3-Aminopropyltrimethoxysilane and organic electron donors mediated synthesis of functional amphiphilic gold nanoparticles and their bioanalytical applications

Abstract

Precise control over functionality and nanogeometry during synthesis of gold nanoparticles (AuNPs) and dispersibility of the same in a variety of solvents has been a challenging requirement for practical applications. Organic reducing agents i.e. 3-glycidoxypropyltrimethoxysilane (3-GPTMS), tetrahydrofuran hydroperoxide (THF–HPO) and cyclohexanone have shown potential for meeting these requirements during the conversion of 3-aminopropyltrimethoxysilane (3-APTMS)-capped gold ions into AuNPs. The reaction products of these reducing agents with 3-APTMS during AuNPs synthesis are catalytic in nature for THF–HPO and cyclohexanone due to the formation of inorganic–organic hybrids and non-catalytic for 3-GPTMS. The presence and absence of such reaction products justify the difference in catalytic ability of the AuNPs as a function of organic reducing agents, which profusely affects the inherent properties of AuNPs for specific applications. The use of cyclohexanone in place of 3-GPTMS or THF–HPO together with 3-APTMS outclasses the other two in imparting better stability to amphiphilic AuNPs with reduced silanol content. The as-synthesized AuNPs enable the formation of nanocomposite (PBNPs–AuNPs) dispersion with Prussian blue nanoparticles (PBNPs). Further, PBNPs–AuNPs may also be converted to homogeneous nanocomposite suspensions with ruthenium bipyridyl solution, justifying even better catalytic ability than that of HRP. The resulting nanomaterial suspensions in one way, efficiently probe the glucose oxidase catalyzed reactions based on peroxidase mimetic ability and, in another way, display excellent electrocatalytic activity during the electrochemical sensing of H₂O₂. The peroxidase mimetic ability of the nanomaterials has been found to vary as a function of 3-APTMS concentration, which confirms the potential role of functional gold nanoparticles in bioanalytical applications.

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Introduction

The requirements for practical usability of gold nanoparticles in a variety of organic and aqueous solvent have directed the use of bipolar organic reagents in the synthesis of functional nanoparticles. Further, functionalization of noble metal nanoparticles with organic amines has received considerable attention from many angles, e.g.,: (1) amine-capped nanocrystals are nearly as stable as their thiol-capped counterparts, however, unlike thiols, amines are weakly bound on Au surface;¹ (2) their functional use in cancer therapy as drug carriers, photothermal agents, contrast agents and radiosensitizers;² (3) susceptibility for the formation of biocompatible linkage;^3,4 and (4) organic amines act as potential stabilizers for nanoparticles.^5,6 These advantages of organic amines have directed us to investigate their role during the synthesis of noble metal nanoparticles.^7,8 The use of 3-APTMS has been very well documented as a potential stabilizer for noble metal nanoparticles.⁹ We investigated in detail the role of 3-APTMS during the synthesis noble metal nanoparticles that required the participation of 3-APTMS compatible organic reducing agents, like 3 glycidoxypropyltrimethoxysilane (3-GPTMS) and tetrahydrofuranhydroperoxide (THF–HPO).^10,11 During the course of AuNPs synthesis, these reducing agents interact in different methods with 3-APTMS, resulting in the formation of organic moiety dependent reaction products that may alter the inherent properties of as synthesized AuNPs. Such products may also influence the dispersion ability of the resulting AuNPs in different polar and non-polar solvents for specific application, due to the bipolar nature of 3-APTMS and the polarity/non-polarity of the organic reducing agents. Earlier findings on the use of 3-GPTMS along with 3-APTMS during nanoparticles synthesis in methanolic medium resulted in AuNPs mostly dispersible in organic solvents.¹² Similarly, the use of THF–HPO with 3-APTMS during such synthesis provided AuNPs mostly dispersible in aqueous medium.¹¹ The dependence of the reducing agent on the solvent required for the synthesis of AuNPs resulted in differential kinetic limitation when allowed to interact with 3-APTMS capped Au³⁺ ions. Such findings revealed the central role of 3-APTMS during noble metal nanoparticles synthesis and the direct major contribution of organic functionalities (3-GPTMS or THF–HPO) for the precise control of desired properties for practical applications. Recently, we have reported the synthesis of Prussian blue nanoparticles mediated by 3-APTMS and cyclohexanone.¹³ Such findings revealed that 3-APTMS capped hexacyanoferrate ions undergo controlled conversion into polycrystalline Prussian blue with excellent electrochemical behaviour for practical applications.¹³ Similar processes also enable the synthesis of polycrystalline mixed metal hexacyanoferrate with nearly all combinations of transition metal ions.¹⁴ Cyclohexanone in aqueous media shows a biphasic system, and even in such conditions the controlled synthesis of nanoparticles are recorded.^13–15 Cyclohexanone mediated synthesis of AuNPs has been reported by Uppal et al.¹⁵ The biphasic system of nanoparticles resulted in transition in nanogeometry of AuNPs with time, suggesting the need for a stabilizer and directed the detailed examination of the role of 3-APTMS during nanoparticle conversion.^9,10 Further, the use of cyclohexanone with 3-APTMS might prove effective in enhancing catalytic activity through the formation of an organic–inorganic hybrid because the interaction of functional alkoxysilane and small organic molecules results in the formation of such catalytic material.¹⁶ The hydrophobic behaviour of cyclohexanone and micellar activity of 3-APTMS may allow the synthesis of functional AuNPs, suitable for the formation of nanocomposites with a variety of known catalytic materials (e.g. metal hexacyanoferrate, ruthenium bipyridyl), dispersible in suitable solvents for practical bioanalytical application. Accordingly, a detailed investigation on the role of 3-APTMS mediated conversion of gold cations in the presence of cyclohexanone is sought from the following angles: (1) conversion of 3-APTMS capped gold ions to respective nanoparticles in the presence of cyclohexanone, (2) characterization and application of 3-APTMS and cyclohexanone mediated AuNPs as a function of functional ability and nanogeometry, (3) checking the dispersibility of as synthesized cyclohexanone mediated AuNPs at different ratios of 3-APTMS and cyclohexanone, (4) the ability of AuNPs to form nanocomposites displaying peroxidase mimetic ability for probing glucose oxidase catalyzed reactions. Further, the rate of 3-APTMS mediated synthesis of AuNPs may be the function of organic reducing agents and accordingly, comparisons of the roles of 3-GPTMS, THF–HPO and cyclohexanone on the following points are also undertaken: (a) time required during nanoparticles conversion as a function of organic moieties, (b) catalytic ability of nanoparticles based on functional ability and nanogeometry, (c) stability of as synthesized nanoparticles for practical applications, and (d) dispersibility of as synthesized nanoparticles in different solvents as a function of organic moieties. The results on these lines are reported in this communication.

