A system for detecting physical or chemical changes in an environment is disclosed. The system comprises means for conducting a chemical reaction; an active biological molecule whose physiological function is noncatalytic which is capable of catalyzing a chemical reaction wherein at least one reactant is converted to one product; and means for sensing and processing information relating to changes in said environment. A method for detecting physical or chemical changes in an environment is also disclosed.
BACKGROUND OF THE INVENTION
The development of chemical sensors began in 1906 when it was discovered that the electrochemical potential at certain thin glass membranes depended upon the hydrogen ion concentration in the bathing solution (1). As a result of this discovery, the first pH electrode was reported in 1909 (2). Since that time, development of chemical sensors has been considerable with blood gas and electrolyte measurement systems having undergone substantial development. In comparison, development of sensors for the detection of metabolites and biomolecules has not progressed as far.
The term "biosensor" usually denotes a sensor which is specific for biological substances. However, the term is often used to specify a sensor which uses biological substances to detect other chemicals. A biosensor is a monitoring device whose selectivity in detecting an analyte is the result of the binding specificity of a biological molecule, e.g., antibody, enzyme or membrane receptor. Analyte concentrations are determined by "transducing" these analyte binding events into a measurable quantity such as an electronic or optical signal. Thus, the basic components of a biosensor are a biological molecule, e.g., antibody, enzyme or membrane receptor, and a transducer.
Biosensors generally fall into three basic categories: electrochemical; optical; or physical. These biosensors incorporate transducers which are well known to the skilled artisan and include calorimetric, piezoelectric, amperometric, optical fiber, optical waveguide, lipid membrane, potentiometric and electrochemical capacitance/impedance devices.
While the aforementioned biosensor transduction techniques and devices may be employed in the invention, future improvements in miniaturization and in other analytical techniques such as mass spectroscopy, gas chromatography and nuclear magnetic resonance spectroscopy may allow such other techniques and systems to also be used in the invention.
The biological component of preexisting biosensors is either an enzyme, an antibody, a membrane receptor, whole cell or tissue. Enzymes, antibodies and membrane receptors are all biological macromolecules whose function is to bind target molecules in a highly specific manner. While integration of enzymes and receptors into sensors appeared to have great potential, their commercial application is often limited.
Sensors incorporating enzymes can detect many chemicals. However, the chemicals which can be detected are limited to those for which stable enzymes are available and still further to those enzymes which either consume or produce measurable molecules. Yet a large number of chemicals are consumed or produced by chemical reactions for which there are no known enzyme catalysts. Biosensors which rely upon enzymes are also limited to the extent that the enzymes are limited in their function by allosterism. Allosteric enzymes are enzymes which are stimulated or inhibited by a modulator molecule which may be the substrate, the product or some other molecule. As a result of allosterism, the kinetic behavior of such enzymes is greatly altered by variations in the concentration of the modulator. A relatively simple example of allosteric behavior is where the enzyme is subject to feedback inhibition, i.e., where the catalytic efficiency of the enzyme decreases as the concentration of an intermediate or subsequent product increases. Use of such enzymes in many biosensor applications is thus limited and requires continuous removal of product.
A further major disadvantage of enzyme sensors is attributable to the fact that the binding affinity of the analyte is determined by its biological role. Most enzymes possess binding affinities in the 10.sup.3 -10.sup.6 M.sup.-1 range and the binding affinity determines the sensitivity of the enzyme sensor. Thus most enzyme sensors have, at best, nanomolar detection limits.
As to membrane receptors, there is difficulty in isolating sufficient quantities of protein. Moreover, the commercial application of membrane receptors in sensors is often limited by their inherent instability and by the need for highly ordered and easily degraded environments.
Sensors incorporating antibodies as the biological component, like enzymes, have high selectivity but can have substantially improved detection limits as compared to enzymes due to the higher binding affinities of antibodies. Antibodies have binding affinities of 10.sup.5 -10.sup.13 M.sup.-1 and thus sensors incorporating antibodies can have detection limits in the picomolar range. Antibodies are limited, however, by the fact that the recognition and identification of antigens by antibodies via immunological reactions is usually a one-time event. As antigen saturates antibody, the development of a specific antigen-antibody complex can be measured. However, whether monoclonal or polyclonal antibodies are used for antigen capture, this reaction (with high affinity bindings and hence improved detection limits) is usually not readily reversible. The antibody-based sensor thus becomes saturated and the presence of analytes in a subsequent sample cannot be detected. The inability to repeatedly use such sensors is a major limitation.
A common method for dissociating antigen-antibody complexes involves incubating the antibody in either low pH solutions or high concentrations of salt, urea or thiocyanate. While such methods can regenerate an antigen binding site, each procedure requires careful control of the reaction conditions and may cause partial protein denaturation, especially after several regeneration cycles. The absence of a reliable method for regenerating an antigen binding site immediately after each binding event has frustrated the development of a sensing system capable of monitoring successive exposures to a given analyte.
It can be readily appreciated that a durable sensing system with high sensitivity which is capable of repeatedly detecting and measuring the concentration of an analyte over large concentration ranges and which is capable of being calibrated without antigen saturation would be a highly desirable advance over the current state of the art in sensing systems.
OBJECTS, FEATURES AND ADVANTAGES OF THE INVENTION
It is therefore a general object of the invention to provide a sensing system which incorporates an antibody capable of reversibly binding with an antigen.
It is a further object of the invention to provide a highly sensitive sensing system capable of detecting extremely low concentrations of analyte.
It is another object of this invention to provide a highly sensitive sensing system capable of detecting analyte over large concentration ranges.
It is another object of the invention to provide a reversible sensing system having the ability to measure both increases and decreases of the concentration of an analyte without the need for a separate regeneration process between samples.
It is another object of the invention to provide a sensing system which incorporates an antibody which permits the system to be calibrated without antigen-combining site saturation.
It is yet another object of the invention to provide a sensing system having the ability to employ new catalytic binding sites for novel analytes.
It is another object of the invention to provide a sensing system with catalytic combining sites having properties similar to membrane receptors.
It is yet another object of the invention to provide a sensing system capable of detecting substances consumed or generated by reactions for which there are no known enzyme catalysts.
It is another object of the invention to provide a sensing system which can detect physical or chemical changes in an environment as the result of the consumption or generation of an analyte by a chemical reaction.
It is another object of the invention to provide a sensing system which incorporates a catalytic antibody which can reversibly bind to an antigen.
