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Peroxidase-like activity of gold nanoparticles stabilized by hyperbranched polyglycidol derivatives over a wide pH range

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Nanotechnology Nanotechnology 26 (2015) 495101 (15pp)

doi:10.1088/0957-4484/26/49/495101

Peroxidase-like activity of gold nanoparticles stabilized by hyperbranched polyglycidol derivatives over a wide pH range Marcin Drozd1, Mariusz Pietrzak1, Paweł Parzuchowski2, Marta Mazurkiewicz-Pawlicka3 and Elżbieta Malinowska1 1

Department of Microbioanalytics, Institute of Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland 2 Chair of Polymer Chemistry and Technology, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland 3 Materials Design Division, Faculty of Materials Science, Warsaw University of Technology, Wołoska 141, 02-524 Warsaw, Poland E-mail: [email protected] Received 29 July 2015, revised 25 September 2015 Accepted for publication 15 October 2015 Published 16 November 2015 Abstract

The aim of this work was to carry out comparative studies on the peroxidase-like activity of gold nanoparticles (AuNPs) stabilized with low molecular weight hyperbranched polyglycidol (HBPG-OH) and its derivative modified with maleic acid residues (HBPG-COOH). The influence of the stabilizer to gold precursor ratio on the size and morphology of nanoparticles obtained was checked, and prepared nanoparticles were characterized by means of transmission electron microscopy and UV-Vis spectroscopy. The results indicated the divergent effect of increasing the concentration of stabilizers (HBPG-OH or HBPG-COOH) on the size of the nanostructures obtained. The gold nanoparticles obtained were characterized as having intrinsic peroxidase-like activity and the mechanism of catalysis in acidic and alkaline mediums was consistent with the standard Michaelis–Menten kinetics, revealing a strong affinity of AuNPs with 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 3, 3′, 5, 5′tetramethylbenzidine (TMB), and significantly lower affinity towards phenol. By comparing the kinetic parameters, a negligible effect of polymeric ligand charge on activity against various types of substrates (anionic or cationic) was indicated. The superiority of steric stabilization via the application of tested low-weight hyperbranched polymers over typical stabilizers in preventing salt-induced aggregation and maintaining high catalytic activity in time was proved. The applied hyperbranched stabilizers provide a good tool for manufacturing gold-based nanozymes, which are highly stable and active over a wide pH range. S Online supplementary data available from stacks.iop.org/NANO/26/495101/mmedia Keywords: gold nanoparticles, nanozymes, heterogeneous catalysis, peroxidase mimetics, hyperbranched polyglycidol (Some figures may appear in colour only in the online journal) 1. Introduction

the point of view of industry or environmental and analytical chemistry. Despite the unsurpassed substrate specificity and high activity offered by natural enzymes, their use is limited due to their protein origin, which is reflected by considerable pH and temperature sensitivity, inhibitor dependent activity

The development of catalysts, which are characterized as having a long life, high stability, high efficiency and appropriate selectivity towards chosen substrates, is crucial from 0957-4484/15/495101+15$33.00

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© 2015 IOP Publishing Ltd Printed in the UK

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stability, but at the expense of large dimensions and a complex preparation procedure [28, 29]. One of the most typical approaches is based on electrostatic repulsion between AuNPs whose surfaces were modified accordingly. However, in this case a major drawback is strong pH and ionic strength dispersion dependence. Interesting prospects are offered by the application of multidentate stabilization with the usage of linear and low molecular weight hyperbranched polymers (e.g. polyethyleneimine) [30–33] or dendritic structures (e.g. poly(amidoamine)) [34–36]. Besides their stabilizing role, hyperbranched polymers additionally provide a great number of functional groups, which may allow for further functionalization of AuNPs via covalent chemistry or electrostatic interactions [7, 10]. Among the most popular strategies of preventing AuNPs from aggregation, citrate capping, utilization of thiol or surfactant self-assembly may be listed [23, 37, 38]. These approaches have, however, a major drawback, which is a substantial deterioration in the catalytic activity of nanoparticles due to the formation of a dense monolayer which occupies the catalytically active sites on the surface [39]. So, to elaborate the efficient and stable nanocatalyst, a trade-off between maintaining the high activity and surface stability should be taken into account. To the best of our knowledge, the relationship between stabilization by means of hyperbranched polyethers and the HRP-like catalytic activity of hereby obtained AuNPs has not yet been thoroughly investigated, although their use, in our opinion, promises nanoparticles with good and interesting properties. Hyperbranched polyglycidol (HBPG) has been proven to be a biocompatible polyether, which can be synthesized via simple, one-pot ring opening multibranching polymerization from commercially available glycidol [40]. Due to the globular topology of HBPG, hydroxyl groups may act as multidentate ligands exhibiting an affinity to the gold surface, at the same time guaranteeing the excellent solubility and stability of AuNPs. Its functional groups’ abundance provides a very convenient possibility for further functionalization in bioanalytical applications [30, 41]. Moreover, hyperbranched polyglycidol prevents protein adsorption in a similar way to poly(ethylene glycol), so the obtained nanocrystals should characterize themselves with a resistance to unspecific protein adsorption, which may be beneficial from the point of view of applications, for instance, in a role of catalytic labels in immunoassays [42–44]. The most widely described type of HBPG-modified nanoparticles are metal oxide-based NPs (e.g. Fe3O4, MnO), which have superparamagnetic properties that enable them to be used for in vivo magnetic resonance imaging [45–47]. Examples of nanomaterials stabilized by HBPG derivatives, which act as catalysts, remain limited to only a few reports [41, 48]. Herein, we described a simple methodology to prepare gold nanoparticles stabilized with hyperbranched polyglycidol (HBPG-OH) and its carboxylated derivative (HBPGCOOH), which possess intrinsic peroxidase-mimicking activity. The relevance of stabilizer type and concentration applied during synthesis, which affect the morphology and catalytic activity of AuNPs, was emphasized and optimal

and a high cost of acquisition, purification and preparation [1]. Therefore, the search for catalysts of high activity which can mimic enzyme activity and overcome the limitations listed above is still an issue. The most intensive research in this field focuses on oxidoreductase mimetics development. Various materials, like molecular imprinted polymers [2], DNA-zymes [3], hemin [4], manganese porphyrins [5] and a wide range of nanomaterials, so-called nanozymes, have been proposed as catalysts, which offer activities similar to those of peroxidases, oxidases and catalases [6]. Oxidation by means of hydrogen peroxide catalyzed by horseradish peroxidase (HRP) or its mimetics is a useful tool in analytical and clinical chemistry, especially in enzymelinked immunosorbent assays and immunosensors with an indirect detection mode. In their case, the oxidation of chromogenic, fluorogenic or electroactive substrates in the presence of H2O2 by catalytic label is typically applied as a source of analytical signal. It requires the application of a label retaining the stable and high activity, even in samples of complex composition. The drawbacks exhibited by enzymes may be eliminated by replacing them with more robust nanocatalysts characterized by increased resistance to inactivation in harsh pH conditions and high activity in the presence of heavy metals or other enzyme inhibitors e.g. azides. Thanks to the unique properties offered by nanozymes, the application of nanozyme-receptor conjugates may contribute to the extension of the applicability of bioassays [7, 8]. Within a large variety of nanozymes, which exhibit peroxidase-like activity, such as noble metal (Ag, Au, Pt, Pd) nanostructures as well as bimetallic structures of various shapes [6–12], metal oxide nanoparticles (e.g. MnO2, V2O5, CuO, Fe3O4, Co3O4) [13–17] and carbon-based nanomaterials like graphene oxide sheets or carbon nanotubes [18, 19], gold nanoparticles seem to be particularly interesting due to their well-known surface chemistry, the simplicity of their preparation and their facility to function with a variety of biomolecules [20]. In contrast to the chemical inertness of bulk gold surfaces, the phenomenon of catalytic activity is strongly related to the nanometric size, unique morphology and the surface chemistry of gold nanoparticles [21]. AuNPs are characterized by a high surface to volume ratio and substantial surface curvature, resulting in the presence of a large number of dangling, highly energetic atoms, which can act as active sites of heterogeneous catalysts [22]. Numerous works in the literature indicate that the activity of AuNPs is size dependent. For instance, nanoparticles of about 13–15 nm reveal the highest activity per area towards the reduction of various substrates like p-nitrophenolate or eosin, which was reported by Fenger and Sau [23, 24]. Equally important in the context of AuNPs’ catalytic activity is maintaining stability and good dispersion, which are necessary to enable the access of substrates to near proximity of superficial gold atoms. Based on reports in the literature, a few general approaches to retaining the dispersion of AuNPs may be listed. The deposition of nanostructures on special templates, e.g. in composites with polymers [25], inorganic frameworks [26, 27], as well as the application of AuNP decorated graphene nanosheets or carbon nanotubes provide their good 2

