FOCUS
Optical and Piezoelectric Biosensors Research advances on fiber-optic, bulk-wave piezoelectric, and surface acoustic-wave devices improve prospects for commercialization One of the most significant applications of analytical chemistry involves its use in the determination of biologically important analytes. In recent years, considerable research in analytical biochemistry has centered on the development of small, portable, and (in some cases) disposable monitoring devices that are collectively referred to as biosensors. Biosensors are based on a number of different operating principles, including amperometric and potentiometric electrochemistry (see the Sept. 15 FOCUS, p. 1091 A), fiber optics, and piezoelectricity or microacoustics. In this second of a two-part FOCUS, optical and piezoelectric sensors are discussed. Fiber-optic biosensors Fiber-optic sensing (1-3) is based on the absorption, scattering, or fluorescence of light by a sample located at the end of an optical fiber, with both the excitation source and detection device located remotely at the beginning of the fiber. The analytical signal may be either direct (absorption, scattering, or fluorescence originating with the analyte species itself) or indirect (a reagent acting as an intermediary by exhibiting a change in spectral properties when analyte is present). Fiber-optic sensors have already been developed for pH measurements and for the detection of the partial pressure of blood gases such as 0 2 and CO2 (2). The first successful commercial realization of fiber-optic sensors was an instrument from Cardiovascular Devices, Inc. (Irvine, Calif.) for the determination of pH, C0 2 , and O2 in blood bypass loops during open-heart surgery. This company has also described a fiber-optic device for in vivo blood gas sensing in critical care patients that has not yet been released commercially (4). Other companies are rumored to be
working on fiber-optic biosensors, but Cardiovascular Devices appears to have a commercial edge at this time. According to W. Rudolf Seitz, who conducts research on fiber-optic sensors at the University of New Hampshire, "Several years down the road there may be several products available for in vivo blood gas measurements. This is a big area right now that really dominates potential applications." One major problem that must be faced in the development of in vivo sensors is that of selectivity—ensuring that a sensor measures the analyte(s) of interest without interference from other components of the biological sample. One approach to the improvement of selectivity that has been adopted by Frank Bright of the State University of New York at Buffalo and Gary Hieftje of Indiana University involves the collection of multidimensional fluorometric data by fiber-optic sensors. "Most of the work that has been done to date involves just wavelength selectivity," explains Bright. "However, fluorescence lifetimes, radiation polarization, and rotational correlation times are also parameters of interest, and these can be used to resolve components that are spectrally similar." The multidimensional approach involves the use of several data channels, each of which contains information that is independent of that found in the other dimensions. According to Bright, "If you have four selectivity parameters—excitation wavelength, emission wavelength, fluorescence lifetime, and polarization—and if you consider that each of those parameters has 100 resolution elements, it turns out that if you use all four parameters simultaneously you will have 108 resolution elements. This is analogous to a chromatographic system with 100 million theoretical plates, which doesn't exist in this day and age. The next step is to try to use
this technique to develop fiber-optic sensors for in vivo work, because multidimensional sensing will place all the selectivity you could ever imagine at your fingertips, at the end of an optical fiber." Another approach to solving the selectivity problem involves the use of unusually selective probe reagents, such as immunoagents. Several types of immunoassays are possible (5), the most straightforward of which involves direct measurement of the signal obtained upon the binding of a naturally fluorescent antigen with an immobilized antibody at a fiber-optic tip. The
.. multidimensional sensing will place all the selectivity you could ever imagine at your fingertips, at the end of an optical
fiber. 9 9
direct measurement technique is exemplified by a procedure (6) in which antibodies to the carcinogen benzo(a)pyrene (BaP) produced by polyclonal techniques are covalently attached to a fiber-optic probe. When the probe is incubated in a sample solution containing BaP (analyte), BaP molecules in solution conjugate with immobilized antibodies on the sensor tip. After the sensor tip is removed from the solution and rinsed, laser excitation radiation is directed to it by opening a shutter; fluorescence from the BaP molecules is then measured by a detection system located at the other end of the fiber. For nonfluorescent analytes, detection can be accomplished by a sandwich technique in which a fluorophor-labeled second antibody is bound to antigen that has been bound to immobilized antibody in a preceding step. A third type of scheme involves com-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987 · 1161 A
FOCUS
SULFITE ANALYSES 232 ppm Sulfite (as S 0 2 )
TOTAL SULFITE* IN WHITE WINE \ Column: Cat. No. 26&051 Eluant5mMH 2 SOA Flow: 0.85 ml/minute I Sample: White Wine, 1)250 Detector: WESCAN SULFITE ANALYZER Potential: 0.6V Range: 100nAFS
