Electrochemical strategies for the label-free detection of amino acids, peptides and proteins

Electrochemical methods for the detection of amino acids, peptides, and proteins in a variety of media are reviewed. Label-free strategies in which the detection is based on the inherent electrochemical properties of the analyte are discussed. Various processes such as direct or mediated (in solution or immobilised) redox processes and interfacial ion transfers have been employed for the electrochemical detection and determination of the target analytes. The various methods covered encompass voltammetry at uncoated and modified electrodes and at immiscible liquid-liquid interfaces, potentiometry at polymer membrane electrodes and electrochemical impedance spectroscopy. The determination of the target analytes in complex biological matrices is discussed. The various approaches highlighted here illustrate the rich capabilities of electrochemical methods as simple, low-cost, sensitive tools for the determination of these important biological analytes at trace and ultra-trace levels.     

Electrochemical detection of amino acids at carbon nanotube and nickel-carbon nanotube modified electrodes

The oxidation and enhanced detection of traditionally 'non-electroactive' amino acids at a single-wall carbon nanotube (SWNT) surface and at a nickel hydroxide film electrochemically deposited and generated upon the SWNT layer is reported. Different CNT are compared, with Nafion-dispersed SWNT offering the most favorable layer for constant-potential amperometric detection. Factors affecting the oxidation process, including the pH or applied potential, are assessed. The response of the SWNT-Nafion coated electrode compares favorably with that of copper and nickel disk electrodes under flow injection analysis (FIA) conditions. The electrodeposition of nickel onto the SWNT-Nafion layer (Ni-CNT) led to a dramatic enhancement of the analytical response (vs. that observed at the SWNT or nickel electrodes alone). The oxidative process at the Ni(OH)(2) layer has been studied and the increase in sensitivity rationalized. In the presence of amino acid the Ni-CNT layer undergoes an electrocatalytic process in which the amino acid reduces the newly formed NiO(OH) species. Furthermore, the attractive response of both the CNT and Ni-CNT layers has allowed these electrodes to be used for constant-potential FIA of various amino acids and indicates great promise for monitoring chromatographic effluents. Once again an improved signal was observed at the Ni-CNT electrode compared to nickel deposited upon a bare glassy carbon electrode (Ni-GC).     

Electrochemical detection of amino acids based on cucurbit[7]uril-mediated three-dimensional gold nanoassemblies

In this paper, cucurbit[7]uril (CB[7])-mediated three-dimensional gold nanoassemblies were successfully prepared to increase the loaded amount of CB[7] and enhance the electrochemical detection of amino acids. Particle sizes of gold nanoparticles (AuNPs) significantly affect stability and detection sensitivity of nanoassemblies. The volume of gold nanoassemblies first increased and then decreased with the increase of CB[7] concentration. The 3D gold nanoassemblies composed of 16 nm AuNPs and 100 µmol/L CB[7] had excellent stability and maximum volume, exhibiting more sensitive detection for a variety of amino acids. And the detection limits of aromatic amino acids are lower in virtue of the higher binding constant between aromatic amino acids and CB[7]. This study will develop and deepen our understanding of molecular recognition in amino acids detection.     

Ultraviolet thermal lensing detection of amino acids

Thermal lensing (TL) permits ultra-sensitive measurements of optical absorption of analytes in very small liquid volumes. We report the construction and use of a TL detector based on pulsed ultraviolet (UV) laser excitation (266 nm). We applied this detector to quantitate amino acids using capillary electrophoresis (CE) as a means of separation. Sixteen individual amino acids are readily detected, but the signal has a complex dependence on intensity caused by the combination of (1) one-photon absorption; (2) two-photon absorption (TPA); and (3) photodestruction of amino acid molecules in the focus of the laser beam. An aqueous solution containing tyrosine, tryptophan, and cysteine is electrophoretically separated and the individual amino acids are detected by UV TL. The estimated limit of detection is 7 microM for tyrosine, 2.5 microM for tryptophan and 33 microM for cysterine, which translates into 0.35 fmol for tyrosine, 0.125 fmol for tryptophan, and 1.65 fmol for cysteine in the 140pL detection volume. It is found that two-photon absorption of water and the formation of color centers in the fused silica walls of the flowcell can contribute a significant, drifting background signal, but this interference can be minimized by selecting an appropriate focus condition and excitation-detection geometry. We suggest that as UV laser sources become available, UV TL may become a method of choice for measuring the concentrations of many analytes in different separation formats in which the volume is highly limited.     

