SPECIAL REPORT
RincpncnpQ I—J\\^J\—A
il Ji )\^JL ι J
Garry A. Rechnitz, University of Delaware
In science, it is generally true that what we cannot measure, we cannot understand. Nowhere is this tru ism more valid than in the rapidly evolving fields of biotechnology and medicine, where progress is often measurement limited. Biosensors, although still in a developmental stage, promise to meet some important measurement needs, especially for drugs, metabolites, and other biomolecules. Although many other types of analytical instru
mentation have similar goals, biosensors are unique in being discrete devices that actually incorporate biolog ical—even living—components as part of a sensor or probe. Research on biosensors is growing rapidly, with new investigators entering the field worldwide and new advances being reported almost weekly. Publica tions on fundamental and applied aspects of biosensors are appearing at a rate approaching 400 papers per year, and numerous conferences have recently been devoted to this topic. Some of the most exciting research is not just an
A crab whose antennules will provide chemoreceptive nerve fibers for use in a biosensor is examined by Garry A. Rechnitz at the University of Delaware; the micropipet lying beside the crab serves as the instrument's transducing elemen t; behind it are a storage oscilloscope and an audio tape recorder for data handling 24
September 5, 1988 C&EN
Crab antennule provides molecular recognition element for biosensor l
15cm
1
Electron micrograph of antennule (at 160X) shows area where hairlike sensory elements join main antennule
Sensing antennule removed from blue c r a b . . .
I
3 mm
1
. . . is dissected to expose chemoreceptive nerve fibers . . .
. which are coupled to a micropipet electrode . . .
. . . to produce a neurosignal of electrical pulses for display as an oscilloscopic trace
extension of earlier techniques but is a truly novel synthesis of biological and analytical concepts. Besides its practical benefits, biosensor research also offers new ideas for measurement techniques that, by focusing on environmentally benign natural materials, are in close harmony with nature. Such an emphasis is appealing and may help restore the popularity of chemistry with students and the general public. As biosensors are developed and improved, major applications can be expected in at least five areas: • Medicine and veterinary medicine. • Biotechnology. • Food and agriculture. • Environmental studies. • Military applications. Biosensors have two principal components: a molecular recognition element (the biological component) and a transducing or signal-generating element (the instrumental component). Much of the art of biosensor research involves optimizing the union of these components and creating new designs for use as biosensors. In doing so, biosensor research brings together
molecular biology, chemistry, materials science, physics, and bioengineering. Finding and exploiting new strategies for biosensor design is by no means a trivial problem. Fortunately, nature is a guide. The sensory abilities of natural species, ranging from bacteria to man, are remarkable with respect to their sensitivity, selectivity, and speed. Few if any biosensors constructed in the laboratory can match these properties; however, biosensors can be devised that have no counterpart in nature by bringing together novel combinations of components or by using synthetic materials.
Biosensors using chemoreceptors A striking illustration of these points can be found in receptor-based biosensors. Chemoreceptors are the biomolecular assemblies involved in the chemical senses, such as olfaction and taste, in metabolic and neural biochemical pathways, and in numerous other physiological functions. Consequently, chemoreceptors are excellent candidates as molecular recognition elements to test ideas for biosensor design, provided they can be coupled to instrumental components that will yield a measurable signal in response to the biomolecule to be sensed. Preferably, such a response should be both dependent on concentration and reversible. Adequate coupling is technically challenging because chemoreceptors are fragile and difficult to handle. One approach to receptor-based biosensors, first developed in my laboratory by Stuart Belli (now at Vassar College), uses intact chemoreceptor structures from natural sources, such as crustaceans and fish. Such organisms use their chemical senses to find food, locate a mate, define territory, recognize danger, or undertake other activities, based upon the detection of certain chemical cues. Such cues can be simple salts, sugars, amino acids, or complex molecules, including steroids or pheromones. Because aquatic organisms live in fresh or seawater, they have the physiological arrangement necessary for solution sensing. Thus they are excellent candidates as biosensors for dissolved materials. Moreover, such animals survive by doing their sensing in "real time" and their sensory apparatus must provide sensitive and selective measurements with exceptionally rapid response and recovery times. These are, of course, just the characteristics that are desirable in biosensors if nature is to be mimicked in the laboratory. Studies in my laboratory show that portions of the sensing antennules of the blue crab Callinectes sapidus can act as molecular recognition elements in constructing biosensors by joining the chemoreceptive nerve fibers to a micropipet electrode. This can be done readily under a dissecting microscope, using micromanipulators. The resulting device has been termed a "recepSeptember 5, 1988 C&EN
25
Special Report trode"—although some of my irreverent colleagues refer to such plant- or animal-based systems, because of their source, as "supermarket biosensors." In response to the interaction of stimulant biomolecules with the chemoreceptive sites on the sensory structures, a signal is produced consisting of electrical pulses or spikes whose frequency is a function of the stimulant concentration. These pulses can be displayed on an oscilloscope and easily collected with automated counting equipment for data analysis. For measurement purposes, a calibration plot of the dose/response relationship is made with known standards and then compared with data from unknown samples. Because the neuronal pulses are produced on a millisecond time scale, sufficient analytical data usually can be collected within a few seconds, although counting can be made more precise for highly quantitative analysis by sampling for a slightly longer time. The fast response times observed are due to two factors. First, the small free energy of binding is converted to an easily detectable electrical signal aided by the active signal transport in the nerves. Thus, a change in potential corresponding to the movement of only 107 sodium ions is too small to be carried by passive transport and yet is actively carried along the nerves. Second, the receptor cell is held far from equilibrium, both in ion concentration and potential across the cell membrane. Triggering of an action potential requires only the movement of about 6000 ions, too few to appreciably change the physiological concentration gradient. Therefore, the potential pulse is not slowed by an approach to equilibrium and the resting potential is re-established within 2 to 3 milliseconds. This is the ultimate limit in response times. The response, R, is related to the concentration, C, of the stimulant biomolecule by the equation
