METHOD FOR THE DETECTION OF POLYCYCLIC AROMATIC HYDROCARBONS

Abstract

A method for the detection of polycyclic aromatic hydrocarbons including: a) a step of contacting an aqueous solution including at least one polycyclic aromatic hydrocarbon with a catalyst for the oxidation of polycyclic aromatic hydrocarbons and with an electrode made of a conductive porous material, and b) a step of detecting the anthraquinone that is formed during the previous step through the oxidation of the polycyclic aromatic hydrocarbons.

Claims

1. A method for the detection of polycyclic aromatic hydrocarbons comprising: a) a step of contacting an aqueous solution comprising at least one polycyclic aromatic hydrocarbon with a catalyst for the oxidation of polycyclic aromatic hydrocarbons and with an electrode made of a conductive porous material, and b) a step of detecting the anthraquinone that is formed during the previous step through the oxidation of the polycyclic aromatic hydrocarbons.

2. The method of claim 1, wherein the catalyst for the oxidation of polycyclic aromatic hydrocarbons is selected from the group consisting of: laccases and related enzymes, electrocatalysts of PAH oxidation, molecular catalysts, polyoxometallates based on ruthenium, vanadium, tungsten or molybdenum, and mixtures thereof.

3. The method of claim 1, wherein the catalyst for the oxidation of polycyclic aromatic hydrocarbons is a laccase.

4. The method of claim 1, wherein the conductive porous material is selected from the group consisting of: carbon-based conductive materials, conductive polymers, and metals and corresponding metal oxides.

5. The method of claim 1, wherein the electrode made of a conductive porous material is an electrode made of carbon nanotubes.

6. The method of claim 1, wherein step a) further comprises the addition of a redox compound.

7. The method of claim 6, wherein the redox compound is selected from the group consisting of: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), (2,2,6,6-tetramethylpiperidin-1-yl)oxy (TEMPO), osmium complexes, quinones and metallocenes.

8. The method of claim 1, wherein the catalyst for the oxidation of polycyclic aromatic hydrocarbons is immobilized on the electrode.

9. A kit for the detection of polycyclic aromatic hydrocarbons comprising: an electrode made of the conductive porous material, and the catalyst for the oxidation of polycyclic aromatic hydrocarbons wherein the conductive porous material and catalyst are as defined in claim 1.

10. A biosensor for polycyclic aromatic hydrocarbons comprising an electrode made of the conductive porous material, on which the catalyst for the oxidation of polycyclic aromatic hydrocarbons is immobilized. wherein the conductive porous material and the catalyst are as defined in claim 1.

Description

BRIEF DESCRIPTION OF FIGURES

[0051] FIG. 1. Principle of the PAH sensor according to the disclosure based on (A) catalyst (such as laccase) in solution and (B) immobilized on the electrode (such as an electrode made of carbon nanotubes).

[0052] FIG. 2. (A) Electrochemical detection of different concentrations of anthracene solutions (a: 0, b: 0.1 fM, c: 0.5 fM, d: 1 fM, e: 10 fM, f: 50 fM, g: 0.1 pM, h: 1 pM, i: 10 pM, and j: 50 pM) obtained with laccase in solution by SWV (0.1 M McIlvaine buffer pH 5, 25? C., ref Ag/AgCl, pulse height=25 mV, pulse width=0.5 s, step height=?5 mV); (B) corresponding linear logarithmic scale between 0.1 fM and 50 pM; (C) Electrochemical detection of different concentrations of anthracene solutions (a: 0, b: 0.1 nM, c: 1 nM, d: 10 nM, e: 0.1 ?M, f: 1 ?M, g: 10 ?M and h: 100 ?M) obtained with immobilized laccase by SWV (0.1 M McIlvaine buffer pH 5, 25? C., ref Ag/AgCl, pulse height=25 mV, pulse width=0.5 s, step height=?5 mV); and (D) corresponding linear logarithmic scale between 0.1 nM and 0.1 ?M.

[0053] FIG. 3. (A) Electrochemical detection of different concentrations of pyrene, naphthalene and benzo(a)pyrene solutions (a: 0.001, b: 0.1 c: 1, and d: 10 pM) obtained with laccase in solution by SWV (0.1 M McIlvaine buffer pH 5, 25? C., ref Ag/AgCl, pulse height=25 mV, pulse width=0.5 s, step height=?5 mV) and (B) corresponding linear logarithmic scale between 1 fM et 10 pM.

EXAMPLES

[0054] Materials and Methods

[0055] Reagents

[0056] All reagents were purchased from Sigma-Aldrich (Saint Louis, Missouri, USA) and were used without further purification. All chemicals employed were of analytical grade. Distilled water was passed through a Milli-Q water purification system to obtain 18.2 M? cm.sup.?1 ultrapure water. Phosphate/citrate (Mcilvaine) and Tris-HCl buffer solutions ware prepared from Milli-Q water.

[0057] Electrochemical Measurements

[0058] The electrochemical experiments were carried out in a three-electrode electrochemical cell using a Biologic VMP3 Multi Potentiostat. The saturated calomel electrode (SCE) served as the reference electrode, a Pt wire was used as the counter electrode and MWCNT bioelectrodes were used as working electrodes. All experiments were conducted at room temperature. All simulated curves were obtained via Origin Pro 9.0. Error bars were estimated from three measurements recorded per sample.

[0059] Preparation of the Glassy Carbon-Modified MWCNT Electrode

[0060] The working electrodes were glassy carbon electrodes (3 mm diameter). 5 mg/mL NMP dispersions of MWCNTs (Multi-Walled Carbon Nanotube, purity >99% Sigma-Aldrich) were prepared by 30 min in ultrasonic bath (Fisher scientific FB 15050) until homogeneous black suspension was obtained. Then 20 ?L of the MWCNTs solution were drop-casted on a GCE and NMP was removed under vacuum obtaining a 5-?m-thick film.

[0061] Laccase Enzymes

[0062] The laccase activity was assayed at room temperature, monitoring the oxidation of ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) at 420 nm (?420 nm=3.6?10.sup.4 M.sup.?1 cm.sup.?1)the assay mixture contained 2 mM ABTS and 50 mM phosphate/citrate buffer, pH 3.0.

[0063] PAH Biosensing

[0064] Two different strategies have been set up:

[0065] Laccase in Solution (FIGS. 2A and B, FIG. 3)

[0066] A reaction solution was prepared with 2 U of laccase, 0.01 mM ABTS as a redox mediator in phosphate/citrate (2 mL at pH=5) at increasing PAH concentrations (from 0 to 1 mM) and incubated at room temperature for 1 hour. MWCNT electrodes were immersed in the reaction solution for 10 minutes, and the detection of as-produced and adsorbed oxidized products were measured by SWV in a phosphate/citrate buffer solution pH 5.

[0067] 40? C./No ABTS, Immobilized Enzyme (FIGS. 2C and 2D)

[0068] In the second developed assay to detect PAHs, the modified MWCNT electrodes were incubated in 20 ?L of laccase (100 U/mL) for 2 h at room temperature. Electrodes were then rinsed with 50 mM Tris-HCl buffer solution at pH=8 and stored at 4? C. The modified bioelectrodes were immersed in a phosphate/citrate buffer solution (2 mL at pH=5) at increasing concentrations of Anthracene (from 0 to 1 mM) and incubated at 40? C. for 2 h. Then, the detection of as-produced and adsorbed oxidized products were measured by SWV in a phosphate/citrate buffer solution pH 5.