Octacalcium phosphate crystalline film and method for producing same

Abstract

An electrode made of a conductive material such as gold is used as the cathode (gold sensor), and a multilayer deposition film of octacalcium phosphate (OCP) crystals is formed on the cathode by electrophoresis of an ethanol dispersion of OCP crystals. This multilayer deposition film is then ultrasonically cleaned to form a single layer deposition film of OCP crystals on the cathode. The OCP crystal film formed on the gold sensor surface has a layer structure in which hydrated and apatite layers are regularly repeated, and is a stable crystalline film in which specific crystal planes are oriented in specific directions, with the hydrated layer as the top layer.

Description

[Technical Field]

[0001] The present invention relates to a crystalline film of octacalcium phosphate and a method for producing the crystalline film of octacalcium phosphate. [Background technology]

[0002] When measuring and quantifying the mass of a substance to be measured in a sample, a quartz crystal microbalance (QCM) sensor has traditionally been used, which performs measurements based on electrical properties such as the oscillation frequency and impedance of a quartz oscillator exposed to the sample.

[0003] In addition to the functions of the QCM, there is also the QCM-D (Quartz Crystal Microbalance with Dissipation) sensor, which can measure, for example, the relationship between weight changes and viscoelasticity changes on the sensor surface from the frequency shift and amplitude attenuation (dissipation) of the quartz oscillator.

[0004] On the surface of these QCM sensors and QCM-D sensors, an electrode made of, for example, gold is provided, and other metals, oxide films, polymer films, etc. are formed on the gold electrode. For example, hydroxyapatite (Ca 10 There is a hydroxyapatite (HA) sensor coated with a particle film of (PO4)6(OH)2:HA. Hydroxyapatite is the main inorganic component of biological bone, and can be used as an artificial bone filling material. Therefore, by utilizing the hydroxyapatite (HA) sensor as a simulated surface for artificial bone and teeth and reproducing the reactions of substances in biological fluids, it is being applied to the development of medical materials such as those that promote bone regeneration, and oral care products such as those that remove tooth stains.

[0005] As a technique for forming a coating on the surface of a substrate of a medical material such as an artificial bone, for example, Patent Document 1 discloses a method for forming a bioabsorbable coating mainly composed of Mg-containing calcium phosphate on the surface of a substrate made of magnesium or a magnesium alloy. Patent Document 2 discloses a medical device material in which multiple calcium phosphate layers with different compositions are provided on the surface of the substrate, and the surface side of the calcium phosphate layers is made of a substance that is more biodegradable in the body than the substrate side and is replaced by bone tissue. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2018-202074 [Patent Document 2] Japanese Patent Publication No. 2020-130766 Summary of the Invention [Problem to be solved by the invention]

[0007] Calcium phosphate has various crystalline phases, but the crystalline phases of calcium phosphate in Patent Documents 1 and 2 are multiphase (e.g., amorphous phase, HAp phase, OCP phase, etc.), and a single-phase crystalline film has not yet been achieved for octacalcium phosphate (OCP), a type of calcium phosphate.

[0008] On the other hand, focusing on the crystal layer, the coating in Patent Document 1 is a porous coating to serve as a drug carrier, while Patent Document 2 has a structure in which the surface of the calcium phosphate layer on the substrate side remains uneven, and the surface side of the calcium phosphate layer is a porous film. In other words, the calcium phosphate coating films formed on the substrate surface by the film formation methods in Patent Documents 1 and 2 are both multilayers of stacked crystals, and formation of a monolayer film in which single crystals exist in parallel has not yet been achieved. In addition, the surface roughness (unevenness) is rough on the order of μm, resulting in poor smoothness and poor adhesion to the substrate, resulting in problems of lack of mechanical and chemical stability.

[0009] Furthermore, conventional techniques have not been able to control the exposure (orientation) of the crystal planes of calcium phosphate crystals.On the other hand, for octacalcium phosphate, which has high biocompatibility and is used as a bone filling material in the biomedical field, a film formation technology, especially a stable thin film formation technology, has not been developed, and research has not progressed to date.

[0010] Therefore, the inventors of the present application focused on octacalcium phosphate, which has high biocompatibility among known calcium phosphate ceramics, and after extensive research, discovered that a stable single-layer film of octacalcium phosphate crystals can be formed on the surface of a substrate.

[0011] The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a stable single-layer crystalline film of octacalcium phosphate and a method for producing the same. [Means for solving the problem]

[0012] As a means for achieving the above object and solving the above problem, the present invention has the following configuration. Akira is , A crystalline film, The crystal film is characterized in that plate-like crystals of octacalcium phosphate are arranged in a single layer, the distribution range of the major axis of the plate-like crystals is 50 nm to 10 μm, the distribution range of the minor axis is 5 to 1000 nm, the aspect ratio, which is the value obtained by dividing the major axis by the minor axis, is 2 to 100, a specific crystal face of the plate-like crystals is oriented in a specific direction, the specific direction is the surface direction of the crystal film, the specific crystal face is exposed on the surface of the crystal film in preference to other crystal faces other than the specific crystal face, and the crystal film is formed on the surface of a conductive substrate.

[0013] exampleFor example, the conductive substrate is made of a conductive material selected from at least gold, titanium, chromium, and silicon. For example, the specific crystal plane is exposed in the thickness direction of the crystal film, in the direction opposite to the conductive substrate. Furthermore, for example, the mean square roughness of the crystal film in a measurement range of 5 μm × 5 μm on the surface of the crystal film is 200 nm or less. Below It has a flat and smooth surface and a film thickness of 200 nm or more. Under a Preferably, the crystal film has a surface smoothness in which the mean square roughness of the crystal film in a measurement range of 5 μm×5 μm is 100 nm or less, and the film thickness is 50 nm or less. The crystal film is characterized by having adhesion properties that prevent it from peeling off from the conductive substrate even after ultrasonic cleaning in ethanol at a frequency of 40 kHz for 1 minute. Furthermore, the crystals in the crystal film have a layer structure in which hydrated layers and apatite layers alternate, and the hydrated layer is exposed as the uppermost layer on the side of the crystal film opposite the conductive substrate in the thickness direction. For example, the specific crystal plane is a (100) plane.

[0014] The method for producing a crystal film of the present invention is characterized by comprising the steps of: synthesizing octacalcium phosphate crystals (OCP crystals) by wet synthesis; preparing an ethanol dispersion of the OCP crystals; forming a multilayer deposition film of the OCP crystals on a cathode made of an electrode made of a conductive material selected from at least gold, titanium, chromium, and silicon by electrophoresis of the ethanol dispersion; and ultrasonically cleaning the multilayer deposition film to form a single layer deposition film of the OCP crystals on the cathode.

