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Abstract
Biological microfibers are remarkable materials with diversity in their chemistry, structure and functions that provide a range of solutions for photonic structures. Here we proposed and demonstrated a humidity detection technique for spectral tuning of whispering gallery modes (WGMs) in a cylindrical microresonator formed by a piece of spider egg sac silk (SpEss) from Araneus Ventricosus. We launched a supercontinuum laser into the SpEss via a tapered single-mode fiber to excite WGMs. When the ambient humidity changed, the profile diameter and effective refractive index of the SpEss changed, which caused the WGM resonant dips to shift. The experimental results showed that when the relative humidity (RH) changed from 20% to 75% RH, the average testing sensitivity of the proposed sensor was 389.1 pm/%RH and the maximum testing sensitivity was 606.7 pm/%RH in the range of 60% to 75% RH. Also, the proposed SpEss-based humidity sensor showed a fast response time of 494 ms and good repeatability with fluctuations less than 8% compared with the initial test values. The SpEss-based sensor expanded the application of spider silk as a biodegradable and biocompatible material in biochemical sensing.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Optical fiber sensors have been extensively studied as biochemical sensors [1–4], which play a critical role in various applications including environmental monitoring, chemical process, food inspection, and biological engineering fields [5–8]. Miniaturization, responsiveness, and biocompatibility are the most critical factors for biochemical sensors [9]. However, common silica fibers exhibit chemical-inertness and biological incompatibility, restricting the implementations to miniaturization of devices and the development of in-vivo bio-sensing. People have to coat special sensing materials on the fiber probe for temperature, humidity, or PH measurement [10–12]. Complicated preparation process limits the practical applications of the fiber-based biochemical sensor. Promising alternatives to optical fibers are natural biomaterials, compared to synthetic materials, many examples of biomaterials in nature show superior structure and properties. For instance, the iridescent scales of the Morpho butterfly gave a different optical response to different vapors as an optical gas sensor [13]; the silk fibroin from Bombyx mori could sense temperature due to the large thermal expansion coefficient [14]. Also, silk from the orb-weaver spider has fascinated human, due to its excellent physical and biomedical properties, such as flexibility, tensile strength, toughness and most importantly, eco-friendly [15,16].
The spider egg sac silk is well known as a semi-crystalline biopolymer which is composed mainly of the amino acids alanine (Ala, 24.44%), serine (Ser, 27.61%) and glycine (Gly, 8.63%), and the amino acid sequences share a number of distinctive features [17]. The molecular structure of SpEss consists of randomly oriented crystalline regions separated by less organized protein chains and embedded in an amorphous matrix [18]. β-sheets as the secondary structure of silk fibroin form through natural physical cross-linking of amino acid sequences, which in SpEss consist of polyalanine (An), alternating glycine and alanine ((GA)n) sequences that form crystallites. These crystalline β-sheets are believed to confer the high tensile strength of SpEss. The amorphous regions of SpEss are commonly made up of the repeat motifs GPGXX and GGX (G, glycine; P, proline; X, subset of residues), which adopt helical structure [17,19]. When combined with β-sheets regions as anchors, these amorphous regions provide SpEss with elasticity. Therefore, a SpEss can be seen as a protein threads comprised two primary parts: the crystalline parts organized in β-sheet, composed of hydrophobic (GA)n/An sequences, and the amorphous parts organized in α-helices, composed of hydrophilic amino acid sequences, hydrogen bonds play a crucial role that link these parts into the fiber and make the silk fibroin more sensitive to changes of temperature and humidity in the environment [20,21].
In this paper, we employed a spider egg sac silk (SpEss) from Araneus Ventricosus to perform the humidity sensing characteristic. We employed a section of SpEss to configure a cylindrical microcavity to excite the WGMs in the profile of the SpEss. The change of ambient humidity would cause the change of the diameter and the effective refractive index of the SpEss. The change of the diameter and effective refractive index would cause the WGM resonant dips to shift, performing the humidity-sensing characteristic. Compared with the dragline silk, the surface of the SpEss was much smoother, and the profile diameter of the SpEss was much larger [22]. The SpEss was suitable for a WGM-based sensor. The average testing sensitivity was 389.1 pm/%RH in the humidity range of 20%-75%, which was higher than some fiber-based sensors [23,24]. Also, the native spider silk is stable at room temperature, and the application of spider silk makes it possible to develop a novel biocompatible, biodegradable and highly sensitive optical-based chemical and biological sensor [25,26].
