1. Introduction

Biosensing is a fast-growing field driven by many potential applications in pharmaceutical research, clinical diagnosis, (bio)-chemical warfare agents detection, food quality control, etc.. In this context, label-free detection is gaining more and more attention since it allows for recognition of target molecules that are captured in their natural forms as opposed to fluorescence-based detection [1]. Moreover, this type of detection is relatively easy and cheap to perform since it does not require laborious labelling chemistry. The possibility to perform such unlabelled biochemical assays relies upon the availability of extremely sensitive refractive index transducers able to convert minute refractive index changes occurring at their biofunctionalized surface. Long Period Gratings (LPGs) arisen in the optical communication field as band pass filters [2] are by now popular sensing devices [3] especially employed in a number of chemical applications for their intrinsic sensitivity to surrounding medium refractive index (SRI) changes [4–6]. However bare LPGs are not attractive devices to perform biochemical detection in aqueous solutions where they show a rather low sensitivity [7]. In fact there are few examples of bare LPGs employed to this purpose and only with a much higher technological effort it has been possible to achieve remarkable sensitivities. Cladding etching, fluid pushed through holes of photonic crystal fiber LPG, coupling to higher order modes near their dispersion turning points are the explored methods to perform biosensing [8–11].

The weakness of the basic device can be overcome by exploiting the enhancement of sensitivity produced by the deposition of a thin layer of suitable refractive index onto the cladding over the grating region [12,13]. This sensitivity gain is produced by the so-called modal transition [14–19]. This is a physical phenomenon enabled by the presence on the silica cladding of a thin overlay whose thickness is of a few hundreds of nanometers and whose refractive index is higher than the cladding. It has been experimentally demonstrated that coated LPGs attenuation bands can shift thousands of nanometers for a unitary change of SRI, making them a competitive technology for the development of biosensors [19]. Moreover the overlay thickness can be used to tune the maximum sensitivity of the coated device around the specific refractive index of the solution in which the detection has to be performed [15,18]. The modal transition can enhance hundreds of times the sensitivity of the bare device around the water refractive index depending on the considered cladding mode [18].

Essential condition to fully exploit the benefits of the LPGs working in transition mode is that the attenuation bands would not loose visibility while transitioning. This is not verified for some materials and related deposition techniques [16,20] and it is achieved only with a good optical quality of the overlay (low absorption and roughness). Up to now the best results in this sense have been obtained with polymer layers deposited by dip-coating.

Ordinary atactic polystyrene (PS) is one of the most common plastics in every day life, it can be deposited on optical fibers in the form of thin films by dip-coating from a solution and it is also a very common substrate material for biological elements immobilization as well as a model system in protein adsorption studies [21]. For instance, microtiter plates made of polystyrene are commonly used as a solid support for antibody immobilization in enzyme-linked immunosorbent assays (ELISAs) which is a well established biochemical technique used as diagnostic tool of medical conditions or as a quality check in food industries [22].

Moreover thin films of PS are an ideal choice from the point of view of the device optical design since they are easily deposited by dip-coating, have excellent optical quality with low losses and have a suitable high refractive index that enables the modal transition [17–19]. At the same time PS is largely used for biomolecules immobilization. All these features make it the right choice for an intermediate layer aimed to fulfill the function of material for the optical design and of biomaterial interface.

Bovine serum albumine (BSA) is a protein which is known to adhere strongly on polystyrene through hydrophobic interactions [23]. Moreover, if the BSA is also functionalized it can be used for a further bio-specific recognition. In this work we have identified adsorption of biotinylated BSA (BBSA) as a simple strategy to perform biofunctionalization of PS coated LPGs. The strong binding between streptavidin (SA) and biotin [24] was then used as a benchmark to test the capabilities of the proposed technological platform for label-free biosensing. Finally, it is worth noting that we used a commercial LPG without any customization of the grating. This implies that there are broad perspectives of further optimizations.

In the following we will briefly recall the basic properties of a bare LPG for refractive index sensing and how these are modified by the presence of a thin high refractive index (HRI) overlay and by the phenomenon of the modal transition. Subsequently we will experimentally characterize the SRI sensitivity of the coated LPG with different overlay thicknesses. In this way we show how it is possible to achieve a sensitivity around the water refractive index exceeding 1000 nm per unitary change of refractive index by working in transition mode and by properly selecting the overlay thickness and the interrogated cladding mode order. The realized and characterized devices will be tested against the adsorption of BBSA and the binding of SA. Finally an investigation of the PS coated optical fibers surface after the biochemical treatments will provide a further proof of the successful protein immobilization.

