Electrochemical method and apparatus

Abstract

Herein is disclosed a method for detecting a material comprising a thiol group (e.g. homocysteine, cysteine, glutathione) in a sample, the method comprising: providing an electrode comprising a carbon material; contacting said electrode with said sample in the presence of catechol or more generally a precursor of ortho-quinone; performing cyclic or square wave voltammetry to convert catechol to ortho-quinone and wherein said ortho-quinone reacts with the material comprising a thiol group to form an electrochemical reaction product.

Claims

1. A method of detecting a material comprising a thiol group in a sample, the method comprising: a) providing a carbon electrode; b) contacting said electrode with said sample in the presence of an optionally substituted catechol; c) carrying out an electrochemical procedure to convert catechol to ortho-quinone and monitoring the conversion wherein said ortho-quinone reacts with the material comprising a thiol group to form an electrochemical reaction product, wherein the method comprises determining a concentration of homocysteine in the presence of at least one of, or any combination of, glutathione, cysteine and ascorbic acid; wherein the electrochemical procedure comprises a voltammetry technique to determine the presence of the electrochemical reaction product; and wherein the voltammetry technique comprises: (i) applying a first scan rate of about 200 mVs.sup.1 to about 1500 mVs.sup.1 to preclude significant signal for a product peak of the glutathione-catechol reaction, to selectively determine the concentration of homocysteine in the sample, and (ii) applying a second scan rate of about 50 mVs.sup.1 or less for the appearance of an adduct peak of the catechol reaction with both glutathione and homocysteine.

2. The method according to claim 1, wherein the electrochemical reaction product is the 1,4-Michael addition reaction product of the ortho-quinone and the material comprising a thiol group, wherein the electrochemical reaction product comprises a disulfide and the regeneration of catechol.

3. The method according to claim 1, wherein the electrochemical reaction product is the 1,4-Michael addition reaction product of the ortho-quinone and the material comprising a thiol group.

4. The method according to claim 1, wherein the electrochemical reaction product comprises a disulfide and the regeneration of catechol.

5. The method according to claim 1, wherein the method comprises determining the total concentration of homocysteine and cysteine, optionally in the presence of ascorbic acid and glutathione.

6. The method according to claim 5, wherein the total concentration of homocysteine and cysteine is determined simultaneously.

7. The method according to claim 1, wherein the method comprises determining the total concentration of glutathione, cysteine, homocysteine and ascorbic acid.

8. The method according to claim 1, wherein the method comprises determining the concentration of homocysteine and glutathione.

9. The method according to claim 1, wherein the method comprises determining the concentration of homocysteine and glutathione in the presence of cysteine and/or ascorbic acid.

10. The method according to claim 1, wherein the method comprises determining the concentrations individually of homocysteine and of glutathione in a sample.

11. The method according to claim 1, wherein the method comprises determining the concentrations individually of homocysteine and of glutathione in a sample, wherein said sample further comprises cysteine and/or ascorbic acid.

12. The method according to claim 1, wherein the voltammetry technique comprises a cyclic voltammetry technique and/or a square wave voltammetry technique.

13. The method according to claim 12, wherein the voltammetry technique consists of a square wave voltammetry technique.

14. The method according to claim 12, wherein the voltammetry technique consists of a cyclic voltammetry technique.

15. The method according to claim 1, wherein the electrode is selected from the group consisting of a glassy carbon electrode (GCE), a carbon nanotube modified glassy carbon electrode (CNT-GCE), a nanocarbon modified glassy carbon electrode (NC-GCE) and a nanocarbon paste electrode.

16. The method according to claim 1, wherein the first scan rate is about 500 mVs.sup.1 to about 1500 mVs.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The invention will now be described, by way of example only and without limitation, with reference to the following Figures and Examples, in which:

(2) FIG. 1: Resulting voltammograms of a solution containing catechol (I.sub.catechol) and thiol (I.sub.catechol+thiol) via: (A) 1,4-Michael addition and (B) Electrocatalytical reaction.

(3) FIG. 2: Cyclic voltammograms of CNT-GCE in 0.1 mM catechol (PBS, pH 7.0) at 20 C. a.) 25 mV s.sup.1 b.) 50 mV s.sup.1 c.) 100 mV s.sup.1 d.) 200 mV s.sup.1 e.) 300 mV s.sup.1 f.) 400 mV s.sup.1. Inset: peak current, i.sub.pa, vs. square root of scan rate, v.sup.1/2. .square-solid. CNT-GCE and GCE.

(4) FIG. 3: Cyclic voltammograms (50 mVs.sup.1, pH 7.0 phosphate buffer) illustrating the 0.1 mM catechol response in an absence (dotted) and presence of 0.1 mM homocysteine (solid) at the i.) CNT-GCE and ii.) GCE.

(5) FIG. 4: Cyclic voltammograms (50 mVs.sup.1, pH 7.0 phosphate buffer) illustrating the 0.1 mM catechol response to homocysteine concentrations ranging from 0-0.1 mM. Inset: peak current of the new peak plotted against the concentration of homocysteine. .square-solid. CNT-GCE and GCE.

(6) FIG. 5: Square wave voltammetry response of 0.1 mM catechol at the CNT-GCE with varying concentration of homocysteine (PBS, pH 7.0) ranging from 0-0.1 mM. Inset: Peak current at ca. 0.20 V (vs. SCE) plotted against concentration of homocysteine. .square-solid. CNT-GCE and GCE.

(7) FIG. 6: Cyclic voltammograms (50 mVs.sup.1, pH 7.0 PBS) for 0.1 mM catechol in an absence (a) and presence (b) of 0.1 mM concentration of i.) glutathione ii.) cysteine iii.) ascorbic acid at CNT-GCE and GCE.

(8) FIG. 7: Cyclic voltammograms (50 mVs.sup.1, pH 7.0 PBS) at CNT-GCE of 0.1 mM catechol containing (a) 0.1 mM homocysteine (b) 0.1 mM glutathione and (c) 0.1 mM homocysteine and glutathione.

(9) FIG. 8: Cyclic voltammograms at i.) GCE (at 1.5 Vs.sup.1) and ii.) CNT-GCE (at 500 mVs-1) of 0.1 mM catechol containing (a) 0.1 mM glutathione and (b) 0.1 mM homocysteine (PBS, pH 7.0).

(10) FIG. 9: Square wave voltammograms of CNT-GCE in solution containing 0.1 mM glutathione-cysteine-ascorbic acid-catechol with varying homocysteine concentration (0-0.1 mM) at 20 C. Inset: Homocysteine peak current at ca. 0.20 V (vs. SCE) plotted against concentration of homocysteine. .square-solid. CNT-GCE and GCE.

(11) FIG. 10: Calibration plot of detection of homocysteine (pH 7.0, PBS at 20 C.) with 0.1 mM catechol present in solution at .square-solid. CNT-GCE. Homocysteine detection in the presence of cysteine, glutathione, and ascorbic acid (PBS, pH 7.0 at 20 C.) at .circle-solid. CNT-GCE, .box-tangle-solidup. CNT-SPE.

(12) FIG. 11: Cyclic voltammograms of NC-GCE in 0.1 mM catechol (PBS, pH 7.0) at 20 C. a.) 25 mV s.sup.1 b.) 50 mV s.sup.1 c.) 100 mV s.sup.1 d.) 200 mV s.sup.1 e.) 300 mV s.sup.1. Inset: peak current, i.sub.p, vs. scan rate, v, of CNTs-GCE.

(13) FIG. 12: Cyclic voltammograms (50 mVs.sup.1) NC-GCE in PBS (pH 7.0) at 20 C. of a.) 0.1 mM catechol in solution b.) dipped in 0.1 mM catechol solution prior to test.

(14) FIG. 13: Cyclic voltammogram (50 mVs.sup.1) of CAT-NC-GCE in a.) PBS (pH 7.0) (dotted) and b.) 0.1 mM homocysteine (solid) (pH 7.0) at 20 C.

(15) FIG. 14: Cyclic voltammogram (50 mVs.sup.1) of CAT-NC-GCE in PBS (pH 7.0 at 20 C.) with varying concentration of homocysteine. Inset: peak current of homocysteine product peak, i.sub.p HCys, vs. homocysteine concentration.

(16) FIG. 15: Square wave voltammetry (freq. 25 Hz, amp. 50 mV, and step pot. 8.0 mV) response of CAT-NC-GCE with varying concentration of homocysteine, pH 7.0, ranging from 0-0.1 mM. Inset: Peak current at ca. 0.30 V (vs. SCE) plotted against concentration of homocysteine.

