Nano structures and polymers: Emerging nanocomposites for plasmonic resonance transducers

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Nano Structures and Polymers: emerging nanocomposites for plasmonic resonance transducers S. Scarano , M.G. Manera , A. Colombelli , M. Minunni , R. Rella PII: DOI: Reference:

S0040-6090(20)30074-2 https://doi.org/10.1016/j.tsf.2020.137859 TSF 137859

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Thin Solid Films

Received date: Revised date: Accepted date:

7 June 2019 10 February 2020 11 February 2020

Please cite this article as: S. Scarano , M.G. Manera , A. Colombelli , M. Minunni , R. Rella , Nano Structures and Polymers: emerging nanocomposites for plasmonic resonance transducers, Thin Solid Films (2020), doi: https://doi.org/10.1016/j.tsf.2020.137859

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Highlights   

Synthesis of gold nanoparticles embedded in Polydimethylsiloxane (PDMS) films. Plasmonic properties of gold embedded in PDMS for sensing applications. Modelling to describe optical and functional properties of gold nanoparticles embedded in PDMS

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Nano Structures and Polymers: emerging nanocomposites for plasmonic resonance transducers

S. Scarano1, M.G. Manera2, A. Colombelli2, M. Minunni1, R. Rella2* 1

Department of Chemistry ‘Ugo Schiff’ and CSGI, University of Florence, via della Lastruccia 3-13, Sesto Fiorentino, 50019, Firenze, Italy

2

CNR-IMM-Institute for Microelectronic and Microsystems, Unit of Lecce, Via per Monteroni, 73100 Lecce, Italy

*Corresponding authors: [email protected]

Abstract Herein, we report polydimethylsiloxane (PDMS) based optical sensing platforms and describe how they can be tuned, using nanomaterials, to exhibit plasmonic properties that can be exploited for sensing applications. The platforms include a colorimetric-based sensor realized on gold nanoparticles grown on PDMS films. A theoretical model based on finite element analysis was developed in order to describe the optical behavior and the functional properties of these plasmonic transducers. We demonstrate the ability of these systems to detect even small changes in the refractive index of the external environment, predicting the evolution of their sensing performance. We have experimentally explored the modulation of the plasmonic properties of these platforms by monitoring the shift of the typical localized surface plasmon resonance peak in order to explore potential plasmon-enhanced functionality.

Keywords: Plasmonic nanostructures, Polydimethylsiloxane; Gold; Nanoparticle array; Optical sensors

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1.

Introduction

The race towards low cost, miniaturized, disposable, and all-integrated biochips for point-ofcare (POC) testing devices of new generation is started [1-2]. These ideal POC tests should combine such features to high analytical performance, i.e. sensitivity, specificity and reproducibility, while remaining user friendly and robust tools to be used in resource limited environments or emergency. In this framework, nanobiosensors based on optofluidics are displaying great perspectives [3-5]. Nanobiosensors generally refer to sensing devices able to exploit fundamental nanoscopic effects in order to detect specific biomolecular interactions, even in label free manner as in the case of optical-based nanobiosensors [6,7]. Application of localized surface plasmon resonance (LSPR) to optical-based nanobiosensors is rapidly growing, resulting in impressive number of publications and diversified research fields. Emerged as alternative to traditional SPR biosensing, LSPR-based approach exploits plasmonic properties of metallic or semi-metallic nanomaterials under optical interrogation [8-12]. The near field elicited is strongly dependent on the inherent features of the nanomaterial employed, i.e. metal type, size and aspect ratio of nanoparticles (NPs), their spatial distribution and the surrounding environment [13-16]. Advantages of LSPR-based bioassays are undoubtedly sensitivity, selectivity, multiplexed format and the simplicity of the optical arrangement required. Analytical information [17-19], can easily be collected by using simple optical setup based on transmittance or reflectance configuration [20,21]. In fact, LSPR of metal nanoparticles is easily observed by measuring changes in their adsorption spectrum, both in terms of wavelength shift and absorbance intensity. As a whole, LSPR-point of care (POC) devices are considered one of the most promising platforms in this sense, also by considering miniaturization possibilities accessible with portable spectrophotometers [22-25]. The first generation of LSPR bioassays was essentially based 3