Experimental section

Materials

3-Aminopropyltrimethoxysilane (3-APTMS), 3-glycidoxypropyltrimethoxysilane, chloroauric acid and o-dianisidine were obtained from Aldrich Chem. Co.; cyclohexanone was obtained from S.D. Fine-Chem. Pvt. Ltd.; toluene, hydrogen peroxide, ethyl acetate, dichloromethane and acetonitrile were obtained from Merck, India. All other chemicals employed were of analytical grade. Aqueous solutions were prepared by using double distilled-deionized water (Alga water purification system). All the experiments were performed at room temperature unless otherwise mentioned.

The absorption spectra of nanoparticles were recorded using a Hitachi U-2900 Spectrophotometer. Transmission electron microscopy (TEM) studies were performed using Morgagni 268D (Fei Electron Optics) and electrochemical measurements were made with electrochemical workstation CHI 660B (CH Instrument, USA).

3-APTMS and cyclohexanone mediated synthesis of AuNPs and its nanocomposite with prussian blue and ruthenium bipyridyl

In a typical procedure 1 ml sol of AuNPs was prepared by adding 100 μl of desired concentration of 3-APTMS to 100 μl of 0.025 M HAuCl₄. The mixture was stirred using a cyclomixer for 2 minutes followed by the addition of 500 μl of cyclohexanone and stirred again on the vortex mixer. Methanol was added to make up the desired volume. The reaction mixture was then left undisturbed in the dark for 1–3 h. The appearance of red, purple or blue colour of the resulting sol indicated the formation of AuNPs. The nanocomposite of PBNPs–AuNPs was made by mixing as synthesized AuNPs and Prussian blue nanoparticles (PBNPs) prepared as described earlier.¹³ AuNPs (100 μl) and PBNPs (50 μl) were mixed by stirring and incubated for 5 minutes resulting in the formation of a homogeneous PBNPS–AuNPs sol. An aqueous solution of ruthenium bipyridyl Ru(bpy) (5 mM, 5 μl) was added to 15 μl of PBNPs–AuNPs sol to obtain a homogeneous dispersion of PB–AuNPs–Ru(bpy) nanocomposite.

Electrochemical measurement

AuNPs of two sizes AuNP₁ (blue) and AuNP₂ (red) were sonicated with a Prussian blue Nanoparticles (PBNPs) suspension, which was made by controlled mixing of ferrous sulphate and potassium ferricyanide following a standard process. The PBNPs–AuNPs suspension was allowed to be adsorbed thoroughly on graphite particles (1–2 μm) and dried at 90 °C overnight. The adsorbed nanocomposite on graphite particles was incorporated within a graphite paste electrode having composition as follows: PBNPs–AuNPs 2.5% (w/w), graphite powder 67.5% (w/w), Nujol oil 30% (w/w). The well of the electrode body (MF-2010 obtained from Bioanalytical Systems, West Lafayette, IN, USA) was filled with active graphite paste. The paste surface was manually smoothed on a clean paper. Electrochemical measurements were performed in a three-electrode configuration equipped with the graphite paste electrode as the working electrode, an Ag/AgCl reference and a platinum plate counter electrode with a working volume of 3 ml. All electrochemical experiments were performed in 0.1 M phosphate buffer solution (pH 7.0) containing 0.5 M KCl.

