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An imaging surface plasmon resonance sensor using polarisation modulation
Sensors and Actuators B 151 (2010) 186–190
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
An imaging surface plasmon resonance sensor using polarisation modulation David Graham, Lionel R. Watkins ∗ Department of Physics, University of Auckland, P.B. 92019, Auckland, New Zealand
a r t i c l e
i n f o
Article history: Received 25 May 2010 Received in revised form 8 September 2010 Accepted 9 September 2010 Available online 18 September 2010 Keywords: Surface plasmon resonance Imaging SPR sensors
a b s t r a c t We describe an imaging SPR instrument that combines the inherent sensitivity of ellipsometric detection methods with the multiplexing ability of camera-based systems. Our approach is to electronically scan the input linear polarisation with the aid of a Faraday rotator and search for the minimum intensity transmitted through an output polariser held at a fixed azimuthal angle. The input polarisation angle at which this occurs is very sensitive to the surface plasmon resonance conditions. Single channel experiments with salt solutions of known refractive index indicate a standard deviation due to noise of 1.7 × 10−6 RIU. Imaging measurements are demonstrated with a patterned sensor and show a similar noise performance over a measurement range of 0.01 RIU and a potential 128 × 48 channels. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Optical sensors based on surface plasmon resonance (SPR) have found wide applications in the measurement and analysis of chemical and biological quantities [1,2]. SPR biosensors, in particular, provide real time, label-free information about reaction kinetics and biological processes [3]. Surface plasmons, which are collective oscillations of the conduction electrons of a metal and their associated evanescent electromagnetic waves, can be excited using diffraction gratings or wave guides [1], but are commonly generated via attenuated total internal reflection in the Kretschmann configuration [4]. The coupling of p-polarised light to this resonance depends strongly on the refractive index of the medium in contact with the metal and is the transduction mechanism for SPR sensors. Measuring the reflectivity of the surface as a function of angle of incidence or wavelength allows small refractive index changes to be detected and was the basis of SPR sensors first demonstrated by Liedberg [5]. Sensitivity to a specific analyte can be achieved by overcoating the metal film with a suitable binding layer and in this way a variety of SPR sensors have been developed with typical resolutions of 10−6 refractive index units (RIU) [1,2]. It is well known that the magnitude and phase of p-polarised light changes sharply through the resonance, while that of s-polarised light does not. Thus, measuring changes in the polarisation ellipse or phase on reflection can lead to sensors with high sensitivity [6,7]. For example, Hooper and Sambles [8] reported an SPR instrument that measured changes in the azimuth of the reflected polarisation ellipse and demonstrated high accuracy
∗ Corresponding author. Tel.: +64 9 373 7599; fax: +64 9 373 7445. E-mail address: [email protected] (L.R. Watkins). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.09.023
refractive index measurements with resolutions (2 standard deviations) of 2 × 10−7 RIU for a silver coated prism with gas samples [8] and better than 5 × 10−7 for gold surfaces with aqueous solutions [9]. Imaging or multi-channel SPR measurements, made with a sensor surface spotted with different sensitising spots, can allow the detection of a large number (100–1000 s) of different interactions in parallel [10–12]. In addition, this allows the use of an unsensitised reference area to enable drifts due to changes in the bulk refractive index or temperature to be corrected. The simplest and most commonly used method for SPR imaging is to monitor the change in intensity of the reflected p-polarised light with a camera. Resolutions of ∼10−5 RIU [13] are typically achieved with this technique, making it significantly less sensitive than single channel angle or wavelength scanned systems. There is therefore considerable interest in developing more sensitive imaging SPR systems [14]. As with single channel systems, the pursuit of greater sensitivity leads to instruments that measure changes in phase or polarisation on reflection. One such approach, using polarisation control to increase the contrast and pairs of multilayer sensing spots, demonstrated a resolution of 2 × 10−6 RIU for 64 independent channels [15,16]. Other approaches have included phase sensitive interferometers [17,18], an array of diffraction gratings [19] and an acousto-optic deflector to modulate the angle of incidence [14]. This last instrument achieved a resolution of 2.6 × 10−8 RIU for 110 channels with a 1 s measurement time. Here we present a novel SPR instrument in which we combine the throughput advantages inherent to imaging with the high sensitivity of ellipsometric measurement. We scan the input polarisation electronically with a Faraday rotator and for each group of pixels in the image, determine the azimuthal angle at which minimum transmission through a fixed analyser occurs. The camera-based
D. Graham, L.R. Watkins / Sensors and Actuators B 151 (2010) 186–190
2. Theory The technique chosen to make high sensitivity imaging SPR measurements was to measure changes in polarisation due to the resonance. Hooper and Sambles [8] have previously demonstrated that single channel SPR measurements can be made by measuring changes in the azimuth of the polarisation ellipse of light reflected from an SPR surface. The minimum transmission through a polarisation analyser occurs when the transmission axis of the analyser is aligned with the minor axis of the ellipse. As Hooper’s instrument achieved high sensitivity and theoretical modelling suggested that good sensitivity could be obtained using a camera, a similar technique was selected for this imaging instrument. For our imaging instrument, light linearly polarised at an azimuthal angle from p-polarised is reflected from a Kretschmann configuration SPR prism. The light is then incident on a polarisation analyser with its transmission axis at an angle from p-polarised and the transmitted light is imaged onto a camera. The input polarisation is scanned and the point at which the transmitted intensity is a minimum, m , is found. This is equivalent to fixing the input polarisation at and finding the setting of the analyser for minimum transmission, m . Consider linearly polarised light with azimuthal angle reflected from the surface of an SPR sensor and then incident on an analyser with its transmission axis at angle . All angles are measured with respect to the horizontal or p-polarised direction. The transmitted electric field amplitude is [8]: T = rp cos cos
+ rs sin sin
.
