JP2007212236A - Film formation method and measuring chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring chip capable of measuring accurately a reaction between a physiologically active substance and a specimen material, and to provide a film formation method for forming an immobilization film of the measuring chip. <P>SOLUTION: This measuring chip 10 is equipped with a dielectric block 12, a thin film 14 and the immobilization film 16. The dielectric block 12 is constituted of a transparent resin which is transparent with respect to light beam, and the section has a trapezoid rod shape. The thin film 14 is formed on the upper surface, on the wider side of two mutually parallel planes of the dielectric block 12, and the immobilization film 16 is formed on the thin film 14. A monitor part 18 is constituted on the surface on the immobilization film 16 side of the measuring chip 10. The monitor part 18 is a part irradiated with a light for monitoring the film formation state, when the immobilization film 16 is formed, and is constituted at a position different from a measuring part E. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a measuring chip having an immobilizing film capable of immobilizing a physiologically active substance, and a film forming method for forming the immobilizing film of the measuring chip.

  Currently, many measurements using intermolecular interactions such as immune reactions are performed in clinical examinations, etc., but conventional methods require complicated operations and labeling substances, so measurement without the need for labeling substances Several techniques that can detect a change in the amount of a substance bound with high sensitivity are used. For example, surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, and measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles. SPR measurement technology detects adsorption and desorption near the surface by measuring the refractive index change in the vicinity of the organic functional film in contact with the metal film of the chip by measuring the peak shift of the reflected light wavelength or the change in the amount of reflected light at a fixed wavelength. It is a method to do. The QCM measurement technique is a technique capable of detecting the adsorption / desorption mass at the ng level from the change in the frequency of the oscillator due to the adsorption / desorption of a substance on the gold electrode (device) of the crystal oscillator. In addition, by functionalizing the surface of gold ultrafine particles (nm level), immobilizing a physiologically active substance on the surface, and performing a specific recognition reaction between the physiologically active substances, it is possible to obtain a living body from the sedimentation and arrangement of gold fine particles. Related substances can be detected.

  In any of the above techniques, the surface on which the physiologically active substance is immobilized is important. Hereinafter, the surface plasmon resonance (SPR) most used in this technical field will be described as an example.

  A commonly used measurement chip includes a transparent substrate (for example, glass), a metal film deposited on the transparent substrate, and a thin film (an immobilized film) having a functional group capable of immobilizing a physiologically active substance thereon. A physiologically active substance is immobilized on the metal surface via a functional group. Then, by supplying the specimen substance to the immobilized physiologically active substance, irradiating the metal film with light, and utilizing the attenuated total reflection of this light, specific binding between the physiologically active substance and the specimen substance is performed. The reaction is measured to analyze the interaction between biomolecules.

  By the way, as a measurement chip having an immobilized film capable of immobilizing a physiologically active substance, a functional group capable of binding to a metal, a linker having a chain length of more than 10 atoms, and a compound having a functional group capable of binding to a physiologically active substance are used. Have been reported (see Patent Document 1). In addition, a measurement chip comprising a metal film and a plasma polymerization film formed on the metal film has been reported (see Patent Document 2). Furthermore, a method of suppressing physical adsorption by immobilizing a hydrophilic hydrogel on the metal surface via a linker is also used (see Patent Document 1, Patent Document 3 and Patent Document 4).

These immobilization films are preferably formed with a desired film thickness in order to accurately measure the reaction between the physiologically active substance and the sample substance. Therefore, it is conceivable to monitor the state of the immobilized membrane. For example, as a method for monitoring the state of an immobilized film, it can be measured by the principle of ellipsometry. However, since the film thickness is not directly measured as a physical quantity, there is a problem that non-linear calculation is required and a measurement error occurs (Patent Document) 5, 6, 7).
Japanese Patent No. 2815120 JP-A-9-264843 US Pat. No. 5,436,161 JP-A-8-193948 WO01 / 59403 EP1261840B1 US2005 / 0025676A1

  The present invention has been made to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a measurement chip capable of accurately measuring a reaction between a physiologically active substance and a sample substance, and a film forming method for forming an immobilized film of the measurement chip.

  In order to solve the above problems, a film forming method of the present invention is a film forming method for forming an immobilized film capable of immobilizing a physiologically active substance on a thin film formed on a flat surface. The first film forming material for forming the immobilized film on the thin film is supplied while monitoring the film forming state of the film, and the film forming state on the thin film is determined as the desired film forming state by the monitor. Then, the first film-forming material is replaced with a second film-forming material for forming the immobilization film, and the immobilization film is formed in multiple stages.

  In the film forming method, the film forming state is monitored, and after the first film forming material is supplied and the desired film forming state is obtained, the second film forming material is supplied. Accordingly, it is possible to prevent the next process from being performed in a state where the film formation by the first film forming material is insufficient, and it is possible to form the immobilization film in multiple stages with high accuracy.

  In the film forming method of the present invention, as described in claim 2, it is preferable that the film forming state is monitored by a non-electrochemical method.