on aggregation effects of NPs in solution elicited by target analytes under investigation, either directly or not [26,27]. However, looking for new sensing platforms exploiting LSPR on all-integrated, flexible and low cost POC devices, innovative approaches to fabricate NPs directly in contact to transparent substrates (silica NPs, glass, Indium Tin Oxide, polydimethylsiloxane (PDMS) etc.) have emerged. Among possible substrates suitable for NPs assembly, PDMS combines its excellent optical transparency in the visible range to the advantages of microfabrication. PDMS-based microfluidics is therefore still the widest used technology in the field of optofluidic applied to sensing and biosensing [28,29]. Lately, after the discovery of spontaneous reduction of Au(III) or Ag(I) ions by applying a simple wetting process based on the immersion of cured PDMS films into metal salt solutions, several papers start dealing with the fabrication of these composite substrates for (bio)sensing purposes appeared [30-35], eventually integrated to microfluidic design [36,37]. Metallic NPs dispersed in PDMS have been also obtained by mixing metal salts solutions with PDMS, before polymerization [38-40]. The NPs formation, observed at PDMS surface occurring without the need of additional reducing/capping agents (i.e. gold and silver NPs, hereafter indicated Au(Ag)NPs embedded PDMS surface), and is attributable to the activity of residual curing agent present in PDMS matrix itself after partial polymerization. A possible mechanism has been first proposed by Zhang et al. in 2008 [34]. Despite some successful attempts in this direction, the one-step growth of Au(Ag)NPs embedded PDMS surface and their investigation by LSPR, remains limited to few works and in some case, with unsatisfactory results, because the obtained absorbance spectra of such nanomaterials embedded PDMS surface display wide and weak resonance bands with limited sensitivity, not adequate for sensitive analytical applications. In particular, two limiting factors can be identified i.e. the intrinsic RIS (refractive index sensitivity) of spherical AuNPs grown at PDMS surface, and the partial embedding of the nanostructures in PDMS causing an important RIS decrease [41,42]. The polymer has to 4

provide an appropriate environment for the metal nanoparticles in order to be able to interact with the analyte (i.e. biomolecules) and this ability is related to their distribution in its matrix. In this paper some promising ways to increase the mobility of Au nanoparticles are reported. The goal is to achieve high concentration of metal NPs on the surface of the PDMS film, to make them accessible to the surrounding environment containing the biomolecule of interest. Furthermore, AuNP-embedded PDMS surfaces transducers characterized by a final moderate refractive index sensitivity (RIS~70 nm RIU−1) should be realized avoiding expensive and time-consuming post-processing treatments such as thermal annealing and/or swelling/shrinking cycles. This work represents an easy, low cost, fast and reproducible approach in fabrication of AuNP-embedded PDMS chips for (bio)sensing purposes. Stable, spectrally well-defined, and efficiently distributed gold nanoparticles, ranging between 10 and 30 nm (depending to the synthesis parameters) on partially cured PDMS films were obtained and tested. In particular the attention was focused on the effect on NPs formation by Au(III) solution concentration and time of growth, explored by scanning electron microscopy (SEM) and localized surface plasmon resonance (LSPR) measurements. Moreover, in order to investigate the optical behavior and the functional properties of these innovative transducers, theoretical analysis and numerical simulations have been performed. In particular, the Finite Element Method was used to predict the optical and functional properties of metal NPs synthetized on PDMS substrate. The optical absorption spectra of the samples have been calculated, providing a deep physical insight of the analyzed system and in agreement with experimental results. As proof of concept, the sensing performances of these systems were investigated, performing a theoretical evaluation of their bulk sensitivities to changes of the optical properties (refractive index) of the environment. Furthermore, the possible partial embedding of AuNPs inside the polymeric substrate was taken in account, in order to investigate its influence on their optical and functional properties, and achieve a deeper 5

understanding of the real sample structure. Numerical results found a confirmation in experimental refractometric measurements realized in the laboratory. 2. 2.1

Materials and methods Materials

Ultrapure deionized water was employed throughout all experiments. A SYLGARD184 Silicone Elastomer kit for PDMS fabrication was purchased from Dow Corning (Midland, MI, USA). All glass labware was washed with aqua regia before use. Tetra-chloroauric acid trihydrate, HAuCl4·3H2O and 11-mercaptoundecanoic acid were purchased from SigmaAldrich (Milan, Italy). All other chemicals employed in this work were reagent grade and commercially available.

