Crystal film of octacalcium phosphate and method for producing same

Abstract

In the present invention, electrophoresis of an ethanol dispersion of octacalcium phosphate (OCP) crystals is performed by using an electrode made of a conductive material, such as gold, as a cathode (gold sensor) to form a multilayer deposition film composed of the OCP crystals on the cathode. The multilayer deposition film is subjected to ultrasonic cleaning to form a single-layer deposition film composed of the OCP crystals on the cathode. The OCP crystal film formed on the surface of the gold sensor has a layer structure in which a hydration layer and an apatite layer are regularly repeated, and is a stable crystal film in which a specific crystal plane is aligned in a specific direction with the hydration layer as the uppermost layer.

Description

Octacalcium phosphate crystalline film and method for producing same

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

[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 characteristics such as the oscillation frequency and impedance of a quartz crystal 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 an Mg-containing calcium phosphate coating on the surface of a substrate made of magnesium or a magnesium alloy. Patent Document 2 discloses a medical device material in which a plurality of calcium phosphate layers with different compositions are provided on the surface of the substrate, and the surface side of the calcium phosphate layer is made of a substance that is more biodegradable in the body than the substrate side and is replaced by bone tissue.

[0006] JP 2018-202074 A JP 2020-130766 A

[0007] Although calcium phosphate has various crystalline phases, 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), which is 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 unevenness remains on the surface of the calcium phosphate layer on the substrate side, 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 of Patent Documents 1 and 2 are both multilayers in which crystals are stacked, and formation of a monolayer film in which single crystals exist in parallel as a single layer 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 lacking 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.

[0012] As a means for achieving the above-mentioned object and solving the above-mentioned problems, the crystal film of the present invention has the following configuration: Namely, the crystal film of the present invention is characterized in that 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.

[0013] The crystal film of the present invention is characterized, for example, in that a specific crystal plane of the plate crystal is oriented in a specific direction. For example, the specific direction is the surface direction of the crystal film, and the specific crystal plane is preferentially exposed on the surface of the crystal film over other crystal planes other than the specific crystal plane. For example, the crystal film is formed on the surface of a conductive substrate. Furthermore, for 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 crystal film has a surface smoothness in which the mean square roughness of the crystal film is 200 nm or less, preferably 100 nm or less, within a measurement range of 5 μm × 5 μm, and a film thickness is 200 nm or less, preferably 50 nm or less. For example, the crystal film has 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 crystalline film have a layer structure in which hydrated layers and apatite layers are alternately repeated, and the hydrated layers are exposed as the uppermost layers on the side of the crystalline film opposite to 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 the cathode by electrophoresis of the ethanol dispersion, using an electrode made of a conductive material selected from at least gold, titanium, chromium, and silicon as the cathode, 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 OCP crystals have a major axis distribution range of 50 nm to 10 μm and a minor axis distribution range of 5 to 1,000 nm, and an aspect ratio, which is the value obtained by dividing the major axis by the minor axis, of 2 to 100; the monolayer 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 film thickness is 200 nm or less, preferably 50 nm or less; and the film has adhesion 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. Furthermore, for example, 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 to 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.