(1)
where rp , rs are the amplitude Fresnel reflection coefficients for the glass-metal-dielectric layers of a typical Kretschmann configuration. The azimuthal angle m of the input linear polarisation for which the transmitted intensity I = |T|2 is a minimum (or a maximum) is readily found by differentiating T and solving ∂T/∂ = 0: tan 2
m
=
2|rp ||rs | cos sin cos |rp |2 cos2 − |rs |2 sin2
,
20
Input polarisation ψm (degrees)
approach allows flexibility in flow-cell design while the electronic approach to polarisation modulation means that the instrument has no moving parts. Imaging measurements made with a polymer film suggest a sensitivity of around 1.7 × 10−6 RIU over a 0.01 refractive index range.
187
15 10 5 0 −5 −10 −15 −20 −25 −30 −0.005
0
0.005
Δn
0.01
Fig. 1. Input polarisation m vs change in the refractive index of the sensed medium for a typical SPR configuration: BK7 substrate (n = 1.51), 3 nm Cr (N = 3.07 + 3.36i) and 46 nm Au (N = 0.161 + 3.64i) [21] with water as the sensed medium (n = 1.33) and = 680 nm. The angle of incidence is 71.5◦ and the output polariser is at = − 45 ◦ .
is labelled as and that this is not related to the tan measured in conventional ellipsometry. However, if the input polarisation for minimum transmission m is measured for two settings of the output polariser , the ellipsometric angles could be determined with this instrument [20]. 3. Experimental apparatus Fig. 2 shows a schematic of the experimental apparatus used for our SPR imaging measurements. The light source was a superluminescent diode (SLD Q Photonics QSDM680-9, center wavelength 677 nm, spectral width 7.5 nm) chosen because its spectral width was sufficient to suppress spurious interference fringes in the experiment. A Glan-Thompson polariser mounted in a Newport SR50CC rotation stage set the initial polarisation state. The beam passed through a Faraday rotator consisting of a 30 mm long, 5 mm diameter rod of terbium doped borosilicate glass with a Verdet constant V = − 96 rad T−1 m−1 . To increase the available rotation, two small mirrors were used to reflect the beam so that it passed
(2)
where is the phase difference between rp and rS . The |rp | and the relative phase difference change sharply through the resonance and so therefore, does the azimuthal angle m for minimum intensity. We find this angle by electronically scanning the input polarisation with the analyser azimuthal angle fixed and finding the point of minimum intensity. Fig. 1 plots m against changes in refractive index of the the sensed medium n when the analyser is set at = − 45 ◦ for a typical SPR system. It is clear that small changes in refractive index cause large changes in m and it is this relationship that is the basis of our imaging sensor. As the analyser azimuth approaches zero, the relationship between m and n becomes steeper, implying that greater sensitivity might be achievable, albeit for a smaller range of refractive index. However, for small values of , the transmitted intensity does not change very much as is varied, so this increase in polarisation change may not result in a concomitant increase in sensitivity if the system is limited by optical noise. Conventional ellipsometry measures the relative amplitude and phase of the reflection coefficients for p- and s-polarised light. Our technique is simpler and gives a measurement that is approximately linearly related to the refractive index of the sensed medium. Note that the input polarisation azimuthal angle
0.015
Fig. 2. Schematic of the experimental apparatus.
D. Graham, L.R. Watkins / Sensors and Actuators B 151 (2010) 186–190
through the rod three times. The rod was placed within a 700 turn coil of 0.45 mm insulated copper wire, wound around a plastic spool. A 0.3 resistor was placed in series with the coil, enabling the current to be monitored via a 12-bit data acquisition card. The coil was driven by ramp generated by a 12-bit digital-to-analogue convertor, and connected to the coil through an amplifier. The magnetic field within the coil, and thus the polarisation rotation, were proportional to the current in the coil. The average power dissipated by the coil was typically around 20 W, so cooling with the aid of a water jacket around the spool was essential. After the modulator, the beam was expanded and collimated using two lenses with focal lengths of 35 and 70 mm, respectively. The expanded beam was coupled, via a high index prism, to a Kretschmann arrangement consisting of a microscope slide coated on one side with a thin gold film in contact with a flow cell. The reflected beam passed through an analyser (a polarising cube beam splitter) before being imaged onto a CMOS camera via a further pair of lenses. A rectangular aperture between the prism and the analyser blocks light reflected at the prism-slide interface. Microscope slides (n = 1.50) were cleaned with aqua regia, rinsed with distilled water and then wiped with methanol and lens tissue. Following this, they were coated with a 3 nm adhesion layer of chromium and approximately 42 nm of gold by thermal evaporation in a vacuum coater. A slide was clamped between a high index, SF2 glass equilateral prism and a flow cell. Index matching oil between the prism and the slide was used to reduce unwanted reflections from this interface. The flow cell, which was machined from aluminium, was mounted on a temperature controlled baseplate. The entire assembly was mounted on a Newport SR50CC rotation stage, allowing the angle of incidence to be precisely controlled. The flow cell was connected through a selector valve to up to seven 50 ml syringes that contained solutions with slightly differing refractive indices. The volume of the flow cell was approximately 0.2 ml and when changing solutions, at least 5 ml of the next solution was flowed through the cell. The camera was a Silicon video 642 M monochrome CMOS camera (Micron MT9V403 sensor) with 640 × 480 resolution at 8 or 10 bits per pixel and up to 204 frames per second (fps) for the full frame. The 8 bit mode was used for our measurements as the intensity was generally such that the shot noise was larger than the quantisation noise. For the results presented, the camera was run at 640 × 240 resolution to increase the available frame rate. Two 200 mm focal length doublet lenses were positioned to give a focused image and collimated beam on the camera. A circular aperture at the beam waist sets the resolution (R) and depth of field (dof) of the optics. For the imaging results presented here, a 3.5 mm diameter aperture was used, giving R = 47 m and dof = 9 mm. This large depth of field was needed as a tilted surface was being imaged. Since the camera pixel size is 10 m square, the sampling spatial frequency is well above the Nyquist limit and thus aliasing was not a problem. Polarisation measurements were made by averaging 5 × 5 pixel square areas that were approximately the same size as the resolution of the imaging optics. A Matlab program was used to acquire images and to control the experiment. The program calls a function implemented in C++ that records a sequence of 280 images from the camera while the polarisation is scanned by changing the current in the modulator. During each 1 ms exposure, the voltage across the current measuring resistor is recorded. 5 × 5 groups of pixels are summed to give a image with 128 × 48 possible channels. For each channel, the resistor voltage for minimum intensity is found by a quadratic least mean squares fit to a plot of intensity vs voltage. Fig. 3 shows the results of a typical scan. Points for which the intensity is more than 0.6 of full scale are ignored as the camera becomes non-linear near full scale. To convert the resistor voltage into the azimuthal angle of the light leaving the modulator, separate calibration measure-
250 Fitted quadratic
Intensity (Camera output)
188
200 Intensity 150
100
50
0 −0.5 −0.4 −0.3 −0.2 −0.1
0
0.1
0.2
0.3
0.4
0.5
Resistor voltage (V) Fig. 3. Measured intensity vs resistor voltage for a typical scan and the fitted quadratic. The input polarisation was scanned ±20◦ , centered on −10◦ with the output polariser set at 45◦ . The intensity is the mean value for a 5 × 5 group of pixels.
