JP2007225521A - Metal ion sensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a metal ion sensor capable of simply and accurately measuring the amount of metal ions. <P>SOLUTION: The metal ion sensor 1 is constituted so as to measure the amount of the metal ions in a liquid sample 20 and has a sample housing space 2 for housing the liquid sample 20, the metal ion receiving layer 6 at least partially exposed to the sample housing space 2 and containing metal storage protein capable of taking in metal ions, the acting electrode 3 provided on the side opposite to the sample housing space 2 of the metal ion receiving layer 6, a reference electrode 5 at least partially exposed to the sample housing space 2 and applying voltage across the acting electrode 3 and the reference electrode 5 and a counter electrode 4 for detecting electrons discharged accompanied by the oxidation reaction of the metal ions taken in the metal storage protein as the value of the current flowing across the acting electrode 3 and the counter electrode 4. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a metal ion sensor.

In recent years, there has been an increasing demand for sensors that can detect biological reactions involving biomolecules such as enzymes, DNA, and antibodies in real time, not in vivo, in vitro.
In particular, after the completion of genome analysis, the specific gravity has shifted from genome analysis to functional analysis of DNA strands. In particular, functional analysis of proteins such as enzymes and antibodies expressed from DNA strands, and optimal drug discovery targets in line with those functions Is becoming important.

In order to efficiently perform these analyses, it is effective to use a sensor such as a DNA chip or a protein chip. This sensor is required to have a function of selecting and amplifying useful information parameters from biological reaction information, converting them into detection parameters, and transmitting them to detection means.
As one of such sensors, an electrochemical detection device using an enzyme has been developed. For example, a blood glucose measuring device in which a reaction layer containing an oxidase (glucose oxidase or glucose dehydrogenase) that catalyzes an oxidation reaction of glucose is fixed to an electrode substrate has been proposed (for example, see Patent Documents 1 and 2).

In this blood glucose measurement device, glucose in the blood is diffused into the reaction layer and oxidized by the oxidase, and at this time, the generated electrons are detected as a current value, thereby measuring the glucose concentration in the blood.
As described above, a sensor capable of detecting various substances in a liquid sample such as blood has been developed. However, a sensor capable of detecting metal ions simply and accurately (metal ion sensor) has not yet been developed. Not.

Japanese Patent Laid-Open No. 6-78791 JP-A-6-90754

  An object of the present invention is to provide a metal ion sensor that can easily and accurately measure the amount of metal ions.

Such an object is achieved by the present invention described below.
The metal ion sensor of the present invention is a metal ion sensor that measures the amount of metal ions to be measured in a liquid sample,
A sample storage space for storing the liquid sample;
A metal ion receiving layer containing a metal storage protein that is at least partially exposed to the sample storage space and capable of taking in the metal ions to be measured;
A first electrode provided on the opposite side of the metal ion receiving layer from the sample storage space;
A second electrode that is at least partially exposed to the sample storage space and applies a voltage to the first electrode;
Electrons emitted as the valence of the metal ion to be measured incorporated into the metal storage protein changes from the first valence to the second valence, And a third electrode for detecting a current value flowing therebetween.
Thereby, the metal ion sensor which can measure the quantity of a metal ion simply and correctly can be obtained.

In the metal ion sensor of the present invention, the metal storage protein includes a metal ion of the same type as the metal ion to be measured, and the valence of the included metal ion is the second valence or the second valence. A third valence different from the valence of 1 and the second valence, and when measuring the amount of the metal ion to be measured between the first electrode and the second electrode It is preferable that at least a part of the encapsulated metal ions is released by applying a voltage opposite to the above voltage.
Thereby, if a reverse voltage is applied after completion | finish of the measurement of the quantity of metal ion, a metal ion acceptance layer can be returned to the state before a measurement. That is, the metal ion sensor can be returned to the initial state. For this reason, a metal ion sensor can be used repeatedly.

In the metal ion sensor of the present invention, the metal ion to be measured is Fe ²⁺ .
It is preferable that the metal storage protein is mainly composed of ferritin.
Ferritin contains an extremely large amount of Fe ³⁺ and can efficiently take in Fe ²⁺ and change it into Fe ³⁺ . Further, when the ^{Fe 3+} changes to ^{Fe 2+,} reliably discharged outside the molecule ^{Fe 2+.} In this ferritin, iron ions with different valences are preferably taken in and out very quickly and with high accuracy.

In the metal ion sensor of the present invention, the content of the metal storage protein in the metal ion receiving layer is preferably 10 to 60 wt%.
Thereby, when the metal ion of a measuring object is contained in a liquid sample, this metal ion can be efficiently taken in into a metal storage protein, and the quantity can be measured correctly.

