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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Gold nano-islands on FTO as plasmonic nanostructures for biosensors Vera Cantale a , Felice C. Simeone a , Roberto Gambari b , Maria A. Rampi a,∗ a b
Dipartimento di Chimica, Univeristà di Ferrara, via Borsari 46, 44121 Ferrara, Italy Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, via Fossato di Mortara 74, 44121 Ferrara, Italy
a r t i c l e
i n f o
Article history: Received 2 August 2010 Received in revised form 16 November 2010 Accepted 4 December 2010 Available online xxx Keywords: Localized surface plasmons Metal nano particles Bio-sensors FTO
a b s t r a c t Metal nanoislands (NIs) deposited on transparent surfaces can be a convenient plasmonic material for bio/organic sensors, under the condition that a stable morphology is reached. Plasmonic materials suitable for the fabrication of low cost biosensors based on localized surface plasmon resonance (LSPR) UV–Vis spectroscopy, are fabricated by a simple methodology based on thermal evaporation of Au on commercially available, transparent fluorine-doped tin oxide (FTO) surfaces. The LSPR UV–Vis spectroscopy performed in transmittance mode reveals: (i) a small energy shift, max , of the LSPR band under immersion both in organic solvent, and significantly in aqueous media, and (ii) a sensible and reproducible max under formation of organic SAMs on the NIs surface. These data indicate that the Au NIs when deposited on FTO substrate exhibit (i) strong adhesion and a high stability, and (ii) a good sensitivity to molecular interaction. The samples also show that the LSPR bands recover the original feature after being exposed to different type of SAMs. Significantly, the absorption maximum, max of the Au-NIs LSPR spectra shows a red shift when SAMs incorporating single strands DNA are exposed to the complementary strands. The plasmonic system based on Au NI deposited on FTO surfaces because of (i) the inexpensive fabrication of stable NIs, (ii) the easy way to detect the molecular interaction occurring at their surface, and (iii) the sensitivity of their LSPR to molecular interaction represents a convenient platform for biosensors. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In the search of label-free, easy to use sensors for bio/organic molecular interactions, we have focused our study on plasmonic systems based on the optical properties of metal nanoparticles deposited on a transparent support. In conventional surface plasmon resonance (SPR) spectroscopy, the response of surface plasmons polaritons (electromagnetic waves propagating at metal surfaces coupled with electron motion) to changes in the refractive index of the surrounding medium are used for monitoring and sensing a variety of phenomena occurring at the interface. The optical properties of thin metal layer surfaces due to the excitation of SPP, have been widely exploited for the development of label-free sensing devises [1–6]. This technology is very versatile, but still expensive and of not trivial implementation. The recently highly improved ability to synthesize and to study nano-sized metal particles, marked a breakthrough in the development of SPR-based techniques, and provided an efficient and inexpensive approach to the fabrication of sensing devises. Nanosized clusters of noble metals (especially Au, Ag) exhibit unique
∗ Corresponding author. Tel.: +39 0532 455165; fax: +39 0532 240709. E-mail address:
[email protected] (M.A. Rampi).