2. Fabriction and characterization
2.1 Sample preparation
We caught the female Araneus Ventricosus (Fig. 1(a)) in nature and raised it in the lab for 3-4 months by feeding mealworms twice a week. Female Araneus Ventricosus produced the SpEss (Fig. 1(b)) by tubuliform glands used as a continuation to protect the eggs against the predators like birds and ants [27,28]. We extracted the SpEss from the egg sac manually with a pair of tweezers under the microscope, and then we winded the SpEss on a reel with controllable speed and horizontal space. We conserved the prepared reel with the SpEss in a chamber where the humidity and temperature could be adjusted and controlled.
Fig. 1 (a) Image of an Araneus Ventricosus and a bunch of SpEss. (b) Scanning electron microscope (SEM) image of the SpEss.
2.2 Optical setup
To improve mechanical stability, we used two drops of UV curable epoxy (Apollo UV, JiGA) to fix both ends of the SpEss on a self-made slide at the height of 3 mm from the surface of the glass slide. The SpEss with a total length of 2.7 cm was retained between the cured epoxy droplets, and the extra parts on both sides were cut off to prevent winding. The profile diameter of the SpEss was 8.7 μm. Standard single-mode fiber (SMF-28, Corning) with 8.3 µm core diameter and 125 µm cladding diameter was used to fabricate the tapered fiber used in the experiment. The tapered fiber was fabricated through the fiber heating fused tapered technique described in the previous report [29]. In this experiment, the tapered waist diameter was measured to be approximately 2.5 µm, and the waist length was about 3 mm (Figs. 2(a) and 2(b)).
Fig. 2 (a) Image of the tapered fiber with the waist diameter of ~2.5 µm. (b) Image of the tapered fiber under the 1000x magnification. (c) Experimental setup scheme for the proposed SpEss-based RH sensor.
Then, we used two drops of UV curable epoxy to fix both ends of the fabricated tapered fiber on another self-made slide at the height of 3 mm from the surface of the glass slide. The fiber with a total length of 3.2 cm was retained between the cured epoxy droplets. We mounted the slide with the tapered fiber on a micromanipulator (MBT616D, Thorlab, the resolution was 62.5 nm) to adjust and control the distance between the tapered fiber and the SpEss. The gap between the fiber taper and SpEss influenced the coupling efficiency of the sensor significantly. In this experiment, we chose to directly contact the fiber taper with the surface of the SpEss in order to ensure sufficient stability of the structure during the experiment. We launched a supercontinuum laser (SuperK Compact, NKT) with the wavelength range of 450-2400 nm into one end of the tapered fiber and received the transmission spectrum from the other end of the fiber with an optical spectrum analyzer (AQ6317C, Yokogawa, resolution was 0.02 nm), in addition, maximum light coupling efficiency could be achieved by using a polarization controller to optimize the polarization state of the incident light field (Fig. 2(c)).
2.3 WGM characterization
Figure 3(a) provided the experimental results of the transmission spectrum from the SpEss-based microresonator when the environment humidity was 20%RH. The transmission spectrum performed WGM resonance information. The free spectral range (FSR) was 56.6 nm, and the resonance wavelength was 1518.8 nm. The Q-factor of the WGM resonator was calculated as 1.6 × 102 using the formula of Q = λr/ΔλFWHM, where λr is the resonance wavelength, and ΔλFWHM is the full width at half-maximum corresponding to λr [30]. However, the rough surface of the SpEss and the high absorption of spider silk protein enhanced light loss and scattering. In addition, the circulating beam was evanescently coupled into the micro-cylinder (SpEss) and experienced the total internal reflection radiating along the cylinder main-axis, which affected by the attenuation and propagation constants of SpEss (Fig. 3(b)) [31]. For those reasons, this optical micro-cylinder resonator should not exhibit very narrow (high Q-factor) transmission resonances.
Fig. 3 (a) Transmission spectrum of the SpEss-based WGM resonator, where the diameter of the SpEss was 8.7 µm. (b) Enlarged schematic diagram showing the connection between the SpEss and the tapered shape fiber.