ments were made. For a given, arbitrary sample, minimum intensity occurs when the light leaving the modulator has azimuthal angle m . If we then step the input polariser by some angle, the modulator current must be adjusted in order to maintain the same output polarisation state and hence minimum intensity. Repeating this procedure for a range of input polariser steps enabled the resistor voltage to be converted to the output polarisation azimuthal angle. The acquisition time is 1 s and it takes approximately another second to perform the calculations, yielding a total measurement time of 2 s. It is expected that with better programming the calculation time could be significantly reduced and that it could be done in parallel with the acquisition. 3.1. Refractive index measurements Refractive index measurements were made using the SPR imaging system described above in order to establish its sensitivity. The input polariser was set at 10◦ and the modulator arranged to give ±30◦ of polarisation scan. For these measurements, the output polariser was fixed at +45◦ . Samples of solutions of known refractive index were prepared by mixing 99.9% pure sodium chloride with distilled water to concentrations of 1.00, 2.00, 3.01, 4.04, 5.01 and 6.03% (w/w). The refractive index difference between these solutions and pure water was found by linear fitting to data from the CRC Handbook [22] and were 0.0018, 0.0035, 0.0052, 0.0071, 0.0088 and 0.0106 RIU, respectively. Fig. 4 shows experimental values of m for a typical channel from the center of the 128 × 48 image. Before a different salt solution was introduced, the flow cell was flushed out with pure distilled water. It is clear from the figure that m returns to the same value each time the flow cell is flushed, demonstrating the repeatability and stability of the setup. The small spikes evident each time a new solution is introduced into the flow cell are due to small changes in temperature. Fig. 5 shows, for the same channel, the change in m plotted against the change in refractive index. The linear response of the sensor is clearly evident. For the total 0.0106 RIU change, the polarisation change, for all 128 × 48 channels, was −25.3 ± 2.3 ◦ , yielding a sensitivity of ( − 2380 ± 220) ◦ RIU−1 . The variation in sensitivity is believed to be due to variations in the thickness and roughness of the gold surface. The noise was calculated for each channel from the standard deviation of the first 1 min of measurement and gave a mean of 0.0041◦ or 1.7 × 10−6 RIU. If the first 10 min of measurement are used to calculate the noise, this gave 0.0084◦ or 3.51 × 10−6 RIU, with the difference being ascribed to slow drifts.
D. Graham, L.R. Watkins / Sensors and Actuators B 151 (2010) 186–190
−4.5
0 −5
n=nw
Solution 1
n=nw −5
Input polarisation ψm (degrees)
Input polarisation ψm (degrees)
10 5
n=nw+0.0018 n=n +0.0035 w
n=nw+0.0052
−10
n=nw+0.0071
−15
n=n +0.0088 w
−20
n=n +0.0106 w
−25 0
189
10
20
30
40
50
60
Uncoated
−5.5
−6
Δ n =0.0006 −6.5
−7
Solution 2
−7.5
Time minutes
Coated
Fig. 4. m for a typical channel with water (nw ) and a sequence of salt solutions of varying concentration.
−8
0
2
4
6
8
10
Fig. 6. Sensor response for coated and uncoated channels with two salt solutions.
3.2. Imaging measurements Measurements were made using a patterned slide to demonstrate that the instrument can image a spatially varying refractive index change. In order to create an area of constant refractive index, drops of fluoropolymer solution (FluoroPel 802A from Cytonix) were placed on the gold surface of one of the microscope slides with a syringe and the slide dried in an oven at 80 ◦ C for 15 min. This left a thin film with refractive index n ∼ 1.37 on circular areas of the sample surface of a thickness sufficient to ensure that the surface plasmon decayed entirely within this layer. We found that best results were obtained if the fluoropolymer solvent was allowed to evaporate until its volume had reduced to 1/5 of the original volume before it was applied to the surface. Imaging measurements were made by alternately flowing one of two different salt solutions through the flow cell. The solutions were 21.7% and 22.0% NaCl (w/w), respectively. From a linear fit to data from the CRC handbook [22], the refractive indices of these solutions were 1.3715 and 1.3721 RIU. When the salt solutions are exchanged, the refractive index and thus m for the uncoated areas will change while that for the coated areas will stay constant. For these measurements, the input polariser was set at 10◦ , the output polariser at 45◦ and the modulator provided ±20◦ of polarisation scan.
