JP2007093300A - Measuring apparatus, and measuring program - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently measure the interaction between a physiologically active substance and a compound by excluding an improper measurement. <P>SOLUTION: In a step S10, the sample data of the same measuring condition as an inputted measuring condition is extracted and, in a step S12, a compound above 5RU in reference data G2 is extracted from the compounds of the sample data. In a step S14, the extracted compound is excluded from a compound group becoming an investigation target. By this arrangement, a compound much in non-specific adsorption quantity is removed from a measuring target. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a measurement device and a measurement program, and more particularly to a measurement device and a measurement program for measuring an interaction between a physiologically active substance and a predetermined compound.

  Various biosensors have been proposed as devices for measuring the interaction between a physiologically active substance and a predetermined compound. Among them, a surface plasmon sensor is known as one of measuring devices using evanescent waves (see Patent Document 1). In general, a surface plasmon sensor includes a prism, a metal film disposed on one surface of the prism, an immobilization film disposed on the metal film to which a physiologically active substance is fixed, a light source that generates a light beam, An optical system for making the light beam incident on the prism at various angles so that a total reflection condition is obtained at the interface between the prism and the metal film; and a light detection means for detecting the intensity of the light beam totally reflected at the interface. And measuring the physiologically active substance based on the detection result of the light detection means.

  In the measurement using the surface plasmon sensor, a compound solution is supplied to the physiologically active substance immobilized on the immobilized membrane, and light is applied to the surface opposite to the side on which the immobilized membrane is supplied. The interaction between the physiologically active substance and the compound in the compound solution is measured based on the refractive index information obtained from the reflected light incident on the beam.

  By the way, in the measurement by the surface plasmon sensor, a reference part to which the physiologically active substance is not fixed is provided separately from the measurement part to which the physiologically active substance is fixed, and the refractive index of the measurement part is obtained based on the refractive index information obtained from the reference part. The rate information is corrected. The correction here cancels the signal change between the compound and the immobilized membrane due to different types and other conditions, and measures only the signal change due to the specific binding between the physiologically active substance and the compound. This is the main purpose.

However, when the compound to be studied is adsorbed in large quantities on the immobilized membrane (such adsorption is hereinafter referred to as “nonspecific adsorption”), the specific binding between the physiologically active substance and the compound is accurately measured. It is likely not.
Japanese Patent No. 3294605

  The present invention has been made in consideration of the above-mentioned facts, and provides a measuring apparatus and a measuring program for efficiently measuring an interaction between a physiologically active substance and a compound by eliminating inappropriate measurement. For the purpose.

  In order to solve the above problems, the measurement apparatus of the present invention provides measurement data obtained by supplying a compound to be examined to a measurement region in which a physiologically active substance is immobilized on an immobilized membrane, and the physiologically active substance. Based on reference data including information on the amount of non-specific adsorption of the compound to the immobilized membrane obtained by supplying the compound to a reference region composed of the immobilized membrane not immobilized, the physiological activity A measuring apparatus for measuring an interaction between a substance and the compound, the storage means storing the reference data for each measurement condition and each compound, and the measurement condition input means for inputting the measurement condition for the measurement And a compound extraction means for extracting a compound corresponding to reference data exceeding a predetermined non-specific adsorption level among the reference data corresponding to the input measurement conditions, Supplying the measurement area and the reference area with a compound excluding the compound extracted by the compound extraction means from the substance group, obtaining measurement data from the measurement area and reference data from the reference area; Measuring means for measuring the interaction between the active substance and the supplied compound, output means for outputting interaction data relating to the interaction measured by the measuring means, and reference data obtained by the measuring means Feedback means for storing in the storage means together with the measurement conditions at the time of the measurement.

