Multiamino polymeric capping of fluorescent silver nanodots as an effective protective, amphiphilic and pH/thermo-responsive coating

Abstract

Herein the in situ photochemical synthesis of silver nanodots (AgNDs) coated with new smart multi-amino polymers is reported. This fast, clean and highly efficient process provides new amphiphilic highly photoluminescent nanohybrids and smart properties (thermal, pH and light response). Furthermore, the polymeric coating over the silver nanoclusters avoids the strong tendency of silver nanodots to aggregate, gives protection against quenchers, tunes fluorescence emission and facilitates handling, storage and maintenance of the hybrids for long periods of time without loss of their photophysical properties. Therefore, a new family of multi-amino copolymers based on 2-amino-ethyl methacrylate protected with the t-butoxycarbonyl (Boc) group (Boc-AEMA) with a majority thermoresponsive monomer, 2-(2-methoxyethoxy)ethyl methacrylate (MEO₂MA), is described. The copolymers with well-defined structures were synthesized by atom transfer radical polymerization (ATRP) and once unprotected, were used for the efficient surface functionalization of silver.

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Introduction

Silver nanodots (AgNDs) are a new class of luminescent hybrid nanoparticles with photophysical properties similar to quantum dots but with much less overall size and without any toxicity associated with heavy atoms. Silver nanodots, also named silver nanoclusters, silver clusters, silver nanocrystals or silver quantum dots, are few-atom clusters of reduced silver atoms (from tens to hundreds of atoms) which show high luminescence quantum yields, large molar extinction coefficients and excellent photostability.^1,2 However, the term nanodot should only be applicable to these well-defined fluorescent nanoclusters encapsulated or free and stable in a colloidal solution as the first one described by Zheng and Dickson in 2002.³

The synthesis of stable, water soluble individual silver nanodots will greatly facilitate the use of these photoactivated nanomaterials, both as optical storage elements and as small, extremely bright, photostable new fluorophores, while simultaneously expanding the accessibility of single molecule methods³ and other important applications as biological analysis, imaging and therapeutic field due to their promising photophysical properties.^4–6 However, the challenge today is to obtain high stable, monodispersed and well-defined silver nanodots, preventing the oxidation pathway both by atmospheric oxygen and by electron acceptor in solution and, also, avoiding its irreversible agglomeration tendency due to thermodynamically favored large silver nanoparticle generation losing their special photophysical properties. This difficult task could be achieved by the correct choice of a suitable stabilizing ligand attached to the nanoparticle surface. Moreover, the nanodot wavelength emission tunability depends of the nature of the ligands used for their synthesis and stabilization. Until date, as ligand have been used: synthetic polymers,^7–11 small molecule ligands,^12–14 enzymes,¹⁵ peptides and single-stranded DNA.^14,16–19 Thus, in the most part of the cases carboxylic acid groups are present and the silver nanodot emits in the red region; meanwhile, when only amine groups are in the reaction media the emission is shifted to yellow.^3,12,20 In fact, in the case of synthetic polymers as ligand the poly(meth/acrylic acid) (PMAA or PAA) and copolymer based on acrylic acid¹¹ are the most used to silver nanodot stabilization being so in general red emitters.

Previous studies by our research group about copolymers based on a methacrylic monomer with pendant protected thiol group (AcSEMA) synthesized by ATRP have allowed the multidentate functionalization of several type of nano-objects such as gold metal nanoparticles,²¹ quantum dot (QDs),^22,23 up-conversion nanoparticles (UCNPs),²⁴ and even graphene.²⁵

