JP2016156705A - Substrate binding-power regulator, molecular sensor using the same, and using method of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new substrate binding-power regulator for changing the binding power of a substrate and a molecular sensor, in changing the detection range of the molecular sensor having a specificity to the substrate.SOLUTION: A molecular sensor 2 having a specificity to a predetermined substrate 1 includes: a factor 3 having a specificity to the substrate 1; and a substrate binding-power regulator 4 for regulating the binding power of the substrate 1 and the factor 3. The substrate binding-power regulator 4 is inert to the substrate 1 and factor 3, is dissolved in a solution containing the factor 3 at a predetermined concentration, and includes compounds for changing the association constant (Ka/M) of the factor 3 to the substrate 1 at a rate of at least three times or more, or a salt or derivative of them.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a substrate binding force regulator that adjusts the binding force between a carbohydrate or other substrate and a molecular sensor having specificity for the substrate, and in particular, to change the binding force between the substrate and the molecular sensor. And a molecular sensor using the same, and a method for using the same.

Molecular sensors that detect carbohydrates (such as glucose) and other substrates are already widely known (for example, Patent Documents 1 and 2).
In Patent Document 1, a molecular identification unit in which an enzyme having substrate specificity or the like is immobilized is combined with a detection unit that senses a physical or chemical change on the measurement target side by the molecular identification unit and converts it into an electrical signal. In the biosensor, an aspect is disclosed in which the detection means is provided with a suction mechanism for promoting a response that draws an object to be detected to a sensor sensitive part of the detection means.
Patent Document 2 discloses a biosensor in an embodiment using a detection device that binds a completely synthesized phenylboronic acid compound having a predetermined chemical formula.

Japanese Patent Laid-Open No. 5-18931 (Example, FIG. 1) JP 2012-26839 A (form for carrying out the invention, FIG. 2)

T. D. James, K. R. A. S. Sandanayake, R. Iguchi, S. Shinkai, J. Am. Chem. Soc., 117, 8982-8987 (1995)

In the system using the enzyme reaction of Patent Document 1, the detection performance of saccharides decreases due to protein denaturation, and in many cases, long-term storage is difficult.
On the other hand, Patent Document 2 uses a completely synthetic phenylboronic acid compound that does not contain a protein, so that it is possible to eliminate the problem of protein denaturation and realize stable detection performance of saccharides.
In general, in the diagnosis of diabetes, it is necessary to detect a wide concentration range of several mg / dL to several thousand mg / dL, assuming that human blood glucose levels range from normal to diabetic.
However, the biosensor of the method shown in Patent Document 2 can only detect a range narrower than the human blood glucose concentration range, and is not suitable for detection unless the sample is diluted.
However, when a sample is diluted, it may be necessary to dilute several hundred times at the maximum, resulting in a dilution error or adjustment of the dilution factor depending on the blood glucose level. There is a strong demand for the development of biosensors that enable detection in the region. There is a similar demand for molecular sensors that detect other substrates.
In view of such a demand, the technical problem to be solved by the present invention is to provide a novel substrate binding force regulator capable of changing the binding force between a substrate and a molecular sensor, and a molecule using the same. A sensor and a method of using the same are provided.

The invention according to claim 1 is added to a solution containing a molecular sensor having a factor specific to a predetermined substrate and adjusts the binding force between the substrate and the molecular sensor. An agent that is inactive with the substrate and the factor, dissolves at a predetermined concentration in a solution containing the factor, and has an association constant (Ka / M ⁻¹ ) of the factor with respect to the substrate of at least It is a substrate binding force regulator characterized by containing the compound or its salt or derivative | guide_body which changes by a ratio 3 times or more.

  The invention according to claim 2 is the substrate binding strength adjusting agent according to claim 1, characterized in that it comprises a compound represented by the following general formula (1) or a salt or derivative thereof: It is.

ここで、Ｒ１からＲ３は均一であっても不均一であってもよく、炭素数が１以上１２以下、好ましくは炭素数が２以上８以下、さらに好ましくは炭素数が３以上７以下のアルキル基、アルケニル基、アルキニル基又はアリール基のいずれかであり、その総炭素数が３以上２４以下、好ましくは総炭素数が６以上１８以下、さらに好ましくは総炭素数が１２以上１５以下であり、また、Ｒ６は炭素数が１以上１０以下、好ましくは炭素数が１以上１０以下のアルキレン基、アルケニレン基、アルキニレン基又はアリーレン基のいずれかである。

Here, R1 to R3 may be uniform or non-uniform, and an alkyl having 1 to 12 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 3 to 7 carbon atoms. Any of a group, an alkenyl group, an alkynyl group or an aryl group, the total carbon number of which is 3 to 24, preferably the total carbon number is 6 to 18 and more preferably the total carbon number is 12 to 15 R6 is an alkylene group, alkenylene group, alkynylene group or arylene group having 1 to 10 carbon atoms, preferably 1 to 10 carbon atoms.