3. Experimental measurement and discussion
To investigate the humidity response of the SpEss-based resonator, we placed the sensor and the tapered coupling fiber inside the temperature and humidity controllable chamber (5518, ETS). The accuracy of the humidity setting was ± 1.5%RH. A calibrated humidity and temperature controller was employed to control the RH value in the chamber with a resolution of ± 0.1%RH. Figure 4(a) provided the experimental results of the SpEss-based humidity sensing with the temperature of 22 °C, where the relative humidity ranged from 20% to 75% RH. The experimental results indicated that the transmitted light intensity decreased along with the increase of the humidity. The main reason might be the occurrence of over-coupling when the tapered fiber was subjected to mechanical stress and bended due to the expansion of SpEss. Another possible reason for the increased loss was the increase of absorption/scattering by the SpEss in a humid environment. Inset in Fig. 4(b) provided the zoom-in spectrum of the resonance dip, and the wavelength shift was 21.4 nm. According to the results in Fig. 4(c), the WGM resonance dip red shifted along with the increase of RH. The shift became large as the increase of RH. The relationship between the RH (x) and the resonance dip (y) could be fitted as y = 0.0039x2 + 0.0046x + 1517.5, which indicated that the higher the RH, the higher the testing sensitivity, and the response was nonlinear. The SpEss-based sensor performed high sensitivity in relatively high humidity environment. In the humidity range of 20%-75% RH, the average testing sensitivity was 389.1 pm/%RH, which was significantly better than those fiber-based resonance wavelength detection sensors reported [23,24]. In the humidity range of 60%-75% RH, the testing sensitivity was as high as 606.7 pm/%RH. The high sensitivity in the humidity range of 60%-75% RH was caused by the nonlinear change of the SpEss refractive index and profile diameter. When the SpEss absorbed water from the surrounding air, the water molecules would interact with the hydrophilic amino acids sequences in the amorphous region, combined themselves with the random coils and break the relatively weak hydrogen bonds that hold the protein chains together which could affect the refractive index of silk fiber [32]. Always, the refractive index of protein decreases/increases because of the uptake/loss of water molecules. The significant expansion occurred in a direction perpendicular to the axis due to silk fiber hydration also contributes to the high sensitivity of the sensor [33].
Fig. 4 (a) Experimental results of the SpEss-based WGM resonator in the humidity range of 20% to 75% RH at the temperature of 22 °C. (b) The zoom-in spectrum of the resonance dip. (c) The experimental results of the relationship between the RH and the resonance wavelength.
The resonant condition for the SpEss-based WGM was 2πneffr≈lλr [34], where neff was the effective refractive index of the SpEss, r was the profile radius of the SpEss, and l was an integer representing the order number of the resonance. The above equation holds for l>>1. Effective refractive index (neff) and radius (r) were closely related to the resonance wavelength, and any change in either neff or r would result in a shift in the WGM resonance wavelength. This shift could be expressed as the formula [35]:
To measure the diameter of SpEss, we packaged the sample in a transparent chamber under a microscope (DSY2000X, COIC) and acquired the images by using a CCD (UOP0510, COIC) with a resolution of 2592 × 1944 pixel (Fig. 5(a)). We controlled the same exposure time for each image to ensure the same background noise. We changed the humidity in the chamber and monitored the real-time humidity with a hygrometer. We changed the distance d between the lens and the sample and recorded a series of images of the SpEss at each humidity. During this process, along with the increase of d, the images were not in focus, then in focus, and finally not in focus (Fig. 5(b)), where we took the measuring results of the SpEss at 65%RH as an example. Finally, we employed an image-processing program supported by the commercial calculated software Matlab to extract the boundary of the SpEss. We obtained different diameter results with different d. The minimum value, which was from the image of the right focus, indicated the right result of the SpEss diameter.
Fig. 5 (a) Experimental setup scheme for measuring the SpEss diameter. (b) The measurement results of the SpEss diameter at 65%RH. (c) The relationship between the SpEss diameter, effect refractive index and the relative humidity. (d) Comparison results of the experimental and calculated results of Δλr.
By using the images from the CCD and the image processing method (Figs. 5(a) and 5(b)), we obtained the profile diameter (d) of the SpEss with different humidity (labelled with ● in Fig. 5(c)). According to the definition of FSR (FSR≈(λr)2/(2πrneff), λr was the resonance wavelength, we might calculate the neff of the SpEss (labelled with ■ in Fig. 5(c)) with different humidity based on the results of the profile radius. Consequently, we obtained the calculated results of the Δλr (labelled with ● in Fig. 5(d)). The experimental results and the calculated results showed a good agreement with the RH range of 20%-75%.
We obtained a series of pictures of the SpEss with different humidity (see the inserts in Fig. 5b). The pictures indicated that the SpEss had a clear boundary, and we employed a commercial calculated software Matlab to program and analyze the changing of the diameter. We could distinguish the boundary of the spider silk by converting the initial image into a grayscale image. The contrast of the silk was quantified as the difference between the background grayscale intensity level and the minimum grayscale intensity level on both sides. Then, the change of the boundary was calculated by scanning the image pixels column by column. Provided the illumination conditions were not changed during the measurement. In addition, a standard single mode fiber SMF-28 with 125 µm diameter was used as a reference to calibrate the diameter of the SpEss.
