Focus
Bioanalytical Applications of Fiber-Optic Chemical Sensors Fiber-optic sensors show potential for in vivo determination blood gases, glucose, and various biochemicals As G. H. Morrison pointed out in last month's editorial (p. 993), advances in analytical instrumentation are increasingly being applied to the solution of bioanalytical problems. The fiber-optic sensor represents one such advance, and recent developments in this field were described by John Peterson of the National Institutes of Health (NIH) at the Workshop on Bioanalytical Chemistry held last February on NIH's Bethesda campus. Peterson, along with Seth Goldstein, first described the use of a chemical indicator sensing package at the end of an optical fiber in the midseventies. This first fiber-optic biosensor was used for in vivo pH monitoring, and it sparked development of many other biosensors, including pC>2 and p C 0 2 sensors, glucose sensors, an alkali metal sensor, and sensors to detect various biochemicals and drugs. Fiber-optic sensors are also being developed for various remote-sensing applications by a group led by Tomas Hirschfeld at the Lawrence Livermore National Laboratory. (Editor's note: We regret to report that we learned after this article was written that Tomas Hirschfeld died unexpectedly April 24.) The concept of a fiber-optic chemical sensor is simple; such a sensor is really just a miniature spectroscopic cell on the end of an optical fiber. Light from a suitable source travels along an optically conducting fiber to a sensor at its end, where it interacts with the sample and then returns to a detector along either the same or another fiber. Fiber-optic sensing differs from traditional spectroscopy in that it allows greater miniaturization and localization of optical measurements. The early sensors were based on indicator dye chemistry and essentially provided a miniaturized spectrophotometric analysis. This approach to sensor development is still often used, and sen-
sors based on fluorescence, luminescence, and other optical processes have also been developed. The utility of all of these sensors has been demonstrated in the laboratory, but most have not been used for actual in vivo sensing. Instrumentation and indicator systems Fiber-optic instrumentation involves conventional light sources, detectors, etc., and so it is not an area requiring significant development, although work on refining both optical
Fiber-optic sensing differs from traditional spectroscopy in that it allows greater miniaturization and localization of optical measurements. probe design and means of immobilizing the indicator on the end of the fiber is continuing. And if a probe is to be used for medical purposes, the sensor components must also be biocompatible and sterilizable, making their design even more challenging. One of the most frustrating aspects of fiber-optic sensor design, according to Peterson, is that ideal fibers for chemical sensing have not been developed. Plastic fibers have a large aperture and are very flexible and easy to work with, but they cannot tolerate elevated temperatures (and therefore cannot be easily sterilized), have high attenuation, and transmit only in the visible range. Glass fibers are available in very small sizes and have very low attenuation, but they have a small aperture, and like plastic fibers, glass fi-
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bers are suitable only for use in the visible range. Fused-silica fibers have recently become available with good UV transmission, but the aperture is not much better than glass, and these fibers tend to be fragile and difficult to work with. "The problem is that fiber-optic chemical sensors do not represent a sufficient market to generate the development and fabrication of suitable fibers," says Peterson (1). The indicator systems used in fiberoptic sensors are based on reversible reactions in which the reagent phase is not consumed by its interaction with the analyte. (Sensors based on nonreversible reactions have not been developed, primarily because the relative consumption of reagent must be small in such sensors, or there must be provision for renewing the reagent. Peterson believes that the idea of a onetime throw-away sensor based on a nonreversible reaction has merit, but so far no one has pursued such a line of research.) A wide variety of reversible indicator systems have been considered, including those based on direct measurement, absorbance, fluorescence (both emission and quenching), and reflectance. Peterson believes that chemiluminescent systems may also be useful, but such systems have not yet been considered. Absorbance and fluorescence sensors The first fiber-optic sensors were based on absorbance measurements, and this technique is still being used to design new sensors. For example, Michael Sepaniak and his co-workers at the University of Tennessee have developed a new spectrophotometric sensor that contains a sample chamber with a well-defined optical path length, making it possible to obtain true absorbance values as well as absorbance spectra (2). The new sensor has been used for measuring bilirubin in blood serum, and Sepaniak hopes the sensor will eventually be used for 0003-2700/86/0358-766A$01.50/0 © 1986 American Chemical Society