(17) FIG. 16: Cyclic voltammogram (50 mV s.sup.1) of CAT-NC-GCE in a.) PBS (pH 7.0) (dotted) and b.) 0.1 mM cysteine (solid) (pH 7.0) at 20 C.

(18) FIG. 17: Cyclic voltammogram (50 mVs.sup.1) of CAT-NC-GCE in PBS (pH 7.0) with varying concentration of cysteine. Inset: peak current of cysteine product peak, i.sub.pCys, vs. cysteine concentration.

(19) FIG. 18: Square wave voltammetry (freq. 25 Hz, amp. 50 mV, and step pot. 8.0 mV) response of CAT-NC-GCE with varying concentration of cysteine, pH 7.0, ranging from 0-0.1 mM. Inset: Peak current at ca. +0.30 V (vs. SCE) plotted against concentration of cysteine.

(20) FIG. 19: Cyclic voltammograms (50 mVs.sup.1, pH 7.0) for CAT-NC-GCE in an absence a.) and presence b.) of 0.1 mM concentration of i.) glutathione ii.) ascorbic acid.

(21) FIG. 20: Cyclic voltammetry (200 mVs.sup.1) of CAT-NC-GCE in a.) 0.1 mM GSH, Cys, and AA b.) 0.1 mM HCys, Cys, and AA (pH 7.0) at 20 C.

(22) FIG. 21: Square wave voltammogram (200 mVs.sup.1) of CAT-NC-GCE in PBS solution (pH 7.0) with presence of homocysteine, cysteine, glutathione, and ascorbic acid. (freq. 25 Hz, amp. 50 mV, and step pot. 8.0 mV).

(23) FIG. 22: Calibration plot of independent and simultaneous detection of a.) homocysteine and b.) cysteine in the presence of glutathione, and ascorbic acid PBS (pH 7.0) 20 C. at the CAT-NC-GCE.

(24) FIG. 23: Cyclic voltammetric responses for 100 M catechol in PBS (pH=7.5) at different scan rate (from 50 to 800 mVs.sup.1) at nanocarbon electrode. Inset: plot of peak current against square root of scan rate. The current was normalized by electrode area. E.sup.0=+0.17V and D=7.010.sup.6 cm.sup.2/s.

(25) FIG. 24: Cyclic voltammetric responses for 100 M catechol in PBS (pH=7.5) in the absence (a) and presence of glutathione (A), homocysteine (B), cysteine (C) and ascorbic acid (D) using nanocarbon paste electrode. Different concentrations were added (b) 40 and (c) 100 M. v=100 mVs.sup.1.

(26) FIG. 25: Calibration curve for (a) cysteine, (b) homocysteine, (c) glutathione and (d) ascorbic acid using a nanocarbon paste electrode and catechol in solution. [Catechol]=100 M.

(27) FIG. 26: Cyclic voltammetric responses for 100 M catechol in PBS (pH=7.5) in the absence (a) and presence of total antioxidants at different concentrations (b) 5 M, (c) 10 M, (d) 20 M, (e) 40 M, (f) 80 M and (g) 100 M using nanocarbon paste electrode. v=100 mVs.sup.1. Inset: analytical curve.

(28) FIG. 27: Cyclic voltammetric responses for 0.05% (A) and 0.3% (B) catechol dissolved in the nanocarbon paste in PBS (pH=7.5) at 100 mVs.sup.1. Three scans with the same paste.

(29) FIG. 28: Calibration curve for (a) cysteine, (b) homocysteine, (c) glutathione and (d) ascorbic acid using nanocarbon-catechol paste electrode.

(30) FIG. 29: Cyclic voltammetric responses in PBS (pH=7.5) in the absence (a) and presence of 40 M mix (A) and 80 M mix (B) using 0.3% catechol dissolved in nanocarbon paste electrode. Curves b, c and d: mix A, C and I, respectively.

(31) FIG. 30: Calibration curve for total antioxidants using nanocarbon-catechol paste electrode.

EXAMPLES

Example 1: Selective Electrochemical Detection of Homocysteine in the Presence of Glutathione, Cysteine, and Ascorbic Acid Using Carbon Electrodes

(32) Reagents

(33) All reagents were purchased through Sigma-Aldrich and Lancaster Synthesis at their highest available purity and were used as received without any further purification steps; catechol (99%, Aldrich), glutathione (98%, Sigma-Aldrich), D,L-cysteine (97%, Lancaster Synthesis), D,L-homocysteine (95%, Sigma), and ascorbic acid (99%, Aldrich). The bamboo-like multi-walled carbon nanotubes, MWCNT (3010 nm diameter, 5-20 M length, >95% purity) were purchased from Nanolab, Waltham, Mass., USA. The bamboo-like carbon nanotubes were characterized by the manufacturer using transmission electron microscopy (TEM). All solutions were prepared with deionized water at a resistivity of no less than 18.2 M cm.sup.1 at 25 C. (Millipore, UK). The buffer solutions, 0.15 M, were prepared using potassium monohydrogen phosphate (K.sub.2HPO.sub.4) (98%, Sigma-Aldrich), potassium dihydrogen phosphate (KH.sub.2PO.sub.4) (99%, Sigma-Aldrich), and potassium hydroxide (KOH) (85%, Sigma-Aldrich) accordingly to the required pH range. All buffer solutions were freshly made prior to experiments with supporting electrolyte of 0.10 M potassium chloride (KCl) (99%, Sigma-Aldrich) added to each solution.

(34) Apparatus

(35) The electrochemical experiments in Example 1 were carried out in a three electrode system using a saturated calomel electrode, SCE, reference electrode (Hach Lange, UK), a platinum mesh 99.99% (Goodfellow, UK) counter electrode, and a glassy carbon electrode, GCE, (CH Instruments, USA) working electrode. The GCE was used as the basis for preparing the modified electrode which will be discussed later herein. The surface area of the bare glassy carbon electrode was 0.071 cm.sup.2. All experiments were conducted using a computer controlled potentiostat, PGSTAT 101 (ECO-chemie, NL). A temperature controlled bath was also used to ensure that all electrochemical experiments were carried out at (202) C. in a Faraday cage. All pH measurements were conducted using a pH213 Microprocessor pH meter (Hanna instruments, UK). The pH meter was calibrated using Duracal buffers of pH 4.010.01, pH 7.000.001, and pH 10.010.01 (Hamilton, CH).

(36) Preparation of Modified Carbon Nanotube Glassy Carbon Electrode (CNT-GCE)

(37) The modification of the GCE electrode was freshly undertaken at the start of each experiment. The GCE was first polished with sequentially 3.0, 1.0, and 0.1 m diamond spray (Kemet, UK) then rinsed with ethanol and de-ionized water. Afterwards, the carbon nanotubes were immobilized onto the surface of the glassy carbon electrode through the drop casting method. The drop casting method consisted essentially of dropping an aliquot of a CNT-ethanol suspension (0.1 mg/mL) over the surface of the GCE. This allowed the ethanol to evaporate at room temperature thus leaving a layer of CNT at the electrode surface. To ensure a full suspension of the CNT-ethanol, the solution was briefly sonicated using a sonication bath prior to drop casting. A total of 6.0 g of CNT was drop casted onto the GCE during the modification of the electrode. The surface area of the modified CNT-GCE was obtained by cyclic voltammetry at different scan rates, ranging from 25 mVs.sup.1 to 400 mVs.sup.1, in 1.0 mM hexaammineruthenium(III) chloride and 0.1 M potassium chloride solution. The calculated average surface area for the CNT-GCE was (0.230.02) cm.sup.2.

(38) Screen Printed Electrodes

(39) The use of multi-walled carbon nanotube screen printed electrodes, CNT-SPE, and graphite screen printed electrodes, G-SPE, were undertaken. The disposable screen printed electrodes were acquired from DropSens (Spain) which has a ceramic substrate consisting of a multi-walled carbon nanotube or graphite working electrode, a carbon counter electrode and a silver reference electrode. The characterization using scanning electron microscopy (SEM) of these screen printed electrodes can be found on the DropSens website.

(40) Electrochemical Characterisation of Catechol

(41) Cyclic voltammograms of the system were taken at different scan rates ranging from 25 mVs.sup.1 to 400 mVs.sup.1 in PBS, pH7.0 at 20 C. (FIG. 2) to initially characterize the electrochemical behaviour of 0.1 mM catechol using both CNTs-GCE and GCE. The voltammogram shows the redox process of catechol at E.sub.1/2=+0.15 V (vs. SCE). This is attributed to the two electron, two proton oxidation of the catechol to the corresponding ortho-quinone species:

(42) ##STR00003##

(43) The inset in FIG. 2 shows that there is a linear correlation when the peak current, i.sub.p, is plotted with the square root of scan rate, v.sup.1/2, suggesting a diffusional process of catechol at either electrode. The diffusion coefficient was estimated using the Randle-evik equation, as being (7.01.0)10.sup.6 cm.sup.2 s.sup.1 for the CNTs-GCE and 7.510.sup.6 cm.sup.2 s.sup.1 for GCE.