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NMR System spectrophotometer (500 MHz) and D2O as a solvent and tetramethylsilane as an internal standard. Comparative studies of nanozymes activity were performed on a Sunrise Magellan microplate reader (TECAN Instruments) at a temperature of 25 °C with the use of disposable, polystyrene 96-well plates. Zeta-potential measurements of AuNPs were obtained with the use of a Zetasizer Nano ZS (Malvern) with a He-Ne laser (633 nm) at a temperature of 25 °C, with the use of quartz cuvettes.

ratios of stabilizer to gold precursor determined. The essential objective of this study was to examine the influence of the surface stabilization strategy on the horseradish peroxidaselike activity of prepared AuNPs towards both anionic (ABTS, phenolate) and cationic (TMB) substrates. According to our experience the loose, globular structure of HBPG on the AuNP’s surface attenuate their catalytic activity to a lesser extent than dense monolayers e.g. thiol-based ones. To elucidate the effect of polymeric surface modifiers on HRP-like activity, comparative studies of kinetic parameters based on the Michaelis–Menten model were conducted.

2.3. General procedures 2.3.1. HBPG-OH modification. To a mixture of HBPG-OH

2. Experiment

(5 g, 700 mmol of hydroxyl units) and 0.9 ml of triethylamine (6.46 mmol), placed in a round-bottomed flask equipped with a reflux condenser, a thermometer and a magnetic stirrer, a solution of 10 g of maleic anhydride (102 mmol) in anhydrous THF was added dropwise over 30 min. The resulting reaction mixture was stirred at reflux for 18 h. Then, the solvent was distilled off at atmospheric pressure. The viscous oil obtained was then heated at 80 °C for an additional 1 h. The crude product was cooled down, transferred into dialysis tubing (MWCO=2000) and extensively dialyzed against distilled water until no sign of maleic acid (λmax=210 nm) in the UV-spectrum of the obtained dialysate was observed. Residuals of triethylammonium counterions in the crude product were removed after 8 h of dialysis against 1 M HCl. After dialysis, water was evaporated in vacuo. As a final result, a yellowish, viscous product of hyperbranched polyglycidol modification with maleic acid was obtained. The degree of the hydroxyl groups’ functionalization was estimated to be about 40% (by means of 1H NMR—see figure S1 in Supporting Information).

2.1. Materials

Gold(III) chloride trihydrate, sodium borohydride, triethylamine, 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 4-aminoantipyrine (4-AAP), phenol (PhOH), 3, 3′, 5, 5′-tetramethylbenzidine (TMB), N-(morpholino)ethanesulfonic acid (MES), sodium citrate trihydrate, maleic anhydride, 3-mercaptopropionic acid (3MPA), carbon disulfide, 2-tris(hydroxymethyl)aminomethane (Tris-base), 2-aminoacetic acid and horseradish peroxidase, type VI, from Amoracia rusticana (HRP) were purchased from Sigma–Aldrich and used as received. Hydrogen peroxide (30 %), nitric acid (68.5 %), hydrochloric acid (37 %) and tetrahydrofuran were purchased from POCh (Poland). Hyperbranched polyglycidol (HBPG-OH) of average molecular weight about 3.2 kDa was synthesized via ring-opening multibranching polymerization in the Department of Polymer Chemistry and Technology, Faculty of Chemistry, Warsaw University of Technology [40]. Dialysis tubing made of cellulose membrane (MWCO=2000 Da) was purchased from Sigma–Aldrich. All glassware used to synthesise AuNPs was rinsed with aqua regia (a mixture of concentrated hydrochloric acid/nitric acid 3:1 (v/v)) prior to use. All solutions used for nanoparticle preparation were filtered with the use of syringe filters (0.45 μm, Corning). All nanoparticle solutions were stored in darkness at 4 °C. The following buffer solutions for kinetic studies were prepared: borate buffer (0.2 M, pH 8.5), phosphate buffer (0.2 M, pH 3.0 and 4.5), Tris-HCl buffer (0.2 M, pH 8.5), acetate buffer (0.2 M, pH 4.0), MES buffer (0.2 M, pH 6.0).

2.3.2. Preparation of unmodified and polymer-stabilized AuNPs. 5 ml of HBPG-OH or HBPG-COOH aqueous

solutions (set of solutions in concentrations ranging from 0.025 to 20 mg ml−1) or 5 ml of deionized water was added to a glassy vial followed by the addition of a freshly prepared 10 mM aqueous solution of HAuCl4 (0.625 ml). The mixture was sealed and magnetically stirred for 10 min. Subsequently, a freshly prepared 50 mM aqueous solution of NaBH4 (0.625 ml) was rapidly injected to the vigorously stirred solution in the dark. The reduction of gold(III) and the AuNPs’ formation was observed as a color change. Then, AuNP solutions were stored in a refrigerator and magnetically stirred to finalize the nanocrystals’ growth for a week, until no visible changes in the UV-Vis spectra were recorded. Independently of the modifier type and concentration used (except for the highest concentration of HBPG-COOH), the prepared AuNPs stored in the dark at 4 °C revealed high stability (with no signs of precipitation or changes in absorption spectrum) for at least three months. For comparative purposes, nonstabilized nanoparticles werealso

2.2. Instrumentation

The average diameter and morphology of synthesized nanoparticles were estimated by transmission electron microscopy (TEM) using a JEOL S5500 electron microscope operating at an accelerating voltage of 30 kV. TEM samples were prepared by dripping diluted suspension in acetone on a carboncoated grid. The UV-Vis spectra of gold nanoparticles were recorded using a Perkin–Elmer Lambda 25 spectrophotometer and quartz microcuvettes with 1 cm pathlength (Hellma Analytics). 1H NMR spectra were recorded using a Varian 3

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prepared in the same manner, with the use of distilled water instead of the polymer solution.

equations: K 1 1 1 = M ⋅ + v0 vmax [S ] Vmax

Synthesis of citrate-stabilized AuNPs. Citratestabilized AuNPs were prepared according to the Turkevich–Frens method [37]. (A more detailed description of AuNP synthesis can be found in Supporting Information.)

k cat =

vmax [E ]

2.3.3.

The molar extinctions coefficients of products at the applied wavelengths were as follows: εquinoneimine = 13 300 M−1 cm−1, εTMB(ox) = 39 000 M−1 cm−1 εABTS −1 cm−1 [50, 51]. The optical pathlength (ox) = 36 000 M was normalized for a total volume of 200 μL for each substrate. The molar concentration of catalysts was estimated using calculations based on TEM micrographs, by assuming sphericity of nanoparticles and the complete reduction of the precursor with the use of the mean diameter.