with crabs, crayfish, lobster, and catfish has demonstrated quantitative responses to amino acids, hormones, nucleotides, drugs, and toxins. In some cases, such responses are highly selective for specific compounds, such as glutamic acid, in the presence of structurally similar compounds. It is still not yet clear whether intact chemoreceptor structures or isolated receptors will be most advantageous as molecular recognition elements for biosensor design. Most of the work on isolated receptors has involved the nicotinic acetylcholine receptor from electric eels and fish. Isao Karube and his coworkers at Tokyo Institute of Technology in Japan recently developed a biosensor in which an acetylcholine-receptorcontaining lipid layer was placed on an ion-sensitive field effect transistor (ISFET). In this work, which is still very preliminary, two coated ISFETs were operated in a differential mode, with one containing the receptor preparation from the electric fish Torpedo cdlifornica and the other a blank membrane. Karube has observed limited acetylcholine responses in the micromolar concentration range. Lemuel B. Wingard of the University of Pittsburgh has investigated the purification, structural character, and reconstitution of receptors for 7-amino butyric acid (GABA), a neurotransmitter, for possible immobilization on piezoelectric crystals or electrical capacitance measuring devices. The GABA receptor recognition site is associated with the opening of a chloride ion channel and has binding sites for picrotoxin, benzodiazepines, and barbiturates. Although work on GABA receptors has not yet reached the stage of sensor construction, informal reports suggest that a capacitive biosensor using acetylcholine receptors is being developed under Army sponsorship.
R = Rmax/fl + ( K / C H
Although a number of laboratories are currently active in the immunosensor field, no fully satisfactory biosensor of this type has yet emerged. Problems with interferences remain to be solved and response is slow. In principle, antibodies are ideal candidates for use as molecular recognition elements in biosensor design. They have the ability to bind antigens quite selectively and with binding constants (which indicate the ability
where K is a constant, Rmax is the maximum response, and n the cooperativity coefficient (a measure of receptor diversity in responding to differing stimuli). For the simplest case, in which n = 1, assuming receptors with identical properties, this equation is identical to the well known Michaelis-Menten equation for enzyme kinetics. The expression predicts an analytical range, between threshold and saturation, of two or three orders of magnitude. The fact that a much wider range of concentrations is actually detected (we have demonstrated ranges of 10~~2 to 10~9 M) is attributed to the presence of receptors of varying sensitivity ranges. In each crab aesthestasc (hairlike structures), as many as 6000 to 8000 sensory endings may converge to 300 to 500 receptor cells. Behavioral studies of live crabs show that they respond to amino acids at levels as low as 10~15 M; however, laboratory biosensors acquire only one channel of information whereas the living organism monitors thousands. Array sensors with multichannel response may be feasible (if technically challenging) and may offer a means for both qualitative and quantitative measurement of responding biomolecules with a single device. Work to date at the University of Delaware 26 September 5, 1988 C&EN
Immunological biosensors
Field effect transistor with chemoreceptors on membrane responds to acetylcholine
D-
Sample solution
7 -Reference electrode
Membrane coated with isolated chemoreceptors Insulator iSourcel
I Drain
^(n-type silicon^ Substrate (p-type silicon)
CIRCLE 18 ON READER SERVICE CARD
M A T T S O N
I N S T R U M E N T S ,
Manufacturers of FT-IR Instruments for the Research, Scientific and Industrial Laboratory
1
9
y
8
8
PROGRESS REPORT TO OUR CUSTOMERS
I N C .
PROGRESS REPORT During the time Mattson Instruments made its last progress report to customers we have made tremendous gains. Mattson Instruments has taken big strides toward our goals of an increased presence in the world FT-IR marketplace, improved system reliability, an expanded product offering, enhanced company stability, and most importantly, the ability to better serve our customers. This report describes that progress.
Gains In Market
Presence
1. Since receiving our first order in September, 1983, Mattson Instruments has delivered over $40 million in FT-IR instruments worldwide. Projections for the current year put the total at more than $50 million. Cumulative FT-IR Sales (millions) 50 40
3. To date, Mattson Instruments has installed over 400 instruments in the United States. Included among our industrial customers in the United States are: Alcoa Amoco AT&T Baxter Laboratories Bio-Rad
Schering Plough
International Paper Company
Shell Oil Company
Kraft
Smith Kline Beckman
Rohm & Haas
Lever Brothers Merck Company
Sun Refining Company
Corning Glass
Mobil
Texas Instruments
Dow Chemical
Northrup King
TRW
DuPont
Owens Corning
Union Carbide
Eastman Kodak
Parke Davis
UNISYS Corporation
Exxon
Pepsico
Jim Walter Corporation
General Motors Corporation
Polaroid PPG Industries Procter and Gamble
Warner Lambert Westinghouse
Mattson Instruments has a long list of university and college customers, including:
10
Massachusetts Institute of Technology Middlebury College
University of Pennsylvania Rensselaer Polytechnic Institute
Cornell University
University of Missouri
Rice University
University of Connecticut
North Carolina State University
University of Alabama Brown University Carnegie-Mellon
1983
1984 1985 1986 1987 1988* * Includes projections for 1988.
