JP2009168487A - Particulate concentration measuring chip, measuring apparatus, and measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a particulate concentration measuring chip which does not require an apparatus to detect a liquid sample or an apparatus and control to control supply of a liquid sample, but allows a predetermined minute amount of liquid sample to certainly pass through an aperture part. <P>SOLUTION: In a particulate concentration measuring chip 1: a microchannel 4 connected to a sample introduction part is formed; an aperture part 5 is provided in a part of the microchannel 4 which is narrower than the other parts of the microchannel so as to detect the number of particulates when a liquid containing the particulates passes through it; a passage part 4b connected downstream of the aperture part 5 has a predetermined capacity; a stop valve 6 is connected to the aperture part 5 via the passage part 4b; and the liquid containing the particulates is stopped at the stop valve 6. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fine particle concentration measurement chip, a fine particle concentration measurement method, and a measurement device for measuring a fine particle concentration in a liquid containing fine particles such as blood cells and inorganic particles, and more specifically, a fine particle-containing liquid passes through. The present invention relates to a particle concentration measuring chip having a structure in which an aperture is provided in a flow path and counting the number of particles passing through the aperture, and a particle concentration measuring apparatus and a measuring method using the particle concentration measuring chip.

  Conventionally, a Coulter counter is widely known as an apparatus for measuring the number of fine particles in a liquid. In the coulter counter, the fine particle-containing liquid is supplied to the flow path by a pump from a liquid container provided inside the apparatus. For example, a narrow aperture portion through which only one fine particle can pass is provided in a part of the flow path. The change in impedance when the fine particles pass through the aperture portion confirms the passage of the fine particles, and the number of fine particles that have passed through the fine particles is counted. This type of Coulter counter can measure the number of cells such as red blood cells and fine particles such as latex particles or inorganic particles.

  Patent Document 1 discloses a cell number measuring apparatus as an example of such a Coulter counter. Here, the cartridge provided with the micro flow path and the aperture part is detachable from the measuring apparatus main body having the measuring part. A microchannel provided with the aperture part and a pair of electrodes for measuring impedance changes in the aperture part are arranged on the cartridge to be attached and detached. More specifically, the downstream portion of the portion provided with the aperture portion of the micro channel is branched into the first and second branch channels. The electrodes are arranged in the first and second branch flow paths, respectively. When the cartridge is attached to the measurement apparatus main body, the pair of electrodes are electrically connected to the measurement unit. And a cell is counted by the impedance change between a pair of electrodes when a cell passes an aperture part.

  In the cell number measuring apparatus described in Patent Document 1, cells can be detected by the impedance change and the number of cells can be counted. However, in order to know the concentration of cells in a liquid containing cells, that is, the concentration of fine particles in a liquid sample, the number of fine particles for a certain amount of sample must be detected.

  In an ordinary fine particle counter capable of flowing a large amount of liquid specimen, the concentration of fine particles can be known based on the number of counted fine particles if the amount of supplied liquid is constant. However, for example, in a system in which a very small amount of a liquid specimen of about 10 nL to 500 μL is supplied, that is, in a system in which a small channel generally known as a microchannel is formed, a microchannel It was very difficult to control the amount of the liquid specimen in the inside. In addition, the flow velocity at the upstream end and downstream end of the flow path is difficult to be constant due to the influence of surface tension and pressure loss. Therefore, even if a small amount of liquid weighed in advance is introduced into the fine flow path and the number of fine particles is counted in the aperture section, the supplied liquid and the number of counted fine particles can be accurately matched. It was difficult.

Therefore, in Patent Document 1, a liquid detection unit that detects that a liquid sample has reached a specific position downstream of the aperture unit is provided. Here, the capacity of the flow path from the aperture section to the specific position is a constant capacity. Therefore, when the detection unit detects that the sample has reached the specific position, the supply of the liquid is stopped by the control from the measuring instrument main body. As a result, the amount of the liquid specimen that has passed through the aperture portion is set to a constant amount.
JP 2007-17303 A

  In the cell counting device described in Patent Document 1, it is detected by the liquid detection unit that the liquid sample has reached a specific position, and the measuring instrument main body passes the aperture unit by stopping the supply of the liquid. The amount of liquid is controlled to a constant amount.

  Therefore, it is possible to detect the ratio of the number of cells in the liquid sample, that is, the concentration, in a very small cell number measuring apparatus in which a small amount of liquid sample is supplied to the microchannel.

