JPH0875629A - Continuous measuring element of adsorbate amount in fluid and coating method of material layer - Google Patents

Abstract

PURPOSE: To provide an element used for a measuring apparatus by which a vital reaction such as a cell bonding operation, a blood coagulating operation or the like is measured simply and continuously under a fluid condition close to that inside a living body. CONSTITUTION: A polymer substratum layer 82 which is composed of polycarbonate, polystyrene or polyethylene is formed on the surface of a sheetlike quartz oscillator 50, a protein material layer 84 such as collagen, fibrinogen, a von Willenbrand factor or the like is formed on it, and a measuring element is formed. The measuring element is incorporated into a flow cell, a fluid such as blood or the like is brought into contact with the protein layer for the measuring element so as to flow, and an adsorbate amount is measured on the basis of a change in the resonance frequency of the quartz oscillator.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an element used in a measuring device for easily measuring biological reactions such as cell adhesion and blood coagulation in a fluidized state.

[0002]

2. Description of the Related Art In the development of medical materials and drugs, it is unavoidable to measure biological reactions such as cell adhesion or blood coagulation due to the materials or drugs. For example,
When the artificial material is brought into contact with blood, the living body recognizes the material as a foreign substance, and the blood component is adsorbed on the surface of the material to form a thrombus. The material having excellent antithrombogenicity is a material that does not change the hemostatic mechanism of blood itself and does not form a thrombus even when contacted with blood. From this, the material to be tested can be brought into contact with blood, the change in the amount of blood component adsorbed from the material to the material can be measured, and the antithrombotic property of the material can be determined by the magnitude of the change.

In this case, it is important to perform the test under the same blood flow conditions as in the living body from the viewpoint of the reliability of the judgment. However, until now, there has been no convenient means for conducting a test under flowing conditions, so conventionally the test was carried out in the state where the blood was allowed to stand.

[0004]

As described above, conventionally, since the test material is tested by placing it in the static blood different from the actual in-vivo environment, the phenomenon that occurs on the surface of the test material is also actual. This is very different from the in-vivo phenomenon, and there is a disadvantage that the determination result and the clinical result do not always match. Therefore, there has been a demand for the development of an apparatus capable of performing a test easily under a flow condition very close to that in a living body. The present invention, cell adhesion under flow conditions close to in vivo,
It is an object of the present invention to provide an element used in a measuring device for simply and continuously measuring a biological reaction such as blood coagulation.

[0005]

In the present invention, a material layer for evaluating an adsorption reaction is formed on the surface of a plate-shaped crystal oscillator to form a continuous adsorbate amount measuring element in a fluid. This element for continuously measuring the amount of adsorbed substances in a fluid is incorporated in a flow cell, brought into contact with a fluid such as blood, and the change in its resonance frequency is measured. The resonance frequency change is related to the amount of adsorbed substance in a known relationship.

As the material layer, if silicone, polystyrene, polyethylene, polycarbonate, TiO ₂ or ZrO ₂ or the like is used as an implantable device such as an artificial blood vessel or artificial valve or a candidate material for extracorporeal circulation, blood flow of those materials is used. Blood compatibility under conditions (antithrombogenicity) can be tested. Further, in the case of evaluating the adsorption reaction of blood components and the like to the protein material, a polymer underlayer is formed on the surface of the plate crystal oscillator, and the protein material layer is formed thereon. The polymeric underlayer is preferably polycarbonate, polystyrene or polyethylene, and the protein material layer can be collagen, fibrinogen, von Willebrand factor or the like.

From the viewpoint of the measurement sensitivity of the crystal unit, the weight of the material layer formed on the surface of the crystal unit is the same as that before the change in the resonance frequency of the crystal unit due to the formation of the material layer forms the material layer. It is preferable to set it within 10% of the resonance frequency of the crystal unit. When a polycarbonate layer is formed as a polymer underlayer on the surface of the crystal unit and a protein material layer is formed thereon to prepare the element, the crystal unit is washed with an alkaline detergent, and then ethanol is added. After cleaning with, form a polycarbonate layer,
A protein material layer is formed on it. The formation of the polycarbonate layer is carried out by adding 0.5 to 20% by weight of polycarbonate to tetrachloroethane, more preferably 2 to 8% by weight,
More preferably, a solution in which 6% by weight is dissolved can be used, and spin coating can be performed.

