JPH01500054A - biochemical detector - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] biochemical analyte The present invention relates to a device used for the measurement of biochemical analytes in liquid samples.

Regular monitoring of various chemical analytes in body fluids can be used to detect certain diseases. For example, in the case of diabetes, glucose ml in the fluid Several methods have already been proposed for measuring . One of those ways is due to the change in the fluorescence spectrum caused by the enzymatic reaction of glucose. be done. In other methods, the oxygen electrode is coated with glucose oxidase, and the coating layer The decrease in the W element due to the enzymatic reaction between oxygen and glucose within the tube is measured using electrodes.

However, in both cases, it is relatively difficult to operate and requires special equipment with high silver content. , it is not possible to perform the interfoot movement of L. or Guru T1-su under limited circumstances.

An object of the present invention is to eliminate or alleviate some of the above-mentioned disadvantages. Ru.

The present invention provides a predetermined method for releasing at least one of prone and hydrogen peroxide. Used to detect analytes in liquid samples that are reactive under enzymatic reaction conditions The present invention provides an apparatus for conjugating a conjugated heptanomerconite. is proton permeable and the analyte produces hydrogen peroxide under enzymatic reaction conditions. is hydrogen peroxide permeable, and the conductivity changes depending on the amount of protons or hydrogen peroxide. A conductive polymer element is connected to the conductive polymer element by joining means provided at intervals on the polymer element. to directly or indirectly detect changes in electrical resistance within the polymer element at step electrical circuit and under predetermined enzymatic reaction conditions with at least one enzyme. of an analyte-permeable, non-conductive enzyme support element containing any necessary auxiliary factors; A first surface is in contact with said conductive polymer element and a second surface is in contact with said liquid. Constructed in direct or indirect contact with the sample.

The device preferably includes a filter member selectively permeable to the analyte; The filter member is configured to filter at least two different species in a liquid sample used in the device. Enzyme-supporting elements are used to screen components, preferably all foreign components. on the second surface of the child.

When generating and detecting protons with the device of the present invention, the sensitivity of the device can be increased. In order to will be reduced. Reduction can be carried out by any suitable means, preferably dilute sodium hydroxide. This is done by immersing it in an alkaline aqueous solution such as aluminum.

According to the device of the present invention, the amount of the analyte present is observed as a change in conductivity; The conductivity of the conductivity can be measured using easily available and/or By connecting polymer elements to various relatively inexpensive devices using the above-mentioned bonding means, can be measured.

It should be noted here that, unlike previous detection systems, the device of the present invention Generally, no special external operating conditions are required.

The devices of the present invention can be constructed in a variety of shapes and sizes, but generally are unilateral or is a multi-layer membrane device made of thin-film polymer elements coated on both sides with enzyme-supporting elements. It's in the shape. If only one side is coated with the enzyme support element, the other side is coated with the polymer The element is sealed with an impermeable coating to prevent unwanted effects. filter part If provided, it is generally in the form of a coating over the enzyme support element.

The bonding means of the present invention are typically made of highly conductive metal electrodes, such as platinum, silver, gold or copper. It is in the form of a metal electrode that extends into the polymer element. These electrodes are poly It is suitable for use as a support for polymer elements, in which case the polymer elements are It becomes a film-like coating that covers part of the electrode.

Various conductive polymers containing conjugated monomer units can be used as polymer elements of the present invention. Available for children. Suitable polymers generally range from 10-4 to 102SC11 It has a conductivity of . Such polymers include polypyrrole and aniline and pyrrole. Includes polymers based on lysine or other quaternizable conjugated monomer units be done. In the development of devices that use enzyme systems that generate protons, polymers are A monomer that has a nitrogen atom that can be quaternized and can obtain protons, as shown in the example below. – Must contain units.

The polymers of the present invention can be manufactured into any suitable shape with the desired electrical conductivity by well known methods. can be done. In general, the polymer is electrochemically stable and does not release ionic species into the liquid. A suitable amount of S in a polar solvent that can be retained and thus maintain the conductivity of the solution. Dissolve the conductivity-imparting dopant and the 7-mer and place the solution in an electrochemical cell. It can be produced by electrochemical polymerization. various suitable dopa Agents are well known in the art and include, for example, sulfonic acids, perchloric acids and Included are strong protic acids such as fluoroboric acid.

These dopants are characterized by their properties and their relationship to conductivity, stability and/or ease of handling. and can be used in polymers in various proportions. Usually repeating polymers The dopant is used in a molar ratio of 4 to 2, preferably 4 to 3, to the dopant units. used.