  However, a sensor for detecting that a liquid sample has reached a specific position has to be provided as the liquid detection unit. Further, it has been necessary to control the main body of the measuring instrument to stop the supply of the liquid by the output from the sensor. Furthermore, a device for stopping the supply of the liquid was also necessary. For this reason, the entire apparatus tends to be complicated.

  In addition, even if the supply of the liquid specimen is stopped as described above, a certain amount of time is required until the pressure gradient in the microchannel is relaxed. For this reason, even if the supply of the liquid specimen is stopped by the control from the measuring instrument main body, the actual liquid flow in the aperture section does not stop immediately, and a time delay has to be generated. Therefore, it is also difficult to accurately control the amount of liquid passing through the aperture portion to a constant amount.

  The object of the present invention is to eliminate the above-mentioned drawbacks of the prior art, and does not require a device for detecting a liquid sample, a device for controlling the supply of a liquid sample, and a control, and more reliably a certain amount of a small amount of liquid. It is an object of the present invention to provide a fine particle concentration measurement chip, a fine particle concentration measurement device using the fine particle concentration measurement chip, and a fine particle concentration measurement method that allow the specimen to pass through the aperture section.

  According to the present invention, there is provided a fine particle concentration measurement chip for measuring a fine particle concentration of a fine particle-containing liquid, the chip main body including a sample introduction part into which the fine particle-containing liquid is introduced, and the sample main body includes the sample A flow channel connected to the introduction portion is formed, and an aperture portion that is narrower than other portions of the flow channel in order to detect the number of fine particles when a part of the flow channel passes therethrough And a stop valve connected to the aperture section through a constant volume flow path section connected to the downstream side of the aperture section, and further comprising a stop valve for stopping the fine particle-containing liquid. A particle concentration measuring chip is provided.

  The stop valve can be formed by various valves. Preferably, the stop valve is a passive liquid stop valve using a Laplace pressure. In this case, the stop valve can be constituted by a narrowed diaphragm, a slit, a partial overlap of narrow tubes, and a weir structure.

  The stop valve may be a valve body made of a flexible material having one end connected to the inner wall of the flow path and deformed in the flow path by the pressure of the fine particle-containing liquid to close the flow path. In this case, since it is only necessary to provide the valve body made of the flexible material in the flow path, the stop valve can be formed at a low cost and with a simple configuration. In that case, a configuration such as a cantilever valve, a doubly supported valve, a slit, or an unbonded flexible obstacle can be employed.

  Furthermore, the stop valve may be made of a porous body disposed in the flow path. In this case, the stop valve can be easily formed simply by disposing the porous body in the flow path. As the porous body, materials such as a sintered body, an open cell body, and a particle aggregate can be used.

  In a specific aspect of the fine particle concentration measurement chip according to the present invention, the chip body includes a plate formed by laminating a plurality of sheet-like members, the sample introduction portion is opened on one side of the plate, and the aperture And a stop valve are provided in the plate. In this case, it is possible to provide a fine particle concentration measuring chip having a plate shape that is easy to carry and easy to handle.

  A particle concentration measuring device according to the present invention is obtained from a particle concentration measuring chip configured according to the present invention, a detecting means for counting the number of particles passing through the aperture portion of the particle concentration measuring chip, and the detecting means. The frequency data of the fine particles is calculated from the obtained number of fine particles per unit time, a predetermined stable period satisfying a predetermined stability index is obtained from the frequency data, the frequency data during the stable period is calculated, and the total frequency The ratio of the stable period to the time at which fluctuation data is obtained, the product of the constant volume of the flow path portion between the aperture section and the stop valve, and the number of fine particles counted in the frequency data during the stable period And a control device for determining the concentration of the fine particles.

  Further, in a specific aspect of the particulate concentration measuring apparatus according to the present invention, the stable period is a longest section among sections in which the CV of the second-order difference variation of the frequency data is 5% or less. In this case, in the above control device, the stable period can be easily achieved simply by selecting the longest inner section while the variation in the data obtained by taking the second-order difference of the frequency data is below a certain threshold value. Can be sought.

  The fine particle concentration measurement method according to the present invention includes a step of detecting the number of fine particles passing through an aperture portion of a fine particle concentration measurement chip configured according to the present invention to obtain frequency data per unit time, and the frequency variation data is predetermined. Calculating a stable period that satisfies the stability index, a ratio of the time of the stable period to a time at which all frequency data is obtained, and the constant capacity of the flow path portion between the aperture section and the stop valve. A step of obtaining a fine particle concentration from the product and the number of fine particles counted in the frequency data during the stable period.

