AU644059B2 - Method and apparatus for amperometric diagnostic analysis - Google Patents

Description

OPI DATE 05/10/89 WOR AOJP DATE 02/11/89 APPLN. ID 33436 89

PCT

PCT NUMBER PCT/US89/01057 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY "Il (51) International Patent Classification 4 C12Q 1/00, 1/26, C12M 1/34 G01N 27/26 (11) International Publication Number: Al (43) International Publication Date: 21 S WO 89/ 08713 September 1989 (21.09.89) (21) International Application Number: PCT/US89/01057 (22) International Filing Date: (31) Priority Application Numbers: (32) Priority Dates: 14 March 1989 (14.03.89) 168,295 Not furnished 15 March 1988 (15.03.88) 13 March 1989 (13.03.89) (33) Priority Country: (74) Agent: YAHWAK, George, Yahwak Associates, Skytop Drive, Trumbull, CT 06611 (US).

(81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, FI, FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), NO, SE, SE (European patent), SU.

Published With international searre

AUST'RAIUA

5 OCT ,59 PATENT OF",-CE (71) Applicant: LIFE-CHEK LABORATORIES [US/US]; 4900 Perry Highway, Pittsburgh, PA 15229 (US).

(72) Inventors: POTTGEN, Paul, A. 2335 Big Rock Road, Allison Park, PA 15101 SZUMINSKY, Neil, J.

1427 Hillsdale Avenue, Pittsburgh, PA 15216 (US).

TALBOTT, Jonathan, L. 165 Woodhaven Drive, Mars, PA 16046 JORDAN, Joseph 1007 Glenn Circle North, State College, PA 16803 (US).

(54) Title: METHOD AND APPARATUS FOR AMPEROMETRIC DIAGNOSTIC ANALYSIS (57) Abstract The present invention relates to a.novel method and apparatus for the amperometric determination of an analyte, and in particular, to an apparatus (10) for amperometric analysis utilizing a novel disposable electroanalytical cell (20) for the quantitative determination of biologically important compounds from body fluids.

WO 99/08713 PCT/LIS8910 1 0 5 7 METHOD AND APPARATU3S FOR. AMPr-OMETRIC DIAGNOSTIC ANALYSIS F=-ED 07 THE~ !NVENTION: The Dresent i.vetion relates to a c-sosaz'eleczroanalvtical cell and a ne.hod and anaraz---s guanzitatively determining the presence of bioloci"call.y ~mnrt-an ccm,:,unds such as glucos-e; TSH; T4; hor-mones su:as HCG; cardiac glycosides such as Digoxin; ant-iarrhvzh--mil-s such as Lidocaine; antiepileptics such as phenobarbitzal; lo antibictics such as Gent4,-amicin; choclesterol; non-therapeutIc drucs and t"he like from body fluids.

Alithough the present invention has broad applications, for tur----oses of: illustration of the invention ec:.

ernah Ls is Wj T* be p~laced up~bn i-zs a 1ic a on in.

quanzitativelv det-ermining the presence of two biologicallv, 4nncr-ant -ompounds glucose and cholesterol.

WZTS RESPECT TO C-luco sE: Diabetes, and specifically diabet!-es mellitus, i s a metabolic disease characterized by deficient insu 1 -i 2produc=Aion by the pancreas which results in abnormal levels of blood glucose. Although t"his disease afflics only WO 89/08713 2 PCT/US89/01057 approximately 4% of the population in the United States, it is the third leading cause of death following heart disease and cancer. With proper maintenance of the patient's blood sugar through daily injections of insulin, and strict control of dietary intake, the prognosis for diabetics is excellent. However, the blood glucose levels must be closely followed in the patient either by clinical laboratory analysis or by daily analyses which the patient can conduct using relatively simple, non-technical, methods.

At the present, current technology for monitoring blood glucose is based upon visual or instrumental determination of color change produced by enzymatic reactions on a dry reagent pad on a small plastic strip. These colorimetric methods which utilize the natural oxidant of glucose to gluconic acid, specifically oxygen, are based upon the reactions: B-D-Glucose 02 H 2 0 D-Gluconic Acid H 2 0 2

H

2 0 2 Reagent H 2 0 color.

WITH RESPECT TO CHOLESTEROL: Current technology for the determination of cholesterol is also based upon similar methods. In the case of cholesterol, the methods presently used are based upon the generalized reactions: Cholesterol H 2 0 Cholestenone +H 2 0 2 H202 Reagent H 2 0 color WO 89/08713 PCT/US89/01057 3 In all present techniques, Dioxygen is the only direct oxidant used with the enzyme cholesterol oxidase for the determination of both free and total cholesterol. Using conventional test methods, oxygen must diffuse into the sensor solution during use from the surrounding air in order to provide sufficient reagent for a complete reaction with the analyte cholesterol in undiluted serum and whole blood speciments.

In both instances, .the presence of the substance is determined by quantifying, either colorometrically or otherwise, the presence of hydrogen peroxide. The present methods of detection may include direct measurement of the hydrogen peroxide produced by either spectroscopic or electrochemical means and indirect methods in which the hydrogen peroxide is reacted with various dyes, in the presence of the enzyme peroxidase, to produce a color that is monitored.

While relatively easy to use, these tests require consistent user technique in order to yield reproducible results. For example, these tests require the removal of blood from a reagent pad at specified and critical time intervals. After the time interval, excess blood must be removed by washing and blotting, or by blotting alone, since the color measurement is taken at the top surface of the reagent pad. Color development is either read immediately or after a specified time interval.

WO 89/08713 PCT/US89/01057 4 These steps are dependent upon good and consistent operating technique requiring strict attention to timing.

Moreover, even utilizing good operating technique, colorimetric methods for determining glucose, for example, have been shown to have poor precision and accuracy, particularly in the hypoglycemic range. Furthermore, instruments used for the quantitative colorimetric measurement vary widely in their calibration methods: some provide no user calibration while others provide secondary standards.

Because of the general lack of precision and standardization of the various methods and apparatus presently available to test for biologically important compounds in body fluids, some physicians are hesitant to use such equipment for monitoring levels or dosage. They are particularly hesitant in recommending such methods for use by the patients themselves. Accordingly, it is desirable to have a method and apparatus which will permit not only physician but patient self-testing of such compounds with greater reliability.