(44) Catechol Electrochemical Characterisation in the Presence of Homocysteine

(45) Cyclic voltammetry (50 mVs.sup.1) was used to observe the electrochemical response of 0.1 mM catechol (pH 7.0, PBS) in the presence of HCys. FIG. 3 shows the comparison of the voltammetric response of the catechol in the absence (dotted line) and presence (solid line) of 0.1 mM HCys at the CNTs-GCE (i) and GCE (ii). In the presence of HCys, the voltammogram shows the forward peak increases as the back peak decreases and a new product peak emerges at ca. 0.20 V (vs. SCE). This peak is due to the reduction of substituted catechol molecule, as described above in Scheme (2).

(46) Electrochemical Detection of Homocysteine

(47) To observe the electrochemical behaviour of catechol with different concentrations of homocysteine, cyclic voltammetry (scan rate of 50 mVs.sup.1) was carried out with a solution containing 0.1 mM catechol at varying homocysteine concentrations ranging from 0-0.1 mM. FIG. 4 shows that as the concentration of homocysteine increases, the forward and new product peak, ca. 0.20 V (vs. SCE), increases as the back peak decreases. When the peak current of the new product peak is plotted with concentration of homocysteine (FIG. 4 inset), the linear trend increases up to 60 M and then decreases at 0.1 mM homocysteine. The systematically increasing trend indicates that homocysteine may be detected.

(48) To increase the sensitivity of HCys detection in the presence of 0.1 mM catechol (PBS, pH 7.0), square wave voltammetry was utilized. The parameters were optimized for CNT-GCE and GCE at frequency 50 Hz, step potential 4.0 mV, and amplitude 50 mV. FIG. 5 shows the square wave voltammograms of the catechol response to different concentrations of homocysteine at the CNT-GCE as a similar response at GCE was observed. The results obtained with square wave voltammetry were consistent with the results obtained with cyclic voltammetry for both electrodes; where the catechol peak (ca. +0.14 V vs. SCE) decreases and the new product peak at ca. 0.20 V (vs. SCE) emerges and grows with increasing homocysteine concentration. There is a linear relationship when the peak current of the product, ca. 0.20 V (vs. SCE), is plotted with concentration of homocysteine. For CNT-GCE, the linear relationship is I(A)=0.2[HCys](M) with concentrations up to 80 M (FIG. 5 inset) and the limit of detection (LOD) was determined to be 120 nM. For GCE, the linear relationship is I(A)=0.2[HCys](M) at homocysteine concentration up to 40 M and a determined LOD of 90 nM.

(49) Interference Studies

(50) In connection with the use of homocysteine detection in authentic biological samples and media, the selectivity of the system was next investigated at each electrode. First, an individual assay with 0.1 mM catechol (PBS, pH 7.0) was done with the separate additions of 0.1 mM of each antioxidant: glutathione (GSH), cysteine (Cys), and ascorbic acid (AA) at each CNT-GCE and GCE. These antioxidants were chosen because they are commonly found in biological samples at high concentrations. In addition, 0.1 mM of each analyte was used to present the worst-case scenario of possibly having abnormally high concentrations present in biological samples.

(51) Interference Study at the Glassy Carbon Electrode (GCE)

(52) Cyclic voltammograms (50 mVs.sup.1) were taken of 0.1 mM catechol (PBS, pH 7.0) solutions containing 0.1 mM of each GSH, Cys, and AA. FIG. 6 shows a cyclic voltammogram comparison in the absence (curve a) and presence (curve b) of these antioxidants: GSH (i), Cys (ii) and AA (iii) reacting with catechol. For GSH and Cys, the voltammograms show the forward peak increases and back peak decreases but only in the case with GSH, a new peak emerges at ca. 0.20 V (vs. SCE) due to the catechol-thiol interaction favoring the 1,4-Michael addition reaction. In the case with the catechol interaction with Cys at the GCE, the favoring reaction appears to be electrocatalytic at the GCE. With AA, the voltammogram shows that the forward peak increases slightly and a new peak emerges ca. 0 V (vs. SCE) indicating that it is the oxidation of pure ascorbic acid at the GCE. Upon examining all the voltammograms, there can be difficulties measuring HCys when in the presence of GSH at GCE because the peak potentials of their adduct with oxidized quinones are close to each other.

(53) Interference Study at the Carbon Nanotube Modified Carbon Electrode (CNT-GCE)

(54) FIG. 6 shows the electrochemical response at each electrode of 0.1 mM catechol (dotted line) in a presence of 0.1 mM of each antioxidants (solid lines): GSH (i), Cys (ii), and AA (iii) at the CNT-GCE. Voltammograms show an increase in forward peak with a decrease in the back peak for catechol reacting with GSH (i) and Cys (ii), with an introduction of a new product peak at ca. 0.200 V, and +0.300 V respectively. This introduction of a new product peak indicates a 1,4-Michael addition reaction is favoured and occurs with the thiols at the CNT-GCE. While there was no new product peak for the presence of ascorbic acid, the voltammograms show a slight increase in the forward peak which is similarly seen with GCE. By examining the peak potentials of the new product peak, the presence of glutathione can be a possible interference towards the detection of homocysteine as the product peak potentials are close to each other. For the case with cysteine, the product peak emerges at a different peak potential further away from the reaction with homocysteine and glutathione. It is considered that the catechol reaction with each different thiol reacts to form a new electrochemical species different from each other thus having different peak potentials.

(55) Homocysteine Selectivity

(56) FIG. 7 shows the behaviour of catechol in the presence of homocysteine (curve a), glutathione (curve b), and both (curve c) at 50 mVs.sup.1, similar behaviour is also seen at GCE. In the presence of both HCys and GSH (FIG. 7c); the new product peaks for both analytes are close which makes it difficult to determine changes in peak current between the two analytes, if it should occur. As an attempt to optimize the single homocysteine signal, one proposed method can be to take advantage of the different molecular size and reaction rates of either analytes with catechol. A higher scan rate may be applied to outrun the glutathione-catechol reaction which still allows the homocysteine-catechol interaction to take place.

(57) FIG. 8 shows cyclic voltammetry at an optimum scan rate at 1.5 Vs.sup.1 for GCE (i) and 500 mVs.sup.1 for CNTs-GCE (ii) was found and applied to a catechol solution with the presence of glutathione (curve a) and homocysteine (curve b) (PBS, pH 7.0) separately to see the possibility of homocysteine selectivity. There is no significant signal for the product peak of the glutathione-catechol reaction (curve a) while for the homocysteine-catechol reaction (curve b), the product peak (ca. 0.20 V vs. SCE) emerges for both systems. This indicates that homocysteine may be detected in the presence of glutathione at the higher scan rate. AA and Cys were not interferences to the homocysteine product signal and homocysteine may be detected in the presence of AA, Cys and GSH using cyclic voltammetry.

(58) Square wave voltammetry (optimized for CNT-GCE at frequency 50 Hz, amplitude 50 mV, and step potential 10 mV and GCE at frequency 50 Hz, amplitude 75 mV, and step potential 30 mV) was applied to a solution containing various HCys concentrations, 0-0.1 mM, in the presence of 0.1 mM for each of catechol, GSH, Cys, and AA. FIG. 9 shows the square wave voltammograms of different homocysteine concentrations in the presence of cysteine, glutathione and ascorbic acid at the CNT-GCE. The inset to FIG. 9 shows the homocysteine-catechol product current peak increases with homocysteine concentration at both electrodes. Homocysteine selectivity was not achieved at GCE under the optimized square wave voltammetry parameters presented because the result shows a signal in the absence of homocysteine due to catechol-glutathione product. While the selectivity of homocysteine was successfully achieved at CNT-GCE as no signal appeared in the absence of homocysteine when the other antioxidants are present. The differences in selectivity can be rationalized by the diffusion changes at the electrode surfaces, bare glassy carbon electrode versus porous layer of carbon nanotube modified electrode. Therefore, the CNT-GCE is the best electrode to obtain selective homocysteine detection in the presence of glutathione, cysteine and ascorbic acid.

(59) Sensitivity of homocysteine at CNT-GCE obtained in the presence of these analytes, at the range 0-10 M, is (0.200.02) A M.sup.1 and the limit of detection is determined to be 660 nM. The narrow working range may be due to the antioxidants present; including homocysteine, undergoing a competition reaction with the available catechol in solution. In spite of the antioxidant present undergoing a reaction we can still observe no change in peak current up to 10 M homocysteine in the presence of 0.1 mM analytes. However, there is a possibility that the dynamic range might be extended if those concentrations were lower.

