Aqueous mercuric ion detection with microsphere optical ring resonator sensors

Sensors and Actuators B 120 (2006) 207–212

Aqueous mercuric ion detection with microsphere optical ring resonator sensors Niranjan M. Hanumegowda, Ian M. White, Xudong Fan ∗ Department of Biological Engineering, 240 D Life Sciences Center, 1201 E. Rollins Street, University of Missouri-Columbia, Columbia, MO 65211, United States Received 16 June 2005; received in revised form 10 January 2006; accepted 5 February 2006 Available online 15 March 2006

Abstract We have developed a novel optical sensor for mercuric ion Hg(II) detection in an aqueous environment by utilizing a fused silica microsphere ring resonator. Due to the high Q-factor associated with the whispering gallery modes (WGM) (>105 ) at the surface of the microsphere, the light interaction with the analytes on the sphere surface is significantly increased, resulting in enhanced sensitivity. The spectral position of the WGM of a microsphere shifts in response to the binding of Hg(II) to the surface of the microsphere, which is activated by the thiol group. Our experimental results show that the Hg(II) detection limit of the microsphere sensor is approximately 50 ppb (w/w) and that mercuric ions bind to thiol groups with 1:3 stoichiometry. Control experiments with zinc ions and 2-mercaptobenzothiazole (2-MBT) were also performed to ensure that Hg(II) binds selectively to thiol groups. © 2006 Elsevier B.V. All rights reserved. Keywords: Aqueous mercuric ion detection; Microsphere optical ring resonator sensor; WGM

1. Introduction Mercury (Hg) has been well known as an environmental pollutant for several decades. The general population may be exposed to mercury compounds through inhalation of ambient air, the soil, or consumption of contaminated food and water. Thus, detection of the presence of mercury is of high importance for environmental monitoring and human health protection [1,2]. Standard methods, such as cold vapor atomic fluorescence detection, for detecting the presence of mercury in water are being employed today [3]. However, these methods require bulky and expensive detection equipment, and require a large sample volume and a long process. Optical sensors that are capable of directly measuring the mercury concentration in water samples can have the advantages of simplicity, lower cost, fast measurement time, and compactness. However, only a few examples have been demonstrated to date, including fiber

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optic and surface plasmon resonance (SPR) based sensors [4–7], which are operated under either fluorescence-based detection or label-free detection schemes. In the label-free scheme where no fluorescence is involved, detection limits of 0.1 ppm and nanomolar were achieved respectively by a commercialized SPReetaTM system and by a lab SPR system with assistance of split-field photodiode detection technology [6,7]. Sub-ppm detection of mercury in nitrogen has also been demonstrated with cantilever-based sensors [8]. Optical ring resonators based on fused silica microspheres have recently been under intensive investigation for label-free bio/chemical sensor development [9–14]. The size of microspheres ranges from a few tens to a few hundreds of microns in diameter. Light propagates around the surface of a ring resonator in the form of whispering gallery modes (WGMs). The WGM is the surface mode and its evanescent field extends into the surrounding medium with the characteristic decay length d on the order of 100 nm. As a result, the WGM is sensitive to inhomogeneous refractive index changes, such as when bio/chemical molecules bind to the sphere surface with a layer thickness of t  d (see Fig. 1), and homogenous refractive index changes, such as when the surrounding bulk solution changes.

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2. Experimental 2.1. Materials HgCl2 (>99.5%), ZnCl2 (>98%), anhydrous ethanol, 3-mercaptopropyltrimethoxysilane (3-MTS), and 2-mercaptobenzothiazole (2-MBT) were purchased from Sigma–Aldrich (Milwaukee, WI). The Hg(II) (Zn(II)) stock solution was prepared immediately before each experiment by adding the appropriate amount of de-ionized (DI) water to HgCl2 (ZnCl2 ). The 0.6 mM 2-MBT solution was prepared by pre-dissolving 0.0016 g 2-MBT in 1 mL anhydrous ethanol before further dilution with 15 mL DI water. 2.2. System setup Fig. 1. The WGM of a sphere is capable of sensing both inhomogeneous refractive index changes such as molecule capture, which occurs when t  d, and homogeneous refractive index change such as bulk solution change, in which case t  d. t, Thickness of the layer where the refractive index changes; d, characteristic decay length of the WGM in surrounding medium. Illustration is not to scale.

