JP2007047105A - Biosensor and molecular weight fractionation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor and a molecular weight fractionation method capable of performing molecular weight fractionation of liquid with a simple constitution. <P>SOLUTION: A molecular weight fractionation layer 70 is formed on the surface of a metal film 50. The molecular weight fractionation layer 70 is constituted of gel having a concentration gradient in the longitudinal direction of a dielectric block 42, and fractionation domains E1-E6 are constituted by the gel having the six-stage concentration wherein the concentration on the supply port 45A side is high and the concentration on the discharge port 45B side is low. Only a material having a small molecular weight is allowed to enter the molecular weight fractionation layer 70 on the supply port 45A side, and a material having a larger molecular weight is allowed to enter the molecular weight fractionation layer 70, gradually toward the discharge port 45B side. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a biosensor that fractionates molecules contained in a liquid sample and detects the fractionated molecules, and a molecular weight fractionation method using the biosensor.

As a method for analyzing a physiologically active substance, a method of performing molecular weight fractionation of substances having different molecular weights to specify a target substance is known (see Patent Document 1). In this electrophoresis method, the molecular weight fractionation of the supplied sample is performed by disposing electrodes on both ends of a gel having a concentration gradient and applying a voltage.
JP-A-5-203621

  However, in general electrophoresis, it is necessary to dispose electrodes at both ends of the gel, which complicates the apparatus configuration.

  The present invention has been made in consideration of the above-described facts, and an object thereof is to provide a biosensor and a molecular weight fractionation method capable of fractionating a liquid with a simple configuration.

  In order to solve the above-mentioned problem, the biosensor according to claim 1 includes a liquid channel in which a supply port to which a liquid sample is supplied, a discharge port from which the liquid sample is discharged, and the liquid channel. And a molecular weight fractionation layer for allowing molecules having different molecular weights to enter at different portions in the traveling direction of the liquid sample, and a detection means for detecting the molecules that have entered the molecular weight fractionation layer. ing.

  In the biosensor of the present invention, by supplying a buffer solution in the liquid channel in advance and supplying the liquid sample from the supply port to the liquid channel filled with the buffer solution, the molecules in the liquid sample are It diffuses to the outlet side. Further, the molecules in the liquid sample enter the molecular weight fraction layer from the surface of the molecular weight fraction layer exposed to the liquid flow path and are diffused. Therefore, molecules having a molecular weight that enter the molecular weight fractionation layer on the supply port side are difficult to diffuse to the discharge port side, and molecules having a molecular weight that cannot enter the molecular weight fractionation layer on the supply port side are discharged from the outlet of the liquid channel. It becomes easy to diffuse to the side.

  In this way, by utilizing the diffusion of molecules in the liquid sample from the supply port side to the discharge port side and the diffusion from the surface of the molecular weight fractionation layer into the molecular weight fractionation layer, no electrode is used. Molecular weight fractionation can be performed with a simple configuration.

  The biosensor of the present invention can be characterized in that, as described in claim 2, the detection means is non-electrochemical detection.

  In this way, since non-electrochemical detection is used, there is no interaction (electron transfer, etc.) between what is detected at the time of measurement and the detection unit, so that operations such as recovery of the detected substance are facilitated. be able to. In addition, it is possible to measure the charge of the measurement object while remaining closer to reality.

  The biosensor according to claim 3 is characterized in that the molecular weight fractionation layer is formed of a gel having a concentration gradient in the traveling direction of the liquid sample.

  In this way, molecules having different molecular weights can be fractionated by forming the molecular weight fractionated layer with a gel having a concentration gradient.

  The concentration gradient of the gel is such that, as described in claim 4, by making the concentration on the supply port side higher than the concentration on the discharge port side, molecules having a low molecular weight are introduced into the gel on the supply port side. It can be made to enter, and a molecule having a large molecular weight can be diffused to the discharge port side in the liquid channel.

  In addition, the molecular weight fractionation layer can be configured to include an agarose dielectric or an acrylamide dielectric as described in claim 5.