The present invention addresses the concerns of the physician by providing enzymatic amperometry methods and apparatus for monitoring compounds within whole blood, serum, and other body fluids. Enzymatic amperometry provides several advantages for controlling or eliminating operator dependant techniques as well as providing a greater Wo 89/09713 Pcr[US9Iol057 linear dynamic range. A system based on this type of method could address the concerns of the physician hesitant to recommend self-testinc for his patients.

Enzvmatic amperometry methods have been applied to the laboratory based measurement of a number of analyvtes including glucose, blood urea nitrogen, and lactate.

Traditionally the electrodes in these systems consist of bulk metal wires, cylinders or disks imbedded in an insulating material. The fabrication process results in individualistic characteristics for each electrcde necessitating calibration of each-sensor. These electrodes are also too costly for disposable use, necessitating mePiculous attention to electrode maintenance for continuous reliable use. This maintenance is not likely to be performed properly by untrained ersonnel (such as patients) therefore to be successful, an enzyme amperometry method intended for self-testing (or non-traditional site testing) must be based on a disposable sensor that can be pmroduced in a manner that allows it to give reproducible S' .*o0 cutput from sensor to sensor and at a cost well below that of traditional electrodes.

S. .i The present invention address these requirements by providing miniaturized disposable electroanalytic sample cells for precise micro-aliquote sampling, a self-contained, automatic means for measuring the electrochemical reduction of the sample, and a method for using the cell and apparatus according to the present invention..

\P0 Thus, in accordance with a first aspect of the present invention, there is provided a method for measuring the amount of a selected compound in body fluids comprising: a) providing a measuring cell having at least a first and second electrode and said cell containing an oxidant and a buffer, b) placing a sample of fluid to be tested in said cell, c) reconstituting said oxidant and buffer with said sample fluid to generate a predetermined reaction, d) allowing said reaction to proceed substantially to completion, e) applying a potential across said electrodes and sample, and f) measuring the resultant Cottrell current to determine the concentration of said selected compound present in said sample.

In accordance with a second aspect of the present invention, there is provided a method for measuring the amount of glucose in blood, comprising: a. providing a measuring cell having at least a first and second electrode and said cell containing an oxidant, a buffer and an enzyme, b. placing a blood sample to be tested in said cell, c. reconstituting said oxidant, buffer and enzyme with said blood sample to generate a predetermination reaction, ieto d. immediately applying a potential across said electrode and blood sample, and e. measuring the resultant Cottrell current when the reaction has proceeded to completion to determine the concentration of said glucose present in said blood sample.

In accordance with a third aspect of the present invention, there is provided a sample cell for determining the concentration of a selected compound in a sample aqueous fluid, 3' 5 comprising: SI 4 a) a metallized first electrode which acts as a working electrode, b) a metallized second electrode which acts as a reference electrode, said second electrode being operatively associated with said first electrode; and c) at least one non-conducting layer member having an opening therethrough, said layer member being disposed in contact with at least one of said electrodes and said layer member being sealed against at least one of said first and second electrodes to form a known electrode area within said opening such that said opening forms a well to receive said sample aqueous fluid and to place said fluid in said known electrode area in contact with said first electrode and second electrode, whereby substantially the entire contents of said well is capable of being substantially simultaneously subjected to a predetermined reaction.

In accordance with a fourth aspect of the present invention, there is provided an apparatus for measuring compounds in a sample aqueous fluid, comprising: a. a housing having an access opening therethrough, b. a sample cell receivable into said access opening of said housing, said sample cell being composed of a metallized first electrode which acts as a working electrode, a metallized second electrode which acts as a reference electrode, said second electrode being operatively associated with said first electrode, at least one non-conducting layer member having an opening therethrough, said layer member being disposed in contact with at least one of said electrodes and said layer 3: member being sealed against at least one of said electrodes and said layer member being sealed against at least one of said first and second electrodes to form a known electrode area within said opening such that said opening forms a well to receive said sample fluid and to place said fluid in said known electrode area in contact with said first electrode and said 7second electrode, c. means for applying an electrical potential to said first electrode and said second electrode, means for creating an electrical circuit between said first electrode and said second electrode through said sample aqueous fluid, e. means for measuring Cottrell current through said sample aqueous fluid, and f. means for visually displaying results of said measurement.

The disposable cells according to the present invention are preferably laminated layers of metallizced plastic and nonconducting material. The metallized layers provide the working and reference electrodes, the areas of which are reproducibly defined by the lamination process. An opening through these layers is designed to provide the samplecontaining area or cell for the precise measurement of the sample. The insertion of the cell into the apparatus according to the present invention, automatically initiates the measurement cycle.

To better understand the process of measurement, a presently preferred embodiment of the invention is described which involves a two-step reaction sequence utilizing a chemical oxidation step using other oxidants than oxygen, and an electrochemical reduction step suitable for quantifying the reaction 2. product of the first step. One advantage of utilizing an oxidant other than dioxygen for the direct determination of an analyte is that they may be prepositioned in the sensor in a large excess of the analyte and thus ensure that the oxidant is not the limiting reagent (with dioxygen, there is normally 3'*V insufficient oxidant initially present in the sensor solution for a quantitative conversion of the' analyte).

i• a

I,

WO 89/08713 PCT/US89/01057 7 In the oxidation reaction, a sample containing glucose, for example, is converted to gluconic acid and a reduction product of the oxidant. This chemical oxidation reaction has been found to precede to completion in the presence of an enzyme, glucose oxidase, which is highly specific for the substrate B-D-glucose, and catalyzes oxidations with single and double electron acceptors. It has been found, however, that the oxidation process does not proceed beyond the formation of gluconic. acid, thus making this reaction particularly suited for the electrochemical measurement of glucose.

In a presently preferred embodiment, oxidations with one electron acceptor using ferricyanide, ferricinium, cobalt (III) orthophenantroline, and cobalt (III) dipyridyl are preferred. Benzoquinone is a two electron acceptor which also provides excellent electro-oxidation characteristics for amperometric quantitation.

