- Email: [email protected]
Cysteine sensing by plasmons of silver nanocubes
Journal of Solid State Chemistry 241 (2016) 110–114
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Journal of Solid State Chemistry journal homepage: www.elsevier.com/locate/jssc
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Cysteine sensing by plasmons of silver nanocubes Eitan Elfassy, Yitzhak Mastai, Adi Salomon n BINA Institute for Nanotechnology and Advanced Materials, Department of Chemistry, Bar-Ilan University, Ramat-Gan 5290002, Israel
art ic l e i nf o
a b s t r a c t
Article history: Received 8 March 2016 Received in revised form 25 May 2016 Accepted 5 June 2016 Available online 7 June 2016
Noble metal nanoparticles are considered to be valuable nanostructures in the field of sensors due to their spectral response sensitivity to small changes in the surrounding refractive index which enables them to detect a small amount of molecules. In this research, we use silver nanocubes of about 50 nm length to detect low concentrations of cysteine, a semi-essential amino acid. Following cysteine adsorption onto the nanocubes, a redshift in the plasmonic modes was observed, enabling the detection of cysteine down to 10 mM and high sensitivity of about 125 nm/RIU (refractive index units). Furthermore, we found that multilayer adsorption of cysteine leads to the stabilization of the silver nanocubes. The cysteine growth onto the nanocubes was also characterized by high-resolution transmission electron microscopy (HR-TEM). & 2016 Elsevier Inc. All rights reserved.
Keywords: Localized surface plasmon resonance Cysteine Silver nanoparticles Chiral plasmonic systems Molecule-plasmon hybrid systems
1. Introduction Recent progress in metallic nanoparticle synthesis [1], as well as nanofabrication [2], has boosted the use of nanoparticles in a new kind of sensor called a “localized surface plasmon resonance” (LSPR) sensor. The synthesis of metallic nanoparticles has been improved to the extent that control over their geometrical parameters and distribution can be achieved [3–6]. The size, morphology, surrounding dielectric environment and metallic type determine the plasmonic frequencies [7] and the relative intensities of the LSPR modes [8]. Noble metal nanoparticles display strong LSPR [9,10] and high EM (electromagnetic) enhancement, and thus enable both chemical- and bio-sensing [11,12]. However, due to the difficulties inherent in colloidal stability, LSPR sensors usually require immobilization of the nanoparticles to a substrate [2]. Among the plasmonic-based sensors, the surface plasmon resonance (SPR) sensor is the leading biosensor which is commercially used in fields such as food safety and medical diagnostics [13]. In the last decade, noble metal nanoparticles have been investigated as a new promising LSPR sensor. Though their bulk sensitivity is considered to be smaller than that of SPR or holearray-based sensors, LSPR sensors still exhibit an advantage due to the fact that their EM field is spatially localized around the particle. Consequently, an adsorbed film of molecules, typically a few nanometers long, occupies a large fraction of the plasmonic field compared with SPR, and thus increases their sensitivity. Indeed, n
Corresponding author. E-mail addresses: [email protected] (E. Elfassy), [email protected] (Y. Mastai), [email protected] (A. Salomon). http://dx.doi.org/10.1016/j.jssc.2016.06.002 0022-4596/& 2016 Elsevier Inc. All rights reserved.
