JP4900690B2 - Biosensor composition and method for producing the same - Google Patents

Description

The present invention relates to a new biosensor component having a feature such as rapid analysis of in vivo reaction of bioceramics and a method for producing the same.

Examples of existing methods for bioceramic coating include laser ablation and sputtering (Patent Document 1). According to such a conventional method, it is difficult to form a uniform thin film on a complex material or a uniform nano thin film on a biosensor substrate because of poor throwing power. On the other hand, the electrophoretic deposition method was able to uniformly coat bioceramic crystals even in complex shapes, but because it is a thick film of 10 μm or more, there is a problem of generation of cracks and peeling from the substrate. (Non-Patent Document 1).
JP 2005-283550 A Journal of Materials Science 16, 319-324 (2005)

The present invention is based on the background as described above, eliminates the conventional problems, takes advantage of the characteristics of the method of forming a bioceramic crystal by electrophoretic deposition, and improves it, thereby inconveniences such as generation of cracks and peeling of the substrate. Provided is a calcium phosphate compound biosensor composition capable of forming a thin ceramic crystal film capable of suppressing the occurrence of oxidization, and useful for analysis of various reactions occurring in vivo, and a method for producing the same. It is an issue.

The present invention is characterized by the following in order to solve the above problems.

First: nanomembrane by nanocrystalline calcium phosphate compound on a conductive substrate is formed, the size of the nanocrystals is at 200nm or less, the thickness of the nano thin film and characterized der Rukoto below 50nm A biosensor construct.

Second: The biosensor composition described above, wherein the conductive substrate is gold or titanium.

Fifth: the In the method of any one of the biosensor, after the crystals of the calcium phosphate compound is electrophoretic deposition on a conductive substrate, and ultrasonic irradiation, to form a nano thin film by nanocrystalline calcium phosphate compound A method for producing a biosensor composition, comprising:

According to the present invention as described above, in order to quickly perform in vivo reaction analysis of bioceramics, a nanothin film of bioceramic nanocrystals is provided on the surface of a metal substrate as a biosensor. In particular, by combining electrophoretic deposition and ultrasonic treatment, it is possible to manufacture a biosensor that can be applied to various surface reaction analyzes such as a quartz crystal (QCM).

According to the biosensor composition of the present invention, it can be applied to analysis of a crystal resonator (QCM), a surface plasmon, an ellipsometer, and the like. It can be used for analysis of various reactions occurring in the living body, environmental conservation, and catalyst material design.

The present invention has the features as described above, and an embodiment thereof will be described below.

In the present invention, in order to overcome the problems of the prior art, bioceramic nanocrystals are used, and a biosensor having a uniform nanothin film having a thickness of 100 nm or less is obtained by a combination of electrophoretic deposition and ultrasonic treatment. First successful production. In the prior art, there is no report about the production method and utilization method of such a biosensor composed of nano thin films and nano crystals. Ceramics present in a living body exist in a form in which nanocrystals interact with an extracellular matrix in a complex manner. Therefore, the sensor material produced by the conventional technique has a large crystal, and it is difficult to detect and compare detailed reactions in the living body. In this method, a nano thin film is produced using living bone-like nanocrystals, and reaction analysis between biomolecules generated in the living body and nanocrystals becomes possible.

The terms “nanocrystal” and “nanothin film” in the present invention have a size scale (dimension) of less than 100 μm and are used as technical concepts belonging to a so-called nanometer (nm) region. The “nanocrystal” in the present invention is more preferably 200 nm or less as its size is confirmed by a normal observation means. The “nano thin film” preferably has a thickness of 50 nm or less. In the present invention, the nanocrystalline thin film means that the nanoscale crystalline calcium phosphate compound is formed in a continuously bonded state.

The deposited nanocrystal is a crystal of a calcium phosphate compound, which may be of various types, for example, typically hydroxyapatite (HA _p : Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ). Is exemplified. This hydroxyapatite is used as a biomaterial with high biocompatibility.

