Organic–inorganic nanohybrids and their applications in silver extraction, chromogenic Cu²⁺ detection in biological systems, and hemolytic assay

Abstract

Fluorescent organic nanoparticles (FONPs) were prepared from an organic tripodal Schiff base ligand (OTL) in pure water by implementing the re-precipitation method. The FONPs were perfectly spherical in shape and their size increased with the increase in the amount of OTL. Bigger FONPs were more fluorescent than the smaller ones and their fluorescent emission showed a strong temperature dependence due to their amorphous nature. They demonstrated specific binding towards Ag⁺ ions which resulted in a strong reducing ability from Ag(I) to Ag(0) to produce organic–inorganic hybrid nanoparticles (i.e. Ag@FONPs). FONPs and Ag@FONPs were characterized by UV-visible, fluorescence, transmission electron microscopic (TEM), NMR, IR, and XRD studies. We showed that the FONPs can be repeatedly used for the extraction of Ag in a systematic manner, while Ag@FONPs can be used for the chromogenic detection of Cu²⁺ in biological systems even in the presence of other metal ions. A detailed hemolytic analysis demonstrated that the smaller Ag@FONPs can also be suitable drug release vehicles in systematic circulation in comparison to the bigger ones.

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Introduction

Several pharmaceutical and consumer products use a range of organic compounds for their diverse requirements. To improve the performance of organic products especially in the aqueous phase while keeping environmental concerns under consideration, researchers use formulations with surfactants, other composite mixtures, and surface decoration. The conversion of organic compounds into organic nanoparticles (ONPs) dramatically increases their applications in diverse environments including biological systems due to their interesting medicinal, electronic, and optical properties.^1–5 Fluorescent organic nanoparticles (FONPs) are known to have markedly improved optical and photo-physical properties compared to their parent organic molecules, and hence can be used as chemosensors for the detection of biologically important ions, biomolecules, molecular imaging probes etc.^6–9 Recently, Petkau et al. reported the controlled bifunctionalization of fluorescent π-conjugated oligomer nanoparticles, which is a novel approach with high applicability to multi-targeted imaging and sensing in biology and medicine.¹⁰ In another approach, Jana et al. utilized FONPs in the monitoring of anticancer drug release.¹¹

Although organic nanoparticles provide advantages like wider applicability and flexibility in materials synthesis and nanoparticle preparation, advances in this field are rather slow due to several inherent problems associated with their synthesis and isolation.^12–16 It is only in past few years that considerable efforts have been put into the development of new methods to synthesize the FONPs with tuneable sizes, morphologies, and dispersion. Recent developments in this regard involve synthesis by precipitation,¹⁷ dilution of oil-in-water microemulsions in water,¹⁸ laser ablation,¹⁹ thermal evaporation,²⁰ and reverse microemulsions.²¹ Among all these methods, re-precipitation is one of the simplest and least time consuming methods, which has sparked the interest of chemists in the synthesis of FONPs. This method not only facilitates the formation of FONPs but also enhances the fluorescent quantum yield by many times.^22,23 FONPs are prepared by simply injecting the non-aqueous solution of organic compounds in the aqueous phase under vigorous stirring at constant temperature. The aqueous phase insolubility of the organic compound compels the molecules to aggregate in roughly spherical particles whose size can be very well controlled by the concentration, temperature, and stirring rate.

Herein, we have selected an organic tripodal Schiff base ligand, OTL (Fig. 1), to prepare FONPs by a re-precipitation method. This is an analogue with slight changes to a molecule with prominent fluorescence properties which demonstrates ‘PET’ based ‘off–on’ sensing for Ag⁺ recognition.²⁴ We believe that ONPs of this molecule will have even better photophysical properties which should show size dependent behaviour. Since its analogue has shown specificity towards Ag⁺ ions, this therefore opens up a new direction of synthesizing organic–inorganic hybrid nanomaterials by reducing Ag⁺ ions on the surface of ONPs. This will allow Ag NPs to grow on the surface of ONPs resulting in the formation of core–shell hybrid morphologies with interesting material properties.^25–27 We can prepare different sizes of ONPs simply by controlling the precursor concentration which will allow us to determine the size-dependent photophysical properties. We further plan to exploit their ratiometric recognition of biologically important Cu²⁺ ions in an aqueous medium which will allow the possibility of their use as drug release vehicles.