The invention according to claim 3 is the substrate binding force adjusting agent according to claim 1, characterized in that it comprises a tetraalkylammonium salt or derivative.
The invention according to claim 4 is the substrate binding force regulator according to claim 1, wherein polyethylene glycol, polyvinyl pyrrolidone or a derivative thereof, polypropylene glycol, polyacrylic acid, polyacrylamide, poly-N, N-dimethylacrylamide or a combination thereof. It is a substrate binding force regulator characterized by including a polymer.
The invention according to claim 5 is the substrate binding force adjusting agent according to any one of claims 1 to 4, wherein the substrate is a carbohydrate.
The invention according to claim 6 is the substrate binding force regulator according to claim 5, wherein when the substrate is glucose as a carbohydrate, the association constant of the molecular sensor is adjusted at a ratio of 3 to 100 times. Is a substrate binding force regulator.
The invention according to claim 7 is a molecular sensor having specificity for a predetermined substrate, wherein a factor having specificity for the substrate and a binding force between the substrate and the factor are adjusted. A substrate binding force adjusting agent, wherein the substrate binding force adjusting agent is inactive with the substrate and the factor, and is dissolved at a predetermined concentration in a solution containing the factor, and the factor of the factor A molecular sensor comprising a compound or a salt or derivative thereof that changes an association constant (Ka / M ⁻¹ ) for a substrate at a ratio of at least 3 times.
The invention according to claim 8 is the molecular sensor according to claim 7, wherein the substrate is a carbohydrate.
In the invention according to claim 9, when the molecular sensor according to claim 7 is used as a reagent for detecting a substrate, the factor of the molecular sensor and the substrate binding force adjusting agent are allowed to coexist in the reagent solution, and then the reagent A method of using a molecular sensor, wherein the sample is mixed in a solution and stirred so as to reach an equilibrium state, and a binding degree between the factor and a substrate in the sample is calculated.

According to the first to fourth aspects of the invention, it is possible to change the substrate concentration range detectable by the molecular sensor by changing the binding force between the substrate and the molecular sensor.
According to the fifth aspect of the present invention, the sugar concentration range detectable by the molecular sensor can be changed by changing the binding force between the carbohydrate as the substrate and the molecular sensor.
According to the invention which concerns on Claim 6, the glucose concentration range which can be detected with a molecular sensor can be changed by changing the binding force of glucose which is a saccharide | sugar as a substrate, and a molecular sensor.
According to the invention which concerns on Claim 7, the molecular sensor which can change the binding force with a substrate and can change the detection range of a substrate easily can be provided.
According to the invention which concerns on Claim 8, the molecular sensor which can change the binding force with the saccharide | sugar as a substrate and can change the detection range of saccharide | sugar easily can be provided.
According to the ninth aspect of the present invention, when the molecular sensor is used as a reagent for detecting a substrate, the binding force between the substrate and the molecular sensor is changed by a simple operation of adding a substrate binding force adjusting agent to the reagent solution. The detection range of the substrate can be changed.

(A) is explanatory drawing which shows the outline | summary of embodiment of the substrate binding force adjusting agent which concerns on this invention, and a molecular sensor using the same, (b) is based on the presence or absence of use of the substrate binding force adjusting agent shown to (a). It is explanatory drawing which shows the output of a molecular sensor typically. (A) is explanatory drawing which shows the molecular sensor used in Example 1, (b) is explanatory drawing which shows the operation principle which the molecular sensor shown to (a) does not show fluorescence in the absence of carbohydrate, (c) is It is explanatory drawing which shows the operation | movement principle in which the molecular sensor shown to (a) shows fluorescence in presence of saccharide | sugar. FIG. 3 is an explanatory view showing the structure of a substrate binding force adjusting agent used in Example 1. In Example 1, it is a graph which shows the fluorescent carbohydrate titration plot of the molecular sensor in the solution which contains 0.5M of betaines 1-5 as a substrate binding force regulator. In Example 1, it is a graph which shows the fluorescent carbohydrate titration plot of the molecular sensor in the solution which contains TBAC, acetamide, DME, and DMF 0.5M as a substrate bond strength adjusting agent. In Example 1, it is a graph which shows the fluorescent carbohydrate titration plot of the molecular sensor in the solution which contains PEG600, PEG6000, and PVP 0.5M in monomer concentration conversion as a substrate binding strength adjusting agent. (A) is a graph showing a fluorescent carbohydrate titration plot of a molecular sensor when the betaine type additive used in Example 1 is not added, and (b) is a graph showing a Benesi-Hildebrandt plot in (a). . (A) is a graph showing a fluorescent carbohydrate titration plot of a molecular sensor when the betaine type additive (betaine 4 in this example) used in Example 1 is added, and (b) is a Benesi-Hildebrandt in (a). It is a graph which shows a plot. In Example 2, it is a graph which shows the fluorescence carbohydrate titration plot of the molecular sensor in the solution which contains betaine 1 to the density | concentration of 0-0.75M as a substrate bond strength adjusting agent. In Example 2, it is a graph which shows the fluorescent carbohydrate titration plot of the molecular sensor in the solution which contains betaine 5 in the density | concentration of 0-0.75M as a substrate binding force regulator. In Example 4, the calibration curves for the glucose concentration of the molecular sensor in a solution containing 1.0 × 10 ⁻⁴ M each of fructose and galactose as contaminants in the absence of a substrate binding force regulator, respectively. It is explanatory drawing shown. In Example 4, a molecular sensor in a solution containing betaine 4 as a substrate binding force adjusting agent at a concentration of 0.25 M, glucose alone, and fructose and galactose as contaminants at 1.0 × 10 ⁻⁴ M, respectively. It is explanatory drawing which shows the calibration curve with respect to glucose concentration, respectively.

