Evanescent Wave Fiber Optic Biosensors

Chris Rowe Taitt, Ph.D. and Frances S. Ligler, D.Phil., D.Sc.

Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, Washington, DC 20375 USA

When light is launched down a waveguide placed in contact with a lower refractive index material, if conditions are present to allow total internal reflection of this light, an electromagnetic component of the light extends out from the surface of the waveguide into the lower index medium. This electromagnetic field, the evanescent wave, has a limited penetration depth and can be used to specifically excite fluorophores bound to, or in close proximity to, the waveguide surface. Evanescent wave fiber optic biosensors have been developed utilizing the limited penetration depth of the evanescent wave to detect a variety of analytes. These sensors are able measure optical events at the fiber's surface with relatively little interference from the bulk solution. The ability of these instruments to detect analytes rapidly and specifically, even in the presence of complex sample matrices, has been demonstrated both under laboratory conditions and in the field.

1. Technical Concept

Fiber optic biosensors utilize two distinct assay configurations for signal generation and measurement: the optrode configuration, discussed in exquisite detail in the preceding chapter, and the evanescent wave configuration, the focus of this chapter. Both configurations rely on the same principle of total internal reflection (TIR) for light propagation and guiding. However, while optrodes use the light shining out the end of the fiber to generate a signal either at the distal face of the fiber or in the medium near the fiber's end, evanescent wave sensors rely on the electromagnetic component of the reflected light at the surface of the fiber core to excite only the signal events localized at that surface. The penetration depth of the light into the surrounding medium is much more restricted than for optrodes while the surface area interrogated is much larger in comparison to optrodes of equal diameter. The result is that evanescent wave biosensors require immobilization of the biological recognition molecules onto the longitudinal surface of the optical fiber core, primarily measure binding events, and are relatively immune from interferents in the bulk solution.

1.1. The evanescent wave

As described in more detail in Chapter 1, TIR is observed at the interface between two dielectric media with different indices of refraction. TIR is described by Snell's law:

where ni and n2 are the refractive indices of the fiber optic core and the surrounding medium, respectively; 0! is the incident light angle through the fiber optic core; and 82 is the angle of either the light refracting into the surrounding medium or the internal reflection back into the core. Total internal reflection requires that nj > n2 and occurs when the angle of incidence is greater than the critical angle, 0C, defined as

This parameter must be considered when designing any biosensor based on optical fibers. However, while Snell's law describes the macroscopic optical properties of waveguides, it does not account for the electromagnetic component of the reflected light, known as the evanescent wave. The evanescent wave is an electric field that extends from the fiber surface into the lower index medium and decays exponentially with distance from the surface, generally over a distance of 100 to several hundred nanometers (Figure 1). For multimode waveguides, the penetration depth, dp, the distance at which the strength of the evanescent wave is He of its value at the surface, is approximated by:

where nj and n2 are refractive indices of the optical fiber and surrounding medium, respectively, and 8 is the angle of incidence (Harrick, 1967).

The importance of the evanescent wave is its ability to couple light out of the fiber into the surrounding medium, thereby providing excitation for fluorophores bound to or in close proximity to the fiber core surface. This confined range of excitation is one of the major factors responsible for the relative immunity of evanescent wave-based systems to the effects of matrix components or interferents beyond the reaction surface.

Evanescent Wave Fiber Optic Biosensors O

Cladding

Analyte Q O /excited!

Core

Evanescent wave

Evanescent wave

Figure 1. Evanescent wave biosensor using partially clad fiber.

A crucial factor in both evanescent excitation and coupling of fluorescence emission back into the fiber core is the waveguide parameter of the optical fiber, or V-number. The Y-number is a dimensionless factor that determines how many modes a fiber can support, and is defined as:

where r is the radius of the optical fiber, ni is the refractive index of the fiber, and n2 is the refractive index of the surrounding medium or cladding. For uniform mode distribution, the power present in the evanescent wave (i.e., coupling out of the fiber) decreases with increasing V-number. On the other hand, the coupling of fluorescence emission back into the fiber from the surface increases with V-number (Thompson, 1991). Love and Button (1988) originally suggested that dipoles close to the surface could emit approximately 2% of their radiated power (fluorescence) into modes coupled back up the fiber; however, Polercky et al. (2000) have more recently analyzed films of dipoles and concluded that a much higher proportion of the radiation can couple into guided modes.