optical properties which are not present in the spectrum of the bulk metal. Absorption and scattering of incident light, occurring when the photon frequency is resonant with collective oscillations of conduction electrons, results in strong UV–Vis bands. This effect is known as non-propagating or localized surface plasmon resonance (LSPR) [7]. A rigorous mathematical description of this phenomenon was achieved by Mie [7]. In his formulation, the extinction (scattering + absorption) E() of a spheroid metal nanoparticle, is related to the properties of the system by the equation:
E() ∝
εi () 2
(εr () + εmed ) + εi ()
2
(1)
where εmed is the dielectric constant of the medium surrounding the NP, is a factor related to the geometry (for a sphere, = 2) (although the formalism is not valid for any arbitrary shaped nanoparticles), εi and εr are the imaginary and real part of the dielectric function of the NP. The Mie theory has provided a deep understanding of the interactions of light with metal nanoparticles, but it has been developed for ideal systems and results to be inadequate for most practical uses. A less rigorous approach is based on the assumption that the equations describing the extinction of the SPR of large metal surfaces [8] hold also for the NP [9]. In this case, in analogy with the
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SPR, the LSPR bands are expected to show a spectral shift () qualitatively described by: = m(nads − nmedium ) (1 − e−2d/l )
(2)
where m is the sensitivity factor (nm/RIU (refractive index unit)), nads and nmedium are respectively the refractive index of the adsorbed film and of the medium, d is the thickness of the adsorbed film, and l is the characteristic electromagnetic decay of the evanescent electromagnetic field [10–13]. According to Eq. (2), the binding of organic molecules to nanoparticles induces an increase of the local refractive index and, in turn, a red shift of the extinction spectrum, as experimentally observed [14,15]. It is thus evident, that LSPR response of plasmonic nanoparticles acts as transducer of changes in the refractive index occurring at the surfaces of the NPs. Although SPPs are characterized by a sensitivity factor m 20–100 times higher than LSPR, the evanescent electromagnetic field at the surface of nanoislands has a shorter decay length l. This property implies that the space surrounding the nanoisland can be probed with electromagnetic fields 20–50 times more intense than that of SPP. This issue makes the LSPR very competitive with the SPP [9]. Hence, the sensitivity of the LSPR to the presence of adsorbates can provide an effective and easy route for monitoring in real time the binding of molecules or molecular interactions occurring at the nanoparticles surface. In fact, LSPR of nanoparticles dispersed in a solution has been exploited in a variety of applications for sensing and monitoring of molecular interaction [14,16]. However, the use of metal nanoparticles in solution requires (i) a careful synthetic procedure to obtain monodisperse NPs, (ii) a fine control of ionic strength, pH, and temperature in order to avoid the flocculation, and (iii) a stabilization of the synthesized nanoparticles with a proper molecular shell which can limit a further functionalization for sensing purpose [17–19]. As an alternative to colloidal dispersions, metal nanoparticles deposited as nanoislands (NIs) on a transparent support result to be a promising plasmonic material. We believe that this surfacebased approach allows for the development of chip-based sensors, and gives the opportunity to design arrays of plasmonic material which can be used in multiplex bio-recognition [20,21]. A series of recent studies have shown that monodisperse nanoislands (NIs) of Ag and Au deposited on transparent substrates can provide a convenient approach to detect metal-surface/molecule interaction [13,22–38]. According to Eq. (1) and to a number of experimental results [14,39,40], the LSPR band energy results to depend not only on the refractive index of the absorbed molecules, but also on the geometrical characteristics of the nanoislands (term in Eq. (1)). Therefore, metal NIs can be a valuable plasmonic material for bio/organic sensors, only under the condition that a stable NIs morphology is reached so that the LSPR band features are well set. The stability can be related both to the adhesion properties of the NIs to solid surfaces—a critical issue in aqueous solutions, and to the restructuration processes of the metal surface. It is worthwhile to observe that while SAMs on Ag are more stable towards oxidation than SAMs on Au, Ag surfaces, contrary to Au, undergo a fast oxidation [70]. Both these phenomena are responsible for large and difficult-to-control variations of their optical properties, and specifically of the LSPR bands feature [41–43]. The widely used approach for increasing the adhesion of Au based on pre-deposition of Ti and Ni layers would require expensive apparatuses. On the other hand, the more accessible deposition of Cr inter-layers [44], can affect and attenuate the response because of Cr diffusion into the Au NIs [45–47]. The relevance of the NIs stability problem is well represented by the large number of studies suggesting different approaches to reach “stable” metal NIs. Layers of mercapto-silane have been used for improving the adhesion of NIs to glass [48,49].