Outline of Embodiment FIG. 1A is an explanatory diagram showing an outline of an embodiment of a molecular sensor to which the present invention is applied.
In the figure, the molecular sensor 2 has specificity for a predetermined substrate 1 and has a factor 3 having specificity for the substrate 1 and a binding force between the substrate 1 and the factor 3. A substrate binding force adjusting agent 4 to be adjusted, and the substrate binding force adjusting agent 4 is inactive with the substrate 1 and the factor 3 and is dissolved in a solution containing the factor 3 at a predetermined concentration. 3 or a compound or a salt or derivative thereof that changes the association constant (Ka / M ⁻¹ ) of 3 to the substrate 1 at a ratio of at least 3 times.
In the present embodiment, the substrate 1 is mainly assumed to be a carbohydrate (such as glucose) for diabetes diagnosis, but is not limited to this. Ion components, umami components, nutrient components, environmental components , Bioactive ingredients, disease-related ingredients, scent ingredients, and the like.
Further, the molecular sensor 2 is required to include at least a factor 3 having specificity for the predetermined substrate 1, and the factor 3 is attached to the predetermined substrate 1 as shown in FIG. On the other hand, since it reacts specifically and changes to the conjugate 5, it is possible to detect the abundance of the substrate 1 by quantifying the appearance degree of the conjugate 5. At this time, the quantification method of the appearance degree of the binding substance 5 is determined from changes in the fluorescence spectrum, absorption spectrum, chemiluminescence spectrum, circular dichroism spectrum, electrochemical signal, etc. in consideration of the characteristics of the molecular sensor 2. Is possible.

Further, the substrate binding force adjusting agent 4 is required to have the following requirements.
That means
(1) Inactive with substrate 1 and factor 3;
(2) dissolving at a predetermined concentration in a solution containing factor 3;
(3) It is a compound that changes the association constant (Ka / M ⁻¹ ) of factor 3 to substrate 1 at a ratio of at least 3 times, or a salt or derivative thereof,
Cost.
Regarding (1), if the substrate binding force adjusting agent 4 is active with respect to the substrate 1 or the factor 3, the substrate binding force adjusting agent 4 affects the binding reaction between the substrate 1 and the factor 3, and is based on the molecular sensor. This is not preferable in that an error is included in the detected amount of the substrate.
Regarding (2), it is not preferable that the substrate binding force adjusting agent 4 is insoluble in the solution because it is difficult to uniformly distribute the solution in the solution. As for the concentration of the substrate binding strength adjusting agent 4 with respect to the solution, the binding strength of the substrate binding strength adjusting agent 4 to the substrate 1 is adjusted according to the concentration of the factor 3 of the molecular sensor 2 and the amount of the substrate 1 to be detected. If the concentration is set too high depending on the characteristics of the substrate binding force adjusting agent 4, the viscosity of the solution increases and the response speed of the molecular sensor 2 decreases. There are concerns.

Further, the association constant (Ka / M ⁻¹ ) is determined by the predetermined substrate 1, the molecular sensor 2 having the factor 3 specific to the substrate 1, and the conjugate 5 that is a complex of the substrate 1 and the factor 3. When the respective concentrations are [A], [B], and [AB] in the equilibrium state in the solution, the following equation (A) is established.
Ka = [AB] / [A] ・ [B] (b)
At this time, when the substrate binding force adjusting agent 4 is not added, the association constant Ka is uniquely determined by the combination of the molecular sensor 2 and the substrate 1 as long as the solution physical properties such as temperature, pH, and viscosity do not change.
Under such circumstances, when the substrate binding strength adjusting agent 4 is added to the solution, the binding strength of the substrate 1 and the factor 3 is indirectly changed by dissolving the substrate binding strength adjusting agent 4 in the aqueous solution. It is presumed to have an effect.
In the present embodiment, if the ratio of the association constant Ka to the value α when the substrate binding force adjusting agent 4 is not used is changed as a result of the addition of the substrate binding strength adjusting agent 4, the binding force between the substrate 1 and the factor 3 is changed. However, when the rate of change of the association constant Ka is small, the rate of change of the binding force by the substrate binding force adjusting agent 4 is also small. Therefore, in this example, the ratio between the association constant Ka and α is It was decided to select it as a guideline to become 3 times or more.

Now, according to the comparative embodiment in which the substrate 1 and the factor 3 of the molecular sensor 2 are bound with the association constant Ka = α without adding the substrate binding force adjusting agent 4, for example, the one-dot chain line I in FIG. As shown, the output signal corresponding to the amount of the combined substance 5 of the substrate 1 and the factor 3 changes according to the concentration of the substrate 1. At this time, the range (for example, the range between the detection lower limit value and the detection upper limit value of the output signal) in which a change corresponding to the concentration of the substrate 1 can be detected in the output signal corresponding to the amount of the bound substance 5 is shown in FIG. ), For example, an area indicated by S ′.
On the other hand, in the present embodiment, by adding the substrate binding force adjusting agent 4, the association constant Ka between the substrate 1 and the factor 3 of the molecular sensor 2 (assuming a ratio of 3 times or more) Changes, and the amount of the binding product 5 between the substrate 1 and the factor 3 is suppressed. For example, as shown by the solid line II in FIG. The output signal corresponding to the amount is shifted to a region indicated by S in FIG.

