DD283683A5 - METHOD AND APPARATUS FOR AMPEROMETRIC DIAGNOSTIC ANALYSIS - Google Patents

Abstract

The invention relates to a method for measuring the amount of a selected compound in body fluids and a sample cell, characterized in that a) a liquid sample cell to be tested is accommodated in a sample cell having a first and a second electrode; b) the sample is mixed with an oxidizing agent and a buffer; c) applying a potential to the electrode and the sample; d) measuring the resulting current to determine the concentration of selected compound present in the sample. {measurement; Links; biologically important; Body fluids; amperometric; Sample cell novel}

Description

Field of application of the invention

The invention relates to a disposable electroanalytical cell and a method for the quantitative determination of the presence of biologically important conditions such as glucose; THS; T4; Hormones like ECQ; Oiglycis glycosides such as digoxion; antiarrhythmic agents such as Lldocaln; antiepleptic agents such as phenobarbital; Antibiotics such as gentamicin; Cholesterol; Nontherapeutic drugs and the like In body fluids.

Although the invention has a wide field of application, for the purposes of illustrating the invention, particular attention is paid to its application in the quantitative determination of the presence of two biologically important prerequisites - glucose and cholesterol.

Characteristic of the known state of the art

The invention is a partial extension of our previously filed application, United States Patent Application Serial No. 168,295, filed March 15, 1988

 In the case of glucose:

Diabetes, and especially diabetes mellitus, is a metabolic disease characterized by poor insulin production of the pancreas, causing abnormal blood glucose levels. Although this disease is present in only about 4% of the United States population, it is the third leading cause of death from heart disease and cancer. Proper maintenance of the patient's blood sugar level through daily injections of insulin and strict control of food intake makes the prognosis for diabetes excellent. However, the blood glucose level in a patient must be closely monitored either by clinical laboratory analysis or by a daily analysis that the patient can perform himself using relatively simple non-technical procedures. Currently, the common technology for monitoring blood glucose is based on the visual or instrumental determination of the color change caused by aromatic reactions on a dry reagent pad on a narrow strip of plastic. These colorimetric methods, which use the natural oxidant for glucose to gluconic acid, especially oxygen, are based on the following reactions:

BD-glucose + O ₂ + H ₂ O- * D-gluconic acid + H ₂ O ₂ H ₂ O ₂ + Reagent - »H ₂ O + color

In case of cholesterol:

Current technology for the determination of cholesterol is also based on similar methods. In the case of cholesterol, the methods currently used are based on the generalized reactions:

Cholesterol + H ₂ O + O ₂ - * Cholestenone + H ₂ O ₂ H ₂ O ₂ + Reagent -> H ₂ O + Color

In all of the techniques currently used, dioxygen is the only direct oxidizing agent used with the enzyme cholesterol oxidase for the determination of both free and total cholesterol. In carrying out conventional testing procedures, the oxygen must diffuse from the ambient air into the sensor solution during use in order for sufficient reagents to be available for complete reaction with the analyte cholesterol in undiluted serum and whole blood samples.

In both cases, the presence of the substance is determined either by colorimetric or other quantification of the hydrogen peroxide present. Current detection methods may involve the direct measurement of the hydrogen peroxide produced either by spectroscopic electrochemical measures and indirect methods in which the hydrogen peroxide is reacted with various dyes in the presence of the enzyme peroxidase to produce a color which is observed.

Although these tests are relatively simple to perform, they still require a consistent user technique to achieve reproducible results. For example, this test requires the collection of blood from a febril layer at specified and critical time intervals. After this time interval, excess blood must be removed by washing or dabbing, or by dabbing only, because the color measurement is made on the surface of the reagent agent layer. The color development is read either immediately or at a later time.

Liese steps require a good and consistent working technique as well as the strict adherence to the time specification. Moreover, even with the use of good working techniques, colorimetric methods for the determination of exemplary glucose have shown that they do not have good precision and accuracy, especially in the hyperglycemic range. In addition, the instruments used for the quantitative colorimetric measurement deviate very much in their calibration methods; Some do not provide user calibration while others provide secondary standards.

Because of the general lack of accuracy and standardization of the various methods and devices currently available for testing biologically important compounds in body fluids, some physicians are reluctant to provide such devices for the observations. Quantities and dosages to use. Above all, they have reservations about recommending such methods for use by the patients themselves. Therefore, it would be desirable if there was a method and device that would not only enable the physician to test such compounds with better reliability, but the patient could do it themselves.

Methods of enzymatic amperometry have been used on a laboratory basis for the measurement of a range of analytes, including glucose, blood urea nitrogen and lactate. Conventionally, the electrodes in these systems consist of non-metallic metal wires, cylinders or discs embedded in an insulating material. The manufacturing process results in individualistic properties of each electrode so that calibration of each sensor is required. These electrodes are also too expensive for the disposable application and require very careful maintenance of the electrodes for continuous and reliable use. This maintenance will be difficult to carry out properly by untrained personnel (such as patients), therefore a successful enzyme amperometry method intended for self-testing (or non-traditional random testing) must be based on a disposable sensor made in a manner which enables each individual sensor to provide reproducible results at costs well below those of conventional electrodes.