McFarland et al. were able to detect a monolayer of 1-hexadecanethiol on silver nanoparticles using dark field microscopy [14], and Riboh et al. reported the detection of 700 pM anti-biotin, a very large molecule [15]. The LSPR sensitivity is related to the plasmonic peak width which is determined by the nanoparticle size, size-distribution and geometry. Particle sizes between 20 nm and 100 nm offer the best compromise. In smaller particles, the plasmonic lifetime is reduced due to electron scattering, and in larger ones, radiative damping starts to dominate resulting in broadening of the plasmonic peak [8]. The effect of the nanoparticle geometry on the plasmon line width of triangular silver nanoprism was investigated by single particle darkfield spectroscopy correlated with electron microscopy [16,17]. Corner truncation and roundness of the silver nanoprism were shown to strongly affect the plasmon line width [16]. Finite-difference time-domain (FDTD) calculations were performed to take into account the effects of particle geometry and almost reproduced the values of the observed line widths [17]. In this study, we chose to work with silver nanocubes mainly due to their two main advantages for sensing applications. First, their cubic symmetry induces splitting of the plasmon modes and a specific distribution of the electromagnetic field around the nanoparticles which enhance the plasmon sensitivity [18]. Secondly, their plasmon modes can be excited in the UV, closer to the optical transitions of organic molecules, thereby increasing their sensitivity. Cysteine is a sulfur-containing amino acid that has been evidenced to play a role in many physiological processes and diseases. For instance, the plasma cysteine/sulphate ratio is increased by a factor of five for patients with Parkinson's disease [19]. In
E. Elfassy et al. / Journal of Solid State Chemistry 241 (2016) 110–114
consequence, many efforts have been made to develop methods for the selective detection of cysteine, such as colorimetric assays based on the aggregation of silver nanoparticles and Ca2 þ ions as cross-linking agents [20]. However, in these colorimetric studies, the effect of the aggregation on the UV–vis. spectrum is used for the detection of cysteine, rather than the shift in the LSPR induced by the adsorption of the cysteine molecules. In this work, we used 50-nm silver nanocubes for cysteine sensing down to 10 μM using the LSPR shift induced by adsorption with no need to immobilize the silver nanoparticles prior to detection. By fitting the experimental results to a simple model [21], a sensitivity of 125 nm/RIU (refractive index units) was deduced. The self-assembly process of the cysteine on the silver nanocubes was studied by high-resolution transmission electron microscopy (HR-TEM) as well, and was found to be in agreement with the model used.
2. Materials and methods 2.1. Materials Ethylene glycol (AR, 107-21-1) was purchased from Bio-Lab Ltd. (Israel). Silver nitrate (7761-88-8), polyvinylpyrrolidone (PVP, 40,000 average molecular weight, 9003-39-8), L-cysteine (52-904) and methanol (99.8%, 67-56-1) were purchased from SigmaAldrich.
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showed that the silver nanoparticles after cysteine adsorption are stable for at least 48 h (see figs. SI1 and SI2 of Supplementary Information). 2.4. UV–vis. measurements UV–vis. measurements were performed with a Jasco V-530 spectrophotometer. Clean methanol was used as a reference baseline. Vials of nanoparticles with different cysteine concentrations were used for the measurements. Before taking measurements, the vials were subjected to one minute of sonication. The cuvettes for the measurements contained 200 μL of nanoparticles each, taken from the vials, in addition to 2.5 mL of methanol. Standard glass cuvettes were used for the measurements. 2.5. TEM and high-resolution TEM measurements Transmission electron microscopy (TEM) measurements were performed with a JEM-1400 (JEOL), with an accelerating voltage of 120 kV. Standard copper grids with carbon film were used. For that purpose, 15 μL of the sample in methanol were dropped on the grid and air dried. For further investigation of the core and the shell of nanoparticles, high-resolution TEM measurements were performed (JEM-2100 JEOL), with an accelerating voltage of 200 kV. Conventional nano-beam diffraction analysis (NBD) and Fourier transform analysis were used for the characterization of the nanoparticles. Elemental analysis was obtained by energy dispersive spectroscopy (EDS) from a Thermo Noran System 7.
