JP5483616B2 - Flow cell and flow cell feeding method - Google Patents

Description

  The present invention relates to a flow cell for feeding a liquid by capillary force.

  Measurements using advanced biomolecule identification functions such as antigen-antibody reactions and DNA fragments (DNA probes) and DNA binding are important technologies for clinical tests, biochemical measurements, and environmental pollutant measurements. It has become. Examples of this measurement include micro TAS (Total Analysis Systems), micro combinatorial chemistry, chemical IC, chemical sensor, biosensor, trace analysis, electrochemical analysis, QCM measurement, SPR measurement, and ATR measurement. In such a measurement field, the sample solution to be measured is often in a very small amount.

  For this reason, in the measurement as described above, a sample cell capable of holding a sample solution is used (for example, see Patent Document 1). Then, a very small amount of sample solution is supplied to the sample cell, and the sample solution is flowed and transferred to a detection unit for measuring the sample cell. Thereby, the measurement is performed with higher sensitivity and higher efficiency without lowering the concentration of the sample such as DNA or antibody dissolved or dispersed in the sample solution. The sample cell that allows the sample solution to flow to the detection unit in this way is called a flow cell.

  The following method has been proposed as a technique for realizing the transfer of a small amount of sample solution in the flow cell. That is, while providing a flow channel facing the detection part of the flow cell, a method of transferring a sample solution by an external pressure such as a syringe pump, a method of transferring by electrostatic force, an electrowetting method, a volume change due to heating, There are a method of transferring a sample solution by generation and a method of using electroosmotic flow.

  In recent years, a technique has been proposed in which a flow channel or a region serving as a pump capable of exhibiting a capillary phenomenon with respect to a sample solution is formed in a flow cell (see, for example, Non-Patent Document 1). A flow cell manufactured by this technique includes a supply unit having an introduction port into which a sample solution is introduced, a transfer unit having a capillary pump for sucking the introduced sample solution, and an introduction port and a capillary pump. The provided flow channels for measurement are formed side by side along a plane direction of the plate-like flow cell. When the sample solution is supplied to the flow cell, the sample solution reaches the capillary pump from the introduction port through the flow path, and is sucked by the capillary pump and continuously flows through the flow path.

  In general, when measuring specimens of sample solutions in fields such as immunoassay, the measurement results of the sample solution to be measured are compared with the measurement results of a reference solution having properties similar to the sample solution, and the difference The amount of the specimen in the sample solution is measured by the above. If this measurement is to be realized by the flow cell using the above-described capillary pump, it is not easy to provide the sample solution system and the reference solution system on the same cell. Therefore, each of the reference solution and the sample solution is caused to flow through one flow cell. Specifically, first, the reference solution is supplied to the flow cell to perform the first measurement of the reference solution, and then the sample solution is supplied to the flow cell to perform the first measurement of the sample solution. Next, after supplying the reference solution to the flow cell again and performing the second measurement of the reference solution, the measurement result of the sample solution is obtained using the first and second measurement results of the reference solution. Here, the second measurement of the reference solution can be omitted.

  It should be noted that the introduction of the reference solution first into the detection unit is effective for immunoassay using a flow cell in which the antibody is immobilized in advance. That is, it is desirable that the flow cell be stored in a dry state. However, in a measurement in which a sample is suddenly introduced from the dry state, sufficient time for restoring the antibody activity cannot be taken. In addition, since the change due to the activity of the antibody and the change due to the antigen-antibody reaction are superimposed, measurement of the antigen becomes difficult. For this reason, the measurement accuracy can be greatly improved by first introducing the reference solution into the detection unit and measuring the sample solution after restoring the antibody activity with the reference solution without the antigen.

  By the way, in the measurement as described above, the solution is supplied to the supply unit using a pipette or the like. In this case, after supplying the reference solution from the introduction port to the supply unit, the operator continues to wait until the reference solution passes through the flow path and is sucked into the capillary pump and transferred from the introduction port. Need to supply. Similarly, it is necessary to supply a second reference solution in succession when the supplied sample solution is completely transferred. That is, when supplying different solutions sequentially to the supply unit, the solution supply timing and solution supply are performed so that the solutions do not mix as much as possible and no gap is formed between the different solutions flowing through the flow path. It is necessary to pay attention to the amount.

  At this time, if the next solution is supplied in a state where the solution is sufficiently stored in the supply unit, the solutions are mixed with each other, so that it is difficult to ensure measurement accuracy. In addition, if the next solution is supplied to the supply unit in an empty state where all of the solution in the supply unit has been transferred, a gap is formed between the solutions, and a large change in the measurement result called so-called injection shock occurs. Will occur. For this reason, it is difficult to compare a minute amount of change between the measurement result of the reference solution and the measurement result of the sincerity solution, and the measurement accuracy cannot be ensured. For this reason, an operator's skill was required for the solution supply operation.

  Therefore, as a method for sequentially supplying the reference solution and the sample solution to the flow cell, for example, a method using a plurality of syringe bombs has been proposed. Specifically, these syringe pumps are each connected to the liquid switch inlet side with a tube, etc., and the liquid switch outlet side is connected to the flow cell, and the liquid switch is switched for each solution, thereby sequentially referring to the flow cell. In this method, a solution and a sample solution are supplied. However, in this method, a large number of parts such as a syringe pump, a liquid switch, and a tube are used, and these are connected in a complicated manner, resulting in a problem that the configuration of the apparatus becomes complicated and the equipment cost increases. Further, in order to ensure measurement accuracy, each time the sample solution is measured, the syringe pump, the liquid switch, the tube, and the like must be replaced, washed, and dried, which is troublesome and the work efficiency is poor.