Amperometric determination of glucose, for example, j,n accordance with the present invention utilizes Cottrell current micro-chronoamperometry in which glucose plus an oxidized electron acceptor produces gluconic acid and a reduced acceptor. This determination involves a preceding chemical oxidation step catalyzed by a bi-substrate biproduct enzymatic mechanism as will become apparent throughout this specification.

~-J~J.Jju~uu In this method of o,.antification, the measurement off a diffusion controlled current at an accurately specified time 20, 30, or 50 second&t, for example) aft"ler the instzant of aD9,lication of a potential has the applicable equazion for anperometry at a controlled potential (E=constant) of: i COTTRELL 4 t) C Y-TA;,BOLITE t>O 0tt 0 where i denotes current_, nF is the number of co~ulomb.-'s per mole, A is the area of the electrode, D is the diffusion coefficient of the reduced form of the reagent, t is the preset time at which the current is measured, and C is the concentration of the metabolite. Measurements by the method accordinr to the present invention of the current due to the reoxidation of the acceptors were found to be proportional to the glucose concentration in the sample.

The-i method and apparatus of the presenz lnvenzion Per-.1t, 2.n preferred embodiments, dir-ect measurement.s of blood guecholest erol and the like. Furthermore, t-he sam.=-e cell according to the present invention, p~rovides the testi-ng of controlled volumes of blood without rrmeasuring.

lnsert:ion *of the sampling cell into t.e a-zara-tus thus ner~:ts automatICic functiJoning and ti'ming of the reaction 2 3. allowing for patient selff-testZing with a very high degree o: preci sion and accuracy.

S..

WO 89/08713 PCT/UIS89/0 I057 One of many of the presenrc-ly pref erred embodiments o-F the invention for use in measuring B-D gluco!9e is described in detail to better understand the nature and scope of the invention. In particular, the method and aoaazus according to this embodiment are designed to pz-ovide clinical self-monitoring of blood glucose levels by a diabetic Datient. The sample cell cof the invention is used to control the sampling' volume and reaction media and act-s as t he electrochemical sensor.. In this described eibodi.ment, benzoquinone is used as the electron acceotoor.

The basic chemical binary r-eac-t:on utilized by the -me--hod according to the present invention is: B-D-glucase Bern==ui4norie H,0O >G1uccr.icAci;d Fverocruiznn zHvdcoouinone >benzcaui_,nane 2e 2H-t The f irst reaction is an oxidation reaction which, proceeds to completion in 'the presence of the enzyme glucose oxidase. Electrochemical oxidation takes place in the second parz. the reaction' an-d provides the means o quant ifying the amount of hydroquinone produced in the oxida tion reaction. This holds true whether catalytic oxidation is conducted with two-electron accemtors or one electr:on acceptors such as ferricyanide [wherein the redox cum1e would be Fe(CN)j6/F (CN) 6 eriiicbl rthophenantro line and cobalt cimyridyl.

catalytic oxidation by glucose oxidase is highlv m f orec -D-glucose, but iJ~ nonsel.ective as to the

'IN.

4 4 f a WO 89/08713 PCT/US89/01057 oxidant. It has now been discovered that the preferred oxidants described above have sufficiently positive potentials to convert substantially all of the B-D-glucose to gluconic acid. Furthermore, this system provides a means by which amounts as small as 1 mg of glucose (in the preferred embodiment) to 1000 mg of glucose can be measured per deciliter of sample results which have not previously been obtained using other glucose self-testing systems.

The sensors containing the chemistry to perform the desired determination, constructed in accordance with the present invention, are used with a portable meter for selftesting systems. In use the sensor is inserted into the meter which turns the meter on and initiates a wait for the application of the sample. The meter recognizes sample application by the sudden charging current flow that occurs when the electrodes and the overlaying reagent layer are initially wetted by the sample rluid. Once the sample application is detected, the meter begins the reaction incubation step (the length of which is chemistry dependent) to allow the enzymatic reaction to reach completion. This period is on the order of 15 to 90 seconds for glucose, with incubation times of 20 to 45 seconds preferred. Following the incubation period, the instrument then imposes a known potential across the electrodes and measures the current at specific time points during the Cottrell current decay.

Current measurements can be made in the range of 2 to WO 9/08713 PCT/US89/01057 11 seconds following potential application with measurement times of 10 to 20 seconds preferred. These current values are then used to calculate the analyte concentration which is then displayed. The meter will then wait for either the user to remove the sensor or for a predetermined period before shutting itself down.

The present invention provides for a measurement system that eliminates several of the critical operator dependant variables that adversely affect the accuracy and reliability and provides for a greater dynamic range than other selftesting systems.

These and other advantages of the present invention will become apparent from a perusal of the following detailed description of one embodiment presently preferred for measuring glucose and another for measuring cholesterol which is to be taken in conjunction with the accompanying drawings in which like numerals indicate like components and in which: FIG. 1 is an exploded view of a portable testing apparatus according to the present invention; FIG. 2 is a plan view of the sampling cell of the present invention; FIG. 3 is an exploded view of the sample cell shown in Figure 2; FIG. 4 is an exploded view of another embodiment of a sample cell according to the invention; rk- I /Ub8 /U1057 12 FIG. 5 is a plan view of -the cell shown in Figure 4; FIG. 6 is still another embodiment of a. sample cell; FIG. 7 is a graph showing current as a function of glucose concentration; FIG. 8 is a graphical presentation of Cottrell current as a function of glucose concentration; and 9 is a presently preferred circuit diagram of an electrical circuit for use in the a-raratu-,-s shown in Ficure is a nreferred embodimentL of the elec-zrocheraical cell.

With snecific ref-erence to Figure 1, a portable e~ecrocn~caltesting apparatus 10 is shown for use in Datient self -testing, such as for example, for blood glucose levels. Apparatus 10 comrises a -front and back :::housing 2.1 and 1.2, respectively, a front panel 12 and a 'circu I board Front panel !3 includes graphi c di splay 16 for providing infomation and instructions to the 2~ 5 ,at _4ent',, and di--rt read-out of the test results. Whiile a sa button 18 is provided to initiate an analysis, it is preferred that the system begin operation when a sample cell (Figure 2) in inserted into the window 19 of the apparatus.