(60) Homocysteine Detection Using Carbon Nanotube Screen-Printed Electrodes

(61) The use of commercially available carbon nanotube screen-printed electrodes, CNT-SPE, was applied to this system. CNT-SPE was tested in a solution containing 0.1 mM of catechol and all of the other analytes mentioned above while varying the concentration of homocysteine (pH 7.0, PBS) at 20 C. To ensure the same potential and conditions used previously, SCE was used as the reference electrode in the testing for comparison to the CNT-GCE. FIG. 10 shows a calibration curve of the tested CNT-SPE plotted in comparison with the other calibration curves of homocysteine concentration up to 10 M. The figure shows the linear range, 0-10 M, using CNT-SPE, is similar to CNT-GCE in the presence of the other analytes. The sensitivity for HCys at CNT-SPE is (0.200.02) A M.sup.1 which is the same in the absence and presence of the analytes at CNT-GCE. Graphite screen-printed electrode was also applied to the same system. However, a signal appeared in the absence of homocysteine due to catechol-glutathione product showing that homocysteine selectivity is not possible under these conditions. The commercially available carbon nanotube screen printed electrodes was therefore shown to be applicable towards homocysteine detection.

(62) Example 1 illustrates that the use of an electrochemically generated ortho-quinone facilitates the reaction of the thiol containing compound, homocysteine, on carbon electrodes. The detection of pure homocysteine may take place using two different carbon electrodes, bare glassy carbon electrode and carbon nanotube modified carbon electrode. The selective detection of homocysteine was achieved using a carbon nanotube modified electrode with a sensitivity of (0.200.02) A M.sup.1 and a limit of detection 660 nM at a linear range of 0-10 M in the absence and presence of other antioxidants: glutathione, ascorbic acid, and cysteine. In addition, the use of commercially available carbon nanotube screen printed electrodes was applied and it was shown that these can be applicable towards facile, fast and disposable electrodes for homocysteine detection.

Example 2: Simultaneous Electrochemical Detection of Homocysteine and Cysteine in the Presence of Ascorbic Acid and Glutathione Using a Nanocarbon Modified Electrode

(63) Reagents

(64) All reagents were purchased through Sigma-Aldrich and Lancaster Synthesis at their highest available purity and were used as received without any further purification steps; catechol (99%, Aldrich), glutathione (98%, Sigma-Aldrich), D,L-cysteine (97%, Lancaster Synthesis), D,L-homocysteine (95%, Sigma), and ascorbic acid (99%, Aldrich). The nanocarbon (1410 nm in diameter) was obtained from Cabot Corporation (Billerica, Mass., USA). The characterization of the nanocarbon was previously characterized using scanning electron microscopy (SEM). All solutions were prepared with deionized water at a resistivity of no less than 18.2 M cm.sup.1 at 25 C. (Millipore, UK). The buffer solutions, 0.15 M, were prepared using potassium monohydrogen phosphate (K.sub.2HPO.sub.4) (98%, Sigma-Aldrich), potassium dihydrogen phosphate (KH.sub.2PO.sub.4) (99%, Sigma-Aldrich), and potassium hydroxide (KOH) (85%, Sigma-Aldrich) accordingly to the required pH range. All buffer solutions were freshly made prior to experiments with supporting electrolyte of 0.10 M potassium chloride (KCl) (99%, Sigma-Aldrich) added to each solution.

(65) Apparatus

(66) The electrochemical experiments were carried out in a three electrode system using a saturated calomel electrode, SCE, reference electrode (Hach Lange, UK), a glassy carbon electrode, GCE, (CH Instruments, USA) working electrode and a platinum mesh 99.99% (Goodfellow, UK) counter electrode. The surface area of the GCE was 0.071 cm.sup.2. All experiments were conducted using a computer controlled potentiostat, PGSTAT 101 (ECO-chemie, NL). A temperature controlled bath was also used to ensure that all electrochemical experiments were carried out at 20 C.2 C. in a Faraday cage. All pH measurements were conducted using a pH213 Microprocessor pH meter (Hanna instruments, UK). The pH meter was calibrated using Duracal buffers of pH 4.010.01, pH 7.000.001, and pH 10.010.01 (Hamilton, CH).

(67) Preparations of Modified Nanocarbon Modified Glassy Carbon Electrode (NC-GCE)

(68) The modification of the nanocarbon modified glassy carbon electrode (NC-GCE) was done prior to each experiment. The GCE was polished with diamond spray (Kemet, UK) in descending order, 3.0, 1.0, and 0.1 M respectively. Next, the polished electrode was rinsed with ethanol followed by de-ionized water. The immobilization of the nanocarbon onto the surface of the GCE is done via the drop cast method. The drop cast method is essentially taking an aliquot of a nanocarbon-ethanol suspension (1:1) and dropping onto the surface of the GCE. A layer of carbon material remains at the surface of the carbon electrode after allowing the volatile solvent to dry at room temperature. A total of 50 g nanocarbon was used to modify the NC-GCE. The use of a sonication bath (Fisher Scientific, 230 V, 50 Hz) is recommended to ensure a full suspension of the nanocarbon-ethanol solution prior to drop casting.

(69) Electrochemical Characterisation of Catechol on Nanocarbon Modified Glassy Carbon Electrode (NC-GCE)

(70) Initial characterization of catechol using a NC-GCE was performed using cyclic voltammetry to determine the electrochemical behaviour. Cyclic voltammograms of the modified electrode were taken in a solution containing 0.1 mM catechol (PBS, pH 7.0) at 20 C. at varying scan rates, ranging from 25 mVs.sup.1 to 300 mVs.sup.1 (shown in FIG. 11). The figure shows that the catechol undergoes a redox process ca. +0.14 V (vs. SCE). This is attributed to the two electron, two proton oxidation of the catechol to the corresponding othro-quinone species, (refer to the first step in scheme (2)). The voltammograms in FIG. 11 show the catechol peak, ca. +0.14 V (vs. SCE), splits with increasing scan rate. Inspection of the inset to FIG. 11 shows that peak current, i.sub.p, increases apparently linearly with voltage scan rate, v, which suggests behaviour consistent with surface confined voltammetry.

(71) The porous structure of nanocarbon on the NC-GCE was investigated in relation to the electrochemical response of catechol. The electrode was pretreated by dipping the NC-GCE into a solution containing 0.1 mM catechol (PBS, pH 7.0) for ca. 43 seconds. Next, the dipped electrode, CAT-NC-GCE, was transferred into PBS (pH 7.0) and a cyclic voltammetry was obtained and to compare to the NC-GCE when catechol is in solution (pH 7.0), shown in FIG. 12. The cyclic voltammograms of the NC-GCE with catechol present in solution (curve a) compared to in CAT-NC-GCE in PBS (pH 7.0) (curve b) are similar to one another. The measured peak current of the catechol in solution at the NC-GCE is (2.00.4) A, and the catechol dipped NC-GCE is (2.00.3) A are in good agreement with each other. Without wishing to be bound by theory, this suggests that the catechol is physisorbed onto the porous nanocarbon, which further indicates that the catechol on the NC-GCE is surface bound. It may be that the electrode can have the option of having the catechol present in solution or immobilized onto the surface of the electrode. The catechol-nanocarbon modified glassy carbon electrode, CAT-NC-GCE, was chosen as the presented electrode.

(72) Electrochemical Characterization of Homocysteine (HCys)

(73) FIG. 13 shows cyclic voltammograms (50 mVs.sup.1) of the electrochemical behaviour of the catechol (curve a) when 0.1 mM homocysteine is present in the solution (curve b). The figure illustrates that when homocysteine is added to the solution, an introduction of a new peak potential emerges (ca. 0.16 vs. SCE). This is due to the addition reaction of a thiol-containing molecule to the ortho-quinone mentioned earlier. Next, various concentration of homocysteine were added to PBS (pH 7.0), ranging from 0 to 0.1 mM (FIG. 14). The inset to the figure shows that the adduct peak (ca. 0.16 vs. SCE) increases linearly, I(A)=+0.033[HCys](M), with homocysteine concentration.

(74) Square wave voltammetry was applied to increase the sensitivity towards homocysteine detection. The parameters were optimized at frequency 25 Hz, amplitude 50 mV and step potential 8.0 mV. FIG. 15 shows the product peak current of homocysteine (ca. 0.16 vs. SCE) increases with increasing homocysteine concentration. The inset to FIG. 15 indicates that there is a linear response, I(A)=+1.13[HCys](M), when the current of the homocysteine product peak, i.sub.p HCys, is plotted with homocysteine concentration. The limit of detection (LOD) was determined to be ca. 8.0 nM, using 3SD/S where SD is the standard deviation at zero analyte concentration and S is the sensitivity given by the gradient of the calibration curve. Using square wave voltammetry, the sensitivity of homocysteine detection, 1.13 A M.sup.1, was increased about thirty times compared to cyclic voltammetry.