  Another method for sequentially supplying the reference solution and the sample solution to the flow cell is to store the reference solution in the flow cell in advance, measure the reference solution first, and then supply the sample solution to the flow cell. A method for measuring a sample solution has also been proposed. However, in this method, for example, the detection part configured in the flow path such as the antibody application part is exposed to the reference solution for a long period of time, so that the detection part deteriorates and affects the measurement accuracy. was there.

  Therefore, in order to solve such a problem, a plurality of capillaries constitute a suction pump, the capillaries are sealed on the outside air side with a plurality of sealing members, and the seal is opened for each solution at any timing. Has been proposed (see, for example, Patent Document 2).

JP 2002-148187 A International Publication No. 2009/145172

Martin Zimmermann, Heinz Schmid, Patrick Hunziker and Emmanuel Delamarche, “Capillary pumps for automous capilally systems”, The Royal Society of Chemistry 2007. Lab Chip. 2007. 7.p.119-125, First published as an Advance Article on the web 17th October 2006.

  However, in the above-described flow cell, when the solution reaches the inlet of each capillary and a capillary force is generated, the flow velocity changes greatly, so that instantaneous fluctuations, so-called pulse fluctuations, are superimposed on the flow velocity at the detection unit. End up. Then, in a pump in which a large number of capillaries are integrated, a pulse-like flow velocity fluctuation occurs for each capillary, so that a pulse is always generated. Since this becomes flow velocity noise during sample measurement, there was a limit to improvement in measurement accuracy.

  Moreover, in the flow cell provided with the capillary pump as described above, the flow rate of the sample solution is the fastest at the initial stage of suction by the capillary pump, and decreases as the suction proceeds. For example, the linear velocity v at which the liquid travels by capillary force in a capillary having a circular cross section with a radius r can be obtained by the following equation (1). In the following equation (1), γ is a surface tension, η is a viscosity coefficient, θ is a contact angle, and t is time.

  For this reason, it has been proposed to suppress a decrease in the flow velocity by processing the shape of the inner surface of the capillary so that the contact area with the liquid is increased or by branching a plurality of capillaries one after another. However, if there is a part where the geometric shape changes, a large flow velocity fluctuation occurs, and in this case as well, there is a limit to improving the measurement accuracy. Furthermore, in the capillary pump having such a structure, since it is necessary to perform fine processing on a large area, the manufacturing cost is high.

  Accordingly, an object of the present invention is to provide a flow cell and a flow cell feeding method capable of improving measurement accuracy.

  In order to solve the above-described problems, a flow cell according to the present invention includes a supply unit to which a sample solution is supplied, and one end connected to the supply unit, and transfers the sample solution supplied to the supply unit by capillary force. A first flow path, a detection unit provided in the middle of the first flow path, and the other end of the first flow path are connected, and the sample solution transferred from the first flow path is capillaryed A flow cell comprising: a second flow channel that is transferred by force; and a plurality of tubes that are connected at one end to the second flow channel and transfer a sample solution in the second flow channel by capillary force. The absolute value of the capillary force by the tube is smaller than the absolute value of the capillary force of the first channel and the absolute value of the capillary force of the second channel. Here, the absolute value of the capillary force of the first channel is larger than the smallest capillary force among the absolute values of the capillary forces of the plurality of tubes.

  The flow cell further includes a substrate, the supply unit includes a recess formed on the upper surface of the substrate, the first flow path includes a tube formed inside the substrate, and the second flow path extends to the inside of the substrate. The plurality of tubes may be formed of holes that communicate with the upper surface of the substrate and the cavities.

  In addition, the flow cell liquid feeding method according to the present invention includes a supply unit to which a sample solution is supplied, and a first flow in which one end is connected to the supply unit and the sample solution supplied to the supply unit is transferred by capillary force. A first portion that is connected to the other end of the first channel and the sample solution that has been transferred from the first channel by capillary force. 2 and one end connected to the second channel, and a plurality of tubes for transferring the sample solution in the second channel by capillary force, the absolute value of the capillary force by the plurality of tubes is: A flow cell feeding method that is smaller than the absolute value of the capillary force of the first flow path and the absolute value of the capillary force of the second flow path, and sequentially supplies the first liquid and the second liquid. The volume of the first solution is not less than the sum of the volumes of the first channel and the second channel. Flow path, and is characterized in that it is less than the volume sum of the second channel and a plurality of tubes.

  According to the present invention, since the absolute value of the capillary force by the plurality of tubes is smaller than the absolute value of the capillary force of the first channel and the absolute value of the capillary force of the second channel, the first channel Since the sample solution is not transferred by the tube until the second flow path is filled with the sample solution, it is possible to prevent the transfer by the tube from occurring every time the sample solution reaches the inlet of each tube. As a result, it is possible to prevent a flow rate fluctuation from occurring for each tube and to greatly change the flow rate of the sample solution, and as a result, the measurement accuracy can be improved.