2.3.4. Polymer, thiol and dithiocarbamate modification of nanoparticles. The influence of the polymers used and the

sulfur-containing modifiers selected on the stability and catalytic activity of nanoparticles was examined. For this purpose, bare and citrate-capped AuNP solutions (0.5 ml, [Au]=1 mM) 0.5 ml of various surface-active ligands (3mercaptopropionic acid, glycine dithiocarbamate) solutions in water (C=100 μM) or hyperbranched polymers HBPG-OH and HBPG-COOH (C=1 mg ml−1) were added and solutions vortexed. The obtained nanoparticles were incubated in the dark for four days to facilitate ligand exchange. Glycine dithiocarbamate was prepared by mixing equal volumes of 1 mM of carbon disulfide and 1 mM of glycine in a 1 mM phosphate buffer of pH 10.5 and finally diluted with water. The occurrence of DTC derivative formation was confirmed via UV spectroscopy.

3. Results and discussion 3.1. HBPG-OH functionalization with maleic anhydride

Polyglycidol was modified using maleic anhydride with the aim of introducing carboxyl-terminated residuals to the polymer structure. The introduction of terminal maleate moieties allows us to obtain a new polymer of polyionic character under studied conditions (pKa1 of unmodified maleic acid is 1.9), whereas hydroxyl groups of glycerol remain uncharged (pKa=14.3). A schematic illustration of the HBPG-OH modification reaction with the use of maleic anhydride is shown in figure 1. Acylation of HBPG-OH was confirmed by 1H NMR analysis (figure S1). In the spectrum of HBPG-COOH the characteristic signals at 6.40 to 6.56 ppm appeared, which are assigned to be of uneven vinyl protons of maleic acid attached to the polyglycidol core. As a result of acylation, a significant part of the hydroxyl group was modified with the moieties capable of relatively easy functionalization by means of wellknown carbodiimide chemistry, which is compatible with the vast majority of bioreceptors [8, 13]. The carboxylic moietiesintroduced also allow for the application of noncovalent, electrostatic interactions with target receptors [7, 10]. The efficiency of HBPG-COOH purification was confirmed by UV-absorption spectroscopy (see figure S2, Supporting Information). The degree of modification was estimated to be 40% (an average of 18 maleic moieties on one HBPG-OH molecule) on the basis of 1H NMR analysis.

2.3.5. Studies of catalytic activity. Studies of AuNPs’

catalytic activity in the oxidation of chromogenic substrates by hydrogen peroxide were carried out with several buffer solutions: 0.2 M borate buffer of pH 8.5 for PhOH/4-AAP assay; 0.2 M phosphate buffer of pH 4.5 for TMB assay; 0.2 M phosphate buffer of pH 3.0 for ABTS assay. Catalyst concentration, expressed as the concentration of gold precursor, was constant (100 μM) in all experiments. In the case of horseradish peroxidase-based experiments, concentrations of enzyme and buffer solutions were as follows: 0.5 μg ml−1 HRP, 0.2 M Tris-HCl of pH 8.5 for PhOH/4-AAP assay; 0.02 μg ml−1 HRP, 0.2 M acetate buffer of pH 4.0 for TMB assay; 0.05 μg ml−1 HRP, 0.2 M acetate buffer pH of 4.0 for ABTS assay. Molar concentrations of HRP in solutions were determined spectrophotometrically at 404 nm using the molar absorptivity value of 102 000 mol−1 dm3 cm−1 [49]. The total reaction volume of 150 μl consisted of 500 mM H2O2 and chromogenic substrates as follows: (PhOH/4-AAP) 100 mM of phenol, 2.5 mM 4-AAP, (TMB) 1.0 mM of TMB, (ABTS) 1.0 mM of ABTS, unless otherwise stated. Michaelis–Menten catalytic parameters were calculated from apparent steady-state kinetic curves with the use of a double reciprocal plot, where v0 is the initial velocity (computed as change of substrate concentration during first 150 s of reaction, from the slope of the nearly-linear kinetic curve), KM is Michaelis constant, vmax is the maximal reaction velocity, [S] is substrate concentration, [E] is catalyst concentration and kcat is turnover number according to

3.2. Characterization of the morphology of AuNPs dependent on stabilizer type and concentration

Polymer-stabilized gold nanoparticles (AuNPs@HBPG-OH and AuNPs@HBPG-COOH) were obtained via direct reduction of tetrachloroaurate ions by sodium borohydride. In parallel preparations two sets of nanoparticles from solutions of fixed concentrations of reducing agent and gold chloride, and varied concentrations of polymeric stabilizers were prepared. As observed, the rate of gold nanoparticle synthesis was dependent on the type of stabilizer. The reduction of the tetrachloroaurate ions, resulting in the formation of zerovalent 4

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Figure 1. Scheme of hyperbranched polyglycidol modification with maleic anhydride.

gold nanoparticles in the presence of HBPG-OH, occurred quickly, which manifested itself in the immediate appearance of a stable, red-brownish (tea-like) solution of well-dispersed nanoparticles. The synthesis of stable HBPG-COOH-coated AuNPs was much slower. The addition of sodium borohydride resulted in the appearance of purple mixtures, which gradually turned ruby red. The rate of the observed process was concentration-dependent. The formation of AuNPs was substantially faster for HBPG-COOH concentrations above 0.4 mg ml−1. Finally, after several days a set of colloidalstable solutions was obtained (no visible changes in absorption spectra were observed for several weeks). Hyperbranched, glycidol-based polyethers, containing carboxyl and hydroxyl (HBPG-COOH) or only hydroxyl (HBPG-OH) functional groups substantially influence the diameters and morphology of the obtained AuNPs (see figure 2). As resulted from TEM micrograph analysis, the size of the nanostructures was controlled by the concentration ratio of polymer to gold precursor (see table 1). Increasing the HBPG-OH concentration in solutions resulted in a monotonous decrease in the size of the obtained nanostructures, down to 2.6 nm in the case of polymer concentration, which amounted to 16 mg ml−1. The influence of HBPG-COOH polymer concentration on the dimensions of the nanostructures was substantially different. In the range of low polymer concentrations, up to 80 μg ml−1, while increasing the concentration the diameter of the nanostructures increased. Interestingly, for concentrations above 0.2 mg ml−1, nanoparticles of smaller diameter and better dispersion (with minimum mean diameter of 4.7 nm for 0.8 mg ml−1 of HBPG-COOH) were formed. Despite differences in the concentrations of obtained nanoparticles, an increment of the polymer concentration caused an increase in the modifier concentration available per AuNP surface area unit. To evaluate the effect of the presence ofligands on the properties of AuNPs, bare nanoparticles, which exhibited sufficient stability for comparative purposes, were also

prepared. Molar concentrations of prepared AuNPs were calculated assuming a perfect sphericity of the nanostructures as well as a complete reduction of a gold precursor. The possible presence of residual, unreacted tetrachloroaurate in the solutions was tested indirectly by means of its oxidizing properties.39 The lack of visible changes in ABTS oxidation in the absence of external oxidant after the addition of AuNP samples confirmed a quantitative reduction. Zeta potential values of −8,0±2.8 mV for AuNPs@HBPG-OH4000, −14,2±0.6 mV for AuNPs@HBPG-COOH4000 and −7.8±0.7 mV for bare AuNPs were obtained, respectively. Despite relatively low potential zeta values, polymer-stabilized AuNPs displayed high colloidal stability due to steric dispersion. Applied polymers contributed to the stabilization of the nanoparticles, acting as a steric hindrance, which very effectively prevents the formation of aggregates. Such a feature is especially desirable when taking the catalytic properties of nanoparticles into account. This study revealed very good stability of AuNPs stabilized with polyglycidol of low molecular weight, contrary to reports in the literature, where only hyperbranched polyglycidols with high (over 20 kDa) molecular weight were considered as good stabilizers of nanoparticles [30]. Due to the relatively small size of the applied polyglycidol molecules, compared to the diameter of the obtained nanoparticles, the architecture of the separate nanocrystals covered by a polymeric shell, rather than of AuNPs encapsulated in a globular polymer, as in the case of dendrimers of high generation, is proposed in our case [34]. According to reports in the literature, the position and shape of the localized surface plasmon resonance (LSPR) band may act as a marker of gold nanoparticles’ size, their dispersion and their interaction with ligands [52, 53]. For the small AuNPs, stabilized by relatively highly concentrated HBPG-OH, (over 0.8 mg ml−1) the recorded plasmon resonance band was significantly interrupted (figure 3). In turn, the bigger HBPG-COOH-stabilized AuNPs reflected as slight 5

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Figure 2. Representative TEM micrographs of selected AuNPs stabilized with the use of various types and concentrations of hyperbranched

polyglycidol-based stabilizers: (a) bare AuNPs, (b)–(d) AuNPs stabilized with 0.8 mg ml−1 (AuNPs@HBPG-OH800), 4.0 mg ml−1 (AuNPs@HBPG-OH4000) and 16.0 mg ml−1 HBPG-OH (AuNPs@HBPG-OH16000), respectively, (e)–(g) AuNPs stabilized with 0.8 mg ml−1 (AuNPs@HBPG-COOH800), 4.0 mg ml−1 (AuNPs@HBPG-COOH4000) and 16.0 mg ml−1 of HBPG-COOH (AuNPs@HBPG-OH16000), respectively.

