Gold nanoparticles possess unique chemical and physical properties, which can be harnessed for drug delivery [1
], the development of sensors [3
], biological imaging [5
], and the detection of biomarkers [6
], among other applications. More specifically, in sensor applications they have been deployed for chemical and biological sensing [7
], diagnostics [9
] and more recently in environmental monitoring [10
] because AuNPs based sensors can be fabricated with the potential to detect a wide variety of molecules (organic and inorganic), proteins, cancerous cells and metal ions. The key properties of AuNPs include electronic, magnetic and optical characteristics, with the latter arising from localized surface plasmon resonance (LSPR), which is the collective effect of the oscillations of free electrons at the surface of the metal [12
]. Gold nanoparticles are rich in conduction electrons, which can be easily polarized under the influence of an electromagnetic field to produce non-linear optical phenomena [13
]. Self-assembled monolayers (SAMs) have been used to modify the surface of gold nanoparticles, as they influence the stability of the colloidal solution, and provide the platform on which the reactivity of the particles can be altered [14
]. Changes in the SPR band can be used to determine whether functionalization of the AuNPs has occurred, while the effects of such changes can provide information on the nature of the interactions between the particles [15
]. Exploitation of these interactions can be used in the design of SAMs with different functional groups, which offer platforms that can selectively interact with appropriate analytes, resulting in the development of highly sensitive and versatile colorimetric sensors.
In the last few years, gold nanoparticles have been commercially available in a range of sizes (5–250 nm), and dispersed in a variety of stabilizers such as citrate buffer, phosphate-buffered saline (PBS), H2
O and D2
]. Published literature is replete with studies in which different preparation methods have been used to produce AuNPs in a variety of shapes, sizes, and in different stabilization media [20
]. However, the deployment of a myriad of preparation methods makes it difficult to compare results from different studies, particularly when little attention is paid to the impact of the experimental conditions on the surface behavior of the synthesized AuNPs. Only recently, a study by Havaldar et al. [23
] compared the functionalization behavior of AuNPs prepared following three different preparation methods, where it was suggested that the citrate-stabilized AuNPs prepared with the Turkevich et al. [17
] method did not show any potential for conjugation with any of the 22 l
-amino acids that were used. Conversely, the particles synthesized using plant-mediated and bacteria-mediated methods were more suitable for functionalization, highlighting for the first time the effect of the synthetic method on the surface properties of the AuNPs. The in situ synthesis introduced by Turkevich et al. [17
] is one of the most frequently used methods for production of citrate-stabilized AuNPs with sizes of about 20 nm. Over the years, a plethora of modifications have been made to this method to obtain AuNPs of different sizes (15–150 nm) by altering the trisodium citrate to HAuCl4
], changing the pH of the solution [25
], controlling the reaction temperature [26
], adding the use of fluorescent light irradiation [27
] or high-power ultrasound [28
], and substituting water with D2
O as the solvent [18
]. However, it has been suggested that increasing the citrate concentration could result in unstable nanoparticles overtime [29
], while adjusting the pH can have a significant effect on the size distribution and potentially on the shape of the particles [30
]. In a study following the method proposed by Turkevich et al. [17
] it was shown that decreasing the molar ratio between citrate and gold during synthesis, the size of the particles increased, while they became more polydisperse and less spherical [31
One of the most extensively studied methods to alter the surface properties of gold nanoparticles has been the use of SAMs based on sulfur, due to their binding affinity for the surface of gold. Selenium-based SAMs are to be preferred to those of sulfur because of their bonding preference and higher packing density on gold compared to thiol monolayers [32
]. As regards the classical method used for the production of AuNPs by citrate reduction and subsequent stabilization by the anion, there is now evidence to suggest that not all of the citrate is displaced from the surface of gold upon functionalization with thiols [34
]. Wei et al. [36
] monitored the kinetics of citrate desorption from the surface of AuNPs and showed that thiol-containing molecules were the most efficient at displacing citrate compared to amine- and carboxylate-containing molecules, but without complete displacement. The explanation for this observation was attributed to different binding energies of citrate on the gold surface. In contrast, Perera et al. [37
] have demonstrated that citrate residues can be displaced by thiols from the surface of AuNPs. Similarly, in a study using gold nanoparticles in thio-compound nanomedicine, citrate was displaced by the thiol-based drug thioabiraterone, which displayed interaction with the AuNPs [38
]. In another study [39
], the ability of three amino acids cysteine, arginine and glutamic acid to replace citrate from the surface of AuNPs was assessed, and it was demonstrated that cysteine containing a thiol functional group was the most effective at replacing citrate. These contradictory findings illustrate the importance of establishing the effect of the presence of the adlayer of citrate or solution contaminants on the functionalization behavior of synthesized AuNPs using both thiolates and selenides. Employing nanoparticles produced following procedures which meet certain standards, would allow better comparison of the results between different research investigations, and at the same time would ensure the quality and consistency of the observations.