FIG. 1 is a perspective view schematically showing the configuration of a flow cell according to an embodiment of the present invention. FIG. 2A is a diagram for explaining the operation of the liquid in the flow cell according to the embodiment of the present invention. FIG. 2B is a diagram for explaining the operation of the liquid in the flow cell according to the embodiment of the present invention. FIG. 2C is a diagram for explaining the operation of the liquid in the flow cell according to the embodiment of the present invention. FIG. 2D is a diagram for explaining the operation of the liquid in the flow cell according to the embodiment of the present invention. FIG. 2E is a diagram for explaining the operation of the liquid in the flow cell according to the embodiment of the present invention. FIG. 2F is a diagram for explaining the operation of the liquid in the flow cell according to the embodiment of the present invention. FIG. 3A is a perspective view showing a simulation result in the first example of the flow cell according to the exemplary embodiment of the present invention. FIG. 3B is a perspective view showing a simulation result in the first example of the flow cell according to the exemplary embodiment of the present invention. FIG. 3C is a perspective view showing a simulation result in the first example of the flow cell according to the exemplary embodiment of the present invention. FIG. 3D is a perspective view showing a simulation result in the first example of the flow cell according to the embodiment of the present invention. FIG. 3E is a perspective view showing a simulation result in the first example of the flow cell according to the embodiment of the present invention. FIG. 3F is a perspective view showing a simulation result in the first example of the flow cell according to the embodiment of the present invention. FIG. 4A is a plan view showing a simulation result in the first example of the flow cell according to the exemplary embodiment of the present invention. FIG. 4B is a plan view showing a simulation result in the first example of the flow cell according to the exemplary embodiment of the present invention. FIG. 4C is a plan view showing a simulation result in the first example of the flow cell according to the exemplary embodiment of the present invention. FIG. 5 is a graph showing changes in flow velocity in the first example of the flow cell according to the embodiment of the present invention. FIG. 6A is a diagram illustrating a simulation result in a comparative example. FIG. 6B is a diagram illustrating a simulation result in the comparative example. FIG. 6C is a diagram illustrating a simulation result in the comparative example. FIG. 7 is a graph showing changes in flow velocity in the first example and the comparative example. FIG. 8A is a perspective view showing a simulation result in the second example of the flow cell according to the exemplary embodiment of the present invention. FIG. 8B is a perspective view showing a simulation result in the second example of the flow cell according to the exemplary embodiment of the present invention. FIG. 8C is a perspective view showing a simulation result in the second example of the flow cell according to the exemplary embodiment of the present invention. FIG. 8D is a perspective view showing a simulation result in the second example of the flow cell according to the exemplary embodiment of the present invention. FIG. 8E is a perspective view showing a simulation result in the second example of the flow cell according to the exemplary embodiment of the present invention. FIG. 8F is a perspective view showing a simulation result in the second example of the flow cell according to the exemplary embodiment of the present invention. FIG. 8G is a perspective view showing a simulation result in the second example of the flow cell according to the exemplary embodiment of the present invention. FIG. 8H is a perspective view showing a simulation result in the second example of the flow cell according to the exemplary embodiment of the present invention. FIG. 9 is a graph showing changes in flow velocity in the second example of the flow cell according to the embodiment of the present invention.

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

<Configuration of flow cell>
As shown in FIG. 1, the flow cell according to the present embodiment includes a lower substrate 101 and an upper substrate 102 disposed on the lower substrate 101. The lower substrate 101 and the upper substrate 102 are disposed between a supply unit 103 to which the sample solution formed on the upper substrate 102 is supplied and the opposing surfaces of the lower substrate 101 and the upper substrate 102. Disposed between the flow path 104 to which one end is connected, the detection unit 105 provided in the middle of the flow path 104, and the opposing surfaces of the lower substrate 101 and the upper substrate 102, and the other end of the flow path 104 is connected. The connection region 106 and a transfer unit 108 including a plurality of through holes 107 that penetrate the upper substrate 102 and reach the connection region 106 are formed. Further, a concave portion is formed on the outer surface of the upper substrate 102 corresponding to the region where the through hole 107 is formed (the region of the connection region 106), thereby forming the discharge unit 109. In addition, the discharge part 109 does not need to be formed. In addition, in the detection unit 105 of the channel 104, when applied to SPR measurement, a metal thin film made of gold (Au) or the like is formed on the surface exposed to the channel of the lower substrate 101. Note that the metal thin film may be formed over the entire area of the lower substrate 101, for example. In this case, the metal thin film is partially exposed in the entire region of the flow path 104 and the connection region 106.

  Here, the lower substrate 101 and the upper substrate 102 are made of a material that transmits light, such as glass or acrylic resin. Thereby, SPR measurement etc. are realizable.

The supply unit 103 has a cylindrical shape (circular tube) whose upper part is formed of a recess opening from the upper substrate 102, and specifically, whose bottom part is sealed.
The flow path 104 functions as a first flow path and is a tube having a rectangular cross section, and the cross-sectional dimension thereof is set in a range in which a capillary phenomenon appears with respect to the liquid.

  The connection region 106 functions as a second flow path and is formed of a rectangular parallelepiped cavity, and the height thereof, that is, the distance between the lower substrate 101 and the upper substrate 102 in the facing direction (vertical direction) is higher than that of the flow path 104. It is set as a range in which no gap is formed in the up-down direction when the liquid enters. In other words, it is only necessary for the liquid that has entered to be in contact with the upper and lower surfaces of the connection region 106 at the same time. In the present embodiment, the width of the connection region 106 in the direction perpendicular to the direction in which the lower substrate 101 and the upper substrate 102 face each other is formed wider than the flow path 104, and the plurality of through holes 107 extend in this width direction. Are arranged, and this row is arranged in the flow channel direction perpendicular to the width direction.

