JP6280877B2 - Biosensor and manufacturing method thereof - Google Patents

Description

  The present invention relates to a biosensor and a manufacturing method thereof.

  Biosensors are sensors that make use of the fact that biological substances such as enzymes and immuno-antibodies react with specific chemical substances in a sensitive manner, and are used to detect trace amounts of organic substances that have been difficult with conventional physical and chemical sensors. Mainly used.

  In recent years, attention has been paid to a quartz crystal microbalance method (QCM method) that can measure a very small mass change on the order of nanograms as a biosensor method. The QCM method uses the piezoelectric effect of quartz, and measures the mass of the target substance from the change in the resonance frequency of the quartz crystal when a sample gas or sample solution is adsorbed on the surface of the working electrode on the quartz crystal. .

  A QCM-type biosensor needs to adsorb an object to be measured on the electrode surface during measurement. Therefore, in order to improve the adsorptivity of the electrode, a biosensor is known in which the electrode surface is coated with hydroxyapatite that exhibits an extremely high adsorption capacity for many chemical substances (Patent Document 1).

International Publication No. 2006/025358

  However, since gold is generally used as an electrode for a biosensor, when the electrode surface is to be coated directly with a hydroxyapatite film, the bond between the gold and the hydroxyapatite is not good. In some cases, the hydroxyapatite film was not well bonded to the surface, cracking occurred in the hydroxyapatite film, or the hydroxyapatite film was peeled off.

  Therefore, an object of the present invention is to provide a biosensor in which the electrode surface is satisfactorily coated with a hydroxyapatite film and a method for producing the same.

  A biosensor according to the present invention is a biosensor having an electrode made of gold on a crystal resonator and measuring the mass of an object adsorbed on the electrode, and titanium is formed on the surface of the electrode. A first intermediate layer, a second intermediate layer made of hydroxyapatite doped with titanium on the first intermediate layer, and a hydroxyapatite film crystallized on the second intermediate layer, It is characterized by providing.

  Hydroxyapatite (hydroxyapatite, HAp) used in the present invention is a main constituent of human bones and teeth, and is widely used as an implant material including artificial bones and artificial tooth roots because of its high biocompatibility. It has been. In addition, it is known to exhibit an extremely high adsorption ability for many chemical substances such as proteins, amino acids, lipids, and sugars.

  Therefore, according to the configuration of the present invention, since the electrode surface is coated with a hydroxyapatite film having excellent adsorption capability, even a small amount of substance can be adsorbed appropriately, and the detection accuracy of the sensor can be improved. . Further, since the hydroxyapatite film is formed through the first intermediate layer made of titanium and the second intermediate layer made of hydroxyapatite doped with titanium, the hydroxyapatite film has good adhesion to the electrode surface. As a result, cracks and peeling of the hydroxyapatite film can be suppressed, and the durability of the hydroxyapatite film can be improved.

  Here, when the electrode surface of the biosensor is coated with a hydroxyapatite film, the thickness of the hydroxyapatite film is important. Since the QCM biosensor measures the mass of the object by changing the resonance frequency of the quartz resonator, if the hydroxyapatite film is too thick, the weight of the hydroxyapatite film affects the resonance of the quartz crystal and measures it. The accuracy may deteriorate. On the other hand, if the hydroxyapatite film is too thin, the object to be measured cannot be properly adsorbed, or it may not be bonded well to the electrode surface, which may cause problems of cracks and peeling.

  Therefore, in the above configuration, the thickness of the hydroxyapatite film is preferably 200 nm to 400 nm.

  Since the hydroxyapatite film in such a range is not too thin, it is possible to avoid problems such as failure to adequately adsorb an object, or cracking or peeling due to poor bonding with the electrode surface. Moreover, since it is not too thick, it is possible to prevent the sensor accuracy from deteriorating by affecting the resonance of the crystal resonator due to the weight of the hydroxyapatite film.

In the above configuration, the thickness of the first intermediate layer is preferably 40 to 50 nm, and the thickness of the second intermediate layer is preferably 20 to 50 nm.