Table 1. Values of mean diameters of prepared AuNPs calculated from representative TEM images (at least 150 particles were taken into

account). average diameter of AuNPs [nm]

Cpolymer −1

16.0 mg ml (AuNPs@HBPG16000) 4.0 mg ml−1 (AuNPs@HBPG4000) 0.8 mg ml−1 (AuNPs@HBPG800) 80 μg ml−1 (AuNPs@HBPG80) 20 μg ml−1 (AuNPs@HBPG20) unmodified (bare AuNPs)

6

HBPG-OH

HBPG-COOH

2.6±0.6 3.0±0.6 5.0±0.8 not calculated not calculated 8.1±1.3

8.3±2.2 5.8±1.3 4.7±1.4 6.4±1.1 5.2±1.0

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number of groups involved in the interaction are sufficient to ensure the steric stabilization of nanostructures [31, 44]. According to our observations, highly hydrophilic, uncharged polyglycidol promotes rapid nucleation and prevents aggregation of seeds at early stages of AuNP formation. Rapid injection results in quick nucleation and nanoparticle formation. The presence of a higher concentration of flexible and highly hydrophilic polymer also alters the rheology of the synthesis medium, which may alsobe of great importance for the the reduction reaction. The presence of HBPG-COOH in precursor solutions causes their acidification, which may substantially affect the nucleation and growth process, resulting in mean diameter increase as compared with other strategies of AuNP preparations [54–56]. Rapid nucleation caused by the addition of a strong reducing agent (NaBH4) in acidic medium of low HBPG-COOH concentration, in combination with its relatively rigid structure, resulted in a weaker and slower adsorption, thus relatively large gold nanoparticles were formed. An increase in HBPG-COOH concentration made the stabilization more effective and resulted in the preparation of the smallest HBPGCOOH-stabilized AuNPs for about 0.8 mg ml−1 of this polymer. A further increase in stabilizer concentration did not affect the stabilization efficiency, but it only caused further acidification of the nucleation environment, which contributed to the formation of bigger nanoparticles. A similar trend concerning the increase of AuNP size when increasing the concentration of chitosan, which was used as a stabilizer and as a reducing agent, was also reported by Huang [57]. The UV-Vis spectra analysisconducted broadly confirms conclusions regarding AuNP size changes based on TEM micrographs, and it can be successfully used as a cheap and handy tool for the fast, comparative characterization of gold nanostructures. Research carried out revealed that an appropriate choice of gold(III) precursor and hyperbranched polymer concentration ratio enables the possibility of AuNP size tailoring.

Figure 3. Influence of modifier concentrations on the UV/Vis

absorption spectra of (a) HBPG-OH, (b) HBPG-COOH-stabilized AuNPs. Absorption spectra were normalized at 400 nm.

red shift and a more explicit LSPR maximum. Illustrations of the LSPR band position and ASPR to A450nm ratio, dependent on the concentration of the modifier, which are often used for comparative studies of AuNP sizes are depicted in S3 (Supporting Information). The blue shift of the LSPR band and the monotonic decrease of the ASPR to A450 ratio with the increase in the HBPG-OH concentration indicates the decreasing diameter of the obtained nanostructures. Due to the small size of the nanostructures formed (smaller than 5 nm) and the presence of the polymer preventing them from aggregation, oscillations resulting in the emergence of the characteristic resonance band are substantially suppressed by the inelastic scattering of conduction electrons. Interestingly, application of HBPG–COOH as a stabilizing agent resulted in two characteristic minima of particles’ size. The lowest wavelength of the LSPR peak position and the lowest ASPR to A450 ratio were obtained for HBPG-COOH concentrations, which amounted to 20 μg ml−1 and 0.8 mg ml−1. LSPR peak broadening in the case of AuNPs@HBPGCOOH16000 confirms the lower stability of these nanostructures under applied conditions. Our experiments revealed that the stabilizing capability of HBPG-COOH is weaker than that of HBPG-OH, which manifests as a slower kinetics of nanostructure formation, as well as their final larger size. Differences in the physical properties of the obtained nanostructures stabilized with various polymers may result from their different chemical properties, revealing a diverse affinity of uncharged hydroxyl groups and anionic carboxylate to gold precursor and zerovalent gold surface. Weak, noncovalent interactions of free electron pairs (oxygen atoms of hydroxyl groups as well as polyether backbone) with nanostructures in connection with polymer flexibility and a large

3.3. The influence of the size of the HBPG-stabilized nanoparticles on their catalytic activity

Severalreports in the literature show that the catalytic activity of a nanomaterial is very strongly dependent on its specific surface area. Therefore, the use of highly concentrated solutions of nanoparticles of small diameters is preferred, which maximizes the availability of catalytically active sites. Our studies of the catalytic activity of obtained AuNPs were based on the oxidation of the phenolate anion in slightly basic pH=8.5, and the subsequent coupling of the obtained quinone-derivative with 4-aminoantypirine and the oxidation of TMB in acidic pH=4.5, by means of hydrogen peroxide. Comparative studies revealed that a NP’s morphology is significantly dependent on its activity. As depicted in figure 4, the relative reaction rate, expressed as the slope of the initial, linear part of the kinetic curve varies according to the polymer to gold precursor ratio. The observed effects were dependent on the size of the NPs with no substantial interference from used polymers, which was confirmed in separate experiments. 7

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Figure 4. Comparison of the peroxidase-like activity of AuNPs prepared with the use of a fixed concentration of gold precursor and various

concentrations of: (a) HBPG-OH, (b) HBPG-COOH modifier towards phenolate (red points) and TMB (blue points) oxidation.

The values of the catalytic reaction rates in the presence of both sets of nanocatalysts, against TMB and PhOH/4-AAP oxidation, are summarized in table S1 (supplementary information). The activity changes presented in figure 4 show a similar trend to the size dependence as a function of the polymer amount described above, especially in the case of the PhOH/ 4-AAP system. TMB oxidation was less sensitive to the size of the applied AuNPs. This observation is consistent with several reports dealing with the activity of nanoparticles of a high surface to volume ratio [7, 23, 24]. In general, it is believed that nanoparticles of small diameter have the best performance, however, the impact of the molar concentration of the catalyst is often underestimated. In our opinion, taking both parameters in question into account is a better indicator of the activity of nanostructures. For a more comprehensive comparison, the relative activities of the selected nanoparticles stabilized with hyperbranched polymers towards phenolate and TMB oxidation were calculated. Activity values were expressed per gold unit area, assuming their monodispersity and spherical shape. The obtained results confirm that, with increasing the diameter of the prepared nanoparticles (2.6–8.3 nm), the relative activity increases. For example, the increase in diameter of the nanoparticles from 5.8 nm to 8.3 nm (induced by increasing the HBPG-COOH concentration from 0.8 to 16.0 mg ml−1) brought a relative increase in activity per unit area at 28.1 % and 15.3 % against TMB and PhOH/4-AAP, respectively. The diameter decrease of HBPG-OH-stabilized AuNPs from 5.0 nm to 2.6 nm (due to increasing the concentration of the modifier) resulted in a decrease in the relative activity per area unit, which amounted to 27.8 and 14.5 % against TMB and PhOH/4-AAP. Our observations lead to the surprising conclusion that, in the range of several nanometers, greater activity per surface unit is exhibited by larger particles. This

phenomenon is similar to the one observed by Fenger and Sau, where nonmonotonic dependence of activity in dye reduction reactions against particle size (with maximum at diameter of 13–15 nm) was also observed [23, 24]. However, the cause of the noticeable loss of peroxidase-like activity per unit area of the smaller nanoparticles remains unclear.