  The through-hole 107 which comprises the transfer part 108 is cylindrical shape (circular pipe), for example, and connects the connection area | region 106 and the exterior of a flow cell. The hole diameter of the through-hole 107 is set to a range in which capillary action occurs with respect to the liquid. Here, the transfer unit 108 is set so that the total capillary force acting on the through-hole 107 is smaller than the capillary force acting on the flow path 104 and the capillary force acting on the connection region 106.

<Operation principle of flow cell>
Next, the operation principle of the flow cell according to the present embodiment will be described with reference to FIGS. 2A to 2F from the viewpoint of capillary force and flow path resistance.
In the following, as shown in FIG. 2A, the radius of the supply portion 103 made of a circular pipe is r _i , the width of the flow path 104 having a rectangular cross section is w, the height is h, the length is l, and the through hole 107 radius of a description as r _c. Here, it is assumed that the radius r _i of the supply unit 103 is larger than the radius r _c of the through hole 107.

First, the capillary force and the channel resistance acting on a general capillary will be described.
The pressure difference P possessed by the ellipsoids having the curvature radii r ₁ and r ₂ is expressed by the following equation (2) from the Young-Laplace equation. In the following formula (2), γ is a surface tension.

P = γ (1 / r ₁ + 1 / r ₂ ) (2)

A capillary force P _c by a capillary having a circular cross section with a radius r is expressed by the following formula (3).

P _c = − (2γ cos θ / r) (3)

A capillary force P _c by a capillary having a rectangular cross section having a width w and a height h is expressed by the following equation (4).

P _c = −γ {(cos θ _r + cos θ _l ) / w + (cos θ _t + cos θ _b ) / h}
... (4)

In the above equation (4), θ _r , θ _l , θ _t , θ _b are contact angles with the right surface, left surface, top surface, and bottom surface of the rectangular capillary tube in order. When each surface of the rectangular capillary has the same contact angle θ, the capillary force P _c is expressed by the following equation (5).

P _c = −2γ cos θ (1 / w + 1 / h) (5)

  A flow path resistance R of a capillary having a circular cross section with a radius r and a length l is expressed by the following equation (6).

R = 81 / πr ⁴ (6)

  A flow path resistance R of a capillary having a rectangular cross section having a width w, a height h, and a length l is expressed by the following expression (7).

R = 4 l / {h ² w ² F (h / w)} (7)

  Here, the function F (x) is expressed by the following equation (8).

Next, based on these, the liquid transfer in the flow cell according to the present embodiment will be described. For simplicity of explanation, the flow cells according to the present embodiment are made of the same material, and the contact angles with the liquid are all the same and θ.
When the liquid is supplied to the supply unit 103 of the flow cell, it is assumed that the radius r _i of the supply unit 103 having a circular cross section is sufficiently longer than the short side h of the flow path 104 having a rectangular cross section. 3) From the comparison of (5), since the force to draw the liquid toward the flow path 104 is stronger than the force to keep the liquid inside the supply section 103, the liquid flows into the flow path 104 from the supply section 103. That is, if the relationship of the following formula (9) is satisfied, the liquid flows into the flow path 104.

1 / r _i <1 / w + 1 / h (9)

  At this time, even when a small amount of liquid droplet is placed in the inlet of the flow channel 104 in the supply unit 103 as in the case of supplying a small amount of reference solution in the measurement of the specimen, the above equation (2) is positive pressure, Since the expression (5) means a negative pressure, the liquid also flows into the flow path 104 side. In the following, the capillary force is a negative pressure, but the magnitude comparison is performed with absolute values.

When the liquid that has flowed into the flow path 104 reaches the connection region 106 having a rectangular cross section, the capillary force that is received is reduced because the width is wider than that of the flow path 104 that also has a rectangular cross section, but the capillary tube of the supply unit 103 still remains. Since it receives a capillary force larger than the force, it proceeds in the connection region 106. And reaches the bottom of the through hole 107 in this state progresses the connection region 106, if the radius r _c of the through hole 107 satisfies a relation of the following equation (10), as shown in Figure 2B, a liquid α Is not sucked into the through-hole 107 and is filled in the connection region 106 except for the portion immediately below the through-hole 107.

1 / r _c <1 / w ₂ + 1 / h (10)

Here, w ₂ indicates the width of the liquid in the connection region 104, and changes in accordance with the liquid surface shape as it advances in the connection region 106. When the through-holes 107 are sufficiently separated from each other, w ₂ increases, so the contribution of the first term on the right side of the above equation (10) decreases. That is, as long as at least the following expression (11) is satisfied, the liquid is not sucked into the through-hole 107 and fills the connection region 106 except for the portion immediately below the through-hole 107.