2.2

Fabrication of the in-situ synthesis of Au-PDMS nanocomposite

AuNP-embedded PDMS surfaces composite arrays were prepared in a two-steps procedure as follows: PDMS monomer and the curing agent were firstly mixed in a proportion of 10:1 (w:w). The PDMS compound was well mixed and then degassed for a duration of 20 minutes. Then known weights of PDMS 1.00 and 5.00g, respectively, were cast in 60 and 90mm diameter Petri dishes, and let to polymerize at 25°C for 24 h. Aqueous solutions of tetrachloroauric acid with different concentrations were prepared, ranging from 1 mM to 50 mM just before use. HAuCl4 volumes 5L were dropped onto PDMS films in different array shapes, varying gold chloride concentration, size of the spots, growth time. The substrates were incubated for increasing time intervals (1÷96 hours) at 25°C. In order to avoid evaporation of HAuCl4 drops, PDMS disks were kept in sealed humidity chambers. Next, the realized chips were carefully washed with deionized water, dried under nitrogen flow, and stored at 4°C until use.

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2.3

Morphological and optical characterization

The morphology and size of the AuNP-embedded PDMS surfaces arrays synthetized onto the surface of the PDMS were characterized by Scanning Electron Microscopy, (ZeissIGMA FE-SEM, Carl Zeiss Microscopy GmbH, Germany).

2.4

Optical characterization

The optical absorption of the gold nanostructures was characterized by a Cary500 UVVisible spectrophotometer. Investigation on the nanoparticle optical properties by monitoring the LSPR peak as a function of the growth parameters was performed by measuring the absorption spectra in the UV-Vis spectral range. In order to characterize the plasmonic transducers in liquid phase, a compact optical fiber system was used. This setup was equipped by a tungsten halogen light source (LS-1, wavelength range 360–2000 nm), a portable spectrophotometer (USB2000 UV–Vis, wavelength ranging between 250–1100nm), and a bifurcated optical fiber probes (R-400-7 UV–Vis, fiber core diameter = 400 m, wavelength range = 250–850 nm). White light emerging from the optical fiber was perpendicularly collimated onto the AuNP-embedded PDMS surfaces spot chip. The same bundle was used as a detection probe. When the experiments require the use of fluids, the fiber tip was entirely immersed in the liquid, in order to minimize multiple light reflection onto the liquid surface. All the spectra were acquired from 400 to 800 nm at room temperature. The experimental set-up allows acquiring the dynamic responses at different fixed spectral range. In our case the sensing response was monitored following the shift in the typical LSPR peak of the gold nanoparticles. Controlled room temperature conditions during all the sensing experiments were guaranteed. The ability of AuNP-embedded PDMS surfaces transducers to act as refractive index sensors was investigated by recording LSPR spectra in solutions with increasing refractive indexes. In particular, the sensitivity of the proposed transducer was 7

evaluated by recording the LSPR spectra in air (n = 1.00), water (n = 1.33), pure glycerol (n = 1.47), and several water/glycerol solutions with increasing glycerol concentrations. 2.5

Modeling and simulation

The radio frequency Module of COMSOL Multiphysics was used to calculate the optical response of metal nanoparticles synthetized on polymeric substrates. In particular, planar distributions of gold NPs on PDMS films were considered, taking into account the morphological characteristics of the AuNP-embedded PDMS surfaces composite system. In order to understand how the LSPR conditions can be affected by the optical properties of the local environment, several key parameters have been explored in the model. The ability of the system to detect even small changes in the refractive index of the external environment was investigated, evaluating the bulk sensitivity of the plasmonic transducer. Also, the possible embedding of AuNPs inside the polymeric substrate was taken in account, in order to investigate the influence on the optical properties of metal nanostructures and predict the variation of their sensing performances.

3. Results and discussion Development of cheap, sensitive, AuNP-embedded PDMS surfaces transducers was our first aim, possibly avoiding tedious post-processing treatments for detecting biomolecules which have to get in close contact to the sensing surface i.e. AuNPs. To that some preliminary considerations have to be done. Because of PDMS hydrophobicity, it is difficult for hydrophilic biomolecules to penetrate into its the inner domains, but by varying the crosslinker concentration, the free volume of the film can be changed facilitating their penetration [43]. Nowadays, is well known how LSPR based sensors enable the detection of very thin layer of adsorbate molecules, owing to the great enhancement of the local electric field near the particle surface when the resonance conditions are satisfied. The penetration depth of the 8