3.4. Influence of surface ligand on catalytic activity of AuNPs

To demonstrate the superiority of AuNPs’ stabilization using polymeric ligands used over typical monolayer-forming sulphur-based stabilizers, the activity of nanoparticles modified with various ligands was examined. To exclude the influence of size and the concentration of AuNPs on catalytic activity, the bare NPs were prepared, modified and subsequently investigated. To the samples of AuNPs from the same batch, aliquots of water or aqueous solutions of HBPG-OH or HBPG-COOH (both 1 mg ml−1), 3-MPA and glycine-dithiocarbamate (both 100 μM) were added. This way, samples of NPs of the same origin and concentration, but with different capping ligands, were obtained. Figure 5 depicts kinetic curves showing the influence of surface modifiers on nanozymes’ activity. Almost complete depletion of peroxidaselike activity for AuNPs stabilized with thiol and DTC ligands was observed. In turn, high catalytic activity of AuNPs after the addition of hyperbranched polymers of both types was retained. The observed effects were caused by chemisorption of sulphur-containing ligands on nanoparticles’ surfaces, thus blocking their active sites, rather than the aggregation of nanoparticles caused by ligands. Almost no change in morphology was noticed, only slight changes in the UV-Vis spectrum, without signs of substantial aggregation being recorded (see figure 5). Despite the relatively large 8

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Figure 5. (a) The UV-Vis absorption spectra of bare AuNPs before and after incubation with solutions of various polymeric and sulfur

containing surface-active ligands, (b) and (c) HRP-like activity (expressed as change of absorbance in time) of selected AuNPs-based nanozymes with different surface coating: HBPG-OH (green), unmodified (black), HBPH-COOH (purple), 3-MPA (red), glycine dithiocarbamate (yellow) towards PhOH/4-AAP and TMB system as peroxidase substrates.

discrepancies in the literature, sulphur-containing ligands negatively affect peroxidase-like activity in general [50, 58]. Densely packed SAMs (self-assembled monolayers) substantially restrict access to the catalytically active surface, while the oxidation requires physical adsorption of hydrogen peroxide on the surfaces of nanoparticles. Asimilar effect was also observed in the presence of other substances with an affinity to the gold surface, such as surfactants. This phenomenon significantly reduces the applications of one of the simplest strategies of AuNP stabilization, which is based on thiolate–gold affinity, when preparing efficient nanozymes. The results of a parallel experiment, where citrate-capped AuNPs were used for further modification with the ligands listed above, are consistent with those obtained for unmodified ones. This shows that nanoparticles stabilized by hyperbranched polymers of both types, as compared to sulfurcontaining ligands, allow them to maintain high activity. The superiority of hyperbranched polymers results from the fact that they contribute to the NPs’ surface blockage to a small degree, while assuring their colloidal stability. It is reflected as only a small deterioration in catalytic activity in comparison with bare AuNPs.

oxidation reaction, which is often assigned to an intrinsic feature of nanozymes. To verify the pH-dependence of the activity of prepared AuNPs, besides the two substrates listed above, a PhOH and 4-AAP system wasalso utilized. The relative activities in time (based on the slopes of absorbance, proportional to the concentration of the product of the catalytic reaction) for three types of chromogenic substrates in the presence of the selected catalyst (AuNP@HBPG–OH4000 or HRP, respectively) are depicted in figure 6. Regardless of catalyst origin, TMB and ABTS cannot be oxidized above pH 6.0, whereas the PhOH/4-AAP substrate system retains a high efficiency over a wide range of pH. Our studies revealed a negative influence of H2O2 on HRP activity against TMB and ABTS, which was observed when its concentration exceeded 4 mM. This was caused by ‘suicide’ substrate inhibition resulting in an irreversible oxidation of iron in the heme cofactor [1]. It should be emphasized that all types of polymer coated Au-based nanozymes (bare, HBPG-OH and HBPG-COOHstabillized) examined retain a high activity even at H2O2 concentrations exceeding 100 mM. The comparative study of enzymatic activity of miscellaneous catalysts of different origin (nanozymes and native HRP) performed clearly depicts that optimal activity in acidic solutions is caused by properties of substrates (probably low solubility of TMB and inadequate susceptibility of ABTS to oxidation in neutral and basic solutions), rather than a feature of the obtained nanozymes. A non-protein origin and a

3.5. pH dependence of peroxidase-like catalysts’ activity towards selected substrates

Most of the literature indicates high peroxidase activity of nanozymes below pH 5.5 based on the TMB or ABTS 9

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Figure 6. Catalysts’ activity against various chromogenic substrates over wide pH range. (ABTS—green bars, TMB—blue bars, PhOH/4-

AAP—red bars.) As catalyst (a) native horseradish peroxidase, (b) HBPG-OH-coated gold-based nanozyme (AuNPs@HBPG-OH4000) was utilized. Phosphate buffer of pH 10.5, 7.4, 4.5, 2.5, borate buffer of pH 9.0 and MES buffer of pH 6.0 were used.

catalytic mechanism, which is not based on metalloporphyrin cofactor, provides greater resistance against extreme pH values and high peroxide concentration. An important advantage of the developed nanoparticles is their high activity at low pH. Our experiments proved the robustness of goldbased nanozymes and assures their compatibility with a wide range of available substrate systems, where native peroxidase activity is substantially deteriorated.

protonated in acidic pH, where oxidation occurs most efficiently. In the case of phenol and 4-aminoantipyrine, only the phenolate ion is involved in the catalytic reaction. In fact, it is a two-step reaction, and its mechanism is based on the catalytic oxidation of phenolate to a quinone derivative and the subsequent, non-catalytic reaction of the product with 4-AAP, creating quinoneimine dye. Due to the fact that reaction kinetics is limited by catalytic steps, not by a subsequent coupling reaction product with 4-aminoantypirine (the coupling reaction rate is faster by about three orders of magnitude than catalytic oxidation), the Michaelis–Menten model was in this case also utilized [59]. A series of apparently steady-state kinetic parameters were determined with the use of selected substrates in acidic, as well as mildly basic media. In all the experiments, a mechanism similar to hyperbolic Michaelis–Menten kinetics was observed (see figure 7). Prepared nanocatalysts exhibit KM values of about one order of magnitude lower than native horseradish peroxidase, which proves their high affinity with all types of substrates. For all examined nanoparticles, regardless of the presence and type of polymer coating, KM values for TMB proved to be somewhat lower than the corresponding values for ABTS. Phenolic compounds reveal a significantly lower affinity to peroxidase, which is a well-known phenomenon; however, due to their wide working pH range, they were also selected for the comparative study. A relationship between the size and the value of KM of individual nanoparticles was noticed. With decreasing diameter, the affinity of individual substrates to surface decreased, regardless of the type and concentration of the modifier (KM changed in order: bare AuNPs (d=8.1 nm)>Au/HBPG-COOH4000 (5.8 nm)>Au/ HBPG-COOH20 (5.2 nm)>Au/HBPG-COOH4000 (3.0 nm). The type of surface coating did not significantly affect its affinity for examined substrates. This can prove the fact that the use of a hyperbranched polymer does not significantly affect the thermodynamic affinity of the substrate to