1 / r _c <1 / h (11)

  Further, when the liquid is supplied to the supply unit 103, there is no liquid suction portion competing with the through hole 107, so that the portion immediately below the through hole 107 in the connection region 106 and the lower portion of the through hole 107 are filled with the liquid. In this way, the liquid having a volume substantially the same as the volume sum of the flow path 104 and the connection area 106, or the flow volume 104, the connection area 106, and the plurality of through holes 107 are equal to or larger than the volume sum of the flow path 104 and the connection area 106. When the supply of the liquid from the supply unit 103 stops after the liquid having a volume less than the sum is supplied, if the following equation (12) is satisfied, the capillary force by the transfer unit 108 including the plurality of through holes 107 is satisfied. Since the capillary force by the flow path 104 is larger than that, a meniscus is formed at the entrance of the flow path 104, and the meniscus does not move. For this reason, as shown in FIG. 2C, a state is formed in which the flow path 104 is filled with the liquid α as well as the connection region 106.

1 / r _c <1 / w + 1 / h (12)

  This state is stable and the liquid does not move. Therefore, for example, in the case of measuring a specimen, it is assumed that if a reference liquid having a refractive index equivalent to that of the specimen liquid is supplied as the liquid α as described above, the antibody activity can be restored, and evaporation does not occur. The reference liquid can be stably stored in the flow cell for an arbitrary time before the measurement of the sample liquid. This also means that it is possible to arbitrarily take the time until the activity of the antibody of the detection unit 105 provided in the flow path 104 is restored.

  Then, when the liquid β made of the sample liquid is supplied to the supply unit 103 following the liquid α made of the reference liquid, a meniscus is formed on the inner surface of the supply unit 103 instead of the meniscus at the entrance of the flow path 104. From the relationship of Expression (13), the capillary force due to the plurality of through-holes 107 becomes larger than the capillary force due to the meniscus formed in the supply unit 103. Then, as shown in FIG. 2D, the liquid is sucked through the plurality of through holes 107, and the liquid β flows through the flow path 104.

1 / r _i <1 / r _c (13)

The flow of the liquid β made of the sample liquid as described above continues until the sample liquid is exhausted from the supply unit 103 or until all the through holes 107 of the transfer unit 108 finish the suction. Thus, if the sample liquid is supplied after the antibody activity is sufficiently restored by the reference liquid, the liquid surface is not fused when the connection region 106 is filled, and a new meniscus is not formed in the through-hole 107. The flow velocity noise of the flow of the sample liquid at 105 is reduced.
Further, even in a measurement that does not require pretreatment for restoring antibody activity, a predetermined value that is greater than or equal to the volume sum of the channel 104 and the connection region 106 and less than the volume sum of the channel 104, the connection region 106, and the plurality of through holes 107 If the measurement is started from the stage where the connecting region 106 and the lower part of the through-hole 107 of the transfer unit 108 are filled, the measurement in the time region where the flow velocity decrease is small and the flow velocity noise is small. Therefore, a highly accurate measurement result can be obtained.

  On the other hand, in the flow cell that does not satisfy the above formula (10) or the above formula (11), as shown in FIG. 2E, the liquid α is sucked by some of the through holes 107 before the connection region 106 is filled. , It will be a large flow of flow velocity noise. In this case, as shown in FIG. 2F, flow velocity noise is generated even when the sample liquid β is supplied.

  The flow of the sample liquid supplied next to the reference liquid has a smaller flow rate fluctuation (lower flow rate) and smaller flow velocity noise than the above-described mechanism. The flow rate at this time can be estimated by the following formula (14).

v = (ΔP) / (ηSR) (14)

  Here, η is a viscosity coefficient, S is a cross-sectional area (wh) of the flow path 104, R is a flow path resistance (the above formula (7)), and ΔP is a pressure difference between the through hole 107 and the supply section 103 in the transfer section 108. And is represented by the following formula (15).

ΔP = 2γ cos θ (1 / r _c −1 / r _i ) (15)

  Further, the time T during which the sample liquid flows is expressed by the following formula (16).

T = (n _c πr _c ² h _c ) / (vS) (16)

<Examples and Comparative Examples>
Next, examples of the flow cell according to the present embodiment and comparative examples will be described. These are simulation results using specific numerical values. The liquid supplied to the flow cell is water (viscosity coefficient η = 1.002 × 10 ⁻³ [Pa · s], and the surface tension γ is 72.75 × 10 ^−3. [N / m], the contact angle is calculated as 60 degrees, and in each of the examples and the comparative example, as a common geometric parameter, the radius r _i of the supply unit 103 is 1.5 [mm]. , Height h _i of the supply unit 103 = 4 [mm], width w of the channel 104 = 0.6 [mm], length l of the channel 104 = 2.5 [mm], through hole in the transfer unit 108 The height h _{c of} 107 is 4 [mm], the number of through holes 107 is n _c = 15, the radius r _c of the through holes 107 is 0.1 [mm], and the through holes 107 in the transfer unit 108 are arranged in 3 rows × 5. Arranged in a row.

[First embodiment]
In the first embodiment, the height h of the flow path 104 is 0.075 [mm], and the simulation results at this time are shown in FIGS. 3A to 3F and 4A to 4C. 3A to 3F are perspective views showing the movement of the liquid at each time, and FIGS. 4A to 4C are plan views showing the movement of the liquid in the connection region 106 at each time.

  In this example, as shown in FIG. 3A, the calculation was started with time 0.0 seconds when the supply unit 103 was filled with liquid.