  In the fine particle concentration measurement method according to the present invention, preferably, the longest interval among the intervals where the CV of the second-order difference variation of the frequency data is 5% or less is used as the stable period.

  In the fine particle concentration measuring chip of the present invention, the fine particle-containing liquid that has passed through the aperture portion stops when it reaches the stop valve, and the flow path portion between the aperture portion and the stop valve has a constant capacity. Therefore, the amount of the fine particle-containing liquid that has passed through the aperture portion can be reliably set to a constant amount. In addition, in order to make the amount of the liquid that has passed through the aperture section constant, a sensor such as a detection device is not required, and a control device that invests the supply of the liquid according to the output from the detection device, and the supply of the liquid There is no need for equipment to stop. Accordingly, a fine particle concentration measurement chip that can reliably make the amount of liquid passing through the aperture portion constant, has a simple structure, can be easily manufactured, and provides an inexpensive fine particle concentration measurement chip. It becomes possible.

  In the fine particle concentration measurement apparatus and measurement method according to the present invention, the fine particle concentration measurement chip of the present invention is used to detect the frequency of fine particles passing through the aperture section, and the frequency variation data per unit time and the frequency variation data are predetermined. Find the stable period that satisfies the stability index, and count by the product of the ratio of the stable period to the total frequency data appearance time, the above-mentioned constant capacity of the flow path part between the aperture part and the stop valve, and the frequency data during the stable period From the number of fine particles thus obtained, the fine particle concentration can be measured with high accuracy.

  Hereinafter, the present invention will be clarified by describing specific embodiments of the present invention with reference to the drawings.

  FIG. 1A is a perspective view showing an appearance of a fine particle concentration measurement chip according to an embodiment of the present invention. The fine particle concentration measurement chip 1 includes a rectangular plate-like base plate 2 and a plate material 3 stacked on the base plate 2. The base plate 2 and the plate material 3 are integrated by an appropriate method such as adhesion.

  As shown in an exploded perspective view in FIG. 1B, a groove opened on the upper surface side is formed on the upper surface of the base plate 2. This groove is closed by a flat plate material, and a microchannel 4 described later is formed. If reference numbers are assigned to both the grooves and the flow paths, the description will be complicated. Therefore, hereinafter, the flow paths formed by closing the grooves will be described with reference numerals.

  The plate material 3 is formed with through holes 3a and 3b. The through hole 3a is a sample introduction part, and the through hole 3b is an exhaust port.

  As shown in FIG. 1B, a micro flow path 4 is formed on the upper surface of the base plate 2. The microchannel 4 has dimensions of a width of about 5 μm to 500 μm and a depth of about 5 μm to 500 μm. In the present embodiment, the amount of liquid whose fine particle concentration is measured is about 10 nL to 500 μL. The micro flow path 4 has a sample introduction side end 4a continuous with the through hole 3a.

  The micro flow path 4 extends along the length direction of the rectangular base plate 2 from the sample introduction side end 4a. An aperture 5 is provided in the middle of the microchannel 4. The aperture 5 is narrower than the portion of the microchannel 4 other than the aperture 5. The gap of the flow path in the aperture portion 5 has such a size that one fine particle can pass and a plurality of fine particles cannot pass. Therefore, the size of the gap is appropriately determined depending on the fine particles to be measured.

  In addition, although the magnitude | size of the microparticles | fine-particles which this invention makes object is not specifically limited, For example, the diameter of 500 micrometers or less can be mentioned. Needless to say, the width and depth of the microchannel are selected so as not to clog the microchannel depending on the size of the microparticles.

  The microchannel 4 reaches the downstream side through the aperture portion 5 and is connected to the stop valve 6. The flow path portion 4b between the aperture portion 5 and the stop valve 6 is formed to have a certain capacity. Here, the flow path portion 4b has a meander shape, but may have a straight shape. As will be described later, in measuring the fine particle concentration, the amount of the fine particle-containing liquid is controlled to be a constant amount, and the fine particle concentration is measured by counting the number of fine particles in the constant amount of the fine particle-containing liquid. Therefore, the flow path portion 4b has a constant capacity for measuring the fine particle concentration.

  The volume of the flow path portion 4b is not particularly limited. For example, when the width of the micro flow path 4 is about 5 μm to 500 μm, or when measuring suspended polymer particles as a fine particle-containing liquid, The size is about 10 nL to 500 μL.

  When counting the number of fine particles in a fine particle-containing liquid having a very small volume and measuring the fine particle concentration as described above, it is difficult to control the volume of the fine particle-containing liquid to be constant as described above. It was.