Peroxidase like catalytic activity of AuNPs

The peroxidase like activity of as synthesized nanoparticles was determined spectrophotometrically by measuring the formation of oxidized product of o-dianisidine at 430 nm (11.3 mM⁻¹ cm⁻¹) using a Hitachi U-2900 spectrophotometer. Typically, the o-dianisidine (20 mM, 10 μl) oxidation activity was measured in water (2 ml) in the presence of Hydrogen peroxide (10 μl). AuNPs (5 μl) were added to start the reaction. The experiment was also conducted replacing o-dianisidine with 3,3′,5,5′-tetramethylbenzidine (TMB).

Glucose detection was performed as follows: (a) 40 μl of 10 mg ml⁻¹ Glucose Oxidase and 200 μl of glucose at different concentrations in 0.1 M phosphate buffer (pH 7.0) were incubated at 35 °C for 45 min; (b) 50 μl of o-dianisidine (0.5 mM), 15 μl of the PBNPs–AuNPs–Ru (bpy) and 1695 μl of 0.1 M phosphate buffer (pH 7.0) were added to the above reaction solution; (c) the reaction mixture was incubated at 45 °C for 30 min followed by measurement of absorption at 430 nm.

Results and discussion

Requirements of organic reducing agents during 3-APTMS mediated synthesis of AuNPs

The synthesis of AuNPs either in aqueous or organic systems have their own limitations specifically for the transfer of newly formed nanoparticles from a polar to a non-polar environment or vice versa. Such requirements may involve the use of some phase transfer reagents, ionic liquid, etc.^17,18 These limitations can be eliminated by the use of suitable reagents having bipolar behaviour, together acting as both reducing and stabilizing agents during the conversion of noble metal salts into respective nanoparticles. The use of organic amines has shown their potential as reagents and stabilizers for the nanomaterial.^9,11,19,20 We have recently demonstrated that 3-APTMS acts as a potential reagent for the conversion of noble metal cations into respective nanoparticles but requires the participation of additional organic reagents during nanoparticles synthesis. Accordingly, the role of 3-GPTMS and THF–HPO for real time synthesis (i.e., within less than 2 h) of AuNPs from 3-APTMS capped Au³⁺ has been demonstrated.^10,11 3-APTMS alone does not enable the synthesis of AuNPs under similar conditions even after 24 h (ESI Fig. S1†). The findings along these lines have shown the organic moieties (3-GPTMS and THF–HPO) dependent behaviour in the resulting nanoparticles with significant variation in their catalytic ability despite the use of 3-APTMS as one of the reagents in both cases. This difference in catalytic behaviour of AuNPs as a function of 3-GPTMS or THF–HPO is due to the formation of an organic–inorganic hybrid in the later case that enhances the catalytic ability of the AuNPs. The major differences in both synthetic protocol and catalytic behaviour are found as follows: (1) 3-GPTMS allows the synthesis of AuNPs only in methanolic medium, whereas the same with THF–HPO takes place in water. (2) The nanoparticles made through 3-APTMS and 3-GPTMS are mostly dispersible in organic solvent with limited dispersibility in water under specific ratio of 3-APTMS/3-GPTMS. (3) THF–HPO and 3-APTMS mediated synthesis result in nanoparticles that are mostly dispersible in relatively more polar solvent and not dispersible in non-polar media. The limited dispersibility of 3-GPTMS and THF–HPO mediated AuNPs led to the search for another reducing agent with dispersibility in a variety of solvents. Uppal et al., have observed the synthesis of AuNPs only in the presence of cyclohexanone, which is a biphasic system with transition in nanogeometry as a function of time.¹⁵ Accordingly, attempts on the synthesis of AuNPs utilizing cyclohexanone and 3-APTMS in a monophasic system have been performed. These findings revealed the significant role of organic reagents during 3-APTMS mediated conversion of noble metal nanoparticles and required a comparative investigation. Fig. 1A shows the real time synthesis of AuNPs mediated by 3-APTMS and THF–HPO, whereas Fig. 1B shows the similar result replacing THF–HPO with cyclohexanone. The results, as shown in Fig. 1, clearly demonstrate the real time conversion of AuNPs with significant variation in the use of 3-APTMS concentration when the reducing agents are changed. Cyclohexanone requires low concentration of 3-APTMS under similar conditions. It is to be noted that the synthesis of AuNPs require methanolic medium when cyclohexanone is used as reducing agent, whereas THF–HPO enables the synthesis in water.