Van Duyne reported on structural changes of Ag NIs in organic solvents, prior to molecular surface modification [50]. Rubinstein has proposed (i) to coat Au-NIs by a robust and transparent layer of ultra thin films of silica [51], or (ii) to sink Au NIs into the glass by a controlled thermal treatment [52]. Stable Au nanorods and Au NIs were obtained by embedding them respectively into glass [53], and into an Al2 O3 matrix [54]. A high stability due to an enhanced adhesion of Au NIs at rough surfaces has been reported when ITO (indium-doped tin oxide) is used as support [55]. We stress that, besides the high cost of ITO substrates, the diffusion of indium at the interface could preclude the right functioning of ITO-based sensor [56–58]. In this article we show that it is possible to obtain “stable” AuNIs by conventional thermal deposition of gold onto fluorine-doped tin oxide (FTO), a transparent and commercially available substrate. The rational of this choice is based on a strong adhesion of Au onto FTO substrates, enhanced by the porosity of SnO2 where Au can penetrate during thermal annealing [59–61]. In the first part, we report a study on the “stability” of these NIs evaporated on FTO (Au NIs/FTO) when immersed in different media, in particular in (i) water, (ii) aqueous buffers commonly used in biology as sodium chloride-tris-EDTA (STE) and phosphatebuffered saline (PBS) solutions, and (iii) ethanol, as one of the most common solvent used for self assembled monolayers (SAMs) formation [62]. In the second part we test the sensing capability of the LSPR bands of Au-NIs/FTO to the formation of organic SAMs. We have measured and compared the spectral shift of max of the LSPR bands under formation of dodecanethiol (C12), hexadecanethiol (C16), and 4-terphenylthiol (SAMs) in order to (i) compare the response of the Au NIs/FTO system to those reported in literature, (ii) to compare the shift induced by SAMs with same refractive index and different thickness, (iii) to compare the shift induced by SAMs of the same thickness and different electronic structure. We also measured the response of the LSPR band to the formation of SAMs of mercaptohexanol (MCH), a compound widely used to intercalate DNA strands on a metal surface [63]. To prove the reversibility of the LSPR response, we have recorded the LSPR bands when alkanethiols forming a SAM on the NIs surfaces are replaced by aromatic molecules, and vice versa. Finally, and importantly we have tested the bio-recognition capability of the Au NIs/FTO, by measuring the change of LSPR bands upon hybridization of single strand of DNA. 2. Materials and methods 2.1. Materials 1-hexadecanethiol, 1-dodecanethiol and 6-mercapto-1hexanol, ethanol, 2-propanol and sodium chloride-tris-EDTA were purchased from Sigma Aldrich, Germany. 4-terphenylthiol from Frinton Laboratories Inc. (Vineland, NJ) and phosphate-buffered saline (PBS) solutions from Lonza Walkersville Inc. (Walkersville, MD). The FTO (TEC 8) surfaces were purchased from Pilkington North America Inc. (Toledo, OH). The modified oligonucleotide (5 thiolated—CAG TGA GGC GTG GCC AGG G-3 ), the complementary (5 -CCC TGG CCA CGC CTC ACT G-3 ) and the non complementary (5 -AAG TGC ATC GTG ATT CAT A-3 ) sequences were purchased from Eurofines MWG Operon (Ebersberg, Germany). 2.2. Nano-islands fabrication The FTO surfaces were cleaned by a solution 1:1 of acetone and 2-propanol in ultrasounds bath for 15 min, then repeatedly rinsed with copious amounts of water MilliQ and lastly gently dried with a pure nitrogen stream. The gold nanoislands are deposited on the
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Fig. 1. SEM images and respective T-LSPR bands of the Au NIs deposited on FTO with nominal thickness 5 nm; A and B images show the morphology of the Au NIs, respectively, immediately after the thermal evaporation and after annealing process for 10 h at 200 ◦ C; C and D represent the respective T-LSPR spectra.
