Improvement of electrochemical detection of transthyretin synthetic peptide and its amino acids on carbon electrodes: Glassy carbon versus amorphous carbon nitride a-CNx
Introduction
The detection and the quantification of small peptides such as transthyretin peptide (PN), a tryptic fragment of transthyretin (TTR), both constitute a great challenge for the diagnosis of transthyretin-related familial amyloïd polyneuropathy (ATTR) [[1], [2], [3], [4], [5], [6]]. In order to enhance the detection sensitivity of this biomarker, likely to be useful for the detection of T49A ATTR mutation which is one of the most frequently reported ones in France [1,7] the common approach consists in the covalent derivatization of the amino acids (AA) residues of its sequence. For instance, one of the most fluorescent label widely used for this purpose is naphthalene-2,3-dicarboxyaldehyde (NDA). Moreover, capillary electrophoresis (CE) coupled with optical detection such as laser induced fluorescence (CE-LIF) remains a powerful approach to analytically evaluate the derivatization reaction efficiency [8]. In brief, the derivatization reaction in presence of cyanide permits to form fluorescent N-2-substituted-1-cyanobenz-[f]-isoindole derivative (CBI) [9].
As CBI and NDA molecules are also electroactive, a property which was recently used to investigate the tagging efficiency of labelled CBI-PN and CBI-AA [5,6], a new challenge for the development of an innovative detection module in chip electrophoresis would be to link up in a single microchip the electrochemical and optical detections [[10], [11], [12], [13]].
Several electrode materials can be used for the electrochemical detection and in particular the carbon based ones which appeared as promising sensors. Among them, carbon nitrides and their nanotubes (CNTs) have been developed as sensors for the detection of organic or biologic molecules (e.g. melamine [14], Chlorpyrifos [15], phenylethanolamine [16], ractopamine [17], and bilirubin [18]). In those examples, CNTs were associated with compounds such as graphene quantum dots or PolyOxoMetallates and they were inserted in thin films of molecular imprinted polymer. Specific cavities formed by the analytes to be detected (these latter were preliminary inserted and then removed before detection) allowed low detection limits in the picomolar range with an obvious selectivity. However, such films are generally deposited on Glassy Carbon (GC) electrodes, which prevents their use in the applications aimed in this work. In fact, the optical detection associated to the electrochemical one requires the mutual use of transparent wafer onto which a thin layer of a transparent and conductive material behaving as a polarizable electrode can be deposited. For this, the oxidation and/or reduction of the aqueous solvent should be minimized within a wide potential window and the electrode material has to present a fair electrochemical reactivity with respect to the targeted analytes to be detected. Therefore, transparent and conductive ITO-coated glass slides appeared to be the most appropriate for electrochemical measurements [[19], [20], [21], [22], [23], [24], [25], [26]]. However, the width of the potential window is at most 2 V for ITO.
For the last decades, other carbon materials were purposely developed so as to widen the potential window in aqueous solvent (∼3 V), to improve the electrode reactivity and to lower the parasitic currents, and thus to become suitable for analytical applications [[27], [28], [29], [30], [31]]. In particular, diamond-like carbon (DLC), boron-doped diamond (BDD) or amorphous carbon nitride (a-CNx) materials, all elaborated as thin films, have shown excellent electroanalytical performances, e.g. stripping analysis for heavy metals detection [[32], [33], [34], [35], [36]], or organic analytes detection [[37], [38], [39]] below micromolar concentrations. However, these electrodes require electrochemical pre-treatments to improve their surface reactivity and to increase the width of the potential window. For instance, cathodic electrochemical pre-treatments carried out in an acidic medium, substantially improved the a-CNx reactivity as compared to as-grown samples or to anodically pre-treated ones [[27], [28], [29]]. In the case of organic analytes detection, it has been shown that an anodic pre-treatment in alkaline medium may afford better peaks separation as it worsens the reactivity towards one component of the mixture, e.g. of ascorbic acid and dopamine [38,39] while a cathodic pre-treatment in a slightly acidic medium allows a fine detection of furosemide using the Square Wave Voltammetry (SWV) method [40].
Until now, most of the a-CNx layers were grown on doped silicon [27,30,31,[33], [34], [35], [36], [37],[41], [42], [43], [44], [45]], possibly as a consequence of their strong adhesion on this substrate that results probably from the formation of an intermediate layer of mixed composition [46]. Titanium was also shown to be a suitable substrate [28,43,45], as well as stainless steel [29,[38], [39], [40]], as both appear to be more appropriate for practical applications. For these three substrates, adhesion of a-CNx was strong enough even for low nitrogen content (x low) materials in which high internal mechanical stresses could have provoked delamination. Recently, ITO covered glass plates were also tested as substrates but the film properties were only investigated in air [46]. More recently, a further study of spectroscopic, microscopic and electrochemical characterization of several ITO/a-CNx electrodes was carried out both in air and in blocking electrode conditions, i.e. in the presence of the supporting electrolyte only [47].
In this applicative work, a-CN0.26 thin films were synthesized on ITO substrates for electrodes elaboration according to the protocol described in Ref. [47]. Then, a soft electrochemical pre-treatment was developed to activate these electrodes instead of hard pre-treatment which could provoke delamination. Here “soft pre-treatment”, as opposed to “hard pre-treatment”, means that the potential excursion during the pre-treatment remains within the potential window in the former case, while it extends beyond it and is accompanied with gas evolution in the latter one. Finally, a derivatization reaction protocol leading to the formation of the electroactive CBI was used to compare the performances of the electroanalytical peak obtained during the detection of PN and of four specific amino acids of PN sequence on three different carbon electrode materials: as-grown a-CNx, pre-treated a-CNx and GC.
Section snippets
Reagents and chemicals
NDA, potassium cyanide, boric acid, lysine and serine were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France). Methanol and sodium hydroxide were obtained from VWR (Fontenay-sous-Bois, France) and PN peptide (GPS1344), a synthetic peptide mimicking a 22-AA tryptic fragment of interest for the diagnosis of T49A ATTR mutation, was purchased from Genepep (Prades le Lez, France). Histidine and threonine were obtained from Alfa Aesar (Karlsruhe, Germany).
The labelling step was performed
Anodic electrochemical pre-treatment of a-CN0.26 electrodes
As mentioned in the introduction, a-CNx electrodes were generally electrochemically pre-treated in a 0.5 M H2SO4 acidic aqueous medium or with 0.1 M KOH alkaline aqueous medium [38]. These protocols have been tested but they were shown to be detrimental in our case as they often created some holes in the a-CN0.26 layer and sometimes its delamination from the glass/ITO wafer (see Fig. S1 in the supplementary information file). As a consequence, a soft anodic electrochemical pre-treatment was
Conclusion
Amorphous carbon nitride (a-CN0.26) thin films were deposited on conductive glass/ITO slides by using the DC magnetron sputtering technique, a graphite target and a plasma containing a 15% N2 partial pressure. The stoichiometry of the a-CN0.26 thin film was determined with the help of XPS measurements. It was demonstrated that a soft anodic electrochemical pre-treatment on glass/ITO/a-CN0.26 electrode performed in a KCl aqueous solution can drastically improve characteristics of the oxidation
Acknowledgements
This research was supported by the French ANR (Agence Nationale de la Recherche) in the context of the P2N “DIMIPOLE” project. The authors are also grateful to A. Pallandre for fruitful discussions.
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