Ultra-small gold nanoparticles synthesized in aqueous solution and their application in fluorometric collagen estimation using bi-ligand functionalization

Abstract

An effective, rapid and facile hydrosol approach was developed to synthesize monodisperse ultra-small gold nanoparticles (∼2 nm) using lithium borohydride (LiBH₄) as a reducing agent. These lithium borohydride gold nanoparticles (LBH-AuNPs) are highly stable at pHs ranging from 3 to 10.6. We have subjected these LBH-AuNPs to bi-ligand co-functionalization with FITC (fluorescent isothiocyanate) and fluorescent lysine molecules. It was observed that these particles exhibit enhanced tolerance of FITC and lysine bi-ligand functionalization. The fluorescence resonance energy transfers (FRET) from the FITC and lysine to the AuNPs, and the replacement of these fluorophores by collagen as ligands have been exploited for the sensitive fluorometric detection of rat tail collagen. Linearity in the reappearance of FITC and lysine fluorescence was observed at 2 to 10 μg ml⁻¹ of extracted rat collagen, which demonstrated the successful use of these bi-ligand surface functionalised gold nanoparticles as a probe for the sensitive fluorometric estimation of rat tail collagen.

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1. Introduction

Gold nanoparticles (AuNPs) are used in various applications like biosensors, diagnosis, imaging systems, etc.^1,2 In particular, the use of AuNPs for various applications in the biomedical field is very common due to their potential to form complexes with various biomolecules.^3,4 Smaller particles are preferred for these purposes, because they have a higher surface area/volume ratio,⁵ so a greater concentration of a range of biomolecules can be delivered, conjugated or sensed. Extensive work has been carried out to achieve stable and size controlled AuNPs by selecting appropriate reducing agents for the reduction of gold chloride salts.⁶ Colloidal AuNPs of size 10–20 nm have been commonly produced using the citrate method described by Frens, in which the size depends upon the citrate/HAuCl₄ ratio.⁷ Furthermore, easy and rapid methods for the production of small AuNPs (<5 nm) have been achieved by the reduction of AuCl₃ or HAuCl₄ with reducing agents like sodium borohydride (NaBH₄), hydrazine, etc.^8,9 Recently, ultra-small nanoparticle (1–4 nm) synthesis has been achieved in organic solvents.¹⁰ In these syntheses, mono-dispersity and precise size were achieved with the capping of alkane thiol-tethered polymeric ligands.^11,12 However, the capping of these alkane thiols is toxic in most biological systems, and also the use of organic solvents is not preferred due to environmental issues.^13a Moreover, organic based AuNPs show poor water miscibility, which limits their use in biological systems. Hence, various approaches have been developed, but achieving hydrosol AuNPs with tuneable sizes is still a challenge.

The unique size-dependent physical and chemical properties of nanomaterials have been exploited to generate and improve colorimetric and fluorometric based analytical methods.¹⁴ In general, dispersed AuNPs exhibit a red or ruby colour in solution, whereas aggregated and larger AuNPs display a blue or purple colour in solution.¹⁵ In contrast to the dispersed AuNPs, the aggregated AuNPs have a substantially smaller interparticle distance compared to their average diameter.^16,17 As a result, these AuNPs display a colour change from red to blue in colloidal solution. Thus, the change in the colour of the solution is highly sensitive to the size, shape and refractive index of the medium, capping agents, and the degree of aggregation of the AuNPs.¹⁸ In colorimetric based assays, the change in colour can be estimated by the shift in SPR (Surface Plasmon Resonance). This principle has been utilized for ligand–metal ion complexes,¹⁵ antigen–antibody reactions,¹⁹ etc.

Furthermore, AuNPs have a greater molar extinction coefficient than organic dyes.²⁰ As a result, when fluorophore tagged molecules or fluorophores interact with AuNPs, their fluorescence will be quenched through the process of fluorescence resonance energy transfer (FRET). In FRET, the electron and/or energy transfer from the fluorophores to the AuNPs leads to fluorescence quenching.²¹ When the targeted analytes are added to the fluorophore associated AuNPs, the fluorophores can be displaced or removed from the surface. As a result, the number of targeted analytes with restored fluorescence from the removal or displacement of fluorophores from the AuNPs can be estimated. The intensity of fluorescence depends upon the concentration of the targeted analytes. This phenomenon has been used for various FRET based assays, like the competitive immunoassay for bio-molecules,¹⁹ the detection of DNA hybridisation,²² and ligand replacement induced fluorescence turn-on for the detection of pesticides,²³ etc. However, its sensitivity for these applications depends on various factors, like the stability of the nanoparticles against the tolerant concentration of the targeted analyte, size, capping, monodispersity, solvent effects, etc. For biological applications, there is a need for a hydrosol based synthesis of ultra-small monodisperse AuNPs which display stability against the above mentioned factors, to achieve increased sensitivity of the FRET based reaction.