Object of the invention

The invention provides enzymatic-amperometric methods for the monitoring of compounds in whole blood, serum and other body fluids. Enzymatic amperometry offers several advantages for the control or elimination of user-dependent techniques, and it has a larger linear dynamic range.

Compared to already known self-test systems, precise and reliable analytical results are obtained with the methods according to the invention.

Explanation of the essence of the invention

It is an object of the present invention to develop suitable methods for the quantitative analysis of selected compounds in body fluids as self-test systomas.

According to the invention, there is provided a method for measuring the amount of a selected compound in body fluids using miniaturized disposable electroanalytical sampling cells for precise micro-aliquot sampling and an independent automatic device for measuring the electrochemical reduction of the sample. Enzymatic amperometry offers several advantages for the control or elimination of user-dependent techniques and has a larger linear dynamic range. A system based on this approach could eliminate the physician's concerns about recommending his patients to try solbs themselves. Enzymatic amperometry methods have been used on a laboratory basis for the measurement of a range of analytes, including glucose, blood urea nitrogen and lactate.

Conventionally, the electrodes in these systems are bulky. Metal wires, cylinders or discs embedded in an insulating material. The manufacturing process results in individualistic properties of each electrode, so calibration of each sensor is required. These electrodes are also too expensive for the disposable application and require very careful maintenance of the electrodes for continuous and reliable use. This maintenance is unlikely to be properly performed by untrained personnel (such as patients), therefore a successful enzyme amperometry method intended for self-testing (or non-traditional random testing) must be based on a disposable on sensor manufactured in a manner which enables it to provide each individual sensor with reproducible results at a cost far below that of conventional electrodes.

The invention addresses these needs by providing miniaturized disposable electroanalytical sampling cells for precise micro-aliquot sampling, an independent automatic apparatus for measuring the electrochemical reduction of the sample, and a method of using the cell and apparatus of the present invention , The disposable cells according to the invention are preferably composed of laminated layers of metallized plastic and non-conductive material. The metallized layers represent the working and reference electrodes whose surfaces have been reproducibly designed by the lamination process. In these layers is an opening that forms the area for receiving the sample or the cell for accurately measuring the sample. By inserting the cell in the device according to the invention, the measuring cycle is automatically set in motion.

For a better understanding of the method of measurement, a presently preferred embodiment of the invention is described, which comprises a two-stage reaction sequence in which a chemical oxidation step using other oxidants instead of oxygen and an electrochemical reduction step for the quantification of the reaction product of the first step are used. The use of another oxidizing agent instead of dioxygen for the direct determination of an analyte has the advantage that it can be pre-loaded into the sensor in a large excess with respect to the analyte and thereby ensures that the oxidant is not the limiting reagent ( in the case of dioxygen, initially insufficient oxidant is normally present in the sensor solution for quantitative conversion of the analyte).

In the oxidation reaction, a sample containing, for example, glucose is converted into gluconic acid and a reduction product of the oxidizing agent. It has been found that this chemical oxidation reaction proceeds to completion in the presence of an enzyme, glucose oxidase, which is highly specific to the substrate B-D-glucose and catalyzes oxidations with single and double electron acceptors. However, it has been found that the oxidation process does not proceed beyond the formation of gluconic acid, making this reaction particularly suitable for the electrochemical measurement of glucose.

In a presently preferred embodiment, electron acceptor oxidations using ferricyanide, ferrinolum, cobalt dld-orthophene, and cobaltdlD-diphyryl are preferred. Benzochlone is a two-electron acceptor that also provides excellent electro-oxidation properties for amperometric quantification.

According to the invention, for example, for the amperometric determination of glucose Cottrell current microchronoamperometry is applied, generate at the glucose plus an oxidized electron acceptor gluconic acid and a reduced acceptor. This determination involves a preliminary chemical oxidation step catalyzed by a double substrate enzymatic double-product mechanism, as is apparent from the specification. In this quantification method, the measurement of a controlled diffusion current occurs at a precisely prescribed time (eg 20, 30 or 50 seconds) after the moment of application of a potential according to the equation applicable to the amperometric equation at a controlled potential (E = constant) of:

'COTTRELL''f*' ⁷ '**''' Cme & Wit at t> 0

* Formula illegible (practice)

where i denotes the current, nF is the number of coulombs per mole, D is the diffusion coefficient of the reduced form of the reagent, t is the fixed cell at which the current is measured, and C is the concentration of the metabolite. The measurements of the current attributed to the reoxidation of the acceptors using the method of the invention proved to be proportional to the glucose charge in the sample.

