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Non-Enzymatic Hydrolysis of Acetylthiocholine by Silver Nanoparticles Belen Hernandez, Santiago Sanchez-Cortes, Pascal Houzé, and Mahmoud Ghomi J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b09196 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 5, 2019

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The Journal of Physical Chemistry

Non-Enzymatic Hydrolysis of Acetylthiocholine by Silver Nanoparticles

Belén Hernández,a,b Santiago Sanchez-Cortes,c Pascal Houzé,d,e Mahmoud Ghomi*a,b

aLaboratoire

Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), UMR 7369, Université de Reims,

Faculté des Sciences, Moulin de la Housse, 51687 Reims Cedex 2, France bSorbonne

Paris Cité, Université Paris 13, Groupe de Biophysique Moléculaire, UFR Santé-Médecine-Biologie

Humaine, 74 Rue Marcel Cachin, 93017 Bobigny cedex, France cInstituto

dUnité

de Estructura de la Materia, IEM-CSIC, Serrano 121, 28006-Madrid, Spain

de Technologies Chimiques et Biologiques pour la Santé (UTCBS), CNRS UMR 8258-U1022, Faculté de

Pharmacie Paris Descartes, Université Paris Descartes, 75006 Paris, France eLaboratoire

de Biochimie, Hôpital Universitaire Necker-Enfants malades, Assistance Publique-Hôpitaux de Paris (AP-HP), 75015 Paris, France

*Corresponding author: M. Ghomi, Tel: +33-1-48388928, E-mail: [email protected]

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ABSTRACT: Since five decades, acetylthiocholine, an OS substituted synthetic analogue of the natural neurotransmitter acetylcholine, has become a key element in various assays used for probing the presence and activity of a highly important enzyme, i.e., acetylcholinesterase, in different biological media. A large number of these assays are now using plasmonic nanostructures because thiocholine, issued from the enzymatic hydrolysis of acetylthiocholine, is able to bind to the surface of both silver and gold nanoparticles by its both end groups (trimethylammonium and thiol groups). Herein, by following the characteristic thiocholine surface-enhanced Raman scattering markers, it is shown that a nonenzymatic hydrolysis of acethylthiocholine is also possible at the surface of silver nanoparticles, presumably because of (i) the silver reactivity toward sulfur atom, and especially to the chemical bonds in which it is involved, (ii) the conformational flexibility of acetylthiocholine for giving the adequate orientation to its scissile S-C bond with respect to the silver surface in order to facilitate its cleavage. Nevertheless, being less efficient than the enzymatic degradation, the non-enzymatic one appears to be incomplete and concentrationdependent, and occurs within the time interval generally required for sample preparation and data accumulation in acetylcholinesterase assays. Therefore, precaution should be used to avoid any distortion of the acquired data by selecting adequate protocols and substrate concentrations.


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INTRODUCTION The positively charged natural agent, acetylcholine (AcCh), is the first discovered neurotransmitter, contributing to the nervous signal propagation by binding to post-synaptic receptors of nicotinic and muscarinic types. Just after its action, AcCh should be degraded by one of the most dynamic enzyme, acetylcholinesterase (AcChE, Figure 1A). The inhibition of AcChE leads to AcCh accumulation, hyperstimulation of its receptors, and finally to disrupted neurotransmission.1-3 As concerns the background on the use of acethylthiocholine (AcSCh), the cyloxy OS substituted analogue of AcCh, as the substrate of AcChE (Figure 1B), we should refer to the original work of Ellman’s group in the mid years of the last century.4,5 A colorimetric assay was elaborated at that time, which was based on the disulfide bond breakdown of bis(pnitrophenyl) at basic pH (~8) by an aliphatic thiol anion, making appear a brilliant yellow colored compound, p-nitrophenyl anion in solution.4 Ellman further suggested to use thiocholine (SCh) (Figure 1E),5 resulting from the hydrolysis of AcSCh by AcChE, as a reactant to detect the presence of AcChE in different media, i.e., biological fluids, cell suspensions, tissues, etc. Because of their unique size- and shape-dependent optical properties, the continuously increasing progress in nanotechnology places plasmonic nanoparticles (NPs), principally made of noble elements (Ag and Au), at a high level of application. In particular, the plasmonic NPs aggregation, leading to the formation of narrow inter-particle spaces (named hot spots) produces a considerable amplification of electromagnetic radiation through the socalled surface plasmon resonance (SPR) effect.6-10 Surface-enhanced Raman scattering (SERS) technique takes particularly advantage of this property through the enhancement by several orders of magnitude the Raman signal arising from the adsorbed molecules on the surface of plasmonic NPs. This fact places SERS as a method of choice for measuring low 3 ACS Paragon Plus Environment


