JP4966752B2 - Fluid measurement substrate, analyzer, and analysis method - Google Patents

Description

  The present invention relates to a fluid measurement board used for monitoring a substance in a fluid, an analysis apparatus including the same, and an analysis method using the analysis apparatus.

  In recent years, a point of care (POC) test, that is, a clinical test performed near a patient in a clinical practice, has attracted attention from the viewpoint of efficiency and speed of diagnosis and treatment. In addition, there is an increasing need for monitoring environmental pollutants in the air, water, and soil, and for making a quick diagnosis and analysis in a short time for environmental impact assessment and food safety inspection. Thus, devices such as microchips have attracted attention as simple analytical instruments that can be easily inspected in the fields of health management and the environment. One such device is μ-TAS (Micro Total Analysis System) (see, for example, Non-Patent Document 1).

  μ-TAS is a small total chemical analysis system that combines a detector with a microchannel, valve, reaction vessel, membrane separation mechanism, electrophoresis column, chromatographic column or a combination of these integrated on a chip. It is. Examples of the detector include an induction plasma (ICP), a mass spectrometer (MS), a thermal lens microscope, a fluorescence microscope, a spectrometer, a surface plasmon resonance (SPR), or an electrochemical measurement device. With μ-TAS, it is possible to improve the portability of measuring instruments, to analyze with a small amount of sample, and to expect higher sensitivity and shorter time of measurement. For example, liquid chromatography, a detector using an SPR phenomenon, an electrochemical detector, and a simple blood glucose meter are used for measuring the concentration of a specific molecule in a biological sample. The simple blood glucose meter can perform measurement only by touching a biological sample to the detection unit.

  In liquid chromatography, a response solution (baseline) obtained from a detector is stabilized by flowing a reference solution such as a pH buffer solution with a pump for feeding liquid, and then a measurement sample is introduced to increase the response. Measure accurately. The detector using the SPR phenomenon quantifies the concentration from the difference in refractive index change before and after the introduction of the measurement sample. When the measurement sample is directly introduced, the baseline is not stable and the reference refractive index is unknown, so the detector using the SPR phenomenon also needs to acquire the baseline of the reference solution in advance for concentration determination. There is. Also, in the case of an electrochemical detector, since the response changes with time as shown in FIG. 1, it is difficult to capture only the change in current or potential based on the electrochemical reaction to be observed. Therefore, the electrochemical detector also needs to obtain a baseline of the reference solution in advance for quantifying the concentration.

  On the other hand, with a simple blood glucose meter, measurement can be performed even if the collected measurement sample (blood) is directly introduced. This is because the glucose concentration in the blood is sufficiently high, and the concentration of the molecule to be measured is low. Is diffusion limited and lacks sensitivity. In order to solve this, it is necessary to carry out the measurement while continuously feeding the liquid and forcibly avoid the diffusion-controlled state.

FIG. 2 shows a schematic diagram of an apparatus capable of continuously feeding a solution to be measured that is adopted in a simple blood glucose meter. As shown in FIG. 2, the measurement target solution LQ _T is packed in a separate container such as a syringe, and the measurement target solution LQ _T is continuously introduced into the sealed flow path 16 using the solution push-out means 111 such as a syringe pump. (For example, see Non-Patent Document 1). Moreover, the schematic of the apparatus which measures the solution of a measuring object after the reference | standard solution measurement employ | adopted with the detector using an SPR phenomenon and an electrochemical detector is shown in FIG.3 and FIG.4. As shown in FIG. 3, the reference solution LQ _B and the measurement target solution LQ _T are packed in two containers, respectively, and the reference solution LQ _B or the measurement is stored in the channel 16 sealed using the two solution pushing means 111. introduces a target solution LQ _T alternately (e.g., see non-Patent Document 2.). In addition, as shown in FIG. 4, the flow path switching valve 25 is incorporated in the fluid measurement substrate 103, the solution in the flow path 16 is sucked using the solution suction means 121 such as a syringe pump, and the valve 25 is switched. The reference solution LQ _B or the measurement target solution LQ _T is measured alternately (see, for example, Non-Patent Document 1). A method using electroosmotic flow without using a valve is also known (see, for example, Non-Patent Document 1).
Micro Chemical Analysis System Shuichi Shoko Transactions of the Institute of Electronics, Information and Communication Engineers C-II vol. J81-C-II N0.7 pp591-599 July 1998 "Selective detection of L-glutamate using a microfluidic device integrated with an enzyme-modified pre-reactorand an electrochemical detector" Katsuyoshi Hayashi, Ryoji Kurita, Tsutomu Horiuchi, Osamu Niwa, Biosensors and Bioelectronics 18 pp1249-1255 2003 years