  Further, the measurement program of the present invention includes measurement data obtained by supplying a compound to be examined to a measurement region in which a physiologically active substance is immobilized on an immobilized membrane, and the immobilization of the physiologically active substance that is not immobilized. Based on the reference data obtained by supplying the compound to a reference region composed of a membrane and containing information on the amount of nonspecific adsorption of the compound to the immobilized membrane, the physiologically active substance and the compound A measurement program for measuring an interaction between the measurement data, the step of storing the reference data for each measurement condition and the compound, the step of inputting the measurement condition for the measurement, and the input measurement condition And extracting the compound corresponding to the reference data exceeding a predetermined non-specific adsorption level from the reference data corresponding to Supplying the compound excluding the compound extracted by the product extraction means to the measurement region and the reference region, obtaining measurement data from the measurement region and reference data from the reference region, and supplying the physiologically active substance A step of measuring an interaction between the measured compound, a step of outputting interaction data relating to the measured interaction, and storing reference data obtained by the measurement together with a measurement condition at the time of the measurement Step.

  Here, the physiologically active substance is a substance described as “Natural Polymer” (symbols 11001 to 11025) in Japanese Industrial Standard (JIS) K3611, in the Biological Data Encyclopedia (Asakura Shoten). Sugar, amino acid, nucleotide, lipid, heme, quinone, protein, RNA, DNA, phospholipid, polysaccharide, bioscience dictionary (Asakura Shoten) described in “1. It means a substance containing any one or more of proteins, amino acids, nucleic acids, lipids, carbohydrates and enzymes described in “Metabolism”.

  The interaction is the speed of the binding reaction, the binding amount, or the speed of the dissociation reaction in a physical, chemical, or biochemical reaction, or the speed of the binding reaction, the binding amount, and the dissociation. It means a combination of two or more of the reaction speeds.

  The non-specific adsorption amount of the compound differs between the immobilized membrane on which the physiologically active substance is immobilized and the immobilized membrane on which the physiologically active substance is not immobilized. Therefore, when the amount of nonspecific adsorption of the compound to the immobilized membrane is large, that is, when the reference data exceeds a predetermined nonspecific adsorption level, there is a possibility that the interaction between the physiologically active substance and the compound cannot be measured correctly. high.

  Therefore, in the present invention, attention is paid to reference data exceeding a predetermined non-specific adsorption level among reference data corresponding to measurement conditions input for measurement, and a compound corresponding to this reference data is extracted. Then, a compound obtained by removing the extracted compound from the group of compounds to be examined is supplied to the measurement region and the reference region, measurement data from the measurement region and reference data from the reference region are obtained, and a physiologically active substance and The interaction between the supplied compounds is measured.

  In the present invention, since an unsuitable compound is excluded in relation to measurement conditions before measurement, wasteful measurement is not performed and measurement can be performed efficiently.

  In the present invention, since the reference data obtained by measurement is stored together with the measurement conditions at the time of measurement, the reference data is accumulated for each measurement, and appropriate measurement is performed based on many reference data by repeating the measurement. It can be carried out.

  In the measuring apparatus of the present invention, the immobilization film is formed on a metal film, and the measurement means is generated when a light beam is incident on a side of the metal film where the immobilization film is not formed. The interaction between the compound and the physiologically active substance and the immobilized membrane on which the physiologically active substance is immobilized may be measured using total reflection attenuation.

  In the measurement program of the present invention, the measurement may cause total reflection attenuation that occurs when a light beam is incident on a side of the metal film having the immobilization film formed on one side of the metal film on which the immobilization film is not formed. Utilizing it, the interaction between the physiologically active substance and the immobilized membrane on which the physiologically active substance is immobilized and the compound can be measured.

  Examples of the measurement as described above include measurement using a surface plasmon sensor and a leakage mode sensor.

  According to the above-described invention, it is possible to efficiently measure the interaction between a physiologically active substance and a compound by eliminating inappropriate measurement.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  The biosensor 10 as the measuring apparatus of the present embodiment is a so-called surface plasmon sensor that measures the interaction path between the physiologically active substance D and the compound using surface plasmons generated on the surface of the metal film.

