Materials Science and Engineering C 32 (2012) 1437–1442
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Comparative study of Ag and Au nanoparticles biosensors based on surface plasmon resonance phenomenon M. Lismont ⁎, L. Dreesen Laboratory of Biophotonics, Department of Physics, B5, University of Liège, Sart-Tilman, Belgium
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Article history: Received 20 October 2011 Received in revised form 27 January 2012 Accepted 17 April 2012 Available online 24 April 2012 Keywords: Localized surface plasmon resonance Colloidal nanoparticles UV–visible spectroscopy Biosensor
a b s t r a c t The specific sensitivity of surface plasmon resonance to changes in the local environment of nanoparticles allows their use as platforms to probe chemical and biochemical binding events on their surfaces without any labeling [1–4]. In this paper, we perform a comparative study of gold and silver nanoparticle based biosensors, prepared within the same conditions, in order to determine which metal seems the best for biological sensing. The prototypical biocytin–avidin interaction is used to study gradual changes over time and with avidin concentration in the absorption spectra bands of biocytinylated 10 nm silver and gold nanospheres. First, the Ag nanoparticles plasmon resonance absorbance signal is about 10 times larger than the Au one. Secondly, for an equivalent concentration of avidin, the optical property modifications are more pronounced for silver nanoparticles than for gold ones of the same geometry. These observations attest the superiority of Ag on Au nanoparticles when optical considerations are only taken into account. Finally, with both biosensors, the specificity of the interaction, checked by replacing avidin with bovine serum albumin, is relatively poor and needs to be improved. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Since a few years, biomedical research is pushing forward the development of biosensors able to rapidly monitor the biological activity of biomolecules found in the blood stream. Biosensor research has been mainly devoted to the evaluation of the relative merits of various signal transduction methods including optical, radioactive, electrochemical, magnetic, etc. Although each of these methods has its individual strengths and weakness, the recent progress in highly sensitive optical transducers combined with high specificity, affinity and versatility of biomolecular interactions has driven the development of a wide variety of optical biosensors with applications in various fields including clinical diagnosis [5] and therapy, biomolecular engineering, targeted delivery of drugs, and biological imaging [6–10]. The nanoparticle (NP)-based biosensor relies on the remarkable optical properties of noble metal NPs, not present in bulk metal, including large optical field enhancements and intense colors resulting from the strong scattering and absorption of light. These properties are due to the collective oscillation of the conduction electrons of metallic nanostructures induced by the incident light field, giving rise to the so-called localized surface plasmon resonance (LSPR) [2,10,11]. Among metals, silver and gold NPs are of particular interest not only because they are relatively air-stable but also because their LSPR absorption bands are in the visible and near-ultraviolet spectral regions, which appear as the most appropriate for technological applications ⁎ Corresponding author. Tel.: + 32 4 366 37 12. E-mail address: [email protected] (M. Lismont). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.04.023
[12]. Additional advantages of these metal NPs include simple preparation methods [8,9,12–14], synthesis over a wide range of sizes and shapes [12,15] and easy surface conjugation to various ligands [16,17]. It is well-established that the extinction peak of the LSPR spectrum and its line-width are dependent upon the size, shape, composition, and spacing of the NPs as well as the dielectric properties of the local environment [3]. Plasmonic NPs can therefore act as transducers that convert small changes in the local refractive index and in the inter-particle distance into spectral shifts and broadening in the absorption spectra bands [17]. The biotin–avidin complex is usually used as a model system to check the efficiency of new biosensors. Biotin is a water soluble vitamin and avidin is a tetrameric protein (63200 Da). Avidin can coordinate to four biotin ligands adsorbed on different colloidal particles leading to their cross-linking and aggregation [18,19]. Several works account the study of biotin–avidin interaction on colloidal gold NPs. Aslan et al. [20] used BSA-biotin coated colloidal 20 nm gold NPs to analyze the molecular recognition process between biotin and streptavidin by using a ratiometric approach of the light scattering. Kohut et al. [21] used 18 nm colloidal gold NPs covered with a monolayer of octadecanethiol. This monolayer, which was modified with synthesized alkyl biotin molecules of particular length, strongly altered the absorption spectrum of gold NPs. Li et al. [22] studied biotin–streptavidin interaction with 20 nm colloidal gold NPs. In their work, polyelectrolyte-functionalized gold NPs were synthesized and then conjugated with biotin. Many works are devoted to gold NPs based biosensors while only a few relate to the use of colloidal silver NPs as physical transducer of
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biochemical interactions. The reasons stem from their weaker stability due to oxidation and their poorer biocompatibility [23,24]. Sastry et al. [18,19] monitored the biotin–avidin interaction on the surface of 7 nm silver and 13 nm gold NPs. They improved the stability of the silver NPs by co-adsorbing the biotin disulfide molecules with an aromatic bifunctional molecule, 4-carboxythiophenol. Although papers related to Au or Ag NPs biosensors are numerous, none of them provide a clear comparison of their particular benefits and drawbacks when they are prepared under the same conditions. The goal of this paper is to address this lack of available data. We used citrate-stabilized and biocytin coated gold and silver colloidal NPs to determine which one seems to be the best to highlight the biomolecular recognition processes. The two types of colloidal NPs have identical geometry and size (nanospheres of 10 nm of diameter) and were prepared with the same experimental conditions. Gradual changes with time in the absorption spectra bands of biocytinylated silver and gold NPs were studied as a function of added avidin amount. We deduced, on one hand, a minimal protein concentration, giving rise to SPR shifts superior to the precision on the SPR maximum location just after protein addition and on the other hand, a critical avidin concentration, which corresponds to the observed LSPR maximum shift. One of the most important characteristics of a biosensor is its specificity. In our case, the specificity of the biocytin coated colloidal silver and gold NPs was checked by replacing avidin by bovine serum albumin (BSA, 66430 Da) which is known for its ability to bind to a wide variety of ligands. BSA has no particular affinity for biocytin [25]. 2. Experimental details 2.1. Chemicals DI water was used in all the experimental steps. Water soluble gold nanospheres [AuNP: 10 nm diameter (citrate)] were obtained from STREM Chemicals Inc. Water soluble NanoXact silver nanospheres with 10 nm diameter were purchased from nanoComposix. Biocytin (C16H28N4O4S, MW 372.48 g/mol) was obtained from Sigma-Aldrich. Biocytin, a water-soluble complex, is the product of the covalent bonding of biotin to the amino group of a lysine (C6H14N2O2) residue [25]. Because biocytin has the same reaction group as biotin, it can react with avidin to form the strongly bonded biocytin–avidin complex. We used biocytin instead of biotin because the former molecule has a longer carbon chain length allowing for better accessibility of the reaction group for the avidin molecules. Biocytin also reduces the steric hindrance, which can affect the amount of avidin molecules adsorbed on the colloidal NPs and, consequently, the optical properties. Avidin and bovine serum albumin (BSA) were kindly provided by the Centre d'Ingénierie des Protéines (University of Liège).