Herein, we report a rapid and facile hydrosol method for the synthesis of monodisperse colloidal AuNPs with average diameter ∼2 nm. To the best of our knowledge, this is the first study where LiBH₄ is used as reducing agent for the aqueous synthesis of gold nanoparticles. In AuNP synthesis, stronger reducing agents result in smaller particle size.^7–9 According to literature, lithium based salts are stronger reducing agents due to their higher negative reduction potential.^24,26 Therefore, LiBH₄ is considered to be a stronger reducing agent compared to NaBH₄.²⁵ Hence, we have used LiBH₄ for the synthesis of ultra-small AuNPs rather than NaBH_4. Furthermore, LBH-AuNPs (lithium borohydride-gold nanoparticles) were subjected to bi-ligand co-functionalization with the fluorescent dye fluorescent isothiocyanate (FITC) and lysine molecules to obtain AuNP LBH–FITC–Lysine nanoparticles (AFL NPs) for the sensitive estimation of rat tail collagen through FRET analysis (Fig. 1). By virtue of their small size, these particles can tolerate a higher concentration of ligand molecules (here, FITC and lysine) compared to the previous study.²⁴ Furthermore, these particles can be used as a probe for the FRET based assay of bio-macromolecules like collagen. In this study, we selected collagen estimation, because only a few colorimetric and fluorometric methods are available for collagen estimation, such as the Sirius red test²⁷ and the detection of collagen by intrinsic fluorescence.²⁸ Upon the addition of collagen to the bi-ligand functionalised AFL NPs, the collagen induced the replacement of the FITC and lysine ligands attached to the AuNPs, and restored their fluorescence (Fig. 1). This turn on fluorescence of FITC and lysine was found to be sensitive for collagen estimation with a limit of detection (LOD) of 2 μg ml⁻¹.

Fig. 1 Schematic representation showing LiBH₄ synthesized AuNPs, bi-ligand surface modified AuNPs with FITC (Ex/Em: 488/520) and lysine (Ex/Em: 355/∼435) for the formation of AFL nanoparticles, and the fluorometric estimation of rat tail collagen by the induction of FITC and lysine fluorescence from AFL nanoparticles. The release and restored fluorescence of FITC and lysine were attributed to the controlled nucleation of the AFL nanoparticles with collagen.

2. Experimental

2.1. Materials

Gold chloride (AuCl₃), lithium borohydride (LiBH₄), collagen, L-lysine and fluorescent isothiocyanate (FITC) were purchased from Sigma-Aldrich. The different pH solutions were prepared by mixing different volumes of NaOH and HCl. All of the experiments were performed in Milli-Q water (Merck-millipore, USA). The average diameter of the LBH AuNPs, frequency distribution of the size of the LBH-AuNPs and standard deviation were calculated for each sample of nanoparticles by averaging at least 100 nanoparticles from a TEM image using ImageJ software (developed by the National Institute of Health).^29,37

2.2. Instruments

A double beam Perkin Elmer Lambda 35 spectrophotometer was used to obtain UV-Visible absorption spectra. Fluorescence emission spectra were obtained with a Hitachi 4500 Japan spectrophotometer with a slit width of 5 nm. The excitation and emission wavelengths of FITC and L-lysine are 488 nm and 355 nm, and 520 nm and ∼435 nm, respectively. The TEM micrographs of the AuNPs and modified AuNPs with lysine, FITC and collagen were obtained using a 200KV transmission electron microscope, from JEOL, Japan. All measurements were conducted in triplicate. The median value and standard deviation (S.D.) were considered when plotting the graphs.