Fig. 1 Molecular structure of OTL.

Results and discussion

FONPs

OTL in pure THF shows an absorbance maximum around 348 nm in the UV region due to a n–π* transition in –C=N– bond of OTL which red shifts to 375 nm as the amount of water increases and ONPs’ formation sets in (Fig. 2a). It makes the solution slightly turbid with a uniform distribution of NPs while no stabilizer such as an appropriate surfactant is required. A prominent red shift in the absorbance happens due to the conformational changes in OTL from the isolated twisted to planar²² when they are stacked together in an ONP with a compact amorphous state. Likewise, their fluorescence emission around 530 nm increases and reaches its maximum in pure water (Fig. 2b). See a comparison in the fluorescence emission in pure THF and water when exposed to UV (Fig. 2c). The little fluorescence in pure THF can be attributed to a rapid non-radiative decay due to the presence of free OTL which may adopt any conformation in contrast to restricted decay when OTL aggregates in the form of FONPs in the aqueous rich environment and produces radiative decay which makes ONPs fluorescent. DLS analysis provides an estimate of the particle size which increases with increases in the amount of OTL used (Fig. 2d). Thus, a controlled precipitation with predominant non-polar interactions among the larger amounts of OTL generates larger NPs. The larger FONPs systematically produce prominent absorbance peaks (Fig. 2e) and thus depict the involvement of a stacking arrangement in the FONP formation. Likewise, fluorescence emission also increased with increases in the concentration or increased particle size (Fig. 2f). To have better understanding of the increased fluorescence intensity the quantum yield was calculated and found to be 0.62, 0.63, 0.60, 0.64, 0.63 for OTL1, OTL2, OTL3, OTL4 and OTL5 respectively. Therefore the increase in fluorescence intensity is related only to an increased concentration from OLT1–OLT5. Both Fig. 2e and f do not show any shift in the absorbance or fluorescence maximum with size which can be attributed to a similar kind of stacking arrangement of OTL molecules in FONPs of different dimensions as well as to their uniform dispersions in the aqueous phase. The stacking arrangement of organic molecules in a NP formation can be attributed to the H-type and J-type^28–30 where H-type molecules are aligned parallel to each other with strong intermolecular interactions and are mainly responsible for the non-radiative decay. On the other hand, J-type molecules are arranged in a head-to-tail direction and are responsible for the high fluorescence efficiency with the bathochromic shift in the UV absorbance. Probably, the latter arrangement also induces the fluorescence in the present FONPs. The fluorescence emission of FONPs also closely depends on the temperature variation as well as the size of the NPs. Increases in the temperature decrease the fluorescence intensity (Fig. 3a). An intensity versus temperature plot for FONPs of different sizes shows a linear dependence on temperature (Fig. 3b). In general, the fluorescence intensity decreases with increasing temperature due to increased molecular collisions that occur more frequently at higher temperatures especially for species in the solution phase. Collisions deplete energy from the excited state that produces fluorescence and thus converts the radiative decay into a non-radiative decay with the result fluorescence intensity decreases with temperature. Herein, organic molecules (OTL) are already packed in the amorphous FONPs and their packing arrangement in fact made them fluorescently active. That is why, bigger FONPs show greater fluorescence in comparison to the smaller ones (Fig. 2f). However, the temperature effect demonstrates a more pronounced effect with a greater negative slope for bigger FONPs in comparison to the smaller ones (Fig. 3b). A linear decrease in the fluorescence intensity with temperature shows that the self quenching of OTL molecules is proportional to the temperature. Since a larger number of OTL molecules are involved in the formation of bigger ONPs, hence they depict steeper slope in comparison to smaller ONPs. In addition, an increase in the temperature induces molecular ordering due to the annealing effects which cause the excited state species to loose their energy through non-radiative decay due to the thermal collisions and thus causing fluorescence quenching. This effect is expected to be more prominent for bigger FONPs in comparison to the smaller FONPs. Fig. 3c and d show the lifetime decay profiles at different temperatures, which were fit to a multi-exponential function with an average lifetime around 1 ns (see ESI, Fig. S1†). In each case, the lifetime decreases with the increase in temperature which was obviously expected in view of the increasing non-radiative decay with temperature. An annealing effect at 75 °C causes a sufficient phase transformation from amorphous to crystalline and few multiple peaks in a narrow range of 2θ ≈ 32° indicates the growth of {111} crystal planes (Fig. S2†).

