BIOSENSOR DEVICE AND METHOD

Abstract: A biosensor apparatus for detecting a binding event between a ligand and receptor. The apparatus includes an electrode substrate coated with a high-dielectric hydrocarbon-chain monolayer, and having ligands attached to the exposed monolayer surface. Binding of a receptor to the monolayer-bound ligand, and the resultant perturbation of the monolayer structure, causes ion-mediated electron flow across the monolayer. In one embodiment, the monolayers have a coil-coil heterodimer embedded therein, one subunit of which is attached to the substrate, and the second of which carries the ligand at the monolayer surface.

Description: BIOSENSOR DEVICE AND METHOD Field of the Invention The present invention relates to biosensors, in particular, to a biosensor for measuring a binding event between a ligand and a ligand-binding receptor, and to methods employing such biosensor. Background of the Invention To a great extent, diagnostic tools used for detecting or quantitating biological analytes are based on ligand-specific binding between a ligand and a receptor. Ligand-receptor binding pairs used commonly in diagnostics include antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, and complementary nucleic acid strands, wherein said ligand is typically the smaller of the two binding pair members. The analyte to be detected may be either member of the binding pair; alternatively, the analyte may be a ligand analog that competes with the ligand for binding to the complement receptor. A variety of methods for detecting ligand/receptor interactions have been developed. The simplest of these is a solid-phase format employing a reporter-labeled ligand whose binding to or release from a solid surface is triggered by the presence of analyte ligand or receptor. In a typical solid-phase sandwich type assay, for example, the analyte to be measured is a ligand wiύi two or more binding sites, allowing ligand binding both to a receptor, e.g., antibody, carried on a solid surface, and to a reporter-labeled second receptor. The presence of analyte is detected (or quantitated) by the presence (or amount) of reporter bound to solid surface. In a typical solid-phase competitive binding assay, an analyte ligand (or receptor) competes with a reporter-labeled analyte analog for binding to a receptor (or ligand) carried on a solid support. The amount of reporter signal associated with the solid support is inversely proportional to the amount of sample analyte to be detected or determined. The reporter label used in both solid-phase formats is typically a visibly detectable particle or an enzyme capable of converting a substrate to an easily detectable product. Simple spectrophotometric devices allow for the quantitation of the amount of reporter label, for quantifying amount of analyte. Detecting or quantitating ligand-specific binding events is also important in high- throughput methods being developed for combinatorial library screening. In a typical method, a large library of possible effector molecules (ligands) is synthesized. The library members are then screened for effector activity by their ability to bind to a selected receptor. The approach has the potential to identify, for example, new oligopeptide antigens capable of high- specificity binding to disease related antibodies, or small-molecule compounds capable of interacting with a selected pharmacological target, such as a membrane bound receptor or cellular enzyme. High-throughput screening methods typically employ simple ligand displacement assays to detect and quantitate ligand binding to a receptor. Displacement assays have the advantage of high sensitivity, e.g., where the displaced ligand is radiolabeled, and also allow for the determination of ligand-receptor binding affinity, based on competitive displacement of a binding agent whose binding affinity to the target receptor is known. In both diagnostics and high-throughput screening, there is increasing interest in developing electrochemical biosensors capable of detecting and quantifying ligand-receptor binding events. Such biosensors are designed to produce electrical signals in response to a selected analyte-specific event, such as a ligand-receptor binding event. The interest in biosensors is spurred by a number of potential advantages over strictly biochemical assay formats, such as those discussed above. First, biosensors may be produced, using conventional microchip technology, in highly reproducible and miniaturized form, with the capability of placing a large number of biosensor elements on a single substrate. Secondly, because small electrochemical signals can be readily amplified (and subjected to various types of signal processing if desired), biosensors have the potential for measuring minute quantities of analyte, and proportionately small changes in analyte levels. A consequence of the features above is that a large number of different analytes can be detected or quantitated by applying a small sample volume, e.g., 10-50 μl, to a single multi- sensor chip. Heretofore, electrochemical biosensors have been more successfully applied to detecting analytes that are themselves electrochemical species, or can be participate in catalytic reactions that generate electrochemical species, than to detecting ligand-receptor binding events. This is not surprising, given the more difficult challenge of converting a biochemical binding event to an electrochemical signal. One approach to this problem is to provide two separate reaction elements in die biosensor: a first element contains a receptor and bound enzyme-linked ligand, and the second element, components for enzymatically generating and then measuring an electrochemical species. In operation, analyte ligand displaces the ligand-enzyme conjugate from the first element, releasing the enzyme into the second element region, thus generating an electrochemical species which is measured in the second element. Two-element biosensors of this type are relatively complicated to produce, particularly by conventional silicon-wafer memods, since one or more biological layers and permselective layers must be deposited as part of the manufacturing process. Further, enzymes or receptors in the biosensor can denature on storage, and the device may have variable "wetting" periods after a sample is applied. Biosensors mat attempt to couple electrochemical activity directly to a ligand-receptor binding event, by means of gated membrane electrodes, have been proposed. For example, U.S. Patent Nos. 5,204,239 and 5,368,712 disclose gated membrane electrodes formed of a lipid bilayer membrane containing an ion-channel receptor that is either opened or closed by ligand binding to the receptor. Electrodes of this type are difficult to make and store, and are limited at present to a rather small group of receptor proteins. Alternatively, direct ligand/receptor binding may be measured electrically by embedding the receptor in a thin polymer film, and measuring changes in the film's electrical properties, e.g., impedance, due to ligand binding to the receptors. U.S. Patent No. 5,192,507 is exemplary. Since ligand binding to the receptor will have a rather small effect on film properties, and since no amplification effect is achieved, die approach is expected to have limited sensitivity. It would thus be desirable to provide a biosensor capable of detecting and quantifying ligand-binding events and characterized by: (i) direct electrochemical conversion of the binding event to electrical signal; (ii) a high electron flow "turnover" from each binding event; (iii) adaptable to substantially any ligand, and (iv) good storage characteristics and rapid wetting with sample application. In addition, the device should be easily produced, and preferably amenable to manufacture using standard microchip technologies. Summary of the Invention One aspect of the invention is a biosensor apparatus for detecting a binding event between a ligand and ligand-binding receptor. An electrode in the apparatus includes an