2.2. Synthesis of the silver nanocubes The silver nanocubes were prepared using a variant of the polyol method described by Xia et al. [22,23] During the synthesis, the solution was stirred vigorously at 130 ⁰C. First, 25 mL of ethylene glycol were poured into a triple-neck round-bottom flask and heated for one hour. Then, 5 mL of 3 mM of hydrochloric acid in ethylene glycol were added. After 15 min, polyvinylpyrrolidone (PVP, 15 mL, 150 mM in ethylene glycol) and AgNO3 (15 mL, 95 mM in ethylene glycol) were simultaneously added, drop by drop, through each of the flask's necks. At that point, the reaction mixture was left overnight. The change in the color of the reaction solution from yellow to transparent proved that only nanoclusters were present. These nanoclusters, in the presence of PVP, grew into single-crystal nanocubes. The flask was then closed and left for 10 h until the solution turned brown-red and the silver nanocubes were obtained. The silver nanocubes were separated by centrifugation at 6000 rpm and were re-dispersed by sonication in water or organic solvents. The nanocubes were washed first with water and then twice with a mixture of acetone and water (2:1 volume ratio). Acetone was used to remove excess PVP from the nanoparticles [24]. Finally, the silver nanocubes were re-dispersed in methanol and the washed nanoparticles were mixed to obtain a homogeneous batch of clean silver nanoparticles in methanol. This clean batch of nanoparticles (with concentration of about 1 mg/mL) was further used for the experiments. 2.3. Self-assembly of cysteine multilayers onto silver nanocubes A stock solution of 1 mM of cysteine in methanol was prepared for the self-assembly processes. Stirring and slight heating were applied until all the cysteine crystals were dissolved and a transparent solution was obtained. Next, clean silver nanoparticles in methanol were inserted in vials with different concentrations of cysteine. The cysteine concentrations ranged between 0 μM (for the control) and 500 μM and the vials were finally closed and left for 24 h before analysis. The UV–vis. and TEM measurements
3. Results and discussion Silver nanocubes were characterized both by TEM and UV–vis. measurements. The size distribution of the nanoparticles was measured as 50 nm75 nm, as can be clearly seen in the TEM image (Fig. 1(A)), in agreement with their measured UV–vis. spectrum shown in Fig. 1(B). Three characteristic plasmon resonance modes appeared: a weak peak at 350 nm, a shoulder at 390 nm and an intense peak at 450 nm, as reported elsewhere for 50-nm silver nanocubes [1,25]. The peaks at 350 nm and 390 nm are due to quadrupole and dipole scattering, whereas the broad band at 450 nm is due to dipole scattering [26,27]. The full width at half maximum (FWHM) of about 75 nm is evidence of nanoparticles with narrow size dispersion [22]. The self-assembly and growth of cysteine onto the silver nanocubes was studied both by TEM and UV–vis. spectroscopy. TEM images of coated silver nanocubes with different concentrations of cysteine are shown in Fig. 2. A thin uniform film of about 1 nm is observed for the concentration of 10 mM cysteine, suggesting coverage of about 1–2 layers. At the concentration of 0.5 mM, a layer of about 5 nm is observed. Further investigations of the structure of the core and the shell of the nanoparticles were carried out by TEM diffraction methods. The core of the coated nanoparticles was characterized by nano-beam diffraction (NBD) analysis (Fig. SI3B of Supplementary Information). According to the NBD, the sets of reflections were clearly found to belong to the face-centered cubic (FCC) structure of silver (a ¼4.067 Å). The shell of the nanoparticles was characterized by fast Fourier transform (FFT) analysis, and the sets of the reflections corresponded to the orthorhombic structure of cysteine (inset of Fig. SI3A). The presence of cysteine was finally confirmed by energy dispersive spectroscopy (EDS). The elemental analysis showed more sulfur in the coating region than in the core of the nanoparticles. The detection of a small amount of cysteine was achieved by a relatively simple, fast and inexpensive method – UV–vis. measurements. The spectra of the bare and cysteine-coated silver
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Fig. 1. (a) TEM image of silver nanocubes confirmed a side length of about 50 nm. (b) UV–vis. spectrum of 50-nm silver nanocubes suspended in methanol, showing the three characteristic plasmon resonance modes at 350, 390 and 450 nm.
Fig. 2. TEM images of silver nanocubes coated with multilayers of cysteine: 10 μM (a), 15 μM (b) and 0.5 mM (c,d). L-cysteine was used for A, B and C and D-cysteine was used for D.
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Fig. 3. (a) UV–vis. spectra of 50 nm silver nanocubes coated with cysteine at different concentrations. Dashed lines: bare silver nanocubes. Solid lines: nanocubes after selfassembly of cysteine for different cysteine concentrations, the trend for increasing concentrations is shown by the arrows. (b) Dependence of the intense plasmonic mode resonance wavelength as a function of cysteine concentration. The plasmon mode of the bare nanoparticles (451 nm) was taken as reference. The dashed line is a fit to the equation: Δλ ¼a(1 exp( b*C)), with a¼ 20.17 nm, b ¼0.031*106 L mol 1 and r2 ¼ 0.98. The physical interpretation of the parameters a and b is discussed in connection with Eq. (2) where a ¼m Δn and b ¼ 2α/δ. The standard deviation was calculated for three parallel samples at high cysteine concentration, assumed to be similar for all the concentrations. (c) Dependence of the intense plasmon peak absorbance as a function of cysteine concentration.