  For the POC, an analysis apparatus that can measure a biomolecule concentration simply and quickly is desired because a fluid measurement substrate is inexpensive. However, in the methods of FIGS. 2 and 3, the collected solution to be measured has to be taken in another instrument such as a syringe, and there are problems of foreign matter contamination and noise generation. Further, in the method of FIG. 4, even if the measurement target solution is directly dropped into the fluid measurement substrate and the measurement target solution can be introduced into the flow channel, a flow path switching mechanism such as a valve is provided in the fluid measurement substrate. Therefore, there is a problem that the price of the fluid measurement board becomes expensive. Furthermore, when the electroosmotic flow is used, a separate power source is required, and it is necessary to separately install an electrode and a decabra in the fluid measurement board, which causes a problem of complicating the structure of the fluid measurement board.

  The present invention has been made in order to solve the above-described problems, and a fluid measurement substrate that can directly introduce a collected solution to be measured, has a simple structure, is inexpensive, an analysis apparatus including the same, and an analysis method using the analysis apparatus The purpose is to provide.

  In order to achieve the object, a fluid measurement board according to the present invention includes a solution introduction port for supplying a reference solution, a solution introduction port for directly dropping a measurement target solution, and discharging the reference solution and / or the measurement target solution. In addition, each solution introduction port and the solution discharge port are connected by a sealed flow path. Moreover, each solution introduction port is arrange | positioned so that a reference | standard solution and a measurement object solution may flow from the same direction with respect to a solution discharge port.

Specifically, the fluid measurement board included in the analyzer according to the present invention is provided with at least one solution introduction port to which a solution is supplied and at least one of the solution introduction ports and connected to an external device. A mouth connection portion, at least one solution discharge port for discharging the solution supplied from the solution introduction port, a discharge port connection portion attached to at least one of the solution discharge ports and connected to an external device; A solution introduction port and the solution discharge port are coupled to each other, and a channel having a common part through which the solution supplied from any of the solution introduction ports passes, and the solution is arranged in the common part of the channel and is in contact with the solution And three metal thin films for electrochemical detection.

  The fluid measurement board according to the present invention can be provided at low cost because of its simple structure. Further, if the solution pushing means for the reference solution is connected to the inlet connection portion of the fluid measurement board according to the present invention and the solution suction means is connected to the discharge port connecting portion, the solution pushing means can be turned ON / OFF only. It is possible to switch the solution to be liquid from the reference solution to the solution to be measured.

  More specifically, by driving the solution push-out means and the solution suction means at the same time and feeding the reference solution, the reference solution flows to the solution discharge port without flowing into the other solution introduction ports. After the common part of the flow path is filled with the reference solution, if the solution pushing means is stopped, the solution suction means operates, so that only the solution at the other solution introduction port can be introduced into the flow path. . There is no need to take the solution to be measured in an instrument such as a syringe, and it is only necessary to directly drop the solution to be measured using a pipette or the like to a solution introduction port to which no solution pushing means is connected.

  Therefore, the present invention can directly introduce the collected measurement target solution, and can provide a fluid measurement substrate that has a simple structure and is inexpensive.

In addition, the flow path of the fluid measurement substrate provided in the analyzer according to the present invention has three metal thin films that perform electrochemical detection in contact with the solution at the common portion. Since the metal thin film and the solution in the flow channel are in contact with each other, electrochemical detection is possible.

  More specifically, the detector using the SPR phenomenon quantifies the concentration from the difference in minute refractive index change before and after introduction of the solution to be measured. When the fluid measurement substrate according to the present invention is used, the reference solution can be replaced with the solution to be measured while the flow path is filled with the solution. For this reason, after measuring the refractive index of the reference solution, the refractive index of the solution to be measured can be continuously measured, and the measurement time can be shortened. Furthermore, continuous liquid feeding can prevent substances other than the solution to be measured from being mixed, and is effective in reducing noise.