resonant electric field for LSPR sensors, is strictly related to the geometrical (size, shape, distribution) and compositional properties of nanoparticles [10]. However, this characteristic length does not exceed few tens of nanometers. Therefore, if gold NPs are deeply embedded into the polymer substrate, their sensing volume can be too far from the target biomolecules. Furthermore, if the interaction between metal nanoparticles and the surrounding polymer chains is too strong, their mobility may be restricted, resulting with an inconvenient entanglement of the particles in the polymer network. Since the biosensing properties are determined by the spatial distribution of the Au nanoparticles, a key point is in the preparation and optimization of the plasmonic transducers. The best solution is to increase the mobility of Au nanoparticles in order to concentrate them at the PDMS interface just in contact with the surrounding environment that contains the bio-molecule of interest. We describe the development of sensitive AuNPembedded PDMS surfaces by avoiding such a cost and time-consuming procedures as such as thermal annealing and/or swelling/shrinking cycles reported for moderate refractive index sensitivity (RIS ~ 70 nm RIU−1).

3.1

Influence of Au(III) concentration on AuNP-embedded PDMS surface and optical behavior of the LSPR chip

The relation between gold (III) precursor concentration and AuNPs obtained on PDMS was serially investigated by varying this parameter from 1 mM to 50 mM. In fact, preliminary tests in array format and recent results obtained in similar conditions [35] demonstrated that Au(III) concentration is a key parameter to tune the final morphology of these nanocomposites. In particular, as for the solution synthesis of AuNPs, the molar ratio between the reducing agent and Au(III) greatly influences the final nanoparticles population. This aspect becomes more intriguing in the case of PDMS as reducing substrate for Au(III), since the formation of NPs at the polymer surface is attributed to the presence of residual curing agent in the PDMS matrix after the polymerization process [34,41-42]. Therefore, slightly 9

different preparations of PDMS substrates may lead to significant variability of AuNPs embedded PDMS surfaces, even upon the same Au(III) concentration and growth time applied. In the perspective of exploiting this affordable and low-cost approach to fabricate LSPR-active composite nanomaterials, here we optimized an advanced and array-based format that minimizes inter-samples variability. As reported in Fig. 1A, the fabrication of small (1÷5 mm2 diameter) AuNP-embedded PDMS surfaces spots can be suitably arrayed on Petri dishes by drop casting on pre-polymerized PDMS films. 5 L Au(III) deposition with the desired spatial resolution allows to obtain tens of LSPR-active transducers at different Au(III) concentration and optical features (depending on the Au(III) and/or growth time). Afterward, the transducers can be either interrogated as they are, or conveniently cut (singularly or grouped) and mounted into customized optofluidic set up, able to scan the array in a fast and suitable way to collect plasmon profiles (Fig. 1B, left). Following this latter format, we evidenced a dependence of the plasmon features from starting Au(III) concentration (Fig. 1B, right). In particular, as reported in Fig. 2A, typical progressive red shift is evoked for equivalent spots array by Au(III) concentration decrease, from (5342)nm to (5591)nm, with a marked change for samples prepared with Au(III) below 12.5 mM. Typically, the red shift of a plasmon band is informative about nanoparticles size, indicating the gradual increase of their diameter. At the same time, the absorbance intensity variation is informative about AuNPs density on the substrate and a progressive decrease of the plasmon depth is observed down to 12.5 mM Au(III). Afterward, a sudden increase of intensity and broadening of the band is recorded for the last two concentration points (6.250 and 3.125 mM). This likely suggests that the strong decrease of Au(III) concentration favors the formation of larger and denser AuNPs distribution at the polymer surface, as also reported elsewhere [34,35].