Next, typical examples of the substrate binding force adjusting agent 4 used in the present embodiment include the following.
(A) A compound represented by the following general formula (1) or a salt or derivative thereof.

  Here, R1 to R3 may be uniform or non-uniform, and an alkyl having 1 to 12 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 3 to 7 carbon atoms. Any of a group, an alkenyl group, an alkynyl group or an aryl group, the total carbon number of which is 3 to 24, preferably the total carbon number is 6 to 18 and more preferably the total carbon number is 12 to 15 R6 is an alkylene group, alkenylene group, alkynylene group or arylene group having 1 to 10 carbon atoms, preferably 1 to 10 carbon atoms.

(B) Tetraalkylammonium salt or derivative.
Here, the alkyl group introduced into the ammonium group may be uniform or heterogeneous and has 1 to 11 carbon atoms, preferably 2 to 8 carbon atoms, and more preferably 3 carbon atoms. The total carbon number is 4 or more and 32 or less, preferably 8 or more and 24 or less, and more preferably 12 or more and 20 or less. For example, tetramethylammonium chloride, tetraethylammonium chloride, tetrapropylammonium chloride, tetrabutylammonium chloride, tetrapentylammonium chloride, tetrahexylammonium chloride, tetraheptylammonium chloride, tetraoctylammonium chloride, tetradecylammonium chloride, tetraundecylammonium There are chloride, dibutyldimethylammonium chloride, dioctyldimethylammonium chloride, didecyldimethylammonium chloride, ethyltripropylammonium chloride, tributylmethylammonium chloride, trioctylmethylammonium chloride and the like. The counter anion may have any structure, and instead of chloride, fluoride, bromide, iodide, hydroxide, tetrafluoroborate, tetraphenylborate, hexafluorophosphate, perchlorate ion, chlorate Ions, chlorite ions, hypochlorite ions, acetate ions, nitrate ions, nitrite ions, sulfate ions, sulfite ions, and the like may be used.

(C) Polyethylene glycol, polyvinyl pyrrolidone or a derivative thereof, polypropylene glycol, polyacrylic acid, polyacrylamide, poly-N, N-dimethylacrylamide or a copolymer thereof.
Here, the molecular weight of the polymer suitable for use is, for example, 1000 or more and 4000000 or less, preferably 1000 or more and 20000 or less, and more preferably 2000 or more and 8000 or less in the case of polyethylene glycol. Moreover, in the case of polyvinylpyrrolidone, it is 1000 or more and 360000 or less, Preferably it is 1000 or more and 1300000 or less, More preferably, it is 2000 or more and 80000 or less. All of these values can be changed depending on the water solubility of the polymer, and it is necessary to appropriately adjust and examine the solubility of the polymer.

  Further, when the molecular sensor 2 according to the present embodiment is used as a reagent for detecting the substrate 1, the factor 3 of the molecular sensor 2 and the substrate binding force adjusting agent 4 are allowed to coexist in the reagent solution, and then the reagent solution. The sample may be mixed and stirred so as to reach an equilibrium state, and the degree of binding between the factor 3 and the substrate 1 in the sample may be calculated.

Hereinafter, the present invention will be described in detail based on embodiments shown in the accompanying drawings. The following examples are merely examples, and are presented to demonstrate the effect of the substrate binding force adjusting agent of the molecular sensor, and are not limited thereto.
Example 1
Various molecular sensors have been developed depending on the type of target substrate. In this embodiment, a carbohydrate sensor that detects a carbohydrate will be described as an example.

  The carbohydrate sensor used in this example is a 9,10-bis [[N-methyl-N- (o--) represented by the following general formula (2), which is a fluorescent molecular sensor having specificity for glucose. Boronobenzyl) amino] methyl] anthracene (hereinafter referred to as a carbohydrate sensor) was used. Moreover, glucose, galactose, and fructose were used for the target carbohydrate.

This carbohydrate sensor is described in detail in, for example, Non-Patent Document 1 (paragraph [0003]). The principle of operation will be briefly described with reference to FIG.
As shown in FIG. 2A, this carbohydrate sensor is a molecular sensor that expresses high glucose affinity when two boronic acids act cooperatively. In this example, fluorescence sensing of glucose using a carbohydrate sensor uses the mechanism of PET (Photo-induced Electron Transfer) shown in FIGS. In the absence of a saccharide (glucose in this example), as shown in FIG. 2 (b), when a fluorescent anthracene molecule is irradiated with light, electrons are excited from HOMO to LUMO and at the same time adjacent amines. Since the electrons in the non-bonding orbital of the electron transfer to the HOMO of the anthracene molecule, it does not exhibit fluorescence. On the other hand, in the presence of a saccharide (glucose in this example), as shown in FIG. 2C, the Lewis acidity of the boron atom increases with the formation of a complex between the saccharide and the boronic acid. As a result, BN The interaction becomes stronger and the nonbonding orbitals of the amine are stabilized. This eliminates quenching due to electron transfer in the non-bonding orbitals of the amine and restores the fluorescence of the anthracene molecule.