  In the present embodiment, by making the volume of the flow path portion 4b constant, it is possible to make the volume of the fine particle-containing liquid that is the measurement target constant.

  The stop valve 6 is a valve that allows only gas to pass without passing liquid. The structure of the stop valve is not particularly limited as long as it fulfills such a function, but in this embodiment, the stop valve has a flow path having a very small cross-sectional area as compared with the flow path portion 4b, and is a passive type using Laplace pressure. It is constituted by a liquid stop valve. The specific structure and modification of the stop valve 6 will be described in detail later.

  A flow path portion 7 through which gas passes is connected downstream of the stop valve 6, and a downstream end of the flow path portion 7 is connected to the above-described through hole 3 b.

  The base plate 2 and the plate material 3 can be formed of an appropriate synthetic resin, glass, ceramic or the like, and the materials are not particularly limited.

  FIG. 2 is a schematic block diagram of the fine particle concentration measurement apparatus of the present embodiment including the fine particle concentration measurement chip. The fine particle concentration measurement device 11 includes the fine particle concentration measurement chip 1, a detection unit 12, and a control device 13. The detection means 12 includes an appropriate sensor that detects fine particles passing through the aperture unit 5 in the fine particle concentration measurement chip 1. The control device 13 starts feeding the liquid containing the fine particle concentration in the fine particle concentration measuring chip 1, and starts the operation of detecting the number of fine particles passing through the aperture unit 5 in the detection unit 12, and is obtained from the detection unit 12. Based on the results, the fine particle concentration in the supplied fine particle-containing liquid is measured as will be described later.

  In the present embodiment, the detection means 12 includes an LED (light emitting diode) 12a as a light emitting element, a light receiving sensor 12b, and lenses 14 and 15, as shown in FIG. The LED 12 a is disposed above the fine particle concentration measurement chip 1 and irradiates the aperture unit 5 with light. The light receiving sensor 12 b is disposed below the aperture unit 5 and is disposed so as to receive light transmitted through the fine particle concentration measurement chip 1. That is, the light receiving sensor 12b is disposed at a position for receiving light emitted from the LED 12a, converged by the lens 14, and transmitted through the fine particle concentration measuring chip 1.

  In addition, since it is necessary to permeate | transmit light, in this embodiment, the base plate 2 and the plate material 3 are made translucent in the part in which the aperture part 5 is provided at least.

  However, the detection means 12 is not limited to the one using the above-described light, and a pair of electrodes facing each other with the aperture portion interposed therebetween is provided as in the measurement apparatus described in Patent Document 1 described above, and impedance change between the electrodes. The number of fine particles may be counted by detecting. In that case, the base plate 2 and the plate material 3 do not necessarily need to be formed of a light-transmitting material.

  Next, an example of a fine particle concentration measurement method using the fine particle concentration measurement apparatus of the present embodiment will be described. FIG. 3 is a flowchart showing each step of the fine particle concentration measurement method of the present embodiment.

  First, in step S1, the feeding of the fine particle concentration-containing liquid is started. The liquid feeding is started by supplying the fine particle concentration-containing liquid from the through hole 3a opened on the upper surface of the fine particle concentration measuring chip 1. For supplying the fine particle concentration-containing liquid, the user may supply the fine particle concentration-containing liquid to the through hole 3a from a syringe or the like. Alternatively, a liquid feed pump may be connected to the through hole 3a, and the fine particle concentration-containing liquid may be supplied by the liquid feed pump. When a liquid feed pump is used, the liquid feed pump may be started to be driven by a measurement start signal from the control device 13. Further, the introduction of the fine particle dispersion liquid and the function of liquid feeding in the cartridge may be separated. For example, a chamber may be provided in the introduction part, and after introducing the fine particle dispersion liquid, the introduction part may be covered with an adhesive seal or the like, and the fine particle dispersion liquid in the chamber may be pushed out using a different gas source. The gas source may be a capillary-connected gas reservoir or a micropump built in the cartridge.

  Next, in step S2, the detection means 12 is driven by a signal from the control device 13, and detection is started. This detection is performed by receiving light emitted from the LED 12a by the light receiving sensor 12b. The light receiving sensor 12b gives the control device 13 an electrical signal corresponding to the intensity of the transmitted light. Compared with the case where fine particles do not exist in the fine particle-containing liquid, when the fine particles pass, the light intensity is significantly reduced. In the aperture portion 5, a gap is set so that only one fine particle can pass through. Accordingly, the fine particles pass through the aperture part one by one. Each time the fine particles pass, the light receiving intensity in the light receiving sensor 12b decreases, and as a result, the electric signal shown in FIG. In FIG. 4A, the vertical axis represents the reciprocal of the received light intensity. Since the received light intensity is remarkably lowered every time the fine particles pass, one pulse rises as one fine particle passes.