The electrophoretic deposition method and the ultrasonic irradiation method in the production method of the present invention are known means, and conditions for producing a predetermined biosensor constituent are appropriately determined. For example, with hydroxyapatite as a representative example, electrophoretic deposition varies depending on the solution, solvent concentration, and the like, but is generally performed at a voltage of 1 mV to 200 V and at a temperature at which the solvent can hold a liquid. The

Application of ultrasonic waves can be processed after this deposition, for example, at a frequency of 20,000 Hz or higher. Of course, it is not limited to these.

Therefore, an example will be shown below and will be described in more detail.

An example of a method for producing a biosensor having a hydroxyapatite thin film which is a kind of bioceramics is shown below. Hydroxyapatite nanocrystals are synthesized by dropping phosphoric acid (0.5 mol / l) at a constant rate into a calcium hydroxide (0.6 mol / l) suspension and adjusting the end point pH to 8.0. did. At that time, the synthesis temperature condition was set to 80 ° C. to obtain a hydroxyapatite nanocrystal having good crystallinity. FIG. 1 shows an X-ray diffraction pattern of a hydroxyapatite nanocrystal. From the result of identification, it was confirmed to be a hydroxyapatite single phase. The diffraction peak is sharp and the crystallinity is very good.

Next, the obtained hydroxyapatite suspension was completely replaced with ethanol, and the weight ratio of hydroxyapatite to ethanol was adjusted to 1%. The suspension was subjected to ultrasonic treatment to obtain a suspension of hydroxyapatite crystals having a positive charge and good dispersibility. Using this, a hydroxyapatite film was formed on a gold substrate for QCM measurement by the electrophoretic deposition method shown in FIG. Hydroxyapatite nanocrystals charged with a positive charge by ultrasonic treatment are attracted to the gold surface on the negative electrode side to form a hydroxyapatite layer. Then, after preparing the hydroxyapatite film, most of the hydroxyapatite layer on the substrate surface was peeled off by ultrasonication in ethanol to prepare a hydroxyapatite nanothin film. From the results of atomic force microscope (AFM) observation shown in FIG. 3 and Table 1, the coating conditions (voltage, electrodeposition time) were optimized, and the ultrasonic treatment time was optimized from the contact angle measurement shown in Table 2. FIG. 3 shows the result of observing the surface of a gold substrate coated with hydroxyapatite nanocrystals by AFM while changing the voltage (10, 50, 100 V) and the electrodeposition time (2, 5, 10 minutes). Hydroxyapatite crystals can be observed to form a uniform coating under high voltage conditions of 100V. In the coating at 10 V, hydroxyapatite crystals are sparsely present and uniform coating formation cannot be observed.

In Table 1, the voltage (10, 50, 100 V) and the electrodeposition time (2, 5, 10 minutes) were changed, and the abundance of hydroxyapatite crystals was expressed as a percentage. Those coated at 100V showed high coverage regardless of the electrodeposition time.

Table 2 shows the results of contact angle measurement with respect to sonication time. When the treatment is continued for 3 minutes or more, the contact angle increases and approaches the contact angle of the gold substrate. This indicates that the hydroxyapatite crystals were desorbed with the ultrasonic treatment.

The presence of the hydroxyapatite layer was confirmed by a high sensitivity Fourier transform infrared spectrophotometer shown in FIG. The thickness of the hydroxyapatite thin film was obtained by measuring the height difference between the hydroxyapatite layer and the gold surface shown in FIG. In FIG. 4, absorption of phosphoric acid is observed at a position of 900 to 1200 cm −1. This indicates the presence of a hydroxyapatite layer on the gold surface.

In FIG. 5, the thickness of the hydroxyapatite thin film prepared under the optimum conditions was obtained by preparing a hydroxyapatite pattern on a gold substrate and measuring the height difference between the hydroxyapatite layer and the gold surface by AFM.

The AFM image shows the result of the hydroxyapatite coating on the surface of the gold sensor subjected to the patterning process.

