Anal. Chem. 2007, 79, 4233-4236
Enhancement of the Detection Power of Surface Plasmon Resonance Measurements by Optimization of the Reflection Angle Alexander Zybin,*,† Daniel Boecker,† Vladimir M. Mirsky,‡ and Kay Niemax†
ISAS-Institute for Analytical Sciences at the University of Dortmund, Bunsen-Kirchhoff-Strasse 11, D-44139 Dortmund, Germany, and Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany
The detection limit of surface plasmon resonance (SPR) measurements has been improved by a factor of ∼2-3.5 if the angle of incidence was near to the reflection minimum of the SPR resonance curve instead at the position of the steepest slope, the standard alignment in SPR imaging. The enhancement of the detection power, a result of signal-to-noise optimization, is demonstrated by applying a photodiode and a CCD camera for SPR detection. The experimental data are compared with data expected from theory. In recent years, analytical instruments based on the surface plasmon resonance (SPR) effect have become routine tools for label-free affinity measurements. The high detection power delivered by sophisticated instrumentation and the possibility of solving various bioanalytical problems provided a rapid increase of application areas of this technique.1 Currently, imaging SPR methods are in the focus of development since there is an increasing demand for bioanalytical high-throughput measurements. Several instrumental arrangements for SPR imaging have been proposed and tested.2-4 In most cases, the SPR surface is illuminated by a collimated monochromatic laser beam. The incidence angle is tuned to the slope of the SPR resonance curve so that any shift of the curve causes a variation of the reflected intensity. Binding reactions on the surface can be simultaneously characterized in many areas if the reflected light is measured by a CCD camera. The strongest signal variation caused by a shift of the resonance curve can be observed if the incidence angle has been tuned to the steepest part of the SPR curve where the sensitivity, defined as s ) dR/dn (R, reflectivity; n, refractive index), has its maximum value. Therefore, this angle is usually used for SPR measurements.4,5 It provides the lowest detection limits if the noise is independent of the position on the reflectivity curve. This is * To whom correspondence should be addressed. E-mail:
[email protected]. † University of Dortmund. ‡ University of Regensburg. (1) Homola, J. Springer Series in Chemical Sensors and Biosensors; SpringerVerlag: Berlin, 2006; Vol. 4. (2) Ro ¨thenha¨user, J. B.; Knoll, W. Nature 1988, 332, 615. (3) http://www.ibis-spr.nl. (4) Shumaker-Parry, J. S.; Campbell, C. T. Anal. Chem. 2004, 76, 907. (5) Nelson, B. P.; Grimsrud, T. E.; Liles, M. R.; Goodman, R. M.; Corn, R. M. Anal. Chem. 2001, 73, 1. 10.1021/ac070074u CCC: $37.00 Published on Web 04/24/2007
© 2007 American Chemical Society
the case, for instance, if the digitalization of the photodetector current is determining the noise level. However, very often the noise depends on the reflected intensity, in particular when shot noise or fluctuations of the laser intensity are the limitations. Therefore, the noise dependence of the reflected intensity on the incidence angle should be taken into account for optimization of the detection limit when, e.g., the shot noise limits the measurement. The present experiment applying alternatively a photodiode or a CCD camera as detectors will show that the optimum angle is different if the signal-to-noise ratio (S/N ratio) instead of the signal alone is taken into account. The influence of intensity fluctuations of the laser and the dynamic range of CCD application will be discussed. It will be demonstrated that the detection power of SPR analysis is significantly enhanced by the optimization of the angle of incidence. Furthermore, the dynamic ranges of the calibration curves measured at the S/N optimized incidence angle and at the angle providing the maximum signal are investigated and compared with theory. The possibility of improving the detection limit of SPR analyses by reflection angle optimization was already pointed out in a very recent paper in this journal.6 However, no experimental verification was given in that publication. EXPERIMENTAL SECTION Two laser diodes operating at λ ) 780 (GHO781JA2C, Sharp) and 850 nm (SDL-5411-G, Spectra Diode Labs) were applied for the measurements with the photodiode (ORC-25CL) and the CCD camera (MV 14-285 by Soliton, Gilching, Germany), respectively. The laser diodes were used to illuminate the gold-coated SPR prism (gold layer: 48 nm with additional chromium adhesion layer of 2-nm thickness). The reflected light was measured either by the photodiode or by the CCD camera. In the latter case, a lens expanded the laser beam to a diameter of ∼3 cm (fwhm) allowing imaging measurements with the CCD. The laser diode was driven by a commercial power supply (type ITC-502, Profile). Two polarizers placed directly before and behind the prism were used for polarization correction of the light. Pure water and a test analyte (NaCl salt solution) were alternately pumped through a flow cell attached to the SPR prism. (6) Boecker, D.; Zybin, A.; Horvatic, V.; Grunwald, C.; Niemax, K. Anal. Chem. 2007, 79, 702.
