Surface-enhanced Raman scattering on colloidal nanostructures

Advances in Colloid and Interface Science 116 (2005) 45 – 61 www.elsevier.com/locate/cis

Surface-enhanced Raman scattering on colloidal nanostructures R.F. Aroca a,*, R.A. Alvarez-Puebla a, N. Pieczonka a, S. Sanchez-Cortez b, J.V. Garcia-Ramos b a

Materials and Surface Science Group, University of Windsor, Windsor, Ontario, Canada N9B 3P4 b Instituto de Estructura de la Materia, CSIC, Serrano, 123, 28006 Madrid, Spain Available online 6 October 2005

Abstract Surface-enhanced Raman scattering combines extremely high sensitivity, due to enhanced Raman cross-sections comparable or even better than fluorescence, with the observation of vibrational spectra of adsorbed species, providing one of the most incisive analytical methods for chemical and biochemical detection and analysis. SERS spectra are observed from a molecule-nanostructure enhancing system. This symbiosis molecule-nanostructure is a fertile ground for theoretical developments and a realm of applications from single molecule detection to biomedical diagnostic and techniques for nanostructure characterization. D 2005 Elsevier B.V. All rights reserved. Keywords: SERS; Colloids; Nanostructure; Raman; Vibrational spectroscopy; Single molecule detection

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preparation and stability of colloidal nanostructures . . . . . . . . . . . . . . . . . . . . . . . 2.1. Colloidal nanospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Mixed silver/gold colloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Colloidal nanostructures of single metal with non-spherical geometries . . . . . . . . . . 2.4. Aggregation of metal nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Colloidal nanostructures supported on thin solid films . . . . . . . . . . . . . . . . . . 3. Characterization of colloidal nanostructures . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Average colloidal SERS of small molecules and their spectral interpretation. . . . . . . . . . . 5. Application of colloidal nanostructures to ultrasensitive analysis and single molecule detection . 6. Biological and biomedical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Surface-enhanced Raman Scattering (SERS) was first observed [1] and later explained [2,3] in spectroelectrochemical experiments. The initial reports were all carried

* Corresponding author. E-mail address: [email protected] (R.F. Aroca). 0001-8686/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cis.2005.04.007

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45 46 46 49 50 50 50 51 51 54 55 59 59

out on silver electrodes with modified roughened surfaces. This new phenomenon was clearly seen as a window of opportunity for surface Raman studies, and it was. However, from the very beginning transmission electron microscopy revealed the underlying nanostructure of active SERS substrates. Later, with the advent of scanning probes microscopy and the ability to resolve at the atomic scale, it became clear that the SERS is indeed a ‘‘nanostructureenhanced Raman scattering’’ rather than a surface enhance-

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R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

ment. Since the first reported SERS on silver and gold sols in 1979 [4], metal colloids have become the most commonly used nanostructures for SERS. Colloidal metal particles have served as a testing ground for the most thorough of theoretical modeling [5,6]. As well they have become central to single-particle, single-molecule Raman spectroscopy with the achievement of the single molecule detection (SMD) [7,8]. In practice, is it now important to distinguish two types of SERS signals when using colloidal nanoparticles. (a) SERS spectra that are the product of an ‘‘average SERS’’ enhancement, the product of SERS from an ensemble of colloidal particles and aggregates and characterized by a stable average spectrum with welldefined frequency and bandwidth. (b) SERS spectra obtained from a single particle or single aggregate of particles that encompasses ‘‘hot spot’’, a spatial region where extremely high enhancement factors are observed which permit single molecule detection. The analytical applications of the average SERS are a mature field, and the work today is turning to the specifics of tuning the experimental condition for a given analyte. The SMD is just beginning and faces both theoretical and experimental challenges. There are several reviews of SERS with emphasis on metal colloids, in particular, the ultra-high enhancement SERS and SMD using colloids is discussed by Kneipp et al. [9], while the applications of metal colloids to provide average SERS enhancements for the detection of biological molecules has been reviewed by Dou and Ozaki [10]. Specific aspects of SERS with colloids are also found in the review by Pettinger [11], while the review of optical properties of metal particles of differing size and shape as those found in metal sols can be found in the reports of Kelly et al. [12], Mulvaney [13] and El-Sayed [14]. In the present review, we attempt to present a glimpse into the expanding world of nanostructure fabrication where the starting point is the colloidal sols used in surface-enhanced spectroscopy. A discussion of their characterization and examples of their applications in ultrasensitive chemical analysis and the detection of biological molecules is given. There are now many examples of SMD using metal nanostructures, illustrating the impressive potential these simple particles have.

2. Preparation and stability of colloidal nanostructures There is a large variety of SERS substrates other than colloids, including electrodes, and metal island films. However, wet chemistry provides an inexpensive and versatile approach to metal nanoparticle fabrication. Colloidal nanoparticles are commonly used for SERS/SERRS studies either in a suspension as a sol, or the analyte – colloid system is cast onto a sustaining surface (commonly a treated glass) and let to dry on the solid support forming particles of a variety of shapes, nanowires and aggregates as shown in Fig. 1. The use of colloidal dispersions for the detection of an

average SERS enhancement has several advantages. The presence of a solution tends to minimize the burning of the sample allowing the use of higher powers, and the use of more energetic laser lines. As well, the use of sols permits the acquisition of an average spectrum due to the Brownian motion that governs the colloidal dispersions. This averaging of signal can be further improved upon by the use of systems that force the recirculation of the sample [15]. Since Faraday’s pioneering work [16], several methods have been developed to synthesize colloidal nanostructures. The number of methods for preparation of colloidal nanostructures with SERS activity is numerous and even today is a very active field of research. Therefore, this section is necessarily restricted to some of the most widely used methods based on chemical reactions in solution – wet chemistry – that yield metal nanoparticle colloids. Within the limits of wet chemistry metal colloids can be prepared by a variety of different procedures: chemical reduction, laser ablation and photoreduction are those most frequently employed. By far the most universally used method for the preparation of metal nanoparticles in suspension for SERS is by chemical reduction. It is usually performed by using a starting metal salt, which is reduced by a chemical agent to produce colloidal suspensions containing nanoparticles with variable sizes depending on the method of production. Generally, the size regime relevant to SERS experiments is between 10 and 80 nm. These particles will thus exhibit different plasmon resonances depending on the size, shape and the dielectric constant of the metal. The two first parameters can be partially controlled by appropriate choice of the method of preparation. The most important parameters in this regard are the nature of the metal, the reducing reagent, the temperature and the metal ion concentration. A number of metal nanostructures prepared by wet methods have been tested for SERS activity such as: Cu [17 – 19], AuPt [20 – 23], AgRh [24], Pd [25] or AuPd [26]. However, the best results continue to be achieved with the coinage metals, gold and silver. 2.1. Colloidal nanospheres Just a cursory glance of the literature will show that the most popular method to prepare nanospheres of silver or gold, for their use as SERS enhancers, are the reduction of the metal cations from their salts. For silver those would be AgNO3 or Ag2SO4, and in the case of gold, HAuCl4 or KAuCl4, with sodium citrate or sodium borohydride. The reduction of Ag+ in a boiling solution by addition of sodium citrate is the most common method for the fabrication of Ag sols to be used in SERS. This method simply consists of adding 2 mL of 1% sodium citrate solution to a 100 mL boiling solution of Ag+ 10
3 M, and keeping it boiling for 1 h. The resulting colloids (Fig. 1a) show a turbid gray-green color with a surface plasmon absorption maximum at 406 nm (Fig. 2). The reduction of Ag with NaBH4 is also widely used [4,27]. In this case, 10 mL Ag+ 10
3 M are added