2.3. Preparation of LiBH₄ synthesised gold nanoparticles (LBH-AuNP) and their pH stability

A series of LiBH₄ solutions were prepared with final concentrations of (a) 0.33 mM, (b) 0.66 mM, (c) 1.32 mM, (d) 2.64 mM, (e) 5.28 and (f) 7.92 mM in 248 ml Milli Q water. To this, 2 ml of 1% (w/v) AuCl₃ solution was added, and the mixture was vigorously stirred for a few minutes, and colloidal gold nanoparticles were formed. The gradual change from a blue to red colloidal solution was observed with LiBH₄ concentrations ranging from 0.33 mM to 7.92 mM. We considered the AuNPs to be optimally synthesized with 2.64 mM of LiBH₄, which was used for all of the experiments. The concentration of the AuNPs was found to be 1.3 μM (ref. 30) (detailed calculation is given in ESI: 1).† The particles synthesized in Milli Q water with 2.64 mM of LiBH₄ had pH ∼7.4. Furthermore, the stability of the optimised LBH-AuNPs was studied by gradually changing the pH using 1 N HCl and 1 N NaOH from pH 3 to 10.6.

2.4. Preparation of the bi-ligand functionalized AuNP LBH–FITC–Lysine (AFL NPs) and mono functionalized AuNP LBH–FITC (AF) and AuNP LBH–lysine (AL) nanoparticles

The bi-ligand functionalised AFL NPs were synthesised in two steps. (a) To 5 ml of the 1.3 μM solution of AuNPs, 50 μl of 500 μM FITC solution (dissolved in 95% ethanol) was added, giving a final concentration of 5 μM FITC in AuNPs, and the solution was incubated for 30 min. Next, 100 μl of 100 mM lysine (solution (b)) was added, giving a final concentration of 2 mM lysine in solution (a), and this was incubated for 30 min. In both reactions (a) and (b), saturated concentrations of FITC (500 μM) and lysine (100 mM) were used. Similarly, for the preparation of the AF and AL solutions, 5 ml of 1.3 μM AuNP solution contained final concentrations of 5 μM FITC and 2 mM lysine, respectively. All of the reactions were incubated for 30 min at room temperature.

2.5. Fluorometric assay of collagen using AFL, AF and AL nanoparticles

A series of collagen solutions with final concentrations of 2 to 10 μg ml⁻¹ were prepared in 2 ml of AFL, AL and AF nanoparticles from a 100 μg ml⁻¹ stock collagen solution. For the real time collagen estimation, rat tail collagen was extracted using methods detailed by Navneeta Rajan et al.,³¹ and the concentration was adjusted to 1 mg ml⁻¹ by the Sirius red test.³² The respective collagen solutions containing AFL, AL and AF were incubated overnight at 4 °C. These reactions were analysed and characterized by fluorescence spectroscopy.

3. Results and discussion

3.1. Synthesis of ultra-small LBH-AuNPs by LiBH₄ reduction

UV-Vis spectra of LBH-AuNPs synthesized with different concentrations of LiBH₄ ranging from 0.33 mM to 7.92 mM are shown in Fig. 2A. The increase in LiBH₄ concentration caused a shift in the SPR peak from 573 nm to 512 nm (a–f). The corresponding colour change from a blue to red colloidal solution was observed as shown in Fig. 2B.

Fig. 2 (A) UV-Vis spectra and (B) colloidal LBH-AuNPs synthesised at LiBH₄ concentrations of (a) 0.33 mM, (b) 0.66 mM, (c) 1.32 mM, (d) 2.64 mM, (e) 5.28 mM and (f) 7.92 mM.

According to Martin et al., NaBH₄ naked synthesis of stabilizer free colloidal AuNPs may result in the anion capping of boron based ions.³³ However, this is not yet confirmed. In this synthesis, where LiBH₄ was used, a similar negatively charged capping of the AuNPs is expected, which renders the colloidal nature of these particles. The gradual blue shifts observed in the SPR spectra with increases in LiBH₄ are indicative of the corresponding reduction in the size of the AuNPs. In agreement with the literature, the SPR shifts towards longer wavelengths with increased particle size.³³ To estimate the particle size from the SPR of these AuNPs, we used an established experimental model based on multipole scattering to estimate the particle size from spectroscopic observation of the AuNPs.³⁰ However, in this study, the model results for the estimation of the large and polydisperse LBH-AuNPs did not correlate with the observed size of the AuNPs in the TEM image with a large % error, although it showed consistency with small AuNPs. From the UV-Visible spectra, the LBH-AuNPs synthesised in conditions (d–f) have a greater blue shift compared to those synthesised in conditions (a–c). Thus, a smaller and narrower distribution of particles can be expected in the former rather than the latter. The diameter of the smaller nanoparticles was estimated by measuring the absorbance at λ_spr/λ₄₅₀, as described by Haiss et al.³⁰ The observed λ_spr of the LBH-AuNPs synthesised at LiBH₄ concentrations of 2.64 mM (d), 5.28 mM (e) and 7.92 mM (f) were 512 nm, 510 nm and 520 nm, respectively. The estimated sizes of the corresponding LBH-AuNPs were ∼4 nm, ∼4 nm and ∼5 nm. Furthermore, TEM analysis was carried out to accurately estimate the size of the AuNPs synthesized at various LiBH₄ concentrations and the results are shown in Fig. 3(a–f).