FTO surfaces by thermal evaporation at a pressure of 10−6 mB and at deposition rate of 0.0016 nm s−1 . The deposition process of NIs is controlled by measuring a nominal thickness of 5 nm with a builtin quartz balance. The samples were then annealed for 10 h at to 200 ◦ C. 2.3. UV–Vis spectroscopy The UV–Vis measurements were carried out by using a Jasco V-570 UV–Vis-NIR spectrophotometer in transmittance mode by placing the samples into the spectrophotometer cavity (scan speed 200 nm/min, band width 1 nm). All the spectra were recorded in air and by using air as reference. A series of measurements performed by repetitive removing and replacing the same sample into the spectrophotometer cavity showed an error in max values of 1 nm. 2.4. Scanning electron microscopy (SEM) The Au NIs/FTO images were obtained using a scanning electron microscopy (SEM FEI XLF30-SFEG of FEI Company Eindhoven) equipped with ultra high resolution lens and in-lens SE (secondaryelectron) detector. 2.5. Stabilization of the nano-islands The samples of Au NIs/FTO were immersed in different media (H2O, STE buffer, PBS buffer and ethanol) for 24 h. Then the samples were rinsed with water MilliQ or ethanol and dried with pure nitrogen. LSPR spectra were recorded before and after immersion in the various media. 2.6. Nano-islands functionalization Self-assembled monolayers (SAMs) of 1-dodecanethiol (C12), 1-hexadecanethiol (C16), 4-terphenylthiol and mercaptohexanol
(MCH), were formed by incubating the Au NIs/FTO samples into respectively 1 mM ethanol and 1 mM water solutions for 16 h. 2.7. Reversibility The C12 SAM formed on Au NIs/FTO has been replaced by a 4terphenylthiol SAM upon immersion of the sample carrying the C12 SAM in 1 mM ethanolic solution of 4-terphenylthiol for 16 h. The 4terphenylthiol SAM has been thus replaced by a new C12 SAM, by incubating the sample in 1 mM ethanol solution of 1-dodecanethiol SAM for 16 h. T-LSPR spectra have been recorded after the formation of each SAM. 2.8. DNA hybridization The DNA hybridization was performed according to the literature protocols [63–66]. In the present case, the samples have been incubated with a PBS buffer solution containing the 5 -thiolatedCAG TGA GGC GTG GCC AGG G-3 -oligonucleotide (ssDNA) for 16 h at room temperature. After incubation the sample was rinsed with PBS buffer and MilliQ water, in order to take away residual thioled ssDNA sequences. Thus, the samples have been incubated for 1 h in an aqueous solution of mercaptohexanol (MCH) according to the protocol [63]. MCH is used as spacer between the ssDNA in order to minimize nonspecific adsorption of the ssDNA and to facilitate hybridization [63]. To perform the hybridization of the ssDNA, the samples have immersed into a PBS solution of the complementary sequence for 16 h a room temperature. After incubation, the samples were rinsed with PBS buffer and MilliQ water and dried with nitrogen. After each step, the T-LSPR spectra of the samples were recorded. A control experiment was done by incubating for 16 h the MCH-DNAss carrying samples in a PBS solution containing the not complementary strand 5 -AAG TGC ATC GTG ATT CAT A-3 [63]. After incubation, the samples were rinsed with PBS and MilliQ water, then dried with nitrogen.
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Fig. 2. Localized surface plasmon resonance (LSPR) spectra measured in transmission mode of a number of different Au NIs/FTO samples of 5 nm nominal thickness and after annealing at 200 ◦ C for 10 h.
3. Results and discussion To characterize the plasmonic response of Au NIs/FTO, and in particular (i) the “stability”, (ii) the “sensitivity” to organic SAMs formation, (iii) the size dependent properties, and (iv) the reversibility, and (v) the biorecognition ability we have performed UV–Vis measurements in transmittance mode of the LSPR bands features (max and ODmax ). 3.1. Fabrication of the NIs on FTO Fig. 1A and B reports the SEM image of the Au-NIs obtained by depositing Au, by using the simple described protocol (see Experimentals) respectively before and after the annealing process. Fig. 1C and D shows the respective LSPR bands. The annealing process of NIs deposited on quartz or glass leads to a sharper LSPR band, with a max shifted towards the blue, according to what reported in literature, indicating that the aspect ratio (a/b, where a is height and b is width) decreases [13]. Therefore the annealing process results to be a key step also for the fabrication of the plasmonic substrate on FTO. Fig. 2 shows a number of LSPR bands recorded for different samples of annealed Au-NIs. The spectra show a relatively large bell shape, as expected for NIs of different aspect ratio, with ODmax values that span from 0.39 to 0.42 and the max values that span between 555 and 585 nm. These data indicate that the samples resulting from such a simple fabrication differ in average morphology. The stability and the sensitivity of a large number of samples are analyzed in the next session. 3.2. Stability of the Au-NIs The “stability” of a large number of Au NIs/FTO samples (showing different values of max ) has been tested both in organic and aqueous solvents, in particular by incubation for 24 h in pure H2 O, STE and PBS aqueous buffers and in ethanol. Importantly, the LSPR spectra measured before and after incubation do not show significant decrease in OD, indicating a good adhesion of the NIs to FTO surfaces. Fig. 3A and B reports the max of samples with different morphology (different values of max ) after incubation respectively in aqueous media and in ethanol. The results indicate that the shift of max is very small. In particular, incubation in pure water and PBS buffer yields max of few nanometers, while a bigger shift was detected for STE, max,STE = 6.75 ± 1.5, as shown in Fig. 3A and summarized in Table 1. The reported max shifts could be related both to gold atom restructuration processes of the NIs or to contam-
Fig. 3. (A) shift of max (max ) of the LSPR spectra measured for different samples of Au NIs versus max values. Different max indicate that the samples have different average morphology. The shift is measured after immersion of 24 h in water (o), STE () and PBS (). (B) max shift measured for different samples of Au NIs versus max after immersion in ethanol for 24 h.
