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Abstract
To date, there is clear experimental evidence that gold-coated tilted fiber Bragg gratings (TFBGs) are highly sensitive plasmonic biosensors that provide temperature-compensated detection of analytes at concentrations in the picomolar range. As most optical biosensors, they bring an evanescent wave in the surrounding medium, which makes them sensitive to both surface refractive index variations (= the useful biosensing signal) and to bulk refractive index changes (= the non-useful signal for biosensing). This dual sensitivity makes them prone to drift. In this work, we study partially gold-coated TFBGs around their cross-section. These gratings present the ability to discriminate both volume and surface refractive index changes, which is interesting in biosensing to enhance the signal-to-noise ratio. The effects induced in the TFBGs transmitted amplitude spectra were analyzed for surrounding refractive index (SRI) changes in the range 1.3360–1.3370. Then, the gold film was biofunctionalized with human epidermal growth factor receptor (HER2) aptamers using thiol chemistry. The detection of HER2 proteins (a relevant cancer biomarker) at 10−9 g/mL, 10−8 g/mL and 10−6 g/mL demonstrated the advantage to identify environmental perturbations through the bare area of the TFBGs, which is left not functionalized. The non-specific drifts that could exist in samples are eliminated and a wavelength shift only related to the surface modification is obtained.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Fiber Bragg gratings (FBGs) are narrow bandpass filters around the so-called Bragg wavelength that are usually photo-inscribed in standard single-mode optical fibers. FBGs are not sensitive to surrounding refractive index changes and can only be used to measure temperature and strain. Their demodulation is based on tracking the Bragg wavelength shift resulting from a change of temperature and/or from the application of an axial or transversal strain [1–3]. Hence, other configurations have been developed to enable light interactions with the fiber surroundings [4–6]. Tilted fiber Bragg gratings (TFBGs) are one of the configurations reported towards this objective. TFBGs are short-period gratings for which the core refractive index modulation is slightly angled (usually < 12°) with respect to the perpendicular to the fiber axis [7,8]. In single-mode fibers, a small tilt angle induces a strong coupling from the core to the cladding at discrete wavelengths below the Bragg wavelength. TFBGs feature several tens of narrow-band cladding mode resonances in their transmitted amplitude spectrum. As they propagate along the cladding/surrounding medium interface, these bands are sensitive to temperature and strain but also to bending and surrounding refractive index changes [9–11]. The resonances at lower wavelengths correspond to cladding modes with increasing evanescent field extending in the surrounding medium. When the environment around a TFBG slightly changes in refractive index, the wavelength and amplitude of the corresponding cladding modes change accordingly [12]. The largest resonance shift occurs around the so-called cut-off wavelength, which corresponds to the mode whose effective refractive index (RI) is the closest to the surrounding refractive index (SRI) [13]. For wavelengths below the cut-off wavelength, the corresponding cladding modes leak out of the fiber. These leaky modes do not shift in wavelength in response to the surrounding refractive index but only in amplitude. The boundary between guided and leaky cladding modes is then the cut-off mode, characterized by the maximum extend of evanescent field in the external medium. This explains the largest refractometric sensitivity compared to other modes, usually of the order of 25 nm/RIU (refractive index unit). This sensitivity can be further enhanced thanks to a thin metal coating deposited on the fiber surface at the TFBG location. This gives birth to the excitation of surface plasmon polaritons (SPP).
Surface plasmon resonance (SPR) involves a resonant transfer of the grating-outcoupled light energy to a surface plasmon in the form of collective electrons oscillations in the metal layer [14,15]. Phase matching conditions indicate that the effective RI and the state of polarization of the modes must coincide with those of the plasmon wave to enable its excitation [16]. It turns out that to allow this coupling light must be polarized in the plane of incidence (TM- or P-polarized) and the metal thickness must be several tens of nanometers. As a result, the corresponding cladding mode resonance strongly decreases, as a result of energy transfer with the surface plasmon [17,18]. The SPR phenomenon enables refractometric sensors to effectively measure modifications close to the metal surface [19]. Upon grafting of bioreceptors on the metal surface, biosensing based on affinity mechanisms can be achieved. As a result, similarly to the Kretschmann prism configuration, SPR-TFBGs provide label-free and sensitive detection amenities [20,21].