With the method according to the invention and the device according to the invention, direct measurements of blood glucose, cholesterol and the like are possible in preferred exemplary embodiments. In addition, the sample cell according to the invention allows the testing of controlled blood volumes without prior measurement. Introducing the sampling cell into the device thus allows automatic functioning and timing of the reaction and allows the patient to self-test with a very high degree of precision and accuracy.

One of the many current preferred embodiments of the invention for use in measuring B-D glucose will be described in detail for a better understanding of the nature and scope of the invention. The method and apparatus according to this embodiment are intended primarily for the clinical self-monitoring of blood glucose levels by a diabetic patient. The sample cell according to the invention is used for the control of the sample volume and the reaction medium and acts as the electrochemical sensor. In this described embodiment, benzoquinone is used as the electron acceptor. The basic chemical binary reaction used in the process of the invention is as follows:

BD-glucose + benzoquinone + H ₂ O- ^ gluconic acid -I hydroquinone hydroquinone - »benzoquinone + 2 e + 2H ⁺

The first reaction is an oxidation reaction which proceeds to completion in the presence of the enzyme glucose oxidase. In the second part of the reaction, an electrochemical oxidation takes place, which provides the opportunity for the quantification of the amount of hydroquinone generated in the oxidation reaction. This is the case, whether the catalytic oxidation with two-electron or one electron acceptor as ferricyanide (wherein the redox couple Fe (CN) (T ³ / Fe (CN) ₈ "would ^8), ferricinium, CobaltdlD-trisorthophenantrolin and CobaltdlD- The catalytic oxidation by glucose oxidase is very specific for BD-glucose, but is nonselective with respect to the oxidizing agent It has now been discovered that the preferred oxidizing agents described above have sufficiently positive potentials to produce substantially all of the BD-glucose in . convert gluconic Furthermore d ^<tse system offers the possibility that with its help, only 1 mg of glucose can be per deciliter of sample was measured (in the preferred embodiment) to 1000 mg glucose ^- results that were previously in the application of other self-test systems could not be achieved for glucose.

The sensors incorporating the chemical means for carrying out the required determination and made in accordance with the invention are used with a portable meter for the self-test systems. In use, the sensor is inserted into the meter, which turns on the meter and initiates a wait for the sample to be used. The meter recognizes the sample insert by the sudden charge current flow that occurs when the electrodes and overlying reagent layer are initially wetted by the sample fluid. Once the sample insert is detected, the meter begins the reaction incubation step (the length of which depends on the chemical agent) to allow the enzymatic reaction to complete. This period is on the order of 15 to 90 seconds for glucose, with incubation times of 20 to 45 seconds being preferred. After the incubation period, the instrument then applies a known potential to the electrodes and measures the current at specific times during Cottrell current decay. Current measurements can be made in the range of 2 to 30 seconds after application of the potential, with measurement times of 10 to 20 seconds being preferred. These current values are then used to calculate the analyte concentration, which is then displayed. The meter will then wait for the user to either remove the sensor or for some time to pass before turning itself off. The present invention relates to a measurement system that eliminates various of the critical user-dependent variables that adversely affect accuracy and reliability and provides a greater dynamic range than other self-test systems.

These and other advantages of the invention will become apparent from the following detailed description of one currently available for measurement

Of glucose preferred Ausfüührungeform and a change for the measurement of cholesterol, which in conjunction with the accompanying drawings, in which like numbers represent identical components, is to be read. In the drawing represent:

Fig. 1: an exploded view of a portable test device according to the invention; 2 shows a top view of the sampling cell according to the invention; Fig. 3 is an exploded view of the sample cell shown in Fig. 2; 4 shows an exploded view of another embodiment of the sample cell according to the invention; Fig. 5 is a plan view of the cell shown in Fig. 4; Fig. 6: yet another embodiment of the sample cell; Fig. 7 is a graph showing the current as a function of glucose concentration; FIj. 8th; a plot of Cottrell current as a function of Glucosekonientration; FIG. 9 is a presently preferred block diagram of an electrical circuit for use with that shown in FIG

Device;

10 shows a preferred embodiment of the electrochemical cell.

In Fig. 1, an electrochemical test device is shown for use by the patient for self-testing, such as the blood glucose level. The device 10 consists of a front and rear housing 11 or 1 2, a front panel 13 and a circuit board 15. The front panel 13 includes graphic display panels 16, which provide information and instructions to the patient and a direct display of the test results. Although there is a start button 18 to initiate the analysis, it is preferred that the system begin operation when a sample cell 20 has been inserted into the window 19 of the device.

