Effects of Reabsorption due to Surface Concentration in Highly

thus performing a spectroscopic separation of frequency components. 2.3 Optical setup. The optical setup used to carry out the reflectance and fluores...
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C: Physical Processes in Nanomaterials and Nanostructures

Effects of Reabsorption due to Surface Concentration in Highly Resonant Photonic Crystal Fluorescence Biosensors Alberto Sinibaldi, Antonio Fieramosca, Norbert Danz, Peter Munzert, Agostino Occhicone, Claudia Barolo, and Francesco Michelotti J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b09095 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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The Journal of Physical Chemistry

Effects of Reabsorption due to Surface Concentration in Highly Resonant Photonic Crystal Fluorescence Biosensors

Alberto Sinibaldia, Antonio Fieramoscab, Norbert Danzc, Peter Munzertc, Agostino Occhiconea, Claudia Barolod, and Francesco Michelottia,*

a

Department of Basic and Applied Science for Engineering, Sapienza University of Rome, Via A.

Scarpa 16, 00161 Rome, Italy b

CNR NANOTEC Institute of Nanotechnology, via Monteroni, 73100 Lecce, Italy

c

Fraunhofer Institute for Applied Optics and Precision Engineering IOF, Albert-Einstein-Str. 7, Jena

07745, Germany d

Department of Chemistry and NIS Interdepartmental Centre and INSTM Reference Centre and ICxT

Interdepartmental Centre, University of Turin, Via Pietro Giuria 7, 10125 Turin, Italy

Corresponding author: * Francesco Michelotti: [email protected]

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Abstract: Photonic crystal enhanced fluorescence biosensors have been proposed as a novel immunodiagnostic tool, due to the increased fluorescence excitation rates and angular redistribution of the emission. Among these, purely dielectric one-dimensional photonic crystals (1DPC) sustaining Bloch surface waves (BSW) at their truncation edge, have recently attracted much interest. We report for the first time on the time resolved experimental study of the effects of excess reabsorption of the BSW coupled fluorescence in the near infrared range around 800 nm. Temporally and angularly resolved measurements of the BSW coupled fluorescence emission permit to put into evidence a strong reabsorption of the fluorescence emission when using highly resonant 1DPC. The results suggest that, when designing 1DPC sustaining BSW for quantitative diagnostic assays, it is necessary to choose a compromise quality factor, to exploit the features arising from the electromagnetic field enhancement while avoiding reabsorption.

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1. Introduction

During the last decade, the field of medical diagnostics has faced a growing need for improved sensors for the detection of trace amounts of chemical and biological analytes with reduced limit of detection and shortened assay duration1. Fluorescence-based sensing of labelled analytes has been established as a highly sensitive and specific method to achieve multiplexed detection. In particular fluorescence excitation and emission in proximity of photonic crystals surfaces2 can achieve large enhancements for the labelled detection of either DNA3 or protein4 biomolecules. Recently, among photonic crystal enhanced fluorescence sensors5, those based on the excitation of a particular type of surface electromagnetic waves, Bloch surface waves (BSW)6, at the truncation edge of purely dielectric one dimensional photonic crystals (1DPC) have attracted much interest. Increased excitation rates and angular redistribution of the fluorescence emission have been demonstrated in a number of experimental configurations7–10. In comparison to fluorescence biosensors exploiting enhanced excitation via surface plasmons and directional surface plasmon coupled emission11,12, the absence of any undesired energy transfer to the metal has been presented as a strong argument in favor of BSW sustained by 1DPC13,14. The quest for the largest excitation field enhancement led to the design and fabrication of 1DPC characterized by extremely large quality factors and long BSW propagation lengths15–18. However, no reports published on BSW for fluorescence biosensors until now discuss the effect of electromagnetic coupling, neither weak nor strong, of fluorophores to the highly resonant BSW modes. On the other hand, whereas the role of absorption for guided modes and label-free sensing has been tackled19, strong coupling of organic emitters and BSW modes has been reported without any reference to sensing applications18,20. Because the presence of coupling leads to an excess reabsorption of fluorescence and can affect the linearity and dynamic range of BSW based fluorescence biosensors, a deeper investigation and understanding is due. 3 ACS Paragon Plus Environment