  The biosensor according to claim 6 is connected to a liquid chromatography.

  Thus, by connecting with liquid chromatography, fractionation by other attributes of the liquid sample possible by liquid chromatography and molecular weight fractionation by molecular weight can be combined and carried out.

  The biosensor according to claim 7, wherein the molecular weight fractionation layer is formed on a metal film, and the detection means is generated by making a light beam incident on a surface of the metal film opposite to the molecular weight fractionation layer. And detecting a molecule that has entered the molecular weight fractionation layer using total reflection attenuation.

  As described above, surface plasmon resonance and leakage mode detection can be used as detection using the total reflection attenuation on the metal film surface.

  The metal film is formed on a light reflecting surface of a dielectric block as described in claim 8, and the detection is performed by making a light beam incident and incident on the metal film through the dielectric block. It is characterized by being performed by reflecting the reflected light beam.

  According to the above configuration, since the metal film is directly formed on the dielectric block, optical loss can be reduced as compared with the case where the metal film and the dielectric block are separated. . Further, when the metal film and the dielectric block are separated, it is necessary to inject refractive index matching oil or the like between the dielectric block and the plate on which the metal film is formed. In this case, it is not necessary to inject refractive index matching oil or the like, and the configuration of the recovery device can be simplified, and the handling is simplified and the convenience is enhanced.

  The molecular weight fractionation method according to claim 9 is a molecular weight fractionation method for performing molecular weight fractionation of a liquid sample using the biosensor according to any one of claims 1 to 8, comprising: A liquid sample supplying step for supplying a liquid sample in an amount smaller than the volume of the liquid channel to the liquid channel supplied with a predetermined buffer solution from the supply port; and after the liquid sample is supplied, the detection And a detecting step of detecting the entered molecules at a plurality of locations of the molecular weight fraction layer having different traveling directions.

  In the molecular weight fractionation method of the present invention, first, a liquid sample having an amount smaller than the volume of the liquid channel is supplied from the supply port. As a result, the buffer solution supplied in advance is pushed out to the outlet side, but since the supplied liquid sample is smaller than the volume of the liquid channel, the buffer solution remains in the liquid channel. Therefore, the molecules of the supplied liquid sample enter the molecular weight fractionation layer and are also diffused to the buffer solution side, that is, the discharge port side.

  By the way, since the molecular weight fractionation layer is a layer that can fractionate molecules having different molecular weights, the molecular weight molecules diffused into the molecular weight fractionation layer on the supply port side are not easily diffused to the discharge port side. Molecules having a molecular weight that cannot enter the molecular weight fractionation layer on the side are likely to diffuse to the outlet side of the liquid channel. Therefore, molecules having different molecular weights enter the molecular weight fraction layer on the supply port side and the molecular weight fraction layer on the discharge port side, and molecular weight fractionation can be performed.

  Since the present invention has the above-described configuration, the analyte adsorbed non-specifically on the flow path member is not collected, and unnecessary substances can be prevented from being mixed into the collected substance.

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

  The biosensor of the present invention is a so-called surface plasmon sensor that detects whether or not a substance in a liquid exists on a measurement surface using surface plasmon resonance generated on the surface of a 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. A sensor stick 40 is stored in the tray T. The sensor stick 40 is a chip having a flow path for flowing a sample, and details 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. 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, a molecular weight fractionation layer 70 is formed as shown in FIG. The molecular weight fractionation layer 70 is made of a gel having a concentration gradient in the longitudinal direction of the dielectric block 42. As the gel, acrylamide, agarose, cellulose acetate, starch, dextran and the like can be used. The concentration gradient has a high concentration on the supply port 45A side, which will be described later, of the flow path member 44, and a low concentration on the discharge port 45B side. Accordingly, only a substance having a low molecular weight can enter the molecular weight fractionation layer 70 on the supply port 45A side, and a substance having a large molecular weight gradually enters the molecular weight fractionation layer 70 toward the discharge port 45B side. be able to.

  In addition, it is preferable that the density | concentration of the above-mentioned gel is the range of 1%-15% (mass%).

