JPS61500706A - Nucleic acid measurement - 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] Nucleic acid measurement The present invention relates to the measurement of nucleic acids, said measurement being concerned with the detection and characterization of the presence of nucleic acids. Both include specific identification of fixed nucleic acid molecules.

The presence of nucleic acids in samples can be determined in many areas of biochemical research and commerce; Furthermore, there is a special need to be able to measure the presence of specific sequences in said nucleic acids. exist.

A hybrid between two single-stranded DNA or RNA molecules with complementary sequences. Utilizing lid formation has been one approach to this problem. Kaka D Molecules that can bind to sequences of NA or RNA are known as DNA probes. It is being

Known DNA (RNA) probe technology uses DNA (RNA) polymers to share similarities in that they are not easy to detect by their biochemical activity. ing. Therefore, the polymer can be labeled with a signal-generating chemical or biochemical species. The following prior art methods are currently known: Ru: Conventional technology Avidin-biotin reaction: This technique uses the biotin of avidin, a glycoprotein in egg white. Depends on affinity for Chin. Biotin (vitamin H) is a DNA polymer can be covalently bonded to nucleotide residues of monomeric subunits.

The denatured subunit still undergoes the classical binding reaction between complementary strands of the duplex [INA]. can be incorporated into synthetic NA probes.

Currently, such proteins with short double-stranded regions formed after exposure to complementary sample DNA To detect the presence of probe, first transfer the unbound probe to the sample DNA/bound probe. It needs to be separated from the complex. This usually involves the sample DNA being immobilized on a substrate. This is done by performing a binding reaction under specific conditions and washing, but centrifugation can have the same effect. The bound probe is a fluorescently labeled antibody, but not an enzyme. Detection is performed by adding avidin conjugated to .

One problem with the method described above is that small oligonucleotide floes (20 nucleotide) is bound to biotin (biotinylated 5it e), and the amount of avidin that can be bound is limited. be. Adding a long [tail J] consisting of several thousand bases or less to the probe DNA Attempts have been made with some success to add tails. Only sections need to be marked. This method has the ability to analyze 10-13g of DNA ( resolution) or 105 copies of a single gene. be able to. Initially, the label in avidin was horseradish peroxidase (longevity). (alkaline phosphatase), but this method currently uses alkaline phosphatase It has been expanded to include

Unfortunately, it may be difficult to produce probe DNA that is bound to biotin. This method should not be used for new diagnostic plans because the labeled tails interfere with sensitivity. Generally difficult to establish.

Use of mesoieta The applicant's European patent application is for at least one of mesoieta and enzymes. is chemically linked to the sequence of a nucleic acid probe, thereby producing a single-stranded nucleus to be investigated. The specific binding of the probe sequence to the target sequence in acidic substances occurs in the presence of the enzyme substrate. The electricity of chemically bound species detected by the sensor electrode This affects chemical availability. Discloses a method by which the presence of a target sequence can be detected by.

As disclosed in the specification, the nucleic acid sequence is RNA.

For example, it can be metsenger RNA, but it is usually DNA. Ru.

Suitable methods for implementing this technique are: (a) providing a single-stranded nucleic acid substance to be investigated for a predetermined target sequence; (b) Select a probe substance that has a nuclear sequence complementary to the target sequence death, (c) The following method: (i) chemically bonding the probe to an enzyme, the enzyme's substrate and mesoieta; (ii) adding a probe bound to the above enzyme to a solution containing both The probe is chemically coupled to the mesoieter, and both the substrate and the enzyme for the substrate are bonded. Add the above-mentioned probe bound to the mesoieter to a solution containing the mesoieter (ii i) chemically combining said probe with a mesoieter/enzyme combination; Add the denatured probe as described above to a solution containing the substrate for the enzyme. Select one of the methods, (d') contacting said solution containing a chemically bound probe sequence with a sensor electrode; Then, the mesoieter generates a charge on the electrode from the substrate reaction catalyzed by the enzyme. move the (e) bringing the solution into contact with the -weighted substance, thereby causing a change in the amount of charge transfer; Enzyme, mesoieta: affect the availability of the combination This is an indication of the specific binding reaction between the probe and target.

The probe substance may be a naturally occurring DNA fragment or a synthetically produced substance. be able to.

Changes in the process order can be easily made. Also, the sensor electrode itself is a mesoieator. or enzymes, but usually probe sequences and targets in solution Preferably both sequences are present.

The mesoieter can be indirectly attached to the probe sequence by a linker group. It is possible to have a substance on the electrode that is reactive towards the linker group. I can do it. In this case the entire complex is present on the electrode and the target sequence is If present and bound to the probe sequence, a change occurs in the electrode current.

Mesoieta disclosed The use of ruthenium complexes on graphite electrodes has been proposed by Koval et al. and Anson (Analytical Chemistry, Vol. 50, No. No. 2, February 1978, page 223]. In this, the following Nori Ten Um complexes are discussed: Ru(NHs)s””” RLI (NH3) sP”+・ha Ru(NHs)sL"2+ However, L is pyridine, 4-aminomethylpyridine (AMP), or H-(4 -Picolinik)-benzamide (PBA).

RU(NH,),OH,”=”◆ Ru (NL) scl”ha metabolic disorder One purpose of conventionally developed DNA probe technology and enzyme detection/measurement technology is a variety of “genetic diseases” and inborn metabolic abnormalities that lead to inherited diseases. The goal was to detect the usual situation. Such diseases include: familial thyroid. tumor (iodotyrosine halogenase deficiency), maple syrup urine disease (alpha keto decarboxylase deficiency), xanthineuria (xanthine oxidase deficiency) deficiency) and methemoglobinemia (methemoglobin reductase deficiency). Lack A complete list of 3,500 conditions with negative genes is available at Mackusick. - “Mendelian Inheritance in Humans” ce in Man)”.