The LSPR shift and the LSPR peak broadening were measured by recording the absorption spectrum of each sample using an Uvikon XS spectrometer equipped with a deuterium tungsten-halogen light source. 2.3. Reproducibility and precision The precisions on the positions of the band maximum are ±0.5 nm and ±1 nm for Ag and Au NPs, respectively. The relative standard deviations on the wavelength shifts are typically 22% and 26% of the values reported in Figs. 4 and 8 for Ag and Au NPs, respectively. They do not significantly depend on protein concentrations. 3. Results and discussion Fig. 1 shows the absorption spectra of 0.086 mM gold and 0.046 mM silver NP solutions prepared as described in the experimental section. The spectra are normalized with the aforementioned metallic NPs concentration to facilitate comparison. Silver nanospheres in the 10 nm-size range have a strong absorption maximum around 395 nm in water. This occurs around 522 nm for gold nanospheres of equivalent size. The silver NPs spectrum is characterized by a narrow Lorentzian absorption peak, whereas gold NPs curve has an asymmetric absorption peak due to the presence of interband transitions. Indeed, in gold, the energy threshold for interband transitions lies at ~2.4 eV (516 nm) and is preceded by an absorption tail starting at about 1.8 eV (688 nm) [26]. On the contrary, the LSPR of silver NPs has no contribution from interband transitions which start at ~ 3.7 eV (335 nm) [26]. Silver NPs therefore allow a more accurate localization of the LSPR resonance and seem to be welladapted to record accurately LSPR shift and broadening. Fig. 1 also clearly shows that silver NPs have a more intense resonance than gold NPs. Indeed, after baseline subtraction on NPs gold spectrum, the absorbance of silver NPs is more or less 10 times higher than the gold one. This is an additional argument in favor of NPs silver based biosensors when compared to gold ones. Fig. 2 shows UV–visible absorption spectra of biocytin coated silver and gold NPs prepared as explained in Section 2.2. LSPR resonances of uncoated-NPs are located at 395 nm and 522 nm for silver and gold, respectively. When NPs are coated with biocytin, LSPR resonances are slightly blue-shifted by 1 nm. These shifts are always close to the precision on the LSPR band maximum location which is typically 1 nm. Their discussion is therefore highly speculative. Such shifts in resonance wavelength have been reported in the literature for NPs of diameter less than 20 nm. The direction of the shift seems to strongly depend on the reactive chemical moiety adsorbed on the NP surface [27]. These displacements are also accompanied by small 14
2.2. Preparation–characterization–measurements
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First, 250 μL of gold or silver colloidal NPs were diluted in 250 μL of DI water, and an aqueous stock biocytin solution of 10–4 M was prepared. The colloidal particles were then capped with the biocytin molecule by adding 500 μL of the biocytin stock solution. The NP solutions were incubated at 4 C during one night to allow the functionalization. The NPs solutions were then centrifuged at 12,000 rpm for 30 min (Au NPs) and 60 min (Ag NPs) to remove extra biocytin. Finally, biocytin coated gold and silver NPs were resuspended in DI water. To set up the aggregation assay, a set of 200 μL samples of biocytin coated gold and silver NPs were prepared in plastic cuvettes and mixed with increasing concentrations of avidin. Aqueous stock avidin and BSA solutions of 10 –6 M were prepared in DI water. Serial avidin dilutions were prepared and added to gold and silver colloidal solutions.
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Wavelength (nm) Fig. 1. Normalized UV–vis spectra of 10 nm gold (continuous line) and silver (dashed line) colloidal NPs. Each spectrum is normalized by the corresponding NPs concentration.
M. Lismont, L. Dreesen / Materials Science and Engineering C 32 (2012) 1437–1442
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Fig. 2. UV–visible spectra of gold and silver colloidal NPs (continuous lines) and biocytin coated gold and silver NPs (dashed lines). Each spectrum is normalized by the corresponding LSPR band maximum.