nanocubes are shown in Fig. 3(A). Two main trends are observed: a redshift in the plasmon resonance peaks due to cysteine multilayer growth, together with an increase in the plasmonic mode amplitudes, as shown in Fig. 3(B) and (C), respectively. The redshift in the intense plasmon resonance peak is about 15 nm, whereas saturation is observed at about 50 μM of cysteine (Fig. 3(B)), probably due to the decay length of the plasmonic field in this wavelength range. In order to estimate the sensitivity of these nanocubes to cysteine detection, we adopted a quantitative simple model which is based on the variation in the refractive index, Δn, due to the adsorbed dielectric medium. The model was developed for metallic surfaces [21], but can also be used for nanoparticles [2]:
where Δλ is the shift of plasmonic mode wavelength, d is the dielectric layer thickness, δ is the electromagnetic field decay length into the dielectric medium and m is the bulk refractive index sensitivity of the plasmon frequency upon change in the refractive index. Assuming that d is linearly proportional to the concentration C, we obtain the following equation:
d⎤ ⎡ Δλ = m Δn ⎢ 1 − e−2 δ ⎥ ⎣ ⎦
1 The refractive index of methanol is n¼1.33 and by taken n¼ 1.49 for cysteine (refractive index of the similar 3-mercaptopropionic acid reported by Sigma) we get Δn¼ 0.16.
(1)
αC ⎤ ⎡ Δλ = m Δn ⎢ 1 − e−2 δ ⎥ ⎣ ⎦
(2)
where α ¼d/C. Substituting Δn ¼0.16 , and fitting this equation to the experimental results, we obtained m ¼125 nm/RIU. We disregarded the data point for C¼ 50 μM at which the plasmonic 1
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modes are barely shifted. In order to check the validity of this model we compared the thickness of the cysteine layer obtained from both TEM and this model. From fitting the model to the experimental results we get α ¼3.1 10 7 m4/mol. Substituting C ¼15 μM and δ ¼ 20 nm (the decay length of silver at λ ¼450 nm [18]), we get d∼5 nm, which is consistent with the TEM image analysis. A similar sensitivity of 145 nm/RIU, using 30 nm silver nanocubes was reported by Sherry et al. using dark field microscopy [18] and a slightly higher sensitivity of 197 nm/RIU was obtained by McFarland et al. using triangular single silver nanoparticles [14]. This relatively high sensitivity enables them to detect 60,000 hexadecanethiol molecules using dark field microscopy. Another important consequence of the cysteine adsorption is the improvement in nanoparticle stability. As can be clearly observed from the UV–vis. measurements (Fig. 3(c)), the plasmonic peaks amplitude increases with the cysteine concentration up to 50 μM in agreement with the detection saturation (Fig. 3(b)). We relate it to stabilization of the silver nanocubes due to the formation of thiol-Ag bonds following cysteine adsorption, and coating of the nanoparticles with molecules. Therefore, their tendency to aggregate dramatically decreases and they become relatively more stable. The chemical adsorption of cysteine onto the silver nanoparticles leads to a reduction in their surface energy which further decreases by the self-assembly process. Furthermore, we note that cysteine is barely soluble in methanol and therefore is more likely to adhere to the silver nanoparticle. For example, in aqueous solutions silver nanoparticles were unstable upon addition of cysteine. Thus, we postulate that methanol was necessary in order get a uniform cysteine coating onto the nanoparticles and to run LSPR measurements under the reported experimental conditions. We mention that the role of PVP polymer was not only essential for nanocubes synthesis but also for initializing the silver nanocube stability prior to cysteine adsorption.
4. Conclusions For a long time, both the chemistry and biology communities have been seeking ultra-sensitive detection systems, with the ability of chiral recognition. We demonstrate here a method for detecting cysteine, a chiral molecule which possesses a vital role in physiological processes and diseases, that is different from the colorimetric assays based on nanoparticle aggregation. High sensitivity of about 125 nm/RIU using UV–vis. measurements is obtained, based on the red shift of the plasmonic modes upon cysteine growth on silver nanocubes. Although higher sensitivity can be achieved using dark-field microscopy [14,18], here we demonstrate a useful method that is effective on a very small molecule. Additionally, by adsorption of chiral molecules on highly symmetric silver nanocubes, we have formed hybrid materials with potential chiroptical properties [28,29]. Such hybrid materials are also interesting for the study of strong coupling between chiral molecules and plasmonic systems [30], a field which has not yet been explored.
Acknowledgment This research was supported by a Grant from the GIF, the German-Israeli Foundation for Scientific Research and Development (number 203785).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jssc.2016.06.002.