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47

Fig. 1. TEM images of (a) silver citrate [69], (b) gold borohydride colloids [146], and (c) gold nanorods [42], (d) Au nanosquares and their diffraction pattern [147]. AFM images of (e) typical film of gold nanoparticles embedded into chitosan film [56] and (f) silver nanowire/G5 DAB-Am dendrimer LBL film [57].

dropwise to 30 mL of ice-cold aqueous solution of sodium borohydride 2  10
3 M with vigorous stirring. The yellowish colloidal suspension has a surface plasmon absorption maximum routinely measured at 391 nm (Fig. 2). The reduction of HAuCl4 (or KAuCl4) is analogous to that with silver; 2 mL of 1% sodium citrate solution is added to a 10 mL boiling solution of Au3+ 10
3 M [28]. The formation of Au nanoparticles is revealed by a deep wine red color observed after 10 min, with a surface plasmon absorption maximum at about 520 nm, and average sizes of 10 –100 nm. For the NaBH4 reduction, 100 mL of a 5  10
3 M in HAuCl solution is added dropwise to 300 mL of vigorously stirred ice-cold 2  10
3 M NaBH4 solution. This solution is unstable so, usually, it is stabilized by adding 50 ml of poly (viny1 alcohol) 1%. The mixture is boiled for 1 h to decompose excess of NaBH4, and the final

volume is adjusted to 500 mL [29]. The colloidal suspension is wine red-violet with a surface plasmon absorption maximum at 535 nm (Fig. 2), with average sizes between 20 and 70 nm (Fig. 1b). Copper colloidal nanospheres can be prepared by adding 5 mL of a Cu2+ 10
2 M solution to 60 mL of sodium citrate 5.6  10
3 M. Because the facility of Cu0 to be oxidized to Cu (II), E Cu(II)/Cu0 = 0.34 V, the colloids are only stable in strongly reductive media. In order to avoid the oxidation of the Cu colloids 30 mL of solution 2  10
2 M in NaBH4 and 2  10
2 M in NaOH are added [30]. The resulting colloidal suspension is yellow-brown but changes to darkred after 1 h, with a maximum surface plasmon absorption measured at about 560 nm (Fig. 2). Considerably effort is now being directed to control the size of Ag and Au colloidal particles, Brust et al. [31] have

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R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61 Ag-citrate

of spherical particles provided by the classic citrate and borohydride sols. Metal colloids prepared by the conventional chemical reduction procedures usually described in the literature are highly stable for months or even years, although the use of old colloids is not convenient after several days, since the chemical properties of the surface may significantly change. The relative high stability of freshly prepared metal colloids prepared by chemical reduction is mainly due to the counter ions existing on the metal surface coming from the ionic species added to the mixture during the preparation. These ions are normally adsorbed on the metal surface and confer to the nanoparticles enough charge to maintain them suspended in the dispersion medium (generally water) thanks to the inter-particle repulsion forces. The stability of colloidal suspensions of silver citrate and borohydride colloids and gold citrate have been studied in a recent paper by using photon correlation spectroscopy as a function of pH (Fig. 3) [33]. The zeta potential of these suspensions varies from 6.5 mV at pH 2, to
56 mV at pH 11 and
20 to
52 mV, in the same interval, for silver borohydride and citrate, respectively. The gold-citrate colloids are slightly more negative from
44 at pH 2 to
61 mV at pH 11. These data indicate that the most unstable colloids are those of silver with borohydride followed by silver and gold citrate ones. This instability is due to a lower electrostatic barrier that makes easier the approaching of different colloids to form aggregates and flocculate. However, this lower electrostatic barrier also helps the chemisorption of analytes; the net result being that the silver borohydride colloids seems to be the best for SERS with negative analytes. Some papers recommend the addition of poly(viny1 alcohol) [29,34] in order to stabilize it for weeks or even months. However, these kind of treatments results in an increase of

Ag-NaBH4

Au-citrate

Au-NaBH4 Cu-citrate Ag-nanowire

400 500 600 700 800 900

Wavelength/nm Fig. 2. Some examples of surface plasmon resonance for silver and gold nanocolloids prepared by different ways.

proposed a biphasic reduction method for gold. Basically, HAuCl4 is dissolved in water and subsequently transported into toluene by means of tetraoctylammonium bromide, which acts as a phase transfer agent. The toluene solution is then mixed and thoroughly stirred together with an aqueous solution of sodium borohydride, in the presence of thioalkanes or aminoalkanes, which readily bind to the Au nanoparticles formed. Depending on the ratio of the Au salt and capping agent (thiol/amine), the particle size can be tuned to between ¨ 1 nm and ¨ 10 nm. Several refinements of the preparative procedure, including the development of analogous methods for the preparation of Ag particles, have been reported [32]. For SERS applications though, none of these methods offers a clear advantage over the distribution 10

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R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

the electrostatic barrier and a decrease in the adsorptivity of analytes. This is why we recommend the use of freshly synthesized Ag borohydride colloids with no stabilizer for SERS experiments. There have been many experimental reports of slightly different methods for the preparation of silver and gold colloids in water media. In particular, a number of acids [35,36] have been tried as a reducing agents since it is well known the capacity of carboxilate groups to reduce ions of noble metals according to the equation: D