Experimental values for m for two typical channels, one from an uncoated area and the other coated, are shown in Fig. 6. As expected, m for the coated area stays almost constant but changes significantly for the uncoated area when the solutions in the flow cell are exchanged. As noted earlier, the spikes in the plot are produced by temperature changes when the solutions are exchanged. Fig. 7 shows an image of the sensor surface with salt solution 1 in the flow cell. The gray scale indicates values of m , as shown in the side bar. Not much difference in refractive index can be seen between the coated and uncoated regions since the fluoropolymer’s refractive index closely approximates that of the salt solution. The roughness of the gold film, which was thermal evaporated, is apparent and is believed to have been caused by baking the slide. A similar image was obtained with the second salt solution in the flow cell and the two images subtracted from each other. The result of this can be seen in Fig. 8. Here the gray scale indicates changes in m with black, which corresponds to m = 0, coinciding with the coated areas of the sensor. Noise measurements were made in a similar manner to that described earlier and, in a one minute interval, was typically 1.3 × 10−6 RIU for the uncoated channels and 1.0 × 10−6 RIU for the coated channels.
Input polarisation change Δ ψm (degrees)
5 0 −5 −10 −15 −20 −25 −30 0
0.002
0.004
12
Time minutes
0.006
0.008
0.01
0.012
Refractive index change Fig. 5. Change in m vs refractive index change for one channel from the center of the image. The straight line fit demonstrates the linear nature of the sensor response.
Fig. 7. Image measured with solution 1: 21.7% NaCl (w/w).
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D. Graham, L.R. Watkins / Sensors and Actuators B 151 (2010) 186–190
References
Fig. 8. Change in subtracted.
m
for salt solutions 1 and 2 when the corresponding images are
4. Conclusion We have demonstrated an imaging SPR instrument that uses ellipsometric methods for refractive index sensing. We fix the output polariser and scan, via a Faraday rotator, the input linear polarisation. The input polarisation azimuthal angle at which minimum transmission occurs changes rapidly through resonance and is a sensitive function of refractive index. While camera based detection schemes typically have lower quantum efficiencies and poorer signal to noise ratios than photodiodes, they nonetheless provide a straight forward way of implementing an array of sensors. Our camera was an inexpensive CMOS device, offering 640 × 480 pixels at up to 204 frames per second. 5 × 5 groups of pixels were averaged together to form a single measurement channel, yielding a potential 128 × 48 possible channels. Using standard solutions of salt with known refractive indices, the experimentally determined sensitivity of our imaging instrument was ( − 2380 ± 220) ◦ RIU−1 with a standard deviation of 1.7 × 10−6 RIU over the 0.0106 RIU linear range of our salt solutions. Imaging measurements were demonstrated through the use of a patterned slide which contained areas of constant refractive index, created by depositing relatively thick films of a fluoropolymer. Images were acquired when each of two different salt solutions were flowed over the sensor. The difference in these images showed no change in m for those areas coated with fluorpolymer, as expected, and approximately 2.4◦ change for the uncoated areas. The corresponding sensitivity was 3900◦ RIU−1 , with a refractive index noise of 1.7 × 10−6 RIU for a potential 128 × 48 channels. Our polarisation imaging instrument has demonstrated good accuracy over a large number of channels using a simple optical system. It was found that little reduction in the refractive index noise could be obtained by averaging groups of channels. An obvious direction for future work is to analyse the cause of this noise limit and to improve the accuracy achievable.
[1] J. Homola, S. Yee, G. Gauglitz, Surface plasmon resonance sensors: review, Sens. Actuators B 54 (1999) 3–15. [2] J. Homola, Present and future of surface plasmon resonance biosensors, Anal. Bioanal. Chem. 377 (3) (2003) 528–539. [3] T. Morton, D. Myszka, Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors, Method Enzymol. 295 (1998) 268–294. [4] E. Kretschmann, H. Raether, Radiative decay of non radiative surface plasmons excited by light, Z. Naturforsch Pt A 23 (1968) 2135–2136. [5] B. Liedberg, C. Nylander, I. Lundström, Surface plasmon resonance for gas detection and biosensing, Sens. Actuators 4 (1983) 299. [6] A. Kruchinin, Y. Vlasov, Surface plasmon resonance monitoring by means of polarization state measurement in reflected light as the basis of a DNA-probe biosensor, Sens. Actuators B 30 (1996) 77–80. [7] H.M. Cho, W. Chegal, Y.J. Cho, Y.P. Kim, H.S. Kim, Enhancement of biomolecular detection sensitivity by surface plasmon resonance ellipsometry, Proc. SPIE 6008 (2005). [8] I.R. Hooper, J.R. Sambles, Sensing using differential surface plasmon ellipsometry, J. Appl. Phys. 96 (2004) 3004–3011. [9] C.E. Stewart, I.R. Hooper, J.R. Sambles, Surface plasmon differential ellipsometry of aqueous solutions for bio-chemical sensing, J. Phys. D: Appl. Phys. 41 (2008) 105408. [10] G. Steiner, Surface plasmon resonance imaging, Anal. Bioanal. Chem. 379 (2004) 328–331. [11] G.J. Wegner, A.W. Wark, H.J. Lee, E. Codner, T. Saeki, S. Fang, R.M. Corn, Real-time surface plasmon resonance imaging measurements for the multiplexed determination of protein adsorption/desorption kinetics and surface enzymatic reactions on peptide microarrays, Anal. Chem. 76 (2004) 5677–5684. [12] C.T. Campbell, G. Kim, SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics, Biomaterials 28 (2007) 2380–2392. [13] J.S. Shumaker-Parry, C.T. Campbell, Quantitative methods for spatially resolved adsorption/desorption measurements in real time by surface plasmon resonance microscopy, Anal. Chem. 76 (2004) 907–917. [14] G.D. VanWiggeren, M.A. Bynum, J.P. Ertel, S. Jefferson, K.M. Robotti, E.P. Thrush, D.M. Baney, K.P. Killeen, A novel optical method providing for high-sensitivity and high-throughput biomolecular interaction analysis, Sens. Actuators B 127 (2007) 341–349. [15] M. Piliarik, H. Vaisocherová, J. Homola, A new surface plasmon resonance sensor for high-throughput screening applications, Biosens. Bioelectron. 