Fig. 2 (a) UV-visible, (b) fluorescence spectra of OTL in pure THF, FONPs in pure water, and their mixture. (c) Photographs of FONPs in pure THF and pure water under the effect of UV light. (d) Variation of the size of FONPs with the concentration of OTL as evaluated from DLS measurements. Variation of the (e) UV-visible and (f) fluorescence spectra of FONPs samples of different sizes.

Fig. 3 (a) Dependence of the fluorescence intensity of N aqueous suspension of FONPs on temperature from 20–70 °C. (b) Variation of fluorescence intensity maximum with temperature for FONPs of different sizes. (c) Time resolve fluorescence of aqueous suspension of FONPs with temperatures from 20–70 °C. (d) Variation of fluorescence lifetime with temperature for FONPs of different sizes.

Ag@FONPs

The cation binding ability of FONPs in the aqueous phase was explored in dilute regimes of various metal nitrate salts (50 μM) in HEPES buffered in DMSO–H₂O (7 : 3, v/v). Both UV-visible (Fig. 4a) as well as fluorescence (Fig. 4b) spectra show a marked Ag⁺ ion recognition behaviour of the FONPs. The UV-visible spectra of the FONPs showed significant interactions with Ag⁺ ions (Fig. 4a) and produces another broad band around 500 nm. This results in a dramatic color change of the sample from turbid to dark brown (Fig. 4c) which indicated the formation of Ag NPs because of the reduction of Ag(I) to Ag(0) by OTL. Similar results were reported for the formation of Au NPs in the presence of spherical micelle assemblies of block copolymers.^31–33 A colloidal suspension of tiny Ag NPs produces their characteristic absorbance around 400 nm in the visible region. However, a significant red shift in absorbance of the Ag NPs is due to their expected growth on the surface of FONPs due to the selective binding ability of FONPs for Ag⁺ ions. Likewise, the emission spectrum of Ag@FONPs completely eliminates the band at 530 nm due to the deactivation of the FONPs surface by the growth of Ag NPs. We will further explain the growth of Ag NPs on the surface of FONPs from TEM studies.

Fig. 4 (a) UV-visible, (b) fluorescence spectra of an aqueous suspension of FONPs and their corresponding Ag@FONPs and (c) a typical photo of an aqueous suspension of FONPs and dark brown colored aqueous suspension of Ag@FONPs, (d) variation in the absorbance of FONPs in aqueous suspension in the presence of different concentrations of Ag salt.