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

[0018] 1 is a flowchart showing the synthesis of OCP crystals and the preparation of an OCP crystal dispersion. 2 is an FE-SEM image of OCP crystal powder. 3 is a flowchart showing the process of forming a deposited film by electrophoresis. 4 is an FE-SEM image of a planar view of a multilayer film of octacalcium phosphate crystals. 5 is an FE-SEM image of residual particles in a residual liquid. 6 is an FE-SEM image showing the relationship between the deposition time of a film by electrophoresis and the plate-like crystals. 7 is a diagram comparing the X-ray diffraction patterns (XRD patterns) of particles before and after deposition. 8 is an ATR-FTIR spectrum, which is the light absorption spectrum of the surface of each particle before and after deposition, measured by Fourier transform infrared spectroscopy (FTIR). 9 is a schematic diagram of an apparatus for forming a deposited film by electrophoresis. 10 is a schematic diagram showing the electrophoretic deposition mechanism of OCP. 11 is a flowchart showing the process of converting a multilayer film of octacalcium phosphate crystals into a single layer. 12 is an AFM image of a single-layer OCP crystal film and its cross-section. 13 is an AFM image of the surface of a gold sensor without a coating and its cross-section. 19A and 19B are AFM images of a hydroxyapatite film and its cross section. This is a diagram showing the particle size distribution of crystals forming a hydroxyapatite film. This is a diagram showing the particle size distribution of crystals forming a single-layer OCP crystal film. This is a spectrum waveform measured by ATR-FTIR of a multilayer film and a single-layer OCP crystal film before ultrasonic cleaning. This is a diagram showing a comparison of the ATR-FTIR spectrum waveforms of a single-layer OCP crystal film and a hydroxyapatite film. This is a diagram showing the results of XPS analysis of a multilayer film and a single-layer OCP crystal film for Ca, P, and O. This is a diagram showing the results of spectral separation for phosphorus (P) in FIG. 19A. This is a flowchart showing the process of evaluating the behavior of a single-layer OCP film when a PBS solution is pumped for a predetermined time. This is a diagram showing the weight change, etc. of a single-layer OCP film in a PBS solution. This is a diagram showing the weight change, etc. of a hydroxyapatite film in a PBS solution. This is a flowchart showing the process of evaluating the behavior of a single-layer OCP film when the single-layer OCP film is immersed in a PBS solution. 1 shows the change in film thickness of a single-layer OCP film with respect to the immersion time in a PBS solution, 2 shows the spectral waveform of a single-layer OCP crystal film measured by ATR-FTIR with respect to the immersion time in a PBS solution, and 3 shows the results of XPS analysis of Ca, P, and O of a single-layer OCP film before and after immersion in a PBS solution.28 is a diagram showing the results of spectral separation of phosphorus (P) in FIG. 27. FIG. 29 is a diagram showing weight changes over time of a monolayer OCP film in a citrate buffer solution. FIG. 30 is a diagram showing weight changes over time of a hydroxyapatite film in a citrate buffer solution. FIG. 31 is an AFM image of a monolayer OCP crystal film immersed in a PBS solution.

[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 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 such a manner that a specific crystal plane of the OCP platelet crystals deposited on a substrate is arranged parallel to the substrate, and that this 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 having 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 step, octacalcium phosphate (OCP) crystals were synthesized by wet synthesis, and an OCP dispersion was prepared. FIG. 1 is a flowchart showing the synthesis of OCP crystals and the preparation of an OCP crystal dispersion. In step S11 of FIG. 1, 25 ml of an acetate buffer solution (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 steps is heated and stirred. Here, the heating temperature is set to 60°C, and stirring is performed at 1,200 revolutions per minute for 3 hours. In this heating and stirring step, the dissolution process of α-TCP shown in formula (1) and the transition process to OCP 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, where the sample is washed once with ultrapure water and three times with ethanol.

[0026] The washed sample was heated and dried (60°C, 48 hours) in step S17 to produce an OCP crystal powder, which was then recovered and 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 synthesis of the OCP crystals described above contains a mixture of plate-like crystals 1 (shown surrounded by a dashed line in Figure 2) that are the target of the octacalcium phosphate crystal coating formation according to this embodiment, and amorphous crystals 2 (shown surrounded by a dotted line in Figure 2).

[0028] <Second Step> In this step, a deposited film (multilayer film) of octacalcium phosphate crystals is formed by electrophoresis. Fig. 3 is a flowchart showing the process of forming an electrophoretic deposited film. In step S21 of Fig. 3, as shown in Fig. 9, 20 ml of a dispersion containing OCP particles 4, i.e., the OCP crystal ethanol dispersion 10 prepared in the first step 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 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 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, the film was formed by varying 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 sample thus obtained 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 4(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 the gold sensor by electrophoresis, viewed in plan (viewed from the direction toward the surface of the gold sensor). Figures 5(a) and 5(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. Meanwhile, 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 findings 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 a film by electrophoresis (also referred to as the electrophoresis time or voltage application time) and the plate-like crystals. Figure 6(a) is an FE-SEM image obtained when the deposition time was 1 minute, (b) is an FE-SEM image obtained when the deposition time was 2 minutes, (c) is an FE-SEM image obtained when the deposition time was 3 minutes, (d) is an FE-SEM image obtained when the deposition time was 4 minutes, and (e) is an FE-SEM image obtained when the deposition time was 5 minutes. In Figures 6(a) to 6(e), the images within the dashed lines are partially enlarged images of each obtained image.