  In the case of an electrochemical detector, when the fluid measurement substrate according to the present invention is used, the inside of the flow path remains filled with the solution, so that the electric double layer on the electrode can be held. For this reason, when the reference solution is replaced with the solution to be measured, the charging current for forming the electric double layer does not flow again. The time taken from the injection of the solution to the measurement is roughly the time until the measurement current amount reaches the steady state, but the measurement time can be shortened because there is no contribution of the charging current. In addition, as shown in FIG. 11, when a solution to be measured is introduced after obtaining a stable baseline, the response current quickly becomes a steady state, and highly sensitive quantification is possible from the increase in current value.

  In order to achieve the above object, an analysis apparatus according to the present invention includes the fluid measurement substrate, a solution pushing means, and a solution suction means.

Specifically, the analyzer according to the present invention includes at least two solution introduction ports to which a solution is supplied, an introduction port connection unit attached to at least one of the solution introduction ports and connected to an external device, At least one solution discharge port for discharging the solution supplied from the solution introduction port, a discharge port connection unit connected to at least one of the solution discharge ports and connected to an external device, and the solution introduction port and the solution discharge port A fluid measurement substrate having a common channel through which the solution supplied from any of the solution introduction ports is coupled, and connected to the introduction port connection portion, and the solution is introduced from the solution introduction port. Solution extruding means for supplying to the flow path, and solution sucking means connected to the solution discharge port for sucking the solution in the flow path from the solution discharge port. The amount of the solution to be discharged is set to the amount of the solution sucked per unit time of the solution suction means, and the solution push-out means pushes the first solution from one solution introduction port into the flow path, and the solution suction After the first solution in the flow path is sucked from the solution discharge port by means, the amount of the solution pushed out per unit time of the solution push-out unit is set to zero, and the solution suction unit causes the solution discharge port to by sucking the first solution of the flow path, and controlling the second solution from the other of said solution inlet as no write pull the flow path.

  By connecting the solution extrusion means for the reference solution to the inlet connection portion of the fluid measurement substrate and connecting the solution suction means to the discharge port connection portion, as described above, only ON / OFF of the solution extrusion means, It is possible to switch the solution to be sent from the reference solution to the solution to be measured. There is no need to take the solution to be measured in an instrument such as a syringe, and it is only necessary to directly drop the solution to be measured using a pipette or the like to a solution introduction port to which no solution pushing means is connected.

  Therefore, the present invention can directly introduce the collected solution to be measured, and can provide an analyzer with a simple structure and low cost. In addition, since it is not necessary to take the solution to be measured in an instrument such as a syringe, foreign matter contamination and noise generation can be reduced.

The amount of the solution the solution extrusion means of the fluid measuring board according to the present invention is extruded in a unit time is equal to the amount of solution the solution suction means for sucking the unit time Kusuru. Accordingly, the reference solution flows to the solution discharge port without flowing into the solution introduction port where the measurement target solution is dropped.

  In order to achieve the object, the analysis method according to the present invention is synchronized so that the solution pushing means and the solution suction means connected to the fluid measurement substrate are driven according to a predetermined measurement order.

  Specifically, the analysis method according to the present invention is a solution analysis method using the analyzer, wherein the amount of the solution to be pushed out in a unit time of the solution pushing means is sucked in the unit time of the solution sucking means. The amount of the solution is set, and the first solution is pushed out from the one solution introduction port to the flow path by the solution pushing means, and the first solution in the flow path is drawn from the solution discharge port by the solution suction means. A first step of analyzing the first solution in the flow path, and after the first step, the amount of the solution pushed out per unit time of the solution pushing means is set to zero, A second step of drawing the second solution into the flow path from the other solution introduction port by sucking the first solution in the flow path from the solution discharge port by the solution suction means; After the step, the solution is sucked by the solution suction means. A third step of analyzing the second solution in the flow path while sucking the second solution in the flow path from the outlet, and analyzing the analysis result of the first solution and the analysis of the second solution It is characterized by analyzing the difference with the result.

