the invention is a composite thin film electrode material useful in fabricating electrochemical biosensors . it is composed of a conducting or semiconducting hydrous metal oxide . disposed in the bulk or on the surface of the oxide is one or more biomolecules and cofactors . the biomolecules react with analytes or substrates to give electroactive products which can be measured electrochemically , either amperometrically or potentiometrically . the metal oxide matrix mediates the electron transfer from these products to the electrode surface and into the external measuring circuit . the hydrous metal oxide is derived substantially from ir , ru , pd , pt , zr , ti and rh , or mixtures thereof . these oxides are both electrically conductive / semiconductive and stable over a wide range of electrochemical polarizations and ph conditions , including those supportive of biomolecules . the preferred oxide is ir oxide because it has ideal electronic and morphological properties and because it is readily grown on the native metal under physiological and other biocompatible conditions . it is also a well - known physiological electrode . hydrous ir oxide may be formed by cycling an ir electrode between positive and negative potential limits in aqueous electrolytes . electrochemical oxide growth is observed in acid , neutral and basic solutions . oxide growth is typically monitored by cyclic voltammetry , which reveals an increasing charge capacity during activation . this capacity results from at least a 1 electron ir ion valence change throughout the bulk of the oxide . the reaction is related to the interconversion of the ir hydrous oxides with structures tentatively assigned by burke and whelan , journal of electroanalytical chemistry v . 162 , pp . 121 - 141 ( 1984 ): 2 iro . sub . 2 ( oh ). sub . 2 . 2h . sub . 2 o !. sup . 2 - + 2e . sup .- ⃡ ir . sub . 2 o . sub . 3 ( oh ). sub . 3 . 2h . sub . 2 o !. sup . 3 - + 3oh . sup .- the open circuit potential of the oxide films undergo a ph dependence of ˜ 50 - 90 mv / ph unit indicative of a dynamic acid - base equilibrium that may include several oxide species . the low density , spongy , amorphous morphology of the films reflects a hydrous structure with hydroxylated inner surfaces . the ir atoms are connected along some directions by bridging oxygen bonds , while in other directions they are terminated by -- o - , oh and oh 2 + . this forms a polyoxometallate network with voids shown by transmission electron microscopy to range from 25 å to 100 å . the presence of microvoids throughout the film , observed in published work using transmission electron microscopy ( mcintyre et al ., journal of the electrochemical society vol . 127 , pp . 1264 - 1268 , 1984 ), provides a feasible means for inclusion of large biomolecules at all stages of growth , i . e ., that the molecules can become adsorbed , then further hydrous oxide can grow around it . this could occur by dissolution / precipitation of the oxide , or by field - assisted diffusion of the macromolecules into the low density hydrous oxide matrix . the mechanism of microvoid formation in ir oxide has not been determined . in addition , hydrous oxides frequently have a very low density and high internal surface area which can promote molecular incorporation by adsorption or ion exchange . oxides of ir and related metals formed by processes such as anodization , electrodeposition , thermal decomposition of precursor salts , sol gel and vacuum evaporation can be made to exhibit a porous low density morphology by appropriate variation of the conditions of formation . the porosity and hydroxylated inner surfaces of these layers are conducive to enabling large biomolecules to enter into the matrix by adsorption . adsorption may be further assisted by application of an electrode potential of polarity opposite from the net surface polarity of the biomolecule . thin film hydrous , conductive / semiconductive oxides , exemplified by ir oxide , serve as highly effective and stable matrices for encapsulating enzymes and other biomolecules . the trapped enzyme is shown schematically in fig1 . the oxide matrix may be used to oxidize or reduce the primary product of the enzyme - substrate reaction , e . g ., h 2 o 2 in the case of the enzyme glucose oxidase ( gox ) and many other oxidases . the small molecule product is produced within the protein globule , but then can easily diffuse out . a three dimensional conductive matrix contacting both the electrode and the protein surface provides a means of immediate interception of the oxidizable species . it is a feature of this invention in this regard that ir oxide and the other subject oxides provide a highly inert conductive matrix that does not degrade in the presence of oxidizing products like h 2 o 2 , a problem noted for conductive polymer matrices employed in prior art . thus , ir oxide can be used to encapsulate enzymes that react with the substrate and oxygen to produce an oxidized product and h 2 o 2 . these enzymes include oxidases of glucose , hexose , cholesterol , aryl alcohol , gluconolactone , galactose , glycoxylate , lactate , glutamate , pyranose , sorbose , pyridoxine , primary alcohols , catachol , 1 , 2 hydroxyacids , ecdysone and choline . a more complete listing of oxidoreductase enzymes is to be found in enzymes by m . dixon et al ., 3rd edition , academic press , 1979 . thus , sensors for these substrates can be fabricated with the subject oxide / enzyme composites operating at greater than about 0 . 