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Recently, we demonstrated by means of steady state fluorescence experiments that the use of BSW sustained by highly resonant 1DPC does not necessarily improve fluorescence emission in the visible range21. Such results permitted to design a class of BSW fluorescence biosensors that could be efficiently used for cancer biomarker detection at low concentration22. Here we report for the first time on the real-time experimental study of the effect of reabsorption of the BSW coupled fluorescence in the near infrared range around 800 nm. Temporally and angularly resolved measurements of the BSW coupled fluorescence emission permit to put into evidence a strong reabsorption effect that needs to be considered in quantitative diagnostic assays.

2. Materials and Methods

2.1 Materials A cyanine molecule Cy7-VG2023, with chemical structure shown in Fig. 1, is used as fluorescent dye in the present work. Cy7-VG20 is characterized by a broad infrared emission spectrum in ethanol solution at low concentration, which is peaked at MAX = 851 nm as shown in Fig. 1. Fluorescence emission can be observed also in methanol and in 1-propanol, whereas it is strongly reduced in water solution. The Cy7-VG20 dye was prepared through a microwave condensation of a quaternized 6carboxybenz[e]indolenine

with

N-[5-anilino-3-chloro-2,4-(propane-1,3-diyl)-2,4-pentadiene-1-

ylidene]anilinium chloride24, in presence of potassium acetate, in EtOH25. The obtained crystalline powder was characterized by Nuclear Magnetic Resonance and Mass Spectrometry. Hydrogen peroxide (30% in water), ethanol (99.8%) and 1-propanol (anhydrous, 99.7%) were purchased from Sigma-Aldrich. Sulfuric acid (95-97%) and methanol (p.a., 99.7%) were purchased from Merck. Water was purified by means of a Merck-Millipore deionizer. All materials were used as purchased. 4 ACS Paragon Plus Environment

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Figure 1 –Absorption (left, blue) and emission (right, red) spectra of Cy7-VG20 in ethanol solution at low concentration. (black curve) Coupling length LC as a function of the wavelength. Inset: chemical structure of the Cy7-VG20 dye.

2.2 Photonic crystals The 1DPC used in the experiments were deposited by plasma ion-assisted evaporation under high vacuum conditions26 on glass cover slides (thickness 170 m). As sketched in Fig. 2a, the 1DPC are characterized by a periodic dielectric stack constituted by the repetition of three Ta2O5 / SiO2 bilayer units (dTa2O5 = 120 nm, dSiO2 = 340 nm) and by two Ta2O5 / SiO2 top layers (dTa2O5= 20 nm, dSiO2= 20 nm). In order to promote stack adhesion to the substrate we deposited a first silica layer of 275 nm. The complex refractive indices of the layers were determined by reflectance / transmittance experiments at 0 = 804 nm: nSiO2= 1.443 + i5E-6, nTa2O5= 2.051 + i5E-5. The 1DPC were designed to operate with the TOPAS substrate (ns = 1.527) and a liquid external cladding (1.33  nc  1.36) at 0.

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Figure 2 (a) Sketch of the 1DPC geometry. (b) Band diagram of the 1DPC. The red line is the TE polarized BSW dispersion. LLc and LLs are the cladding and substrate light lines, respectively. The experimentally accessible region is evidenced in orange (c) Enlarged view of the orange region: (left axis) Cy7-VG20 emission spectrum corrected by the CCD spectral sensitivity, (top) TE reflectance at

0 plotted in blue, (bottom) angular emission pattern in the TOPAS substrate calculated for a Cy7VG20 emitter at the surface of the 1DPC. The inset shows the TE BSW transverse intensity distribution at 0 and at resonance.