petition between fluorophor-labeled and unlabeled antigen (analyte) for a limited number of receptor binding sites. An example of this competitive type of assay is a technique developed by S. M. Angel of Lawrence Livermore National Laboratory in which a competition is set up between untagged antigen (analyte) and tagged antigen for binding sites on the surface of an optical fiber. Displacement of the tagged antigen results in a decrease in the intensity of the fluorescence signal. According to Angel, detection limits as low as 10~12 M have been accomplished with this technique. In a similar effort by Michael J. Sepaniak, Tuan Vo-Dinh, and colleagues at the University of Tennessee and Oak Ridge National Laboratory (3), rabbit immunoglobulin-G (IgG) is covalently immobilized on the distal sensing tip of a quartz optical fiber. The sensor is then exposed to fluorescein isothiocyanate-labeled and unlabeled antirabbit IgG. As expected, the sensor response to fluorescence emission at the optical fiber sensing tip is once again inversely proportional to the amount of unlabeled antigen (anti-IgG) in the sample. According to Seitz, one important goal in future research on fiber-optic sensors involves the development of reversible indicators for ionic species such as K + , Na + , and Cl~. "We're trying to develop a chemical system that will enable us to use the kinds of ionophores found in ion-selective electrodes for optical instead of electrochemical readout," Seitz explains. Piezoelectric devices
0
minutes
5
with the WESCAN Sulfite Analyzer • Excellent correlation with Monier-Williams • Detects to sub-ppm levels • Less than 12 minutes per sample • Automated systems available WESCAN INSTRUMENTS 2051 Waukegan Road Deerfield, IL 60015 PHONE: 312-392-2670
Piezoelectric devices are sensitive mass-to-frequency transducers that originated in the electrical engineering community and are now of interest for potential analytical chemistry applications. There are two types of piezoelectric sensors: bulk-wave devices and surface acoustic-wave (SAW) devices. SAWs are capable of fundamental oscillations at higher frequencies than are bulk-wave devices, and some people believe that this makes SAW devices capable of lower detection limits, although this is still a matter of some controversy. A piezoelectric device is sensitive to changes in the mass, density, or viscosity of samples in contact with its active surface. The most straightforward application of piezoelectric sensors involves the microweighing of substances deposited on the active surface of the device in a nonspecific manner. At the next level of sophistication, piezoelectric devices are used for surface reaction sensing (e.g., the real-
CIRCLE 173 ON READER SERVICE CARD 1162 A · ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
time monitoring of coating processes or of chemical reactions involving covalent bonding). In addition, they are used to measure the viscosity of liquids in contact with the sensor surface, and they are applicable to the monitoring of electrochemical reactions based on their sensitivity to mass changes associated with redox processes. Potential analytical applications depend on efforts to enhance this capability for simple mass detection with tech-
If you want to make a sensor you can s e l l . . . , you have to think in terms of a great deal of information going far beyond what one or two or even an array of several sensors will give you. % tt niques to improve selectivity for analytes of interest. "If you can place an antibody or other specific material on a surface, resulting in a selective addition, a wide variety of commercial applications are possible," says Glenn Bastiaans of Integrated Chemical Sensors Co. (Newton, Mass.). "The most immediate market would be for clinical assays, especially in doctors' offices or other locations where you would prefer to avoid a large, sophisticated instrument. Home health care applications may also be important down the line." Bastiaans and his colleagues have already developed SAW devices for the determination of antihuman IgG (using a surface coating containing human IgG), and human chorionic gonadotropin (HCG, the hormone that serves as an indicator of pregnancy). These two devices are now in the proof-of-principle stage of commercialization. Work also is proceeding in a number of other laboratories to develop specific surface coatings t h a t will enhance piezoelectric selectivity while simultaneously achieving device stability, rapid response time, and high sensitivity. Although there have been significant successes, such as the IgG and HCG sensors, this effort is proving to be quite a challenge. "I hope that people will not be led down the primrose path, thinking that all of this is very simple and trivial," explains Hank Wohltjen of Microsensor Systems, Inc. (Fairfax, Va.). "It's really not. There are some
Reversible Polymeric Gels and Related Systems Reveals
T
his comprehensive new book presents the powerful techniques and sophisticated data analyses used to study gel systems. The editor has compiled leadingedge work in this area from physicists, biochemists, medical technologists, and small-molecule and macromolecular chemists worldwide. You'll find an excellent introduction to gels, along with a detailed description of synthetic systems in nonaqueous media. You'll study in-depth discussions on gels made from rodlike polymers, gelation of DNA and gelatin, and a kinetic theory pertaining to the cytoskeleton. You'll gain new insights into the physics and chemistry of gelation in these important areas of research: • • • • • •
Phase Relationships Kinetics and Mechanisms of Gelation Structural Investigations Viscoelastic Properties Short-Range Dynamics Biogels
This volume also includes an in-depth report on a promising copolymer for biomedical applications. All polymer scientists, colloid and biophysical chemists, and physicists working in both experimental and theoretical studies will find this far-reaching book a significant addition to the literature of reverse gelation. Paul S. Russo, Editor, Louisiana State University Developed from a symposium sponsored by the Division of Polymer Chemistry, Inc. of the American Chemical Society ACS S y m p o s i u m Series N o . 3 S 0 3 2 4 pages (1987) Clothbound ISBN 0 - 8 4 1 2 - 1 4 1 5 - 8 IX 87-20305 US & Canada $ 6 4 . 9 5 E x p o r t $ 7 7 . 9 5 Order from: American Chemical Society Distribution Office Dept. 68 IfSS Sixteenth St., N.W. Washington, DC 20036 or CALL TOLL FREE
800-227-5558 and use vour credit card!