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Contents lists available at ScienceDirect
Journal of Solid State Chemistry journal homepage: www.elsevier.com/locate/jssc
Rapid communication
Cysteine sensing by plasmons of silver nanocubes Eitan Elfassy, Yitzhak Mastai, Adi Salomon n BINA Institute for Nanotechnology and Advanced Materials, Department of Chemistry, Bar-Ilan University, Ramat-Gan 5290002, Israel
art ic l e i nf o
a b s t r a c t
Article history: Received 8 March 2016 Received in revised form 25 May 2016 Accepted 5 June 2016 Available online 7 June 2016
Noble metal nanoparticles are considered to be valuable nanostructures in the field of sensors due to their spectral response sensitivity to small changes in the surrounding refractive index which enables them to detect a small amount of molecules. In this research, we use silver nanocubes of about 50 nm length to detect low concentrations of cysteine, a semi-essential amino acid. Following cysteine adsorption onto the nanocubes, a redshift in the plasmonic modes was observed, enabling the detection of cysteine down to 10 mM and high sensitivity of about 125 nm/RIU (refractive index units). Furthermore, we found that multilayer adsorption of cysteine leads to the stabilization of the silver nanocubes. The cysteine growth onto the nanocubes was also characterized by high-resolution transmission electron microscopy (HR-TEM). & 2016 Elsevier Inc. All rights reserved.
Keywords: Localized surface plasmon resonance Cysteine Silver nanoparticles Chiral plasmonic systems Molecule-plasmon hybrid systems
1. Introduction Recent progress in metallic nanoparticle synthesis [1], as well as nanofabrication [2], has boosted the use of nanoparticles in a new kind of sensor called a “localized surface plasmon resonance” (LSPR) sensor. The synthesis of metallic nanoparticles has been improved to the extent that control over their geometrical parameters and distribution can be achieved [3–6]. The size, morphology, surrounding dielectric environment and metallic type determine the plasmonic frequencies [7] and the relative intensities of the LSPR modes [8]. Noble metal nanoparticles display strong LSPR [9,10] and high EM (electromagnetic) enhancement, and thus enable both chemical- and bio-sensing [11,12]. However, due to the difficulties inherent in colloidal stability, LSPR sensors usually require immobilization of the nanoparticles to a substrate [2]. Among the plasmonic-based sensors, the surface plasmon resonance (SPR) sensor is the leading biosensor which is commercially used in fields such as food safety and medical diagnostics [13]. In the last decade, noble metal nanoparticles have been investigated as a new promising LSPR sensor. Though their bulk sensitivity is considered to be smaller than that of SPR or holearray-based sensors, LSPR sensors still exhibit an advantage due to the fact that their EM field is spatially localized around the particle. Consequently, an adsorbed film of molecules, typically a few nanometers long, occupies a large fraction of the plasmonic field compared with SPR, and thus increases their sensitivity. Indeed, n
Corresponding author. E-mail addresses: [email protected] (E. Elfassy), [email protected] (Y. Mastai), [email protected] (A. Salomon). http://dx.doi.org/10.1016/j.jssc.2016.06.002 0022-4596/& 2016 Elsevier Inc. All rights reserved.