  With the analysis method according to the present invention, it is possible to switch the solution to be fed from the reference solution to the measurement target solution only by turning on / off the solution pushing means. After measuring the reference solution, the measurement target solution is continuously Can be measured. Moreover, it is not necessary to take the solution to be measured in an instrument such as a syringe, and it is only necessary to directly drop the solution to be measured using a pipette or the like to a solution introduction port to which no solution pushing means is connected.

  INDUSTRIAL APPLICABILITY The present invention can provide a fluid measurement substrate that can directly introduce a collected solution to be measured, has a simple structure, is inexpensive, an analysis device including the fluid measurement substrate, and an analysis method using the analysis device.

  Embodiments of the present invention will be described with reference to the accompanying drawings. The embodiment described below is an example of the configuration of the present invention, and the present invention is not limited to the following embodiment. In the present specification and drawings, the same reference numerals denote the same components.

(Embodiment 1)
A schematic diagram of the analyzer 801 of the present embodiment is shown in FIG. The analysis device 801 includes a fluid measurement substrate 501, a solution push-out unit 111, and a solution suction unit 121. A top view of the fluid measurement substrate 501 is shown in FIG. The fluid measurement substrate 501 includes a substrate 51 on which a solution introduction port 53, a solution introduction port 54, a solution discharge port 55, and a flow path 56 are formed. Furthermore, the fluid measurement substrate 501 has a lid 52 on which an inlet connection part 57 and an outlet connection part 58 are formed. In FIG. 6, the lid 52 is omitted.

  The substrate 51 and the lid 52 are made of, for example, silicon, quartz, glass, metal, or organic polymer material. Organic polymer materials include, for example, polydimethylsiloxane (PDMS), acrylic resin (PMMA), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyvinylidene chloride, polystyrene (PS), polyvinyl acetate And general-purpose plastics such as fluororesin, polytetrafluoroethylene (PTFE), ABS resin and AS resin. Organic polymer materials include polyamide (PA), polyacetal (POM), polycarbonate (PC), modified polyphenylene ether (m-PPE, modified PPE), polybutylene terephthalate (PBT), glass fiber reinforced polyethylene terephthalate (GF-). There are general-purpose engineering plastics of PET and cyclic polyolefin (COP). Organic polymer materials include super engineering plastics, polyethylene sulfide (PPS), polysulfone (PSF), polyethersulfone (PES), amorphous polyarylate (PAR), liquid crystal polyester (LCP), polyetheretherketone (PEEK). And polyimide (PI) general-purpose engineering plastics.

  The organic polymer material may be a thermosetting resin. Examples of the thermosetting resin include epoxy resins, urethane resins (polyurethanes), silicon resins (silicones), phenol resins, unsaturated polyesters, alkyd resins, urea resins (urea resins), melamine resins, and ionomers.

  The organic polymer material may be a photocurable resin. Examples of the photocurable resin include those containing a photocationic polymerizable compound and a photocationic polymerization initiator. Examples of the photocationically polymerizable compound include compounds having at least one photocationically polymerizable functional group such as epoxy group, oxetanyl group, hydroxyl group, vinyl ether group, episulfide group, and ethyleneimine group in the molecule. However, a compound having at least one epoxy group in the molecule is preferable because photocuring property is high and photocuring proceeds efficiently even with a small amount of light. The properties (molecular weight) of these photocationically polymerizable compounds are not particularly limited, and any of monomers, oligomers, and polymers may be used. Moreover, these photocationic polymerizable compounds may be used independently and 2 or more types may be used together. Any of the above organic polymer materials may be used, and for example, epoxy resin, urethane resin, silicon resin, acrylic resin, protein, rubber, or a composite material thereof can be used.

  The solution introduction port 53 and the solution introduction port 54 are spaces to which a solution is supplied. The introduction port connecting portion 57 is attached to the solution introduction port 53 and can be connected to an external device that supplies the solution. The solution discharge port 55 is a space for discharging the solution supplied from the solution introduction port 53 or the solution introduction port 54. The discharge port connection portion 58 is attached to the solution discharge port 55 and can be connected to an external device that discharges the solution. The flow path 56 has a common portion 56 a that connects the solution introduction port 53, the solution introduction port 54, and the solution discharge port 55, and the solution supplied from either the solution introduction port 53 or the solution introduction port 54.