  By using a non-electrochemical method, it is possible to suppress the formation of the immobilization film from being inhibited by the monitor.

  In the film forming method of the present invention, as described in claim 3, the flat surface is formed on the surface of a transparent member, and the film forming state monitor irradiates light to the thin film from the transparent member side. Then, this is performed by utilizing the total reflection attenuation of the light.

  Thus, the film formation of the immobilization film can be monitored by utilizing the total reflection attenuation of light.

  In the film forming method of the present invention, as described in claim 4, it is preferable that the light is irradiated to a position different from the measurement unit when the physiologically active substance is fixed.

  Since some immobilization films to be formed are affected by light irradiation, the position where light is irradiated for monitoring is preferably different from the position used as the measurement unit.

  In the film forming method of the present invention, as described in claim 5, the area irradiated with the light is preferably 10% or less of the total area of the fixed film.

  The measuring chip according to claim 6 has a thin film formed on a flat surface of a transparent member and a monitor unit on the upper surface, and is formed on the thin film while being monitored by light irradiation to the monitor unit. An immobilization membrane capable of immobilizing an active substance, and a measurement unit configured to be fixed at a position different from the monitor unit on the immobilization film and to which a physiologically active substance is immobilized are configured.

  Here, the thin film can be made of platinum, gold, silver or the like. According to the above configuration, the monitor unit irradiated with light for monitoring and the measurement unit where the physiologically active substance is fixed and the measurement is performed are arranged at different positions. Therefore, there is no influence on the measurement part due to the irradiation of the monitoring light, and the reaction between the physiologically active substance and the sample substance can be measured with high accuracy.

  In addition, as for the said monitor part, it is preferable that it is 10% or less of the total area of the said fixed film | membrane as described in Claim 7.

  Since the present invention has the above-described configuration, it is possible to provide a measurement chip capable of measuring the reaction between a physiologically active substance and a sample substance with high accuracy, and a film forming method for forming an immobilization film on the measurement chip.

  Hereinafter, embodiments of the present invention will be described.

  The measurement chip 10 of this embodiment is for fixing a physiologically active substance LG and measuring an interaction with a specimen substance. The physiologically active substance LG is configured on the surface of the measurement chip 10 as shown in FIG. Hereinafter, this position where the physiologically active substance LG is fixed is referred to as “measurement unit E”.

  As shown in FIG. 1, the measurement chip 10 of this embodiment includes a dielectric block 12, a thin film 14, and an immobilization film 16. The dielectric block 12 is made of a transparent resin or the like that is transparent to the light beam, and has a trapezoidal bar shape in cross section. A thin film 14 is formed on a wide upper surface of two parallel surfaces of the dielectric block 12, and an immobilization film 16 is formed on the thin film 14. A monitor unit 18 is formed on the surface of the measurement chip 10 on the fixed film 16 side. The monitor unit 18 is a portion that is irradiated with light to monitor the film formation state when the immobilization film 16 is formed. The monitor unit 18 is configured at a position different from the measurement unit E. The measurement unit E is a region where the physiologically active substance LG is fixed, and a flow path for supplying a sample to the physiologically active substance LG is formed on the measurement unit E.

  The thin film 14 of the measurement chip 10 may be formed by a conventional method, for example, a sputtering method, a vapor deposition method, an ion plating method, a plating method, or the like. The material is selected from metals or metal oxides, semiconductors, and organic substances. Preferably, it is a metal or metal oxide, more preferably a free electron metal selected from the group consisting of gold, silver, copper, platinum or aluminum. For example, when considering use for a surface plasmon resonance biosensor, there is no particular limitation as long as surface plasmon resonance can occur. Preferred examples include free electron metals such as gold, silver, copper, aluminum, and platinum, with gold being particularly preferred. These metals can be used alone or in combination. In consideration of adhesion to the dielectric block 12, an intervening layer made of chromium or the like may be provided between the dielectric block 12 and the layer made of metal.

  Although the film thickness of the thin film 14 is arbitrary, for example, when considering the use for a surface plasmon resonance biosensor, it is preferably 0.1 nm or more and 500 nm or less, and particularly preferably 1 nm or more and 200 nm or less. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 0.1 to 10 nm.

  In this embodiment, the immobilization film 16 is formed while monitoring the film formation state. As the monitoring method, a non-electrochemical method such as a surface plasmon resonance (SPR) measurement technique, a crystal oscillator microbalance ( QCM) measurement technology, measurement technology using a functionalized surface from colloidal gold particles to ultrafine particles, and the like can be used.

  Here, an example using a surface plasmon resonance biosensor will be described.

  The biosensor for surface plasmon resonance measures the interaction between a physiologically active substance and a test substance by utilizing the phenomenon of surface plasmon resonance.

  The phenomenon of surface plasmon resonance is due to the fact that the intensity of light reflected at the boundary between an optically transparent substance such as glass and the metal thin film layer depends on the refractive index of the sample on the metal side. The sample can be analyzed by measuring the intensity of the emitted light.