In previous works [22,23], we have successfully implemented SRI measurements using either fully bare or fully gold-coated TFBGs. Both configurations show rapid and sensitive response to SRI changes, with a circa tenfold enhancement in sensitivity for the gold-coated configurations compared to the bare ones. However, both configurations reveal the total refractive index change, corresponding to changes at the surface and changes in the volume (along the depth of the evanescent field penetration, which corresponds to a fraction of the operating wavelength). In environmental sensing or biomedical sensing, it is interesting to discriminate both effects as the sensor can be affected by unwanted fluctuations of the volume refractive index (such as those arising from temperature changes as it is known that 0.1 °C shift induces a refractive index change of 10−5 RIU). Such a discrimination can increase the signal-to-noise ratio (SNR) while minimizing drifts that are present when the interrogation accounts for both modifications.
To meet this requirement, several options are possible for the sensor preparation and some have already been reported. One can use two TFBGs either in two fibers or cascaded in a single fiber and cover only one of them with gold. This configuration requires a larger spectral range (at least 200 nm) or two channels in case of two fibers to interrogate the gratings. Ideally, both gratings should behave identically, which is not easy to ensure in practice. Another option consists in shallow gold coating so as to excite the SPR and stay sensitive to the volume refractive index changes thanks to the cut-off mode. This solution has been presented in Ref. [24]. A thin gold layer coating induces a more limited sensitivity of the SPR mode compared to the standard configuration with a thickness close to 50 nm [25–27]. Another option, as a trade-off between previous ones, consists in partially coating a TFBG with a gold layer. This relevant configuration has not been investigated before and is the subject of this experimental work. Part of the grating was left bare to enable volume sensing only while the remaining was gold-coated and biofunctionalized (with a specific surface biochemistry) to measure surface changes upon analyte binding in addition to volume refractive index sensing.
First, we analyzed the effects induced in the TFBGs transmitted amplitude spectrum for SRI changes in the range 1.3360–1.3370. Then, we biofunctionalized the gold film with single-strand DNA aptamers as bioreceptors to turn the platform into a biosensor [28–30]. We have been able to assess the sensing performances of this TFBG platform for human epidermal growth factor receptor (HER2) protein, a relevant cancer biomarker [31,32]. A bio-detection experiment was performed to show the metrological performance of this hybrid platform.
2. Bare/gold-coated TFBGs and statement of work
As aforementioned, the objective here is to use both the intrinsic properties of both bare and gold-coated TFBG sensors to discriminate volume and surface refractive index changes. In a bare grating, the cut-off mode presents the highest sensitivity to the surrounding refractive index changes. An example is shown in Fig. 1(a) for a bare TFBG immersed in calibrated salted aqueous solutions with different SRI values ranging from 1.3359 to 1.3371 (measured by a hand-held Reichert refractometer). The usual calibration of such a sensor consists in tracking the wavelength shift of the cut-off mode versus SRI change (Fig. 1(b)). The determined sensitivity of this mode is ∼23 nm/RIU, as determined from a linear regression of the measured data (Fig. 1(c)). We use the optical vector analyzer (OVA) LUNA CTe that can provide 1525–1610 nm broadband light and measure the transmission spectrum, simultaneously. The resolution of the OVA is 1.25 pm. For all experiments, TFBGs are well fixed during the sensing process to avoid any unwanted polarization fluctuations.
Fig. 1. Transmitted amplitude spectrum of a bare TFBG immersed in different aqueous media with a very slight SRI change (10−3 RIU) (a). Zoom on the cut-off mode behavior (b) and wavelength sensitivity of the cut-off mode as a function of the SRI value (c).
For gold-coated TFBGs, the corresponding transmission amplitude spectrum exhibits an attenuation of specific modes impacted by the SPR excitation [19]. Figure 2(a) shows the typical transmitted amplitude spectrum of a gold-coated TFBG immersed in different calibrated refractive index liquids. Here the modes have radially-polarized evanescent fields (corresponding to P-modes), as they are excited by core-guided light that is P-polarized relative to the tilt plane. Also, the modes located between the position of the SPR (the most highly attenuated mode) and the cut-off mode (the highest mode in amplitude at the left side of the SPR) are the most relevant to track. Figure 2(b) presents a zoom around the selected cladding mode resonance whose wavelength shift is the highest as a function of the SRI value. It shows a sensitivity of ∼64 nm/RIU, as computed from the linear regression of the raw data (Fig. 2(c)).