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traces of molecular species in solution with the aim of achieving the stage of single molecule detection.11 Various methods have been suggested to induce the aggregation of negatively charged gold and silver colloids in aqueous solution, for instance (i) surface charge screening of NPs by the addition of positive counterions,12 (ii) self-assembly of Ag NPs by aliphatic diamines,8 or by Lys- and Arg-rich cationic peptides.9-11,14-16 During the last decade, it has been shown that switching “on” and “off” the fluorescent and Raman signals from the analytes adsorbed on plasmonic NPs can become a powerful method to achieve biosensing/biorecognition-based assays.17-20 For instance, we can mention the strategies used for the detection of low traces of thrombin,14,18 or the N-arginine phosphorylation as a posttranslational process in proteins.15 In relation with the present work, plasmonic NPs have also been used to detect the presence/activity of AcChE.21-31 In fact, SCh molecules liberated by the enzymatic reaction (Figure 1) produces a strong aggregation of negatively charged Au- and AgNPs by binding to their surfaces through their both end groups, i.e., thiol32 and positively charged trimethylammonium. Such induced NPs aggregation have led to the development of sensitive AcChE assays based on “turn-on”/“turn-off” of enzymatic reaction leading to start/stop SCh production in the medium. A few applications in this field can be briefly summarized as follows: (i) the AcChE assay showing a red-to-gray color change of AuNPs;21 (ii) the amperometric biosensor made of biosilicificated AcChE-functionalized AuNPs, with a high sensitivity to small substrate (AcSCh) concentrations;22 (iii) the pathogen (enterovirus) detection assay based on the red-to-purple color change of the samples containing small size AuNPs;23 (iv) the dual colorimetric/fluorimetric protocol using rhodamine B-covered AuNPs for organophosphorous and carbamate pesticides detection;24 (v) the fluorescence turn-on of gold nanoclusters-Cu2+ ensemble in the presence of SCh used to probe AcCh activity;25 and (vi) the organophosphorous pesticide detection by graphene oxide-Au nanocomposites-based


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AcCh biosensor.26 Furthermore, In the beginning of the present decade, after a series of preliminary investigations27,28 Liron’s group suggested to use the middle wavenumber SERS markers resulting from the binding of SCh to silver surfaces in turn-on AcChE assays.29 Inversely, they have shown that the absence of the corresponding SERS markers can serve in a turn-off assay, revealing the enzymatic inhibition by a reference insecticide, paraoxon.29 More recently, high wavenumber SERS markers from metal carbonyl-AuNPs conjugates, were identified as a probe to detect organophosphorous pesticides.30 SCh SERS markers on AgNPs

were

recently used as

indicators

of a

less

reputed enzyme activity,

butyrylcholinesterase.31 In the course of our preparatory turn-on/turn-off SERS experiments related to the activation/inhibition of AcChE enzymatic reaction, we observed that the substrate AcSCh can also be hydrolyzed non-enzymatically when it is simply found in contact with AgNPs. It should be mentioned that silver surfaces are known for their high reactivity toward sulfur atom containing compounds.32,33 It is also worth emphasizing that two previous investigations had reported on the non-enzymatic hydrolysis of the natural neurotransmitter (AcCh) in bulk at basic pH (>10) using classical Raman markers,34 as well as on the concentration-dependent disulfide bond breakdown of cystine (Cys-Cys dimer stabilized by a S-S bridge) by SERS markers on AgNPs.35