University of Florida
2. A recently published survey illustrates the growing recognition and acceptance of Mattson Instruments by FT-IR buyers. Percent of FT-IR Buyers Naming Mattson Instruments As A Choice 31%
Georgia Institute of Technology John Hopkins University Kansas State University University of Kentucky University of Maine
30%
Northeastern University N.Y.U. University of North Carolina Ohio University Ohio State University University of Oklahoma
Rutgers University Syracuse University S.U.N.Y. Temple University University of Texas Tulane University Virginia Commonwealth University University of Wisconsin
Pennsylvania State University
20%-
Research and government facilities using Mattson*! FT-IR spectrometers include: 11%
10%
IBM
Colgate Palmolive
General Electric
20
R.J. Reynolds
Bristol-Myers
Ford Motor Company
30
Grumman Aerospace Corporation
11%
LI 1984
1985
1986 1987
Source: Market Position Study, Analytical Chemistry
Aberdeen Proving Grounds
Maryland Department of Health
U.S. Department of Energy
Bell Labs Consumer Products Safety Commission
NASA National Bureau of Standards
U.S. Environmental Protection Agency U.S. FDA
Federal Bureau of Investigation
National Gallery of Art Naval Research Labs Smithsonian Institution
Woods Hole Oceanographic Institute
Lockheed Space Operations
PROGRESS REPORT A Broad Product
Offering
Today, Mattson Instruments offers its customers a broad product line, one that can meet just about any FT-IR requirement: • Polaris™. A versatile and affordable FT-IR spectrometer with selectable resolution to 0.5 wavenumbers (cm""1), by no means a lightweight. Available as a stand-alone data collection system or use the Polaris with Mattson Instruments' outstanding ICON™ FT-IR analytical software. • Cygnus ™, A real workhorse of a spectrometer, with selectable resolution to 0.125 wavenumbers, highstability cube corner retroreflectors, and kinematically mounted optics and sample holder. An external sample beam is included for GC/FT-IR and FT-IR microscopy applications. Cygnus operates with Mattson Instruments' UNIX®-based EXPERT-IR™ data acquisition and analysis software. • Nova Cygni™. Mattson Instruments' top-of-theline spectrometer system provides selectable resolution to 0.09 wavenumbers, Mattson's research-grade optics, a versatile and spacious sampling compartment, and external sample beam control. EXPERT-IR software and two operator terminals make Nova Cygni a powerful multi-user, multi-tasking data collection and analysis system. • ICON FT-IR Analytical Software. Mattson Instruments' ICON software, operating in the MSDOS® environment on the IBM®AT (and compatibles), provides complete control of spectrometer operations and access to a wide array of spectral analysis procedures. ICON supports the latest 80386 microprocessor technology and is also available for the new PS/2 personal computer. • EXPERT-IR Analytical Software. EXPERT-IR, Mattson Instruments' UNIX-based analytical software for advanced IR applications, provides complete spectrometer control, access to standard and advanced analytical procedures and multi-user, multi-tasking operation. EXPERT-IR utilizes the power and speed of the new 68030 microprocessors. • FT-IR Accessories. Mattson Instruments' complete line of FT-IR accessories includes the AccuLoad™ Attenuated Total Reflectance (ATR) Sampler, the Bach-Shearer™ FT-IR microscope,
diffuse and specular reflectance samplers, the LYRA™ lightpipe for on-the-fly FT-IR analysis of gas chromatograph fractions and more. * Cryolect When it comes to achieving both the sensitivity of mass spectroscopy and the specificity of infrared analysis - with just one GC injection - Mattson Instruments' Cryolect is the star of the show. (More information on the next page.)
Significant Progress In Customer Service, System Reliability 1. As a result of the substantial expansion of our field service organization, we believe Mattson Instruments has the highest ratio of field service engineers to FTIR instruments in the United States. 2. Mattson Instruments provides a guaranteed response to each customer's request for assistance within just 60 minutes. 3. Only Mattson Instruments offers a 98% UPTIME warranty on new instruments - a first in the FT-IR industry!
Sound Financial
Position
1. Mattson Instruments has matured into a profit maker, with consistent monthly profits. 2. The Company has a strong current ratio (the ratio of current assets to current liabilities) of 2.2 to 1.0. 3. Mattson Instruments' credit line with one of the largest banks in the United States has been expanded by over $1 million. 4. Also, Mattson Instruments has completed a private equity placement of $2 million with its original investor group to finance the Company's long-term growth plans. 5. Mattson Instruments' total assets are approaching $10 million.
PROGRESS REPORT
Mattson Instruments9 Cryolect For Matrix Isolated GC/FT-IR Mattson Instruments' Cryolect is a complete turnkey GC/FT-IR system designed for the infrared analysis of complex mixtures at sensitivities equal to those achieved by mass spectrometery. This remarkable IR sensitivity and specificity is made possible by matrix isolation (MI), a technique in which individual molecules of the gas chromatograph fraction are isolated in a cryogenically cooled argon matrix. Isolation in an inert matrix eliminates intermolecular band broadening and, thereby, significantly improves upon the inherent resolving power and sensitivity of the infrared analysis method. In recent months, approximately 20 research papers have been published to report results obtained using the matrix isolation technique. Specificity has been shown to include identification of isomers, conformers, and tautomers. Sensitivity has been shown to be improved by a factor of 1000 as compared to nonmatrix isolated GC/FT-IR techniques. Please contact Mattson Instruments to receive a bibliography of
Our
Thanks
We at Mattson Instruments would like to take this opportunity to thank you, our customers for your trust and confidence through the past five years. We value your loyalty and support; our many repeat customers make us especially honored.
