Method and apparatus for amperometric diagnostic analysis.

Method and apparatus for measuring the concentration of a compound in a sample - Test method and apparatus for measuring the concentration of a compound in a sample

This invention relates to a method and apparatus for quantifying the presence of biologically important compounds, such as glucose, TSH,

T4, hormones such as HCG, cardiac strengthening glycosides such as digoxin, antiarrhythmic such as lidocaine, epi10 antipsychotic such as phenobarbital, antibiotic such as gentamicin, cholesterol, non-therapeutic drugs and the like, body fluids.

Although the present invention has a wide range of applications, in order to describe the invention, its application in the quantitative determination of glucose and cholesterol of two biologically important compounds will be particularly emphasized.

Diabetes, especially diabetes, is a metabolic disease characterized by underproducing insulin, which results in abnormal blood glucose levels. Although this disease affects only about four percent of the population • ««

In the US, it is the third most common cause of death after heart disease • *. * · And cancer. With the right treatment of the patient's blood sugar with daily insulin injections and careful eating, the prognosis for diabetes is excellent. However, • · · ·: ***: the patient's blood sugar level should be closely monitored either • · · f · *; clinical laboratory analysis or daily analyzes that the patient can perform using relatively simple non-technical methods.

• · · • · · • · · • · · • · ·

Currently, the current technique for monitoring blood glucose is based on visual or instrumental monitoring of color change by enzymatic means. '·. 35 reactions produce a dry reagent pad with a small strip of plastic. These colorimetric methods, which utilize a natural oxidant, especially oxygen, to convert glucose to gluconic acid, are based on reactions:

BD-glucose + O₂ + H₂O -> D-glucose acid + H₂O₂B₂O₂ + reagent - H₂O + color

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

cholesterol + H₂O + O₂ > cholestenone + H₂O₂

B₂O₂ + reagent> H₂O + color

In the prior art, diacid is the only direct oxidant used in the enzyme cholesterol oxidase to determine both free and total cholesterol. When using conventional test methods, oxygen should be mixed into the test solution during use from ambient air to provide sufficient reagent for a complete reaction with the cholesterol to be analyzed in the undiluted serum and whole blood sample.

In either case, the presence of the compound is determined by a quantitative determination of hydrogen peroxide, either colorimetrically or otherwise. Current detection methods may include direct measurement of hydrogen peroxide either spectroscopically or by electrochemical means, and indirect methods in which hydrogen peroxide is allowed to react with various dyes, in the presence of an enzyme peroxidase, to produce a color that is monitored.

Although relatively easy to use, these experiments require consistent user technique to obtain reproducible results. For example, these experiments require the removal of blood from the reagent pad at specified and critical intervals35. After this time, excess blood must be removed by washing and soaking, or by soaking alone, as the color measurement is taken from the top surface of the reagent pad.

Color development is read either immediately or after a specified period of time.

These steps depend on good and consistent 5 operating techniques that require strict timing monitoring. In addition, although good operating techniques are used, colorimetric methods, e.g., for the determination of glucose, have been shown to have poor accuracy and accuracy, especially in the hypoglycemic region. In addition, the calibration methods for devices used for quantitative measurement vary widely, which is not possible for the user to calibrate, while some have secondary standards.

Because of the shortcomings in the accuracy and standardization of various methods and devices currently used to test biologically important compounds in body fluids, a few physicians are reluctant to use such equipment to monitor levels and dosage. They are particularly reluctant to recommend such methods for use by patients themselves. Similarly, it is desirable to have a method and apparatus that allows not only the physician but the patient himself to test such compounds with high reliability.

This invention responds to the physician's concerns by providing enzymatic amperometric methods and an apparatus for monitoring compounds in whole blood, serum, and other body fluids. Enzymatic amperometry offers several advantages for controlling or eliminating user-dependent technology as well as providing a larger linear dynamic range. A system based on this type of method provides a solution to a physician’s concerns who are hesitant to recommend self-testing to their patients.

<img file="FI101021B_D0001.tif" />

Enzymatic amperometric methods have been applied to the laboratory-based measurement of numerous analytes such as glucose, blood urea nitrogen, and lactate. Traditionally, the electrodes in these systems comprise wires, cylinders or discs of base metal embedded in an insulating material. The manufacturing process results in individual characteristics for each electrode, making it necessary to calibrate each sensor. These electrodes are also too expensive to be disposable, which makes it unnecessarily necessary to monitor electrode maintenance for continuous reliable use. This maintenance can hardly be performed by properly untrained personnel (such as patients), therefore, in order to be successful, an enzyme amperometric method for self-testing (or non-traditional site testing) should be based on a disposable sensor that can be fabricated to allow reproducible output. from one probe to another, and at a price well below the price of traditional electrodes.