In Fig. 3, the sample cell 20 is a metallized plastic substrate having a specially sized opening forming a volumetric well 21 after assembly of the cell for receiving the reagent agent layer and the blood to be analyzed. The sample cell 20 includes a first substrate 22 and a second substrate 23, which may preferably be made of styrene or other non-conductive plastic material. From the second substrate 23 is the reference electrode 24. The reference electrode 24 may preferably be made such that the electrode is evaporated, for example, on a substrate made of a material such as polyimide-Kapton. In the preferred embodiment, the reference electrode 24 is a silver-silver chloride electrode. This electrode can be made by first applying a silver layer of silver chloride, either by chemical or electrochemical means, before using the substrate to make the cell. The silver chloride layer may also be formed in situ on a silver electrode if the reactant layer contains certain oxidants such as ferricyanide and chloride, as shown in the following reactions:

Ag + Ox -> Ag ⁺ + Red Ag ⁺ -Cl "- * AgCl

Alternatively, the Silbor silver chloride electrode may be prepared by applying a silver oxide layer (by reactive sputtering) to the silver film. This silver oxide layer is then converted to silver chloride in situ at the time of the test by the following reaction:

Ag ₂ O + H ₂ O + 2Cr -> 2AgCl + 2 (OH) "

when the sensor is wetted by the sample liquid and restores the reagent layer containing the chloride. The silver electrode has a silver chloride-containing layer. The reference electrode may also be of the type commonly known as a "pseudo" reference electrode based on the large excess of oxidizing agent to provide a known potential at a noble metal electrode. In a preferred embodiment, two electrodes of the same noble metal are used, but one generally has a larger effective surface area and is used as the reference electrode. Due to the large excess of the oxidized means and the larger effective surface of the reference electrode, a resistance to a shift of the potential of the reference electrode is created. The indicator or working electrode 26 may be present either as a strip of platinum, gold, palladium or metallized plastic on the reference electrode 24, or alternatively, the working electrode 26 and the reference electrode may be fabricated as a coplanar unit with the working electrode 26 between the material of the co-planar reference electrode 24 is embedded. The sample cell 20 is preferably fabricated by embedding or laminating the electrodes between the substrate to form a composite unit.

As can be seen in Fig. 2, the first substrate 22 has a slightly shorter length so that the end portion 27 of the reference electrode 24 and working electrode 26 is exposed to allow electrical contact to be made with the test circuit provided in the device. In this embodiment, after a sample has been placed in the well 21, the sample cell 20 is pushed into the window 19 of the faceplate to initiate the test. In this embodiment, a reactant may be added to the well 21 or, preferably, a layer of dry reagent is placed therein and a sample (drop) of the blood is added to the well 21 containing the reagent.

FIGS. 4 to 6 show alternative embodiments of the sample cell 20. In Fig. 4, the sample cell 120 is shown having first substrate 122 and second substrate 123. The reference electrode 124 and the working electrode 126 are laminated between the first substrate 122 and second substrate 123. The opening 121 is sized to receive the sample for testing. The end 130 is adapted to be inserted into the device and the electrical contact with the respective electrodes is made through the cutouts 131 and 132 on the cell. The reference electrode 124 also has the cutout 133 for making electrical contact with the working electrode 126. In Fig. 6, the working electrode 226 is folded, thereby providing a larger, more effective surface around the opening 221 to achieve higher sensitivity or specificity. In this case, the reference electrode 224 is below the working electrode 226. The working electrode has the cutout 234 for making electrical contact with the reference electrode 224 through the cutout 231 in the substrate 222. The end 230 of substrate 222 also has the cutout 232, through which electrical contact with the working electrode 226 can be created. The sample cell according to the invention is pushed through window 19 to initiate the test procedure. Once inserted, a potential is applied across the reference electrode 24 and working electrode 26 to the end portion 27 of the sample cell to detect the presence of the sample. Once the presence of the sample is determined, the potential is removed and the incubation period initiated. Optionally, during this period, a vibrator device for the movement of the reactants has been initiated to aid in its dissolution (a 20-45 second incubation period is normally provided for the determination of glucose, and generally no vibration is required). Next, an electric potential is applied to the end portion 27 of the sample cell to the reference electrode 24 and the working electrode 26, and the current flowing through the sample is measured and displayed on the panel 16. In order to be able to use the device according to the invention with full advantage, the necessary chemical means for the self-test systems are contained in a dry reagent layer which is located on the disposable cell and forms a complete sensor for the relevant analyte. The disposable electrochemical cell is assembled by laminating metallized plastic and non-conductive materials in such a manner as to provide a well-defined working electrode area. The reactant layer is either applied directly to the cell or preferably incorporated (or coated) into a support matrix such as filter paper, membrane filter, fabric, nonwoven fabric, which is then placed in the cell. When a carrier matrix is used, its pore size and pore volume can be selected to provide the required accuracy and mechanical support. In general, membrane filter and nonwoven fabric represent the best materials for the underlayer of the reactant layer. Pore sizes of 0.45 to 50μπι and intermediate pore volume of 50 to 90% are appropriate. The coating formulation generally contains a binder such as gelatin, carrageenan, methyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, etc., which serves to retard the dissolution of the reactants until the reactant layer has absorbed most of the liquid from the sample. The concentration of the binder is generally on the order of 0.1 to 10%, with 1 to 4% being preferred.