McFarland et al. were able to detect a monolayer of 1-hexadecanethiol on silver nanoparticles using dark field microscopy [14], and Riboh et al. reported the detection of 700 pM anti-biotin, a very large molecule [15]. The LSPR sensitivity is related to the plasmonic peak width which is determined by the nanoparticle size, size-distribution and geometry. Particle sizes between 20 nm and 100 nm offer the best compromise. In smaller particles, the plasmonic lifetime is reduced due to electron scattering, and in larger ones, radiative damping starts to dominate resulting in broadening of the plasmonic peak [8]. The effect of the nanoparticle geometry on the plasmon line width of triangular silver nanoprism was investigated by single particle darkfield spectroscopy correlated with electron microscopy [16,17]. Corner truncation and roundness of the silver nanoprism were shown to strongly affect the plasmon line width [16]. Finite-difference time-domain (FDTD) calculations were performed to take into account the effects of particle geometry and almost reproduced the values of the observed line widths [17]. In this study, we chose to work with silver nanocubes mainly due to their two main advantages for sensing applications. First, their cubic symmetry induces splitting of the plasmon modes and a specific distribution of the electromagnetic field around the nanoparticles which enhance the plasmon sensitivity [18]. Secondly, their plasmon modes can be excited in the UV, closer to the optical transitions of organic molecules, thereby increasing their sensitivity. Cysteine is a sulfur-containing amino acid that has been evidenced to play a role in many physiological processes and diseases. For instance, the plasma cysteine/sulphate ratio is increased by a factor of five for patients with Parkinson's disease [19]. In
E. Elfassy et al. / Journal of Solid State Chemistry 241 (2016) 110–114
consequence, many efforts have been made to develop methods for the selective detection of cysteine, such as colorimetric assays based on the aggregation of silver nanoparticles and Ca2 þ ions as cross-linking agents [20]. However, in these colorimetric studies, the effect of the aggregation on the UV–vis. spectrum is used for the detection of cysteine, rather than the shift in the LSPR induced by the adsorption of the cysteine molecules. In this work, we used 50-nm silver nanocubes for cysteine sensing down to 10 μM using the LSPR shift induced by adsorption with no need to immobilize the silver nanoparticles prior to detection. By fitting the experimental results to a simple model [21], a sensitivity of 125 nm/RIU (refractive index units) was deduced. The self-assembly process of the cysteine on the silver nanocubes was studied by high-resolution transmission electron microscopy (HR-TEM) as well, and was found to be in agreement with the model used.
2. Materials and methods 2.1. Materials Ethylene glycol (AR, 107-21-1) was purchased from Bio-Lab Ltd. (Israel). Silver nitrate (7761-88-8), polyvinylpyrrolidone (PVP, 40,000 average molecular weight, 9003-39-8), L-cysteine (52-904) and methanol (99.8%, 67-56-1) were purchased from SigmaAldrich.
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showed that the silver nanoparticles after cysteine adsorption are stable for at least 48 h (see figs. SI1 and SI2 of Supplementary Information). 2.4. UV–vis. measurements UV–vis. measurements were performed with a Jasco V-530 spectrophotometer. Clean methanol was used as a reference baseline. Vials of nanoparticles with different cysteine concentrations were used for the measurements. Before taking measurements, the vials were subjected to one minute of sonication. The cuvettes for the measurements contained 200 μL of nanoparticles each, taken from the vials, in addition to 2.5 mL of methanol. Standard glass cuvettes were used for the measurements. 2.5. TEM and high-resolution TEM measurements Transmission electron microscopy (TEM) measurements were performed with a JEM-1400 (JEOL), with an accelerating voltage of 120 kV. Standard copper grids with carbon film were used. For that purpose, 15 μL of the sample in methanol were dropped on the grid and air dried. For further investigation of the core and the shell of nanoparticles, high-resolution TEM measurements were performed (JEM-2100 JEOL), with an accelerating voltage of 200 kV. Conventional nano-beam diffraction analysis (NBD) and Fourier transform analysis were used for the characterization of the nanoparticles. Elemental analysis was obtained by energy dispersive spectroscopy (EDS) from a Thermo Noran System 7.