[0036] These FE-SEM images confirm plate crystals at all deposition times. The abundance of plate crystals at each deposition time was determined using image processing software called ImageJ. The results showed that the abundance of plate crystals was 80% when the deposition time was 1 minute, 60% when it was 2 minutes, 55% when it was 3 minutes, 52% when it was 4 minutes, and 40% when it was 5 minutes. Since the abundance of plate crystals varies depending on the deposition time, subsequent experiments and evaluations used samples obtained with a deposition time of 1 minute, at which the abundance of plate crystals reached 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 plate-like crystals, and residual particles) before and after deposition shown in the XRD patterns of FIG.

[0039]

[0040] From the three XRD patterns shown in Figure 7 for the OCP crystal powder, the deposited plate-like crystals, and the residual particles, the crystalline phase is the same before and after deposition and in the undeposited particles, and all are single-phase OCP. Since the crystalline phase of the OCP particles does not change before and after deposition, it can be seen 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 plate-like crystals is larger than that of the other particles, indicating 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]

[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 width (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 of the surfaces of the particles (OCP crystal powder, deposited plate-like 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 (wave number) 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 spectral waveform in FIG. 8(b), the second derivative waveform 8b of the residual particles has a wave number of 1123 cm -１ and 1104 cm -１It can be seen that there are no peaks like those in the other waveforms (second-order differential waveform 6b of the OCP crystal powder and second-order differential waveform 7b of the sedimentary platelet 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 (plate-like crystals) 13 with a regular layer structure and irregular particles 15 with a disrupted layer structure. The plate-like crystals 13, which are composed 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 hydrated layer of the plate crystal 13 bonds with the hydrogen ions, causing the crystal as a whole to be 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 of the gold sensor to form plate-like crystals.

[0052] According to the DLVO theory, which explains the dispersion and aggregation phenomenon 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-order minimum) on a potential curve (not shown) that represents the aggregation force between particles. This also shows that, as shown in Figure 10, in an ethanol dispersion containing counter ions, 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] The octacalcium phosphate crystal deposition film formed in the second step is a multilayer film. In this step, the surface layer of the multilayer film is removed, leaving only the deeper 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 divided 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 on which the multilayer film has been deposited is ultrasonically cleaned. Here, pure ethanol is used as the cleaning solution, and the gold sensor on which the octacalcium phosphate crystals formed in the second step have been deposited 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 sample after the cleaning was immersed in 20 ml of ethanol. In the following step S33, the sample was pulled out of the ethanol and dried by nitrogen blowing. In step S34, a gold sensor with a single-layer deposited film adhered thereto, i.e., an OCP crystal film with a single-layer deposited film, was formed. 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. Here, surface measurements and other methods were performed to evaluate the single-layered OCP crystal film. Figure 12(a) is an AFM (atomic force microscopy) image of the single layer OCP crystal film formed in this process. Figure 12(b) is a cross-sectional view of the single layer OCP crystal film of Figure 12(a) cut along the line A-A'.

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

[0058] 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 to 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)). When the surface 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 the coverage and RMS is linear from actual measurements, even when the edges of the plate crystals overlap (not shown). From this linearity, it can be estimated that the RMS at a coverage of 100% is approximately 71 nm.

[0061] As mentioned above, the AFM image showed an RMS value of 43 nm at a coverage rate of 60%, and 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 a maximum thickness of 100 nm is adopted, taking into account the OCP crystal thickness range (20 to 100 nm) described in other literature, the RMS value may be greater than the above 71 nm. Therefore, here, the preferred RMS value for a monolayered OCP crystal film is set to 100 nm or less.

[0062] On the other hand, when the ends of the OCP crystals overlap, the RMS value also becomes large. Therefore, for the same reason as when specifying the upper limit of the film thickness of the OCP crystal film described below, if we assume a wide range and set it at twice 100 nm, the upper limit of the RMS value is thought to be 200 nm.