(75) Electrochemical Characterization of Cysteine (Cys)

(76) A solution containing 0.1 mM cysteine, Cys, (pH 7.0) was initially tested using cyclic voltammetry (50 mVs.sup.1) at the CAT-NC-GCE. FIG. 16, shows a result that a product peak appears ca. +0.30 V (vs. SCE). Cysteine, being a thiol-containing compound, can also react with catechol via 1,4-Michael addition reaction as in scheme (2). To see the influence of different cysteine concentrations, cyclic voltammetry (50 mVs.sup.1) was applied to different concentrations ranging, 0-0.1 mM (FIG. 17). The inset to FIG. 17 shows that the cysteine product peak (ca. +0.30 V vs. SCE) increases also linearly with cysteine concentration, I(A)=+3.96[Cys](nM).

(77) The same square wave voltammetry parameters as optimized during the homocysteine detection in this example (see above) were also applied to the electrochemical system of cysteine because the aim is to detect both homocysteine and cysteine in the same experiment. FIG. 18 shows the voltammogram of the CAT-NC-GCE at varying cysteine concentrations, 0-0.1 mM. The figure illustrates there is small product peak that emerges at ca. +0.28 V (vs. SCE) with increasing cysteine concentration; this is a result of the thiol containing molecule, cysteine, reacting with catechol (as described above). The inset to FIG. 18 further shows that there is a linear relationship, I(A)=+9.71[Cys](nM), when cysteine concentration is plotted with product peak current of cysteine, i.sub.p Cys. The determined LOD is ca. 6.0 nM. However, sensitivity, 9.71 nM A.sup.1, towards cysteine detection using square wave voltammetry is four times less.

(78) Interference Studies

(79) The selectivity of homocysteine and cysteine detection was investigated in PBS (pH 7.0) with the presence of other antioxidants, such as glutathione (GSH) and/or ascorbic acid (AA), as they can interfere by possibly interacting with the catechol. The interaction between catechol and the possible interfering analytes, GSH and AA, was investigated first. FIG. 19 shows the voltammograms of CAT-NC-GCE in PBS (pH7.0) (curve a) with the presence of 0.1 mM antioxidants (curve b): GSH (i), and AA (ii). In the presence of glutathione (FIG. 19i), there is an introduction of a new product peak ca. 0.20 V (vs. SCE). While in FIG. 11ii, the peak at ca. +0.30 V (vs. SCE) is due to the oxidation of AA at the electrode. Under inspection, GSH can interfere with the detection of HCys given that their peak positions are close to each other; thus making it difficult to quantify HCys in the presence of GSH.

(80) One method for homocysteine selectivity in the presence of glutathione is to take advantage of their reaction rates and molecular size by increasing the scan rate. The aim is to possibly outrun the catechol reaction with, the larger molecule, glutathione at a higher scan rate but be able to allow the catechol to react with homocysteine. In FIG. 20, an optimum faster scan rate of 200 mVs.sup.1 was applied to a solution containing a mixture of 0.1 mM Cys and AA with the presence of 0.1 mM GSH (curve a) and 0.1 mM HCys (curve b). There is no observable peak in the presence of glutathione when a higher scan rate is applied (curve a). When the same scan rate was applied to a solution containing homocysteine, there is slight indication of the homocysteine product peak (curve b). These voltammograms show that a peak at ca. 0.20 V (vs. SCE) at the higher scan rate corresponds to the reaction of homocysteine-catechol. Consequently, the cysteine product peak (ca. +0.30 V vs. SCE) current decreases significantly as a result of the higher scan rate. This value is consistently seen at the square wave voltammograms mentioned above (scan rate at 200 mVs.sup.1) for the cysteine-catechol product peak.

(81) The same square wave parameters mentioned above were also applied to obtain simultaneous electrochemical detection of both homocysteine and cysteine in the presence of ascorbic acid and glutathione. Glutathione and ascorbic acid have been reported to be as high as 0.1 mM in biological samples and/or media, this concentration is considered to be the abnormal range for high risk for the diseases mentioned earlier. Therefore, testing at these high concentrations presents the likely worst-case scenario in a sample. FIG. 21 shows a reductive square wave voltammogram of a solution containing 0.1 mM of each of cysteine, homocysteine, glutathione, and ascorbic acid (pH 7.0) at the CAT-NC-GCE. The figure shows peaks with potentials at ca. +0.30 V, +0.10 V, and 0.20 V (vs. SCE) for cysteine product, catechol and homocysteine product respectively. The different peak potentials make it possible to detect both homocysteine and cysteine in the presence of all of the analytes mentioned above simultaneously.

(82) An experiment was carried out in order to detect homocysteine and cysteine independently and simultaneously in the presence of both AA and GSH. A concentration of one analyte was held constant at 0.1 mM while the other varied, ranging from 0 to 0.1 mM. First, FIG. 22 shows a linear trend of peak current versus concentration of homocysteine (22a) and cysteine (22b) when in the presence of other analytes. The linear relationship of homocysteine with peak current in the presence of the other analytes is I (A)=+0.88[HCys](M) with a determined LOD of 10 nM. Cysteine has a linear relationship at I (A)=+7.50[Cys] (nM) with a LOD of ca. 6.0 M. These values are similar to the sensitivity of pure homocysteine and cysteine. The horizontal line drawn across each plot indicates the median of the analyte that was held constant. The error of the analyte being held constant is due to limited reproducibility of the nanocarbon loading done to each electrode surface (minimum error, 30%) and not due to the detection method. Nonetheless, these values are comparative to these seen when GSH and AA are absent from the solution.

(83) From Example 2 it can be concluded that catechol was successfully immobilized onto a nanocarbon modified glassy carbon electrode to facilitate the reaction of oxidized catechol with two thiol-containing molecules, homocysteine and cysteine. A simultaneous electrochemical detection of both homocysteine and cysteine was achieved separately first and then second in the presence of glutathione and ascorbic acid to determine their calibration curves. The sensitivity of homocysteine detection was (0.880.296) A M.sup.1 with a LOD of 10 nM and the sensitivity of cysteine detection was (7.500.20) A nM.sup.1 with a LOD of 6.0 M. These values are well within the concentration range of analytes, 0-0.1 mM, reported in biological samples. This demonstrates the possibility of having simultaneous quantitative detection of homocysteine and cysteine in the presence of glutathione and ascorbic acid.

Example 3: Detection of Total Antioxidant Concentrations of Glutathione, Cysteine, Homocysteine and Ascorbic Acid Using a Nanocarbon Paste Electrode

(84) In this example, the electrocatalytic reaction between catechol and the antioxidants, glutathione, cysteine, homocysteine and ascorbic acid is studied at a nanocarbon paste electrode and used to measure the total antioxidant concentration in aqueous solution. Two different approaches are described: one in which catechol is dissolved in solution and the second in which catechol is dissolved into the nanocarbon paste electrode. Similar limits of detection of 2.0 M and 1.9 M and sensitivities of 8.810.sup.3 A/M and 0.11 M.sup.1 are reported, respectively at nanocarbon and nanocarbon-catechol paste electrodes. Three different commercial multivitamin drug samples were analysed and the results were in a good agreement with those from independent analysis.

(85) Reagents

(86) Catechol (C.sub.6H.sub.6O.sub.2, Aldrich), L-cysteine (C.sub.3H.sub.7NO.sub.2S, Sigma-Aldrich), DL-homocysteine (C.sub.4H.sub.9NO.sub.2S, Sigma-Aldrich), glutathione (C.sub.10H.sub.17N.sub.3O.sub.6S, Sigma-Aldrich), ascorbic acid (C.sub.6H.sub.8O.sub.6, Sigma-Aldrich), potassium phosphate dibasic (K.sub.2HPO.sub.4, Aldrich), potassium phosphate monobasic (KH.sub.2PO.sub.4, Sigma), nanocarbon particles (diameter 2710 nm, Monarch 430, Cabot Performance), mineral oil (Aldrich) were used as received without further purification. Phosphate buffer solution (PBS) was prepared using the adequate amount of K.sub.2HPO.sub.4 and KH.sub.2PO.sub.4 salts. All solutions were prepared using deionised water of resistivity not less than 18.2 M cm at 25 C. (Millipore, Billerica, Mass., USA). Prior to experiments, all solutions were purged through nitrogen (N.sub.2, BOC, Surrey) to remove oxygen from the system.

