JPH0772060A - Method for detecting biochemical material and biochemical-material detecting sensor used in the method - Google Patents

Abstract

PURPOSE:To detect biochemical material quantitatively with high accuracy by forming probe DNA and hybrid for a sample for detecting DNA, and measuring the binding constant based on the time change of the frequency of a frequency converting element caused by the formation. CONSTITUTION:Probe DNA comprising the base sequence peculier to the DNA of biochemical material and the complementary base sequence is chemically bonded and foxed on the electrode of a frequency converting element. The converting element is dipped into water, and the probe DNA and hybrid are formed with a sample as target DNA. As the chemical material, there are nucleic acid and the like such as the DNA and RNA. The probe DNA is obtained by a DNA synthesizer by a phosphoramidite method. Tiol is introduced into the end of the base sequence of the DNA. The converting element is dipped into the aqueous solution of the synthesized material and made to react. The probe DNA and the converting element are bonded. The change with the lapse of time of the converting element caused by the change in weight in hybridization is measured, and the binding constant in hybridization is measured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biochemical substance detection method and a biochemical substance detection sensor used in the method.

[0002]

2. Description of the Related Art Conventionally, for example, Japanese Patent Laid-Open No. 3-2
As described in Japanese Patent Publication No. 10198, after the DNA hybridization reaction, separation by electrophoresis or analysis and separation / quantification of the DNA hybridization reaction by a method of labeling with radioisotope and quantifying is performed. . However, these methods can be performed only in a limited place for labeling, require special technology to handle radioisotopes, and require time for operation, processing, and measurement for labeling. There are drawbacks such as this.

For example, Japanese Patent Laid-Open No. 3-210198 discloses a method of detecting a product of an enzymatic reaction by reacting an enzyme-modified antibody after hybridizing a DNA modified with an antigen having a complementary base sequence to a target DNA. Is listed. However, these methods have drawbacks such as poor quantification due to inactivation of enzyme activity, and time-consuming operations, treatments, and measurements for producing enzyme-modified antibodies. Conventionally, the calculation of binding constants for analyzing various phenomena peculiar to DNA such as hybridization of biochemical DNA with DNA and intercalation of chemical into DNA has been performed by changing the absorbance of the ultraviolet spectrum or circular dichroism. Although it is performed by using a spectroscopic method such as measuring a sex spectrum, there are problems such as expensive equipment and poor quantitativeness.

The present invention is free from the drawbacks of the prior art such as the need for special techniques, poor quantitativeness, long-time measurement, and the use of expensive measuring equipment.
The DNA formed was analyzed simply by direct analysis of the DNA hybrid formation reaction, and the binding constant in the DNA hybrid formation was measured for kinetic analysis.
A method for separating and quantifying A hybrid, which comprises:
It is an object of the present invention to provide a method for detecting a biochemical substance by facilitating the formation of a hybrid with high accuracy, quantitatively and easily, and a biochemical substance detection sensor used in the method.

[0005]

According to the present invention, a probe DNA having a base sequence complementary to a base sequence peculiar to DNA of a biochemical substance is chemically bonded and immobilized on an electrode of a frequency conversion element to be immobilized. The converted frequency conversion element is immersed in water, a sample for detecting the DNA is injected into the water, and the sample is hybridized with the probe DNA as a target DNA having a base sequence specific to the probe DNA. A method for detecting a biochemical substance, characterized by measuring the change over time in the frequency of a frequency conversion element due to the weight change due to the hybridization to measure the binding constant (K) in the hybridization; A biochemical substance detection sensor is provided.

The biochemical substance detected by the method of the present invention can include those having a specific base sequence and those having a specific base constituting the biochemical substance. A nucleic acid that constitutes a microorganism such as a virus and has a specific base sequence, and a base or a component containing the base that constitutes the nucleic acid can be included. Examples of such nucleic acids include DNA and RNA
Can be given.

The frequency conversion element used in the present invention can include, for example, a crystal oscillator and a surface acoustic wave element (SAW).

The probe DNA in the present invention is composed of a base sequence complementary to the base sequence peculiar to the biochemical substance DNA, and can be obtained by a DNA synthesizer by the phosphoramidite method. Or, D of biological origin
It can also be obtained by using the phosphoramidide method for NA.

The target DNA in the present invention is the above-mentioned biochemical substance DNA having a base sequence specific to the above-mentioned probe DNA.
It has a unique base sequence complementary to the base sequence of A.