Thanks to the use of a living polymerization procedure (ATRP), the polymers had low polydispersity and this together with the multidentate functionalization allowed achieving a homogeneous and compact coating with only a small increase of the hydrodynamic radius. Besides, the use of polymer capping in the design of nanohybrid materials has the advantage that the polymer endows with its own properties to the whole material. This strategy permitted to transfer the inherent properties of the polymer to the hybrid, such as water solubility, thermo- and pH-responsiveness and so on. Based on these previous works, in this contribution we develop a new approach for the synthesis and functionalization of luminescent silver nanodots based on the use of aminated thermoresponsive copolymers as reducing agent and stabilizing ligand. In a first step, copolymers based on amino protected methacrylic monomer (Boc-AEMA) and a thermosensitive monomer (MEO₂MA) synthesized by ATRP are described and furthermore, after deprotection, copolymers with the free pendant amine group are used for the functionalization and stabilization of Ag nanodots. So, the in situ photochemical synthesis of silver nanoclusters in presence of the new copolymers based on MEO₂MA and AEMA (2-amino ethyl methacrylate) offers the possibility to attain yellow emitter nanohybrids with excellent stability in the medium and also new colloidal properties in aqueous medium (amphiphilic properties, pH and temperature responsiveness). Until our knowledge, this is the first example in the literature of silver nanodots which emission properties can be modulated with the temperature in colloidal solutions.

Experimental section

Synthesis of 2-(tert-butoxycarbonylamino)ethyl methacrylate (Boc-AEMA) monomer

The synthesis was done following a procedure similar to that described in the literature.²⁶ To an ice-cooled solution consisting of 1.3 mL of triethylamine (0.94 g, 9.3 mmol) and 0.96 mL of Boc-ethanolamine (1 g, 6.2 mmol) in 10 mL of anhydrous dichloromethane, methacryloyl chloride (0.67 mL, 6.3 mmol) was dropwise added while the solution was maintained under constant agitation. After that, the ice bath was removed and the reaction was kept at room temperature (Scheme 1). The reaction was monitored by thin layer chromatography (TLC-hexane/ethyl acetate 8/2).

Scheme 1 Synthesis of 2-(tert-butoxycarbonylamino)ethyl methacrylate (Boc-AEMA).

When the reaction was completed, the solution was filtered and the product washed several times with water. The organic phase was dried over anhydrous magnesium sulphate, filtered and the excess of solvent was removed by rotary evaporation obtaining a yellowish-white solid which was purified by recrystallization from hexane/dichloromethane (9 : 1 v/v) mixture. 650 mg, yield 65%. ¹H-NMR: (400 MHz, CDCl₃) δ (ppm) 1.45 (s, 9H, (CH₃)₃–C), 1.95 (s, 3H, CH₃–C), 3.44 (m, 2H, CH₂–N), 4.2 (t, 2H, CH₂–O), 4.76 (s, 1H, NH–C=O), 5.59 (m, 1H, CHH=C), 6.12 (m, 1H, CHH=C).

Synthesis of p(MEO₂MA-co-Boc-AEMA)-x

The copolymerization of the synthetized amino-protected methacrylic monomer (Boc-AEMA) in different molar proportions (x = 10 and 25 mol%) with a thermoresponsive monomer, (MEO₂MA) was carried out by ATRP in benzonitrile (50% w/w) at 70 °C with a constant monomer/initiator/ligand/catalyst ratio of 65/1/1/1 (Scheme 2). A typical procedure is described for sample with 10 mol% of AEMA: the degassed monomers, MEO₂MA (1.76 g, 7.68 mmol) and Boc-AEMA (0.24 g, 1.94 mmol), the ligand, 1,1,4,7,10,10-hexamethyltriethylenetetramine HMTETA (36.5 μL, 0.134 mmol) and the solvent benzonitrile (2 mL) were added to a dry pyrex tube with CuCl (13 mg, 0.134 mmol). After this mixture was degassed by bubbling nitrogen for 20 min, the initiator ethyl-2-bromoisobutyrate, EBrⁱB (19.7 μL, 0.134 mmol) was introduced into the ampoule using degassed syringe to start the polymerization. The ampoules were immediately placed in a thermostatic oil bath at 70 °C, regulated with a precision of ±0.1 °C. At a selected time (47 min), the polymerization was stopped, cooled, and quenched with chloroform. Then, the crude was passed through a neutral alumina column to remove the catalyst. The solution was concentrated by rotary evaporation and the polymer was precipitated adding the solution to a large excess of n-hexane under stirring. The precipitated products were decanted and dried under high vacuum until a constant weight was reached. Total monomer conversions were measured gravimetrically.