  The molecular weight fractionated layer 70 can be formed as follows. First, as shown in FIG. 5A, a spacer plate 72 is disposed outside the metal film 50 portion of the dielectric block 42, and a lid member 74 is laminated thereon, so that the metal film 50, the lid member 74, A space is formed between the two. Then, as shown in FIG. 5B, the dielectric blocks 42 are directed in the vertical direction, and gels having different concentrations are sequentially filled into the spaces. The molecular weight fractionation layer 70 can be formed by hardening the gel at a predetermined time and removing the spacer plate 72 and the lid member 74.

  The crosslinking agent is described in “Electrophoresis” 1989, 2,220-228. In the acrylamide gel, a divalent polymerization crosslinking agent represented by N, N′-methylenebisacrylamide, a hydroxyl group-containing gel is used. If present, a polyepoxy compound may be used.

  In the present embodiment, six regions in which gels having six levels of concentration are sequentially filled to allow molecules with different molecular weights to enter (hereinafter, each region is referred to as “fractionation region E1 to E6”, see FIG. 6). Thus, a molecular weight fraction layer 70 is formed. Light beams L1 to L6 are incident on the fractional areas E1 to E6, respectively.

  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. A vertical convex portion 42D formed on the extension is formed. 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 disposed on the molecular weight fractionation layer 70 of the dielectric block 42 as shown in FIG. 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 between the molecular weight fractionation layer 70 and the liquid channel. 45 is configured. 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.

  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 hole 46 </ b> C and the engagement protrusion 42 </ b> C engaged with the flow path member 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 45A, and evaporation of the liquid supplied to the liquid channel 45 is prevented.

  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 sample plate 17, a waste liquid collection container 18, and a buffer liquid container 19 are placed on the container mounting table 16. Various samples are stocked on the sample plate 17. The waste liquid recovery container 18 includes recovery containers 18A to 18H, and the recovery containers 18A to 18H are formed with an opening K into which a pipette tip CP described later can be inserted. The buffer container 19 includes a plurality of stock containers 19A to 19EH, and an opening K into which the pipette tip CP can be inserted is formed in the same manner as the recovery container.

  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 cross 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. 7, 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. 8, 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 six light beams L1 to L6 via the first optical system 54B and is incident on the fractional regions E1 to E6 of the dielectric block 42 arranged in the measurement unit 56, respectively. In the fractionation regions E1 to E6, the light beams L1 to L6 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 to L 6 are totally reflected at the interface between the dielectric block 42 and the metal film 50. The totally reflected light beams L1 to L6 are also reflected with various reflection angle components. The totally reflected light beams L1 to L6 are received by the light receiving unit 54D through the second optical system 54C, are each 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 total reflection attenuation angle data (hereinafter referred to as “total reflection attenuation angle data”) of the fractional areas E1 to E6. The total reflection attenuation angle data is 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. 9, the control unit 60 includes a CPU 60A, a ROM 60B, a RAM 60C, a memory 60D, and an interface I / F 60E that are connected to each other via a bus B, and a display unit 62 that displays various types of information. These are connected to an input unit 64 for inputting various instructions and information.

  Various programs for controlling the biosensor 10 and various data are recorded in the memory 60D.

  Next, a procedure for molecular weight fractionation in the biosensor 10 will be described.

  On the mounting table 12A of the biosensor 10, a tray containing the sensor stick 40 in which the liquid channel 45 is filled with the buffer solution C is set. A sample TA is set in the sample plate 17, and a buffer solution is set in the buffer solution container 19.

  First, one sensor stick 40 is pushed up to the position of the stick holding member 14C by the push-up mechanism 12D, and is held by the stick holding member 14C. The stick holding member 14 </ b> C moves along the lower guide rail 14 </ b> B while holding the sensor stick 40, and conveys the sensor stick 40 to the measuring unit 56. The sensor stick 40 conveyed to the measurement unit 56 is positioned at a predetermined measurement position and is pressed and fixed from above by a pressing member 58.