  However, the intensity of light received by the light receiving sensor 12b may be lowered by a contamination source other than the fine particles to be measured. Therefore, the control device 13 stores a predetermined constant threshold value TH. If the pulse is higher than the threshold value TH, it is determined as a fine particle, and the threshold value is determined. Pulses lower than TH are judged as contamination sources other than fine particles. Then, the number of pulses determined to be fine particles is counted in step S3. For example, in FIG. 4B, the number of fine particles within the unit time T0 is five.

  That is, the number of fine particles per unit time T0 is counted in FIG. Thereafter, after counting the number of fine particles per unit time, in step S4, the frequency data of fine particles comprising the number of fine particles per unit time shown in FIG. 5A is obtained.

  Next, in step S5, as shown in FIG. 5B, the frequency data is first-order differentiated to obtain frequency data first-order difference, and in step S6, the frequency data is obtained by second-order differentiation. The obtained frequency data second floor difference is calculated. As described above, the second-order differentiation of the frequency data is for the following reason.

  In the frequency data, the frequency tends to decrease as the measurement time elapses. This is because the pressure gradient in the fluid becomes gentle and the flow velocity decreases due to the flow tip moving to a position away from the pressure source at the time of liquid feeding. Therefore, the second-order difference of the frequency data is used in order to eliminate such fluctuation factors such as fluctuations in flow velocity. It should be noted that a very small amount of fluid passing through the microchannel 4 has a particularly large flow rate at the start and end of the liquid supply, so the time ta shown in FIG. There is a section in which the second-order difference of the frequency data is likely to fluctuate even during the period from the time to the time tb, that is, the time when the appearance of fine particles ends. Therefore, in order to eliminate the influence of such variation in frequency data, in this embodiment, the particle concentration is detected based on the stable period Ts shown in FIG. 5A in step S7.

  The stable period Ts is determined by obtaining the longest interval that falls within a certain threshold with respect to the result of second-order differentiation of the frequency data per unit time. That is, the first-order difference of the frequency variation data shown in FIG. 5B is further differentiated, and the longest period among the periods where the CV of the variation in the second-order difference shown in FIG. Ts. However, 5% is a standard, and can be set to an appropriate value depending on the type of the fine particle-containing liquid to be measured, the size of the microchannel 4, the size of the gap in the aperture section 5, the liquid feeding speed, and the like.

  Since there may be a plurality of sections with CV of 5% or less, the longest section of sections with CV of 5% or less is set as the stable period.

  In any case, the stable period Ts can be easily obtained by the control device 13 by defining the period in which the variation of the second-order difference is equal to or less than a certain threshold value as the stable period Ts shown in FIG. it can.

  In this embodiment, the ratio of the obtained stable period Ts to the total frequency data appearance time Ta, that is, Ts / Ta, and the capacity V of the flow path portion 4b between the aperture section 5 and the stop valve 6 are calculated. The fine particle concentration is determined from the product (Ts / Ta) · V and the number of fine particles counted in the frequency data during the stable period Ts. That is, in step S8, when the number of fine particles counted in the frequency data during the stable period Ts is N, the fine particle concentration is obtained by N × (Ts / Ta) / V.

  Since the fine particle concentration obtained as described above is obtained based on the number of fine particles that have passed through the aperture section 5 during the stable period Ts, the flow velocity at the time of starting or stopping the liquid supply changes greatly. It is possible to eliminate the influence of variation in the portion. Therefore, the concentration of fine particles in a small amount of microfluid can be detected with high accuracy.

  In addition, when the fine particle concentration measurement chip 1 of the present embodiment is used, the liquid supply is stopped by the stop valve 6 described above, and no device for stopping the liquid supply is required outside the fine particle concentration measurement chip. . Moreover, the control operation for stopping liquid feeding is not required. Therefore, the structure of the particle concentration measuring device is hardly complicated, and the entire structure of the particle concentration measuring device can be simplified to reduce the cost.

  Details of the stop valve 6 will be described.

  FIGS. 6A and 6B are schematic diagrams for explaining the principle of a stop valve including a passive liquid stop valve using a Laplace pressure provided with a narrow throttle portion.