Fig. 3 TEM images of LBH-AuNPs synthesised at LiBH₄ concentrations of (a) 0.33 mM, (b) 0.66 mM, (c) 1.32 mM, (d) 2.64 mM, (e) 5.28 mM and (f) 7.92 mM. Insets show frequency distribution graphs.

From the TEM analysis, the particles synthesised at lower LiBH₄ concentrations (a–c) showed larger and/or aggregated AuNPs (∼5 nm to ∼35 nm). This is evident from the shifted and broadened SPR peaks of the corresponding AuNPs. As a result, the above mentioned model cannot be used for these polydisperse AuNPs. On the contrary, particles synthesised with increased LiBH₄ concentrations (d–f) exhibit smaller average particle sizes of 2.2 ± 0.4 nm, 3.4 ± 0.3 nm and 3.7 ± 1.3 nm, respectively. These data are accumulated from the frequency distribution graphs produced using ImageJ software which are shown as the inset in Fig. 3(d–f). Thus, the particles synthesized at higher LiBH₄ concentrations showed very good consistency in their diameters, which were obtained from TEM and spectroscopic observations. Here, we found that the particles synthesised with 2.64 mM LiBH₄ showed remarkable stability at room temperature for more than six months, whereas others precipitated. In general, the stable colloidal AuNPs were achieved at an optimum ratio of metal precursor to reducing agent.⁷ Particles synthesised using these optimised conditions were used to perform the below mentioned experiments.

3.2. Stability of LBH-AuNPs with different pH

The initial pH of the colloidal solution of these particles synthesized with 2.64 mM of LiBH₄ was ∼7.4. The stability of these LBH-AuNPs at different ionic strengths was estimated by changing the pH from 3 to 10.6. The pH was adjusted using 1 N HCl and 1 N NaOH. As shown in Fig. 4A, the LBH-AuNPs showed a slight red shift overall in the SPR peak of ∼3 nm at pHs of 3, 5, 7, 9 and 10.6 (a–e). Furthermore, these particles maintain their colloidal nature at these pHs, as shown in Fig. 4B(a–e). These observations confirm that these particles are more stable under both acidic and basic conditions.

Fig. 4 (A) UV-Visible spectra of LBH-AuNPs in different pH solutions: (a) pH 3, (b) pH 5, (c) pH 7, (d) pH 9 and (e) pH 10.6. (B) Optical images of corresponding colloidal LBH-AuNP solutions from (a)–(e).

3.3. Bi-ligand functionalization of LBH-AuNPs with lysine and FITC

The attachment of lysine and FITC to sodium borohydride synthesised AuNPs was explained by Shukla et al.²⁴ In this surface functionalization, the NCS group of the FITC interacts with the AuNPs,² whereas at physiological pH, the α-NH₂ of lysine interacts with the AuNPs.^13b Here, a similar functionalization was performed with LBH-AuNPs. However, in the previous study, the conjugations of lysine and FITC to the AuNPs were explained by using FRET of FITC. In this study, we are for the first time introducing FRET of lysine (Ex/Em: 355/∼410–435 nm) and FITC (Ex/Em: 488/520 nm) to explain the bi-ligand functionalization of the LBH-AuNPs with FITC and lysine to produce LBH-AuNP–FITC–Lysine nanoparticles (AFL nanoparticles). In this study, we selected lysine and FITC molecules for a few reasons; firstly, lysine and FITC are fluorescent molecules, and functionalization of these molecules can be monitored by FRET and ligand replacement reactions. Secondly, fluorescence of these molecules can be used for the fluorometric estimation of bio-macromolecules. As reported in the literature, lysine shows a broader fluorescence peak at ∼435 nm at higher concentrations (>10 mM),³⁴ which overlaps with the Raman water peak at 405 nm. However, at lower lysine concentration (≤2 mM), a broad lysine fluorescence shoulder peak at ∼435 nm can be distinctly observed next to the water Raman peak, as shown in Fig. 5II.