2.2. Synthesis of the silver nanocubes The silver nanocubes were prepared using a variant of the polyol method described by Xia et al. [22,23] During the synthesis, the solution was stirred vigorously at 130 ⁰C. First, 25 mL of ethylene glycol were poured into a triple-neck round-bottom flask and heated for one hour. Then, 5 mL of 3 mM of hydrochloric acid in ethylene glycol were added. After 15 min, polyvinylpyrrolidone (PVP, 15 mL, 150 mM in ethylene glycol) and AgNO3 (15 mL, 95 mM in ethylene glycol) were simultaneously added, drop by drop, through each of the flask's necks. At that point, the reaction mixture was left overnight. The change in the color of the reaction solution from yellow to transparent proved that only nanoclusters were present. These nanoclusters, in the presence of PVP, grew into single-crystal nanocubes. The flask was then closed and left for 10 h until the solution turned brown-red and the silver nanocubes were obtained. The silver nanocubes were separated by centrifugation at 6000 rpm and were re-dispersed by sonication in water or organic solvents. The nanocubes were washed first with water and then twice with a mixture of acetone and water (2:1 volume ratio). Acetone was used to remove excess PVP from the nanoparticles [24]. Finally, the silver nanocubes were re-dispersed in methanol and the washed nanoparticles were mixed to obtain a homogeneous batch of clean silver nanoparticles in methanol. This clean batch of nanoparticles (with concentration of about 1 mg/mL) was further used for the experiments. 2.3. Self-assembly of cysteine multilayers onto silver nanocubes A stock solution of 1 mM of cysteine in methanol was prepared for the self-assembly processes. Stirring and slight heating were applied until all the cysteine crystals were dissolved and a transparent solution was obtained. Next, clean silver nanoparticles in methanol were inserted in vials with different concentrations of cysteine. The cysteine concentrations ranged between 0 μM (for the control) and 500 μM and the vials were finally closed and left for 24 h before analysis. The UV–vis. and TEM measurements
3. Results and discussion Silver nanocubes were characterized both by TEM and UV–vis. measurements. The size distribution of the nanoparticles was measured as 50 nm75 nm, as can be clearly seen in the TEM image (Fig. 1(A)), in agreement with their measured UV–vis. spectrum shown in Fig. 1(B). Three characteristic plasmon resonance modes appeared: a weak peak at 350 nm, a shoulder at 390 nm and an intense peak at 450 nm, as reported elsewhere for 50-nm silver nanocubes [1,25]. The peaks at 350 nm and 390 nm are due to quadrupole and dipole scattering, whereas the broad band at 450 nm is due to dipole scattering [26,27]. The full width at half maximum (FWHM) of about 75 nm is evidence of nanoparticles with narrow size dispersion [22]. The self-assembly and growth of cysteine onto the silver nanocubes was studied both by TEM and UV–vis. spectroscopy. TEM images of coated silver nanocubes with different concentrations of cysteine are shown in Fig. 2. A thin uniform film of about 1 nm is observed for the concentration of 10 mM cysteine, suggesting coverage of about 1–2 layers. At the concentration of 0.5 mM, a layer of about 5 nm is observed. Further investigations of the structure of the core and the shell of the nanoparticles were carried out by TEM diffraction methods. The core of the coated nanoparticles was characterized by nano-beam diffraction (NBD) analysis (Fig. SI3B of Supplementary Information). According to the NBD, the sets of reflections were clearly found to belong to the face-centered cubic (FCC) structure of silver (a ¼4.067 Å). The shell of the nanoparticles was characterized by fast Fourier transform (FFT) analysis, and the sets of the reflections corresponded to the orthorhombic structure of cysteine (inset of Fig. SI3A). The presence of cysteine was finally confirmed by energy dispersive spectroscopy (EDS). The elemental analysis showed more sulfur in the coating region than in the core of the nanoparticles. The detection of a small amount of cysteine was achieved by a relatively simple, fast and inexpensive method – UV–vis. measurements. The spectra of the bare and cysteine-coated silver
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Fig. 1. (a) TEM image of silver nanocubes confirmed a side length of about 50 nm. (b) UV–vis. spectrum of 50-nm silver nanocubes suspended in methanol, showing the three characteristic plasmon resonance modes at 350, 390 and 450 nm.
Fig. 2. TEM images of silver nanocubes coated with multilayers of cysteine: 10 μM (a), 15 μM (b) and 0.5 mM (c,d). L-cysteine was used for A, B and C and D-cysteine was used for D.
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Fig. 3. (a) UV–vis. spectra of 50 nm silver nanocubes coated with cysteine at different concentrations. Dashed lines: bare silver nanocubes. Solid lines: nanocubes after selfassembly of cysteine for different cysteine concentrations, the trend for increasing concentrations is shown by the arrows. (b) Dependence of the intense plasmonic mode resonance wavelength as a function of cysteine concentration. The plasmon mode of the bare nanoparticles (451 nm) was taken as reference. The dashed line is a fit to the equation: Δλ ¼a(1 exp( b*C)), with a¼ 20.17 nm, b ¼0.031*106 L mol 1 and r2 ¼ 0.98. The physical interpretation of the parameters a and b is discussed in connection with Eq. (2) where a ¼m Δn and b ¼ 2α/δ. The standard deviation was calculated for three parallel samples at high cysteine concentration, assumed to be similar for all the concentrations. (c) Dependence of the intense plasmon peak absorbance as a function of cysteine concentration.