Mnþ þ nR
COO
Y M0 þ nR þ nCO2 However, for SERS applications, the resulting colloids do not present any advantage over the methods previously discussed. Recently, and in order to obtain new substrates for SERS with different surface charges, we produced silver colloids using the biological amino acid glycine. This process was very simple, 6 mL of 1% glycine (Gly) solution, previously adjusted at pH 9 to ensure that it is in the carboxilate form, and is added to 100 mL of boiling solution of Ag+ 10
3 M. The solution is kept boiling during 30 min. This method gives rise to a yellow-gray solution with surface plasmon absorption at 420 nm (Fig. 4b). Fig. 4a shows the glycine spectrum, a background spectrum of the colloid, and SERS of salicylic acid on glycine and citrate silver colloids. Even though the background spectra of glycolloids appear very clean, the SERS quality is much better when salicylic acid is adsorbed onto citrate colloids. This illustrates one big advantage of colloids obtained with both,

49

citrate and borohydride methods; they tend to give higher quality spectra than other methods. Though some spurious Raman bands may appear in a SERS spectra obtained using gold or silver citrate colloids, they have been flagged in detail by Sanchez-Cortes and Garcia-Ramos [37]. However, one can eliminate the weak interference of these bands using other chemical reducing agents such as hydrazine [38] or hydroxylamine [39]. This also has the advantage of the eliminating residual oxidation products, which, in latter case, are molecular nitrogen and nitrogen oxides (in the case of hydroxylamine). The disadvantage is that one is using toxic and instable reducing agents. 2.2. Mixed silver/gold colloids Given the differences in optical properties of Ag and Au, the corresponding colloids display different plasmon absorption maxima which, in keeping with the electromagnetic mechanism of SERS, would govern the local field enhancement [40]. Ag sols display absorption maximum at about 420 nm while Au sols show a maximum above 500 nm [41]. Therefore, the preparation of Ag/Au mixed colloids allows for the combination of the SERS activities of both metals allowing the use of a broader interval of the electromagnetic spectrum. Ag-coated Au colloids and Au-coated Ag colloids can be prepared by growing Ag or Au on pre-formed citratereduced Au or Ag nanoparticles by chemical reduction of the corresponding metal salt [41]. The mechanism and

a

b

Gly-powder

Ag-Gly colloids 400

600

800

Wavelength/nm SERS of Salicylic acid on Ag-Gly colloids

c

SERS of Salicylic acid on Ag-citrate colloids

400

600

800

1000

Raman

1200

1400

d

1600

1800

Shift/cm-1

Fig. 4. (a) Raman spectra of glycine (Gly) and Ag-Gly silver colloids. SERS spectra of salicylic acid on Ag-Gly and on Ag-citrate colloids. (b) Surface plasmon absorption for Ag-Gly colloids. (c) Chemical structures of Gly and, (d) salicylic acid.

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R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

morphology of the resulting mixed particles depends on the relative amount of the depositing metal. In the deposition process, factors such as the depositing metal atoms to surface ratio must be taken into account so to control the deposition process. Primarily this allows control over the metal ion diffusion and subsequent epitaxial growth on the pre-formed metal particle. For Ag-coated Au colloids, low Ag fractions lead to core-shell particles. On the contrary, higher Ag fractions lead to the formation of independent particles of each metal. In the preparation of Au-coated Ag colloids, a low Au fraction leads to the formation of individual particles of each metal. These particles tend to migrate to the Ag surface and are immobilized upon it. Only when the Au fraction is increased to 3% actual Au-coated Ag particles are obtained. At higher Au fractions (7%) the deposition mechanism seems to be a combination of the two. In general, the SERS activity of the mixed colloids is placed at an intermediate position between Ag and Au. The coverage of Au with Ag induces an increase of the enhancement factor as compared to the pure Au, although it does not reach the value achieved with Ag. The Ag coverage by Au represent an improvement of the SERS activity of the deposited metal. In the last case a high portion of the Ag is covered by Au as indicated by the SERS spectrum profile of pyridine [41]. 2.3. Colloidal nanostructures of single metal with nonspherical geometries In order to increase the electromagnetic field enhancement factor [12], some non-spherical geometries have been created and tested. The morphology of the prepared colloids can be dramatically affected by the experimental conditions followed during the metal reduction. In particular, the temperature, reactant concentrations, counter ions and the total volume are of great importance in relation to the size, shape and stability of these systems. Jana et al. [42] proposed a method to obtain gold nanorods. In this method, 9 mL of a solution, consisting of 2.5  10
4 M HAuCl4 and 0.1 M cetyltrimethylammonium bromide, are mixed with 0.05 mL of 0.1 M ascorbic acid. Next, 1.0 mL of Au-citrate colloid solution (seed solution) is mixed with this solution. The result is a red colloidal suspension (Fig. 1c) with two plasmons at ¨ 525 and 885 nm due to the excitation of the transversal and longitudinal plasmons, respectively. These particles had been used for SERS by Nikoobakht and ElSayed with optimal results [43]. The production of nanowires and nanoprisms is also in full swing. Silver nanowire synthesis is carried out employing the poliol process modified according to Sun [44] and Tao [45]. Twenty-five milliliters of solution of poly (vinyl pyrrolidone), PVP, 0.36 M in anhydrous ethylene glycol is heated to 160 -C. Then, 12 mL of AgNO3 0.12 M in ethylene glycol are added drop-wise into the hot PVP. The solution is kept for 30 min with constant stirring at the same