Fig. 2. Transmission amplitude spectrum of a gold-coated TFBG immersed in different aqueous media with a very slight SRI change (10−3 RIU). Zoom on the most sensitive cladding mode resonance (b) and wavelength sensitivity of this mode as a function of the SRI value (c).
In order to precisely detect the small refractive index changes on the grating surface that could be screened by bulk variations, we used here partially-coated TFBGs, as sketched in Fig. 3. The gold layer is deposited along one side of the fiber at the grating location while the other side of the fiber is left bare. This hybrid configuration will combine the sensing properties of both bare and gold-coated gratings through the cut-off mode and SPR excitation, respectively.
The transmitted amplitude spectrum of partially-coated TFBGs is hybrid between bare and gold-coated gratings. The demodulation process therefore considers both the cut-off and the most sensitive modes. Figure 4 displays the transmitted amplitude spectrum (P-polarization) of a partially-coated TFBG, highlighting the two resonances of interest to be tracked for the bulk-compensated surface sensing. In the following section, we present the experiments that we have performed to produce partially-coated TFBGs in telecommunication-grade single-mode optical fibers.
Fig. 4. Transmitted amplitude spectrum of partially-coated TFBGs in a calibrated salted aqueous solution (n = 1.3367).
3. Manufacturing of partially-coated TFBGs
TFBGs used in this study were manufactured into hydrogen-loaded single mode fiber SMF-28 by means of the phase mask technique and a 193 nm excimer laser beam with 50 Hz repetition rate and 5 mJ pulse power energy. The inscription setup is the Noria System from NorthLab Photonics. We used a phase mask with a tilt angle of 8 degrees with respect to the plane perpendicular to the incident laser beam and a slit to inscribe 1 cm long gratings (static exposure).
After the inscription process, gratings were annealed at 100 °C for 12 h to remove the residual hydrogen and to stabilize their spectral properties. To obtain a partial coating around the fiber cross-section, the annealed TFBGs were put in a dedicated V-groove contained on a metal plate. The latter is the HFV001 model from Thorlabs that has the capability to host fibers with diameters ranging from 150 µm to 341 µm. They were also connected to a red laser source to determine the direction of their tilt angle, as shown in Fig. 5(a). The fiber was rotated until the strongest emitting position was reached (Fig. 5(b)), ensuring that the grating planes are well oriented prior to the gold covering. The well oriented fiber was then fixed in the V-groove and subject to a sputtering process (Fig. 5(c)). Using a sputtering chamber from Leica, a ∼50 nm gold deposition was made on top of the V-groove plate containing the grating. This value was chosen according to previous works [32–35], as a good trade-off between enhanced refractive index sensitivity and good spectral quality.
Fig. 5. Gold deposition process. Side view showing light outcoupling from a TFBGs (a), picture of a TFBG on the V-groove for orientation optimization before gold deposition (b) and sketch of the fiber cross-section on the V-groove after gold deposition (c).
Since the bare part of each TFBG is not perfectly sealed during the sputtering process, there is a need to clean the residual gold particles on the fiber surface. Selective chemical cleaning is very difficult over the curved surface of the fiber. Therefore, we used 800 nm femtosecond laser pulses to scratch the gold layer from the surface of the fiber. The laser system is from Spectra-Physics and is composed of a Mai Tai oscillator and a Spitfire pro. amplifier. The pulses have 120 fs duration, 1 kHz repetition rate. We used a variable attenuator to select the required energy (25 nJ up to 30 nJ) so as to remove gold without modifying the refractive index of the silica fiber cladding. The experimental setup is described in [33] and a picture is shown in Fig. 6. This setup is conventionally used for FBGs inscription. The optical fiber is positioned on a two-axis translation stage from Aerotech. A high numerical aperture (0.5) and long working distance (17 mm) microscope objective (Mitutoyo) is used for laser focusing few microns (5µm) below the surface of the optical fiber to remove larger lines of gold. The fiber is moved perpendicularly to its optical axis with a speed of 100 µm/s to scratch one line of about 50 µm. Then, it is translated by 5 µm to scratch the second line. This inter-distance between the lines is used because we noticed that with these experimental parameters (energy and translation speed), the gold is removed on an average width of 7 µm for each scanned line.