6v versus ag / agcl . thus , the following representation of the redox behavior is consistent with the mixed valence hydrous oxide structure , using the ir oxide example : 1 . in reduction , an electron is injected into the oxide network from the electrode . 2 . the electron is transported through the polyoxometallate network by a hopping mechanism from ir + 3 to ir + 4 sites . 3 . the electron becomes localized as an ir + 3 site . 4 . anions and cations move freely through the porous matrix to compensate the charge , which may also be accompanied by changes in the ir -- o -- ir bonding . a reverse scenario occurs for oxidation . the electrode is highly reversible . note that in the case of ir oxide , ir + 3 or ir + 4 sites near to the enzyme active region can act as donors or acceptors , respectively . this is illustrated in fig2 . an ir + 4 acceptor , which is placed at about 0 . 2v versus ag / agcl in a typical hydrous oxide at neutral ph , can thus under some conditions take the place of o 2 or other co - factor in a reaction involving an oxidoreductase enzyme . thus , in cases where the active site is sufficiently close to the enzyme surface for electron tunneling to occur to the surrounding metal oxide matrix , the oxide also acts as the electron transfer mediator . this transfer is shown in fig2 as an electron transfer from a reduced acceptor , a - , to an ir + 4 site . it is well established in prior art that the ir + 3 / ir + 4 redox reactions in ir oxide films are both rapid and reversible for many cycles in physiological stimulation electrodes , so highly reversible enzyme electrodes are possible . co - immobilization of two or more enzymes can be brought about by growing the oxide in contact with the appropriate enzyme mixture . for example , ir oxide can be grown in a mixture of acetylcholinesterase ( ache ) and choline oxidase to sense acetylcholine . in this case , acetylcholine interacts with the esterase to produce choline . the choline then interacts with the oxidase to produce betaine aldehyde and h 2 o 2 when o 2 is an acceptor , or another quantifiable reduced species when another co - factor is used . as indicated above , the co - factor can be the matrix itself . additional electron transfer mediators can be co - immobilized into the oxide matrix by electrostatic binding . useful electron transfer shuttles need to be reversible redox moieties . they must further be capable of transferring electrons to or from the active redox site of the oxidoreductase enzyme . active redox centers within these enzymes are typically fad / fadh , nad + / nadh , nadp + / nadph and porphyrin derivatives . electron transfer mediators useful for these centers are ferrocenes , quinones , and metal complexes of ir , ru and os . derivatives of these mediators may be prepared to enhance the ionostatic binding in the metal oxide matrix , for example ferrocene with carboxylate substituents . similarly , electron transfer shuttle bearing polymers or oligomers can be formed in situ electrochemically in the electrode &# 39 ; s porous matrix . amperometric sensors based on oxidoreductase enzymes have numerous applications . for example , glucose oxidase electrodes can be used to monitor glucose concentration in the blood of diabetics . cholinesterase electrodes can be used to assay for acetylcholine activity and inhibition by nerve agents used in chemical warfare or by pesticides . serum cholesterol can be determined using cholesterol oxidase electrodes . blood alcohol level is able to be measured by an amperometric response of blood or blood plasma to an alcohol oxidase electrode . the electrodes may be used in many of the electrochemical biosensor apparatus described in prior art . one preferred arrangement is for the enzyme electrode to be the working electrode member of a three electrode configuration in an electrochemical cell . the cell also contains a counter electrode and a reference electrode such as saturated calomel or ag / agcl . in one mode of operation the working electrode is held at a fixed potential versus the reference using a potentiostat . a sample containing the analyte is added to the electrolyte and a current is registered in the external circuit that is proportional to the analyte concentration . in order for this current to be measured , the potential must be fixed at a value sufficient to oxidize ( or reduce ) the electroactive product of the enzyme - substrate reaction . the enzyme can also be provided in a two electrode configuration in which the second