¹H NMR titrations help us to determine the binding sites of Ag⁺ coordination to the receptor pseudo-cavity of the organic host (i.e. FONP) (Fig. S3†). The amorphous nature of the FONPs leaves several surface cavities which subsequently become the active sites for the entrapment of Ag⁺ ions and their reduction into tiny Ag NPs. ¹H NMR titrations can help us to investigate this process by varying the mole ratio between Ag⁺ and FONPs from 0 to 5 (Fig. S3A–G,†). A mole ratio of 0.5 (Fig. S3B,†) induces a shift of Δδ = 0.06 in the signal of –CH=N (8.67 ppm) while aromatic protons (7.54–6.82 ppm) also demonstrate a shift of Δδ = 0.02–0.07. The shift in the –CH=N signal is quite expected as the imine linkage bears the sp² nitrogen, which acts as a soft centre for the coordination of soft metal ions like Ag⁺. Increases in the mole ratio to 1.5 (Fig. S3D,†) even shifted the signal of –CH=N up to Δδ = 0.09, with relatively little shift in the aromatic protons. A mole ratio of 2.0 (Fig. S3E,†) induces a split in the signal of –CH=N and a merger in the aromatic signals, which were otherwise quite prominent between δ = 7.3–7.4 at low mole ratios. The splitting in the signals indicates that the organic receptor is not offering symmetric binding sites from all the three pods to the Ag⁺ ions. The solubility profile of both host and metal salt compelled us to use a DMSO(d₆)–D₂O solvent system, and the presence of D₂O limits us to following the –OH signal. However, the above titrations demonstrate the most significant role of imine linkages for the formation of the metal complex which is further confirmed from the IR measurements in the absence and presence of Ag⁺ as depicted in Fig. S4.† The peaks located at 1641, 1550, 1279 and 1073 cm⁻¹ in the case of FONPs shift to 1646, 1541, 1283 and 1078 cm⁻¹ in the case of Ag@FONPs, respectively. The Ag⁺ ions have a good affinity for electrons and are known to bind well with ligands containing lone pair electrons. As good electron donors, both –OH and N of OTL can coordinate with the Ag⁺ ions to form stable complexes which induce the red-shift in the IR spectra and this is previously reported by other groups as well.³⁴ In addition, the band for imine linkages originally at 1446 cm⁻¹ shows a shift of 5 cm⁻¹ which confirms their participation in the complexation with Ag⁺ ions. Thus, the collective analysis of NMR and FTIR spectra of OTL and Ag@FONPs clearly demonstrates the interactions of Ag⁺ with the sp² nitrogen atom of the –CH=N linkage, which produces no additional signals that could be related to some oxidized species in OTL. This is also not even observed from the IR band due to the C=O group or change/cleavage in the structure of OTL. In the light of the above results, we purpose the following mechanism for the formation of Ag NPs:

where OTL ligands on the surface of ONPs first interact with Ag⁺ ions through the sp² hybridised nitrogen atom to form Ag(OTL)⁺ complex. This complex in the presence of light initiates a photoreduction of Ag⁺ to Ag⁰. A similar type of mechanism has also been proposed by Hada et al.³⁵ and Balzani et al.³⁶ for the formation of Ag from Ag(dipy)₂²⁺ complex in aqueous solution. Once the nucleating centres are created on the surface of ONPs, they become active sites for subsequent autocatalytic process in the presence of Ag⁺ ions and eventually grow into prominent Ag NPs to produce organic–inorganic hybrid morphologies. We will further discuss this point in the light of TEM studies later in the text.

Effect of silver concentration

Growth of the Ag NPs on the surface of ONPs is directly related to the amount of the Ag⁺ ions reduced into Ag(0) and is closely monitored from the UV-visible behavior. A variation in the concentration of Ag⁺ ions from 0.2 mM to 4 mM at constant concentration of FONPs (10 μM) after equilibrating the samples for 12 h is depicted in Fig. 4d. In the absence of Ag⁺ ions, FONPs give a characteristic absorbance around 375 nm. However, increasing the amount of Ag⁺ produces a new band around 500 nm whose intensity increases up to 1.6 mM, thereafter, it decreases as the concentration further increases. The first increments in the absorbance can be assigned to the increased number of Ag NPs formed on the surface of FONPs which later on agglomerate to produce larger particles and thus cause a decrease in the absorbance. We will further explain this from the TEM studies.