As a result of the cross-sectional analysis, it can be seen that the thickness of the hydroxyapatite layer is 15-20 nm.

In the synthesis of the hydroxyapatite nanocrystal of Example 1, crystals having a smaller crystal size were synthesized by setting the synthesis temperature conditions to 4 ° C. and 21 ° C. FIG. 6 (additional figure; change in figure number) shows the results of XRD measurement. Crystals synthesized at low temperature clearly have broad diffraction lines and small crystal size.
A biosensor having a hydroxyapatite nano thin film was produced from the obtained crystal by the same production method as in Example 1. FIG. 7 shows an AFM image of the substrate surface produced using crystals synthesized at 4 ° C. and 21 ° C.

The thickness of the hydroxyapatite nano thin film prepared in Examples 1 and 2 was measured with an ellipsometer. Table 3 shows the measurement results. The film thickness of the thin film layer produced by any crystal also showed 10-20 nm, and it was confirmed that it was a nano thin film layer.

FIG. 8 shows protein adsorption behavior when the gold substrate having a hydroxyapatite thin film prepared in Example 1 is used as a biosensor. The frequency when the substrate surface was filled with a 1 mg / ml human serum albumin solution (pI 4.7, MW 69 kDa, 10 mM phosphate buffer solution) under the temperature condition maintained at 22 ° C. was measured. After immersion in the albumin solution, the frequency decreased rapidly. This curve shows the adsorption behavior of albumin molecules on the hydroxyapatite surface, and it was confirmed that the gold substrate having the hydroxyapatite thin film of Example 1 can be used as a protein adsorption behavior measurement.

FIG. 9 shows protein adsorption behavior with respect to changes in ionic strength when the gold substrate having a hydroxyapatite thin film prepared in Example 1 is used as a biosensor. Under the temperature condition maintained at 22 ° C., the substrate surface is immersed in a 1 mg / ml fibrinogen solution prepared using a phosphate buffer solution (10 to 200 mM) having different concentrations, and vibrations generated as fibrinogen adsorbs. The decrease in number was measured. As a result, it was found that the amount of fibrinogen adsorbed on hydroxyapatite decreased with the increase of phosphate ions. This shows that the protein adsorption amount of hydroxyapatite depends on the phosphate ion concentration.
[Comparative Example 1]

As a comparative example of Example 4, FIG. 10 shows the measurement results of the amount of fibrinogen adsorbed on the gold surface with respect to ionic strength change. Compared with the results of Example 4, it can be seen that the substrate having the hydroxyapatite thin film strongly depends on the phosphate ion concentration. This phenomenon is a prominent feature in protein adsorption of hydroxyapatite and is not observed on other substrates, indicating that the substrate prepared in Example 1 has a hydroxyapatite thin film. As described above, it was shown that the biosensor construct produced by this method is useful for analyzing protein adsorption behavior in an environment simulating the inside of a living body. Furthermore, effectiveness against various proteins is shown below. The proteins used are classified into acidic proteins (human serum albumin, catalase), neutral proteins (hemoglobin, immunoglobulin), and basic proteins (cytochrome c, lysozyme). Q-Sense D300 (Q-Sense AB, Gothenburg, Sweden) was used for the measurement.

The adsorption | suction behavior of the protein at the time of using the gold | metal | money base which has the hydroxyapatite thin film (crystal | crystallization synthesized at 21 degreeC) produced in Example 2 as a biosensor (the center made the electrophoresis volume by 100V) is shown. The frequency when the substrate surface was filled with 0.1, 0.5 and 1.0 mg / ml human serum albumin solution (pI4.7, 10 mM phosphate buffer solution) under the temperature condition maintained at 22 ° C. The extinction value (D value) was measured. The result is shown in FIG. The initial protein concentration increased the frequency decrease, with an increase in the extinction value. This indicates that human serum albumin molecules were adsorbed on the hydroxyapatite thin film and changed the viscoelastic properties of the adsorbed layer.