electrode substrate with a detection surface covered by a monolayer of hydrocarbon chains. The chains are anchored at their proximal ends to the detection surface, and are sufficiently close-packed and ordered to form an effective barrier to electron flow across die monolayer mediated by a redox ion species in an aqueous solution in contact with the monolayer. The ligand whose binding to a receptor is to be detected is attached to me distal ends of a portion of die monolayer chains, such mat binding of a ligand-binding receptor to ligand perturbs the monolayer sufficiently to measurably increase electron flow across the monolayer mediated by such redox ion species. The aqueous solution of redox species in contact wim the monolayer is held in a chamber that is also designed to receive sample receptor, to bring the receptor into contact witii ligand on me monolayer. Ion-mediated electron flow across said monolayer, in response to binding events occurring between said receptor and ligand, is measured in an electrical circuit in die apparatus. In a preferred embodiment, me monolayer is composed of 8-22 carbon atom chains attached at their proximal ends to the detection surface, e.g., a gold surface, by a thiolate linkage. The chains have a preferred molecular density of about 3 to 5 chains/nm 2 . The dielectric constant of me monolayer in the presence of me solution of redox species, but in me absence of die binding receptor, is preferably less man about 2, with a change in me dielectric constant of 10% or more, by receptor binding to the ligand, being readily detectable. Exemplary ligand-receptor pairs include antigen-antibody, hormone-receptor, drug- receptor, cell-surface antigen-lectin, biotin-avidin, substrate/antibody and complementary nucleic acid strands, where the ligand is typically the first-named of tiiese pairs. Where die apparatus is used to detect a ligand or analog of me ligand, die apparatus may further include a receptor which competes wim the analyte ligand or analog for binding to me ligand on the monolayer. One exemplary ligand is an oligosaccharide ligand, and one exemplary receptor, the Verotoxin receptor, also known "Shiga-like toxin". The electrode employed in die biosensor may be prepared, in accordance wim another aspect of the invention, by (i) subjecting the conductive metal surface of the electrode substrate to mild oxidation conditions, (ii) adding to the substrate, a solution of hydrocarbon chains having lengtiis between 8-22 carbon atoms and derivatized at one chain end with a thiol group, and (iii) applying a positive potential to die electrode. The potential placed on me electrode is preferably at least 250 mV vs NHE (normal hydrogen electrode), in a solution containing the alkyl tiiiol to be deposited, and electrolytes including lidiium ion and perchlorate anions. A selected portion of me hydrocarbon chains are derivatized at tiieir ends opposite me tiiiol group , with the ligand of interest. The oxidative conditions applied to die electrode surface are such as to produce deposition of a monolayer of close-packed, oriented chains on die substrate, as evidenced by me ability of the electrode to form an effective barrier to electron ion flow across the monolayer mediated by a redox ion species in an aqueous solution in contact witii the monolayer. In another general embodiment of the biosensor apparatus, ligand molecules are attached to die hydrocarbon chains forming die monolayer in the electrode through a heterodimer- subunit complex composed of first and second peptides mat togemer form α-helical coiled-coil heterodimer, where: (i) the first peptide is covalently bound to the electrode surface through a spacer, such as an oligopeptide or hydrocarbon chain; (ii) the ligand is covalently attached to the second peptide; (iii) binding of die second peptide to the first peptide, to form such complex, is effective to measurably reduce the electron flow across the monolayer mediated by such redox ion species, relative to electron flow observed in the presence of the first peptide alone; and (iv) binding of a ligand-binding receptor to die ligand, witii such forming part of said complex, is effective to measurably increase the electron flow across of the monolayer mediated by such redox species. Also contemplated is an electrode for use in a biosensor apparatus of this type, composed of a substrate having a detection surface and ligand molecules attached to surface through an α-helical coiled-coil heterodimer of the type detailed above. The electrode just described can be produced, in accordance with anotiier aspect of the invention, by contacting together: (a) a detection surface having attached thereto, a first heterodimer-subunit peptide, and (b) a second heterodimer subunit capable of binding to the first subunit to form an α-helical heterodimer, and having a covalently attached ligand capable of binding specifically to such ligand-specific receptor. These and other objects and features of die invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings. Brief Description of the Drawings Fig. 1 is a simplified, partly schematic view of the a biosensor apparatus constructed in accordance with the invention; Fig. 2 is an enlarged view of a region the electrode in the biosensor shown in Fig. 1; Figs. 3A-3C illustrate three methods for forming a biosensor electrode having a lipid monolayer and attached ligand molecules, in accordance wim the invention; Fig. 4. is a plot of monolayer thickness as a function of applied voltage in an electrode monolayer formed in accordance witii the method illustrated in Fig. 3B; Fig. 5 illustrates me triggering of conductance by receptor-ligand interaction on a biosensor electrode, in accordance wim die invention; Figs. 6 A and 6B illustrate tiie perturbation of lipid monolayer structure with binding of PAK peptide to disaccharide ligands on a monolayer; Fig. 7 shows plots of changes in oxidation (solid circles) and reduction (open squares) current of Fe(CN)/7 4' as a function of time after addition of PAK peptide to die monolayer illustrated in Figs. 6A and 6B; Figs. 8A-8C illustrate the perturbation of lipid monolayer structure witii binding of Verotoxin to trisaccharide ligands on a monolayer; Fig. 9 shows plots of changes in oxidation (solid circles) and reduction (open squares) current of Fe^N)^/ 4" as a function of time after addition of Verotoxin to the monolayer illustrated in Figs. 8A and 8B; Fig. 10 is a plot of electrode current of Fe(CN) 6 * ' f*' as a function of temperature in a monolayer electrode constructed in accordance with the invention; Figs. 11A and 1 IB demonstrate ion gating effects with a negatively charged ligand in an electrode monolayer; Figs. 12A and 12B demonstrate ion gating effects with a positively charged ligand in an electrode monolayer; Figs. 13A and 13B illustrate the structure of an electrode monolayer having an embedded K coil peptide subunit (13A), and an embedded K coil/E coil heteroduplex; Fig. 14 shows the change in oxidation (solid circles) and reduction (open squares) current as a function of time after addition of E coil peptide subunit to an electrode of die type illustrated in Fig. 13A containing an embedded K coil peptide subunit; Fig. 15 shows changes in oxidation of Fe^N)^/ 4 " (open circles) and reduction (open squares) as a function of time after addition of PAK peptide to an electrode containing di¬ saccharide ligands on a K coil/E coil lipid monolayer; Fig. 16 shows changes in oxidation of Fe^N^ 3" / 4 " (open circles) and reduction (open squares) as a function of time after addition of Verotoxin peptide to an electrode containing trisaccharide ligands on a K coil/E coil lipid monolayer; Figs. 17A-17E show a synthetic patiiway used for producing a trisaccharide-hydrocarbon conjugate employed in the monolayer shown in Figs. 8A-8C; and Fig. 18 shows a syntiietic pathway used in producing a disaccharide-hydrocarbon conjugate employed in die monolayer shown in Figs. 6A and 6B. Detailed Description of the Invention A. Biosensor Annaratus Fig. 1 is a