One particular disease of interest is heelocyte anemia, in which a specific chain of One of the normal globin gene codons corresponding to amino acids 5, 6 and 7 However, it is not normal CCT-GAG-GAG, but CCT-GTG-GAG. , glutamic acid is replaced by valine at position 6. synthetic oligonucleotide Otide has been manufactured to address sequence variations during prenatal diagnosis of diseases. It can act as a probe to differentiate between normal and abnormal genes. can be distinguished well. A similar approach has been taken using point displacement. , antitrypsin deficiency has been reported to occur.

However, as can be seen from the above explanation, the detailed metabolic measurement methods currently available are: have the disadvantage of requiring some or all of the following: Short-lived reagents (radioactive: air-sensitive or photosensitive), highly trained personnel (for autoradiography, εSR measurement or light level detection) expensive equipment (for commercial use).

Known measurements for antigen/antibody and DNA/complementary DNA binding reactions The methods are usually similar to those described above and have similar problems.

One aspect of the present invention provides a method for measuring nucleic acids, which method comprises: (a) (i) a nucleic acid sequence complementary to a given target sequence; and (ii) It is chemically bonded to this and can perform a specific binding reaction with the antiligand. providing a probe substance containing a first ligand capable of (b) the probe substance; (i) When the enzyme is catalytically active, it can transfer charge to the electrode surface. suitable mesoieta, enzyme, substrate systems, and (ii) chemically combined with any one of said mesoieta, enzyme or substrate; a second ligand capable of performing a competitive binding reaction with the antiligand; Beauty (iii) the antiligand The anti-rigger is brought into contact with an assay system containing in the specific binding reaction with the first (C) system and the target sequence. Contact the solution suspected of containing any of the target sequences present. The availability of the first ligand is increased by binding to the probe. It is characterized by influencing and thus changing the rate of charge transfer to the electrodes.

By using the method described above, a measurement method having an amplification step can be carried out to obtain a measurement method. can increase the analytical power of

Furthermore, the method of the invention is useful for short-lived radioactive measurements used in other types of measurement methods. No ingredients required.

The electrochemical behavior when combined with mezoieta compounds was determined by the present inventors. Enzyme/substrate pairs studied include: enzyme base Pyruvate oxidase pyruvate amino acid oxidase amino acid aldehyde oxidase aldehyde xanthine oxidase xanthine glucose oxidase glucose Glycolate oxidase Glycolate sarcosine oxidase Sarcosine Galactose oxidase Galactose diaphorase oxidase NADH Glutathione reductase NADPHPQQ enzyme Glucose dehydrogenase Glucose methanol dehydrogenase Methanol alcohol and other alcohols Methylamine dehydrogenase Enzyme that binds to methylamine cytochrome B Lactic acid oxidase lactate metal flavoprotein Carbon monoxide oxidoreductase - carbon oxide In the present invention, these enzymes - Although either pair of substrates can be used in combination with mesoieta, this technique There are some limitations to the measurement conditions that will be obvious to those of ordinary skill in the field. is considered to be imposed on Of these pairs, the behavior has been determined in detail; and an enzyme/substrate that provides a good and preferably linear response over the expected measurement range. It is clearly advantageous to use pairs.

Ferrocene (bisucyclopentangenyl iron and its derivatives) is a charge transfer It has advantages over other mesoietors used during enzyme/substrate reactions for this purpose.

Ferrocene (bis7s cyclopentagenyl iron: Pe CF2) and its derivatives The unique structure and properties of the body have generated a considerable amount of theoretical and experimental research. first Ferrocene, synthesized in 1951, is the best metallocene compound currently known. is also an early example.

Ferrocene has low solubility in aqueous solution and low extinction coefficient, resulting in a low spectrophotometric value. Although found to limit the value of the measurement method, ferrocene is I realized that I was missing the point.

Ferrocene has the following advantages: (a) A cyclopentadienyl ring that can be functionalized A wide range of redox potentials easily obtained by substitution; (b) Electrochemically reversible one-electron redox property; (c) pH-independent oxidation Reduction potential and slow autoxidation of reduced forms.

The inventors have identified such common types of ferrocene, i.e. rotations of one or both rings. Some of the derivatives of monomers or polymers substituted with found individual ferrocenes of: Ferrocene derivative Eo Solubility E l,1'-dimethyl-1001,0- Acetic acid 124 S 370 Hydroxyethyl-1613- Ferrocene 165 1. D 335 1.1'-bis(hydroxy 224 S 385 methyl)- Monocarboxylic acid 275 S 420 1.1'-dicarboxylic acid 285 S-chloro-3451,D- Methyl-trimethylamino-400S-3 indicates only soluble; I , D are insoluble and solubilized with detergent in 3% Tween solution. rgent 5olublize). Eo is standard sweet potato It is shown in ITlv with respect to the pole, and E is measured in Cl11-'M-'.

of the various ferrocenes shown in the table above in phosphate buffer at pH 7.0. The E° values span the potential range of E” = 100-400 mV vs. SCE. This trend is consistent with that expected based on the effects of substituents.

Generally, electron-donating groups stabilize the positive charge and promote oxidation more than electron-withdrawing groups. do.