Scheme 2 (A) Synthesis of p(MEO₂MA-co-Boc-AEMA) copolymers via ATRP and (B) deprotection to obtain p(MEO₂MA-co-AEMA) amino functionalized copolymers.

Synthesis of p(MEO₂MA-co-AEMA)-x copolymers by deprotection with TFA

Boc-protecting groups of the side chains of the copolymers can be easily cleaved under mild acidic conditions, using a solution of trifluoroacetic acid (TFA) in dichloromethane (DCM), to obtain polymers with primary amine groups (Scheme 2).²⁷ In a typical example, a solution of protected polymer in dichloromethane (50 mg mL⁻¹) was prepared. The solution was stirred for at least 10 min to ensure that the mixture was fully homogeneous. Then, in an ice-water bath, 0.5 mL TFA (100 μL mg⁻¹) were added dropwise and the solution stirred for 2 h to ensure complete deprotection of the amino groups. The reaction was washed with saturated sodium hydrogen carbonate to neutralize the acidic solution of TFA and, subsequently, washed several times with water. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent removed at reduce pressure until complete evaporation. Finally, the resulting Boc-deprotected p(MEO₂MA-co-AEMA) copolymer was isolated by precipitation from hexane and dried under vacuum.

Synthesis of silver nanodots functionalized with the amino copolymers, AgNDs@p(MEO₂MA-co-AEMA)-x

Luminescent AgNDs functionalized with multi-amino thermoresponsive copolymers were obtained photochemically using a photoreactor irradiation source (UVP, CL100-UV-crosslinker model) equipped with four lamps of UVA irradiation with light emission centered at 365 nm which generate a power of 29 W m⁻², experimentally measured with a spectroradiometer v2.2 (LuzChem). We have followed the procedure previously described by Maretti et al.,¹² but now using the multi-amino copolymers as unique stabilizers for the synthesis of hybrid AgNDs. The experiments were performed in quartz cuvettes of 2 mm of optical path (Hellma Analytics), which allow monitoring the reaction until the end and prevent outside scale signals in the absorption spectrum by the rapid growth of the concentration of the nanoclusters in the medium with the irradiation time. The experimental conditions for obtaining highly fluorescent and stable AgNDs were optimized varying several parameters that can influence the reaction, such as photoinitiator (type and concentration), incident light (wavelength, intensity and irradiation time), concentration of silver precursor and amine content in the copolymer. It has been concluded that Irgacure 2959® (I-2959) was the best initiator when irradiated in the photoreactor with four UVA lamps at 365 nm (29 W m⁻²), with appropriate kinetics, and maintaining the reactants at an equimolar concentration in toluene.

Reactions were prepared in the dark by adding all starting reagents: the photoinitiator (Irgacure 2959®, I-2959), the silver salt as precursor (silver trifluoroacetate, CF₃COOAg) and the p(MEO₂MA-co-AEMA)-x multi-amino copolymer as ligand. Reaction was studied maintaining equimolecular concentration of Ag/I-2959/amine groups at 2 mM and 5 mM, using toluene as solvent. In a typical experiment toluene solutions containing, for example, 2 mM CF₃COOAg, 2 mM I-2959 and 2 mM of final molar concentration of amino groups in the copolymer [thus 0.2 mM concentration of copolymer in the case of p(MEO₂MA-co-AEMA)-10 or 0.08 mM for p(MEO₂MA-co-AEMA)-25] were deoxygenated by purging with argon for 20 min followed by exposure to UVA irradiation at the photoreactor. The reaction evolution was monitored by UV/Vis spectroscopy at the Ag plasmon absorption band (437 nm) and fluorescence spectroscopy of the emission signal (520 nm) in the front-face mode by orienting the sample at angles of 35° and 55° with respect to the excitation and emission beams by using 2 mm path length quartz cuvette. Once the AgNDs have been formed, reaching the maximum value in absorbance/emission signals, the reaction was open to air. The nanohybrids synthesized in this manner are stable for at least one year, even in the presence of air and/or without any protection from ambient light.

In order to evaluate thermo- and pH-responsiveness of the new hybrids the toluene solutions of the hybrids synthesized were concentrated by rotary evaporation and transferred to aqueous buffer solutions at different pHs.