With this type of carbohydrate sensor, glucose can be quantified in a wide pH range of pH 4-11. However, it is the detection range that becomes a problem when using the carbohydrate sensor. In general, the blood glucose level of normal humans is 80 to 100 mg / dl (about 4.4 to 5.5 mM) on an empty stomach, and the highest blood glucose level is over 200 mg / dl in diabetic humans.
Since the glucose detection range of this carbohydrate sensor is about 1.8 to 18.1 mg / dl, it is lower than the blood glucose concentration range of humans and is not suitable for detection unless the sample is diluted. However, dilution of the specimen may require a dilution of several hundreds at the maximum, resulting in a dilution error or adjustment of the dilution factor depending on the blood glucose level. Therefore, it is indispensable to individually develop multiple types of carbohydrate sensors with association constants that fall within the detection range suitable for diabetes diagnosis, but molecular design of boronic acid carbohydrate sensors that allow for association constants is difficult. I will master it.

Therefore, in this example, a substrate binding force adjusting agent that adjusts the binding force between the substrate and the carbohydrate sensor is used.
In this example, as shown in FIG. 3, as the substrate binding force adjusting agent, betaines 1 to 5, tetrabutylammonium chloride (TBAC), acetamide, N, N-dimethylformamide (DMF), 1,2-dimethoxy Ethane (DME), polyethylene glycol (PEG) (molecular weight 600 and 6000) (hereinafter referred to as PEG 600 and PEG 6000) and polyvinyl pyrrolidone (PVP) (molecular weight 40000) were used. Any of the substrate binding force adjusting agents is inactive with a boronic acid group that is a factor of a molecular sensor. Since boronic acid groups reversibly form ester bonds with diols, molecules having such functional groups do not serve as substrate binding force modifiers.

<Evaluation of structure dependence of substrate binding force modifier>
In order to examine a substrate binding force regulator having a high effect on the carbohydrate sensor in this example, a binding experiment between a carbohydrate sensor and glucose was performed under the following conditions.
First, a phosphate buffer solution (pH 7.5), a sugar sensor, a substrate binding force adjusting agent, and glucose are added to a 1.5 ml plastic tube at final concentrations of 0.05 M, 1.2 × 10 ⁻⁵ M, 0, respectively. .5M (in the case of polymer, adjusted as monomer unit concentration) 1.0 × 10 ^{−6 to} 3.0 × 10 ⁻¹ M are blended, and 34% (volume fraction) of methanol is added. An aqueous solution containing was prepared. The prepared sample solution was transferred to a 96-well multiplate for fluorescence measurement, allowed to stand for 30 minutes at 25 ° C. in a light-shielded state, and then the fluorescence intensity (excitation wavelength: 379 nm, fluorescence wavelength: 425 nm) of each sample was measured. However, when PVP was used as a substrate binding force adjusting agent, the equilibrium was not reached in 30 minutes. Therefore, the measurement was performed after allowing to stand at 25 ° C. for 60 minutes in the dark.
The obtained fluorescence intensity was plotted against the glucose concentration to prepare a fluorescent carbohydrate titration plot. For comparison, the measurement was also performed under the condition (control) in which the substrate binding force adjusting agent was not added.
In this experiment, the binding of glucose as a substrate to the carbohydrate sensor appears as an increase in fluorescence intensity at 425 nm.
The evaluation of the structure dependence of the substrate binding force regulator was performed using the detection range of the carbohydrate sensor and the association constant Ka obtained from the fluorescent carbohydrate titration plot.

-Creation of fluorescent carbohydrate titration plots-
In this example, the fluorescent carbohydrate titration plot was created with the horizontal axis representing the glucose concentration in logarithmic terms and the vertical axis representing the relative fluorescence intensity.
FIG. 4 shows a fluorescent carbohydrate titration plot of a carbohydrate sensor in a solution containing 0.5M betaines 1 to 5 as a substrate binding force adjusting agent.
FIG. 5 shows a fluorescent carbohydrate titration plot in a solution containing 0.5 M TBAC, acetamide, DME, and DMF as a substrate binding force adjusting agent.
Further, FIG. 6 shows a fluorescent carbohydrate titration plot in a solution containing 0.5 M of PEG600, PEG6000, and PVP as a substrate binding force adjusting agent in terms of monomer concentration.

-Detection range and association constant of carbohydrate sensor-
The glucose detection range by the carbohydrate sensor is obtained by converting the vertical axis of the fluorescent carbohydrate titration plot into the amount of change in fluorescence intensity, setting the lower limit value to 0% and the saturation value to 100%, and creating a calibration curve concentration range that specifies the glucose concentration ( The amount of change in fluorescence intensity was 20 to 80%.
On the other hand, the association constant Ka of the carbohydrate sensor was calculated using the Benesi-Hildebrandt equation (b).