The ferrocene derivatives that can be used in the method of the present invention are limited to the specific examples shown in the table above. For example, the present inventors should not think that ferroceneboronic acid (bor onicacid), polymeric ferrocene, borontetraferrocene and ethyl It was confirmed that amine ferrocene exhibits mesoieta properties. The ferrite shown in the table above Rocene derivatives are known to those of ordinary skill in the art. For example, iodine, nitrene or dia can be further modified by treatment with chloride precursors or mercuric chloride. can.

The applicant's European Patent Application No. 82305597 consists of electrically conductive materials and includes transfers electrons to the electrode when the enzyme is catalytically active on its outer surface A sensor electrode having a combination of a mesoieta compound and an enzyme is It is listed in the range.

The purpose of such an electrode is to be able to initiate the reaction catalyzed by the enzyme. detecting the presence of the species or two or more selected components, measuring the amount thereof, and/or or to monitor its levels.

Examples of electrode structures, mesoiators and uses are described in the above-mentioned patent applications.

In this specification, the mesoieter and and its derivatives.

This specification also includes the structure and structure of the electrode disclosed in the earlier application mentioned herein by reference. and materials such as gold, platinum, silver, carbon or one-dimensional d imens 1ona 1) Use of electrode material selected from the group consisting of conductors do.

Examples of mesoieta compounds mentioned above generally refer to mesoieta of the ferrocene group. Although limited, non-ferrocene mesoieters can also be used in the present invention. . Several types of compounds exhibit mesoietic action, i.e., transfer from enzymes to the electrode surface. It can act to transfer charge (in some cases to other enzymes).

The system studied contained the following mesoieta compounds: Carbon-boron compounds (including carborane) Viologen (N,N'-dialkyl or diaryl derivative of 4,4'-dipyridyl) conductor) Metal carbonyl (including chromium carbonyl) one-dimensional conductor (TCN phenazine dyes (including salts of Q) (phenazine methosulfate and phenazine (including ethosulfate) and metalloporphyrins (including cytochrome-C).

However, the preferred types of mesoieta are transition metal complexes, especially mesoieta-poor has at least one, preferably two, organic rings, and the rings or polycycles are conjugated by a metal atom in covalent electron contact with at least two double bonds and a polycyclic ring The reason for this is that such complexes have stability and rapid exhibits electron transfer reactions, has a wide redox potential range and is often oxygen sensitive This is because. Usually preferred mesoietors include iron, nickel or nickel as the transition metal. or ruthenium.

A preferred specific binding reaction in the method of the present invention is avidin (or streptavidin). gin) and biotin, but the present invention is limited to this specific binding reaction. and should not be interpreted as , but extends to pairs with the following specific binding partners: antigen and antibody hormones and receptors lectins and carbohydrates cofactors and enzymes, and Nucleic acids and complementary nucleic acids A preferred method for measuring mesoieta in solution is described in the applicant's above-mentioned patent application. Cyclic portas such as those described in This is done by using metric. Complete cyclic alteration It is not necessary to use a voltammogram in all cases; If the cell is stabilized at a particular potential and all current and/or It is sufficient to measure voltage and/or voltage (as exemplified later). ).

As stated above, those of ordinary skill in this technical field will be familiar with such technology. Therefore, I will not discuss it in detail here.

In a second aspect of the invention - the presence of a predetermined sequence in the heavyweight nucleic acid molecule and and/or a nucleic acid sequence probe for determining copy number; The nucleic acid sequence of the probe is: a) the mesoieta compound or the mesoieta compound is electrochemically bonded; and b) biothione. is connected to binding of the probe nucleic acid sequence to the target nucleic acid sequence characterized in that the electrochemical bond between the eta compound and the enzyme changes Ru.

In a third aspect of the present invention, a method for measuring nucleic acids is provided, and the method uses an enzyme and an electrode. and a substrate for the enzyme, and the mesoieta compound is combined with the enzyme. As the conversion of substrate to product progresses, charge is transferred from the enzyme to the electrode. the nucleic acid sequence of the probe to the enzyme or the mesoieta compound. The activity of said enzyme and said mesoieta compound, and therefore to reduce the amount of charge transferred to the electrode from the complementary target bound to the nucleic acid sequence of the probe. It is characterized by being changed depending on the presence or absence of a target nucleic acid sequence.

The properties, enzymes, methodologies and equipment of mesoieters described in relation to the first aspect of the invention. The general statements regarding location apply equally well to these second and third aspects. , these aspects need to be understood accordingly.

The basis of the present invention is the applicant's earlier patent application, in particular the applicant's European patent application No. 82. No. 305,597, the electrochemically active The flow of electrical current in response to the presence of matter or "mesoieters." flowing The magnitude of the current depends on the amount of (a) DNA probe/target) DNA complex produced. proportional or (b) DNA probe/target) Increases in proportion to the amount of DNA complex produced. or as a perturbation of the existing steady-state current that decreases. fluctuate.

In one preferred embodiment of the present invention, the electrode uses biotin-labeled cytochrome- Therefore, it shows reversible electron transfer. Biotin-labeled cytochrome - 〇 is mesoieta Acts as a compound, but also combines with mesoieta, adding to the general description above to reduce charge transfer to the electrodes. Biotin-labeled nucleic acid (DNA or RNA) is added to the reaction mixture, while biotin cytochrome- Competitive relationship between Gando mezoieta) complex and avidin (antiligand) Specific binding reaction, on the other hand, avidin (antiligand) complex and biotin labeling Competitive specific binding reaction with a nucleic acid (DNA or RNA) (second ligand) is started.