Quenching experiment

In order to know the amount of thiophenol (quencher) that the functionalized nanoclusters are able to support before losing their emission properties, experiments were conducted by adding small aliquots (5 μL) of thiophenol (TPSH, 50 μM) to toluene solutions of both AgNDs@p(MEO₂MA-co-AEMA)-x hybrids. After each addition, absorbance at 437 nm was measured and compared with that of AgNDs coated with a molecular amine ligand, cyclohexylamine (CHA).

Results and discussion

A new strategy for the functionalization of silver fluorescent nanodots (AgNDs) was designed based on the synthesis of a new family of p(MEO₂MA-co-Boc-AEMA) copolymers with defined structure through controlled radical polymerization by ATRP. For the synthesis, amino functionalities should be protected to prevent undesirable side reactions. After deprotection, amino groups provide the polymer with capability of acting as a ligand for the synthesis of AgNDs and also offer the inherent properties of new copolymers to the final hybrid (solubility and stability in water and ability to response to the pH and/or temperature changes of the medium) without prejudice to their photoluminescent properties.

To obtain polymers with thermo-responsiveness in water, Boc-AEMA was copolymerized with MEO₂MA which belongs to a family of monomers for the synthesis of polymers with thermal response. Among these new thermoresponsive polymers, p(MEO₂MA) has become one of the most studied having an T_cp around 26 °C.²⁸ This value can be easily modulated by copolymerization of MEO₂MA with other monomers.²⁹ Furthermore, polymers based on MEO₂MA combine thermoresponsive properties with others such as not toxicity or anti-immunogenicity. In order to study the possible differences and the effect of the content of amine on the functionalization of AgNDs two different copolymers were synthesized by varying the percentage of Boc-AEMA. The experimental ATRP conditions used in the preparation of these novel multi-protected amino copolymers and their characterization are shown in Table 1.

Table 1 Data obtained from the characterization of the copolymers p(MEO₂MA-co-Boc-AEMA)-x with different techniques^a

Sample	FBOC-AEMA	Conv. (%)	Mw (g mol^−1)	Mw/Mn	Tcp (°C)
a FBOC-AEMA, experimental molar fraction composition in the copolymer (calculated by ^1H-NMR), conversion (%), Mw, molecular weight average (calculated by SEC), Mw/Mn polydispersity index. The cloud point temperature (Tcp) was measured in water at neutral pH.b Values recalculated for the unprotected polymers in function of their molar composition.
p(MEO2MA-co-Boc-AEMA)-10	10	72	12 230	1.12	21
p(MEO2MA-co-Boc-AEMA)-25	25	81	15 060	1.24	11
p(MEO2MA-co-AEMA)-10	—	—	11 700^b	1.12	45
p(MEO2MA-co-AEMA)-25	—	—	13 415^b	1.24	68

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The chemical structure and the molar fraction composition of p(MEO₂MA-co-Boc-AEMA)-x copolymers (where x is referred to the mol% of Boc-AEMA) were analyzed by ¹H-NMR and FTIR. Fig. 1 depicts the ¹H-NMR spectra where the signals corresponding to both monomers are indicated. As it can be observed, the intensity of the signal associated with AEMA increases when increasing its feed molar fraction (f_BOC-AEMA), and allows quantifying the experimental molar composition of the copolymers (F_BOC-AEMA), which is in good agreement with the feed molar fraction, indicating similar reactivity between both monomers during polymerization.

Fig. 1 ¹H-NMR spectra for p(MEO₂MA-co-Boc-AEMA)-x copolymers (above) and the deprotected p(MEO₂MA-co-AEMA)-x copolymers (below), with 10 mol% (red lines) and 25 mol% (blue lines) of aminated comonomer.

In the FTIR spectrum of p(MEO₂MA-co-Boc-AEMA)-x [Fig. S1 in ESI†], the absorption peaks at 1366 and 1523 cm⁻¹ attributed to the symmetric bending vibration of t-butyl group and NH deformation (amide II band) disappeared for the deprotected copolymer, at the time that a broad band at 1600 cm⁻¹ appears due to the free NH₂ functionalities.