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Focus monitoring drugs, toxins, and naturally occurring biochemicals in various body fluids such as cerebral spinal fluid, amniotic fluid, or even the interstitial fluids of soft tissue. Other researchers are designing fiber-optic biosensors that use enzymatic, immunochemical, or other biocatalytic reactions instead of the traditional dye indicator reactions. For example, Mark Arnold of the University of Iowa has designed a fiber-optic chemical sensor in which an isolated enzyme is immobilized at the surface of a bifurcated optical fiber bundle (3). Response of the sensor is based on directly measuring the enzymatic generation of a spectrophotometrically detectable product. Immunosensors
Researchers are designing fiber-optic biosensors that use enzymatic, immunochemical, or other biocatalytic reactions instead of the traditional dye indicator reactions. are being developed by both Sepaniak and by Hirschfeld's group, and like the enzyme-based sensors, these are highly specific and have the potential for determining many different biomedically important analytes. Fluorescence-based fiber-optic chemical sensors have been aimed primarily at the determination of pH and blood gases (CO2 and O2), although Sepaniak's group is also working on an optical-fiber fluoroprobe for clinical determination of drugs in body fluids or tissue. Blood gas sensors have been developed by several researchers, including Peterson, Hirschfeld, and Otto Wolfbeis of the Institut fur Organische Chemie in Graz, Austria. This is the only type of fiber-optic sensor that is commercially available. Cardiovascular Devices of Irvine, Calif., currently the only company selling fiber-optic sensing devices (although it is likely to have some competition in the near future), offers two such sensors: an extracorporeal sensor for use in an externally diverted bloodstream (such as during openheart surgery) and a catheter for actual in vivo use. Affinity sensors
The newest trend in fiber-optic sensor development is that of affinity sensors, which combine fluorescence
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detection with competitive-binding reactions. In addition to their inherent high selectivity, these sensors can use analytical reactions that don't directly produce an optical change. A competitive-binding affinity sensor for glucose was developed several years ago by J. S. Schultz (4). In this sensor, the specific glucose-binding reagent concanavalin A is immobilized on the inner wall of a hollow fiber, and glucose competes for binding sites with fluorescein-labeled dextran. In the absence of glucose, the dextran is bound to the concanavalin A substrate, but when the glucose concentration increases, some of the dextran is driven off into the optical path, and there is an increase in the fluorescence intensity proportional to the glucose concentration. At the 1986 Pittsburgh Conference held last March in Atlantic City, Rudolf Seitz of the University of New Hampshire presented preliminary work on the development of a sensor that uses fluorescence energy transfer as an alternative to visualization of the dextran displacement by glucose. In Seitz's approach, both the dextran and concanavalin A are labeled with fluorophores, one with a donor and one with an acceptor. As with Schultz's sensor, dextran is bound to the concanavalin A in the absence of glucose, and the distance between donor and acceptor is short enough that the excited donor transfers energy to the acceptor. Added glucose displaces the dextran from the concanavalin A and causes a decrease in the efficiency of energy transfer. Efforts are under way to develop conditions in which the energy transfer process not only quenches donor emission but also leads to significant acceptor emission intensities. This would allow a single ratio measurement of donor-to-acceptor emission intensity, and such a ratio measurement would be inherently more stable than a single intensity measurement. Also at this year's Pittsburgh Conference, Seitz introduced a competitive-binding sensor designed to determine fluoride, chloride, and nitrite anions using fluorescein-labeled dextran and Texas-Red-labeled polyethylenimine confined by a dialysis membrane. Seitz is also developing a sensor for determination of alkali metal ions based on competition of the metal ions and fluorescein-labeled polyethylenimine for crown ether binding sites. Future fiber-optic sensor development is expected to make use of increasingly sophisticated optical techniques. For example, in a REPORT published in the January 1984 issue of
Focus ANALYTICAL C H E M I S T R Y , Seitz pre-
dicted that the use of multiwavelength and temporal information could allow fiber-optic sensors to determine two or more analytes simultaneously. Multiwavelength measurements could also be used to monitor reagent-phase stability or to relate analyte concentrations to intensity ratios at two wavelengths. And if the sensor involves luminescence, time resolution can also be used. Comparison of fiber-optic sensors with microelectrodes and CHEMFETs Recently there has been a great deal of interest in sensors of all types, and other approaches to biosensing, including development of microelec-