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broadenings arising from local modifications of the refractive index due to the adsorption of biocytin on the metallic surfaces. Indeed, it has been demonstrated that for gold and silver NPs of radius R b 10 nm, an additional damping process, termed chemical interface damping, must be considered [2]. This one results from the modified electronic properties of the NP surface due to the presence of adsorbed species [28] and, more precisely, from the dynamic charge transfer of electrons into and out of adsorbate or surface states. Indeed, due to the statistical nature of this process, the electrons lose their phase coherence giving rise to a broadening of the plasmon absorption band [29]. Biocytin coated silver and gold NPs were utilized as an avidin sensor by monitoring the red-shift in the LSPR band which results from the aggregation of NPs and from the local refractive index variations. Fig. 3A and B show the optical absorption spectra of the biocytin coated silver and gold NPs at different times after addition of 10 nM avidin solution. The spectra of the uncapped (dashed lines) biocytin coated silver and gold NPs are also drawn for comparison. We observe that the spectral changes begin quickly after avidin addition and become more pronounced as a function of time. 140 min after the addition of protein solution, strong broadenings of the resonances and LSPR-shifts of 13.9 nm and 17.3 nm for gold and silver colloidal NPs, respectively, are recorded. We can also distinguish a progressive increase of the spectral intensity as a function of time for both types of NPs. Similar experiments were conducted with different avidin concentrations lying between 4 nM and 100 nM. The optical properties of these different samples were monitored every 5 min for the first 50 min and every 10 min for the next 90 min. Fig. 4A and B show the time evolution of the LSPR shift, defined as the difference between the bands maxima after and before protein addition. It is clear that the addition of avidin to biocytin silver and gold NPs induces immediate cross-linking of the particles and therefore strong and easily visible LSPR shifts. For a 4 nM avidin concentration, they are equal to 2 nm and 3 nm for gold and silver NPs, respectively. In all curves, the LSPR shift increases progressively to reach its final value approximately 60 min after avidin addition. After this time, the shift evolution does not appear as significant. For a same avidin concentration, the LSPR shift is always larger for colloidal silver NPs than for gold ones. This difference between silver and gold NPs comes from the higher dielectric sensitivity of Ag NPs compare to Au one [30]. The highest LSPR shift is equal to 26 nm for silver NPs, with 6 nM of avidin, and 14 nm for gold NPs, with 10 nM of avidin. We can also observe on Fig. 4 that the LSPR shift is not maximum for the highest avidin concentration (10 − 7 M) for both types of colloidal NPs. This observation can be relied on the number of avidin molecules present in solution (Fig. 5), and on the dielectric sensitivity of silver and gold
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Wavelength (nm) Fig. 3. Optical absorption spectra of biocytin coated Ag NPs (A) and Au NPs (B) after addition of 10 nM of avidin. Spectra a, b and c are recorded after 10, 80 and 140 min, respectively. Vertical lines are guides for the eyes.
NPs. Indeed, for a 10 − 7 M avidin concentration, we can suppose that every avidin molecule has one of its four reactive sites that is complexed with a biocytin molecule adsorbed on the NPs surface (Fig. 5C). The major part of biocytin molecules being bounded to avidin and taking into consideration the steric impediment of avidin molecules, the aggregation of NPs resulting from cross-linking by the tetrameric protein is therefore compromised. Thus, we can suppose that the observed LSPR shift for 10 − 7 M avidin mainly results from a variation of the local refractive index around the NPs. When the avidin concentration is lower (typically 4 nM, as illustrated in Fig. 5A), changes in local refractive index caused by the binding of avidin with biocytin are weak and involve small LSPR shifts. With this protein amount, the NPs aggregation takes place slowly for both types of NPs. An intermediate avidin concentration (Fig. 5B) gives rise to larger LSPR shifts resulting from the combination of variations of the local refractive index and NPs aggregation. These observations lead to the introduction of a critical avidin concentration, which corresponds to the protein concentration giving rise to the maximum LSPR shift. In our case, these concentrations are equal to 6 nM and 10 nM for silver and gold NPs, respectively. The fact that the measured critical avidin concentration is lower for silver NPs than for gold ones confirms the higher dielectric sensitivity of silver NPs and is an additional argument suggesting the superiority of silver over gold for biosensor applications [30]. In order to study the linearity of both biosensors, we plotted the maximum LSPR shift as a function of the avidin concentration for
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Time (min) Fig. 4. LSPR shifts for biocytin coated silver (A) and gold (B) colloidal NPs as a function of time and for 4 nM (diamonds), 6 nM (pentagons), 8 nM (squares), 10 nM (circles), 50 nM (stars) and 100 nM (triangles) avidin concentrations.
