Tuning Dynamic Range and Sensitivity of White-Light, Multimode

This increases the flexibility of a white-light SPR sensor by increasing the ..... David Monzón-Hernández , Joel Villatoro , Dimas Talavera , Donato...
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Anal. Chem. 1999, 71, 5116-5122

Tuning Dynamic Range and Sensitivity of White-Light, Multimode, Fiber-Optic Surface Plasmon Resonance Sensors Louis A. Obando and Karl S. Booksh*

Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287

This paper presents the advantages of modifying the geometry of the sensing tip of a white-light, multimode optical fiber SPR sensor to optimize the dynamic range and sensitivity. By selectively beveling the distal end of the fiber probe, the wavelength of resonance can be redshifted by more than 100 nm and blue-shifted by more than 30 nm. This increases the flexibility of a white-light SPR sensor by increasing the dynamic range of accessible RIs and by shifting the resonance to the most sensitive regions of the detector. Sensitivity, measured in wavelength shift per RI change, can be increased by a factor of 4. Also, multiple-wavelength regions of SPR activity can simultaneously be observed on the same probe, thus increasing the information content of a SPR spectrum. Surface plasmon resonance (SPR) spectroscopy has been employed for quantitative and qualitative analysis in analytical chemistry,1-3 biochemistry,4-7 physics,8,9 and engineering10-13 applications. Being a surface technique that is sensitive to changes of 10-5-10-6 refractive index (RI) units within approximately 200 nm of the SPR sensor/sample interface, SPR spectroscopy is becoming increasingly popular for monitoring the growth of thin organic films deposited on the sensing layer.14-17 As little as 0.01 nm of average film deposition can be detected when the RI (1) Brecht, A.; Gauglitz, G. Anal. Chem. Acta 1997, 347, 219-233. (2) Marco, M.-P.; Gee, S.; Hammock, B. D. TrAC, Trends Anal. Chem. 1995, 14, 341-350. (3) Berger, C. E. H.; Beumer, T. A. M.; Kooyman, R. P. H.; Greve, J. Anal. Chem. 1998, 70, 703-706. (4) Alfthan, K. Biosens. Bioelectron. 1998, 13, 653-663. (5) Knibiehler, M.; Goubin, F.; Escalas, N.; Johnsson, Z. O.; Maraguil, H.; Hubscher, U.; Ducommun, B. FEBS Lett. 1996, 391, 66-70. (6) Karlsson, R.; Michaelsson, A.; Mattsson, L. J. Immunol. Methods 1991, 145, 229-240. (7) Jordan, C. E.; Corn, R. M. Anal. Chem. 1997, 69, 1449-1456. (8) Lerme, J.; Palpant, B.; Prevel, B.; Cottancin, E.; Pellerin, M.; Treilleux, M.; Vialle, J. L.; Perez, A.; Broyer, M. Eur. Phys. J. D 1998, 4, 95-108. (9) de Brujin, H. E.; Kooyman, R. P. H.; Greve, J. Appl. Opt. 1990, 29, 19741978. (10) Bender, W. J.; Dessey, R. E.; Miller, M. S.; Claus, R. O. Anal. Chem. 1994, 66, 963-970. (11) Ruiz, E. G.; Garces, I.; Aldea, C.; Lopez, M. A.; Mateo, J.; Chamarro, J. A.; Alegret, S. Sens. Actuators A 1993, 37-38, 221-225. (12) Matsubara, K.; Kawata, S.; Minami, S. Appl. Spectrosc. 1988, 42, 13751379. (13) Weiss, M. N.; Srivastava, S.; Groger, H. Electron. Lett. 1996, 32, 842-843. (14) Jung, L. S.; Campbell, C. T.; Chinowsky, T. M.; Mar, M. N.; Yee, S. S. Langmuir 1998, 14, 5636-5648. (15) Hausch, M.; Beyer, D.; Knoll, W.; Zentel, R. Langmuir 1998, 14, 72317216.