  Since the first intermediate layer and the second intermediate layer in such a range are not too thin, they do not appropriately bond to the electrode surface or appropriately bond to the hydroxyapatite film formed on the electrode surface. Can be avoided. Moreover, since it is not too thick, it is possible to prevent the sensor accuracy from deteriorating due to the weight of these intermediate layers affecting the oscillation of the crystal resonator.

  As described above, according to the biosensor of the present invention, since the electrode surface is coated with a hydroxyapatite film, even a small amount of substance can be adsorbed appropriately, and the detection accuracy of the sensor can be improved. it can. Further, since the hydroxyapatite film is formed through the first intermediate layer made of titanium and the second intermediate layer made of hydroxyapatite doped with titanium, the hydroxyapatite film has good adhesion to the electrode surface. The formed hydroxyapatite film can be prevented from cracking and peeling, and the durability can be improved.

It is the figure which showed typically the manufacturing method of the biosensor concerning 1st Embodiment. It is the figure which looked at the biosensor concerning a 1st embodiment from one direction. It is a figure explaining the mass measuring method of the target object by a biosensor. It is a figure which shows the result of having verified the performance of the biosensor concerning an experiment example. It is a figure which shows the result of having verified the performance of the biosensor concerning a comparative example.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. However, all of these embodiments are illustrative and do not give a limited interpretation of the present invention. In the drawings, the same or corresponding parts are denoted by the same reference numerals.

  FIG. 1 is a diagram schematically illustrating a method for manufacturing a biosensor according to the present embodiment. Hereafter, the process is demonstrated in order. FIG. 1 and FIG. 2 to be described later schematically show a biosensor manufacturing method for explanation, and the electrodes, the first intermediate layer, and the second intermediate shown in these drawings. The technical scope of the present invention is not limited by the formation position and formation range of the layer and the hydroxyapatite film.

(1) Biosensor preparation process First, as shown to Fig.1 (a), the biosensor 10 is prepared. The biosensor 10 is a QCM type biosensor that has an electrode 12 made of gold on a crystal resonator 11 and measures the mass of a measurement object adsorbed on the electrode 12 by a change in the resonance frequency of the crystal resonator 11. is there. In addition to gold, platinum, titanium, or the like can be used as the electrode 12, but gold is preferably used to ensure sufficient sensor accuracy.

(2) 1st intermediate | middle layer formation process Next, as shown in FIG.1 (b), the 1st intermediate | middle layer 13 is formed in the surface of the electrode 12 of the biosensor 10. FIG. Since the first intermediate layer 13 contains titanium, it is stably bonded to the electrode 12 made of gold. The first intermediate layer 13 may be formed only on the electrode 12 on the side where the measurement object is to be adsorbed.

  Examples of the method for forming the first intermediate layer 13 include a dipping method, a sputtering method, a plasma spraying method, a laser ablation method, and the like. A dense film can be formed with a desired film thickness and is suitable for mass production. From the viewpoint of being present, it is preferable to use a sputtering method.

  Moreover, it is preferable that the film thickness of the 1st intermediate | middle layer 13 is 40-50 nm. If the thickness is smaller than this, the electrode 12 may not be coupled well or may not be coupled well with the second intermediate layer 14 formed thereon, and if it is thicker than this, the resonance of the crystal unit 11 is affected and the sensor accuracy is increased. May get worse.

  In this specification, “titanium” is not only pure titanium but also titanium compounds such as titanium oxide and titanium hydride, and titanium alloys in which aluminum, chromium, nickel, etc. are added to the main component titanium. Is also widely included.

(3) Second Intermediate Layer Formation Step Next, as shown in FIG. 1C, the second intermediate layer 14 is formed on the first intermediate layer 13. The second intermediate layer 14 is made of hydroxyapatite doped with titanium, and improves the adhesion between the first intermediate layer 14 and the hydroxyapatite film.

  Since titanium constituting the first intermediate layer 13 has good bonding properties with both gold and hydroxyapatite, the second intermediate layer 14 is not formed, but only through the first intermediate layer 13 made of titanium. It is also possible to form the hydroxyapatite film 15a. On the other hand, when the second intermediate layer 14 made of hydroxyapatite doped with titanium is formed between the first intermediate layer 13 made of titanium and the hydroxyapatite film 15a, the second intermediate layer 14 makes the first Therefore, the intermediate layer 13 and the hydroxyapatite 15a can be bonded more favorably, so that cracking and peeling of the hydroxyapatite film 15a can be further suppressed and durability can be further improved. In addition, elution of hydroxyapatite during use of the sensor can be suppressed, and sensor accuracy can be improved.