temperature and then cooled to room temperature and diluted 1:10 with acetone. The suspension is centrifuged and the precipitate is re-dispersed in ethanol and then sonicated. These nanowires suspensions have a similar color to that of silver citrate nanospheres but presents absorption bands at 348 and 381 nm, most likely, corresponding to the transversal plasmons (Fig. 2). This method of the poliol can be used also to generate nanoprisms [46]. However, recently, in our group a new method was developed to produce gold nanoprism, nanospheres or nanotriangles as a function of pH and the ratio of a mixture between Au3+ 0.01% (w/v) solution and a fulvic acid dispersion ranging from 25 to 250 mg L
1 [47]. The mixture reacts at room temperature, and an increase in temperature results in an increase in the reaction kinetics. The nanotriangles prepared at pH 5 with an equivolumetric mixture of gold and fulvic acid solutions give a SERS signal which is comparable to that obtained with gold island films. 2.4. Aggregation of metal nanoparticles The electromagnetic interpretation of SERS is based on the excitation of the surface plasmon resonance [40] of particles and aggregates, and a general understanding of the reported SERS phenomena can be achieved from the analysis of the complex energy distribution and spatial localization of surface plasmon resonances in SERS active media [48]. In many instances, the experimental data indicate partial aggregation of metal nanoparticles, and that aggregation leads to an increase in SERS activity [49,50]. Several aggregation agents can be employed in SERS experiments, perchlorate, nitrate and chloride being the most frequent [51 – 53]. Organic polymers such as polylysine can also be employed for such purpose [54]. Inorganic aggregation agents which do not include chloride are preferred because of the complex effects that chloride can have on these systems [55], as well to avoid the interference effects of organic molecules. Clearly, we are considering only partial aggregation of the nanoparticles [50]. Partial aggregation of the colloidal particles can be induced by controlling the final concentration of the added adsorbate or aggregation agent. The addition of halides to Ag colloid has drastic consequences on the morphology of the particles and the chemical properties of the corresponding surfaces. For example, the addition of chloride induces a significant change in the surface properties by induction of atomic roughness, with the creation of atomic clusters that passivate surface reaction centers [52]. 2.5. Colloidal nanostructures supported on thin solid films The use of colloids for SERS is not limited to suspensions. There is an upsurge in the recent published work to produce colloidal metal-nanoparticles of different shapes and or sizes embedded in different thin solid films forming suitable substrates for SERS applications. These

R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

are, in practice, portable SERS sensors for specific analytical applications. For instance, we have reported films of gold nanospheres embedded in the biopolymer chitosan [56], acting as a non-active organic matrix (Fig. 1e), and in a parallel development, silver nanowires in a dendrimer matrix were transferred to a glass slide support using the layer by layer technique [57] (Fig. 1f). Several groups have reported the preparation of thin films containing SERS active colloidal nanostructures by a variety of different techniques. Transferring silver nanospheres by silinization of a glass slide [58]. Spontaneous 2-dimensional reassembling of multilayer interfacial films on H2O surface and a subsequent deposition, forming hexagonally packed arrays of Au nanoparticles [59]. Self-assembled film by alternative deposition with a cationic polyelectrolyte [60,61] and selfassembly of monodisperse metal colloid particles into monolayers on polymer-coated substrates [62]. The use of these systems has some advantages. The transference of the colloids to solid supports stabilizes them, avoiding the aggregation/flocculation processes that usually occur with time. Some of the films are self-sustained and formed portable SERS active substrates. Many of these films are water insoluble, thus allowing the transfer of analytes by means of Langmuir-Blodgett technique in a similar way as it has been carried out with metal island films [63] for ultrasensitive chemical analysis and single molecule spectroscopy.

3. Characterization of colloidal nanostructures Characterization of nanostructures is in itself a growing field of research [64]. In particular, linear optical techniques form a group of methods used for the characterization of nanomaterials and they are vital to forecasting a given nanoparticle’s probability as a successful field enhancer. A complete overview of nanomaterial characterization can be found in the works edited by Wang [65]. When focusing on a particle’s SERS potential, the properties that are particular important to the SERS phenomena are morphology, size, shape, crystal structure, tertiary structures, dipole plasmon absorption, surface potential, chemical composition and what materials are present at the surface be it stabilizers or contaminates. Morphological properties are attained primarily from electron microscopes such as TEM [66,67] (Fig. 1a – d), where size and shape can be inferred. As well Scanning Probe Microscopy’s, Atomic Force and Scanning Tunneling Microscopy (AFM and STM) can determine particle sizes for carefully prepared samples (Fig. 1e, f). On the other hand, direct information of size and shape in solution also can be obtained by means of dynamic (Fig. 3b) or static light scattering techniques [68,69]. Crystal structures can be attained readily with X-ray diffraction (Fig. 1d). Material compositions of nanoparticles can be measured by XPS or EDAX. The characterization of the chemical species adsorbed on the metal surface can be done by a number of

51

techniques employing electrons (EELS, Auger spectroscopy), ions (SIMS) [70] and most commonly photons. Between the last techniques those based on X-rays (XAS, XAFS, XPS) [71] and vibrational spectroscopies (RAIRS, SERS, SEIRA) [40,72] are the most frequently used. The optical properties of nanoparticles are dependent on a particle’s electronic states which have been shown, for materials in the nano-regime, to be strongly dependent on a particles size, morphology, its interactions with other particles, and the dielectric constant of the solvent or substrate the colloids reside in. The optical responses of nanomaterials are easily determined in the UV – Visible spectral region. These absorptions are primarily due to collective electron resonances and are commonly referred to as dipole particle plasmon resonance (DPPR) (Fig. 2) [12]. While in general these are ensemble measurements, techniques exist to measure the plasmon of single metal nanoparticles for example the measurements for an isolated silver particle have been attempted using Near Field Scanning Probe techniques (NSOM) [73]. SERS itself can be used to attain a measure of the magnitude of photonic fields at the surface of a nanostructures for a given excitation frequency. The theoretical models developed for the electrodynamics of nanoparticles and aggregates are becoming even more sophisticated and SERS measurements are a testing ground for computational models. The optical properties of spheroidal particles are described in excellent monographs [74,75]. Further refinement to the theory takes into account the break down of the macroscopic dielectric properties at these nanosize scales where the mean free path of the electron is on par with the surface lengths of the particles [14]. The theory has expanded to account for the fields of aggregated particle both as fractals and as isolated interacting particles [76]. As well, the effects of solvents and other local environmental factors have been addressed and nicely summarized [13]. Finally, an important property for a metal colloid in solution is its surface potential, or surface charge, which drastically effects a given molecules ability to get to the enhancing surface. This property is routinely determined through zeta potential measurements (Fig. 3a) which is attained by photon correlation spectroscopy, and should be used to optimize the enhancement of metals colloids in average SERS measurements [33].

4. Average colloidal SERS of small molecules and their spectral interpretation In principle, SERS is applicable to all molecules including anions and cations. Therefore, the analytical applications of SERS encompasses the study of molecules of different size, ranging from macromolecules like proteins [77] or DNA [78] and RNA [79] to medium/small molecules of diverse chemical nature: amphetamines [80], aromatic thiols [81], cancer drugs [82], p-benzosemiquinone radical anions [83], phthalimides [84] or pyrazoles [85].

