Electrogenerated chemiluminescence detection of amino acids based on precolumn derivatization coupled with capillary electrophoresis separation

A novel method for highly sensitive detection of primary and secondary amino acids with selective derivatization using acetaldehyde as a new derivatization reagent was proposed by capillary electrophoresis (CE) coupled with electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(II). The precolumn derivatization of these amino acids with acetaldehyde was performed in aqueous solution at room temperature for 1 h. Upon optimized derivatization, the ECL intensities and detection sensitivities of the amino acids were significantly enhanced by 20-70 times. Using four amino acids, arginine, proline, valine, and leucine, as model compounds, their derivatives could be completely separated by CE and sensitively detected by ECL within 22 min. The linear ranges were 0.5-100 microM for arginine and proline and 5-1000 microM for valine and leucine with the detection limits of 1 x 10(-7) (0.5 fmol, arginine), 8 x 10(-8) (0.4 fmol, proline), 1 x 10(-6) (5 fmol, valine), and 1.6 x 10(-6) M (8 fmol, leucine) at a signal-to-noise ratio of 3. The derivatization reactions and ECL process of amino acids were also proposed based on in situ Fourier transform infrared and ultraviolet spectrometric analyses.     

Batch-type chemiluminescence detection cell for sensitization and simplification of capillary electrophoresis

A simple and convenient chemiluminescence detection cell was designed for capillary electrophoresis. The detection cell easily combined with capillary electrophoresis equipment. Luminol chemiluminescence was adapted for use with the detection cell. Detailed analysis and testing of the system revealed that luminol could be determined over a range of 2.5 x 10(-10)-6.5 x 10(-7) M (correlation coefficient, 0.999), with a detection limit (S/N = 3) of 2.5 x 10(-10) M (7 amol). Furthermore, each component in a mixture of glycine, glycylglycine, and glycylglycylgycine, which were labeled with isoluminol isothiocyanate, was baseline separated and sensitively detected. Moreover, the stacking procedure was applied to postcolumn detection in capillary electrophoresis. When acetonitrile stacking was used under certain conditions in the present system, chemiluminescence intensities of luminol and labeled compounds were about 1 order of magnitude higher than those obtained without stacking. The detection limit for luminol was 1.5 x 10(-11) M (S/N = 3), representing the highest sensitivity of luminol yet reported. Finally, the effect of p-iodophenol as an enhancer of luminol chemiluminescence was examined under weak alkaline conditions. The chemiluminescence intensity of luminol was approximately 2 orders of magnitude higher than that in the unenhanced reaction. A preliminary immunoassay using horseradish peroxidase-labeled anti-mouse IgG was also developed.     

Sensitive and universal indirect chemiluminescence detection for capillary electrophoresis of cations using cobalt(II) as a probe ion

Highly sensitive and universal indirect chemiluminescence detection for capillary electrophoresis of cations was described. This novel method is based on use of the ultrasensitive cobalt(II) as a probe ion in the running buffer. A strong and stable background chemiluminescent signal can be generated by the luminol-hydrogen peroxide reaction catalyzed by cobalt(II) ion. Displacement of the cobalt(II) probe ion in the running buffer by a migrating sample cation results in a quantifiable decrease in the background signal. The conditions for electrophoresis and the chemiluminescent reaction were systematically investigated using a commercial capillary electrophoresis instrument with an in-house-built chemiluminescence detector. Under the optimal conditions, the detection limits of the concentration for manganese(II), cadmium(II), nickel(II), lead(II), and 14 lanthanides were (3.0-6.0) x 10(-9) mol/L (S/N = 3), which was approximately 3 orders of magnitude better than indirect UV detection and 2 orders better than indirect laser-induced fluorescent detection. A mixture of 18 metal ions including 14 lanthanides was efficiently separated within 3.5 min using lactate to partially complex the metal ions. Our data demonstrated that CE with indirect CL detection was a powerful and universal tool for analysis of inorganic and organic cations.     