The SEC analysis of the copolymers indicated that the polymerizations were well controlled. The molecular weight distributions were narrow and therefore, polydispersity indexes were lower than 1.25 (Table 1). The deprotection of p(MEO₂MA-co-Boc-AEMA)-x with TFA yield a new family of copolymers, p(MEO₂MA-co-AEMA)-x, with free amine groups. The complete deprotection was confirmed by ¹H-NMR by the disappearance of the tert-butyl signal at 1.4 ppm in the ¹H-NMR spectra (Fig. 1).

Moreover, the synthesized polymers are thermoresponsive and exhibit cloud point temperature, T_cp, in water solution. This property is characterized by a sudden decrease of the polymer solubility when temperature is raised above a critical temperature.

Experimentally this phase separation can be followed determining the cloud point temperature (T_cp) by measuring absorbance changes under visible light. In Table 1, T_cp values in aqueous solutions at pH 7 (1 mg mL⁻¹), for copolymers with protected amine and after deprotection, are collected. A decrease of the cloud point compared to p(MEO₂MA) homopolymer (∼26 °C) is observed, which can be attributed to the increase of the hydrophobicity due to the t-butyl groups of Boc.

Therefore, after hydrolysis, T_cp values of both p(MEO₂MA-co-AEMA)-x copolymers exhibit a noticeable increase because amino groups enhance polymer hydrophilicity. In addition, the thermoresponsive behavior of the new copolymers becomes dependent on the pH due to the protonation of amino groups at low pH. The T_cp value can be modulated by varying the pH of the aqueous solution, as it is shown in Fig. S2 in ESI.† In the case of a 10 mol% of amine groups, the increase of pH decrease the cloud point while the copolymer with higher amino groups content, becomes completely soluble at acid pH, only at basic pH the cloud point is detectable. At low pH there is a greater amount of hydrophilic protonated amino groups (–NH₃⁺), which provides the polymer of a higher solubility and even suppresses the LCST. With increasing pH, a modulation of the T_cp as function of pH is attained for p(MEO₂MA-co-Boc-AEMA)-10.

The unprotected polymers have been used for the photochemically in situ synthesis and functionalization of fluorescent silver nanodots with polymeric coating [AgNDs@p(MEO₂MA-co-AEMA)-x], taking advantage of the great affinity of amino groups for the surface of metal nanoparticles, following the method described by Scaiano group for molecular amines coating as shown in Scheme 3.¹²

Scheme 3 Schematic representation of the in situ photochemical synthesis of new AgNDs@p(MEO₂MA-co-AEMA)-x hybrids.

The procedure involves the photochemical activation of the photoinitiator (I-2959) in toluene with UVA light (at 365 nm) to yield free radicals, namely ketyl radicals which are known as strong reducing agents toward metal ions,^30,31 that reduce the silver salt precursor, CF₃COOAg, in the presence of the amino groups of the polymer. Then, AgNDs can be generated, stabilized and coated by the polymer in one step.

The first experimental evidence that reflects the formation of silver nanoclusters in our systems can be displayed on the absorbance and emission spectra of the samples obtained with the irradiation time (Fig. 2).

Fig. 2 Evolution of (A) absorption and (B) emission spectra with the irradiation time with four lamps at 365 nm (29 W m⁻²) of a toluene solution of Ag/I-2959/p(MEO₂MA-co-AEMA)-25 5/5/0.2 mM under argon. Reaction directly performed and monitorized in a 2 mm path length quartz cuvette.

In all cases, a characteristic absorbance peak centered on 437 nm is observed together with an intense emission band located at 520 nm that can be attributed to the formation of Ag⁰ nanoclusters stabilized by the amino functional groups.^12,20 Fig. 2A shows the growth of the absorbance band centered at 437 nm with irradiation time. The band that appears is clean, without occurrence of other bands due to formation of any other kind of silver nanoparticles or aggregates. The fluorescence emission provides the same behavior, emission intensity increases with the irradiation time. Any displacement of the emission band occurs until 300 s of irradiation time was reached, time in which begins to drop due to the formation of not fluorescent silver nanoparticles (Fig. 2B).