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SERS practitioners have recognized that setting the SERS experiment for chemical analysis requires fine tuning of several variables such as laser excitation line, adsorption optimization and aggregation. Most of the SERS reports using common colloids (i.e. silver and gold colloids obtained by reduction with sodium citrate or sodium borohydride) are carried out using laser lines ranging from 442 nm to 830 nm. Typical colloids show plasmons ranging from 380 to 440 nm for silver and 510 to 540 nm for gold. However the colloidal suspension usually shows a broad distribution of sizes and shapes (Fig. 1a, b), implying also a broad distribution of surface plasmons. On the other hand, due to the interaction of the analyte with the colloids there is a tendency to form aggregates that shift the plasmon to the red. Experimentally, it was noticed very early on that the addition of electrolytes like NaCl or NaNO3 to the colloidal solution affect positively the enhancement of the signal [50,86]. The effect of the addition electrolytes is to decrease the electrostatic barrier which inhibits both the adsorption of the analyte on the colloid and the aggregation of the sols [33,69]. The visible region of the electromagnetic spectrum is where most of the colloidal SERS is carried out; however, the applications also extend to the near infrared region. Extending the method to the other end of the spectrum is not as common, to date there only one paper reporting SERS in the UV region [87] and it was done by using rhodium and ruthenium electrodes. Near-IR SERS provides excellent discrimination against fluorescent interference and is out of the electronic resonances with most molecules allowing the use of higher laser power without photobleaching or destruction of the analyte. Fourier-transform (FT)-SERS uses a solid state Nd: YAG laser line at 1064 nm [88 – 92] has the advantage of avoiding fluorescence interference and the low probability for photobleaching of the sample. However, the sensitivity of the detection system is much lower than that of the CCD

instruments and compounded to that the enhancement factors obtained in that spectral region are very modest compared with those obtained with higher energy of excitation. While molecular resonance can be a minus it also can be an enormous plus. When working in the visible region of the spectrum, it is possible to use a laser line that is in double resonance, that is in resonance with both the nanostructure plasmon and the molecular electronic absorption (Resonance Raman Scattering [93]), given rise to the surface-enhanced resonance Raman scattering (SERRS). SERRS has developed into an extremely powerful analytical tool that yields information about the molecular structure of the analyte with sensitivity comparable or even better than that achieved using fluorescence spectroscopy [94,95]. In the case of chemisorption, SERRS may be the result of electronic resonances with new excited electronic states of the chemisorbed species having absorption band overlapping with the wavelength of the exciting laser line. The latter is commonly referred to in the literature as the Fchemical_ SERS effect [96]. The first example illustrates the SERS of a small molecule chemically adsorbed onto the metal colloid. Fig. 5 shows the SERS spectra of the p-nitrothiophenol on silver colloids for three laser lines, 514.5, 633 and 780 nm [97]. The relative intensities in the spectrum recorded with the 514.5-nm laser line are different to those obtained at 633 and 780 nm. There can be two explanations for the variation of the relative intensities in the spectra of chemisorbed species on silver when moving to higher energies for excitation. First, for a well oriented chemisorbed species, the perpendicular and tangential component of the local field on the silver colloids could be quite different in magnitude for laser lines on different sides of the plasmon resonance [40]. Therefore, relative intensities will vary according to the orientation of the polarizability derivatives and the magnitude of the local field at the surface. The

Ag-pNTP

LL: 514 nm

300

LL: 633 nm

500

700

Wavelength/nm

LL: 780 nm

600

800

1000

Raman

1200

1400

1600

shift/cm-1

Fig. 5. SERS spectra of p-nitrothiphenol on silver colloids excited with three laser lines. The inset shows the absorption spectrum of the Ag-p-nitrothiphenol complex [97].

R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

alternative explanation considers the possibility of a charge transfer Ag complex with an electronic absorption in the spectral region of the exciting laser lines. The UV – Vis spectrum of the isolated Ag-complex shows a strong absorption at 223 nm with a broad shoulder in the 400-nm region as shown in Fig. 5. The 514.5-nm laser line is in preresonance with the absorption of the complex explaining the variation in relative intensities observed with this particular laser line. Since the variation in relative intensities was also observed with the isolated complex, it can be concluded that a charge-transfer absorption band is responsible for the peculiar relative intensity pattern observed in the SERS of p-nitrothiophenol. A second example of colloidal SERS is the planar molecule of 1,8-naphthalimide, that belongs to the C2v point group symmetry, with a total irreducible representation C = 21a 1 + 8a 2 + 20b 2 + 11b 1 [98]. Following the IUPAC recommendations of the report on notation for the spectra of polyatomic molecules, the x-axis was chosen perpendicular to the molecular plane and the C2 axis was selected along the z-axis; thereby the molecular plane was the yz plane. Also, lower case letters are used for the normal vibrational modes. The solid state Raman spectrum obtained with the 325 nm laser line gives a pre-resonant Raman spectrum with a typical pattern of relative intensity shown at the top of Fig. 6. The spontaneous Raman spectrum is represented by the inelastic scattering obtained with the 633nm excitation (bottom spectrum in Fig. 6). It can be seen that the strong ring stretching vibration band at 1583 cm
1 is seen in both spectra at 325 nm and 633 nm and is a good reference for the discussion of the dispersion of relative intensities. As the molecular electronic resonance is approached the in-plane ring stretching modes completely dominates the spectrum and the relative intensity of vibra-

LL = 325 nm, powder SERS at 442 nm

SERS at 633 nm

LL = 633 nm, powder

500

1000

1500

Raman shift/ cm-1 Fig. 6. Raman and SERS spectra on silver colloids of 1,8-naphthalimide obtained by using different laser lines (325, 442 and 633 nm).