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3.2

Morphology of AuNP-embedded PDMS surfaces by SEM analysis

The morphology of the AuNP-embedded PDMS surfaces plasmonic transducers was first investigated by plan view SEM analyses. Fig. 3 reports an overview of the gold NPs distribution inside the PDMS matrix under different growth conditions. Highly dense and disordered synthetized gold nanoparticles distribution can be observed. The different colored spots, evidenced in Fig. 3A, are corresponding to different Au(III) concentrations starting from 50 mM down to 3.125 mM. By decreasing the Au(III) concentration, an increase in the gold nanoparticles size is evidenced. SEM images (Fig. 3B-D) confirm this finding where size dimension ranging between 10÷15 nm are obtained with Au(III) concentration between 50 mM and 12.5 mM. On the contrary, SEM images reported in Fig. 3E and F, relative to lower Au(III) concentration, showed a marked different morphology of the nanostructured surface. In detail, not only size dimension of AuNPs are larger than 20 nm, but the surface density is so high that can be assimilated to a packed nanostructured film. Cracks on the surface underlined this feature that fits well with the observed optical behavior previously discussed for these samples, i.e. the remarkable red shift and broadening of the LSPR absorption band. Table I shows statistical results obtained by the analysis of the SEM images related to AuNP-embedded PDMS surfaces plasmonic transducers performed onto 5 equivalent spot array obtained with Au(III) different concentrations. As reported in the literature, particles of size ranging between 10 and 15 nm typically generate optical absorption effects; while, if the size of the particles increases, the scattering effect is dominant. The optical analysis of our samples shows that both effects are present. In particular, the intensity of the plasmonic peak varies for particles below 15 nm when exposed different refractive indexes solutions. On the contrary, a shift in wavelength maximum is obtained for nanoparticles greater than 20 nm.

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3.3

Optical characterization and refractometric measurements

Effect of Au(III) concentration. The ability of AuNP-embedded PDMS surfaces transducers to act as refractive index sensors was further investigated by recording LSPR spectra in solutions with increasing refractive indexes by testing glycerol/water solutions at different ratio. The results in Figs. 4a and 4b are obtained onto AuNP-embedded PDMS surfaces transducers synthetized at different Au(III) concentrations. The results put in evidence both a variation in the intensity of the LSPR peak and a red shift, due to the variation in the chemical environment close to the surface of gold NPs. In particular, the variation in the intensity of the peak is evidenced in the case of Au nanoparticles size ranging around 10 nm as estimated by SEM image and confirm the results obtained by Scarano et al. [35]. On the contrary, for particles size upper to 20 nm the behavior shows a red shift and an increasing in the intensity. In order to better define the sensing performance of our materials, we also consider the figure of merit (FOM), defined as sensitivity divided by the LSPR line [38]. This parameter is widely used to characterize nanostructures sensing potential independently of their shape or size. For our AuNP-embedded PDMS surfaces plasmonic transducers we find FOM values and sensitivities relative to the different morphology of sensing layer. In particular, in Table II the sensitivity and FOM values relative to the different

starting

Au(III) concentrations are reported. ΔλLSPR/RIU, ΔextLSPR/RIU, and relative FOMext and FOMλ of the analyzed transducers are shown. The sensing performances are comparable to those obtained by NPs which exploit the effects of very exotic shapes and, in most cases, ordered spatial distribution. Both of these properties require many technological efforts for a fine control of the fabricated structures [45]. The results reported in Table II, underline the effect of the nanoparticles size onto the refractive index sensitivity. Different Au(III) concentrations give rise to different size of the Au nanoparticles inside the PDMS during the synthesis process. The overall result obtained stresses that, contrarily to what one may suppose, great benefit on both the RIS (wavelength and absorbance sensitivity) may be 12

obtained by working with low Au(III) concentrations. In particular, ΔλLSPR/RIU shows about a four-folds enhancement from 50 to 3.125 mM, together with a slight increase of Δ extLSPR/RIU.

This is evident by observing the FOM trends, which significantly improve for

samples obtained within 6.25 and 3.125 mM Au(III). Effect of time growth. The time growth parameter was explored on two different AuNPembedded PDMS surfaces substrates in terms of chip sensitivity. To this aim, the highest (50 mM) and the lowest (1 mM) Au(III) concentrations, able to produce gold nanostructures at PDMS surface, were tested. The nano-particles growth was performed for 24, 48, 72, and 96 hours, respectively. By increasing the growth time, a positive effect on both wavelength and absorbance sensitivities is observed (Table III). A modeling approach has been designed to flank the experimental evidences, aiming to develop an in silico approach for AuNP-embedded PDMS surfaces chips optimization.

3.4

Modelling and simulation results

Owing to the spherical symmetry of the structures, a 2D simulation has been developed to significantly reduce the computational cost. The simulation domain, reported in Fig. 5a, is composed by different layers with optimized thicknesses, and represents the unit cell of the analyzed system. Starting from the top of the geometry, the first domain represents the PDMS substrate, on which the AuNPs have been synthetized. A linear array of hemispherical particles with an average diameter of 20 nm has been considered. This NPs size represents AuNP-embedded PDMS surfaces chips which experimentally showed the best sensitivity, eventually obtained with the lowest Au(III) concentration. A thin PDMS layer, characterized by increasing thickness, was inserted in the model in order to simulate the partial or complete embedding of the structures inside the polymer. The last domain at the bottom represents the external environment. In the model, a free triangular mesh has been used for all computational domains with a local refinement near the particles 13