In the metal ion sensor of the present invention, in the metal ion receiving layer, the metal storage protein is preferably unevenly distributed on the sample storage space side.
Thereby, a liquid sample and a metal storage protein can be made to contact reliably, and the metal ion in a liquid sample can be taken in more efficiently by a metal storage protein.
In the metal ion sensor of the present invention, it is preferable that the metal storage protein is fixed by a fixing substance in the metal ion receiving layer.
As a result, the metal storage protein can be prevented from diffusing into the liquid sample, and the amount of metal ions can be measured more accurately.

In the metal ion sensor of the present invention, the immobilizing substance may be a compound having a first binding group that binds to the first electrode and a second binding group that binds to the metal storage protein. preferable.
Thereby, the metal storage protein can be reliably separated from the upper surface of the first electrode by the chain length of the compound. As a result, it is possible to suitably prevent the metal storage protein from contacting with the first electrode and being denatured or deactivated.
In the metal ion sensor of the present invention, it is preferable that the immobilizing substance is mainly composed of a biological polymer or a modified product thereof.
By using these polymers as a fixing substance, it is possible to suitably prevent the metal storage protein from being easily denatured and inactivated.

In the metal ion sensor of the present invention, it is preferable that the metal ion accepting layer includes a mediator that mediates transfer of electrons between the metal storage protein and the first electrode.
Thereby, electrons can be efficiently transferred between the metal storage protein and the first electrode.

In the metal ion sensor of the present invention, it is preferable that a part of the mediator is fixed to the first electrode via a spacer molecule.
In such a configuration, by using spacer molecules having a relatively short difference length, it is possible to ensure that some mediators are positioned in the vicinity of the first electrode, and the remaining mediators are made of metal. It can be in a dispersed state in the ion receiving layer. As a result, for example, electrons emitted from the metal storage protein are transferred to a mediator dispersed in the layer, and further to a mediator located in the vicinity of the first electrode, and transferred to the first electrode more smoothly. Can be made. For this reason, the response speed of the metal ion sensor can be improved.
In the metal ion sensor of the present invention, the content of the mediator in the metal ion receiving layer is preferably 0.1 to 10 wt%.
Thereby, an electron can be moved reliably and rapidly between metal storage protein and a 1st electrode.

Hereinafter, a metal ion sensor of the present invention will be described in detail based on a preferred embodiment shown in the accompanying drawings.
1 is a longitudinal sectional view schematically showing the configuration of the metal ion sensor of the present invention, FIG. 2 is a schematic diagram for explaining the operation of the metal ion sensor shown in FIG. 1, and FIG. 3 is shown in FIG. 4 is a schematic view showing an example of a metal storage protein provided in the metal ion sensor, FIG. 4 is a longitudinal sectional view for explaining a method for producing the metal ion sensor shown in FIG. 1, and FIG. 5 is another configuration of the metal ion receiving layer. It is a schematic diagram which shows an example. In the following description, the upper side in FIGS. 1, 2, 4 and 5 is referred to as “upper”, and the lower side is referred to as “lower”.

  A metal ion sensor 1 shown in FIG. 1 includes a sample storage space 2 for storing a liquid sample 20, a working electrode (first electrode) 3, a counter electrode (third electrode) 4, and a reference electrode (second electrode). Electrode) 5 and a metal ion receiving layer 6, and each of these parts is laminated on a substrate 8. The metal ion sensor 1 detects the amount of metal ions (measurement target ions) in the liquid sample 20 supplied to the sample storage space 2 as a current value flowing between the pair of electrodes 3 and 4.

Here, examples of the liquid sample 20 include body fluids such as blood, urine, sweat, lymph, cerebrospinal fluid, bile, and saliva, treated fluids obtained by performing various treatments on these body fluids and foods, food wastes, and household wastewater. And groundwater.
Examples of metal ions to be measured include iron (Fe) ions, copper (Cu) ions, manganese (Mn) ions, cobalt (Co) ions, ruthenium (Ru) ions, nickel (Ni) ions, and zinc (Zn). ) Ions, palladium (Pd) ions, chromium (Cr), and the like, and any other heavy metal ions.

The substrate 8 supports each part constituting the metal ion sensor 1.
Examples of the constituent material of the substrate 8 include various resin materials such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate (PET), polyacrylic acid, and epoxy resin, various glass materials, and various ceramic materials. These can be used alone or in combination of two or more. In addition, as the board | substrate 8 which consists of composite materials, the flame-retardant printed circuit board etc. which are comprised with glass fiber and an epoxy resin are mentioned, for example.

A working electrode 3 is provided on the substrate 8.
As the constituent material of the working electrode 3, the counter electrode 4 and the reference electrode 5, which will be described later, for example, a metal material such as gold, silver, copper, platinum, platinum or an alloy containing them, or a metal oxide such as ITO, respectively. Examples thereof include physical materials and carbon-based materials such as carbon.
A metal ion receiving layer 6 is provided on the working electrode 3.

The metal ion accepting layer 6 includes a metal storage protein 61 and is provided so that at least a part (upper surface) thereof is exposed to the sample storage space 2.
Here, the metal storage protein 61 has a function of taking in the metal ions contained in the liquid sample 20 and oxidizing the taken-in metal ions. And an electron (e < ^- >) is discharge | released with the oxidation reaction of this metal ion.