  As a surface plasmon measuring apparatus for analyzing the characteristics of a substance to be measured by utilizing a phenomenon in which surface plasmons are excited by light waves, an apparatus using a system called a Kretschmann arrangement (for example, JP-A-6-167443) can be cited. See the official gazette). A surface plasmon measuring apparatus using the above system basically has a dielectric block (corresponding to the dielectric block 12 of the present embodiment) formed in a prism shape, for example, and a sample formed on one surface of the dielectric block. A metal film (corresponding to the thin film 14 of the present embodiment) brought into contact with a substance to be measured such as a liquid, a light source that generates a light beam, and the light beam with respect to the dielectric block, the dielectric block and the metal film Detect the surface plasmon resonance state, that is, the state of total reflection attenuation by measuring the intensity of the light beam totally reflected at the interface and the optical system that makes incident at various angles so that the total reflection condition is obtained at the interface Photodetecting means is provided.

  In order to obtain various incident angles as described above, a relatively thin light beam may be incident on the interface by changing the incident angle, or a component incident on the light beam at various angles is included. As described above, a relatively thick light beam may be incident on the interface in a convergent light state or a divergent light state. In the former case, a light beam whose reflection angle changes according to the change in the incident angle of the incident light beam is detected by a small photodetector that moves in synchronization with the change in the reflection angle, or the direction in which the reflection angle changes Or an area sensor extending along the line. On the other hand, in the latter case, it can be detected by an area sensor extending in a direction in which each light beam reflected at various reflection angles can be received.

  In the surface plasmon measuring apparatus having the above configuration, when a light beam is incident on a metal film at a specific incident angle that is greater than the total reflection angle, an evanescent wave having an electric field distribution is generated in the measured substance in contact with the metal film. The evanescent wave excites surface plasmons at the interface between the metal film and the substance to be measured. When the wave number vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon. The intensity of the reflected light decreases sharply. This decrease in light intensity is generally detected as a dark line by the light detection means. The resonance described above occurs only when the incident beam is p-polarized light. Therefore, it is necessary to set in advance so that the light beam is incident as p-polarized light.

  If the wave number of the surface plasmon is known from the incident angle at which this total reflection attenuation (ATR) occurs, that is, the total reflection attenuation angle (θSP), the dielectric constant of the substance to be measured can be obtained. In this type of surface plasmon measurement apparatus, as shown in Japanese Patent Application Laid-Open No. 11-326194, in order to measure the total reflection attenuation angle (θSP) with high accuracy and a large dynamic range, It is considered to use detection means. This light detection means is provided with a plurality of light receiving elements arranged in a predetermined direction, and arranged so that different light receiving elements receive light beam components totally reflected at various reflection angles at the interface. Is.

  In that case, there is provided differential means for differentiating the light detection signals output from the light receiving elements of the arrayed light detection means with respect to the arrangement direction of the light receiving elements, and based on the differential value output by the differential means. In many cases, the total reflection attenuation angle (θSP) is specified to obtain a characteristic related to the refractive index of the substance to be measured.

  Moreover, as a similar measuring device using total reflection attenuation (ATR), for example, “Spectroscopic Research” Vol. 47, No. 1, (1998), pages 21 to 23 and pages 26 to 27 are described. Devices are also known. This leakage mode measuring device is basically a dielectric block formed in a prism shape, for example, a clad layer formed on one surface of the dielectric block, and formed on the clad layer to be in contact with the sample liquid. Optical waveguide layer to be generated, a light source for generating a light beam, and the light beam to the dielectric block at various angles so that a total reflection condition is obtained at the interface between the dielectric block and the cladding layer. The optical system includes an incident optical system and light detection means for detecting the excitation state of the waveguide mode, that is, the total reflection attenuation state by measuring the intensity of the light beam totally reflected at the interface.

  In the leakage mode measuring apparatus having the above-described configuration, when a light beam is incident on the cladding layer through the dielectric block at an incident angle greater than the total reflection angle, the light waveguide layer transmits a specific wave number after passing through the cladding layer. Only light having a specific incident angle having a wave length propagates in the waveguide mode. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer, resulting in total reflection attenuation in which the intensity of light totally reflected at the interface is sharply reduced. Since the wave number of guided light depends on the refractive index of the substance to be measured on the optical waveguide layer, knowing the specific incident angle at which total reflection attenuation occurs, the refractive index of the substance to be measured and the measurement object related thereto The properties of the substance can be analyzed.

  In this leakage mode measuring apparatus, the above-mentioned array-shaped light detecting means can be used to detect the position of the dark line generated in the reflected light due to the total reflection attenuation, and the above-described differentiating means is applied in conjunction therewith. Often done.

  In addition, the surface plasmon measurement device and the leakage mode measurement device described above may be used for random screening to find a specific substance that binds to a desired sensing substance in the field of drug discovery research. In this case, the thin film layer A sensing substance is fixed on the sensing substance on the sensing substance (a metal film in the case of a surface plasmon measuring apparatus, a clad layer and an optical waveguide layer in the case of a leakage mode measuring apparatus), and various analytes are placed on the sensing substance. A sample solution dissolved in a solvent is added, and the total reflection attenuation angle (θSP) is measured every time a predetermined time elapses.