It is yet another object of the invention to provide a sensing system which incorporates a catalytic monoclonal antibody which can reversibly bind to an antigen.
It is yet another object of the invention to provide a sensing system containing an antibody wherein the antigen binding site is regenerable immediately after each binding event.
It is another object of the invention to provide a sensing system containing an antibody for detection of an analyte which inhibits the catalytic effectiveness of the antibody.
These and other objects, features and advantages of the invention are achieved by the invention.
SUMMARY OF THE INVENTION
The invention is broadly directed to a system for detecting physical or chemical changes in an environment. The system comprises means for conducting a chemical reaction, an active biological molecule whose physiological function is noncatalytic which is capable of catalyzing a chemical reaction in the environment, a transducer capable of detecting physical or chemical changes in the environment caused by the binding of the antibody or the subsequent chemical reaction and further capable of generating information relating to those changes and means for processing the information generated by the transducer in response to those changes. Desirably, the active biological molecule is one which has been rationally transformed into, synthesized, or identified as a catalyst.
The invention is also directed to a method for detecting physical or chemical changes in an environment wherein the changes are the result of a chemical reaction (or a plurality of reactions) taking place in the environment in which at least one reactant is converted to at least one product. The method comprises the steps of causing the chemical reaction to take place in the environment by contacting the reactant with an active biological molecule under conditions which are conducive to the conversion of the reactant to the product, sensing physical or chemical changes taking place in the environment as a result of the chemical reaction and transducing those changes into information related thereto and processing the information generated by the transducer in response to the physical or chemical changes.
In another aspect, the invention is directed to a system for detecting the presence of an analyte of interest in an environment wherein the analyte of interest or some other component of the environment is consumed or generated during the course of the chemical reaction, i.e., a component which is a coreactant, coproduct, intermediate, precursor or other species which is formed or consumed due to one or more reactions in the environment. The system comprises means for conducting a reaction, an active biological molecule which is capable of catalyzing the chemical reaction, a transducer capable of detecting the presence or absence of a component of the environment, typically but not necessarily, a reactant or coreactant or a product or coproduct, and further capable of generating information relating thereto and means for processing the information generating by the transducer. Desirably the active biological molecule is one which has been rationally transformed into, synthesized, or identified as a catalyst.
The invention is also directed to a method for detecting an analyte of interest in an environment wherein the analyte or another component of the environment is consumed or generated during the course of the chemical reaction. The method comprises the steps of causing the chemical reaction to take place in the environment by contacting a reactant of the chemical reaction with an active biological molecule under conditions which are conducive to the conversion of the reactant to a product, sensing the presence or absence of a component of the environment as defined above, generating information relative thereto and processing the information generated by the transducer to determine the presence of the analyte of interest.
In another specific embodiment, the invention is directed to a system for detecting an analyte of interest in an environment wherein the analyte of interest or another component of the environment is consumed or generated during the course of a chemical reaction and wherein the transducer is capable of detecting binding of the analyte to the antibody and further capable of generating information related to that binding. The system comprises means for conducting a reaction, an active biological molecule which has been rationally transformed into, synthesized, or identified as being catalytic for the reaction and which is capable of binding a component of the environment, a transducer capable of detecting binding of the analyte to the antibody and generating information relative thereto and means to process that information to determine the presence of the analyte of interest.
In another embodiment the invention is a method for detecting an analyte of interest or other component in an environment which comprises causing the component to bind to a catalytic antibody, detecting the binding of the component to the antibody, transducing the data obtained from the binding event and generating information relating to that binding event, processing the information generated by the transducer and releasing the bound component from the antibody by allowing the antibody to catalyze a chemical reaction involving the bound component. The analyte or related component of the environment may be released from the antibody in the same environment or the complex may be removed to a separate environment where the analyte or related component is released.
Still other embodiments of the invention include systems and methods as described above for detecting the presence of chemicals which act as inhibitors of a chemical reaction catalyzed by an antibody. In such systems and methods the physical or chemical change in the environment or the presence or absence of a component of the environment is caused by inhibition of the antibody-catalyzed reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention, as well as other objects, features and advantages thereof will be understood more clearly and fully from the following description of certain preferred embodiments, when read with reference to the accompanying drawings, in which:
FIG. 1 is a schematic diagram of a pH-based catalytic antibody potentiometric biosensors;
FIG. 2 is a schematic diagram of a catalytic antibody immunochemically-sensitive field-effect transistor;
FIG. 3 is a schematic diagram of a catalytic antibody amperometric biosensor;
FIG. 4 is a schematic diagram of a catalytic antibody impedance biosensor;
FIG. 5 is a schematic representation of a catalytic antibody optical fiber sensor;
FIG. 6 is a schematic representation of an optical fiber-based sensing system;
FIG. 7 is a schematic diagram of one configuration of a catalytic antibody electrogenerated chemiluminescent (ECL) biosensor;
FIG. 8 is a schematic diagram of a catalytic antibody calorimetric biosensor;
FIG. 9A shows a top view, FIG. 9B shows a bottom view and FIG. 9C shows a side view of crystals used as a bulk acoustic wave (BAW) chemical sensor; view (B) of crystals used as a bulk acoustic wave (BAW) chemical sensor;
FIG. 10 depicts a plexiglas housing used for mounting crystals in a catalytic antibody BAW biosensor;
FIG. 11 is a schematic representation of a catalytic antibody lipid-bilayer biosensor;
FIGS. 12A-D show schematic representations of possible configurations for catalytic antibodies immobilized to a lipid-bilayer membrane; (A) catalytic antibodies covalently immobilized to natural or synthetic lipids; (B) Fab fragments of catalytic antibodies immobilized to lipids; (C) Fab fragments of catalytic antibodies covalently attached to membrane associated domain of a second protein; and (D) Fab fragments of catalytic antibodies covalently bonded to synthetic hydrophobic polypeptide;
FIG. 13 is a schematic diagram of a pH-based catalytic antibody sensing system;
FIG. 14 graphically depicts the differential response of two separate additions and dilutions of methyl p-nitrophenylcarbonate (MpNPC), expressed as a function of time (min) and response (volts) for the biosensor of FIG. 1;
FIG. 15 graphically depicts the differential response of electrodes to addition and dilution of MpNPC in the presence of transition state analog (TSA), methyl p-nitrophenylphosphate as a function of time (min) and response (volts) for the biosensor of FIG. 1;
FIG. 16 graphically depicts the differential response of the biosensor of FIG. 1 to addition and dilution of MpNPC after flushing TSA from the biosensor;
FIG. 17 graphically depicts the response of two separate but identically prepared optical fiber sensors as shown in FIG. 5 to the addition of 250 .mu.M MpNPC; the square symbols represent the response of the noninhibited 48G7 sensor; the plus symbols are the response of the TSA inhibited sensor; and the diamond symbols represent the differential signal; and
FIG. 18 graphically depicts the sequential response to additions of 250 .mu.M MpNPC; the plus symbols represent the response in the presence of TSA inhibitor; the square symbols represent the response after flushing away of the TSA inhibitor; and the diamond symbols represent the differential signal.