  When an instruction to start measurement is input from the input unit 64, the control unit 60 executes a measurement process shown in FIG. In the measurement process, total reflection data in a state where the liquid channel 45 is filled with the buffer solution is acquired as correction total reflection data, and then the sample TA is supplied to profile the total reflection data.

  First, in step S12, an output instruction signal for the light beam L is output to the light source 54A. Thereby, the light beam L is emitted from the light source 54A. The emitted light beam L becomes six light beams L1 to L6 by the first optical system 54B and is incident on the fractional regions E1 to E6 of the liquid flow path 45, respectively. In step S14, an operation instruction signal is output to the light receiving unit 54D and the signal processing unit 54E. As a result, the light beams L1 to L6 that are totally reflected in the fractional areas E1 to E2 and have passed through the second optical system 54C are received by the light receiving unit 54D, and the received light is photoelectrically converted for each area to generate a light detection signal. Is output to the signal processing unit 54E. In the signal processing unit 54E, predetermined processing is added to the light detection signal, and correction total reflection attenuation angle data is generated and output to the control unit 60.

  In step S16, the control unit 60 determines whether or not a predetermined time has elapsed. After the predetermined time has elapsed, the control unit 60 stores the input correction total reflection attenuation angle data in the memory 60D in step S18.

  Next, in step S20, a sample TA supply instruction signal is output. Thereby, the sample TA is supplied to the liquid channel 45 by the head 24. Specifically, the sample TA is supplied as follows. First, the head 24 moves to the upper part of the sample plate 17 on which the sample TA is set, and a step is formed so that the pipette portion 24A is long downward and the pipette portion 24B is short (see FIG. 7C). Then, the head 24 is lowered, and only the tip of the pipette tip CPA attached to the pipette portion 24A is inserted into the cell in which the sample TA is stored, and the sample TA is sucked into the pipette tip CPA. Next, the head 24 is raised and moved to the upper part of the measurement unit 56, and the length is adjusted so that the pipette unit 24A and the pipette unit 24B have the same height (see FIG. 7A). Then, the head 24 is lowered to insert the tip of the pipette tip CPA on the pipette portion 24A side into the supply port 45A of the liquid channel 45, and the tip of the pipette tip CPB on the pipette portion 24B side is the discharge port of the liquid channel 45 It is inserted into 45B (see FIG. 14A). Then, the sample TA is injected into the liquid channel 45 from the pipette tip CPA.

  At this time, the sample TA having the same volume as the volume from the supply port 45A of the liquid channel 45 to the fractionation region E1 is injected (FIGS. 11A, 12A, and 13A). reference). This is because the sample TA is gradually diffused in the liquid channel 45 from the fractionation area E1 to the fractionation area E6 side.

  Note that the buffer liquid pushed out from the discharge port 45B by the supply of the sample TA is sucked by the pipette tip CPB (see FIG. 14B).

  Next, in step S22, the total reflection attenuation angle data obtained from the light detection signals from the fractional areas E1 to E6 is acquired, and in step S24, the total reflection attenuation angle data is corrected with the correction total reflection attenuation angle data. Thus, the entry state data indicating the entry state of the substance Y in the sample TA into the fractionation regions E1 to E6 is generated. The entry state data can be obtained as a value corresponding to a change in the refractive index of the molecular weight fraction layer 70 by the substance Y entering the molecular weight fraction layer 70.

  In step S26, the approach state data is stored, and in step 28, the approach state data is output to the display unit 62. Thereby, approach state data for every predetermined time is stored in the memory 60 </ b> D and displayed on the display unit 62.

  Next, in step S30, it is determined whether or not a predetermined time required for molecular weight fractionation has elapsed from the sample TA. If this predetermined time has elapsed, this processing is terminated. If the predetermined time has not elapsed, the process returns to step S22. The required time here is a time required for the sample TA to be sufficiently diffused to the fractionation area E6 and further to be sufficiently diffused into the fractionation areas E1 to E6.

  By the measurement process, it is possible to profile the state of entry of the substance contained in the sample TA into each of the fractional areas E1 to E6.