  Further, the metal storage protein 61 contains the same kind of metal ion having a different valence from the metal ion to be measured, and the amount of metal ion is set between the working electrode 3 and the reference electrode 5 as described later. It is preferable to use one that releases a metal ion having the same valence as the metal ion taken in by applying a voltage opposite to the voltage at the time of measurement. Thereby, if a reverse voltage is applied after the measurement of the amount of metal ions is completed, the metal ion receiving layer 6 can be returned to the state before the measurement. That is, the metal ion sensor 1 can be returned to the initial state. For this reason, the metal ion sensor 1 can be used repeatedly.

When the metal ion to be measured is Fe ²⁺ (iron divalent ion), for example, an iron storage protein such as ferritin or hemosiderin can be used as the metal storage protein 61. In particular, ferritin is the main component. It is preferable to use those that do.
This ferritin is a protein having a molecular weight of about 450,000, which is widely present in hepatocytes, bone marrow and the like. As shown in FIG. 3, ferritin has a spherical structure with a diameter of about 13 nm, and an outer shell is formed by 24 polypeptide subunits. There are two types of subunits, H chain and L chain.

Ferritin contains an extremely large amount of Fe ³⁺ and can efficiently take in Fe ²⁺ and change it into Fe ³⁺ . Further, when the ^{Fe 3+} changes to ^{Fe 2+,} reliably discharged outside the molecule ^{Fe 2+.} In this ferritin, iron ions with different valences are preferably taken in and out very quickly and with high accuracy.
In addition, when the metal ion to be measured is other than Fe ²⁺ , the metal storage protein 61 converts the iron ion stored in the iron storage protein into the same kind of metal ion having a different valence from the metal ion to be measured. What is necessary is just to substitute and use. Thereby, the iron storage protein substituted by other metal ions selectively takes in the metal ions corresponding to the substituted metal ions.

The content of the metal storage protein 61 in the metal ion receiving layer 6 is preferably about 20 to 80 wt%, and more preferably about 40 to 60 wt%. Thereby, when the metal ion of a measuring object is contained in the liquid sample 20, this metal ion can be efficiently taken in into the metal storage protein 61, and the quantity can be measured correctly.
In the metal ion receiving layer 6, the metal storage protein 61 is preferably fixed by a fixing substance. Thereby, the metal storage protein 61 can be prevented from diffusing into the liquid sample 20, and the amount of metal ions can be measured more accurately.
As this fixed substance, it is preferable to use a compound having a first binding group that binds to the working electrode 3 and a second binding group that binds to the metal storage protein 61. In the configuration shown in FIG. 2, the metal storage protein 61 is immobilized on the upper surface of the working electrode 3 through such a compound.

By fixing the metal storage protein 61 in this manner, the metal storage protein 61 can be reliably separated from the upper surface of the working electrode 3 by the chain length of the compound. As a result, it is possible to suitably prevent the metal storage protein 61 from contacting with the working electrode 3 and being denatured or deactivated.
In addition, since the metal storage protein 61 is unevenly distributed on the side of the sample storage space 2 in the metal ion receiving layer 6, the liquid sample 20 and the metal storage protein 61 can be reliably brought into contact with each other. Metal ions can be efficiently taken up by the metal storage protein 61.
The compound used as such a fixing substance is preferably a compound having a linear structure having 50 to 100 (particularly 60 to 80) carbon atoms. Thereby, the metal storage protein 61 can be sufficiently separated from the upper surface of the working electrode 3, and the metal storage protein 61 can be more reliably prevented from coming into contact with the working electrode 3 and being denatured and deactivated. .

  Here, the first binding group is appropriately selected according to the constituent material of the working electrode 3 and is not particularly limited, but the constituent material of the working electrode 3 is gold (Au), silver (Ag), copper ( In the case of Cu) or the like, a thiol group, a thiosulfonate group or the like can be mentioned. When the constituent material of the working electrode 3 is a metal oxide (for example, ITO), a thiol group, a halogen group, an alkoxysilyl group, a halogen Silyl group, phosphoric acid group and the like.

On the other hand, examples of the second binding group include an N-succinimide ester group, an amino group, a disulfide group, and tyrosine (hydroxyl group).
For example, the N-succinimide ester group is substituted with the amino group of the metal storage protein 61, whereby the metal storage protein 61 and the compound are bonded. Further, the amino group forms an amide bond with the carboxyl group of the metal storage protein 61, whereby the metal storage protein 61 and the compound are bonded.
Specific examples of the compound as described above include the following polyethylene glycol compounds.