Since the avidin (or streptavidin) concentration is constant, avidin-bi, The concentration of free biotin cytochrome-0 species increases by increasing the concentration of f\n-nucleic acid complexes. This results in a corresponding increase in the rate of charge transfer from the enzyme to the electrode surface. There will be an increase in

The above examples of the invention were described with respect to a first ligand coupled to a mesoieter. However, the present invention also comprises a mesoieter bound to an enzyme or substrate. It is necessary to understand that this can be considered.

In another preferred embodiment of the invention, the electrode surface is catalyzed by an enzyme in the presence of a mesoieter. In a more preferred example, the mesoieter reacts with a nucleic acid probe. It is a covalently bonded cytochrome-〇. Electrochemical Availability of Mezoieta The property is a homologous bond between a nucleic acid probe and target DNA. It is affected by the degree of US binding.

In yet another preferred embodiment of the invention, the electrode comprises an enzyme complex in the presence of mesoieta. detect and measure reactions catalyzed by In special cases, enzyme complexes are It consists of a tin-peroxidase-avidin complex, which is further enriched with biotin or can bind to biotin-labeled nucleic acids. Steady state current is peroxide group Obtained when using quality. This current transfers the biotin-labeled nucleic acid into the measurement mixture (a s6ay　mlxture).

In each of the three examples above, the probe-containing species and mesoieta, enzyme and an electrochemical system consisting of a substrate. The charge transfer speed is perturbs by reducing the availability of probe-containing species. subordinate Therefore, when a sample containing a target nucleic acid is added to the measurement system, the probe nucleic acid The degree of homologous binding between reflected in a decrease in stability and a corresponding change in charge transfer rate.

In yet another preferred embodiment of the invention, the mesoieter is a ruthenium compound.

Ruthenium compound pentaamine-isonicotinamine-ruthenium (II) The reduction rate of I) was first shown by T'aube in 1970 to be high. was discovered.

Since this time, ruthenium compounds have been widely used both in liquid systems and on electrodes. Many studies have been conducted on incorporating ruthenium compounds into charge transfer electrodes. is a first in the present invention.

Particular ruthenium compounds preferred in embodiments of the invention include: CRu(NHs)sPy〕” CRu(NH3)gPy)’◆・2◆[Ru(N)I-)sL]”” However, L is 4-aminomethylpyridine (AMP), or L is (4-Picoluc)-bezuzamide (PBA) [Ru(NHs)sOH2)””+ and [:RLI(NH3)SCI) ”’　”　.

A method for bonding the first compound described above to a graphite electrode was described by Kobal and Anson. (see above).

The present invention also extends to a novel complex of DNA or RNA and ruthenium. Ru. It is known to produce adducts of ruthenium compounds with xanthines ( Lark and Taub: Journal of the American Chemical Society 97.6 March 1975), therefore, the method of the present invention can be applied to nucleic acids which are ruthenium adducts. It is believed that this can extend to otide derivatives.

In order that the invention may be better understood, reference is made to the drawings by way of example. I will explain further. ; Figure 1 shows a measurement method (protocol) in which nucleic acid binds to an enzyme. ) is shown.

FIG. 2 shows a measurement method in which nucleic acid binds to mesoieta.

Figure 3 shows a measurement method in which a nucleic acid binds to a linker that has an affinity for the electrode surface. vinegar.

Figure 4 a shows the biotin of 2 and button g in the presence of Western horseradish peroxidase. - shows a cyclic portammogram of cytochrome-C.

Figure 4 shows the same cyclic reaction as in Figure 4a when 300 μm of hydrogen peroxide was added. Kuvolta modalum is shown.

Figure 5 shows biotin-cytochrome-C at 1.4 mg/m1, 105 μg/m1 All working electrodes were surface modified with BPS in the presence of horseradish peroxidase. This figure shows the recording of the current generated by the Y pre-recording needle.

Figure 6 shows the results obtained in the presence of 6 μM biotin-peroxidase avidin enzyme complex. Cyclic cycle of I"Ru(NHs)sPy" when gold is used as the electrode for A Cuportamogram is shown.

FIG. 7 shows FIG. 6 in more detail.

Figure 8 shows the catalytic effect when biotin-bound DNA is added to the measurement mixture. In Figure 1, E is an enzyme, e.g. , glucose oxidase, which is the product of a substrate S (e.g. glucose), P (e.g. gluconic acid), releasing an electron, e, and The more oxidized form of mesoieta Mox (e.g. ferridinium ion) Reduced to reduced form, Mred (e.g. ferrocene). Mred is the electrode When oxidized with G, the flowing current was measured and was proportional to the amount of substrate S present.

DNA fragments that can be derived from naturally occurring DNA sequences or synthesized Fragment D was coupled to enzyme E by any suitable method. In the presence of excess substrate S, Steady state current was obtained. A specific target that is complementary to the sequence of the DNA fragment Convert the DNA sample whose sequence needs to be measured into single strands by any suitable method. , then added to the reaction mixture.

If a target sequence that is complementary to the DNA fragment is present in the mixture, this It bound to the fragment and interfered with the enzymatic reaction between enzyme E and substrate S. Therefore, the substrate S The amount of product P produced was reduced and, coupled with this, the reduction of Mox was reduced. M Changes in the reduction rate of ox were reflected in a decrease in the current at electrode G.

The change in current depends on the amount of fragmented DNA D currently bound to the DNA. proportional to the amount of target present.