Herein the conditions for the functionalization of citrate-stabilized gold nanoparticles with the short chain aliphatic compounds: cysteamine (Cys), 3-mercaptopropionic acid (3-MPA) and l
-selenocystine (SeCyst), respectively are investigated. The selection of these thiolate and diselenide compounds, which are of great interest for their utilization in SAMs, was based on their ability to provide platforms that can be utilized to link their terminal groups with other structures, thus producing materials with desirable properties for sensor applications. Two types of citrate-stabilized AuNPs were chosen; a purchased solution and an in-house synthesized based on a slightly modified version of the Turkevich method [20
]. The functionalization and purification methods were kept the same for both types of AuNPs, in order to assess the effect of the solution characteristics on the functionalization behavior. The presence of adsorbed citrate or other species on the surface of purchased and in-house prepared gold nanoparticles that could potentially affect the extent to which the particles are functionalized was investigated. For this purpose, UV-vis spectroscopy, 1
H nuclear magnetic resonance (1
H-NMR), attenuated total reflectance-Fourier-transform infrared (ATR-FTIR), and transmission electron microscopy (TEM) measurements were employed.
2. Experimental Section
2.1. Materials and Methods
The following reagents and materials were all purchased from Sigma-Aldrich Co., Ltd. (Gillingham, UK): gold nanoparticles (AuNP) stabilized suspension in citrate buffer (stated average diameter, 10 nm), cysteamine hydrochloride (NH2CH2CH2SH, ≥98%), HEPES buffer sodium salt (0.01 M), sodium bicarbonate (≥99.7%), hydrochloric acid (reagent grade, 37%), nitric acid (ACS reagent 70%), deuterium oxide deuteration degree min. 99.96% for NMR spectroscopy, l-selenocystine (CO2HCH(NH2)CH2(Se)2CH2CH(NH2)CO2H, 95%), 3-mercaptopropionic acid (HSCH2CH2CO2H, ≥99%), phosphate buffer solution (1.0 M, pH 7.7), gold standard for ICP (1000 mg/L Au in hydrochloric acid), hydrogen tetrachloroaurate (III), HAuCl4.3H2O, 99.99% pure, sodium hydroxide solution (0.05 M, ≥97%), sodium citrate monobasic, HOC(COONa)(CH2COOH)2, ≥99.5%. Trisodium citrate dihydrate, Na3C6H5O7.2H2O, 99% was purchased from Fischer Scientific (Loughborough, UK).
2.2. Synthesis and Characterization of Citrate-Capped AuNPs
All glassware was washed in aqua regia (3:1 HCl:HNO3
), rinsed with deionized water, and then oven-dried prior to use. Citrate-stabilized AuNPs were prepared using citrate reduction of HAuCl4
]. Briefly, HAuCl4
(100 mL, 0.506 mM) was brought to the boil under vigorous stirring, and then trisodium citrate dihydrate (10 mL, 19.4 mM, pH 8.5) was quickly added. The color of the solution turned from light yellow to colorless, black and finally wine red. After boiling and stirring for 10 min, the solution was set aside and allowed to cool to room temperature, followed by filtering through a 0.22 μm pore diameter syringe filter (Merck Millipore, Watford, UK) to remove any large particulates. The resulting solution was stored in the dark at 4 °C until required. The UV-vis absorption spectrum showed that λmax
the peak maximum of the SPR band was at 520 nm. Transmission electron microscopy (TEM) indicated that the average size of the formed nanoparticles was 13.8 ± 1.2 nm. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis was performed to determine the concentration of the synthesized AuNPs, and was found to be 10.80 mg L−1
The purchased AuNPs used in this study were acquired from Sigma-Aldrich Co., Ltd. According to the information supplied, the solution was characterized by a core size range of 8–12 nm (average diameter of 10 nm) and an absorption maximum of 510–525 nm, stabilized in citrate buffer. The measured absorbance maximum was 524 nm, and the concentration of gold in the solution determined by ICP-OES was 60.3 ± 0.2 mg L−1 or (0.307 mM). This result corresponds to an average nanoparticle diameter of 8 nm, which was confirmed by TEM measurements indicating an average diameter of 7.3 ± 1.2 nm.