Fig. 1 Real time synthesis of AuNPs mediated by 3-APTMS–THF–HPO (A) and 3-APTMS–cyclohexanone (B).

Cyclohexanone and 3-APTMS mediated synthesis of AuNPs

Cyclohexanone efficiently enables the synthesis of AuNPs in the presence of 3-APTMS in methanolic medium with excellent nanogeometry under specific ratios of 3-APTMS/cyclohexanone within 0.5–3 h at room temperature. The concentrations of both the reagents significantly control the synthesis of AuNPs. First, we investigated the synthesis of AuNPs under two conditions: (a) varying the concentrations of 3-APTMS while keeping the concentration of cyclohexanone constant, (b) keeping 3-APTMS concentration constant while changing the concentrations of cyclohexanone. Fig. 2 shows the visual photographs along with the respective absorption maxima of AuNPs synthesized at different concentrations of 3-APTMS (3–12 mM), while keeping the concentration of cyclohexanone constant (4.8 M), displaying gradual variations in SPR and showing the significance of 3-APTMS during AuNPs synthesis. We chose two different concentrations of 3-APTMS (5 mM and 10 mM) to witness the effect of cyclohexanone concentration on the AuNPs. The higher concentration of 3-APTMS (10 mM) resulted in blue nanoparticles, whereas lower concentration (5 mM) largely gave red nanoparticles as shown in Fig. 3A and B, respectively.

Fig. 2 UV-VIS spectra showing change in λ_max of AuNPs made using a constant concentration of cyclohexanone (4.8 M) and varying the concentration of 3-APTMS; (i) 3 mM (ii) 4 mM (iii) 6 mM (iv) 8 mM (v) 9 mM (vi) 10 mM (vii) 12 mM.

Fig. 3 UV-VIS spectra showing change in λ_max of AuNPs made using (A) constant concentration of 3-APTMS (10 mM) and varying cyclohexanone concentration (i) 1.0 M, (ii) 1.5 M, (iii) 2.0 M, (iv) 3.0 M (v) 4.0 M; (B) constant concentration of 3-APTMS (5 mM) and varying cyclohexanone concentration; (i) 4.0 M (ii) 5.0 M (iii) 6.0 M (iv) 7.0 M (v) 8.0 M.

The morphology and nanogeometry of as synthesized AuNPs were examined by transmission electron microscopy. Fig. 4 shows the TEM images of AuNPs revealing the range of average size of 3–45 nm corresponding to 3-APTMS concentrations of 8 mM, 10 mM, 12 mM and 15 mM at 4.8 M cyclohexanone. These findings show that there is a maximum and minimum limit of 3-APTMS (3–15 mM) and cyclohexanone (1.0–7.0 M) concentrations, beyond which the synthesis AuNPs does not occur. Such specific requirements of 3-APTMS and cyclohexanone could be possible due to micellar characteristics and the requirement of critical micellar concentration of the analytes. Below 3 mM and above 15 mM there was no sign of AuNPs formation at any cyclohexanone concentration. Such limitation may also be linked to specific CMC of cyclohexanone during 3-APTMS mediated conversion of nanoparticles. An optimum concentration of each component is required for AuNPs due to micellar behaviour of 3-APTMS. The proposed mechanism for the 3-APTMS and cyclohexanone mediated synthesis is shown in Scheme 1. Cyclohexanone in the prevailing medium undergoes keto–enol tautomerism. The enolate ion acts as an electron donor to the 3-APTMS capped Au³⁺ ion, which in turn acts as a Lewis acid, leading to the formation of AuNPs. It is important to compare the role of other reducing agents like 3-GPTMS and THF-HPO with cyclohexanone required for the synthesis of AuNPs. Organic moieties (3-GPTMS, THF–HPO and cyclohexanone) used for the synthesis of 3-APTMS and Au³⁺ mediated AuNPs differ in the following respects: (i) 3-GPTMS and cyclohexanone have an affinity for organic medium, whereas THF–HPO have affinity for aqueous medium, (ii) 3-GPTMS is a bulky group, whereas cyclohexanone and THF–HPO are comparatively smaller moieties, (iii) tendency of the reducing agents to form organic–inorganic hybrids during AuNPs synthesis and (iv) 3-GPTMS, THF–HPO and cyclohexanone have different CMC, allowing variable interaction with 3-APTMS capped gold ions during nanoparticle synthesis. The difference in properties of as-synthesized AuNPs using different reducing agents is born out of these differences in their nature and discussed vide infra.

Fig. 4 TEM images of AuNPs made at constant cyclohexanone (4.8 M) and varying 3-APTMS concentrations of 8 mM (red), 10 mM (purple), 12 mM (blue), and 15 mM (violet).