(87) Apparatus

(88) All electrochemical experiments were conducted at (251) C. using an Autolab (Eco Chimie, Utrecht, The Netherlands), with a standard three-electrode configuration consisting of nanocarbon or nanocarbon-catechol paste as a working electrode, a graphite rod as a counter electrode and a saturated calomel electrode (SCE) as reference electrode. All experiments were performed at least three times.

(89) Preparation of Nanocarbon and Nanocarbon-Catechol Paste Electrode

(90) Nanocarbon:

(91) The carbon paste was prepared by hand pasting nanocarbon with mineral oil (55:45) using a pestle and mortar. The pastes were kept at room temperature until used.

(92) Nanocarbon-Catechol:

(93) Catechol solution was prepared with a certain amount of solid catechol dissolved in acetone. Catechol is not soluble in mineral oil so the acetone is employed to initially dissolve it. The modified nanocarbon-catechol paste electrode was prepared by hand pasting nanocarbon with mineral oil and an aliquot of catechol solution using a pestle and mortar. The resulting paste was left for at least 30 mins in nitrogen atmosphere to evaporate the acetone. After the evaporation, solid catechol is assumed to be distributed within in the paste. The pastes were kept in nitrogen atmosphere at room temperature until used to avoid catechol oxidation.

(94) For both pastes, unmodified and catechol-modified, the material was packed into the well of the working electrode to a depth of 1 mm. The surface exposed to the solution was polished using a weighing paper to give a smooth finish before use. The body of the working electrode was a Teflon tube tightly packed with the carbon paste. The electrical contact was provided by a copper wire.

(95) Preparation of Samples

(96) The antioxidants content of multivitamin drugs was determined by using the proposed method at unmodified and modified nanocarbon paste electrode. Five tables were weighed and triturated. The sample stock solutions were prepared by dissolving approximately 20 mg of powder in PBS in a 10 mL volumetric flask. Working solutions were prepared by the dilution of suitable volumes from the stock. Sample 1 contains ascorbic acid, cysteine and glutathione. Samples 2 and 3 contain ascorbic acid and glutathione.

(97) Electrochemical Behaviour of Catechol in a Nanocarbon Paste Electrode

(98) Initially, voltammetric studies at different scan rates (from 50 to 800 mVs.sup.1) involving the electrochemical behaviour of catechol in phosphate buffer solution (pH=7.5) using the nanocarbon paste electrode were performed (FIG. 23). The voltammograms show a chemically reversible redox process with a formal potential at ca +0.17 V (vs SCE). This process is attributed to the two electron oxidation of catechol to the corresponding o-quinone species in Scheme (3).

(99) The inset in FIG. 23 shows the peak current increased linearly with the square root of scan rate, suggesting a diffusional process of catechol at this electrode. The catechol diffusion coefficient value was estimated as being 7.010.sup.6 cm.sup.2/s.

(100) Catechol in SolutionDetection of the Antioxidants Glutathione, Cysteine, Homocysteine and Ascorbic Acid

(101) The characterization of the reaction between the electrochemically generated o-quinone and the antioxidants, glutathione, cysteine, homocysteine and ascorbic acid, was carried out at a nanocarbon paste electrode using cyclic voltammetry (100 mVs.sup.1). The corresponding voltammetric responses of 100 M catechol (pH=7.5) in the absence (curve a) and presence (curves b and c) is of glutathione (A), homocysteine (B), cysteine (C) and ascorbic acid (D) are detailed in FIG. 24. FIG. 24, curve a, shows the oxidation of catechol and the addition of antioxidants led to an increase in the height of the oxidation peak and a decrease in the magnitude of the reduction peak as shown in all the cases (curves b and c) indicating an electrocatalytic reaction (the same behaviour detailed at FIG. 1B) rather than 1,4-Michael addition (FIG. 1A). In all cases, an analytical curve was obtained by plotting oxidation peak current (I.sub.o) versus concentration (FIG. 25). In some situations it was not possible to measure the reduction current peak, because at high concentration of the analytes, it decreases to zero. The sensitivity for cysteine is 0.026 A/M, for glutathione is 0.014 A/M, for homocysteine is 0.023 A/M and for ascorbic acid is 0.016 A/M. All these sensitivities were obtained in pure solution. The sensitivities differ a little from compound to compound. The data show that the system is more sensitive to cysteine than glutathione. Homocysteine and cysteine show similar sensitivity. The detection limits for each individual compound are: 1.7, 3.2, 2.0 and 2.8 M, respectively, for cysteine, glutathione, homocysteine and ascorbic acid.

(102) The nanocarbon paste electrode is not selective. All of the analytes showed similar responses. However, this observation enables a strategy for the detection of the total concentrations. Accordingly, cyclic voltammograms were carried out in mixtures of glutathione, homocysteine, cysteine and ascorbic acid. Different total concentrations were chosen from 5 to 100 M. For each one, different mixtures were prepared as shown in Table 1. FIG. 26 shows the cyclic voltammograms response for different total antioxidants mixtures and the analytical curve (inset). The linear equation obtained was I.sub.o (A)=2.409+0.0088[antioxidants] (M). The reproducibility for each different total concentration was better than 5% (n=3) and the detection limit was 2.0 M, demonstrating that the nanocarbon paste electrode is efficient for the detection of total antioxidant concentrations. These values reflect the analytical results for the range of composition studied in Table 1: 10%<[analyte]<40%, this in turn reflects the use in the medicine analysis reported below. Using the equation obtained for total antioxidants (I.sub.o (A)=2.409+0.0088[antioxidants] (M)) to calculate the concentration of 100% glutathione (100 MI.sub.o=4.11) or 100% cysteine (100 MI.sub.o=5.35), values of 193 M and 334 M are, respectively, obtained. Both values are over estimates because the sensitivities of pure individual molecules (cysteine 0.026 A/M, glutathione 0.014 A/M) and total antioxidants (0.0088 A/M) are not similar as each molecule contributes differently in the absence or presence of other compounds. However, in the application to biological samples or multivitamin drugs, the antioxidants are found mixed not isolated and analyses to within a certain tolerance are still valuable.

(103) TABLE-US-00001 TABLE 1 Different mixtures and the oxidation current obtained from cyclic voltammetry using nanocarbon paste electrode. [Gluta- [Homo- thione]/ cysteine]/ [Cysteine]/ [Ascorbic [Total]/ Mix M M M acid]/M M Io/A A 25.0 25.0 25.0 25.0 100 3.41 B 25.0 25.0 35.0 15.0 100 3.29 C 25.0 25.0 15.0 35.0 100 3.16 D 25.0 35.0 15.0 25.0 100 3.32 E 25.0 15.0 35.0 25.0 100 3.10 F 35.0 15.0 25.0 25.0 100 3.28 G 15.0 35.0 25.0 25.0 100 3.39 H 15.0 25.0 25.0 35.0 100 3.49 I 35.0 25.0 25.0 15.0 100 3.32 A 20.0 20.0 20.0 20.0 80.0 2.88 B 20.0 20.0 30.0 10.0 80.0 3.04 C 20.0 20.0 10.0 30.0 80.0 3.12 D 20.0 30.0 10.0 20.0 80.0 3.17 E 20.0 10.0 30.0 20.0 80.0 3.14 F 30.0 10.0 20.0 20.0 80.0 3.13 G 10.0 30.0 20.0 20.0 80.0 3.19 H 10.0 20.0 20.0 30.0 80.0 3.05 I 30.0 20.0 20.0 10.0 80.0 3.26 A 10.0 10.0 10.0 10.0 40.0 2.66 B 10.0 10.0 15.0 5.00 40.0 3.04 C 10.0 10.0 5.00 15.0 40.0 2.76 D 10.0 15.0 5.00 10.0 40.0 2.73 E 10.0 5.00 15.0 10.0 40.0 2.83 F 15.0 5.00 10.0 10.0 40.0 2.71 G 5.00 15.0 10.0 10.0 40.0 2.72 H 5.00 10.0 10.0 15.0 40.0 2.76 I 15.0 10.0 10.0 5.00 40.0 2.71 A 5.00 5.00 5.00 5.00 20.0 2.64 B 5.00 5.00 7.50 2.50 20.0 2.57 C 5.00 5.00 2.50 7.50 20.0 2.57 D 5.00 7.50 2.50 5.00 20.0 2.55 E 5.00 2.50 7.50 5.00 20.0 2.58 F 7.50 2.50 5.00 5.00 20.0 2.52 G 2.50 7.50 5.00 5.00 20.0 2.56 H 2.50 5.00 5.00 7.50 20.0 2.65 I 7.50 5.00 5.00 2.50 20.0 2.61 A 2.50 2.50 2.50 2.50 10.0 2.50 B 2.50 2.50 3.75 1.25 10.0 2.49 C 2.50 2.50 1.25 3.75 10.0 2.50 D 2.50 3.75 1.25 2.50 10.0 2.45 E 2.50 1.25 3.75 2.50 10.0 2.46 F 3.75 1.25 2.50 2.50 10.0 2.54 G 1.25 3.75 2.50 2.50 10.0 2.55 H 1.25 2.50 2.50 3.75 10.0 2.51 I 3.75 2.50 2.50 1.25 10.0 2.42 A 1.25 1.25 1.25 1.25 5.00 2.41 B 1.25 1.25 2.00 0.500 5.00 2.46 C 1.25 1.25 0.500 2.00 5.00 2.47 D 1.25 2.00 0.500 1.25 5.00 2.42 E 1.25 0.500 2.00 1.25 5.00 2.52 F 2.00 0.500 1.25 1.25 5.00 2.45 G 0.500 2.00 1.25 1.25 5.00 2.45 H 0.500 1.25 1.25 2.00 5.00 2.40 I 2.00 1.25 1.25 0.500 5.00 2.41
Catechol in Nanocarbon PasteDetection of the Antioxidants Glutathione, Cysteine, Homocysteine and Ascorbic Acid