TEM analysis

TEM studies can help us to evaluate the shape and size of the FONPs as well as the nature of Ag@FONPs hybrid morphologies. Fig. 5a shows a low magnification TEM image of small FONPs in the form of fine spheres made from 1.1 × 10⁻⁴ M of OTL which mostly exist as aggregates in a dried state on the copper grid. Aggregation happens due to the predominance of the close van der Waal's interactions among the aromatic rings and may be to some extent due to the hydrogen bonding among the hydroxyl groups in the amorphous state. The average size of the spherical FONPs of this sample is close to 175 nm as observed from DLF studies (Fig. 2d) though some larger spheres of more than 200 nm are also visible due to the presence of polydisperse behaviour. The latter behaviour is the consequence of the budding mechanism which allows the larger aggregates to split into smaller ones as dehydration sets in and is clearly depicted in Fig. 5b. Fig. 5c shows another typical chain like arrangement of FONPs of the same sample. Such kinds of aggregation behaviour are usually demonstrated by the NPs which lack stabilizing agents. A colloidal particle can be properly stabilized in the form of a colloidal sol if an appropriate amphiphilic stabilizing agent like a surfactant is used. Since in the present study, we focus on the synthesis and properties of FONPs, we preferred not to use any surfactant which might impede their fluorescence behavior. A further increase in the concentration of OTL to 4.5 × 10⁻⁴ M increases the size of the FONPs to around 250 nm as depicted by the DLS studies in Fig. 2d which suggests a good correlation between the DLS and TEM studies. However, increases in the concentration also induces the deformation in the spherical shape. See the deformed NPs indicated by the black arrows in Fig. 5d and e. The number of deformed FONPs is much lower than the number of spherical FONPs and there is no marked effect on the overall fluorescence behavior of the FONPs. As FONPs specifically bind to Ag⁺ ions as depicted in Fig. 4 and 5, TEM analysis demonstrates the growth and formation of small Ag NPs on the surface of FONPs. Fig. 5f and g show TEM micrographs of Ag@FONPs of the samples of Fig. 5a–c. Both images show several small black Ag NPs of 5–10 nm grown on the spherical FONPs. A much darker contrast of the Ag NPs in comparison to that of FONPs is simply due to their metallic nature which causes much greater diffraction. Black arrows indicate some larger Ag NPs in comparison to several smaller ones. It seems that they are the result of inter-particle fusion among the smaller Ag NPs when they happen to grow in close vicinity of each other. Fig. 5h and i show the images of Ag@FONPs of sample of Fig. 5d and e with larger sizes in comparison to the previous sample. Again, small Ag NPs grown on the surface of FONPs are clearly visible. XRD patterns show a fine fcc crystalline geometry of Ag with prominent peaks at 27.7°, 32.2°, 38.0°, and 46.2° (Fig. 5j). The images also demonstrate that no independent Ag NPs exist in the solution and the surface of the FONPs constituted by the J-type packing arrangement of OTL molecules is responsible for the reduction of Ag(I) into Ag(0) with the consequence that Ag NPs only grow on the surface of the FONPs. Thus, the growth of the Ag NPs in fact impedes the absorption in addition to the radiative decay of FONPs as depicted in Fig. 4a and b, respectively, which happens due to the quenching effects. TEM images were also recorded after dissolving the organic matter in THF–H₂O. Slightly agglomerated Ag nanoparticles were clearly observed which were earlier adsorbed on the surface FONPs (Fig. S5†).

Fig. 5 (a) TEM micrographs of fine spherical FONPs made with OTL = 1.1 × 10⁻⁴ M in pure water in low resolution. (b) This image shows the budding process of larger NPs into smaller ones, which later on arrange themselves in chain like arrangements (c). Images (d) and (e) show the presence of deformed NPs (indicated by arrows) along with the spherical NPs when FONPs are prepared with OTL = 4.5 × 10⁻⁴ M. TEM micrographs (f) and (g) of Ag@FONPs prepared with OTL = 1.1 × 10⁻⁴ M and AgNO3 = 0.2 mM. See the growth of Ag NPs as dark dots at the surface of the spherical FONPs. Arrows indicate some of the large size Ag NPs which are produced most probably from the inter-particle fusion of among the smaller ones at the surface of the FONPs. Images (h) and (i) depict similar images of Ag@FONPs made with OTL = 4.5 × 10⁻⁴ M and AgNO₃ = 0.2 mM. (j) XRD patterns of the FONPs sample dried at 75 °C demonstrating the fcc crystal structure of the Ag NPs.

Application of FONPs in Ag extraction and Cu recognition

Formation of Ag@FONPs (Fig. 6(a)i) suggests that FONPs can be repeatedly used for the extraction of Ag⁺ ions from aqueous samples with the ability of FONPs to work for any given number of times. In order to demonstrate this, we dissolved Ag@FONPs in THF–H₂O (8 : 2, v/v) mixture that produced a bright yellow colored solution (Fig. 6(a)ii) due to tiny Ag NPs. Because of the favorable solubility of FONPs in THF–H₂O (8 : 2, v/v), the solution is practically left with the colloidal suspension of Ag NPs which can be easily extracted from the solution by centrifugation. The remaining transparent solution (Fig. 6(a)iii) containing mainly soluble OTL can further be used for the formation of FONPs by increasing the amount of water and hence can be repeatedly used for the same purpose.