electrode is both the counter and the reference electrode . such a configuration is convenient for analysis of a drop of solution , such as blood , which may be placed to bridge two electrodes , as in glucose test strips for blood glucose monitoring . the metal oxides also function as ph electrodes with their open circuit potentials varying monotonically with ph . enzymes which give products that alter the ph of the local environment may also be trapped within the oxide matrix . the potential of the electrode may then be calibrated as a function of substrate concentration , providing a potentiometric enzyme electrode . enzymes reacting with substrate to yield acidic or basic products that are suitable for potentiometric analysis include acetylcholinesterase , butyrylcholinesterase , uricase , urease , penicillinase , adenosine deaminase and methionine lyase . a more complete listing of such enzymes is to be found in enzymes by m . dixon et al ., 3rd edition , academic press , 1979 . in principle , the hydrous oxides can be used to immobilize any biomolecule in the hydrous metal oxide , such as antibodies . an immobilized antibody will attract an antigen in solution forming a strongly bound complex . in one scheme , a &# 34 ; sandwich assay &# 34 ;, the antibody is exposed to a solution containing an unknown concentration of an antigen , thus blocking some fraction of the antibody binding sites . next the electrode is exposed to a solution of the antibody that binds specifically to the antibody - antigen complex and which is labeled with an electroactive group such as an oxidoreductase enzyme . the surface concentration of the labeled complex may then be read out amperometrically or potentiometrically by exposing the electrode to a solution of the substrate specific to the label , e . g ., glucose in the case of a gox label . other schemes for electrochemical immunosensing are possible , for example allowing labeled antigen to compete for binding sites with the unlabeled antigen of unknown concentration (&# 34 ; competitive binding immunoassay &# 34 ;). immobilizing enzyme substrates in the hydrous metal oxide matrix is also possible . thus , an electrode for detecting the concentration or activity of an enzyme in solution may be made by interaction with the electrode - bound substrate to form a detectable product ( amperometric or potentiometric ). for example , a potentiometric sensor for acetylcholinesterase activity in blood may be made using a hydrous metal oxide electrode impregnated with a acetylcholine . the amount of acetic acid formed , and thus the potentiometric response , will be a function of the amount of active enzyme in the blood analyte solution . nucleic acid strands may also be immobilized onto the oxides , thus forming highly specific recognition sites for strands of complementary bases . when electrochemically active labels are used to detect the double strands such sensors may be used in dna - based diagnostics . the following specific examples illustrate how the invention may be exercised but should not be construed to limit the scope of the invention as delineated in the claims . glucose oxidase / ir oxide electrode for determining glucose . a three electrode electrochemical cell with 10 ml of electrolyte was employed for oxide growth and for sensor measurements . a 1 mm diameter ir wire encapsulated in epoxy and polished flat was used as the working electrode . a bioanalytical systems ag / agcl single junction reference electrode and a 2 cm 2 pt flag counter electrode were also employed . potential control was achieved with a solartron si 1287 electrochemical interface and associated data acquisition and control software . oxide growth was carried out in 0 . 1m phosphate buffered saline ( pbs , ph 7 . 2 ) by pulsing between - 0 . 6 v and 0 . 8 v at 1 hz for 800 cycles . this procedure provides a layer with a charge capacity of 30 - 35 millicoulombs / cm 2 . enzyme incorporation into the electrode was achieved by dissolving the enzyme into the electrolyte immediately before activation . after activation and impregnation , electrodes were rinsed several times with distilled deionized water and stored at 4 ° c . in 0 . 1m pbs . glucose oxidase ( sigma , 15 - 25 u / mg solid ) was dissolved to the level of 1000 u / ml in 0 . 1m pbs and the electrode activated according to the above protocol . amperometric measurements of electrode activity were conducted in 0 . 1m pbs which had been purged for 10 minutes with ar . the electrode was maintained at the measurement potential , typically 0 . 