Generally, an electrochemical cell is used with two electrodes, usually platinum or gold. , a highly conductive metal such as silver is used. The voltage applied between the electrodes The range of oxidation-reduction potential is ±0.5■.

In fact, in order to form the polymer in the desired range on the electrode, for example, a standard is placed inside the cell. A reference electrode, such as a quasi-calomel electrode (SCE), is used to monitor and adjust the electrode voltage. inserted. If an SCE reference electrode is used, the electrode on which the polymer is formed is typically maintained at a voltage between 0.5V and 1.5V with respect to SCE.

The applied voltage, temperature, solvent, monomer and dopant concentration, polymerization rate, electrode structure, etc. These electrochemical polymerization conditions produce polymers of suitable shape, typically in the form of thin films. The ability to improve health on a large scale or on a small scale is worthy of recognition. suitable polymer ~, especially polypyrrole am, e.g. Cambridge, Massachusetts, United States It is commercially available from Volaloid Corporation. acetonitrile and for polymers soluble in organic solvents such as dimethylformamide, the polymer can be initially prepared in another form, for example in granules, which is then dissolved in a suitable solvent. The solution is then applied to a suitable substrate on which, for example, conductor tracks are formed. , a printed circuit board with electrical connections from the polymer to a suitable resistance measuring device It can be applied to etc. In this case, if desired, it is possible to It has the ability to incorporate dust resistance measurement equipment.

Electrochemical polymerization conditions can also be varied to tune the conductivity of the polymer. Ru. In general, polymers are used to determine the concentration of the analyte to be measured and the resistance measurement equipment used in the device. Depending on factors such as the sensitivity of the It is desirable that the material has electrical conductivity. Details of suitable methods for producing conductive polymers The details are, for example, F, H, AI-Arrayed. It is described in Material Forum 1986, 9.209-216.

The device of the present invention can be used to monitor the electrical conductivity of a polymer when it comes into contact with an analyte-containing solution. Various devices can be used to directly or indirectly measure resistance/IJ conductivity. example For example, a standard ohmmeter gauge that measures the resistance of a device and converts that resistance to an absolute value. Resistive bridge circuits can be used. Generally, in response to the change in conductivity that occurs, Measurements that can indicate analyte concentration directly on a display or other output data It is advantageous to use fixed equipment. Preparation of certain polymer films takes time It should be noted that it depends on In addition, the entire polymer element must reach an equilibrium state. The response speed of the device of the present invention with respect to varies considerably depending on the surface area relative to the volume of the polymer element in contact with the support element . Therefore, when using the device, on the one hand, the analyte-containing solution must be introduced into the device; In addition to measuring the initial conductivity before contact with the analyte-containing solution, the conductivity is measured at an appropriate time after contact with the analyte-containing solution. On the one hand, it is necessary to account for the relative change in conductivity, and on the other hand, the time course of conductivity after the initial exposure. It will be necessary to monitor the change. In such cases, use a known analyte solution as a standard. to correlate changes in conductivity to analyte concentration according to pre-established correlations. Using the device of the invention in conjunction with suitable data recording or processing equipment such as It is convenient.