Example 2: Mesoieta bound to a DNA probe In Example B, the redox- The active mesoieta M (substituted ferrocene in this example) should be used as a probe. Directly bound to fragmented DNA. Form of DNA-probe bound to mesoieta The composition does not interfere with the amperometric response of Mesoieta M and also Mutual binding between the target sequence and the complementary target sequence contained in the DNA being measured It did not interfere with the action.

mesoiator - a current droplet produced by adding a DNA probe to the measurement mixture The constant response was measured. If there is a single-stranded form of genetic material to which the probe is complementary, In this case, the probe bound to a complementary sequence in the sample DNA. This is the amperometric response significantly reduced or completely blocked, i.e., mesoieter-DNA probes. (Tagera) complexes with DNA were not effective in amperometric titrations. Initial amperometric response The decrease in mesoieter-DNA probe/target) DNA complex formation amount, and therefore a sequence that is complementary to the known sequence of the mesoieter DNA probe. directly proportional to the amount of genetic material it contains.

The target DNA probe also contains one or more linker groups L (e.g. For example, biotin can be used).

In this example method, a mesoiator-linker-DNA probe is attached to a DNA probe. treatment with a sample thought to contain single-stranded genetic material, which is the target for Zoieta-Linker-DNA probe is also attached to a complementary sequence to that present. Ta.

Electrochemically active substances R1 compatible with linker group L, e.g. The electrode, on which the labeled avidin was present, was then immersed into the reaction mixture.

The current measured when a potential is applied is that the electrochemically active substance R is a mesoieter. -Linker-DNA Probe/Target-Decreased when bound to DNA complex It was a little. This decrease in current also occurs when the target is complementary to the known sequence of the DNA-probe. was proportional to the amount of single-stranded sequence added when using a single-stranded sequence.

Example 4: Biotin-Cytochrome-○ and Veroxita In this particular example, the electrodes Reversible electron transfer was demonstrated when othine-labeled cytochrome-○ was used. bio Chin-labeled cytochrome-〇 acts as a mesoieta compound, but also It could also be conjugated to avidin as described in the general description.

Adding a biotin-labeled nucleic acid (DNA or RNA) to a mixture, on the other hand, Specific competitive binding reaction between othine-cytochrome-C complex and avidin, etc. In one method, a specific competitive binding reaction between an avidin complex and biotin-labeled DNA is used. response has begun.

Since the concentration of avidin (or streptavidin) is constant, the avidin-vidin The concentration of free biotin-cytochrome-0 species increases by increasing the concentration of otin-nucleic acid complexes. There is a corresponding increase in the rate of charge transfer from the enzyme to the electrode surface. A corresponding increase occurred.

- For example, using a gold electrode surface modified with 4,4-dipyridyl or a similar substance. used. In this example method, biothione-labeled cytochrome-○ is an enzyme, in this example It acted as a mesoieter against horseradish peroxidase.

The enzyme is catalyzed by the catalytic action that occurs when H2O2 is added as illustrated in Figure 4 a'J6 and Figure 4. As shown by the current caused by Charge was transferred between cytochrome-○) and substrate (1,02).

Both enzyme and substrate were present in excess. This method sets the potential of the cathode to an appropriate negative level. The current due to catalysis was obtained by equilibrating at the value of It depended on biothione-cytochrome-C concentration.

Avidin or streptavidin was then added to the system. some mesoie ta formed a complex with avidin, thus reducing the catalytic current. Biochi When DNA or RNA containing protein is added, some biotin-citrochrome-C is released. This was because the former competed with the existing avidin. As a result, As shown in Figure 5, the current due to the mediating action is caused by the DNA bound to the added biotin. increased at a rate of

Experimental conditions A conventional three-electrode system is used, with 4,4-bipyridyl sulfur as the working electrode. A doped coated gold electrode was used. Other components of the measurement mixture are 0.05M phosphate buffer , pH 6,2; biotin-citrochrome-C (SIGMA>0.14-2. 8mg; LO23oo~80μM; Horseradish peroxidase 210μs /ml; avidin 5-50 μg; biotin polyuridyl (BRL) 1-10 It was μI.

Cyclic portammogram of biotin-cytochrome-〇 (scanning range - 200 ~ +200 mV), as shown in Figure 4a, is the enzyme horseradish peroxide. There was no change when sidase was added to the cells.

As a result of adding the substrate (H2O2), as shown in Figure 4, the current due to catalytic action A significant change occurred.

The electrodes were equilibrated at a reducing potential as generally shown in Figure 5 and Addition of substrate generates a catalytic current, which is a biotin-cytochrome - It depended on the C concentration.

In the second step, avidin was added to the measurement system; as a result of adding the same amount of substrate, Figure 5 As shown in Figure 2, the catalytic current decreased, but this was due to the biotin-cytochrome This is because some of Mu-C formed a complex.

As shown in Figure 5C, in the third step, biotin-DNA and RNA are added to the system and the same Adding an amount of 820□ increases the catalytic current, which causes the probe to It was found that there is a dependency on

In this example, a reaction catalyzed by an enzyme is detected and measured in the presence of mesoieta. using the electrode surface, in this case as a mesoieter, to covalently bind DNA or RNA. covalently bonded with biotin-cytrochrome-C or DNA or RNA Citrochrome-C was used.

A catalytic current in the presence of excess enzyme or substrate is generated in an electrochemical cell. Biotin cytochrome C-DNA (BCDNA) or cytochrome C-DNA present in It became a measure of ROM-DNA (CDNA).

Biotin was attached to DNA using a cytochrome-〇 bridge (Manning (M ann ing) etc.). Biotin-labeled cytochrome-C (available from SIGMA) Activation of 5 mg in O,LM triethanolamine (TEA) pH 7,8 Calf thymus DNA (Pharmacia PLC Biochemicals) 4 Added to mg.