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The cell was made out of a Teflon block with engraved channels, which was tightly pressed against the gold layer. A peristaltic pump provided the flow through the cell. The SPR prism was mounted on a rotating platform for precise angle adjustment. The reflected light from the SPR surface was imaged onto the Soliton 1024 × 1360 pixels CCD camera using a f ) 5 cm lens. The charge capacity of a single pixel of the camera was 36 000 electrons. No imaging lens was used when the silicon photodiode was used as detector. Here, the laser beam was modulated by a chopper (modulation frequency, 1 kHz), and the detector signal was amplified by a lock-in amplifier (SR 830, Stanford Research). THEORETICAL CONSIDERATIONS Limitations of SPR Analyses by Noise. In SPR measurements, the signal caused by a change of the index of refraction ∆n can be expressed as
S ) η(d(I0R)/dn) ∆n ) ηI0(dR/dn) ∆n
(1)
where η is the quantum efficiency of the photodetector and I0 is the illumination intensity. Depending on the experimental parameters, there are different noise sources that determine the limit of detection. These sources will be discussed below. (a) Shot Noise Limitation. The shot noise can be written as
N ) xηI0R
(2)
Taking (1) and (2) into account, the S/N ratio is given by
S/N ) xηI0/R (dR/dn) ∆n
(3)
Both signal and noise are considered in terms of the photoelectron numbers generated in the photodetector. Since both, R and dR/dn, are wavelength and angle dependent,
dR/dn xR
(4)
has to be optimized with regard to the wavelength or the reflection angle for a given illumination intensity. (b) Noise Limitations with CCD Detectors. Applying a CCD detector, the laser intensity can only be increased until the pixel capacities are saturated by the reflected light. A few microwatts of radiation power distributed over the area of 106 pixels is sufficient to saturate any modern CCD chip. It means that the illumination intensity of CCDs has to be adapted to the reflectivity of the SPR surface layer. In case the reflection is low, the intensity can easily be increased to a value not far from pixel saturation in order to find the optimum detection power. Let us assume that the optimum illumination intensity is
Iopt 0 ) ICCD/R
(5)
where ICCD should be below the CCD saturation intensity, e.g., 50% of saturation. 4234
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The optimum S/N ratio is given by
S/N ) xηICCD
dR/dn ∆n R
(6)
where
E)
dR/dn R
(7)
is the parameter that has to be optimized by variation of the laser wavelength or the incidence angle. In the following, E is named relative sensitivity. (c) Noise due to Light Intensity Fluctuations. The same value E as in (7) should be used for optimization of the detection power if the noise is directly proportional to the intensity. This is the case, for example, when the dominating noise is due to fluctuations of the radiation source. In this case, the noise can be written as N ) RηI0R (R, proportionality coefficient), and
S/N ) R-1
dR/dn ∆n R
(8)
is depending only on E for both types of noise sources. (d) Practical Aspect. Current fabrication technologies of goldcoated glasses for SPR applications cannot provide ideal reproducibility of the layer, which has some influence on the optimum angle. Since the procedure to find the optimum conditions for each layer is very time-consuming, there is also the option to obtain the optimum angle from the measured reflectivity curve in dependence on the angle. Taking into account that
dθ/dn ) β ) const one derives
E)
dR/dn dR dθ 1 dR/dθ ) )β R dθ dn R R
(9)