Fig. 6. Picture of the focused femtosecond pulses laser set-up used to remove gold (left) and schematic of the setup with a close-up of the fiber cross-section and side view after partial surface cleaning from gold layer (right).
Figure 7 depicts the microscopic image of a partially-coated TFBG surface. The right part of the fiber shows the bare section of the TFBGs after femtosecond laser modification. We can appreciate some transparency. The left part of the picture shows the TFBG section surrounded by the gold coating where the fiber surface appears darker due to the presence of the gold layer.
To make sure that the TFBGs are sensitive enough for biosensing purposes, a refractometric characteristic test was first conducted. The biofunctionalization and experimental tests towards biosensing were then implemented on the partially-coated TFBGs. The bulk refractive index interference was well discriminated by this configuration, as shown in section 5.
4. Bio-functionalization of the gratings
TFBGs were first biofunctionalized with HER2 aptamers. To this aim, they were immersed into phosphate buffer saline (PBS) buffer for 10 min for rinsing and to get a baseline. Afterwards, the samples were immersed in HER2 aptamers solution diluted in PBS buffer for 60 minutes to be biofunctionalized. As the HER2 aptamer modification on partially-coated TFBGs is based on thiol chemistry with sulfhydryl group (-H-S), the bounding of sulfhydryl group is strong on gold surface while only adsorption occurs on silica. Therefore, the thorough rinsing performed in buffer prior to use of the functionalized sensor allows the removal of any aptamer or mercapto-hexanol that could be anchored on the bare part. After the bio-functionalization step, TFBGs were immersed in mercapto-hexanol solution for blocking process for 30 min. Finally, TFBGs were again rinsed in PBS buffer for 10 min.
After the bio-functionalization process, TFBGs were put into HER2 protein solutions for biosensing experiments. The concentrations of HER2 proteins solutions were set to 10−9 g/ml, 10−8 g/ml and 10−6 g/ml while the incubation in each solution lasted several minutes. Finally, TFBGs were immersed in PBS buffer for 10 min again.
5. Experimental results
In this section, we present the experimental results obtained with partially-coated TFBGs. They include both the volume refractometric characterization in calibrated refractive index solutions and the surface refractive index measurements through HER2 protein bio-sensing.
5.1 Partially-coated TFBG RI characterization
The RI characterization was implemented with 6 different calibrated solutions. The time spent in each solution was 5 min and the results are displayed in Fig. 8. The transmitted amplitude spectra of a partially-coated TFBG are depicted in Fig. 8(a), with a vertical offset in the vertical axis (for readability). With the bulk RI changing from 1.3338 to 1.3503, the cut-off and the most sensitive modes experienced a shift towards longer wavelengths that can be easily seen on the spectra, since the RI change is quite broad (> 10−2 RIU). Figure 8(b) shows the tracking of the cut-off mode and the most sensitive mode in the 6 tested solutions. Over this large RI change, the bulk RI response of the cut-off mode and the most sensitive mode are similar, which was expected. For the cut-off mode, the determined bulk sensitivity is 413 nm/RIU while it is 401 nm/RIU for the most sensitive mode.
Fig. 8. Bulk RI response of partially-coated TFBGs: (a) transmission spectra of partially-coated TFBG with 6 bulk RI changing from 1.3338 to 1.3503. (b) tracking of cut-off mode and most sensitive mode in 6 bulk RI for 5 minutes each.
As shown in Fig. 9, we then tested the RI sensitivities of the cut-off and most sensitive mode for small RI changes in the range 1.3361–1.3370, which is close to the RI of the PBS buffer. Figure 9 was generated from the spectrum reported previously in Fig. 4. By tracking the wavelength shift of the cut-off and the most sensitive modes when the RI of the solutions changes from 1.3360 to 1.3370, we determine their respective sensitivities. Figure 10 shows the linear fitting of the experimental results. We report sensitivities of 21 nm/RIU for the cut-off mode and 67 nm/RIU for most sensitive mode, which confirms the plasmonic enhancement provided by the metal coating. Hence, for slight SRI variations, the differential sensitivity between the cut-off and most sensitive mode clearly appears and will be exploited in biosensing experiments to compensate for unwanted drifts.