ination of gold by some impurity of the media (solvents/buffers). Possibly the larger shift measured for incubation in STE, consisting of concentrated salt solutions, could be related to impurity effects of the buffer salts. Significantly, the data of Fig. 3 show that the max shifts are randomly distributed respect to the relative values of max , that is the adhesion/restructuration process is small regardless the morphology of the samples. In Fig. 3 we observe a trend in the LSPR feature towards red shift. The small value suggests that a possible restructuration process confined to the surface occurs upon immersion in a solvent. It is well known that under thermal treatment Au surfaces reconstruct to more dense, contracted structures [69]. The contact with solvents stabilizes the unreconstrucuted surface which relaxes to the bulk lattice leading to a red shift of the LSPR maximum. When using PBS, which is a buffer and not a pure solvent, the interaction of Table 1 max shifts () of the LSPR band of Au NIs/FTO samples after stabilization and functionalization with different SAMs in the same solvents (water and ethanol). Stabilization Solvent Water
max (nm) 1 ± 2.6
Ethanol
1.8 ± 1.8
Functionalization SAM MCH C12 C16 TerPh
max (nm) 10.2 ± 2 9.8 ± 2.7 11.7 ± 2.2 21 ± 2.1
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Fig. 4. Surface plasmon (SP) bands after formation of organic SAMs on Au NIs. (A) dodecanethiol (C12); (B) hexadecanethiol (C16); (C) terphenylthiol (TP); (D) mercaptohexanol (MCH).
the dissolved ions with the Au surface can be the reason for the observed slightly different behaviour. 3.3. Sensitivity of the Au-NIs LSPR band to SAMs formation Fig. 4A–D shows respectively T-LSPR spectra of representative Au NIs samples, before and after incubation in ethanol solution of C12, C16, and terphenylthiol, and in aqueous solution of MCH. The average max shifts, calculated over more than 100 samples (see Supporting information, Fig. S2), are respectively max,C12 = 9.8 ± 2.7 for C12, max,C16 = 11.7 ± 2.2 nm for C16, max,terph = 21 nm ± 2.1 for terphenylthiol, and max,MCH = 10.2 ± 2 nm for MCH, as summarized in Table 1. In the present case the reported standard deviations represent the distribution of over samples with different average morphology, and not, as usually, the error distribution of experimental measurement. In all experiments, always an higher shift was recorded after functionalization with C16 than after binding C12. The results indicate that the LSPR bands of the Au NIs on FTO show a good sensitivity to the presence of organic SAMs, that allows to disentangle between aliphatic and aromatic electronic structure. In fact, the different max,C12 = 9.8 ± 2.7 for C12 SAMs and max,terph = 21 nm ± 2.1 for terphenyl SAMs, are related to two SAMs having the same thickness [67,68] but different refractive index. Importantly, the small standard deviations reported in Table 1 show that the max are independent on the sample morphology (see Supporting information, Fig. S2). By comparing the data of Table 1, we observe that the max measured after stabilization is in the error of the max measured after SAM formation. These data indicate that the Au NIs/FTO samples do not need to undergo any stabilization process.
In order to confirm this conclusion we have compared the spectral shift measured after direct immersion in the thiol solution (max,d ), with that obtained after two processes, that is (i) stabilization in pure solvents (max,s ), and (ii) subsequent incubation in the thiol solutions (max,SAM ). The results show that max,d = max,s + max,SAM for a large number of samples (>70) with different initial max . These data represent a further confirmation that the Au NIs/FTO samples do not need to undergo any stabilization process in contrast to other plasmonic Au or Ag NIs reported in the literature [50].