20 (2005) 2104–2110. [16] M. Piliarik, H. Vaisocherová, J. Homola, Towards parallelized surface plasmon resonance sensor platform for sensitive detection of oligonucleotides, Sens. Actuators B 121 (2007) 187–193. [17] A.G. Notcovich, V. Zhuk, S.G. Lipson, Surface plasmon resonance phase imaging, Appl. Phys. Lett. 76 (13) (2000) 1665–1667. [18] S.-J. Chen, Y.-D. Su, F.-M. Hsiu, C.-Y. Tsou, Y.-K. Chen, Surface plasmon resonance phase-shift interferometry: real-time DNA microarray hybridization analysis, J. Biomed. Opt. 10 (3) (2005) 034005. [19] J. Dostálek, J. Homola, Surface plasmon resonance sensor based on an array of diffraction gratings for highly parallelized observation of biomolecular interactions, Sens. Actuators B 129 (2008) 303–310. [20] S.C. Som, C. Chowdhury, New ellipsometric method for the determination of the optical constants of thin films and surfaces, J. Opt. Soc. Am. 62 (1972) 10–15. [21] E.D. Palik, Handbook of Optical Constants of Solids, Academic Press, San Diego, 1998. [22] D.R. Lide, CRC Handbook of Chemistry and Physics, 88th ed., CRC Press/Taylor and Francis, 2008.
Biographies David Graham received an MSc degree in physics from the University of Auckland in 2005. He is currently a PhD candidate at the University of Auckland having worked on SPR sensors. Lionel Watkins received his PhD from the University of Wales, UK. His research interests include ellipsometry and optical interferometry.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
An imaging surface plasmon resonance sensor using polarisation modulation David Graham, Lionel R. Watkins ∗ Department of Physics, University of Auckland, P.B. 92019, Auckland, New Zealand
a r t i c l e
i n f o
Article history: Received 25 May 2010 Received in revised form 8 September 2010 Accepted 9 September 2010 Available online 18 September 2010 Keywords: Surface plasmon resonance Imaging SPR sensors
a b s t r a c t We describe an imaging SPR instrument that combines the inherent sensitivity of ellipsometric detection methods with the multiplexing ability of camera-based systems. Our approach is to electronically scan the input linear polarisation with the aid of a Faraday rotator and search for the minimum intensity transmitted through an output polariser held at a fixed azimuthal angle. The input polarisation angle at which this occurs is very sensitive to the surface plasmon resonance conditions. Single channel experiments with salt solutions of known refractive index indicate a standard deviation due to noise of 1.7 × 10−6 RIU. Imaging measurements are demonstrated with a patterned sensor and show a similar noise performance over a measurement range of 0.01 RIU and a potential 128 × 48 channels. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Optical sensors based on surface plasmon resonance (SPR) have found wide applications in the measurement and analysis of chemical and biological quantities [1,2]. SPR biosensors, in particular, provide real time, label-free information about reaction kinetics and biological processes [3]. Surface plasmons, which are collective oscillations of the conduction electrons of a metal and their associated evanescent electromagnetic waves, can be excited using diffraction gratings or wave guides [1], but are commonly generated via attenuated total internal reflection in the Kretschmann configuration [4]. The coupling of p-polarised light to this resonance depends strongly on the refractive index of the medium in contact with the metal and is the transduction mechanism for SPR sensors. Measuring the reflectivity of the surface as a function of angle of incidence or wavelength allows small refractive index changes to be detected and was the basis of SPR sensors first demonstrated by Liedberg [5]. Sensitivity to a specific analyte can be achieved by overcoating the metal film with a suitable binding layer and in this way a variety of SPR sensors have been developed with typical resolutions of 10−6 refractive index units (RIU) [1,2]. It is well known that the magnitude and phase of p-polarised light changes sharply through the resonance, while that of s-polarised light does not. Thus, measuring changes in the polarisation ellipse or phase on reflection can lead to sensors with high sensitivity [6,7]. For example, Hooper and Sambles [8] reported an SPR instrument that measured changes in the azimuth of the reflected polarisation ellipse and demonstrated high accuracy
∗ Corresponding author. Tel.: +64 9 373 7599; fax: +64 9 373 7445. E-mail address: [email protected] (L.R. Watkins). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.09.023
refractive index measurements with resolutions (2 standard deviations) of 2 × 10−7 RIU for a silver coated prism with gas samples [8] and better than 5 × 10−7 for gold surfaces with aqueous solutions [9]. Imaging or multi-channel SPR measurements, made with a sensor surface spotted with different sensitising spots, can allow the detection of a large number (100–1000 s) of different interactions in parallel [10–12]. In addition, this allows the use of an unsensitised reference area to enable drifts due to changes in the bulk refractive index or temperature to be corrected. The simplest and most commonly used method for SPR imaging is to monitor the change in intensity of the reflected p-polarised light with a camera. Resolutions of ∼10−5 RIU [13] are typically achieved with this technique, making it significantly less sensitive than single channel angle or wavelength scanned systems. There is therefore considerable interest in developing more sensitive imaging SPR systems [14]. As with single channel systems, the pursuit of greater sensitivity leads to instruments that measure changes in phase or polarisation on reflection. One such approach, using polarisation control to increase the contrast and pairs of multilayer sensing spots, demonstrated a resolution of 2 × 10−6 RIU for 64 independent channels [15,16]. Other approaches have included phase sensitive interferometers [17,18], an array of diffraction gratings [19] and an acousto-optic deflector to modulate the angle of incidence [14]. This last instrument achieved a resolution of 2.6 × 10−8 RIU for 110 channels with a 1 s measurement time. Here we present a novel SPR instrument in which we combine the throughput advantages inherent to imaging with the high sensitivity of ellipsometric measurement. We scan the input polarisation electronically with a Faraday rotator and for each group of pixels in the image, determine the azimuthal angle at which minimum transmission through a fixed analyser occurs. The camera-based