Fiber optic probes have been constructed from both unclad and partially clad fiber cores. Additional light may be lost from long, partially clad fibers, due to a V-number mismatch. Light propagating through a waveguide is limited to specific modes that are functions of the wavelength and the physical characteristics of the waveguide. If the refractive index of the cladding is different from that of the medium surrounding the declad sensing region, according to Equation 4, a V-number mismatch occurs, and the fluorescent emission is not guided into the core but enters the clad region and is not transmitted to the detection optics. In order for the light guided in the unclad region of the core to be propagated in the clad region of the core, the following constraint must be satisfied:

where nc0 is the refractive index of the fiber core, ncl is the refractive index of the cladded fiber, naq is the refractive index of the aqueous medium, raq is the radius of the declad sensing region, and rd is the radius of the cladded region.

The mechanism for holding an unclad fiber in place can have the same effect as cladding. Einink et al. (1990) described the phenomenon discussed above in terms of skew rays (higher order modes). By using a holder that minimized contact with the fiber core, the sensitivity of the fiber optic biosensor increased. Lackie (1992) patented a mounting system for an unclad fiber where the holder at one end of the fiber had the same refractive index as the fiber itself in order to avoid a V-number mismatch. Glass (1989) patented a similar mounting system for unclad fiber probes with the same goal in mind. Erb and Downward (1998) coated the ends of an unclad fiber with a Teflon having the same refractive index as the aqueous samples (n = 1.3) to prevent the V-number mismatch and subsequent loss of light in higher order modes where the fiber probe touched the holder.

Using partially clad fibers, it was demonstrated that decreasing the fiber radius to eliminate the V-number mismatch increased the light coupling efficiency from the sensing region into the clad region and subsequent transmission of the fluorescence through the rest of the fiber (Thompson and Kondracki, 1990; Thompson and Villaruel, 1991; Golden et al., 1992, 1994a,b; Anderson et al., 1994a,b; Anderson and Golden, 1995; Gao et al., 1995). Whereas lower order modes are concentrated near the center of the fiber core, higher order modes are distributed more towards the outer edge of the core and penetrate further into the surrounding medium. Thus, higher order modes will effect greater evanescent excitation of surface-bound fluorophores. However, emitted fluorescence coupled into back into the fiber is also carried in higher order modes. In the case of the untapered fibers, the fluorescence in these higher order modes is preferentially lost into the cladding. With tapering, the higher order modes are converted into the lower order modes; these lower order modes concentrate near the center of the fiber core and can easily become propagated modes which reach the detector. By altering the geometry of the sensing region (Figure 2), immunoassay sensitivities could also be increased up to 80-fold (Anderson et al., 1993).

Figure 2. Schematic of untapered and tapered fibers. A) Fiber probe with cladding stripped away from core. B) Step-tapered core to create V-number match between sensing and cladded regions. C) Continuously tapered core to maximize excitation light at surface. D) Combination tapered core to reach a V-number match quickly and subsequently maintain maximum excitation light at the fiber surface.

Figure 2. Schematic of untapered and tapered fibers. A) Fiber probe with cladding stripped away from core. B) Step-tapered core to create V-number match between sensing and cladded regions. C) Continuously tapered core to maximize excitation light at surface. D) Combination tapered core to reach a V-number match quickly and subsequently maintain maximum excitation light at the fiber surface.