However, in the formula (b), Δ: change amount Δt: change amount at saturation Ka: association constant [G] t: total guest concentration (in this example, the guest is glucose concentration, and the host described below is sugar Quality sensor).
This equation can be used only when [G] t >> [H] t holds (G: guest, H: host).
An approximate straight line based on the above equation can be obtained by taking a Benesi-Hildebrandt plot with the horizontal axis 1 / [G] t and the vertical axis 1 / Δ. The association constant was calculated from the slope of the approximate line.
More specifically, for example, as shown in FIG. 7 (a), a fluorescent carbohydrate titration plot of a carbohydrate sensor under the condition that a substrate binding force adjusting agent is not added is prepared, and based on this, FIG. As shown in b), a Benesi-Hildebrandt plot was taken and the association constant Ka = 3230M ⁻¹ was calculated.
Further, for example, as shown in FIG. 8 (a), a fluorescent carbohydrate titration plot of a carbohydrate sensor under the condition that betaine 4 (0.5M) is added as a substrate binding force adjusting agent is prepared. As shown in FIG. 8B, a Benesi-Hildebrandt plot was taken to calculate an association constant Ka = 421M ⁻¹ .

-Evaluation results-
Table 1 shows the results of the glucose detection range and the association constant Ka by the carbohydrate sensor under the conditions in which the substrate binding strength adjusting agent was not added or in the conditions where each substrate binding strength adjusting agent was added.

Table 1 shows the detection range and association constant of the saccharide (glucose) of the saccharide sensor in a solution containing 0.5 M of the binding force adjusting agent.
According to Table 1, when a substrate binding force regulator that does not bind to a boronic acid group, which is a factor introduced into a carbohydrate sensor, is added, the association constant for glucose of the carbohydrate sensor changes, and accordingly, the glucose detection range. Changes. It can be seen that not all substrate binding force modifiers are effective, and the effect varies depending on the chemical structure of the substrate binding strength modifier. Further, in this example, a great change is induced particularly in the betaine derivative. If the alkyl chain introduced into the ammonium group in the chemical structure of betaine is elongated, the influence on the detection range of glucose becomes strong. In particular, a remarkable effect appeared when the alkyl chain had 3 or more carbon atoms and 9 or more carbon atoms. The shift in the detection range of glucose is not limited to the betaine structure, and the same effect is produced even with TBAC, which is a tetraalkylammonium salt having an intermolecular counterion structure.
On the other hand, low-molecular-weight amide compounds and ether compounds such as acetamide, DMF, and DME have little effect. Polymerized products have little effect on low-molecular-weight PEG600, and high-molecular-weight PEG6000 and PVP have remarkable effects. It was confirmed.
In addition, about polymerized PEG6000 and PVP, when there is too much addition amount, since there exists a possibility that the viscosity of a solution will rise and the response speed of a carbohydrate sensor may fall, it needs to pay attention to this point.

  Thus, in this example, in order to greatly change the detection range of the carbohydrate sensor, the ammonium bond has 3 or more carbon chains (total carbon number of 9 or more), preferably 4 or more (as a substrate binding force adjusting agent). A betaine derivative (betaine 3-5) having an alkyl chain having a total carbon number of 12 or more, more preferably 5 or more (total carbon number of 15 or more), a tetraalkylammonium salt, PEG6000, or PVP is effective. It was confirmed.

Example 2
<Evaluation of substrate concentration adjustment agent dependence on concentration>
In Example 1, a betaine having an alkyl chain with 3 or more carbon atoms (total carbon number 9 or more), preferably 4 or more (total carbon number 12 or more), more preferably 5 or more (total carbon number 15 or more) in the ammonium group. It has been verified that derivatives (betaines 3 to 5), tetraalkylammonium salts and the like strongly influence the substrate binding property of a carbohydrate sensor as a molecular sensor.
In Example 2, betaine 5 which strongly influenced the substrate binding property of the carbohydrate sensor among the substrate binding force adjusting agents was used, and the concentration of the substrate binding force adjusting agent exerted on the substrate binding property of the carbohydrate sensor. The impact was examined. In the experiment, betaine 1, which had a small change in substrate binding, was used as a control for comparison.

The addition concentration of the substrate binding force adjusting agent and the glucose binding property of the carbohydrate sensor were evaluated under the following conditions.
First, a phosphate buffer solution (pH 7.5), a sugar sensor, betaine 1 or betaine 5 as a substrate binding force adjusting agent, and glucose are added to 0.05 ml, 1.2 × 10 ^{− in} a 1.5 ml plastic tube, respectively. ^It mix | blended so that it might become 5M, 0-0.75M, 1.0 * 10 < ^-6 > -6.0 * 10 < ^-1 > M, and the aqueous solution containing 34% (volume fraction) of methanol was prepared. The prepared sample solution was transferred to a 96-well multiplate and allowed to stand at 25 ° C. for 30 minutes in the dark. After that, the fluorescence intensity (excitation wavelength: 379 nm, fluorescence wavelength: 425 nm) was measured in the same manner as in Example 1, A plot was created.
In the same manner as in Example 1, from the fluorescent carbohydrate titration plot, the abscissa glucose concentration was logarithmically displayed, and the ordinate was created as relative fluorescence intensity.
Specifically, FIG. 9 shows a fluorescent carbohydrate titration plot of a carbohydrate sensor in a solution containing betaine 1 at a concentration of 0 to 0.75 M as a substrate binding force adjusting agent.
In addition, FIG. 10 shows a fluorescent carbohydrate titration plot of a carbohydrate sensor in a solution containing betaine 5 at a concentration of 0 to 0.75 M as a substrate binding force adjusting agent.

