulfide-containing peptides was demonstrated with a tryptic digest of ribonuclease A.

The extremely low sample volumes required for capillary electrophoresis and the high sensitivity and selectivity of electrochemical detection make capillary electrophoresis/electrochemistry (CEEC) a very useful method for bio-analysis. In this paper, two types of dual-electrode detectors for CEEC are described. The first employs a ring-disk microelectrode placed in a wall-jet configuration and is used for the selective detection of substances undergoing chemically reversible oxidations. Collection efficiencies obtained for catecholamines with this configuration were between 25 and 35%. The second electrode design consists of two adjacent carbon fibers embedded in an epoxy matrix and is analogous to the parallel dual-electrode configuration used in liquid chromatography/electrochemistry. This configuration can be used to confirm peak identity and purity by operating the electrodes at two different potentials. Alternatively, it is possible to perform simultaneous oxidative and reductive electrochemical detection.

A separation-based biosensor has been developed that is capable of near-real-time analysis of aspartate and glutamate with a temporal resolution of less than 2 min in anesthetized or awake, freely moving animals. The instrument consists of a microdialysis sampling system, an on-line reactor, an injection interface, and a CE-LIF system. Primary amine analytes are derivatized with NDA/CN following microdialysis sampling using an on-line reactor to produce fluorescent CBI derivatives. The reaction takes approximately 1 min. The derivatized sample then travels to a microinjection valve which alternately sends CE running buffer and reacted microdialysis sample to the CE column via an injection interface. The interface allows a controllable volume of 10-20 nL to be injected onto the CE separation capillary. Separation of aspartate and glutamate from the other amino acids present in the microdialysis sample was achieved within 70 s. Detection limits for glutamate and aspartate using laser-induced fluorescence detection were 0.1 [[micro]molar]. The linear dynamic range was acceptable for the determination of aspartate and glutamate in dialysate samples where the levels are between 1 and 10 [[micro]molar]. Full automation of the system was achieved by computer control of the valve, the interface, and the data collection system. The performance of this system was demonstrated in an anesthetized rat by monitoring ECF levels of aspartate and glutamate released in brain after stimulation with high concentrations of [K.sup.+].

One of the drawbacks of chemically modified electrodes for electrochemical detection in conjunction with capillary electrophoresis (CE) is that the optimal run buffer for the CE separation is often not compatible with the electrolyte conditions necessary for maximum detector response. This paper reports the development of a membrane-based on-column mixer for CE which makes it possible to perform the separation and detection using different buffer conditions. The mixer consists of a cellulose acetate tube ca. 2-mm length and 50-mm i.d. The pH and/or ionic strength of the CE run buffer can be altered at the detector end by placing the mixer in a cathodic buffer reservoir containing a modifying solution of either HCl or NaOH. Ions from the modifying solution diffuse through the membrane into the detector end of the capillary and cause the desired change in pH. The use of this system was demonstrated for the detection of several biologically important compounds by capillary electrophoresis-electrochemistry (CEEC) using chemically modified electrodes. This included the use of a CoPC electrode for the detection of ribonucleosides, a D-amino acid oxidase electrode for the detection of amino acids, and a mixed-valent RuCN electrode for the detection of disulfides. Detection limits and detector stability were improved over those reported previously for unoptimized systems.

A system has been developed for reductive electrochemical detection with capillary electrophoresis. The instrumental setup is much simpler than that of reductive liquid chromatography/electrochemistry (LCEC). The new method also possesses major advantages over LC-based methods in terms of a less complicated deoxygenation system and shorter deoxygenation times. Concentration detection limits were similar to those reported for reductive LCEC. Mass detection limits for anthraquinone-2-carboxylic acid and (dinitrophenyl)-[Gamma]-aminobutyric acid were 1.3 and 1.6 fmol, respectively, at a S/N of 2. The reproducibility of the signal for a 9.8-fmol anthraquinone-2-carboxylic acid injection yielded a relative standard deviation of 6.2%. The high selectivity of reductive electrochemical detection was demonstrated by the detection of mitomycin C directly in human serum without prior extraction procedures.

Microdialysis sampling has become an important means of continuously monitoring reactions in vivo. This sampling technique places a constraint on the analysis method because of the very small sample volume provided. On the other hand, microdialysis provides the advantage of clean samples that do not require cleanup prior to analysis. An on-line coupling of microdialysis sampling to capillary electrophoretic (CE) analysis is described that uses the advantages of microcolumn separations to overcome the small volume limitation. An interface was designed which converts the continuous microdialysis sample stream into discrete 60-nL sample plugs and then injects a portion of this plug into the CE system. The on-line interface provided precision of 2.6% with minimal band broadening or peak height loss relative to off-line sampling. Using a high-speed micellar electrokinetic chromatography (MEKC) separation, resolution of the investigational anti-neoplastic SR 4233 from its main metabolite SR 4317 was achieved in less than 60 s. This allowed the on-line system to achieve a 90-s temporal resolution for determining the pharmacokinetics of SR 4233 in vivo.

In this paper, we report the design and application of two chemically modified microelectrodes for capillary electrophoresis. The electrodes are based on the immobilization of two different types of modifiers into a carbon paste matrix. The first design incorporated the electrocatalyst cobalt phthalocyanine, which was used to catalyze the oxidation of several analytes including the thiols cysteine and glutathione. These thiols could be detected to levels of 3.1 X 10 to the -8 and 3.0 X 10 to the -7 M, respectively. This electrode was also employed for the analysis of cystein in urine, illustrating the increase in selectivity for these types of electrodes. The second design consisted of immobilized glucose oxidase and was employed for the selective detection of glucose. These modified carbon paste microelectrodes were found to be very stable and reproducible when used as detectors for capillary electrophoresis.