As a method of chemically binding and immobilizing the probe DNA on the electrode of the frequency conversion element in the present invention, for example, when the frequency conversion element is a crystal oscillator and the electrode is a gold electrode, the probe DNA A method of synthesizing a thiol group introduced at the end of a complementary base sequence, immersing and reacting a crystal oscillator in the aqueous solution for a certain period of time, then taking out the crystal oscillator from the aqueous solution and drying it . The thiol group is S-trityl-3-
Mercaptopropyloxy-β-cyanoethyl-N, N
-Diisopropylaminophosphoramidide and the like are included, and the introduction of a thiol group to the terminal of the complementary base sequence of the probe DNA can be performed by the phosphoramidite method.

In a preferred embodiment of the method for immobilizing probe DNA on an electrode in the method of the present invention, the electrode is a gold electrode, the probe DNA is single-stranded, and a thiol is added to the end of its base sequence. Synthesize what introduced the group,
Further, DNA having a complementary nucleotide sequence is hybridized to synthesize Japanese chain DNA, the frequency conversion element is immersed in and reacted with an aqueous solution of the double stranded DNA, and then the melting point of the double stranded DNA or higher is reached. The single strand that is not immobilized on the electrode is heated to be desorbed, and then the frequency conversion element is taken out of the aqueous solution and dried to be chemically bonded and immobilized on the electrode. According to this immobilization method,
The probe DNA can be uniformly immobilized on the electrode, and the efficiency of hybrid formation with the target DNA can be improved.

In the method of the present invention, according to the first preferred embodiment of the method for measuring the binding constant (K), after the time-dependent change in frequency associated with hybridization reaches adsorption equilibrium, the crystal oscillator is placed in the water. Then, it was immersed in another large amount of water and the change in frequency with time was measured, and the binding rate constant k ₁ was obtained from the initial rate of decrease in frequency or the initial rate of increase in adsorbed weight V _{1 due} to hybridization. , The dissociation rate constant k ₂ from the rate of increase in the initial frequency or the rate of decrease in the initial adsorption weight V ₂ when immersed in a large amount of water as described above.
Then, the following equations (1) to (6): [probe] + [target] ← → [ds-DNA] (1) V ₁ = k ₁ [probe] [target] (2) k ₁ = V ₁ / [Probe] [Target] (3) V ₂ = k ₂ [ds-DNA] (4) k ₂ = V ₂ / [ds-DNA] (5) K = k ₁ / k ₂ (6) [wherein [Probe]: Probe concentration (mol /
l) [Target]: target concentration (mol / l) [ds-DNA]: hybridized double-stranded DN
A concentration (mol / l) V ₁ : initial adsorption rate (mol / s) K ₁ : adsorption rate constant (M ^-1 s ^-1 ) V ₂ : initial dissociation rate (mol / s) k ₂ : dissociation rate constant ( s ⁻¹ ) K: binding constant (M ⁻¹ )], the binding constant (K) is obtained.

According to the second preferred embodiment of the method for measuring the binding constant (K) in the method of the present invention, the probe DNA
A fixed amount of target DNA is injected into water in which the immobilized frequency conversion element is immersed to form a hybrid,
The operation for obtaining the adsorption amount of the target DNA on reaching adsorption equilibrium by changing the injection amount of the target DNA repeated, the concentration of the target DNA during injection of the target DNA, the initial concentration of the target DNA [Target] ₀
And the adsorption amount of the target DNA when the adsorption equilibrium is reached is defined as the saturated adsorption amount (Δm) with respect to the initial concentration [target] ₀ of the target DNA, and the correlation between the initial concentration [target] ₀ and the saturated adsorption amount (Δm) The relationship is calculated and the following equation (I): [target] ₀ / Δm = (1 / Δm _max ) [target] ₀ + (1 / Δm _max K) (1) (wherein, [target] ₀ , Δm And K are as described above, and Δm _max is the probe DNA of the target DNA
Is the saturated adsorption amount for
The correlation between ₀ and [target] ₀ / Δm was plotted by plotting [target] ₀ as the X axis and [target] ₀ / Δm as the Y axis, and calculating the reciprocal of the slope of the straight line connecting these plots to obtain the target DNA The saturated adsorption amount Δm _max for the probe DNA is obtained, and the binding constant (K) is obtained from the obtained Y-axis intercept value.