The role of the periodic stack is to provide the photonic band gaps (PBG) needed to confine the BSW at the 1DPC surface. In Fig. 2b, we show the PBG structure calculated for the infinite 1DPC by means of an iterative plane-wave eigensolver27, for radiation polarized along the 1DPC layers (transverse electric – TE). In the PBG (light grey), radiation at frequency  and with a parallel component of the wave-vector  cannot propagate in the bulk of the 1DPC. However, as shown in Fig. 2b, a TE polarized BSW appears (solid red line) in a restricted region of the lowest PBG (white), between the light dispersions (dashed) in the cladding (LLc) and substrate (LLs). The BSW is confined by Bragg reflection on the 1DPC side (inside a PBG) and total internal reflection (TIR) on the cladding side

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(between the two LL). The role of the two top layers is to tune the BSW dispersion (Ta2O5 layer) and to provide a silica termination (SiO2 layer) for easy chemical functionalization in biosensing applications. Since its dispersion lays beyond the LLc, the BSW can be excited or out-coupled only from the substrate side, in the Kretschman-Raether configuration. For such a reason the 1DPC coated cover slides were coupled by means of a contact oil to molded plastic (TOPAS®) substrates with prismatic windows, as shown in Fig. 2a. The substrate allows both the excitation of BSW when illuminated from the bottom and the BSW out-coupling through the windows. In Fig. 2b, the dotted lines delimitate the interval  that is relevant for the present work. The region filled in orange is magnified in Fig. 2c and plotted in terms of the angle  and of the wavelength , respectively. The angular reflectance spectrum for illumination at 0 (top curve) is expected to show a resonant dip at 0 in correspondence of the excitation of the BSW. The BSW transverse intensity distribution at resonant excitation is depicted in the inset of Fig. 2c and is characterized by an evanescent tail in the external liquid, with a penetration depth Lp ~ 140 nm. On the other hand, a Cy7VG20 fluorescent molecule with an emission spectrum peaked at MAX (left curve) is expected to produce an angularly dispersed emission pattern in the substrate (bottom curve), as calculated by a Green’s function approach21, in which each angle corresponds to a different wavelength in the emission spectrum. In such a configuration, the dispersive properties of the BSW play the same role of a grating, thus performing a spectroscopic separation of frequency components.

2.3 Optical setup The optical setup used to carry out the reflectance and fluorescence measurements was previously described elsewhere23 and used here with minor modifications. As shown in Fig. 3, the setup can be used in two configurations. It can illuminate at 0 (=2.5 nm, spectrally filtered LED) the 1DPC from 7 ACS Paragon Plus Environment

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the substrate side in a wide angular range and measure the reflectance by means of a CCD array sensor (Fig. 3a). The curved prismatic input and output windows of the TOPAS substrate are used to focus the illumination beam and to perform Fourier imaging on the CCD, respectively. Alternatively, the 1DPC can be illuminated from top (not resonantly) with a slightly focused unpolarized excitation laser beam at EXC = 632.8 nm. Great care was taken to check that the laser intensity was low enough to avoid photo-bleaching of the dye. The fluorescence emission that is coupled to the BSW modes is radiated into the substrate and collected by means of the same CCD, but operated at a different gain and integration time (Fig. 3b). In both cases the CCD detects radiation in the [63.2 °,67.2 °] angular range above the total internal reflection (TIR). In this work, the external liquids were methanol (nmet=1.323), ethanol (net=1.357) and 1-propanol (nprop = 1.379), with the TIR edge situated at 60.06 ° , 62.73 ° and 64.57 °, respectively.

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Figure 3 Sketches of the configurations used for: (a) reflectance measurements at 0, (b) fluorescence measurements, with excitation at EXC. Collection is performed by means of the same CCD array sensor. The bottom part of the chip is blackened to avoid scattering of the excitation beam.