Fig. 9. Evolution of the cut-off mode and most sensitive mode for a RI characterization in a small variation range.
Fig. 10. Sensitivity of cut-off mode (left) and most sensitive mode (right) of a partially-coated TFBG resulting from the measurements performed in Fig. 9.
5.2 Partially-coated TFBG bio-sensing
In this section, we analyzed the experimental biosensing results of partially-coated TFBG. For these assays, we tracked the wavelength shift of the cut-off and the most sensitive modes when bio-functionalized TFBGs were immersed first in PBS buffer and then in HER2 proteins solutions at growing concentrations.
Figure 11 displays the wavelength shift of the cut-off and the most sensitive modes in each tested solution. One can observe that the cut-off mode (black curve) keeps a similar level with a slight drift in HER2 proteins solutions. Oppositely, the most sensitive mode contains sudden jumps when passing from one solution to another and drifts in whole experiments. Let us note that jumps have been reported in many previous similar experiments and are attributed to unwanted pressure/surface tension changes when dipping the optrodes in the different tested media. More interestingly, in solutions containing HER2 proteins, the most sensitive mode presents an exponential behavior that is increasing with the protein’s concentration.
Fig. 11. Wavelength shift of cut-off and most sensitive modes when the hybrid TFBG platform is immersed in the tested HER2 solutions.
The drifts recorded for the cut-off and the most sensitive modes in PBS buffer depict the bulk fluctuations. Considering the calibration performed in Fig. 8, we subtracted the cut-off wavelength shift from the most sensitive one to compensate for the bulk effect. The subtracted result in PBS buffer is considered as the baseline for the HER2 proteins detection. Figure 12 depicts the compensated results in HER2 proteins solutions with the 3 tested concentrations. From the referenced results, one can observe that the signal increases exponentially in the first minutes upon immersion and then saturates for each concentration.
To determine the pure effects of HER2 proteins binding, we computed the deviations between HER2 proteins detection and PBS buffer and plotted the result in Fig. 13. With the HER2 proteins concentration increase from 10−9 g/mL to 10−6 g/mL, the useful signal (i.e., the most sensitive mode’s wavelength shift) increases from 3 to 20 pm, while the error remains close to 2 pm. The latter was computed from an adjustment of the data with the best fitting curve. At 10−9 g/mL, the signal and error amplitudes are comparable. It is therefore much more reliable to consider the limit of detection to be of the order of 10−8 g/ml, which remains more than tenfold the minimum concentration found in practical applications [34] and confirms the very good sensitivity of the developed hybrid platforms.
Fig. 13. Bar chart showing the compensated sensor response for the 3 tested HER2 proteins concentrations.
6. Conclusion
The spectral characteristic of partially-coated TFBGs has been experimentally investigated in order to verify the interesting perspectives for biochemical sensing applications. The results showed that the cut-off mode and the most sensitive mode are both excited and are visible in the transmitted amplitude spectrum of the partially-coated TFBGs. This dual sensing mechanism promotes a possibility to measure the bulk refractive index and the surface refractive index changes, simultaneously. The sensitivity of the cut-off mode and the the most sensitive mode have been determined to be 21 nm/RIU and 67 nm/RIU, respectively.
In the study of HER2 proteins biochemical sensing, we succeeded in measuring the bulk refractive index and surface refractive index changes by using HER2 aptamers immobilized on the gold coating. As it was expected, the partially-coated configuration shows the advantage to identify the environmental perturbation detected by the bare part of the TFBGs. The drifts that exist in blank solutions are eliminated and an increase in terms of wavelength are detected in biochemical sensing. HER2 proteins are detected at concentration of 10−8 g/mL and 10−6 g/mL. with a compensated wavelength shift of 8 pm and 20 pm, respectively. This is high enough considering the resolution of the device, on the one hand, and the requirements of the target application, on the other hand.
Funding
Fonds De La Recherche Scientifique - FNRS.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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