The newest trend in fiber-optic sensor development is that of affinity sensors, which combine fluorescence detection with competitivebinding reactions. trodes and chemically sensitive field effect transistors (CHEMFETs), are being pursued by bioanalytical researchers. Although electrodes offer small size, low cost, and sensitivity to a variety of analytes, their performance has been disappointing because of various stability problems. But there are indications that this may be changing. "Because of the new technologies and ideas that have come out in terms of all types of electroactive polymers that can be used both as coatings for the electrodes and as membrane materials," says Peterson, "you can now do things with electrodes that you couldn't do just a few years ago. It has become a very interesting field." CHEMFETs, which combine an electrode with a solid-state charge indicator, are attractive for in vivo chemical monitoring because of their small size and versatility, but although they have gotten a great deal of publicity, they have not worked out satisfactorily so far. There is, however, still a great deal of interest in CHEMFET development. of proven value in most applications, Peterson believes that fiber-optic devices can be as small as electrosensors and that they offer several advantages
for medical applications. They are safe (because no electrical connection with the body is involved), the optical leads are flexible, and materials suitable for long-term implantation, such as plastic, can be used. Fiber-optic biosensors are also easily made, and some are even sufficiently simple in design to be disposable. And because the measurement is equilibrium based rather than diffusion rate dependent, as is the case with some electrodes, fiberoptic sensors exhibit particular advantages in long-term stability and simplification of calibration. But optical sensors are also subject to some limitations relative to microelectrodes. Because the reagent and the analyte are in different phases, there is necessarily a mass transfer step before constant response is reached, and this leads to relatively slow response times for fiber-optic sensors. According to Peterson, the response time of a fiber-optic sensor is related to the design of the sensor; a response time of 30 s is typical for 90% response. To some extent fiber optics is in direct competition with microelectrodes and CHEMFETs, but, says Peterson,
"Each has its own unique advantages, and all three are important because they offer the capability of on-line, in situ analysis." Because optical techniques are the workhorse of the analytical lab (it has been estimated that 80% of all laboratory measurements are done by optical methods), researchers developing fiber-optic sensors have a broad base of known analytical reagent systems from which to derive new sensor designs. "Reversible indicators are available for much of what is of interest in analytical measurements," says Peterson, "and their adaptation t o fiber optics is limited only by one's ingenuity." M.D.W. References (1) Peterson, J. I. Proceedings of the Symposium on Biosensors; Sponsored by the IEEE Engineering in Medicine and Biology Society and the National Science Foundation, Los Angeles, Calif., Sept. 15-17, 1984; pp. 35-39. (2) Coleman, J. T.; Eastham, J. F.; Sepaniak, M. J. Anal. Chem. 1984, 56, 2246-49. (3) Arnold, M. A. Anal. Chem. 1985, 57, 565-66. (4) Schultz, J. S.; Mansoure, S.; Goldstein, I. J. Diabetes Care 1982, 5, 245-53.
New Monochromator Attracts Attention of IR Spectroscopists "There's a long dry spell between new technologies in some fields of instrumentation, and this is something quite radically new—a whole new technology on how to do spectroscopic measurements in the field of infrared [IR] spectroscopy. However, it does have a number of limitations and, in my opinion, it's probably not likely to replace gratings and interferometers for general spectroscopic use." The instrument engineer quoted above (he requested anonymity) was referring to a device called an acoustooptic tunable filter, or AOTF (photo). The AOTF was introduced commercially at this year's Pittsburgh Conference by the Westinghouse Electric Corporation Combustion Control Division. The device drew a significant amount of interest at this year's Pittsburgh Conference exhibit, and, despite some assertions to the contrary, the question as to whether or not the AOTF will one day be used in general spectroscopic applications is still very much an open one.
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Essentially, the AOTF is a new type of monochromator. Historically, four types of devices—filter wheels, prisms, diffraction gratings, and interferometers—have been used to separate IR wavelengths spatially or temporally for molecular spectroscopy. Westinghouse is promoting its AOTF as a possible fifth major type of spectroscopic analyzer. At its Pittsburgh