Enzymatic reaction of the analyte itself or fermentation between the analyte and a derivative obtained by the enzymatic reaction. Various enzymatic reaction systems that generate protons or hydrogen peroxide through elementary reactions are known in the art. It is being In a suitable enzymatic reaction system for measuring alcohol analytes, the aid of NAD The reaction to oxidize alcohol, for example ethanol, to acetaldehyde using Alcohol dehydrogenase is used which catalyzes and releases protons. For measuring lactic acid In a suitable reaction system, the oxidation of lactic acid to pyruvate can be carried out with the help of NAD. mediated lactate dehydrogenase is used. In addition, when measuring glutamate, NAD aids. A group that catalyzes the reaction of oxidizing glutamic acid to 2-oxyglutamic acid. Lutamic acid dehydrogenase is used. Enzyme systems suitable for measuring glucose are Glucose catalyzes the reaction that oxidizes cose to gluconic acid and releases hydrogen peroxide It is an oxidative enzyme. Other reaction systems of this type use the enzyme cholesterol oxidase. It can also be used to measure cholesterol using Releases hydrogen. Certain amines and amino acids may be similarly treated with suitable amines or amino acids. Can be detected using amino acid oxidase. Furthermore, other subjects The binding between the analyte and the antibody is detectable using an enzyme that binds to the antibody. This one changes the enzyme from a catalytically inactive form to a catalytically active form. Reactions that release protons or hydrogen peroxide are catalyzed by appropriate reactants. Therefore , cytotoxic drugs such as methotrexate, peanuts, corn, small Food poisons such as aflatoxins produced by molds that grow on wheat, etc., dioxane, etc. Analytes such as industrial effluents and toxic products can also be measured using the device of the invention. can do. Preferably, protons or hydrogen peroxide are released by suitable reagents. For example, the enzyme that catalyzes the oxidation can be conjugated with a carboxyl group by a suitable protein denaturation reaction. using carbodiimide or sodium cyano resulting in the attachment of mino groups. Using reductive amination of aldehydes with borohydrides and amines, Alternatively, acylation of the 7-mino group with anhydride or other suitable methods known in the art may be used. Techniques can also be used to utilize systems conjugated to analyte samples. use In the ready device, the analyte-enzyme conjugate is bound with an antibody against the enzyme. Ru. Therefore, when the device comes into contact with a solution containing analyte molecules, these previously bound The enzyme-analyte conjugate is replaced by a proton- or hydrogen peroxide-releasing assay. Frees the enzyme-analyte conjugate for drug catalysis. -For example, glucose acid Glucose oxidase is a glucose oxidase that catalyzes the oxidation of glucose to gluconic acid. Can be used with glucose reagents that release hydrogen peroxide.

As mentioned above, enzyme support elements and polymers are isolated from foreign substances present in liquid samples. To protect the enzyme support element, the enzyme support element is preferably coated with a filter material. It will be done. If the liquid sample is a biochemical liquid, e.g. When the liquid sample is blood or urine, it is especially recommended to use a filter material. desirable. Filter components generally contain proteinaceous and/or macromolecular substances. Not qualitatively transparent. Suitable filter materials include polyacrylamide and polysaccharide. Includes highly cross-linked wettable polymer gels such as gels.

The filter material is secured by forming a layer on the outer surface of the conductive polymer film. If the filter material is a polymerizable gel, it is preferably made of a conductive material. can be polymerized on a synthetic polymer film to form a filter layer thereon. , and the filter layer is placed on the II conductive polymer film with an external mechanical support member. It can also be secured. Therefore, the device of the present invention is suitable for use with plastics such as polyvinyl chloride. The film is placed in an inert, non-permeable, electrically insulating housing, such as a plastic material. Conveniently, the outer surface of the filter member is fitted with a window in the central area. of the present invention In the device, an unknown solution that is believed to contain the analyte is placed in the second part of the enzyme support element. directly or through said filter member, and conductive between the electrical junctions. The change in electrical conductivity is measured with a resistance measuring device.

Therefore, from another point of view, the present invention can detect a predetermined analyte in a liquid sample. Provided is a method for detecting enzyme support elements of the device of the present invention. The second surface of the liquid sample is directly contacted or selectively transmitted through the analyte. a step of contacting through a filter member, and a joint portion of the polymer element of the device; and measuring a change in resistance between the steps.

Further preferred features and advantages of the invention will be explained by way of example and preferred embodiments with reference to the accompanying drawings. This will become clear from the following description. Figure 1 shows the ethanol content of liquid sample L. 1 is a longitudinal sectional view of a device according to the invention used for measuring quantities; FIG. Figure 1 is of glass. The present invention has a conductive polypyrrole layer 2 on the bottom 3 of an inert and insulating container 1. It shows a bright device. The ethanol permeable and substantially non-conductive enzyme support layer 4 is , located on and in contact with the polypyrrole layer 2 at the first surface 5, and a second The surface 6 is covered with a filter element layer 7.

Two spaced electrical connections in the form of platinum wires 9 extend through the bottom wall 10 of the container 1. extends to the polypyrrole layer 2, whose end 13 has a conductivity of 112 Ω and an ohmmeter Ω. connected to.

The enzyme support layer 4 is a highly cross-linked material such as polyacrylamide or polysaccharide gel. It consists of a wettable polymer gel in which the enzyme is immobilized together with the necessary auxiliary factors.

In the case of the device described here, a polymer having a molecular weight in the range of 1000 to 2000 is first used. A prepolymer of lyacrylamide was prepared and combined with ethanol dehydrogenase. Coenzyme NAD is added. This prepolymer then immobilizes the enzyme and coenzyme. Therefore, it can be crosslinked using a conventional method.