Separation and detection of phosphorylated and nonphosphorylated peptides in liquid chromatography-mass spectrometry using monolithic columns and acidic or alkaline mobile phases

Efficient chromatographic separation is a prerequisite for the sensitive analysis of complex peptide mixtures using liquid chromatography-mass spectrometry. This is especially true for the analysis of mixtures of unmodified and posttranslationally modified peptides, for example, phosphorylated peptides in the presence of their unmodified analogues. Applying monolithic capillary columns based on poly(styrene/divinylbenzene), the influence of acidic eluents based on trifluoroacetic and heptafluorobutyric acid as well as an alkaline eluent based on triethylamine-acetic acid (pH 9.2) on the separation of synthetic phosphopeptides was evaluated. Heptafluorobutyric acid offered the longest retention times and highest selectivities and, hence, the most effective separation. Application of the alkaline eluent in conjunction with detection in negative ion mode electrospray ionization mass spectrometry, on the other hand, allowed the detection of phosphorylated peptides with significantly lower limits of detection, as compared to acidic eluents in combination with detection in positive ion mode. Pairs of phosphorylated and nonphosphorylated synthetic peptides, ranging from 7- to 16-mers, as well as phosphorylated peptides form a tryptic protein digest could be separated both at acidic and alkaline pH. Utilizing a 60 x 0.20-mm-i.d. capillary column, the limit of detection in negative ion detection mode for a 4-fold phosphorylated peptide in a beta-casein digest was 10 fmol. Together with the capability for fast separation of protein digests, monolithic columns, thus, facilitate the effective and sensitive analysis of this important posttranslational modification.     

On-column coaxial flow chemiluminescence detection for underivatized amino acids by pressurized capillary electrochromatography using a monolithic column

A new method for pressurized capillary electrochromatography (pCEC) coupling with chemiluminescence (CL) detection using a modified on-column coaxial flow detection interface was developed. To evaluate the feasibility and reliability of the experimental setup, the typical CL compounds luminol and isoluminol were separated and detected by using this pCEC-CL system. A detailed investigation of CL detection interface and postcolumn CL reagent flow rate parameters was described. The excellent resolution and detection sensitivity was achieved by using 3-microm ODS-C18 packed column with 30% ACN (v/v), 5 mmol/L phosphate buffer (pH 8.0). Moreover, with the presence of Co(II) (1.0 x 10(-4) mol/L) in the mobile phase, the linear range of the concentration for luminol was 2.0 x 10(-9)-2.0 x 10(-6) mol/L with a detection limit (S/N = 3) of 2.0 x 10(-10) mol/L, and 2.5 x 10(4) theoretical plates was achieved. In addition, separation and detection of the underivatized amino acids (l-threonine and l-tyrosine) were accomplished by using a polymerized monolithic column based on the principle of the luminol-H2O2-Cu(II)-amino acid CL system. Under the optimum conditions, the mixture of amino acids was efficiently separated with satisfactory results.     

Characterization of an EDTA bonded conducting polymer modified electrode: its application for the simultaneous determination of heavy metal ions

An EDTA bonded conducting polymer modified electrode (EDTA-CPME) was fabricated by polymerization of 3',4'-diamino-2,2';5',2'-terthiophene monomer on a GCE, followed by the reaction with EDTA in the presence of catalyst. The surface of the resulting modified electrode was characterized with EQCM, ESCA, SEM, Auger electron spectroscopy, scanning Auger microscopy, and electrochemical methods. The amounts of polymer and EDTA attached on the polymer film were determined. Simple immersing of the EDTA-CPME into a sample solution led to the chemical deposition through the complexation with Pb2+, Cu2+, and Hg2+ ions, simultaniously. Various experimental parameters that affect the simultaneous analysis of the metal ions, e.g., EDTA amount, pH, deposition time, and deposition temperature, were optimized. Calibration plots for the EDTA-CPME with square wave voltammetry were obtained in the concentration range between 5.0 x 10(-10) and 1.0 x 10(-7) M for Cu(II) and between 7.5 x 10(-10) and 1.0 x 10(-7) M for Pb(II) and Hg(II). The detection limits for Pb(II), Cu(II), and Hg(II) ions were determined to be about 6.0 x 10(-10), 2.0 x 10(-10), and 5.0 x 10(-10) M, respectively. Interference effects from other metal ions were studied at various pHs and it was found that there was little or no effect on the simultaneous determination. The stability of the EDTA-CPME was remarkably improved by coating the surface with the Nafion film, and the electrode can be used for more than one month. Analytical availability of the EDTA-CPME was demonstrated by the application for the certified standard urine reference material and tap water.     