  As shown in FIG. 1, the biosensor 10 includes a tray holding unit 12, a transport unit 14, a container mounting table 16, a liquid suction / discharge unit 20, a pressing unit 26, an optical measurement unit 54, and a control unit 60.

  The tray holding unit 12 includes a mounting table 12A and a belt 12B. The mounting table 12A is attached to a belt 12B spanned in the arrow Y direction, and is movable in the arrow Y direction by the rotation of the belt 12B. Two trays T are positioned and placed on the mounting table 12A. In the tray T, eight sensor sticks 40 are stored. The sensor stick 40 is a chip to which the physiologically active substance D is fixed, and details thereof will be described later. Under the mounting table 12A, a push-up mechanism 12D that pushes up the sensor stick 40 to a position of a stick holding member 14C described later is disposed.

  As shown in FIGS. 2 and 3, the sensor stick 40 includes a dielectric block 42, a flow path member 44, a holding member 46, an adhesive member 48, and an evaporation preventing member 49.

  The dielectric block 42 is made of transparent resin or the like that is transparent to the light beam, and has a prism portion 42A having a trapezoidal cross section, and the prism portion 42A integrally with both ends of the prism portion 42A. The formed held portion 42B is provided. A metal film 50 is formed on the upper surface on the wider side of the two parallel surfaces of the prism portion 42A as shown in FIG. On this metal film 50, a physiologically active substance D to be analyzed by the biosensor 10 is fixed. The dielectric block 42 functions as a so-called prism. When the measurement is performed by the biosensor 10, a light beam is incident from one of two opposing non-parallel sides of the prism portion 42A, and a metal film is formed from the other. The light beam totally reflected at the interface with 50 is emitted.

  On the surface of the metal film 50, as shown in FIG. 4, an immobilization film 50A is formed. The immobilization film 50 </ b> A is for immobilizing the physiologically active substance D on the metal film 50. The immobilization film 50A is selected according to the physiologically active substance D to be immobilized. Examples of the immobilized membrane 50A include hydrogels such as agarose, dextran, carrageenan, alginic acid, starch, and cellulose, or derivatives thereof such as carboxymethyl derivatives, or water-swellable organic polymers such as polyvinyl alcohol, polyacrylic acid, and polyacrylamide. Polyethylene glycol or the like can be used. In particular, polyethylene glycol derivatives and dextran derivatives are preferably used from the viewpoint of maintaining physiological activity.

  On the immobilization film 50A, a measurement region E1 in which the physiologically active substance D is immobilized and a reference region E2 in which the physiologically active substance D is not immobilized and a reference signal for signal measurement in the measurement region E1 is formed. Is done. The reference region E2 is formed when the above-described immobilization film 50A is formed. As a formation method, for example, a surface treatment is performed on the immobilization film 50 </ b> A to deactivate a binding group that binds to the physiologically active substance D. Thereby, half of the immobilized film 50A becomes the measurement region E1, and the other half becomes the reference region E2.

  As shown also in FIG. 5, the physiologically active substance D is fixed to the portion of the immobilizing film 50 </ b> A exposed to the liquid channel 45 other than the reference region E <b> 2. The physiologically active substance D is not fixed in the reference region E2. Light beams L2 and L1 are incident on the reference region E2 and the measurement region E1 located on the upstream side of the reference region E2, respectively. The reference area E2 is an area provided for correcting data obtained from the measurement area E1 where the physiologically active substance D is fixed.

  On both side surfaces of the prism portion 42A, engaging convex portions 42C that are engaged with the holding member 52 along the upper edge are virtual surfaces perpendicular to the upper surface of the prism portion 42A along the lower edge. Vertical protrusions 42D configured on the extension are formed at seven locations, respectively. Further, an engagement groove 42E is formed in the center portion along the longitudinal direction of the lower surface of the dielectric block 42.