A Q-factor in excess of 106 and 109 can be obtained when the sphere is in water and air, respectively. Such strong light circulation effects significantly enhance the light–matter interaction, rendering an effective sensing length of 10–100 cm. Theoretical analysis shows that a detection limit of 10−8 to 10−9 RIU (refractive index unit) is achievable [10,11]. Recently, the detection limit on the order of 10−7 RIU was demonstrated [14]. This result is comparable to or better than the waveguide based and SPR based sensors [15,16]. In addition to the high sensitivity, the small size of the microsphere sensor system makes it appealing for a commercial bio/chemical sensor. Compared to the bulkier SPR sensors, ring resonators are smaller in size, and the circulation of the light results in a very small required surface area for light–matter interaction, which reduces the consumed sample volume. Furthermore, microsphere ring resonators are compatible with fiber optic and waveguide technologies and capable of performing multiplexed detection [17–20]. As a consequence, microsphere ring resonators are regarded as a promising candidate for nextgeneration sensor applications. In this article, we report, to our knowledge, the first demonstration of detection of the presence of mercuric ions (Hg(II)) in water with a microsphere optical ring resonator. The microspheres were submersed in water during the experiment and treated with a mercapto-silane solution, which has a high affinity for mercury [7,21]. The WGM spectral position shifted upon Hg(II) binding to thiol groups. We showed that the microsphere sensor was capable of detecting aqueous Hg(II) on the order of 10–100 ppb (w/w). Concentration dependent results further revealed that mercuric ions bound to thiol groups with 1:3 stoichiometry. In addition, we performed control experiments with Zn(II) and 2-mercaptobenzothiazole (2-MBT) to verify the selective binding of Hg(II) to thiol groups.

Fig. 2 shows the sensor configuration. Fused silica microspheres of approximately 200 ␮m in diameter were fabricated by melting the end of an optical fiber with a CO2 laser. A small fluidic cell was built around the sensor, allowing for injection and withdrawal of liquid samples. The sphere was immersed in DI water and brought in contact with a fiber prism. The fiber prism was made by polishing a single-mode-fiber at a desired angle. For the experiment in this article, the angle was 74◦ to enable the excitation of the second radial mode of the WGM [14]. The excitation light from a tunable diode laser (980 nm, New Focus, linewidth <300 kHz) was coupled into the microsphere via frustrated total internal reflection through the fiber prism. The laser, controlled by a 16-bit DAQ board (National Instruments 6052E), scanned in wavelength with the scanning rate of 3–10 Hz and its wavelength change was calibrated with a wavemeter. The reflected light was collected by a detector from the other side of the fluidic cell. A spectral “dip”, indicative of the WGM spectral position, occurred in the reflected light when the light was on resonance with the WGM, as illustrated in Fig. 3. The WGM spectral position, recorded by the same DAQ board, shifted in response to the immobilization of thiol groups onto the sphere surface and the subsequent binding of Hg(II).

Fig. 2. (A) Configuration of a microsphere ring resonator sensor. Excitation light from a tunable diode laser could be coupled into the WGM through a fiber prism. (B) The fiber prism was obtained by polishing an optical fiber at 74◦ . The whole sensor was immersed in DI water.

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processes can be approximately modeled in the frame of the first-order Langmuir adsorption, i.e.: dθ(t) = k[1 − θ(t)], dt

(1)

where θ denotes the normalized thiol activated sites and hence the WGM spectral shift. The adsorption rate, k, is estimated to be 0.036 s−1 . Generally, we observed that saturation occurred 1–5 min after the silane solution was introduced. As a result, we could conclude that the 30 min incubation time used was sufficient for attaching 3-MTS on the sphere surface. 3. Results and discussion

Fig. 3. WGM spectral positions before (A) and after (B) Hg(II) bound to thiol groups on the sphere surface. The full-width-at-half-maximum linewidth (δλ) of the WGM was 4 pm, corresponding to a Q-factor (λ/δλ) of 2.5 × 105 . Curves are vertically shifted for clarity.

2.3. Surface activation The sphere sensor was activated with thiol groups by attaching 3-MTS to the sphere surface. Spheres were first placed in boiling DI water for 30 min after fabrication, followed by 30 min incubation at room temperature in 1% 3-MTS solution prepared in 90/10 (v/v) ethanol/water mixture. The spheres were then rinsed with anhydrous ethanol and DI water five times each and baked in an oven at 100 ◦ C for 30 min before being immersed in DI water for subsequent Hg(II) sensing. In separate experiments, we studied the kinetics of 3-MTS immobilization onto the sphere surface by monitoring the incubation processes. One percent 3-MTS was achieved by using a digital syringe to inject a controlled amount of 3-MTS to the fluidic cell where the sphere was immersed. Fig. 4 shows, as an illustration, one of the binding curves. The immobilization