2. Theoretical background

Long period fiber gratings are in-fiber diffraction gratings obtained by inducing a periodic refractive index modulation (typ. 100-500 μm period) of the core of a single mode optical fiber along few centimeters of its length (typ. 2-3 cm). LPGs act coupling the fundamental guided core mode to discrete forward propagating cladding modes and to each of them at a distinct wavelength where the following phase matching condition is satisfied [2]:

 

Fig. 1 Pictorial description of mode coupling in LPGs and spectral dependence on SRI (not to scale): a) spectrum of incoming white light; b) spectrum of transmitted light at the output of the bare LPG, also showing the shift of the attenuation bands for increasing SRI; c) LPG structure.

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LPGs are sensitive to a number of environmental parameters (temperature, strain, bending, external refractive index) which affect the phase matching condition changing, in turn, the attenuation bands spectral position [3]. However the dependence of the LPG spectral features on the SRI changes is what makes them particularly attractive for chemical sensing applications [4]. The effective refractive indices of the cladding modes increase by increasing the SRI and therefore the attenuation bands shift to shorter wavelengths according to Eq. (1). This happens as long as the SRI is smaller than the cladding refractive index. The matching between the SRI and the cladding index determines coupling to radiation modes, producing as a consequence broadband losses in the transmission spectrum, and marks the beginning to leaky modes coupling for even higher SRIs. The sensitivity characteristic of a bare LPG to SRI changes has an increasing (in modulus) non-linear monotone trend. The result is that the maximum sensitivity is achieved for SRIs close to that of the cladding while for SRIs around the water refractive index the LPG is scarcely sensitive. The behavior is very different when a thin layer of sub-wavelength thickness (ranging in hundreds of nanometers) and with higher refractive index than the cladding is deposited onto the latter [14,17]. The HRI overlay draws the optical field towards the external medium extending its evanescent wave. As a result there is an increased sensitivity of the device to the SRI changes. Moreover the HRI overlay is a waveguide itself and allows mode propagation depending on its thickness, refractive index (RI) and SRI. For a given material (fixed RI) and overlay thickness, when the SRI is varied in a certain range the lowest order cladding mode is gradually and completely sucked into the overlay. Its effective index becomes close to the overlay RI leaving a vacancy in the effective index distribution of the cladding modes. At the same time all the higher order cladding modes effective indices shift to recover the previous effective indices distribution. This is reflected through the phase matching condition in the shift of each attenuation band toward the next lower one. In the middle of this modal transition the attenuation bands can exhibit a sensitivity $(|\partial {\lambda }_{\mathrm{res}}/\partial \mathrm{SRI}|)$ of thousands of nanometers per refractive index unit (RIU) [17,18]. The sensitivity characteristic of the coated LPG is drastically modified compared to the bare device. It has a resonant-like shape whose resonant peak can be tuned around the desired SRI by changing the overlay thickness [18]. Therefore the coated device can be used as biochemical sensor if the overlay surface is properly functionalized in the way to specifically concentrate biochemicals which in turn produce localized refractive index changes.

3. Coated LPGs working in transition mode: fabrication and characterization

A commercial UV-written LPG (Λ = 460 μm, L = 3 cm) was used for the experiments. The dip-coating technique was used to deposit thin films of atactic polystyrene (m.w. 280.000, Sigma Aldrich), whose refractive index is about 1.59, onto the grating region. This deposition technique consists mainly of immersing the fiber substrate into a chloroform solution of the polymer and then of withdrawing it with a well-controlled speed [25]. Different overlay thicknesses were obtained for different experiments by changing the solution concentration, namely 8.5, 9.5 and 10.5% by weight of polystyrene. Before each deposition the LPG was thoroughly cleaned in boiling chloroform. The dip-coating was performed by means of an automated system at an extraction speed of 10 cm/min. After solvent evaporation, the coated device was fixed straight, under constant strain and at controlled room temperature in a bowl (see Fig. 2 ). The investigation of the thin film coated LPG under test consisted of recording its transmission spectra for different SRIs. The optoelectronic set-up used for SRI characterization comprises a white light source of 400-1800 nm wavelength range (Ando AQ4303C) and an optical spectrum analyzer (Yokogawa AQ6319). The analysis was focused on the spectral range 1200-1700 nm. The SRI was changed by using aqueous glycerol solutions whose refractive indices were measured by an Abbe refractometer at 589 nm. Figure 3.a) shows the spectral position of the attenuation band related to the fourth cladding mode for the bare device and for the coated device. In particular the resulting overlay thicknesses after dip-coating from the 8.5, 9.5 and 10.5% solutions were $\cong 270$ nm, $\cong 320$ nm and $\cong 370$ nm, respectively. They were inferred by previous deposition process characterizations through atomic force microscopy (AFM) (for more details see for example [26]).