D. Graham, L.R. Watkins / Sensors and Actuators B 151 (2010) 186–190
2. Theory The technique chosen to make high sensitivity imaging SPR measurements was to measure changes in polarisation due to the resonance. Hooper and Sambles [8] have previously demonstrated that single channel SPR measurements can be made by measuring changes in the azimuth of the polarisation ellipse of light reflected from an SPR surface. The minimum transmission through a polarisation analyser occurs when the transmission axis of the analyser is aligned with the minor axis of the ellipse. As Hooper’s instrument achieved high sensitivity and theoretical modelling suggested that good sensitivity could be obtained using a camera, a similar technique was selected for this imaging instrument. For our imaging instrument, light linearly polarised at an azimuthal angle from p-polarised is reflected from a Kretschmann configuration SPR prism. The light is then incident on a polarisation analyser with its transmission axis at an angle from p-polarised and the transmitted light is imaged onto a camera. The input polarisation is scanned and the point at which the transmitted intensity is a minimum, m , is found. This is equivalent to fixing the input polarisation at and finding the setting of the analyser for minimum transmission, m . Consider linearly polarised light with azimuthal angle reflected from the surface of an SPR sensor and then incident on an analyser with its transmission axis at angle . All angles are measured with respect to the horizontal or p-polarised direction. The transmitted electric field amplitude is [8]: T = rp cos cos
+ rs sin sin
.
(1)
where rp , rs are the amplitude Fresnel reflection coefficients for the glass-metal-dielectric layers of a typical Kretschmann configuration. The azimuthal angle m of the input linear polarisation for which the transmitted intensity I = |T|2 is a minimum (or a maximum) is readily found by differentiating T and solving ∂T/∂ = 0: tan 2
m
=
2|rp ||rs | cos sin cos |rp |2 cos2 − |rs |2 sin2
,
20
Input polarisation ψm (degrees)
approach allows flexibility in flow-cell design while the electronic approach to polarisation modulation means that the instrument has no moving parts. Imaging measurements made with a polymer film suggest a sensitivity of around 1.7 × 10−6 RIU over a 0.01 refractive index range.
187
15 10 5 0 −5 −10 −15 −20 −25 −30 −0.005
0
0.005
Δn
0.01
Fig. 1. Input polarisation m vs change in the refractive index of the sensed medium for a typical SPR configuration: BK7 substrate (n = 1.51), 3 nm Cr (N = 3.07 + 3.36i) and 46 nm Au (N = 0.161 + 3.64i) [21] with water as the sensed medium (n = 1.33) and = 680 nm. The angle of incidence is 71.5◦ and the output polariser is at = − 45 ◦ .
is labelled as and that this is not related to the tan measured in conventional ellipsometry. However, if the input polarisation for minimum transmission m is measured for two settings of the output polariser , the ellipsometric angles could be determined with this instrument [20]. 3. Experimental apparatus Fig. 2 shows a schematic of the experimental apparatus used for our SPR imaging measurements. The light source was a superluminescent diode (SLD Q Photonics QSDM680-9, center wavelength 677 nm, spectral width 7.5 nm) chosen because its spectral width was sufficient to suppress spurious interference fringes in the experiment. A Glan-Thompson polariser mounted in a Newport SR50CC rotation stage set the initial polarisation state. The beam passed through a Faraday rotator consisting of a 30 mm long, 5 mm diameter rod of terbium doped borosilicate glass with a Verdet constant V = − 96 rad T−1 m−1 . To increase the available rotation, two small mirrors were used to reflect the beam so that it passed
(2)
where is the phase difference between rp and rS . The |rp | and the relative phase difference change sharply through the resonance and so therefore, does the azimuthal angle m for minimum intensity. We find this angle by electronically scanning the input polarisation with the analyser azimuthal angle fixed and finding the point of minimum intensity. Fig. 1 plots m against changes in refractive index of the the sensed medium n when the analyser is set at = − 45 ◦ for a typical SPR system. It is clear that small changes in refractive index cause large changes in m and it is this relationship that is the basis of our imaging sensor. As the analyser azimuth approaches zero, the relationship between m and n becomes steeper, implying that greater sensitivity might be achievable, albeit for a smaller range of refractive index. However, for small values of , the transmitted intensity does not change very much as is varied, so this increase in polarisation change may not result in a concomitant increase in sensitivity if the system is limited by optical noise. Conventional ellipsometry measures the relative amplitude and phase of the reflection coefficients for p- and s-polarised light. Our technique is simpler and gives a measurement that is approximately linearly related to the refractive index of the sensed medium. Note that the input polarisation azimuthal angle
0.015
Fig. 2. Schematic of the experimental apparatus.