simplified schematic view of a biosensor apparatus 20 for detecting a binding event between a ligand and a ligand-binding receptor or agent, in accordance with the invention. The apparatus includes a working electrode 22 having a conductive detection surface 24, and a hydrocarbon-chain monolayer 26 formed on die detection surface. In die embodiment shown, the detection surface is die upper surface of a conductive film 28 deposited on an electrode substrate 30, which may be non-conductive material. Details of the monolayer formed on die detection surface, and the method of forming die monolayer on die surface, are discussed below. A cover 32 in die apparatus has an upper wall 34, and side walls, such as wall 34, which are joined to edge regions of the electrode substrate to form a closed chamber 38 tiierewith. The chamber serves to hold an aqueous electrolyte solution required for biosensor operation, as will be described. Liquid may be introduced into or withdrawn from die chamber through a valved port 39 as shown. Although not shown, the chamber may include a second port or vent to facilitate liquid flow through the port. A reference electrode 40 and a counter electrode 42 in die apparatus are carried on the chamber-facing surface of wall 34, as shown, and are mus both in conductive contact witii electrode 22 when die chamber is filled with electrolyte solution. The reference electrode, which is held at ground, serves as die voltage potential reference of die working electrode, when a selected potential is placed on die working electrode by a voltage source 44. This potential is measured by a voltage meaning device 46 which may additionally include conventional circuitry for maintaining the potential at a selected voltage, typically between about -500 to +800 mV. Voltage source 44 is connected to counter electrode 42 tiirough a current measuring device 48 as show, for measuring current flow between the two electrodes during biosensor operation. The reference and counter electrodes are Pt, Ag, Ag/AgCl, or other suitable electrodes. The reference and working electrodes, and die circuitry connecting them to die working electrode, are also referred to herein, collectively, as means for measuring ion-mediated electron flow across me working-electrode monolayer, in response to ligand-receptor binding events occurring at the monolayer surface. Fig. 2 is an enlarged view of a portion of the working electrode, including die electrode monolayer, showing individual hydrocarbon chains, such as chains 50, forming the monolayer, and ligand molecules, such as molecules 52, covalently attached to distal ends of the hydrocarbon chains. The ligand employed in die biosensor is a selected binding partner in a ligand/receptor binding pair, where the analyte to be detected is related to one of the two binding partners. Ligand-receptor binding pairs used commonly in diagnostics include antigen- antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, and complementary nucleic acid strands, where the ligand is typically die smaller of the two binding pair members. The analyte to be detected may be either member of the binding pair, or alternatively, a ligand analog mat competes witii the ligand for binding to the complement receptor. The ligand molecules are attached to distal ends of the chains through conventional derivatization reactions, e.g., ester, etiier, amide, or sulfhydryl linkages, according to standard methods. The number of chains in the monolayer carrying distal-end ligands is preferably about 1 to 10 mole percent of die total chains, but may range from 0.01 to 100% . The chains forming the monolayer are typically 8-22 carbon, saturated hydrocarbon chains, although longer chains, chains with some unsaturation, chains with non-carbon chain atoms, such as lipid ethers, and/or chains witii minor branching, such as by non-chain metiiyl groups, may be employed, within die constraint tiiat the chains, at a sufficient packing density, form a sufficiently close packed and ordered monolayer to be effective as a barrier to electron flow, under biosensor operating conditions, as discussed below. This density is calculated to be between 3-5 chains/nm 2 . As an example of the variation in chain composition allowed, die embodiment of die invention shown in Fig. 13B has a hydrocarbon-chain monolayer mat includes coil-coil peptide heterodimers embedded in die planar chain matrix, while still retaining a low dielectric barrier to ion flow tiirough die monolayer. In the embodiment shown, the chains are coupled to die electrode detecting surface tiirough sulfhydryl linkages, although other suitable coupling groups may be employed. Methods for producing monolayers having suitable hydrocarbon chain densities will now be discussed. B. Electrode Monolaver Production Figs. 3A-3C illustrate three metiiods for forming hydrocarbon chain monolayers suitable for use in die biosensor electrodes. One approach, illustrated in Fig. 3 A, involves passive diffusion of chains, such as hydrocarbon chains 54 and ligand-derivatized chains, such as chains 56, onto die surface an electrode 58, under conditions effective to couple die diffused chains to die electrode detection surface. The diffusion method illustrated in 3A is a two-step process. In die first step, hydrocarbon chains alone (in the absence of ligand-derivatized chains) are allowed to react witii the detected surface over an extended period, e.g. , 24-48 hours, until a selected packing density less than full packing density is achieved. The diffusion reaction is carried out under conditions suitable for coupling the derivatized chains to die detection surface. Where the chains have tiiiol coupling groups, and die electrode surface is gold, the surface is subjected to mild electro-chemically oxidizing conditions, with a perchlorate salt present in solution, then reacted witii the chains under mildly oxidizing conditions. The extent of packing can be monitored, for example, by ellipsometry measurements to determine the thickness of die layer on the detection surface. At maximum density, i.e., saturation, a given chain length will produce a given monolayer thickness. As a guide, CQ chains produce a maximum monolayer thickness of about 3θA, and shorter length chains, proportionately thinner monolayers. Thus, in the case of a monolayer formed of C^ chains, die passive buildup of die monolayer may be stopped when a 25A monolayer diickness is observed. The second diffusion step involves die passive diffusion of ligand-derivatized tiiiol-chains 56 onto the partially formed monolayer, indicated at 60, again under suitable thiolate coupling conditions, until a high-density monolayer 62 is achieved, as evidenced, for example, by me measured diickness of the monolayer and/or a plateauing of the thickness/time curve. Although this approach has been applied successfully to monolayer production in die invention, it suffers from two limitations. First, ratiier long diffusion times- on the order of one to several days- are required to reach maximum packing density. Secondly, the percent chains containing attached ligands is difficult to control reproducibly, so mat die final monolayers will have variable mole percentages of ligands, and mus, different performance characteristics. These limitations are substantially overcome in the method illustrated in Fig. 3B, in accordance with another novel aspect of the invention. In this approach, a mixture of free and ligand-carrying hydrocarbon chains, such as chains 66, 68, respectively, at a desired mole ratio, are actively driven to die surface by applying a positive voltage potential to the substrate, here indicated at 64. In practice, the hydrocarbon chain mixture (about 1 mM hydrocarbon chains) in an ethanolic solution of 100 mM Li perchlorate, neutral pH, is added placed over die electrode, and a selected potential is applied to the electrode. The buildup of die monolayer can be monitored by increase in layer diickness, as above. Preferably, however, monolayer formation is monitored by measuring electron flow across