  As shown in FIG. 6 (a), a stop valve 22 is formed at the end 21 a of the micro flow channel 21 by providing a narrow throttle portion having a very small cross-sectional area relative to the micro flow channel 21. A flow path 23 is connected to the other end side of the stop valve 22. The flow path 23 is a part through which gas escapes, and the cross-sectional area thereof is not particularly limited, but is larger than the cross-sectional area of the stop valve 22.

  As shown in FIG. 6B, since the cross-sectional area of the flow path of the stop valve 22 is very small, when the liquid 24 flows into the micro flow path 21, the fine particle-containing liquid 24 and the micro flow path 21 and When the Laplace pressure caused by the surface tension of the inner wall of the stop valve 22 is balanced with the back pressure of the fluid, the liquid does not flow from the microchannel 21 into the stop valve 22, and only the gas passes through the stop valve 22. Accordingly, when the fine particle-containing liquid 24 reaches the inlet of the stop valve 22, the liquid feeding is stopped.

  6A and 6B schematically show a structure in which a cylindrical stop valve 22 is connected to a cylindrical microchannel 21, but the particle concentration measurement chip 1 shown in FIG. In the case of having a simple plate structure, as shown in a schematic perspective view in FIG. 7, a stop valve 6 and a channel portion connected to the downstream side of the stop valve 6 may be formed in the micro channel 4.

  In FIG. 7, a part of the channel portion 4 b of the microchannel 4 is illustrated as having a rectangular planar shape. The stop valve 6 is formed by providing a channel portion having a rectangular cross section that is much narrower than the rectangular planar channel portion 4b, that is, a narrow throttle portion. Further, the planar shape of the flow path portion connected to the downstream side of the stop valve 6 is also rectangular. A groove for forming the flow path portion 4 b constituting the micro flow path 4 is formed on the upper surface of the base plate 2. On the other hand, a groove is formed on the lower surface of the plate material 3 in order to form the stop valve 6 and the flow path portion. The stop valve 6 and the flow path portion 4b only need to communicate with each other so that only the gas passes through the stop valve 6.

  Further, as shown in a schematic plan view in FIG. 8, when forming the stop valve 6 composed of the narrow throttle portion provided at the end of the flow path portion 4 b, a plurality of narrow throttle portions 6 a to 6 c may be provided. .

  As described above, the stop valve used in the present invention is not limited to the one provided with the narrow throttle portion as described above, and the inner wall is water-repellently processed on the downstream side of the flow path portion 4b of the micro flow path. It may be a passive liquid stop valve using pressure.

  The stop valve may be formed of a flexible valve body. An example of a stop valve using such a flexible valve body will be described with reference to FIG.

  In FIG. 9, only the stop valve component in the fine particle concentration measurement chip 31 having a three-layer plate structure is schematically shown in an exploded perspective view. In this modification, an intermediate plate 34 is laminated between the base plate 32 and the plate material 33. A channel portion 4 b of the microchannel 4 is formed in the base plate 32. Here, the flow path part 4b is formed in the base plate 32, and the end of the flow path part 4b is connected to the opening 4c opened upward. A cantilever-like flexible valve element 34a is formed on the intermediate plate 34 so as to face the opening 4c. In other words, the intermediate plate 34 is formed with a cantilever-like flexible valve element 34a whose front end is a free end. The valve body 34a can be deformed so that the tip can move in the vertical direction by the pressure of the fine particle-containing liquid. Therefore, the intermediate plate 34 and the valve body 34a are formed to have such a material and thickness that can be deformed. As such a material, an appropriate material such as a synthetic resin or a polymer gel can be used.

  A recess 33 a is formed on the lower surface of the plate material 33. The recess 33a has a size that allows the valve body 34a to enter. A downstream channel portion 33b is connected to the recess 33a. The flow path portion 33b has an opening 33c that opens to the recess 33a. The tip of the valve body 34a is moved upward by the pressure of the fine particle-containing liquid to close the opening 34c. In this way, the passage of liquid is stopped.

  The planar shape of the cantilever-like flexible valve element is not limited to the rectangular valve element 34a, but may be various shapes as shown in FIGS. 10 (a) to 10 (c). That is, as shown in FIG. 10A, a cantilevered flexible valve element 41 whose width gradually increases toward the tip 41b as compared with the fixed side end 41a may be used. Further, as shown in FIG. 10B, a planar valve body 42 in which a rectangular region and a semicircular region are connected and the free end side is a semicircular region may be used. Further, as shown in FIG. 10 (c), a valve body 43 having a width gradually increasing from the fixed end 43a toward the free end 43b and having a curved outer periphery at the free end 43b is used. May be.