DETAILED DESCRIPTION OF THE INVENTION
Broadly, the invention relates to systems and methods for detecting physical or chemical changes in an environment or for detecting the presence of analytes of interest is an environment wherein an active biological molecule is employed to cause a environment reaction to take place, changes in the environment are sensed and the sensory data are transduced into information which can be processed to provide the measurements sought. By active biological molecule is meant, broadly, all biological molecules or aggregations thereof whose physiological function is noncatalytic. These are desirably species which have been rationally transformed into, synthesized, or identified as catalytic. The active biological molecules may be contained in or on cells, components of cells or tissue, and may be in purified or unpurified form.
The active biological molecules include, but are not limited to, catalytic antibodies, naturally occurring and synthesized species thereof, fragments of such antibodies, synthetic peptides derived therefrom and antibodies which have been rendered catalytic by chemical, physical or genetic modification, e.g., single chain antibodies. Rationally synthesized monoclonal antibodies are most highly preferred.
As used in the following detailed description of the invention, the term "detection" includes the concepts of measurement and quantitation. Such measurement and quantitation can be with respect to the total quantity of analytes of interest and/or the concentration thereof, and such measurements can be one-event, intermittent, or continuous as is known in the art.
The terms "reaction" and "means for conducting reaction" are used in their broadest sense. Reactions include, without limitation, oxidations, reductions, additions, transformations, condensations, eliminations, substitutions, cleavages, rearrangements, and other phenomena where a chemical species is consumed or transformed. By "consumed" is meant any phenomenon where the original species is changed, modified, or otherwise converted to a different chemical or physical species including an isomer. By "means for conducting a reaction" is broadly meant any vessel for containing an environment wherein an analysis is to be carried out, including containers, vessels, tanks, analysis equipment, probes, samples, conduits, and any other known means as are available or may become available in the art.
As used herein, the term "active biological molecule" means a biological molecule whose physiological function is noncatalytic. As will be apparent from the following discussion, such active biological molecules are preferably those which have been rationally transformed into, synthesized, or identified as catalytic for a given chemical reaction. Methods for such synthesis are described in application Ser. No. 674,253, filed on Nov. 27, 1984, and application Ser. No. 556,016, filed Nov. 29, 1983, the priority of which applications is claimed. A further description of the methods for rationally transforming, synthesizing, or identifying active biological molecules, particularly monoclonal antibodies, are known in the literature.
A "biological molecule" is broadly defined as a molecule which has been constructed from the compounds from which organisms are formed. Such compounds can be amino acids, nucleic acids, saccharides, membrane lipids, or biological cofactors.
Amino acids include the twenty essential amino acids and other amino acids which can be incorporated into proteins. Molecules constructed from amino acids also include peptides, i.e., chains composed of amino acids linked together through peptide bonds. These can be molecules such as neurotransmitters, hormones, and/or peptides derived from the functional parts of larger peptides. Molecules construced from amino acids also include proteins, i.e., longer chained peptides which may have one of many noncatalytic functions such as electron transfer proteins (e.g., ferredoxins and flavodoxins), immune protection proteins (antibodies), proteins that generate or transmit nerve impulses (e.g., acetylcholine, dopamine, and the rhodopsin membrane receptors), structural proteins (e.g., collagen, fibrin, glycoproteins, elastin, etc.), other binding proteins (e.g., histones), and mass transport proteins (e.g., ferritin, hemoglobin).
Saccharides include monosaccharides (e.g., glucose, fructose, etc.), oligosaccharides (e.g., sucrose, raffinose, etc.), and polysaccharides (e.g., starch--a fuel storage molecule; and cellulose--a structural molecule). Membrane lipids include molecules such as phospholipids (e.g., lipid bilayer membranes and other fatty acids), glycolipids, cholesterol and its derivatives, and prostaglandin and its derivatives. Nucleic acids include the five common nucleotides (adenine, guanine, cytosine, uracil, and thymine), oligonucleotides, and polynucleotides or nucleic acids such as DNA, m-RNA, and t-RNA. Cofactors are biological molecules whose catalytic function may not be generated or specifically directed until associated with a polypeptide chain. Examples are riboflavin derivatives, porphyrins, thiamin pyrosphosphate and nicotinamide adenine dinucleotide, etc.
As used herein, the term "antigen" means any molecule which is specifically bound by an active biological molecule as defined above and further in the specific description of the invention.
The preferred active biological molecules used in the sensors of the invention are catalytically active antibodies, fragments thereof, or synthetic peptides derived therefrom. Still further preferred are monoclonal antibodies which have been synthesized as catalysts according to the methods taught in the priority applications and further described below.
The active biological molecules may be used in the sensors of the invention per se or may be contained in or on cells or cellular components or on tissue, and they may be in purified or unpurified form.
The detection systems of the invention generally include an active biological molecule, typically referred to hereinafter as an antibody capable of catalyzing a chemical reaction (a "catalytic antibody"), a transducer capable of detecting events taking place as a result of the chemical reaction and further capable of generating information relating to those events and means for measuring the information so generated. In general, the events which are detected by the transducer may be the appearance or disappearance of chemical species including, inter alia, reactants, coreactants, products, coproducts, precursors, intermediates or such species as may be involved in follow-on reactions or may be physical or chemical changes in the environment due to the reaction or binding of the catalytic antibody and one or more of such species. The transducer detects these events by sensing any number of changes in physical or chemical phenomena including but not limited to changes in pH, temperature, ionic state, impedance/capacitance properties, mass, emission characteristics (e.g., fluorescence and electrogenerated chemiluminescence), absorption characteristics and reflection characteristics of light of various wavelengths, changes in electrical potential, wave propagation, substrate binding, etc. These events are associated with a chemical reaction or reactions taking place in the environment.