Following this experimental procedure a study of the influence of the concentration of the reactants on the kinetics of formation of AgNDs was done (detailed in Fig. 3).

Fig. 3 Growth of the 437 nm absorption band (A) and 520 nm emission band (B) corresponding with the formation of AgNDs@p(MEO₂MA-co-AEMA)-x hybrids upon light irradiation time (365 nm, four lamps, 29 W m⁻²) as a function of different millimolar proportions of the reactives: silver trifluoro acetate/I-2959/p(MEO₂MA-co-AEMA)-x. All reactions performed in toluene in 2 mm quartz cuvette.

For both polymers, it is observed that increasing silver and/or polymer concentration, the absorbance and fluorescence increases until a value, above this value a decay of absorbance and fluorescence is detected. From these results it follows that the optimal reagent concentration for synthesis would be 5/5/0.5 mM for reaction mixtures Ag/I-2959/p(MEO₂MA-co-AEMA)-10 and 5/5/0.2 mM for reaction mixtures Ag/I-2959/p(MEO₂MA-co-AEMA)-25. From the above results is also observed as the content of the amino groups presents in the polymer exerts a marked influence on emission properties and the kinetics of obtaining fluorescent hybrids. On the one hand, the fluorescent quantum yields of the AgNDs@p(MEO₂MA-co-AEMA)-25 hybrids ranging from 1.3–2.1%, while AgNDs@p(MEO₂MA-co-AEMA)-10 hybrids reach values of 4.8%, all values expressed relatives to Fluorescein as standard. Moreover, high contents of amino groups in the polymer cause a lower rate of AgNDs formation but also the fluorescence loss occurs much later with the irradiation time.

These results led us to study the stability of hybrids under light irradiation at 365 nm depending on the type of polymeric coating employed in the synthesis. It is well known that silver nanoclusters are highly reactive in solution and very sensitive to UV light.³¹

The study of irradiation stability of the hybrids is shown in Fig. 4A and B, which shows both absorption evolution and fluorescence evolution under irradiation, respectively. In both cases there are an increase, maintenance for sometimes and then a fall down. The first increase obeys to the silver nanodot formation while the plateau is the irradiation time where the nanodots are stables before the formation of new non fluorescence species. Fig. 4B depicts that for AgNDs@(MEO₂MA-co-AEMA)-10 hybrids the emission begins to fall after around 30 s of light irradiation, while in the case of AgNDs@(MEO₂MA-co-AEMA)-25 hybrids, the emission properties remain intact until 240 s of irradiation time with UV light.

Fig. 4 Photostability study of AgNDs@p(MEO₂MA-co-AEMA)-x hybrids (irradiation with four lamps at 365 nm, 29 W m⁻²). (A) Absorption spectra evolution for AgNDs@p(MEO₂MA-co-AEMA)-25 in toluene and (B) emission properties versus irradiation time for AgNDs@p(MEO₂MA-co-AEMA)-10 (red circles) and AgNDs@p(MEO₂MA-co-AEMA)-25 (black squares).

The decrease of the fluorescence with the irradiation time is attributed to the formation of large non-fluorescent silver nanoparticles from the aggregation of silver nanoclusters. As show in the Fig. 4A, the appearance of a new band centered at 495 nm in the absorption spectrum reveals experimental evidence of the formation of these non-fluorescent silver NPs.³¹

Since the photostability of the hybrids is influenced by the nature of the polymer coating, we wonder whether this factor will also influence its chemical stability. Experiments of fluorescence quenching of both hybrids have been conducted. As AgNDs are highly sensitive to the presence of other reactive nucleophilic groups (e.g., NH₃, SH, and CN),³¹ a study of the variation of fluorescence emission versus the presence of a quenching agent, thiophenol (TPSH), has been done (Fig. 5A).

Fig. 5 (A) Evolution of the fluorescence emission of AgNDs@p(MEO₂MA-co-AEMA)-10 hybrids in toluene with the addition of thiophenol (TPSH). (B) Stern–Volmer representation for AgNDs@p(MEO₂MA-co-AEMA)-x hybrids and AgNDs@CHA quenched by TPSH (where F₀ = fluorescence area emission without quencher and F = fluorescence area emission the presence of an amount of quencher). Inset: detailed experiment at the start of the reaction.