  Examples of the method for forming the second intermediate layer 14 include a dipping method, a sputtering method, a plasma spraying method, and a laser ablation method. A dense film can be formed with a desired film thickness, and is suitable for mass production. From the viewpoint of being present, it is preferable to use a sputtering method.

  The second intermediate layer 14 can be formed by sputtering using a target in which titanium and hydroxyapatite are mixed at a predetermined ratio. Here, from the viewpoint of satisfactorily bonding the first intermediate layer 13 and the hydroxyapatite film 15a, the ratio (Ca / Ti) of calcium atoms to titanium atoms in the second intermediate layer 14 is 2.3 to 2.5. It is preferable that

(4) Hydroxyapatite Film Formation Step Next, as shown in FIG. 1 (d), a hydroxyapatite film 15 a is formed on the second intermediate layer 14. If the first intermediate layer 13 or the second intermediate layer 14 is exposed, the measurement object may be adsorbed on the first intermediate layer 13 or the second intermediate layer 14 and sensor accuracy may deteriorate. It is preferable that the second intermediate layer 14 is formed on almost all of the first intermediate layer 13, and the hydroxyapatite film 15 a is formed on almost all of the second intermediate layer 14.

  Examples of the method for forming the hydroxyapatite film 15a include a dipping method, a sputtering method, a plasma spraying method, and a laser ablation method. A dense and uniform film can be formed with a desired film thickness, and is suitable for mass production. From the viewpoint of being present, it is preferable to use a sputtering method. In the case where all of the first intermediate layer 13, the second intermediate layer 14, and the hydroxyapatite film 15a are formed by sputtering, it is possible to manufacture in a single chamber by using a multi-chamber apparatus, and productivity is increased. Can be improved.

The following conditions can be used as conditions for forming the hydroxyapatite film 15a by sputtering.
・ RF sputtering method ・ Sputtering pressure: 0.06 Pa
・ Sputtering power: 300W
Under the above conditions, the hydroxyapatite film 15a could be formed at a film formation rate of 6.7 nm / min.

  Moreover, it is preferable that the film thickness of a hydroxyapatite film | membrane is 200 nm-400 nm. If it is thinner than this, the second intermediate layer 14 will not be sufficiently bonded, and there is a risk of cracking or peeling. If it is thicker than this, the resonance of the crystal unit 11 will be affected and the sensor accuracy may be deteriorated.

  At this point, the hydroxyapatite film 15a is in an amorphous state and is not crystallized.

(5) Crystallization Step Next, as shown in FIG. 1D, the hydroxyapatite film 15a formed on the second intermediate layer 14 is crystallized. Examples of the crystallization method include hydrothermal treatment and heating with an electric furnace. By this crystallization process, hydroxyapatite that has been in an amorphous state is crystallized. When the hydroxyapatite film is crystallized, the surface is charged, so that the measurement object is easily adsorbed. Thus, the biosensor 20 in which the surface of the electrode 12 is coated with the crystallized hydroxyapatite film 15b can be obtained. Note that crystallization by hydrothermal treatment is desirable because crystallization by heating with an electric furnace may make the biosensor unstable and may not provide sufficient accuracy.

  According to the biosensor 20 manufactured as described above, since the surface of the electrode 12 is coated with the hydroxyapatite film 15b having an extremely high adsorption ability with respect to many chemical substances, even a small amount of substance is appropriately adsorbed. In the measurement of various gases, ions, alcohols, enzymes, proteins, DNA, etc., the detection accuracy of the sensor can be improved. In particular, since hydroxyapatite is excellent in biocompatibility and is a main constituent material of bone and teeth, the biosensor 20 can be used as a bone quality simulator or a tooth quality simulator.