7v versus ag / agcl in these studies , and the electrode &# 39 ; s capacitative current allowed to decay to & lt ; 1 μa / cm 2 . the analyte was then added incrementally and the steady state current measured after each addition . as shown in fig3 this electrode responded with a calibration curve that increases at low substrate concentration , then levels off to a saturation value . this behavior can be described with simple michaelis - menton enzyme kinetics and is typical of gox enzyme electrodes . the dynamic range for the electrode and the saturation current densities obtained ( 180 - 200 μa / cm 2 ) are large compared to many other gox electrodes reported in prior art . this is a direct result of the uniquely high surface area ir oxide morphology . the closest analogy to the present system is the use of conductive polymers to entrap gox , e . g ., polypyrrole . a report by cho et al . ( sensors and actuators b 30 , 137 ( 1996 )) on gox / polypyrrole electrodes formed by electroadsorption of gox into the preformed polypyrrole matrix exhibited a saturation current of 11 μa / cm 2 at 100 mm glucose , 0 . 7v polarization . since o 2 was removed from the electrolyte , the results in this example indicate that reduced fadh 2 in the enzyme is transferring its electron directly to the metal oxide matrix . direct transfer between adsorbed gox ( fadh 2 ) and an electrode surface has been observed in prior art ( lin and bocarsley ( 1996 ), anal . chem . 68 : 796 - 806 ). if the electrolyte is purged with o 2 , then h 2 o 2 is formed as the end product , which can also be detected amperometrically by the ir oxide matrix electrode . electrodes impregnated with gox were stored in 0 . 1m pbs at room temperature . these conditions are more extreme than the usual storage conditions of 4 ° c . frequently reported in the prior art for gox electrodes . before each measurement a blank was recorded to establish any residual background current . responses were then measured to 100 mm glucose . in the electrode tested there was an initial decline in activity over the first 14 days , followed by some regaining of response . after one month of storage the electrodes are still active but only with about 20 % of the fresh electrode response . nevertheless , the current densities of the aged electrodes are close to or exceed those reported for many gox electrodes in prior art . an interpretation is that some of the gox is buried deeper in the ir oxide matrix and is more protected . also , there is some evolution of the oxide morphology which may enhance the access of the enzyme by glucose . table 1______________________________________storage of gox impregnated ir oxide electrode in 0 . 1 m phosphatebuffered saline ( pbs ) at room temperature . response to 90 . 9mm glucose at 0 . 7 v . electrode area = 7 . 85 × 10 . sup .- 3 cm . sup . 2 days na______________________________________ 0 190 1 60 2 48 3 35 9 32 10 34 14 12 16 19 21 47 23 37______________________________________ table 2 presents several electrode loading procedures for preparing gox - impregnated electrodes . activities of each different electrode were measured in ar - purged solutions by polarizing the electrode at 0 . 7v in 0 . 1n pbs electrolyte , allowing the capacitative current associated with the porous electrode structure to minimize , then adding glucose to 100 mm . the responses for the different preparations in table 2 are summarized in table 3 . responses to glucose in the absence of enzyme were negligible . all protocols in table 2 produced enzyme - active electrodes . the most active electrodes were produced by growing the oxide in the presence of the gox . table 2______________________________________protocols for incorporating enzymes into ir oxide electrodes . scheme procedure______________________________________a activate electrode in a solution containing the enzyme . p polarize the activated electrode at 0 . 7 v for 20 minutes in an electrolyte containing the enzyme to permit anodic adsorption of the negatively charged enzyme . c cycle the activated electrode in an electrolyte containing the enzyme between - 0 . 6 v and 0 . 8 v versus ag / agcl reference . w wet the dried , activated electrode with an enzyme solution and allow water to evaporate . rinse electrode with distilled h . sub . 2 o . d dip the activated electrode in an enzyme solution for 10 minutes , then rinse with distilled h . sub . 2 o . e electrodeposit ir oxide in solution containing enzyme . ______________________________________ table 3______________________________________results for limiting response of several oxidase electrodes ( 1 mmdiameter disks ) prepared by trapping the enzyme in an anodiciridium oxide matrix . the background current is given as i . sub . 0and the current density above background is given as δi . sub . sat . all measurements made at 0 . 7 v . solutions purged for 15 min in arbefore measurements . adsorption charge capacity gox ! protocol ( mc / cm . sup . 2 ) u / ml i . sub . 0 , μa / cm . sup . 2 δi . sub . sat , μa / cm . sup . 2______________________________________ 1a 30 1000 9 120c 14 1000 2 25p 29 100 1 . 66 6 . 24w 16 7116 21 24 . 5d 35 1000 5 12 . 1e 16 . 3 1000 7 12 . 9______________________________________ the experimental evidence indicates that the enzyme gox becomes trapped in the oxide matrix if the ir is activated in its presence at ph 7 . the enzyme has a 60 × 52 × 77 å spheroid shape , within the range of the anodic oxide void size . the isoelectric point of gox is 4 . 