As shown in Fig. 5B, there is a clear difference in the behavior of the hybrids against the quencher agent (TPSH). The coating with p(MEO₂MA-co-AEMA)-10 show higher protection against the quencher, as their emission is stable even after adding higher amounts of TPSH. Anyway, both polymeric hybrids are much more stable, in the presence of the TPSH quencher, than the AgNDs stabilized with a molecular amine (cyclohexylamine, CHA). The photoluminescent stabilization improvement achieved with the multidentate polymeric coating versus the use of monofunctional ligands in brush type coverage that are more accessible to outsiders agents and was consistent with previous results obtained by our research group for other photoluminescent nanoparticles such as QDs.²³ The resistance against the environment, photo- and chemical stability, seems to be related to both the polymer architecture coating of the nanoclusters and the content of amino groups present in the copolymer.

Additionally, the functionalization of these AgNDs with multi-amino smart polymers provides to the new nanohybrids with the inherent properties of the polymer to the nanoparticle. Thus, both synthetized hybrids of AgNDs@p(MEO₂MA-co-AEMA)-x present on one hand, an advantageous amphiphilic nature which makes them soluble in both aqueous and organic solvents and, on the other hand, keep their capacity to respond to external stimuli, thermo-responsive and pH-responsive, as discussed below.

Highlight that these hybrids retained their fluorescence emission properties in water. Fig. 6 collect the comparison between the spectroscopic properties of the AgNDs in both types of solvents, toluene and water, where we can observe as hybrids exhibit a blue shift of the absorption spectrum in water relative to toluene, while, on the contrary, the emission bands undergoes an abrupt bathochromic shift (ca. 625 nm, Fig. 6), which translates to these AgNDs reach Stokes shifts over 100 nm.

Fig. 6 Normalized absorbance (dashed lines) and fluorescence emission (solid lines) of AgNDs@p(MEO₂MA-co-AEMA)-10 both in toluene (pink) and in water at pH-5.7 at 4 °C (blue) or 60 °C (red).

In both cases, hybrids exhibit a behavior that resembles the observed for the parent copolymers (Fig. S2 in ESI†) excepting that the hybrids show cloud points about 5 °C lower than those exhibited by copolymers. If we keep in mind that amino groups in the copolymers resulted in an increasing of the hydrophilicity and in consequence increasing of the cloud point, this fact should indicate a decrease of the free amino groups because a part of them are attached to the silver surface. Thus, the hybrid with 10 mol% of amino groups (AgNDs@p(MEO₂MA-co-AEMA)-10) still presents a good hydrophobic/hydrophilic balance and exhibit a nice modulation of its solubility as function of pH and temperature. In this case, almost no variation of absorbance with temperature at acid pH is observed because the solubility is high in the temperature range studied. However the thermal response is remarkable at basic pH, where the hybrid solubility suddenly decreases above 40–45 °C (Fig. 7A). On contrary, the AgNDs@p(MEO₂MA-co-AEMA)-25 does not exhibit that is called thermoresponsiveness at any pH (Fig. 7B), the absorbance does not experience sharp changes with temperature, but gradually increases. Therefore, the collapse of the polymer is not detected with the temperature which indicates a loss of both thermo- and pH-response of these hybrids due to high content of no thermosensitive monomer present in the copolymer.

Fig. 7 Variation of the absorbance at 600 nm with the temperature of the medium at different pHs for the hybrids (A) AgNDs@p(MEO₂MA-co-AEMA)-10 and (B) AgNDs@p(MEO₂MA-co-AEMA)-25 respectively.

The thermal behavior of the hybrids in aqueous solution has also been analyzed by DLS and sTEM (Fig. 8A). It is clearly seen that as at temperatures below the T_cp the hybrid nanoparticles are small, while a temperature above T_cp the hybrids form large aggregates as measured by sTEM and DLS performed for each hybrid in water.