A data processing unit is used to convert the resistance measurements obtained from the ohmmeter Ω. P is connected to an ohmmeter Ω. This unit measures resistance continuously Record the difference in values and compare this with pre-established correlations in known solutions. is processed to provide an output signal corresponding to the ethanol concentration, which is It is then displayed on a suitable display device.

Example 1 - Preparation of ethanol sensor Polypyrrole film (2,5X 1.I CI, thickness 0.2-) was A suitable non-conductive resin is used to secure the metal conductor legs to the film at intervals. Adhesives, such as ``Araldite'' from DiverGeigy in Bazelle, Switzerland. It was attached to a glass slide support using a commercially available adhesive. child The resin is also used to insulate the legs between the slide and the film. . A suitable conductive lead for connecting to an ohmmeter is attached to the metal on the slide. Connected to the conductor leg. This film element is then placed in a container and activated. 1.50% polyacrylamide prepolymer (PAN) was added. 1.5g This PAN contains about 1000 μmol of active ester, but this one is white. Side's method (A, Po11ak et al., J. Chea+, Soc, 1980. 102, p. 6324), Hepes buffer, pH 7.5. 0.3M, magnesium chloride 30mM and NADo, 3sM (81). It was prepared by dissolving it in a buffer solution for 2 to 3 minutes. Dithiothreitol (0,1 st), then add triethylenetetramine (0.85 ml) to initiate a crosslinking reaction. started. After 2.5 minutes, add alcohol obtained from horse liver to the above buffer (11). A solution containing 40 mg of dehydrogenase was added. After 2 minutes, poly7- gelatinized. We started with polypyrrole film coated with enzyme-containing polyacrylamide gel. I took it out. The coated film was then dried in air for 1 hour at room temperature and treated with ammonium sulfate. Washed for 20 min in the above buffer solution containing 50 mM and finally washed with buffer solution. Dry the cleaned coating polymer overnight and attach it to the sensor. - prepared.

Example 2-I no Kan Yoni, Otogun ♂ Koji (7) All IFj Bolibi Using another sample of film, we replaced the alcohol dehydrogenated mulberry obtained from horse liver. using glucose oxidase and then nicotinamide nucleotide from the buffer. (Procedure described above for 1-tanol sensor preparation except that NAD> was omitted) A glucose sensor was prepared by repeating the steps.

Example 3 - Detection of ethanol The ethanol sensor of Example 1 was immersed in a J-thorium hydroxide aqueous solution (1M) for 5 minutes. (If you want to extend the soaking time to about 30 minutes, For example, the concentration can be as low as 0.5H).

The ethanol sensor is then equilibrated in deionized water, removed from the water, and Shake off the water on top. Next, the conductivity of the sensor was measured using a Solartron Schlumberger 1186 Electrochemical Interface (Hampshire, UK) 7-Mbala Sora (manufactured by Lutron Limited) was measured. Add ethanol (100 μm) to deionized water. The ethanol concentration was then adjusted to 10 mM, and the sensor was immersed in this solution. 2 minutes The operation of dipping and measuring conductivity was repeated.

Results After 10 minutes, the current increases by 0.9ak and after 30 minutes it reaches a high value of 2.0ak. did. Add ethanol sample (100μm) to the colander to bring the concentration of the solution to 20o+ The experiment was repeated as H.

After 10 minutes the current increased to 0.9111A and after 30 minutes it was 2.8ak. difference Add ethanol sample (100μ)) to make the concentration of the solution 30a+M. The experiment was repeated. The current increased to 1.2ak after 10 minutes and 2.8ak after 30 minutes. became.

The second step [initial deprotonation] is virtually optional, but it is important to consider the responsiveness and sensitivity of the sensor. There is a benefit in increasing the

The polypyrrole film of the sensor is a substantially deprotonated film. Can be used from the beginning, such films are overvoltage, preferably SCE 4.5 to 3. It can be prepared by electrochemical polymerization in OV.

Example 4 - Detection of glucose Example 3: The glucose sensor was immersed in deionized water to equilibrate, and the surface water was removed. The conductivity was measured in the same manner.

Glucose (100 mg) was added to prepare a solution with a concentration of 30 mH, and the sensor was added to this solution. - immersed in water. The operation of dipping for 2 minutes and measuring the conductivity was repeated.

Results After 4 minutes the increase in current is 30mA and after 20 minutes it reaches a maximum value of 40+aA. Ta.

A similar operation was performed using two slightly thinner films prepared separately under the same conditions. When done, the current increase for a solution containing 25a+H glucose is 37 after 20 minutes. +aA and 40-A.