  Further, as shown in FIGS. 11A and 11B, the stop valve may be constituted by a both-sided flexible valve body. As shown in FIG. 11B, in the fine particle measurement chip 51 of the present embodiment, a base plate 52, intermediate plates 53 to 55, and a plate material 56 are laminated. The intermediate plates 53 and 55 are provided with stop valves.

  The details of the stop valve formed on the intermediate plate 55 are shown enlarged in FIG. In the stop valve 57, a circular opening 55 a is formed in the intermediate plate 55. A double-sided valve body 58 is formed in the opening 55a. The valve body 58 is connected to the main body portion of the intermediate plate 55 by bridges 59a and 59b. The valve body 58 has a valve main body 58a having a figure smaller in diameter than the opening 55a. Here, the valve body 58a is deformed so as to move in the vertical direction by the pressure of the liquid flowing upward from the through hole provided in the intermediate plate 54. In this case, although the liquid is not allowed to pass, the valve body 58a always seals the through hole so that the gas can pass. In this way, the stop valve may be constituted by a flexible valve body that is both supported.

  Moreover, as shown in FIG. 12, the downstream end of the channel portion 4b of the microchannel 4 may be filled with a porous body 71 to constitute a stop valve. Examples of such a porous body 71 include an appropriate porous material that does not transmit the fine particle-containing liquid but allows gas to pass therethrough. That is, an appropriate material such as a foam having continuous pores or an inorganic fine particle aggregate can be used.

  Furthermore, as shown in FIG. 13, a stop valve 81 due to an unbonded flexible obstacle may be provided at the lower end of the flow path portion 4b. The stop valve 81 has a flow path portion 82 that is continuous with the flow path portion 4b. An unbonded region 83 indicated by hatching is provided around the flow path portion 81a. And the flow-path part 7 which isolate | separates from the flow-path part 82 and escapes gas is provided. The flow path portion 82 and the flow path portion 7 are disposed in the unbonded region 83. In the unbonded region 83, the liquid does not reach the channel portion 7 from the channel portion 82, but the gas reaches the channel portion 7 from the channel portion 82. Therefore, only the gas that has passed through the non-bonded region 83 passes and functions as a stop valve.

  As described above, the stop valve used in the present invention can be variously modified, and is not limited to the modified examples. In any case, the volume of the flow path portion 4b is constant, and when the stop valve is reached, the feeding of the fine particle-containing liquid is automatically stopped. Therefore, the number of fine particles in the fine particle-containing liquid is counted, and the fine particle concentration can be measured with high accuracy based on the fine particle count number in the fine particle-containing liquid controlled to a constant amount.

  Examples of the fine particle-containing liquid measured by the present invention include various liquids, such as blood, human or animal body fluids, or inorganic particle-containing liquids. As for the fine particles, suitable cells such as erythrocytes in blood or inorganic particles in a liquid containing inorganic particles can be used.

  In addition, although the magnitude | size of the microparticles | fine-particles which this invention makes object is not specifically limited, For example, the thing of the magnitude | size of 5 micrometers-500 micrometers or less can be mentioned.