3.6. Comparison of Michaelis–Menten kinetic parameters for enzyme mimics and native enzymes

Michaelis–Menten steady state kinetic parameters are a convenient tool for characterizing the properties of catalysts like enzymes and enzyme-mimics. A set of nanoparticles, AuNPs@HBPG-COOH4000 and AuNPs@HBPG-COOH20, bare AuNPs and AuNPs@HBPG-OH4000, characterized by sufficient stability and high activity, was chosen. The studies aimed at comparison of polymer concentration on catalytic properties of AuNPs@HBPG-COOH. Results were also compared with those for AuNPs stabilized with HBPG-OH and for native HRP, to assess the influence of catalyst origin and ligand type on kinetic parameters. Different surface compositions of nanoparticles (lack of modifier, neutral or anionic surface ligand) may influence the catalyst–substrate affinity (KM), as well as the substrates and products transportation to the active surface of the catalyst, which is typically reflected by changes in vmax and kcat values. For direct comparison of the maximum efficiency of catalysts, the value of kcat, which is expressed as the rate per single nanozyme particle, was utilized. In our study to examine the significance of the polyanionic character of HBPG-COOH on the catalytic activity of AuNPs, three different types of peroxidase substrates were applied. ABTS is the anionic substrate containing two sulfonate groups in the deprotonated form, even in relatively acidic pH, which is optimal for ABTS oxidation by hydrogen peroxide. In turn, TMB, as the aromatic amine, tends to be 10

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nanocatalysts of large surface area and significantly higher dimensions (5.2·102–1.4·104 s−1) stem from small sizes of the catalysts, and hence a smaller surface of a single particle [7]. Nevertheless, smaller nanoparticles have a large surface area to volume ratio, and hence larger surface active sites per single particle. The use of nanoparticles of a small diameter can be beneficial in bioanalytical applications due to the increased compatibility with typical bioreceptors. 3.7. pH and ionic strength dependent stability of AuNPs

The stability of the obtained nanocatalysts in solutions of relatively high ionic strength during kinetic studies is crucial from the point of view of further quantitative assessment of their activity, which is directly dependent on their surface condition. The effect of buffers acting as the catalysis medium on the optical and catalytic properties of AuNPs was examined. In the experiment, the tendency to salt-induced aggregation of sterically stabilized AuNPs (with the use of HBPGCOOH and HBPG-OH of different concentrations), unmodified nanoparticles as well as electrostatically stabilized ones (using sodium citrate), was studied. Figure 8 shows the effects of four-day incubation of the chosen nanoparticles in 0.2 M buffers (chosen to further study enzymatic activity—borate buffer, pH 8.5; phosphate buffer, pH 4.5) on their absorption spectra. In the case of nanoparticles that are unmodified and stabilized with low concentrations of hyperbranched polymers (AuNPs@HBPG-COOH20 and AuNPs@HBPGOH20), as well as nanoparticles electrostatically stabilized with citrate anions, prolonged interaction with buffers resulted in a broadening and shift of the LSPR band towards longer wavelengths, caused by aggregation (see figure 8). No visible changes in the LSPR band after incubation in both examined buffer solutions for AuNPs@HBPG-COOH4000 were noticed, which is evidence of their high stability achieved through steric stabilization by polymer molecules of globular topology. Unmodified hyperbranched polyglycidol as a AuNP’s surface stabilizer exhibits very similar properties to its carboxylated derivative in ensuring the resistance to pH and high ionic strength. In the case of the pH 8.5 buffer for all tested nanoparticles, regardless of the type of ligand surface and the stabilization strategy, the relative drop in the original catalytic activity in comparison to the particles incubated in water did not exceed 13%. A much greater effect on optical properties and activity was caused by incubation in the phosphate buffer of pH 4.5. The acidic medium appeared to be more destructive for gold nanoparticles because it resulted in a faster and more developed aggregation of nanoparticles without a polymer shell, which was confirmed by the loss of their characteristic optical properties (see insets of figure 8). This was also reflected as a more significant decrease in catalytic activity towards the oxidation of TMB. The observed decrease in activity of unmodified AuNPs reached 40%, while for AuNPs@HBPG-COOH4000, the activity drop after a four-day incubation amounted to only 11%. These results confirm the high stability and robustness of the particles obtained with the use of stabilizers proposed by us. The

Figure 7. Exemplary kinetics analysis of HBPG-COOH@AuNPs.

Steady state reaction rates were obtained from initial slopes of absorbance curves in time for varied TMB concentrations and fixed H2O2 concentration of 375 mM and catalyst concentration of 3.55 nM at pH 4.5 and room temperature. Inset shows the double reciprocal plot of reaction rate versus substrate concentration respectively.

the surface of the catalyst due to the loose, globular topology of the modifier. No clear relationship between the type of substrate (cationic or anionic) and the values of KM can indicate the low impact of surface charge derived from the carboxyl group-containing polymer due to the relatively low density of anionic groups (see table 2). It was observed that the kcat values for both series of nanoparticles with large amounts of polymer are closer to each other and lower than it is in the case of nanoparticles prepared with use of a low concentration of modifier (or in the case of no modifier being present). This may prove that the maximum rate of the reaction is mainly dependent on the rate of substrate diffusion to the catalyst surface, rather than the AuNP’s type and charge. Reports in the literature describing the influence of nanocatalyst modifications on KM values against TMB and ABTS pointed to the existence of a clearer relationship between the charge of the surface and KM in the case of the modification of low-molecular compounds or linear homopolymers [51]. In this study, modification with the use of only partially functionalized HBPG-COOH, of a generally globular and rigid structure, does not result in the creation of a coating dense enough to substantially affect the affinity to substrates. In contrast, changes in the value of kcat can be explained to some extent by different sizes, and thus differences in active surface, directly influencing the obtained turnover number values. In the case of particles modified with different concentrations of the same polymer, AuNPs@HBPG-COOH20 showed higher values of kcat than AuNPs@HBPG-COOH4000, probably due to a lower number of stabilizing polymer molecules on their surface, which is beneficial from the point of view of substrates and products transportation. Low turnover values compared to 11

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Table 2. Michaelis–Menten parameters of peroxidase-like nanozymes and native HRP.

vmax [10−8 M s−1]

KM [μM]

12

HBPG-COOH@ AuNPs 4000 HBPG-OH@AuNPs 4000 HBPG-COOH@ AuNPs 25 AuNPs unmodified HRP

Ccatalyst [nM]

ABTS

TMB

3.55 26.1 5.27 1.42 0.114 (TMB) 0.284 (ABTS) 2.84 (PhOH)

38.0 47.9 45.8 33.0 472

35.4 44.4 40.7 23.5 421

PhOH/4-AAP 2 7 3 2 16

901 051 299 943 952

kcat[s−1]

ABTS

TMB

PhOH/4-AAP

ABTS

TMB

PhOH/4-AAP

0.855 1.65 2.03 0.81 55.0

1.84 3.06 6.67 2.15 55.8

1.46 6.56 3.11 0.94 4.40

2.41 0.63 3.86 5.67 4 837

5.19 1.17 12.6 15.1 1 965

4.11 2.51 5.89 6.59 15.5

kcat was expressed as a ratio of vmax to the molar concentration of nanoparticles obtained from TEM micrograph analysis and further calculations. The mass concentration of AuNP catalysts was 25 μg ml−1 and HRP was 0.05 mg ml−1 for ABTS, 0.02 μg ml−1 for TMB and 0.50 μg ml−1 for PhOH/4-AAP, respectively.