Scheme 1 Proposed mechanism of 3-APTMS and cyclohexanone mediated synthesis of AuNPs.

Effect of organic reducing agents on the dispersibility of AuNPs

Dispersibility of AuNPs largely depends on the medium, which in turn is determined by the apparent polar or nonpolar behaviour of the organic moieties (3-GPTMS/THF–HPO/cyclohexanone). First we discuss the dispersibility of AuNPs made using 3-APTMS and cyclohexanone both in aqueous and organic solvents, viz. water, methanol and dichloromethane (DCM) based on absorption spectra and visual photographs. AuNPs are profusely dispersible in DCM and methanol at all concentrations of 3-APTMS and cyclohexanone. However, the dispersibility of the same in water is affected at higher concentrations of cyclohexanone. Dispersibility of AuNPs in different solvents is shown in Fig. 5(A)–(C). In order to make a detailed investigation on the dispersibility of AuNPs in organic solvents, the absorption spectra of the same as a function of AuNPs concentrations in water, dichloromethane, acetonitrile and ethyl acetate were recorded, as shown in Fig. 6(A). The results show a linear relationship between the absorbance and the concentration of AuNPs with significant variation in λ_max as a function of the nature of the solvents. The dependence of λ_max on the refractive index and polarity index of these solvents is shown in Fig. 6(B). These results justified the use of AuNPs both in aqueous and non-aqueous media. THF–HPO enables the dispersibilty of AuNPs in aqueous and methanolic media. Solvents like DCM and toluene reject the nanoparticle completely, resulting in a two phasic mixture. Dispersibility of AuNPs made with THF–HPO in different media has been presented in ESI (Fig. S2†). Dispersibility of AuNPs with 3-GPTMS as the organic reducing moiety in different media has been reported earlier where the nanoparticles were mostly dispersible in the organic phase with limited dispersibility in water.¹² Dispersibility of as synthesized AuNPs as a function of 3-GPTMS, THF–HPO and cyclohexanone is mainly due to micellar behaviour of 3-APTMS and CMC of the reaction products generated in the reaction medium.

Fig. 5 UV-VIS spectra of AuNPs and their dispersibility in DCM, methanol and water, made using constant concentration of (A) cyclohexanone (4.8 M) and varying concentrations of 3-APTMS: (g) 5 mM, (h) 7 mM, and (i) 9 mM; (B) cyclohexanone (3.8 M) and varying concentrations of 3-APTMS: (j) 5 mM, (k) 7 mM, and (l) 9 mM; (C) 3-APTMS (10 mM) and varying concentrations of cyclohexanone: (m) 0.9 M, (n) 1.8 M, and (o) 3.8 M. Images of corresponding AuNPs sol in the same solvents are shown in insets of (A)–(C).

Fig. 6 (A) UV-VIS spectra of AuNPs in (i) acetonitrile (ii) dichloromethane (iii) ethyl acetate and (iv) water. Inset shows the dependence of absorption maxima (λ_max) on relative concentration. (B) Dependence of λ_max on polarity index (i) and refractive index (ii).

Effect of organic reducing agents on the catalytic ability of AuNPs

One of the important properties as predicted in Scheme 1 is the formation of an organic–inorganic hybrid between the organic amine linked to alkoxysilane and cyclohexanone. The formation of such a hybrid material enhances the catalytic properties of AuNPs compared to those in absence of the same. Therefore, an increase in 3-APTMS concentration will allow an increase in catalytic activity. Cyclohexanone mediated AuNPs synthesized at different 3-APTMS concentrations were checked for their catalytic behaviour as peroxidase mimetics that allow the catalytic oxidation of o-dianisidine in the presence of H₂O₂ as shown in eqn (1).

The catalytic behaviour of AuNPs can be examined from the absorption maxima of the oxidized product of o-dianisidine close to 430 nm. It is important here to understand the catalytic behaviour of 3-APTMS itself. The findings as shown in ESI S3,† justify that 3-APTMS does not enable the formation of the oxidized reaction product of o-dianisidine and confirm its non-catalytic activity. Catalytic behaviour of AuNPs is found to increase with increasing 3-APTMS concentration irrespective of their size, as shown in Fig. 7(A)–(D) recorded in methanol and acetonitrile, justifying the practical usability of nanomaterial in different solvents. In order to confirm the catalytic ability as a function of functional ability, we also examined the peroxidise mimetic behaviour of AuNPs made with increasing concentrations of cyclohexanone maintaining constant concentrations of 3-APTMS yielding AuNPs of increasing nanogeometry. The results of catalytic behaviour of AuNPs recorded in methanol are shown in Fig. 7E. The results reveal an increase in catalytic ability with an increase in nanogeometry when functional ability of the nanomaterial remains consistent.