Fig. 5 Functionalization of AuNPs with L-lysine, FITC, and FITC and L-lysine. (I) FITC fluorescence (Ex/Em: 488/520 nm) of (a) FITC, (b) AuNP–FITC (AF) and (c) AuNP–FITC–Lysine (AFL). Inset shows magnified spectra of (b) and (c). (II) Lysine fluorescence (Ex/Em: 355/∼435 nm) of (a) lysine, (b) LBH-AuNP–Lysine (AL) and (c) LBH AuNP–FITC–Lysine (AFL).

In the FRET study of the AFL nanoparticles, the individual fluorescence spectra of the FITC (5 μM) and lysine (2 mM) molecules (Fig. 5I(a) and II(a)) showed overall fluorescence quenching with the AuNPs (1.3 μM) to achieve AF and AL nanoparticles, as shown in Fig. 5II(a) and II(b), respectively. The FRET of lysine and FITC with the AuNPs were observed separately to gain better understanding of the bi-ligand functionalization of the LBH-AuNPs. In the preparation of the AFL nanoparticles, lysine was added to the AF nanoparticles. This resulted in the functionalization of the AF particles with lysine to produce bi-ligand AFL nanoparticles. Upon the addition of lysine to the AF nanoparticles, the restored fluorescence of the replaced FITC and the unbound residual lysine fluorescence were observed as shown in Fig. 5I(b) and I(b), which correspond to 2.3% (0.115 μM) and 4.3% (85 μM) of the concentrations of free and/or replaced FITC and lysine, respectively. Hence, more than 95% co-functionalization of both molecules was achieved with LBH-AuNPs. The concentrations of residual FITC and lysine were calculated using the standard graphs given in ESI: 2.†

Moreover, the above functionalizations were confirmed by UV-Visible spectroscopy. As shown in Fig. 6I(a–d), a red shift in the SPR peak from 512 nm to 545 nm was observed with corresponding functionalization, namely, (a) solely AuNPs, (b) AuNP–FITC (AF), AuNP–Lysine (AL) and AuNP–FITC–Lysine (AFL). The inset shows the corresponding colour change in colloidal solution from red to blue (a–d). This shift towards a longer wavelength can be attributed to an increase in the size and/or aggregation of the particles. This result was confirmed by TEM analysis. The TEM images of the corresponding samples are shown in Fig. 6II(a–d).

Fig. 6 (I) UV-Visible spectra of the functionalized nanoparticles: (a) AuNPs, (b) AuNP–FITC (AF), (c) AuNP–Lysine (AL), (d) AuNP–FITC–Lysine (AFL) with the inset showing the corresponding optical pictures. (II) TEM images of the corresponding functionalised particles from (a) to (d).

From comparison of the TEM images shown in Fig. 6II(a–d), compared to the LBH particles alone, AuNP–FITC (AF) showed a meagre change in particle size, whereas the AuNP–Lysine (AL) functionalised particles showed open chain string morphology. This observed morphology was due to the formation of interparticle hydrogen bonding between the lysine functionalised AuNPs.²⁴ Co-functionalized AuNPs with lysine and FITC showed an even greater degree of aggregation and/or increase in particle size. This gradual change in the size and aggregation of the particles explains the gradual shift of the SPR and the broadening of peaks for the corresponding functionalised AuNPs Fig. 6I(a–d). Therefore, UV-Visible spectroscopy and TEM analysis explain the single and bi-ligand functionalization of the LBH-AuNPs with lysine and FITC.

3.4. Fluorometric estimation of rat tail collagen using bi-ligand functionalised AFL nanoparticles

According to recent literature, AuNPs crosslink and immobilize the collagen by a nucleation process which has been used to build bio-active surfaces for tissue engineering.³⁵ In this study, the nucleation properties of the AuNPs with collagen were used for the rapid and sensitive fluorometric based estimation of collagen using AFL nanoparticles. Herein, we used the co-functionalized AFL particles for the quantification of collagen by a ligand replacement reaction. Commercial type-I rat tail standard collagen (P) was used, with varying concentrations ranging from 2 to 10 μg ml⁻¹. The FITC and lysine fluorescence recovery was observed to be highly linear with corresponding collagen (P) concentrations of 2 to 10 μg ml⁻¹, as shown in Fig. 7(A) and (B).