(104) An alternative method is the use of catechol mixed with nanocarbon paste so as to create a reagentless sensor. In this case, the experiment requires only the presence of the target to provide a signal and after the paste is polished and washed free of analyte, the sensor is ready for reuse.

(105) Different percentages of catechol dissolved in the paste (0.03, 0.05, 0.1, 0.3, 0.6 and 1%) were used to make the modified electrode. FIG. 27 shows cyclic voltammetric responses for 0.05 and 0.3% nanocarbon-catechol paste in PBS (pH=7.5) at 100 mVs.sup.1. In both cases, similar electrochemical behaviour to catechol in solution was observed. Three scans with the same paste are presented in FIG. 27, showing that the electrode surface area is not reproducible (standard deviation is about 53%); this is probably due to the low amount of catechol generating limited homogeneity in the paste. Because of this irreproducible signal from electrode to electrode the absolute height of the oxidation or reduction peak cannot be used (without standard additions). To solve this problem the ratio between the oxidation (I.sub.o) and reduction (I.sub.r) peak currents may be used. The standard deviation in this case was 3.8% (n=3).

(106) Using pastes with percentages of catechol 0.03, 0.05 and 0.1%, the sensor can quantify less than 20 M of antioxidants and using pastes with percentages of catechol 0.3, 0.6 and 1%, the electrode can detect up to 80 M of antioxidants with different sensitivity. Nanocarbon pastes with high percentages of catechol lose sensitivity because low concentrations of antioxidants do not affect significantly the signal of catechol. For further studies, the modified electrode containing 0.3% of catechol was chosen because this electrode is more sensitive to the antioxidants and can detect a large range (up to 80 M) of antioxidants.

(107) Cyclic voltammograms were recorded at 100 mVs.sup.1, using the nanocarbon-catechol paste chosen before, in different concentrations of (a) cysteine, (b) homocysteine, (c) glutathione and (d) ascorbic acid separately in order to obtain individual analytical curves. The calibration curves (FIG. 28) shows a linear relationship for all individual antioxidants. The sensitivity for cysteine is 0.108 M.sup.1, for glutathione is 0.076 M.sup.1, for homocysteine is 0.088 M.sup.1 and for ascorbic acid is 0.066 M.sup.1. The data show that the nanocarbon-catechol paste electrode is more sensitive to cysteine, having a similar behaviour to the nanocarbon paste electrode. The detection limits for each individual analyte are: 1.9, 1.2, 2.7 and 2.3 M, respectively, for cysteine, glutathione, homocysteine and ascorbic acid.

(108) As the nanocarbon-catechol paste electrode is not totally selective, various analytes which might interfere with the measurement of the antioxidants (urea, uric acid, creatinine, glucose and hydrogen peroxide) were tested and the results are shown in Table 2. The data show that the ratios obtained for all the tested analytes (creatinine: 1.80, glucose: 1.91, hydrogen peroxide: 1.76, urea: 1.86 and uric acid: 1.89) are very similar to that obtained only in pure PBS (1.850.07), indicating that these analytes do not react significantly with the o-quinone.

(109) TABLE-US-00002 TABLE 2 Ratio between I.sub.o and I.sub.r for different analytes Analytes Concentration/M Io/Ir Creatinine 100 (1.80 0.08) Glucose 100 (1.91 0.08) Hydrogen peroxide 100 (1.76 0.08) Urea 100 (1.86 0.08) Uric acid 100 (1.89 0.09) Nanocarbon-catechol paste in PBS: I.sub.o/I.sub.r = (1.85 0.07)

(110) To detect the total concentration of glutathione, cysteine, homocysteine and ascorbic acid, cyclic voltammograms were carried out in various mixtures of glutathione, homocysteine, cysteine and ascorbic acid. Different total concentrations (glutathione, homocysteine, cysteine and ascorbic acid) were chosen from 5 to 80 M. For each one, different mixtures were prepared as shown in Table 3. FIG. 29 shows the cyclic voltammograms response for three mixtures for two different total concentrations. The data show that regardless of the combination of the different concentration, the ratio is almost the same (standard deviation <5%), indicating that in a mix the individual behaviour of each molecule does not significantly influence the amount output which is rather controlled by the total concentration of the species studies. FIG. 30 shows the calibration curve for the total concentration of antioxidants. The linear equation obtained was Io/Ir (A)=1.79+0.11[antioxidants] (M). The reproducibility for each different total concentration was between 3-5% (n=3) and the detection limit was 1.93 M, demonstrating the good performance of the modified electrode. Table 4 shows the final comparison between the two systems. Both paste electrodes were efficient to quantify total antioxidants based in a reaction with electrochemically generated o-quinone. The nanocarbon-catechol paste electrode has the advantage that is a reagentless sensor in comparison with nanocarbon paste electrode. Preferably, it should be kept in a nitrogen atmosphere to avoid catechol oxidation.

(111) TABLE-US-00003 TABLE 3 Different mixtures and the ratio I.sub.o/I.sub.r obtained from cyclic voltammetry using nanocarbon-catechol paste electrode [Gluta- [Homo- thione]/ cysteine]/ [Cysteine]/ [Ascorbic [Total]/ Mix M M M acid]/M M Io/Ir A 20.0 20.0 20.0 20.0 80.0 10.71 B 20.0 20.0 30.0 10.0 80.0 10.65 C 20.0 20.0 10.0 30.0 80.0 10.53 D 20.0 30.0 10.0 20.0 80.0 10.43 E 20.0 10.0 30.0 20.0 80.0 10.86 F 30.0 10.0 20.0 20.0 80.0 10.86 G 10.0 30.0 20.0 20.0 80.0 10.67 H 10.0 20.0 20.0 30.0 80.0 10.49 I 30.0 20.0 20.0 10.0 80.0 10.30 A 10.0 10.0 10.0 10.0 40.0 6.53 B 10.0 10.0 15.0 5.00 40.0 6.19 C 10.0 10.0 5.00 15.0 40.0 6.16 D 10.0 15.0 5.00 10.0 40.0 6.33 E 10.0 5.00 15.0 10.0 40.0 6.16 F 15.0 5.00 10.0 10.0 40.0 6.36 G 5.00 15.0 10.0 10.0 40.0 6.20 H 5.00 10.0 10.0 15.0 40.0 5.85 I 15.0 10.0 10.0 5.00 40.0 6.12 A 5.00 5.00 5.00 5.00 20.0 3.99 B 5.00 5.00 7.50 2.50 20.0 3.77 C 5.00 5.00 2.50 7.50 20.0 3.81 D 5.00 7.50 2.50 5.00 20.0 3.76 E 5.00 2.50 7.50 5.00 20.0 4.06 F 7.50 2.50 5.00 5.00 20.0 3.89 G 2.50 7.50 5.00 5.00 20.0 3.65 H 2.50 5.00 5.00 7.50 20.0 3.87 I 7.50 5.00 5.00 2.50 20.0 3.72 A 2.50 2.50 2.50 2.50 10.0 2.88 B 2.50 2.50 3.75 1.25 10.0 2.95 C 2.50 2.50 1.25 3.75 10.0 2.81 D 2.50 3.75 1.25 2.50 10.0 2.96 E 2.50 1.25 3.75 2.50 10.0 2.88 F 3.75 1.25 2.50 2.50 10.0 2.76 G 1.25 3.75 2.50 2.50 10.0 2.88 H 1.25 2.50 2.50 3.75 10.0 2.95 I 3.75 2.50 2.50 1.25 10.0 2.76 A 1.25 1.25 1.25 1.25 5.00 2.10 B 1.25 1.25 2.00 0.500 5.00 2.09 C 1.25 1.25 0.500 2.00 5.00 2.14 D 1.25 2.00 0.500 1.25 5.00 2.30 E 1.25 0.500 2.00 1.25 5.00 2.37 F 2.00 0.500 1.25 1.25 5.00 2.36 G 0.500 2.00 1.25 1.25 5.00 2.18 H 0.500 1.25 1.25 2.00 5.00 2.24 I 2.00 1.25 1.25 0.500 5.00 2.13