the monolayer, e.g., employing the circuit configuration shown in Fig. 1. In tiiis case, formation of the monolayer, indicated at 70, will be characterized by a steady drop in electrode current, until a stable low current flow is reached, at which point maximum chain packing has been achieved. The time required to achieve saturation packing density will vary witii applied voltage, and can be a short as 10 seconds ~ that is, about 4 orders of magnitude faster than monolayer formation by diffusion. Fig. 4 is a plot of monolayer thickness formed using a thiol-group C n hydrocarbon chain under coupling conditions like those above, after 10 minutes at the electrode voltage indicated. As seen, complete or nearly complete monolayer formation (30 A thickness) occurs within 10 minutes at about IV (vs. NHE) potential and above. At lower positive voltages, additional reaction time is required. Preferably die voltage applied to the electrode is at least voltage between about +250 mV relative to a normal hydrogen electrode (+250 vs. NHE) and 1.2V (vs. NHE). Not only are rapid monolayer formation times achieved, but die percentages of ligand- and non-ligand chains present in the reaction mixture are precisely represented in the monolayers, giving highly reproducible electrode characteristics. Fig. 3C shows a modification of the Fig. 3B method, where the hydrocarbon-chain mixture reacted witii die electrode (indicated at 71) includes non-ligand chains, such as chains 72, and peptide subunit conjugates, such as indicated at 74, containing a peptide subunit 76 mat is capable of forming a stabilized, alpha-helical peptide heterodimer with an oppositely charged, complementary subunit. Such heterodimer subunits are described in PCT patent application WO CA 95/00293, for "Heterodimer Polypeptide Immunogen Carrier Composition and Metiiod", publication date 23 November 1995, which is incorporated herein by reference. Exemplary subunits are referred to herein as K coils, referring to a positively charged subunits whose charge is provided dominantly by lysine residues, and E coils, referring to negatively subunits whose charge is provided dominantly by glutamic acid residues. In the embodiment shown, subunit 76 is attached to the distal end of a hydrocarbon chain 78 (end opposite die chain's tiiiol group) by suitable lipid-to-peptide conjugation, e.g. , by ester linkage to a hydrocarbon fatty acid. Alternatively, and as described below, the peptide subunit may be linked to die electrode surface tiirough a peptide spacer, e.g., tripeptide spacer that extends from one end of the subunit and includes cysteine as a terminal residue, for sulfhydryl attachment to the electrode surface. In both cases, the peptide subunit conjugate is mixed with the hydrocarbon chains, at a selected mole ratio, then driven into a monolayer formation by applying a positive voltage to the electrode, as above, until a densely packed monolayer 80 is formed. A suitable ligand is then attached to die monolayer by contacting the monolayer with a ligand-coil conjugate 82 composed of the oppositely charged complement of the monolayer coil, indicated at 84, coupled to a selected ligand 86. The two oppositely charged subunits spontaneously self-assemble into heterodimers, effectively coupling die ligand to die monolayers with the high affinity constant of the two heterodimers. The method provides, in addition to the advantages mentioned above witii respect to Fig. 3B, a "universal" biosensor substrate which can be modified to include one of a large number of different ligands in die substrate monolayer, simply by contacting die universal substrate witii a conjugate of the oppositely charged peptide subunit and die selected ligand. In die example shown in Fig. 3C, a universal substrate monolayer 80 is converted to a ligand-specific monolayer 88 by addition of die ligand-specific conjugate 82. C. Biosensor Characteristics: Directly Attached Ligand This section examines die dielectric properties of die biosensors of the invention, as evidenced by the conductance properties of die biosensor monolayer membranes in the presence and absence of ligand-receptor binding. The present section considers membranes having directly attached ligands of the type described witii respect to Figs. 3 A and 3B. The next section examines similar electrical properties in biosensor membranes in which die ligand is attached tiirough heterodimer peptide subunits, as described with respect to Fig. 3C. The basic operational features of the biosensor are illustrated in Fig. 5. The figure shows a biosensor electrode 90 in a biosensor apparatus of the type described in Fig. 1, where an electrode monolayer 92 is formed, as above, of a densely ordered array of hydrocarbon chains containing ligand molecules, such as molecule 94, attached to die distal ends of some of die chains. The electrode is in contact witii a solution of ionic species, indicated at 98, capable of undergoing a redox reaction, i.e., losing or gaining an electron, at a suitably charged electrode. Exemplary redox species are FeζCN),, 3" '* " , as a negatively charged species, and Ru(NH 3 ) 6 2+/3+ as a positively charged species. Otiier probes which can be used include MoζCN)/" (E Q = + 800 mV), W(CN) 6 * (E„= +580 mV), Fe(CN) 4 ' (E„= +580 mV), Ce 4+/3+ , (E„= + 1.4V), and Fe +3/2+ (Eo= +666mV). Typical redox ion concentrations are between 0.01 and 10 mM. The redox solution is contained in chamber, like chamber 38 in Fig. 1, and is in contact witii reference and counter electrodes. The voltage potential placed on me electrode, i.e., between die electrode and reference electrode, is typically at least 90 mV above die electrochemical potential (βg) value of the redox species, for oxidation, and at least 90 mV below the electrochemical potential, for reduction of die species. Consider, for example, FeζCN^ 3" ' 4" , witii an En of 450 mV (vs. NHE). Above about 550 mV electrode potential, any Fe2+ species is oxidized to Fe3 + , and at an electrode potential below about 350 mV, and Fe+3 is reduced to Fe+2. Similarly, Ru(NH 3 ) 6 2+/3+ has an E„ of +50 mV (vs. NHE), so oxidation is achieved at an electrode potential above about + 150 mV, and reduction, below about -50 mV. In the absence of receptor binding to die ligand, die monolayer retains its dense ordered packing, forming an effective barrier to electron flow across the monolayer mediated by me redox ion species, when a suitable oxidizing or reducing potential is placed across the monolayer. This is reflected by a low or zero measured current across die membrane. The dielectric constant of die monolayer in mis condition is typically about 1-2. Witii binding of a receptor 96 to a ligand on a monolayer, as shown at the right in the figure, the ordered structure of die monolayer is perturbed sufficiendy to allow die movement of redox species tiirough tiie monolayer, producing electron flow tiirough the electrode. Measurements performed in support of the invention indicate that one triggering event leads to IO 2 to 10* ionic and electron transfer events per second, and tiius is highly multiplicative. The biosensor records tiiis binding event as an increase in current across the electrode, i.e., between the working and counter electrodes. By analogy to a transistor, die redox solution serves as die "source", the monolayer as the "gate", and die underlying electrode as die "drain". Current flow in a transistor is initiated by applying a threshold voltage to die gate. In die biosensor of die invention, current flow is initiated by a stimulus— in mis case, a ligand-receptor binding event- to die monolayer "gate" . A biosensor electrode 100 constructed in accordance with the invention, and having a disaccharide ligand indicated at 102 is shown before and after receptor binding in Figs. 6 A and 6B, respectively. Synthesis of the disaccharide-hydrocarbon chain used in the membrane is described in Examples ID and IE. The electrode was prepared as described with reference to Fig. 3B, employing a ratio of non-ligand to ligand-chains of about 4 to 