  For example, when the substance Y1 contained in the sample TA is a low molecular weight substance that can enter the fractionation region E1, as shown in FIG. 11 (A), from the supply port 45A of the liquid channel 45. When the sample TA is supplied while reaching the fractionation area E1, as shown in FIG. 11B, the substance Y1 enters the fractionation area E1. In addition, the substance Y1 is gradually diffused in the liquid flow path 45, moves to the fraction areas E2 and E3, and enters the fraction areas E2 and E3. Thereby, the substance Y1 in the liquid flow path 45 decreases as it approaches the fractionation region E6. Therefore, as shown in FIG. 11C, the entry state data of each of the fraction areas E1 to E6 has the highest degree of entry into the fraction area E1 and the fraction area E6 has a low entry degree. It is done. As the entry data, data after a predetermined time when the degree of entry into each fraction area is most characteristically recognized is adopted.

  When the substance Y2 contained in the sample TA is a substance having a medium molecular weight that cannot enter the fractionation areas E1 and E2 and can enter the fractionation areas E3 to E6, FIG. As shown in FIG. 12B, when the sample TA is supplied from the supply port 45A of the liquid flow path 45 to the fractionation area E1, the substance Y2 enters the fractionation areas E1 and E2, as shown in FIG. Without entering, the liquid channel 45 is diffused from the fraction area E1 side to the fraction area E3 side, and enters the fraction area E3. Furthermore, the substance Y2 is diffused in the liquid channel 45, moves to the fraction areas E4 and E5, and enters the fraction areas E4 and E5. Thereby, the substance Y2 in the liquid flow path 45 decreases as it approaches the fractionation region E6. Therefore, as shown in FIG. 12C, the entry state data of each of the fraction areas E1 to E6 has a low degree of entry into the fraction areas E1 and E2, and the highest degree of entry into the fraction area E3. Thus, the fractional areas E4 and E5 and those having a gradually decreasing degree of entry are obtained.

  When the substance Y3 contained in the sample TA is a high molecular weight substance that cannot enter the fractionation areas E1 to E4 and can enter the fractionation areas E5 and E6, FIG. As shown in FIG. 13B, when the sample TA is supplied from the supply port 45A of the liquid channel 45 to the fractionation area E1, the substance Y3 is separated into the fractionation areas E1 to E4 as shown in FIG. Without entering, the liquid channel 45 is diffused from the fraction area E1 side to the fraction area E5 side, and enters the fraction area E5. Furthermore, the substance Y3 is diffused in the liquid channel 45, moves to the fraction area E6 side, and enters the fraction area E6. Therefore, as shown in FIG. 13C, the entry state data of each of the fraction areas E1 to E6 has a low degree of entry into the fraction areas E1 to E4 and a degree of entry into the fraction areas E5 and E6. A high one is obtained.

  As described above, according to the biosensor of the present embodiment, the diffusion of the substance Y in the liquid flow channel 45 and the diffusion of the substance Y into the molecular weight fractionation layer 70 are utilized, so that voltage application can be performed. Therefore, the molecular weight fractionation can be performed with a simple configuration, and the molecular weight distribution of the substance in the sample TA can be analyzed.

  Although only the molecular weight fractionation in this embodiment was performed, the molecular weight fractionation was performed after separation by other properties of the sample TA, such as hydrophilicity, by liquid chromatography, as shown in FIG. Dimensional information can be obtained.

  In this embodiment, the sensor stick 40 is used in which the metal film 50 is formed on the dielectric block 42 that functions as a prism. However, as shown in FIG. 16, the metal film 80A is a transparent flat plate 82. A sensor chip 84 formed on one surface and having the molecular weight fractionation layer 70 laminated on the metal film 80A may be used. In this case, the optical prism P is brought into close contact with the surface of the lithographic plate 82 where the metal film 80A is not formed, and the light beam L is incident on the metal film 80A via the optical prism P and the incident light beam L is incident on the surface. Reflect. According to this configuration, the flat plate 82 on which the metal film 80A is formed can be separated from the optical prism P, and the sensor chip 84 can be simplified.