The optimum angle corresponds to the maximum of the absolute value of (dR/dθ)/R. It can be found measuring the reflectivity curve and fitting the data to the theoretical curve. Theoretical Enhancement of Detection Power. In order to illustrate the enhancement of the detection power theoretical simulations, based on the SPR 4 Phase Fresnel Reflectivity OnlineCalculation,7 were performed. For example, Figure 1a shows the theoretical dependence of the relative sensitivity E on the incident angle for the laser wavelength 780 nm and a gold layer of 48 nm. The curve was calculated for ∆n ) 6 × 10-5. The relative sensitivity E has a maximum at about 59.68°, 0.07° from the minimum of the SPR reflectivity curve at 59.75° (see Figure 1c). The relative sensitivity at this angle is a factor of ∼3.5 larger than at the angle that provides the maximum change of the signal as shown in Figure 1b. Note that the enhancement of the detection power depends on the reflectivity in the minimum. It can be further increased if the reflection in the minimum is reduced by (7) http://unicorn.ps.uci.edu/calculations/fresnel/fcform.html.
Figure 2. SPR signal data (9) measured by a photodiode applying a salt solution (∆n ) 6 × 10-5) and 780-nm laser light in dependence on the reflection angle (SPR gold layer, 48 nm). The dependence of the noise (b) on the angle was measured with pure water in the flow cell. The dashed and dotted lines connecting the experimental data points should help to guide the eyes.
Figure 1. Theoretical SPR reflectivity (c), sensitivity (b), and relative sensitivity (a) in dependence on reflection angle taking into account a 48-nm gold layer, a laser wavelength of 780 nm, and a index of refraction difference of dn ) 6 × 10-5. The dotted lines indicate the angle position where the SPR signal and the detection power have their maximum values. The software for the calculation is provided by ref 7.
optimizing the thickness of the gold layer. However, it is difficult to obtain a reflectivity smaller than 1% experimentally. In the particular case shown in Figure 1, the reflectivity in the minimum was ∼1.1% which is near to the experimental value measured with the photodiode (1.5%). Using the CCD camera and the laser diode at 850 nm, 5% reflectivity was measured in the SPR minimum although 0.1% is expected from theory taking into account the gold layer thickness of 48 nm. The difference is most likely due to the nonperfect quality of the expanded beam and the roughness of the gold surface. In order to fit the theoretical SPR curve to the experimental data points, a gold layer thickness of 54.5 nm instead of 48 nm was used in the calculations. RESULTS AND DISCUSSION The relative sensitivity E ) (dR/dn)/R of the SPR arrangement with photodiode and CCD detector was measured with a 0.03% solution of NaCl in water. The salt solution and pure water were alternately pumped through the flow cell providing a refractive index difference of ∼6 × 10-5. The photodiode data are presented in Figure 2. The dominating noise came from fluctuations of the laser intensity. The noise dependence on the angle of incidence was measured by pumping pure water through the flow cell, while the signal was recorded by alternating pumping of water and 0.03% NaCl solution. The
Figure 3. Relative SPR sensitivity E in dependence on the reflection angle measured with a photodiode, 780-nm laser light, a salt solution of ∆n ) 6 × 10-5, and a gold layer of 48-nm thickness. The experimental uncertainty of the data point is ∼15%. The full curve is a theoretical prediction using the experimental wavelength, index of refraction difference, and gold layer thickness. The free software for the calculation is provided by ref 7.