[0015] In the method for producing a crystal film according to the present invention, for example, the monolayer deposited film is a crystal film in which the (100) plane of the OCP crystal is oriented in the surface direction of the deposited film and is exposed on the surface of the deposited film preferentially over other crystal planes other than the (100) plane, the distribution range of the major axis of the OCP crystal is 50 nm to 10 μm, the distribution range of the minor axis is 5 to 1000 nm, and the aspect ratio, which is the value obtained by dividing the major axis by the minor axis, is 2 to 100, the monolayer deposited film is formed on the surface of the conductive substrate as the cathode, and the mean square roughness of the surface of the deposited film in a measurement range of 5 μm × 5 μm is 200 nm or more. Below It has a flat and smooth surface and a film thickness of 200 nm or more. Under can be, Preferably, the deposited film has a surface smoothness in which the root mean square roughness in a measurement range of 5 μm × 5 μm is 100 nm or less, and the film thickness is 50 nm or less; The film is characterized by having adhesion properties that prevent it from peeling off from the conductive substrate even after ultrasonic cleaning in ethanol at a frequency of 40 kHz for 1 minute. Furthermore, for example, the crystals in the single-layer deposited film have a structure in which hydrated layers and apatite layers alternate, and the hydrated layer is exposed as the uppermost layer on the side of the deposited film opposite the conductive substrate in the thickness direction. For example, the electrophoresis time of the multilayer deposited film by electrophoresis is at least 1 minute.

[0016] The QCM sensor of the present invention is a quartz crystal microbalance sensor comprising a conductive substrate coated with the crystal film of the present invention, characterized in that the (100) plane of the octacalcium phosphate plate crystals constituting the crystal film is exposed on the surface of the crystal film in preference to other crystal planes other than the (100) plane. [Effects of the Invention]

[0017] According to the present invention, the technology for forming a stable crystalline film of octacalcium phosphate on the surface of a substrate can be applied to the general surface coating technology of medical instruments and devices. [Brief explanation of the drawings]

[0018] [Figure 1] 1 is a flowchart showing the steps of synthesizing OCP crystals and preparing a dispersion of OCP crystals. [Figure 2] FE-SEM image of OCP crystal powder. [Figure 3] 10 is a flowchart showing a process for forming a deposited film by electrophoresis. [Figure 4] This is a FE-SEM image showing a planar view of a multilayer film of octacalcium phosphate crystals. [Figure 5] FE-SEM image of residual particles in the residual liquid. [Figure 6] FE-SEM images showing the relationship between deposition time of electrophoretic films and platelet crystals. [Figure 7] FIG. 1 is a diagram showing the results of comparing the X-ray diffraction patterns (XRD patterns) of particles before and after deposition. [Figure 8] These are ATR-FTIR spectra, which are the light absorption spectra on the surface of each particle before and after deposition, measured by Fourier transform infrared spectroscopy (FTIR). [Figure 9] FIG. 1 is a diagram schematically illustrating an apparatus for forming a deposited film by electrophoresis. [Figure 10] FIG. 1 is a diagram illustrating a schematic diagram of an electrophoretic deposition mechanism of an OCP. [Figure 11] 1 is a flowchart showing the process of converting a multilayer film of octacalcium phosphate crystals into a single layer. [Figure 12] AFM images of a single-layer OCP crystal film and its cross section. [Figure 13] AFM images of the surface and cross section of an uncoated gold sensor. [Figure 14] AFM images of a hydroxyapatite film and its cross section. [Figure 15] FIG. 1 is a diagram showing the particle size distribution of crystals forming a hydroxyapatite film. [Figure 16] FIG. 1 is a diagram showing the distribution of particle sizes of crystals forming a single-layer OCP crystal film. [Figure 17] The figure shows the spectral waveforms measured by ATR-FTIR for a multilayer film and a single-layer OCP crystal film before ultrasonic cleaning. [Figure 18] FIG. 1 is a diagram showing a comparison of the ATR-FTIR spectrum waveforms of a single-layer OCP crystal film and a hydroxyapatite film. [Figure 19] FIG. 10 shows the results of XPS analysis of Ca, P, and O in a multilayer film and a single-layer OCP crystal film. [Figure 20] FIG. 20 is a diagram showing the results of spectral separation of phosphorus (P) in FIG. 19. [Figure 21] 10 is a flowchart showing the steps for evaluating the behavior of a single-layer OCP film when a PBS solution is pumped for a predetermined time. [Figure 22]FIG. 10 is a graph showing the weight change of a single-layer OCP film in a PBS solution. [Figure 23] FIG. 1 is a graph showing the weight change of a hydroxyapatite film in a PBS solution. [Figure 24] 1 is a flowchart showing the steps for evaluating the behavior of a single-layer OCP film when the single-layer OCP film is immersed in a PBS solution. [Figure 25] FIG. 1 shows the change in film thickness of a single-layer OCP film with respect to the immersion time in a PBS solution. [Figure 26] This shows the spectral waveform measured by ATR-FTIR of a monolayer OCP crystal film as a function of immersion time in a PBS solution. [Figure 27] FIG. 1 shows the results of XPS analysis of Ca, P, and O before and after immersion of a single-layer OCP film in a PBS solution. [Figure 28] FIG. 28 is a diagram showing the results of spectral separation of phosphorus (P) in FIG. 27. [Figure 29] FIG. 10 is a graph showing the weight change over time of a monolayer OCP film in a citrate buffer solution. [Figure 30] FIG. 10 is a graph showing the weight change over time of a hydroxyapatite film in a citrate buffer solution. [Figure 31] This is an AFM image of a single-layer OCP crystalline film immersed in a PBS solution. DETAILED DESCRIPTION OF THE INVENTION

[0019] An embodiment of the present invention will be described in detail below with reference to the accompanying drawings and tables. As described below, in this embodiment, the first step involves synthesizing octacalcium phosphate crystals, the second step involves forming a multilayer deposition film (multilayer film) of octacalcium phosphate crystals on a substrate, and the third step involves converting the multilayer film of octacalcium phosphate crystals into a single layer (single layer film formation). Then, in the fourth step, the sensor on which the single layer was formed was evaluated for its sensing capability and its reaction characteristics in biological fluids. Note that octacalcium phosphate will hereinafter be abbreviated as OCP where appropriate.