  On the other hand, when the metal film 50 is formed on the dielectric block 42 as in the present embodiment, optical loss can be reduced. Further, when the metal film and the dielectric block are separated, it is necessary to inject refractive index matching oil or the like between the prism and the plate on which the metal film is formed. As described above, if the metal film 50 is formed on the dielectric block 42, it is not necessary to inject refractive index matching oil and the like, and the configuration of the biosensor can be simplified and the handling is also easy. This increases convenience.

  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. In addition, for example, quartz crystal microbalance (QCM) measurement technology, optical measurement technology using a functionalized surface from colloidal gold particles to ultrafine particles, and the like. Can be applied.

  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.

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 explanatory drawing of the manufacturing method of the molecular weight fractionation layer of this embodiment. It is a figure which shows the state in which the light beam is injecting into the fraction 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 flowchart of the measurement process of this embodiment. (A) (B) of the low molecular weight substance of this embodiment is a figure which shows the approach state of the substance to a molecular weight fraction layer, (C) is a graph which shows an example of the approach data of each fraction layer. (A) (B) of the core substance of this embodiment is a figure which shows the approach state of the substance to a molecular weight fraction layer, (C) is a graph which shows an example of the approach data of each fraction layer. (A) (B) of the high molecular substance of this embodiment is a figure which shows the approach state of the substance to a molecular weight fraction layer, (C) is a graph which shows an example of the approach data of each fraction layer. It is a figure explaining the sample supply operation | movement to the liquid flow path of this embodiment. It is the example which plotted the measurement result on the two-dimensional map which combined the molecular weight fraction in this embodiment, and the hydrophilic property by liquid chromatography. It is a schematic sectional drawing of the sensor chip of the other form by which this invention is implemented.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Biosensor 20 Liquid absorption / extraction part 40 Sensor stick 42 Dielectric block 44 Channel member 45 Liquid channel 45A Supply port 45B Discharge port 50 Metal film 54 Optical measurement part 70 Molecular weight fractionation layer 84 Sensor chip

Claims (9)

A liquid channel formed with a supply port for supplying a liquid sample and a discharge port for discharging the liquid sample;
A molecular weight fractionation layer having a surface exposed to the liquid flow path and allowing molecules having different molecular weights to enter in different portions in the traveling direction of the liquid sample;
Detection means for detecting molecules entering the molecular weight fractionation layer;
Biosensor equipped with.   The biosensor according to claim 1, wherein the detection means is non-electrochemical detection.   The biosensor according to claim 1 or 2, wherein the molecular weight fractionation layer is formed of a gel having a concentration gradient in the traveling direction of the liquid sample.   The biosensor according to claim 3, wherein the concentration gradient of the gel is such that the concentration on the supply port side is higher than the concentration on the discharge port side.   The biosensor according to any one of claims 1 to 4, wherein the molecular weight fractionated layer includes an agarose dielectric or an acrylamide dielectric.   The biosensor according to any one of claims 1 to 5, wherein the liquid channel is connected to a liquid chromatography. The molecular weight fractionated layer is formed on a metal film,
The detection means detects a molecule that has entered the molecular weight fractionation layer by using a total reflection attenuation generated by making a light beam incident on a surface of the metal film opposite to the molecular weight fractionation layer. ,
The biosensor according to any one of claims 1 to 6, wherein: The metal film is formed on the light reflecting surface of the dielectric block,
8. The biosensor according to claim 7, wherein the detection is performed by causing a light beam to be incident on the metal film through the dielectric block and reflecting the incident light beam. A molecular weight fractionation method for performing molecular weight fractionation of a liquid sample using the biosensor according to any one of claims 1 to 8,
A liquid sample supply step of supplying a liquid sample in an amount smaller than the volume of the liquid channel from the supply port to the liquid channel to which a predetermined buffer solution is supplied in advance;
After the liquid sample is supplied, a detection step of detecting the entered molecules at a plurality of different positions in the traveling direction of the molecular weight fractionated layer by the detection means;
A molecular weight fractionation method comprising:

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