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difference between the film and bulk solution is 0.1 RI unit.14 Thus, a submonolayer of adsorbed protein-like substance (RI 1.4) from an aqueous solution (RI 1.3) can easily be observed. However, there is an inherent lack of selectivity of the SPR effect for quantitative molecular analyses that must be overcome by functionalizing the metal surface with analyte-specific receptors (i.e., antibodies).14,18-21 Depending on the binding constant of the receptor-ligand system, a reversible probe can be constructed with nanomolar sensitivity. The physics of the SPR effect has been extensively described.22,23 The evanescent field of a photon can optically excite a standing charge-density wave on a thin, metal surface. The wave function of this SP wave is governed by the angle and energy of the incident photon as well as the complex dielectric constants of both the metal film and the substrate on which the film is deposited. If the wave function of the SP matches the wave function of the environment at the metal surface, largely determined by the complex dielectric constant (refractive index) of the medium in contact with the metal layer, the energy of the photon is transferred from the SP wave to the medium. As the RI of the medium in contact with the SP wave increases, the wavelength and angle from normal for optimal SPR coupling increases. To exploit the SP effect for analytical applications, two types of SPR sensors have been employed: constant-angle sensors and constant-wavelength sensors. Constant-angle SPR sensors employ the traditional “Kretschmann configuration” where a metal-coated BK7 glass prism is used for the sensing area. Monochromatic light is passed through the prism and reflected off the metal-coated sensing area. The normalized intensity of reflected light (versus an air or water reference) is plotted against the incident angle of the photon.17,24 This system is easily modified to perform white(16) Lawrence, C. R.; Martin, A. S.; Sambles, R. Thin Solid Films 1992, 208, 269-273. (17) Frutos, A. G.; Corn, R. M. Anal. Chem. 1998, 70, 449A-455A. (18) Sasaki, S.; Nagata, R.; Hock, B.; Karube, I. Anal. Chem. Acta 1998, 368, 71-76. (19) Lofas, S.; Johnsson, B. J. Chem. Soc., Chem. Commun. 1990, 1526-1528. (20) Lofas, S.; Johnsson, B.; Edstrom, A.; Hannson, A.; Lindquist, G.; Hillgren, R.-M. M.; Stigh, L. Biosens. Bioelectron. 1995, 10, 813-822. (21) Berger, C. E. H.; Beumer, T. A. M.; Kooyman, R. P. H.; Greve, J. Anal. Chem. 1998, 70, 703-706. (22) Raether, H. Surface Plasmons on Smooth and Rough Surfaces and on Gratings; Springer-Verlag: Berlin, 1988. (23) Liebsch, A. Electronic Excitations at Metal Surfaces; Plennum Press: New York, 1997. (24) Lenferink, A. T. M.; Kooyman, R. P. H.; Greve, J. Sens. Actuators B 1991, 3, 261-265. 10.1021/ac990470f CCC: $18.00

© 1999 American Chemical Society Published on Web 10/09/1999

Figure 1. Simulation showing how λSPR increases nonlinearly with increasing θinc and the wavelength of light. Reflection intensity is lowest (maximum energy transfer) at the darkest areas. The two smaller graphs show how λSPR is red-shifted when θinc is increased by 10°.