53

tional modes below 1300 cm
1 is negligible. There are three different optical properties that can be extracted from the observed spectra. First, the differences in the intensity pattern and minor frequency differences between the SERS spectrum at 633 nm and the spontaneous Raman at the same frequency hint to chemical adsorption of the phthalimide onto the silver particle. Secondly, the results previously reported [98], clearly illustrated the wavelength dependence of SERS intensities for each irreducible representation of the chemisorbed molecule and it was confirmed that SERS spectrum obtained with excitation to the red of the plasmon resonance contains mainly totally symmetric modes. The latter is in agreement with the presence of a predominant perpendicular electric field component and a preferentially head-on molecular orientation. The new SERS spectrum obtained with 442 nm laser line also agrees with the published spectrum at 488 nm in that it contains a large number of vibrational modes corresponding to species of symmetry other than the totally symmetric ones. Thirdly, although we were not able to obtain SERS with the 325-nm laser line, the pre-resonant trend in relative intensities can be clearly seen in the SERS spectrum obtained with the 442 nm laser line shown in Fig. 6. The relative intensity pattern in the SERS spectrum at 442 nm shows the tendency to give prominence to the ring modes, however, it is also apparent the contribution of several modes belonging to species of symmetry other than the a1 type. The naphthalimide case illustrates the variables that should be considered when analyzing and interpreting the SERS spectra of chemisorbed species, they are: (1) the polarization (direction) of the local electric field at the molecular adsorption site. (2) The molecular orientation with respect to the nanostructure surface. (3) The tuning of the excitation laser line into the excited electronic state of the adsorbate that could give rise to three regimes, off-resonance, pre-resonance or resonance Raman scattering. Studies where these variables are analyzed for specific adsorbate are found with increasing frequency in the literature [81,84,99– 102]. SERS on colloids also allows to gather valuable information on molecular properties and reactivity [103 – 105] structural studies on macromolecules [79,95,106] and live microorganisms [77,107,108] or to extract differences within molecular systems [109]. It is a powerful quantitative analytical tool [110] for the detection of molecules in the zeptomol regime [81,111] and at the single molecule level [8,112,113]. In a related application SERS spectroscopy provides an excellent tool to study photochemical reactions as part of the field of enhanced-photochemistry, or can simply be used to determine the chemical changes in a target molecule. For example, photoreduction of methyl viologen [114], as well as photodecomposition of azo compounds [115], have been confirmed to occur on silver. Moskovits’ group used a capillary flow method, and SERS, to quantify the photochemical kinetics of various molecules adsorbed on colloidal silver surfaces. This group has successfully examined the photodescomposition of 2-

R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

The SERS spectrum of 4-nitrobenzenethiol is dominated by a strong peak at 1336 cm
1 assigned to the symmetric stretching vibration of the nitro group, which indicates that the molecule is not using this group in its interaction with silver colloids. The induced photoreaction of 4-nitrobenzenethiol to give 4-aminobenzenethiol when is irradiated with a laser line of 514.5 nm in a stationary condition (without recirculation) is evident since this spectrum (Fig. 7 c) shows the same spectral pattern than that corresponding to the amino-derivative (Fig. 7b).

c b a 1650

1500

1350

Raman Shift

1200

1050

900

(cm-1)

Fig. 7. SERS spectra of (a) 4-nitrobenzenethiol and (b) 4-aminobenzenethiol in silver sol, taken while flowing the sample solutions through a glass capillary to minimize photoinduced reaction. (c) SERS spectrum of 4nitrobenzenethiol in silver sol taken in static condition. All of them were collected by using a 514 nm laser line [15].

Apart from the average SERS, with enhancement factors up to seven orders of magnitude, is the ability of SERS/ SERRS to provide ultrasentitive detection, commonly, in the atto and zeptomol regime as well as the ultimate limit of the single molecule detection (SMD). The high enhancement factors (1010 or more) required to observe the SMD has led to the presence of the ubiquitous ‘‘hot-spots’’ [48], the explanation [48,121] and fabrication [122] of which is inspiring many research projects. Metal colloids were part of the first reports on single molecule experiments by Nie [112] and Kneipp [8] and the activity around SMD is flourishing using metal colloids and nanostructure fabricated by physical methods [63]. Fig. 8 show the reported spectrum of ¨ 80 molecules and those of a single molecule for a protein on silver citrate 1620 1550

pyrazinecarboxylic acid [116], 2-aminopyridine [117] and diazanaphthalenes [104], the photoreaction of phthalazine [118], the photodesorption of 4-vinylbenzoic acid [119], and the photoisomerization of maleic acid [120]. Fig. 7 shows the SERS spectra of 4-nitrobenzenethiol (Fig. 7a) and 4-aminobenzenethiol in silver sol (Fig. 7b), taken while flowing the sample solutions through a glass capillary to minimize photoinduced reaction, and the static SERS spectrum of 4-nitrobenzenethiol on silver sol [15].

5. Application of colloidal nanostructures to ultrasensitive analysis and single molecule detection

c

1800

1600

1400

1200

1000

Raman Shift / cm-1

800 800

1153

1012

1161

1400

1022 1022

15 - 20 s

10 - 15 s

1017

1319

1169

1311 1274 1229 1265

1375 1331 1279

20 - 25 s

1227 1159

1379

1379 1311

1600

25 - 30 s

1528

1564 1530

1626 1582 1554 1526 1634 1580 1628 1578

1149 1084 1022

1272 1230

1450 1373

b

1618 1572 1528

1708

1718

1720

1621

1721

1618 1560

1271

a

1014

1800

1014

Intensity (arb. unit)

h

1151

54

1200

1000

5 - 10 s

0-5s

800

Raman Shift / cm-1

Fig. 8. SERRS spectra of a single green fluorescent protein molecule (5 s integration time, 500 nW excitation power) adsorbed on a silver colloid. (a) Averaged spectrum built up by 80 different individual molecules. (b,c) The spectral time series from two individual green fluorescent protein molecules [95].

R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

colloids cast on a polylysine coated glass surface [95]. The single molecule spectra show better resolution due to the decrease in the fluorescence in the interactions between different molecules. Also, the series with time show sudden frequency jump from 1524 to 1562 cm
1. This frequency jump can be interpreted in terms of a conversion of the chromophore from the deprotonated to the protonated form. This reversible conversion is commonly accepted to occur in green fluorescence and related proteins. This is an example of the potential of the technique to gain structural evidence for photophysical processes thought to occur in fluorescent proteins at the single molecule level. Single molecule studies have a huge potential in exploring molecular interactions, particularly molecule – surface interaction. Much work has been done to examine the dynamics of a single molecule interaction with nanomaterials [123 –126]. This SM spectroscopy holds much promise and the relatively simple metal colloid is central to the developing science. Ultrasensitive detection is usually achieved by casting the colloidal – analyte system onto a glass surface. Fig. 9 shows the results obtained by casting 1-naphthalenethiol diluted in silver citrate colloids on a silver island film. Micro-Raman SERS spectra were recorded using a 50  objective, with a spatial resolution of about 1 Am2. Assuming an average distribution of molecules onto the surface area covered by the aliquot on the silver island film, one can estimate the average number of molecules under the illuminated area, which can be equated with the number of molecules contributing to the scattered signal detected in the far field. Under these conditions of the results obtained for 1-naphtalenethiol show that SERS is detected from an average of 10 molecules/Am2. These spectra are obtained with a good signal/noise ratio for concentrations of 103 molecules/Am2 or higher. On the other hand, ultradetection can be also achieved directly on the colloidal suspension by using indirect measurements. Moore et al. had achieved the

zeptomol regime for 14 enzymes with biological interest by coupling the catalytic capacity of the enzymes to break determined chemical bonds with the capacity to give SERRS of some dyes. In this case, the complexation sites for metals of a benzotriazole dye, a well known SERRS active species, were masked avoiding the complexation sites for metals by substituting the nitrogen proton by derivatives of the 3-phenyl butyric acid and using a methylene linker group (Fig. 10). The ‘‘complex’’ dye was then unable to be chemisorbed on the silver colloids surface and so, unable to give SERRS. The complex acts as a substrate for the enzymes; they react with this substrate giving rinse to the free dye which is able to be complexed on the metal colloid and give SERRS. By using this method, not only the zeptomolar detection is achieved but also it is possible to follow the enzymatic kinetics and activity.