  The solution introduction port 53 or the solution introduction port 54 is formed so that the supplied solution flows into the common portion 56 a of the flow channel 56 from the same direction toward the solution discharge port 55. In the fluid measurement substrate 501, as shown in FIG. 6, the solution introduction port 54, the solution introduction port 53, and the solution discharge port 55 are arranged in this order from the left, and are connected by a linear flow path 56.

  7 may be a T-shaped channel 56 like the fluid measurement substrate 502 and a Y-shaped channel 56 like the fluid measurement substrate 503 in FIG. 6 to 8 show the fluid measurement board having two solution inlets and one solution outlet, but there may be three or more solution inlets, and there may be a plurality of solution outlets. May be.

  The flow path 56 has at least one metal thin film in contact with the solution at the common portion 56a. In the case of the fluid measurement substrate 501 in FIG. 5, the flow path 56 has the reference electrode 31, the enzyme-attached working electrode 32, and the counter electrode 33 as a metal thin film. The reference electrode 31, the working electrode 32 with the enzyme, and the counter electrode 33 are formed so as to be exposed in the flow path 56 between the solution introduction port 53 and the solution discharge port 55, and the order in which they are formed is the solution introduction port. Reference electrode 31, working electrode 32 with enzyme, and counter electrode 33. The reference electrode 31, the enzyme-attached working electrode 32, and the counter electrode 33 are each connected to an electrode pad 34 formed on the substrate 51.

  A material having high conductivity is used for each electrode. Such materials include gold, silver, copper, palladium, platinum, platinum black, iron, aluminum, titanium, tin, zinc, nickel, tungsten or iridium single metal, stainless steel, brass, bronze, carbon steel or duralumin alloy. Or carbon is mentioned. The enzyme electrode 32 is fixed with an enzyme that specifically reacts with the substance to be measured. In the case of the fluid measurement substrate 501, since the solution always flows from the solution introduction port 53 side, the enzyme is not necessarily fixed to the electrode, but is fixed in the flow channel upstream of the working electrode. May be.

  The lid 52 is formed with an introduction port connection portion 57 and a discharge port connection portion 58. Although FIGS. 6 to 8 omit the lid 52, the inlet connection part 57 and the outlet connection part 58 are shown for the sake of explanation. The substrate 51 and the lid 52 are joined so as to seal the flow path 56 of the substrate 51. The introduction port connecting portion 57 is formed at a position such that it is above the solution introduction port 53 when the substrate 51 and the lid 52 are joined. The discharge port connecting portion 58 is formed at a position such that it is above the solution discharge port 55 when the substrate 51 and the lid 52 are joined. The lid 52 is provided with a hole on the solution inlet 54 so that liquid can be dropped from the outside.

  As shown in FIG. 5, the solution push-out means 111 is connected to the introduction port connecting portion 57 with a tube 42, and supplies the solution from the solution introduction port 53 to the flow path 56. The solution suction means 121 is connected to the discharge port connecting portion 58 via the tube 42 and sucks the solution in the flow path from the solution discharge port 55. The solution pushing means 111 and the solution suction means 121 are, for example, a syringe pump, a hydrogen pump, a diaphragm pump, or a micro piezoelectric pump.

  The electrode pad 34 on the substrate 51 and the electrochemical measuring device 151 are connected. The analyzer 801 operates by an electrochemical measurement method (amperometry) using a flow-type enzyme.

  Next, an analysis method using the analyzer 801 will be described. Specifically, in the analysis method using the analyzer 801, the amount of the solution pushed out per unit time of the solution pushing means 111 is set to the amount of the solution sucked per unit time of the solution sucking means 121, and the solution pushing means 111 is set. The first solution in the channel 56 is pushed out from the solution inlet 53 to the channel 56 and the solution suction means 121 sucks the first solution in the channel 56 from the solution outlet 55. After the first step, and after the first step, the amount of the solution pushed out per unit time of the solution push-out means 111 is set to zero, and the solution suction means 121 uses the solution discharge port 55 through the first flow path 56. The second step of drawing the second solution into the flow path 56 from the solution inlet 54 by sucking one solution, and the flow path 56 from the solution discharge port 55 by the solution suction means 121 after the second step. Aspirate the second solution of One, and a third step of analyzing the second solution in the channel 56, and characterized by analyzing the difference between the analysis result of the analysis result and the second solution of the first solution.