M Drozd et al

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Figure 8. UV-Vis absorption spectra of (a) AuNPs/HBPG-COOH4000, (b) AuNPs/HBPG-COOH20, (c) bare AuNPs, (d) citrate-stabilized AuNPs in water (blue line), borate buffer pH 8.5 (red line) and phosphate buffer pH 4.5 (green line). Insets show images of AuNPs samples after incubation in water (1), borate buffer (2) and phosphate buffer (3).

presence of both the examined polymers in a concentration of 4 mg ml−1 allows us to obtain nanoparticles which maintain optical properties and catalytic activity in solutions of high ionic strength and the broad pH range. This may be beneficial from the point of view of potential applications eg. in bioconjugates or optical nanosensors, for which the increased stability of AuNPs in solutions of high ionic strength is required. Moreover, the effect of hydrogen peroxide on the stability of colloidal AuNPs was evaluated in the framework of the studies conducted. The presence of high amounts of hydrogen peroxide (up to 1 M) did not influence the optical properties of nanoparticles. In the case of bare AuNPs, the formation of bubbles was observed at pH 8.5, as the evidence of hydrogen peroxide decomposition (catalase-like activity of AuNPs) [7, 12]. The presence of an even smaller amount of polymeric modifier leads to this undesirable kind of activity being disabled.

the obtained nanoparticle in the range of 2.5 to 8.3 nm. When increasing the concentration of HBPG-OH, the average diameter of the prepared AuNPs decreased, while for HBPGCOOH quite the opposite trend was noticed. The obtained nanoparticles exhibited an intrinsic catalytic activity towards typical peroxidase substrates in both acidic (for TMB and ABTS) and slightly basic pH (for PhOH/4-AAP). A set of experiments which were carried out showed a significant dependence of the catalytic activity on a number of parameters, such as: the size and morphology of the nanoparticles, the presence of sulphur-containing ligands, and pH (especially in the case of ABTS and TMB). In the framework of the presented work, the impact of polyglycidol and its derivative as nanozymes’ stabilizers was exhaustively tested. The examined polymers exhibited a negligible interfering influence on the catalytic activity of AuNPs, at the same time providing high dispersion stability, even in solutions of high ionic strength. The kinetics of oxidation of HRP substrates using HBPG-stabilized AuNPs were fitted according to the typical Michaelis–Menten model. Prepared nanozymes revealed a high affinity with the examined substrates, manifesting as average KM values which were an order of magnitude smaller than those calculated for their natural counterpart, horseradish peroxidase. Our observations confirmed the versatility of gold-based nanozymes in a wide range of pH. It was found

4. Conclusion Both polyglycidol and its carboxylated derivative exhibited the capacity to stabilize AuNPs. Varying the concentration and kind of hyperbranched polymer during gold precursor reduction by means of NaBH4 allowed us to tailor the size of 13

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[12] Li J, Liu W, Wu X and Gao X 2015 Mechanism of pH-switchable peroxidase and catalase-like activities of gold silver platinum and palladium Biomaterials 48 37–44 [13] Wan Y, Qi P, Zhang D, Wu J and Wang Y 2012 Manganese oxide nanowire-mediated enzyme-linked immunosorbent assay Biosens. Bioelectron. 33 69–74 [14] Andre R, Natálio F, Humanes M, Leppin L, Heinze K, Wever R, Schröder H–C, Müller E and Tremel W 2011 V2O5 nanowires with an intrinsic peroxidase-like activity Adv. Funct. Mater. 21 501–9 [15] Chen W, Chen J, Liu A, Wang M, Li G and Lin X 2011 Peroxidase-like activity of cupric oxide nanoparticles Chem. Catal. Chem. 3 1151–4 [16] Gao L et al 2007 Intrinsic peroxidase-like activity of ferromagnetic nanoparticles Nat. Nanotechnology 2 577–83 [17] Mu J, Wang Y, Zhao M and Zhang L 2012 Intrinsic peroxidase-like activity and catalase-like activity of Co3O4 nanoparticles Chem. Commun. 48 2540–2 [18] Song Y, Qu K, Zhao C, Ren J and Qu X 2010 Graphene oxide: intrinsic peroxidase catalytic activity and its application to glucose detection Adv. Mater. 22 2206–10 [19] Song Y, Wang C, Zhao C, Qu K, Ren J and Qu X 2010 Labelfree colorimetric detection of single nucleotide polymorphism by using single-walled carbon nanotube intrinsic peroxidase-like activity Chem. Eur. J. 16 3617–21 [20] Daniel M–C and Astruc D 2004 Gold nanoparticles: assembly supramolecular chemistry quantum-size-related properties and applications toward biology catalysis and nanotechnology Chem. Rev. 104 293–346 [21] Haruta M 1997 Size- and support-dependency in the catalysis of gold Catal. Today 36 153–66 [22] Cui H, Zhang M, Shi M, Xu Y and Wu Y 2005 Light emission of gold nanoparticles induced by the reaction of Bis[2,4,6trichlorophenyl] oxalate and hydrogen peroxide Anal. Chem. 77 6402–6 [23] Fenger R, Fertitta E, Kirmse H, Thunemann A F and Rademann K 2012 Size dependent catalysis with CTABstabilized gold nanoparticles Phys. Chem. Chem. Phys. 14 9343–9 [24] Sau K T, Pal A and Pal T 2001 Size regime dependent catalysis by gold nanoparticles for the reduction of eosin J. Phys. Chem. B 105 9266–72 [25] Chang C–P, Tseng C–C, Ou J–L, Hwu W–H and Ger M–D 2010 Growth mechanism of gold nanoparticles decorated on polystyrene spheres via self-regulated reduction Colloid Polym. Sci. 288 395–403 [26] Sun Z, Zhao Q, Zhang G, Li Y, Zhang G, Zhang F and Fan X 2015 Exfoliated MoS2 supported Au–Pd bimetallic nanoparticles with core–shell structures and superior peroxidase-like activities RSC Adv. 5 10352–7 [27] Liu Y, Fu W, Li C, Huang C and Li Y 2015 Gold nanoparticles immobilized on metal–organic frameworks with enhanced catalytic performance for DNA detection Anal. Chim. Act. 861 55–61 [28] Chen X, Tian X, Su B, Huang Z, Chen X and Oyama M 2014 Au nanoparticles on citrate-functionalized graphene nanosheets with a high peroxidase-like performance Dalton Trans. 43 7449–54 [29] Zhang Y, Xu C, Li B and Li Y 2013 In situ growth of positively-charged gold nanoparticles on single-walled carbon nanotubes as a highly active peroxidase mimetic and its application in biosensing Biosens. Bioelectron. 43 205–10 [30] Zhou L, Gao C, Hu X and Xu W 2011 General avenue to multifunctional aqueous nanocrystals stabilized by hyperbranched polyglycerol Chem. Mater. 23 1461–70 [31] Fisher W, Quadir M A, Barnard A, Smith D K and Haag R 2011 Controlled release of DNA from photoresponsive

that differences in HBPG-OH and its carboxylated derivative concentration, and their ability to stabilize the nanostructures, significantly affect the kinetic parameters against all types of substrates. However, the influence of the stabilizers’ charge was negligible. The explanation, associated with the relatively low charge concentration resulting from the bulky, globular structure of hyperbranched polymer, was suggested. For bioanalytic purposes, e.g. bioconjugation, a high density of carboxylic groups is not necessary, whereas a low density of negative surface charge is beneficial from the point of view of catalysis. We believe that the studies presented will imply that low molecular weight hyperbranched polyglycidol-stabilized AuNPs may be used as an efficient platform, revealing peroxidase-like activity and enabling further noninvasive functionalization for bioanalytical applications.

Acknowledgments This work has been financially supported by Warsaw University of Technology.