[0020] In this description, a "single layer film" refers to a film formed in which a specific crystal plane of the OCP platelet crystals deposited on a substrate is aligned parallel to the substrate, and the crystal plane is exposed to the outside in the layer direction at the top surface of the layer without being covered by other crystals. A film with such an arrangement of OCP platelet crystals is also called a "single layer" film.

[0021] On the other hand, a "multilayer film" is a film in which plate-like crystals are stacked in layers perpendicular to the substrate, and various crystal planes other than the specific crystal plane are exposed on the outermost surface, making it impossible to observe the specific crystal plane on the outermost surface. Regarding the crystalline film, it is appropriate to call it a single-layer film from the viewpoint of film thickness, and to call it a single-phase crystalline film from the viewpoint of crystalline phase.

[0022] <First step> In this process, octacalcium phosphate (OCP) crystals were synthesized by wet synthesis, and an OCP dispersion was prepared. Figure 1 is a flowchart showing the synthesis of OCP crystals and the preparation process of the OCP crystal dispersion. In step S11 of Figure 1, 25 ml of acetate buffer (acetate buffer) with a pH adjusted to 3.6 was prepared. In the subsequent step S12, 0.6204 g of α-tricalcium phosphate (α-TCP:Ca3(PO4)2) was added to the acetate buffer.

[0023] In step S13, the sample obtained in the above process is heated and stirred. Here, the heating temperature is set to 60°C, and stirring is performed at 1200 rpm for 3 hours. During this heating and stirring process, the α-TCP dissolution process shown in formula (1) and the transition to OCP process shown in formula (2) occur, followed by the phase transition from α-TCP to OCP shown in reaction formula (3).

[0024] Ca3(PO4)2+2H + ←→ 3Ca 2+ +2HPO4 2- (1) 8Ca 2+ +2HPO4 2- +4PO4 3-+5H2O → Ca8H2(PO4)6·5H2O (2) 3Ca3(PO4)2+7H2O→Ca8H2(PO4)6·5H2O+Ca 2+ +2OH - (3)

[0025] The heated and stirred sample is centrifuged in step S14, and the centrifuged sample is washed in step S15, here washing with ultrapure water once and washing with ethanol three times.

[0026] The washed sample was heated and dried (60°C, 48 hours) in step S17 to produce and recover OCP crystal powder, which was then evaluated in step S18. Meanwhile, in step S19, the washed sample was dispersed in ethanol (2 wt%) to produce an OCP crystal ethanol dispersion. The OCP crystal ethanol dispersion produced here was used for film formation in the second step described below.

[0027] Figure 2 is an image taken with a field emission scanning electron microscope (FE-SEM) of the OCP crystal powder produced by heating and drying in step S17 for evaluation in step S18 in Figure 1. As shown in Figure 2, the OCP crystal powder obtained by the above-described synthesis of OCP crystals contains a mixture of plate-like crystals 1 (shown surrounded by a dashed line in Figure 2) that are the target of coating formation of the octacalcium phosphate crystals according to this embodiment, and amorphous crystals 2 (shown surrounded by a dotted line in Figure 2).

[0028] <Second process> In this process, a deposited film (multilayer film) of octacalcium phosphate crystals is formed by electrophoresis. Figure 3 is a flowchart showing the process for forming an electrophoretic deposited film. In step S21 of Figure 3, as shown in Figure 9, 20 ml of a dispersion containing OCP particles 4, i.e., the OCP crystal ethanol dispersion 10 prepared in the first process described above, is placed in a glass container 14.

[0029] In step S22, a gold electrode (also referred to as a gold sensor) 18 serving as a cathode and a counter electrode 17 serving as an anode were introduced, spaced 1 cm apart, into the OCP crystal ethanol dispersion liquid in the glass container 14. Then, in step S23, a direct current of 100 V was applied between the anode and cathode, and the charged OCP particles 4 in the OCP crystal ethanol dispersion liquid 10 were moved by the electric field and deposited on the substrate (gold sensor).

[0030] As the conductive material used for the cathode, other than gold, for example, a conductive base material selected from titanium, chromium, and silicon can be used.

[0031] In this process, to examine the relationship between the deposition time of the film by electrophoresis and the crystal shape, film formation was performed by changing the application time of the voltage between the anode and the cathode. That is, in step S24 of Fig. 3, the application time of the voltage (deposition time) was measured, and in step S25, it was determined whether the application time reached a predetermined time (target deposition time). Here, five patterns of film deposition time were used: 1 minute, 2 minutes, 3 minutes, 4 minutes, and 5 minutes.

[0032] In step S26, the sample deposited on the gold sensor was heated and dried (60°C, 48 hours). In the following step S27, the heated and dried sample was evaluated. Meanwhile, in step S28, the remaining liquid that could no longer be deposited by electrophoresis was centrifuged, and the resulting sample was heated and dried (60°C, 48 hours) in step S29 to obtain residual particles. The residual particles were then evaluated in step S30.

[0033] Figures 4(a) and (b) are FE-SEM images of the sample obtained in step S26 of Figure 3, i.e., a multilayer film of octacalcium phosphate crystals deposited on a gold sensor by electrophoresis, viewed from the direction toward the gold sensor surface. Figures 5(a) and (b) are FE-SEM images of residual particles in the remaining liquid obtained in step S29 of Figure 3. In each of Figures 4 and 5, (b) is an enlarged image of (a).

[0034] The FE-SEM image shown in Figure 4 reveals that electrophoresis has resulted in the formation of a multilayer film consisting of only plate-shaped octacalcium phosphate crystals on the gold sensor. In Figure 4(b), particularly prominent plate-shaped crystals are shown enclosed by dashed lines. On the other hand, the FE-SEM image shown in Figure 5 reveals that the remaining particles that were not deposited by electrophoresis consist of amorphous particles (i.e., amorphous crystals). These results demonstrate that electrophoresis can selectively deposit only plate-shaped crystals on the gold sensor (in other words, the crystal shape to be deposited can be selected).

[0035] Figure 6 shows FE-SEM images showing the relationship between the deposition time of the electrophoretic film (also called the electrophoresis time or voltage application time) and the platelet crystals. Figure 6(a) is an FE-SEM image obtained after a deposition time of 1 minute, (b) after a deposition time of 2 minutes, (c) after a deposition time of 3 minutes, (d) after a deposition time of 4 minutes, and (e) after a deposition time of 5 minutes. In Figures 6(a) to 6(e), the images within the dashed lines are enlarged partial images of each obtained image.