  First, at time 0.085 seconds shown in FIG. 3B, the liquid passes through the flow path 104 and reaches the center of the connection region 106. Further, at the time of 0.150 seconds, as shown in FIG. 3C, the portion where the liquid does not fill the connection region 106 remains in a slight state. Here, as can be seen from FIG. 4A, which is the same time as FIG. 3B, it can be seen that the fluid inside the connection region 106 flows avoiding the portion directly below the through hole 107. 4B, which is the same time as FIG. 3C, clearly shows that the fluid inside the connection region 106 flows avoiding the portion directly below the through hole 107. Further, at the time of 0.175 seconds shown in FIG. 4C, the liquid is filled over the entire connection region 106, and some through holes 107 start sucking the liquid.

  Then, suction through each through hole 107 is continued at time 0.258 seconds (FIG. 3D) and 0.490 seconds (FIG. 3E), and at time 0.630 seconds, as shown in FIG. The suction is finished.

  Here, at the time of 0.258 seconds and 0.490 seconds shown in FIG. 3D and FIG. 3F, paying attention to the through-hole 107a located in the center, the height of sucking up the liquid is higher than the time of 0.258 seconds. .490 seconds is lower. That is, the liquid level is lowered as time advances. This is because when the other through-hole 107 sucks up the liquid, the supply amount of the liquid from the flow path 104 is insufficient, so that the other through-hole 107 itself becomes a supply source of the liquid to the other through-hole 107. Thus, each through-hole 107 realizes the suction of the liquid at a constant flow rate as a whole by operating in a coordinated manner so as to make the inflow from the flow path 104 constant.

FIG. 5 is a graph showing changes in the flow velocity (center speed v ₀₀ ) at 0.0375 [mm] above the center of the flow path 104 (center in the cross section of the flow path 104, 0.5 h). Here, up to about 0.17 seconds, the liquid surface moves in a complicated manner to fill the connection region 106, so the flow velocity greatly fluctuates, and the baseline decreases greatly as the connection region 106 is advanced. Yes. However, since the cooperative suction of the through-hole 107 has started since about 0.17 seconds, the flow velocity noise is extremely small and the baseline is almost flat, that is, the flow velocity fluctuation is small and the flow velocity noise is small. It turns out that small liquid feeding is performed. Further, in the last period of liquid feeding, since a rapid speed reduction occurs, the end of liquid feeding can be easily detected.

Here, the molecule n _c πr _c ² h _c in the above formula (16) is the sum of the capillaries' inner volume, specifically, 1.9 × 10 ⁻⁹ [m ³ ]. The denominator Sv represents the volume flow velocity, which is the average flow velocity multiplied by the cross-sectional area. The relationship between the average flow velocity v and the central flow velocity v ₀₀ is expressed by the following equation (17).

In the above equation (17), F (x) is given by the above equation (8), and x is the aspect ratio h / w of the rectangular cross section. In this embodiment, the specific value on the right side is about 0.61. From the graph of FIG. 5, the central flow velocity of the liquid in the state in which the through-hole 107 sucks the liquid is about 0.14 [m / s], and the volume flow velocity is 3.8 × 10 ⁻⁹ [m ^3. / S], the suction time T of the through-hole 107 is about 0.5 seconds. This substantially coincides with the suction time by the through hole 107 read from the graph of FIG. In this way, it can be seen that accurate measurement can be performed if measurement is performed during a period of stable flow velocity after a period when the initial flow velocity fluctuation is large.

[Comparative example]
In this comparative example, the height h of the flow path 104 is set to 0.1 mm, and the other configuration of the flow cell is the same as that of the first embodiment. Simulation results in this case are shown in FIGS. 6A to 6C. 6A to 6C, the left figure is a perspective view showing the movement of the liquid at each time, and the right figure is a plan view showing the movement of the liquid in the connection region 106 at the same time.
Also in this comparative example, as in the first example described above, the calculation was started with time 0.0 seconds when the supply unit 103 was filled with liquid.

  At the time 0.050 seconds shown in FIG. 6A, the portion immediately below the through hole 107 in the connection region 106 is not filled with liquid, but at the time 0.100 seconds shown in FIG. Sucking is starting. Furthermore, at the time of 0.200 seconds shown in FIG. 6C, suction is performed in many through-holes 107 even though part of the connection region 106 is not filled.

  FIG. 7 is a time change of the flow velocity 0.05 [mm] above the detection unit 105 at the center of the channel 104 (center in the channel cross section, 0.5 h). The result of the flow cell (h = 0.075 [mm]) is also described. In the flow cell of h = 0.1 [mm] which is a comparative example, since the cooperative suction by the plurality of through holes 107 and the complicated movement of the meniscus in the connection region 106 are performed simultaneously, the flow velocity noise is large. It has become. Further, since the height h of the flow path 104 and the connection region 106 is larger than that in the first embodiment, the flow path resistance is decreased, and as a result, the flow velocity is increased.

[Second embodiment]
Similar to the first example, the second example is the same as the reference solution after restoring the antibody activity with the reference solution in the flow cell having the height h of the flow path 104 of 0.075 [mm]. It is a simulation result when the sample liquid assumed to be a physical property is supplied. This is illustrated in FIGS. 8A-8H and FIG. Here, FIG. 8A is a perspective view showing the configuration of the entire flow cell, and FIGS. 8B to 8H are perspective views showing the movement of the liquid at each time, showing only the liquid level. FIG. 9 is a graph showing changes in the flow velocity (center speed v ₀₀ ) at 0.0375 [mm] above the center of the flow path 104 (center in the cross section of the flow path 104, 0.5 h). .

