JP6228088B2 - Blood coagulation test chip - Google Patents

Description

  The present invention relates to a blood coagulation test chip used for measuring blood coagulation ability.

  Coagulation activity is an important item in clinical examinations, and prothrombin time (blood clotting time: PT), which is one of the indicators of clotting activity, is considered to be highly sensitive to exogenous clotting factors. It is an index used for screening for deficiency of exogenous coagulation factors, abnormal liver function, and monitoring of oral anticoagulant therapy.

  Conventionally, methods such as a stirring resistance method, a light scattering method, a heat conduction method, and a crystal resonator method have been invented for measuring the blood coagulation time. Generally, the stirring resistance method and the light scattering method are often used. (For example, refer nonpatent literature 1).

  The stirring resistance method is a method in which a sample is introduced together with an activator and stirred with fins, and the blood coagulation time is measured from the increase in stirring resistance.

  In the light scattering method, a reagent containing a component that promotes clotting activation is mixed in plasma in a test container, light is incident on the container, and the change in the amount of scattered light is measured to measure the blood clotting time. Is the method. As a method of obtaining the blood coagulation time from the scattered light amount, there are a method of using the scattered light amount as it is, a method of using a differential value of the scattered light amount, and a method of obtaining a time until the scattered light amount reaches a certain value.

  Recently, a method for measuring the coagulation ability of blood using a refractive index change has been proposed. In this method of measuring the coagulation ability of blood using this change in refractive index, the flow rate is calculated from the change in refractive index at different points in the microchannel, the time, and the distance traveled by the solution, and the flow rate is the amount of plasma clotting activity. The blood coagulation is inspected by utilizing the change due to the difference (see, for example, Patent Documents 1 to 3).

  In addition, conventional blood coagulation tests have been performed by capturing physical and optical changes caused by fibrin, which is the final product of the blood coagulation cascade. It has been reported that there is a correlation between the rate and the coagulation ability of blood (for example, Non-Patent Documents 1 and 2).

JP 2011-232137 A JP 2013-053959 A JP 2013-053960 A

Inoue et al., “SUB-SECOND DETERMINATION OF BIOGENIC PROTEIN POLYMERIZATION ACTIVITY USING FLOW INDUCED REFRACTIVE INDEX“ VALLEY ””, Micro TAS 2013, pp.230-232. Hayashi et al., `` POLYMERIZATION OF BIOLOGICAL MOLECULES IN A MICROCHANNEL GENERATES BOTH HIGH AND LOW-REFRACTIVE INDEX INGREDIENTS '', Micro TAS 2013, pp.898-900.

  In the method of measuring the coagulation ability of blood using refractive index change, the flow rate is calculated from the refractive index change at different points in the microchannel, the time, and the distance traveled by the solution, and the flow rate is the difference in plasma clotting activity. The blood coagulation is examined by utilizing the change due to the above. In this case, a blood coagulation test chip having a microchannel is used, and a change in refractive index is observed by a surface plasmon resonance (SPR) measurement method while flowing plasma and a coagulation reagent (coagulation activator) through the channel.

  In a conventional blood coagulation test chip, a coagulation reagent is first flowed as a coagulation activation solution in the flow path, then plasma is flowed as a solution to be tested, and the plasma and the coagulation reagent are contacted in series in the flow path. The surface where the coagulation reagent and the coagulation reagent contacted was used as an interface, and a coagulation reaction was caused at this interface. And the change of the refractive index which arises in the vicinity of the interface was observed by the SPR measuring method.

  When observing a change in the refractive index by the SPR measurement method, a substrate made of glass or the like in which the entire bottom surface of the channel is made of a gold thin film is generally used. ), The sensing part is contaminated by the contact of plasma or fibrin, which is a product of a coagulation reaction, and the sensitivity changes. In addition, since a solidification reaction occurs in the cross-sectional direction of the flow path, fibrin is formed in the cross-sectional direction of the flow path, and there is a problem that the flow path is easily contaminated and clogged. For these reasons, the conventional blood coagulation test chip has a problem in that the measurement accuracy decreases as the measurement of blood coagulation ability is repeated.

  The present invention has been made to solve such a problem, and the object of the present invention is to provide a blood coagulation test chip capable of suppressing contamination in the sensing portion and the flow path and suppressing a decrease in measurement accuracy. Is to provide.