  If the analyte in the sample liquid binds to the sensing substance, the refractive index of the sensing substance changes with time due to this binding. Therefore, by measuring the total reflection attenuation angle (θSP) every predetermined time and measuring whether or not the total reflection attenuation angle (θSP) has changed, the binding state of the analyte and the sensing substance is determined. It is possible to determine whether or not the analyte is a specific substance that binds to the sensing substance based on the result. Examples of the combination of the specific substance and the sensing substance include an antigen and an antibody, or an antibody and an antibody. Specifically, rabbit anti-human IgG antibody can be immobilized on the surface of the thin film layer as a sensing substance, and human IgG antibody can be used as the specific substance.

  Note that in order to measure the binding state between the subject and the sensing substance, it is not always necessary to detect the angle of total reflection attenuation (θSP) itself. For example, a sample solution can be added to the sensing substance, and the amount of change in the total reflection attenuation angle (θSP) thereafter can be measured, and the binding state can be measured based on the magnitude of the angle change. If the above-described arrayed light detecting means and differentiating means are applied to a measuring device using total reflection attenuation, the change amount of the differential value reflects the angle change amount of the total reflection attenuation angle (θSP). Therefore, the binding state between the sensing substance and the analyte can be measured based on the amount of change in the differential value (see Japanese Patent Application No. 2000-398309 by the present applicant). In such a measurement method and apparatus using total reflection attenuation, a sample liquid consisting of a solvent and an analyte is placed on a cup-shaped or petri-shaped measuring chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface. The amount of change in angle of the total reflection attenuation angle (θSP) described above is measured.

  Furthermore, a measuring apparatus using total reflection attenuation capable of measuring a large number of samples in a short time by sequentially measuring a plurality of measuring chips mounted on a turntable or the like is disclosed in JP-A-2001-2001. No. 330560.

  The measurement chip 10 of this embodiment is used for a biosensor for surface plasmon resonance, and the dielectric block 12 functions as a prism. In measurement, light is emitted from one of two opposing side surfaces that are not parallel to each other. A beam is incident, and a light beam totally reflected at the interface with the thin film 14 is emitted from the other side.

  Further, in the present embodiment, as shown in FIG. 2, the measuring device includes a light source 54A, a first optical system 54B, a second optical system 54C, a light receiving unit 54D, and a signal processing unit 54E as monitoring means. 54 is used. A divergent light beam L is emitted from the light source 54A. The light beam L is incident on the monitor unit 18 of the dielectric block 12 via the first optical system 54B (see FIG. 6). In the monitor unit 18, the light beam L is incident on the interface between the thin film 14 and the dielectric block 12 with various incident angle components and at an angle greater than the total reflection angle. The light beam L is totally reflected at the interface between the dielectric block 12 and the thin film 14. The totally reflected light beam L is received by the light receiving unit 54D through the second optical system 54C, is photoelectrically converted, and a light detection signal is output to the signal processing unit 54E. The signal processing unit 54E performs predetermined processing based on the input light detection signal, and obtains data of the total reflection attenuation angle of the monitor unit 18 (hereinafter referred to as “total reflection attenuation data”). The total reflection attenuation data is output to the control unit 61.

  Next, the immobilization film 16 will be described.

As the immobilization film 16, a compound that generates a chemical bond with the surface of the thin film 14 is used, and preferably has a hydrophilic functional group in the molecule. This is because a hydration layer is formed when the surface of the thin film 14 is present in the aqueous solution, and this has the effect of preventing non-specific adsorption of substances in the aqueous solution. Examples of the hydrophilic functional group include —OH, —SH, —COOH, —NH ₂ , —NR ¹ R ² (wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group), —CHO , Polyethylene oxide, polypropylene oxide and the like.

  The compound that forms a chemical bond with the surface of the thin film 14 made of a material selected from platinum, gold, and silver is not particularly limited, but any functional group having the chemical formula shown in [Chemical Formula 1] is present in the molecule. And the like. Particularly preferred are compounds having —SH, —S—, or —S—S—. A disulfide compound is mentioned.

  These compounds form a chemical bond by bringing the solution into contact with the surface of the thin film 14, and terminate the reaction with an optimum reactivity while monitoring the degree of chemical modification (film formation state) during the reaction. As the monitoring means, the above-described measuring device 54 is used.

  The surface of the thin film 14 is preferably surface-modified with a polymer compound. The polymer compound may be a hydrophilic compound or a hydrophobic compound. Examples of hydrophilic polymer compounds include proteins such as albumin and casein, sugar derivatives such as agar, sodium alginate and starch derivatives, cellulose compounds such as carboxymethyl cellulose and hydroxymethyl cellulose, polysaccharides such as chitin and chitosan, polyvinyl alcohol, poly Synthetic hydrophilic polymers such as -N-vinylpyrrolidone, polyacrylamide, and polyacrylic acid. Examples of the hydrophobic polymer compound include polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, polymethyl methacrylate, polyester, and nylon.