 

Fig. 2 Schematic of the experimental set-up.

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When an HRI overlay is deposited onto the grating the effective refractive index of the cladding modes is increased, as a consequence the attenuation bands experience a blue shift that is bigger for thicker overlays [15]. The spectral characterization to SRI changes with an overlay thickness of 270 nm for the fourth and fifth cladding modes is reported in Fig. 3.b) and 3.c) respectively. As abundantly reported in literature, the attenuation bands move from their initial position to that initially related to the next lower cladding mode by increasing the SRI [15,17]. It is worth noting that the attenuation bands are clearly visible during the transition, due to the optical quality of the overlay, making possible the device interrogation also in the most sensitive region of its transfer characteristic. Figure 3.d) resumes the SRI characterizations in terms of attenuation bands minima position for the fourth and fifth cladding modes and for the three different overlay thicknesses. Experimental data were fitted with a Lorentian-Cumulative function (best fit) [18]. The position of the attenuation band related to the fifth cladding mode in air can be only inferred by the fitting since it was out of the OSA spectral window. The sensitivity characteristics (extrapolated from the experimental data) of the coated devices are reported in Fig. 3.e) and 3.f) for the fourth and fifth cladding modes respectively. As a matter of fact, the presence of the nanocoating drastically modifies the sensitivity characteristics of a bare LPG in response to SRI changes. The well-known sublinear monotone trend with maximum sensitivity for SRI close to that of the cladding is changed into a resonant-like characteristic. The final sensing properties of the coated devices are determined by the following key parameters: 1) the maximum sensitivity$(|\partial {\lambda }_{\mathrm{res}}/\partial \mathrm{SRI}|)$, 2) the transition index (SRI at maximum sensitivity), and 3) the bandwidth (SRI range evaluated at half maximum sensitivity). All these parameters are strongly dependent on the overlay thickness and the mode order [18].

From the reported data, it is apparent how by changing the overlay thickness it is possible to tune the resonant peak of the sensitivity characteristic for the considered cladding mode in the desired SRI range and in particular to achieve remarkable sensitivities around the water refractive index. It is also obvious that one should wish to interrogate always the highest order cladding mode tuned in full transition in order to exploit the highest sensitivity. However an important constraint that should be taken into account is the spectral position of the interrogated attenuation band that should fall in a region where inexpensive light sources are available.

 

Fig. 3 a) Effect of the overlay deposition on the fourth order cladding mode attenuation band; spectral characterization to SRI changes for the fourth (b) and fifth (c) cladding modes with an overlay thickness of 320 nm; d) SRI characterization in terms of attenuation bands minima position for the fourth and fifth cladding modes and for three overlay thicknesses; sensitivity (| ∂λ_res/∂SRI|) of the fourth (e) and fifth (f) cladding modes for three overlay thicknesses.

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In light of these considerations in Fig. 4 is reported the attenuation band related to the fourth order cladding mode when the overlay thickness is 320 nm and to the fifth order one when the overlay thickness is 370 nm. In both cases the surrounding medium is water. The attenuation band of the fourth order cladding mode for the bare LPG in air was kept as a reference.

 

Fig. 4 Spectral position of the fourth order cladding mode attenuation band for the bare device in air, with an overlay thickness of 320 nm in water and of the fifth order cladding mode with an overlay thickness of 370 nm in water.

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With the considered overlay thicknesses it is possible to exploit the sensitivity enhancement given by the phenomenon of the modal transition and to interrogate two different cladding modes while using the same SLED around 1550 nm. It is worth noting that the peak SRI sensitivity is always achieved when an attenuation band is in the middle way between its initial position and that of the next lower attenuation band in the bare device. For this reason if one wish to interrogate a cladding mode in full transition at a specific wavelength it is also necessary to tune the attenuation bands position of the bare device by properly choosing the grating period or by changing the cladding diameter [27].