D. Graham, L.R. Watkins / Sensors and Actuators B 151 (2010) 186–190
through the rod three times. The rod was placed within a 700 turn coil of 0.45 mm insulated copper wire, wound around a plastic spool. A 0.3 resistor was placed in series with the coil, enabling the current to be monitored via a 12-bit data acquisition card. The coil was driven by ramp generated by a 12-bit digital-to-analogue convertor, and connected to the coil through an amplifier. The magnetic field within the coil, and thus the polarisation rotation, were proportional to the current in the coil. The average power dissipated by the coil was typically around 20 W, so cooling with the aid of a water jacket around the spool was essential. After the modulator, the beam was expanded and collimated using two lenses with focal lengths of 35 and 70 mm, respectively. The expanded beam was coupled, via a high index prism, to a Kretschmann arrangement consisting of a microscope slide coated on one side with a thin gold film in contact with a flow cell. The reflected beam passed through an analyser (a polarising cube beam splitter) before being imaged onto a CMOS camera via a further pair of lenses. A rectangular aperture between the prism and the analyser blocks light reflected at the prism-slide interface. Microscope slides (n = 1.50) were cleaned with aqua regia, rinsed with distilled water and then wiped with methanol and lens tissue. Following this, they were coated with a 3 nm adhesion layer of chromium and approximately 42 nm of gold by thermal evaporation in a vacuum coater. A slide was clamped between a high index, SF2 glass equilateral prism and a flow cell. Index matching oil between the prism and the slide was used to reduce unwanted reflections from this interface. The flow cell, which was machined from aluminium, was mounted on a temperature controlled baseplate. The entire assembly was mounted on a Newport SR50CC rotation stage, allowing the angle of incidence to be precisely controlled. The flow cell was connected through a selector valve to up to seven 50 ml syringes that contained solutions with slightly differing refractive indices. The volume of the flow cell was approximately 0.2 ml and when changing solutions, at least 5 ml of the next solution was flowed through the cell. The camera was a Silicon video 642 M monochrome CMOS camera (Micron MT9V403 sensor) with 640 × 480 resolution at 8 or 10 bits per pixel and up to 204 frames per second (fps) for the full frame. The 8 bit mode was used for our measurements as the intensity was generally such that the shot noise was larger than the quantisation noise. For the results presented, the camera was run at 640 × 240 resolution to increase the available frame rate. Two 200 mm focal length doublet lenses were positioned to give a focused image and collimated beam on the camera. A circular aperture at the beam waist sets the resolution (R) and depth of field (dof) of the optics. For the imaging results presented here, a 3.5 mm diameter aperture was used, giving R = 47 m and dof = 9 mm. This large depth of field was needed as a tilted surface was being imaged. Since the camera pixel size is 10 m square, the sampling spatial frequency is well above the Nyquist limit and thus aliasing was not a problem. Polarisation measurements were made by averaging 5 × 5 pixel square areas that were approximately the same size as the resolution of the imaging optics. A Matlab program was used to acquire images and to control the experiment. The program calls a function implemented in C++ that records a sequence of 280 images from the camera while the polarisation is scanned by changing the current in the modulator. During each 1 ms exposure, the voltage across the current measuring resistor is recorded. 5 × 5 groups of pixels are summed to give a image with 128 × 48 possible channels. For each channel, the resistor voltage for minimum intensity is found by a quadratic least mean squares fit to a plot of intensity vs voltage. Fig. 3 shows the results of a typical scan. Points for which the intensity is more than 0.6 of full scale are ignored as the camera becomes non-linear near full scale. To convert the resistor voltage into the azimuthal angle of the light leaving the modulator, separate calibration measure-
250 Fitted quadratic
Intensity (Camera output)
188
200 Intensity 150
100
50
0 −0.5 −0.4 −0.3 −0.2 −0.1
0
0.1
0.2
0.3
0.4
0.5
Resistor voltage (V) Fig. 3. Measured intensity vs resistor voltage for a typical scan and the fitted quadratic. The input polarisation was scanned ±20◦ , centered on −10◦ with the output polariser set at 45◦ . The intensity is the mean value for a 5 × 5 group of pixels.
ments were made. For a given, arbitrary sample, minimum intensity occurs when the light leaving the modulator has azimuthal angle m . If we then step the input polariser by some angle, the modulator current must be adjusted in order to maintain the same output polarisation state and hence minimum intensity. Repeating this procedure for a range of input polariser steps enabled the resistor voltage to be converted to the output polarisation azimuthal angle. The acquisition time is 1 s and it takes approximately another second to perform the calculations, yielding a total measurement time of 2 s. It is expected that with better programming the calculation time could be significantly reduced and that it could be done in parallel with the acquisition. 3.1. Refractive index measurements Refractive index measurements were made using the SPR imaging system described above in order to establish its sensitivity. The input polariser was set at 10◦ and the modulator arranged to give ±30◦ of polarisation scan. For these measurements, the output polariser was fixed at +45◦ . Samples of solutions of known refractive index were prepared by mixing 99.9% pure sodium chloride with distilled water to concentrations of 1.00, 2.00, 3.01, 4.04, 5.01 and 6.03% (w/w). The refractive index difference between these solutions and pure water was found by linear fitting to data from the CRC Handbook [22] and were 0.0018, 0.0035, 0.0052, 0.0071, 0.0088 and 0.0106 RIU, respectively. Fig. 4 shows experimental values of m for a typical channel from the center of the 128 × 48 image. Before a different salt solution was introduced, the flow cell was flushed out with pure distilled water. It is clear from the figure that m returns to the same value each time the flow cell is flushed, demonstrating the repeatability and stability of the setup. The small spikes evident each time a new solution is introduced into the flow cell are due to small changes in temperature. Fig. 5 shows, for the same channel, the change in m plotted against the change in refractive index. The linear response of the sensor is clearly evident. For the total 0.0106 RIU change, the polarisation change, for all 128 × 48 channels, was −25.3 ± 2.3 ◦ , yielding a sensitivity of ( − 2380 ± 220) ◦ RIU−1 . The variation in sensitivity is believed to be due to variations in the thickness and roughness of the gold surface. The noise was calculated for each channel from the standard deviation of the first 1 min of measurement and gave a mean of 0.0041◦ or 1.7 × 10−6 RIU. If the first 10 min of measurement are used to calculate the noise, this gave 0.0084◦ or 3.51 × 10−6 RIU, with the difference being ascribed to slow drifts.