Fig. 7 UV-VIS spectra of o-dianisidine–H₂O₂ system catalyzed by AuNPs in methanol (A and B) acetonitrile (C and D), justifying the role of functional ability; curves E and F show results as a function of nanogeometry.

Thus, the catalytic behaviour of AuNPs primarily depends on the functional ability of 3-APTMS rather than nanogeometry. Similar properties of AuNPs made from the use of THF–HPO have been observed as reported earlier.¹¹ 3-GPTMS does not result in the formation of an organic–inorganic hybrid, thus catalysis largely depends on nanogeometry irrespective of 3-APTMS concentration. Investigating the use of AuNPs as a peroxidase mimetic resulted in a very high K_m value, which means it is too weak to be used as a peroxidase mimetic. For a material to work as a potential substitute of peroxidase mimetic, it should be stable enough at room temperature and its peroxidase mimetic ability should be equivalent to, or higher than that of the peroxidase enzyme under feasible experimental conditions. In addition to that, the resulting nanocomposite should have homogeneous dispersion in the reaction medium as per the requirements for homogeneous catalysis. Such requirements restrict the practical usability of many such catalytic materials due to poor solubility in the working solvent. Accordingly, there is need to improve the mimetic ability of AuNPs by making nanocomposites of the same having dispersibility for homogeneous catalysis. 3-APTMS and cyclohexanone mediated Prussian blue nanoparticles (PBNPs), behave as artificial peroxidases, the nanocomposite with as-synthesized AuNPs may be a perfect peroxidase replacement because the resulting PBNP–AuNPs nanocomposite is dispersible in aqueous medium. The possibility of nanocomposite formation may be further increased because the reacting systems of both PBNPs and AuNPs possess similar reaction products, resulting in the formation of monophasic dispersions useful for homogeneous catalysis. The PBNP–AuNPs nanocomposite has been characterized by transmission electron microscopy. The microstructure and diffraction pattern, as shown in Fig. 8, confirm the formation of polycrystalline PBNPs–AuNPs nanocomposite.

Fig. 8 TEM image of PBNP–AuNPs nanocomposite. Inset shows the respective diffraction pattern.

It is also noteworthy that if the performance of the nanocomposite could further be enhanced in the presence of other suitable material, manipulating the catalytic ability for desired application could only be possible on the processability of the PBNP–AuNPs suspension. Tris (2,2-bipyridyl) ruthenium [Ru(bpy)] is an important functional optical material, which displays capping affinity with 3-APTMS and retains inherent photoactivity.^21,22 This property of Ru(bpy) prompted us to use it to form a nanodispersion with the 3-APTMS functionalized nanocomposite of PBNP and AuNPs, and indeed the excellent monophasic nanomaterial for homogeneous catalysis was obtained. The schematic representation of peroxidise mimetic activity is shown in Scheme 2, depicting the ratio of various components participating in the formation of the nanocomposite dispersion. It is to be noted from the details given in Scheme 2 that when 3-APTMS (B) is added in methanolic gold salt solution (A), a dark yellow coloured product (C) is formed, justifying the specific interaction of gold ions with 3-APTMS.

Scheme 2 Schematic representation of various stages of nanocomposite formation for homogeneous catalysis.

The formation of the nanocomposite dispersion of as synthesized AuNPs was examined by mixing each component under ambient conditions. Fig. 9A shows the UV-VIS spectra of AuNPs, PBNPs, PBNPs–AuNPs and PBNPs–AuNPs–Ru(bpy). The results demonstrate the formation of a nanocomposite dispersion for practical applications as a peroxidase mimetic. It should be noted that PBNPs–AuNPs–Ru(bpy) show characteristic absorbance between 400–460 nm (Fig. 9A), however, their use as a peroxidase mimetic at 430 nm provides valuable information on probing the oxidized product of o-dianisidine. We have examined the peroxidase mimetic ability of the AuNPs, AuNPs–PBNPs and the AuNPs–PBNPs–Ru(bpy) nanocomposite as shown in Fig. 9B(i)–(iii) respectively for the o-dianisidine–H₂O₂ system. The K_m value for AuNPs was calculated from the results shown in Fig. 9B(iv) and for that of AuNPs–PBNPs and AuNPs–PBNPs–Ru (bpy) from Fig. 9B(v). The K_m value for AuNPs is found to be 30.4 mM, which means it is too weak to be used as a peroxidase mimic and the K_m for PBNPs–AuNPs comes out to be 4.6 mM, which is comparable to that recorded on using HRP under practical experimental conditions.²³ Addition of Ru(bpy) to the nanocomposite further enhances the mimetic ability with a K_m to the order of 2.9 mM. The mimetic ability of the nanocomposite has been very effective in the precise probing of the glucose oxidase catalysed reaction. Based on the peroxidase like property of PBNP–AuNPs–Ru(bpy), we intended to detect the glucose content by utilizing o-dianisidine as a chromogenic substrate analogous to that reported earlier based on the measurement of H₂O₂ as a function of a GOx catalyzed reaction.^24–26 The H₂O₂ formed as a function of the GOx catalyzed oxidation of glucose was monitored spectrophotometrically using the oxidation product of o-dianisidine to indirectly measure the concentration of glucose. A typical glucose concentration–response curve is shown in Fig. 9(C) and the inset shows the corresponding calibration plot. The linear range and lowest detection limit for glucose were found to be 0.5–3 mM and 0.5 mM, respectively.