4. Biofunctionalization of PS coated LPGs

In order to perform a quantitative detection of target molecules, antibodies (or other bioreceptor like elements) should be immobilized on the sensor surface exposing their functional binding sites toward the analytes in free solution. Therefore the immobilization step plays a crucial role in biosensors development [28]. The antibody immobilization by direct passive adsorption onto immunoassay well surfaces have been reported to orient the antibodies randomly. Further on, this physical adsorption can lead to their conformational changes with a consequent possible loss of functionality [29]. A common way to control antibodies orientation on a polystyrene substrate is to exploit a first layer of immobilized avidin followed by the binding of a biotinylated antibody [30]. The avidin-biotin complex as a model receptor-ligand pair has drawn much interest in light of its high specificity, high affinity, multiple binding and general applicability as an immobilization method [31]. Moreover the strong binding between avidin and biotin is commonly used as a benchmark for biosensors to test their performance against the specific binding of a biotinylated sample to an avidin-modified substrate. In fact for this high affinity non-covalent system it is possible to assume complete attachment.

(Strept)avidin (SA), is a globular protein and is the smaller and slightly acidic bacterial (secreted from Streptomyces avidinii) counterpart of avidin. Its lower isoelectric point makes it preferable to avidin since it shows less non-specific binding. As avidin, SA consists of four identical subunits each containing a single biotin (small water soluble molecule also known as vitamin H) binding site. The binding sites are on opposite sides so that after immobilization it becomes bivalent. Therefore, streptavidin-biotin pair act as crosslinking agent or as binding mediator between molecular layers. However also the directly adsorbed streptavidin is slightly unfolded and flattened since thermodynamics opens the hydrophobic protein core to press it against the polystyrene surface [32]. Therefore, a better strategy would be to use a biotinylated sublayer to bind streptavidin indirectly.

Bovine serum albumin (BSA) is a globular protein which adheres with a strong force and almost without conformational change on the hydrophobic polystyrene, but with very weak force on glass [23]. Its blocking abilities have been extensively reported [33]. Therefore, adsorption of biotinylated BSA was a good candidate for a simple biofunctionalization of polystyrene overlayered LPGs aimed to bind streptavidin as a biorecognition element. In fact the resulting outer streptavidin layer provides a generic binding matrix for a further detection of any biotinylated sample or for the immobilization of biotynilated antibodies (with well-retained activity) in an immunoassay perspective.

Chemicals used in our expertiments are: streptavidin (SA, protein, m.w. 52.8 kDa, Invitrogen); albumin, biotinylated labeled bovine (BBSA, protein, ≥ 80%; 8-16 mol biotin per mol albumin, m. w. 66.5 kDa, Sigma Aldrich); phosphate-buffered saline solution (PBS, 137mM NaCl, total ionic strength 150 mM, pH 7.4, Sigma Aldrich). SA and BBSA were received as lyophilized powders and were used as 0.1 mg/mL protein solutions in 10 mM PBS. PBS was also used as rinsing buffer throughout the experiment and was freshly prepared in sterile double distilled water (ddH₂O) for each experiment. Chemicals were added and withdrawn from the bowl by carefully pipetting. Dynamics of protein adsorption were continuously monitored through automated spectral acquisition of the attenuation bands related to the fourth and fifth cladding mode in transition (around 1550 nm). The LPG was fed by a superluminescent light emitting diode (SLED, 10 mW, 1550 nm central wavelength) and the spectra were recorded each 5 seconds from a computer controlled optical spectrum analyzer (see Fig. 2).