[0036] These FE-SEM images confirm platelet crystals at all deposition times. Using image processing software called ImageJ, we calculated the platelet crystal abundance at each deposition time. The results showed that the platelet crystal abundance was 80% at 1 minute, 60% at 2 minutes, 55% at 3 minutes, 52% at 4 minutes, and 40% at 5 minutes. Since the platelet crystal abundance varied depending on the deposition time, subsequent experiments and evaluations used samples obtained with a deposition time of 1 minute, at which the platelet crystal abundance was 80%.

[0037] Next, we will explain the results of a study from a different perspective on the differences in particles before and after deposition of octacalcium phosphate crystals by electrophoresis. Figure 7 shows the results of comparing the X-ray diffraction patterns (XRD patterns) of particles before and after deposition. Specifically, in Figure 7, the pattern indicated by reference numeral 3a is the XRD pattern of the as-synthesized OCP crystal powder (OCP crystal powder produced in step S17 of Figure 2), reference numeral 4a is the XRD pattern of the plate-like crystals (crystals of the multilayer film) deposited on the gold sensor by electrophoresis, and reference numeral 5a is the XRD pattern of the remaining particles that were not deposited by electrophoresis.

[0038] Table 1 shows an example of the crystallite size and crystallinity for each of the particles (OCP crystal powder, deposited platelet crystals, and residual particles) before and after deposition shown in the XRD patterns of FIG.

[0039] [Table 1]

[0040] Figure 7 shows the XRD patterns of the OCP crystal powder, the deposited platelet crystals, and the residual particles. The crystalline phase is the same before and after deposition, as well as in the undeposited particles, and all are single-phase OCP. This lack of change in the crystalline phase of the OCP particles before and after deposition indicates that the crystals are stable. Figure 7 and Table 1 show that there is no difference in the crystallinity of any of the particles before and after deposition (crystallinity is maintained), and the crystallite size of the deposited platelet crystals is larger than that of the other particles. This indicates that crystals with large crystallite sizes are selectively deposited by electrophoresis.

[0041] The crystallite size was calculated using the following formula (4) (Halder-Wagner formula), and the crystallinity was calculated using the following formula (5).

[0042]

number

[0043] In equation (4), D is the crystallite size (nm), K is the Scherrer constant, λ is the wavelength of the X-ray (nm), ε is the strain, β is the broadening of the diffraction line (rad), and θ is the Bragg angle (rad). Here, the diffraction peak near the diffraction angle 2θ = 4° was used.

[0044] Crystallinity = crystalline peak area / crystalline and amorphous peak areas (5)

[0045] Figure 8 shows ATR-FTIR spectra, which are the light absorption spectra on the surfaces of the particles (OCP crystal powder, deposited platelet crystals, and residual particles) before and after deposition, measured by Fourier transform infrared spectroscopy (FTIR). Figure 8(a) shows the ATR-FTIR spectral waveform of each particle. Figure 8(b) shows the spectral waveform obtained by second-order differentiation of the spectral shape shown in Figure 8(a). In Figure 8(b), the frequency (wavenumber) of 1050 to 1150 cm shown by the dashed line in Figure 8(a) is 1050 to 1150 cm. -1 The second derivative waveform in the Kaiser range is shown.

[0046] That is, the waveform obtained by second-order differentiation of the ATR-FTIR spectrum waveform 6a of the as-synthesized OCP crystal powder in Figure 8(a) is waveform 6b in Figure 8(b), the waveform obtained by second-order differentiation of the ATR-FTIR spectrum waveform 7a of the plate-like crystals deposited by electrophoresis in Figure 8(a) is waveform 7b in Figure 8(b), and the waveform obtained by second-order differentiation of the ATR-FTIR spectrum waveform 8a of the residual particles in Figure 8(a) is waveform 8b in Figure 8(b).

[0047] Focusing on the spectrum waveform in Figure 8(b), the second derivative waveform 8b of the residual particles has a wave number of 1123 cm -1 and 1104cm -1 It can be seen that there are no peaks like those in the other waveforms (second-order differential waveform 6b of OCP crystal powder, second-order differential waveform 7b of sedimentary plate crystal). This is because hydrogen phosphate ions (HPO4 2-) stretching vibration is absent in the residual particles.

[0048] That is, even though the residual particles were crystalline, the layer structure was destroyed, and it is believed that only plate-like crystals having a layer structure were deposited on the gold sensor.

[0049] Figure 10 shows a schematic diagram of the electrophoretic deposition mechanism of OCP in this process. As shown in Figure 10, an ethanol dispersion 10 of as-synthesized OCP crystals contains a mixture of crystals with a regular layer structure (plate-like crystals) 13 and irregular particles 15 with a destroyed layer structure. The plate-like crystals 13, which consist of alternating hydration layers 11 and apatite layers 12, have a specific charge due to the charge-up of the hydration layers 11, which causes the OCP particles to move by electrophoresis.

[0050] In FIG. 10, when a predetermined voltage is applied between two electrodes (counter electrode 17 and gold sensor 18) connected to a DC power supply 19, ethanol is electrolyzed to produce hydrogen ions and ethoxide ions (CHO - ), and the hydration layer of the plate-like crystal 13 bonds with the hydrogen ions, causing the crystal as a whole to become positively charged (+). - ) becomes negatively (-) charged and becomes a counter ion, creating a positive and negative electric double layer.

[0051] Therefore, it is thought that electrophoresis enables the positively charged OCP particles to move to the cathode, and the positive calcium ions present in large quantities in the apatite layer interact with the negative ions in the gold sensor to form plate-like crystals.

[0052] According to the DLVO theory, which explains the dispersion and aggregation phenomena between charged particles, the van der Waals force, which is the attractive force between particles, is maximized at the position where the interparticle distance is short (first minimum) on the potential curve (not shown) that represents the aggregation force between particles. This also shows that in an ethanol dispersion containing counterions, as shown in Figure 10, the van der Waals force F increases in the order F0 (= electrophoretic force Fe) → F2 → F1, and the plate-like crystals become more stable as they approach the electrode (gold sensor 18).

[0053] <Third process> The octacalcium phosphate crystal deposition film formed in the second step is a multilayer film. Therefore, in this step, the surface layer of the multilayer film is removed, leaving only the deep layer on the gold sensor side. In other words, to leave only the crystalline film firmly attached to the substrate, the multilayer film of octacalcium phosphate crystals is converted into a single layer.