When polystyrene is used as the base layer, it is preferable to apply a solution of tetrachloroethane dissolved in 0.5 to 20% by weight, more preferably 2 to 8% by weight, by spin coating. When polyethylene is used as the underlayer, it is preferable to apply a solution by dissolving it in chloroform, chlorobenzene or the like in an amount of 0.5 to 20% by weight, more preferably 2 to 8% by weight by spin coating.

The protein material layer can be formed by immersing the crystal oscillator having the underlayer in a protein material solution. The protein concentration in the solution is preferably 10 μg / ml to 5 mg / ml. The element for continuously measuring the amount of adsorbed substances in the fluid which has been once measured and used can be reused by immersing it in a tetrachloroethane solution and wiping it away.

[0010]

[Operation] The plate crystal unit incorporated in the resonance circuit
It vibrates at a resonance frequency (f ₀ ) perpendicular to the thickness direction.
At this time, when a substance in the fluid is adsorbed on the surface of the crystal unit and a mass load (ΔW) is generated by the adsorption layer, a change Δf in the resonance frequency as shown in the following equation occurs. Therefore, the mass load ΔW, that is, the amount of adsorption can be determined from the resonance frequency change Δf. Δf = −f ₀ ² ΔW / NAρ where ΔW: mass change, A: electrode area, ρ: specific gravity of crystal, and N: frequency constant of crystal.

Silicone, polystyrene, polyethylene, polycarbonate, Ti on the surface of the plate crystal oscillator
By forming a material layer of O ₂ , ZrO ₂ or the like, it is possible to compare the biological response to each material. Further, by forming a protein material layer of collagen, fibrinogen, von Willebrand factor, etc. on the surface of the crystal oscillator, it is possible to compare biological reactions to each protein.

If the protein material layer is formed directly on the surface of the crystal unit where a thin film metal electrode is partially formed, the protein material layer peels off during measurement. Form a polymer underlayer such as polystyrene, polyethylene, or polycarbonate that has adhesion to both the crystal exposed on the surface of the crystal unit and the metal electrode material such as gold, platinum, nickel, and the protein material, By forming the protein material layer thereon, it is possible to stably form the protein material layer on the surface of the crystal unit.

[0013]

The present invention will be described in detail below with reference to examples. [Embodiment 1] FIG. 1 is a perspective view of a flow cell in which the measuring element of the present invention is incorporated and used, FIG. 2 is a sectional view thereof, and FIG. 3 is an exploded view.

The flow cell 100 comprises a lower member 10, an upper member 20, an external seal member 30, and a cell chamber seal member 40.
And plate crystal unit 50, and the 4 corners are bolt 64
And it is assembled by tightening with a nut. Upper member 20
Consists of vinyl chloride coated with silicon, for example,
As well shown in FIG. 4 which is a perspective view seen from the back side and FIG. 5 which is a plan view seen from the back side, a frame-shaped convex portion having a circular outer peripheral portion in the central portion of the lower surface and a rectangular concave region inside 21 is provided. Further, two holes 22 and 23 are provided halfway in parallel with the member surface from one side surface, and slits 24 and 25 are formed from the side portions of the holes 22 and 23 toward the area surrounded by the frame-shaped convex portion 21. It is open. Upper member 2
In the holes 22 and 23 provided on the side surface of 0, for example, Teflon (registered trademark), vinyl chloride coated with silicone, or the like,
Tube 6 made of a material that does not easily absorb substances in the fluid
1, 62 are connected.

The lower member 10 is made of, for example, Teflon and has a circular recess 11. A cable 71 connected to an oscillator (not shown), a frequency counter, etc. is watertightly fixed to the lower member, and the electrical contacts 72 and 73 connected to the conductor of the cable 71 are exposed on the surface of the recess 11. ing. The electric contact 7 is provided in the recess 11 of the lower member 10.
The plate-shaped crystal unit 50 is detachably arranged so that the electrodes 51 are in contact with 2, 73.

