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Abstract
Surface plasmon resonance sensor based on gold-coated tilted fiber Bragg gratings (SPR-TFBGs) are perfectly suited for fine refractometry. Thanks to the functionalization of the gold layer, they can be used for label-free biosensing. They have been largely used for the specific detection of proteins and cells. In this work, we experimentally demonstrate that they are enough sensitive to detect a very small entity like an environmental pollutant. In this context, we report here a bio-functionalization of the SPR-TFBG with thrombin aptamers for lead ion detection. We used aqueous solutions of lead ions with increasing concentrations from 0.001 ppb to 10 ppb. Based on the affinity bending of Pb2+ ions to the thrombin aptamer, we experimentally demonstrated low detection level of lead ion concentration (0.001 ppb) while the saturation limit is meanly fixed by the physical dimension of the sensor and the binding efficiency.
© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Optical fiber sensors have been widely used for in situ detection with the advantages of small-size, anti-electromagnetic interference, remote and real-time monitoring ability. They have been successfully used in many applications [1,2], including healthcare monitoring [3,4], food safety [5], environmental sensing [6,7], among others. Among all possible configurations, tilted fiber Bragg gratings (TFBGs) have the refractive index modulation of the fiber core slightly angled with respect to the perpendicular to the fiber axis [8,9]. When a thin metal film is coated on the fiber surface, the evanescent wave couples with the surface plasmon wave under phase matching condition and generates a surface plasmon resonance (SPR) [10–13]. The SPR phenomenon enables sensors to measure the effective refractive index of plasmons and their changes in response to the modifications in a thin layer close to a metal surface [14–17]. Since TFBGs in single mode fiber (SMF) do not require to etch away the cladding layer of the fiber before depositing the coating, the use of the pristine fibers improves the reproducibility of the sensor [18]. Importantly, the SPR-TFBG provides an analysis method that is label-free, real-time, and sensitive [9], which brings it as an ideal platform for in situ monitoring in harsh environments.
With the modernization and industrialization of the world in the past decades, environmental concerns, especially the water quality, have become increasingly serious. Industrial pollutants, such as metal waste, are directly discharged in open environment and in rivers and severely impact the human health [19]. Lead is a heavy metal that is among the most toxic elements in the environment. It can result in weakness, kidney problems and brain damage [20,21]. In the human body, these ions interact with the thiol group of proteins and affect their life cycles, even at very low concentration (µg/L, or ppb) [22–24]. The directive of European Parliament on the quality of water intended for human consumption released in 2020 which is also recommended by World Health Organization (WHO) indicates that the limitation of lead value should be 10 µg/L (10 ppb) and finally reached 5 µg/L (5 ppb) in 15 years [25]. Therefore, a real-time monitoring and highly sensitive environmental sensor is highly required for lead ion tracing. The conventional analytical methods like spectroscopic techniques, anodic voltammetry and chromatography are sensitive for lead ion detection but are not practical for quick and field use. For recent years, many researchers put their efforts to develop methods based on optical fiber platform. In 2015, R. Verma et al. presented an SPR based multimode optical fiber (MMF) sensor using conducting polymer and chitosan for detection of Cd2+, Pb2+ and Hg2+ in contaminated water and reached a detection limit (LOD) of 0.440 ppb [26]. In 2018, A. M. Shrivastav et al. presented an SPR-based sensor with the imprinted nanoparticles coated on cladding removed fiber surface for simultaneous determination of Pb(II) and Cu(II) in aqueous samples. The detected concentration of Pb(II) reached 4.06 × 10−6 ppb [27]. In 2022, S. Ghosh et al. proposed a hybrid optical fiber grating sensor system by concatenating a Long period grating (LPG) and a fiber Bragg grating (FBG) for lead ion detection. The optical gratings were functionalized with a chemically synthesized ammonia-doped graphene oxide and cross-linked chitosan-based nanocomposite material (CCS-NGO) and poly (acrylic acid) (PAA) polymer. The sensitivity of the sensor achieved to 2.547 nm/nM, with a detection limit of 0.5 nM [19]. Another sensor based on the D-shaped optical fiber was demonstrated by R. Biswas et al. in 2022. The D-shaped optical fiber was functionalized with gold nanoparticles with further accompaniment of aspartic acid and the sensor reached the detection concentration down to 5 ppb [28]. M. Gomaa et al. presented a unclad multimode fiber coated with gold nano particles and graphene for lead ion sensing. The fiber sensor reached a sensitivity of 0.21 nm/ppm with a LOD 0.48 ppm [29]. In the same year, K. N. Vajresh et al. demonstrated a etched fiber Bragg grating sensor for lead sensing. The maleic acid (MA) functionalized gold nanoparticles are coated on the etched grating and the sensor reached a sensitivity of 56 pm/10−1 M with a LOD of 2 × 10−7 ppb [20]. In 2023, G. Wang et al. demonstrated a polydopamine-maleic acid (PDA-MA) functional membrane coated Mach-Zehnder interferometer for lead ion detection and reached a detection limit of 0.1678 ppb [30]. In the same year, G.L. Xiong et al. presented a Michelson interferometer constitute of a three-core fiber (TCF), a SMF, and a no-core fiber (NCF) for lead ion sensing. The optical fiber sensor is integrated with Ti3C2Tx (MXene) and reached detection concentration of 0.286 ppb [31]. On the other hand, it has been demonstrated that DNA sequences possess high binding affinity and specificity for lead ions [32–34] named Pb2+-binding DNA aptamers. Various articles report on the DNA-based lead ion sensors [35–37]. However, only few studies have been reported that combine optical fiber platforms and Pb2+-binding DNA aptamers, e.g. [38].