However, in said formula, X shows a 1st bonding group and n shows an integer greater than or equal to 1.
Such a polyethylene glycol-based compound has an unshared electron pair in the oxygen atom contained therein. For this reason, between the adjacent molecules, the unshared electron pair affects each other and the steric hindrance (rotation barrier) increases. Thereby, the rigidity of the fixing substance is increased, and as a result, the film strength of the metal ion receiving layer 6 is relatively increased. As a result, the metal storage protein 61 and the working electrode 3 can be reliably prevented from coming into contact with each other, and the metal storage protein 61 can be more reliably prevented from being denatured and deactivated. The amount of the target metal ion can be accurately measured. Such an effect is more prominent when n is 11 or more.
In addition, since the polyethylene glycol compound itself is excellent in biocompatibility, the denaturation and inactivation of the metal storage protein 61 can be prevented.

The metal ion accepting layer 6 preferably includes a mediator 62 that mediates the movement of electrons between the metal storage protein 61 and the working electrode 3. Thereby, an electron can be efficiently moved between the metal storage protein 61 and the working electrode 3.
Examples of the mediator 62 include potassium ferricyanide, ferrocene or ferrocene derivatives, nickelocene or nickelocene derivatives, quinone or quinone derivatives (for example, p-benzoquinone, pyrroloquinoline quinone, etc.), flavin derivatives such as flavin adenine dinucleotide (FAD), nicotine Amidoadenine dinucleotide (NAD), nicotinamide derivatives such as nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate, 2,6-dichlorophenolindophenol, hexacyanoferrate (III), octacyanotungsten An ion etc. are mentioned, Among these, it can use combining 1 type (s) or 2 or more types.

When the metal ion receiving layer 6 includes the mediator 62, the content thereof is preferably about 0.01 to 5.0 wt%, and more preferably about 0.1 to 1.0 wt%. By setting the content of the mediator 62 within the above range, electrons can be reliably and rapidly moved between the metal storage protein 61 and the working electrode 3.
Further, it is preferable that a part of the mediator 62 is fixed to the working electrode 3 through spacer molecules. In such a configuration, by using spacer molecules having a relatively short difference length, a part of the mediators 62 can be surely positioned in the vicinity of the working electrode 3, and the remaining mediators 62 are made to be metal ions. It can be in a dispersed state in the receiving layer 6. As a result, for example, electrons emitted from the metal storage protein 61 are transferred to the mediator 62 dispersed in the layer 6 and further to the mediator 62 located in the vicinity of the working electrode 3, and move to the working electrode 3 more smoothly. Can be made. For this reason, the response speed of the metal ion sensor 1 can be improved.

From this point of view, the spacer molecule is preferably one whose chain length is shorter than the chain length of the compound used as the fixing substance. Specifically, the spacer molecule preferably includes a linear structure having 5 to 40 carbon atoms (particularly 10 to 25).
As the mediator 62 to which such spacer molecules are bound, for example, the following polyethylene glycol compounds are preferably used for the same reason as described above.

In the above formula, X represents a binding group to the working electrode 3, and n represents an integer of 1 or more.
Further, the metal ion receiving layer 6 having the above-described configuration may contain a compound containing a hydroxyl group or a carboxyl group at the terminal and bound to the working electrode 3. By including such a compound, nonspecific protein adsorption to the metal ion accepting layer 6 can be prevented (blocked). As a result, when measuring the amount of metal ions, it is possible to prevent or reduce the detection of currents other than the electrons emitted from the metal storage protein 61, and more accurate current values (the amount of metal ions). ) Can be measured.
As specific examples of such a compound, for the same reason as described above, for example, the following polyethylene glycol compounds are preferably used.

In the above formula, X represents a binding group to the working electrode 3, and n represents an integer of 1 or more.
The counter electrode (third electrode) 4 is disposed so as to face the working electrode 3 so that at least a part (in this embodiment, the lower surface) of the counter electrode (third electrode) is exposed to the sample storage space 2. The counter electrode 4 is an electrode for detecting electrons emitted as a result of the oxidation reaction of metal ions taken into the metal storage protein 61 as a current value flowing between the working electrode 3 and the counter electrode 4.
The reference electrode (second electrode) 5 is fixed to the sealing part (partition wall part) 10 so that a part thereof is located in the sample storage space 20. The reference electrode 5 is an electrode that applies a voltage to the working electrode 3.

In the metal ion sensor 1 as described above, the value of the voltage applied between the working electrode 3 and the reference electrode 5 is changed at a constant speed, for example, while the liquid sample 20 is supplied into the sample storage space 2. (Voltage sweep) and CV (cyclic voltammetry) measurement is performed.
At this time, when the metal sample (hereinafter represented by Fe ²⁺ ) is contained in the liquid sample 20, this Fe ²⁺ is incorporated into the metal storage protein 61 (hereinafter represented by ferritin) of the metal ion receiving layer 6. In the protein shell, it is oxidized to Fe ³⁺ and electrons are emitted.
The emitted electrons move to the working electrode 3 through the mediator 62 in the metal ion receiving layer 6 and are taken out to an external circuit connecting the working electrode 3 and the counter electrode 4, and have a current (oxidation current) value. As measured.