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.     

Photothermal deflection determination of iron(II) with ferrozine with sorption preconcentration on Silufol plates

Photothermal deflection spectroscopy was applied to the selective detection of iron(II) chelate with ferrozine by its sorption preconcentration on Silufol plates. The linearity range was 1 x 10(-11) - 6 x 10(-8) mol cm(-2) of chelate at the plate surface, which corresponded to 1 x 10(-9) -4 x 10(-6) M of chelate in solution. The limits of detection and quantification are 8 x 10(-12) and 2.5 x 10(-11) mol cm(-2) at the plate from 15 microL of test solution (0.5 nM and 1.5 nM in solution, respectively), and the absolute detection limit is 8 fmol in the whole spot applied to a plate. Characteristics and features of photothermal deflection detection are discussed.     

Flow injection analysis of L-lactate with enzyme amplification and amperometric detection

A flow injection analysis method for the determination of the lactate anion with enzyme amplification and amperometric detection is described. The system utilizes an oxygen electrode for measurement of changes in the oxygen concentration in the flow stream. Two enzymes, lactate oxidase and lactate dehydrogenase, were randomly coimmobilized on aminopropyl controlled-pore glass (AMP-CPG) and packed into a reactor. beta-NADH was used as a coenzyme for the regeneration of lactate from pyruvate. The experimental conditions for the determination of the lactate anion were studied for this system by the simplex and the univariant methods. The results obtained under these two conditions were compared. The simplex experimental condition yielded a calibration curve whose linear portion had a slope that was 1.2 times greater than that of the linear portion of the curve obtained under univariant conditions. The limit of detection under simplex condition was 1.19 x 10(-7) M vs 3.29 x 10(-7) M lactate under univariant conditions. The relative standard deviation obtained for this system at 6 x 10(-6) M lactate (n = 10) was about 2.5% under simplex conditions and 3.6% under univariant maximization conditions.     

Photocatalytic dehalogenation coupled on-line to a reversed micellar-mediated chemiluminescence detection system: application to the determination of iodinated aromatic compounds

The effects of illumination time, temperature, catalyst concentration, and pH on the on-line photocatalytic dehalogenation of iodinated aromatic compounds in a near-UV-illuminated titanium dioxide (anatase type) aqueous suspension were monitored via the iodine-luminol chemiluminescence (CL) reaction in a reversed micellar medium, and a new, automated, rapid, and efficient method was developed. A water-cooled, 400-W high-pressure Hg lamp was used as an internal light source. The flow procedure involved the following: (1) photocatalytic dehalogenation/degradation of the iodinated compound by the near-UV-illuminated titanium dioxide and the production of iodide species, (2) oxidation of iodide into iodine, (3) extraction of iodine into cyclohexane, (4) membrane separation of the iodine-containing organic phase from the aqueous phase, and (5) the detection of iodine using the luminol CL reaction in the reversed micellar solution of cetyltrimethylammonium chloride in 6:5 (v/v) chloroform-cyclohexane/water buffered with sodium carbonate. Results for the dehalogenation of the iodinated compounds, o-iodobenzoic acid and L-thyroxine (3,5,3',5'-tetraiodothyronine) sodium, were compared with a standard inorganic iodide solution. After establishing the optimum chemical and instrumental conditions, detection limits of 0.8 x 10(-9) and 0.2 x 10(-9) M and linear calibration graphs were obtained with dynamic ranges from 0.79 x 10(-7) to 7.9 x 10(-7) M and from 0.20 x 10(-7) to 2.0 x 10(-7) M for o-iodobenzoic acid and L-thyroxine, respectively. A precision of approximately 4% relative standard deviation (n = 6) was provided at an o-iodobenzoic acid concentration of 0.79 x 10(-7) M. The method developed was applied to the on-line determinations of iodinated aromatic compounds such as L-thyroxine sodium and iopamidol ((S)-N,N'-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2-hydroxy-1-oxopropyl)amino]-2,4,6-triiodoisophthaldiamide) in pharmaceuticals.     