  The flow path member 44 has a rectangular parallelepiped shape slightly narrower than the dielectric block 42, and is arranged side by side on the metal film 50 of the dielectric block 42 as shown in FIG. 3. A channel groove 44 </ b> A is formed on the lower surface of each channel member 44, communicated with the supply port 45 </ b> A and the discharge port 45 </ b> B formed on the upper surface, and the liquid channel 45 between the metal film 50. Composed. Accordingly, six independent liquid channels 45 are formed in one sensor stick 40. On the side wall of the flow path member 44, a convex portion 44 </ b> B is formed that is press-fitted into a concave portion (not shown) inside the holding member 46 to ensure adhesion with the holding member 46.

  In addition, since it is assumed that the liquid flow path 45 is supplied with a liquid containing protein, in order to prevent the protein from adhering to the flow path member 44, the material of the flow path member 44 is a non-protein. It is preferable not to have specific adsorptivity.

  The holding member 46 is long and includes an upper plate 46A and two side plates 46B. The side plate 46B is formed with an engagement hole 46C that is engaged with the engagement convex portion 42C of the dielectric block 42. The holding member 46 is attached to the dielectric block 42 with the engagement holes 46 </ b> C and the engagement protrusions 42 </ b> C engaged with the six flow path members 44 interposed therebetween. Thereby, the flow path member 44 is attached to the dielectric block 42. In the upper surface plate 46A, a tapered pipette insertion hole 46D that narrows toward the flow path member 44 is formed at a position facing the supply port 45A and the discharge port 45B of the flow path member 44. A positioning boss 46E is formed between the adjacent pipette insertion hole 46D and the pipette insertion hole 46D.

  An evaporation preventing member 49 is bonded to the upper surface of the holding member 46 via an adhesive member 48. A pipette insertion hole 48D is formed at a position facing the pipette insertion hole 46D of the adhesive member 48, and a positioning hole 48E is formed at a position facing the boss 46E. Further, a slit 49D, which is a cross-shaped cut, is formed at a position facing the pipette insertion hole 46D of the evaporation preventing member 49, and a positioning hole 49E is formed at a position facing the boss 46E. By inserting the boss 46E into the holes 48E and 49E and bonding the evaporation preventing member 49 to the upper surface of the holding member 52, the slit 49D of the evaporation preventing member 49 and the supply port 45A and the discharge port 45B of the flow path member 44 are formed. Configured to face each other. When the pipette tip CP is not inserted, the slit 49D covers the supply port 45B, and the liquid supplied to the liquid channel 45 is prevented from evaporating.

  As shown in FIG. 1, the transport unit 14 of the biosensor 10 includes an upper guide rail 14A, a lower guide rail 14B, and a stick holding member 14C. The upper guide rail 14 </ b> A and the lower guide rail 14 </ b> B are horizontally disposed in the arrow X direction perpendicular to the arrow Y direction, above the tray holding unit 12 and the optical measurement unit 54. A stick holding member 14C is attached to the upper guide rail 14A. The stick holding member 14C can hold the held parts 42B at both ends of the sensor stick 40 and can move along the upper guide rail 14A. The engagement groove 42E of the sensor stick 40 held by the stick holding member 14C and the lower guide rail 14B are engaged, and the stick holding member 14C moves in the arrow X direction, so that the sensor stick 40 is placed on the optical measurement unit 54. To the measurement unit 56. The measuring unit 56 is provided with a pressing member 58 that presses the sensor stick 40 during measurement. The pressing member 58 is movable in the Z direction by a driving mechanism (not shown), and presses the sensor stick 40 disposed in the measurement unit 56 from above.

  A compound solution plate 17, a buffer solution stock container 18, and a waste solution container 19 are placed on the container mounting table 16. The compound solution plate 17 is partitioned into 96 so that various compound solutions can be stocked. The buffer liquid stock container 18 is composed of containers 18A to 18E, and an opening K into which a pipette tip CP (to be described later) can be inserted is formed in the containers 18A to 18E. As the buffer solution, for example, based on a known buffer solution (Chemical Handbook Application 2nd edition, pages 1312 to 1320), salt, surfactant (Tween 20 etc.), metal ions (magnesium ions, potassium ions, calcium ions) Etc.), stabilizers (DTT, etc.) and the like can be used. In particular, PBS buffer (phosphate buffer saline), Tris buffer, HEPES buffer and the like are preferably used.