The cell chamber seal member 40 is mounted along the inside of the frame-shaped convex portion 21 on the lower surface of the upper member 20, and the frame-shaped convex portion 21 is attached.
After mounting the external seal member 30 on the outer side of the frame,
1 is inserted into the recess 11 of the lower member 10, and the upper and lower members 10,
The flow cell is assembled by tightening 20 with bolts 64 and nuts. At this time, a region surrounded by the plate crystal oscillator 50 and the frame-shaped convex portion 21 of the upper member 20 becomes a cell chamber. The fluid introduced from the tube 61 into the flow cell enters the cell chamber through the slit 24 connected to the hole 22 provided in the upper member 20, flows as a laminar flow on the surface of the crystal oscillator 50, and the other end of the cell chamber Flows into the hole 23 from the other slit 25 located at, and is discharged through the tube 62.

The size of the cell chamber is appropriately determined according to the viscosity and the flow velocity of the fluid, but when the fluid to be flown into the flow cell is blood, the cross-sectional area of the passage is 50 mm in order to flow the fluid in a laminar state.
^It should be ² or less. In the case of the present embodiment, the size of the cell chamber is 0.3 mm × 15 mm × 15 mm (channel cross-sectional area 4.5.
mm ² ). The fluid is circulated in the flow cell while being immersed in a constant temperature bath maintained at 37 ° C., and the adsorption amount is continuously measured.

The crystal oscillator 50 preferably oscillates at a fundamental resonance frequency of 5 to 10 MHz from the viewpoint of easy handling and measurement sensitivity. The slits 24 and 25 having a width of 1 mm and a length of 15 mm for introducing and discharging the fluid as a laminar flow into the cell chamber have a positional relationship with the plate crystal oscillator 50 as shown in FIG. It is formed on the crystal unit 50. The cell chamber sealing member 40 was a 1 mm thick silicone resin sheet, and the external sealing member 30 was a 0.3 mm thick silicone resin sheet.

Basic resonance frequency 5 MHz with a diameter of 2.5 cm
AT-cut disc-shaped crystal unit (plate thickness 350μ
m) is an alkaline detergent (MERCK, EXTRAN M)
AO1) It was immersed in a 2% aqueous solution, ultrasonically cleaned for 3 minutes, immersed in ethanol, and wiped with a cloth. Commercially available polycarbonate or silicone resin dissolved in a solvent was cast thereon to form a film having a thickness after drying of about 10 nm.

The polycarbonate film is formed by using commercially available polycarbonate beads (Mitsubishi Kasei, Novarex) with an alkaline detergent (MERCK, EXTRAN MAO1).
% Aqueous solution and dried at 100 ° C. for 1 hour or more, then dissolved in a concentration of 6% by weight using tetrachloroethane as a solvent, and the solution was applied to a plate-shaped crystal resonator whose surface was previously washed at 1000 rpm. 10 seconds at 2000 rpm
Spin coating for 10 seconds at 75 ° C for 1 second
It was carried out by drying for 1 hour at 150 ° C. for 1 hour.

The silicone resin film is formed by using a commercially available silicone resin (Iwaki glass, silicone coating agent).
After diluting 40 to 50 times with distilled water at 0 ° C. and immersing the plate-shaped crystal resonator whose surface has been washed in this for about 10 seconds,
It was carried out by pulling up and rinsing with water, and then naturally drying for about 24 hours or heating and drying at 100 to 150 ° C. for about 30 minutes.