regions. All the materials introduced in the model are characterized by their complex and frequency-dependent dielectric functions (or refractive index). In the visible spectral range, nanostructured gold can be modeled as having a complex valued dielectric constant, with real and imaginary components. In the model, these components are described by an interpolated version of the often-used experimental data from Johnson and Christy [48]. The polymeric substrate, realized with a specific weight ratio (10:1) of the base to the curing agent, was characterized by a wavelength dependent refractive index derived from Cai et al. [49]. In this model, the LSPR activation was performed using the wavelength modulation technique, simulating an incident light beam coming from the top of the geometry. Port boundary conditions were set for both the upper and lower edges of the simulation domain, in order to calculate the reflection and transmission coefficients of the system. In order to simulate an infinite array of plasmonic NPs, Floquet periodic boundary conditions were set on the sides of the unit cell. The ability of the system to act as a sensor, detecting even small variations in the RI of the external environment was numerically investigated by monitoring the spectral shift of the calculated LSPR absorption band. In order to evaluate the bulk sensitivity of the AuNP-embedded PDMS surfaces as transducer, the exposure of the system to solutions characterized by increasing refractive indexes was simulated. Furthermore, the influence of a few nanometers thick PDMS layer on the sensing performances of the system was considered, in order to demonstrate the presence of free metal NPs available for possible chemical binding events. The plasmonic properties of the AuNP-embedded PDMS surfaces system were theoretically analyzed, considering thus the possible influence of a thin layer of PDMS on the optical response of gold nanoparticles. The LSPR activation was simulated using the wavelength modulation technique in transmission configuration, with an incident angle  = 0° and multiple wavelength in the visible spectral range (= 500˗700 nm). 14

In Figure 5 the simulation output is shown. Owing to the excitation of the localized surface plasmon resonance, the normalized absorbance signal of free gold nanostructures, exhibits a pronounced peak around  543 nm. Owing to the NPs size selected in the model, this result can be compared with the optical absorbance of AuNP-embedded PDMS surfaces chips obtained with lower Au(III) concentration. In particular, a comparison with the normalized absorbance spectra of the sample prepared with Au(III) concentration of 6.25 mM is reported in Figure 5. The small differences between simulated and experimental absorbance spectra may be attributed to the geometrical approximation adopted in the simulation model. All the metal NPs have been considered homogeneous with a specific diameter of 20 nm, however the optical response of real sample could easily be affected by dimension inhomogeneity. The PDMS layer influence was considered to take in account the possible NPs embedding in polymer. A layer characterized by increasing thickness (from 1 to 20 nm) was simulated. As expected, the presence of a PDMS layer affects the optical response of the system, inducing an evident redshift of the resonance conditions. The inset reported in Fig. 5, describes the progressive red shift of the LSPR absorbance peak as function of the PDMS thickness. The numerical results relative to a distribution of free metal nanostructures present features very similar to those experimentally found, thus suggesting the presence of uncovered plasmonic nanostructures in the AuNP-embedded PDMS surfaces system. However, in order to have a deeper understanding of the real sample structure, a numerical evaluation of the bulk sensitivity of this system was performed, considering three different configurations. Free metal nanostructures as well as AuNPs covered by a 3 and 5 nm thick PDMS layer, were analyzed. For each configuration, the ability of the system to detect even small changes in the refractive index of the environment was investigated by monitoring the variations of the relative absorbance spectra. As an example, the effect of the high-index dielectric surrounding on the optical response of free AuNPs is reported in Fig. 6, where an evident 15