The invention also is in a method for detecting various events taking place in an environment. These methods employ the detection systems of the invention and generally comprise either causing a chemical reaction to take place in an environment by contacting a reactant with an active biological molecule, e.g., a catalytic antibody or causing a catalytic antibody to bind to a component of the environment and then using the detection systems of the invention to detect and/or quantify the events taking place in the environment as a result of the chemical reaction or the antibody binding.
Catalytic antibodies are usually produced by immunizing animals with molecules which are designed to mimic the structure and charge distribution of the transition state for the reaction to be catalyzed. The animals make many different antibodies for the transition state analog (TSA) having different amino acid sequences and, consequently, different binding affinities and catalytic activities. Careful screening of the various antibodies for binding and catalytic activity yields antibodies which catalyze the reaction of interest.
It will be understood that the binding of a catalytic antibody to its reactant (antigen) is different than the binding of a noncatalytic antibody to its antigen. While both kinds of antibodies bind by ionic and Van der Waals forces and by hydrophobic partition forces, the binding of a normal high affinity (10.sup.9 -10.sup.14 M.sup.-1) antibody to an antigen is very slow to reverse under the binding conditions and is practically irreversible.
In contrast to noncatalytic antibodies, catalytic antibodies bind reversibly to an antigen. Catalytic antibodies catalyze chemical reactions in which the reaction products are not permanently bound by the antibody and the free antibody combining site is regenerated as the product is formed and diffuses away from the antibody. The catalytic antibody thus is regenerated without altering the environmental conditions and is ready for another cycle of catalysis. Further, because catalytic antibodies bind reversibly, the sensors of the invention respond reproducibly and continuously to decreasing as well as increasing concentrations of analyte.
The reversible binding ability of catalytic antibodies is especially significant in view of the fact that recognition and identification of antigens by antibodies is a one-time event. Typically, as antigens saturate the antibody, the development of a specific antigen-antibody complex can be measured. However, whether monoclonal or polyclonal antibodies are used for antigen capture, this reaction is usually not readily reversible. As a result, the presence of specific analytes in the same or in a subsequent sample cannot be detected. Their inability to repeatedly recognize analytes is a major limitation in using noncatalytic antibodies as components of sensing systems.
The recent identification of antibodies having catalytic activity offers new methods of regenerating antigen binding sites. Integration of catalytic antibodies into sensor systems not only avoids the usual problems of dissociating an antigen-antibody complex, creating a reversible sensor, but also permits a sensor to detect analyte concentrations over large concentration ranges and to be calibrated without antibody site saturation. In addition, the ability to form new catalytic antibodies and hence new catalytic sites for novel analytes or to form catalytic sites with binding properties similar to membrane receptors opens important new areas for development of catalytic antibody sensors. Since a catalytic antibody can potentially be engineered for any analyte, a sensor incorporating a catalytic antibody specific for a certain analyte can detect that analyte under circumstances where it might otherwise be undetectable.
Sensors according to the invention have applications in several different areas. An important area is that of clinical medicine, where many different analytical instruments and techniques are currently employed to determine the concentration of clinically important chemicals. Until recently, radioimmunoassay techniques were used for most chemical analyses but these require typically large and expensive instruments designed for large centralized hospital or clinical laboratories. Pressure to cut health care costs is now creating more demand for less expensive, smaller analyzers which can be used in decentralized organizations, e.g., in individual hospital wards, outpatient departments, and physicians' offices. The biosensors of the invention, as a result of their small size, low cost, selectivity, and sensitivity, may well serve that need. Reliable chemical sensors already exist for potassium, sodium, hydrogen, lithium, and calcium ions but have yet to be developed for most proteins, hormones, metabolites, and organic drugs.
Veterinary health care is another area having needs similar to those of the human health care field. Biosensors may have many potential applications in the diagnosis and monitoring of animal health problems.
Another potentially large area of use for biosensors is that of fermentation control (3). There are many industrial applications of biochemical and microbiological processes in fields such as the production of food, pharmaceuticals, wastewater treatment and energy production. Fermentation reactions also have an important role in such biotechnological processes. It is necessary to carefully control the systems involved to optimize production. Rapid and sensitive on-line monitoring and control of reactant and product concentrations, reaction conditions and the like call for sensors specific to the substrates and products of fermentation.
Environmental monitoring is another growing area wherein biosensors are needed (4). Rising concerns over atmospheric, water, and soil pollution are creating a demand for chemical sensors to monitor substances such as pesticides, phenols, phenoxyacids, nitrilotriacetate, heavy metals, nitrate, phosphate, sulphate, and urea.
The defense industry also has a need for sensitive chemical sensors to monitor trace levels of chemical and biological warfare agents. Other applications for chemical sensors include food and feed process and quality control, agricultural diagnostics and monitoring, industrial hygiene, and toxicology testing.
Catalytic Antibodies
Antibodies capable of catalyzing a chemical reaction are defined as "catalytic antibodies." Catalytic antibodies are identified and described in Schochetman and Massey, application Ser. No. 674,253 filed Nov. 27, 1984, referred to above in the "Statement of the Invention."
During the course of a chemical reaction, the reactants undergo one or more transitions through structures which are energetically less favorable than either the reactant or product. In molecular terms, these transition states (or intermediate structures) reflect changes in bond lengths and bond angles as well as bond formations and breakages. The energy required to achieve a transition state is denoted as the activation energy, which may also be considered as the difference in energy between the energy of the transition state and the energy of the reactants.
Catalysts increase chemical reaction rates by lowering the activation energy of a reaction. Antibodies elicited to a hapten or antigen, which immunogens are chosen because, inter alia, they resemble the presumed transition state structure (i.e., a transition state analog) can catalyze reactions. The antibody thus produced should stabilize the energy of the transition state relative to reactants and products. This approach has been successfully demonstrated in the generation of several catalytic monoclonal antibodies.
Thus, the specific application of this approach requires immunization of mice with a transition state analog, reactant, or product compound, or these compounds after covalently bonding them to a carrier protein such as bovine serum albumin or keyhole limpet hemocyanin. Once an immune response is detected in the mouse serum, polyclonal antibodies can be isolated from that serum by a combination of ammonium sulfate fractionation, molecular radius and affinity chromatographic techniques. Because this preparation of antibodies is polyclonal, a variety of proteins will be present which bind the transition state structure with differing specificity, avidity, and isotype. The distribution of antibody molecules will change as the animal's immune response matures.