The reactant layer absorbs a certain amount of the sample liquid as it is applied to the surface of the layer, eliminating any need for prior measurement of the sample volume. In addition, due to the measurement of current flow, instead of the reflected light, it is not necessary to remove the blood from the surface of the reactant layer prior to measurement, as is the case with relfaxon spectroscopy systems. Although the liquid sample could be applied directly to the surface of the reactant layer, the sensor preferably includes a disperser spreading or wicking layer for better spreading of the blood over the entire surface. This layer, generally a nonwoven fabric or a suction paper, is located above the reactant layer and serves to rapidly distribute the blood on the reactant layer. In some applications, the disperser may not contain additional reactants.

For the determination of glucose, cells were prepared in a coplanar design whose reaction medium layer contains the following formulations:

glucose oxidase 600p / ml potassium 0.4 M phosphate buffer 0.1M potassium chloride 0,5 m gelatin 2.0 g / dl

This was prepared by coating a membrane filter with a solution of the above composition and air-drying. The reactant layer was then cut into strips 1 which just fitted into the window opening of the cells, and these strips were placed on the electrodes exposed in the windows. A wicking layer of rayon fiber nonwoven fabric was then placed over this layer of reactant and held in place with the help of an overlying baffle. In order to prove the applications of the technology according to the invention, a large number of examples in an aqueous solution at 25 ⁰ C was performed. The electrolyte consisted of a phosphate buffer with a pH of 6.8 and had approximately 0.1 moles of total phosphate and 0.5 M potassium chloride reagent. The potentials were compared with a normal hydrogen electrode (NHE). In these tests, it was found that any potential between approximately +0.8 and 1.2 volts (vs. NHE) is suitable for the quantification of hydroquinone when benzoquinone is used as the oxidant. The limiting currents are proportional to the hydroquinone concentrations in the range of 0.0001 M and 0.050 M. The determination of glucose by Cottrell-Strom (i,) - Mikrochronoamperometrie using the method according to the invention results from the reaction of hydroquinone to benzoquinone. The decrease of the Cottrell currents over time follows the equation:

i,., V2 = const.

The main difference between these two techniques is the application of the corresponding controlled potential after the glucose-benzoquinone reactivity has ended and the correlation of the glucose concentrations with the Ccttrell currents measured at a certain later time. The current-time representation is shown in FIG. The proportionality between the glucose concentrates and the Cottrell currents (recorded at t = 30 seconds after application of the potential) is shown in FIG.

It should be noted that Cottrell chronoamperometry of metabolites requires dual assays of enzymatic catalysis and controlled potential electrolysis. Gluconic acid anions of 99.9 +% were obtained in the presence of glucose oxidase. At the same time, equivalent amounts of benzoquinone were reduced to hydroquinone, which was easily quantitated on stationary palladium thin wells or sample cells in quiescent solutions. The results of these many tests demonstrate the microchronoamperometric methodology of the invention and its practical application to self-monitoring of glucose by diabetics. In a presently preferred application example of the invention, a series of tests were carried out using ferrocyanide, which showed some improved working possibilities.

FIG. 9 shows a schematic diagram of a preferred circuit 15 for use in device 10. The circuit board 15 comprises a microprocessor and the LCD Ta (el 16. The work and reference electrode on the sample cell 20 make contact at the contacts W (working electrode) and R (reference electrode), respectively The voltage source 41 is through the analog power switch 43 is connected to the battery 42. The current from the electrodes W and R is converted by the adjustable resistor 44, and the voltage of the frequency converter 46 is electrically connected to the microprocessor Other circuits known to the skilled technician may of course be used to obtain the advantages to be applied to the invention.

In Fig. 10, the cell 400 consists of the coplanar working electrode and the reference electrode 424 laminated between upper 422 and lower 426 nonconductive material. The laminate is on an adhesive layer 425. The upper material 422 includes a punched-out opening 428 which, along with the width of the working electrode material, is the working electrode surface and forms (with an overlapping reaction layer, not shown) the cell's top recess , At one end of the cell 400 is an open area 427 similar to the end portion 27.

The efficiency that results from using the device of the invention as a self-testing device for patients such as diabetics in the preferred embodiment at home can be seen from the following table, which compares the technology of the invention with four available devices becomes. As can be seen, the invention is simpler, and in this case simplicity means consistency in the results.