  The waste liquid container 19 includes a plurality of containers 19A to 19E, and an opening K into which the pipette tip CP can be inserted is formed in the same manner as the containers 18A to 18E.

  The liquid suction / discharge section 20 is configured to include a cross rail 22 and a head 24 that are bridged in the arrow Y direction above the upper guide rail 14A and the guide rail 16B. The crossing rail 22 can be moved in the arrow X direction by a drive mechanism (not shown). The head 24 is attached to the cross rail 22 and is movable in the arrow Y direction. The head 24 is also movable in the vertical direction (arrow Z direction) by a drive mechanism (not shown). As shown in FIG. 6, the head 24 includes two pipette portions 24A and 24B. Pipette tips CP are attached to the tip portions of the pipette portions 24A and 24B, and the length in the Z direction can be individually adjusted. Many pipette tips CP are stocked in a pipette tip stocker (not shown) and can be replaced as necessary.

  In this embodiment, the liquid is supplied to the sensor stick 40 by the pipette tip CP. Instead of the pipette tip, one end is connected to the solution plate and the other end is connectable to the sensor stick 40. An injection tube may be provided, and the liquid may be supplied by a liquid feed pump.

  As shown in FIG. 7, the optical measurement unit 54 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. A divergent light beam L is emitted from the light source 54A. The light beam L becomes two light beams L1 and L2 via the first optical system 54B, and is incident on the measurement region E1 and the reference region E2 of the dielectric block 42 arranged in the measurement unit 56. In the measurement region E1 and the reference region E2, the light beams L1 and L2 include various incident angle components with respect to the interface between the metal film 50 and the dielectric block 42 and are incident at an angle greater than the total reflection angle. The light beams L 1 and L 2 are totally reflected at the interface between the dielectric block 42 and the metal film 50. The totally reflected light beams L1 and L2 are also reflected with various reflection angle components. The totally reflected light beams L1 and L2 are received by the light receiving unit 54D through the second optical system 54C, are photoelectrically converted, and a light detection signal is output to the signal processing unit 54E. In the signal processing unit 54E, predetermined processing is performed based on the input light detection signal, and measurement data G1 in the measurement region E1 and reference data G2 in the reference region E2 are obtained. The measurement data G1 and reference data G2 are output to the control unit 60.

  The control unit 60 has a function of controlling the entire biosensor 10, and is connected to a light source 54A, a signal processing unit 54E, and a drive system (not shown) of the biosensor 10 as shown in FIG. As shown in FIG. 8, the control unit 60 includes a CPU 60A, a ROM 60B, a RAM 60C, a memory 60D, and an interface I / F 60E, which are connected to each other via a bus B, and display various types of information. And an input unit 64 for inputting various instructions and information.

  The memory 60D stores various programs for controlling the biosensor 10, various data, and a non-specific adsorption database H.

  As shown in FIG. 9, in the nonspecific adsorption database H, for each measurement condition specified by the type of immobilized membrane, the type of buffer, and the compound concentration, reference data G2 for a plurality of compounds under this measurement condition is stored. It is recorded. The data for each measurement condition is hereinafter referred to as sample data SP. The sample data SP is recorded and increased for each measurement by the biosensor 10. The reference data G2 includes the non-specific adsorption amount of compound A on the immobilized membrane 50A, and the reference point required when the running buffer is supplied onto the immobilized membrane 50A on which the physiologically active substance D is not immobilized. And the amount of change RU between the signal obtained when the compound A is supplied.

  The memory 60D stores a group of compounds to be studied. The compound group is a plurality of compounds whose interaction should be examined with respect to the physiologically active substance D, and is registered in advance by the user.

  Next, measurement with the biosensor 10 will be described.