1. The disaccharide is specifically reactive wim a Pseudomonas PAK peptide, indicated at 104, forming a ligand- receptor pair wim the peptide. The increase in biosensor electrode current, when PAK peptide receptor is added to die biosensor chamber, is seen in Fig. 7 for both oxidation (solid circles) and reduction (open squares) current from Fe(CN) 6 3" / 4' . The increase over time presumably reflects the kinetics of binding, demonstrating that the biosensor is useful as well in measuring the rate ligand-receptor binding events. Fig. 6B illustrates the perturbation of the hydrocarbon chain structure witii receptor binding. As anotiier example, the biosensor electrode illustrated in Figs. 8A-8C (electrode 22 from Fig. 2) has a trisaccharide ligand 52 which is shown before and after receptor binding in Fig. 6 A and Figs. 6B and 6C, respectively. Synthesis of the trisaccharide-hydrocarbon chain used in the membrane is described in Examples IB and IC. The electrode was prepared as described witii reference to Fig. 3B, employing a ratio of non-ligand to ligand-chains of about 4 to 1. The disaccharide is specifically reactive witii a Verotoxin, indicated at 106, forming a ligand-receptor pair. Verotoxin was prepared as described in Example 2. Figs, 8B and 8C illustrate two possible binding configurations. The configuration in Fig, 8B has little effect on the monolayer structure, and hence on biosensor current, because binding is "remote" from the membrane surface; the configuration illustrated in Fig. 8C, by contrast, produces significant perturbation of die monolayer structure, and tiius would be expected to significantly enhance biosensor current. The oxidation and reduction current plots shown in Fig. 9 demonstrate that Verotoxin binding to die membrane does in fact produce a major change in monolayer structure. As seen, bom oxidation and reduction current increase from near-zero levels, in the absence of Verotoxin, to a level in die μAmp range an hour after Verotoxin is introduced into die biosensor. In die examples above, die stimulation of biosensor current by receptor binding may be the result of (i) steric perturbation of die monolayer chains, as indicated in Figs. 6B and 8C, (ii) charge effects on the monolayer surface due to charged groups on die receptor, or (iii) a combination of die both effects. Studies conducted in support of die invention indicate tiiat both effects can be operative. The effect of hydrocarbon-chain disruption in die biosensor monolayer, was examined by plotting biosensor current as a function of electrode temperature. If lipid-chain disruption leads to greater electron flow in me biosensor, raising the temperature of the monolayer, and thus die motion of the lipid chains, should increase measured electron flow mediated by redox carriers. This was in fact observed, as seen in Fig. 10. The current/temperature plot has a peak corresponding to the phase transition temperature of the monolayer chains (about 55 C C), consistent with the idea mat maximum lipid disruption occurs at the point of maximum extent of phase boundaries in the hydrocarbon chains. The effect on conductance of charge on die monolayer surface can be seen from Figs. 11 and 12. In die study represented in Fig. 11 A, a negatively charged ligand was attached to the distal ends of a portion of die chains forming the monolayer. In tiie figure, die electrode is indicated at 108, tiie monolayer, at 110, chains forming the monolayer, at 112, and chain- attached ligands, at 114. Electrode current was measured for die negatively charged redox species Fe(CN) 6 >/4" , and independently, with the positively charged species Ru(NH 3 ) 6 2+ ' 3+ , at oxidation potentials indicated above. As seen in Fig. 11B, die oxidation current for die positively charged species shows die ion-dependent behavior expected for ion migration through the monolayer, indicating tiiat the monolayer is conductive to positively charged redox species. In this figure, the electrode is indicated at 116, die monolayer, at 118, chains forming the monolayer, at 120, and chain- attached ligands, at 122. Conversely, no significant electron flow was observed with the negatively charged redox species. Similar results were obtained witii a monolayer designed to contain a positively charged surface ligand, as illustrated in Fig. 12A. In tiiis case, ion-dependent current was observed for oxidation of die negatively charged iron redox species, but not die positively charged ruthenium species. D. Biosensor Characteristics: Heterodimer Attached Ligand In anotiier embodiment, the ligand in die biosensor is anchored to biosensor surface, i.e., embedded witiiin the hydrocarbon-chain monolayer, by a coiled-coil heterodimer complex formed of two subunit peptides.The heterodimer-subunit peptides employed in the biosensor invention are two non-identical, preferably oppositely charged polypeptide chains, typically each about 21 to about 70 residues in length, having an amino acid sequence compatible with their formation into two-stranded α-helical heterodimeric coiled-coils. They are designated herein as HSP1 (heterodimer-subunit peptide 1), and HSP2 (heterodimer-subunit peptide 2). In die discussion below, HSP1 will refer to die peptide attached to die biosensor surface in the biosensor, and HSP2, to the peptide having an attached ligand. It will be understood that these designations refer to die functional role played by die subunit peptide, not die actual peptide sequence. In aqueous medium, die isolated heterodimer-subunit peptides are typically random coils. When HSP1 and HSP2 are mixed togetiier under conditions favoring the formation of α-helical coiled-coil heterodimers, they interact to form a two-subunit α-helical coiled-coil heterodimeric complex. Peptides in an α-helical coiled-coil conformation interact witii one anotiier in a characteristic manner that is determined by die primary sequence of each peptide: The tertiary structure of an α-helix is such tiiat 7 amino acid residues in the primary sequence correspond to approximately 2 turns of die α-helix. Accordingly, a primary amino acid sequence giving rise to an α-helical conformation may be broken down into units of 7 residues each, termed heptads. The heterodimer-subunit peptides are composed of a series of heptads in tandem. When die sequence of a heptad is repeated in a particular heterodimer-subunit peptide, die heptad may be referred to as a "heptad repeat", or simply "repeat". Specific types of amino acid residues at defined positions in each heptad act to stabilize die two-stranded α-helical coiled-coil heterodimeric structure or complex. The heterodimer peptides may also contain residues tiiat can be reacted (eitiier intra- or inter-helically) to stabilize the α-helical or coiled-coil nature of die polypeptides. One example of a stabilizing modification is die incorporation of lactam bridges in the first and last (terminal) repeats of heterodimer-subunit peptides, as detailed in PCT application WO CA95/00293 for "Heterodimer Polypeptide Immunogen Carrier Composition and Metiiod", publication date 23 November 1995, which is incorporated herein by reference. The dimerization of HSP 1 and HSP2 is due to the presence of a repeated heptad motif of conserved amino acid residues in each peptide's primary amino acid sequence. Repeating heptad motifs having appropriate amino acid sequences direct die HSP1 and HSP2 polypeptides to assemble into a heterodimeric α-helical coiled-coil structure under permissible conditions. The individual α-helical peptides contact one anotiier along their respective hydrophobic faces. HSP1 and HSP2 may assemble into a heterodimer coiled-coil helix (coiled-coil heterodimer) in either parallel or antiparallel configurations. In a parallel configuration, tiie two heterodimer-subunit peptide helixes are aligned such tiiat they have the same orientation (amino-terminal to carboxyl-terminal). In an antiparallel configuration, the helixes are arranged such that the amino-terminal end of one helix is aligned with the carboxyl-terminal end of die