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.     

Elution-modified displacement chromatography coupled with electrospray ionization-MS: on-line detection of trace peptides at low-femtomole level in peptide digests

Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between 1:10(5) and 1:10(6). In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest. We found that EMDC facilitated rapid detection and sequence analysis of trace peptides at levels of approximately 0.5 fmol/microL in complex peptide mixtures with a wide dynamic concentration range. Accordingly, the detection of kemptide by EMDC was found to be 3-4 orders of magnitude more sensitive than that attained in conventional linear elution chromatography separations performed with the same peptide loads. Kemptide was phosphorylated in vitro and was detected along with its neutral loss product in peptide mixtures at low femtomole levels. EMDC enabled both detection and amino acid sequence determination on trace levels of phosphorylated and other posttranslationally modified peptides, suggesting that the technique may be useful for proteomics applications where detection and analysis of trace level peptides are problematic.     

Determination of electrophoretic mobility distributions through the analysis of individual mitochondrial events by capillary electrophoresis with laser-induced fluorescence detection.

Here we report on the analysis of mitochondrial preparations by capillary electrophoresis with postcolumn laser-induced fluorescence detection. Individual mitochondria are detected by fluorescent labeling with the mitochondrion-selective probe, 10-nonyl acridine orange. Interactions between the organelles and the capillary walls are controlled by derivatization of the capillaries with poly(acryloylaminopropanol). As expected from the presence of charged groups in their outer membranes, isolated mitochondria have intrinsic electrophoretic mobilities. This property may be influenced by variations in size, morphology, membrane composition, and damage caused during the isolation procedure. The mobility distributions of mitochondria isolated from NS1 and CHO cells ranged from -1.2 x 10(-4) to -4.3 x 10(-4) cm2 V(-1) s(-1) and -0.8 x 10(-4) to -4.2 x 10(-4) cm2 V(-1) s(-1), respectively. Furthermore, there seems to be no correlation between the density of the mitochondrial fraction and the resultant electrophoretic mobility distribution. These results suggest a new method for characterization of organelle fractions and for counting individual organelles.     

Chromatographic detection using tris(2,2'-bipyridyl)ruthenium(III) as a fluorogenic electron-transfer reagent

Tris(2,2'-bipyridyl)ruthenium can be excited to fluorescence by visible light (lambda abs 454 nm, lambda em 607 nm) when in the M(II) oxidation state, but not in the M(III) state. A novel chromatographic detection method using the non-fluorescent M(III) form of the complex as a postcolumn fluorogenic reagent is demonstrated. The M(III) form is a powerful oxidizing agent (E degree = 1.27 V vs NHE, 1.05 V vs Ag/AgCl). The M(III) reagent is generated on-line from the M(II) form of the complex by a highly efficient porous carbon electrode and then reacted briefly with chromatographic effluent; the M(II) created by electron transfer from oxidation-susceptible analytes is then detected by fluorescence. The fluorescence detector can be calibrated for number of electrons transferred by injection of either M(II) or an oxidative standard such as ferrocyanide. It is hoped that this redox-based detection scheme will provide an alternative to electrochemical detection. Among the advantages are freedom from surface fouling and the potential for extremely low detection limits. The scheme was applied to detection of the peptide dynorphin A and several of its fragments. Dynorphin A contains tyrosine at the N-terminus (position 1) and tryptophan in position 15; these amino acid residues are susceptible to oxidation and peptides containing them can be detected on that basis. Flow injection testing of the model compounds Tyr-Gly-Gly-Phe-Leu and Gly-Gly-Trp-Gly indicated that tyrosine transferred approximately 1 electron to the M(III) reagent and that tryptophan transferred approximately 4 electrons. Similar results were obtained from the chromatographic runs. Dynorphin A and six dynorphin A fragments containing the N-terminal tyrosine were detected easily at 100 nM concentration (14 pmol) using laser-induced fluorescence. As expected, one fragment that did not contain tryptophan or tyrosine was not detected. A mass detection limit of 80 fmol was estimated for the tyrosine-containing fragments.     