In contrast, a monoclonal antibody cell line produces a single antibody which binds to the transition state analog structure at a defined idiotope and with a defined affinity. These cell lines are produced by fusing a specific B cell (isolated from the mouse which demonstrated an immune response) with an appropriate myeloma tumor cell. This cell line can be maintained by tissue culture or in vivo passage of the tumor in mice.
Thus, catalytic antibodies may be either monoclonal or polyclonal antibodies. The advantage of monoclonal catalytic antibodies is the ability to screen a large number of cell lines and specifically identify an antibody which not only binds tightly to the eliciting transition state analog, but also displays maximal catalytic activity. In addition, hybridoma cell lines provide a constant, inexpensive source of chemically identical antibodies. Methods for producing monoclonal antibodies are well known in the art. (Koprowski, H. et al., U.S. Pat. No. 4,196,265, issued Apr. 1, 1980.)
Major limitations are associated with traditional chemical and enzymatic catalysts for both synthesis and sensor applications. Chemically catalyzed processes often require high temperatures and pressures and may not produce high yields of the desired product. This is because harsh reaction conditions and the lack of reaction specificity often result in the formation of undesirable side reactions. Furthermore, chemical catalysts are unknown for many important reactions and the rational design of future catalysts is extremely difficult. Other limitations existing with commercial chemical catalysts are their relatively high cost, a requirement for chemical activation, their inability to function at atmospheric pressure and/or in the presence of trace quantities of water, and potential problems with flammability or explosion in the presence of atmospheric oxygen.
While both enzymes and antibody catalysts can be used effectively in aqueous solutions at room temperature, industrial applications of enzymatic catalysts in synthesis and sensors are limited by the requirement that naturally occuring enzymes with the desired reaction specificity be identified. Thus, many enzymes which catalyze industrially important reactions have yet to be isolated. Other limitations of enzymatic catalysis are protein instability, difficulty in isolation, the requirement for exotic reactants (e.g., redox or transaminase compounds), as well as allosteric inhibition of enzymatic catalysis (which is a fundamental part of an enzyme's physiological function).
In contrast, rationally designed monoclonal antibodies are ideal for many synthetic and sensor applications. Large scale production of these proteins is well defined, detection specificity can be as broad or as specific as the eliciting hapten, immunoglobins are extremely stable biomolecules with respect to temperature and time, allosteric inhibition is not a problem, and both substrate binding affinities and turnover numbers can be selected from a family of catalytic monoclonal antibodies elicited by a single transition state analog. In addition, because an antibody catalyst can be designed to accelerate a reaction by a different pathway than an existing enzyme, requirements for expensive coreactants may be alleviated. The advantage can also be of particular value in defining new reaction conditions (e.g., pH requirements), or in identifying mechanisms for controlling the rate of antibody binding site regeneration (e.g., esterase antibody catalysts demonstrate a first order dependence on hydroxide ion, esterase enzymes do not). While all of these examples demonstrate the significant advantages of using antibody catalysts in either synthetic or sensor applications, the most important advantage is that a catalyst can be designed for almost any analyte.
Catalytic antibodies may also be used to increase the rate or site and stereospecificity of reactions catalyzed by nonproteinaceous molecules such as biological cofactors and coenzymes or nonbiologically derived cofactors. Examples of the former class of compounds include, but are not limited to, porphyrins, flavins, nicotinamide adenine dinucleotide, pyridoxal phosphate, biotin, or tetrahydrofolate derivatives. Examples of nonbiologically based cofactors include, but are not limited to, aluminum porphyrins, 4-ethylpyridine , and platinum or palladium catalysts.
Catalytic antibodies described heretofore are useful in accelerating the reaction rates for a variety of chemical transformations. These reactions may be unimolecular, bimolecular, or even more complex in nature. They may catalyze reactions similar to those catalyzed by enzymes such as oxidations, reductions, isomerizations, additions or eliminations from double bonds, condensations, substitutions, hydrolyses, or rearrangements. They may also catalyze nonbiologically relevant reactions such as synthesis of organometallic molecules and polymers, meta-alkylation of aromatics and/or stereospecific hydrogenations. It is this wide diversity of reaction mechanisms that will allow catalytic antibody sensors to detect the presence of an extremely large number of analytes by a variety of transducer methods.
The conditions for reaction and subsequent detection of analytes are as broad as those which allow the formation of antibody-reactant complex. It will be appreciated that these conditions may vary depending on the particular reactant and the particular antibody which is employed. For example, one set of conditions suitable for complex formation includes solution phase and emulsion reaction systems which employ a protic solvent, preferably water, maintained at a pH value between pH 3.0 and 10.0 and at a temperature between 4.degree. C. and 60.degree. C. Catalysis may be carried out at reduced or elevated pressures, but usually is performed at ambient pressure. In addition to solution phase and emulsion reaction systems, suitable conditions also include the use of support materials to which the antibody is physically or chemically bonded. Such support materials are well known to those of ordinary skill in the art as are methods for attaching or immobilizing antibodies to them.
In practice, the reactant(s) is (are) contacted with an appropriate antibody under conditions suitable for the formation of a complex between the antibody and its substrate. The complexed reactant(s) is (are) converted to product(s), and the product(s) released from the complex. Thus, the sensors of the invention are automatically regenerated by the reaction catalyzed in the antibody binding site. In addition, because the catalytic activity found in each antibody may result in a tight binding of the transition state structure, these proteins usually bind transition state analogs far more tightly than the reactants. Consequently, very small concentrations of transition state analog compounds in the assay solution will decrease the catalytic activity of the antibody by binding competitively to the antigen binding site. Accordingly, concentrations of transition state analogs can be determined by monitoring a decrease of the antibody catalyzed reaction rate in the presence of a known quantity of substrate.
Transducers
The transducers used in prior art biosensors generally fall into three basic categories: electrochemical; optical; and physical. The transducers used in the invention likewise fall into the same three basic categories. These include calorimetric, piezoelectric, amperometric, optical fiber, optical waveguide, lipid membrane, potentiometric and electrochemical capacitance/impedance devices. These transducers are described below as they are used in sensing systems incorporating noncatalytic antibodies, enzymes and protein receptors. However, it will be understood that these same transducers and the principles of their operation are applicable with catalytic antibodies in the sensing systems of the invention.