The Q-factor of the spheres as prepared in the previous section ranged from 105 to 106 . Fig. 3 shows the WGM spectra of the particular sphere (215 ␮m in diameter) used in our experiment. The linewidth of the WGM was 4 pm, which corresponds to a Q-factor of approximately 2.5 × 105 and provides a detection limit in spectral shift on the order of 0.08 pm [10,11,14]. The Q-factor remained constant throughout the experiment. After immersion of the sphere in the fluidic cell filled with DI water, the sensor baseline was established when the sphere and water reached thermal equilibrium. Different concentrations of HgCl2 were then injected into the cell through a digital syringe. The amount of Hg(II) solution was controlled so that the final Hg(II) concentration in the cell varied from 140 nM to 11 mM. According to the previous studies, the maximum density of binding sites on the sphere for silane agents is approximately 5 × 1014 cm−2 [22]. Therefore, even at the lowest concentration, the number of Hg(II) ions is still 100 times more than the maximum binding sites on the sphere surface. As a result, concentration depletion could be ignored in our experiment and the concentration of Hg(II) in the cell after each injection was regarded as constant during the binding process. Upon the injection of HgCl2 , the WGM spectral position shifted to a higher wavelength in response to the binding of Hg(II) to thiol groups on the sphere surface, as shown in Fig. 3. Sufficient time was given to ensure that equilibrium was reached between the mercury ions in water and thiol groups. Then, a higher concentration of Hg(II) was injected, resulting in a new equilibrium that further shifted the WGM to the longer wavelength. The WGM spectra were monitored throughout the experiment at a rate of 3 Hz. One of the spectral “dips” was used to plot the ladder-like sensorgram, a part of which is shown in Fig. 5. The binding process between Hg(II) and thiol groups on the sphere surface can be described by mHg(II) + nS ⇔ Hgm S n .

(2)

The dissociation constant Kd is therefore given by Kd = Fig. 4. Sphere was activated with thiol group when 3-MTS bound to the sphere surface. Theoretical simulation based on the first-order Langmuir model and experimental results are shown in solid and dashed curves, respectively. Both curves are normalized to the saturation values. k is estimated to be 0.036 s−1 .

[Hg(II)]m [S]n [Hgm Sn ]

(3)

If we define the normalized WGM shift, δ, as the ratio of occupied thiol sites to the total binding sites [S]0 , i.e.

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Fig. 5. Sensorgram showing binding of Hg(II) of various concentrations to thiol groups on the sphere surface. Arrows indicate the time when HgCl2 was added. Inset: normalized sensorgram for [Hg(II)] = 2.67 ␮M. Simulated curve is obtained by use of the first-order Langmuir model with k = 0.012 s−1 .

δ = n[Hgm Sn ]/[S]0 , Eq. (1) can be applied to describe the binding kinetics, as only one layer of Hg(II) will form through the Hg–thiol binding. The inset in Fig. 5 shows the detail of the binding process for [Hg(II)] = 2.67 ␮M. The binding rate of 0.012 s−1 obtained from the simulated curve using Eq. (1) indicates that the detection can be completed in a few minutes. This detection time, even at a much lower Hg(II) concentration, is still at least five times shorter than that in SPR-based mercury sensors [7]. The normalized WGM spectral shift at equilibrium, δeq , is derived from Eqs. (2) and (3) and expressed as follows: δeq n [S]n−1 [Hg(II)]m = γ[Hg(II)]m . n = (1 − δeq ) Kd 0

(4)

Experimental results for the normalized and absolute WGM shift as a function of Hg(II) concentration can be obtained from Fig. 5 and are plotted in Fig. 6. In order to determine the binding stoichiometry, we fit the experimental results with different integer combinations of m and n. It is found the best fit occurs when m:n = 1:3 and when γ = 2.5 × 104 . This 1:3 stoichiometry binding was also observed previously in Hg(II) detection with SPR, and was attributed to the formation of bulky mercury chloride–hydroxyl complexes HgCl4 (OH)3− [7]. According to Fig. 6, the microsphere sensor starts to show saturation at approximately 80 ␮M Hg(II) and the linear log–log response range covers from 2 × 10−7 to 6 × 10−5 M (inset B in Fig. 6). The detection limit can be deduced by considering the minimum spectral resolution and sphere saturation level. As shown in Fig. 6, saturation level for the sphere under study is 13.5 pm whereas the sensor spectral resolution obtained from the WGM linewidth is 0.08 pm. Using m, n, and γ provided above and Eq. (4), the detection limit of our sensor is estimated to be [Hg(II)]min = 240 nM, or 50 ppb (w/w). This detection limit is close to what was observed experimentally. In fact, the two lowest detectable concentrations used in our experiment were 140 and 380 nM, corresponding to 28 and 76 ppb, respectively.

Fig. 6. Triangles are the experimental results for normalized (left axis) and absolute (right axis) sensor response as a function of Hg(II) concentration. The sensor saturation level is approximately 13.5 pm. Solid curves are the fit using Eq. (4). (1) m:n = 1:1, γ = 8.33 × 103 ; (2) m:n = 1:2, γ = 1.67 × 104 ; (3) m:n = 1:3, γ = 2.5 × 104 . For clarity, the same results are also plotted in the linear–linear and log–log scales in inset (A) and (B), respectively.