  Further, the hydroxyapatite film 15 b is formed on the electrode 12 via the first intermediate layer 13 and the second intermediate layer 14. Here, the first intermediate layer 13 is made of titanium having a good bondability with gold or hydroxyapatite, and the second intermediate layer 14 is made of titanium that bonds the first intermediate layer 13 and the hydroxyapatite film 15b well. Since it is made of doped hydroxyapatite, the hydroxyapatite film 15b is firmly bonded to the surface of the electrode 12, which can prevent cracks and peeling and improve the durability of the hydroxyapatite film 15b. In addition, since the elution of hydroxyapatite when using the sensor can be suppressed, the sensor accuracy can be improved.

  In addition, according to the biosensor manufacturing method described above, the first intermediate layer 13, the second intermediate layer 14, and the hydroxyapatite film 15a are formed by the sputtering method. Can be formed with a thickness. As a result, the first intermediate layer 13, the second intermediate layer 14, and the hydroxyapatite film 15a having desired film thicknesses that do not adversely affect the resonance of the crystal resonator can be formed. Can be prevented. Moreover, since the sputtering method is suitable for mass production, the productivity of the biosensor 20 can be improved.

  FIG. 2 is a view of the biosensor 20 manufactured by the above-described manufacturing method as seen from one direction. The biosensor 20 includes electrodes 12 a and 12 b on both surfaces of the crystal unit 11. The first intermediate layer 13, the second intermediate layer 14, and the crystallized hydroxyapatite film 15b are formed on at least one electrode surface. Since the hydroxyapatite film 15b is formed through the first intermediate layer 13 and the second intermediate layer 14, the surface of the electrode 12 of the biosensor 20 is well coated with the hydroxyapatite film 15b.

(Measuring method)
A method for measuring the mass of an object by the biosensor 20 manufactured by the above-described manufacturing method will be described with reference to FIG.

  When an AC voltage is applied to the electrode 12 of the crystal resonator 11 using a power source, an oscillation circuit or the like (not shown), the crystal resonator 11 resonates due to the piezoelectric effect. Before dropping the object to be measured on the surface of the electrode 12, the resonance frequency f1 of the quartz crystal resonator 11 at this time (point a in FIG. 3) is measured.

  Next, when the measurement object is dropped onto the surface of the electrode 12 and is adsorbed, the resonance frequency of the crystal unit 11 is lowered according to the amount of adsorption as shown in FIG. When a certain time elapses, the resonance frequency of the crystal unit 11 converges, so the resonance frequency f2 at this point (point b in FIG. 3) is measured.

ΔＦ：共振周波数変化量
Ｆ：水晶振動子の固有周波数
Ａ：電極の表面積
Δｍ：電極表面の質量変化量
The relationship between the change in the mass of the electrode surface and the resonance frequency of the crystal resonator is expressed by the following equation.
[Formula 1] Sauerbrey's formula

ΔF: Resonance frequency change amount F: Natural frequency of crystal resonator A: Surface area of electrode Δm: Change amount of mass on electrode surface

  Accordingly, the mass change amount Δm of the surface of the electrode 12 is obtained by the above-described Sauerbrey equation using the resonance frequency change amount Δf (f2−f1) of the crystal unit 11 generated by the adsorption of the measurement object to the surface of the electrode 12. Thereby, the mass of the measuring object adsorbed on the electrode 12 surface can be measured.

  In addition, in order to adjust the pH of the measurement object to obtain a stable measurement condition, a buffer solution may be dropped on the surface of the electrode 12 before the measurement object is dropped. Examples of the buffer solution include HEPES buffer solution, PBS buffer solution, CAPS buffer solution, TRIS buffer solution and the like.

  When a buffer solution is used, the resonance frequency of the crystal unit 11 is changed even when the buffer solution is dropped. Therefore, the object to be measured is dropped after the frequency fluctuation converges, and the resonance frequency of the crystal unit 11 is reduced. The change amount ΔF is obtained by subtracting the resonance frequency (f1) converged after dropping the buffer solution from the resonance frequency (f2) converged after dropping the measurement object. If this ΔF is used to determine the mass change amount Δm on the surface of the electrode 12 using the Sauerbrey equation, the mass of the measurement object adsorbed on the surface of the electrode 12 can be measured.

(Experimental example)
In this experimental example, a biosensor in which a hydroxyapatite film is formed on a gold electrode through a first intermediate layer made of titanium and a second intermediate layer made of hydroxyapatite doped with titanium is manufactured. The performance was verified. The result is shown in FIG.