light SPR measurements.14,25-28 Collimated white light is reflected off the metal layer and dispersed across an array detector. The collected spectrum is normalized (versus an air or water reference) and plotted as a function of wavelength. Both the laser and whitelight systems are readily conformed to automated, benchtop, flowthrough instruments. In fact, such a laser-based instrument is marketed and optimized for bioanalytical analyses.19,20 However, these prism or planar substrate-based sensors are not well-suited for field or in situ analysis; to do this, optical fiber-based SPR sensors are needed. Consequently, fiber-optic-based SPR sensors have recently attracted much attention.10,13,29-32 Numerous sensor geometries have been proposed for the fiber sensors. Some have attached prisms to the end of an optical fiber probe.14,26,33 In general, singlemode optical fibers have been employed. Either a small patch of cladding is removed from the center of a length of optical fiber to expose the sensing area10,13,30,31 or a mirror is affixed to the distal (25) Johnston, K. S.; Chinowsky, T. M.; Yee, S. S. Proc. SPIE 1996, 2836, 178185 (Chemical, Biochemical, and Environmental Chemical Sensors VIII). (26) Homola, J.; Schwotzer, G.; Lehman, H.; Willsch, R.; Ecke, W.; Bartelt, H. Proc. SPIE 1995, 2508, 324-333 (Chemical, Biochemical, and Environmental Fiber Sensors VII). (27) Johnston, K. S.; Yee, S. S.; Booksh, K. S. Anal. Chem. 1997, 69, 18441851. (28) Jory, M. J.; Bradberry, G. W.; Cann, P. S.; Sambles, J. R. Meas. Sci. Technol. 1995, 6, 1193-1200. (29) Fontana, E.; Dulman, H. D.; Doggett, D. E.; Pantell, R. H. IEEE Trans. Instrum. Meas. 1998, 47, 168-173. (30) Ronot-Trioli, C.; Trouillet, A.; Veillas, C.; El-Shaikh, A.; Gagnaire, H. Anal. Chem. Acta 1996, 319, 121-127. (31) Alonso, R.; Villuendas, F.; Tornos, J.; Pelayo, J. Sens. Actuators A 1993, 37-38, 187-192. (32) Jorgenson, R. C.; Yee, S. S. Sens. Actuators B 1993, 12, 213-220. (33) Garces, I.; Aldea, C.; Mateo, J. Sens. Actuators B 1992, 7, 771-774.

tip of the optic fiber and the exposed sensing area is near the tip.29,32 Single-mode fibers have the advantage of maintaining the polarization of the exciting light and propagating only a single angle of reflection through the fiber. Together this maintains a sharp SPR dip without an excessive reflective background. Concurrently, multimode optical fibers have been largely avoided for true SPR fiber-based sensors. The mixing of modes and loss of polarization leads to relatively shallow, broad reflectance dips compared to prism-based sensors.27 However, any loss of calibration precision stemming from inability to reliably determine the exact minimum of the SPR spectra can be overcome by employing multivariate calibration.27,34 The location of λSPR can be predicted for a given incident angle, and vice versa, using a simulation that calculates the reflection coefficients for a multilayer structure based on Fresnel and Maxwell equations (Figure 1).35-37 It was assumed that the incident angle at the fiber interface was the average propagation angle in the fiber and it was not necessary to include the effects of the angular distribution of light rays in multimode fibers. The simulation predicts that an increase in the incident angle will excite SPR at a longer wavelength. By tapering the probes, the angle of incidence is increased by the degree of the angle of taper. SPR minimum are increasingly red-shifted as the amount of the taper is increased. The simulation also shows that the number of (34) Johnston, K. S.; Booksh, K. S.; Chinowsky, T.; Lee, S. S. Sens. Actuators B 1999, 54, 80-88. (35) Ishimaru, A. Electromagnetic Wave Propagation, Radiation, and Scattering; Prentice Hall: Englewood Cliffs, NJ, 1991. (36) Griffiths, D. J. Introduction to Electrodynamics, 2nd ed.; Prentice Hall: Englewood Cliffs, NJ, 1981. (37) Johnston, K. S. Characterization of Thin Films Using Surface Plasmon Resonance. Master Thesis, University of Washington, 1995.