  Coating of the hydrophilic polymer compound on the thin film 14 can be performed by a conventional method, for example, spin coating, air knife coating, bar coating, blade coating, slide coating, curtain coating, spray method, vapor deposition method, casting method. It can be performed by a dipping method or the like. Alternatively, it may be chemically bonded via a molecule that is chemically bonded to the surface of the thin film 14.

  These polymer materials can be surface modified from chemical treatments using chemicals, coupling agents, surfactants, surface deposition, etc., physical treatments using heating, ultraviolet rays, radiation, plasma, ions, etc. Is possible.

In the polymer compound coating layer formed as described above, it is preferable that the immobilization film 16 has a functional group capable of forming a covalent bond with a physiologically active substance. Preferred functional groups capable of forming a covalent bond with a physiologically active substance include —OH, —SH, —COOH, —NR ¹ R ² (wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group) ), —CHO, —NR ³ NR ¹ R ² (wherein R ¹ , R ² and R ³ independently represent a hydrogen atom or a lower alkyl group), —NCO, —NCS, epoxy group, or vinyl group Etc. Here, the number of carbon atoms in the lower alkyl group is not particularly limited, but is generally about C1 to C10, preferably C1 to C6.

  After forming the polymer compound coating layer, by chemically modifying the surface of the thin film 14 so that the physiologically active substance can be immobilized, the interaction between biomolecules is converted into a signal such as an electrical signal, It is possible to measure and detect the target substance, which is useful.

  This chemical modification can be performed by introducing a functional group by bringing the surface of the thin film 14 into contact with an aqueous solution or an organic solvent. Again, the reaction is terminated with an optimum reactivity while monitoring the degree of chemical modification (film formation state). As the monitoring means, the above-described measuring device 54 is used.

  The number of reaction stages for forming the immobilization film 16 of the measurement chip 10 is not particularly limited, but the control of film formation becomes more important as the number of reaction stages increases. For this reason, it is preferable to perform film formation while monitoring the film formation state as the number of film formation reactions increases. The immobilization film 16 is formed as follows.

  In order to form the immobilization film 16, a reactor 20 for storing liquid is prepared as shown in FIG. The reactor 20 is integrally formed of rubber, and has a flow path member 22 that is long and forms a space R that is open on the bottom side. As shown in FIG. 4, the flow path member 22 is attached to the dielectric block 12 by the holding member 48, and as shown in FIG. 6, the flow path member 22 is formed between the thin film 14 and the flow path 21 along the thin film 14. Constitute.

  As shown in FIG. 3, a plurality of injection holes 24 (12 in this embodiment) are perforated in the longitudinal direction on the upper surface of the flow path member 22 into which the pipette tip CP can be inserted. In addition, a supply port 26 communicated with the space R is provided at one end of the flow path member 22, and a discharge port 28 communicated with the space R is provided at the other end of the flow path member 22.

  On the inner side of the flow path member 22, ribs 23 protruding from the upper part of the flow path member 22 to the space R are formed at equal intervals along the longitudinal direction. The strength of the flow path member 22 is reinforced by the ribs 23. Further, the lower side of the side surface of the flow path member is provided with a convex frame 22A protruding in the horizontal direction from the upper side.

  As shown in FIG. 4, the reactor 20 is fixed on the dielectric block 12 by a holding member 48. As shown in FIG. 5, the holding member 48 includes a pressing plate 48 </ b> A and a mounting plate 48 </ b> B. 48 C of joining pillars with the pressing board 48A are provided in the corner | angular part in the peripheral part of the mounting board 48B.

  A long opening 49 along the reactor 20 is formed in the holding plate 48A. As shown in FIG. 4, the upper side of the flow path member 22 protrudes from the opening 49. Further, a communication hole 51 into which the supply port 26 and the discharge port 28 of the reactor 20 can be inserted is formed on the side surface of the pressing plate 48A. As shown in FIG. 2, the pressing plate 48 </ b> A comes into contact with the convex frame 22 </ b> A of the flow path member 22 to bring the reactor 20 into close contact with the dielectric block 12. The reactor 20 is held by placing the dielectric block 12 on the placement plate 48B, covering the reactor 20 thereon, and covering the presser plate 48A from above the supply port 26 and the discharge port 28 to the communication hole 51. Do this by plugging in. As a result, the reactor 20 is held on the dielectric block 12, and a flow path 21 is formed between the thin film 14 and the flow path member 22, as shown in FIG.

  A buffer liquid supply device for supplying / discharging the buffer liquid BA is connected to the flow path 21. As shown in FIG. 7, the buffer liquid supply apparatus includes a supply tube 60, a switching valve 62, a storage part 64, a discharge tube 66, a peristaltic pump 68, and a waste liquid bottle 69. The supply tube 60 and the discharge tube 66 are respectively connected to the supply port 26 and the discharge port 28 of the reactor 20. The storage unit 64 includes a plurality of tanks, and the switching valve 62 switches a tank that communicates with the supply tube 60 by switching the valves. The peristaltic pump 68 is connected to the discharge tube 66, and causes the cleaning liquid in the storage unit 60 to flow into the flow path 21 from the supply port 26 by suction, and the buffer solution BA that has flowed in flows out from the discharge port 28 to be discharged into the waste liquid bottle 69. .