The Mattson Instruments Cryolect Matrix Isolated GC/FT-IR analysis system provides the sensitivity ofmass spectroscopy with the specificity ofinfrared analysis.
these important publications. No other analytical technique available today can match the sensitivity and specificity of Mattson Instruments' Cryolect. To learn more about this and other advances in FT-IR technology being pioneered by Mattson Instruments, please call or write today.
Mattson Instruments' Commitment Mattson Instruments' commitment to its customers is not simply to maintain a high level of quality in the instruments we design and manufacture. Rather, Mattson Instruments is continually striving to provide you with a better and better FT-IR instrument. After all, FT-IR is our business - our only business!
Mattson Instruments, Inc. 1001 Fourier Court Madison, Wl 53717 Telephone: 608/831-5515 FAX: 608/831-2093
William J. Kennedy President - CEO
David R. Mattson Chairman - Chief Technical Officer
Polaris, Cygnus, Nova Cygni, ICON, and EXPERT-IR are trademarks ofMattson Instruments, Inc. UNIX is a registered trademark of AT&T Bell Laboratories. MS-DOS is a registered trademark of Microsoft Corporation. IBM is a registered trademark of International Business Machines Corporation. Motorola is a registered trademark of Motorola, Inc. Copyright © 1988, Mattson Instruments, Inc.
Piezoelectric crystal sensor changes frequency when exposed to antigens Oscillator
Piezoelectric crystal coated with antibodies
Frequency counter
Data collection system
of an antigen to interact with an antibody) that are neither too high nor too low. The introduction of monoclonal antibodies in recent years has further improved selectivity and has provided more uniformity in binding constants, as well. Such antibodies can now be raised to numerous biomolecules, drugs, viruses, and cellular materials at an acceptable cost. In practice, however, because of the high molecular weight of antibodies it is difficult to couple their reaction with corresponding antigens to a transducer in such a manner that the observed signal reflects the antibody-antigen interaction, at least in a quantitative way. George G. Guilbault of the University of New Orleans takes an ingenious approach to this problem. He coats piezoelectric crystals with antibodies to make biosensors for gaseous pollutants, such as the pesticide parathion. In this case, antiparathion antibodies are coated on quartz piezoelectric crystals, using bovine serum albumin/glutaraldehyde for immobilization. When mounted in a suitable apparatus, such crystals undergo changes in frequency if exposed to the antigen parathion. Guilbault has reported sensitivity in the parts per billion to parts per million range with response times of two to three minutes and sensor lifetimes of more than two weeks. Such an approach also may be extended by work now under way to solution samples. Because such frequency monitoring techniques really just involve changes in mass or pressure on the piezoelectric surface, no special coupling of antigen to antibody is needed and the technique may be generally applicable, provided interferences (due to chemisorption or physisorption) can be minimized. Ranald M. Sutherland and coworkers at Battelle Research Center in Geneva have described various modifications of internal reflection spectroscopy techniques relevant to the design of fiber optic immunosensors. This very interesting work indicates that the interaction of a coated antibody with its antigen can be monitored optically on a microscale. This biosensor has been used to measure concentrations of the important drug methotrexate. In favorable cases, immunoreactions can be coupled to electrochemical transducers for biosensor construction. Masuo Aizawa and his coworkers at Tokyo Institute of Technology have shown, for example, that antibodies to the Wasserman antigen (which is measured CIRCLE 18 ON READER SERVICE CARD
in testing for syphilis) can be immobilized on a cellulose acetate membrane, and potential changes occur when syphilis-positive serum is added to the sample. Working in my laboratory, Toshio Yao (now at the University of Osaka Prefecture) extended this concept to low-molecular-weight ligands such as riboflavin (vitamin B2) by using aporiboflavin-binding proteins. As early as 1978, ionophores (ion carriers) were used as a means of coupling antibody-antigen reactions with potentiometric membrane electrodes. The concept is simple; if an antigen can be coupled to an ionophore, then the reaction of the antigen with its antibody should modulate the ion carrier properties of the ionophore and produce a potential change. This effect has been extensively demonstrated for antibody responses but only in the past year was a reversible and reusable biosensor for antigen measurements developed using such an approach. The antigen sensor is a layered arrangement, with a small fixed quantity of antibody trapped in the space between the sensing membrane electrode and an outer barrier membrane of collagen chosen to permit passage of the low-molecular-weight antigen but not the bulky antibody. With this arrangement, the entire antibodyantigen reaction takes place within the tip of the sensor, where antigen bound in the sensing membrane competes for the trapped antibody with free antigen in the sample. This is equivalent to a classical competitive inhibition experiment, except that some of the reagents are immobilized at the sensor tip. This type of biosensor has two attractive analytical features. First, the antibody is retained within the sensor tip for repeated reuse, so that even expensive mono-
Antigen biosensor has antibody layer between sensing and barrier membranes Internal reference electrode
Antigen-containing sensing membrane
Internal solutior
Sp^éefr
A b
, Ab Ag A l g
-Ab
Spacer
Ah
Ab
— A g — Ag A
Collagen / membrane permeable to antigen
9
Ag
Antigen-containing sample
Trapped antibody layer
Ag = Antigen Ab = Antibody
September 5, 1988 C&EN
31
Special Report clonal antibodies can be used economically. Second, because the dynamic antibody-antigen competition is limited to the sensor tip region, the probe is reversible and can be employed for continuous antigen monitor ing, either to follow changes in antigen concentration in a given sample or to map out antigen levels in a succession of samples or regions. Such sensors so far are limited to low-molecular-weight antigens, such as dinitrophenol, and to antibody-antigen systems hav ing binding constants in the rapidly reversible range. Nonspecific interference effects can be a problem in biological fluids. Jerome S. Schultz and his coworkers at the Universi ty of Michigan have developed a highly interesting biosensor for glucose. This reversible affinity sensor operates on the basis of competition between glucose and dextran for the binding sites on concanavalin A, a commonly used biochemical reagent. Future prospects for immunological biosensors in clude possible use of catalytic antibodies and "Fab" antibody fragments. The latter are enzymatically cleaved portions of antibodies of lower molecular weight but similar binding ability; they are beginning to be commercially available and hold special promise for biosensor application because interference from re ceptor sites is reduced.