  In this embodiment, the state in which the flow channel 104, the connection region 106, and the lower portion of the through-hole 107 are filled with the reference liquid is an initial state (time 0.0 seconds), and the sample liquid is detected at time 0.199 seconds. Is introduced into the supply unit 103 and the calculation is performed.

  At times 0.001 seconds and 0.198 seconds shown in FIGS. 8A to 8C, a reference liquid having a volume substantially the same as the volume sum of the flow path 104 and the connection area 106 is supplied, and the flow path 104, the connection area 106, and The lower portion of the through hole 107 is filled. At this time, a meniscus is formed in the vicinity of the connection portion between the flow path 104 and the supply unit 103, and a capillary force due to the meniscus acts on the reference liquid. A meniscus is also formed below each through-hole 107 of the transfer unit 108, and a capillary force due to the meniscus acts on the reference liquid. Among these capillary forces, the capillary force due to the meniscus formed in the vicinity of the connection portion between the flow channel 104 and the supply portion 103 is larger, and therefore the reference liquid is not transferred, so the flow channel 104, the connection region 106 and the flow cell of the flow cell The state where the lower part of the through hole 107 is filled with the reference liquid is continued, and the liquid level hardly changes.

As shown in FIG. 9, at about 0.02 seconds, a meniscus is formed at the bottom of the through hole 107 and at the entrance of the flow path 104, so that a pulsed flow velocity is generated. Once the meniscus is formed in this way, the condition of the above equation (12) is satisfied, and a stable state is obtained. That is, the detection unit 105 continues to have a flow velocity of 0. As described above, since the sample liquid is introduced into the supply unit 103 at time 0.199 seconds, the stable state continues until time 0.198 seconds immediately before that.
The introduction time of the sample liquid can be arbitrarily set. Further, during this time, the reference solution stays on the detection unit 105. Therefore, the time for restoring the antibody activity can be arbitrarily set by arbitrarily setting the introduction time of the sample solution.

  After realizing such a state, when the sample solution is supplied to the supply unit 103 next, the meniscus formed at the connection portion between the flow path 104 and the supply unit 103 disappears, and a meniscus is formed on the inner surface of the supply unit 103. Is done. However, since the capillary force due to the meniscus is small, the reference liquid and the sample liquid are sucked up in each through-hole 107 by the capillary force due to the meniscus formed in the lower part of each through-hole 107. As a result, the sample liquid supplied to the supply unit 103 passes through the flow path 104 and the connection region 106 and is transferred to the through hole 107 as shown in FIGS. 8D to 8H.

  The sample liquid supplied to the supply unit 103 at time 0.199 seconds (FIG. 8D) has a cylindrical shape as an initial shape for convenience of calculation. As a result, since a meniscus is formed on the wall surface of the supply unit 103 as described above, the upper surface of the sample liquid vibrates at a very initial stage. Due to this influence, as shown in FIG. 9, the flow velocity fluctuates from time 0.2 seconds to 0.23 seconds. Thereafter, when the vibration of the upper liquid surface of the sample liquid stops, the flow velocity noise is constantly small and the flow rate is small. The actual supply of the sample liquid is assumed to be supplied by a pipette, but the initial flow rate fluctuation time of the sample liquid due to this is considered not to be significantly different from the present embodiment. A highly accurate result can be obtained by measuring the antigen-antibody reaction while avoiding the initial flow rate fluctuation time of the sample liquid. Further, since a meniscus has already been formed in the lower part of each through-hole 107 of the transfer part 108, flow velocity noise due to this meniscus formation does not occur. Therefore, since the flow rate of the sample solution can be prevented from changing greatly, the measurement accuracy can be improved.

In the first and second embodiments, the calculation is performed with the number of through-holes 107 being 15 because of the calculation processing capability of the computer. However, even when a larger number of through-holes 107 are provided, 1, the same result as in the second embodiment can be obtained.
For example, in the first embodiment, when the number of through holes 107 is 660, the suction time through the through holes 107 is 22 seconds. At this time, if the length of the flow path 104 is set to 10 [mm], the flow path resistance increases, so that the suction time is 88 seconds, and a longer measurement can be performed. In this way, a period in which the flow velocity noise is small and the flow velocity decrease is small can be set in accordance with the measurement time and timing.

  As described above, according to the present embodiment, the absolute value of the capillary force by the plurality of through-holes 107 is smaller than the absolute value of the capillary force of the flow path 104 and the absolute value of the capillary force of the connection region 106. Since the sample solution is not transferred through the plurality of through holes 107 until the channel 104 and the connection region 106 are filled with the sample solution, each time the sample solution reaches the inlet of each through hole 107, the through hole 107 The transfer can be prevented from occurring. As a result, it is possible to prevent the flow rate fluctuation from occurring in each through-hole 107 and greatly change the flow rate of the sample solution, and as a result, the measurement accuracy can be improved.