Fig. 6 Photographs of ((a)i) aqueous suspension of Ag@FONPs, ((a)ii) Ag NPs in THF + water mixture, and ((a)iii) the solution after extraction of Ag NPs. See details in the text. (b) UV-visible absorbance of Ag@FONPs in the presence of various ions. (c) A variation in the UV-visible absorbance of Ag@FONPs in the presence of varying amounts of Cu²⁺ ions. (d) A variation in the intensity of absorbance of Cu²⁺ complex with the amount of Cu²⁺ ions, and this is compared in (e) to blood serum. (f) Variation of percentage hemolysis with the amount of Ag@FONPs for NPs of different sizes.

Likewise, Ag@FONPs can be used as sensors for metal ions’ and anions’ recognition. A change in the absorption profile of Ag@FONPs dissolved in aqueous THF (8 : 2, v/v) upon addition of a particular metal ion (50 μM) in HEPES buffered DMF–H₂O (8 : 2, v/v) gives direct evidence of the binding behaviour (Fig. 6b). The addition of Cu²⁺ resulted in a significant change in the absorption spectrum of FONPs. The binding of FONPs to Cu²⁺ furnished a new band at 421 nm. On the addition of any other metal ion (i.e. K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Sr²⁺, Mn²⁺, Ni²⁺, Co²⁺, and Zn²⁺) no such significant binding was seen. The sensing of Cu²⁺ is of immense importance because Cu²⁺ is third most abundant transition metal in the human body. It plays a significant role in various physiological processes ranging from hemoglobin biosynthesis to nerve function regulation.^37–39 The excessive use of copper due to its applications in industrial, pharmaceutical and agricultural purposes has led to serious threats to the environment and human health.^40,41 The misregulation of Cu²⁺ is related to many diseases such as Alzheimer's, Prion, Menkes, and Wilson’s diseases, lipid metabolism, and inflammatory disorders.^42,43 These diverse applications of Cu²⁺ have led to a strong interest in the development of selective Cu²⁺ probes.^44–47 To gain more insight into the Cu²⁺ recognition behavior, titration of Ag@FONPs dissolved in aqueous THF (8 : 2, v/v) with Cu²⁺ was performed (Fig. 6c). A successive increase in the amount (0–50 μM) of Cu²⁺ results in a continuous reduction of absorbance at 350 nm and a simultaneous increase in absorbance at 421 nm with one isobestic point at 376 nm (Fig. 6c). The detection limit (LOD) was measured to be 0.24 μM. The linear range is observed between 0–4 μM concentration of Cu²⁺ (Fig. S6†). To test the practical applicability of Ag@FONPs as a Cu²⁺ selective chromogenic sensor the interference of other metal ions in the sensing of Cu²⁺ by Ag@FONPs was studied. Competitive experiments were carried out in the presence of Cu²⁺ (50 equiv.) mixed with one of K⁺, Ca²⁺, Mg²⁺, Ba²⁺, Sr²⁺, Mn²⁺, Co²⁺, Ni²⁺, and Zn²⁺ (50 equiv.). As shown in Fig. S7,† no significant variation in the intensity was found by comparing the profile with and without the other metal ions, which means that Ag@FONPs dissolved in aqueous THF (8 : 2, v/v) can selectively sense the Cu²⁺ at μM level. Fig. 6d demonstrates the complete saturation of all binding sites on the surface of Ag@FONPs by 4 μM Cu²⁺ that provides about 1 : 25 mole ratio between the OTL and Cu²⁺. It is possible to calculate the binding constant of Cu²⁺ ions with OTL by using log[A_max − A]/[A − A_min] = −log[Cu²⁺] − log β (where A_max and A_min are the maximum and minimum absorbance, A is the measured absorbance, and β represents the binding constant), an equation previously used by other authors⁴⁸ for related systems. The inset shows the application of this relation on the data of Fig. 6d, which gave the binding constant = 6.9. In addition, a variation in the pH did not show any marked effect on the absorption profile of Ag@FONPs (Fig. S8†) and hence it can be utilized over a wide pH range. There is no effect on the stability of Ag@FONPs in the absence and presence of Cu²⁺ ions. This has been determined by using EDTA, the presence of which diminishes the peak observed at 421 nm and eventually generated a spectrum similar to that before the addition of Cu²⁺ ions (Fig. S9†). Also the UV-vis spectral changes were observed on addition of Cu²⁺ ions to Ag@FONPs solution as function of time for a period of ten days. No significant changes were observed as shown in Fig. S10.† Hence, it can be concluded that AgNP@FONPs are quite stable before and after interaction with Cu²⁺ ions. Ag@FONPs also do not show any selectivity against Cu⁺ (Fig. S11†).