In order to achieve such an object, the present invention is introduced from the first introduction port into which the solution to be inspected is introduced, the second introduction port into which the coagulation activating solution is introduced, and the first introduction port. A first flow path through which the solution to be tested flows, a second flow path through which the coagulation activation solution introduced from the second introduction port flows, and a solution to be tested and a second flow through the first flow path A coagulation activating solution flowing through the channel, and a third channel that flows in contact with the sample solution to be tested and the coagulation activating solution that are in contact with each other along the flow direction; A solution and a lead-out port through which the coagulation activation solution is led out, and the solution to be inspected introduced from the first introduction port and the coagulation activation solution introduced from the second introduction port are sucked from the lead-out port As a result, the solution to be inspected and the coagulation activating solution that flowed into the third flow path and flowed into the third flow path A contact is made at the center of the third flow path, and the contacted surface serves as an interface. While causing a coagulation reaction at this interface, the third flow path flows toward the outlet, and the bottom surface of the third flow path is A region in which a thin gold film is formed as a region for observing a change in refractive index generated near the interface, and at least a region other than a region for observing a change in refractive index on the bottom surface of the first channel and the bottom surface of the third channel In addition, a titanium oxide thin film is formed as a thin film of a material that hardly adsorbs biomolecules .

In the present invention having such a feature, the solution to be inspected introduced from the first inlet and the coagulation activation solution introduced from the second inlet are sucked from the outlet to the third flow path. When infused, the solution to be inspected and the coagulation activating solution that have flowed into the third flow path come into contact with each other at the center of the third flow path , and the contacted surface serves as an interface while causing a coagulation reaction at this interface. Then, it flows through the third channel toward the outlet.

  In the present invention, the product resulting from the coagulation reaction generated at the interface between the solution to be inspected and the coagulation activating solution is generated and flows only at the center of the third flow path. As a result, the region where the change in refractive index observed near the interface between the solution to be inspected and the coagulation activation solution (sensing part) is touched only by a part of the product due to the coagulation reaction, and the sensing part is contaminated. Is suppressed. Further, contamination of the inner wall of the flow path is also suppressed. In addition, if the sensing portion is formed on the surface of the bottom surface of the third flow path where the coagulation activating solution flows, the sensing solution does not touch the sensing portion.

In the present invention, a region for observing a change in refractive index that occurs near the interface is formed in a part of the bottom surface of the third channel, and at least the bottom surface of the first channel and the bottom surface of the third channel are refracted. A thin film of a material that is difficult for biomolecules to adsorb is formed in a region other than the region where the rate change is observed . This reduces the amount of biomolecules and coagulum adsorbed on the bottom surface of the flow path, reduces the thickness of biomolecules and coagulum stacked on the bottom surface of the flow path, Contamination can be further suppressed. Examples of materials that are difficult to adsorb biomolecules include titanium, titanium alloys, and cobalt-chromium alloys . In the present invention, a region in which a change in refractive index is observed is a gold thin film, and a region in which a change in refractive index is observed. A titanium oxide thin film is formed in the other region.

Further, in the present invention may be modified polymer having high biocompatibility that can suppress the adsorption of fibrinogen or the like is formed on a region to observe the change of refractive Oriritsu.

  According to the present invention, the solution to be inspected is introduced from the first inlet and flows into the first channel, the coagulation activating solution is introduced from the second inlet and flows into the second channel, The solution to be tested flowing through the first channel and the coagulation activating solution flowing through the second channel are caused to flow into the third channel, and the solution to be tested and the coagulation activating solution are passed through the third channel. Since it is made to flow while contacting along the flow direction, a product due to a coagulation reaction occurring at the interface between the solution to be inspected and the coagulation activating solution is generated only at the center portion of the third flow path, and the sensing portion In addition, it is possible to suppress contamination in the flow path and to suppress a decrease in measurement accuracy.

It is a figure explaining the principle of this invention. It is a figure which shows a mode that plasma and the coagulation reagent flow in contact with the center part of the 3rd flow path in the principle of this invention. It is a figure which shows the flow-path board | substrate which comprises the blood coagulation test | inspection chip | tip of embodiment. It is a figure which shows the board | substrate for a measurement which comprises the blood coagulation test | inspection chip | tip of embodiment. It is a sectional side view which shows the state which combined the flow-path board | substrate and the measurement board | substrate. It is a figure which shows a mode that plasma and the coagulation reagent flow in contact with the center part of the 3rd flow path in the blood coagulation test chip | tip of embodiment. It is a figure which shows the refractive index of a certain time when plasma and a coagulation reagent are introduce | transduced in the flow path of the blood coagulation test chip | tip of embodiment.