  Further, when a reference liquid that is equal to or larger than the volume sum of the flow channel 104 and the connection region 106 and less than the sum of volumes of the flow channel 104, the connection region 106, and the plurality of through holes 107 is supplied to the supply unit 103, the flow channel 104, the connection region 106. And the lower part of each through-hole 107 of the transfer part 108 is filled with the reference liquid. At this time, a meniscus is formed in the vicinity of the connection portion between the flow path 104 and the supply unit 103, and a capillary force due to the meniscus acts on the reference liquid. A meniscus is also formed below each through-hole 107 of the transfer unit 108, and a capillary force due to the meniscus acts on the reference liquid. Among these capillary forces, the capillary force due to the meniscus formed in the vicinity of the connection portion between the flow channel 104 and the supply portion 103 is larger, and therefore the reference liquid is not transferred, so the flow channel 104, the connection region 106 and the flow cell of the flow cell The state where the lower part of the through hole 107 is filled with the reference liquid is continued, and the liquid level hardly changes. After realizing such a state, when the sample solution is supplied to the supply unit 103 next, the meniscus formed at the connection portion between the flow path 104 and the supply unit 103 disappears, and a meniscus is formed on the inner surface of the supply unit 103. Is done. However, since the capillary force due to the meniscus is small, the reference liquid and the sample liquid are sucked up in each through-hole 107 by the capillary force due to the meniscus formed in the lower part of each through-hole 107. Thereby, the sample liquid supplied to the supply unit 103 passes through the flow path 104 and the connection region 106 and is transferred to the through hole 107. At this time, since a meniscus has already been formed in the lower part of each through-hole 107 of the transfer unit 108, flow rate noise due to meniscus formation does not occur, so it is possible to prevent the flow rate of the sample solution from changing greatly, As a result, measurement accuracy can be improved.

  Moreover, since the geometric positional relationship from the flow path 104 is not the same for each through-hole 107, the start time to suck up the liquid is not simultaneous. For this reason, a situation is formed in which each capillary tube sucks up at a different liquid level. In general, the liquid level rising speed (sucking speed) due to the capillary force is higher and the higher the suctioned speed is, the smaller it is. When the through hole 107 having a high liquid level and the through hole 107 having a low liquid level are adjacent to each other, a situation in which the high liquid level is lowered may occur. For this reason, since each through-hole 107 sucks up the liquid cooperatively, it is possible to realize a state in which the liquid is fed at a substantially constant speed as the transfer unit 108.

  In addition, since the liquid is sucked up cooperatively in each through hole 107, the time at which the suction is finished in each through hole 107 is also gathered. Thereby, since the flow velocity noise generation period at the end of siphoning can be shortened, it is possible to provide a long period during which the flow velocity noise is small during measurement.

  Further, in the flow cell, it is possible to provide a capillary-driven liquid feeding mechanism and a liquid feeding method that can suppress fluctuations in the flow speed to a small level, have a constant flow speed, and have a long period of low flow velocity noise. Simplification, cost reduction, improved measurement accuracy, and higher sensitivity can be realized. In particular, in the immunoassay, since the reference solution can be stored for a certain period of time before the measurement of the sample solution, the time for restoring the antibody activity can be sufficiently long. Furthermore, in an immunoassay using an SPR surface plasmon resonance (SPR) device, an antigen-antibody reaction can be accurately detected by using a reference solution having a refractive index close to that of a sample solution.

  The present invention can be applied to, for example, a liquid feeding mechanism that is used in a flow analysis, a chemical sensor, a biosensor, and the like and that realizes a flow with a constant flow velocity and small flow velocity noise without requiring an external driving force.

  DESCRIPTION OF SYMBOLS 101 ... Lower board | substrate, 102 ... Upper board | substrate, 103 ... Supply part, 104 ... Flow path, 105 ... Detection part, 106 ... Connection area | region, 107, 107a ... Through-hole, 108 ... Transfer part, 109 ... Discharge part.

Claims (3)

A supply unit to which a sample solution is supplied;
A first flow path having one end connected to the supply unit and transferring the sample solution supplied to the supply unit by capillary force;
A detector provided in the middle of the first flow path;
A second channel connected to the other end of the first channel and transferring the sample solution transferred from the first channel by capillary force;
A flow cell having one end connected to the second flow path, and a plurality of tubes for transferring the sample solution in the second flow path by capillary force,
The absolute value of the capillary force by the plurality of tubes is smaller than the absolute value of the capillary force of the first channel and the absolute value of the capillary force of the second channel. Further comprising a substrate,
The supply unit includes a recess formed on the upper surface of the substrate,
The first flow path comprises a tube formed inside the substrate,
The second flow path consists of a cavity formed inside the substrate,
2. The flow cell according to claim 1, wherein the plurality of tubes include holes that communicate the upper surface of the substrate and the cavity. A supply section to which a sample solution is supplied, a first flow path having one end connected to the supply section, for transferring the sample solution supplied to the supply section by capillary force, and a middle of the first flow path A second flow path for transferring the sample solution transferred from the first flow path by capillary force, and a second flow path connected to the other end of the first flow path. One end of which is connected to the second flow path, and a plurality of tubes for transferring the sample solution in the second flow path by a capillary force. The absolute value of the capillary force by the plurality of tubes is the first flow rate. A flow cell feeding method that is smaller than the absolute value of the capillary force of the path and the absolute value of the capillary force of the second flow path,
Supplying the first liquid and the second liquid to the supply unit in order,
The volume of the first solution is equal to or greater than the volume sum of the first flow path and the second flow path and less than the volume sum of the first flow path, the second flow path, and the plurality of tubes. A flow cell feeding method characterized by the above.