Specifically, the measurement of the amount of Fe ²⁺ contained in the liquid sample 20 is performed as follows, for example.
First, prior to actual measurement, a preparation solution (buffer solution) is supplied into the sample storage space 2 to perform CV measurement. Thereby, the background current is measured.
Next, the actual liquid sample 20 is supplied into the sample storage space 2 and CV measurement is performed. The amount (concentration) of Fe ²⁺ in the liquid sample 20 can be estimated by comparing the CV curve appearing at this time with the background CV curve (see FIG. 4).
It is preferable to fix the voltage at which the peak value appears in the CV curve and compare the current value measured at the voltage value to estimate the amount (concentration) of Fe ²⁺ . Thereby, since the difference in current value becomes clearer, the amount of Fe ²⁺ can be measured more accurately.

Thus, according to the metal ion sensor 1 of the present invention, the amount of Fe ²⁺ (metal ion) can be measured easily and accurately.
When a voltage opposite to the voltage for measuring the amount of Fe ²⁺ is applied between the working electrode 3 and the reference electrode 5, Fe ³⁺ inside ferritin is injected from the working electrode 3 through the mediator 62. It is reduced by the remaining electrons and released out of ferritin as Fe ²⁺ .
For this reason, the applied voltage between the working electrode 3 and the reference electrode 5 is preferably about 0.6 V on the plus side and about 0.3 V on the minus side when measuring the amount of Fe ²⁺ .

After the measurement is completed, an excess Fe ³⁺ incorporated in ferritin is ^removed from Fe by applying a predetermined negative voltage (about −0.5 V) between the working electrode 3 and the reference electrode 5 for several minutes. ^Reduce to ²⁺ and release, and return the amount of Fe ³⁺ in ferritin to the standard level. In addition, this excludes the liquid sample 20 after completion of the measurement, and again supplies the preparation liquid into the sample storage space 2 to perform CV measurement, so that the CV curve at this time almost coincides with the background curve. You can confirm that.
By setting it as such a state, the metal ion sensor 1 can be reused.

Such a metal ion sensor 1 can be manufactured as follows, for example.
[1] First, as shown in FIG. 5A, a substrate 8 is prepared.
Then, the working electrode 3 is formed on the substrate 8 as shown in FIG. The working electrode 3 can be formed as follows.
First, a metal film (metal layer) is formed so as to cover the upper surface (electrode formation surface) of the substrate 8. This includes, for example, chemical vapor deposition (CVD) such as plasma CVD, thermal CVD and laser CVD, dry plating methods such as vacuum deposition, sputtering and ion plating, and wet plating such as electrolytic plating, immersion plating and electroless plating. It can be formed by a method, a thermal spraying method, a MOD method, a metal foil bonding, or the like.

Next, a resist material is applied (supplied) on the metal film and then cured to form a resist layer having a shape corresponding to the shape of the working electrode 3.
Next, unnecessary portions of the metal film are removed using the resist layer as a mask. For the removal of the metal film, for example, one or more of physical etching methods such as plasma etching, reactive ion etching, beam etching, and optical assist etching, and chemical etching methods such as wet etching are used. They can be used in combination.

Thereafter, the working electrode 3 is obtained by removing the resist layer.
Note that, when the patterning of the metal film is unnecessary, the resist layer forming step, the metal film removing step, and the like can be omitted.
In addition, the working electrode 3 is, for example, applied (supplied) on the substrate 8 with a conductive material containing conductive particles, and then subjected to post-processing (for example, heating, infrared irradiation, It can also be formed by applying ultrasonic waves.

  Here, the coating method includes, for example, a spin coating method, a casting method, a micro gravure coating method, a gravure coating method, a bar coating method, a roll coating method, a wire bar coating method, a dip coating method, a spray coating method, and a screen printing method. , Flexographic printing method, offset printing method, ink jet method, microcontact printing method and the like can be used alone or in combination.

[2] Next, the metal ion receiving layer 6 is formed on the working electrode 3.
[2-1] First, as shown in FIG. 5C, a monomolecular film is formed using the polyethylene glycol-based compound as described above.
This is performed, for example, as follows.
First, a polyethylene glycol compound is dissolved in a solvent to prepare a liquid material.

Examples of the solvent include anhydrous ethanol, anhydrous dichloromethane, anhydrous chloroform, anhydrous THF, anhydrous DMF and the like, and these can be used alone or as a mixed solution.
Further, a mediator 62 is added to the liquid material as necessary.
The concentration (content) of the polyethylene glycol-based compound in the liquid material is preferably about 0.1 to 30 mM, and more preferably about 1 to 15 mM.

Next, the liquid material is allowed to stand for a certain period of time in contact with the upper surface of the working electrode 3, and then washed and dried. Thereby, a monomolecular film of a polyethylene glycol compound is obtained on the upper surface of the working electrode 3.
As a method for bringing the liquid material into contact with the upper surface of the working electrode 3, a method (immersion method) in which the substrate 8 on which the working electrode 3 is formed is immersed in the liquid material is suitable.