1.2 ml of 6% formaldehyde was added to the same buffer for crosslinking and the mixture The mixture was incubated at 37°C for 1 hour.

The formaldehyde was then removed by extensive dialysis.

Thorium chloride was added to this sample to give a final concentration of IM (not covalently bonded). to dissociate which cytochrome C).

This solution was passed through a FPLC gel filtration column using Sepharose 6. It was fractionated using B. Both of these components were detected at 254 nm. At 260nm Extinction coefficient for DNA at 25 cm'/mg and biotin at 410 nm ・Nucleotide pairs using the extinction coefficient of 7.7 cm2/mg for cytochrome C When the ratio of cytochrome-C was calculated, it was found to be approximately 20:1 in the product. I started it.

BCDNA or CDNA is a mesoieter in an electrochemical cell, e.g. Acted as a horseradish peroxidase. catalytic in the presence of a substrate Current is a measure of BCDNA or CDNA abundance or hybrid abundance It became.

The electrodes were equilibrated at reduction potential. The catalytic current is biotin-cytochrome-C Presence of DNA (or RNA) or cytochrome-CDNA (or RNA) showed that.

4. Standard Sanden using gold coated with 4-bipyridyl sulfide as working electrode The polar system was used in 0.05M phosphate buffer at pH 6.2. In this example, Mezoi Biotin-cytochrome-DNA (BCDNA) was used as a protein, but Western A cytochrome bridge mediates between horseradish peroxidase and the electrode. It is contemplated that any other electrochemical label attached to the Ru.

If cytochrome-C is used, its concentration must be kept below 300μ. However, this is because cytochrome-C bleaches (bl) at higher concentrations. This is because there was a risk of being subject to

gAI 6: Biotin-labeled phage lambda DN using the method in this example detects reactions catalyzed by enzyme complexes in the presence of mesoieta. or using an electrode surface that can be measured. In this example the enzyme complex is Chinperoxidase - Avidin is added with biotin or biotin-labeled D the biotin in a proportion that allows it to bind to biotin-containing substances such as NA or RNA. Constructed from peroxidase-avidin.

This biotin peroxidase-avidin complex was combined with H2O2 and mesoieta. , for example cytochrome C, ferrocene compounds or [Ru( Steady-state current was obtained by using in the presence of NH3)sPy]2+ . The electrodes were equilibrated at negative potential. The substrate, H2O2, was present in large excess. biotin The current decreased when labeled DNA or RNA was added to the cell. Biochi The bound DNA could be in solution or bound to the membrane.

Mesoie of the critical concentration and excess amount of biotin peroxidase-avidin complex At the critical concentration of substrate and substrate, the electrodes are placed at a suitable potential (in this particular example saturation The steady-state voltage is proportional to the enzyme concentration when equilibrated at I got the flow.

From this point on, the experiment illustrated by this example can be carried out in two different experimental formats: This was done by the following addition method: a) Add biotin DNA or biotin RNA as a solution b) Addition of immobilized biotin DNA or biotin RNA.

In both cases the nucleic acid reacts with the enzyme complex, producing an increase in current that is Biotin was proportional to and acted as a measure of added DNA or RNA. .

Ultrasonicated phage lambda DNA was transferred to BVRI technical information. Biotin-11- Nick translation in the presence of dUTP, using the following reagents: Used: 1) Tris 500mM at pH 7,8, MgC1z 50mM, 2-mel Captoethanol 100mM and nuclease-free B5A 100mg/m 10μ of a solution of 0.2μM dATP, dC’TP, dGTP mixture in Il β 2) EDTA 0.1nA, 10mM Tris pH 7.5 and sodium chloride Lambda phage DNA 4μg in 100mM Tris pH 7.5 10μl of 0.4mM biotin-11-dUTp in 100mM The volume was brought to 90 μβ and the reaction mixture was carefully mixed.

4) Tris 50mM at pH 7.5, magnesium acetate 5mM, 2-merkabuto Ethanol 1mM, phenylmethylsulfonyl fluoride 0.1mM, 5 0% glycerin and nuclease-free BSA 100 t-tg in 1 ml DNAase 140 pg/ml mixture 0.4-L 27 h 10 μl of DNA polymerase was added to t(l).

This mixture was incubated at 15° C. for 90 minutes. The reaction was carried out in 300mM EDTA 10 It was stopped by adding μl.

Purification of biotin-labeled DNA was carried out using the “spin” method of Maniatis et al. It was carried out according to the column method [Mantiati, Fritsall and Samburg Author, [Molecular Cloning A Laboratory Manual J 198 2. Published by Cold Spring Harbor Lapse, pp. 466-467].

A 1+nj2 disposable syringe capped with a small amount of glass wool was used. bed A volume of 0.9 ml of Sephadex was added to 0.15 M of sodium chloride. Equilibrate with thorium and inject 0.015M of sodium citrate with a pt of 17.0. Fill the gun and spin it several times in a bench centrifuge until the filling volume is 0.9 m2. I set it. Then add 0.1 mj2 of buffer to the syringe and repeat at the same speed for the same number of times. I turned it over.

Finally, add 0.1 ml of the nick translation mixture and at exactly the same rate. Centrifuge again the same number of times and transfer 100 μl of the eluate into an Ebbendorf tube without a stopper. The effluent contained biotin-labeled phage lambda DNA. there was. Biotin standard probed and nick-translated as described above. Identified phage lambda DNA BRL and biotin-cytochrome-〇 labeled material activation By heating the sexed calf thymus DNA at 80°C for 2 hours in a vacuum oven. bound to a nitrocellulose membrane.