Electrochemical transducers include those having enzyme electrodes (5, 6), immunochemical-mediated ion transport electrodes (7a), and electrochemically-linked immunochemical electrodes. Enzyme electrodes typically consist of an enzyme-containing layer on the surface of either a potentiometric ion-selective or redox electrode or an amperometric electrode. The detection of either the consumption of a reactant or the formation of a product of the enzymatically catalyzed reaction forms the basis for the detection of enzyme substrates.
A typical example is the glucose sensor in which glucose oxidase, which is immobilized in a gel matrix on the surface of the detector, catalyzes the oxidation of glucose according to the reaction: ##STR1##
An amperometric electrode can be employed to sense either the depletion of oxygen or the production of hydrogen peroxide in order to detect changes in the concentration of the substrate glucose. A similar electrode can also be used in a potentiometric mode in which the consumption and/or production of electroactive species alters the redox potential of the electrode. The electrode material may be any inert metal or other inert conductive material. Platinum, gold and glassy carbon are commonly employed. Alternatively, a potentiometric ion-selective electrode sensitive to hydrogen ions can be employed to detect changes of pH in the enzyme-containing layer to determine the glucose concentration. A variety of chemicals can be detected using analogous sensing mechanisms with different enzymes; however, these enzyme-based detection systems limit the number of chemicals which can be detected to those for which stable enzymes are available. Nevertheless, the same transducing devices may be used in the catalytic antibody sensing systems of the invention without encountering the limitations associated with enzyme systems, the difference being that catalytic antibodies are used in place of the enzyme.
Immunochemical-mediated ion transport electrodes are based on well known ion-selective electrodes. These electrodes rely on hydrophobic ionophores to facilitate the solution of hydrophilic ions into a low dielectric medium, often a plasticized PVC membrane. The ionophores are either macrocyclic or acyclic but form cyclic structures upon complexation with specific ions. Natural antibiotics such as nigericin and valinomycin are often employed as ionophores. Ion-selective electrodes based on these sensing mechanisms are well established in the literature (7b) and are now used for greater than 50% of the clinical assays of sodium and potassium ions (8). These sensors use an antigen-labeled ionophore in a low dielectric membrane in a configuration similar to an ion-selective electrode (7a). The ionophore allows a relatively small exchange current density of several ionic species across the membrane-solution interface. Binding of the antibody to the antigen changes the exchange current density of each ionic species differently, resulting in a change in the mixed-potential at the interface.
In one example of an immunoelectrode biosensor, the antigen is the drug digoxin which is coupled to the ionophore benzo-15-crown-5 (7a). The electrode is used to detect antibodies to digoxin which bind to the digoxin antigen at the surface of the membrane and which affect the kinetics of ionic transport across the interface, altering the equilibrium potential. The electrode may also be used in a competition assay to detect digoxin. Detection limits for the antibody are in the .mu.g/ml range and for digoxin are in the nM range.
Electrochemically linked immunochemical sensors have been discussed in the literature but a reliable sensor which relies on the direct detection of immunochemical reactions as the mechanism for operation has not been described. In one example of such an immunochemical sensor, enzyme-linked electrodes have been employed in which an enzyme-labeled antibody supplies electroactive species for either potentiometric or amperometric detection (9).
Still another example of an immunochemical electrode is the immunochemically sensitive field effect transistor (IMFET) (10,11,12). This detection scheme relies on the ability of a field effect transistor to measure changes in the interfacial charge at the membrane-solution interface, thus allowing measurement of ionically charged antigen or antibody binding at the interface.
Capacitance of impedance transducers are known in which the electrochemical impedance of an electrode interface is measured. Either antigen or antibody can be immobilized on the surface(s) of the sensor. When the corresponding antibody or antigen binds to its counterpart, its presence at the interface alters the makeup of the ionic double layer changing the interfacial impedance (capacitance and/or conductance).
Optical transducers (exemplified by optical fiber and waveguide sensors) comprise the second category of transducers and they too are known in the art (13,14). Conceptually optical fiber sensors are simple. The optical fiber is merely used as a conduit to carry light to and from a miniaturized spectrophotometric cell in which reactant and/or product concentrations are determined either by light absorption, transmission, or fluorescence techniques. Typically, a reversible indicator system (either colorimetric or fluorometric) is trapped inside a semipermeable membrane at the end of the fiber. Sensors for pH (15), pO.sub.2 (16), pCO.sub.2 (17), and glucose (18) are examples of optical fiber-based sensors.
Alternatively, optical fibers and waveguides can be used in a total internal reflection mode in which the reflected light is altered by changes in a thin layer of the medium surrounding the fiber (e.g., an aqueous sample solution). In this case, the incident and reflected light interact with the surrounding medium via the evanescent field which extends into the surrounding medium from the surface of the fiber, decaying exponentially. The evanescent field extends into the surrounding medium a distance approximately equal to the wavelength of the reflected light. Any absorbing species within the evanescent field can absorb a fraction of the light, causing a decrease in the magnitude of the reflected light. Similarily, fluorophores in the evanescent field region can be excited by the light and the emitted fluorescence efficiently captured by the optical fiber and guided back through the fiber for analysis.
Optical waveguide sensors are used in a manner similar to optical fibers. In this case an optical prism is used to waveguide light rather than an optical fiber. The optical waveguide sensor can be operated either in the evanescent field mode or in the bulk excitation mode in a manner analogous to its optical fiber counterpart.
Optical fibers and waveguides can also be employed to trap light generated by chemiluminescent or electrogenerated chemiluminescent (ECL) reactions, either in the evanescent field or bulk modes of operation. Such ECL transducers offer an extremely sensitive means for detecting ECL-active compounds (TAG) such as, for example, ruthenium trisbypridine, Ru(Bpy).sub.3. Electrochemiluminescent compounds and ECL-TAG assay systems are described in, inter alia, Bard et al. published International Application Serial No. PCT/US85/02153, filed Oct. 30, 1985 and Massey et al. published International Application Serial No. PCT/US87/00987, filed Apr. 30, 1987, which publications are hereby incorporated by reference into this application. Biosensors which couple ECL-TAG systems, catalytic antibodies and optical fiber or waveguide transducers are described in more detail below.