Fig. 7 Reappearance of the fluorescence from FITC and lysine from AuNP–FITC–Lysine (AFL) nanoparticles with different concentrations of collagen varying from 2 μg ml⁻¹ to 10 μg ml⁻¹. (A) and (B) represent the data associated with the reappearance of FITC and lysine fluorescence, respectively. I_F(P)_C is associated with the fluorescence peak intensity in the presence of collagen, and I_F(P)₀ is associated with the fluorescence peak intensity in nanoparticles without collagen. Collagen (P) shows the data associated with pure collagen obtained from Sigma-Aldrich, and collagen (RT) is the data associated with the extracted rat rail collagen.

The complete spectra are given in ESI: 3(a).† However, reaction precipitation was observed at higher concentrations of collagen (20 μg ml⁻¹). This is evident from the TEM images of the AFL NPs at collagen concentrations of 2 and 20 μg ml⁻¹, and colloidal images of these concentrations, as shown in ESI: 3(b).† 2 μg ml⁻¹ collagen remained in colloidal state, whereas 20 μg ml⁻¹ collagen showed precipitation. Furthermore, a similar reaction was performed with extracted rat tail collagen³¹ where the concentration was adjusted to 1 mg ml⁻¹ by the Sirius red method²⁷ (the standard graph of collagen by the Sirius red method is given in ESI: 3(c)†). Linearity in the reappearance of the FITC and lysine fluorescence from 2 to 10 μg ml⁻¹ was observed with the extracted rat tail collagen (RT), as shown in Fig. 7(A) and (B), respectively. The recovery response curves of FITC and lysine with different concentrations of rat tail collagen and pure standard collagen showed similar quantification and detection sensitivity (10.3 ± 0.4 and 0.34 ± 0.04 fluorescence units/(μg ml⁻¹) for FITC and lysine, respectively). The % recovery of different concentrations of rat tail collagen with the concurrent reappearance of lysine and FITC fluorescence is shown in Tables I and II in ESI: 3(d)†, respectively. This linearity in the reappearance of FITC and lysine fluorescence with increased concentrations of collagen could be attributed to the controlled aggregation of the AFL particles with the collagen. This aggregation of the AFL AuNPs with the collagen matrix may have occurred as a result of the interaction between the side chain of lysine, hydroxy-lysine and the hydroxyl-proline residue of the collagen fibrils³⁶ with the AFL particles, which could possibly lead to the controlled replacement of the fluorescent lysine and FITC molecules attached to the AuNP surface. As a result of this, bi-ligand functionalised AuNPs showed a linear reappearance of FITC and lysine fluorescence with increased collagen concentrations. To further demonstrate the usefulness of the bi-ligand functionalised AuNPs, similar experiments were performed with mono-functionalised AuNP–Lysine and AuNP–FITC, as shown in ESI: 3(e).† However, similar linear responses in the reappearance of lysine and FITC fluorescence with standard collagen (P) were not observed with mono-functionalised AuNP–Lysine and AuNP–FITC. Therefore, bi-ligand functionalised LBH-AuNPs were found to be more sensitive and efficient for the fluorometric estimation of collagen. Moreover, the fluorescence reappearance of both lysine and FITC from the AFL AuNPs provides additional confirmation that the method can be used for the estimation of collagen. Thus, these bi-ligand surface modified LBH AuNPs (AFL particles) have been successfully used for the sensitive fluorometric quantification of rat tail collagen.

4. Conclusions

In summary, a unique, rapid and facile hydrosol synthesis of highly dispersed AuNPs with sizes of ∼2 nm has been achieved using LiBH₄ as a reducing agent. The small particle size and stability under different physico-chemical conditions renders these particles suitable for bi-ligand co-functionalization with higher concentrations of fluorescent lysine and FITC molecules. Collagen induced controlled aggregation of the AFL particles was successfully used for the fluorometric quantification of rat tail collagen. Thus, these uniquely synthesized AuNPs have most of the desired characteristics, and can be used for a wide range of applications.

Acknowledgements

This work was financially supported by the Department of Biotechnology, Ministry of Science and Technology, Govt. of India (BT/PR13243/GBD/27/227/2009). We are thankful to Dr Gopal Pande, CCMB for useful discussion and lab space.

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Footnotes

† Electronic supplementary information (ESI): Calculation of the concentration of optimised LBH-AuNPs, fluorescence standard graphs of lysine and FITC, TEM and optical images of the collagen–AFL nanoparticles and a standard graph of extracted rat tail collagen are available. See DOI: 10.1039/c3ra48047j

‡ Equal contribution.

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