An application of pulsed amperometric detection (PAD) in microelectrode flow stream is discussed. PAD was used as the detection method of capillary electrophoresis for the separation and characterization of several charged carbohydrates and for the detection of glucose in blood. Off-column detection and a gold wire microelectrode were used in the system. The advantages of the method over other carbohydrate analysis approaches were demonstrated.

A thiol-specific detector for capillary electrophoresis which employs a gold/mercury amalgam microelectrode has been developed. The detection method is based on the catalytic oxidation of mercury in the presence of thiols. Detection limits for glutathione are 0.53 fmol (or 2.1 X [10.sup.-8]M). Deoxygenation of the system was important for reproducibility of response. Under deoxygenated conditions, the RSD for 20 injections was 2.1%. The electrode can easily be regenerated in situ by electrodeposition or by removing and dip-coating with mercury. The use of this method for the separation and detection of a number of biologically important thiols is reported., The utilization ofa gold/mercury amalgam in the separation and detection of thiols by capillary electrophoresis/electrochemistry is discussed. Several biologically important thiols, including glutathione were subjected to a detection method based on thecatalytic oxidation of mercury. Reproducibility of response was made possible by the deoxygenation of the system. The method effectively demonstrated its thiol-specificity.

Cyanobenz(f)isoindole amino acid and peptide derivatives have been subjected to cyclic voltammetry experiments. Results indicate that derivatized amino acids exhibit pH-independent oxidation potentials which can be controlled to enhance the selectivity of their detection. Moreover, additional chromatographic and voltammogram data show that peptide derivatives also feature oxidation potential variations. These oxidation potential differences in both amino acid and peptide derivatives can be attributed to the nature of the electron moieties attached to the heterocyclic nitrogen which may either promote stability or instability in the cation.

This review focuses on selected applications of the separation and analysis of peptides and proteins published during the period of 1997-1998. Specific topic areas covered include high-performance liquid chromatography (HPLC), ultrafiltration, capillary electrophoresis (CE), affinity-based methods for protein isolation and separation, mass spectrometry (MS), detection of nonenzymatic posttranslational modifications, nuclear magnetic resonance spectroscopy (NMR), infrared (IR) and Raman spectroscopy, circular dichroism (CD), UV-visible absorption spectroscopy, dynamic light scattering, and calorimetry. The quantification and identification of peptides and proteins by chromatographic methods and MS have become fairly routine, as has the conformational analysis of peptides and small proteins in solution by CD, IR, and NMR. Therefore, these topics are not reviewed in detail here. In this review, we have attempted to highlight new technological developments or unique applications of analytical methods that impact the analysis of peptides and proteins.

The development of a separation-based sensor for catecholamines based on microdialysis (MD) coupled to microchip electrophoresis (ME) with electrochemical (EC) detection is described. The device consists of a pyrolyzed photoresist film working electrode and a poly(dimethylsiloxane) microchip with a flow-gated sample injection interface. The chip was partially reversibly sealed to the glass substrate by selectively exposing only the top section of the chip to plasma. This partially reversible chip/electrode integration process not only allows the reuse of the working electrode but also greatly enhanced the reproducibility of electrode alignment with the separation channel. The developed MD–ME–EC system was then tested using L -DOPA, 3-O-MD, HVA, DOPAC, and dopamine standards, which were separated in less than 100 seconds using a background electrolyte consisting of 15 mM sodium phosphate (pH 7.4), 15 mM sodium dodecyl sulfate, and 2.5 mM boric acid. A potential of +1.0 V vs. Ag/AgCl was used for amperometric detection of the analytes. The device was evaluated for on-line monitoring of the conversion of L -DOPA to dopamine in vitro and for monitoring dopamine release in an anesthetized rat in vivo following high K+ stimulation. The system was able to detect stimulated dopamine release in vivo but not endogenous levels of dopamine. [ABSTRACT FROM AUTHOR]

Microchip electrophoresis coupled with amperometric detection is more popular than voltammetric detection due to the lower limits of detection that can be achieved. However, voltammetry provides additional information about the redox properties of the analyte that can be used for peak identification. In this paper, two dual electrode configurations for microchip electrophoresis are described and evaluated for obtaining voltammetric information using amperometry. The dual-series electrode configuration was first evaluated to generate current ratios in a single run by applying two different potentials to the working electrodes placed perpendicular to the separation channel. However, it was found that it is difficult to obtain realistic current ratios with this configuration, primarily due to the relative placement of electrodes with respect to the channel end of the simple-t microchip. Correction factors were needed to obtain current ratios similar to those that would be obtained for sequential injections at two different potentials using a single electrode. A second approach using a dual-channel chip with two parallel electrodes was then developed and evaluated for obtaining voltammetric identification. The newly developed microchip permitted the injection of same amount of sample into two unique separation channels, each with an electrode at a different detection potential. Migration times and current ratios for several biologically important molecules and potential interferences including nitrite, tyrosine, hydrogen peroxide, and azide were obtained and compared to the responses obtained for analytes found in macrophage cell lysates. [ABSTRACT FROM AUTHOR]