In this work, we used thrombin aptamers immobilized on the gold-coated TFBG as one of the identified Pb2+-binding DNA aptamers. Thrombin aptamer is a man-made single stranded oligonucleotides that bind to specific targets [34–40]. In the presence of lead ions, the deoxyguanosine units in the thrombin aptamer will specifically bind with the Pb2+ and the thrombin aptamer induces a conformational change from a random coil structure to a folded quadruplex structure [33,34,41–42]. As a result, the surrounding refractive index of the TFBGs will be changed and detected by the sensor.
2. Statement of the work
TFBGs were manufactured into hydrogen-loaded single-mode fiber SMF28 by means of a pulsed excimer laser at 193 nm (NORIA system from Northlab Photonics) [43]. A phase mask was mounted to apply an 8 degrees tilt angle in the plane perpendicular to the incident laser beam. The grating length was chosen equal to 9 mm with a grating period of 550 nm. After the inscription process, the gratings were annealed at 100 °C for 12 h to remove the residual hydrogen and to stabilize the grating. A ∼50 nm thick gold layer was deposited on TFBGs surface using a sputtering chamber from Leica for further functionalization [44,45].
For the biofunctionalization process, gold-coated TFBGs were first immersed into phosphate-buffered saline (PBS) buffer for rinsing and get a baseline for 10 min. Then TFBGs were immersed for 60 min in thrombin aptamer solution diluted with PBS buffer. After the thrombin immobilization, TFBGs were again immersed in PBS buffer to rinse out the unbounded particles. Since the gold surface cannot be fully covered by thrombin aptamers, a blocking process is required in order to block the gold surface from unwanted non-specific direct binding during the detection. Therefore, TFBGs were immersed in mercapto-hexanol solution diluted with PBS buffer for 30 min. Finally, TFBGs were rinsed again in PBS buffer for 10 min. The bio-functionalized TFBGs are then ready for lead ion sensing. A schematic of the detection principle of the sensor is reported in Fig. 1, where we can see one of the possible Pb2+ structures of the thrombin binding aptamer (the lead ion captured by thrombin aptamer) [33,36,39,42,40].
Fig. 1. Schematic of the operating principle of biofunctionalized gold-coated TFBGs for the detection of lead ions.
For the preparation of controlled lead ions solutions, we diluted the Pb(NO3)2 with ultrapure water (also named as milli-Q water) to get 5 solutions with Pb2+ concentration of 0.001 ppb, 0.01 ppb, 0.1 ppb, 1 ppb and 10 ppb, respectively. For Pb2+ detection, the bio-functionalized TFBG was first immersed into milli-Q water for 2 min to get a baseline, then it was immersed in lead ion solution with concentration of 0.001 ppb for 3–5 min. After each test in lead ions solution, the TFBG was immersed again into milli-Q water for rinsing and determining the changes in the measurand related to the Pb2+ thrombin binding aptamer.
The experimental set-up is depicted in Fig. 2. An optical vector analyzer (OVA) LUNA CTe (Maximum rated output power 2.4 mW) operating in the wavelength range 1525–1610 nm is used to measure the transmitted amplitude spectrum [46]. The output light propagates first through a linear polarizer to select the required polarization for clean SPR excitation. Then the polarized light is launched into the TFBGs before reaching the input detection unit of the OVA. The TFBGs sensors were immersed into 600 µL solution which was hold in a plastic container. The experiments in this study were implemented at room temperature.