[0054] Figure 11 is a flowchart showing the process of converting the multilayer film of octacalcium phosphate crystals into a single layer. In step S31 of Figure 11, the gold sensor with the multilayer film deposited thereon is ultrasonically cleaned. Here, pure ethanol is used as the cleaning solution, and the gold sensor with the octacalcium phosphate crystals deposited thereon in the second step is ultrasonically cleaned in pure ethanol for 1 minute at an ultrasonic oscillation frequency of 40 kHz and an ultrasonic output of 130 W.

[0055] In step S32, the cleaned sample was immersed in 20 ml of ethanol. In the following step S33, the sample was removed from the ethanol and dried by nitrogen blowing. In step S34, a gold sensor with a single-layer deposited film was formed, i.e., an OCP crystal film with a single-layer deposited film. Then, in step S35, the OCP crystal film with the multilayer film formed into a single layer was evaluated.

[0056] As described above, even when the ultrasonic cleaning frequency was 40 kHz and the ultrasonic output was 130 W during the process of converting the multilayer structure into a single layer, a single layer film remained on the substrate, demonstrating that the single layer OCP film has strong adhesion to the substrate. Surface measurements were performed to evaluate the single-layered OCP crystalline film. Figure 12(a) is an AFM (atomic force microscopy) image of the single layer OCP crystalline film formed in this process. Figure 12(b) is a cross-sectional view of the single layer OCP crystalline film in Figure 12(a) cut along the line A-A'.

[0057] Figure 12(c) is an enlarged AFM image of the area H enclosed by the dotted line in Figure 12(a), Figure 12(d) is a cross-sectional view of the single-layer OCP crystal film of Figure 12(c) cut along the line B-B', Figure 13(a) is an AFM image of the surface of a gold sensor without a coating, and Figure 13(b) is a cross-sectional view of the gold sensor of Figure 13(a) cut along the line C-C'.

[0058] When compared with the AFM images of the uncoated gold sensor (Fig. 13(a) and (b)), the AFM images and cross-sectional views in Fig. 12(a) to (d) show that a single layer of deposited film is fixed on the gold sensor by this process. It is also clear that the (100) plane of the OCP crystal is exposed (bare) to the outside in the layer direction, as shown in Fig. 12(c).

[0059] The coverage of the single-layer OCP crystal film formed in this process was calculated using AFM analysis software and found to be in the range of 20–95%, averaging approximately 60% (the coverage of the uncoated gold sensor was 0%). Furthermore, the surface smoothness (the surface roughness, or root mean square roughness (RMS) calculated from the AFM image) of the single-layer OCP crystal film was measured over a 5 μm × 5 μm area (the area of ​​the image shown in Figure 12(a)). The RMS values ​​were 16 nm, 38 nm, and 43 nm for coverages of 35%, 46%, and 60%, respectively. Since the root mean square roughness of the uncoated gold sensor surface (the base substrate) over a 5 μm × 5 μm area was less than 2 nm, the RMS of the single-layer OCP crystal film formed in this process was found to be greater than 2 nm, approximately 20 times rougher than the gold sensor surface.

[0060] The relationship between coverage and RMS is linear when the edges of the platelet crystals overlap, although this is not shown in the figure. From this linearity, it can be estimated that the RMS value at 100% coverage is approximately 71 nm.

[0061] As mentioned above, the AFM image showed an RMS value of 43 nm at a coverage rate of 60%. Based on this result, the theoretical RMS value for a coverage rate of 100% can be calculated to be 71 nm. However, since the thickness of the OCP crystal shown in Figure 12(b) is in the range of approximately 30 to 70 nm, if we adopt a maximum thickness of 100 nm, taking into account the range of OCP crystal thicknesses (20 to 100 nm) reported in other literature, the RMS value may be greater than 71 nm. Therefore, here, we set the preferred RMS value for a monolayer OCP crystal film to be 100 nm or less.

[0062] On the other hand, if the edges of the OCP crystals overlap, the Rms value also increases. Therefore, for the same reason as in specifying the upper limit of the thickness of the OCP crystal film described below, if we assume a wide range and set the upper limit to 200 nm as double 100 nm, the upper limit of the Rms value is thought to be 200 nm.

[0063] Furthermore, we compared the monolayer OCP crystal film formed in this process with a hydroxyapatite particle film coated on a gold electrode in a hydroxyapatite (HA) sensor. Figure 14(a) is an AFM image of the hydroxyapatite film, and Figure 14(b) is a cross-sectional view of the hydroxyapatite film in Figure 14(a) cut along the line D-D'.

[0064] Figure 15 shows the particle size distribution of a hydroxyapatite film. Here, when the long axis (major diameter) and short axis (minor diameter) of a particle are defined as shown in Figure 15(a), the long axis, short axis, and aspect ratio (long axis divided by short axis) are distributed as shown in Figure 15(b).

[0065] Figure 16 shows the particle size distribution of a single-layer OCP crystal film. When the long axis (major diameter) and short axis (minor diameter) of the particle are defined as shown in Figure 16(a), the long axis, short axis, and aspect ratio obtained by dividing the long axis by the short axis are distributed as shown in Figure 16(b).

[0066] Table 2 shows the results of a comparison between a hydroxyapatite film and a single-layer OCP crystal film in terms of the particle size distribution range, etc.

[0067] [Table 2]

[0068] A comparison of the AFM images of the single-layer OCP crystal film and the hydroxyapatite film taken at the same magnification, Figure 12(c) and Figure 14(a), and a comparison of the crystal sizes (distribution ranges of the major and minor axes) shown in Table 2, revealed that the single-layer OCP crystal film clearly differs from the hydroxyapatite film in terms of crystal shape and size, and that there is also a significant difference in film thickness between the two.

[0069] Furthermore, as shown in Table 2, a significant difference was observed in the aspect ratio of the particle size between the single-layer OCP crystal film and the hydroxyapatite film.

[0070] The thickness of a single-layer OCP crystal film is approximately 20 to 50 nm, as seen in the cross-sectional AFM image of Figure 12(d), but other literature suggests that the thickness of the OCP crystal is in the range of approximately 20 to 100 nm. Taking the range described in the literature into account, if we adopt a maximum thickness of 100 nm, the average film thickness will be 100 nm when the coverage of the single-layer OCP crystal film reaches 100%. However, since the thickness doubles when the edges of the OCP crystals overlap in the crystal film, we assume a theoretical upper limit of 100 × 2 = 200 nm, and set the film thickness here to 200 nm or less.