Comparisons of the glucose system

steps 1 2 3 4 invention Turn instrument on X X X X X Oak instrument X X finger pressure X X X X X Apply blood X X X X X Trigger the timing sequence X X X wipe off X X X Inserting the reading strip X X X X Read results X X X - X X Total steps per test 8th 8th 7 5 4 detection system RS » RS RS RS polarography Range (mg / dl) 10-400 40-400 25-450 40-400 0-1000 CV "hypoglycemic 15% 15% 5% euglycämisch 10% 10% 3% hyperglycämisch 5% 5% 2% correlation 0.921 0.862 0.95

* RS = reflection spectroscopy "coefficient of variation

In particular, when the determination of cholesterol is considered using the invention, the generalized chemical process can be represented as follows:

Cholesterol ester + H ₂ O + CE - »Cholesterol + fatty acid (1) Cholesterol + OX + CO - »Cholestenone + Red (2)

Red -> Ox + e "(3)

wherein the enzymes cholesterol esterase (CE) and cholesterol oxidase (CO) catalyze reactions 1 and 2, respectively, and CO facilitates electron transfer with a variety of electroactive pairs (Ox and Red). Reaction 2 is novel in that other electron acceptors can be used instead of dioxygen to oxidize cholesterol in the presence of the enzyme cholesterol oxidase. Reaction 1 is known to those skilled in the art, and it is for the purpose of determination

-8- 283 633

of total cholesterol (free cholesterol and cholesterol esters) required. Reaction 3 is the electrooxidation process for the study and quantification of cholesterol.

Using alternative oxidants according to the invention, the specific reactions become: A: Reaction 1 above Cholesterol + 2Ferr! Cyanide - CO-> cholestenone + 2ferrocyanide

Ferrocyanide * ferricyanide +1 e "B: Reaction 1 above

Cholesterol + benzoquinone - CO-> cholestenone + hydroquinone Hydroquinone - »Benzoquinone + 2H ⁺ + 2e ~

Cholesterol oxidase (CO) from a variety of sources will catalyze the electron transfer of cholesterol to a variety of oxidants, including benzoquinone, benzoquinone derivatives such as methylbenzoquinone, ethylbenzoquinone, chlorobenzoquinone, orthobenzoquinone (oxidized form of catechol), benzoquinone sulfonate and potassium ferricyanide. It is also envisaged that the enzyme will facilitate electron transfer with other alternative oxidants. As can be seen from reaction 3, the reduced product can then be followed by amperometry for the quantitative determination of cholesterol.

Sources of the enzyme that catalyzes the oxidation of cholesterol with alternative oxidants include CO of Nocardia, Streptomyces, Schizophyllum, Peeudomanas and Brevibacterium; the experimental conditions under which it is capable of rapidly catalyzing the oxidation of cholesterol by benzoquinone or any of the other oxidants depend somewhat on the source of the enzyme. For example, CO of Streptomyces rapidly catalyzes substrate oxidation with benzoquinone in phosphate buffer in the presence of one of numerous surfactants, such as octylgluconopyranoside and CHAPSO; the same reaction proceeds more slowly under identical conditions with CO of Brevibacterium or Nocardia. However, Nocardia and Brevibacterium sources are active catalysts for cholesterol oxidation by alternative oxidants under other conditions.

It also plays the role of the oxidizing agent in which the enzyme is most active. For example, does Nocardia cholesteroloyidase catalyze substrate oxidation with benzoquinone in 0.? Moles of TRIS buffer and 3g / dl CHAPSO fast, but slower with ferricyanide under identical conditions; the enzyme derived from the Brevibacterium source is relatively inactive with ferricyanide in TRIS buffer with a variety of surfactants, but when benzoquinone is used as the oxidizing agent, the reaction is very fast. Alternatively, the oxidation of cholesterol in phosphate buffer with either ferricyanide or benzoquinone and with a variety of surfactants as activators is very rapidly catalyzed by enzyme CO from the schizophyllum source.

As has been shown, cholesterol oxidase will catalyze the oxidation of cholesterol by ferricyanide. Other examples in which CO catalyzes cholesterol oxidation by ferricyanide include a Nocardia source in TRIS buffer with a variety of surfactants such as sodium deoxycholate, sodium taurodeoxycholate, CHAPS, thesit and CHAPSO. In addition, Nocardia's CO will also catalyze substrate oxidation with ferricyanide in phosphate buffer with sodium dioctyl sulfosuccirtate, sodium deoxycholate, sodium taurodeoxycholate, and Triton X-100. The buffer concentration is from 0.1 to 0.4 moles. The concentration of the surfactant for maximum activity of the oxidase enzyme varies with each detergent. For example, the enzyme with deoxycholate or taurodeoxycholate is most active in 0.2 M TRIS with detergent ranging from 20 to 90 millimoles. However, the catalytic enzyme activity can be observed up to and through a concentration of 10%. With octylgluconopyranoside, the maximum activity of the enzyme with the oxidant ferricyanide occurs at a detergent concentration of approximately 1.2%; however, the enzyme also retains 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 glycadeoxycholate, CHAPS of -chloramidopropyldimethylammonio-i-propanesulfonate), CHAPSO (3 (-chloramidopropyl) dimethylammonio-2-hydroxy-1-propanesulfonate, octylgluconopyranoside, octylthiogluconopyranoside, nonylglyconopyranoside, dodecylgluconopyranoside, Triton X-100, Dioctylsulfosuccinate, thesit (hydroxypolythoxydodecane), and lecithin (phosphatidylquinone) Buffers acceptable for the course of reaction with the enzyme include phosphate, TRIS, MOPS, MES, HEPES, tricine, bicine, ACES, CAPS, and TAPS An alternative generalized Reaction scheme for the measurement of cholesterol in serum and other biological fluids is given as follows:

Scheme II

Cholesterol ester - CE- »cholesterol + fatty acid (1) Cholesterol + Ox ₍ -CO- * Cholestenone + Redi (4) Red, + Ox ₂ - * Ox ₁ + Red ₂ (5)

Red ₂ - »Ox _} + e ~ (6)

where Ox and Red _{2 act} as an electron accelerator pair between cholesterol and the electroactive pair Ox ₂ / Red ₂ . In this case, Ox ₁ and Redt need not be electroactive because they do not have to be involved in the Elektrooxydationsprozeß (reaction 6). However, from a thermodynamic and kinetic point of view, this pair, with the aid of the enzyme cholesterol oxidase, must be able to pick up electrons from cholesterol and pass them on to the electroactive couple (Ox ₂ / Red ₂ ). Specific examples of these chemical processes are:

example 1

Reaction 1 above

, Cholesterol + benzochlone -CO- * cholestenone + hydroquinone

Hydroquinone + 2 ferricyanide »benzochlone + 2-ferrocyanide

Ferrocyanide - »Ferricyanld + 1 e ~

Scheme II is favorable when the reaction rate of cholesterol with the electroactive oxidizer of Scheme I is so low that it precludes its use in a practical sensor. As stated above, Scheme II is also favorable if the electron accelerator itself (Οχι / Red,) is either not electroactive or has poor electrochemistry under the conditions of enzyme chemistry. Under these conditions, Scheme II is particularly applicable. Other electron accelerators (Ox, / Red ₍ ) between cholesterol and ferricyanide can be used for Scheme II, such as phenazineethosulfate, phenazine methosulfate, tetramethylbenzidine, derivatives of benzoquinone, naphthoquinone and naphthoquinone derivatives, anthraquinone and anthraquinone derivatives, catechol, phenylenediamine, tetramethylphenendiamine and other derivatives of phenylenediamine.

In addition, while it is intended that the oxidized form of the electron linker will receive electrons from cholesterol, either the oxidized or the reduced form of the accelerator may be incorporated into the sensor, provided that a rapid reaction with cholesterol and ferricyanide occurs. If the reduced form is sufficiently stable and the oxidized form is not, then a reducing agent can be incorporated into the sensor in a relatively small amount (as compared to the analyte to be determined) and still form the electron linker. However, this creates a corresponding background signal that must be taken into account. The reducing agent must also be isolated from ferricyanide in the sensor by incorporating it into a separate reactant layer. Various formulations of the above chemical agents, which play a role in Schemes I and II, have been prepared as dry films on membranes. These membranes are housed in the sensor, which can then be used for the determination of cholesterol. A preferred formulation of the reagents used for Scheme II consists of the following:

Cholesterol esterase about 400 units / ml

Cholesterol oxidase from Streptomyces about 200 units / ml

0.05 mole of potassium ferricyanide

0.5 mole of potassium chloride

0.2 mole of phosphate, pH 6.9

3g / dl CHAPSQ

2g / dl gelatin and

0.0001 moles of hydroquinone (in the distribution or absorbent layer).

The intended concentrations are those of the solutions applied to the porous pads, filter paper of the membranes; these concentrations are restored when the membrane absorbs the serum or the whole blood sample. For the determination of cholesterol, larger pore sizes are required in the filter pad than are used for glucose. The reason for this is that the cholesterol in the serum is present in large lipoproteins (chylomicron, LDL, VLDL and HDL) which must penetrate the various layers of the sensor before they reach the reactants. The surfactants largely break this natural micelle into smaller micelles, providing a larger effective total area on which the enzymes catalyze the reaction. Due to the instability of benzoquinone, a small amount of hydroquinone, which is more stable due to its lower vapor pressure, is i. don sensor inserted to support electron acceleration between cholesterol and ferricyanide. After inserting the serum sample into the sensor, the hydroquinone is oxidized to benzoquinone; the benzoquinone is then free to pick up electrons from the substrate and transfer them to ferricyanide. Under these conditions, the reaction rate of cholesterol with a small amount of benzoquinone is higher than that of a large excess of ferricyanide.