In order to perform measurements with liquids, the 1DPC was topped with a metal plate, which was sealed to the surface by means of a VITON o-ring, thus defining a cell located above the sensitive area. During the measurements, the cell was always kept closed by means of a glass window to prevent solvent evaporation and cooling, which might give rise to refractive index thermal drifts and resonance shifts. The depth of the liquid layer (>1 mm) justifies the assumption that the BSW evanescent tail is fully contained in the liquid itself.

Figure 4 Experimental BSW resonance position at 0 for three different liquids: (red) methanol, (black) ethanol, (blue) 1-propanol.

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As an example, in Fig. 4 we show the angular reflectance spectrum recorded at 0, for three different liquids on top of the 1DPC: methanol, ethanol, 1-propanol. The excitation of a BSW at the interface 1DPC/liquid is revealed by the appearance of a very narrow dip in the reflectance spectrum. The experimentally found resonant angle in ethanol (~65.8 °) is shifted with respect to the theoretical prediction (~63.8 °) shown in Fig. 2c, most likely due to a difference of the two top layers’ thicknesses from the values given above for the design structure. The fabrication tolerances are in fact not small enough to guarantee that such thicknesses are effectively 20 nm28. As expected, the BSW dip shifts when different liquids are used, owing to their different refractive index. This demonstrates the sensitivity of the BSW sensors when used in a label-free mode of operation23, which however is not the main focus of the present work. From Fig. 4, we fitted each dip with a Lorentz curve in a restricted interval around the minimum, retrieving the values of the resonant angle BSW, the full width half maximum FWHM and the depth RMAX. It is then possible to evaluate the following quality factors Q = BSW / FWHM of the resonances: Qmet = 2085, Qet = 2474, Qprop = 1627. The large Q values demonstrate that the 1DPC can strongly localize the electromagnetic field21.

3. Results

3.1 Experimental procedure The procedure adopted to carry out the measurements was divided in three parts. The first step consists of cleaning the 1DPC sensitive surface by a piranha solution (3:1 sulphuric acid and hydrogen peroxide) for 15 min. Then the 1DPC was rinsed several times with deionised water and dried in a nitrogen flux. For every series of measurements, we used always the same BSW bio-chip that did not show any degradation after repeated piranha cleaning (up to 20 times). 10 ACS Paragon Plus Environment

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The second step is to expose the sensitive area of the 1DPC to 200 l of ethanol. A reference (background) signal for the reflectance and fluorescence measurement was acquired under this condition. The third step is the removal of pure ethanol and injection of a solution of the Cy7-VG20 dye in ethanol at several different concentrations C ranging from 10 nM to 1 M. During the incubation of the solution (30-40 min) we measured either the reflectance spectrum or the fluorescence emission spectrum as a function of time. Similarly to our previous work with different molecular species29, we observe experimentally that the incubation time is sufficient to obtain an organic Cy7-VG20 layer assembled at the 1DPC surface, bound to the hydroxy-groups generated after piranha by Van der Waals forces. We observed much weaker effects if the 1DPC were not treated with the piranha solution before incubation. We point out that we did not make use here of any functionalization strategy to covalently attach the organic dye onto the photonic stack.

3.2 Real-time reflectance measurements In Fig. 5, we show the temporal dependence of BSW, FWHM and RMAX extracted from the real-time measurement of the reflectance.

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Figure 5 Temporal dependency of BSW, FWHM and RMAX for a 10 nM solution of Cy7-VG20 in ethanol.