[0071] On the other hand, since the film thickness of the single-layer OCP crystal film measured by QCM (QCM film thickness) was basically 50 nm or less, a more preferable film thickness of the single-layer OCP crystal film is set to 50 nm or less as shown in Table 2.

[0072] Note that with single-layer OCP crystalline films, due to individual sample differences, experimental conditions, and other factors, it is possible that particles larger than those obtained in actual experiments may be formed, and that when OCP particles are synthesized, the particle size may exceed the range shown in Figure 16(b). Therefore, Table 2 lists the range of the major axis of single-layer OCP crystalline films that is expected to exceed the particle size shown in Figure 16(b) as a distribution range. The same applies to the aspect ratios shown in Table 2.

[0073] In evaluating the single-layer OCP crystal film formed in this process, in addition to the evaluation using the AFM images described above, the crystal surface was measured using ATR-FTIR and X-ray photoelectron spectroscopy (XPS) as a method to highlight the characteristics of the single layer, i.e., the differences from multilayers.

[0074] In the analysis of multilayer films by ATR-FTIR, both the interactions between OCP crystals and the bonding of individual OCP crystals can be observed because the penetration depth of infrared light into the crystals is 0.5 to 2 μm. In contrast, in the analysis of monolayer films by ATR-FTIR, the interactions between OCP crystals are eliminated and functional groups derived from the individual crystals can be detected, so only the bonding of individual OCP crystals within the monolayer film can be observed.

[0075] Figure 17 shows the spectral waveforms measured by ATR-FTIR for the multilayer film and the single-layer OCP crystal film before ultrasonic cleaning. As shown in Figure 17, there is a clear difference in shape between the ATR-FTIR spectral waveform 21 of the multilayer film and the ATR-FTIR spectral waveform 23 of the single-layer OCP crystal film. Note that waveform 20 is the ATR-FTIR spectral waveform of the gold sensor.

[0076] When we focus on the change in the ATR-FTIR spectrum waveform 23 of the single-layer OCP crystalline film shown in Figure 17, we can see that the light absorption intensity is at a wavenumber of 1195 cm -1 ,628cm -1 and increases to a wavenumber of 1035 cm -1 The wave number is 1195 cm -1 ,628cm -1 The increase in the hydrated layer of the OCP crystal film is due to the hydrogen phosphate ions (HPO4 2- ), which means an increase in H2O absorption, and the wavenumber is 1035 cm -1 The decrease in 3- ) resulting from decreased absorption.

[0077] That is, the wave number is 1195 cm -1 ,628cm -1 The increase in the light absorption intensity at 1035 cm indicates that the hydration layer is increasing throughout the monolayer OCP crystal film. -1 The decrease in light absorption intensity at PO4 3- This decrease in absorption indicates that the hydrated structure of the entire monolayer OCP crystal film is stronger than that of the apatite layer.

[0078] Furthermore, when the spectrum waveforms in Figure 17 are examined in detail, the ATR-FTIR spectrum waveform 23 of the single-layer OCP crystalline film has a wavenumber of 1118 cm -1 At 1125cm -1 and 1108cm -1 The peak splits into two and the wavenumber is 1035 cm -1 1038cm -1 and 1028cm -1This peak splitting is due to the monolayer formation of the OCP crystal film, and indicates that the monolayer OCP crystal film formed has high crystallinity and a solid hydration layer.

[0079] 18 shows a comparison of the ATR-FTIR spectrum of the single-layer OCP crystal film according to this embodiment and that of a hydroxyapatite film. As shown in FIG. 18, the spectrum 27 of the single-layer OCP crystal film contains a peak at 1195 cm -1 and 1123cm -1 In the spectrum of hydroxyapatite film, there are two peaks (significant light absorption points) that are not seen in the spectrum of hydroxyapatite film.

[0080] These two peaks indicate that a hydration layer is definitely formed on the single-layer OCP crystal film, and therefore, the presence or absence of these peaks in the spectral waveform makes it easy to distinguish between a single-layer OCP crystal film and a hydroxyapatite film.

[0081] Next, we will explain the results of measuring the crystal surface by X-ray photoelectron spectroscopy (XPS), where narrow scan analysis was employed to examine the energy range of specific elements.

[0082] Figure 19 shows the results of XPS analysis of calcium (Ca), phosphorus (P), and oxygen (O) in a multilayer film and a single-layer film (the single-layer OCP crystal film according to this embodiment). Figure 19 shows that there are differences in the waveform shape and emitted photoelectron intensity for each of the above elements between the multilayer film and the single-layer film.

[0083] Figure 20 shows the results of spectral separation for phosphorus (P) shown in Figure 19. Figure 20 (a) shows the peak separation results for phosphorus (P) in a multilayer film, and (b) shows the results in a single-layer film. Table 3 below shows the proportions of ions in the multilayer film and the single-layer film.

[0084] [Table 3]

[0085] In XPS analysis, the escape depth of photoelectrons is 2 to 6 nm, making it possible to detect the chemical state at the outermost surface of the film. From FIG. 20(b) and Table 3, it can be seen that in the single-layer OCP crystalline film according to this embodiment, hydrogen phosphate ions (HPO4 2- ) increases. This suggests that a hydration layer is exposed on the outermost surface of the monolayer film.

[0086] Therefore, on the surface of the substrate (gold sensor) where the multilayer film is monolayered, a crystalline film of OCP is formed, which has a layer structure in which the hydrated layer formed on the surface opposite the substrate in the thickness direction of the crystalline film is the uppermost layer, and the hydrated layer and the apatite layer are regularly repeated. In other words, the crystalline structure of the monolayer film formed by monolayering is layered.

[0087] <Fourth step> In this step, we evaluated the sensing capability of the sensor on which the monolayer film was formed through steps 1 to 3, and its reaction characteristics in a biological fluid environment. Specifically, we evaluated the behavior of the monolayer OCP film deposited on the substrate (gold sensor) in phosphate buffer solution (PBS solution).

[0088] Figure 21 is a flowchart showing the process for evaluating the behavior of a single-layer OCP film when a PBS solution is poured (pumped) into the film for a predetermined period of time. In step S41 of Figure 21, the sample (single-layer OCP film) to be evaluated was subjected to ozone cleaning using ultraviolet light (UV / O3) for 5 minutes. This removed organic contaminants from the sample.