In this case, the temperature of the liquid material is preferably about 4 to 40 ° C, and more preferably about 10 to 25 ° C.
The immersion time of the substrate 8 is preferably about 30 to 200 minutes, more preferably about 60 to 150 minutes.
As a method of bringing the liquid material into contact with the upper surface of the working electrode 3, a method of applying the liquid material onto the working electrode 3 (coating method) or a method of spraying (spraying method) can also be used.

[2-2] Next, as shown in FIG. 5D, the metal storage protein 61 is disposed on the monomolecular film.
First, the metal storage protein 61 is dissolved in a solvent to prepare a liquid material.
Examples of the solvent include various kinds of water such as pure water, ultrapure water, ion exchange water, distilled water, and RO water, or various buffers in which salts are dissolved in this water.
The concentration (content) of the metal storage protein 61 in the liquid material is preferably about 0.1 to 30 μM, and more preferably about 1 to 15 μM.

Next, this liquid material is allowed to stand for a certain period of time in contact with the upper surface of the monomolecular film, and then washed and dried. Thereby, the metal storage protein 61 is arrange | positioned on the upper surface of a monomolecular film.
Further, when the polyethylene glycol compound has a binding group with the metal storage protein 61 at the terminal, the compound and the metal storage protein 61 are bonded.
As a method for bringing the liquid material into contact with the upper surface of the monomolecular film, the coating method is suitable.
In this case, the temperature of the liquid material and the temperature of the atmosphere are each preferably about 4 to 40 ° C, more preferably about 10 to 25 ° C.

The standing time is preferably about 10 to 150 minutes, and more preferably about 30 to 100 minutes.
As a method for bringing the liquid material into contact with the upper surface of the monomolecular film, an immersion method or a spray method can also be used.
As described above, the metal ion receiving layer 6 in which the metal storage protein 61 is disposed on the monomolecular film is obtained.

[3] Next, as shown in FIG. 5E, the counter electrode 4 is disposed so as to face the working electrode 3, and the reference electrode 5 is disposed at a predetermined position, for example, an adhesive. Is supplied to the outer peripheral portion and then cured to form the sealing portion 10. Thereby, the sample storage space 2 is defined by the metal ion receiving layer 6, the counter electrode 4, and the sealing portion 10.
Examples of the adhesive include an epoxy adhesive, an acrylic adhesive, and a rubber adhesive.
At this time, an inlet (supply port) 101 for supplying the liquid sample 20 into the sample storage space 2 is formed in the sealing portion 10.
Through the above steps, the metal ion sensor 1 shown in FIG. 1 is obtained.

Next, another configuration example of the metal ion receiving layer 6 will be described.
Hereinafter, the metal ion receiving layer 6 shown in FIG. 6 will be described focusing on the differences from the metal ion receiving layer 6 shown in FIG. 2, and the description of the same matters will be omitted.
The metal ion accepting layer 6 shown in FIG. 6 includes a metal storage protein 61 and a mediator 62 (impregnated) in a matrix (base material) made of a polymer (fixed substance). . Thereby, it is possible to prevent the metal storage protein 61 and the mediator 62 from diffusing into other layers and the liquid sample 20, and to move electrons between the metal storage protein 61 and the working electrode 3 more reliably ( Can be delivered).

  The polymer constituting the matrix is not particularly limited. For example, a natural polymer such as a bio-related polymer (animal-derived polymer) or a plant-derived polymer, a synthetic polymer (synthetic resin), or These modification | denaturation things etc. are mentioned, Among these, it can use combining 1 type (s) or 2 or more types. Among them, the polymer constituting the matrix is preferably a polymer mainly composed of a bio-derived polymer or a modified product thereof. By constituting a matrix using these as a polymer, it is possible to suitably prevent the metal storage protein 61 from being easily denatured and inactivated.

Examples of such bio-related polymers include albumin (for example, bovine serum albumin: BSA), globulin, myoglobin, a mixed polymer of carboxymethyl cellulose and BSA, a mixed polymer of polyvinyl pyrrolidone and BSA, polyethylene glycol and BSA. And a mixed polymer.
Examples of the modified product include those subjected to treatment for breaking the hydrophobic bond, hydrogen bond, and ionic bond of the biological polymer. Examples of such treatment include heat treatment, pressure treatment, pH adjustment treatment, treatment with a denaturing agent, and the like.

The matrix preferably has a cross-linked structure. Thereby, the metal storage protein 61 and the mediator 62 can be firmly held (supported) on the matrix. Moreover, it contributes to the improvement of the mechanical strength of the metal ion receiving layer 6.
As the cross-linking agent that forms a cross-linked structure in the matrix, for example, when a polymer having a peptide as a main component is used, for example, glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, trinitromethane These can be used, and one or more of these can be used in combination.
For such a metal ion receiving layer 6, first, a metal storage protein 61, a mediator 62, and, if necessary, a crosslinking agent is dissolved in a solvent to prepare a liquid material.
Examples of the solvent include various kinds of water such as pure water, ultrapure water, ion exchange water, distilled water, and RO water, or various buffers in which salts are dissolved in this water.