The plate-shaped crystal oscillator having the material layer thus formed, that is, the element for continuously measuring the amount of adsorbed substances in the fluid was incorporated in the above-mentioned flow cell 100. The device temperature was maintained at a constant temperature of 37 ° C. during the measurement. The component blood from which blood platelets had been removed from heparin-collected blood was circulated for about 10 minutes at a shear rate of 350 sec ⁻¹ to wait for protein adsorption saturation. Next, when whole blood was flown, the measuring element coated with polycarbonate showed a frequency change of 200 Hz in about 15 minutes. Plate-shaped crystal resonator used in this embodiment, the resonance frequency change of 1Hz corresponds to a mass change of 18 ng / cm ^2, is 3.6 g / cm ² when converted to the frequency change in the mass of the adsorbent material . On the other hand, the frequency change of the measuring element coated with the silicone resin is 20 Hz or less in 15 minutes, which is 0.36 μg / c when converted to the mass of the adsorbed substance.
It was less than m ² .

From this, it was concluded that the silicone resin is a material having a smaller blood component adsorption amount than polycarbonate and an excellent antithrombotic property. This shows a good correspondence with the results of clinical tests and the like, and it can be seen that this method is appropriate as a method for determining the antithrombotic property of a material. Similarly, a material layer made of polystyrene, polyethylene, TiO ₂ , and ZrO ₂ was also examined.

To form the polystyrene layer, commercially available polystyrene beads (Nippon Steel Chemicals, Estyrene) were washed with a 2% aqueous solution of an alkaline detergent (MERCK, EXTRAN MAO1) and dried at 100 ° C. for 1 hour or more. Then, using tetrachloroethane as a solvent to dissolve to a concentration of 6% by weight,
This solution was applied to a plate-shaped crystal resonator whose surface was cleaned as described above at 1000 rpm for 10 seconds and then 2000 r
It was carried out by spin coating under the condition of pm for 10 seconds and drying at 75 ° C. for 1 hour and subsequently at 150 ° C. for 1 hour. The film thickness measured by an ellipsometer was 10 nm.

The polyethylene layer was formed by washing commercially available polyethylene beads (Mitsubishi Yuka, Mitsubishi Polyethylene) with a 2% aqueous solution of an alkaline detergent (MERCK, EXTRAN MAO1) and drying at 100 ° C. for 1 hour or more. Then, it was dissolved in chloroform at a concentration of 6% by weight as a solvent, and this solution was added to a plate-shaped crystal resonator whose surface was washed at 1000 rp.
m for 10 seconds, then 2000 rpm for 10 seconds, spin coating at 75 ° C. for 1 hour, then 150
It was carried out by drying at 0 ° C. for 1 hour. The film thickness measured by an ellipsometer was 10 nm.

The TiO ₂ film has a film thickness of 100 nm formed by sputtering using TiO ₂ (high purity chemistry) as a target.
It was formed into a film. The ZrO ₂ film is 100 nm thick by sputtering with ZrO ₂ (high purity chemistry) as a target.
It was formed into a film. When the measuring element formed with these material layers was incorporated into the flow cell 100 and the same measurement was performed,
Adsorption amount of blood component is 4.0μ for polystyrene
g / cm ² , 4.7 μg / cm for polyethylene
^2, for the TiO ₂ 0.4 [mu] g / cm ^2, ZrO ₂
Was 2.0 μg / cm ² .

Example 2 An AT-cut quartz resonator having a basic resonance frequency of 5 MHz and a diameter of 2.5 cm was immersed in a 2% aqueous solution of an alkaline detergent (MERCK, EXTRAN MAO1) and ultrasonically cleaned for 3 minutes. Then, it was immersed in ethanol and wiped with a cloth. Commercially available polycarbonate beads (Mitsubishi Kasei, Novarex) are used as an alkaline detergent (MER
CK, Extran MAO 1) washed with 2% aqueous solution,
After drying at 100 ° C. for 1 hour or more, tetrachloroethane was dissolved as a solvent to a concentration of 6% by weight. This solution was added to a plate-shaped crystal oscillator whose surface was
spin coating under conditions of pm for 10 seconds and 2000 rpm for 10 seconds, then at 75 ° C. for 1 hour, then 15
It was dried at 0 ° C. for 1 hour. When the film thickness of the polycarbonate thus formed was measured with an ellipsometer,
It was 10 nm.