The curves were obtained when the Cy7-VG20 solution with lowest concentration, i.e. 10 nM, was injected and incubated for 30 minutes. This provides information about the growth of the thin film (and the genesis of the very first step of the growth). It is clear that the adhesion of the organic layer on top of the sensitive area broadens the resonance and decreases its depth, due to the increased absorption. The BSW resonance position decays initially and reaches a stable position with a slow residual drift towards larger angles. Such a drift is due to a very slow decrease of the temperature of the system, due to slight evaporation of ethanol. The BSW reflectivity signatures dips are not simple Gaussian peaks, whose shape even changes during the dye incubation time. For an accurate determination of small refractive index perturbations it would 12 ACS Paragon Plus Environment

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be necessary to adopt more sophisticated resonance fitting and tracking methods30. However, here the label-free measurements are used to demonstrate dye binding at the surface and not to quantify its surface density, which would need ultimate accuracy of the tracking process.

3.3 Angular fluorescence measurement in steady state conditions After the real-time label-free measurement we removed the solution from the measurement cell and washed three times with ethanol in order to remove the dye in solution completely. We are therefore in the condition in which a very thin dye layer is on top of the 1DPC in pure ethanol environment. Such procedure allowed us to rule out all fluorescence contributions originating from the bulk of the solution and to consider only the thin film contribution to the fluorescence spectra. Fig. 6 shows the three fluorescence spectra measured for the pristine sample (after piranha) in ethanol (grey), after exposition for 30 min to the 10 nM solution (black), and after exposition to the 100 nM solution (before exposing to 100 nM the 1DPC was again cleaned by piranha) (blue). We took particular care to check that the fluorescence emission was always well contained inside the angular detection window of the optical detection setup, for any concentration of the dye solutions used during the experiments.

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Figure 6 Angular emission spectra at two different concentrations of the Cy7-VG20 dye in ethanol: (grey) background, (black) 10 nM, (blue) 100 nM.

Clearly, the intensity of the fluorescence signal grows when passing to a more concentrated solution. However, a shift of the emission peak is observed as well (0.3 °), which is much larger than that found in the label-free measurement (0.03 °). Because the angular intensity distribution is linked to the emission spectrum via the dispersion of the surface wave, this is the first evidence of a fluorescence emission modification due to the increased concentration of dye bound at the 1DPC surface, which will be better evidenced by the real-time fluorescence measurements discussed below. In the Fig. 7 we resume the results of the label-free and fluorescence measurements before and after incubating the 100 nM Cy7-VG20 solution. At the end of incubation, the BSW resonance is shifted towards smaller angles (refractive index is decreased) and a fluorescence signal appears that is well distinguishable from the background. Since 0 lays within the smaller wavelength part of dye main absorption peak, the adsorption of the dye layer leads to a negative change of the average refractive (water and dye layer) probed by the BSW exponential tail.

Figure 7 (red) Reflectance and fluorescence before incubation, (blue) Reflectance and fluorescence after incubation of a 100 nM solution of Cy7-VG20 in ethanol. 14 ACS Paragon Plus Environment

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3.4 Real-time angularly resolved fluorescence In order to get a better insight on the drifts of the fluorescence spectrum and intensity evidenced by the results shown in Fig. 6, we performed temporally resolved fluorescence measurements. Here, instead of monitoring the label-free signal, we monitor the fluorescence angular spectrum as a function of time during the Cy7-VG20 incubation time. We repeated the measurement for several different Cy7-VG20 concentrations in ethanol from 1 nM to 1 M. After each measurement (each incubation) we regenerated the 1DPC by performing a piranha cleaning. In Fig. 8a we show the results of the experimentally measured time dependence of the BSW coupled fluorescence power W, i.e. the intensity integrated over the whole angular spectrum (area of the fluorescence band shown in Fig. 6 and Fig. 7). Clearly, for small concentrations, the integrated intensity grows during the measurement due to the increase of the dye concentration at the surface of the 1DPC. The fluorescence intensity curves approach a constant plateau WPL at the end of the incubation time. We point out that, once reached WPL, the fluorescence intensity was stationary, indicating that the surface density of emitters/re-absorbers was constant under illumination and confirming our assumption, that photo-bleaching was not effective.