otiier helix, and vice versa. Heterodimer-subunit peptides designed in accord witii the guidance presented in die above PCT application typically show a preference for assembling in a parallel orientation vs. an antiparallel orientation. For example, die exemplary peptides identified by SEQ ID NO: 1 and SEQ ID NO:2 in the above CA95/00293 PCT patent application, form parallel-configuration heterodimers as do otiier peptide sequences discussed in die PCT application. When attaching a ligand to HSP2, it is generally desirable to attach the ligand at or near the end of the peptide that will form the distal end of die heterodimer. In particular, where die heterodimer forms a parallel configuration, die HSP1 peptide is preferably anchored to the biosensor surface at its C terminus, and die ligand attached to die HSP2 peptide at its N terminus. As just noted, one of the two subunit peptides (HSP1) in die heterodimer is attached to the biosensor surface, and die second peptide (HSP2) contains a ligand intended to participate in an analyte-dependent Iigand/anti-ligand binding reaction. In botii cases, the peptide is syntiiesized, or derivatized after syntiiesis, to provide the requisite attachment function and ligand, respectively. Considering the modification of HSP 1, the peptide may be synthesized, at eitiier its N or C terminus, to carry additional terminal peptides that can function as a spacer between the biosensor surface and the helical-forming part of the peptide. Alternatively, the HSP1 peptide can be attached to die biosensor surface thorough a high-affinity binding reaction, such as between a biotin moiety carried on die peptide and an avidin molecule covalently attached to the surface. Where the heterodimer is embedded in a hydrocarbon-chain monolayer, as described below, die spacer anchoring die HSP1 peptide to die biosensor surface may be a hydrocarbon chain. The chain is preferably a fractional length of the chains making up the bilayer, such that the distal ends of die heterodimer peptides in die assembled monolayer are at or near the exposed surface of the monolayer. Thus, for example, if the monolayer is made up of 18- carbon chains, die spacer is preferably 2-10 carbons in length, depending on die lengtii of the assembled heterodimer. The hydrocarbon-chain spacer, in the form of a omega-thio fatty acid, may be coupled to a terminal hydroxyl or amine coupling during solid-phase synthesis, as oudined above. The derivatized peptide, in turn, can be attached to a metal surface by standard thiolate coupling (Dakkouri, et al., Langmuir (1996) 12:2849-2852). supra). Considering the ligand-attachment to HSP2, die ligand selected will be determined by die analyte to be tested. Ligand-receptor binding pairs, i.e., ligand/ligand-binding agent pairs used commonly in diagnostics include antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, substrate/enzyme, and complementary nucleic acid strands. The ligand is typically die smaller of the two binding pair members, particularly where the ligand is attached to a hydrocarbon-chain monolayer, as described below. However, attachment of eitiier binding pair is contemplated herein. Where the ligand is a polypeptide, e.g., peptide antigen, die antigen can be syntiiesized by eitiier solid-state or recombinant metiiods, to include the peptide antigen at die end of the HSP2 peptide that will orient distally in the assembled heterodimer. Where die ligand is a non¬ peptide moiety, e.g., a non-peptide hormone, drug, or nucleic acid, die HSP2 peptide can be synthesized to include one or more residues that can be specifically derivatized witii the ligand. The ligand is preferably covalently attached to die N-terminal amino acid residue, or to one or die residues facing the exposed face of the heterodimer. Preferred coupling groups are die tiiiol groups of cysteine residues, which are easily modified by standard methods. Other useful coupling groups include die thioester of methionine, the imidazolyl group of histidine, die guanidinyl group of arginine, the phenolic group of tyrosine and die indolyl group of tryptophan. These coupling groups can be derivatized using reaction conditions known to diose skilled in die art. To attach the ligand-derivatized HSP2 peptide to the surface-immobilized HSP1 peptide, the two peptides are contacted under conditions that favor heterodimer formation. A medium favoring coiled-coil heterodimer formation is a physiologically-compatible aqueous solution typically having a pH of between about 6 and about 8 and a salt concentration of between about 50 mM and about 500 mM. Preferably, the salt concentration is between about 100 mM and about 200 mM. An exemplary benign medium has die following composition: 50 mM potassium phosphate, 100 mM KCl, pH 7. Equally effective media may be made by substituting, for example, sodium phosphate for potassium phosphate and/or NaCl for KCl. Heterodimers may form under conditions outside die above pH and salt range, medium, but some of the molecular interactions and relative stability of heterodimers vs. homodimers may differ from characteristics detailed above. For example, ionic interactions between the ionic groups that tend to stabUize heterodimers may break down at low or high pH values due to die protonation of, for example, Glu side chains at acidic pH, or the deprotonation of, for example, Lys side chains at basic pH. Such effects of low and high pH values on coiled-coil heterodimer formation may be overcome, however, by increasing salt concentration. Increasing the salt concentration can neutralize the stabilizing ionic attractions or suppress the destabilizing ionic repulsions. Certain salts have greater efficacy at neutralizing die ionic interactions. For example, in die case of die K-coil peptide in Fig. 2A, a IM or greater concentration of C10 4 " anions is required to induce maximal α-helical structure, whereas a 3M or greater concentration of Cl " ions is required for die same effect. The effects of high salt on coiled-coil formation at low and high pH also show tiiat interhelical ionic attractions are not essential for helix formation, but rather, control whether a coiled-coil tends to form as a heterodimer vs. a homodimer. Fig. 13A shows a biosensor electrode 124 in which die hydrocarbon chain monolayer, indicated at includes a K coil peptide subunits, such as subunit 128, as described above. In die embodiment shown, each peptide subunit is coupled to the electrode surface via a tripeptide spacer, such as spacer 130 in subunit 128, which is itself attached to die electrode surface tiirough a sulfhydryl linkage, as shown. The peptide, including die peptide spacer, is formed conventionally, e.g. , by solid phase synthesis. The amount of peptide subunit in die monolayer is about 20 mole percent. The monolayer was formed according to die metiiod described above witii respect to Fig. 3C. As indicated above, die peptide subunit may alternatively be coupled to die distal ends of a portion of the hydrocarbon chains in die monolayer, placing the subunit more on the monolayer surface. A hydrocarbon chain-peptide conjugate suitable for tiiis application may be made, for example, by attaching an activated-end hydrocarbon chain to the terminal amino acid of the peptide, as the terminal step in solid phase synthesis. Presumably because of die positive charge imparted to the monolayer by the K coil subunits, the monolayer shows relatively high conductance to negatively charged redox species, such as FeζCN),, 3" , as evidenced by a relatively high oxidation or reduction current witii the redox species. Fig. 13B shows die same monolayer, but after addition of complementary, negatively charged E coil subunits, such as indicated at 130. As shown, oppositely charged subunits pair to form charge-neutral heterodimers in the monolayer. This pairing is effective to reduce monolayer conductance substantially, as evidenced by the time-dependent fall in measured oxidation or reduction current in die presence of FefCN)^ ions (Fig. 14). As shown in Fig. 3C, the second