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incident angle-wavelength combinations that excite SPR increases at longer wavelengths. The resulting SPR spectrum is a sum of those excitations, and consequently, SPR features become broader as the resonant wavelength increases. These features are broader than those produced by techniques that use a single incident wavelength or angle. Coupling multivariate calibration with white-light, multimode fiber-optic SPR systems presents the possibility of constructing small, field-portable SPR sensors. It has been shown that multivariate calibration with 32-nm resolution yields the same analytical accuracy and precision as “minimum-hunt” calibration with 1-nm resolution.27 White-light, fiber-optic systems can be constructed with small battery-powered lamps and low-resolution spectrographs that fit onto PC cards. Readily replaceable and interchangeable sensing areas can be affixed onto the end of the optical fibers with SMA-type connectors. One problem that needs to be addressed is optimizing the dynamic range and sensitivity of these field-portable systems. Currently, the desired wavelength of SPR resonance for a given refractive index is tuned by adding SiOx, or other dielectric coatings, either above or below the metal sensing area.10,13,33 These overlays are not ideal for three reasons. First, coating the metal surface with the dielectric prevents functionalization of the metal with analyte-specific complexing agents (i.e., thiol binding of antibodies to a gold layer). Second, applying a dielectric coating requires expensive equipment such as an electron beam evaporator. And third, overlays can only increase the dynamic range of a sensor at the expense of sensitivity; by the nature of an overlay, the analyte is moved away from the sensor, minimizing the effect of the sample coupling with the SP wave. EXPERIMENTAL SECTION A white-light, fiber-optic SPR refractometer was constructed similarly to that proposed by Jorgenson and Yee.32 Full-spectrum light from a 50-W QTH source (Oriel) was collected by a 0.38 numerical aperture (NA), 400 mm core, low OH optical fiber (3M; FT-400-EMT). Light exiting this optical fiber jumper was collimated and passed through a 50/50 broad-band, nonpolarizing beam splitter (Oriel). Half of the light was then focused into a fiber jumper that led to the SPR probe tip, and half was lost. The distal end of this fiber jumper and the SPR probe tip were each connected with a SMA connector. The jumper and the probe were thus joined by a SMA to SMA adapter (Thor Labs). Light reflected from the tip of the probe returned up the second fiber jumper and passed through the beam splitter with half being lost and half being focused onto a third fiber-optic jumper. The distal end of the third jumper was SMA connected and attached to a f/2.2 holographic spectrograph (Kaiser). The spectrograph provided 400 nm of spectral coverage from 530 to 930 nm. Dispersed light was collected by a 256 × 1024 pixel CCD camera (Princeton Instruments). The camera was thermoelectrically cooled to -38 °C. Rough probe tips were prepared by securing approximately 5.5 cm of optical fiber into the SMA connector with 48-h epoxy. The Tefzel buffer was removed with a mechanical stripper and the cladding dissolved in acetone. This exposed 0.5 cm of fiber core for dual tapering with shaft and straight probes and approximately 0.25 cm for the dual-tapered probes. A polishing chuck was machined that permits polishing the fiber tip 90, 80, 5118 Analytical Chemistry, Vol. 71, No. 22, November 15, 1999

Figure 2. Fiber probe tip archetypes investigated for SPR analyses: (A) straight (ST), (B) dual-tapered (DT), (C) dual-tapered with shaft (DS), and (D) chiseled/truncated chiseled.