As discussed previously, the WGM spectral shift can be induced by either the actual binding process or homogeneous refractive index change in the solution. One of the characteristics of homogeneous index change is that the WGM spectral shift is linearly dependent upon the analyte concentration in the linear–linear scale and does not exhibit saturation, as described in Ref. [14]. Inset A in Fig. 6, however, clearly shows that the response of WGM to the Hg(II) concentration was highly nonlinear and tended to saturate at high Hg(II) concentrations, which suggests the WGM shift was due to the binding of Hg(II) to the thiol groups. To further verify this and to demonstrate that our sensor has a strong binding affinity for Hg(II), control experiments were performed with ZnCl2 , which is known to have much lower binding affinity [7]. Again the sphere was prepared as previously. ZnCl2 solution was first injected into the fluidic cell to make the Zn(II) concentration 120 ␮M; only a small shift was observed, as shown in Fig. 7. In contrast, a much larger shift was induced when Hg(II) was added, even when the concentration of Hg(II) was only 16 ␮M. With this experiment, the possibility that Cl2− is responsible for the WGM shift can also be eliminated. In addition, we used 2-MBT, a chelating agent that forms complexes with Hg(II) [6,23], to confirm the presence of Hg(II) on the sphere surface. To avoid the size dependent WGM response that may interfere with our experiment, two spheres of similar size were chosen [10,14]. After silanization, the first sphere was exposed to 30 ␮M 2-MBT solution. A slight shift in WGM was observed due to the non-specific binding, as seen in Fig. 8. In contrast, the second sphere, after first exposed to 500 nM Hg(II), showed four times larger response to the same concentration of 2-MBT (arrow B in Fig. 8). An even larger shift was observed when the 2-MBT concentration increased. All the evidence supports that the WGM spectral shift originally observed was due to the actual binding between Hg(II) and thiol groups on the sphere surface.

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can be significantly reduced [24]. In addition, using a smaller sphere will further increase the detection limit, as the sensor sensitivity is inversely proportional to the sphere size [10,14]. Moreover, the sandwich type detection like using 2-MBT will provide another method to improve the detection limit, as shown earlier. Given the high sensitivity and quick sensing response demonstrated in this work, along with the potential for improvement, this research will open a door to a series of applications in chemical sensing, especially heavy metal detection using microsphere ring resonator sensors. Acknowledgements

Fig. 7. Control experiment with Zn(II) (concentration 120 ␮M) and Hg(II) (16 ␮M) showed that thiol groups bound selectively to Hg(II).

The authors thank the financial support from 3M NonTenured Faculty Award and University of Missouri Research Board Award (RB 05-013). We would also like to thank the stimulating discussion with Dr. Guoping Mao at 3M Company. References

Fig. 8. Thiol-activated sphere #1 (248 ␮m in diameter) showed a slight spectral shift due to the non-specific binding between silane and 2-MBT of 30 ␮M. For a comparison, large spectral shift occurred in thiol-activated sphere #2 upon additions of 2-MBT of 30 and 55 ␮M (arrows B and C) following the treatment with 500 nM HgCl2 (arrow A). Curves are vertically shifted for clarity.

4. Summary We have demonstrated a novel label-free optical sensor for mercury detection in water using a fused silica microsphere. The detection limit of 50 ppb (w/w) has been achieved, which is comparable to results obtained using SPR. Our experiments have also shown that mercuric ions bind to thiol groups on the sphere surface in 1:3 stoichiometry. Furthermore, the detection time for our sensors was only a few minutes, faster than SPRbased sensors. Various control experiments were also carried out to verify the specific binding of Hg(II). Improvements can be made to the detection limit in future experiments. The Q-factor, which determines the sensor spectral resolution and hence the detection limit, can be enhanced by shifting the operating wavelength to the region near 500 nm where the absorption of the light by water surrounding the sphere

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Biographies N.M. Hanumegowda is currently working towards the master degree at the Department of Biological Engineering at the University of Missouri-Columbia. I.M. White received his PhD degree from Stanford University in 2002. From 2002 to 2005, he was principal member technical staff at Sprint Advanced Technology Labs. He is presently a postdoctoral fellow at the Department of Biological Engineering at the University of Missouri-Columbia. His research interest includes optical bio/chemical sensors and nanophotonics. X. Fan received his PhD degree from the University of Oregon, USA. From 2000 to 2004, he worked at Corporate Research Laboratories at 3M Company. He is presently an assistant professor at the Department of Biological Engineering at the University of Missouri-Columbia. His research interest includes optical sensors based on ring resonators and nanophotonics.