Biocatalytic biosensors More research effort has been invested in biocataly tic biosensors than any other type. Indeed, the entire biosensor field traces its origin to the general develop ment, more than 20 years ago, of so-called "enzyme electrodes," based on immobilized enzymes. Such ear ly biosensors are now a subset of a much larger catego ry involving a range of biocatalysts as molecular recog nition elements. Two principal trends now under way are the use of enzyme conjugates, cycling enzymes, and enzyme sequences in biosensor construction, as well as the substitution of complex biocatalysts, including bac terial cells and plant or animal tissue sections as re placements for isolated enzymes. At the same time, efforts are being made to extend biocatalytic biosensors from electrochemical to fiber optics, thermistors, or other signal transducers; practically all types of trans ducers now have been employed in conjunction with biocatalysts. Initially, researchers tried to couple sensing ele ments with a single isolated enzyme. When the en zyme employed is highly active, is stable for a relative ly long time, has well-defined selectivity, and can be used under conditions compatible with the require ments of the signal transducer, an excellent biosensor can be prepared. Unfortunately, accessible systems meeting these criteria are limited to a relatively small number of substrates, such as urea, glucose, amino ac ids, and nucleotides. Highly stable biosensors possibly can be prepared using artificial enzymes (synzymes), but only one publication on this subject has appeared to date. More commonly, several enzymes or enzymes plus cofactors are needed to convert the desired substrate to products that can be conveniently monitored with a 32
September 5, 1988 C&EN
Enzymes can be used in several ways as recognition elements in catalytic biosensors
Transducer
Transducer
E2 E1 E2 E1 E K E 2 E 1 . •E2^sE1 — P1 P1 P2
Transducer
Ab-(L-E) Ab-(L)
L
Transducer
Rc-(L-E)
Ab-(L) Ab-(L-E)
Rc-(L)
L
L
Rc-(L)
C L E L .R -( - >
Ab = Antibody Ε E = Enzyme L = Ligand Ρ P = Product Re = Chemoreceptor S = Substrate
The earliest enzyme-containing biosensors coupled the sensing element with a single isolated enzyme (upper left), which works well in the relatively few instances when the characteristics of the enzyme are optimum. Usually, however, several enzymes or enzymes plus cofactors, acting in sequence (upper right), must be used to form a product that is suitable for monitoring. However, this often lowers the efficiency of the sensor. Biosensors also can use enzymes conjugated with a ligand coimmobilized with an appropriate antibody (lower left) for the immunoassay of free ligand by means of changes in enzyme activity. The enzyme-ligand conjugate also can be bound with a chemoreceptor (lower right), which serves as the molecular recognition element, to further extend the applicability of the sensor.