*With reference to Figures 2 and 3, sample cell 20 is a metallized plastic substrate having a specifically-sized opening 21 which defines a volumetric well 21, when the cell is assembled, for containing a reagent pad and the blood to be analyzed. Cell 20 comprises a first substrate 22 and a second substrate 23 which may be preferably made from styrene or other substantially non-conducting plastic. Positioned on second substrate 23 is reference electrode 24. Reference electrode 24 may be preferably manufactured, for example, by vapor depositing the electrode onto a substrate made from a material such as the polyamide Kapton. In the preferred embodiment, reference electrode 24 is a silver-silver chloride electrode. This electrode can be produced by first depositing a silver layer on a silver chloride layer by either chemical or electrochemical means before the substrate is used to construct the cells. The silver chloride layer may even be generated in-situ on a silver electrode when the reagent layer contains certain of the oxidants, such as ferricyanide, and chloride as shown in the following reactions: Ag ag+ Red A~ C7 AgCl Alternatively the silver-silver chloride electrode can be produced by depositing a layer of silver oxide (by reactive t i t S VY U by) U6 11.1 PCIJUS89/0io57 14 sputtering) onto the silver film. This silver oxide layer is then converted in-situ at the time of testing to silver ,hljoide according to the reaction:

H

2 0 2C1 2AgCl 2(OH) when the sensor is wetted by the sample fluid and reconstitutes the chloride containing reagent layer. The silver electrode is thus coated with a layer containing silver chloride.

The reference electrode may also be of the tvoe generally known as a "pseudo" reference electrode which relies upon the large excess of the oxidizing species to establish a known potential at a noble metal electrode. In a preferred embodiment, two electrodes of the same noble metal are used, however one is generally of greater surface area and is used as the reference electrode. The large excess of the oxidized species and the larger surface area of the reference resists a shift of the potential of the reference electrode.

Indicator or working electrode 26' can be either a strip of platinum, gold or palladium metallized plas-ic positioned on reference electrode 24 or alternately the working electrode 26 and the reference electrode may be manufactured as a coplanar unit with electrode 26 being 25 sandwiched between coplanar electrode 24 material.

.o S Preferably, sample cell 20 is prepared by sandwiching or laminating the electrodes between the substrate to form a composite unit.

4h,q" wu 89/U 1 PCT/US89/01057 As shown in. Figure 2, first substrate 22 is of a slightly shorter length so as to expose an end portion 27 of electrodes 24 and 26 and allow for electrical contact with the testing circuit contained in the apparatus. In this embodiment, after a sample has been positioned within well 21, cell 20 is pushed into window 19 of the front panel to initiate testing. In this embodiment, a reagent may be applied to well 21, or, preferably, a pad of dry reagent is positioned therein and a sample (drop) of blood is placed into the well 21 containing the reagent.

Referring to Figures 4-6, alternative embodiments of sample cell 20 are shown. In Figure 4, .sample cell 120 is shown having first 122 and second 123 substrates. Reference electrode 124 and working' electrode 126 are laminated between substrates 122 and 123. Opening 121 is dimensioned to contain the sample for testing. End 13C (Figure 5) is designed to be inserted into the apparatus, and electrical contact is made with the respective electrodes through cutouts 131 and 132 on the cell. Reference electrode 124 also includes cut out 133 to permit electrical contact with working electrode 126.

In Figure 6, working electrode 226 is folded, thereby providing increased surface area around opening 221, to achieve increased sensitivity or specificity. In this case, 25 reference electrode 224 is positioned beneath working Sincludes cu out 23 electrode 226. Working electrode includes cut out 234 to i' I I'

I

,/Y s 16 perit electrical contact with reference electrode 224 through cut out 231 in substrate 222. End 230 of substrate 222 also includes cut out 232 to permit electrical contact with working electrode 226.

Referring to Figures 1 and 2, the sample cell according to the present invention is positioned through window 19 (Figure 1) to initiate the testing procedure. Once inserted, a potential is applied at portion 27 (Figure 2) of the sample cell across electrodes 24 and 26 to detect the presence of the sample. Once the sample's presence is detected, the potential is removed and the incubation period initiated. Optionally during this period, a vibrator means 31 (Figure 1) may be activated to provide agitation of the reagents in order to enhance dissolution (an incubation period of 20 to 45 seconds is conveniently used for the determination of glucose and no vibration is normally required). An electrical potential is next applied at portion 27 of the sample cell to electrodes 24 and 26 and the current through the sample is measured and displayed on display 16.

To fully take advantage of the above apparatus, the needed chemistry for the self testing systems is incorprated into a dry reagent layer that is positioned onto the disposable cell creating a complete sensor for the inended analvte. The disposable electrochemical cell is constructed by the lamination of metallized mlastics and nonconducting materials' in such a way that there is a precisely defined working electrode area. The reagent layer t I i

S-

N 3 s e, WO 89/08713 PCT/US89/01057 17 is either directly coated onto the cell or preferably incorporated (coated) into a supporting matrix such as filter paper, membrane filter, woven fabric or non-woven fabric, which is then placed into the cell. When a supporting matrix is used, it pore size and void volume can be adjusted to provide the desired precision and mechanical support. In general, membrane filters or nonwoven fabrics provide the best materials for the reagent layer support.

Pore sizes of 0.45 to 50um and void volumes of 50-90% are appropriate. The coating formulation generally includes a binder such as gelatin, carrageenan, methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc., that acts to delay the dissolution of the reagents until the reagent layer has adsorbed most of the fluid from the sample. The concentration of the 'inder is generally on the order of 0.1 to 10% with 1-4% preferred.

The reagent layer imbibes a fixed amount of the sample fluid when it is applied to the surface of the layer thus eliminating any need for premeasurement of sample volume.

Furthermore, by virtue of measuring current flow rather than reflected light, there is no need to remove the blood from the surface of the reagent layer prior to measurement as there is with reflectance spectroscopy systems. While the fluid sample could be applied directly to the surface of the reagent layer, to facilitate spread of blood across the entire surface of the reagent layer the sensor preferably WO 89/08713 PCT/US89/01057 18 includes a dispersing spreading or wicking layer. This layer, generally a non-woven fabric or adsorbant paper, is positioned over the reagent layer and acts to rapidly distribute the blood over the reagent layer. In some applications this dispersing layer could incorporate additional reagents.