values lower than the critical protein concentration (6 nM and 10 nM for Ag and Au NPs respectively). The results, reported in Fig. 6, are relatively well fitted with linear laws (continuous lines). The slopes are equal to 1.78 nm/nM and 10.18 nm/nM for Au and Ag NPs devices,
respectively. The higher slope of the Ag NPs based biosensor confirms its superiority over the gold one: smaller avidin concentration change gives rise to higher LSPR shift modification. We also evaluated the rate, T, by fitting the LSPR shifts versus time curves (spectra in Fig. 4) with the function y0 þ Aexp − Tt . In this expression, t is the time (x axis). y0 and A are parameters depending on protein concentration and having no particular interest for this manuscript. The rate, T, varies from 35 to 15 min when avidin concentration increases from 4 to 100 nM whatever the NPs under consideration. We also notice that, for most avidin concentrations, the aggregation process of NPs by cross-linking is completed after more or less 60 min of reaction, in agreement with Aslan et al. results [20]. From Fig. 4, we can extract the minimum protein concentration giving rise to SPR shifts superior to the precision on the SPR band maximum location just after protein addition. It is equal to 4 nM for both NP solutions. The corresponding LSPR wavelength shifts are about 2 nm and 3 nm for gold and silver, respectively. In order to check the selectivity of biocytin coated colloidal silver and gold NPs biosensors, we repeated similar biomolecular recognition experiments by replacing avidin by BSA. Fig. 7A and B show UV–vis spectra from the biocytin coated silver and gold NPs, respectively, recorded 30 min after the addition of 10 nM BSA. Absorption curves of the uncapped biocytin coated silver and gold NPs (dashed lines) are also drawn for comparison. Clear LSPR shift and peak broadening are observed for silver NPs. The LSPR shift can only result from a small change of the local refractive index around the NPs due to BSA adsorption. Indeed, due to its chemical configuration, BSA cannot produce aggregation of NPs by cross-linking such as avidin. The associated LSPR resonance broadening is due to chemical interface damping following the BSA adsorption on a metallic surface. In this case, the electron transfer can take place between biocytin and NPs surface but also between BSA and NPs surface. The LSPR absorption band of gold NPs is not clearly shifted but only slightly broadened. The differences between the two biosensors are again due to the lower dielectric sensitivity of gold NPs to local refractive index variations in comparison to the silver one. Kinetic studies were also carried out with BSA. Fig. 8 shows the time evolution of the LSPR shifts induced by 1 nM, 10 nM and 100 nM of BSA addition in comparison to the LSPR shifts due to biomolecular recognition between biocytin and avidin. The time evolutions of the LSPR shifts have the same profile when either avidin or BSA was added. However, for the same protein concentration, the LSPR shift is always at least three times larger with avidin than with BSA whatever the metallic
Fig. 5. Aggregation of metal NPs for 4 nM (scheme A), 10 nM (scheme B) and 100 nM (scheme C) avidin concentrations. The gray spheres, green circles and red squares represent metal NPs, biocytin and avidin molecules, respectively.
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NPs. Moreover, the LSPR shifts due to BSA adsorption are always more pronounced for biocytin coated colloidal silver NPs than for gold ones for equivalent proteins concentrations and time. These observations allow us to conclude that biosensors based on biocytin coated colloidal silver and gold NPs are poorly specific.
We investigated the optical properties evolution and, more precisely, the localized surface plasmon resonance (LSPR) of biocytin coated silver and gold colloidal NPs as a function of avidin concentrations, in order to determine which metal is the most suitable for the development of efficient biosensors. First, we showed the significant difference between the absorption intensity of uncoated silver and gold NPs. The strong absorption of silver NPs is very interesting for potential future in vivo applications. Both biocytin coated colloidal gold and silver NPs showed a clear LSPR shift when avidin was added. These wavelength shifts and broadenings increased with the time of avidin incubation in the samples and reached their final values after around 60 min. The silver NPs showed more obvious optical changes than gold NPs indicating that colloidal silver NPs are more sensitive to detection of avidin than gold ones. The minimal avidin concentration was determined to be 4 nM for both silver and gold colloidal NPs. The corresponding wavelength shifts were about 2 nm and 3 nm for gold and silver NPs, respectively. Finally, the specificity of the biocytin–avidin interaction was checked by replacing avidin by BSA. The observed shifts were two to three times lower than for avidin solutions of same concentrations attesting to a poor biosensor selectivity.
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Fig. 8. Evolution with time of the LSPR shifts for biocytin coated silver (A) and gold (B) colloidal NPs after addition of 4 nM (open squares), 10 nM (open triangles) and 100 nM (open circles) avidin solutions. Black symbols correspond to the time evolutions of the LSPR shifts when 1 nm (squares), 10 nm (triangles) and 100 nm (circles) of BSA were added.