(112) TABLE-US-00004 TABLE 4 Comparison between the nanocarbon paste and nanocarbon-catechol paste electrodes for total antioxidants detection Nanocarbon-catechol Parameters Nanocarbon paste paste Store Room temperature Room temperature in N.sub.2 atmosphere Catechol In solution: 100 M In the paste: 0.3% Total antioxidants - range 5 to 100 M 5 to 80 M Sensitivity 8.8 10.sup.3 A/M 0.11 M.sup.1 Detection limit 2.0 M 1.9 M
Antioxidant Determination in Multivitamin Drug Samples

(113) After the detection of glutathione, cysteine, homocysteine and ascorbic acid using unmodified and modified nanocarbon paste electrode, both paste electrodes were used to determine total antioxidant concentrations in commercial multivitamin drug samples with different formulations. The results were obtained using both the above reported analytical curves: I.sub.o (A)=2.409+0.0088[antioxidants] (M) and Io/Ir (A)=1.79+0.11[antioxidants] (M), respectively, for unmodified and modified nanocarbon paste electrodes. Table 5 reports the results obtained with both paste electrodes compared to those expected as reported on the product. The agreement between results shown in the table confirms the sensors (unmodified and modified) consist of a reliable methodology to determine antioxidants in samples comprising multivitamins.

(114) TABLE-US-00005 TABLE 5 Antioxidants content in commercial multivitamin drug samples with different formulations Nanocarbon- AA/ GSH/ Cys/ Total/ Nanocarbon paste catechol paste Samples mol* mol* mol* mol* electrode/mol electrode/mol 1 2.8 10.sup.3 6.5 10.sup.6 1.6 10.sup.4 3.0 10.sup.3 (3.3 0.1) 10.sup.3 (3.21 0.08) 10.sup.3 2 4.5 10.sup.4 3.2 10.sup.5 4.8 10.sup.4 (4.72 0.05) 10.sup.4 (4.7 0.1) 10.sup.4 3 5.1 10.sup.4 8.1 10.sup.6 5.2 10.sup.4 (5.1 0.2) 10.sup.4 (5.05 0.05) 10.sup.4 AA = ascorbic acid; GSH = glutathione; Cys = cysteine *reported on the product

(115) From Example 3 it can be concluded that the application of nanocarbon paste electrodes for detecting total concentrations of glutathione, homocysteine, cysteine and ascorbic acid based on the reaction with electrochemically generated o-quinone may be achieved. The use of nanocarbon paste electrodes was demonstrated by measurement of the concentration of total antioxidants in multivitamin drug samples and the reliability of the method.

Example 4: Electrochemical Detection of Both Homocysteine and Glutathione Using Carbon Nanotube Modified Carbon Electrode (CNT-GCE)

(116) Experiments were done to investigate the detection of both homocysteine and glutathione using catechol solution at the carbon nanotube modified carbon electrode. Initial results showed that upon applying a low scan rate using cyclic voltammetry (50 mVs.sup.1) to a solution containing 0.1 mM catechol, glutathione and homocysteine, an adduct peak appears containing the catechol reaction with both glutathione and homocysteine thus making it more challenging for quantification when both are present in the solution. This is due to the reaction of catechol taking place with both glutathione and homocysteine at the electrode. Accordingly, the following two step procedure is proposed to address the problem of quantifying either homocysteine or glutathione in the presence of each other.

(117) Homocysteine selectivity was investigated by examining the reaction rates of glutathione and homocysteine with catechol. A higher voltage scan rate was applied with the aim to outrun the glutathione-catechol reaction but allow the homocysteine-catechol reaction to still take place. An optimized scan rate of 500 mVs.sup.1 was applied to cyclic voltammetry of a solution containing 0.1 mM each of catechol, glutathione and/or homocysteine (PBS, pH 7.0) using CNT-GCE. The results show that little to no product peak appears when the high scan rate is applied to a solution containing catechol and glutathione while an analytically useful adduct peak appears in the presence of catechol and homocysteine. This suggests that the higher scan rate was fast enough to preclude the catechol reaction with glutathione but still allow the reaction to take place with homocysteine thus allowing selective determination of homocysteine at the CNT-GCE. This measurement at high scan rate thus allows the measurement of homocysteine.

(118) Though the selective determination of homocysteine is achievable at high scan rate, the further concept of glutathione detection with the same sample is also addressed. For a solution containing both homocysteine and glutathione, a high cyclic voltammetry scan rate of 500 mVs.sup.1 can be first applied and then after agitation of the solution, a low scan rate of 50 mVs.sup.1 can be applied afterwards with cyclic voltammetry being measured at both scan rates. The concentration of homocysteine present in solution is first determined using the peak current at high scan rate using the analytical curve at this scan rate, for example I (A)=(0.04780.0113) [HCys](M), and taking into account the size and shape of the electrode. The homocysteine concentration, was used to calculate the peak current at the lower scan rate using a different curve, for example, I (A)=(0.02500.00077) [HCys](M), taking into account the size and shape of the electrode. This value may be subtracted from the total adduct peak current at the low scan rate thus providing the current for glutathione-catechol reaction. The glutathione concentration can then be determined using another appropriate calibration curve at 50 mVs.sup.1, such as I (A)=(0.01460.00030) [GSH](M).

(119) To provide a preliminary test of this proposed procedure, a number of solutions containing homocysteine and glutathione with 0.1 mM catechol (PBS, pH7.0) at 20 C. at the CNT-GCE were tested. The three examples of analytical curves relate to a GC electrode a radius of ca. 1.5 mm and catechol concentration of 0.1 mM. The results can be seen in the table below (table 6) showing that this procedure provides good agreement with what is present in solution, for concentrations of each homocysteine and glutathione up to 10 M. The proposed method will work generally providing both glutathione and homocysteine are of much lower concentration then the added catechol.

(120) TABLE-US-00006 TABLE 6 Preliminary determination of homocysteine and glutathione with 0.1 mM catechol (PBS, pH 7.0) at 20 C. using CNT-GCE Run 2 (repeated run with Run 1 different electrode) Mixture Calculated Calculated Calculated Calculated HCys GSH HCys @ 500 GSH @ 50 HCys @ 500 GSH @ 50 (M) (M) mVs.sup.1 (M) mVs.sup.1 (M) mVs.sup.1 (M) mVs.sup.1 (M) 10 10 9.3 11.6 11.4 13.2 5 10 4.2 11.0 5.8 12.7 10 5 9.6 4.9 9.8 4.8 10 3 10.5 3.2 9.2 4.8 3 10 3.0 9.2 2.6 11.9 1 10 1.2 10.3 1.1 8.6 10 1 10.3 1.2 9.7 2.6 I.sub.x@y = peak current for catechol in the presence of x at y (mVs.sup.1). [HCys.sub.500 mVs1] = homocysteine concentration at applied scan rate, 500 mVs.sup.1.

(121) The summary of the procedures and calculations used in connection with Example 4 is set out below. 1. Run cyclic voltammetry of mixed solution at 500 mVs.sup.1. (I.sub.Hcys@ 500 mVs-1) 2. Stir solution. Run cyclic voltammetry of mixed solution at 50 mVs.sup.1. (I.sub.Hcys+GSH@ 50 mVs-1) 3. Determine [Hcys] using, I.sub.Hcys@ 500 mVs-1 from step 1 with equation, I (A)=(0.04780.0113) [HCyS.sub.500 mVs1](M). 4. Determine I.sub.p Hcys at 50 mVs.sup.1, I.sub.Hcys@ 50 mVs-1, using I (A)=(0.02500.00077) [HCys.sub.500 mVs-1](M). 5. Subtract I.sub.p Hcys@ 50 mVs-1 (step 4) from I.sub.p Hcys +GSH@ 50 mVs-1 (step 2) to give (I.sub.p GSH @50 mVs-1). 6. Determine [GSH] from current obtained, I.sub.p GSH @50 mVs-1, at step 5 using I (A)=(0.01460.00030) [GSH](M).