75, 70, 60, 30, 20, 15, and 10° from normal. Each polished surface of the fiber was polished on 5-, 3-, 1-, and 0.3-mm lapping films until visually smooth. The probe tips were then sputter-coated with 1 nm of Cr and 50 nm of Au (Cressington). Coating thickness was monitored electronically by a quartz crystal microbalance placed in the sputtering chamber (Cressington). One hundred sucrose (Aldrich) solutions from 0 to 50% by mass were prepared by dilutions from a stock sucrose solution. This resulted in samples having RIs from 1.3298 to 1.4148 as verified by a low-cost refractometer (Fisher Scientific). Although the analyses and solutions could be equivalently presented in terms of RI, the analytical precision of the balance (MettlerToledo), 0.1 mg, is greater than the analytical precision of the available refractometer, 1 × 10-4 RI, for the volume prepared. Spectra were collected using Winspec software included with the CCD camera and controller (Princeton Instruments). A 100-ms integration time was chosen for data collection, and each spectrum represents the average of 25 integrations. House-written code was employed to convert the Winspec files to ASCII text files and consequently to import the text files into Matlab (MathWorks Inc.) All data manipulations were performed in the Matlab operating environment. Reported SPR spectra are determined by taking the ratio of each reflectance spectrum to an air reflectance spectrum collected at the beginning of a sample run. Subpixel wavelength location of each SPR dip was found by the roots of a second-order polynomial fit about the local minima of each dip. RESULTS AND DISCUSSION Initial Investigation of Probe Designs. Three probe designs were selected for presentation in this paper. The traditional straight (ST) probe (Figure 2A) is of the type employed in other white-light, multimode optical fiber applications.27,33 The dualtapered (DT) probe (Figure 2B) has one side and the front mirror polished at complimentary angles to maintain orthogonality between the two surfaces. Thus if angle A were polished 75° from normal to the long axis of the fiber, the front mirror would be polished 90° - 75° ) 15° from normal. The dual-tapered plus shaft (DS) probe (Figure 2C) maintains the tapered sensing area but adds a length of straight, unaltered sensing area to the probe tip. Other probe designs, such as a chisel-tipped probe, where the top and bottom of the probe were polished to the same angle,

Figure 3. Relative SPR spectra of sucrose samples collected with the ST probe design.

and a truncated chisel-tipped probe (Figure 2D), were investigated. It was determined that these probe designs do not return sufficient light to be of use. Incorporating one 90° angle in the probe tip is required to maintain a majority of the reflected light below the acceptance angle of the fiber. More complicated three-dimensional probe tip designs based on conical or truncated conical geometries were rejected. Although these designs may present useful properties, the difficulty of constructing such a probe is prohibitive. Forming a conical tip on an optical fiber would require a pipet puller, a HF acid bath, or an expensive automatic polisher. Instead, the scope of this investigation is limited to designs that can be easily and reproducibly constructed in most laboratories. Straight Probes. A collection of SPR spectra of aqueous sucrose solutions spanning 0-50% sucrose (by weight) is presented in Figure 3. These are the reflection spectra that have been normalized by the reflection spectrum of air. A number of features that are particular of white light SPR sensors are evident in Figure 3. First, the relative reflection intensity never drops below 50%. Only the light p-polarized with respect to the sensor surface is SPR active; thus, half the photon flux down the sensor has no probability of absorbance. Second, the reflectance dips are rather broad compared to prism-based white-light refractometers. This is caused by the multimode nature of the fiber; photons traveling down the fiber will interact with the sensing surface with a distribution of angles equal to the acceptance cone of the fiber. Thus, each angle will have a slightly different wavelength of maximum coupling for a given solution. The resulting SPR spectrum is an ensemble over the range of propagating angles. Third, the information content is most readily visible in the spectral shift, not in intensity changes, across all samples. However, over small RI ranges univariate intensity calibration is possible,10,21,33 but multivariate calibration methods are needed for global intensity-based analyses.27 Fourth, the shift in wavelength coupling is not linear with respect to analyte concentration or RI changes. The SPR spectra shift only 75 nm from 1.329 to 1.372 RI units (0-25% sucrose), but shift 225 nm from 1.372 to 1.415 RI units (25-50% sucrose).

Figure 4. Calibration curve for minimum fit of white-light, fiber-optic SPR probe.