6. Biological and biomedical applications With an understanding of the basic experimental requirements to achieve SERS, and the clear theoretical guidance provided by the electromagnetic interpretation, the application of SERS and SERRS to biological molecules and biomedical applications continues to grow rapidly. Several recent excellent reviews are available, Dou and Ozaki [10] have specifically review the use of metal colloids for SERS of biological molecules. Vo-Dinh [127] provides a review of SERS in biomedical applications, and the volume of publications in this area is such that this section is necessarily very limited in scope and is not comprehensive. Notably, SERS and or SERRS techniques in the visible spectral region can be applied to the vibrational study of fluorescent molecules, due to the fact that at short distances from the surface, fluorescence quenching prevails over the field enhancement. In short, the extremely high crosssections and functional specificity of surface-enhanced

~107 molecules/µm2

~105 molecules/µm2

~103 molecules/µm2

~102 molecules/µm2

~10 molecules/µm2

600

800

1000

55

1200

1400

Raman Shift (cm-1) Fig. 9. Ultrasensitive analysis of 1-naphthalenethiol in citrate silver and cast on 13 nm silver island film (LL ¨785 nm).

56

R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

N

N

N

N

N

O

OH

N

+

Enzyme

N N N H

N N N O

Collapsible linkage

Dye SERRS

O

H

O H

Dye “complex” No SERRS Fig. 10. Alternative strategy to attain zeptomol detection.

Vibrational spectroscopy facilitate the application of this technique in recognition studies of highly fluorescent biological molecules [80,128,129]. Alternatively, FT-Raman with near infrared excitation may be successfully used in the study of very fluorescent materials. To quote just one example, the latter would be the case of hypericin and its analogues [129,130]. However, as previously noted the low sensitivity can be an issue when exciting with 1064 nm. Therefore, most of the work to obtain structural characteristics of different kinds of molecules used as drugs and their interactions with biomolecules such as proteins [131,132] and nucleic acids [78,133 – 135] has been carried out using the SERS and SERRS techniques on silver and gold colloids in the visible. Given the complexity of the biomolecular systems, the study of interactions usually begins with a comprehensive study of the SERS spectra and the interpretation of the vibrational signature of several kinds of very fluorescent molecules, using various excitation lines in the visible and NIR regions [129,136 – 138]. In a second step, the inter-

action of these target molecular systems with different kinds of biomolecules are studied [139 – 141]. The SERS spectra recorded with 782 nm excitation line of the mutagenic 9-amino-acridine (9AA), DNA and their complex is shown in Fig. 11. The spectrum of the complex shows bands corresponding to both the drug and the biopolymer. However, the intensity of DNA bands is very weak in the complex as compared to other drug bands that are still observed in the complex (i.e., those at 1460, 1370, 1168, and 1133 cm
1). The bands that are weakened in the complex spectrum can be attributed to the imino form of 9AA [142], which in absence of biopolymer, can be directly attached to the metal surface. In contrast, the stronger 9AA bands of the complex can be assigned to the amino 9AA tautomer. The enhancement of the Raman features corresponding to the amino form together with the strong decrease of the imino Raman features suggests that 9AA interacts with DNA under the protonated amino tautomer. On the other hand, the SERS spectrum of DNA is generally dominated by adenine bands (at 1646, 1558, and 1440 cm
1

9AA

9AA/ DNA

DNA

1600

1400

1200

800

600

Raman shift/cm-1 Fig. 11. SERS (LL 782 nm) spectra of 9-amino-acridine DNA and their complex.

400

R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

the broad feature centered at 1356, 1216, and 736 cm
1) [143]. These bands undergo marked changes in the complex with the drug along with a further intensification. The one appearing at 1440 cm
1 is shifted slightly upward, while the broad feature centered at 1356 cm
1 appears as a narrower band at 1346 cm
1. Besides, the ring breathing adenine band at 735 cm
1 is downward shifted to 729 cm
1. All these changes can be attributed to the interaction with 9AA. The band observed at 837 cm
1 indicates that the B structure in DNA is maintained in the complex with 9AA. The spectral changes observed for adenine bands upon complexation with the drug suggest an important role of adenine residues in the intercalation of 9AA. On the other hand, the interaction with nucleic bases other than adenine is improbable because it would induce the intensification of bands corresponding to other bases in the SERS of the complex as a consequence of the approaching of the polymer through those parts of the chain richer in other bases. In conclusion, from the SERS spectra of the 9AA/ DNA complex it is possible to deduce that the drug interacts with DNA under the amino form. The interaction of antitumoral drug emodin with human protein serum albumin (HSA) was examined using SERS. SERS spectra of emodin at different pHs are shown in Fig. 12. Usually, the molecular pKa value is shifted downward O

H

O

H

O

O

when the adsorbate is linked to a metal surface. Thus, the spectra of emodin recorded at pH 4, 7, and 12 (Fig. 12a– c) must correspond to the spectra of the neutral, monoanionic, and dianionic emodin species. The main changes observed in these spectra concern the band at 1673 cm
1, which is shifted to 1641 cm
1 at alkaline pH, and the bands at 1360 – 1250 cm
1, which undergo an upward shift. This effect is a consequence of the deprotonation and the electronic resonance increase in the drug. Another interesting effect of the pH is the progressive decrease of the relative SERS enhancement as the pH was raised; the pattern was established by taking the 679 cm
1 band of DMSO as an internal standard. The SERS spectrum of emodin/albumin complexes reveals interesting differences depending on the use of defatted (HSAf) or not defatted (HAS) serum albumina. The 1:4 emodin/HSAf complex displays a very weak spectrum (Fig. 12g). When this ratio is increased to 1:2 the SERS intensity is increased (Fig. 12f). The multiplied SERS of Fig. 6e (Fig. 12f,  3) shows a higher similarity to the SERS of emodin at alkaline pH (Fig. 12c). In contrast, the intensity of the 1:4 emodin/HSA complex is approximately 1 order of magnitude higher (Fig. 12d) than the SERS of the 1:2 emodin/HSAf complex (Fig. 12e), as deduced from the comparison of the 679 cm
1 band of DMSO. In addition, the SERS spectral pattern of the H