Luminol chemiluminescence in unbuffered solutions with a cobalt(II)-ethanolamine complex immobilized on resin as catalyst and its application to analysis.

Using a heterogeneous catalyst, Co(II)-ethanolamine complex sorbed on Dowex-50W resin, the chemiluminescence (CL) of luminol in unbuffered or weakly acidic solution was studied in the presence of H2O2. The maximum luminol CL wavelength at pH 5.7 was 448 nm, 23 nm longer than that in a basic solution (pH 10.5). Three different ligands, mono-, di-, and triethanolamine, and six transition metal ions, Co(II), Cu(II), Ni(II), Mn-(II), Fe(II), and Fe(III) were compared by CL measurements. The CL intensity decreased in the order mono- > di- > triethanolamine and Co(II) > Cu(II) > Ni(II) > Fe-(III) > Mn(II) > Fe(II). This heterogeneous CL system was developed as H2O2 and glucose flow-through sensors. Detection limits (S/N = 3) of H2O2 and glucose using Dowex-50W-X4-Co(II)-monoethanolamine as catalyst are 1 x 10(-7) M and 1 x 10(-6) M, respectively. On the basis of the studies of the CL, fluorescence, UV-vis and ESCA spectra and the effect of dissolved oxygen in luminol solution, a mechanism for CL emission in unbuffered solution was considered as the formation of a superoxide radical ion during the decomposition of H2O2 catalyzed by the Co(II)-ethanolamine immobilized resin. Then the superoxide radical ion acted on luminol and the CL was emitted. The applications of the proposed method to determine H2O2 in rainwater without any special pretreatment and glucose in human urine and orange juice samples give satisfactory results.     

Homogeneous immunoassay for detection of TNT and its analogues on a microfabricated capillary electrophoresis chip

A homogeneous immunoassay for TNT and its analogues is developed using a microfabricated capillary electrophoresis chip. The assay is based on the rapid electrophoretic separation of an equilibrated mixture of an anti-TNT antibody, fluorescein-labeled TNT, and unlabeled TNT or its analogue. The band intensities of the free fluorescein-labeled TNT and of the antibody-antigen complex reveal the relative equilibrated concentrations. Titration of the anti-TNT antibody with a fluorescein-labeled TNT derivative yields a binding constant of (3.9 +/- 1.3) x 10(9) M(-1). The dissociation rate constant of the complex is determined by kinetic capillary electrophoresis using a folded channel and a rotary scanner to interrogate the separation at multiple time points. The dissociation rate constant is found to be 0.035 +/- 0.005 s(-1), and the resulting binding rate constant is (1.4 +/- 0.7) x 10(7) M(-1) s(-1). Binding constants of TNT and five of its analogues are determined by competitive assays: TNT (4.3 +/- 2.6) x 10(8) M(-1); 1,3,5-trinitrobenzene (5.1 +/- 3.3) x 10(7) M(-1); picric acid (7.5 +/- 4.4) x 10(6) M(-1); 2,4-dinitrotoluene (7.9 +/- 4.0) x 10(6) M(-1); 1,3-dinitrobenzene (1.0 +/- 0.7) x 10(6) M(-1); and 2,4-dinitrophenol (5.1 +/- 3.0) x 10(4) M(-1). TNT and its analogues can be assayed with high sensitivity (LOD 1 ng/mL) and with a wide dynamic range (1-300 ng/mL) using this chip-based method.     