5. Biochemical experiments: results and discussion

The first biochemical experiment was performed monitoring the fourth cladding mode after that the grating was dip-coated in a 8.5% solution of PS. The resulting overlay (tov$\cong 270$nm) was too thin to produce an appreciable sensitivity enhancement of the device around the water refractive index (approx. 9-fold sensitivity gain with respect to the same mode in the bare device as estimated from Fig. 3.e) and therefore during the experiment any adsorption dynamic could be clearly noticed. The second test was focused on the same cladding mode when the overlay was deposited by a 9.5% PS solution. The thicker overlay (tov$\cong 320$ nm) determined a better tuning of the sensitivity characteristic peak around the water refractive index with an approx. 50-fold sensitivity gain. It is worth noting that the PBS refractive index as measured by an Abbe refractometer at 589 nm and at 25°C was 1.3342 (+/-5*10-4), while the water refractive index at the same temperature is 1.3324. Moreover the BBSA and SA solutions showed to have a refractive index differing from that of the PBS (at the same temperature and wavelength) for less than the instrument accuracy. After few minutes in PBS the signal (dip central wavelength) became stable, however the grating was monitored in this condition for more than 1 hour. The volume of solution around the grating was kept constant at about 3 mL. In order to add the BBSA solution, 2 mL of PBS were withdrawn being sure that the coated LPG would be still completely covered by the liquid and then 2 mL of the BBSA solution were added. At this point an adsorption kinetic started to be recorded (see Fig. 5 and 6.a). It is worth noting that the coated device is obviously also sensitive to the refractive index changes of the bulk solution, but this effect is almost instantaneous and can be revealed as an abrupt shift of the band, well distinguished by the adsorption dynamic. The BBSA was kept in incubation for more than 2 hours. Afterwards washing cycles served to remove excess proteins not stably bound on the surface. These cycles were performed removing 2 mL of solution from the bowl and adding the same volume of PBS. In the first experiment it was repeated 2 times. The abrupt shift to a higher wavelength when the solution is removed could be partially related to the lower amount of liquid on the grating and partially to the unbound proteins sucked away from the surface. In fact after the PBS is added there is again an adsorption dynamic in the attempts to restore the plateau conditions. However, after few washing cycles the signal converges (as more clearly appreciable in the second experiment) to a stable intermediate value between the starting point and the plateau reached in the first dynamic, thus revealing the formation of a BBSA ad-layer. When the SA is added to the bowl following the aforementioned procedure a second dynamic is observable related to the binding of SA on the biotinylated sites of the BSA. This dynamic is faster, the wavelength shift is bigger and after the washing cycles there is a smaller tendency of the SA to be detached from the surface. Moreover the stable signal after the wash is also closer to the plateau value of the main dynamic. Afterwards the LPG was carefully cleaned in boiling chloroform to remove the proteins and the PS layer as well.A new thicker overlay (tov$\cong 320$nm) was deposited onto the same LPG by dip-coating in a 10.5% PS solution. This time the overlay thickness was such that the fifth cladding mode in transition with water as external medium was placed around 1550 nm and thus could be well interrogated with the same SLED used for the previous experiment. In this way it was possible to achieve the sensitivity enhancement due to both the exploitation of a higher order mode and the modal transition. In fact an approx. 90-fold gain was obtained with respect to the sensitivity of the fourth cladding mode in the bare device and a 1.7-fold gain with respect to the same mode in transition with an overlay of 320 nm. The experiment proceeded in the same way as the first one except for the washing cycles which were in this case 4 between each dynamic (see Fig. 3.b), in order to better remove the excess of unbound proteins and to better identify the plateau value in clean buffer solution with respect to the previous experiment. The blue shift of the wavelength from the beginning of the BBSA adsorption dynamic to the end of the first washing cycle and just before the starting of the SA binding dynamic was 0.85 nm and 1.48 nm for the fourth and fifth cladding modes respectively. The amplitude of the SA binding dynamic was measured to be 1.6 nm and 2.75 nm for the fourth and fifth cladding modes respectively, taken from the end of the first washing cycle to the end of the second. It is therefore apparent the approximately 1.7 fold sensitivity gain between the two experiments is consistent with that expected due to the tuning of the device in different points of its sensitivity characteristic.

 

Fig. 5 Sketch of the overlayered LPG with polymeric coating, biotinylated BSA ad-layer and binding SA (not to scale).

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Fig. 6 a) Time plot of BBSA adsorption and SA binding by monitoring the fourth order cladding mode of the LPG coated with a 320 nm PS overlay; b) time plot of BBSA adsorption and SA binding by monitoring the fifth order cladding mode of the LPG coated with a 370 nm PS overlay.