This experiment uses DNA in solution and DNA bound to nitrocellulose. I did it in two ways.

In both cases the measuring device comprised a gold electrode in a cell with a volume of 0.7 m 12 .

The reagents used in this example were: Phosphate buffer pH 6.2 0.05M mesoieitaitaferrocene monocarbo 0.15mMb) [:RLI(NH3)51 Pyr"" 0.16mM The concentration of biotin-peroxidase-avidin enzyme complex was as described. +2028.8mM as 60nM-0.6μM substrate Generation of the enzyme complex was carried out as follows: Bexstatin ABC (Ceralab (s) era tab)) under suitable conditions; 5 mg/mj2 of biotin-per 11 in 1 mβ assay buffer containing 0.1% Tween 20. It was incubated for 30 minutes with 50 μl of avidin of T1g/ff1l. then 0.1 Further dilutions were made in assay buffer containing % Tween 20.

Example L1 Use of Furosene Monocarboxylic Acid When ferrocene monocarboxylic acid is used as a mesoieter, The current is linear under the condition of enzyme concentration of 60nM to 6nM and is 0.8 to 0.7μ. Catalytic currents in the range of A were produced.

Takumi Kai: Use of ruthenium complexes as mediators [:PyRu(Nl13)s ] In the case where 2+ is used as a mesoiator, the current due to the catalytic action is different from the previous case. This was chosen for the three auxiliary experiments shown in the examples below, as the Ta.

Example 7c: Enzyme concentration 6. OnM In this example, the enzyme concentration was 6 nM. In the presence of H2O2, as shown in Figure 8a, Steady state current due to sea urchin catalysis was observed.

Add 200 mg of bithion-labeled phage lambda DNA to the electrochemical cell. In this case, the steady state due to catalytic action and the injection of H,0□ also result in catalytic action. A current was generated. This result indicates that biotin-DNA has some biotin avidin enzyme complex. The enzyme complex that has formed a complex with biotin but is not bound to the biotin DNA This shows that there is a large excess.

Example 7d: Enzyme concentration 0.6 nM This example follows the same lines as the previous example, with the only difference being the concentration of the enzyme conjugate. In this case, it was set to 0.6 nM.

The catalytic current, which indicates the enzyme complex concentration as shown in Figure 7, is the biotin D After the addition of NA (200 μg), there was a sudden change as shown in FIG. 7C. More H Even after injection of 20□, the current remained at a low level as shown in Figure 7d.

Example 7e: DNA immobilized on membrane When H20□ was used in almost the same manner as described above, a current due to catalytic action was obtained. . The biotin-labeled DNA immobilized on the nitrocellulose membrane was g to 0.2 μg was added to the cells. This cell is combined with this biotin DNA membrane. After heating the mixture for 10 minutes, additional H2O2 was injected as shown in Figure 7e. Since no current was generated due to the catalytic action, the enzyme complex was present on the membrane. It was found that the compound bound to Otin DNA, thereby blocking charge transfer.

international search report 1Me++? @'al mm, pCT/C'B 8410043 2-2-ALNNEX To F-’ INτ3R-NAτl0NAL 5EA RCHR4JORT ON

Claims (47)