  When the sensor stick 40 is conveyed to the measurement unit 56 and an instruction to start measurement is input from the input unit 64 together with the measurement conditions (the type of immobilized membrane, the type of buffer, and the compound concentration), the control unit 60 performs FIG. The measurement process shown in FIG.

  First, in step S10, sample data having the same measurement conditions as the input measurement conditions are extracted, and in step S12, compounds having reference data G2 of 5 RU or more are extracted from the compounds of the sample data. In step S14, the extracted compound is excluded from the compound group under consideration.

  For example, when measurement conditions with an immobilized membrane of CMD, a buffer of PBS, and a compound concentration of 10 μM are input, a sample No. is selected from the non-specific adsorption database H shown in FIG. 1 is extracted. Then, paying attention to the reference data G2 for each compound, a compound whose reference data is 5 RU or more is extracted. Here, Compound No. Since 7 is 19RU, it is extracted. And compound no. 7 is excluded from the measurement target.

  In this way, compounds with a large amount of non-specific adsorption are excluded from the measurement target. Here, the reference data G2 with 5 RU or more is excluded, but it is not necessarily 5 RU, and the user can set an arbitrary threshold value.

  Next, in step S16, the first compound solution to be studied is supplied to the liquid channel 45, and in step S20, the measurement data G1 is acquired from the measurement region E1, and in step S22, the reference data from the reference region E2 is acquired. Get G2. In step S24, the measurement data G1 is corrected with the reference data G2 to calculate interaction data G3. In step S26, the interaction data G3 is output to the display unit 62. Accordingly, the interaction data G3 is displayed on the display unit 62.

  In step S28, the acquired reference data G2 is stored in the memory 60D together with the measurement conditions, and in step S30, it is determined whether or not the measurement of all the compounds to be studied has been completed. When determination is denied, it returns to step S16 and repeats said process. If the determination is affirmative, this process ends.

  According to the measurement process, since a compound having a large amount of non-specific adsorption is excluded from the measurement target, useless measurement is not performed and the measurement can be performed efficiently.

  Further, since the reference data G2 obtained by the measurement is fed back to the memory 60D, the data of the compound having a large amount of non-specific adsorption can be accumulated for each measurement condition, and based on a large amount of reference data by repeating the measurement. Appropriate measurement can be performed.

  In the present embodiment, the surface plasmon sensor is described as an example of the biosensor, but the biosensor is not limited to the surface plasmon sensor. Other, for example, quartz crystal microbalance (QCM) measurement technology, optical measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles, etc. It is also possible to store the reference data in a database in advance, and correct the measured measurement data with the stored reference data.

  In addition, a leaky mode detector can be used as another biosensor using total reflection attenuation. The leakage mode sensor is composed of a dielectric, and a thin film composed of a clad layer and an optical waveguide layer that are sequentially layered thereon. One surface of the thin film serves as a sensor surface, and the other surface receives light. It becomes a surface. When light is incident on the light incident surface so as to satisfy the total reflection condition, a part of the light is transmitted through the cladding layer and taken into the optical waveguide layer. In this optical waveguide layer, when the waveguide mode is excited, the reflected light at the light incident surface is greatly attenuated. The incident angle at which the waveguide mode is excited varies according to the refractive index of the medium on the sensor surface, similarly to the surface plasmon resonance angle. By detecting the attenuation of the reflected light, the reaction on the sensor surface can be measured.

  Next, creation of an immobilized film on the dielectric block described in the above embodiment and measurement of a nonspecific adsorption amount of a compound with respect to the created immobilized film will be described. The preparation of the immobilized membrane and the measurement of the nonspecific adsorption amount were performed with a commercially available surface plasmon sensor.

(1) Creation of immobilized membrane (Creation of immobilized membrane 1)
On the dielectric block formed with the metal film, an immobilization film composed of a hydrogel film was prepared by the following method.

  After the dielectric block was treated with Model-208 UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, it was dissolved in ethanol / water (80/20) and dissolved in ethanol / water (manufactured by Aldrich). A 0 mM solution was added so as to contact the metal film, and surface treatment was performed at 40 ° C. for 30 minutes and further at 25 ° C. for 16 hours. Thereafter, washing was performed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water.