Fig. 8 (A) Variation of hydrodynamic size of the hybrid at pH 7 as a function of temperature. Inset: sTEM images of the sample AgNDs@p(MEO₂MA-co-AEMA)-10 in water at two temperatures, below and above the LCST. (B) Fluorescence emission spectra of hybrid AgNDs@p(MEO₂MA-co-AEMA)-10 in water at pH 5.7 recorded for several consecutive cooling–heating cycles (4 °C ↔ 60 °C). (C) Change in the normalized fluorescence emission in each cycle and (D) changes in normalized fluorescence emission with the pH and temperature for sample AgNDs@p(MEO₂MA-co-AEMA)-10. Inset: photograph of aqueous solutions of hybrid AgNDs@p(MEO₂MA-co-AEMA)-10 at different pHs under visible light.

Keep in mind that the samples for sTEM were always prepared from the same hybrid solution in water, so only the differences can be attributed to the effect of temperature.

Noteworthy that although in the case of AgNDs@p(MEO₂MA-co-AEMA)-25 hybrids the increase of absorbance with temperature is minimized (Fig. 7B), the aggregation also occurs and is detected both by DLS and sTEM (Fig. 8A), but there aggregates are soluble thankfully the large amount of NH₂ groups present in the copolymer. Therefore the fluorescence emission of the hybrids in water has been compared, for each pH, at two selected temperatures (4 °C and 60 °C), above and below the LCST, respectively. The obtained response has always followed the same pattern as showed in Fig. 8D and S3 in ESI,† a significant increase in the fluorescence emission is detecting when the collapse of the polymer occurs regardless of the pH of the medium. This behavior was totally reproducible and the variation of the fluorescence is completely reversible over several consecutive cycles of heating–cooling whenever the pH of the medium was acid; otherwise, working under basic pH conditions, after the first collapse of hybrid irreversible precipitation of the formed aggregates occurs (as shown in Fig. 8C).

So, the multi-amino polymeric coating offers the possibility to attain silver emitter nanohybrids with excellent stability in the medium and also new colloidal properties in aqueous medium (amphiphilic properties, pH and temperature responsiveness). Until our knowledge, this is the first example in the literature of silver nanodots which emission properties can be modulated with the temperature in colloidal solutions. Only similar behavior has been found before by our group for the functionalization of QDs with multi-thiolated methacrylic copolymers also based on MEO₂MA. The QD nanohybrids showed good water dispersability and an abrupt increase of the fluorescence emission when the polymer collapse at acidic media.²²

Conclusions

In summary, in this work thermoresponsive copolymers with amino functionalities have been successfully employed to obtain high yellow emitter and smart silver@polymer nanodots by in situ photochemical synthesis. From our study, it is clear that a suitable concentration of all reagents together with the appropriate synthetic protocol is needed in order to obtain fluorescent nanodots instead non-fluorescent conventional Ag nanoparticles and aggregates. In contrast with low molecular ligands, the multidentate polymer coating offers remarkable protection against quenchers and optical photostability under UV/Vis light that depend on amine content, facilitating the hybrids stabilization, handling, storage and maintenance for long periods of time, up to one year, without any deterioration of their photophysical properties. Moreover, it has been evidenced that amine content in the copolymer as low as 10 mol% is more suitable to obtain high fluorescent silver nanodots than when 25 mol% of amine in the copolymer was used. In addition, colloidal silver nanoclusters protected with copolymers with 10 mol% of amine, presented an amazing luminescent response with changes of temperature and pH in aqueous medium. All this, combined with the change in water solubility as function of temperature and pH that exhibited the hybrids due to the polymer wrapping, envisioned a great promise for their applications in bioimaging, biological analysis and nanobiomedicine for vehiculization and monitoring of bioactive substances and in new therapeutic procedures.

Acknowledgements

This work received financial support from the Ministerio de Economía y Competitividad (MINECO) through the Project MAT2014-57429-R.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Description of materials and characterization method as well as additional figures are included. See DOI: 10.1039/c6ra12024e

‡ Current address: Instituto IMDEA-Energía, Parque Tecnológico de Móstoles, Avda. Ramón de la Sagra, 3, E-28935 Móstoles-Madrid (Spain). E-mail: E-mail: marta.liras@imdea.org

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