signal transducer. Although it is sometimes possible to coimmobilize several enzymes (and/or cofactors), effi ciency tends to decline as a result of necessary compro mises in operating conditions or additional interfer ences. Frieder W. Scheller and his coworkers at the Central Institute for Molecular Biology in Berlin, East Germa ny, have been especially imaginative in coupling multienzyme layers to electrochemical biosensors for am plification or switching and to eliminate interference. Biosensors for such clinical substrates as glucose, lac tate, and uric acid, as well as for enzymes such as cholinesterase, amylase, and creatine kinase, have been developed in this manner. An example of what can be accomplished is an amperometric biosensor for deter-
mining starch that uses a glucoamylase-glucose oxidase enzyme membrane in conjunction with an anti-interference layer containing glucose oxidase and catalase to harmlessly eliminate any endogenous glucose that may be present in the sample. In some cases, enzyme catalysis can be combined with antibody-antigen interactions in the construction of biosensors. The antigen (or hapten or other ligand) to be determined first is conjugated with an enzyme in such a manner that the enzyme activity changes when the conjugated antigen binds with its antibody. If fixed quantities of antibody and antigen-enzyme conjugate are immobilized at or near the sensor, the system can then detect levels of free antigen as a result of shifts in the competitive binding equilibria that cause proportional changes in enzyme activity. Perhaps the newest development in this field has been the use of chemoreceptors in my laboratory as binding reagents with enzyme amplification. This new technique, tentatively entitled "enzyme-amplified receptor assay" (ERA), is based upon the ability of various drugs and other biomolecules to bind sites on receptor molecules. The nicotinic acetylcholine receptor, for example, binds drugs of abuse, anesthetics, and antidepressant drugs. Consequently, free drugs in a sample can be determined in a competitive binding experiment in which the free drug competes with enzyme-labeled drug for a fixed number of receptors. The ERA approach has the powerful advantage of combining receptors as molecular recognition elements with enzyme amplification to enhance sensitivity. Enzyme-catalyzed processes have been coupled to many types of signal transducers in the construction of biosensors. Thus, enzyme thermistors, enzyme-based fiber optic sensors, enzyme transistors, and similar devices have all been derived from the original concept of enzyme electrodes. Some of these variants are of only limited interest, but others offer real advantages in terms of improved operating characteristics, lower cost, or practical applicability. Development of fiber optic biosensors that use coupled enzymes as molecular recognition elements has been pursued by Norbert Opitz at Max Planck Institut fur Systemphysiologie in Dortmund, West Germany; Mark A. Arnold at the University of Iowa; and Otto S. Wolfbeis at Karl-Franzens University in Graz, Austria, among others. In general, some kind of indicator system, often fluorescent, is needed to mediate between the primary enzyme-catalyzed reaction and the optical transducer. W. Rudolf Seitz of the University of New Hampshire considers fiber-optic-based biosensors, which can use one or more fibers and use conventional or laser excitation, to have an advantage compared with electrodebased biosensors of this type in that they don't require a reference electrode, have good calibration stability and biocompatibility, and possibly produce a higher information content because of their multiwavelength operation. On the other hand, they suffer from problems caused by background light and have a limited dynamic range and a slower response time. They also
are subject to photobleaching. Unlike potentiometric sensors, the strength of the signal produced by optical sensors also declines as their size is reduced. In practice, it is the individual application that usually favors one type of sensor over another, because operating factors critical to one situation may be unimportant in another. At present, the development of optical and electrochemical biosensors is proceeding more or less in parallel, with neither type yet emerging as clearly superior. According to John I. Peterson of the National Institutes of Health, the ideal optical fiber for chemical sensing has yet to be developed. Plastic, glass, and quartz fibers all have some drawbacks, and the full range of indicator systems has not been critically evaluated. Several laboratories in Japan, Sweden, and the U.S. are working on transistor devices coupled to immobilized biocatalysts. In general, this work employs conventional biochemical systems that already use a molecular recognition element in electrode-based sensors, modifying the physical arrangement to make it compatible with the transistor device. Noteworthy in this connection is a disposable sensor for freshness in fish, proposed by Karube. It consists of an immobilized xanthine oxidase enzyme layer formed over the gate insulator of an amorphous silicon field effect transistor. The resulting biosensor measures hypoxanthine, which is considered to be a reliable indicator of fish freshness. A second major trend in the development of biocatalytic biosensors focuses on the use of cellular materials and intact structures as biocatalytic alternatives for isolated enzymes. This research had its origin with electrochemical biosensors constructed in the mid-1970s that use immobilized or trapped bacterial cells as mo-
Fiber optic sensor uses coupled enzymes as molecular recognition elements To source and detector Single optical fiber
- Fluorescence emission
Laser • excitation
Immobilized layer containing coupled enzymes and fluorescent transducer species September 5, 1988 C&EN
33
Special Report lecular recognition elements, and it continues to this day as ever more sophisticated natural materials are being employed. Development of this field has literally involved a process of "asking questions of nature/' In retrospect, the idea seems obvious. Instead of immobilizing enzyme sequences and cofactors at sensors, why not use such systems as they exist in nature, that is, in cells? In essence, the use of intact cells simply maintains the enzymatic pathways in their natural environment, resulting in a considerable stabilization of the desired enzymatic activity. Under proper conditions, cells also can synthesize fresh enzymes on a continuous basis or can be induced to generate enzymes not present initially. In consequence, self-perpetuating sensors and sensors with variable selectivity can be prepared. Even without this special feature, cell-based sensors have much longer useful lifetimes (30 to 60 days) compared with the corresponding isolated-enzyme sensor. In addition, the specific catalytic activity of cellular materials remains sufficiently high in determinations where isolated enzymes have failed. These advantages occur without sacrificing overall selectivity in most cases. And strategies for enhancing selectivity, if that is necessary, have been developed. Initially, microorganisms such as bacteria or fungi were used in constructing biosensors, but specialized tissues from higher animals and plants were soon found to offer further advantages. Aside from having less stringent sterility requirements and fewer interferences, tissue-based biosensors have some remarkable biocatalytic properties not found in other systems. A few examples of such biosensors illustrate this point: • A banana-based biosensor can measure the neurotransmitter dopamine. In this // bananatrode, ,/ slices of banana pulp are coupled to an oxygen electrode and a complex biochemical pathway (the browning reaction)