The detection limit (DL) of the sensor is calculated as the ratio of the sensor resolution (R) to the sensitivity (S), which could be expressed as the formula:
The sensor resolution depends on the system noise and can be determined by the variances of different noise sources [36]:
whereσ is the standard deviation of the resulting spectral variation, ΔλFWHM is the full width at half-maximum and is related to the Q-factor by Q = λr/ΔλFWHM. FWHM could be calculated from the transmitted spectrum (Fig. 3(a)) as 9.49 nm at λ = 1518.8 nm. 60 dB is a good signal to noise ratio (SNR) for a typical photonic link and also applied to this system, then was calculated as 66.7 pm. We assumed the standard deviation due to temperature stabilization is 10 fm [36]. The optical spectrum analyzer with a spectral resolution of 20 pm ± 5% was used for our experiment, the error in determining the position of the resonant mode is uniformly distributed between −1 pm and 1 pm, then σspec-res was calculated as 0.58 pm. The sensor resolution was 200.1 pm. And the detection limit of the SpEss-based microresonator was 6.5 × 10-1%RH in the range of 20% to 60% RH and 3.3 × 10-1%RH in the range of 60% to 75% RH.To test the stability of the SpEss-based humidity sensor, we carried out the measurement using the same SpEss sample after 3 days interval followed by one week from its initial tests within the same range of 20% to 75% RH at the constant temperature of 22 °C (Fig. 6(a)). The performance of the sensor was stable throughout one week with less than 8% fluctuations from its initial test values. We tested the repeatability of the sensor by increasing and decreasing the relative humidity in the chamber for ten times within the range of 20% to 75% RH, respectively, and the results showed that the sensor demonstrated good repeatability (Fig. 6(b)).
Fig. 6 (a) The repeatability of the SpEss-based sensor over the period of one week. (b) The repeatability of the SpEss-based sensor during the humidity increasing and decreasing process. (c) The time-dependent response of the SpEss-based sensor. (d) The RH response of the SpEss-based sensor with different temperature.
We moved the SpEss-based humidity sensor from chamber A to chamber B with a rapid change of RH (from 56%RH to 73%RH) to estimate the response time. In our experiment, we employed a photoelectric detection system (2117-FC-M, Newport and 6221, NI) to detect the response time with the incident laser source wavelength of 1530 nm, being close to the resonance dip at an initial relative humidity of 56% (Fig. 4(b)). We first placed the sensor in the chamber A and allowed the sensor to equilibrate at the humidity of 56%RH, and then we moved the sensor to chamber B and introduced a step humidity change of 73%RH. The response time, which represented the time taken for the sensor to reach 90% of the final value was determined from the time-dependent response of the proposed sensor presented in Fig. 6(c) [37]. The response time of the SpEss cylindrical microcavity sensor was estimated as 494 ms, which was better than other fiber-based configurations [38,39].
As a natural biological protein, spider silk also responds to the changes in temperature. Here, we investigated the temperature effect on the SpEss-based humidity sensor by setting a relatively moderate temperature for SpEss in the chamber (5518, ETS) from 22 °C to 42 °C with the step of 5 °C. We controlled the experimental humidity from 20% to 75% RH and the testing results were provided in Fig. 6(d). The WGM resonant wavelength shift over the entire humidity range were 21.4 nm, 17.32 nm, 13.47 nm, 10.24 nm and 7.27 nm for the sensor under 22 °C, 27 °C, 32 °C, 37 °C and 42 °C, respectively. The results demonstrated that the sensitivity of the sensor decreased with the increase of ambient temperature. Opposite to the humidity effect, the diameter of the SpEss decreased and the refractive index increased with the increasing temperature which caused blue shift by the large thermal expansion coefficient of the protein in the SpEss [14]. In addition, the increasing temperature accelerated the loss of water molecules in SpEss and limited the humidity effect, resulting in a decrease in sensitivity. Therefore, it is necessary to calibrate the humidity sensor at different operating temperature and the proposed humidity sensor is suitable to work under the condition which the temperature fluctuation range is small considered the temperature cross sensitivity.
4. Conclusion
A novel spider silk-based whispering gallery mode resonator for humidity sensing is proposed and experimentally demonstrated. Changes in the refractive index and diameter of the SpEss due to changes in the ambient relative humidity led to a spectral shift of the WGM resonances. The proposed sensor had an average sensitivity of 389.1 pm/%RH in the RH range of 20%-75% and a maximum sensitivity of 606.7 pm/%RH in the RH range of 60% to 75%. The proposed SpEss-based humidity sensor performed the good repeatability with the fluctuation of less than 8% and response time of 494 ms. Although the cross-sensitivity of the sensor to temperature was high, the SpEss had proven itself a good candidate for RH sensing application under small temperature fluctuation and promised a way to fabricate biodegradable, bio-absorbable and biocompatible protein-based microresonator for biochemical sensing that used natural materials as their constituent.
Funding
National Key R&D Program of China (2018YFC1503703), National Natural Science Foundation of China (11574061, 61675053, 61775047 and 61705051), the 111 project (B13015), Fundamental Research Funds for Harbin Engineering University of China.
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