[0014]

EFFECTS OF THE INVENTION According to the present invention, special technology is required, quantitativeness is poor, and long-term measurement is required.
Without the drawbacks of the prior art such as the use of expensive measuring instruments, the DNA hybrid formation reaction can be analyzed easily and directly, and further, the binding constant in the DNA hybrid formation can be measured to carry out the kinetic analysis and formation. A method for separating and quantifying the formed DNA hybrids, and a method for detecting a biochemical substance by facilitating the formation of the DNA hybrids with high accuracy, quantitatively, and a biochemical substance detection sensor used in the method. Provided. INDUSTRIAL APPLICABILITY The present invention provides analysis of characteristics specific to biochemical substances such as strength of DNA hybrid, salt concentration effect of measurement solution, observation of intercalation phenomenon, genes attracting attention as drugs, lipozymes and antisenses. It is possible to provide a method that can be used for enhancing the efficiency of transfer of nucleic acid compounds into cells, controlling intracellular distribution, designing drugs, screening drugs, and the like.

[0015]

The present invention will be described in more detail with reference to the following examples.

Examples 1 to 3 10 bases having a thiol group introduced at the 5'end (5'-dG
GGAATTCGT-3 ') and a DNA oligomer having no thiol group and complementary to the DNA oligomer.
DN of base sequence (3'-CCCTTAAGCA-5 ')
The crystal oscillator was immersed in an aqueous solution in which the A oligomer was hybridized, and immobilized on the gold electrode. A crystal oscillator whose complementary chain has been eliminated by aging is immersed in 5.5 ml of pure water, and 500 ng of a DNA oligomer complementary to the DNA oligomer immobilized on the crystal oscillator of 500 ng is injected into pure water for hybridization. After being formed and reaching the adsorption equilibrium, the crystal oscillator was taken out and separately immersed in a large amount of pure water to change the frequency with time at 20 ° C. (Example 1), 30 ° C. (Example 2), and 60. C (Example 3) was measured. Figure 1
The measurement results at 20 ° C. (Example 1) are shown in FIG. In order to investigate the effect of temperature on the hybridization formation of DNA, the binding constant of the DNA double helix at each measurement temperature was calculated. For example, in order to determine the binding constant at a measurement temperature of 20 ° C., in FIG. 1, the initial adsorption rate V _{1 associated} with the hybrid formation is determined from the slope of the tangent line of the initial hybrid formation curve, and the above equation is applied from the V ₁ value The binding rate constant k ₁ is determined, the initial dissociation rate V ₂ is determined from the slope of the initial tangent line of the dissociation curve when immersed in a large amount of pure water, and the above equation is applied from the V ₂ value to determine the dissociation rate constant k 1. ₂
Then, the above equation was applied from the obtained binding rate constant k ₁ and dissociation rate constant to obtain the binding constant (K) at 20 ° C.
I asked. Measuring temperature is 30 ° C (Example 2) or 60 ° C
The same experiment as in the case of 20 ° C. (Example 1) was carried out except that (Example 3) was changed. The results obtained are shown in Table 1.

[0017]

[Table 1]

Example 4 A probe DNA-immobilized crystal oscillator similar to that in Example 1 was placed in 5.5 ml of pure water kept at a constant temperature of 60 ° C., and after stable oscillation, the same target DNA as in Example 1 was applied. 180
Saturated adsorption amount (Δm) of the target DNA when 0 ng was injected and equilibrium was reached was determined. Injection volume is 18
Target DNA obtained by repeating the same operation except that the amounts were changed to 00 ng, 3600 ng and 5400 ng
Adsorbed amount of target DNA (Δm)
The relationship of is shown in FIG. This relationship is because the saturated adsorption amount with respect to the injection amount of the target DNA is extremely small, so that the initial concentration of the target DNA [target] is substantially
_Since it corresponds to the relationship between ₀ and Δm, the relationship of FIG. 2 is applied to the above formula (I), and the relationship between [target] ₀ and [target] ₀ / Δm is expressed as
When the concentration was taken and the target DNA concentration / Δm was taken on the Y axis and plotted, the results shown in FIG. 3 were obtained. From the slope of the straight line connecting the plots shown in FIG. 3, the saturated adsorption amount of target DNA to probe DNA (Δ
m _max ), and the value of the coupling constant (K) obtained from the value of the Y-axis intercept of the straight line in FIG. 2 is 3.4 × 10 ⁵ M
It was ^-1 .