[0089] In the next step S42, a PBS solution was pumped through the single-layer OCP membrane at 37°C and a rate of 77 ml per minute. The PBS solution used had a pH of 7.4±0.2 and contained 1370 mmol / L NaCl, 81 mmol / L NaHPO, 26.8 mmol / L KCl, and 14.7 mmol / L KHPO.

[0090] In step S43, the behavior in the PBS solution was evaluated using QCM-D. Figure 22 shows the weight change of the single-layer OCP film in the PBS solution, with the scale on the left vertical axis representing the frequency shift Δf of the quartz crystal oscillator and the scale on the right vertical axis representing the amplitude attenuation (dissipation ΔD). The harmonic n used in the evaluation was n=5.

[0091] In Figure 22, the scale on the left side of the vertical axis, from bottom to top, corresponds to weight loss, so the change in Δf due to the PBS solution, i.e., the gradual dissolution and peeling of the single-layer OCP film over time, resulting in a loss of weight, is thought to indicate the high reactivity of the single-layer OCP film in the PBS solution. Furthermore, a change in ΔD, as shown in Figure 22, i.e., a loss of resonance energy in the single-layer OCP film, was also confirmed.

[0092] Figure 23 shows the weight change of a hydroxyapatite film in a PBS solution over time. As can be seen from Figure 23, there is no change in Δf or ΔD of the hydroxyapatite film, and in comparison with Figure 22, it is clear that the single-layer OCP film behaves significantly differently from the hydroxyapatite film in a PBS solution, and exhibits superior bioreactivity to the hydroxyapatite film.

[0093] Figure 24 is a flowchart showing the process of evaluating the behavior of a single-layer OCP film deposited on a substrate (gold sensor) when the film is immersed in a PBS solution. In step S51 of Figure 24, the sample (single-layer OCP film) to be evaluated was immersed in a PBS solution. Here, the sample was immersed in 3 ml of PBS solution at 37°C for 0 minutes, 5 minutes, and 120 minutes.

[0094] In step S52, the immersed sample was washed once with ultrapure water and once with ethanol. In the following step S53, the washed sample was dried at 60°C for 24 hours. Then, in step S54, QCM weight measurement was performed to evaluate the behavior of the single-layer OCP film immersed in the PBS solution.

[0095] Figure 25 shows the change in film thickness of a single-layer OCP film versus the immersion time in PBS solution, and Figure 26 shows the spectral waveforms measured by ATR-FTIR for a single-layer OCP crystal film immersed for 0, 5, and 120 minutes.

[0096] From FIG. 25, it can be seen that the single-layer OCP film gradually dissolves and peels off as the immersion time in the PBS solution increases, and the film thickness also gradually decreases.

[0097] From the spectrum waveform in Figure 26, the light absorption intensity of the single-layer OCP crystal film decreased as the immersion time in the PBS solution increased, confirming the dissolution of the crystals. -1 The wave number decreases to 1037 cm -1 Wave number 1112 cm -1 The decrease in the phosphate ions (HPO4 2- ) and the wavenumber is 1037 cm -1 The increase in the phosphate ions (PO4 3- This is due to the increased absorption of apatite. In other words, the apatite layer is thought to increase relatively because it is dissolved from the hydration layer during the immersion process.

[0098] Figure 27 shows the results of XPS analysis of calcium (Ca), phosphorus (P), and oxygen (O) before and after immersion of a single-layer OCP film in a PBS solution. From Figure 27, it can be seen that there are differences in the waveform shape and emitted photoelectron intensity for P due to immersion. Figure 28 shows the results of spectral separation for phosphorus (P) in Figure 26. As can be seen from (a) and (b) of Figure 28, immersion for 5 minutes resulted in a significant increase in the amount of HPO4 2- decreases, H2PO4 - The component of increased.

[0099] Based on the above results, the reason why the ΔD value of the single-layer OCP film increases over time as shown in Figure 22 can be considered. In the PBS solution, surrounding ions and water diffuse into the hydration layer of the single-layer OCP film, causing the layer structure to swell, and the HPO42- It is thought that reacted with water and was converted to H2PO4. The reaction formula is: HPO4 2- +H3O + →H2PO4 - Furthermore, after 120 minutes of immersion, PO4 3- Increased HPO4 2- This is thought to be because the apatite layer becomes the main layer due to dissolution from the hydrated layer.

[0100] Figure 29 shows the weight change over time of a single-layer OCP film in a citrate buffer solution (pH = 6). Figure 30 shows the weight change of a hydroxyapatite film under the same conditions as above. In Figures 29 and 30, the scale on the left vertical axis represents the frequency shift Δf of the quartz crystal resonator, and the scale on the right vertical axis represents the amplitude attenuation (dissipation) ΔD. The harmonic n used in the evaluation was n = 5.

[0101] Figures 29 and 30 show that the monolayer OCP film behaves differently from the hydroxyapatite film. That is, the hydroxyapatite film exhibits an increase in Δf and a decrease in ΔD, indicating that it dissolves in citrate buffer solution. In contrast, the monolayer OCP film differs significantly from the hydroxyapatite film in that both Δf and ΔD show almost no change in citrate buffer solution, confirming that swelling and dissolution hardly occur. This demonstrates that stable sensing can be achieved using a sensor formed with a monolayer OCP film.

[0102] Figure 31 shows AFM (atomic force microscopy) images of a single-layer OCP crystal film immersed in a PBS solution. Figure 31(a) is an AFM image of the single-layer OCP crystal film immersed for 0 minutes, and Figure 31(b) is a cross-sectional view of the single-layer OCP crystal film in Figure 31(a) taken along line E-E'.

[0103] Similarly, Figure 31(c) is an AFM image of a single-layer OCP crystal film immersed in PBS solution for 5 minutes, Figure 31(d) is a cross-sectional view of the single-layer OCP crystal film of Figure 31(c) cut along line F-F', Figure 31(e) is an AFM image of the single-layer OCP crystal film after immersion for 120 minutes, and Figure 31(f) is a cross-sectional view of the single-layer OCP crystal film of Figure 31(e) cut along line G-G'.

[0104] Considering the AFM image of the single-layer OCP crystal film shown in Figure 31 together with the spectral waveform in Figure 26, it is understood that when the single-layer OCP crystal film is thinned by immersion in PBS solution, the crystal film dissolves from the (100) face of the OCP crystal. This is because the plate-like shape of the crystal is maintained even when the crystal film is thinned, and this behavior is understood to be the behavior of the hydration layer and apatite layer peeling off one by one from the (100) face of the crystal in the single-layer OCP film, which has a layered structure.