The adsorption | suction behavior of the protein at the time of using the gold | metal | money base which has the hydroxyapatite thin film (crystal | crystallization synthesized at 21 degreeC) produced in Example 2 as a biosensor (the center made the electrophoresis volume by 100V) is shown. When the substrate surface is filled with 0.1, 0.5, 1.0 mg / ml catalase solution (pI 5.5, MW 240 kDa, 10 mM phosphate buffer solution) under the temperature condition maintained at 22 ° C. The extinction value (D value) was measured. The result is shown in FIG. The initial protein concentration increased the frequency decrease, with an increase in the extinction value. This indicates that catalase molecules were adsorbed on the hydroxyapatite thin film and changed the viscoelastic properties of the adsorption layer.

The adsorption | suction behavior of the protein at the time of using the gold | metal | money base which has the hydroxyapatite thin film (crystal | crystallization synthesized at 21 degreeC) produced in Example 2 as a biosensor (the center made the electrophoresis volume by 100V) is shown. When the substrate surface is filled with an immunoglobulin solution of 0.1, 0.5, 1.0 mg / ml (pI 5.8-7.4, 10 mM phosphate buffer solution) under the temperature condition maintained at 22 ° C. The frequency and the extinction value (D value) were measured. The result is shown in FIG. The initial protein concentration increased the frequency decrease, with an increase in the extinction value. This indicates that immunoglobulin molecules were adsorbed on the hydroxyapatite thin film and changed the viscoelastic properties of the adsorption layer.

The adsorption | suction behavior of the protein at the time of using the gold | metal | money base which has the hydroxyapatite thin film (crystal | crystallization synthesized at 21 degreeC) produced in Example 2 as a biosensor (the center made the electrophoresis volume by 100V) is shown. Frequency and dissipation value when the substrate surface is filled with 0.1, 0.5, 1.0 mg / ml hemoglobin solution (pI 6.9, 10 mM phosphate buffer solution) under the temperature condition maintained at 22 ° C. (D value) was measured. The result is shown in FIG. The initial protein concentration increased the frequency decrease, with an increase in the extinction value. This indicates that hemoglobin molecules were adsorbed on the hydroxyapatite thin film and changed the viscoelastic properties of the adsorption layer.

The adsorption | suction behavior of the protein at the time of using the gold | metal | money base which has the hydroxyapatite thin film (crystal | crystallization synthesized at 21 degreeC) produced in Example 2 as a biosensor (the center made the electrophoresis volume by 100V) is shown. Frequency and dissipation when the substrate surface is filled with 0.1, 0.5, 1.0 mg / ml cytochrome c solution (pI 9.8, 10 mM phosphate buffer solution) under the temperature condition maintained at 22 ° C. The value (D value) was measured. The result is shown in FIG. The initial protein concentration increased the frequency decrease, with an increase in the extinction value. This indicates that cytochrome c molecules were adsorbed on the hydroxyapatite thin film and changed the viscoelastic properties of the adsorbed layer.

The adsorption | suction behavior of the protein at the time of using the gold | metal | money base which has the hydroxyapatite thin film (crystal | crystallization synthesized at 21 degreeC) produced in Example 2 as a biosensor (the center made the electrophoresis volume by 100V) is shown. Frequency and dissipation value when the substrate surface is filled with 0.1, 0.5, 1.0 mg / ml lysozyme solution (pI11.1, 10 mM phosphate buffer solution) under the temperature condition kept at 22 ° C. (D value) was measured. The result is shown in FIG. The initial protein concentration increased the frequency decrease, with an increase in the extinction value. This indicates that lysozyme molecules were adsorbed on the hydroxyapatite thin film and changed the viscoelastic properties of the adsorbed layer.