Fig. 9 (A) UV-VIS spectra of AuNPs (i), PBNPs–AuNPs (ii), PBNPs–AuNPs–Ru(bpy) (iii), inset to (A) shows the visual images of AuNPs (i), PBNP–AuNPs (ii) and PBNP–AuNPs–Ru(bpy) (iii); [B] UV-VIS spectra of the o-dianisidine–H₂O₂ system catalyzed by AuNPs (i), PBNP-AuNPs (ii), and PBNP-AuNPs-Ru(bpy) (iii); kinetic analysis of o-dianisidine–H₂O₂ system catalyzed by AuNPs (iv), PBNPs–AuNPs and PBNPs–AuNPs–Ru(bpy) (v); [C] response curve for glucose detection using glucose oxidase catalyzed formation of H₂O₂ monitored by PBNP–AuNP–Ru(bpy)–o-dianisidine system; inset to [C] shows linear calibration plot for glucose and images show colour change as a function of glucose concentrations.

The findings on peroxidase mimetic activity have been recorded using o-dianisidine as a substrate that shows maximum absorption at 430 nm. This may introduce misleading information due to the absorption maxima of Ru(bpy) falling within a similar range. Accordingly, we used 3,3′,5,5′-tetramethylbenzidine (TMB) as the substrate replacing o-dianisidine. Similar findings as shown in Fig. 10 confirm the catalytic activity of the nanocomposite as a peroxidase mimetic.

Fig. 10 UV-VIS spectra of 3,3′,5,5′-tetramethylbenzidine (TMB)–H₂O₂ system catalyzed PBAuRu(bpy).

Effect of organic reducing agents on the stability of AuNPs

A comparative study on the stability of AuNPs made from the use of 3-APTMS capped gold ions in the presence of 3-GPTMS, THF–HPO and cyclohexanone has been made. The results on the stability of AuNPs made with cyclohexanone are shown in ESI Fig. S4,† justifying that as synthesized AuNPs are stable for >6 months. Although 3-GPTMS mediated AuNPs remained stable for a long time (>90 days), they suffered the problem of hydrolysis due to the Si–O–Si linkage, which was reduced several fold when 3-GPTMS was replaced by cyclohexanone. THF–HPO system is the least stable of the three.

Electrocatalytic ability of AuNPs and their nanocomposite

The results of the peroxidase mimetic ability of AuNPs based on UV-VIS spectroscopy directed us to evaluate their electrocatalytic ability for wider practical application of the nanomaterial. Since AuNPs are not the potential electroactive species required for the precise evaluation of electrocatalytic performance, it was convenient to make a nanocomposite with known potential redox material for such an investigation and the choice of Prussian blue (PB) again seemed to be reasonable. Because the evaluation of electrocatalytic ability for practical application is based on the dynamics of oxidation and reduction of PB–AuNPs and subsequent measurement of electron exchange during electrochemical sensing, the role of 3-APTMS present in the nanocomposite could play a crucial role as 3-APTMS serves as a potential electron donor in polar media (Scheme 1) during AuNPs synthesis. The results shown in Fig. 9B(II) depicted the application of PBNPs made from 3-APTMS and cyclohexanone as reported earlier.¹³ A similar nanocomposite for electroanalytical application may introduce severe problems during efficient electron transfer due to the large concentrations of 3-APTMS and may require detailed investigation. Accordingly, we chose to use conventional Prussian blue (PB) made from potassium hexacyanoferrate and ferrous sulphate to make a nanocomposite with as synthesized gold nanoparticles of two different sizes AuNP₁ (blue) and AuNP₂ (red) in order of increasing nanogeometry. The as prepared PB–AuNPs were adsorbed on graphite particles and incorporated into a graphite paste electrode for understanding heterogeneous electrocatalysis based on probing the electrochemical behaviour of a surface confined redox species. Fig. 11[1](a)–(c) shows the voltammograms of these modified electrodes for PB (a), PB–AuNP₁ (b) and PB–AuNP₂ (c) at different scan rates in 0.1 M phosphate buffer (pH 7.0) containing 0.5 M KCl. The results as seen in Fig. 11[1](a)–(c) show the redox behaviour of Prussian blue as a function of AuNPs nanogeometry and reveal faster electron exchange on increasing the nanogeometry of AuNPs as evaluated from the plots (i) and (ii), shown in insets to Fig. 11[1] of the respective voltammograms. Because PB is used as an artificial peroxidase, the finding of the electrocatalytic reduction of H₂O₂ at the surface of the modified electrodes has also been investigated. Fig. 11[2](a)–(c) shows the voltammograms of these modified electrodes in the absence (1) and the presence (2) of 2 mM H₂O₂ in 0.1 M phosphate buffer, pH 7.0 at the scan rate of 10 mV s⁻¹ for PB (a), PB–AuNP₁ (b) and PB–AuNP₂ (c). The results as shown in Fig. 11[2](a)–(c) clearly reveal subsequent enhancement in electrocatalytic property as a function of AuNPs nanogeometry. Finally, we studied amperometric analysis of H₂O₂ at constant operating potential of 0.05 V vs., Ag/AgCl in 0.1 M phosphate buffer pH 7.0 as shown in Fig. 11[3] for PB (a), PB–AuNP₁ (b) and PB–AuNP₂ (c). The electrocatalytic responses (Fig. 11[3]) again confirm the role of AuNPs nanogeometry on electrocatalysis and justify an increase of the same on increasing nanogeometry.