[0105] As explained above, this embodiment allows the formation of a stable crystalline film on the surface of a substrate (gold sensor) in which octacalcium phosphate is arranged in a single layer, with the hydrated layer being the topmost layer and having a layer structure in which hydrated and apatite layers are regularly repeated. In other words, it is possible to form a monolayer film on the substrate in which the (100) plane of the octacalcium phosphate crystals is exposed to the outside without being covered by other crystals. This will enable us to elucidate the mechanisms of biodegradation and reabsorption of octacalcium phosphate in vivo and its elution behavior in biological fluid environments, enabling a wide range of applications in biomedicine and medicine.

[0106] By peeling off the layered octacalcium phosphate crystalline film from the substrate and using it to cover the bone defect site, the OCP can fuse with bone tissue, and the interaction between the OCP and collagen tissue can lead to the attachment of osteoclasts to the (100) surface of the OCP crystal, promoting osteoblast differentiation and bone regeneration through interactions with proteins. In other words, the ability to elucidate and control the reaction behavior of proteins adsorption in biological fluids on the crystal film surface makes it possible to apply octacalcium phosphate crystalline films to a wide range of biomedical applications.

[0107] In a monolayer OCP crystal film, the (100) face of the crystal exposed on the surface is the largest face of the OCP crystal, has surface active sites where chemical reactions occur, is charged, and has high adsorption capacity. Hydrogen bonds are formed by the hydrated layer on the top, so the monolayer OCP crystal film can be used as a drug carrier.

[0108] Furthermore, by adsorbing a drug onto the (100) surface of a single-layer OCP crystal film and introducing it into a living body, the bond between the OCP and the drug is broken by water molecules in the biological fluid environment, and hydrogen bonds are formed between the drug and water molecules, enabling the drug to be released into the biological fluid in a sustained manner. [Industrial Applicability]

[0109] This method can be applied to the general formation of octacalcium phosphate crystal coatings on conductive solid surfaces such as medical devices. In addition, coating octacalcium phosphate crystals on sensor surfaces can be used to study the reactions of layered OCP compounds, the mechanisms of bone and tooth formation, and the behavior of calcium phosphate phase transitions. [Explanation of symbols]

[0110] 1 Plate-shaped crystals 2. Amorphous crystals 4 OCP particles 10 OCP crystal ethanol dispersion 11 Hydration layer 12 Apatite layer 13 Plate-like crystals 14 Glass containers 15 Irregular particles 17 Counter electrode 18 Gold Sensor 19 DC power supply

Claims

1. octacalcium phosphate platelet crystals are arranged in a single layer, the major axis of the platelet crystals is distributed in a range of 50 nm to 10 μm, the minor axis is distributed in a range of 5 to 1000 nm, and the aspect ratio, which is the value obtained by dividing the major axis by the minor axis, is 2 to 100; a specific crystal plane of the plate-like crystal is oriented in a specific direction, the specific direction is a surface direction of the crystal film, and the specific crystal plane is exposed on the surface of the crystal film preferentially over other crystal planes other than the specific crystal plane; The crystalline film is formed on the surface of a conductive substrate.

2. 2. The crystal film according to claim 1, wherein the conductive substrate is made of a conductive material selected from the group consisting of gold, titanium, chromium, and silicon.

3. 2. The crystal film according to claim 1, wherein the specific crystal face is exposed in a thickness direction of the crystal film, in a direction opposite to the conductive substrate.

4. The crystal film according to claim 1, characterized in that the surface of the crystal film has a surface smoothness such that the root mean square roughness in a measurement range of 5 μm × 5 μm is 200 nm or less, preferably 100 nm or less, and the film thickness is 200 nm or less, preferably 50 nm or less.

5. 2. The crystal film according to claim 1, which has adhesive properties such that it does not peel off from the conductive substrate even after ultrasonic cleaning in ethanol at a frequency of 40 kHz for 1 minute.

6. The crystal film described in claim 3, characterized in that the crystals in the crystal film have a layer structure in which hydrated layers and apatite layers alternate, and the hydrated layer is exposed as the uppermost layer on the side of the thickness direction of the crystal film opposite the conductive substrate.

7. 7. The crystal film according to claim 1, wherein the specific crystal plane is a (100) plane.

8. synthesizing octacalcium phosphate crystals (OCP crystals) by wet synthesis; preparing an ethanol dispersion of the OCP crystals; a step of forming a multilayer deposition film of the OCP crystals on a cathode by electrophoresis of the ethanol dispersion, the cathode being made of a conductive material selected from at least gold, titanium, chromium, and silicon; a step of ultrasonically cleaning the multilayer deposition film to form a single layer deposition film made of the OCP crystal on the cathode; A method for manufacturing a crystal film, comprising:

9. the single-layer deposited film is a crystal film in which the (100) plane of the OCP crystal is oriented toward the surface of the deposited film and is exposed at the surface of the deposited film preferentially over other crystal planes other than the (100) plane; the OCP crystals have a major axis distribution range of 50 nm to 10 μm, a minor axis distribution range of 5 to 1000 nm, and an aspect ratio, which is the value obtained by dividing the major axis by the minor axis, of 2 to 100; The method for producing a crystal film according to claim 8, wherein the single-layer deposited film is formed on the surface of the conductive substrate as the cathode, the deposited film has a surface smoothness such that the root mean square roughness in a measurement range of 5 μm × 5 μm is 200 nm or less, preferably 100 nm or less, the deposited film has a film thickness of 200 nm or less, preferably 50 nm or less, and has adhesive properties such that it does not peel off from the conductive substrate even after ultrasonic cleaning in ethanol at a frequency of 40 kHz for 1 minute.

10. The method for producing a crystal film according to claim 9, characterized in that the crystals in the single-layer deposited film have a structure in which hydrated layers and apatite layers are alternately repeated, and the hydrated layer is exposed as the uppermost layer on the side of the deposited film opposite the conductive substrate in the thickness direction.

11. 9. The method for producing a crystal film according to claim 8, wherein the electrophoresis time for the multilayer deposited film by electrophoresis is at least 1 minute.

12. A quartz crystal microbalance (QCM) sensor comprising a conductive substrate coated with the crystal film according to any one of claims 1 to 6, A QCM sensor characterized in that the (100) plane of the plate-like crystals of octacalcium phosphate constituting the crystal film is exposed on the surface of the crystal film in preference to other crystal planes other than the (100) plane.

Images