Next, the plate-shaped crystal resonator having the polycarbonate layer formed thereon was dipped in a solution of collagen, fibrinogen, and von Willebrand factor adjusted to a concentration of about 20 μg / ml for 8 hours or more to be fixed.
The film thickness of the protein immobilized on the polycarbonate was about 150 nm. The adhesion of the polycarbonate was increased by removing the stains on the surface of the crystal unit by washing with the alkaline detergent and subsequent washing with ethanol. Further, the polycarbonate beads were washed with an alkaline detergent to remove the dirt on the surface, so that the adhesion to the crystal unit was increased. If these washing operations are omitted, the polycarbonate layer peels off along with the protein layer formed thereon during the measurement, making the measurement impossible.

The measuring element having the protein layer thus formed was incorporated into a flow cell in the same manner as in Example 1. The device temperature was maintained at a constant temperature of 37 ° C. during the measurement. The component blood from which blood platelets and white blood cells had been removed from the blood collected from heparin was circulated at a shear rate of 350 sec ^-1 for about 10 minutes to wait for protein adsorption saturation. Then whole blood was circulated. The resonance frequency change of the crystal oscillator is about 1500 Hz for fibrinogen and about 1000 Hz for collagen, and about 1700 for von Willebrand factor.
It was Hz.

As a result, the protein fibrinogen,
It was possible to confirm the strength of the biological response to collagen and von Willebrand factor. The concentration of polycarbonate dissolved in tetrachloroethane is 2-8
Weight percent was preferred. The concentration of polycarbonate is 0.
When the concentration is in the range of 5 to 20% by weight, the layer functions as an underlayer, but when the concentration exceeds 20% by weight, the adhesiveness decreases, and
If the amount is less than 5% by weight, the coating is incomplete and the function as an underlayer for adhering the crystal oscillator and the protein layer cannot be sufficiently exhibited.

[Embodiment 3] A case in which polystyrene is used instead of polycarbonate as the polymer base film will be described. An AT-cut crystal unit with a basic resonance frequency of 5 MHz and a diameter of 2.5 cm is treated with an alkaline detergent (MER
CK, Extran MAO 1) dipping in a 2% aqueous solution,
After ultrasonic cleaning for 3 minutes, it was immersed in ethanol and wiped with a cloth.

Commercially available polystyrene beads (Nippon Steel Chemicals,
Ethylene) is washed with a 2% aqueous solution of alkaline detergent (MERCK, EXTRAN MAO1), and then at 100 ° C for 1
After drying for more than an hour, tetrachloroethane was dissolved as a solvent to a concentration of 6% by weight. This solution was applied to a plate-shaped crystal unit whose surface was previously cleaned at 1000 rpm for 10 seconds,
Further, spin coating was performed at 2000 rpm for 10 seconds, and the coating was dried at 75 ° C. for 1 hour and subsequently at 150 ° C. for 1 hour. The film thickness of the polystyrene thus formed was measured by an ellipsometer and found to be 10 nm.

Then, the protein fibrinogen, collagen, and von Willebrand factor were immobilized on the polystyrene base film in the same manner as in Example 2. When the same measurement as in Example 2 was performed using this measuring element, it was about 150 in the case of fibrinogen.
The resonance frequency change was about 1000 Hz in the case of 0 Hz and collagen, and the resonance frequency change was about 1700 Hz in the case of von Willebrand factor.

The concentration of polystyrene dissolved in tetrachloroethane was preferably 2 to 8% by weight. If the concentration of polystyrene is in the range of 0.5 to 20% by weight, it will function as an underlayer, but if the concentration exceeds 20% by weight, the adhesion will decrease, and if it is less than 0.5% by weight, the coating will be incomplete. As a result, the function as an underlayer for adhering the crystal oscillator and the protein layer to each other could not be sufficiently exhibited.

Example 4 An example in which polyethylene is used as the polymer underlayer will be described. A 2.5 cm diameter AT-cut quartz crystal with a basic resonance frequency of 5 MHz is dipped in a 2% aqueous solution of an alkaline detergent (MERCK, EXTRAN MAO1), ultrasonically cleaned for 3 minutes, and then dipped in ethanol to wipe the cloth. I wiped it off.