Fig. 5. SP bands of bare Au NIs/FTO (solid line); after formation of dodecanthiol SAM (dashed line); after replacement of the dodecanthiol SAM by a terphenylthiol SAM (short dash line): after replacement of the terphenylthiol SAM by the original dodecanethiol SAM (dash dot line).
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Fig. 6. (A) LSPR spectra recorded for: bare Au NIs (solid line), after absorption of the thiolated ssDNA (short dash line), after immersion in a water solution of MCH (dash dot line), and after hybridization with the complementary strand (dash line). (B) LSPR spectra recorded for: bare Au NIs (solid line), after absorption of the thiolated ssDNA (short dash line), after immersion in a water solution of MCH (dash dot line), and after incubation with the non complementary strand (dash line).
3.4. Reversibility of the Au NIs LSPR Fig. 5 reports the LSPR spectra recorded for (i) a sample of bare Au NIs, (ii) after formation of a C12 SAM, (iii) after replacement of the C12 SAM by 4-terphenylthiol SAMs, and (iv) after a second replacement of the 4-terphenylthiol SAM with the original C12 SAM. These results show that the formation of dodecanethiol SAM yields a red shift of about 10 nm of max, in agreement with the results reported in Fig. 4A. By replacing the C12 SAM with a terphenyl SAM, a further red shift of 11 nm is observed. Under restoration of C12 SAM, the value of max goes back exactly to the original value by a blue shift of 11 nm. These results indicate a complete reversibility of the Au NIs/FTO plasmonic response under SAMs exchange and demonstrate that there is neither loss nor restructuration of the NIs gold atoms during the formation of different SAMs and their replacement. We note that by substituting the bulk refractive index for C12 ( = 1.46) and terphenyl ( = 1.55) into Eq. (2) (by using air = 1, dC12 = 1.62 nm, dTerphenyl = 1.76 nm, l = 17 nm [9,11]), we obtain respectively a sensitivity factor m = 116 nm/RIU for C12 and m = 221 nm/RIU for terphenyl. We observe that the order of magnitude of these values of m is in the range of that reported in literature for LSPR [9,11], even though they are not similar. The difference in m cannot be attributed to a change in morphology, as indicated by the complete reversibility of the system; it can be rather related to the fact that the refractive index of SAMs formed by these compounds are certainly different from the bulk, given the well know different architectures, organization and the packing of these molecules in the SAMs are very different. 3.5. Biorecognition by L-SPR of Au-NIs/FTO The sensitivity of the Au NIs LSPR to biorecognition reactions occurring at their surfaces has been tested by measuring max upon DNA hybridization. In the present case, we have exploited the hybridization of 19 bases oligonucleotides in particular a modified oligonucleotide (5 -thiolated–CAG TGA GGC GTG GCC AGG G-3 ) and the complementary (5 -CCC TGG CCA CGC CTC ACT G-3 ). The NIs were functionalized following a standard procedure [63–66] which consists of several steps (see Experimentals). Fig. 6A reports the LSPR bands recorded for each step. The max LSPR band recorded for bare Au NIs shifts of 13 nm after absorption of the ssDNA, remains almost constant, under insertion of MCH, and shifts of 11 nm upon hybridization. The results indicate that the LSPR of the Au NIs is sensitive to the presence of monolayers of ssDNA and more significantly, that the hybridization process induces a shift towards the red. Consider-
ing that this shift is the result of the hybridization of a ssDNA which is diluted into a MCH SAMs, we can conclude that the LSPR of these NIs shows a sensitivity towards the hybridization DNA. We have also performed in parallel a blank experiment following the literature protocol [63] where, instead to utilize the complementary strand of DNA, we have incubated the sample with a non complementary DNA sequence (5 -AAG TGC ATC GTG ATT CAT A-3 ) for 16 h in PBS solution. Fig. 6B reports the LSPR bands, the resulted of the several incubations. Afterwards to the binding of ssDNA to the Au NIs surface we have a red shift. The insertion of MCH into the ssDNA SAM does not lead to any shift of the plasmon band. When the sample is incubated with a non complementary strand, the LSPR band exhibits a red shift of only 1 nm. We can affirm that the biosensor could discern between a complementary and not complementary strand of DNA. This proves the specificity of the sensor to the binding of complementary DNA strand, an important issue for, e.g. the detection of mismatches in DNA strands in the analysis of genetic mutations. 