FIG. 17 shows a graph in which the frequency change (Δf value) and the extinction value change (ΔD value) of various proteins performed in Examples 6 to 11 are plotted. In acidic protein, it was clear that the extinction value changed linearly with frequency change (weight increase). On the other hand, it was found that neutral and basic proteins can draw straight lines with different slopes as the frequency increases, as shown in the figure. This difference in inclination means that the viscoelastic characteristics of the adsorption layer clearly change during the process of protein adsorption. It can be seen that a softer adsorption layer is formed as the inclination is larger. This may be due to changes in the adsorption form accompanying changes in protein density. In addition, it can be seen that the size of the extinction value is clearly different between neutral protein and basic protein, but this also depends on the amount of adsorbed protein.

Protein adsorption behavior when a gold substrate having a hydroxyapatite thin film (crystals synthesized at 4 ° C., 21 ° C., and 80 ° C.) prepared in Example 2 is used as a biosensor (center subjected to electrophoresis volume at 100 V). Indicates. Under the temperature condition maintained at 22 ° C., each substrate surface was coated with 1.0 mg / ml human serum albumin solution (pI 4.7, molecular weight 69,000, 10 mM phosphate buffer solution) and 1.0 mg / ml fibrinogen ( The vibration frequency and the extinction value (D value) when filled with pi5.8, molecular weight 340,000, 10 mM phosphate buffer solution) were measured. The results are shown in FIGS. The initial protein concentration increased the frequency decrease, with an increase in the extinction value. In human serum albumin molecules, the amount of adsorption (difference in frequency change was observed depending on the synthesis temperature, but in fibrinogen, no difference was observed in the amount of adsorption. This is considered to depend on the molecular weight (size) of the protein. In human serum albumin, no difference was found in the slope calculated from the change in frequency (Δf value) and the change in dissipation value (ΔD value), which means that the same viscoelastic layer was applied to the particle surface having different crystallinity. Is formed.

X-ray diffraction pattern of hydroxyapatite nanocrystals Schematic diagram of electrophoretic deposition method AFM image for determining optimum coating conditions High sensitivity Fourier transform infrared spectrophotometer (FT-IR-RAS) analysis spectrum diagram AFM image and thickness measurement diagram of hydroxyapatite thin film X-ray diffraction pattern of hydroxyapatite nanocrystals synthesized at various temperatures AFM image of thin film coating using hydroxyapatite nanocrystals with different crystal sizes Adsorption behavior of albumin on hydroxyapatite Changes in adsorption behavior for hydroxyapatite with changes in phosphate concentration Changes in adsorption behavior on the gold surface with changes in phosphoric acid concentration Adsorption behavior of serum albumin molecules: time plot of frequency change (Δf value) and dissipation value change (ΔD value) Adsorption behavior of catalase molecules: time plot of frequency change (Δf value) and extinction value change (ΔD value) Adsorption behavior of immunoglobulin molecules: time plot of frequency change (Δf value) and extinction value change (ΔD value) Adsorption behavior of hemoglobin molecules: time plot of frequency change (Δf value) and extinction value change (ΔD value) Adsorption behavior of cytochrome c molecule: time plot of frequency change (Δf value) and extinction value change (ΔD value) Adsorption behavior of lysozyme molecules: time plot of frequency change (Δf value) and dissipation value change (ΔD value) Plot of frequency change (Δf value) and extinction value change (ΔD value) of various proteins Graph showing adsorption behavior of human serum albumin using apatite particles with different crystallinity (time plot of frequency change (Δf value) and extinction value change (ΔD value)) Graph showing the adsorption behavior of fibrinogen using apatite particles with different crystallinity (time plot of frequency change (Δf value) and extinction value change (ΔD value))

Claims (3)

On a conductive substrate and nano thin film is formed by nanocrystals of calcium phosphate compounds, wherein is not less 200nm or less the size of the nanocrystals, biosensor thickness of the nano-thin film is characterized in der Rukoto below 50nm Construct. The biosensor structure according to claim 1, wherein the conductive substrate is gold or titanium. A method of manufacturing a biosensor according to claim 1 or claim 2, after the crystals of the calcium phosphate compounds were electrophoresed deposited on a conductive substrate, and ultrasonic irradiation, nano thin film according nanocrystalline calcium phosphate compound A process for producing a biosensor composition, characterized in that