Commercially available polyethylene beads (Mitsubishi Yuka, Mitsubishi Polyethylene) were washed with a 2% aqueous solution of an alkaline detergent (MERCK, EXTRAN MAO1), and the mixture was dried at 100 ° C. for 1 hour.
After drying for more than an hour, it was dissolved at a concentration of 6% by weight using chloroform as a solvent. This solution was spin-coated on a plate-shaped crystal resonator whose surface had been washed previously at 1000 rpm for 10 seconds and further at 2000 rpm for 10 seconds, and dried at 75 ° C for 1 hour and then at 150 ° C for 1 hour. Let When the film thickness of the polyethylene thus formed was measured by an ellipsometer, it was 10 nm.

Then, the protein fibrinogen, collagen, and von Willebrand factor were immobilized on the polyethylene underlayer by the same method as in Example 2. When the same measurement as in Example 2 was performed using this measuring element, it was about 15 in the case of fibrinogen.
In the case of 00 Hz and collagen, the resonance frequency change was about 1000 Hz, and in the case of von Willebrand factor, the resonance frequency change was about 1700 Hz.

The concentration of polyethylene dissolved in chloroform was preferably 2 to 8% by weight. When the concentration of polyethylene is in the range of 0.5 to 20% by weight, it functions as an underlayer, but when the concentration exceeds 20% by weight, the adhesiveness decreases,
If it is less than 0.5% by weight, the coating is incomplete and the function as an underlayer for adhering the crystal oscillator and the protein layer cannot be sufficiently exhibited.

Example 5 The used measuring elements used in Examples 2 to 4 were immersed in a tetrachloroethane solution for 1 minute or more, and then wiped with a cloth to remove the polycarbonate layer, the protein material layer and the adsorbed substance thereon. Was removed. The plate-shaped crystal unit thus regenerated by removing the material layer was immersed in a 2% aqueous solution of an alkaline detergent (MERCK, EXTRAN MAO1), ultrasonically cleaned for 3 minutes, immersed in ethanol, and wiped with a cloth. . Then, in the same manner as in Example 2, a polycarbonate layer was formed by spin coating, and a protein material layer was formed thereon. When the same measurement as in Example 2 was performed using the measuring element in which the material layer was formed on the surface of the regenerated quartz oscillator in this manner, the same result as in Example 2 was obtained.

[0040]

Industrial Applicability According to the present invention, a biological reaction under a fluidized state can be easily measured, and the measurement result is highly reliable, which is extremely useful for the development of medical materials and drugs.

[Brief description of drawings]

FIG. 1 is an external view of a flow cell.

FIG. 2 is a sectional view of a flow cell.

FIG. 3 is an exploded view of the flow cell.

FIG. 4 is a bottom perspective view of an upper member.

FIG. 5 is a bottom view of the upper member.

FIG. 6 is a plan view of a lower member.

FIG. 7 is a view for explaining the relationship between a plate crystal oscillator and a cell chamber.

FIG. 8 is a sectional view of an embodiment of a measuring element according to the present invention.

FIG. 9 is a cross-sectional view of another embodiment of the measuring element according to the present invention.

[Explanation of symbols]

10 ... Lower member, 11 ... Recess, 20 ... Upper member, 21 ...
Frame-shaped convex portions, 22, 23 ... Holes, 24, 25 ... Slits, 3
0, 30a ... External sealing member, 40 ... Cell chamber sealing member, 50 ... Plate crystal oscillator, 51 ... Electrode, 61, 62 ...
Tube, 64 ... Bolt, 71 ... Cable, 72, 73
... electrical contacts, 80 ... material layer, 82 ... base layer, 84 ... protein material layer, 100 ... flow cell

Front page continuation (72) Inventor Yuji Kubo 1618 Ida, Nakahara-ku, Kawasaki-shi, Kanagawa Nippon Steel Co., Ltd. Keio University School of Medicine