  Next, 10 wt% epichlorohydrin solution (solvent: 1: 1 mixed solution of 0.4 M sodium hydroxide and diethylene glycol dimethyl ether) was brought into contact with the surface coated with 11-hydroxy-1-undecanethiol at 25 ° C. The reaction was allowed to proceed for 4 hours in a shaking incubator.

  Thereafter, the surface was washed twice with ethanol and five times with water. Next, 4.5 ml of 1 M sodium hydroxide was added to 40.5 ml of a 25 wt% aqueous dextran (T500, Pharmacia) solution and the solution was contacted on the epichlorohydrin treated surface. It was then incubated for 20 hours at 25 ° C. in a shaking incubator. The surface was washed 10 times with 50 ° C. water. Subsequently, a mixture of 3.5 g of bromoacetic acid dissolved in 27 g of 2M sodium hydroxide solution was brought into contact with the dextran-treated surface and incubated for 16 hours in a shaking incubator at 28 ° C. The surface was washed with water and then the above procedure was repeated once.

(2) Nonspecific adsorption amount of compound to immobilized membrane The nonspecific adsorption property of the compound was evaluated by the following method for the measurement chip having the immobilized membrane prepared as described above.

  The following compounds A to C are dissolved in a PBS buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) / DMSO 5% solution to a concentration of 0.03 mM. A PBS buffer / DMSO 5% solution is allowed to flow through the SPR device as a running buffer, and a reference point is obtained. Next, the prepared compound A solution is allowed to flow for 3 minutes, and then a PBS buffer / DMSO 5% solution is allowed to flow for 3 minutes. The increase (RU) of the SPR signal from the reference point is defined as the nonspecific adsorption amount. The measurement results are shown in Table 1. It is clear that the amount of nonspecific adsorption to the immobilized membrane varies depending on the structure of the compound.

(3) Immobilization of protein A solution of Neutravidin (manufactured by PIERE) 100 μg / ml (acetate buffer pH 5.0) was prepared. A mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was added to the measurement chip, and the activation treatment was performed for 2 minutes or 20 minutes. After washing with 10 mM phosphate buffer, the above Neutr avidin solution was added and allowed to stand for 20 minutes, and Neutr avidin was amine coupled. Further, after washing with 10 mM phosphate buffer, an ethanolamine / HCl solution (1 M, pH 8.5) was added to the measurement chip to block COOH groups remaining without reacting with Neutr avidin.

  Through the above operation, Neutr avidin was covalently fixed to the surface of the measurement chip. Table 2 shows the amount of change in resonance signal (RU value) before Neutravidin addition and after washing as Neutravidin fixed amount (RU value). As is apparent from Table 2, the longer the activation time, the greater the amount of Neutr avidin immobilized.

(4) Dependence of Nonspecific Adsorption of Compound on Protein Fixing Amount The nonspecific adsorption property of the compound was evaluated by the following method on the measurement chip on which Neutravidin prepared as described above was immobilized.

  The following compound A is dissolved in a PBS buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) / DMSO 5% solution to a concentration of 0.03 mM. A PBS buffer / DMSO 5% solution is allowed to flow through the SPR device as a running buffer, and a reference point is obtained. Next, the prepared compound A solution is allowed to flow for 3 minutes, and then a PBS buffer / DMSO 5% solution is allowed to flow for 3 minutes. The increase (RU) of the SPR signal from the reference point is defined as the nonspecific adsorption amount. The measurement results are shown in Table 2.

  It is clear that as the amount of Neutravidin immobilized increases, the amount of nonspecific adsorption of the compound to the immobilized membrane decreases.

  From the above, it is clear that the nonspecific adsorption amount of the low molecular weight compound varies depending on the structure of the immobilized membrane and the compound. In addition, the amount of the compound adsorbed on the immobilized membrane also changes depending on the amount of protein immobilized. Therefore, a database for nonspecific adsorption of compounds is required for each type of immobilized membrane.