1.2. Optical fibers

Optical fibers possess a number of related physical characteristics that can be used to distinguish them, such as radius, refractive index, V-number (described above), and the numerical aperture (NA); the last is a measure of the difference in refractive indices between the fiber core and the cladding and is related to the V-number:

Monomode fibers, such as those used in the telecommunications industry, typically have a core size of 5-10 ¿un and propagate only a single mode at any given wavelength. As they have lower numerical apertures, they have a higher percentage of total power present outside the core; greater than 50% of the power can be in the media surrounding the monomode fibers with low V-numbers (Gloge, 1971) - hence, more power to excite fluorophores via the evanescent field. Single mode fibers have been used for evanescent sensing by a number of groups (Lew et al., 1986; Villaruel et al., 1987; Carlyon et al., 1992; Hale et al., 1996), but are not generally given much attention for sensing purposes; their small core radii render them extremely fragile and difficult to handle. Furthermore, the intensity of power present in their evanescent fields is such that many fluorophores rapidly photobleach. For these reasons, most evanescent wave sensors utilize multimode silica or plastic fibers for light transmission. Multimode fibers possess the advantages of good light transmission over short

Multimode fibers possess the advantages of good light transmission over short and medium distances with a wide variety of optical components. While evanescent excitation of surface-bound fluorophores is not as efficient in the monomode fibers, ease of use, lower rates of photobleaching, and increased coupling efficiency are strong advantages of multi-mode fibers. And most importantly, the larger, multimode fibers produce more surface area for the immobilization of the biological recognition molecules.

Fibers composed of fused silica offer excellent optical transmission from the near UV range to the near-IR range of wavelengths and have low intrinsic fluorescence. In our studies, however, we found that the amount of intrinsic fluorescence in fused silica fibers was a significant problem for the discrimination of fluorescence below 600 nm (Ligler et al., 1995). Photobleaching the fibers immediately prior to conducting a fluorescence assay eliminated the background fluorescence; operating at wavelengths above 600 nm avoided the problem altogether (Shriver-Lake et al., 1995b). Diameters of silica multimode fiber cores generally range from 50 (xm up to 1.5 mm. Furthermore, fibers made from this material are resistant to most biological buffer systems. However, methods required to resolve V-number mismatches (i.e., tapering) require time-consuming and often hazardous procedures that are difficult to reproduce from batch to batch, e.g., etching with hydrofluoric acid.

Plastic fibers, on the other hand, can be injection molded to fit the user's and instrument's requirements (Slovacek et al., 1992; King et al., 1999; Saaski and Jung, 2000). Furthermore, dopants can be added to change the refractive index over a wide range. The most commonly used plastic fibers are composed of polymethylmethacrylate (PMMA) or polystyrene. While plastic fibers have a very limited range of temperatures at which they can be used in comparison to silica fibers, this range of temperatures (-30° to 80°C) is currently sufficient for study of most biological systems. The chief problem with plastic fibers is the limited spectral range for which they can be used. This limitation is due to the high attenuation in the red and near-IR spectrum from -CH absorption bands (Figure 3). This problem can be partially circumvented by doping the plastics with deuterium; deuterium replaces the -CH absorption band with a -CD band, thereby increasing the range of useful wavelengths. These deuterium-doped plastic fibers, however, tend to lose their optical transmission over time (Boisde and Harmer, 1996). In practice, an additional problem with the use of plastic fibers is the variation in the starting materials used for molding; plasticizers and other additives which fluoresce at visible wavelengths may be included in company-proprietary formulations. As a result, each batch of starting material must be screened for the resulting optical properties at the specific wavelengths intended for use.

Figure 3. Spectra of various plastics used in optical fibers. Adapted from Boisde and

Harmer (1996).

Wavelength (nm)

Figure 3. Spectra of various plastics used in optical fibers. Adapted from Boisde and

Harmer (1996).

1.3. Instrumentation

In addition to the optical fiber probe coated with a biological recognition molecule, the fiber optic biosensor consists essentially of a fluorimeter that is capable of discriminating excitation light from emitted fluorescence and a fluid transfer system to move samples and reagents over the fiber probe. The components and configurations for these two systems depend on the geometry of the fiber and the degree of automation of the sensor.