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Our works clearly demonstrate the superiority of Ag NPs biosensors versus gold ones when only optical characteristics are taken into account. Unfortunately, silver is less biocompatible than gold, and for potential medical applications this drawback has to be circumvented. PEG coating and encapsulation in silica shell are two promising ways to achieve better biocompatibility [31–33]. Acknowledgements We acknowledge Belgian Fund for Scientist Research (F.R.S.F.N.R.S.) for financial support. Dr G. Feller (Centre d'Ingénierie des protéines, University of Liège) is thanked for his technical help during the UV–visible measurements. We thank Mr S. Douglas for his rereading. References [1] K. Aslan, J.R. Lakowicz, C.D. Geddes, Anal. Biochem. 330 (2004) 145–155. [2] S.A. Maier, in: Springer (ed.), Plasmonics: Fundamentals and Applications, (Springer Science + Business Media LLC, New York, 2007) pp 65–88. [3] K.L. Kelly, E. Coronado, L.L. Zhao, G.C. Schatz, J. Phys. Chem. B 107 (2003) 668–677. [4] R.J. Green, R.A. Frazier, K.M. Shakesheff, M.C. Davies, C.J. Roberts, S.J.B. Tendler, Biomaterials 21 (2000) 1823–1835. [5] K.K. Jain, Clin. Chem. 53 (11) (2007) 2002–2009. [6] B. Sepulveda, P.C. Angelomé, L.M. Lechuga, L.M. Liz-Marzan, Nano Today 4 (2009) 244–251. [7] M. De, P.S. Ghosh, V.M. Rotello, Adv. Mater. 20 (2008) 4225–4241. [8] P.C. Chen, S.C. Mwakwari, A.K. Oyelere, Nanotechnol. Sci. Appl. 1 (2008) 45–66. [9] E. Katz, I. Willner, Angew. Chem. Int. Ed. 43 (2004) 6042–6108. [10] J. Yguerabide, E.E. Yguerabide, Anal. Biochem. 262 (1998) 137–156. [11] J. Yguerabide, E.E. Yguerabide, Anal. Biochem. 262 (1998) 157–176.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Comparative study of Ag and Au nanoparticles biosensors based on surface plasmon resonance phenomenon M. Lismont ⁎, L. Dreesen Laboratory of Biophotonics, Department of Physics, B5, University of Liège, Sart-Tilman, Belgium
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Article history: Received 20 October 2011 Received in revised form 27 January 2012 Accepted 17 April 2012 Available online 24 April 2012 Keywords: Localized surface plasmon resonance Colloidal nanoparticles UV–visible spectroscopy Biosensor
a b s t r a c t The specific sensitivity of surface plasmon resonance to changes in the local environment of nanoparticles allows their use as platforms to probe chemical and biochemical binding events on their surfaces without any labeling [1–4]. In this paper, we perform a comparative study of gold and silver nanoparticle based biosensors, prepared within the same conditions, in order to determine which metal seems the best for biological sensing. The prototypical biocytin–avidin interaction is used to study gradual changes over time and with avidin concentration in the absorption spectra bands of biocytinylated 10 nm silver and gold nanospheres. First, the Ag nanoparticles plasmon resonance absorbance signal is about 10 times larger than the Au one. Secondly, for an equivalent concentration of avidin, the optical property modifications are more pronounced for silver nanoparticles than for gold ones of the same geometry. These observations attest the superiority of Ag on Au nanoparticles when optical considerations are only taken into account. Finally, with both biosensors, the specificity of the interaction, checked by replacing avidin with bovine serum albumin, is relatively poor and needs to be improved. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Since a few years, biomedical research is pushing forward the development of biosensors able to rapidly monitor the biological activity of biomolecules found in the blood stream. Biosensor research has been mainly devoted to the evaluation of the relative merits of various signal transduction methods including optical, radioactive, electrochemical, magnetic, etc. Although each of these methods has its individual strengths and weakness, the recent progress in highly sensitive optical transducers combined with high specificity, affinity and versatility of biomolecular interactions has driven the development of a wide variety of optical biosensors with applications in various fields including clinical diagnosis [5] and therapy, biomolecular engineering, targeted delivery of drugs, and biological imaging [6–10]. The nanoparticle (NP)-based biosensor relies on the remarkable optical properties of noble metal NPs, not present in bulk metal, including large optical field enhancements and intense colors resulting from the strong scattering and absorption of light. These properties are due to the collective oscillation of the conduction electrons of metallic nanostructures induced by the incident light field, giving rise to the so-called localized surface plasmon resonance (LSPR) [2,10,11]. Among metals, silver and gold NPs are of particular interest not only because they are relatively air-stable but also because their LSPR absorption bands are in the visible and near-ultraviolet spectral regions, which appear as the most appropriate for technological applications ⁎ Corresponding author. Tel.: + 32 4 366 37 12. E-mail address: [email protected] (M. Lismont). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.04.023
[12]. Additional advantages of these metal NPs include simple preparation methods [8,9,12–14], synthesis over a wide range of sizes and shapes [12,15] and easy surface conjugation to various ligands [16,17]. It is well-established that the extinction peak of the LSPR spectrum and its line-width are dependent upon the size, shape, composition, and spacing of the NPs as well as the dielectric properties of the local environment [3]. Plasmonic NPs can therefore act as transducers that convert small changes in the local refractive index and in the inter-particle distance into spectral shifts and broadening in the absorption spectra bands [17]. The biotin–avidin complex is usually used as a model system to check the efficiency of new biosensors. Biotin is a water soluble vitamin and avidin is a tetrameric protein (63200 Da). Avidin can coordinate to four biotin ligands adsorbed on different colloidal particles leading to their cross-linking and aggregation [18,19]. Several works account the study of biotin–avidin interaction on colloidal gold NPs. Aslan et al. [20] used BSA-biotin coated colloidal 20 nm gold NPs to analyze the molecular recognition process between biotin and streptavidin by using a ratiometric approach of the light scattering. Kohut et al. [21] used 18 nm colloidal gold NPs covered with a monolayer of octadecanethiol. This monolayer, which was modified with synthesized alkyl biotin molecules of particular length, strongly altered the absorption spectrum of gold NPs. Li et al. [22] studied biotin–streptavidin interaction with 20 nm colloidal gold NPs. In their work, polyelectrolyte-functionalized gold NPs were synthesized and then conjugated with biotin. Many works are devoted to gold NPs based biosensors while only a few relate to the use of colloidal silver NPs as physical transducer of