  Next, a procedure for forming the immobilization film 16 will be described.

  First, the buffer solution BA is supplied to the flow path 21 configured as described above. The supply of the buffer solution BA is performed by operating the verista pump 68 and causing the cleaning solution in the reservoir 60 to flow into the flow path 21 from the supply port 26. Then, the measuring device 54 is operated to emit light from the light source 54A and enter the monitor unit 18 as shown in FIG. 6, and the reflected light is received by the light receiving unit 54D. Are obtained continuously. From the total reflection attenuation data, the film thickness state of the fixed film 16 can be known.

  Next, the supply of the buffer solution BA is stopped, and the first solution Y <b> 1 containing the first film-forming substance for forming the immobilization film 16 is supplied to the flow path 21. This supply is performed by inserting the pipette tip CP into the injection hole 24. And it waits until it is judged that the predetermined film thickness was obtained from the total reflection attenuation data obtained by the measuring device 54. After the predetermined film thickness is obtained, the buffer solution BA is supplied again to the flow path 21 and washed.

  Next, a second solution Y2 containing a second film forming material for forming the immobilization film 16 is supplied. This supply is also performed by inserting the pipette tip CP into the injection hole 24. And it waits until it is judged by the total reflection attenuation data obtained by the measuring apparatus 54 that the predetermined film thickness was obtained. After the predetermined film thickness is obtained, the buffer solution BA is again supplied to the flow path 21 for cleaning, and the film formation of the immobilized film 16 is completed.

  According to this embodiment, since the film forming state of the immobilizing film 16 is monitored and a predetermined film pressure is obtained and the process proceeds to the next process, the immobilizing film 16 can be formed with high accuracy.

  Further, according to the present embodiment, since the monitor unit 18 is configured at a position different from the measurement unit E, when the immobilization film 16 is affected by light irradiation (for example, light irradiation and heat generation). Even in the case of reaction inhibition caused by this, the influence on the measurement of the interaction between the actual physiologically active substance LG and the specimen substance can be prevented.

  Even when the detection method is QCM, the monitor unit of the measurement chip vibrates at a high frequency and there is a concern about the influence on the reaction. Therefore, the monitor unit and the measurement unit are preferably at different positions.

  Further, as shown in FIG. 8, a plurality of monitor units 18 may be configured at positions adjacent to each measurement unit E. In this manner, by forming a plurality of positions at positions corresponding to the respective measurement units E, the film forming state can be measured on average.

  In the present embodiment, an example in which the measurement chip 10 is provided with a dielectric block that functions as a prism has been described. However, as shown in FIG. 9, a thin film 14 is formed on a flat transparent plate 13. Thus, the measurement chip 11 can also be configured. In this case, the film forming state is monitored using a separate measuring prism.

  Moreover, it is preferable that the monitor part 18 is 10% or less of the total area of the fixed film 16 to be formed. This is because the monitor unit 18 and the measurement unit E are arranged at different positions and the area required for the measurement unit E is taken into consideration.

  By the way, a normal biosensor is composed of a receptor site for recognizing a chemical substance to be detected and a transducer site for converting a physical change or a chemical change generated therein into an electrical signal. In the living body, there are enzymes / substrates, enzymes / coenzymes, antigens / antibodies, hormones / receptors and the like as substances having affinity for each other. Biosensors use the principle that one of these substances having affinity with each other is immobilized on a substrate and used as a molecular recognition substance, thereby selectively measuring the other substance to be matched.

  The bioactive substance can be immobilized on the thin film 14 by covalently bonding the bioactive substance LG via the functional group of the immobilization film 16 on the surface of the measurement chip 10 obtained as described above.

  In order to configure the flow path for supplying the physiologically active substance LG onto the measurement chip 10 in order to immobilize the physiologically active substance LG, as shown in FIG. The flow path member 17 is disposed on the surface on which is formed. A channel groove that is exposed to the immobilization film 16 is formed on the lower surface of the channel member 17, communicated with the supply port 17 </ b> A and the discharge port 17 </ b> B formed on the upper surface, and between the immobilization film 16. A flow path 17C is configured. In order to hold the flow path member 17 on the dielectric block 12, a holding member 19 is attached, and an evaporation preventing member 19B is attached to the upper surface of the holding member 19 via an adhesive member 19A, so that the flow path member 17 is fixed. Is done.

  The physiologically active substance immobilized on the surface of the measurement chip 10 of the present embodiment is not particularly limited as long as it interacts with the measurement object. For example, immune proteins, enzymes, microorganisms, nucleic acids, low molecular organic compounds And non-immune proteins, immunoglobulin-binding proteins, sugar-binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides having ligand-binding ability.