Fig. 11 [1] Cyclic voltammograms of PB (a), PB–AuNP₁ (b) and PB–AuNP₂ (c) in 0.1 M phosphate buffer of pH 7.0, containing 0.5 M KCl at the scan rates of 0.01, 0.02, 0.035, 0.050, 0.070, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50 V s⁻¹; insets to Fig. 11(1) show the plot of anodic and cathodic current density vs. scan rate (i) and square root of scan rate (ii) respectively; [2] cyclic voltammograms of PB, PB–AuNP₁ and PB–AuNP₂ in absence (1) and the presence (2) of 2 mM H₂O₂ in 0.1 M phosphate buffer of pH 7.0, containing 0.5 M KCl at the scan rate of 0.01 V s⁻¹; [3] amperometric response of H₂O₂ analysis at 0.05 V vs. Ag/AgCl in 0.1 M phosphate buffer of pH 7.0, containing 0.5 M KCl, inset to [3] shows the linear range of calibration curves for PB (a), PB–AuNP₁ (b) and PB–AuNP₂ (c).

Conclusions

To summarize, we present a comparative study on the role of organic reducing agents (3-GPTMS, THF–HPO and cyclohexanone) during 3-APTMS mediated controlled synthesis of AuNPs. As synthesized AuNPs differ from each other in properties such as dispersibility, catalysis, stability etc., thus making them available for use in various applications. In a bid to get 3-APTMS mediated AuNPs having better stability and dispersibility than with THF–HPO and 3-GPTMS, we used cyclohexanone, which ensures the formation of an organic–inorganic hybrid and facilitates the peroxidase mimetic ability of AuNPs. AuNPs can be made dispersible in different solvents (organic or aqueous) by adjusting the constituents' (3-APTMS/cyclohexanone) ratio. Thus, the use of different reducing agents having compatibility with different solvents results in AuNPs with significant variation in properties from each other, presenting the option to select AuNPs for specific applications either in aqueous or organic media. These nanoparticles display functional ability to form monophasic nanocomposites for homogeneous catalysis with Prussian blue nanoparticles and ruthenium bipyridyl for specific application as peroxidase mimetics. In addition to that, the nanoparticles also enable the formation of nanocomposites with Prussian blue useful as heterogeneous redox catalysts displaying excellent electrochemical properties as a function of nanogeometry. Both mimetic and electrocatalytic ability could be explored for probing glucose oxidase catalyzed reactions, justifying the potential viability in biomedical applications.

Authors contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

Abbreviations

3-APTMS	3-Aminopropyltrimethoxysilane
3-GPTMS	3-Glycidoxy propyltrimethoxysilane
THF-HPO	Tetrahydrofuran hydroperoxide
DCM	Dichloromethane
PBNPs	Prussian blue nanoparticles
AuNPs	Gold nanoparticles
Ru(bpy)	Ruthenium bipyridyl

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Footnote

† Electronic supplementary information (ESI) available: 3-APTMS alone does not enable the synthesis of AuNPs under similar conditions even after 24 hours (ESI Fig. S1). The dispersibility of gold nanoparticles made with 3-APTMS and THF–HPO in methanol, acetonitrile, dichloromethane (DCM) and toluene is presented in Fig. S2, which clearly indicates better dispersibility in methanolic medium and undergoes a red shift in acetonitrile and results in a biphasic system in DCM and toluene. 3-APTMS is not catalytic in nature as presented in Fig. S3. The results on the stability of AuNPs made with cyclohexanone are shown in ESI Fig. S4. See DOI: 10.1039/c4ra07624a

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