The observed binding affinity of Ag@FONPs for Cu²⁺ is a consequence of unique combination of sulfur and sp² nitrogen donor sites of the FONPs. These types of binding sites also prevail in the naturally occurring copper proteins, highlighting the importance of these binding sites to constitute the optimum coordination spheres in terms of compatibility between host and guest.⁴⁹ Thus, the binding sites of FONPs act as receptor subunits for the Cu²⁺ ions and the occurrence of such a kind of host–guest process in the vicinity of the Ag NPs causes modulation in the photophysical properties of the Ag NPs thereby allowing them to act as signaling subunits of the sensor assembly. In addition, the above mentioned NMR studies also indicated the presence of unsymmetrical binding of the tripodal ligand through three binding pods to Ag⁺, which means that all binding sites are not engaged in associating with Ag⁺ ions, and hence there is equal probability of free binding sites to interact with Cu²⁺ ions. Unfortunately, this association is not clearly depicted by the emission spectroscopy because the fluorescence emission of the ONPs has already been quenched by the interactions with the Ag⁺ ions and its subsequent formation of Ag@FONPs. Therefore, no marked changes are observed in the fluorescence spectrum of AgNP@FONPs in the presence of Cu²⁺ or other metal ions or anions (Fig. S12†). In addition, the binding affinity of the Ag@FONPs to bind Cu²⁺ also works very well in biological systems (Fig. S13†), where no change in the binding affinity from the absorption spectra of Ag@FONPs in the presence as well as in the absence of blood serum is observed (Fig. 6e). This shows that Ag@FONPs are highly specific towards the Cu²⁺ ions even in the presence of a complex biological environment. Taking advantage of this observation, we further tried to understand the hemolytic ability of Ag@FONPs so as to explore their potential as drug delivery vehicles in the systemic circulation. Hemolytic assay of four samples of Ag@FONPs has been presented in Fig. 6f along with the sample photos of 230 nm Ag@FONPs and corresponding absorbances (Fig. S14†). The smallest Ag@FONPs (i.e. 213 nm) show the least hemolysis and this remains less than 5% even up to 200 μg mL⁻¹, while it increases with the increase in the size of the NPs. Similar hemolytic behavior on the basis of the size of the NPs has already been reported by other authors.^50–52 As depicted in the TEM images (Fig. 5), the surface of Ag@FONPs is mainly occupied by the tiny Ag NPs which are considered to be the active sites for interactions with the cell membrane of red blood cells because charged surfaces have greater potential to interact with and rupture the cell membranes.^53,54 Thus, the larger sized Ag@FONPs are expected to induce greater hemolysis than the smaller ones. Therefore, smaller NPs of ∼200 nm with minimum hemolysis can be used as vehicles of drug release in systemic circulation. Additionally, we tried to evaluate the biocompatibility of Ag@FONPs with living cells and performed the MTT assay with the osteosarcoma cancer cell line under standard conditions (incubation at 37 °C for 2 days in CO₂ atmosphere, 5%). Ag@FONPs of largest size (311 nm) show a cell viability as high as 82%, thus Ag@FONPs of smaller size are much better vehicles for drug release as indicated above from the hemolytic assay.