(A) is a perspective view which shows the external appearance of the microparticle density | concentration measuring chip based on one Embodiment of this invention, (b) is the disassembled perspective view, (c) is a model of the flow path comprised inside. FIG. It is a schematic block diagram which shows the fine particle concentration measuring apparatus which concerns on one Embodiment of this invention. It is a flowchart which shows the process of the fine particle measurement method of one Embodiment of this invention. (A) is a figure which shows the pulse waveform obtained from a detection means in the measuring method of one Embodiment of this invention, (b) is fine particle appearance per unit time calculated | required from the pulse waveform of (a) It is a figure which shows data. (A) is the frequency data of the application of the fine particles obtained in one embodiment of the present invention, (b) shows the first-order difference of the frequency variation data of (a), (c) is (a) The second-order difference of the frequency data is shown. (A) And (b) is each schematic diagram for demonstrating the principle of the stop valve which has the structure which provided the narrow aperture part. It is a schematic perspective view for demonstrating the modification provided with the stop valve by a narrow aperture part. It is a schematic plan view for demonstrating the stop valve provided with the some narrow aperture part. It is a typical partial notch disassembled perspective view which shows the structure where the stop valve which consists of a cantilever-like flexible valve body is provided. (A)-(c) is each typical top view for demonstrating the modification of the flexible valve body which comprises a stop valve. (A), (b) is a typical perspective view for demonstrating the stop valve comprised using the flexible valve body of both ends, and the disassembled perspective view of the microparticle measuring chip provided with this stop valve It is. It is a typical plane sectional view showing a stop valve which consists of a porous body. FIG. 10 is a schematic plan view for explaining still another modified example of the stop valve of the particle measuring chip of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Fine particle concentration measurement chip 2 ... Base plate 3 ... Plate material 3a, 3b ... Through-hole 4 ... Micro flow path 4a ... Sample introduction side edge part 4b ... Flow path part 4c ... Opening part 5 ... Aperture part 6 ... Stop valve 6a -6c ... part 7 ... flow path part 11 ... fine particle concentration measuring device 12 ... detection means 12a ... LED
12b ... Light receiving sensor 13 ... Control device 14, 15 ... Lens 21 ... Micro flow channel 21a ... End 22 ... Stop valve 23 ... Channel 24 ... Fine particle-containing liquid 31 ... Fine particle concentration measurement chip 32 ... Base plate 33 ... Plate material 33a ... Recess 33b ... Flow path portion 33c ... Opening 34 ... Intermediate plate 34a ... Valve element 34c ... Opening part 41 ... Flexible valve element 41a ... Fixed side end part 41b ... Tip 42 ... Valve element 43 ... Valve element 43a ... Fixed end 43b ... Tip 51 ... Fine particle measuring chip 52 ... Base plate 53-55 ... Intermediate plate 55a ... Opening 56 ... Plate material 57 ... Stop valve 58 ... Valve body 58a ... Valve body 59a, 59b ... Bridge 71 ... Porous body 81 ... Stop valve 81a ... channel portion 82 ... channel portion 83 ... non-bonded area

Claims (9)

A fine particle concentration measurement chip for measuring the fine particle concentration of a liquid containing fine particles,
A chip body having a sample introduction part into which a fine particle-containing liquid is introduced; and a channel connected to the sample introduction part is formed in the chip body, and a part of the channel passes through the chip body. In order to detect the number of fine particles at the time, an aperture part that is narrower than the other part of the flow path is provided,
A fine particle concentration measuring chip, further comprising a stop valve connected to the aperture part through a constant volume flow path part connected to the downstream side of the aperture part, and stopping the fine particle-containing liquid.   The fine particle concentration measurement chip according to claim 1, wherein the stop valve is a passive liquid stop valve using Laplace pressure.   2. The stop valve is a flexible valve body having one end connected to an inner wall of the flow path, deformed in the flow path by the pressure of the fine particle-containing liquid, and closing the flow path. The fine particle concentration measurement chip according to 1.   The fine particle concentration measurement chip according to claim 1, wherein the stop valve is a porous body disposed in the flow path.   The chip body is made of a plate formed by laminating a plurality of sheet-like members, the sample introduction part is opened on one side of the plate, and the aperture part and the stop valve are provided in the plate. The fine particle concentration measurement chip according to any one of claims 1 to 4. Fine particle concentration measurement chip according to any one of claims 1 to 5,
Detection means for counting the number of fine particles passing through the aperture portion of the fine particle concentration measurement chip;
The frequency data of the fine particles is calculated from the number of fine particles per unit time obtained from the detecting means, a predetermined stable period satisfying a predetermined stability index is obtained from the frequency data, and the frequency data during the stable period is obtained. Calculate and count in the frequency data during the stable period, the product of the ratio of the stable period to the time at which all frequency variation data is obtained, the constant capacity of the flow path portion between the aperture section and the stop valve A fine particle concentration measuring device comprising: a control device that obtains the fine particle concentration from the number of fine particles that have been obtained.   The fine particle concentration measurement apparatus according to claim 6, wherein the stable period is a longest section among sections in which the CV of the second-order difference variation of the frequency data is 5% or less. Detecting the number of fine particles passing through the aperture portion of the fine particle concentration measurement chip according to any one of claims 1 to 5, and obtaining frequency data per unit time;
Calculating a stable period in which the frequency variation data satisfies a predetermined stability index;
Counted in the frequency data during the stable period, the product of the ratio of the time of the stable period to the time over which all frequency data is obtained, the constant volume of the flow path portion between the aperture section and the stop valve. And a step of determining a fine particle concentration from the number of fine particles. The fine particle concentration measurement method according to claim 6, wherein a longest section among sections in which the CV of the second-order difference variation of the frequency data is 5% or less is used as the stable period.

Images