converts dopamine to melanin in reaction steps that consume oxygen. • A pig kidney slice biosensor, for analysis of glutamine, consists of a kidney cortex tissue section that is coupled to a potentiometric ammonia electrode. This biosensor works well in cerebrospinal fluid and has been used as a diagnostic device for Reye's syndrome. • A flower-blossom-based biosensor uses a crosssectional slice of a single magnolia petal coupled to a gas-sensing electrode for amino acid measurements. • A leaf-based biosensor uses cucumber leaves, after removal of the waxy outer layer (cuticle), incorporated in the tip of a potentiometric electrode for measuring cysteine. • A rabbit-muscle biosensor can determine the nucleotide adenosine S'-monophosphate. • A toad-bladder-based biosensor can analyze for vasopressin (a diuretic hormone). Other tissue-based biosensors include: a cabbage leaf-based sensor that is used to measure vitamin C; one based on soybean leaves that can determine metribuzin; a biosensor based on mouse small intestine that responds to adenosine; one based on beef liver that is used to determine hydrogen peroxide; a corn kernel sensor that measures pyruvate; and a squash-based biosensor used in the determination of glutamic acid. A new tissue-based sensor from my laboratory contains slices of mushrooms, freshly grown or from supermarkets, coupled to an oxygen electrode. Such a device can be used for the sensitive and fairly selective measurement of tyrosine because mushrooms are rich in enzymes that preferentially metabolize tyrosine in an aerobic reaction. Thus, oxygen consumption produces a signal when the sensor is exposed to samples containing tyrosine. Bacterial and tissue biosensors have only begun to be
Biological components provide the molecular recognition element for biosensors Biological component
Chemoreceptors Intact structures Preparations containing isolated chemoreceptors Antibodies/antigens Polyclonal antibodies Monoclonal antibodies Antibody fragments Enzyme/antigen conjugates or labeled antibodies Biocatalysts Isolated enzymes Enzyme sequences Microorganisms Plant and animal tissue
34
September 5, 1988 C&EN
Advantages
Disadvantages
Use natural structures that are already in the optimal state for detection Can be prepared just prior to use for freshness, which enhances sensitivity
Fragility, limited lifetime
Relatively inexpensive
Have several binding constants, limited selectivity High cost, limited availability
Good selectivity, binding constant is uniform Low molecular weight Improved amplification
High selectivity and activity Expand accessible pathways Great variety, self-contained, regeneration may be possible High activity, natural configuration
Difficult to store, few types available, limited selectivity
Limited availability, cost uncertain Tedious to prepare
Limited availability and stability Subject to interferences, stringent operating conditions Poor selectivity, limited lifetime Subject to interferences and contamination
Transducers provide the signal-generating element for biosensors Transducer
Electrochemical Potentiometrjc Amperometric Conductimetric Optical Fiber optics
Surface plasmon resonance Interference-effect optodes Total internal reflection fluorescence Field effect transistors Thermistors Piezoelectric crystals
Disadvantages
Advantages
Wide response range, commonly available instrumentation, selective Response to change in dose is linear, sensitive Inexpensive, rugged
Responses to change in dose are logarithmic, slow response, limited selectivity Subject to interferences and protein fouling Nonselective, poor signal-to-noise ratio
Small size, rapid response, no reference cell needed, suitable for sensor arrays, calibration stability Suitable for immunoreactions Can be used for multiplexing and in array sensors, small size Can distinguish free and bound fluophor
Special instrumentation and indicators needed, some problems in turbid samples, losses from photobleaching Geometric factors must be controlled Require specialized instrumentation
Very small size, potentially low cost, suitable for automated systems Simple, linear response Operate over a wide temperature range, sensitive
exploited. The possibility of using stabilized biocatalytic materials that are abundantly available at exceptionally low cost may be attractive to workers in developing countries and as teaching tools in undergraduate laboratories. Even students who are not scientifically inclined appear to be fascinated by the concept that nature itself can be used to measure something, and are easily led to an appreciation that all measurement ultimately uses natural phenomena.
Applications As biosensors pass from the conceptual into the development stage, future work inevitably will be increasingly application-driven. Marketing studies suggest that biosensors may become a major new commercial technology by the end of this century. Although many technical problems need yet to be overcome, biosensors offer a prospect of measuring biomolecules important in medicine, biotechnology, and other areas with convenience and speed, as well as at low cost. Biosensors may bring about significant changes in medical practice and the provision of health care in the future. They may be used, for example, for clinical testing at a patient's bedside or in a physician's office, instead of at centralized laboratories. If the need for specimen collection, transportation, and processing can be reduced through the use of biosensors, considerable savings in time and cost might be realized. Biosensors might even be used in ambulances and hospital emergency rooms. Biosensors also might be used for continuous monitoring during critical care. Although many vital functions already can be monitored, biosensors might extend this function to metabolites, therapeutic drugs, and other analytes. They possibly will be useful during surgery as well, not only to monitor drug or anesthetic levels but also to gauge the effectiveness of surgery by analyzing marker components in vessels draining affected organs.