The present invention meets these requirements by providing miniature disposable electroanalytical sample cells for accurate microparticle sampling, automatic means for measuring the electrochemical reduction of a sample: and a method for measuring the cell of the present invention.

: 25 and to use the device.

• · · • · · · * *: Characteristics of the method and device according to the invention • · · / · ': appear from the claims below.

. . ·. The disposable cells of the present invention are preferably laminated layers of metallized plastic and non-conductive material. Metallized layers form working and reference electrodes with repeated areas • »♦

1. J usually determined by the lamination process. The gap between these: '·. The 35 layers are designed to form a sample area or cell for accurate measurement of the sample.

Placing the cell inside the device according to the present invention automatically starts the measurement period.

For a better understanding of the measurement process, a preferred embodiment of the invention is described which comprises a two-step reaction step utilizing a chemical oxidation step using an oxidant other than oxygen and an electrochemical reduction step suitable for measuring the amount of the first step reaction product. One advantage of utilizing an oxidant other than diacid to directly determine the analyte is that they can be pre-placed in the sensor in large excess over the analyte, thus ensuring that the oxidant is not a limiting reagent (there is normally too little oxidant initially present in the sensor solution for quantitative analyte conversion).

• · · t: • · · • · ·:::

• · · • · · • · · • · · • · ·

In the oxidation reaction, for example, a sample containing glucose is converted to gluconic acid and the reaction product of an oxidant. This chemical oxidation reaction has been found to proceed in the presence of an enzyme, glucose oxidase, which is particularly characteristic of the substrate BD-glucose, and catalyzes oxidations at one- and two-electron acceptors. However, it has been found that the oxidation process does not proceed from the formation of gluconic acid, thus making this reaction particularly suitable for the electrochemical measurement of glucose.

In a preferred embodiment, oxidations with single electron acceptors, using agents such as ferricyanide, ferricinium, cobalt (III) orthophenanthroline, and cobalt (III) dipyridyl, are preferred. Benzoquinone is a two-electron acceptor that also provides excellent electrooxidation properties for amperometric quantification.

<img file="FI101021B_D0002.tif" />

• ·

For example, the amperometric determination of glucose in accordance with this invention utilizes Cottrell current microcronoamperometry in which glucose and an oxidized electron acceptor produce gluconic acid and a reduced acceptor. This determination comprises a pre-chemical oxidation step catalyzed by the enzymatic mechanism of the by-product by-product, as will be apparent throughout this specification.

In this method of measuring quantity by measuring the diffusion-controlled current at a well-defined time (e.g. 20, 30 or 50 sec e.g.) after switching on the voltage, the formula for amperometry at the set voltage (E = constant) is applicable:

- 0.5 i = nFA (-Dt) 'C

COTTRELL METABOLITE when t&gt; 0 when t = 0 where i is 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 preset time at which the current is measured, and C is the concentration of the metabolite. The current measurements due to reactor reoxidation according to the method of this invention were found to be relative to the glucose concentration of the sample.

The method and apparatus of the present invention allow, in preferred embodiments, the direct measurement of glucose cholesterol and the like. In addition, the sample cell of the present invention allows for the testing of adjusted blood volumes without pre-measurement. The insertion of the sampling cell thus allows for the automatic operation and timing of the reaction, allowing the patient to self-test with very high accuracy and accuracy.

One of the many presently preferred embodiments of the invention for use in measuring BD-glucose is described in detail to better understand the nature and scope of the invention. In particular, the method and apparatus of this embodiment are designed for a diabetic patient to allow clinical self-monitoring of blood glucose levels. The sample cell according to the invention is used to control the sampling volume and the reaction medium and acts as an electrochemical sensor. In the embodiment described herein, benzoquinone is used as the electron acceptor. The basic binary chemical reaction utilized by the process of this invention is

BD-glucose + benzoquinone + H₂O -&gt; Gluconic acid + hydroquinone hydroquinone -&gt; benzoquinone + 2e '+ 2H +