The third and fourth points from above are shown in Figure 4 and Table 1, respectively. The calibration curve for 100 sucrose samples is shown in a plot of solution refractive index versus wavelength of minimum. There is a strong, systematic, albeit nonlinear, trend that is easily exploited for calibration. Taking the derivative of the calibration curve demonstrates the differential in sensitivity at lower and higher RI (Table 1). The sensitivity of the spectral shift is approximately 2000 nm/RI unit for the most dilute solutions and almost 10 000 nm/RI unit for the most concentrated solutions analyzed (Table 1). With this probe geometry, refractive indexes above 1.42 (50% sucrose) and below 1.33 (water) are not reasonably accessible. Increasing the refractive index above 1.43 would push the SPR dip outside the range of the spectrograph. Even if a moveable grating spectrograph were employed, the sensitivity of the detector decreases significantly at wavelengths greater than 900 nm. Similarly, the SPR dip of a sample with a RI less than 1.32 would fall in the noisy region of the spectra between 530 and 550 nm or would lie off the detector altogether. Dual-Tapered Probes. Applying a dual taper to the tip of the fiber probe can dramatically shift the wavelength of maximum Analytical Chemistry, Vol. 71, No. 22, November 15, 1999

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Table 1. Sensitivity and Dynamic Range of the SPR Probesa sensitivity [δλ/δRI (×1.0 × 103)] dual taper + shaftb

dual taperb refractive index (% sucrose by mass)

straight

1.3298 (0) 1.3468 (10) 1.3638 (20) 1.3723 (25) 1.3808 (30) 1.3893 (35) 1.3978 (40) 1.4148 (50)

1.15 1.59 2.02 2.45 3.11 4.11 5.51 9.86

80/10 DT

75/15 DT

80/10 DS

80/10 DS

75/15 DS 1

75/15 DS 2

70/20 DS 1

60/30 DS 1

1.38 2.29 3.23 4.35 6.22 9.12

1.52 3.74 5.12 7.04

0.19 1.04 1.68 2.17 2.90 3.99 5.52 10.31

0.81 2.16 2.86 3.52 4.67 6.53

0.29 1.27 1.80 2.21 2.88 3.96 5.56 10.83

0.63 4.89 8.52

0.63 1.24 1.65 2.03 2.66 3.64 5.07 9.65

0.72 1.03 1.46 1.91 2.63 3.71 5.21 9.84

a Sensitivity is given as the change in wavelength (nm) per change of 1 refractive index unit. b DT, dual taper; DS, dual taper shaft; 1, first dip; 2, second dip.

Figure 5. Relative SPR spectra of distilled water for ST and two DT probe designs.

SPR coupling. However, with this red-shift comes a substantial broadening of the SPR dip (Figure 5). An 80°/10° DT shifts the resonance of water 73 nm and a 75°/15° DT shifts the water resonance an additional 36 nm. Applying a 60°/30° dual taper shifted the water resonance to the edge of the detector and the resonance dip was so broad that efficient location of the minimums was difficult. Small-angle tapers, such as 85°/5°, would yield redshifts less than 70 nm and largely maintain the sharp SPR dip. With the increasing red-shift in the SPR dip comes increasing sensitivity and decreasing dynamic range of the SPR probe. Figure 6 presents the derivatives of the nonlinear calibration curves for an ST and two DT probes. The 75°/15° DT probe has 2-3 times the sensitivity of the ST probe over the range of 0-25% sucrose. Concurrently, the ST probe is sensitive to changes above 25% sucrose while the 75°/15° DT probe can monitor changes below 1.33 RI. The dynamic range and sensitivity of the 80°/10° DT probe lies between that of the ST and the 75°/15° DT probes. It is important to note that there is not a straight trade between sensitivity and dynamic range, as there is with most linear calibration applications or with adding a dielectric overlay to the sensing area. Tapering the fiber shifts the dynamic range of the sensor to lower RI regions; the greater the taper, the greater the shift. However, the dynamic range is most sensitive to changes in the tapered angle for small deviations from the ST probe. Also, there is a relative loss of sensitivity at the highest accessible RI with the dual-tapered probes. For each of the probes geometries presented in Figure 6, the most concentrated sucrose solution 5120 Analytical Chemistry, Vol. 71, No. 22, November 15, 1999

Figure 6. Minimum-fit calibration curve for ST and two DT probe designs.