  The analysis method will be described by taking glucose solution measurement by an electrochemical measurement method using an enzyme reaction by glucose oxidase as an example. A phosphate buffer solution was used as a reference solution, a glucose solution was used as a measurement target solution, and glucose oxidase was used as an enzyme to measure the glucose concentration of the measurement target solution.

  The fluid measurement substrate 501 used here is a polydimethylsiloxane (PDMS) substrate 51 having a length of 20 mm, a width of 50 mm, and a thickness of 2 mm. A groove having a width of 1 mm and a depth of 50 μm is patterned as a flow path 56 to introduce a solution. What formed the opening | mouth 54, the solution introduction port 53, and the solution discharge port 55 was used. Gold thin films were formed by vapor deposition as the reference electrode 31, the working electrode 32 with the enzyme, and the counter electrode 33. For the reference electrode, a silver thin film was further deposited on the gold thin film electrode.

First, the reference solution LQ _B is filled in the syringe 41 of the syringe pump that is the solution pushing means 111. The amount of the solution pushed out by the solution pushing means 111 per unit time is equal to the amount of the solution sucked by the solution suction means 121 per unit time, and the syringe pump that is the solution pushing means 111 and the syringe pump that is the solution suction means 121 are To drive. Thereby satisfying the channel 56 at the reference solution LQ _B.

When the flow path 56 is filled with the reference solution LQ _B , the background measurement is started using the electrochemical measuring device 151. The measurement potential is 500 mv vs. Ag was used. After applying the potential, the state changes to a steady state in about 200 seconds. The amount of current (background current) at this time is taken as the analysis result of the first solution. In this measurement, it was about 1 nA.

Next, the solution inlet 54 is filled with the measurement target solution LQ _T that is a glucose solution. For example, a glucose solution is dropped into the solution introduction port 54 using a pipette. After the solution inlet 54 is filled, the solution pushing means 111 is stopped, and the injection of the reference solution LQ _B is stopped. Since the solution suction means 121 is driven, the glucose solution filled in the solution introduction port 54 is sucked into the flow path 56. The channel 56 is displaced from the reference solution LQ _B in test liquid LQ _T.

  As with the background measurement, a current flows when a voltage is applied. Thereafter, when a predetermined time elapses, the current changes to a steady state. The amount of current at this time is adopted as the analysis result of the second solution. In this measurement, an increase in the current value of a current of about 2 nA at a concentration of 0.1 mg / dl, about 20 nA at 1 mg / dl, about 200 nA at 10 mg / dl, about 1500 nA at 100 mg / dl was observed.

  As a comparative experiment, measurement was performed using a similar enzyme-immobilized electrode in a stationary system, but measurement was not possible at a glucose solution concentration of 1 mg / dl or less.

  Using the analysis method of the present invention, it is possible to measure a glucose solution whose concentration is known in advance and to correlate it with the current value, and to quantify the unknown glucose solution concentration from this correlation. The analysis device 801 having a simple structure and a low-cost fluid measurement substrate and a liquid feeding mechanism can perform a highly sensitive and simple analysis by an analysis method based on an electrochemical measurement method.

(Embodiment 2)
A schematic diagram of the analyzer 802 of this embodiment is shown in FIG. FIG. 9 shows a cross section of the fluid measurement substrate 504. The difference between the fluid measurement board 504 and the fluid measurement board 501 shown in FIGS. 5 and 6 is that the fluid measurement board 504 includes a substrate 91 and a lid 92.

  The substrate 91 is formed of a material that transmits light. For example, BK7. Unlike the substrate 51 of FIGS. 5 and 6, the solution introduction port 53, the solution introduction port 54, the solution discharge port 55, and the channel 56 are not formed in the substrate 91. Further, the reference electrode 31, the working electrode 32 with the enzyme, the counter electrode 33, and the electrode pad 34 are not formed. An antibody-attached metal thin film 94 is formed on the substrate 91 at a position where the common portion 56a of the flow path 56 is present when the substrate 91 and a lid 92 described later are joined. For example, the antibody-attached metal thin film 94 is a gold thin film having a thickness of 1000 Å. The antibody is, for example, an IgG antibody. The antibody-attached metal thin film 94 can be formed by vapor deposition. Furthermore, the substrate 91 has a prism 95 on the side opposite to the surface on which the antibody-attached metal thin film 94 is formed.