The first reaction is an oxidation reaction that proceeds to completion in the presence of the enzyme glucose oxidase. The electrochemical oxidation takes place in the second part of the reaction and provides the means to determine the amount of hydroquinone produced in the oxidation reaction. This is true whether the catalytic oxidation is performed with two-electron acceptors or single-electron acceptors such as ferricyanide (with a redox pair) 'would be Fe (CN)₆_₃/ Fe (CN) ₆_₄), ferricinium, cobalt III tris jj · 25 ortho phenanthroline and cobalt (III) trisdipyridyl.

t ** ': Catalytic oxidation by glucose oxidase is very characteristic • · · /: ·. BD-glucose but non-selective for oxidant. It has now been found that oxidants have sufficient positive potentials to change substantially all »· ·

Z BD-glucose to gluconic acid. In addition, this system provides • · · * * *

·. means by which amounts as low as 1 mg (in the preferred embodiment) to 1000 mg of glucose can be measured from a deciliter of sample, such results as before. *. 35 min has not been obtained using other glucose self-testing systems • · · \.

<td></td><td>Detectors containing chemicals to perform the desired assay and constructed in accordance with this invention are used with a portable meter for self-testing systems. On the meter</td>

<td> 5</td><td>an indicator is switched on which switches on the meter and starts waiting for a sample. The meter detects the input of a sample from the sudden charge current that occurs when the electrodes and the overlying reagent layer are initially moistened with the sample fluid. When the sample is detected, the meter starts</td>

<td> 10</td><td>incubation step (the length of which depends on the chemicals) to allow the enzymatic reaction to proceed to completion. This period is on the order of 15 to 90 sec for glucose, with a preferred incubation time of 20 to 45 sec. After the incubation period, the device then sets the known potential</td>

<td> 15</td><td>between the trodes and measures the current at specific times during the attenuation according to the Cottrell equation. Current measurements can be performed in the range of 2-30 sec, followed by potential utilization, whereby the measurement times are preferably 10 to 20 sec. These current values ​​are used to calculate</td>

<td>20 I «1 4</td><td>analyte concentration, which is then displayed. The meter then waits for either the user to remove the probe or a predetermined time before closing itself.</td>

<td> « · 4 1 • · « • &gt; • 4 • 4 -.:: 25 • · · » • • · · • • · · ·</td><td>The present invention provides a measurement system that eliminates a number of critical user-dependent variables that adversely affect accuracy and</td>

<td>• · · t:</td><td>reliability and encompasses a larger dynamic range</td>

<td>• · · • »* a * · «· · •</td><td>than other self-testing systems.</td>

<td> . 30 • · · • · · ··· • · · • · « • · · • •</td><td>These and other advantages of the present invention will become apparent upon consideration of the following detailed description, which shows one presently preferred embodiment of glucose.</td>

<td> » · ·</td><td>and another for measuring cholesterol, which is</td>

<td> ' · ·</td><td>to be understood in connection with the accompanying drawing, in which the same</td>

<td>. 35 t · · • »· t 4 t · fc • ·</td><td>numbers refer to similar components where:</td>

Figure 1 is an exploded view of a portable testing apparatus according to the present invention;

Figure 2 is a plan view of a sampling cell according to the present invention;

Figure 3 is an exploded view of the sample cell shown in Figure 2;

Figure 4 is an exploded view of another embodiment of a sample cell according to the invention;

Figure 5 is a plan view of the cell shown in Figure 4;

Figure 6 is another embodiment of a sample cell;

Fig. 7 is a graph showing a function of current from glucose concentration;

Figure 8 is a graphical representation of the Cottrell current as a function of glucose concentration; and «« r • · · »» «· • · · • * ♦ ·» «·» ·

Figure 9 is a currently preferred block diagram of an electrical circuit for use in the apparatus shown in Figure 1.

Figure 10 is a preferred embodiment of an electrochemical cell.

V «« • · · «··

With particular reference to Figure 1, there is shown a portable electrochemical testing device 10 for use in patient self-testing, such as for blood glucose levels. The device 10 comprises a front and rear housing 11 and 12, a front panel 13 and a circuit board 15, respectively. While the trigger button 18 is arranged to start the analysis, it is preferable that the system starts operating when the sample cell 20 is placed in the window 19 of the device.