For glucose determination, cells utilizing the coplanar design were constructed having the reagent layer containing the following formulations: Glucose oxidase 600u/ml Potassium Ferricyanide 0.4M Phosphate Buffer 0.1M Potassium Chloride Gelatin This was produced by coating a membrane filter with a solution of the above composition and air drying. The reagent layer was then cut into strips that just fit the window opening of the cells and these strips were placed over the electrodes exposed within the windows. A wicking layer of a non-woven rayon fabric was then placed over this reagent layer and held in place with an overlay tape.

In order to prove the application of the technology according to the present invention, a large number of examples were run in aqueous solution at 25 0 C. The electrolyte consisted of a phosphate buffer of pH 6.8 which was about 0.1 molar total phosphate and 0.5 M potassium WO) 89/08713 P'CT/IuS89/010 5 7 3.9 chloride reagent. The potentials are referenced to a normal hydrogen, electrode. (NHE). In these tests it was found tnat any potential between approximately 0.8 and 1.2 vcl.t (vs NXE) is suitable for the quantification of hydroquir-cne when benzoo-uinone is used as the oxidant. The limitin5j current:s are proportional to hydroquinone concentrations in the range between 0.0001 M and 0.050 M.

Determination of glucose by Cottrell curren.t microchronoamperomezry with the pr:esent method is crea-zeo in the react-ion of hvdrocninone to benzcuinone. Cvtzrell current'-s decay wi th time in .accordance with the ecaal-on: t1/2 const w here t denotes the -time. The main difference between these two techniques consists of applying the appropriate' controlled potential after the glucose-benzoquinone reaction is complete and correlating glucose concentrations with Cottrell currents measured at a fixed time thereafter. The current-time readout is shown in Figure 8. Proportionality between glucose concentrations and Cottrell currents (recorded at t 30 seconds after the application of potential) is shown in Figure 7.

it should be noted that Cotrell chronoamveronetry of metabolites needs the dual safecruards of enzvmatic cat7alysis and controlled otential electrolysis. c-luconic acid y--elds 25 of 9.9k Derentwere attained in the vresence of clucose oxidase. Concomitantly, equivalent amounts of benzoau;-Inone

IVI

were reduced to hydroquinone, which was conveniently quantitated in quiescent solutions, at stationary palladium thin film anodes or sample cells.

The results of these many tests demonstrates the microchronoamperometric methodology of the present invention and its practicality for glucose self-monitoring by diabetics.

In a presently preferred embodiment of the invention utilizing ferrocyanide, a number of tests were run showing certain improved operating capabilities.

Referring to Figure 9, a schematic diagram of a preferred circuit 15 for use in the apparatus 10 is shown.

Circuit 15 includes a microprocessor and LCD panel 16. The working and reference electrodes on the sample cell 20 make contact at contacts W (working electrode) and R (reference electrode), respectively. Voltage reference 41 is connected to battery 42 through analogue power switch 43. Current from the electrodes W and R is converted to a voltage by op amp 45. That voltage is converted into a digital signal (frequency) by a voltage to frequency converter 46 electrically connected to the microprocessor 48. The microprocessor 48 controls the timing of the signals. Measurement of current flow is converted by microprocessor 48 to equivalent glucose, cholesterol or other substance concentrations. Other circuits within the skills of a practised engineer can obviously be utilized to obtain the 2 advantages of the present invention.

With regard to Figure 10, cell 400 consists of ."coplanar working 426 and reference 424 electrodes laminated .o between an upper 422 and lower 423 nonconducting material.

SLamination is on an adhesive layer 425. The upper material i It .1.

WW/%jWPCTIS89/O1 057 21 422 includes a die cut opening 428 which, along with the width of the working electrode material defines the working electrode area and provides (with an overlapping reagent layer not depicted) the sampling port of the cell. At one end of cell 400 is an open area 427 similar tCo end position 27 of Figure 2.

The efficiency of using the apparatus according to the Present invention -66o provide a means for in-hone self testing by patients such as diabetics (in the preferred ~0embod~ment) can be seen in the following table in whnich the tech-noloay according to the present invention is cormared to four cornercially available units. As will be seen, the present invention is simpler, and in this instance sinnlicity breeds consistency in results.

0@ 0 S *0 0 I.e.

It 0.

5 0 0

S

S

.00

S.

.6 9 9 9 5 0* $6

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j;?i r'K/ WO 89/03713 WO 8908713PCTJUS89/01057 22 GLUCOSE SYSTEM COMPARISONS Present Inven- 4 tion Steips Turn Instrument On Calibrate Instrument Finger Puncture Apply Bl.ood Initiate Timing Sequence Blot Insert Strip to Read Read Results Total Steps Per Testing Detection System x x x x x x x x 8

RS*

x x x x x x x 8

RS

x x x x x x x x Range (mg/dl) 10-400 40-400 CV*O, Hypoglycemic- 15% 15% Euglycemic 10% 10% Hyperglycemic 5% 5% correlation 0.921 '0.862 (*RS Reflectance Spectroscopy) **Coefficient of variation 7 5 4 'PS RS Polarographic 25-450 40-400 0-1000 3% 2% 0.95 WO 89/08713 PCT/US89/01057 23 With specific regard to the determination of cholesterol utilizing the present invention, the generalized chemistry may be depicted as: Scheme I Cholesterol Esters H 2 0 Cholesterol Fatty acid (1) Cholesterol OX +CO Cholestenone Red (2) Red Ox e- (3) where the enzymes cholesterol esterase (CE) and cholesterol oxidase (CO) catalyze reactions 1 and 2 respectively and CO permits electron transfer with a variety of electroactive couples (Ox and Red). Reaction 2 is novel in that electron acceptors other than dioxygen may be used to oxidize cholesterol in the presence of the enzyme cholesterol oxidase. Reaction 1 is well known to those in the field and is necessary for the determination of total cholesterol (free cholesterl and cholesterol esters). Reaction 3 is an electro-oxidation process for probing and quantitating the cholesterol.