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At this point some useful considerations can be done on the sensorgrams. Although BBSA and SA as proteins have similar refractive index of about 1.45 [34,35] and have also similar dimensions (8 x 8 x 3.8 nm for the BSA and 5.4 x 5.8 x 4.8 nm for the SA) [21,36] the observed difference of wavelength shifts can be explained by different binding conformations. Fundamentally, BSA adsorbs to polystyrene only in side-on orientation with 8 nm x 8 nm side to the surface and experience a slight conformational change which leads to a flattening of the protein on the PS surface below 3.8 nm [33,37] while SA attached via biotinylated linker is in its natural globular form with molecular diameter of 5.2 nm [38]. Therefore, the layer of bound SA is slightly thicker than the layer of pre-adsorbed BBSA and this could explain the different wavelength shift related to the two proteins or layers. With reference to the Fig. 6.b, it is easy to observe that the first part of the BBSA adsorption kinetic, up to 30 min from the beginning of the experiment, can be related to the formation of a strongly bound protein monolayer, afterwards the change of the adsorption kinetic in a linear trend is most likely due to a multilayer adsorption [39]. This is confirmed by the fact that a fraction of loosely bound proteins is removed upon rinsing. The multilayer formation and the consequent removal by wash, although less pronounced, can be noticed also in the SA binding dynamic.Finally we briefly report an AFM analysis of PS coated optical fibers which experienced the same biological treatment as the tested LPG. The images were taken on dry samples in air. The employed apparatus is an AFM-SNOM system, the Multiview 1000 by Nanonics Imaging Ltd., integrated with a conventional optical microscope by Olympus, and equipped with cantilevered optical fiber probes (Nanonics Imaging Ltd.) with a nominal spring constant 1 N/m and a tip radius of curvature 5 nm. All images were obtained using tapping mode operation and a set-point 80% of the free amplitude oscillation. AFM data were not filtered, although the topographic image data were flattened using a first or second order line fit to eliminate sample tilt using WSxM free software downloadable at http:www.nanotec.es. Fig. 7.a shows a representative topography image (1x1 μm²) of an untreated PS coated optical fiber. There can be noticed that except for the curvature of the surface and some isolated imperfections, the surface appears to be quite flat with a roughness (RMS) of only 1.09 nm. The PS coated fiber was then immersed in PBS for 2 hours, rinsed with DI water and then dried with dry nitrogen. The liquids used in our biological modification process change the surface morphology increasing the surface roughness to 1.24 nm, according to other reported AFM analyses (see Fig. 7.b) [32]. The topography and the phase image of the PS coated fiber after the adsorption of BBSA and the following SA binding are reported in Fig. 7.c and 7.d respectively. In this case the surface roughness was found to be 1.62 nm. It is important to outline that during the whole second experiment reported in the paper the attenuation band is reduced just of 0.1 dB. Therefore we can conclude the roughness of the surface doesn’t affect much the quality of the detection with respect to that characterizing overlays obtained with other deposition techniques [14–16].The contrast in the phase images is due to both sharp topography variation and stiffness variation. However in this case, since an homogenous biological coating was expected, the phase image served to better show the contour of the revealed features. The images show nicely and densely packed globular features, indicating a continuous protein coverage of the fiber surface. From the topography image it is possible to retrieve an average height of the globular features of about 7 nm, which is consistent with the expected thickness of the biological coating considered the height of the BSA and of the SA. The cross-sectional analysis revealed diameters of the globular features of few tenths of nanometers up to more than 50 nm. Although the proteins considered here should have much smaller diameters it is well known that the topography images in the lateral dimensions suffer of the convolution effect produced by the AFM tip. In fact the dimensions here reported well agree with those of other AFM studies [32,40].

 

Fig. 7 Topography image of (a) a PS coated optical fiber; b) PS coated optical fiber after PBS for 2 hours; c) optical fiber overlayered with adsorbed BBSA and bound SA; d) phase image related to the topography shown in c).

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5. Conclusions

In this work we demonstrated that overlayered long period gratings working in transition mode can be used as suitable transducers for label-free biochemical detection. The sensitivity of the device could be tailored by acting on the PS overlay thickness and on the interrogated cladding mode to work in aqueous solutions with sensitivity largely exceeding 1000 nm per RIU. The PS thin films as overlays showed to be an ideal choice to achieve a good optical design of the device thanks to their ease of deposition, low losses and higher refractive index than the cladding. At the same time PS thin films offered suitable features for protein immobilization. For these reasons, further developments of the project will focus on the use of PS as basic material and will be aimed to the functionalization of the surface of its thin films for a stable covalent attachment of bioreceptor like elements. Finally, it is worth noting that a commercial LPG was used without any customization of the grating parameters, therefore higher sensitivities are attended if the grating is also carefully designed.