4. Conclusions In general commercial sensors are characterized by low cost, simple fabrication, high sensitivity and reversibility. The LSPR spectroscopy performed with Au NIs supported on transparent substrates is a convenient technique to monitor molecular interactions, provided two mandatory conditions: (i) high “stability” of the Au NIs in different media, and (ii) a good “sensitivity” of their LSPR to molecular interactions occurring at the surface. In this paper we have shown that polydisperse Au nanoislands deposited on FTO surfaces can be a convenient plasmonic material. The fabrication of Au nanoislands consists on a simple standard thermal evaporation of gold on commercial FTO surfaces followed by a mild annealing process. The Au NIs evaporated on FTO are highly “stable” in different organic and more importantly in aqueous solvents. We believe that the high stability of the gold deposits is related to the roughness of commercially available FTO. The LSPR of the Au NIs shows a good “sensitivity” to absorption of different organic SAMs, and are able to disentangle between aliphatic or aromatic SAMs. The simple protocol of fabrication yields samples of Au NIs of different morphology which is reflected by LSPR bands with maximum energy max spanning of 30 nm. Significantly, all the fabricated samples, formed by polydisperse NIs, exhibit the same “stability” and “sensitivity”. Therefore the samples (i) can been used directly after the fabrication, without stabilization processes and (ii) the recorded shift in max is related exclusively to the change in refrac-
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tive index of the SAMs, without any contribution to restructuration processes. Interestingly, the LSPR of the samples Au NIs/FTO recovers the same values after multiple SAM replacements, indicating (i) high stability of the metal nanoislands and (ii) the reversibility of this plasmonic materials as molecular sensors. In addition the red shift of max of the LSPR band recorded under hybridization of single DNA strand anchored to their surface by the complementary strand indicates that this plasmonic material is very sensitive to bio-molecular interactions occurring at their surface. Further experiments will be required to verify whether this approach is suitable to determine hybridization of single DNA strand to gene sequences generated by polymerase-chain reaction. These results encourage further studies aimed at determining whether this strategy is important to detect point mutations. Both these issues are relevant to possible application of this novel strategy to biomedicine. All the characteristics of the system Au NIs/FTO: (i) inexpensive fabrication (ii) high stability, (iii) good sensitivity to biomolecular interactions, and (iv) easy monitoring of the optical properties—meet the requirements of plasmonic materials for the fabrication of convenient, versatile systems for bio-organic sensors based on T-LSPR spectroscopy. Acknowledgments We thank Prof. Luca Benini (Department of Electronics and Informatics, University of Bologna) and Dr. Carlotta Guiducci (EPFL, Lausanne), for helpful discussion. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.snb.2010.12.008. References [1] V. Silin, A. Plant, Trends Biotechnol. 15 (9) (1997) 353–359. [2] A. Szabo, L. Stolz, R. Granzow, Curr. Opin. Struct. Biol. 5 (1995) 699–705. [3] U. Johnsson, L. Fagerstam, B. Ivarsson, R. Karlsson, K. Lundh, S. Liif, B. Persson, H. Roos, I. Ronnberg, S. Sjolander, E. Stenberg, R. Stlhlberg, C. Urbaniczky, H. Ostlin, M. Malmqvist, BioTechniques 11 (1991) 620–627. [4] G. Feriotto, G. Breveglieri, S. Gardenghi, G. Carandina, R. Gambari, Mol. Diagn. 8 (1) (2004) 33–41. [5] G. Feriotto, G. Breveglieri, A. Finotti, S. Gardenghi, R. Gambari, Lab. Invest. 