analyzed with each probe displayed a SPR minimum near 875 nm. However, the sensitivity at 875 nm for the ST probe is 9860 nm/ RI unit, for the 80°/10° DT probe is 9120 nm/RI unit, and is only 7040 nm/RI unit for the 75°/15° DT probe. Dual-Tapered Probes with Shaft. Incorporating a dual taper, while leaving a length of shaft unaltered, constructs a probe that exhibits bimodal SPR spectra. (Figure 7) The first resonance dip is relatively narrow and slightly blue-shifted from the resonance minimum of the ST probe. The second resonance dip is very broad and red shifted relative to the ST probe. The second resonance is easily noticeable in the 80°/10° DS probe. For the 75°/15° DS probe, the second resonance is very broad and location of the minimum is difficult, even in low RI solutions. For fibers with more radical tapers, 70°/20° and 60°/30°, the second minimum is shifted beyond the range of the detector for the most dilute samples. The dynamic ranges of the DS probes are seen in Table 1. The first minimum of the 80°/10° DS probe is blue-shifted by approximately 40 nm relative to the ST probe for the more concentrated samples. This permits analysis beyond 50% sucrose with the 80°/10° DS probe. However, the gain in upper end dynamic range is small; the probe sensitivity of 10 030 nm/RI at 50% sucrose extrapolates to an increase in the dynamic range of 3% or 0.0025 refractive index unit. The second dip has a much shorter dynamic range. For the 75°/15° DS probe, the dip shifts beyond the range of the detector around 20% sucrose. For the 80°/10° DS probe, the dynamic range of the second dip ends at 35% sucrose.

Figure 7. Relative SPR spectra of 5% sucrose solutions collected with the ST and three DS probe designs.

The advantages associated with this class of probe geometry are seen in comparing the sensitivities of the DS probes (Table 1). While the sensitivity of the first dip is constant throughout all geometries, the second dip of the 80°/10° DS probe has approximately twice the sensitivity to changes in RI than the straight, unaltered, probe. The second dip of the 75°/15° DS probe has up to 4 times the sensitivity of the ST probe. The differential sensitivity of one probe increases the information content of the collected SPR spectra. With one SPR dip, or two dips that respond identically, it would be impossible to resolve spectral shifts associated with RI changes from spectral shifts associated with alignment problems. Both shifts would be of constant wavelength throughout the spectrum. However, with two dips, each with a different sensitivity, RI information can be validated by their relative location. Finally, the dual-minimum spectra combine the best of dynamic range and sensitivity from the ST and DT probes. The first minimum has the dynamic range of the ST probes, 0-50% sucrose. The second minimum increases the sensitivity of the probe at the lower RI where the sensitivity of the ST probe is low. Impact On Probe Design and Calibration. Five sundry aspects of modifying the optical fiber geometry for SPR probes merit mention. The ability to tune the wavelength of resonance without dielectric overlays allows customization of SPR probes for antibody-facilitated analyses. First, all modifications exist beneath the metal coating; therefore, the efficient thiol binding of antibodies to the gold layer is readily accomplished. Second, these modifications are simple and easily performed in any laboratory that would make their own SPR probe tips. Unlike SiOx overlays, expensive equipment is not needed to construct custom probe tips. The most expensive consumable is the SMA connector. Third, a finer array of polishing angles is accessible with electric fiber polishers. Electric fiber polishers are capable of polishing the fiber tips at shallower angles than the polishing chuck. (The limitation of the polishing chuck is derived from the inability of drilling 400-mm holes through the chuck at greater than 80° from normal.)