Characterization of prolidase activity using capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection and application to evaluate collagen degradation in diabetes mellitus

A new method for prolidase (PLD, EC 3.4.13.9) activity assay was developed based on the determination of proline produced from enzymatic reaction through capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)3(2+)] electrochemiluminescence detection (ECL). A detection limit of 12.2 fmol (S/N = 3) for proline, corresponding to 1.22 x 10(-8) units of prolidase catalyzing for 1 min was achieved. PLD activity determined by CE-ECL method was in agreement with that obtained from the classical Chinard's one. CE-ECL showed its powerful resolving ability and selectivity as no sample pretreatment was needed and no interference existed. The clinical utility of this method was successfully demonstrated by its application to assay PLD activity in the serum of diabetic patients in order to evaluate collagen degradation in diabetes mellitus (DM). The results indicated that enhanced collagen degradation occurred in DM.     

Influence of tyrosine on the dual electrode electrochemical detection of copper(II)-peptide complexes.

The bluret reaction makes peptides electrochemically active. This is the basis of an electrochemical method for the detection of peptides following their liquid chromatographic separation. This paper discusses the influence of tyrosine, an electroactive amino acid, on the detection of Cu(II)-peptide (bluret) complexes containing it. The dual electrode detector has an upstream anode and a downstream cathode. Tyrosine-containing peptides yield anodic signals that are approximately the sum of the tyrosine signal and the signal from the bluret complex of a nonelectroactive model peptide, phenylalanylglycyl-glycine (FGG). The cathodic signal is depressed in comparison to FGG. This is traced to the presence of an intramolecular reaction between Cu(III) and a reaction product resulting from the oxidation of the tyrosinyl residue. The rate constant for the corresponding intermolecular reaction is significant (10(6)-10(7) M-1 s-1), but in practical analytical situations, the concentrations of the reactants will be small, so the reaction will not be a major factor. Sensitivities for several bioactive peptides are reported. The dependence of the signals on the position of tyrosine in a tripeptide is also studied.     

Nanoparticle-functionalized porous polymer monolith detection elements for surface-enhanced Raman scattering

The use of porous polymer monoliths functionalized with silver nanoparticles is introduced in this work for high-sensitivity surface-enhanced Raman scattering (SERS) detection. Preparation of the SERS detection elements is a simple process comprising the synthesis of a discrete polymer monolith section within a silica capillary, followed by physically trapping silver nanoparticle aggregates within the monolith matrix. A SERS detection limit of 220 fmol for Rhodamine 6G is demonstrated, with excellent signal stability over a 24 h period. The capability of the SERS-active monolith for label-free detection of biomolecules was demonstrated by measurements of bradykinin and cytochrome c. The SERS-active monoliths can be readily integrated into miniaturized micrototal-analysis systems for online and label-free detection for a variety of biosensing, bioanalytical, and biomedical applications.     

Carbon-Pt nanoparticles modified TiO2 nanotubes for simultaneous detection of dopamine and uric acid

The present work describes sensing application of modified TiO2 nanotubes having carbon-Pt nanoparticles for simultaneous detection of dopamine and uric acid. The TiO2 nanotubes electrode was prepared using anodizing method, followed by electrodeposition of Pt nanoparticles onto the tubes. Carbon was deposited by decomposition of polyethylene glycol in a tube furnace to improve the conductivity. The C-Pt-TiO2 nanotubes modified electrode was characterized by cyclic voltammetry and differential pulse voltammetry methods. The modified electrode displayed high sensitivity towards the oxidation of dopamine and uric acid in a phosphate buffer solution (pH 7.00). The electro-oxidation currents of dopamine and uric acid were linearly related to the concentration over a wide range of 3.5 x 10(-8) M to 1 x 10(-5) M and 1 x 10(-7) M to 3 x 10(-5) M respectively. The limit of detection was determined as 2 x 10(-10) M for dopamine at signal-to-noise ratio of 3. The interference of uric acid was also investigated. Electro-oxidation currents of dopamine in the presence of fix amount of uric acid represented a linear behaviour towards successive addition of dopamine in range of 1 x 10(-7) M to 1 x 10(-5) M. Furthermore, in a solution containing dopamine, uric acid and ascorbic acid the overlapped oxidation peaks of dopamine and ascorbic acid could be easily separated by using C-Pt-TiO2 nanotubes modified electrode.