1.3.1. The fluorimeter. The fluorimeter portion of the biosensor can be divided into the excitation and emission collection components. Generally, lenses are used to focus light from the source onto or into the fiber and line filters are included in the excitation path if necessary. The first fiber optic biosensors used halogen lamps (Block and Hirschfeld, 1987; Glass, 1989; Block et al„ 1990) or xenon lamps (Kooyman et al., 1987; Lipson et al., 1992) as light sources; these were soon replaced with lasers with more uniform excitation wavelengths (Thompson and Ligler, 1988; Golden et al., 1992; Hale et al., 1996). The development of hydrophilic, near infrared dyes (Shriver-Lake et al., 1995b, Wadkins et al., 1995) and diode lasers (Golden et al., 1994, 1997; Choa et al., 1996) meant that the advantages of laser excitation could be implemented in small, low cost devices. An additional advantage of the diode lasers is that the mechanical choppers originally included in order to discriminate fluorescence from excitation and ambient light could be eliminated and the diode lasers pulsed to achieve the same effect.

The ability to discriminate a weak fluorescent signal above the background excitation light is the most critical feature of the fluorimeter. Choppers or pulsed lasers provide one mechanism to accomplish that discrimination; excitation line filters and high quality emission filters are also crucial. However, perhaps the most interesting factor in accomplishing the discrimination is the variety of geometries employed by different groups to separate the excitation and emission paths physically. Figure 4 depicts several of these strategies. Strategies A, B, and C rely on coupling the light into the evanescent wave from the center of the core and recovering the fluorescent light from the higher order modes. Appropriate lenses and filters are included with each of these configurations.

While the first fluorescence immunoassays performed on the surface of a waveguide involved collection of the fluorescent signals perpendicular to the waveguide (Kronick and Little, 1975), nearly all fiber optic biosensors collect the emitted light out the end of the fiber. This means that the signal is integrated over the active surface of the fiber core and focused on a single detector, usually either a photomultiplier tube or photodiode. In a notable exception to end-face collection, Fang and Tan (1999) determined that they could detect individual fluorescent molecules using evanescent excitation and signal collection normal to the waveguide using a microscope equipped with an intensified charge coupled device. In this case, there was no intent to integrate the signal from multiple biological recognition events.

1.3.2. Thefluidics. In addition to the fluorometer, the fluidics component for delivering the sample and reagents to the fiber probe is an integral part of the biosensor. Primary considerations include the mechanism for holding the fiber probe in place within the fluid stream, the capillary-type chamber surrounding the probe with its inlet and outlet ports, and the associated pumps and valves for automated assays in biosensors intended for applications other than research.

Figure 4. Strategies for separation of excitation and emission light paths. A) A traditional dichroic mirror is used to separate excitation and emission based on the difference in wavelengths. The excitation light may pass through the mirror while the emission light is reflected onto a detector (left) or, in a scheme that has generally proven more effective, the stronger excitation light is reflected onto the end of the fiber while the emitted light passes straight through. B) The excitation light passes through a hole in an off-axis parabolic mirror while the emission light is reflected by the mirror onto a detector (Thompson and Levine, 1992). C) A fiber bundle is used between a high numerical aperture sensing probe and the optics. The 635 nm excitation light is channeled down the central silica fiber while the emitted higher wavelength fluorescence is coupled back up the surrounding plastic fibers (Golden et al., 1997). D) The fiber is illuminated using a light source normal to the fiber, and the fluorescence is detected at the distal end (Kooyman et al, 1987; Ligler et al, 2002).

Figure 4. Strategies for separation of excitation and emission light paths. A) A traditional dichroic mirror is used to separate excitation and emission based on the difference in wavelengths. The excitation light may pass through the mirror while the emission light is reflected onto a detector (left) or, in a scheme that has generally proven more effective, the stronger excitation light is reflected onto the end of the fiber while the emitted light passes straight through. B) The excitation light passes through a hole in an off-axis parabolic mirror while the emission light is reflected by the mirror onto a detector (Thompson and Levine, 1992). C) A fiber bundle is used between a high numerical aperture sensing probe and the optics. The 635 nm excitation light is channeled down the central silica fiber while the emitted higher wavelength fluorescence is coupled back up the surrounding plastic fibers (Golden et al., 1997). D) The fiber is illuminated using a light source normal to the fiber, and the fluorescence is detected at the distal end (Kooyman et al, 1987; Ligler et al, 2002).