Utilizing alternative oxidants according to the present invention, the specific reactions become:

A:

Reaction I above Cholesterol 2B'erricyanide Cholestenone 2Ferrocyanide Fertocyanide Ferricyanide le- WO 89/08713 PCT/US89/01057 24

B:

Reaction 1 above Cholesterol Benzoquinone Cholestenone Hydroquinone Hydroquinone Benzoquinone 2H+ 2e- Cholesterol oxidase (CO) from a variety of sources will catalyze electron transfer from cholesterol to a variety of the oxidants including benzoquinone, benzoquinone derivatives such as methylbenzoquinone, ethylbenzoquinone, chlorobenzoquinone, ortho-benzoquinone (oxidized form of catechol), benzoquinonesulfonate, and potassium ferricyanide. It is also anticipated that the enzyme will allow electron transfer with other alternate oxidants. As indicated in Reaction 3, the reduced product can then be monitored amperometrically for the quantitative determination of cholesterol.

Sources of the enzyme catalyzing the oxidation of cholesterol with alternate oxidants include CO from Nocardia, Streptomyces, Schizophyllum, Pseudomanas, and Brevibacterium; experimental conditions under which it is able to rapidly catalyze the oxidation of cholesterol by benzoquinone or any of the other oxidants depend somewhat upon the source of the enzyme. For example, CO from Streptomyces rapidly catalyzes substrate oxidation with benzoquinone in phosphate buffer in the presence of any of a variety of the surfactants including octylgluconopyranoside and CHAPSO; the same reaction under WO 9/08713 PCr/us9/0 1057 identical conditions with CO from either Brevibacterium or Nocardia is slower. However, both Nocardia and Brevibacterium sources are active catalysts for cholesterol oxidation by alternate oxidants under other conditions.

The oxidant also plays a role in which the enzyme is most active. For example, cholesterol oxidase from Nocardia rapidly catalyzes substrate oxidation with benzoquinone in 0.2 molar TRIS buffer and 3 g/dL CHAPSO but is slower with ferricyanide under identical conditions; the Brevibacterium source of the enzyme is relatively inactive with ferricyanide in TRIS buffer with a variety of surfactants but when benzoquinone is used as the oxidant the reaction is very fast. Alternatively, the Schizophyllum source of the enzyme CO rapidly catalyzes the oxidation of cholesterol in phosphate buffer with either ferricyanide or benzoquinone and with a variety of surfactants as activators.

As indicated, cholesterol oxidase will catalyze the oxidation of cholesterol by ferricyanide. Additional examples where CO catalyzes cholesterol oxidation by ferricyanide include a Nocardia source in TRIS buffer with a variety of surfactants including sodium deoxycholate, sodium taurodeoxycholate, CHAPS, Thesit, and CHAPSO.

Furthermore, CO from Nocardia will also catalyze substrate oxidation with ferricyanide in phosphate buffer with sodium dioctylsulfosuccinate, sodium deoxycholate, sodium taurodeoxycholate, and Triton X-100. The buffer WO 89/08713 PCT/US89/01057 26 concentration is from 0.1 to 0.4 molar. Surfactant concentration for maximum activity of the oxidase enzyme varies with each detergent. For example, with deoxycholate or taurodeoxycholate, the enzyme in 0.2 M TRIS is most active with detergent in the range from 20 to 90 millimolar.

However, enzyme catalytic activity is observed up to and through a 10% concentration. With octyl-gluconopyranoside, the maximum activity of the enzyme with the oxidant ferricyanide occurs at a detergent concentration of approximately however, the enzyme still maintains activity at higher and lower concentrations of the surfactant.

Both esterase and CO require a surfactant for high activity. Specific surfactants include sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate, CHAPS (3-(3-chlolamidopropyl)dimethylammonio-l-propanesulfonate), CHAPSO (3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-lpropanesulfonate), octyl-gluconopyranoside, octylthiogluconopyranoside, nonyl-gluconopyranside, dodecylgluconopyranoside, Triton X-100, Dioctyl sulfosuccinate, Thesit (Hydroxypolyethoxydodecane), and lecithin (phosphatidylcholine). Buffers acceptable for this reaction to occur with the enzyme include phosphate, TRIS, MOPS, MES, HEPES, Tricine, Bicine, ACES, CAPS, and TAPS. An alternate generallized reaction scheme for the measurement of cholesterol in serum and other biological fluids is given WO 89/08713 PCT/US89/01057 27 Scheme II Cholesterol Esters Cholesterol Fatty Acid 1 Cholesterol Ox, Cholestenone Red I 4 Red 1 Ox 2 Ox Red 2 Red 2 Ox 2 e- 6 where Ox, and Red 2 function as an electron mediator couple between the cholesterol and the electroactive couple Ox 2 /Red 2 In this case, Ox, and Red i need not be electroactive because they do not have to participate in the electrooxidation process (Reaction However, from both a thermodynamic and kinetic perspective, this couple with the assistance of the enzyme cholesterol oxidase must be able to accept electrons from cholesterol and relay them to the electroactive couple (Ox 2 /Red 2 Specific examples of this chemistry include Example 1 Reaction 1 above Cholesterol Benzoquinone Cholestenone Hydroquinone Hydroquinone 2Fericyanide Benzoquinone 2Ferrocyanide Ferrocyanide Ferricyanide le- Scheme II is beneficial when the rate of reaction of cholesterol with the electroactive oxidant as in Scheme I is so slow that it precludes its use in a practical sensor. As mentioned above, Scheme II is also beneficial when the WO 89108713 PCT/US89/01057 28 electron mediator itself (Oxl/Red) is either not electroactive or exhibits poor electrochemistry under conditions of the enzyme chemistry. It is under these conditions that Scheme II is particularly applicable. Other electron mediators (Oxl/Red,) between cholesterol and ferricyanide for use in Scheme II may be possible including phenazine ethosulfate, phenazine methosulfate, tetramethylbenzidine, derivatives of benzoquinone, naphthoquinone and naphthoquinone derivatives, anthraquinone and anthraquinone derivatives, catechol, phenylenediamine, tetramethylphenenediamine, and other derivatives of phenylenediamine.