In the same manner as in Example 1, the detection range for glucose of the carbohydrate sensor and the association constant Ka were calculated from the fluorescent carbohydrate titration plot.
The results are shown in Tables 2 and 3.

Table 2 shows the detection range and association constant of the saccharide (glucose) of the saccharide sensor in a solution containing betaine 1 at a concentration of 0 to 0.75 M as a substrate binding force adjusting agent in Example 2. Table 3 shows the detection range and association constant of the carbohydrate (glucose) of the carbohydrate sensor in a solution containing betaine 5 at a concentration of 0 to 0.75 M as a substrate binding force adjusting agent in Example 2.
According to Table 2, even if the concentration of betaine 1 used as a control was increased, almost no change in the detection range was confirmed. On the other hand, according to Table 3, when the concentration of betaine 5 as a substrate binding force adjusting agent was increased, the upper limit of the glucose detection range was remarkably changed to a high concentration range.
That is, in this example, it was demonstrated that the upper limit of the detection range was expanded to nearly 100 times by adding betaine 5 which is a substrate binding force adjusting agent.
Further, in this example, with respect to the association constant Ka, in the embodiment in which betaine 5 as a substrate binding force adjusting agent was added (the ratio of the association constant was a maximum of 85.0 times), the embodiment in which betaine 1 was added (of the association constant) It is understood that the ratio is sufficiently changed compared to 2.0 times or less).

Example 3
<Evaluation of substrate selectivity of carbohydrate sensor accompanying addition of substrate binding force regulator>
In this example, it was evaluated whether or not a carbohydrate sensor as a molecular sensor having glucose selectivity affects substrate selectivity in the presence of betaine 4 which is a substrate binding force regulator.
As the substrate, in addition to glucose, two types of carbohydrates, fructose and galactose, which are known as impurities in blood glucose level measurement, were used.
The substrate selectivity for the carbohydrate sensor was evaluated under the following conditions.
First, a phosphate buffer solution (pH 7.5), a carbohydrate sensor, a substrate binding force regulator (betaine 4), and a carbohydrate (either glucose, fructose, or galactose) in a final concentration of 1.5 m plastic tube. Is a solution containing 0.05%, 1.2 × 10 ⁻⁵ M, 0M or 0.25M, 1.0 × 10 ^{−6 to} 1.0M, and containing 34% (volume fraction) of methanol. Was prepared. The prepared reaction was transferred to a 96-well multiplate and allowed to stand at 25 ° C. for 30 minutes, and then the fluorescence intensity (excitation wavelength: 379 nm, fluorescence wavelength: 425 nm) was measured to prepare a fluorescent carbohydrate titration plot.
And the association constant Ka with respect to glucose, fructose, and galactose of a carbohydrate sensor was computed from the created fluorescent carbohydrate titration plot. The numerical values in parentheses indicate the association constant ratio (selectivity index) of fructose and galactose to glucose.
The results are shown in Table 4.

According to Table 4, also in fructose and galactose, the change of the association constant by addition of betaine 4 was seen like glucose. This indicates that the substrate binding force modifier acts not only on the substrate glucose, but also on all carbohydrates that can bind to the factor (boronic acid group) introduced into the molecular sensor. .
In addition, it was confirmed that the selectivity for increasing the substrate binding in the order of glucose>flucose> galactose in the carbohydrate sensor was not lost by the addition of betaine 4. It was also found that the ratio of the association constant for glucose and the association constant for fructose and galactose of the carbohydrate sensor was larger under the conditions where betaine 4 was added at 0.25 M than when no betaine was added. Without addition, the ratio of the association constant of glucose and fructose was 5.7 times, but by adding 0.25M betaine 4, it was 9.2 times, and with galactose, the ratio was also increased from 21 times to 32 times. It was confirmed that
Thus, when betaine 4 which is a substrate binding force adjusting agent was added, it was confirmed that fructose and galactose also decreased the association constant in the same manner as glucose. In addition, the substrate selectivity estimated from the association constant ratio proved that the substrate selectivity was higher in the presence of the substrate binding force modifier than in the absence. That is, it is understood that not only the detection range of the carbohydrate sensor as a molecular sensor is expanded but also the substrate selectivity is improved.

Example 4
<Evaluation of influence of impurities on glucose calibration curve>
In this example, a calibration curve for glucose of a carbohydrate sensor as a molecular sensor having glucose selectivity was compared with a solution containing 1.0 × 10 ⁻⁴ M of fructose and galactose as impurities. In order to confirm that the inclusion of the substrate binding force adjusting agent does not affect the calibration curve, a glucose calibration curve was also prepared in the presence of 0.25 M betaine 4.
The substrate selectivity for the carbohydrate sensor was evaluated under the following conditions.
First, phosphate buffer solution (pH 7.5), carbohydrate sensor, substrate binding force regulator (betaine 4), carbohydrate (glucose only, and fructose and galactose to glucose 1.0 in a 1.5 mL Eppendorf tube. Solution containing 10 × ^{4 −4} M at a final concentration of 0.05M, 1.2 × 10 ⁻⁵ M, 0M or 0.25M, 1.0 × 10 ^{−4 to} 3.0 × 10 ⁻³ M, respectively. And an aqueous solution containing 34% (volume fraction) of methanol was prepared. The prepared reaction was transferred to a 96-well multiplate and allowed to stand at 25 ° C. for 30 minutes, and then the fluorescence intensity (excitation wavelength: 379 nm, fluorescence wavelength: 425 nm) was measured to prepare a glucose calibration curve.