2.3. Modification and Purification of Cysteamine, 3-Mercaptopropionic Acid and l-Selenocystine Capped AuNPs
For the functionalization of the AuNPs with either cysteamine (Cys), 3-mercaptopropionic acid (3-MPA) or l-selenocystine (SeCyst), solutions of varying molarities (0.256, 0.506, 1.012, 2.024, 3.036, 4.048 and 5.06 mM) of the three compounds were prepared in different dissolution media. The media used were deionized water, D2O, HEPES buffer (0.01 M, pH 9.7) and phosphate buffer (0.01 M, pH 7.7). Functionalization was performed in 96-well plates, by adding 50 μL of AuNPs and 50 μL of each of the functionalizing agents, dissolved in any of the chosen dissolution media. To obtain a final volume of 1 mL for each solution, which would undergo purification or any later analyses, 10 wells were used each time. Stability assessment of the colloids was performed for all solutions and at various pH values after adjustment with either HCl or NaOH. During preliminary studies, optimization of the stability conditions was performed using the purchased AuNPs, which were subsequently used for the modification of both purchased and synthesized AuNPs. The conditions were kept the same to ensure that any disparity in the observations was not caused by differences in the methods followed. UV-vis measurements of the colloidal solutions were obtained after 2 h and overnight incubation at 4 °C. All samples were stored in the dark.
The modified gold nanoparticles were purified by removing the excess un-adsorbed functionalizing agents (Cys, 3-MPA or SeCyst) before any measurements. After overnight incubation at 4 °C, excess reagents were removed by a cycle of centrifugation using open-top thick wall propylene tubes (TLA-120.2, Beckman Coulter, Brea, CA, USA). 1 mL aliquots of the samples was initially centrifuged at 22,000 rpm for 40 min at 4 °C, the supernatant was carefully removed and 500 μL of phosphate buffer (pH 7.7) was added. The pellets were re-suspended in phosphate buffer after sonication at room temperature for 5 min, and the resulting colloidal solution was then centrifuged at 22,000 rpm for 40 min at 4 °C. Lastly, after removing the supernatant, the precipitate was freeze dried and kept in the freezer for subsequent analyses. The above speeds and times were experimentally optimized, in order to avoid aggregation or the production of tight pellets.
2.4. Characterization of AuNPs Capped with Different Agents
The UV-vis absorption spectra of the samples were recorded using a microplate reader (Infinite M200, Tecan, San Francisco, CA, USA) in Nunclon transparent 96 well microplates (Thermo Scientific, Hempstead, UK), to obtain information about the position and shape of the LSPR band, before and after the various modifications (e.g., functionalization of the AuNPs). Attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectra were obtained using a PerkinElmer spectrum 100 FTIR spectrometer (PerkinElmer, Waltham, CA, USA) equipped with a universal ATR (UATR) accessory. The samples were lyophilized prior to analysis. FTIR results were used to study the conjugation of the AuNP surface. 1H nuclear magnetic resonance (1H-NMR) spectra were recorded on an AVANCE III (400 MHz) NMR spectrometer (Bruker, Billerica, MA, USA) to assess the functionalization and characterize the composition of the synthesized and purchased AuNP solutions. All samples were freeze dried overnight and re-suspended in D2O before analysis. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analyses were performed on an ACTIVA M ICP-OES spectrometer (HORIBA Jobin Yvon IBH Ltd., Glasgow, UK), in order to determine the concentration of gold in the purchased and synthesized AuNP solutions. Appropriate volumes of Au standard to give concentrations of 0, 5, 10, 15 and 20 mg L−1 were transferred into 100 mL volumetric flasks, 1 mL of aqua regia was added in each sample, and the volume was made up to mark with deionized water. The synthesized and purchased AuNP samples were prepared by digestion of 1 mL of each solution with equal volume of aqua regia for 30 min, and then the volume was made up to 10 mL. All samples were analyzed in triplicates, at two wavelengths: 242.795 and 267.595 nm. Inductively coupled plasma mass spectrometry (ICP-MS) measurements were obtained on a NexIon 350X ICP-MS tuned using a NexIon Setup Solution (Perkin Elmer, Buckinghamshire, UK). Semi-quantitative analysis was performed to assess whether the re-suspension of gold in the supernatant had occurred after the centrifugation of the AuNPs. The following Au standards: 0, 0.1 and 1 mg L−1 were prepared in deionized water. Transmission electron microscopy (TEM) measurements were carried out to investigate the size, shape, and confirm the surface modification of the nanoparticles. For the analysis, a few drops of methanol were added to vials of dried samples, and after sonication a drop was placed on a copper TEM grid coated in a continuous carbon film. Following drying, the samples were examined with a FEI Titan3 Themis G2 S/TEM (FEI company, Hillsboro, OR, USA) operating at 300 kV fitted with four energy dispersive X-ray (EDX) silicon drift detectors, a Gatan Quantum ER energy filter (Gatan Inc, Pleasanton, CA, USA) and a Gatan One-View CCD. The size distribution analysis was performed using ImageJ analysis software. Energy-dispersive X-ray (EDX) spectroscopy and mapping was undertaken using Bruker Esprit v1.9 software, and electron energy loss spectroscopy (EELS) were collected and analyzed using Gatan Digital Micrograph V3.01.