Next, this liquid material is allowed to stand for a certain period of time in contact with the upper surface of the working electrode 3, and then washed and dried. Thereby, the metal ion receiving layer 6 is obtained on the upper surface of the working electrode 3.
As the method for bringing the liquid material into contact with the upper surface of the metal ion receiving layer 6, the coating method is suitable.
In this case, the temperature of the liquid material and the temperature of the atmosphere are each preferably about 4 to 40 ° C, more preferably about 10 to 25 ° C.
The standing time is preferably about 10 to 150 minutes, and more preferably about 30 to 100 minutes.
In addition, as a method for bringing the liquid material into contact with the upper surface of the metal ion receiving layer 6, an immersion method or a spray method can also be used.

Further, between the metal ion accepting layer 6 and the working electrode 3, the purpose of preventing the metal storage protein 61 from contacting the working electrode 3 and the adhesion between the metal ion accepting layer 6 and the working electrode 3 are improved. An intermediate layer may be provided for the purpose and the like.
Examples of the intermediate layer provided for the former purpose include a layer having a configuration in which the metal storage protein 61 is removed from the metal ion accepting layer 6 of the present embodiment.
Moreover, as an intermediate | middle layer provided for the latter objective, the layer comprised combining 1 type (s) or 2 or more types in C1-C5 aminoalkanethiol, hydroxyalkanethiol, etc. is mentioned, for example.

The metal ion sensor 1 as described above can easily and accurately measure the amount of metal ions as described above. In particular, in the case of measuring the amount of Fe ²⁺ as a metal ion, it becomes possible to monitor the amount of Fe ²⁺ in the blood over time, which is considered to be a cause of iron deficiency anemia, aplastic anemia, and the like. For this reason, it becomes possible to more easily and accurately determine the progress and therapeutic effect of these disease states.

As mentioned above, although the metal ion sensor of this invention was demonstrated based on embodiment of illustration, this invention is not limited to these.
For example, each part which comprises the metal ion sensor of this invention can be substituted with the thing of the arbitrary structures which can exhibit the same function.
Further, for example, one or more layers for any purpose (adhesion improvement) may be provided between the layers within a range that does not cause deterioration of the characteristics of the metal ion sensor.

Next, specific examples of the present invention will be described.
1. Production of sensor (Example 1)
<1A> First, a glass substrate was prepared, and a working electrode having an average thickness of 200 nm was formed by vacuum deposition.

<2A> Next, the following three compounds of Chemical Formula 5, Chemical Formula 6 and Chemical Formula 7 were prepared and dissolved in anhydrous dichloromethane so as to have a molar ratio of 2: 1: 7 to prepare a solution. The total concentration of the compounds in this solution was 3 mM. A predetermined amount of potassium ferricyanide was added to the solution.
And the glass substrate was immersed in this solution at 20 degreeC for 2 hours. Then, it took out from the solution, washed with pure water, and dried by nitrogen blowing. Thereby, a monomolecular film was obtained.

<3A> Next, ferritin (metal storage protein) was dissolved in pure water to prepare a ferritin-containing solution. The ferritin concentration in the ferritin-containing solution was 10 μM.
Then, this ferritin-containing solution was dropped on the monomolecular film to form a liquid film, which was left at 20 ° C. for 1 hour while preventing the liquid film from drying. Then, after washing with pure water, it was dried by nitrogen blowing. Thereby, metal storage protein was arrange | positioned and the metal ion acceptance layer was obtained.
In the metal ion receiving layer, the ferritin content was 30 wt%, and the total content of ferrocene and potassium ferricyanide was 31 wt%.

<4A> Next, after arranging the counter electrode made of platinum so as to face the working electrode and supplying the epoxy adhesive to the outer peripheral portion with the carbon reference electrode arranged at a predetermined location The sealing portion was formed by curing. Thereby, a sample storage space is defined by the metal ion receiving layer, the counter electrode, and the sealing portion.
At this time, an inlet for supplying the liquid sample into the sample storage space was formed in the sealing portion.
A sensor was obtained as described above.

(Example 2)
<1B> First, a glass substrate was prepared, and a working electrode having an average thickness of 200 nm was formed by vacuum deposition.
<2B> Next, aminoethanethiol was dissolved in anhydrous dichloromethane to prepare a solution. The concentration of aminoethanethiol in this solution was 3 mM.
And the glass substrate was immersed in this solution at 20 degreeC for 2 hours. Then, it took out from the solution, washed with pure water, and dried by nitrogen blowing. Thereby, a monomolecular film was obtained.

<3B> Next, denatured bovine serum albumin (BSA) and glutaraldehyde were dissolved in pure water to prepare a solution.
Then, this solution was dropped on the monomolecular film to form a liquid film, which was left at 20 ° C. for 1 hour while preventing the liquid film from drying. Then, after washing with pure water, it was dried by nitrogen blowing. Thereby, a BSA layer was obtained.