[Brief description of drawings]

FIG. 1 is a graph showing an example of changes over time in frequency in a first preferred aspect of the method of the present invention.

FIG. 2 is a graph showing the relationship between the target DNA concentration and the saturated adsorption amount of target DNA in the second preferred embodiment of the method of the present invention.

FIG. 3 is a graph showing the relationship between the target DNA concentration and the target DNA / Δm in the second preferred embodiment of the method of the present invention.

Claims (6)

[Claims] 1. A probe DNA having a base sequence complementary to a base sequence peculiar to a biochemical substance DNA is chemically bonded and immobilized on an electrode of a frequency conversion element, and the fixed frequency conversion element is submerged in water. A sample for detecting the DNA is injected into the water, the sample is allowed to form a hybrid with the probe DNA as a target DNA having a base sequence specific to the probe DNA, and the weight change due to the hybridization A method for detecting a biochemical substance, which comprises measuring the change over time in the frequency of the frequency conversion element, and measuring the binding constant (K) in hybridization. 2. The measurement of the binding constant (K) is carried out after the time-dependent change in frequency associated with hybrid formation reaches adsorption equilibrium.
The crystal oscillator was taken out of the water, immersed in another large amount of water, and the time-dependent change in frequency was measured. From the rate of decrease in the initial frequency or the rate of increase in the initial adsorption weight V ₁ associated with hybridization, The binding rate constant k ₁ is determined, and the dissociation rate constant k ₂ is determined from the initial rate of increase in frequency when immersed in a large amount of water or the initial rate of decrease in adsorbed weight V ₂ , and the following equation (1) (6): [probe] + [target] ← → [ds-DNA] (1) V ₁ = k ₁ [probe] [target] (2) k ₁ = V ₁ / [probe] [target] (3) V ₂ = k ₂ [ds-DNA] (4) k ₂ = V ₂ / [ds-DNA] (5) K = k ₁ / k ₂ (6) [wherein, [probe]: probe concentration (mol /
l) [Target]: target concentration (mol / l) [ds-DNA]: hybridized double-stranded DN
A concentration (mol / l) V ₁ : initial adsorption rate (mol / s) K ₁ : adsorption rate constant (M ^-1 s ^-1 ) V ₂ : initial dissociation rate (mol / s) k ₂ : dissociation rate constant ( The method according to claim 1, which is carried out by determining the binding constant (K) from s ^-1 ) K: binding constant (M ^-1 ). 3. The probe D is used for measuring the binding constant (K).
The amount of target DNA adsorbed when the adsorption equilibrium is reached is calculated by injecting a fixed amount of target DNA into the water in which the NA-immobilized frequency conversion element is immersed to form a hybrid. Target DN at the time of target DNA injection by changing and repeating
The concentration of A is the initial concentration of the target DNA [target] _0, and the target DNA when the adsorption equilibrium is reached.
Adsorption amount of target DNA is the initial concentration of target DNA [Target]
_As the saturated adsorption amount (Δm) with respect to ₀ , the correlation between the initial concentration [target] ₀ and the saturated adsorption amount (Δm) was calculated, and the following formula (I): [target] ₀ / Δm = (1 / Δm _max ) [Target] ₀ + (1 / Δm _max K) (1) (wherein [target] ₀ , Δm and K are as described above, and Δm _max is the probe DNA of the target DNA.
Is the saturated adsorption amount for
The correlation between ₀ and [target] ₀ / Δm was plotted by plotting [target] ₀ as the X axis and [target] ₀ / Δm as the Y axis, and calculating the reciprocal of the slope of the straight line connecting these plots to obtain the target DNA The method according to claim 1, wherein the saturated adsorption amount Δm _max for the probe DNA is obtained, and the binding constant (K) is obtained from the obtained Y-axis intercept value.
The method described. 4. The method according to claim 1, wherein the frequency conversion element is a crystal oscillator. 5. A frequency conversion element and a DN of a biochemical substance chemically bonded and immobilized on an electrode of the frequency conversion element.
A biochemical detection sensor comprising a probe DNA having a base sequence complementary to the base sequence unique to A and used in the method according to any one of claims 1 to 4. 6. The sensor according to claim 5, wherein the frequency conversion element is a crystal oscillator.