84 (2004) 796–803. [6] Y.J. Li, J. Xiang, F. Zhou, Plasmonics 2 (2007) 79–87. [7] U. Kreibig, M. Vollmer, Optical Properties of Metal Clusters, Springer, Berlin, 1995. [8] L.S. Jung, C.T. Campbell, T.M. Chinowsky, M.N. Mar, S.S. Yee, Langmuir 14 (1988) 5636–5648. [9] A.J. Haes, S. Zou, G.C. Schatz, R.P. Van Duyne, J. Phys. Chem. B 108 (2004) 109–116. [10] K.A. Willets, R.P. Van Duyne, Annu. Rev. Phys. Chem. 58 (2008) 267–297. [11] A.J. Haes, R.P. Van Duyne, Anal. Bioanal. Chem. 379 (2004) 920–930. [12] C.L. Haynes, R.P. Van Duyne, J. Phys. Chem. B 105 (24) (2001) 5599–5611. [13] A.J. Haes, D.A. Stuart, S. Nie, R.P. Van Duyne, J. Fluoresc. 14 (4) (2004) 355–367. [14] M.E. Stewart, C.R. Anderton, L.B. Thompson, J. Maria, S.K. Gray, J.A. Rogers, R.G. Nuzzo, Chem. Rev. 108 (2008) 494–521. [15] B. Sepulveda, P.C. Angelomé, L.M. Lechuga, L.M. Liz-Marzàn, Nano Today 4 (3) (2009) 244–251. [16] R. Elghanian, J.J. Storhoff, R.C. Mucic, R.L. Letsinger, C.A. Mirkin, Science 277 (1997) 1078–1081. [17] M. Brust, M. Walker, D. Bethell, D.J. Schiffrin, R.J. Whyman, Chem. Soc. Chem. Commun. 7 (1994) 801–802. [18] G. Frens, Colloid Polym. Sci. 250 (1972) 736–741. [19] S.D. Perrault, W.C.W. Chan, J. Am. Chem. Soc. 131 (47) (2009) 17042–17043. [20] N. Nath, A. Chilkoti, Anal. Chem. 74 (2002) 504–509. [21] N. Nath, A. Chilkoti, Anal. Chem. 76 (2004) 5370–5378. [22] C.R. Yonzon, E. Jeoung, S. Zou, G.C. Schatz, M. Mrksich, R.P. Van Duyne, J. Am. Chem. Soc. 126 (2004) 12669–12676. [23] A. Haes, L. Chang, W.L. Klein, R.P. Van Duyne, J. Am. Chem. Soc. 127 (2005) 2264–2271.
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Biographies Vera Cantale, after she graduated in Cellular and Molecular Biology at the University of Ferrara, she is currently Ph.D. student at the Department of Chemistry.
Please cite this article in press as: V. Cantale, et al., Gold nano-islands on FTO as plasmonic nanostructures for biosensors, Sens. Actuators B: Chem. (2011), doi:10.1016/j.snb.2010.12.008
G Model SNB-12753; 8
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Her scientific activity involves the development of nanostrucutred materials for biorecongition and molecular electronics. Felice Carlo Simeone graduated in chemistry at the University of Florence. He received his Ph.D. in electrochemistry at the University of Ulm (D) under the supervision of Prof. D.M. Kolb. His research interests are in scanning probe microscopy, in particular scanning tunneling microscopy under electrochemical control, electrochemistry, and integration of electrochemistry with unconventional lithographies. His current activity at the University of Ferrara, besides the exploitation of plasmon resonance for bio-recognition, involves the use of scanning probes for measurements of single molecules conductivity. Roberto Gambari is full professor of Biochemistry at the University of Ferrara. He is an active member of the American Society for Biochemistry and Molecular Biology. Coordinator of several research projects and networks including “Target Project
Genetic Engineering”, “Use of PNA, ribozymes and peptides for the study of gene functions and possible diagnostic and therapeutic applications” and “Applications of a dielectrophoresis-based Lab-on-a-chip to diagnosis and drug research and development”. Partner of the European Projects ITHANET and COCHISE. He is author of more that 220 papers and of more than 8 patents. Maria Anita Rampi is Professor of Chemistry at the University of Ferrara, where she teaches Inorganic Chemistry and Chemistry of Nanostructured Materials. Her work has focused on (i) fast transient spectroscopy, photochemistry and photophysics of supramolecular systems, (ii) photoinduced energy and electron transfer processes in supramolecular systems both in solution and in self assembled monolayers on solid surfaces, and (iii) charge transfer processes in molecular junctions. For several years she was visiting professor at Max Plank Institute in Göttingen, Germany, and is still carrying a collaboration at Harvard University, Cambridge, USA.
Please cite this article in press as: V. Cantale, et al., Gold nano-islands on FTO as plasmonic nanostructures for biosensors, Sens. Actuators B: Chem. (2011), doi:10.1016/j.snb.2010.12.008