To detector

Figure 5. Optical fibers within capillary fluidics chambers. Adapted from Anderson et al. (1994a).

To detector

Figure 5. Optical fibers within capillary fluidics chambers. Adapted from Anderson et al. (1994a).

Several capillary chambers with individual fluid inlets and outlets have been described as part of a fiber optic biosensor system and deliver fluid to the surface of the fiber (Block and Hirschfeld, 1987; Glass, 1989; Slovacek and Love, 1992; Anderson et al., 1993; Meserol, 1996; Neel and Lyst, 1997). Where the capillaries are intended for use with unclad fiber probes, significant attention has been given to the mechanism for holding the probe in the center of the capillary in order to prevent the V-number mismatch problems described above. Using a single fiber in a capillary tube, Oroszlan et al. (1993) reported the first fully automated assay system based on an optical fiber and computerized the operation of over 200 sequential 15-30 minute assays over a single fiber probe.

The simple capillary system described by Anderson and colleagues (1993) (Figure 5) was the first example connecting multiple probes in order to perform simultaneous analyses on a single sample for multiple analytes (Shriver-Lake et al., 1998; Bakaltcheva et al., 1998). The addition of samples and reagents over four fibers in series was automated in a system based on the Analyte 2000 fiber optic biosensor (Research International, Woodinville, WA) and included commercially available pumps and valves. This automated prototype was tested for its ability to collect and identify aerosolized bacteria while airborne in a small, unmanned plane (Ligler et al., 1998; Anderson et al., 1999) (Figure 6). This was the first demonstration that samples could be both collected and tested without manual operations.

The demonstration of a fully automated biosensor was the impetus for the commercial development of an automated portable system. Hitherto fore, the only commercial systems were the ORD device made by Block and his associates and the Analyte 2000. Both devices were basically small fluorimeters designed

Figure 6. Analyte 2000 with automated fluidics for remote sensing (Ligler et al., 1998).

to measure fluorescence emitted from one or four probes, respectively; neither system included an automated fluidics component and both were designed primarily for use by the research community. Research International subsequently developed an automated portable system designed to flow fluids through a disposable coupon containing four optical probes (King et al., 1999; Saaski and Jung, 2000; Saaski, 2000). The fluidics system went through several iterations, primarily due to necessary improvements in the mechanism for fluid transport. The first three prototype instruments, the Mantis, SOF-FOWG and RAPTOR, used a pneumatic fluidic system based on small air pumps, pinch valves, and pressurized fluid reservoirs. In theory, if no samples ever went through the pumps, the pumps could not clog. However, the membranes in the valves proved unreliable and variations in the rate of fluid flow affected the assay performance. In the latest version of the system, the RAPTOR-Plus, the pneumatic fluidics have been replaced with a peristaltic pump-driven system based on small, custom-made pumps. This system is proving to be very reliable in terms of long term operation (> 1 year to date). The disposable coupon that contains the fiber probes has been altered only to remove the on-board valves related to the pneumatic fluidics and to accommodate minor improvements in the fiber probes themselves. The coupon holds four polystyrene probes, which are inserted after being coated with the recognition biomolecule and dried. The probes are designed to integrate a combination tapered sensing region with a lens for signal collection and a tab for gluing the probe into the coupon (Figure 7) (Saaski and Jung, 2000). The coupon automatically aligns the probe so that it is in the middle of the fluid channel (Saaski, 2000) and the light emitted from the end of the fiber is focused onto a collection lens in the permanent portion of the biosensor.

Figure 7. Schematic (A) and photograph (B) of injection-molded optical fiber probes and fluidics coupon for use with the RAPTOR-Plus.

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