References [1] Valderrama B, Ayala M and Vazquez-Duhalt R 2002 Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes Chem. Biol. 9 555–65 [2] Chen Z, Xu L, Liang Y and Zhao M 2010 pH-sensitive watersoluble nanospheric imprinted hydrogels prepared as horseradish peroxidase mimetic enzymes Adv. Mater. 22 1488–92 [3] Pelossof G, Tel-Vered R, Elbaz J and Willner I 2010 Amplified biosensing using the horseradish peroxidase-mimicking DNAzyme as an electrocatalyst Anal. Chem. 82 4396–402 [4] Fruk L and Niemeyer C M 2005 Covalent hemin-DNA adducts for generating a novel class of artificial heme enzymes Angew. Chem. Int. Ed. 44 2603–6 [5] Xie S, Chai Y, Yuan Y and Yuan R 2014 Manganese porphyrin-double stranded DNA complex guided in situ deposition of polyaniline for electrochemical thrombin detection Chem. Commun. 50 7169–72 [6] Wei H and Wang E 2013 Nanomaterials with enzyme-like characteristics [nanozymes]: next-generation artificial enzymes Chem. Soc. Rev. 42 6060–93 [7] He W et al 2011 Au@Pt nanostructures as oxidase and peroxidase mimetics for use in immunoassays Biomaterials 32 1139–47 [8] Wang Z, Yang X, Yang J, Jiang Y and He N 2015 Peroxidaselike activity of mesoporous silica encapsulated Pt nanoparticle and its application in colorimetric immunoassay Anal. Chim. Act. 862 53–63 [9] Liu J, Hu X, Hou S, Wen T, Liu W, Zhu X, Yin J–J and Wu X 2012 Au@Pt core/shell nanorods with peroxidase- and ascorbate oxidase-like activities for improved detection of glucose Sensors Actuators B 166-167 708–14 [10] Nangia Y, Kumar B, Kaushal J and Suri R 2012 Palladium@gold bimetallic nanostructures as peroxidase mimic for development of sensitive fluoroimmunoassay Anal. Chim. Act. 751 140–5 [11] Gao Z, Xu M, Lu M, Chen G and Tang D 2015 Urchin-like [gold core]@[platinum shell] nanohybrids: a highly efficient peroxidase-mimetic system for in situ amplified colorimetric immunoassay Biosens. Bioelectron. 70 194–201 14

Nanotechnology 26 (2015) 495101

[32]

[33]

[34] [35]

[36]

[37] [38]

[39]

[40]

[41]

[42]

[43]

[44]

M Drozd et al

[45] Wang L, Neoh K G, Kang E T, Shuter B and Wang S–C 2009 Superparamagnetic hyperbranched polyglycerol-grafted Fe3O4 nanoparticles as a novel magnetic resonance imaging contrast agent: an in vitro assessment Adv. Funct. Mater. 19 2615–22 [46] Zhao L et al 2012 Hyperbranched polyglycerol-grafted superparamagnetic iron oxide nanoparticles: synthesis characterization functionalization size separation magnetic properties and biological applications Adv. Funct. Mater. 22 5107–17 [47] Thomas A, Bauer H, Schilmann A–M, Fischer K, Tremel W and Frey H 2014 The ‘needle in the haystack’ makes the difference: linear and hyperbranched polyglycerols with a single catechol moiety for metal oxide nanoparticle coating Macromolecules 47 4557–66 [48] Li H and Cooper-White J J 2013 Hyperbranched polymer mediated fabrication of water soluble carbon nanotube– metal nanoparticle hybrids Nanoscale 5 2915–20 [49] Nicell J A and Wright H 1997 A model of peroxidase activity with inhibition by hydrogen peroxide Enzyme. Microbial. Tech. 21 302–10 [50] Liu Y, Wang C, Cai N, Long S and Yu F 2014 Negatively charged gold nanoparticles as an intrinsic peroxidase mimic and their applications in the oxidation of dopamine J. Mater. Sci. 49 7143–50 [51] Yu F, Huang Y, Cole A J and Yang V C 2009 The artificial peroxidase activity of magnetic iron oxide nanoparticles and its application to glucose detection Biomaterials 30 4716–22 [52] Haiss W, Thanh N T K, Aveyard J and Fernig D G 2007 Determination of size and concentration of gold nanoparticles from UV-Vis spectra Anal. Chem. 79 4215–21 [53] Liu X, Atwater M, Wang J and Huo Q 2007 Extinction coefficient of gold nanoparticles with different sizes and different capping ligands Colloid Surf. B 58 3–7 [54] Jimenez-Ruiz A, Perez-Tejeda P, Grueso E, Castillo P M and Predo-Gotor R 2015 Nonfunctionalized gold nanoparticles: synthetic routes and synthesis condition dependence Chem. Eur. J. 21 9596–609 [55] Patungwasa W and Hodak J H 2008 pH tunable morphology of the gold nanoparticles produced by citrate reduction Mater. Chem. Phys. 108 45–54 [56] Goia D V and Matijević E 1999 Tailoring the particle size of monodispersed colloidal gold Colloid Surf. A 146 139–52 [57] Huang H and Yang X 2004 Synthesis of chitosan-stabilized gold nanoparticles in the absence/presence of tripolyphosphate Biomacromolecules 5 2340–6 [58] Jv Y, Li B and Cao B 2010 Positively-charged gold nanoparticles as peroxidase mimic and their application in hydrogen peroxide and glucose detection Chem. Commun. 46 8017–9 [59] Vojinović V, Carvalho R H, Lemos F, Cabral J M S, Fonseca L P and Ferreira B S 2007 Kinetics of soluble and immobilized horseradish peroxidase-mediated oxidation of phenolic compounds Biochem. Eng. J. 35 126–35

hyperbranched polyglycerols with oligoamine shells Macromol. Biosci. 11 1736–46 Pérignon N, Mingotaud A–F, Marty J–D, Rico-Lattes I and Mingotaud C 2004 Formation and stabilization in water of metal nanoparticles by a hyperbranched polymer chemically analogous to PAMAM dendrimers Chem. Mater. 16 4856–8 Kozytskiy A V, Raevskaya A E, Stroyuk O L, Kotenko I E, Skorik N A and Kuchmiy S Y 2015 Morphology optical and catalytic properties of polyethyleneimine-stabilized Au nanoparticles J. Mol. Catal. A 398 35–41 Scott R, Wilson O and Crooks R 2005 Synthesis characterization and applications of dendrimer-encapsulated nanoparticles J. Phys. Chem. B 109 692–704 Esumi K, Miyamoto K and Yoshimura T 2002 Comparison of PAMAM-Au and PPI-Au nanocomposites and their catalytic activity for reduction of 4-nitrophenol J. Colloid Interface Sci. 15 402–5 Endo T, Yoshimura T and Esumi K 2005 Synthesis and catalytic activity of gold–silver binary nanoparticles stabilized by PAMAM dendrimer J. Colloid Interface Sci. 286 602–9 Turkevich J, Stevenson P C and Hillier P 1951 A study of the nucleation and growth processes in the synthesis of colloidal gold Discuss. Faraday Soc. 11 55–75 Niidome T, Nakashima K, Takahashi H and Niidome Y 2004 Preparation of primary amine-modified gold nanoparticles and their transfection ability into cultivated cells Chem. Commun. 19 1978–9 Wang S, Chen W, Liu A–L, Hong L, Deng H–H and Lin X–H 2012 Comparison of the peroxidase-like activity of unmodified amino-modified and citrate-capped gold nanoparticles Chem. Phys. Chem. 13 1199–204 Sunder A, Hanselmann R, Frey H and Mullhaupt R 1999 Controlled synthesis of hyperbranched polyglycerols by ring-opening multibranching polymerization Macromolecules 32 4240–6 Hu X, Zhou L and Gao C 2011 Hyperbranched polymers meet colloid nanocrystals: a promising avenue to multifunctional robust nanohybrids Colloid Polym. Sci. 289 1299–320 Harder P, Grunze M and Dahint R 1998 Molecular conformation in oligo[ethylene glycol]-terminated selfassembled monolayers on gold and silver surfaces determines their ability to resist protein adsorption J. Phys. Chem. B 102 426–36 Zill A, Rutz A L, Kohman R E, Alkikany A M, Murphy C J, Kong H and Zimmermann S C 2011 Clickable polyglycerol hyperbranched polymers and their application to gold nanoparticles and acid-labile nanocarriers Chem. Commun. 47 1279–81 Wang S, Zhou Y, Yang S and Ding B 2008 Growing hyperbranched polyglycerols on magnetic nanoparticles to resist nonspecific adsorption of proteins Colloid Surface B 67 122–6

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