Electrochemical sensor for detection of unmodified nucleic acids

We have developed a nucleic acid (NA) sensor based on mediated electrochemical oxidation of guanine residues. In this method, oligonucleotide probes are bound to a tin-doped indium oxide (ITO) electrode through a self-assembled phosphonate monolayer. The end carboxyl moiety of the monolayer is activated with carbodiimide and reacted with the amine group of a C6 alkyl linker which has been added to the 5'-end of the oligonucleotide probe. Upon hybridization of the complementary target NA, the hybrid is detected using a redox-active mediator, tris(2,2'-bipyridyl) ruthenium(II). We speculate that the monolayer does not impede electron-transfer since it contains many defect sites when assembled on a polycrystalline ITO surface. These defect sites are accessible to the mediator, but not to NA or proteins. The electrocatalytic current was a linear function of the amount of guanine bound at the electrode surface, with a detection limit of 120 amoles of guanine cm(-2) at 0.28 cm(2) ITO electrodes.     

Electrochemical determination of aromatic amino acids in extracts containing hydrophilic solvents

Some regularities of the extraction of aromatic amino acids (phenylalanine, tyrosine) with hydrophilic solvents and their mixtures from aqueous salt solutions were found. The electrochemical (potentiometric, conductometric) determination of amino acids was studied directly in the organic extract without their preliminary separation.     

Electrochemical behavior of the N-nosyl-protected amino acids in N,N-dimethylformamide

The electrochemical reduction of serine, glycine, and leucine protected by the 4-nitrobenzenesulfonyl group in N,N-dimethylformamide at mercury cathode occurs at two steps. The first one at −0.8 V vs. SCE, after a one-electron transfer, leads the anion radical formation that dimerizes and adsorbs at electrode. In the second step at −1.4 V, an instable dianion forms which then cleaves. The mechanism is discussed. Published in Russian in Elektrokhimiya, 2006, Vol. 42, No. 4, pp. 377–381. The text was submitted by the authors in English.     

Colorimetric detection of thiol-containing amino acids using gold nanoparticles

This paper reports findings of an investigation of the unusual colorimetric change of gold nanoparticles in the presence of thiol-containing amino acids such as homocysteine, cysteine and glutathione. The colorimetric change for homocysteine exhibits a rate that is about two orders of magnitude higher than that for cysteine, and at least five orders of magnitude higher than that for glutathione. The reactivity is effectively reduced or suppressed by the coexistence of either cysteine or glutathione. It is believed that the reactivity involves encapsulation of the particles by the thiol-containing amino acids which is followed by crosslinking at the encapsulating shells. In comparison with cysteine and glutathione, homocysteine has a slower encapsulating rate but a faster crosslinking rate. Implications of the findings of the interfacial encapsulation and crosslinking reactivities of gold nanoparticles to potential nanoparticle-enhanced analytical detection of thiol-containing amino acids are also briefly discussed.     

Indirect conductimetric detection of amino acids after liquid chromatographic separation

A liquid chromatographic method using indirect conductimetric detection is proposed for the determination of low levels of organic compounds, which does not require any special functional characteristics of the analyte. The signal detected is proportional to the molar concentration of the analyte and independent of its nature. The detector response is linearly dependent on analyte concentrations over at least three orders of magnitude. The basis of the detection is to create a conducting background, which will decrease on elution of the organic compounds. The theory of the method is discussed, with special reference to the quantitative displacement of the conducting species of the mobile phase from the column by the analyte on sample injection. The proposed method has been applied to study the chromatographic behaviour of twenty-one amino acids, where a 5 -μm Econosil CN column was used as the stationary phase with a mixture of water-acetonitrile-tetrahydrofuran (70:20:3) containing 1 mM perchloric acid or trichloroacetic acid as the mobile phase. The proposed method allows as little as 10 ng of each amino acid to be determined.     

Amperometric detection of amino acids with a passivated copper electrode

The amperometric behaviour of a copper electrode towards amino acids is studied by means of a rotating disc electrode. A theoretical model describing the anodic background current in a buffer solution and the increase of the current caused by amino acids is checked experimentally. The influences of the amino acid concentration, the rotation speed of the electrode and the composition of the buffer solution are studied. It is proved that chemical dissolution of a passivating film on the electrode surface, which is enhanced by the complexation reaction between the amino acid and copper(II) ions, is the principle of the phenomena observed. The applicability to flow systems is demonstrated.     

High-voltage contactless conductivity detection of underivatized amino acids in capillary electrophoresis

High-voltage contactless conductivity detection of underivatized amino acids in both acidic and basic media is demonstrated. The suitability of different acidic buffer solutions at pH values of about 2.5 was investigated with 12 amino acids. Lactic acid as background electrolyte gave the best results in terms of detection limits for arginine, lysine and histidine, which were approximately 2 x 10(-7), 3 x 10(-7) and 4 x 10(-7) M, respectively. However, the sensitivity for other species was not quite as good and the detection limits in the order of 0.5-1 x 10 (-5) M. The use of basic conditions at pH 10-11 generally led to more stable baselines and more consistent sensitivities. A range of 20 amino acids was investigated with alkaline buffers and detection limits were typically about 10(-6) M. Urine and beer samples were analyzed. Nine and eleven amino acids could be identified, respectively.     