Fig. 2. Image and schematic of the experimental set-up where the gold-coated TFBG is connected in transmission mode to an optical vector analyzer.
3. Experimental results
Figure 3 shows the transmission spectrum of the gold-coated TFBGs. The peak-to-peak amplitudes of the modes in the wavelength range 1540–1550 nm are strongly impacted by the SPR. By selecting the few modes neighboring around the SPR mode, we get two crossing lines, as shown in Fig. 3. The intersection of the crossing lines was tracked to evaluate the wavelength shift during the bio-functionalization process, as already done in previous works [45,47].
Fig. 3. Spectral intersection analysis of the bio-functionalized gold-coated TFBGs. The inset shows a zoom around the SPR signature in the spectrum.
Figure 4 shows the evolution of the wavelength shift of the intersection point during the whole biofunctionalization process. The parts in blue background represent the measurements when TFBGs were immersed in PBS buffer. For the first 10 min, the TFBG has a relatively stable signal. In the thrombin solution, the wavelength increased exponentially and relatively stabilized, indicating the binding dynamic. After the thrombin immobilization, the TFBG was again immersed in PBS buffer to remove unbounded aptamers. As mentioned before, a blocking process of the sensor surface is required to avoid non-specific binding. Therefore, the TFBG was immersed in mercapto-hexanol solution. The wavelength showed again a strong red shift, as already observed in our previous works. After 30 min blocking process, the TFBG was finally put back to PBS buffer for 10 min rinsing. The sensor is then ready for lead ion detection. Several sensors were prepared according to this process and used for this experimental study.
Fig. 4. Sensorgram showing the gold-coated TFBG response during the different steps of the biofunctionalization process.
For the first sensing tests, we used three solutions of lead ion with increasing concentrations from 0.001 ppb to 0.1 ppb. Figure 5 presents the sensorgram of aptamer immobilized gold-coated TFBGs during the lead ion detection process. It shows the wavelength shift of the crossing point versus time in different solutions. The parts with blue background show the wavelength shift when the sensor is in milli-Q water, while the parts with green backgrounds represent the wavelength shift of the sensor in lead solutions. We applied a nonlinear fit with a 95% confidence band and a 95% prediction band, to show the effective detection dynamic of the sensor. The first immersion in milli-Q water is performed to stabilize the signal and the level is used as a reference. In the lead ion solutions, the wavelength increased exponentially in the first minutes, then gradually reached a stable value. Hence, the response time (when 95% of the stable state is reached) for 10−3 ppb lead ion concentration is determined to be 0.9 min. This wavelength increase is relative to an increase of the surface refractive index of the gold-coated TFBG sensor due to Pb2+ - thrombin binding aptamers. The binding mechanism is complex. As mentioned before, it induces conformational change of the thrombin aptamer from a random coil structure to a quadruplex structure, which results in a denser molecular layer at the gold surface [33]. After each immersion in lead ion solution lasting 10 min, the TFBGs were immersed into the milli-Q water for rinsing. There, a blue shift was observed so that after each rinsing, the wavelength lies at a lower value compared to the reference (value in the first milli-Q water). We have made investigations to understand this systematic mechanism. In [48], a blue shift has been reported in the SPR signal obtained with the Kretschmann-prism measurement method of a GR-5 DNAzyme biofunctionalized gold-coated chip. They attribute the observed shift to a refractive index decrease due to the removal of gold nanoparticles from the surface of the sensor in the presence of Pb2+ caused by the cleavage of the substrate. Another possible explanation can be provided by the penetration depth of the SPR and the refractive index distribution at the surface and in the volume around the sensor. The aptamers are small in size (less than 5 nm) while the penetration depth of the SPR is around half of the Bragg wavelength [49]. When the sensor is put back into milli-Q water, the effective refractive index of the surrounding medium is dominated by the volume refractive index (water) due to the conformational change of the thrombin aptamers.
Figure 6 shows the results of wavelength shifts after each lead ion solution. As one can see, the final shift value after rinsing lies at around 0.102 nm which is nearly the same as that of 0.1 ppb lead ion solution. This result depicts that the TFBG sensor has reached its saturation, leaving unbound lead ions in the surrounding solution.