It is a whole perspective view of the biosensor of this embodiment. It is a perspective view of the sensor stick of this embodiment. It is a disassembled perspective view of the sensor stick of this embodiment. It is sectional drawing of the 1 liquid flow path part of the sensor stick of this embodiment. It is a figure which shows the state in which the light beam is injecting into the measurement area | region and reference area | region of the sensor stick of this embodiment. It is a side view of the pipette part which comprises the liquid supply part of this embodiment. It is the schematic of the optical measurement part vicinity of the biosensor of this embodiment. It is a schematic block diagram of the control part of this embodiment, and its periphery. It is a figure which shows the nonspecific adsorption | suction database of this embodiment. It is a flowchart of the measurement process of this embodiment.

Explanation of symbols

10 Biosensor 40 Sensor stick 50 Metal film 50A Immobilized film 54 Optical measurement unit 60D Memory 60 Control unit 62 Display unit 64 Input unit D Bioactive substance E1 Measurement region E2 Reference region G1 Measurement data G2 Reference data G3 Interaction data H Non Specific adsorption database

Claims (4)

Measurement data obtained by supplying a compound to be examined to a measurement region in which a physiologically active substance is immobilized on an immobilized membrane, and a reference region composed of the immobilized membrane to which the physiologically active substance is not immobilized A measurement device for measuring an interaction between the physiologically active substance and the compound based on reference data obtained by supplying the compound and including reference data including information on the non-specific adsorption amount of the compound to the immobilized membrane Because
Storage means for storing the reference data for each measurement condition and each compound;
Measurement condition input means for inputting measurement conditions for the measurement;
A compound extraction means for extracting a compound corresponding to reference data exceeding a predetermined non-specific adsorption level among the reference data corresponding to the input measurement conditions;
A compound obtained by removing the compound extracted by the compound extraction means from the compound group to be studied is supplied to the measurement region and the reference region, and measurement data from the measurement region and reference data from the reference region are obtained. Measuring means for measuring an interaction between the physiologically active substance and the supplied compound;
Output means for outputting interaction data relating to the interaction measured by the measuring means;
Feedback means for storing the reference data obtained by the measurement means in the storage means together with the measurement conditions at the time of the measurement;
Measuring device. The immobilization film is formed on a metal film;
The measurement means is configured to fix the physiologically active substance and the physiologically active substance by using total reflection attenuation generated by making a light beam incident on the side of the metal film where the immobilization film is not formed. Measuring the interaction between the immobilized membrane and the compound;
The measuring apparatus according to claim 1. Measurement data obtained by supplying a compound to be examined to a measurement region in which a physiologically active substance is immobilized on an immobilized membrane, and a reference region composed of the immobilized membrane to which the physiologically active substance is not immobilized For measuring an interaction between the physiologically active substance and the compound based on reference data obtained by supplying the compound and including reference data including information on the non-specific adsorption amount of the compound to the immobilized membrane. A measurement program,
Storing the reference data for each measurement condition and each compound;
Inputting measurement conditions for the measurement;
Extracting a compound corresponding to reference data exceeding a predetermined non-specific adsorption level from the reference data corresponding to the input measurement conditions;
A compound obtained by removing the compound extracted by the compound extraction means from the compound group to be studied is supplied to the measurement region and the reference region, and measurement data from the measurement region and reference data from the reference region are obtained. Measuring the interaction between the physiologically active substance and the supplied compound;
Outputting interaction data relating to the measured interaction;
Storing reference data obtained by the measurement together with measurement conditions at the time of the measurement;
A measurement program that causes a computer to execute. The measurement uses the total reflection attenuation that occurs when a light beam is incident on the side of the metal film having the immobilization film formed on one side thereof where the immobilization film is not formed. Measuring the interaction between the immobilized membrane on which the physiologically active substance is immobilized and the compound;
The measurement program according to claim 3.

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