2 , so it is negatively charged at ph 7 . ellipsometric investigations in prior art have shown that gox absorbs at au electrodes as the potential is swept positive of the metal isoelectric point (˜- 0v / nhe ) ( szucs et al ., journal of the electrochemical society 136 ( 12 ), 1989 , pp . 3748 - 3754 ). similarly , the enzyme should be attracted to the positively charged sites at the ir oxide surface during anodization , enhancing the enzyme loading effect above that which would be expected from simple physical trapping . proteins are generally negatively charged in neutral aqueous solution . however , the metal oxides can absorb either anions or cations , depending on their direction of polarization . ache / chox electrode for determining acetylcholine . an amperometric electrode sensitive to acetylcholine was fabricated by co - incorporating acetylcholinesterase ( ache ) and choline oxidase ( chox ) into anodically formed ir oxide . the formation electrolyte contained 1000 u / ml and 100 u / ml of the respective enzymes dissolved in 0 . 1m pbs . activation proceeded as in example 1 . the amperometric response curve ( 0 . 7v ) for the activated chox / ache dual enzyme electrode to solution additions of acetylcholine is shown in fig4 . neither enzyme alone gave a responded amperometrically to acetylcholine . choline oxidase singly immobilized in the electrode gave an amperometric response to choline at this potential . hence , the dual - enzyme electrode is detecting the internal fadh 2 or h 2 o 2 produced by the choline oxidase catalyzed oxidation of choline derived from the interaction of acetylcholine with ache : ## str1 ## acetylcholinesterase potentiometric electrode for determining acetylcholine . a thin film composite acetylcholine sensor was made by activating a 1 cm 2 ir film in 1000 u / ml ache . the ir film was prepared by dc magnetron sputtering onto a glass microscope slide with an intermediate ti adhesion layer . an unbuffered or weakly buffered electrolyte was used to maximize the ph change at the electrode . fig5 shows the potentiometric response of the composite electrode to additions of acetylcholine to unbuffered saline . a positive change in potential was observed which varied with acetylcholine concentration . the positive response indicates that acidic products are being formed at the electrode surface , in agreement with the formation of acetic acid by the enzymatic hydrolysis of acetylcholine . the shape of the calibration curve ( fig5 ) in dicates that michaels - menton kinetics are followed . the largest response is achieved in unbuffered media ( e . g ., 0 . 1m nacl versus 0 . 1m pbs ) or in low buffer concentrations ( e . g ., 2 . 5 - 5 mm hepes buffer ). fig6 shows the rate of response on addition of a 1 mm increment of acetylcholine during the measurement sequence to generate fig5 . it is seen that the full scale response in about 35 seconds . note that this is very short compared to similar experiments reported by tran - minh et al . ( biosensors & amp ; bioelectronics 5 : 461 - 471 ) for ir oxide electrodes coated with polymer layers containing acetylcholinesterase ( 5 - 8 minutes ). electrode for cholinesterase inhibitors . to evaluate the ache electrode response to cholinesterase inhibitors , a 1 cm by 0 . 125 mm ir wire electrode was activated 1000 u / ml ache / 0 . 1m pbs . the charge capacity following activation was 34 mc / cm 2 . the electrode was then rinsed with distilled , deionized water and introduced into an electrochemical cell equipped with a magnetic stirrer , 2 . 5 mm hepes buffer ( ph 7 . 8 ) and a ph electrode . acetylcholine was then added to produce a final concentration of 5 mm . as seen in fig7 a potential response due to the production of acetic acid from the reaction of the electrode - bound enzyme with the solution is observed . the potential response is complete within seconds compared to minutes for ir oxide ph electrodes coated with enzyme - bound polymer layers of prior art , as also seen in fig6 . this is a result of the intimate contact between the product - generating enzyme and the ph sensitive electrode matrix . the electrodes were then rinsed in distilled water and incubated for 12 hours in a 10 - 6 m paraoxon inhibitor solution in 2 . 5 mm hepes buffer . the incubated electrodes were then retested for response to 5 mm acetylcholine . it is seen from fig7 that an instantaneous response is observed , but on mixing the response is suppressed . the total ph change in solution was negligible . then 10 - 3 m pyridine 2 - aldoxime methiodide ( pam ) antidote was added to the solution . the ph of the solution was observed to drop by ˜ 0 . 2 units over a period of 2 hours , indicating a partial reactivation of the electrode . further modifications or alterations to the invention will be apparent to those skilled in the art ; the particular forms of the invention described are to be taken as preferred embodiments . the scope of the invention should be determined solely by the appended claims .