redshift of the absorbance signal toward higher wavelengths as well as a slight increase of its intensity, can be noticed. In Fig. 7, the calibration curves relative to the three analyzed configurations have been reported. Here the wavelength shift of the LSPR absorbance peaks relative to their spectral position in water (RI=1.33) are reported as function of the increasing refractive index of the external environment. The optical response of the three configurations exhibits a linear dependence on the RI of the environment, confirming the ability of these systems to detect even small RI variations. From these curves, a quantitative information about the refractive-index sensitivity can be obtained, considering the wavelength shift of the signal per RI unit (nm RIU-1). As expected, the free metal nanostructures exhibit a higher bulk sensitivity with respect to the PDMS covered AuNPs. The modeling supports that the highest sensitivity is obtained in case of free AuNPs, while it markedly decreases in the presence of PDMS layers. Moreover, this study is in agreement with the experimental results here reported, thus suggesting the presence on the real sample of free metal nanostructures when working with very low metal precursor i.e. Au(III) concentration. In other words, we here demonstrated that simple, low cost, sensitive and post-processing free LSPR active gold nanostructures can be obtained on PDMS, by playing with Au(III) concentration and growth time. The increased sensitivity experimentally observed was further supported by modeling studies and coherent findings were obtained. Moreover, in order to confirm the reproducibility of the optical behavior inside a single spot and between equivalent spots array, fig. 8 and fig. 9 demonstrate the homogeneity of the plasmon resonance wavelength position registered in different point of the spot and between the equivalent spots respectively. The figures report a typical behavior obtained by using 1mM Au(III) concentration with a LSPR=543nm. As regard 5mM Au(III and 10mM Au(III) concentration, a LSPR=540nm and LSPR=538nm respectively were obtained.

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4. Conclusions This paper reports a method for in situ synthesis of Au nanoparticles in free standing PDMS on the basis of reductive properties of the cross-linking agent of PDMS. The proposed method is an environmentally safe synthesis method circumventing the need of using preformed nanoparticles and without requiring any additional reducing/stabilizing agents. The region where the interacting gold nanoparticles are distributed on the surface of the polymer but partially embedded in the bulk, the size of the nanoparticles, as well as the color of the free-standing films, can be simply controlled by optimizing the Au(III) concentration. Modeling of the nanocomposite AuNP-embedded PDMS surfaces transducer and comparison with the morphological, optical and functional features in terms of LSPR sensitivity characterization in liquid phase are here proposed. As whole, we demonstrated the good agreement between experimental characterization of AuNP-embedded PDMS surfaces and modeling approach in describing the sensitivity of this nanocomposite material. This represents an important and encouraging achievement for innovative (bio)sensing applications at the metal/dielectric interface. LSPR-active AuNPs can be obtained in situ simply by drop casting on PDMS films in array format, by employing single reagent at low concentration, room temperature, and static conditions. Moreover, the LSPR chips here realized can be easily coupled to microfluidic systems for a variety of applications from affinity-based sensing, i.e. from DNA- and immuno-based assays to other biochemical analyses on microchips.

Acknowledgment This work was supported by a grant from Ministry of Education, University and Research for the scientific program SIR2014 Scientific Independence of young Researchers (RBSI1455LK). The authors thank G. Montagna for technical support and LSPR optical setup preparation. 17

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Table I: Mean values of the major axis of gold nanoparticles measured by SEM images and their dominant optical behavior [Au(III)] From 50 to 12.5 Below 12.5 Concentration (mM) > 20 nm Average NPs formation of Au NPs Dimension 10÷15 nm cluster aggregates Optical Response

Absorption

Scattering

Table II: Summary of the absorption sensitivity ΔλLSPR/RIU, scattering sensitivity ΔextLSPR/RIU and relative figure of merit FOM ext and FOMλ for AuNPs/PDMS transducers realized by different Au(III) concentration and incubated at 25°C for 96h Au(III) concentration

50 mM 25 mM 12.5 mM 6.25 mM 3.125 mM

SensLSPR

SensextLSPR

FOMλ

FOMext

(nm/RIU)

(a.u./RIU)

21

0.6

0.4

1.0

24

0.6

0.4

1.2

31

0.5

0.6

1.1

65

0.8

1.5

1.7

76

0.7

1.7

1.6

Table III: Summary of the absorption sensitivity λLSPR/RIU and scattering sensitivity extLSPR/RIU for AuNPs/PDMS transducers realized by lower and higher Au(III) concentration and incubated at 25°C for different time windows. Incubation temperature time windows 24 h 48 h 72 h 96 h [Au(III)] SensλLSPR SensλLSPR SensλLSPR SensλLSPR Concentration (nm/RIU) (nm/RIU) (nm/RIU) (nm/RIU) 4 14 27 60 1mM 1 3 9 20 50mM SensextLSPR SensextLSPR SensextLSPR SensextLSPR (a.u./RIU) (a.u./RIU) (a.u./RIU) (a.u./RIU) not evaluable 0.2 0.4 0.8 1mM not evaluable 0.3 0.5 0.5 50mM