plots clearly demonstrate why the optimum detection power is not at the reflection angle where the SPR signal has its maximum. The noise is decreasing faster than the intensity signal when the angle is tuned into the direction of the reflection minimum. In Figure 3, the measured relative sensitivity is plotted versus the angle of incidence. The solid curve represents the theoretical (∆I/ I)/∆n dependence on the angle fitted to the experimental points. The curve corresponds to the theoretical SPR signal obtained with a gold layer of 48 nm and a refractive index difference ∆n ) 6 × 10-5. The relative sensitivity at the maximum at 59.68° is ∼1300 while it is only ∼340 at 59.40° where the signal has its maximum. This means that the optimization of the reflection angle resulted in an improvement of the detection limit of ∼3.5 in comparison with the usually applied angle position where the SPR signal has its largest value. Analytical Chemistry, Vol. 79, No. 11, June 1, 2007
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Figure 4. Relative SPR sensitivity E in dependence on reflection angle measured with the same salt concentration and gold layer as in Figure 3, however, using a 850-nm laser and a CCD camera for detection. The experimental error of the data is ∼15%. The full curve represents a theoretical prediction using the free software provided by ref 7.
The CCD measurements were performed by illuminating relatively small pixel areas (20 × 20 pixels). Here, the shot noise is the limiting factor as discussed above. The intensity of the laser was tuned to ∼50% of the saturation level of the pixels for any incidence angle. The reflectivity at the minimum of the resonance curve was ∼5% as mentioned above. Therefore, the improvement of the detection limit by reflection angle optimization was smaller than in the SPR arrangement with photodiode detection. The S/N ratio measured with the CCD in dependence on incidence angle together with a (∆I/I)/∆n fit is shown in Figure 4. The relative sensitivity was found to be ∼1000 at the optimum reflection angle and ∼500 at the position where the signal had its maximum. Therefore, the improvement of the detection limit was ∼2 in a good agreement with theory. Calibration curves for SPR analyses at optimized reflection angles are not expected to be linear since reflectivity curves are more bent near the minimum of the SPR resonance than in the usually applied working point. In order to examine the linearity and dynamic of a calibration curve at the optimized angle, aliquots of NaCl in water were prepared and the signal dependence on the refractive index variation was measured. Figure 5 shows two calibration curves measured with the 780 nm laser diode and the photodiode as detector. The data obtained at the optimized reflection angle are plotted as full squares. They are compared with the corresponding data measured at the angle where the SPR signal has its maximum (full dots). The theoretical calibration curves were calculated with the software provided by ref 7. The theoretical curves are shown as full and dashed curves, respectively. A deviation from linearity can be seen in the calibration curve measured at optimum angle. However, it is relatively small over the total dynamic range.
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Figure 5. Calibration curves for NaCl measured at the reflection angle of maximum relative sensitivity (9) and maximum SPR signal response (b). The full and dashed curves are fits to the respective experimental data applying the free software provided by ref 7.
It has to be noted that the dynamic range using a CCD camera is limited by the saturation of the pixels at optimum angle of incidence. As a consequence, the illumination intensity has to be reduced if a larger dynamic range is required in SPR analyses. Note that any intensity reduction results in a decrease of detection power because of growing shot noise. In practice, a compromise between detection power and dynamic range has to be found. CONCLUSION The power of SPR detection with photodiode and CCD was studied from the viewpoint of signal-to-noise ratio. In accordance with theoretical considerations, the optimum reflection angle was found to be near to the reflection minimum of the SPR curve instead of the position where the signal has its maximum. The experimental data were in a good agreement with relative sensitivities calculated on the basis of the SPR reflection curve. The detection power was shown to be ∼2 (CCD detection) and ∼3.5 times (photodiode detection) better than at the reflection angles where the SPR signals had their maximum signals. The dynamic range of SPR measurements at optimized angle was found to be comparable with the corresponding dynamic range at the angle producing the maximum signal. The calibration curve at the angle of enhanced detection power was found to be slightly nonlinear. However, this should be no problem in SPR analyses. ACKNOWLEDGMENT The authors gratefully acknowledge the financial support by the Ministry of Innovation, Science, Research, and Technology of the state of North RhinesWestphalia and the Ministry of Education and Research of the Federal Republic of Germany. Received for review February 20, 2007. AC070074U
January
12,
2007.
Accepted