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biochemical interactions. The reasons stem from their weaker stability due to oxidation and their poorer biocompatibility [23,24]. Sastry et al. [18,19] monitored the biotin–avidin interaction on the surface of 7 nm silver and 13 nm gold NPs. They improved the stability of the silver NPs by co-adsorbing the biotin disulfide molecules with an aromatic bifunctional molecule, 4-carboxythiophenol. Although papers related to Au or Ag NPs biosensors are numerous, none of them provide a clear comparison of their particular benefits and drawbacks when they are prepared under the same conditions. The goal of this paper is to address this lack of available data. We used citrate-stabilized and biocytin coated gold and silver colloidal NPs to determine which one seems to be the best to highlight the biomolecular recognition processes. The two types of colloidal NPs have identical geometry and size (nanospheres of 10 nm of diameter) and were prepared with the same experimental conditions. Gradual changes with time in the absorption spectra bands of biocytinylated silver and gold NPs were studied as a function of added avidin amount. We deduced, on one hand, a minimal protein concentration, giving rise to SPR shifts superior to the precision on the SPR maximum location just after protein addition and on the other hand, a critical avidin concentration, which corresponds to the observed LSPR maximum shift. One of the most important characteristics of a biosensor is its specificity. In our case, the specificity of the biocytin coated colloidal silver and gold NPs was checked by replacing avidin by bovine serum albumin (BSA, 66430 Da) which is known for its ability to bind to a wide variety of ligands. BSA has no particular affinity for biocytin [25]. 2. Experimental details 2.1. Chemicals DI water was used in all the experimental steps. Water soluble gold nanospheres [AuNP: 10 nm diameter (citrate)] were obtained from STREM Chemicals Inc. Water soluble NanoXact silver nanospheres with 10 nm diameter were purchased from nanoComposix. Biocytin (C16H28N4O4S, MW 372.48 g/mol) was obtained from Sigma-Aldrich. Biocytin, a water-soluble complex, is the product of the covalent bonding of biotin to the amino group of a lysine (C6H14N2O2) residue [25]. Because biocytin has the same reaction group as biotin, it can react with avidin to form the strongly bonded biocytin–avidin complex. We used biocytin instead of biotin because the former molecule has a longer carbon chain length allowing for better accessibility of the reaction group for the avidin molecules. Biocytin also reduces the steric hindrance, which can affect the amount of avidin molecules adsorbed on the colloidal NPs and, consequently, the optical properties. Avidin and bovine serum albumin (BSA) were kindly provided by the Centre d'Ingénierie des Protéines (University of Liège).
The LSPR shift and the LSPR peak broadening were measured by recording the absorption spectrum of each sample using an Uvikon XS spectrometer equipped with a deuterium tungsten-halogen light source. 2.3. Reproducibility and precision The precisions on the positions of the band maximum are ±0.5 nm and ±1 nm for Ag and Au NPs, respectively. The relative standard deviations on the wavelength shifts are typically 22% and 26% of the values reported in Figs. 4 and 8 for Ag and Au NPs, respectively. They do not significantly depend on protein concentrations. 3. Results and discussion Fig. 1 shows the absorption spectra of 0.086 mM gold and 0.046 mM silver NP solutions prepared as described in the experimental section. The spectra are normalized with the aforementioned metallic NPs concentration to facilitate comparison. Silver nanospheres in the 10 nm-size range have a strong absorption maximum around 395 nm in water. This occurs around 522 nm for gold nanospheres of equivalent size. The silver NPs spectrum is characterized by a narrow Lorentzian absorption peak, whereas gold NPs curve has an asymmetric absorption peak due to the presence of interband transitions. Indeed, in gold, the energy threshold for interband transitions lies at ~2.4 eV (516 nm) and is preceded by an absorption tail starting at about 1.8 eV (688 nm) [26]. On the contrary, the LSPR of silver NPs has no contribution from interband transitions which start at ~ 3.7 eV (335 nm) [26]. Silver NPs therefore allow a more accurate localization of the LSPR resonance and seem to be welladapted to record accurately LSPR shift and broadening. Fig. 1 also clearly shows that silver NPs have a more intense resonance than gold NPs. Indeed, after baseline subtraction on NPs gold spectrum, the absorbance of silver NPs is more or less 10 times higher than the gold one. This is an additional argument in favor of NPs silver based biosensors when compared to gold ones. Fig. 2 shows UV–visible absorption spectra of biocytin coated silver and gold NPs prepared as explained in Section 2.2. LSPR resonances of uncoated-NPs are located at 395 nm and 522 nm for silver and gold, respectively. When NPs are coated with biocytin, LSPR resonances are slightly blue-shifted by 1 nm. These shifts are always close to the precision on the LSPR band maximum location which is typically 1 nm. Their discussion is therefore highly speculative. Such shifts in resonance wavelength have been reported in the literature for NPs of diameter less than 20 nm. The direction of the shift seems to strongly depend on the reactive chemical moiety adsorbed on the NP surface [27]. These displacements are also accompanied by small 14
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First, 250 μL of gold or silver colloidal NPs were diluted in 250 μL of DI water, and an aqueous stock biocytin solution of 10–4 M was prepared. The colloidal particles were then capped with the biocytin molecule by adding 500 μL of the biocytin stock solution. The NP solutions were incubated at 4 C during one night to allow the functionalization. The NPs solutions were then centrifuged at 12,000 rpm for 30 min (Au NPs) and 60 min (Ag NPs) to remove extra biocytin. Finally, biocytin coated gold and silver NPs were resuspended in DI water. To set up the aggregation assay, a set of 200 μL samples of biocytin coated gold and silver NPs were prepared in plastic cuvettes and mixed with increasing concentrations of avidin. Aqueous stock avidin and BSA solutions of 10 –6 M were prepared in DI water. Serial avidin dilutions were prepared and added to gold and silver colloidal solutions.