  The lid 92 is formed of the material described with reference to the lid 52 in FIGS. A solution inlet 53, a solution inlet 54, a solution outlet 55, and a channel 56 are formed in the lid 92. FIG. 10 shows the fluid measurement substrate 504 viewed from the lid 92 side. Since the solution introduction port 53, the solution introduction port 54, the solution discharge port 55, and the flow path 56 are formed on the surface of the lid 92 on the substrate 91 side, these are indicated by broken lines.

  The solution inlet 53 was formed with an inlet connection portion 57 so as to penetrate the lid 92, and the solution outlet 55 was formed with an outlet port connection portion 58 so as to penetrate the lid 92. The lid 92 has a hole as a solution introduction port 54 so that liquid can be dropped from the outside. Like the fluid measurement substrate 501 described in FIG. 6, the solution introduction port 53 or the solution introduction port 54 is configured so that the supplied solution flows into the common portion 56 a of the flow channel 56 from the same direction toward the solution discharge port 55. Is formed. Further, the solution introduction port 54, the solution introduction port 53, and the solution discharge port 55 are arranged in this order from the left, and are connected by a linear flow path 56. It may be T-shaped or Y-shaped as shown in FIGS. Further, the number of solution inlets may be 3 or more, and the number of solution outlets may be plural.

  The lid 92 is placed on the substrate 91 so as to seal the flow path 56 of the lid 92, thereby forming a fluid measurement substrate 504. As described in the analysis apparatus 801 in FIG. 5, the solution pushing means 111 and the solution suction means 121 are connected to the fluid measurement substrate 504. The analyzer 802 analyzes the solution by a detection method using the SPR phenomenon. Therefore, a light source 96, a lens 97, and a photodetector 98 are provided outside. These are arranged so that the light from the light source 96 passes through the lens 97, is collected by the prism 95, is irradiated onto the metal thin film 94 with an antibody, and the reflected light can be received by the photodetector 98.

  Next, an analysis method using the SPR phenomenon using the analyzer 802 will be described. The analysis method will be described by taking measurement of a solution containing IgG as the measurement target solution as an example.

  The fluid measurement substrate 504 used this time is provided with a polydimethylsiloxane (PDMS) lid 92 having a length of 20 mm, a width of 50 mm, and a thickness of 2 mm, and a groove having a width of 1 mm and a depth of 50 μm to be a flow path 56 is patterned. What formed the solution introduction port 53, the solution introduction port 54, and the solution discharge port 55 was used. On the substrate 91, a gold thin film obtained by attaching an IgG antibody to BK7 was formed as a metal thin film 94 with an antibody.

As described in the analysis apparatus 801 in FIG. 5, the solution pushing means 111 and the solution suction means 121 are driven, and the flow path 56 is filled with the reference solution LQ _B (IgG solution 10 ng / ml). Here, light was emitted from the light source 76, and a standard SPR angle was measured by the photodetector 78. The SPR angle at this time is taken as the analysis result of the first solution.

Next, the solution inlet 54 is filled with the measurement target solution LQ _T which is an IgG solution. The solution pushing means 111 as described in the analyzer 801 of FIG. 5 is stopped to suck test liquid LQ _T in the flow path 56. When the reference solution LQ _B in the flow channel 56 is replaced with the measurement target solution LQ _T , the SPR angle gradually changes. The SPR angle at this time is adopted as the analysis result of the second solution. In this example, it changed about 2.3 × 10 ⁻⁴ degrees 10 minutes after the start of the change. It was also confirmed that the SPR angle change increased in proportion to the concentration of the IgG solution, and the lower limit of detection was 1 ng / ml.

  As a comparative experiment, when the measurement target solution was directly introduced into the detection unit, the SPR angle changed greatly. Since the change at this time is a change that is the sum of the change accompanying the introduction of the sample solution and the change accompanying the antigen-antibody reaction, it is difficult and possible to quantify the antigen concentration in the measurement sample. Complicated fluid measurement board configuration and calculation are required.