FIG. 11 shows calibration curves with respect to the glucose concentration of the carbohydrate sensor in a solution containing 1.0 × 10 ⁻⁴ M each of fructose and galactose as impurities in the absence of a substrate binding force regulator. Show.
Further, a carbohydrate sensor in a solution containing betaine 4 at a concentration of 0.25 M as a substrate binding force regulator and containing only glucose and 1.0 x 10 ^-4 M each of fructose and galactose as impurities. FIG. 12 shows calibration curves for the glucose concentration of each.
11 and 12, it is understood that the logarithmic approximation line containing only glucose and the logarithmic approximation line containing glucose and contaminants (fructose, galactose) substantially coincides with each other. That is, it was confirmed that the concentration of fructose and galactose in the human body had no effect on glucose detection by the carbohydrate sensor.

As is clear from Examples 1 to 4 above, for example, betaines 3 to 5, which are betaine-type additives as substrate binding force modifiers, change the association constant of a carbohydrate sensor as a molecular sensor, and perform a dilution operation. It was confirmed that the detection range was shifted to the high concentration side without passing.
In addition, it is understood that the addition of betaines 3-5 increases the substrate selectivity in a concentration-dependent manner. From these results, it was confirmed that the hydration effect of betaines 3 to 5 had some influence on the binding between the carbohydrate sensor as a molecular sensor and the substrate, and caused a change in the substrate binding force.
In addition, it was confirmed that the substrate binding strength adjusting agents other than the betaine type additives (for example, betaines 3 to 5) have the same action as the betaine type additives.
In addition, as an application of betaine-type additives (for example, betaines 3 to 5) to a carbohydrate sensor for diabetes, the detection range is close to the standard value for diabetes 200 mg / dl, and measurement is not affected by impurities. It can be used as a measurement technique. And it is expected that the range including the reference value of diabetes can be measured by changing the solvent condition and the type of betaine type additive.

  DESCRIPTION OF SYMBOLS 1 ... Substrate, 2 ... Molecular sensor, 3 ... Factor, 4 ... Substrate binding force regulator, 5 ... Conjugate

Claims (9)

A substrate binding force adjusting agent which is added to a solution containing a molecular sensor having a factor specific to a predetermined substrate and adjusts the binding force between the substrate and the molecular sensor,
The substrate and the factor are inactive, and are dissolved at a predetermined concentration in a solution containing the factor, and the ratio of the association factor (Ka / M ⁻¹ ) of the factor to the substrate is at least 3 times or more. A substrate binding force adjusting agent comprising a compound or a salt or derivative thereof which is changed by the above. The substrate binding force adjusting agent according to claim 1,
A substrate binding force regulator comprising a compound represented by the following general formula (1) or a salt or derivative thereof.

Here, R1 to R3 may be uniform or non-uniform, and an alkyl having 1 to 12 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 3 to 7 carbon atoms. Any of a group, an alkenyl group, an alkynyl group or an aryl group, the total carbon number of which is 3 to 24, preferably the total carbon number is 6 to 18 and more preferably the total carbon number is 12 to 15 R6 is an alkylene group, alkenylene group, alkynylene group or arylene group having 1 to 10 carbon atoms, preferably 1 to 10 carbon atoms. The substrate binding force adjusting agent according to claim 1,
A substrate binding force regulator comprising a tetrabutylammonium salt or derivative. The substrate binding force adjusting agent according to claim 1,
A substrate binding force regulator comprising polyethylene glycol, polyvinyl pyrrolidone or a derivative thereof, or polypropylene glycol, polyacrylic acid, polyacrylamide, poly-N, N-dimethylacrylamide or a copolymer thereof. The substrate binding force adjusting agent according to any one of claims 1 to 4,
A substrate binding force regulator, wherein the substrate is a carbohydrate. In the substrate binding force regulator according to claim 5,
When the substrate is glucose as a carbohydrate, the substrate binding force adjusting agent is characterized by adjusting the association constant of the molecular sensor at a ratio of 3 to 100 times. A molecular sensor having specificity for a predetermined substrate,
A factor specific for the substrate;
A substrate binding force adjusting agent that adjusts the binding force between the substrate and the factor,
The substrate binding force adjusting agent is inactive with the substrate and the factor, dissolves at a predetermined concentration in a solution containing the factor, and associates the factor with the substrate (Ka / M ^−1). ), Or a salt or derivative thereof, which changes the compound at a ratio of at least 3 times. The molecular sensor according to claim 7,
A molecular sensor, wherein the substrate is a carbohydrate.   When the molecular sensor according to claim 7 is used as a reagent for detecting a substrate, the factor of the molecular sensor and the substrate binding force adjusting agent are allowed to coexist in the reagent solution, and then a sample is mixed into the reagent solution. And a molecular sensor is used to calculate the degree of binding between the factor and the substrate in the sample.

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