  Examples of immunity proteins include antibodies and haptens that use the measurement target as an antigen. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, IgD can be used. Specifically, when the measurement target is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenic E. coli Among them, antibodies against O antigens 26, 86, 55, 111, 157 and the like can be used.

  The enzyme is not particularly limited as long as it shows activity against the measurement object or a substance metabolized from the measurement object, and various enzymes such as oxidoreductase, hydrolase, isomerase , A desorbing enzyme, a synthesizing enzyme and the like can be used. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. In addition, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit specific reactions with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.

  The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.

  As the nucleic acid, one that hybridizes complementarily with the nucleic acid to be measured can be used. As the nucleic acid, either DNA (including cDNA) or RNA can be used. The type of DNA is not particularly limited, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.

  Examples of the low molecular weight organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.

  The non-immune protein is not particularly limited, and for example, avidin (streptavidin), biotin or a receptor can be used.

  As the immunoglobulin-binding protein, for example, protein A or protein G, rheumatoid factor (RF) and the like can be used.

  Examples of sugar-binding proteins include lectins.

  Examples of the fatty acid or fatty acid ester include stearic acid, arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, and ethyl behenate.

  When the physiologically active substance is a protein or nucleic acid such as an antibody or an enzyme, the immobilization is performed by covalently bonding to the functional group of the immobilized membrane 16 using the amino group, thiol group or the like of the physiologically active substance. Can do.

  The measurement chip 10 on which a physiologically active substance is immobilized as described above can be used for detection and / or measurement of a substance that interacts with the physiologically active substance.

  That is, by using the measurement chip 10 on which the physiologically active substance is immobilized and bringing the test substance into contact therewith, a substance that interacts with the physiologically active substance immobilized on the measurement chip 10 is detected and / or measured. can do.

  As the test substance, for example, a sample containing a substance that interacts with the above physiologically active substance can be used.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.

1. Preparation of Measurement Chip A pellet of ZEONEX (manufactured by ZEON Corporation) was melted at 240 ° C., and this melt was molded into an 8 mm long × 120 mm × 1.5 mm substrate by an injection molding machine. A mask with an opening other than the peripheral edge of 1 mm is placed on the sensor side surface of 8 mm long by 120 mm wide on this substrate, and a parallel plate type 6 inch sputtering apparatus (SH-550, ULVAC, Inc.) is used. Then, sputtering was performed so that the gold thickness was 50 nm in the opening, and a gold thin film was formed.

  A flow path for supplying a liquid was formed on the gold thin film forming side of the substrate. The channel is in contact with an area of 1 mm peripheral edge of the surface of the substrate on which the gold thin film is formed, has a solution inlet and outlet at both lateral ends, and holds the solution with a thickness of 1 mm inside. A polydimethylsiloxane resinous member was installed. This substrate was set in an apparatus (hereinafter referred to as a surface plasmon apparatus) shown in FIG. 22 of JP-A-2001-330560.

  The channel was filled with an ethanol / water (80/20) solution, and then the channel was filled with 0.1 mM 16-carboxy-1-hexadecanethiol and 0.9 mM 11-hydroxy in ethanol / water (80/20). Substitution with -1-undecanethiol solution. Surface treatment was performed at 25 ° C. for 1 hour, and the inside of the flow path was again replaced with an ethanol / water (80/20) solution. During this time, detection of the amount of change in the resonance signal (RU value) was continued with the surface plasmon device, and after changing 300 RU in 5 minutes, no change was observed in the value, and 16-carboxy-1-hexadecanethiol and 11-hydroxy-1 -It was confirmed that the surface modification of undecanethiol was saturated.

Next, a 1: 1 mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was filled in the flow path, and allowed to stand for 7 minutes. Furthermore, after replacing with pure water, 0.10 g of NH ₂ -PEG-COOH (Mw 3400, manufactured by NECTER) was replaced with a solution of 1 mL of 5 mN aqueous sodium hydroxide solution. Surface treatment was performed at 25 ° C. for 16 hours, and the inside of the flow path was again replaced with pure water. During this time, the detection of the amount of change in the resonance signal (RU value) was continued with the surface plasmon device, and after 6 hours, after 3000 RU changes, no change was observed in the value, indicating that the surface modification of NH ₂ -PEG-COOH was saturated. confirmed.

  The flow path was removed from the substrate to obtain the measurement chip of the present invention. Hereinafter, the surface subjected to the above surface modification is referred to as a sensor surface.

2. Measurement of non-specific adsorption of low molecular weight compounds Adsorption of non-specific proteins on the sensor surface causes noise during measurement, so it is preferable that the amount be as small as possible. Using the prepared measuring chip, the nonspecific adsorption property of CGP-74514A Hydrochloride (manufactured by Sigma) was measured. Here, by covering a part made of polypropylene at a location different from the portion where the reaction was monitored at the time of creating the measurement chip on the sensor surface (monitor portion), the width (vertical direction) 1 mm, length (horizontal method) 7.5 mm, depth A 1 mm long flow path was formed, and the measurement chip having the flow path was installed in a surface plasmon device. HBS-N buffer (Biacore) was added to the cell and allowed to stand for 20 minutes, and then CGP-74514A Hydrochloride solution (10 μM, HBS-N buffer) was added to the channel and allowed to stand for 10 minutes. The composition of the HBS-N buffer is HEPES (N-2-Hydroxyethylperazine-N'-2-ethanesulfonic Acid) 0.01 mol / l (pH 7.4) and NaCl 0.15 mol / l. No change was observed in the resonance signal (RU value) after standing for 10 minutes, and the nonspecific adsorption was extremely low on the sensor surface.