very exciting prospects that people are just beginning to explore, but I think we have a long way to go on the device end of things, let alone in the immobilization of bioreagents on SAW surfaces and in obtaining useful activities in solution." Despite these challenges, Wohltjen sees a bright future for piezoelectric sensors, "It appears to me," he says, "that piezoelectric devices will be applicable to a very broad range of gasphase and solution analyses. Potential applications include the detection of biospecies with antigen and antibodytype coatings, the determination of ions in solution using chelating compounds, and the sensing of gas-phase species using, for example, sorbent polymers. In addition, they may be applicable to the detection of whole cells and other large species that are not easily handled with other techniques." Bastiaans sees potential SAW applications in clinical chemistry testing, warfare agent detection, environmental monitoring, and process control. George Guilbault of the University of New Orleans has applied enzymes and antibodies onto the active surfaces of bulk-wave piezoelectric devices for the detection of enzyme substrates or inhibitors (in the case of immobilized enzymes) and of antigens (in the case of immobilized antibodies). According to Guilbault, "Sensitivity is at the partsper-billion concentration level, with almost total specificity being observed. The responses are totally reversible, with response times and baseline recovery times of less than one minute." However, Guilbault's contention that enzymes and antibodies immobilized on a quartz surface also maintain bioactivity out of solution has generated considerable controversy among piezoelectric researchers. Focus on applications Many other biosensor principles are being pursued. Aside from amperometric, potentiometric, optical, and piezoelectric sensors, research is proceeding on thermal biosensors (Bengt Danielsson, University of Lund), in vivo monitoring with microdialysis sampling probes coupled to liquid chromatography-electrochemistry (Peter Kissinger, Purdue University and Bioanalytical Systems, Inc.), biosensing using neuroreceptors as selectivity reagents (Lemuel B. Wingard, Jr., University of Pittsburgh), and the influence of the receptor-transducer interface on strategies for biosensor development (Michael Thompson, University of Toronto). "The position I take is that things have reached a state today where a lot
1164 A · ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
of things are going on in sensors that weren't going on a few years ago," says John I. Peterson, a researcher on optical biosensing at the National Institutes of Health. "For example, the electrode people are doing a lot of really interesting things that nobody even thought about a few years ago. So the prospects of really good sensor developments, I think, are very good today. "However," Peterson continues, "anyone who is interested in a particular application should look at electrodes, look at fiber optics, look at other possibilities, and make as good a judgment as possible as to what technology is best for that application. I think everything should be looked at more from an application orientation. There are so many ideas and so much going on, that no single idea, such as field-effect transistors, acoustic-wave devices, or anything else is going to be the wave of the future. You have to think in terms of what type of approach will be best for the application you have in mind." A biosensor researcher from a major corporation, speaking at the recent ACS Division of Analytical Chemistry Summer Symposium on biosensors, contended that "the last person in the world qualified to define sensor is the scientist who works on it. I think the person who can really give you an adequate definition is the user, the customer. We've done enough surveys at our company to know what definition they give. The definition is very interesting as far as the grandmother at home is concerned: A sensor is a thing that tells her what she wants to know. What does she want to know? Basically she wants to know, in the case of biosensors, 'Am I healthy?' To answer that question, you cannot rely on one or two analytes. If you want to make a sensor you can sell to that grandmother, you have to think in terms of a great deal of information going far beyond what one or two or even an array of several sensors will give you. You have to think of sensors not in terms of analytes, but in terms of useful information." Stu Borman References (1) Seitz, W. R. Anal. Chem. 1984,56,16 A34 A. (2) Peterson, J. I.; Vurek, G. G. Science, 13 April 1984,123. (3) Peterson, J. I., Encyclopedia of Medical Devices and Instrumentation; John Wiley and Sons: New York, in press. (4) Gehrich, J. L. et al. IEEE Trans. Biomed. Eng. 1986, BME-33,117. (5) Tromberg, B. J. et al. Anal. Chem. 1987, 59,1226-30. (6) Vo-Dinh, T. et al. Appl. Spectrosc. 1987,47(5), 735-38.