Concluding remarks

We demonstrate a simple method for the synthesis of fine spherical FONPs by using a OTL in aqueous phase. OTL itself is not a fluorescent molecule but shows radiative emission when it aggregates in a specific order into amorphous nanoparticles whose emission increases with increases in size and decreases with rises in temperature. Because of its specific Ag⁺ ions’ binding ability, FONPs also show the same property and possess an ability to reduce Ag(I) into Ag(0) resulting in the growth of Ag NPs on the surface of FONPs to produce organic–inorganic hybrid nanoparticles. Ag@FONPs are not fluorescent due to the quenching of fluorescence emission of FONPs when Ag NPs grow on their surface. However, Ag@FONPs are fine chromogenic agents for the detection of Cu²⁺ even in the presence of blood serum and hence can be used in biological systems. Apart from this, NPs of ∼200 nm show little hemolysis up to 200 μg mL⁻¹, thus making them suitable vehicles for drug release in systemic circulation.

Experimental

Materials

All chemicals were obtained from Sigma and were used as received without further purification. The organic tripodal Schiff base (Fig. 1) was synthesized as reported earlier.⁵⁵

Synthesis of FONPs and Ag@FONPs

The FONPs were synthesized by a re-precipitation method.⁵⁶ Breifly, 1 mL of OTL solution in tetrahydrofuran was injected with a microsyringe at a constant rate to double distilled water (50 mL) under vigorous stirring. The solution was then sonicated for 20 min at a constant temperature to produce FONPs of the desired size which provided a slight turbidity with a greenish tinge (Fig. 4c). The size of FONPs was simply controlled by varying the concentration of OTL in the solution. Organic–inorganic hybrid Ag@FONPs were prepared by taking an aqueous suspension of FONPs of the desired concentration along with 0.2 mM of AgNO₃. Heating this solution at constant temperature of 70 °C initiated the reduction of Ag(I) into Ag(0) resulting in the formation of Ag NPs covalently attached to the surface of FONPs through sulfur linkages. The color of the final suspension was dark brown indicating the formation of Ag@FONPs (Fig. 4c).

Methods

UV-visible absorption spectra of FONPs suspensions were recorded on a Specord 250 Plus Analytik jena spectrometer. Steady state and time resolved fluorescence spectroscopy of ONPs suspensions were carried out by using a PTI QuantaMaster and a PicoMaster 2 TCSPC Lifetime Fluorometer, respectively. Both instruments were equipped with a thermoelectrically temperature controlled cell holder that allowed measurement of the spectrum at a constant temperature within ±1 °C. In order to obtain the fluorescence lifetime, the profile of instrument response function (IRF) (excitation pulse) has been measured in addition to the fluorescence decay. In a typical experiment, two curves are measured: the IRF using a scatterer solution and the decay curve (see ESI, Fig. S1a†). The analysis is then performed by convoluting the IRF with a model function (e.g. a single exponential decay or a double exponential decay or some other function) and then comparing the result with the experimental decay. This is done by an iterative numerical procedure until the best agreement with the experimental decay curve is achieved. ¹H spectra were recorded in DMSO-d₆ on a JEOL II 400 spectrometer (400 MHz with TMS as internal standard; chemical shifts are expressed in ppm). FT IR spectra were recorded on a Bruker Tensor 27 spectrometer in the form of liquid samples. The PANalytical X'PERT PRO diffractometer was used for X-ray diffraction (XRD) with a scan speed of 10° min⁻¹ for 2θ over a range from 10 to 80 (45 kV, 40 mA using Ni-filtered Cu K_α radiations). Transmission Electron Microscopic (TEM) analysis was done on a JEOL 2010F at an operating voltage of 200 kV. The samples were prepared by mounting a drop of the solution on a carbon-coated Cu grid and allowing this to dry in the air. The particle sizes of the FONPs and AgNP@FONPs were measured by using the external probe feature of a Metrohm Microtrac Ultra Nanotrac Particle Size Analyzer (Dynamic Light Scattering).

Acknowledgements

This work is supported by CSIR, New Delhi, [01(2417)/10/EMR-11]. A. S. thanks CSIR for a fellowship. G. K. thankfully acknowledges the financial support provided by the Research and Development Council (RDC) of Newfoundland and Labrador, NSERC, and the Office of Applied Research at CNA. These studies were partially supported by financial assistance under Article 27.8 of the CAS agreement of WLU, Waterloo.

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Footnote

† Electronic supplementary information (ESI) available: UV-vis absorption spectra. See DOI: 10.1039/c4ra00808a

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