MATERIAL AND METHODS Sample Preparation. Acetylcholinesterase (AcChE) from electrophorus electricus (electric eel, lot C3389), as well as powder samples of acetylthiocholine (AcSCh) iodide and acetic acid were provided by Sigma-Aldrich (Saint-Quentin-Fallavier, France). Thiocholine iodide (SCh) was from Boc Sciences (Shirley, USA). To prepare solution samples of AcChE, we have strictly followed the protocol previously reported by Liron et al.29 Stock solution of 5 ACS Paragon Plus Environment


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AcSCh and SCh at 100 mM, i.e., ~29 mg/mL (AcSCh) and ~25 mg/ml (SCh), were prepared by dissolving each of them in water taken from a Millipore filtration system. Sodium acetate solution at pH 7 was obtained by neutralizing acetic acid (1N) by NaOH (1N). All these samples were used for recording bulk (solution) Raman data. Further dilution of AcSCh and SCh samples were performed successively in water, and then in colloidal solution to reach the concentrations needed for SERS measurements. In certain experiments, to increase the ionic strength of solution samples, sodium chloride, purchased from Merck (Fontenay-sous-Bois, France), was added (used at 15 mM concentration, i.e., 0.9 mg/mL).

Silver Nanoparticles. Colloidal suspension was elaborated by adding 300 mL of a sodium hydroxide solution (1 M) to 90 mL of a 6 x 10-2 M hydroxylamine hydrochloride solution. Then 10 mL of a 1.11 x 10-3 silver nitrate aqueous solution were added dropwise to the mixture under vigorous stirring. The solution containing silver colloids turned to brown; it was aged one week at 4 °C before use. This preparation protocol was shown to give rise to polydisperse silver colloids, as described previously.9,10 The measured pH of colloidal solution at preparation was between 5.5 and 6.

Experimental Setup. Bulk Raman and Surface-enhanced Raman scattering (SERS) data were collected on an InVia spectrometer (Renishaw Ibérica S.A., Gavá, Spain) equipped with an electrically cooled CCD camera. Samples were excited by means of the 442 nm and 532 nm lines of diode lasers for recording bulk Raman and SERS data, respectively. The spectral resolution was ~2 cm-1. The total acquisition time was ~300 s in bulk Raman and ~30 s in SERS experiments.

Post-Record Spectroscopic Data Treatment. Buffer subtraction and smoothing of the observed spectra was performed using the GRAMS/AI Z.00 package (Thermo Galactic, Waltham, MA, USA). Final presentation of Raman spectra was done by means of SigmaPlot package 6.10 (SPSS Inc., Chicago, IL, USA). 6 ACS Paragon Plus Environment

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Quantum Mechanical Calculations. Energetic and geometrical data of AcSCh, SCh, acetic acid and acetate ion were estimated by the density functional theory (DFT) approach,36 using the hybrid B3LYP functional.37,38 Polarized triple zeta basis sets, commonly referred to as 6-311++G(d,p), were used. The hydration effect was considered by a purely implicit model, which consists in placing the solute in a polarizable continuum medium (PCM),39 the relative permittivity of which was supposed to be that of water (r=78.39). To check the reliability of the geometrical data, harmonic vibrational calculations were performed, allowing to confirm the correspondence of an optimized geometry to a local minimum through the absence of any imaginary frequency. All quantum mechanical calculations were carried out using the Gaussian09 package.40

RESULTS AND DISCUSSION Bulk Raman Spectra of AcSCh and Its Degradation Products. Raman spectra observed from the aqueous samples containing AcSCh, SCh and acetate in the middle wavenumber region are displayed in Figure 2A-C. It is to be emphasized that acetic acid (pKa=4.7) may fluctuate between its protonated (Figure 1C) and deprotonated (Figure 1D) forms, within the pH range of our experiments (i.e., 5

Non-Enzymatic Hydrolysis of Acetylthiocholine by ... - ACS Publications

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