An alternative and preferred formulation of Scheme II reactants that can be incorporated into the reactant layer of the sensor is as follows:

Cholesterol oxidase from Streptomyces about 200 units / ml

Candida lipase about 500 units / ml

3g / dl CHAPSO

0.2 mol of TRIS, pH 7.5

0.05 mole of potassium ferricyanide

0.5 mole of potassium chloride

0.05 mol of MgCl ₂

2g / dl gelatin and

0.001 mol of hydroquinone (in the distribution layer).

The magnesium salt in this formulation increases the resistance of the esterase enzyme in the phosphate-free reactant layer; Lipase supports the breaking up of lipoproteins. With these dry reagent layers sandwiched into the sensor and using the described evaluation methodology, the following results were obtained:

-10- 283 683 Serum Cholesterol, mg% Average Current, μΑ

91 19.3

182 27.2

309 38.6

These results show the quantitative response of the sensor to serum cholesterol levels. An alternative and preferred embodiment of the sensor in Scheme I is provided by the following roughening agent compositions:

Cholesterol esterase about 400 units / ml Nocardia cholesterol oxidase about 200 units / ml

1 g / dl Triton X-100 0.1 mmol TRIS buffer, / jH8.6 0.2 mol Callum ferrocyanide 0.5 mol Potassium chloride 0.02 mol / lIgCl ₂

2 g / dl gelatin or

Cholesterolesteraäe about 200 units / ml Cholesterol oxidase from Streptomyces about 200 units / ml

0.06 mole of sodium deoxycholate

0,1MolTRIS buffer pH8,6

0.2 mol of potassium ferricyanide

0.5 mole of potassium chloride

2 g / dl gelatin.

While the preferred embodiment of the invention has been illustrated and described, it is intended that the invention be varied and modified, and that it is therefore not intended to limit the scope of the foregoing, it is desirable and intended changes and modifications that may be made to adapt the invention to various uses and conditions. It is therefore intended that such changes and modifications fall within the full scope of equivalents and, thus, within the scope of the following claims. The terms and expressions used in the above specification are used herein as terms of description rather than limitation, and in the application of such terms and expressions, it is not intended to eliminate equivalents of the features shown and described or portions thereof, it being understood in that the scope of the invention is defined and limited only by the appended claims. , It has been fully, clearly, concisely and accurately described the invention and the manner and process of making and its use so that those skilled in the art or people concerned may make and use the same.

Claims (9)

A method for measuring the amount of a selected compound in body fluids, characterized in that a) a liquid sample to be tested is placed in a sample cell having a first and a second electrode; b) the sample is mixed with an oxidizing agent and a buffer; c) applying a potential to the electrodes and the sample; d) measuring the resulting current to determine the concentration of selected compound present in the sample. 2. Method according to claim \, characterized in that the compound of the glucose, cholesterol, TSH, T4 _; Hormones, antiarrhythmic agents, antiepileptic Mittol and non-therapeutic drug group is selected. 3. The method according to claim 1, characterized in that the oxidizing agent from the benzoquinone, ferricyanide, ferricinium, cobalt (lll) -trosorthophenantroline and cobalt (lll) -tripyridyl comprising group is selected. 4. Method according to claim 1, characterized in that the electrodes of the sample cell represent a reference and a working electrode. 5. Sample cell, characterized by a) a first and a second non-conductive substrate, wherein in the first substrate is an opening; a metallized first electrode is accommodated on the second substrate; and a second electrode is mounted on the first electrode, the first electrode overlying a portion of the second electrode to form a laminate with the first and second electrodes therebetween, and the opening exposes the electrode to form a sample well;
or b) first and second non-conductive substrates and first and second metallized electrodes, wherein the first electrode is disposed on the second substrate and the second electrode including a non-conductive substrate is sandwiched between the first and second substrates; an opening through the first substrate and the second electrode, wherein the opening forms a sample well. or c) first and second non-conductive substrates and a working and a reference electrode, the reference electrode having a metallized layer on the second substrate and the working electrode having a metallized layer on a nonconductive pad laminated between the first and second substrates; wherein the working electrode has a folded portion with an opening therethrough that is a sample well, and an opening through the first substrate is aligned with the opening in the working electrode. 6. sample cell according to claim 5a), characterized in that the first electrode is a reference electrode and the second electrode is a working electrode. 7. sample cell according to claim 5 b), characterized in that the first electrode is a working electrode and the second electrode is a reference electrode, and that the opening in the second electrode is smaller than the opening of the first substrate. 8. Sample cell according to claim 5 b), characterized in that the first electrode is a reference electrode and the second electrode is a working electrode. The sample cell of claim 5c), characterized in that the first substrate has a pair of recesses exposing and forming a contact surface on the working electrode and a contact surface on the reference electrode, and a recess in the working electrode lies under the recess in the first substrate to form the contact surface of the reference electrode. For this 6 pages drawings