Furthermore, while it is understood that the oxidized form of the electron relay accepts electrons from cholesterol, in the sensor either the oxidized or the reduced form of the mediator may be incorporated provided it reacts rapidly with both cholesterol and ferricyanide. If the reduced form is sufficiently stable and the oxidized form is not, then reductant, may be incorporated into the sensor in relatively small quantity (in comparison with the analyte to be determined) and still provide the electron relay. However, this causes a corresponding background signal that must be accounted for. The reductant, must also be isolated from ferricyanide in the sensor by incorporation into a separate reagent layer.

WO 39/O8713 PCT/US89/01057 29 Several formulations of the above chemistries encompassing both Schemes I and II have been prepared as dry films on membranes. These membranes are positioned in the sensor which can then be used for the determination of cholesterol. A preferred formulation of the reagents involving Scheme II consists of the following Cholesterol Esterase 400 Units/mL Cholesterol Oxidase from Streptomyces 200 Units/mL 0.05 molar Potassium Ferricyanide 0.5 molar Potassium Chloride 0.2 molar Phosphate, pH 6.9 3 g/dL CHAPSO 2 g/dL gelatin and 0.0001 molar hydroquinone (in the spreading or wicking layer).

The concentrations provided are that of the solutions which are coated onto porous supports, filter paper or membranes; these concentrations are reestablished when the membrane imbibes the serum or whole blood specimen. For cholesterol determinations larger pore sizes in the filter support are necessary than that used for glucose. This is because the cholesterol resides in the serum in large lipoproteins (chylomicrons, LDL, VLDL, an HDL) which must penetrate the various layers of the sensor until they reach the reagents.

The surfactants to a major extent break these natural micelles up into smaller micelles providing a greater total 4F V WO 89/08713 PCT/US89/01057 surface area on which the enzymes catalyze the reaction.

Due to the instability of benzoquinone a small quantity of hydroquinone, which is more stable by nature of its lower vapor pressure, is incorporated into the sensor to assist electron mediation between cholesterol and ferricyanide.

Upon introduction of the serum specimen into the sensor the hydroquinone is oxidized to benzoquinone; the benzoquinone is then free to pick up electrons from the substrate and cycle them to ferricyanide. Under these conditions the rate of the reaction of cholesterol with a small quantity of benzoquinone is more rapid than that with a large excess of ferricyanide.

An alternate and preferred formulation of reagents utilizing Scheme II that may be incorporated into the reagent layer of the sensor is: Cholesterol Oxidase from Streptomyces 200 Units/mL Lipase from Candida 500 Units/mL 3 g/dL CHAPSO 0.2 molar TRIS, pH 0.05 molar Potassium Ferricyanide molar Potassium Chloride 0.05 molar MgC12 2 g/dL gelatin and 0.001 molar hydroquinone (in the spreading layer).

The magnesium salt in this formulation increases stability of the esterase enzyme in the phosphate-free reagent layer; WO 89/08713 PCT/US89/01057 31 Lipase assists the break up of the lipoproteins. With these dry reagent layers incorporated into the sensor and using the evaluation methodology as described, the following results were obtained.

Serum Cholesterol. ma% Average Current, uA 91 19.3 182 27.2 309 38.5 These results demonstrate the quantitative response of the sensDr to serum cholesterol levels.

Alternate and preferred embodiment of the sensor utilizing Scheme I is provided by reagent compositions: Cholesterol Esterase 400 Units/mL Cholesterol Oxidase from Nocardia 200 Units/mL 1 g/dL Triton X-100 0.1 molar TRIS buffer, pH 8.6 0.2 molar Potassium Ferricyanide molar Potassium Chloride 0.02 molar MgC1 2 2 g/dL gelatin

OR

Cholesterol Esterase 200 Units/mL Cholesterol Oxidase from Streptomyces 200 Units/mL 0.06 molar Sodium deoxycholate 0.1 molar TRIS buffer, pH 8.6 0.2 molar Potassium Ferricyanide WO 89/08713 PCT/US89/01057 32 molar Potassium Chloride 2 g/dL gelatin Thus, while we have illustrated and described the preferred embodiment of my invention, it is to be understood that this invention is capable of variation and modification, and we therefore do not wish or intend to be limited to the precise terms set forth, but desire and intend to avail ourselves of such changes and alterations which may be made for adapting the invention of the present invention to various usages and conditions. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview, of the following claims. The terms and expressions which have been employed in the foregoing specifications are used therein as terms of description and not of limitation, and thus there is no intention, in the use of such terms and expressions, of excluding equivalents of the features shown and described or portions thereof, it being recognized that the scope of the invention is defined and limited only by the claims which follow.

Having thus described our invention and the maanner and process of making and using it in such full, clear, concise, and exact terms so as to enable any person skilled in the WO 89/08713 PCT/US89/01057 art to which it pertains, or to with which it is most nearly connected, to make and use the same.

Claims (29)

1. A method for measuring the amount of a selected compound in body fluids comprising: a) providing a measuring cell having at least a first and second electrode and said cell containing an oxidant and a buffer, b) placing a sample of fluid to be tested in said cell, c) reconstituting said oxidant and buffer with said sample fluid to generate a predetermined reaction, d) allowing said reaction to proceed substantially to completion, e) applying a potential across said electrodes and sample, and f) measuring the resultant Cottrell current to determine the concentration of said selected compound present in said sample.

2. The method of Claim 1, wherein the compound is selected from the group consisting of glucose, cholesterol, TSH, T4, hormones, antiarrhythmics, antiepileptics, and non- therapeutic drugs.

3. The method of Claim 1 or 2, wherein the oxidant is a material selected from the group consisting of benzoquinone, ferricyanide, ferricinium, cobalt (III) orthophenantroline, and cobalt (III) dipyridyl.

4. The method of any one of Claims 1 to 3 including providing as said first electrode a working electrode and as said second electrode a reference electrode.

5. The method of any one of Claims 1 to 4 including also providing in said cell an enzyme as a catalyst and said enzyme is an oxidoreductase. S 1I S I S

6. The method of any one of Claims 1 to 5 including selecting said buffer from the group consisting of phosphate, TRIS, MOPS, MES, HEPES, Tricine, Bicine, ACES, CAPS, and TAPS.