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broadenings arising from local modifications of the refractive index due to the adsorption of biocytin on the metallic surfaces. Indeed, it has been demonstrated that for gold and silver NPs of radius R b 10 nm, an additional damping process, termed chemical interface damping, must be considered [2]. This one results from the modified electronic properties of the NP surface due to the presence of adsorbed species [28] and, more precisely, from the dynamic charge transfer of electrons into and out of adsorbate or surface states. Indeed, due to the statistical nature of this process, the electrons lose their phase coherence giving rise to a broadening of the plasmon absorption band [29]. Biocytin coated silver and gold NPs were utilized as an avidin sensor by monitoring the red-shift in the LSPR band which results from the aggregation of NPs and from the local refractive index variations. Fig. 3A and B show the optical absorption spectra of the biocytin coated silver and gold NPs at different times after addition of 10 nM avidin solution. The spectra of the uncapped (dashed lines) biocytin coated silver and gold NPs are also drawn for comparison. We observe that the spectral changes begin quickly after avidin addition and become more pronounced as a function of time. 140 min after the addition of protein solution, strong broadenings of the resonances and LSPR-shifts of 13.9 nm and 17.3 nm for gold and silver colloidal NPs, respectively, are recorded. We can also distinguish a progressive increase of the spectral intensity as a function of time for both types of NPs. Similar experiments were conducted with different avidin concentrations lying between 4 nM and 100 nM. The optical properties of these different samples were monitored every 5 min for the first 50 min and every 10 min for the next 90 min. Fig. 4A and B show the time evolution of the LSPR shift, defined as the difference between the bands maxima after and before protein addition. It is clear that the addition of avidin to biocytin silver and gold NPs induces immediate cross-linking of the particles and therefore strong and easily visible LSPR shifts. For a 4 nM avidin concentration, they are equal to 2 nm and 3 nm for gold and silver NPs, respectively. In all curves, the LSPR shift increases progressively to reach its final value approximately 60 min after avidin addition. After this time, the shift evolution does not appear as significant. For a same avidin concentration, the LSPR shift is always larger for colloidal silver NPs than for gold ones. This difference between silver and gold NPs comes from the higher dielectric sensitivity of Ag NPs compare to Au one [30]. The highest LSPR shift is equal to 26 nm for silver NPs, with 6 nM of avidin, and 14 nm for gold NPs, with 10 nM of avidin. We can also observe on Fig. 4 that the LSPR shift is not maximum for the highest avidin concentration (10 − 7 M) for both types of colloidal NPs. This observation can be relied on the number of avidin molecules present in solution (Fig. 5), and on the dielectric sensitivity of silver and gold
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NPs. Indeed, for a 10 − 7 M avidin concentration, we can suppose that every avidin molecule has one of its four reactive sites that is complexed with a biocytin molecule adsorbed on the NPs surface (Fig. 5C). The major part of biocytin molecules being bounded to avidin and taking into consideration the steric impediment of avidin molecules, the aggregation of NPs resulting from cross-linking by the tetrameric protein is therefore compromised. Thus, we can suppose that the observed LSPR shift for 10 − 7 M avidin mainly results from a variation of the local refractive index around the NPs. When the avidin concentration is lower (typically 4 nM, as illustrated in Fig. 5A), changes in local refractive index caused by the binding of avidin with biocytin are weak and involve small LSPR shifts. With this protein amount, the NPs aggregation takes place slowly for both types of NPs. An intermediate avidin concentration (Fig. 5B) gives rise to larger LSPR shifts resulting from the combination of variations of the local refractive index and NPs aggregation. These observations lead to the introduction of a critical avidin concentration, which corresponds to the protein concentration giving rise to the maximum LSPR shift. In our case, these concentrations are equal to 6 nM and 10 nM for silver and gold NPs, respectively. The fact that the measured critical avidin concentration is lower for silver NPs than for gold ones confirms the higher dielectric sensitivity of silver NPs and is an additional argument suggesting the superiority of silver over gold for biosensor applications [30]. In order to study the linearity of both biosensors, we plotted the maximum LSPR shift as a function of the avidin concentration for
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values lower than the critical protein concentration (6 nM and 10 nM for Ag and Au NPs respectively). The results, reported in Fig. 6, are relatively well fitted with linear laws (continuous lines). The slopes are equal to 1.78 nm/nM and 10.18 nm/nM for Au and Ag NPs devices,
respectively. The higher slope of the Ag NPs based biosensor confirms its superiority over the gold one: smaller avidin concentration change gives rise to higher LSPR shift modification. We also evaluated the rate, T, by fitting the LSPR shifts versus time curves (spectra in Fig. 4) with the function y0 þ Aexp − Tt . In this expression, t is the time (x axis). y0 and A are parameters depending on protein concentration and having no particular interest for this manuscript. The rate, T, varies from 35 to 15 min when avidin concentration increases from 4 to 100 nM whatever the NPs under consideration. We also notice that, for most avidin concentrations, the aggregation process of NPs by cross-linking is completed after more or less 60 min of reaction, in agreement with Aslan et al. results [20]. From Fig. 4, we can extract the minimum protein concentration giving rise to SPR shifts superior to the precision on the SPR band maximum location just after protein addition. It is equal to 4 nM for both NP solutions. The corresponding LSPR wavelength shifts are about 2 nm and 3 nm for gold and silver, respectively. In order to check the selectivity of biocytin coated colloidal silver and gold NPs biosensors, we repeated similar biomolecular recognition experiments by replacing avidin by BSA. Fig. 7A and B show UV–vis spectra from the biocytin coated silver and gold NPs, respectively, recorded 30 min after the addition of 10 nM BSA. Absorption curves of the uncapped biocytin coated silver and gold NPs (dashed lines) are also drawn for comparison. Clear LSPR shift and peak broadening are observed for silver NPs. The LSPR shift can only result from a small change of the local refractive index around the NPs due to BSA adsorption. Indeed, due to its chemical configuration, BSA cannot produce aggregation of NPs by cross-linking such as avidin. The associated LSPR resonance broadening is due to chemical interface damping following the BSA adsorption on a metallic surface. In this case, the electron transfer can take place between biocytin and NPs surface but also between BSA and NPs surface. The LSPR absorption band of gold NPs is not clearly shifted but only slightly broadened. The differences between the two biosensors are again due to the lower dielectric sensitivity of gold NPs to local refractive index variations in comparison to the silver one. Kinetic studies were also carried out with BSA. Fig. 8 shows the time evolution of the LSPR shifts induced by 1 nM, 10 nM and 100 nM of BSA addition in comparison to the LSPR shifts due to biomolecular recognition between biocytin and avidin. The time evolutions of the LSPR shifts have the same profile when either avidin or BSA was added. However, for the same protein concentration, the LSPR shift is always at least three times larger with avidin than with BSA whatever the metallic
Fig. 5. Aggregation of metal NPs for 4 nM (scheme A), 10 nM (scheme B) and 100 nM (scheme C) avidin concentrations. The gray spheres, green circles and red squares represent metal NPs, biocytin and avidin molecules, respectively.