Other types of optical transducers are based on light reflectance techniques. The simplest approach involves monitoring the reflectivity of a smooth surface such as silicon using a p-polarized light beam incident at its Brewster angle. A layer of absorbed or immobilized antibodies results in an increase in the reflectivity and subsequent reaction with antigen results in further reflectivity increases (19). Another approach involves the measurement of surface plasmon resonance using total internal reflection to excite the plasmon in a metal film on a glass prism or diffraction grating (20). The resonance is detected as a sharp minimum in the reflected light at a particular incident angle. Changes in the refractive index of the bathing fluid within fractions of a micrometer from the metal surface result in a change in the critical incident angle. The absorption of proteins (and antigen-antibody binding) at the interface can therefore be detected as a change in the resonance angle.
The third category of transducers is physical transducers. Several detection schemes rely on the measure ment of physical changes such as temperature or mass to monitor biochemical reactions.
Transducer which are based on the principle of measurement of the heat of reaction for a biochemical reaction are known (21). The most common example is the enzyme thermistor pioneered by Danielsson et al. (22) which uses a sensitive thermistor as the transducer to detect temperature changes resulting from enthalpy changes arising from enzyme catalyzed reactions in the vicinity of the thermistor. Enzymatic reactions are characterized by a considerable heat generation, generally in the range of 25-100 kJ/mol. The most straightforward embodiment of the biosensor is the thermal enzyme probe (TEP), in which the enzyme is directly attached to the temperature transducer (a thermistor) either by crosslinking the protein or trapping it in a dialysis tube surrounding the thermistor. Unfortunately, using this geometry, most of the heat generated by the enzymatic catalysis of substrate (analyte) reaction is lost to the surrounding environment and the sensitivity is low. A more efficient arrangement of the components is achieved by employing small columns packed with the enzyme bound to support particles in a flow-through system with the thermistor placed at the exit of the column.
A number of analytes have been detected using the several enzyme sensor devices. The concentration ranges for detection are typically in the range of 0.01 to 10 mM, depending on the enzyme activity and reaction enthalpy. Table I (22) lists the substances analyzed with enzyme thermistors with their concentration ranges.
TABLE I
SUBSTANCES ANALYZED WITH ENZYME THERMISTORS
Concentration
Substance Immobilized enzyme range (mmol/l)
Clinical analysis
Asorbic acid Asorbic acid oxidase 0.05-0.6
AIP Apyrase 1-8
Cholesterol Cholesterol oxidase 0.03-0.15
Cholesterol esters Cholesterol esterase + 0.03-0.15
cholesterol oxidase
Creatinine Creatinine 0.01-10
iminohydrolase
Glucose Glucose oxidase + 0.002-0.8
catalase
Lactate Lactate 0.005-2
2-monooxygenase
Oxalic acid Oxalate oxidase 0.005-0.5
Oxalic acid Oxalate decarboxylase 0.1-3
Triglycerides Lipoprotein lipase 0.1-5
Urea Urease 0.01-500
Uric acid Uriease 0.05-4
Soluble enzyme analysis
Urea Urease (soluble) 0.1-100 units/ml
H.sub.2 O.sub.2 Catalase (soluble) 0.1-100 units/ml
Glucose + ATP Hexokinase (soluble) 0.1-2.5 units/ml
Immunological analysis TELISA
Albumin (antigen) Immobilized antibodies + -10-
enzyme-linked antigen 10
Gentamicin (antigen) Immobilized antibodies + 0.1- g/ml
enzyme-linked antigen
Insulin (antigen) Immobilized antibodies + 0.1-1.0 U/ml
enzyme-linked antigen 0.1-50 g/ml
Fermentation analysis and process control
Cellobiose .beta.-glucosidase + 0.05-5
glucose oxidase +
catalase
Cephalosporin Cephalosporinase 0.005-10
Ethanol Alcohol oxidase 0.01-2
Galactose Galactose oxidase 0.01-1
Lactose Lactase and glucose 0.05-10
oxidase + catalase
Penicillin G Penicillinase 0.05-500
Sucrose Invertase 0.05-100
However, the number of analytes which may be detected by such enzyme-based thermal biosensors is limited to analytes consumed or generated by chemical reactions for which there are known enzyme catalysts.
The piezoelectric acoustic wave transducer represents another example of transducers which sense changes in physical parameters of the environment. It has been shown that the effect of a thin film on the surface of a piezoelectric crystal effects the resonant frequency of the crystal as if the thin film were an equivalent mass change of the crystal itself (23). Thus, acoustic wave sensors effectively measure the change in mass of a piezoelectric substrate as molecules bind to its surface(s). Bulk acoustic wave (BAW) and surface acoustic wave (SAW) transducers have been employed extensively as gas detectors (24) but only recently have they been used to detect chemicals in fluids (25, 26).
Bulk acoustic wave transducers have been used as gas sensors to monitor the absorption of gas molecules into various coatings on the surface of piezoelectric crystals in many different scientific investigations (24). However, the operation of piezoelectric crystals in a fluid medium has received little attention. Results of investigations of BAW devices in aqueous media in which interfacial chemistries were varied by the deposition of lipid films of stearic acid and also of polyacrylamide gel have been reported (26). It has been demonstrated that the binding of antibody to surface-immobilized antigen affects the harmonic frequency of the system. Therefore, the BAW transducer can be operated reliably in an aqueous environment and the immunochemical reactions on the crystal surface can be detected.
The chemical sensing mechanism which nature employs, namely, receptor proteins embedded in lipid bilayer membranes provides an example of a sensitive and selective chemical transducer (27,28). Specifically, it has been suggested that natural receptor proteins are not necessary for transduction applications and that specific chemical events (e.g., antigen-antibody or enzyme-substrate binding) at the membrane-solution interface can perturb the dipole potential of the lipid headgroups to significantly alter the conductivity of the membrane. In other words, the binding of charged or dipolar species near the interface can alter the electrostatic barrier to ion migration across the membrane. Thus, such lipid bilayer membranes can be used as transducers in the catalytic antibody sensing systems of the invention by embedding catalytic antibodies into or on the surface of the membrane instead of noncatalytic antibodies, receptor proteins, or enzymes to achieve a sensitive and selective biosensor which is rapidly reversible in response to changes in the concentration of an analyte of interest.
In addition to detecting various events associated with the chemical reaction, the transducer is also capable of generating information associated with those events by "transducing" or converting the events into a measurable quantity such as an electronic or optical signal. These signals are then chanelled via connections well known in the art to a device which processes the information represented into, e.g., displays, records, and digitized data.