D. Graham, L.R. Watkins / Sensors and Actuators B 151 (2010) 186–190
−4.5
0 −5
n=nw
Solution 1
n=nw −5
Input polarisation ψm (degrees)
Input polarisation ψm (degrees)
10 5
n=nw+0.0018 n=n +0.0035 w
n=nw+0.0052
−10
n=nw+0.0071
−15
n=n +0.0088 w
−20
n=n +0.0106 w
−25 0
189
10
20
30
40
50
60
Uncoated
−5.5
−6
Δ n =0.0006 −6.5
−7
Solution 2
−7.5
Time minutes
Coated
Fig. 4. m for a typical channel with water (nw ) and a sequence of salt solutions of varying concentration.
−8
0
2
4
6
8
10
Fig. 6. Sensor response for coated and uncoated channels with two salt solutions.
3.2. Imaging measurements Measurements were made using a patterned slide to demonstrate that the instrument can image a spatially varying refractive index change. In order to create an area of constant refractive index, drops of fluoropolymer solution (FluoroPel 802A from Cytonix) were placed on the gold surface of one of the microscope slides with a syringe and the slide dried in an oven at 80 ◦ C for 15 min. This left a thin film with refractive index n ∼ 1.37 on circular areas of the sample surface of a thickness sufficient to ensure that the surface plasmon decayed entirely within this layer. We found that best results were obtained if the fluoropolymer solvent was allowed to evaporate until its volume had reduced to 1/5 of the original volume before it was applied to the surface. Imaging measurements were made by alternately flowing one of two different salt solutions through the flow cell. The solutions were 21.7% and 22.0% NaCl (w/w), respectively. From a linear fit to data from the CRC handbook [22], the refractive indices of these solutions were 1.3715 and 1.3721 RIU. When the salt solutions are exchanged, the refractive index and thus m for the uncoated areas will change while that for the coated areas will stay constant. For these measurements, the input polariser was set at 10◦ , the output polariser at 45◦ and the modulator provided ±20◦ of polarisation scan.
Experimental values for m for two typical channels, one from an uncoated area and the other coated, are shown in Fig. 6. As expected, m for the coated area stays almost constant but changes significantly for the uncoated area when the solutions in the flow cell are exchanged. As noted earlier, the spikes in the plot are produced by temperature changes when the solutions are exchanged. Fig. 7 shows an image of the sensor surface with salt solution 1 in the flow cell. The gray scale indicates values of m , as shown in the side bar. Not much difference in refractive index can be seen between the coated and uncoated regions since the fluoropolymer’s refractive index closely approximates that of the salt solution. The roughness of the gold film, which was thermal evaporated, is apparent and is believed to have been caused by baking the slide. A similar image was obtained with the second salt solution in the flow cell and the two images subtracted from each other. The result of this can be seen in Fig. 8. Here the gray scale indicates changes in m with black, which corresponds to m = 0, coinciding with the coated areas of the sensor. Noise measurements were made in a similar manner to that described earlier and, in a one minute interval, was typically 1.3 × 10−6 RIU for the uncoated channels and 1.0 × 10−6 RIU for the coated channels.
Input polarisation change Δ ψm (degrees)
5 0 −5 −10 −15 −20 −25 −30 0
0.002
0.004
12
Time minutes
0.006
0.008
0.01
0.012
Refractive index change Fig. 5. Change in m vs refractive index change for one channel from the center of the image. The straight line fit demonstrates the linear nature of the sensor response.
Fig. 7. Image measured with solution 1: 21.7% NaCl (w/w).
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References
Fig. 8. Change in subtracted.
m
for salt solutions 1 and 2 when the corresponding images are
4. Conclusion We have demonstrated an imaging SPR instrument that uses ellipsometric methods for refractive index sensing. We fix the output polariser and scan, via a Faraday rotator, the input linear polarisation. The input polarisation azimuthal angle at which minimum transmission occurs changes rapidly through resonance and is a sensitive function of refractive index. While camera based detection schemes typically have lower quantum efficiencies and poorer signal to noise ratios than photodiodes, they nonetheless provide a straight forward way of implementing an array of sensors. Our camera was an inexpensive CMOS device, offering 640 × 480 pixels at up to 204 frames per second. 5 × 5 groups of pixels were averaged together to form a single measurement channel, yielding a potential 128 × 48 possible channels. Using standard solutions of salt with known refractive indices, the experimentally determined sensitivity of our imaging instrument was ( − 2380 ± 220) ◦ RIU−1 with a standard deviation of 1.7 × 10−6 RIU over the 0.0106 RIU linear range of our salt solutions. Imaging measurements were demonstrated through the use of a patterned slide which contained areas of constant refractive index, created by depositing relatively thick films of a fluoropolymer. Images were acquired when each of two different salt solutions were flowed over the sensor. The difference in these images showed no change in m for those areas coated with fluorpolymer, as expected, and approximately 2.4◦ change for the uncoated areas. The corresponding sensitivity was 3900◦ RIU−1 , with a refractive index noise of 1.7 × 10−6 RIU for a potential 128 × 48 channels. Our polarisation imaging instrument has demonstrated good accuracy over a large number of channels using a simple optical system. It was found that little reduction in the refractive index noise could be obtained by averaging groups of channels. An obvious direction for future work is to analyse the cause of this noise limit and to improve the accuracy achievable.
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Biographies David Graham received an MSc degree in physics from the University of Auckland in 2005. He is currently a PhD candidate at the University of Auckland having worked on SPR sensors. Lionel Watkins received his PhD from the University of Wales, UK. His research interests include ellipsometry and optical interferometry.