In Fig. 8b we show the experimental dependency of WPL versus C. In case reabsorption would be negligible, W and WPL would be directly proportional to the dye’s surface density 31. Since  is expected to follow a Langmuir’s isotherm model with respect to C32, also the WPL versus C dependence should follow the same model, which predicts a nonlinear dependence with a saturation for large C. From the fit one could evaluate the surface density of binding sites at the1DPC surface and the affinity constant. However, if reabsorption cannot be neglected, direct proportionality between W and  is lost. Consequently, the dependence of WPL versus C is distorted with respect to a simple Langmuir model. Since a theoretical quantitative relationship between WPL and  can be hardly found, being related to 15 ACS Paragon Plus Environment

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the interaction of the fluorescence emission with absorbing molecules next to a highly resonating 1DPC, the whole phenomenon is difficult to model. Nevertheless, in Fig. 8b the first part of the dependency (without the last data point) can be fitted by a modified Hill’s model33: 𝑊𝑃𝐿 =

𝑊′ 1+

𝐶0

𝑚

()

+ 𝑊0

𝐶

where W0 is a residual background, n accounts for the deviation from the Langmuir isotherm model (m=1), W´ and C0 are fitting constants. From the fit we find m=1.9, indicating that we are far from the ideal Langmuir behavior, due to the fact that the WPL values obtained for large C are much lower than expected. This is evident for the last data point, which is well below the fit for the other data points. From the qualitative analysis of Fig. 8 one clear result is therefore emerging. Increasing the concentration of the Cy7-VG20 dye in the solution beyond a threshold leads to a distortion of the WPL vs C dependence, which cannot be adequately described by a simple model (Langmuir). The threshold, for the present experiments, can be qualitatively positioned between 10 and 100 nM. It indicates that using the BSW fluorescence biosensors above such a threshold could lead to a deviation of the response of the sensor from the standard models used in the literature and to a decrease of the dynamic range. Clearly, the threshold might be different for other molecular species and binding strategies. The same effect may affect the performance of other types of highly resonating photonic crystal fluorescence biosensors5.

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Figure 8 (a) Real-time dependence of the fluorescence power W plotted for increasing concentrations C of Cy7-VG20 solutions. (b) Dependence of WPL as a function of C. The solid line is the Hill’s model fit of the first six data points. The dashed line is a guide for the eye.

4. Discussion

The experimental observations can be explained on the base of the properties of the 1DPC, which was designed to obtain a very narrow BSW resonance. Such a condition corresponds to a very large value of the coupling length LC, i.e. the length over which the BSW coupled fluorescence emission remains localized at the 1DPC surface before leaking in the coupling prism. In Fig. 1, we show the plot of LC as a function of the wavelength, calculated for the 1DPC used here by means of the coupling mode plane wave analysis theory of prism coupling developed by R. Ulrich21,34. At MAX, LC is about 0.59 mm. Such a large value enhances the interaction of the radiation emitted in the BSW mode with the dye molecules. However, here we assume that the coupling is weak and that there is just a reabsorption of the radiation by the dye itself. The reabsorption process is influenced by the Stokes shift of the dye. For the Cy7-VG20 dye, the Stokes shift is small (about 20nm) and the absorption and emission bands 17 ACS Paragon Plus Environment

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overlap significantly, as shown in Fig. 1. This plausibly makes reabsorption very efficient. In the case of fluorescent labels with a larger Stokes shift such as quantum dots especially31, the effect will be smaller. With the known Stokes shift of most organic dyes, it would not be, anyhow, negligible. The BSW enhanced reabsorption is responsible for the effects observed experimentally. With reference to Fig. 8, when exposing the 1DPC to the Cy7-VG20 solution with a concentration 1 M, the signal grows immediately with the time constant of the CCD camera and then quickly decreases due to reabsorption. For smaller concentrations the effect is less fast and less strong. With reference to Fig. 6, the increase of the concentration from 10 nM to 100 nM leads to an overall growth of the fluorescence band, which however is cut at the large angles side (lower wavelength according to the scheme shown in Fig. 2c) due to reabsorption in the region of superposition of the absorption and fluorescence spectra. One could argue that strong coupling between the excitons of the dye and the photonic BSW modes is taking place as previously reported for other organic emitters in a solid matrix18,20, leading to the propagation of surface exciton polaritons. In such a condition of strong field confinement, one observes a distortion of the BSW dispersion and a gap opening associated to the absorption band of the dye.