  Using the analysis method of the present invention, it is possible to measure the SPR angle of an IgG solution whose concentration is known in advance, to correlate the SPR angle with the concentration, and to quantify the unknown IgG solution concentration from this correlation. . The analysis device 802 having a simple structure and a low-cost fluid measurement substrate and a liquid feeding mechanism can perform a highly sensitive and simple analysis by an analysis method using the SPR phenomenon.

  The present invention is not limited to detectors using the SPR phenomenon and electrochemical detectors, but can be applied to analyzers using flow paths, and the effects of continuous liquid feeding and impurity contamination prevention can be obtained.

It is the figure which showed the change of the electric current based on an electrochemical reaction. It is the schematic of the conventional analyzer. It is the schematic of the conventional analyzer. It is the schematic of the conventional analyzer. It is the schematic of the analyzer which concerns on this invention. It is a top view of the fluid measurement board | substrate which concerns on this invention. It is a top view of the fluid measurement board | substrate which concerns on this invention. It is a top view of the fluid measurement board | substrate which concerns on this invention. It is the schematic of the analyzer which concerns on this invention. It is a top view of the fluid measurement board | substrate which concerns on this invention. It is the figure which showed the change of the electric current based on the electrochemical reaction in the analyzer which concerns on this invention.

Explanation of symbols

801, 802 Analytical apparatus 101, 102, 103, 501, 502, 503, 504 Fluid measurement substrate 111 Solution pushing means 121 Solution suction means 131 Pipette 151 Electrochemical measuring instrument 25 Valve 31 Reference electrode 32 Working electrode 33 with enzyme Counter electrode 34 Electrode pad 41 Syringe 42 Tube 51, 91 Substrate 52, 92 Lid 53, 54 Solution inlet 55 Solution outlet 16, 56 Channel 56a Common part 57 Inlet connection part 58 Outlet connection part 94 Metal thin film with antibody 95 Prism 96 Light source 97 Lens 98 Photodetector LQ _B Reference solution LQ _T Solution to be measured LQ _D Waste liquid

Claims (2)

At least two solution introduction ports to which a solution is supplied, at least one of the solution introduction ports attached to at least one of the solution introduction ports, connected to an external device, and at least one for discharging the solution supplied from the solution introduction port One solution discharge port, a discharge port connection unit attached to at least one of the solution discharge ports and connected to an external device, and connecting the solution introduction port and the solution discharge port, and from any of the solution introduction ports A fluid measurement substrate having a flow path having a common part through which the supplied solution also passes,
A solution push-out means connected to the introduction port connecting portion and supplying a solution from the solution introduction port to the flow path;
A solution suction means connected to the solution discharge port and sucking the solution in the flow path from the solution discharge port;
With
The amount of the solution pushed out per unit time of the solution pushing means is set to the amount of the solution sucked per unit time of the solution sucking means, and the first solution is fed from the one solution introduction port by the solution pushing means. The solution suction means sucks the first solution in the flow path from the solution discharge port and then sets the amount of the solution pushed out per unit time of the solution push-out means to zero, and the solution suction means by sucking the first solution of the flow path from the solution discharge port in unit, analyzes and controls the second solution from the other of said solution inlet as no write pull the flow path apparatus. A method for analyzing a solution using the analyzer according to claim 1 ,
The amount of the solution pushed out per unit time of the solution pushing means is set to the amount of the solution sucked per unit time of the solution sucking means, and the first solution is fed from the one solution introduction port by the solution pushing means. A first step of analyzing the first solution in the flow path while pushing the first solution in the flow path from the solution discharge port by the solution suction means,
After the first step, the amount of the solution to be pushed out per unit time of the solution pushing means is set to zero, and the solution sucking means sucks the first solution in the flow path from the solution discharge port. A second step of drawing the second solution into the flow path from the other solution introduction port;
After the second step, a third step of analyzing the second solution in the flow path while sucking the second solution in the flow path from the solution discharge port by the solution suction means;
And analyzing the difference between the analysis result of the first solution and the analysis result of the second solution.

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