Fig. 6. Histogram of the effective bounding of lead ions on the functionalized surface of the gold-coated TFBG immersed in three growing concentrations.
To explain again this observation, we have calculated the number of immobilized thrombin aptamers and the number of lead ions in each solution to evaluate the concentration limit of detection. Since the fiber diameter is 125 µm and the TFBG length is 9 mm, the sensing area is 3.533 mm2. The thrombin aptamer can be considered with a diameter of ∼2 nm [50]. Hence, if we consider that the area of a thrombin aptamer attached on the gold layer is a square of 4 nm2, the total number of thrombin aptamers immobilized on the gold surface is maximum 8.831 × 1011. However, a surface is never totally perfectly covered and one can usually consider that the effective coverage is a fraction of the total surface. In addition, the fact that the wavelength shift in blocking process is much bigger than aptamer immobilization (shown in Fig. 4) also indicates this. Looking at the literature on the subject [48,51], an averaged number of 7.6 × 1012 molecules/cm2 of thiol-modified DNA can be considered on gold surfaces. Considering the effective area of our sensor, this leads to a total number of thrombin aptamers of ∼2.685 × 1011 molecules. This is equivalent to an effective coverage of ∼30% of the total surface, which is very likely in practice. Let us estimate the total number of lead ions in the used solutions. The molar mass of Pb is 207.2 g/mol and the used volume is ∼600 µL, so the total number of lead ions in the solutions of 10−3 ppb, 10−2 ppb and 0.1ppb are 1.74 × 109, 1.74 × 1010, and 1.74 × 1011, respectively. Hence, at 0.1 ppb, the number of immobilized aptamers and the number of ions in solutions are of the same order of magnitude, which can justify the trend of saturation of the probe. Subsequently, the saturation limit for lead ions detection can be approximated to ∼0.154 ppb.
In order to consolidate the results, we implemented another experiment using a set of lead ion solutions with higher concentration of 0.1 ppb, 1 ppb and 10 ppb. The TFBG was again first immersed in milli-Q water to get a stable baseline as a reference. Then, as in the previous experiment after each immersion of the TFBG in lead ion solution, it was rinsed in milli-Q water to determine the detection level (wavelength shift value). As shown in Fig. 7, in lead ions solution, the wavelength showed an exponential increase and stabilization, while in milli-Q water the wavelength achieved a blue shift. We can see from Fig. 8 that the wavelength shift relative to 0.1 ppb detection lies around 0.110 nm, which is comparable to the previous experiment (last concentration in Fig. 6). In the following detection with the lead ion concentrations of 1 ppb and 10 ppb, the wavelength shift was computed to be 0.127 nm and 0.131 nm when back to milli-Q water (as shown in Fig. 8). This trend again depicts the saturation of the binding around the lead ions concentration of 0.1 and 1 ppb, which can be explained by the accumulation of unbound lead ions around the TFBGs surface that are released after the milli-Q water rinsing. Therefore, we evaluate the response time only for the first concentration (0.1 ppb) of lead ions to be 3.3 min.
Fig. 8. Histogram of the effective bounding of lead ion on functionalized surface after three concentrations of 0.1 ppb, 1 ppb and 10 ppb.
For comparison, Table 1 reports the sensing performances of the most relevant optical based lead ion sensors reported so far to our knowledge. We note that among the sensors using the optical fiber, our sensor shows promising results with the advantage of using pristine standard telecommunication fiber without modification (etching or splicing).
Table 1. Performance comparison of fiber optic lead ion sensorsa
4. Conclusion
Lead ion sensing has been experimentally investigated using thrombin aptamer functionalized gold-coated TFBGs. The thrombin aptamer was successfully immobilized on the gold layer of the TFBGs. Five lead ion solutions with concentration from 0.001 ppb to 10 ppb were used to confirm the detection capability of the sensor. Our observations have confirmed the capability of the probe to detect a lead concentration as low as 0.001 ppb, which is much less than the 5 ppb limit recommended by WHO for the drinking water directive. We have also confirmed that the sensor response gets saturated with a lead ions concentration above 0.1 ppb. In environmental sensing, TFBGs bring all their inherent advantages, including easy fabrication, remote monitoring, label-free sensing and compatibility with cost-effective telecommunication-grade equipment. This novel platform therefore promotes a great potential in practical applications.
Funding
Fonds De La Recherche Scientifique - FNRS.
Acknowledgement
The authors are grateful to Dr. M. Lobry for providing the intersection demodulation program used in the analysis reported here.
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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