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NPs. Moreover, the LSPR shifts due to BSA adsorption are always more pronounced for biocytin coated colloidal silver NPs than for gold ones for equivalent proteins concentrations and time. These observations allow us to conclude that biosensors based on biocytin coated colloidal silver and gold NPs are poorly specific.
We investigated the optical properties evolution and, more precisely, the localized surface plasmon resonance (LSPR) of biocytin coated silver and gold colloidal NPs as a function of avidin concentrations, in order to determine which metal is the most suitable for the development of efficient biosensors. First, we showed the significant difference between the absorption intensity of uncoated silver and gold NPs. The strong absorption of silver NPs is very interesting for potential future in vivo applications. Both biocytin coated colloidal gold and silver NPs showed a clear LSPR shift when avidin was added. These wavelength shifts and broadenings increased with the time of avidin incubation in the samples and reached their final values after around 60 min. The silver NPs showed more obvious optical changes than gold NPs indicating that colloidal silver NPs are more sensitive to detection of avidin than gold ones. The minimal avidin concentration was determined to be 4 nM for both silver and gold colloidal NPs. The corresponding wavelength shifts were about 2 nm and 3 nm for gold and silver NPs, respectively. Finally, the specificity of the biocytin–avidin interaction was checked by replacing avidin by BSA. The observed shifts were two to three times lower than for avidin solutions of same concentrations attesting to a poor biosensor selectivity.
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Fig. 8. Evolution with time of the LSPR shifts for biocytin coated silver (A) and gold (B) colloidal NPs after addition of 4 nM (open squares), 10 nM (open triangles) and 100 nM (open circles) avidin solutions. Black symbols correspond to the time evolutions of the LSPR shifts when 1 nm (squares), 10 nm (triangles) and 100 nm (circles) of BSA were added.
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Our works clearly demonstrate the superiority of Ag NPs biosensors versus gold ones when only optical characteristics are taken into account. Unfortunately, silver is less biocompatible than gold, and for potential medical applications this drawback has to be circumvented. PEG coating and encapsulation in silica shell are two promising ways to achieve better biocompatibility [31–33]. Acknowledgements We acknowledge Belgian Fund for Scientist Research (F.R.S.F.N.R.S.) for financial support. Dr G. Feller (Centre d'Ingénierie des protéines, University of Liège) is thanked for his technical help during the UV–visible measurements. We thank Mr S. Douglas for his rereading. References [1] K. Aslan, J.R. Lakowicz, C.D. Geddes, Anal. Biochem. 330 (2004) 145–155. [2] S.A. Maier, in: Springer (ed.), Plasmonics: Fundamentals and Applications, (Springer Science + Business Media LLC, New York, 2007) pp 65–88. [3] K.L. Kelly, E. Coronado, L.L. Zhao, G.C. Schatz, J. Phys. Chem. B 107 (2003) 668–677. [4] R.J. Green, R.A. Frazier, K.M. Shakesheff, M.C. Davies, C.J. Roberts, S.J.B. Tendler, Biomaterials 21 (2000) 1823–1835. [5] K.K. Jain, Clin. Chem. 53 (11) (2007) 2002–2009. [6] B. Sepulveda, P.C. Angelomé, L.M. Lechuga, L.M. Liz-Marzan, Nano Today 4 (2009) 244–251. [7] M. De, P.S. Ghosh, V.M. Rotello, Adv. Mater. 20 (2008) 4225–4241. [8] P.C. Chen, S.C. Mwakwari, A.K. Oyelere, Nanotechnol. Sci. Appl. 1 (2008) 45–66. [9] E. Katz, I. Willner, Angew. Chem. Int. Ed. 43 (2004) 6042–6108. [10] J. Yguerabide, E.E. Yguerabide, Anal. Biochem. 262 (1998) 137–156. [11] J. Yguerabide, E.E. Yguerabide, Anal. Biochem. 262 (1998) 157–176.
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