Multiple reflections required to achieve sensitivity Unstable, leakage problems, slow response, biocompatibility problems Nonselective, subject to drift Work best in gas phase, slow recovery
Biosensors might be used, too, in home care to measure drug maintenance levels or to give early warning of abnormal physiological changes. And they may play a role in screening large populations for illegal drugs. Even a modest cost saving might be significant in mass screening. And faster feedback of results to patients would be of benefit. Many of these considerations also would be useful in veterinary medicine. Indeed, it is possible, because of less stringent regulatory requirements, that biosensors will find veterinary application more quickly than in human medicine. Potential uses of biosensors in biotechnology and pharmaceutical production are closely related to and partly overlap biomedical applications. However, present trends in biotechnology suggest that monitoring fermentation processes and antibody production by means of biosensors may especially be of crucial importance. Similarly, the pharmaceutical industry has needs ranging from monitoring the purity and potency of pharmaceutical preparations to pharmacological measurements at the level of therapeutic action. Other logical industrial applications in which biosensors could play a major role include monitoring worker safety, product testing, especially in the personal care and beauty product field, and monitoring raw materials or process intermediates. Environmental and public health applications are still in their infancy. But use of biosensors to detect or measure toxic and mutagenic substances already has been established. Organophosphorus compounds, for example, can be measured by their inhibitory effect on acetylcholinesterase enzyme, and there is no reason that intact chemoreceptor structures could not be used to sense pollutants. Many plants owe their resistance to pesticides to their ability to detoxify such substances, so plant-tissue-based biosensors for pesticides might be devised from leaves or roots. Karube has studied a bacterial sensor for screening September 5, 1988 C&EN
35
Special Report mutagens, using special bacterial strains. His results suggest such sensors have advantages in speed and sensitivity over conventional tests. Similarly, I. John Higgins and his group at Cranfield Institute of Technology in England have devised sensors to detect bacterial contamination of foods and oils. Rapid changes in agriculture likewise generate special needs for techniques or devices for monitoring applications ranging from genetic manipulation of plants and animals through processing of agricultural products to disposal of wastes and by-products. Adequate food production and distribution is an unsolved social problem in many parts of the world. Even where food supplies are plentiful, there is concern about the wholesomeness and nutritional value of food products. Biosensors might help resolve these concerns by providing effective and economical means of monitoring food production, packaging, transportation, and storage. In fact, biosensors already have been proposed for use in agriculture and the food industry for measuring biochemical parameters important to peanuts, cereal grains, fermentation broths, wine, dairy products, and food additives. For example, a ferrocene-based glucose sensor can monitor the freshness of chilled meat, another sensor can detect bacterial contamination of milk, and a peptide sensor can monitor the hydrolysis of milk proteins. Military uses of biosensors have been largely neglected—except by the military. Biosensors might have military applications for detecting chemical and biological warfare agents, for safeguarding the health of military personnel in confined environments like submarines, or for remote monitoring in combat. Information relating to the status of such applications is limited, but the armed services are known to have biosensor research programs in-house as well as on an extramural basis. Because biosensors usually have multiple applications, such efforts may yield general dividends in the long run, especially for environmental and biomedical uses. Although the possibilities for biosensors (real or perceived) appear virtually unlimited, the present growth of biosensor research cannot continue indefinitely. Many of the ideas I have mentioned will remain academic curiosities or will be superseded by better ideas. Experience with other scientific instrumentation has shown again and again that active research and development cannot be sustained unless real advantages over competing technologies can be realized and utilized on a wide basis. Many other instruments and techniques are technically and economically competitive with biosensors for specific measurement situations. And there are formidable obstacles to bringing an idea to reality. Most workers in the biosensor field think that success will be attained first through contributions to medicine. Indeed, exciting possibilities exist for using implantable biosensors in connection with the development of artificial organs and prosthetic implants. The artificial pancreas, which is already beyond the animal-testing stage, would not be possible without 36
September 5, 1988 C&EN
implantable glucose biosensors. Similarly, extracorporeal "user-friendly" biosensors for glucose are highly promising as home testing devices for diabetic monitoring. If problems of biocompatibility and in-situ calibration can be overcome, other analytes and diverse types of biosensors are likely to be targeted for home monitoring. As in any competitive scientific field, the tendency is to draw upon scientific capital accumulated by earlier researchers, rather than to seek new and creative ideas. Yet the biosensor research field, exactly because it requires a fresh synthesis of biological and analytical concepts, should heed the challenge to see what everyone has seen, but think what no one else has thought. • Suggested readings Turner, A. P. F., Karube, I., Wilson, G. S. (eds.), "Biosensors: Fundamentals and Applications," Oxford University Press, 1987. Rechnitz G. A., "Biosensors: An Overview," Journal of Clinical Laboratory Analysis, 1, 308-312 (1987). Ngo, T. T. (ed.), "Electrochemical Sensors in Immunological Analysis," Plenum Press, New York, 1987. Janata, J., Bezegh, A., "Chemical Sensors," Analytical Chemistry, 60, 62R-74R (1988).
Garry A. Rechnitz is Unidel Professor of Chemistry and Biotechnology at the University of Delaware. He received a B.S. degree in chemistry from the University of Michigan in 1958 and a Ph.D. degree from the University of Illinois in 1961. He taught chemistry at the University of Pennsylvania from 1961 to 1966 and then at the State University of New York, Buffalo, until he joined the faculty at the University of Delaware in 1977. During a sabbatical leave in 1987 he was visiting professor of pathology at Scripps Clinic and Research Foundation, La folia, Calif. Rechnitz s research in bioanalytical chemistry and on sensors has resulted in more than 275 publications and has provided research experience for more than 120 graduate and postdoctoral students. His research has been supported by the National Science Foundation and the National Institutes of Health. The assistance of Michael Buch, Susan Hallowell, May Ho, and Susan Mikkelsen in the preparation of this article is gratefully acknowledged.
Reprints of this C&EN special report will be available in black and white at $5.00 per copy. For 10 or more copies, $3.00 per copy. Send requests to: Distribution, Room 210, American Chemical Society, 1155—16th St., N.W., Washington, D.C. 20036. On orders of $20 or less, please send check or money order with request.