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Figure 9 Normalized angular fluorescence pattern (log scale) for Cy7-VG20 placed at the surface of the 1DPC in ethanol. The orange cone marks the angular range in which the TE BSW is radiated.

However, in the present case, we observed very slight changes of the dispersion and no suppression of the BSW mode at 0 inside the dye absorption band, as shown in Fig. 5a and Fig. 7, indicating that the coupling is indeed weak. Moreover, strong coupling cannot be achieved here, because there is an energy dissipating mechanism which is usually neglected in reports on BSW coupled fluorescence, although it is classical in coupled oscillator theory. To clarify this point, in Fig. 9 we plot (log scale) the fluorescence emission pattern generated by a Cy7-VG20 positioned at the surface of the 1DPC in ethanol, calculated with the Green’s function approach21. Fig. 9 shows the strong directionality of the emission into the substrate due to the fluorophore coupling to the BSW mode and its subsequent leakage in the substrate. The energy emitted into the cladding and the substrate are almost equal, being 42.1 % and 53.1 %, respectively. The missing energy is either absorbed in the 1DPC layer or coupled to guided modes that do not leak in the external media. Out of the 53.1% emitted in the substrate, only 14.5% is coupled to the BSW, indicating that the coupling is weak. Such a mechanism justifies the fact that the increase of the Cy7-VG20 concentration at the surface leads to an overall reabsorption of the fluorescence energy. The experimental results reported above demonstrate that the interaction between the dye emitter and the fluorescence emission at the surface of a strongly resonant 1DPC can drive BSW fluorescence based biosensors’ response away from the expected Langmuir behavior and limit the dynamic range. From Fig. 1, we observe that LC is even larger in the visible range (2-3 mm), where normally the BSW enhanced fluorescence biosensors operate, and an even stronger limitation is expected. Such a penalty can be reduced by designing less resonant 1DPC crystals (lower Q), therefore reducing LC. For example, in BSW fluorescence biosensors operating in the visible range, we recently 19 ACS Paragon Plus Environment

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demonstrated that depositing a thin absorbing layer on top of similar 1DPC leads to an increase of BSW and to a decrease of LC. Under such conditions the reabsorption process can be neglected and the biosensors response follows the standard Langmuir model21,22.

4. CONCLUSIONS

A broad analysis of the real-time measurements of BSW_coupled near-infrared emission of cyanine dye molecules at the surface of a 1DPC shows that unexpected limitation of fluorescence due to excess reabsorption may take place. We tentatively assign the phenomenon to the reabsorption of the radiation emitted into the BSW modes, due to their very long propagation length and the strong molecular absorption of the common dyes such as the Cyanine used here. We substantiated this assignment by the concentration dependence of the signal, combined with the study of the angular dependence of signals. This is an issue for practical use since, in a BSW based fluorescence bioassay, binding of labelled species provides the transduction signal. However, if the fluorescent labels concentration at the surface of the 1DPC reaches too large values, it can drive the biosensor away from its calibrated response and reduce the dynamic range. The results indicate that this penalty (distortion and saturation of the basic signal) can be reduced by designing less resonant 1DPC crystals (lower Q) and reducing the BSW propagation length, as previously demonstrated in fluorescence measurements obtained in the visible range with different dye molecules.

ACKNOWLEDGEMENTS This work was funded by the European Commission through the project BILOBA (Grant agreement 318035). 20 ACS Paragon Plus Environment

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