Referring to Figure 3, the sample cell 20 is a metallized plastic material with a specially sized opening 21 that defines a volumetric source 21 when the cell is installed to contain a reagent pad and blood to be analyzed. The cell 20 comprises first and second substrates, which may preferably be made of styrene or other substantially non-conductive plastic. Mounted on the second substrate 23 is a reference electrode 24. The reference electrode 24 may preferably be made e.g. by vapor depositing an electrode on a substrate made of a material such as polyimide capton. In a preferred embodiment, the reference electrode 24 is a silver-silver chloride electrode. This electrode can be manufactured by first depositing a silver layer silver electrode by either chemical or electrochemical means before the substrate is used to construct the cells. The silver chloride layer may even be formed in place on the silver electrode when the reagent layer contains certain oxidants such as ferricyanide and chloride, as can be seen from the following reactions:

Ag + Ox ---&gt; Ag⁺ + red

Ag⁺ + Cl- ---&gt; AgCl

Alternatively, the silver-silver chloride electrode can be prepared by depositing a layer of silver oxide (by reactive metallization) on a silver film. This silver oxide layer is then converted to silver chloride in situ during the experiment according to the reaction:

Ag₂O + H₂0 + 2C1 ----&gt; 2AgCl + 2 (OH) when the detector is moistened with the sample liquid and re-forms the chloride-containing reagent layer.

The reference electrode may also be of the type commonly known as a pseudo-reference electrode, which relies on a large excess of oxidant, to impart a known potential to the noble metal electrode. In a preferred embodiment, two electrodes of the same noble metal are used, however, the other is generally larger in area and is used as a reference electrode.

The large excess of oxidants and the large surface area of ​​the reference resist the change in the potential of the reference electrode.

The indicator of the working electrode 26 may be either a platinum, gold or palladium strip or a metallized plastic placed on the reference electrode 24 or alternatively the working electrode 26 and the reference electrode may be made as a unitary unit with the electrode 26 sandwiched between the material of the same level electrode 24.

Preferably, the sample cell 20 is fabricated by depositing or laminating the electrodes between the substrate to form a composite unit.

As shown in Figure 2, the first substrate 22 is slightly shorter to expose the end portion 27 of the electrodes 24 and 26, and to allow electrical contact with the test circuit included in the device. In this embodiment, after the sample is placed in the source 21, the cell 20 is inserted into the window 19 on the front panel to begin testing. In this embodiment, the reagent may be placed in the source 21, or, preferably, a pad of dry reagent is placed therein, and a sample (drop) of blood is placed in the source 21 containing the reagent.

Referring to Figures 4-6, alternative embodiments of the sample cell 20 are shown. Figure 4 shows a sample cell 120 having first and second substrates 22, 123. Aperture 121 is sized to contain a sample to be tested. The head 130 is designed to be inserted inside the device, and electrical contact with the respective electrodes through the cuts 131, 132 in the cell. Reference 101021 electrode 124 also includes a cut 133 to allow electrical contact with the working electrode 126.

In Fig. 6, the working electrode 226 is folded, thus forming an increased surface area around the opening 221, to achieve the increased sensitivity or specificity. In this case, the reference electrode 224 is located below the working electrode 226. The working electrode includes a cut 234, allowing electrical contact with the reference electrode 224 through its cut 232 in the substrate 222. The end 230 of the substrate 222 also includes a cut 232, allowing electrical contact with the working electrode 226.

The sample cell of the present invention is located in a window

19 through, to start the test method. After placement, the potential is coupled to the sample cell portion 27 between electrodes 24 and 26 to detect the presence of the sample. When the presence of the sample is detected, the potential is removed and the incubation period is started. Optionally, during this period, the vibrator device 31 may be activated to effect mixing of the reagents, to promote dissolution (usually an incubation period of 20 to 45 seconds is used to determine glucose, and vibration is not normally required). The electric potential is next set in the sample cell portion 27 at the electrodes 24 and 26, and the current through the sample is measured and displayed on the display 16.

To take full advantage of the device described above, the necessary chemicals for the self-test system are connected to a dry reagent layer housed in a disposable cell that provides a complete detector for the intended analyte. The disposable electrochemical cell is constructed by laminating metallized plastic and non-conductive materials to provide a well-defined electrode working area. The reagent layer is either coated directly on the cell or attached (coated) to a support matrix, such as filter paper, a membrane filter, a woven fabric, or a non-woven fabric, which is then placed in the cell. When a support matrix is ​​used, its pore size and void space can be adjusted to provide the desired accuracy and mechanical support. In general, membrane filters or non-woven fabrics provide the best materials to support the reagent layer. Pore ​​sizes of 0.45 to 50 micrometers and voids of 50 to 90% are suitable. The formation of the coating generally comprises binders such as gelatin, carrageenan, methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc., which act to delay the dissolution of the reagents until the reagent layer has absorbed most of the liquid from the sample. The concentration of the binder is generally in the order of 0.1 to 10%, preferably 1 to 4%.