In addition to the transduction mechanisms discussed above, many other analytical chemistry techniques could be employed to detect physical or chemical changes resulting from a reaction catalyzed by a catalytic antibody. Table II lists many of the common analytical techniques that could be thus employed.
TABLE II
Some Commonly Used Analytical Chemistry Techniques
ultraviolet and visible photoacoustic spectoscopy
absorption spectoscopy Raman spectoscopy
infrared absorption spectroscopy atomic absorption spectroscopy
fluorescence spectroscopy atomic fluoresence refractometry
atomic emission spectroscopy optical rotatory dispersion
plasma emission spectroscopy nuclear magnectic resonance
polarimetry (NMR) spectroscopy
circular dichroism X-ray fluorescence
electron spin resonance spectroscopy
spectroscopy electron diffraction
X-ray diffraction
Some Commonly Used Analytical Chemistry Techniques
X-ray photoelectron neutron activation analysis
spectroscopy (XPS) secondary ion mass
mass spectrometry spectrometry
electrochemical potentiometry electrogravimetry
eletrochemical polarography electrochemcial voltammetry
electrochemical conductimetry thermogravimetry
differential thermal analysis differential scanning calorimetry
liquid chromatography column chromatography
high performance liquid adsorption chromatrography
chromatography (HPLC) reverse phase chromatography
partition chromatography ion-exchange chromatography
liquid-liquid chromatography gel chromatography gas-liquid
chromatography thin-layer chromatography
paper chromatography electrophoresis
electrochromatography chemiluminescence
electrogenerated Auger spectroscopy
chemiluminescence
Means for Processing Information Generated by the Transducer
Devices used to process the information generated by the transducers are well known in the art. The particular device used depends on the type of physical or chemical phenomenon being detected. Electrochemical, calorimetric and lipid bilayer membrane transducers are often coupled with electrometers, voltmeters, impedance analyzers and capacitance bridges. Photomultiplier tubes, photodiodes, phototransistors, spectrophotometers, monochromators, and photon counters are among the devices commonly used to process information generated by optical and ECL transducers. Frequency counters and delay-line oscillators are devices often used with piezoelectric transducers.
Specific Embodiments of the Invention
The sensing systems of the invention incorporate catalytic antibodies. The catalytic antibodies used in the sensing systems and methods of the invention may be released into the test environment as free floating particles or may be immobilized on a surface of the transducer or on a separate surface which is associated with the transducer. Preferably, the catalytic antibody is immobilized using techniques well known in the art (29), which techniques generally include any one of four different methods (or combinations thereof): adsorption; entrapment; crosslinking; or covalent bonding. For example, in sensing systems wherein the transducer is a lipid membrane device, the catalytic antibody or antibodies are immobilized into or onto the lipid membrane. In systems wherein the transducer is an electrode or probe, the catalytic antibody may be immobilized or trapped on a spacer material such as, for example, a piece of filter paper which is sandwiched between the sensing membrane of the electrode or probe and a dialysis membrane which is fitted and secured over the tip of the electrode or probe. In a number of the sensing systems, the catalytic antibody may be immobilized on a suitable gel matrix (e.g., polyacrylamide, collagen or crosslinked protein), a silicon wafer, a polymer, an oxidized metal surface or other nonconductive surface. Other immobilization schemes include crosslinking, silane coupling and immobilization in vesicles, micelles and reverse micelles.
In one embodiment of the invention, a potentiometric catalytic antibody biosensor may be used to detect physical or chemical changes in an environment as a result of a chemical reaction taking place in that environment. One such reaction is the hydrolysis of methyl p-nitrophenyl carbonate (MpNPC), a reaction which is catalyzed by a known catalytic antibody. The hydrolysis of each MpNPC molecule produces two hydrogen ions causing a change in the reactive environment, namely, lowering of the pH. The reaction is as follows: ##STR2##
A catalytic antibody specific for the above reaction can be synthesized, as described in Example 1 below, so that the antibody catalyzes the reaction. The catalytic antibody so produced, antibody 48G7, is incorporated into the potentiometric biosensor.
In the potentiometric biosensor, a pH electrode (an ion-selective electrode) is used as the transducer to sense and measure the pH change resulting from the hydrolysis. FIG. 1 shows a pH electrode sensor according to the invention. Glass tube 10 comprises the body of the sensor. The 48G7 antibody is immobilized or trapped on a spacer material 12, preferably a filter paper, located behind a first membrane 14 on the surface of a pH sensitive glass membrane 16 so that the first membrane and the pH sensitive glass membrane form a sensing area 18. Preferably, first membrane 14 is a dialysis membrane. First membrane 14 is secured to the end of the glass tube with securing ring 20. The securing ring may be an O-ring or silicone rubber tubing. Glass tube 10 is filled with a solution 22 (e.g., 0.1M HCl) into which is immersed an internal Ag/AgCl electrode 24.
When the sensor is placed into a test solution containing MpNPC the solution passes through first membrane 14 and into sensing area 18. The hydrogen ions produced as a result of the hydrolysis catalyzed by 48G7 in sensing area 18 diffuse toward pH sensitive glass membrane 16 (as well as away from the glass membrane). As more hydrogen ions are produced, the concentration rises in the sensing area which creates a diffusion gradient of ions away from the sensing area into the bulk of the solution. A steady state is reached in which the rate of diffusion out of the sensing area is equal to the catalytic production rate. The rate of hydrogen ion production is proportional to the concentration of the substrate MpNPC, and thus, the steady state pH at the pH sensitive glass membrane is an indicator of the substrate concentration. Based upon stoichiometric considerations, information generated by the pH electrode can be processed to calculate the concentration of p-nitrophenol (PNP), the other product of hydrolysis, as well as MpNPC. Therefore, in addition to detecting changes in the sensing area, the sensor is capable of quantitatively detecting the presence of an analyte and, because a catalytic antibody is incorporated in the sensor, binding of the antibody to its specific antigen is reversible allowing regeneration of the antibody binding site so that the sensor may be used continuously in subsequent analyses. The use of such a pH-based potentiometric catalytic antibody biosensor is described in more detail in the examples below.
The judicious design and production of a variety of different catalytic antibodies capable of catalyzing reactions of analytes of interest to produce or consume ionic species which can be measured with ion-selective electrodes will make it convenient to assay for many analytes of interest using biosensors of the invention. In accordance with the invention, other potentiometric electrodes can alternatively be employed to measure the potential changes associated with the ion-selective membrane. For example, coated wire electrodes or field-eff