[0063] Furthermore, the monolayer OCP crystal film formed in this process was compared 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) taken along the line D-D'.

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

[0065] FIG. 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 FIG. 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 FIG. 16(b).

[0066] Table 2 shows the results of a comparison between the hydroxyapatite film and the single-layer OCP crystal film with respect to the particle size distribution range and the like.

[0067]

[0068] A comparison of the AFM images of the single-layer OCP crystal film and the hydroxyapatite film taken at the same magnification, shown in 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 the single-layer OCP crystal film is approximately 20 to 50 nm, as shown in the cross-sectional view of the AFM image in Figure 12(d), for example. However, other literature suggests that the thickness of the OCP crystal is in the range of approximately 20 to 100 nm. Therefore, if we adopt a maximum thickness of 100 nm in consideration of the range described in the literature, the average film thickness will be 100 nm when the coverage rate 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 for the single-layer OCP crystal film, due to individual differences in samples, experimental circumstances, and the like, particles larger than those obtained in actual experiments may be formed, and it is expected that the particle size will exceed the range shown in Figure 16(b) when the OCP particles are synthesized. Therefore, Table 2 lists the range of the major axis of the single-layer OCP crystal film 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 multilayer films.

[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 single-layer films by ATR-FTIR, the interactions between OCP crystals are eliminated and functional groups derived from the single crystals can be detected, so only the bonding of individual OCP crystals within the single-layer film can be observed.

[0075] Figure 17 shows the spectral waveforms measured by ATR-FTIR for a multilayer film and a 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. Waveform 20 is the ATR-FTIR spectral waveform of the gold sensor.

[0076] When attention is paid to the change in the ATR-FTIR spectrum waveform 23 of the single-layer OCP crystal film shown in FIG. 17, the light absorption intensity is at a wave number of 1195 cm -１ , 628 cm -１ and the wave number increases to 1035 cm -１ The wave number is 1195 cm. -１ , 628 cm -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- ) This is due to decreased absorption.

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

[0078] Furthermore, when the spectrum waveform in FIG. 17 is examined in detail, the ATR-FTIR spectrum waveform 23 of the single-layer OCP crystal film has a wave number of 1118 cm -１ At 1125 cm -１ and 1108 cm -１ The peak splits into two and the wavenumber is 1035 cm -１ 1038 cm -１ and 1028 cm -１ This peak splitting is due to the monolayer formation of the OCP crystal film, and indicates that the formed monolayer OCP crystal film 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 1123 cm -１ , there are two peaks (notable light absorption points) that are not seen in the spectral waveform 25 of the 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 a specific element.

[0082] 19 shows the results of XPS analysis of a multilayer film and a single-layer film (the single-layer OCP crystal film according to this embodiment) for calcium (Ca), phosphorus (P), and oxygen (O). It can be seen from FIG. 19 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]

[0085] In XPS analysis, the escape depth of photoelectrons is 2 to 6 nm, making it possible to detect the chemical state at the top surface of the film. From FIG. 20(b) and Table 3, it can be seen that in the single-layer OCP crystal 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 has been 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] <Step 4> In this step, the sensor on which the monolayer film was formed through Steps 1 to 3 was evaluated for its sensing capability and its reaction characteristics in a biological fluid environment. Specifically, the behavior of the monolayer OCP film deposited on the substrate (gold sensor) in phosphate buffer solution (PBS solution) was evaluated.

[0088] 21 is a flowchart showing a process for evaluating the behavior of a single-layer OCP film when a PBS solution is poured (sent) into the film for a predetermined time. In step S41 of FIG. 21, the sample (single-layer OCP film) to be evaluated is subjected to ozone cleaning by ultraviolet irradiation (UV / O ３) was performed for 5 minutes, thereby removing organic contaminants from the sample.

[0089] In the next step S42, a PBS solution was pumped onto the single-layer OCP membrane at 37°C and a rate of 77 ml / min. 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 of the single-layer OCP film 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 Fig. 22, the scale on the left side of the vertical axis corresponds to weight loss from bottom to top, 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 its weight, is thought to indicate high reactivity of the single-layer OCP film in the PBS solution. Furthermore, the change in ΔD shown in Fig. 22, i.e., the loss of resonance energy of 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] 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 FIG. 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 sample after the immersion was washed once with ultrapure water and once with ethanol. In the subsequent 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 when immersed in the PBS solution.