The reagent layer absorbs a predetermined amount of the sample liquid, thus completely eliminating the need to measure the sample volume beforehand. In addition, by measuring the current rather than the reflected light, there is no need

Removes blood from the surface of the reagent layer before measurement, as in reflection spectroscopy systems. Although the liquid sample could be applied directly to the surface of the reagent layer, in order to facilitate the spread of blood over the entire surface of the reagent layer, the detector preferably includes a dispersion, application or core layer. This layer, which is usually a non-woven fabric or absorbent paper, is placed on top of the reagent layer, and acts to spread blood rapidly over the reagent layer. In some applications, this dispersion layer could comprise additional reagents.

For the determination of glucose, cells with the same level of structure and a reagent layer containing the following substances were constructed:

600 μ / ml

0.4M

0, IM

0.5M

2.0 g / dl glucose oxidase potassium ferricyanide phosphate buffer potassium chloride gelatin

This was prepared by coating the membrane filter with a solution of the above composition and air drying. The reagent layer was then cut into strips that just fit into the window opening of the cells, and these strips were placed on top of the electrodes that were visible in the windows. A core layer of non-woven rayon fabric was then placed on top of this reagent layer and held in place with tape.

To test the technique of this invention, a large number of samples were tested in aqueous solution at 25 °. The electrolyte consisted of phosphate buffer at pH 6.8, about 0.1 molar total phosphate, and 0.5 M potassium chloride reagent. The potentials are compared to a normal hydrogen electrode (NHE). In these experiments, it was found that any potential between about +0.8 and 1.2 V (compared to NHE) is suitable for determining the amount of hydroquinone when benzoquinone is used as an oxidant. The limiting currents are relative to hydroquinone concentrations in the range of 0.0001 M to 0.050 M.

Determination of glucose by Cottrell current (i_t) by microcronoamperometry in this method gives the reaction of hydroquinone to 30a benzoquinone. Cottrell currents attenuate over time according to the following formula:

i_t . t 1/2 = constant

The main difference between the two techniques involves the use of an appropriately adjusted potential after the glucose benzoquinone reaction is complete and the correlation of glucose concentration with Cottrell currents is measured at a specified time thereafter. The current-time readings are shown in Figure 8. The relationship between glucose concentration and Cottrell currents (read at time t = 30 sec after potential generation) is shown in Figure 7.

It should be noted that chronoamperometry of Cottrell metabolites requires double confirmation of enzymatic catalysis and controlled potential electrolysis. Residue 99.9+ of gluconic acid was obtained in the presence of glucose oxidase. In this connection, equivalent amounts of benzoquinone were reduced to hydroquinone, the amounts of which were suitably determined in stationary solutions, stationary, palladium thin film anodes or sample cells.

The results of these many experiments demonstrate the microcronoamperometric methodology of this invention and the practical self-monitoring of glucose in a diabetic.

In a presently preferred embodiment of the invention using ferrocyanide, numerous experiments were performed which showed certain improved possibilities of operation.

Referring to Figure 9, there is shown a schematic diagram of a preferred circuit 15 for use in the device 10. The circuit 15 includes a microprocessor and an LCD panel 16. The working and reference electrodes on the sample cell 20 contact the contacts W (working electrode) and R (reference electrode), respectively. The voltage reference 41 is connected to the battery 42 via an analog current30 switch 43. The current from the electrodes W and R is changed by a control resistor 44, and the voltage to the frequency converter 46 is electrically connected to the microprocessor. Other circuits known to those skilled in the art may well be utilized to achieve the advantages of this invention.

Referring to Figure 10, cell 400 is comprised of coplanar working and reference electrodes 426, 424 laminated between upper and lower non-conductive materials 422, 426. Lamination occurs to the adhesive layer 425. The upper material 422 includes a die-cut aperture 428 that, along the width of the working electrode material, defines the surface area of ​​the working electrode and forms (along with the non-described reagent layer on top) a cell sampling port. At one end of the cell 400 there is an open area 427 which is similar to the terminal 27.