O

H

O

-O

HO

57

O

H

O

O

-

-O O

O

O

a b c d

b f g 1500

e

1000

500

Wavenumber/cm-1 Fig. 12. Structure and acid-basic equilibria of emodin. SERS spectra of emodin (a) at pH 4, (b) 7 and (c) 12, together with the SERS spectra of emodin/albumin complexes at the following conditions: (d) 1:4 emodin/HAS, (e) 1:2 emodin/HSAf, (f) 1:2 emodin/HSAf, and (g) 1:4 emodin/HSAf.

R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

f e d

c b O

a

H

O

H

O

DMSO

dianionic form of DT could correspond to the small, new bands increasing at alkaline pH at 1505, 986, 844 or 580 cm
1. The SERS spectrum at acidic pH (Fig. 13c) shows many differences characteristic of the transition from neutral to monoionized danthron. All these changes are related to deprotonation of the molecule since they involve bands attributed to C –OH bending or CfO stretching motions [145], which are very sensitive to the internal H-bonds. The SERS spectra of DT-BSAf and DT – HSAf complexes (Fig. 13e and f) are similar to those at pH 6.5 and 11.5 (Fig. 13a and b), except for some small differences, indicating that DT interacts with those proteins through its dissociated form. In the DT – HSAf complex (Fig. 13f), further shifts downwards are observed for the bands at 1655 and 1593 cm
1 to 1645 and 1583 cm
1, respectively. In addition, new bands are observed at 1614 and 1348 cm
1. The latter changes could be due to the second ionization of danthron, which could not be observed when the free molecule is adsorbed on Ag, but which can occur when DT is interacting with HSAf. In contrast, the SERS spectrum of the DT –HSA complex (Fig. 13d) resembles that of DT at low pH (Fig. 13c), where DT is in the neutral form. This

DMSO

58

O

f 1500

1000

500

Wavenumber/cm-1 Fig. 13. SERS spectra of danthron at (a) pH 11.5, (b) 6.5 and (c) 3.5, and SERS of the following complexes: (d) danthron/HSA; (e) danthron/BSAf; and (f) danthron/HSAf.

emodin/HSA complex is very similar to the SERS of emodin recorded at acidic pH (Fig. 12a). The above differences between the SERS of emodin/HSAf and emodin/HSA complexes indicate the existence of two different binding sites for emodin in human albumin, which depends on the presence of fatty acids in the protein. In conclusion, SERS spectroscopy revealed important data concerning the interaction of anthraquinone drugs with albumins of different origins and the influence of other ligands, such as fatty acids, on the interaction of drugs with these important blood carrier proteins. The application of SERS to the study of analogous drugs may be valuable for the design of more active ones. Fig. 13 show danthron (DT), an anthraquinone analogous, spectrum as a function of pH [138] and its complexes with not defatted (HSA) and defatted albumins (HSAf and BSAf). In the SERS spectrum of DT at pH 3.5 (Fig. 13c), a set of bands is observed, which corresponds to the neutral form of danthron [144]. On the other hand, the SERS spectra at pH 6.5 and 11.5 (Fig. 13b and a) are similar with respect to the position and relative intensity of the bands attributed to the CfO and C – OH groups, indicating that they mainly correspond to the monoionized form of DT. However, the

e

d

c b O

a

O

1500

H

H

O

O

1000

500

Wavenumber/cm-1 Fig. 14. Micro-SERS spectra of quinizarin at (a) pH 11.5, (b) 6.5 and (c) 3.5, and SERS of the following complexes: (d) quinizarin/HSA; (e) quinizarin/BSAf; and (f) quinizarin/HSAf.

R.F. Aroca et al. / Advances in Colloid and Interface Science 116 (2005) 45 – 61

clearly suggests that the drug is in the neutral form in the complex with non-defatted HSA. SERS spectra of quinizarin (QZ), another anthraquinone analogous, and its different complexes with albumins are shown in Fig. 14. As in the case of DT, the changes observed on decreasing the pH (Fig. 14a– c) are attributed to a change in the protonation state of QZ. In this context, the shift of the CfO stretching band downwards, together with the shift upwards of the two intense bands at 1392 and 1232 cm
1 observed at high pH (Fig. 14a) to 1403 and 1270 cm
1 must be a consequence of deprotonation of QZ. The SERS spectra recorded at neutral and alkaline pH reveal differences between them, which may be due to a second ionization of the molecule. The SERS spectrum of the QZ – HSAf complex (Fig. 14f) in general shows a pattern closer to that of QZ at pH 11.5 (Fig. 14a), indicating that QZ interacts with HSAf through dianionic form. However, the SERS spectral pattern of the QZ –BSAf complex (Fig. 14e) approaches that of QZ at pH 6.5 (Fig. 14b), which suggest a different interaction mechanism of the drug in each case. In contrast, the SERS spectrum of the QZ –HSA complex (Fig. 14d) is more similar to that of QZ at low pH (Fig. 14c), where QZ is assumed to be in the neutral form, as demonstrated by the existence of bands at 1642, 1404, 1368, 1341, 1262 and 1144 cm
1. As in the case of DT, this result indicates that QZ interacts with non-defatted albumins in its neutral form.

7. Conclusion Vibrationsl Spectrosccopy (Raman and infrared) has available a vast body of spectral data, collected in databases as the result of detailed studies of gases, liquids and solids. This represents the great advantages of vibrational spectroscopy for identification, chemical analysis and a realm of applications. The natural extension is surface-enhanced vibrational spectroscopy (SERS and surface-enhanced infrared absorption- SEIRA) with its corresponding database for SERS (and SEIRA) presently under construction. The vast body of SERS data is growing daily establishing the ultimate analytical tool: the ultrasensitive vibrational spectroscopy. SERS, as part of the surface-enhanced spectroscopy field, is presently fueling the field of nanostructure fabrication and characterization, with inroads into the world of chemical analysis and biomedical applications.

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