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

[0096] From FIG. 25, it can be seen that the single-layer OCP film gradually dissolves and peels off with the passage of immersion time in the PBS solution, 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 is due to hydrogen phosphate ions (HPO4 2- ) and the wave number 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 HPO4 2- It is thought that reacted with water and was converted to H2PO4. The reaction formula is: HPO4 2- +H3O + →H2PO4 - ・H2O. 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 indicates the frequency shift Δf of the quartz crystal resonator, and the scale on the right vertical axis indicates the amplitude attenuation (dissipation) ΔD. The harmonic n used in the evaluation was n = 5.

[0101] 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 dissolution in citrate buffer solution. In contrast, the monolayer OCP film differs significantly from the hydroxyapatite film, with almost no change in Δf or ΔD in citrate buffer solution, confirming that swelling and dissolution hardly occur. This demonstrates that stable sensing can be achieved using a sensor formed with an OCP monolayer film.

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

[0103] Similarly, Fig. 31(c) is an AFM image of the single-layer OCP crystal film immersed in PBS solution for 5 minutes, Fig. 31(d) is a cross-sectional view of the single-layer OCP crystal film of Fig. 31(c) taken along line F-F', Fig. 31(e) is an AFM image of the single-layer OCP crystal film after immersion for 120 minutes, and Fig. 31(f) is a cross-sectional view of the single-layer OCP crystal film of Fig. 31(e) taken 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 a 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 therefore, in the single-layer OCP film with a layered structure, the hydration layer and the apatite layer peel off one by one from the (100) face of the crystal.

[0105] As described 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 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 OCP crystal (100) surface, promoting osteoblast differentiation, and bone regeneration through interaction with proteins. In other words, the ability to elucidate and control reaction behaviors such as protein 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.

[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.

[0110] REFERENCE SIGNS LIST 1 Plate-shaped crystal 2 Irregular crystal 4 OCP particle 10 OCP crystal ethanol dispersion 11 Hydration layer 12 Apatite layer 13 Plate-shaped crystal 14 Glass container 15 Irregular particle 17 Counter electrode 18 Gold sensor 19 DC power supply

Claims

1. A crystalline film 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, and the aspect ratio, which is the value obtained by dividing the major axis by the minor axis, is 2 to 100.

2. The crystal film according to claim 1, wherein a specific crystal plane of the plate-like crystal is oriented in a specific direction.

3. The crystal film described in claim 2, characterized in that the specific direction is the surface direction of the crystal film, and 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.

4. The crystal film according to claim 3, wherein the crystal film is formed on the surface of a conductive substrate.

5. The crystal film according to claim 4, wherein said conductive substrate is made of a conductive material selected from at least gold, titanium, chromium, and silicon.

6. The crystal film according to claim 4, wherein the specific crystal face is exposed in the thickness direction of the crystal film and in the direction opposite to the conductive substrate.

7. A crystal film according to claim 4, 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.

8. The crystalline film according to claim 4, 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.

9. The crystal film described in claim 6, 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 of the crystal film opposite the conductive substrate.

10. The crystal film according to any one of claims 1 to 9, wherein the specific crystal plane is the (100) plane.

11. A method for producing a crystal film, 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 the cathode by electrophoresis of the ethanol dispersion, using an electrode made of a conductive material selected from at least gold, titanium, chromium, and silicon as the cathode; and ultrasonically cleaning the multilayer deposition film to form a single layer deposition film of the OCP crystals on the cathode.

12. The method for producing a crystal film according to claim 11, wherein 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, the deposited film has a surface smoothness such that the root mean square roughness in a measurement range of 5 μm x 5 μm is 200 nm or less, preferably 100 nm or less, the film thickness is 200 nm or less, preferably 50 nm or less, and the film has adhesion 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.

13. A method for producing a crystal film as described in claim 12, 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.

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

15. A quartz crystal microbalance (QCM) sensor comprising a conductive substrate coated with the crystal film according to any one of claims 1 to 9, wherein the (100) plane of the octacalcium phosphate platelet 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.

Images