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Figure Captions Fig. 1. A) Sketched representation of the concept here developed for the fabrication of AuNP-embedded PDMS surfaces arrays. Tens AuNPs spots are obtained on PDMS films, previously deposited and polymerized onto Petri dishes, by drop casting. After 24 h, drops are removed leaving well-defined AuNPs spots on the polymer surface. B) The arrays can be conveniently interrogated by optical fiber through a fast scan of the surface, giving information on the plasmon features of each spot. On the left, the optical set up, based on optical fiber, and the reading approach is sketched; on the right, typical plasmon bands recorded accordingly to AuNPs spots grown under different conditions, i.e. Au(III) concentration. Fig. 2 a) Normalized extinction bands of AuNP-embedded PDMS surfaces transducers prepared at different Au(III) concentrations, from 50 to 3.125 mM, evidence the progressive wavelength red shift with precursor decrease. The array of spots is sketched on the right, with colors according to plot series. b) Correlation plot that evidences the red shift of the maximum wavelength of each spot series. Standard deviations were inferred from n=4 independent spot replicates of the array. Fig. 3 A) Optical image and morphological images obtained by SEM characterization of the AuNP-embedded PDMS surfaces transducers realized by using different Au(III) concentration in a drop of 5 l in volume: B) 50 mM, C) 25 mM, D) 12.5 mM, E) 6.25 mM, F) 3.125 mM Fig. 4 Typical optical absorption refractometric tests in water-glycerol solution realized by using AuNP-embedded PDMS surfaces transducers obtained by using a) 50 mM and b) 3.125 mM Au(III) concentration in a drop of 5l in volume Fig. 5 a) Scheme of the model adopted to simulate AuNP-embedded PDMS surfaces transducer and the corresponding LSPR absorption peak of the structure obtained in reflection mode. In the inset the variation in the LSPR absoption peak by increasing the PDMS thickness deposited onto the surface of the simulated transducer. Fig. 6 Simulated variation in the LSPR absorption peak due to a variation in the refractive index of the liquid in contact with the simulated transducer. A red shift is evidenced. Fig. 7 Calibration curves and relative calculated sensitivities of the simulated transducers by considering different PDMS thickness deposited onto the AuNPs layer. Higher sensitivity is obtained for AuNPs not covered by PDMS polymer. Fig. 8 Optical absorption curves measured onto different regions of a typical spot sample (1 mM Au(III) concentration). Fig. 9 Optical absorption measurements performed onto 14 equivalent spot arrays of AuNP embedded PDMS surface obtained by 1mM Au(III) concentration

22

Fig. 1 Rella et al

23

50mM Au(III) 25mM Au(III) 12.5mM Au(III) 6.25mM Au(III) 3.125mM Au(III)

Au(III) decrease

Normalised Absorbance

1.00

0.75

0.50

0.25

0.00

a) 400

500

600

700

800

Wavelength (nm)

LSPR maximum wavelength (nm)

560

550

540

b) 530

0

10

20

30

40

Au(III) concentration (mM)

Fig. 2 Rella et al

24

50

B)

A)

200 nm

C)

50 mM

D)

200 nm

25 mM

E)

200 nm

12.5 mM

200 nm

3.125 mM

F)

200 nm

6.25 mM

Fig. 3 Rella et al

25

0.6

50mM

a) )

Absorbance

0.5

0.4

0.3

0.2 500

H2O GLYC 25% GLYC 35% GLYC 50%

525

550

575

Wavelength (nm)

Absorbance

0.7

3.125mM

b) )

0.6

H2O GLYC 25% GLYC 35% GLYC 50%

0.5

500

525

550

Wavelength (nm) Fig. 4 Rella et al 26

575

Fig 5 Rella et al

27

0.8

n=1.33 n=1.35 n=1.36 n=1.38 n=1.40

0.7

Absorbance

0.6 0.5 0.4 0.3 0.2 550

560

570

580

590

Wavelength (nm)

Fig.6 Rella et al

28

600

Fig. 7 Rella et al

29

Fig. 8 Rella et al

30

Fig. 9 Rella et al

31

Author contributions section

Dr. Simona Scarano was involved in the synthesis of the nanoparticles and embedded in PDMS Dr. Adriano Colombelli was involved in the simulation and optical measurements Dr. Maria Grazia Manera was involved in the morphological analysis and refractometric measurements Prof. Maria Minunni was involved in the supervision of chemical synthesis and preparation of the sensitive samples . Prof. Roberto Rella was involved for the supervision and preparation of the paper

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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