peptide subunit, e.g., die E coil subunit, added to die monolayer may be derivatized with a ligand, producing a monolayer having charge-neutral heterodimers embedded therein (or attached to the monolayer surface), and a ligand exposed on the monolayer surface. The resulting electrode is effective to measure ligand-specific receptor binding events in a biosensor operated in accordance witii the invention. The operating characteristics of such a biosensor are illustrated in Fig. 15. The electrode in tiiis biosensor includes (i) a monolayer with embedded K coils covalently attached to the electrode surface, (ii) complementary E coils forming heterodimers with the K coils in the monolayer, and (iii) surface disaccharide ligands of die type shown in Fig. 6 attached covalently to die E coils and disposed therefore at the monolayer surface. As seen in Fig. 15, addition of th& PAK protein receptor (see Fig. 6B) produces a increase in both oxidation and reduction currents, witii the current increase over time presumably reflecting additional binding events after receptor addition to die biosensor electrode. A similar biosensor having a trisaccharide, rather than disaccharide, ligand attached to the E coil subunit in tiie electrode monolayer was tested with the Verotoxin receptor described above witii respect to Figs. 8A-8C, with the results seen in Fig. 16. The solid lines in die figure show die increase in oxidation and reduction current observed, as a function of time, after addition of Verotoxin. The following examples are intended to illustrate, but in no way limit the invention. Example 1 Synthesis of Receptors in a Form Suitable For Immobilization on a Gold Electrode Selective tosylation of 1, 16-dihydroxyhexane provided the monotosylated alcohol 1 in 42% yield. Trisaccharide 2, obtained as described in die literature (Janson, et al, J. Org. Chem. 52:5629 (1988)), was converted into an anomeric mixture of trichloroacetamidates 3. Glycosylation of alcohol 1 witii glycosyl donor 3 in CH 2 C1 2 in the presence of a catalytic amount of trimethylsilyl trifluorometiianesulfonate gave trisaccharide glycoside ω-tosylate 4, which was used in die next step witiiout purification. The tosyloxy group of compound 4 was displaced by tiiiocyanate to provide the trisaccharide glycoside 5, terminated at die reducing end by spacer-arm containing the masked tiiiol function. Reduction of thiocyanate by the action of sodium borohydride (Olsen, R.K., and Snyder, H.R., J. Org. Chem. 30:184 (1965).) followed by saponification of acetate groups gave trisaccharide receptor 6. The disaccharide imidate 7 was reacted with alcohol 1 in a similar fashion to that described for die trisaccharide 4. Syntiiesis of the disaccharide glycosyl donor 7 is not described here but follows established metiiods that are considered a general art. Nucleophilic substitution of the tosyloxy group by thiocyanate was carried out as described for preparation of 5 to give compound 8. Reduction of thiocyanate accompanies by deacetylation afforded syndietic disaccharide receptor 9. A. lό-φ-Toluensulfonyloxylhexadecanol (Structure li To a solution of 1.1 g of 1,16-dihydroxyhexadecane in 10 ml of dry pyridine 0.8 g of tosyl chloride was added. After 2 h mixture was concentrated diluted witii 20 ml of acetone, 5 g of Si0 2 was added and acetone was removed in vacuum. The solid was applied on Si0 2 and eluted with pentane-etiiyl acetate (2:1) to yield 748 mg (42%) of C-101. B. 2.3.4.6-tetra-Q-acetyl-D-galactopyranasyl('αl→4V6-0-acetyl-2.3-di-0-benzoyl-D- palactopyranosyl(fll→4 )-2.3.6-tri-0-benzoyl-D-glυcopyranosyl(fll→0)-( 16- thiocyano)hexadecanol (Structure 51 A mixture of 277 mg of imidate, 100 mg of C-101 and 0.5 g of mol. sieves (4A) was stirred for 1 h. Then 8 μl of TMSOTf was added. After 2 h 1 ml of EA was added, solid was removed by filtration. Filtrate was concentrated and dried in vacuum. A solution of the residue and 200 mg of KSCN in 6 ml of DMF was stirred at 80°C for 2 hours. Mixture was concentrated, dissolved in 30 ml of CH 2 C1 2 , washed with water and concentrated again. Chromatography of the residue on Si0 2 with pentane-ethyl acetate (3:2) gave 225 ml (73%) of C-105. C. D-Galactopyranosyl( ' αl-*4 > )-D-|galactopyranosyl(<31→4)-D-glucopyranosyl(gl-*OVfl6- thiolhexadecanol (Structure 6) To a solution of 60 mg of C-105 in 4 ml of dry MeOH -40 mg of NaBH 4 was added Ar. After stirring for 2 h at 45 °C mixture was concentrated and dissolved under gentle reflux in a solution of 50 mg of NaOH in 10 ml of water. After stirring overnight at 45°C, the mixture was neutralized with cationite (H + -form) and applied on "Seppak" (C-18). The cartridge was washed with 20, 40, 50, 80, and 100% solution of MeOH in water. Fractions of 100% MeOH was concentrated to give 24.4 mg (81%) of C- 106. D. (16-thiocvano.hexadecanvl 4-0-(2-acetamido-3.4 ή-tri-0-acetyl-2-D-galactopyranosyl)- 2.3.6-O-acetvl-fl-D-galactopvranoside C-108 (Structure 8. Mixture of 100 mg of imidate 7, 68 mg of C-101 and 100 mg of mol. sieves (4A) in 5 ml of CH 2 C1 2 was stirred for 1 h. Then 5 μl of TMSOTf was added. After 2 h 1 ml of EA was added, solid was removed by filtration. Filtrate was concentrated and dried in vacuum. A solution of die residue and 70 mg of KSCN in 3 ml of DMF was stirred at 80 °C for 2 h. Mixture was concentrated, dissolved in 30 ml of CH 2 C1 2 , washed with water and concentrated again. Chromatography of die residue on Si0 2 witii pentane-ethyl acetate (2:1) gave 82 mg (70%) of C-108. E. (lό-thiohvdroxylhexadecanyl 4-Q-(2-acetarnido-2-deoxy-g-D-galactopyranosyl)-g-D- galactopyranoside (Structure 9) A solution of 54.2 mg of 8 (C-108) and 40 mg of NaBH 4 in 3 ml of dry MeOH was refluxed for 2 h then neutralized witii Dowex (H + ), concentrated and chromatographed on C-18 in H 2 0 (50:50):100) to yield 19.7 mg (52%) of C-lll. Example 2 Isolation of Verotoxin Receptor Shiga-like (Vero) toxin I (SLT-I) was purified from Escherichia coli JM101 (pJB128) in a simple, one step procedure using CHROMOSORB-P containing covalently coupled syntiietic analogs of the toxin's αGal(l,4)/3Gal (digalactoside) host cell receptors (SYNSORB-P1). Bacteria were grown in baffled Fernbach flasks at 37 °C in carbenicillin- (50 μg/ml) supplemented tryptic soy broth (TSB) containing SYNSORB-P1 (15 g/L) to bind toxin released from growing cells. Late log phase cultures were treated for 30 minutes at 37 °C with Polymyxin B sulfate (0.1 mg/mL) to release intracellular SLT-I and also allow tiiis to bind to the SYNSORB-P1. Next, the SYNSORB-P1 was collected and washed thoroughly with 250 mM NaCl (pH 3.8) to remove cells and cellular debris. THe SLT-I was eluted from the washed SYNSORB-P1 using 50 mM Tris base (pH 10) containing 250 mM NaCl (TN) and concentrated using an Amicon ultrafiltration unit. The concentrated SLT-I was stable for weeks at 4°C and could be frozen for extended periods of time witiiout appreciable loss of activity. On average, 61% (n = 10, SD mean = 8, range 48% to 76%) of the SLT activity in the original Polymyxin-treated TSB cultures was recovered in die TN fraction eluted from the SYNSORB-Pl. SDS-polyacrylamide gel electrophoretic analysis of the SLT-I preparation revealed two prominent Coomassie blue-stained bands. The molecular weight of these two bands was calculated to be 35,000 and 7,500, respectively. The 7.5 KDz band reacted in western immunoblots with SLT-I but not SLT-II B subunit-specific monoclonal antibody. Amino terminal microsequence analysis of botii bands confirmed tiieir identity as the A and B subunits of SLT-I. Average yield of SLT-I was 0.32 mg/L (n = 8, SD mean = 0.3, range 0.1 to 0.8) of TSB culture and its specific activity in the Vero cytotoxicity assay was 4.4 pg/mL/CDjo. The results demonstrate the utility of SYNSORB in the facile and rapid purification of carbohydrate binding toxins or lectins. Aldiough the invention has been described witii respect to various specific embodiments and methods, it will be appreciated that various modifications and changes can be made witiiout departing from die invention.