The effectiveness of using the apparatus of this invention to provide a means for home testing by patients, such as diabetics (in a preferred embodiment), can be seen in the following table comparing the technique of this invention to four commercially available units. As can be seen, the present invention is simpler, and in this case, simplicity provides consistency in results.

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I this

COMPARISON OF GLU ASSEMBLY SYSTEMS

<td> 5</td><td>Step</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td>invention</td>

<td></td><td>start</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td></td><td>Calibrating</td><td>X</td><td>X</td><td></td><td></td><td></td>

<td></td><td>finger puncture</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td></td><td>blood placement</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td> 10</td><td>timing start</td><td>X</td><td>X</td><td>X</td><td></td><td></td>

<td></td><td>wipe</td><td>X</td><td>X</td><td>X</td><td></td><td></td>

<td></td><td>strip setting</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td>

<td></td><td>reading the results</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td></td><td>number of steps</td><td> 8</td><td> 8</td><td> 7</td><td> 5</td><td> 4</td>

<td> 15</td><td>per test observation-</td><td>RS *</td><td>RS</td><td>RS</td><td>RS</td><td>Polaro-</td>

<td></td><td>system</td><td></td><td></td><td></td><td></td><td>graphic</td>

<td></td><td>range (mg / dl)</td><td> 10-400</td><td> 10-400</td><td> 10-400</td><td> 10-400</td><td> 0-1000</td>

<td></td><td>CV **</td><td></td><td></td><td></td><td></td><td></td>

<td> 20</td><td>hypoglycemic</td><td> 15%</td><td> 15%</td><td></td><td></td><td> 5%</td>

<td></td><td>euglyseeminen</td><td> 10%</td><td> 10%</td><td></td><td></td><td> 3%</td>

<td></td><td>hyperglyseeminen</td><td> 5%</td><td> 5%</td><td></td><td></td><td> 2%</td>

<td></td><td>correlation</td><td> 0,921</td><td> 0,862</td><td></td><td></td><td> 0,95</td>

(* RS = reflectance spectroscopy) ** coefficient of variation

With particular reference to the determination of cholesterol, utilizing this invention, chemistry can be generally described as follows:

Scheme 1 Cholesterol esters + H₂O + CE ---&gt; Cholesterol + Fatty Acid (1) Cholesterol + OX + CO ---&gt; Cholestenone + Red (2)

Red ---&gt; 0x + e- (3) where the enzymes cholesterol esterase (CE) and cholesterol oxidase (CO) catalyze reactions 1 and 2, and CO allows i

electron transfer together with several electroactive pairs (Ox and red). Reaction 2 is novel in that electron acceptors other than diacid can be used to oxidize cholesterol in the presence of the enzyme cholesterol oxidase. Reaction 1 is well known to those skilled in the art and is necessary for the determination of total cholesterol (free cholesterol and cholesterol esters). Reaction 3 is an electrooxidation process to sense and quantify cholesterol.

When utilizing the alternative oxidants of this invention, specific reactions include:

A:

Reaction 1 above cholesterol + 2 ferricyanide -CO ---&gt; cholestenone + ferrocyanide ferrocyanide ---&gt; ferricyanide + leB:

Reaction 1 above cholesterol + benzoquinone -CO ---&gt; cholestenone + hydroquinone hydroquinone ---&gt; benzoquinone + 2H + + 2cholesterol oxidase (CO) from multiple sources catalyzes the electron transfer from cholesterol to various oxidants such as benzoquinone, and its derivatives such as methylbenzoquinone, ethylbenzoquinone, orthobenzoquinone (oxidized form of catechol), benzoquinone sulfonate and potassium ferricyanide. It is also assumed that the enzyme allows electron transfer with other alternative oxidants. As shown in Reaction 3, the reduced product can then be displayed amperometrically for the quantitative determination of cholesterol.

Sources of the enzyme that catalyzes the oxidation of cholesterol, along with alternative oxidants, include CO derived from Nocardia, streptomyzes, schizso101021 filly, pseudomanas, and brevibacterium; the experimental conditions under which the oxidation of cholesterol with benzoquinone or any other oxidants can be rapidly catalyzed depend somewhat on the source of the enzyme.