[Claims] 1. When measuring nucleic acids, (a) (i) a nucleic acid sequence complementary to a given target sequence; and (ii) It is chemically bonded to this and can perform a specific binding reaction with the antiligand. The first ligand capable of providing a probe substance containing; (b) the probe substance; (i) When the enzyme is catalytically active, it can transfer charge to the electrode surface. suitable mediators, enzymes, substrate systems, and (ii) chemically combined with any one of said mediator, enzyme or substrate; a second ligand capable of performing a competitive binding reaction with the antiligand; Beauty (iii) the antiligand in a specific binding reaction with the antiligand. competition between the first ligand and the second ligand; (c) contacting said system with a solution suspected of containing said target sequence; by binding any of the target sequences present to the probe. first affects the availability of the ligand and thus the rate of charge transfer to the electrode. A nucleic acid measurement method characterized by changing. 2. 2. The method according to claim 1, wherein the nucleic acid is deoxyribonucleic acid. 3. 2. The method according to claim 1, wherein the nucleic acid is a ribonucleic acid. 4. Specific binding between either the first or second ligand and the antiligand Reactions include biotin and avidin, antigens and antibodies, hormones and receptors, lectins and carbohydrates. Reagents selected from the group of pairs consisting of substances, cofactors and enzymes, and nucleic acids and complementary nucleic acids. or a specific binding reaction between a pair of derivatives thereof. . 5. Both the first ligand and the second ligand are biotin or a derivative thereof. Claim 4, wherein the antiligand is avidin or a derivative thereof. Method. 6. 2. The method of claim 1, wherein the mediator is an organometallic compound. 7. The mediator has at least one organic ring, and the ring is conjugated. Characterized by at least two double bonds and a metal atom in covalent electron contact with each ring The method according to claim 6. 8. 8. The method of claim 7, wherein the metal is a transition metal. 9. Claim No. 1 in which the metal is selected from the group consisting of iron, chromium and niruthenium. The method described in Section 8. 10. 10. The method of claim 9, wherein the mediator is ferrocene. 11. Mediator: viologen, polyviologen, phenazine, felicyan from compounds and their derivatives, metal porphyrins and one-dimensional conductors The method according to claim 1, wherein the method is selected from the group consisting of: 12. 2. The method of claim 1, wherein the mediator is a carboborane. 13. 2. The method of claim 1, wherein the mediator is a ruthenium compound. 14. The mediator is ferrocene; chloroferrocene; methyl-trimethylamine. Noferrocene; 1,1-dimethylferrocene; 1,1-dicarboxyferrocene ; Carboxyferrocene; Vinylferrocene; Trimethylaminoferrocene; 1 ,1'-dimethylferrocene; polyvinylferrocene; ferrocene monocarvone Acid; hydroxyethylferrocene; acetoferrocene and 1,1'-bishydride The method according to claim 10, wherein the method is selected from the group consisting of roxymethylferrocene. . 15. 12. The method of claim 11, wherein the mediator is a cytochrome. 16. 16. The method of claim 15, wherein the mediator is cytochrome-C. 17. 12. The method of claim 11, wherein the mediator is TCNO. 18. Claims 1 to 17, wherein the mediator is localized to the conductive surface. method described in one of the sections. 19. Claims 1 to 17, wherein the enzyme is a non-oxygen-specific flavoprotein. A method described in one of the sections. 20. The flavoproteins are combined with methanol oxidase and pyruvate oxidase. xanthine oxidase (EC1.2.3.3), xanthine oxidase (EC1.2.3.2. ), sarcosine oxidase (EC1.5.3.1.), ribamide dehydrogenase enzyme (EC1.6.3.4.), glutathione reductase (EC1.6.4.) ．． 2. ), carbon monoxide oxidoreductase, glucose oxidase, glyco acid oxidase (EC1.1.3.1.), L-amino acid oxidase (E C1.4.3.2. ) and lactose oxidase. Scope of Claim 19. The method according to item 19. 21. Any one of claims 1 to 17, wherein the enzyme is a quinone protein. The method described in section. 22. The quinone protein is combined with glucose dehydrogenase and alcohol dehyde. Claims selected from the group consisting of logonase and methanol dehydrogenase The method according to paragraph 21. 23. Any one of claims 1 to 17, wherein the enzyme is localized to the electrically conductive surface. The method described in Section 1. 24. Any of claims 1 to 17, wherein the enzyme is a haem-containing enzyme. or the method described in one of the sections. 25. Heme-containing enzymes such as lactate dehydrogenase, yeast, and cytochrome-C peroxy Claims selected from the group consisting of Dase and Western horseradish peroxidase The method according to paragraph 24. 26. Any one of claims 1 to 17, wherein the enzyme is cuproprotein. The method described in section. 27. Claim 26, wherein the cuproprotein is galactose oxidase The method described in section. 28. Is the electrode a group consisting of gold, platinum, silver, carbon, or one-dimensional conductors? Claims 1 to 17 made of a material selected from How to put it on. 29. Determine the presence and/or copy number of a given sequence in a single-stranded nucleic acid molecule. In the nucleic acid sequence probe for determining If the nucleic acid sequence of the probe is a) the mediator compound or said mediator compound is electrochemically bonded; any of the enzymes and b) Bithion is connected to binding of the probe nucleic acid sequence to the target nucleic acid sequence A nucleus characterized in that the electrical bond between the eta compound and the enzyme is changed. Acid sequence probe. 30. When measuring nucleic acids, The electrode is placed in contact with the enzyme and the enzyme's substrate, and the mediator compound is As the addition of the substrate to the product in combination with the enzyme proceeds, the electricity is removed from the enzyme. The nucleic acid sequence of the probe is said enzyme and said mediator by conjugation to either a mediator compound; The activity of the compound, and therefore the amount of charge transfer to the electrode, is determined by the amount of binding to the nucleic acid sequence of the probe. the presence or absence of a complementary target nucleic acid sequence Features of nucleic acid measurement method. 31. Mediator compounds can be transition metal compounds, organic conductors, phenazines, carbon-phosphates, etc. Claim 29 or The method according to paragraph 30. 32. Claims in which the mediator compound is biothione-labeled cytochrome-C The method according to paragraph 31. 33. Invert either avidin or streptavidin into the reaction mixture and Claims 31 and 31 are combined with a radiator to reduce charge transfer to the electrode. or the method described in paragraph 32. 34. Electrodes catalyze reactions catalyzed by enzyme complexes in the presence of mediators. 33. The method according to claim 31 or 32, wherein the method comprises detecting and measuring. 35. Claim that the enzyme complex consists of a biotin-peroxidase-avidin complex The method according to item 32 or 33. 36. Enzyme complex further binds to biotin- or biotin-labeled nucleic acids 36. The method according to claim 35, wherein: 37. 30. The method of claim 29, wherein the mediator is immobilized on the surface. 38. 38. The method of claim 37, wherein the surface comprises nitrocellulose. 39. In nucleic acid probes, a) Nucleic acid b) a nucleus comprising a mediator chemically bound to said nucleic acid; Acid probe. 40. The mediator compound may be a transition metal compound, an organic conductor, a phenazine, or a carbon-phosphate compound. Claim 39 selected from the group consisting of urin compounds and viologens Probe as described. 41. 41. The probe according to claim 40, wherein the mediator is cytochrome-C. . 42. 41. The probe of claim 40, wherein the mediator is ferrocene. 43. The probe according to claim 40, wherein the mediator is a ruthenium compound. Bu. 44. Claim 4 wherein the mediator is [Ru(NH3)5Py]2+ The probe according to item 3. 45. The accompanying drawings have been described above and are substantially identical to those shown in the accompanying drawings. Nucleic acid measurement method. 46. The accompanying drawings have been described above and are substantially identical to those shown in the accompanying drawings. Probe used for measuring nucleic acids. 47. for carrying out the measuring method as claimed in any of the above claims. Kits of reagents and/or equipment.