Claims: IT IS CLAIMED: 1. A biosensor apparatus for detecting a binding event between a ligand and ligand- binding receptor, comprising an electrode having (i) a detection surface, (ii) formed on said detection surface, a monolayer composed of hydrocarbon chains anchored at tiieir proximal ends to die detection surface, and having free distal ends defining an exposed monolayer surface, said chains being sufficiently close-packed and ordered to form an effective barrier to electron flow across die monolayer mediated by a redox ion species in an aqueous solution in contact witii the monolayer, and (iii) die ligand attached to die monolayer, at the surface tiiereof, such tiiat binding of a ligand-binding receptor to ligand perturbs the monolayer sufficiently to measurably increase electron flow across the monolayer mediated by such redox ion species, a chamber for containing such an aqueous solution of redox species in contact with said monolayer, means for introducing receptor into said chamber, and circuit means for measuring ion-mediated electron flow across said monolayer, in response to binding events occurring between said receptor and ligand. 2. The apparatus of claim 1, wherein the monolayer further includes a heterodimer- subunit complex composed of first and second peptides tiiat together form α-helical coiled-coil heterodimer, where (i) said first peptide is covalently bound to the detection surface, (ii) the ligand is covalently attached to the second peptide; (iii) binding of die second peptide to die first peptide, to form such complex, is effective to measurably reduce die electron flow across die monolayer mediated by such redox ion species, relative to electron flow observed in die presence of the first peptide alone, and (iv) binding of a ligand-binding receptor to the ligand, with such forming part of said complex, is effective to measurably increase the electron flow across of the monolayer mediated by such redox species. 3. The apparatus of claim 2, wherein the first peptide subunit is covalently anchored to the detection surface through an oligopeptide spacer or a hydrocarbon-chain spacer. 4. The apparatus of claims 1 or 2, wherein die electrode has a gold detection surface and said monolayer is composed of 8-22 carbon atom chains attached at their proximal ends to die detection surface by a tiiiolate linkage. > 5. The apparatus of claims 1 or 2, wherein said chains have a molecular density of about 3 to 5 chains/nm 2 . 6. The apparatus of claim 4 wherein the dielectric constant of said monolayer, in the presence of such solution but in the absence of such binding receptor, is less tiian about 2. 7. The apparatus of claim 5, wherein the change in dielectric constant of said monolayer, in the presence of such solution and a detectable amount of such binding receptor, is at least about 10%. 8. The apparatus of claims 1 or 2, for use in detecting me presence, in a body-fluid sample, of a receptor which forms with said ligand, a ligand-receptor binding pair selected from die group consisting of antigen-antibody, hormone-receptor, drug-receptor, cell-surface antigen-lectin, biotin-avidin, oligosaccharide-binding receptor, oligonucleotide-DNA binding protein, and complementary nucleic acid strands, wherein said ligand is selected from the group consisting of antigens, hormones, drugs, cell-surface antigens, and oligonucleotides. 9. The apparatus of claim 8, wherein the ligand is an oligosaccharide. 10. The apparatus of claim 9, wherein the ligand is a trisaccharide, and die receptor is a Verotoxin receptor. 11. The apparatus of claim 10, wherein die ligand is an oligonucleotide having a selected nucleotide sequence, and die receptor is a target polynucleotide having a region of sequence complementary to that of the ligand. 12. The apparatus of claims 1 or 2, for use in detecting die presence, in a body-fluid sample, of a ligand analyte which forms with a receptor, a ligand-receptor binding pair selected from the group consisting of antigen-antibody, effector-receptor, drug-receptor, cell-surface antigen-lectin, biotin-avidin, oligonucleotide sequence-DNA binding protein, and first and second complementary nucleic acid strands, wherein said ligand is selected from die group consisting of antigens, receptor effectors, drugs, cell-surface antigens, and oligonucleotides, and die apparatus further includes, for inclusion in such solution, a receptor whose binding to die analyte prevents its binding to die monolayer-bound ligand. 13. The apparatus of claim 12, for use in detecting an antigen analyte, wherein the ligand is an analyte or analyte analog, and the. apparatus further includes, for inclusion in such solution, an antibody whose binding to die analyte prevents its binding to die monolayer-bound ligand. 14. The apparatus of claims 1 or 2, for use in screening for a compound effective to inhibit binding of a ligand to a receptor, wherein the ligand and receptor form a binding pair selected from the group consisting of effector-receptor, drug-receptor, and oligonucleotide sequence-DNA binding protein, die ligand is attached to the monolayer, and the apparatus further includes such receptor, for inclusion in such solution. 15. A metiiod of forming an electrode for use in measuring binding events between a ligand and a receptor, comprising subjecting an electrode substrate having a conductive metal surface to mild oxidation conditions, adding to the substrate, a solution of hydrocarbon chains having lengths between 8-22 carbon atoms and derivatized at one chain end with a tiiiol group, where a selected portion of said chains are derivatized at their ends opposite the thiol group, with said ligand, applying a positive potential to said electrode, said oxidative conditions being such as to produce deposition of a monolayer of close-packed, oriented chains on said substrate, as evidenced by die ability of the electrode to form an effective barrier to electron ion flow across the monolayer mediated by a redox ion species in an aqueous solution in contact with the monolayer. 16. The method of claim 15, wherein said subjecting includes placing a potential of at least 260 mV on said electrode, in a solution containing the alkyl thiol to be deposited, and electrolytes including litiiium ion and perchlorate anions. 17. The method of claim 15, wherein die hydrocarbon chains are C 8 to C^ chains containing thiol groups at one end, and a portion of die chains contain a chemical functional group at the other end. 18. A mediod of producing a ligand-specific biosensor for use in a biosensor apparatus capable of detecting a binding event between a ligand and ligand-binding receptor, said method comprising contacting together: (a) a biosensor electrode having (i) a detection surface, (ii) formed on said detection surface, a monolayer composed of hydrocarbon chains anchored at their proximal ends to die detection surface, and (iii) embedded in the hydrocarbon-chain monolayer and covalently attached to die detection surface, a first heterodimer-subunit peptide, and (b) a second heterodimer subunit capable of binding to said first subunit to form an α- helical heterodimer, said second peptide having a covalently attached ligand capable of binding specifically to such ligand-specific receptor, and by said contacting forming such heterodimer in said monolayer, where said chains are sufficiendy close-packed and ordered to form an effective barrier to electron flow across die monolayer mediated by a redox ion species in an aqueous solution in contact with the monolayer, in the absence of attachment to the chains of said first peptide, witii (i) attachment of the first peptide to die chain ends being effective to measurably increase such electron flow, relative to electron flow in the absence of such attachment, (ii) binding of the second peptide to die chain-attached first peptide to form such heterodimer being effective to measurably reduce such electron flow, relative to electron flow in the presence of the attached first peptide alone, and (iii) binding of a ligand-binding species to a ligand covalendy attached to the second peptide in such heterodimer being effective to measurably increase such electron flow across the monolayer, relative to electron flow in the presence of the heterodimer alone. 19. The method of claim 18, wherein the electrode has a gold detection surface and said monolayer is composed of 8-22 carbon atom chains attached at their proximal ends to the detection surface by a thiolate linkage. 20. The method of claim 18, wherein said chains have a molecular density of about 3 to 5 chains/nm 2 .

Inventors: HODGES ROBERT S, IRVIN RANDALL T, LENNOX R BRUCE, HODGES, ROBERT, S., IRVIN, RANDALL, T., LENNOX, R., BRUCE
Applicants: PENCE INC, UNIV MCGILL, MCGILL UNIVERSITY, PENCE, INC.
Application year: 1997
Main classification: G01N27
IPC codes: C12Q1, C40B40, G01N27, G01N33