For example, CO obtained from streptomyzes rapidly catalyzes the oxidation of a substrate with benzoquinone in phosphate buffer in the presence of any of a number of surfactants, e.g. octyl gluconopyranoside and CHAPSO; the same reaction under identical conditions where CO is obtained from brevibacterium or nocardia is slower. However, both nocardia and brevibacteria sources are active catalysts for oxidizing cholesterol with alternative oxidants under other conditions.

The oxidant also plays its part, where the enzyme is the most active. For example, cholesterol oxidase from nocardia rapidly catalyzes the oxidation of a substrate with benzoquinone in 0.2 M TRIS buffer and 3 g / dl CHAPSO, but is slower under ferricyanide-identical conditions;

the brevibacterial source of the enzyme is relatively inactive when ferricyanide is present in TRIS buffer as well as various surfactants, but when benzoquinone is used as an oxidant, the reaction is very rapid. Alternatively, the schizophyll source of the enzyme CO rapidly catalyzes the oxidation of cholesterol in phosphate buffer in the presence of ferricyanide or benzoquinone, and with various surfactants as activators.

As shown, cholesterol oxidase catalyzes the oxidation of cholesterol with ferricyanide. Additional examples where CO catalyzes the oxidation of cholesterol with ferricyanide include the source of Nocardia in TRIS buffer as well as various surfactants such as sodium deoxycholate, sodium taurodeoxycholate, CHAPS, tesite, and CHAPSO. Additional CO obtained from nocardia also catalyzes the oxidation of the substrate with ferricyanide in phosphate buffer in the presence of sodium dioctyl sulfosuccinate, sodium deoxycolate, sodium taurodeoxycholate, and Triton X-100. The concentration of the buffer is 0.1 to 0.4 moles. The concentration of surfactant for maximum oxidase enzyme activity varies with each detergent. E.g. with deoxycholate or taurodeoxycholate, the enzyme in 0.2 M TRIS buffer is most active, with the amount of detergent ranging from 20 to 90 mM. However, the catalytic activity of the enzyme is observed below and through 10% concentration. For octyl gluconopyranoside, the maximum activity of the enzyme when the oxidant is ferricyanide is present at a detergent concentration of about 1.2%; however, the enzyme still retains its activity at higher and lower concentrations of surfactant.

Both esterase and CO require a surfactant for high activity. Specific surfactants include sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate, CHAPS, (3- (chloramidopropyl) dimethylammonio-1-propanesulfonate). CHAPSO (3- (3-chlorolamidopropyl) dimethylammonio-2-hydroxy-1-propanesulfonate), octylgluconopyranoside, octylthiogluconopyranoside, nonylglyconopyranoside, dodecylgluconopyranoside, dodecylgluconopyranoside, Triton X-100, dioctyl Buffers that are acceptable for this reaction to occur with the enzyme include phosphate, TRIS, MOPS, MES, HEPES, tricine, bicin, ACES, CAPS, and TAPS. An alternative general reaction scheme for measuring cholesterol in serum and other biological fluids is given below:

Scheme II Cholesterol Esters -CE&gt; Cholesterol + Fatty Acid 1 Cholesterol + Ox-1 -CO&gt; Cholestenone + Red Red-L + Ox₂---&gt; Οχ_χ + red ₂ red₂ ----&gt; Ox₂ + e101021 with Ox and red₂ act as an electron mediator for the cholesterol electroactive pair Ox₂/red₂ between. In this case Οχ_τ and red₁ they do not have to be electroactive because they do not have to participate in the electrooxidation process (reaction 6). However, from both a thermodynamic and kinetic perspective, this pair, enzyme cholesterol oxidase, must be able to receive electrons from cholesterol and transfer them to an electroactive pair (Ox₂/Red₂) .

Specific examples of this chemistry include:

Example 1

Reaction 1 above cholesterol + benzoquinone -CO ---&gt; cholestenone + hydroquinone hydroquinone + 2ferricyanide ----&gt; benzoquinone + 2ferrocyanide ferrocyanide ----&gt; ferricyanide + le20 Scheme II is useful when the reaction rate of cholesterol with an electroactive oxidant, as in Scheme I, is so slow that it prevents its use in a practical detector. As mentioned above, Scheme II is also useful when the electron mediator itself (Ox₁/Red₁) is either not electroactive or shows poor electrochemistry under enzyme chemistry conditions. It is in these circumstances that Scheme II is particularly applicable. Other electron mediators (Ox-1 / red!) Between cholesterol and ferricyanide, for use in Scheme II, may be possible, incl. phenazine ethosulfate, phenazine methosulfate, tetramethylbenzidine, benzoquinone derivatives, naphthoquinone and naphthoquinone derivatives, anthraquinone, and anthraquinone derivatives, catechol, phenylenediamine, tetramethylphenylenediamine and other phenylenediamine derivatives.