3. Measurement of interaction between protein and analyte compound 1-Ethyl-2,3-dimethylamino in a flow path of 1 mm in width (vertical direction), 7.5 mm in length (horizontal method) and 1 mm in depth constructed on the sensor surface The mixture was filled with a mixed solution of propylcarbodiimide (200 mM) and N-hydroxysuccinimide (50 mM) for 10 minutes, and then replaced with 50 mM acetate buffer (pH 4.5, manufactured by Biacore). Next, Protein A (manufactured by Nacalai Tesque) solution (100 μg / mL, 50 mM acetate buffer, pH 4.5) was filled in the cell for 10 minutes, and then replaced with 50 mM acetate buffer (pH 4.5).

  Furthermore, after filling with ethanolamine / HCl solution (1M, pH 8.5) for 10 minutes, the activated COOH group remaining without reacting with Protein A was blocked by replacing with 50 mM acetate buffer (pH 4.5). .

  Further, after filling with NaOH aqueous solution (10 mM) for 1 minute, HBS-EP buffer (HEPES (N-2-Hydroxyethylperazine-N'-2-ethanesulfonic Acid) 0.01 mol / L (pH 7.4), NaCl 0.15 mol / L, EDTA 0.003 mol / L, surfactant P20 0.005 wt%, manufactured by Biacore), protein A adsorbed nonspecifically on the sensor surface was removed. This sample is called a Protein A fixed measurement chip.

  The Protein A fixed measurement chip was set on the surface plasmon measurement device. The flow path was filled with HBS-EP buffer, and measurement was started. The inside of the flow path was replaced with a mouse IgG (Cosmo Bio) solution (10 μg / mL, HBS-EP buffer) and allowed to stand for 5 minutes. After 5 minutes, a signal change of 600 RU was shown, and the interaction between the protein and the sample substance could be detected by using the measurement chip.

It is a perspective view of the measuring chip of this embodiment. It is sectional drawing which shows the state with which light is irradiated to the measuring chip of this embodiment, and the schematic block diagram of a measuring apparatus. (A) of the reactor of this embodiment is the perspective view seen from the upper surface side, (B) is the perspective view seen from the lower surface side. It is a perspective view which shows the state which fixed the reactor using the holding member on the measuring chip of this embodiment. It is a disassembled perspective view which shows the relationship with the holding member in the case of fixing a reactor on the measurement chip | tip of this embodiment. It is a cross-sectional view of the flow path comprised on the measurement chip by the reactor of this embodiment. It is the schematic which shows the mechanism which supplies a buffer liquid to the measuring chip of this embodiment, and the mechanism which supplies the solution for film forming. It is a perspective view which shows the modification of the measuring chip of this embodiment. It is a perspective view which shows the other modification of the measurement chip | tip of this embodiment. It is a disassembled perspective view which shows the member for comprising the flow path for fixing a bioactive substance to the measuring chip of this embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Measuring chip 11 Measuring chip 14 Thin film 16 Immobilization film 18 Monitor part 21 Flow path 20 Reactor 54 Measuring apparatus E Measuring part

Claims (7)

A film forming method for forming an immobilized film capable of fixing a physiologically active substance on a thin film formed on a flat surface,
Supplying a first film-forming substance for forming the immobilization film on the thin film while monitoring a film-forming state on the thin film;
After the film forming state on the thin film is determined to be a desired film forming state by the monitor, the first film forming material is replaced with a second film forming material for forming the immobilization film,
A film forming method for forming the immobilized film in multiple stages.   The film forming method according to claim 1, wherein the film forming state is monitored by a non-electrochemical method. The flat surface is configured on the surface of the transparent member,
The film forming method according to claim 2, wherein the film forming state monitor is performed by irradiating the thin film with light from the transparent member side and utilizing the total reflection attenuation of the light. .   The said light is irradiated to the position different from a measurement part, if the said bioactive substance is fixed, The film forming method of Claim 3 characterized by the above-mentioned.   The film forming method according to claim 4, wherein an area irradiated with the light is 10% or less of a total area of the fixed film. A thin film formed on the flat surface of the transparent member;
An immobilization membrane that has a monitor portion on the upper surface, is formed on the thin film while being monitored by light irradiation to the monitor portion, and is capable of immobilizing a physiologically active substance;
A measurement unit configured to fix a physiologically active substance at a position different from the monitor unit on the immobilized membrane;
Measuring chip with.   The measurement chip according to claim 6, wherein the monitor unit is 10% or less of the total area of the immobilization film.

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