Fourth, probes can be constructed such that the wavelength of resonance for the RI of interest occurs near the wavelength of maximum reflectance signal to noise. This is useful when the probe is employed for screening or alarm applications (i.e. when a decision is to be made at a given RI). Were the resonance to occur near 750 nm (peak intensity), the signal to noise of the ratio spectra around the wavelengths of maximum information would be much greater. This effect can be seen in Figures 2 and 4 where the signal is most noisy at the ends of the spectra and relatively clean in the middle. As the RI approaches the target RI, the signal becomes cleaner and the precision of analysis consequently improves. Fifth, the modified probe designs are not as compatible with traditional minimum-hunt-based calibration compared to the ST probe design. As the reflectance spectra exhibit broader dips, estimation of the true minimums becomes increasingly difficult. For the DS probes, often the first or second minimum becomes an inflection point in the ensemble SPR reflection spectra. However, the effective broadening of the spectra is not a hindrance to multivariate calibration methods. Multivariate methods rely on intensity changes at individual wavelengths for calibration. Therefore, the broadening of the spectra actually improves the information distribution for multivariate analysis. The robustness and applicability of multivariate analysis for calibration of broad SPR spectra has been demonstrated.27,34 In these studies, the effective width of the SRP spectral dip has been increased by a factor of 32-64 without any degradation of predictive performance of the SPR system when multivariate methods were employed. Other Factors Impacting SPR Spectra. In the acquired spectra, SPR features dip from approximately 80 to 65% of the incident light intensity. Two reasons for this are the TE component of white light and stray light at the interface of two SMA connectors. Only the TM component of white light will couple into the surface plasmon wave (SPW), and consequently, the minimum reflection intensity should be 50% of the incident light intensity. Stray light from reflections at the interface of two coupled SMA connectors produced a background that was not accounted Analytical Chemistry, Vol. 71, No. 22, November 15, 1999

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for in the ratio of spectra. These two combined effects account for only 35% of the incident light coupling into the SPW. Not addressed in this study are two factors that impact the dynamic range and sensitivity of white-light, fiber-optic SPR probes. The most important is the optical fiber employed. The RI of the core and cladding of the fiber define the range of angles that propagate down the fiber. Fibers with lower NA propagate modes closer to normal to the fiber-optic interface, on average, down the fiber. Assuming that the decrease in NA is accomplished while preserving the RI of the fiber core, decreasing the NA simultaneously narrows the SPR dip and blue-shifts the ensemble SPR spectra by eliminating the higher-wavelength couplings. Concurrently, increasing the RI of the fiber core blue-shifts the SPR dip. This shifts the dynamic range of the sensor equal to the increase in RI of the fiber core. Therefore, much can be done to optimally tune a SPR probe by also adjusting the manufactured properties of the optical fiber itself; however, it is less expensive to alter the geometry of the probe tip than to engineer specialty fibers for each SPR application. The most economical strategy for optimizing the SPR probe design is to choose a commercial fiber that is most appropriate for a given application, keeping in mind that it is easier to red-shift the SPR than to blue-shift the dip, and to modify the geometry of the commercial fiber. The second issue not addressed is the mode distribution of light propagating down the fiber. It is observed that, when the sensor was out of alignment, the spectra were slightly blue-shifted. Further investigation showed that when the blue-shift cor-

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responded to an increased proportion of skew rays propagating down the fiber. Increasing the contribution of skew rays resulted in a blue shift of up to 15 nm. Conceivably, adjusting the skew ray distribution, along with fiber composition and geometry, will result in even broader control of the sensitivity on dynamic range of the white-light, optical fiber SPR probes. CONCLUSIONS The ability to selectively tune the wavelength of resonance with a white-light, multimode, optical fiber SPR sensor by modifying the geometry of the fiber tip has been demonstrated. Advantages of this method compared to SiOx overlays include maintaining accessibility of the gold surface for antibody bonding and maximizing sensitivity of the probe. Coupling these probe tip modifications with multivariate calibration methods should enable construction of inexpensive, field-portable SPR sensors of environmental contaminants. ACKNOWLEDGMENT This work is supported by the National Science Foundation. Thanks to Nathan Wilken ([email protected]) for developing software used in manipulating file formats.

Received for review May 4, 1999. Accepted August 27, 1999. AC990470F