  The first intermediate layer, the second intermediate layer, and the hydroxyapatite film in this experimental example were all formed by sputtering. The second intermediate layer is formed by uniformly arranging ten smaller titanium chips (size 5 mm × 5 mm × 1 mm) on a 4-inch hydroxyapatite target and sputtering them simultaneously. Formed by.

  FIG. 4A shows the X-ray diffraction analysis result of the hydroxyapatite film. An X-ray diffractometer (manufactured by Shimadzu Corporation, model number XRD-6100) was used for the X-ray diffraction analysis. A peak was confirmed around 2θ = 32 °, and it was confirmed that hydroxyapatite was crystallized.

  FIG. 4B shows the frequency change of the crystal resonator when the measurement object is dropped on the electrode surface. The measurement object was dropped after the HEPES buffer solution was dropped on the electrode as a buffer solution and the frequency fluctuation of the crystal unit was converged.

  As shown in the figure, when 5 ul of albumin was dropped as an object to be measured, the frequency of the crystal resonator varied by about 420 Hz. When this frequency fluctuation amount Δf is converted into the mass fluctuation amount Δm on the electrode using the above-described equation 1, it becomes about 260 ng.

(Comparative example)
As a comparative example, a biosensor in which a hydroxyapatite film was formed on an electrode made of gold via an intermediate layer made of zinc oxide was manufactured, and its performance was verified. The result is shown in FIG. The intermediate layer made of zinc oxide and the hydroxyapatite film were both formed by sputtering.

  As shown in FIG. 5, when a buffer solution (HEPES) was dropped on the electrode of a biosensor provided with an intermediate layer made of zinc oxide, the frequency of the crystal resonator rapidly increased. This is considered to be due to the elution of hydroxyapatite that coats the electrode by the dropping of the buffer solution, and in the biosensor using zinc oxide in the intermediate layer, the adhesion of the hydroxyapatite film is not sufficient. Therefore, it was confirmed that the sensor accuracy could not be secured with the configuration using zinc oxide in the intermediate layer.

10, 20 Biosensor 11 Crystal resonator 12 (12a, 12b) Electrode 13 First intermediate layer 14 Second intermediate layer 15a Hydroxyapatite film (amorphous state)
15b Hydroxyapatite film (crystalline state)

Claims (11)

A biosensor having an electrode made of gold on a crystal resonator and measuring the mass of an object adsorbed on the electrode,
A first intermediate layer comprising titanium on a surface of the electrode;
A second intermediate layer made of hydroxyapatite doped with titanium on the first intermediate layer;
A biosensor comprising a crystallized hydroxyapatite film on the second intermediate layer. The hydroxyapatite film has a thickness of 200 nm to 400 nm.
The biosensor according to claim 1. The film thickness of the first intermediate layer is 40 to 50 nm.
The biosensor according to claim 1 or 2. The biosensor according to any one of claims 1 to 3, wherein the thickness of the second intermediate layer is 20 to 50 nm. The biosensor according to any one of claims 1 to 4, wherein a ratio of calcium atoms to titanium atoms in the hydroxyapatite doped with titanium is 2.3 to 2.5. Preparing a biosensor for measuring the mass of an object adsorbed on the electrode having gold electrodes on a crystal resonator; and
Forming a first intermediate layer containing titanium on the electrode;
Forming a second intermediate layer made of hydroxyapatite doped with titanium on the first intermediate layer;
Forming a hydroxyapatite film on the second intermediate layer;
And a step of crystallizing the hydroxyapatite film. The hydroxyapatite film is formed by a sputtering method.
A method for producing the biosensor according to claim 6. The hydroxyapatite film has a thickness of 200 nm to 400 nm.
The method for producing a biosensor according to claim 6 or 7. The film thickness of the first intermediate layer is 40 to 50 nm.
The manufacturing method of the biosensor of any one of Claims 6-8. The biosensor manufacturing method according to claim 6, wherein the second intermediate layer has a thickness of 20 to 50 nm. The method for producing a biosensor according to any one of claims 6 to 10, wherein a ratio of calcium atoms to titanium atoms in the hydroxyapatite doped with titanium is 2.3 to 2.5.

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