nanocubes are shown in Fig. 3(A). Two main trends are observed: a redshift in the plasmon resonance peaks due to cysteine multilayer growth, together with an increase in the plasmonic mode amplitudes, as shown in Fig. 3(B) and (C), respectively. The redshift in the intense plasmon resonance peak is about 15 nm, whereas saturation is observed at about 50 μM of cysteine (Fig. 3(B)), probably due to the decay length of the plasmonic field in this wavelength range. In order to estimate the sensitivity of these nanocubes to cysteine detection, we adopted a quantitative simple model which is based on the variation in the refractive index, Δn, due to the adsorbed dielectric medium. The model was developed for metallic surfaces [21], but can also be used for nanoparticles [2]:
where Δλ is the shift of plasmonic mode wavelength, d is the dielectric layer thickness, δ is the electromagnetic field decay length into the dielectric medium and m is the bulk refractive index sensitivity of the plasmon frequency upon change in the refractive index. Assuming that d is linearly proportional to the concentration C, we obtain the following equation:
d⎤ ⎡ Δλ = m Δn ⎢ 1 − e−2 δ ⎥ ⎣ ⎦
1 The refractive index of methanol is n¼1.33 and by taken n¼ 1.49 for cysteine (refractive index of the similar 3-mercaptopropionic acid reported by Sigma) we get Δn¼ 0.16.
(1)
αC ⎤ ⎡ Δλ = m Δn ⎢ 1 − e−2 δ ⎥ ⎣ ⎦
(2)
where α ¼d/C. Substituting Δn ¼0.16 , and fitting this equation to the experimental results, we obtained m ¼125 nm/RIU. We disregarded the data point for C¼ 50 μM at which the plasmonic 1
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modes are barely shifted. In order to check the validity of this model we compared the thickness of the cysteine layer obtained from both TEM and this model. From fitting the model to the experimental results we get α ¼3.1 10 7 m4/mol. Substituting C ¼15 μM and δ ¼ 20 nm (the decay length of silver at λ ¼450 nm [18]), we get d∼5 nm, which is consistent with the TEM image analysis. A similar sensitivity of 145 nm/RIU, using 30 nm silver nanocubes was reported by Sherry et al. using dark field microscopy [18] and a slightly higher sensitivity of 197 nm/RIU was obtained by McFarland et al. using triangular single silver nanoparticles [14]. This relatively high sensitivity enables them to detect 60,000 hexadecanethiol molecules using dark field microscopy. Another important consequence of the cysteine adsorption is the improvement in nanoparticle stability. As can be clearly observed from the UV–vis. measurements (Fig. 3(c)), the plasmonic peaks amplitude increases with the cysteine concentration up to 50 μM in agreement with the detection saturation (Fig. 3(b)). We relate it to stabilization of the silver nanocubes due to the formation of thiol-Ag bonds following cysteine adsorption, and coating of the nanoparticles with molecules. Therefore, their tendency to aggregate dramatically decreases and they become relatively more stable. The chemical adsorption of cysteine onto the silver nanoparticles leads to a reduction in their surface energy which further decreases by the self-assembly process. Furthermore, we note that cysteine is barely soluble in methanol and therefore is more likely to adhere to the silver nanoparticle. For example, in aqueous solutions silver nanoparticles were unstable upon addition of cysteine. Thus, we postulate that methanol was necessary in order get a uniform cysteine coating onto the nanoparticles and to run LSPR measurements under the reported experimental conditions. We mention that the role of PVP polymer was not only essential for nanocubes synthesis but also for initializing the silver nanocube stability prior to cysteine adsorption.
4. Conclusions For a long time, both the chemistry and biology communities have been seeking ultra-sensitive detection systems, with the ability of chiral recognition. We demonstrate here a method for detecting cysteine, a chiral molecule which possesses a vital role in physiological processes and diseases, that is different from the colorimetric assays based on nanoparticle aggregation. High sensitivity of about 125 nm/RIU using UV–vis. measurements is obtained, based on the red shift of the plasmonic modes upon cysteine growth on silver nanocubes. Although higher sensitivity can be achieved using dark-field microscopy [14,18], here we demonstrate a useful method that is effective on a very small molecule. Additionally, by adsorption of chiral molecules on highly symmetric silver nanocubes, we have formed hybrid materials with potential chiroptical properties [28,29]. Such hybrid materials are also interesting for the study of strong coupling between chiral molecules and plasmonic systems [30], a field which has not yet been explored.
Acknowledgment This research was supported by a Grant from the GIF, the German-Israeli Foundation for Scientific Research and Development (number 203785).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jssc.2016.06.002.
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