Capillary electrophoresis of FITC labeled amino acids with laser-induced fluorescence detection

FITC labeled amino acids have been separated using a home-huilt capillary electrophoresis with a laserinduced fluorescence detection (CE-LIF) system. Seventeen peaks can now be generated from the twenty common amino acids. The key conditions lie in the optimization of pH, buffer electrolytes and buffer additives.     

Enhanced chemiluminescence detection of some dansyl amino acids in liquid chromatography

Summary The dansylated amino acids alanine, glutamic acid, methionine and norleucine were separated by reversedphase HPLC and detected via chemical excitation using the post-column, TCPO-peroxyoxalate reaction system. Enhancement of the chemiluminescence emission was achieved by including the surfactant Triton X-100 in the eluent.     

Thermovoltaic detection. III. Thermal decomposition of some amino acids

The thermal dissociation reactions of selected amino acids were studied by thermovoltaic detection (TVD), TG and DSC. All of the TVD curves except L-arginine contained a broad EMF output peak in the 200–300°C temperature range. The leading edges of the peak maxima were reproducible to within ± 1–2% while the trailing edges were reproducible to ± 20% or so. The latter was related to the irreproducible nature of the electrode-decomposition products interface. The TVD curves were not unique in that they yielded thermal analysis curves that were not inherently different from other TA techniques.     

Analysis of amino acids by capillary zone electrophoresis with electrochemical detection

The precapillary derivatization of 20 amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) and CN(-) was investigated. All these derivatized amino acids could be oxidized on the carbon fiber microdisk bundle electrode except proline. Capillary zone electrophoresis with electrochemical detection was employed for the analysis of 19 amino acids. The optimum conditions of separation and detection were borate, pH 9.48, for the electrolyte, 18 kV for the separation voltage and 1.15 V versus a saturated calomel electrode for the detection potential. Limits of detection of concentration or mass for individual amino acids were between 1.7 x 10(-7) and 1.8 x 10(-6) mol/L or 84 and 893 amol (according to the signal-to-noise ratio of 3) for the injection voltage of 6 kV and injection time of 10 s. The relative standard deviations were between 0.80 and 2.3% for the migration times and 1.4 and 6.4% for the electrophoretic peak currents. From a mixture of 19 amino acids, 10 amino acids (Arg, Lys, Orn, Try, Ser, Ala, Gly, Cys, Glu, Asp) could be well separated. The other 9 amino acids appeared on three electrophoretic peaks. From the samples, in which the nine amino acids do not exist simultaneously, some of them could also be detected. The method was applied to the determination of amino acids in beer by the standard addition method. The recovery for the amino acids in beer was 91-109%.     

Isocratic ion chromatographic determination of underivatized amino acids by electrochemical detection

An isocratic chromatographic separation with amperometric detection of underivatized amino acids at a copper oxyhydroxide modified glassy carbon electrode is described. A simple and sensitive quantitation procedure of amino acids without the need of tedious and time-consuming derivatization step was achieved by coupling anion-exchange chromatography with electrochemical detection. The effects of hydroxide, nitrate and acetonitrile concentration in the mobile phase on the capacity factors and peak resolution was evaluated. Under the optimized isocratic chromatographic conditions (i.e. 60 mM NaOH) and under constant applied potentials (i.e. 0.55 V versus Ag/AgCl) the detection limit ranged between 4 and 24 pmol injected and the linear dynamic range spanned generally over three or four order of magnitude for all investigated amino acid compounds. Direct determination of several common free amino acids in beer and milk samples were performed.     

Electrochemical oxidation of sulfur-containing amino acids on an electrode modified with multi-walled carbon nanotubes

MWNT-modified electrodes are introduced for the voltammetric determination of sulfur-containing amino acids. The morphology of the electrode surface has been characterized by atomic force microscopy. The MWNT layer consists of deeply intertwined vermicular structures with the average diameter of 25 nm. Cysteine, glutathione and methionine are oxidized on the electrode while only cysteine gives signals on the glassy carbon (GC) electrode. The application of such electrodes leads to a decreased overpotential and increase of oxidation currents for cysteine in comparison with a bare GC electrode. The schemes of oxidation are proposed. A decrease of the lower limit of determination and an enlargement of the analytical range for antioxidants were obtained. A simple, fast and accurate procedure for the voltammetric determination of methionine in pharmaceuticals has been developed and can be recommended for quality control purposes.