Claims (17)

[Claims] 1. An element for continuously measuring the amount of adsorbed substances in a fluid, wherein a material layer for evaluating an adsorption reaction is formed on the surface of a plate-shaped crystal resonator having a thin-film metal electrode formed on at least a part thereof. 2. The continuous adsorbate amount measuring element in a fluid according to claim 1, wherein the material layer is a material layer for an artificial organ. 3. The resonance frequency change of the crystal unit due to the formation of the material layer is within 10% of the resonance frequency of the crystal unit before the formation of the material layer. Item 3. A device for continuously measuring the amount of adsorbed substances in a fluid according to item 1 or 2. 4. An adsorbate amount in a fluid, characterized in that a polymer underlayer is formed on the surface of a plate-shaped crystal resonator having a thin-film metal electrode formed on at least a part thereof, and a protein material layer is formed thereon. Continuous measuring element. 5. The polymer underlayer is polycarbonate,
The continuous adsorbate amount measuring element for a fluid according to claim 4, which is made of polystyrene or polyethylene. 6. The protein material layer is collagen,
The element for continuously measuring the amount of adsorbed substances in a fluid according to claim 4 or 5, comprising fibrinogen or von Willebrand factor. 7. The change in the resonance frequency of the crystal resonator due to the formation of the polymer underlayer and the protein material layer is within 10% of the resonance frequency of the crystal resonator before the formation of each layer. 7. The continuous adsorbate amount measuring element in fluid according to claim 4, 5 or 6. 8. A step of washing a plate-shaped crystal resonator having a thin-film metal electrode formed at least at a part thereof with an alkaline detergent, a step of subsequently washing with ethanol, a step of forming a polycarbonate layer, and a step thereover. And a step of forming a protein material layer on the element. 9. The amount of adsorbate in a fluid according to claim 8, wherein the polycarbonate layer is formed by spin coating using a solution in which 0.5 to 20% by weight of polycarbonate is dissolved in tetrachloroethane. A method for coating a protein material layer on a continuous measurement element. 10. A step of washing a plate-shaped crystal oscillator having a thin-film metal electrode formed at least at a part thereof with an alkaline detergent, a step of subsequently washing with ethanol, a step of forming a polystyrene layer, and a step thereover. And a step of forming a protein material layer on the element. 11. The amount of adsorbate in a fluid according to claim 10, wherein the polystyrene layer is formed by a spin coating method using a solution in which polystyrene is dissolved in tetrachloroethane in an amount of 0.5 to 20% by weight. A method for coating a protein material layer on a continuous measurement element. 12. A protein material layer is formed by the method according to claim 8, after a used element for continuously measuring the amount of adsorbed substances in a fluid is immersed in a tetrachloroethane solution and wiped off. A method for coating a protein material layer on an element for continuously measuring the amount of adsorbate in a fluid. 13. A step of washing a plate-shaped crystal resonator having a thin-film metal electrode formed at least at a part thereof with an alkaline detergent, a step of subsequently washing with ethanol, a step of forming a polyethylene layer, and a step thereover. And a step of forming a protein material layer on the element. 14. The polyethylene layer is formed by adding 0.5 to 2 of polyethylene to chloroform or chlorobenzene.
The method for coating a protein material layer on an element for continuously measuring the amount of adsorbed substances in a fluid according to claim 13, wherein the solution is formed by a spin coating method using a solution in which 0% by weight is dissolved. 15. Adsorption in a fluid, characterized in that a device for continuously measuring the amount of adsorbed material in a used fluid is immersed in a chloroform or chlorobenzene solution and wiped off, and then a protein material layer is formed by the method according to claim 13. A method for coating a protein material layer on a continuous quantity measuring element. 16. The protein material layer for a continuous adsorbate amount measuring element in a fluid according to claim 8, wherein the protein material layer is formed by immersion in a protein material solution. Coating method. 17. The method for coating a protein material layer on an element for continuously measuring an adsorbed amount in a fluid according to claim 8, wherein the protein material is collagen, fibrinogen or von Willebrand factor. .