In addition, while it is understood that the oxidized form of electron transfer receives electrons from cholesterol, either the oxidized • t »··· of the mediator can be connected to the detector.

Λ · * t:

· »· • · V fc ♦ · • · · or a reduced form, provided that it reacts rapidly with both cholesterol and ferricyanide. If the reduced form is sufficiently stable and the oxidized form is not, then a relatively small amount of reducing agent can be attached to the detector (compared to the analyte to be determined) and still provide electron transfer. However, this causes a corresponding background signal that needs to be taken into account. The reducing agent should also be isolated from ferricyanide in the detector by incorporation into a separate reagent layer.

Several preparations of the chemical combinations described above have been prepared according to both Scheme I and Scheme II as dry films for membranes. These membranes are placed in a detector that can then be used to determine cholesterol. A preferred preparation of reagents comprising Scheme II includes the following

Cholesterol esterase 400 units / ml cholesterol oxidase from streptomyces 200 units / ml 0.05 mol potassium ferricyanide 0.5 mol potassium chloride 0.2 mol phosphate, pH 6.9 3 g / dl CHAPSO g / dl gelatin and 0.0001 mol hydroquinone (application or heart floor).

The concentrations given are from solutions coated on porous supports, filter paper or membrane; these concentrations are regenerated when the membrane absorbs serum or whole blood. For cholesterol determinations, larger pore sizes of the filter support are necessary than those used for glucose. This is because cholesterol is present in the serum as a major lipoprotein (chylomicrons, LDL,

VLDL and HDL), which should penetrate the different layers of the detector until they reach the reagents. Surfactants largely break these natural micelles into smaller micelles, which provide a larger total surface area on which the enzymes catalyze the reaction. Due to the instability of benzoquinone, a small amount of hydroquinone, which is more stable in nature with a lower vapor pressure, is attached to the detector to aid in electron transfer between cholesterol and ferricyanide. Upon introduction of a particular serum into the detector, hydroquinone is oxidized to benzoquinone; the benzoquinone is then free to pick up electrons from the substrate and recycle them into ferric cyanide. Under these conditions, the reaction rate of cholesterol with a small amount of benzoquinone is faster than with a large excess of ferricyanide.

An alternative and preferred reagent preparation, utilizing Scheme II that can be incorporated into the reagent layer of the detector, is:

Cholesterol oxidase from streptomyzes, 200 units / 1 lipase candidiasis 500 units / ml

3 g / dl CHAPSO

0.2 molar TRIS, pH 7.5 0.05 molar potassium ferricyanide 0.5 molar potassium chloride 0.05 molar MgCl 2

2 g / dl gelatin and 0.001 mol hydroquinone (in the application layer). The magnesium salt in this formation increases the stability of the esterase enzyme in the phosphate-free reagent layer; lipases help break down lipoproteins. The following results were obtained with these dry reagent layers attached to the detector and using the described evaluation method.

182

309

Mean serum cholesterol mg%. current μΑ 15.3 27.2 38.5

These results indicate a quantitative response of the detector to serum cholesterol levels.

An alternative and preferred embodiment of the detector utilizing Scheme I is obtained with reagent combinations:

Cholesterol erastase 400 units / ml cholesterol oxidase from nocardia 200 units / ml g / dl Triton X-100

0.1 molar TRIS buffer, pH 8.6

0.2 moles of potassium ferricyanide 0.5 moles of potassium chloride 0.02 moles of MgCl₂ g / dl of gelatin

OR cholesterol esterase 200 units / ml cholesterol oxidase from streptomyces 200 units / ml 0.06 mol sodium deoxycholate

0.1 M TRIS Buffer

0.2 moles of potassium ferricyanide

0.5 mol of potassium chloride 2 g / dl of gelatin

Although preferred embodiments of the invention have been described, it is to be understood that the present invention may vary and change, and is not limited to the precise terms set forth, but also includes such changes and variations that may be made to adapt the present invention to various uses and conditions. The terms and expressions used in the foregoing specifications are used herein to be illustrative and not restrictive, and thus the use of such terms and expressions is not intended to exclude other equivalents that fall within the scope of the claims set forth below.