7. A method for measuring the amount of glucose in blood, comprising: a. providing a measuring cell having at least a first and second electrode and said cell containing an oxidant, a buffer and an enzyme, b. placing a blood sample to be tested in said cell, c. reconstituting said oxidant, buffer and enzyme with said blood sample to generate a predetermination reaction, d. immediately applying a potential across said electrodes and blood sample, and e. measuring the resultant Cottrell current when the reaction has proceeded to completion to determine the concentration of said glucose present in said blood sample.

8. The method of Claim 7 including selecting said oxidant from the group consisting of benzoquinone, ferricyanide, ferricinium, Cobalt (III) orthophenantroline, and Cobalt (III) dipyridyl.

9. The method of Claim 7 or 8 including providing as said first electrode a working electrode and as said second electrode a reference electrode.

10. The method of any one of Claims 7 to 9 including adding as said enzyme, glucose oxydase.

11. The method of Claim 7 where in step b) said placing of the blood sample to be tested in the cell generates a current and initiates a timing sequence, and wherein the reaction of step d) is allowed to proceed with an open circuit between said first and second electrodes. s l

12. A sample cell for determining the concentration of a selected compound in a sample aqueous fluid, comprising: a) a metallized first electrode which acts as a working electrode, S*. b) a metallized second electrode which acts as a reference electrode, said second electrode being operatively associated with said first electrode; and c) at least'one non-conducting layer member having an operning therethrough, said layer member being disposed in contact with at least one of said electrodes and said layer member being sealed against at least one of said first and second electrodes to form a known electrode area within said opening such that said opening forms a well to receive said sample aqueous fluid and to place said fluid in said known electrode area in contact with said first electrode and second electrode, whereby substantially the entire contents of said well is capable of being substantially simultaneously subjected to a predetermined reaction.

13. The cell of Claim 12 including a reagent layer positioned within said well created by said opening.

14. The cell of Claim 13 wherein said reagent layer contains an oxidant, a buffer, and a binding agent.

The cell of Claim 14 wherein said reagent layer is a layer of said oxidant, buffer and binding agent coated onto a porous matrix and said matrix is positioned within said cell.

16. The cell of Claim 14 wherein said reagent layer is a mixture of said oxidant, buffer, and binding agent deposited directly into said cell. S.'25

17. The cell of any one of Claims 14 to 16 wherein said oxidant is selected from the group consisting of .benzoquinone, ferricyanide, ferricinium, Cobalt (III) ortho- phenantroline, and Cobalt (III) dipyridyi.

18. The cell of any one of Claims 14 to 17 wherein S3' said buffer is selected from the group consisting of phosphate, TRIS, MOPS, MES, HEPES, Tricine, Bicine, ACES, CAPS and TAPS.

19. The cell of any one of Claims 14 to 18 wherein said reagent layer also includes an enzyme and said enzyme is an oxidoreductase.

The cell of any one of Claims 12 to 19 wherein said first electrode is composed of a non-conducting substrate coated with a metal selected from the group consisting of platinum, gold, palladium, silver and carbon.

21. The cell of any one of Claims 12 to 20 wherein said second electrode is composed of a non-conducting substate coated with a metal selected from the group consisting of platinum, gold, palladium, silver, carbon, silver/silver chloride and silver/silver oxide.

22. The cell of Claim 12 wherein a first non- conducting layer member has an opening therethrough and is positioned on said first electrode, and said first electrode is positioned on said second electrode, and said second electrode being positioned on a second non-conducting layer 'iember.

23. The cell of Claim 12 wherein said first layer member includes a plurality of notches therein exposing and defining an electrical contact area on said first electrode, and said first electrode has a notch therein to expose and define an electrical contact area on said second electrode.

24. The cell of Claim 22 wherein said first electrode is folded a plurality of times with corresponding openings in each fold creating a well in which to hold said sample fluid, said first electrode positioned with respect to said first non- conducting layer member such that the opening in said first *iY isubstrate is juxtaposed with said openings in said first electrode.

*25, The cell of Claim 12 wherein said first and second electrodes are coplanarly positioned on a single substrate.

26. The cell of Claim 12 wherein said second electrode 3a has a circular opening therein and said opening of said layer member is concentric with said openings of said second electrode, and said opening in said second electrode is of smaller diameter than said opening of said layer member whereby 4. /1 a circular functional electrode area is defined on said second electrode, and said first electrode is positioned beneath said second electrode such that said opening in said second electrode "exposes and defines -a functional electrode area on said first electrode.

27. An apparatus for measuring compounds in a sample aqueous fluid, comprising: a, a housing having an access opening therethrough, b. a sample cell receivable into said access opening of said housing, said sample cell being composed of a metallized first electrode which acts as a working electrode, a metallized second electrode which acts as a reference electrode, said second electrode being operatively associated with said first electrode, at least one non-conducting layer member having an opening therethrough, said layer member being disposed in contact with at least one of said electrodes and said layer member being sealed against at least one of said electrodes and said layer member being sealed against at least one of said first and second electrodes to form a known electrode.area within said opening such that said opening forms a well to receive said sample fluid and to place said fluid in said known electrode area in contact with said first electrode and said :second electrode, c. means for applying an electrical potential to said first electrode and said second electrode, d. means for creating an electrical circuit between said first electrode and said second electrode through said S' sample aqueous fluid, e. means for measuring Cottrell current through said sample aqueous fluid, and f. means for visually displaying results of said measurement. II t-T b o 39

28. The apparatus of Claim 27 wherein said means for measuring Cottrell current through said sample aqueous fluid includes a microprocessor.

29. The apparatus of Claim 27 or 28 also comprising means for initiating an electrical potential upon insertion of said sample aqueous fluid to detect the presence of said sample aqueous fluid, and said initiating means also having means for signaling said microprocessor to commence a reaction timing sequence when the presence of said sample aqueous fluid is detected and means for removing said potential during said reaction timing sequence. DATED this 4th day of October, 1993 LIFE-CHEK LABORATORIES, By its Patent Attorneys, E. F. WELLINGTON CO., S. Wellington) 9 i. i* A/RR/2252/9 i *l4-