Example 5 - Preparation of Cholesterol Sensor Alcohol Dehydrogenase and Nicotinase Cholesterol oxidase (1mg) instead of dooradenine dinucleotide Same as Example 1 except that Sigma Chemical Company Limited was used. The sensor was prepared according to the procedure. Gelation time was 2.5 minutes and crosslinking time was 2 minutes. It was.

Example 6 - Detection of cholesterol The cholesterol sensor was equilibrated as in Example 3 for ethanol detection. . 10% Br1j30 containing cholesterol water (available from ICI, UK) The response of this sensor to a non-ionic polyethylene oxide cleaning agent solution was Examined.

Results When immersed in a solution of cholesterol (100 ml), the conductivity was 3 mA in 15 minutes. and reached a maximum of 6 mA in 70 minutes.

international search report m, 1. +A--mls, pcτ/GB 117100192ANNEX T o I+HE rNTERNATIONAL 5EARCHRX? O just τON

Claims (1)

[Claims] 1. Contains conjugable monomer units, is broton permeable, and allows the analyte to react with enzymes. If hydrogen peroxide is produced under reactive conditions, hydrogen peroxide is permeable, and broton or A conductive polymer element (2) whose conductivity changes depending on the amount of hydrogen peroxide is placed in the port. The joining means (9) provided at intervals in the polymer element allow Connected to an electric circuit (12) for directly or indirectly detecting changes in electric resistance. , at least one enzyme and any necessary complements under predetermined enzymatic reaction conditions. The analyte-permeable, electrically non-conductive enzyme support element (4) containing co-factors is shown in its first table. on the surface (5) and in contact with said conductive polymer element (2) and on the second surface (6). The liquid sample is directly or indirectly contacted with the broton and oxidant. reactivity under predetermined enzymatic reaction conditions that release at least one of the hydrogen oxides. device used to detect an analyte in a liquid sample. 2. Screeching at least one other component in the liquid sample used in the device A filter member (7) that selectively transmits the specimen for the purpose of The device according to claim 1, provided on the surface (6) of the device. 3. The filter member comprises at least one of a protein substance and a macromolecular substance. 3. The device of claim 2 which is qualitatively impermeable. 4. The claimed filter element comprises a highly cross-linked wettable polymer gel. The device according to scope 2 or 3. 5. An enzymatic reaction system (4) that generates broton when it comes into contact with the specimen is used. Claim in which the polymer element (2) is used in reduced form to increase the sensitivity of the device. The device according to any one of ranges 1 to 4. 6. At least one side of the thin bell-shaped polymer element (2) is covered with an enzyme support element (4). 6. A device as claimed in any one of claims 1 to 5 in a coated thin film multilayer structure. 7. The bonding means (9) is in the form of a highly conductive metal electrode, which is connected to the polymer element (2). 7. A device as claimed in any one of claims 1 to 6 extending within. 8. Polymer element (2) is a polymer having a conductivity of 10-4 to 102 Scm-1 The apparatus according to any one of claims 1 to 7, comprising: 9. A specific analyte in which the enzyme support element (4) emits protons to catalyze the oxidation of the analyte. 9. The device according to any one of claims 1 to 8, comprising a body dehydrogenase. 10. The enzyme support element (4) releases hydrogen peroxide to catalyze the oxidation of the analyte. The device according to any one of claims 1 to 8, which contains an analyte oxidase. 12. The polymer element (2), the enzyme support element (4) and the filter member (7) ) is incorporated into the container (1) containing the test solution (L), and the test solution is incorporated into the enzyme support element. directly or indirectly with the second surface (6) of the child (4) or via the filter member (7). 11. A device according to any one of claims 1 to 10, which contacts the device. 12. At least one component that is converted from inactive to active by the reaction between the analyte and its antibody. The enzyme support element (4) contains two antibody-conjugated enzymes, and the enzyme support element (4) further comprises: (4) generates at least one of protons and hydrogen peroxide in the presence of the enzyme in an active state; 12. The device according to any one of claims 1 to 11, comprising a reagent for producing one. 13. Claims 1 to 12, comprising the electrical circuit connected to the joining means. Equipment described in any of the above. 14. The second surface ( 6) a filter that allows the liquid sample (L) to pass through directly or selectively through the subject; - indirect contact via the member (7) and the polymer element (1) of the device (1). 2) measuring the resistance change between the joining means (9) in the liquid sample; A method for detecting a predetermined analyte.