<4B> Next, ferritin (metal storage protein), denatured bovine serum albumin (fixed substance), glutaraldehyde (crosslinking agent), and potassium ferricyanide (mediator) were dissolved in pure water to prepare a solution. .
The concentration of ferritin, the concentration of denatured bovine serum albumin, the concentration of glutaraldehyde, and the concentration of potassium ferricyanide in this solution are 0.5 wt%, 2.0 wt%, 0.5 wt%, and 1.0 wt%, respectively. Mixed.
Then, this solution was dropped on the BSA layer to form a liquid film, and left at 20 ° C. for 1 hour while preventing the liquid film from drying. Then, after washing with pure water, it was dried by nitrogen blowing. As a result, a metal ion receiving layer was obtained.

<5B> Next, after arranging the counter electrode made of platinum so as to face the working electrode and supplying the epoxy adhesive to the outer peripheral portion with the carbon reference electrode arranged at a predetermined location The sealing portion was formed by curing. Thereby, a sample storage space is defined by the metal ion receiving layer, the counter electrode, and the sealing portion.
At this time, an inlet for supplying the liquid sample into the sample storage space was formed in the sealing portion.
A sensor was obtained as described above.

2. Evaluation Using a plurality of sensors of each Example, CV measurement was performed on the Fe ²⁺ solution having a known concentration by changing the voltage between the working electrode and the reference electrode.
As a result, it was confirmed that the measured current value was larger than that in the case where CV measurement was performed with the prepared solution, and there was a correlation between the Fe ²⁺ concentration and the current value.
By the method described above, ferritin-containing iron ions were replaced with copper ions, cobalt ions, and nickel ions, and a sensor was prepared in the same manner as in the above examples. As a result of measurement, it was found that the amount of metal ions corresponding to the metal ions contained therein can be measured.

It is a longitudinal cross-sectional view which shows typically the structure of the metal ion sensor of this invention. It is a model for demonstrating operation | movement of the metal ion sensor shown in FIG. It is a schematic diagram which shows an example of the metal storage protein with which the metal ion sensor shown in FIG. 1 is provided. It is a figure which shows the CV curve observed by CV measurement. It is a longitudinal cross-sectional view for demonstrating the manufacturing method of the metal ion sensor shown in FIG. It is a schematic diagram which shows the other structural example of a metal ion acceptance layer.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Metal ion sensor 2 ... Sample storage space 20 ... Liquid sample 3 ... Working electrode 4 ... Counter electrode 5 ... Reference electrode 6 ... Metal ion receiving layer 61 ... Metal storage protein 62 ... Mediator 8 ··········································· 101

Claims (11)

A metal ion sensor for measuring the amount of metal ions to be measured in a liquid sample,
A sample storage space for storing the liquid sample;
A metal ion receiving layer containing a metal storage protein that is at least partially exposed to the sample storage space and capable of taking in the metal ions to be measured;
A first electrode provided on the opposite side of the metal ion receiving layer from the sample storage space;
A second electrode that is at least partially exposed to the sample storage space and applies a voltage to the first electrode;
Electrons emitted as the valence of the metal ion to be measured incorporated into the metal storage protein changes from the first valence to the second valence, A metal ion sensor comprising: a third electrode for detecting a current value flowing therebetween.   The metal storage protein includes a metal ion of the same kind as the metal ion to be measured, and the valence of the included metal ion is the second valence or the first valence and the second valence. And a voltage opposite to the voltage when measuring the amount of the metal ion to be measured is applied between the first electrode and the second electrode. The metal ion sensor according to claim 1, wherein the metal ion sensor emits at least a part of the encapsulated metal ions. The metal ion to be measured is Fe ²⁺
The metal ion sensor according to claim 2, wherein the metal storage protein has ferritin as a main component.   The metal ion sensor according to any one of claims 1 to 3, wherein a content of the metal storage protein in the metal ion receiving layer is 10 to 60 wt%.   5. The metal ion sensor according to claim 1, wherein the metal storage protein is unevenly distributed on the sample storage space side in the metal ion receiving layer.   The metal ion sensor according to claim 1, wherein the metal storage protein is fixed by a fixing substance in the metal ion receiving layer.   The metal ion sensor according to claim 6, wherein the immobilizing substance is a compound having a first binding group that binds to the first electrode and a second binding group that binds to the metal storage protein.   The metal ion sensor according to claim 6, wherein the fixing substance is mainly composed of a biological polymer or a modified product thereof.   The metal ion sensor according to claim 1, wherein the metal ion receiving layer includes a mediator that mediates electron transfer between the metal storage protein and the first electrode.   The metal ion sensor according to claim 9, wherein a part of the mediator is fixed to the first electrode via a spacer molecule.   The metal ion sensor according to claim 9 or 10, wherein a content of the mediator in the metal ion receiving layer is 0.1 to 10 wt%.

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