[Principle of the Invention]
In the present invention, the blood coagulation test chip includes a first introduction port through which a solution to be examined is introduced, a second introduction port through which a coagulation activation solution is introduced, and a test sample introduced from the first introduction port. A first flow path through which the solution flows, a second flow path through which the coagulation activation solution introduced from the second introduction port flows, and a solution to be tested and a second flow path through the first flow path. The coagulation activating solution flows in, the inflowed test solution and the coagulation activating solution flow in contact with each other along the flow direction, the test solution flowing in the third channel and the coagulation. It is set as the structure provided with the outlet port from which activation solution is derived | led-out.

In the present invention having such a configuration, the solution to be inspected introduced from the first inlet and the coagulation activation solution introduced from the second inlet are sucked into the third flow path by being sucked from the outlet. The solution to be inspected and the coagulation activating solution that have flowed into the third channel are brought into contact with each other at the center of the third channel, and the contacted surface serves as an interface, while causing a coagulation reaction at this interface. The solution to be inspected and the coagulation activating solution are allowed to flow through the third channel toward the outlet.

  For example, as shown in FIG. 1, the chip 1 is provided with a first inlet 2, a second inlet 3, and an outlet 4, and the first inlet 2, the second inlet 3, and the outlet 4. Are connected by a Y-shaped flow path (Y-shaped flow path) 5, and a flow path 5-1 communicating with the first introduction port 2 is communicated with the first flow path and the second introduction port 3. 5-2 is a second flow path, and a flow path 5-3 communicating with the outlet 4 is a third flow path. Then, the outlet 4 and the suction pump 6 are connected by a tube 7, and the solution to be inspected from the first inlet 2 using the pipette 8 with the suction pump 6 sucking the flow path 5. The plasma 10 is introduced as follows, and the coagulation reagent 11 is introduced as a coagulation activation solution from the second inlet 3 using the pipette 9.

Then, the plasma 10 introduced from the first inlet 2 flows through the first flow path 5-1, and the coagulation reagent 11 introduced from the second inlet 3 flows through the second flow path 5-2. , Flows into the third flow path 5-3. Then, the plasma 10 and the coagulation reagent 11 that have flowed into the third flow path 5-3 come into contact with each other at the center of the third flow path 5-3 , and the contacted surface serves as an interface 12 (see FIG. 2) . It flows toward the outlet 4. That is, the plasma 10 and the coagulation reagent 11 flow toward the outlet 4 while contacting the inside of the third flow path 5-3 along the flow direction.

  In this case, since the plasma 10 and the coagulation reagent 11 are in contact with each other at the interface 12, a coagulation reaction occurs at the interface 12. Therefore, a region for observing a change in the refractive index generated near the interface 12 is formed as the sensing portion 13 on the bottom surface of the third flow path 5-3, and the change in the refractive index observed in the sensing portion 13 is determined. Based on this, the coagulation ability of plasma 10 is determined. That is, the clotting ability of plasma 10 is high when the change in refractive index is large. Based on this change in refractive index, the coagulation ability of plasma 10 is determined as the coagulation ability of blood.

  For example, a surface plasmon resonance (SPR) measuring device is used as a detector for detecting a change in refractive index. The SPR measurement device is composed of a light source such as an LED, a polarizing plate, a condenser lens, and a CCD camera.

Embodiment
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
FIG. 3 is a view showing a flow path substrate constituting the blood coagulation test chip of the present embodiment, FIG. 3 (a) is a plan view, and FIG. 3 (b) is a cross-sectional view taken along line AA in FIG. 3 (a). FIG.

  The flow path substrate 201 is made of polydimethylsiloxane (PDMS), and has a Y-shaped flow path (Y-shaped flow) connecting the first inlet 202, the second inlet 203, and the outlet 204. A channel 205 is formed on the lower surface of the channel substrate 201.

  The flow path 205 has a height h of 50 μm and a width w of 0.5 mm. Further, the first introduction port 202, the second introduction port 203, and the outlet port 204 are opened on the upper surface of the flow path substrate 201.

  FIG. 4 is a measurement substrate constituting the blood coagulation test chip of the present embodiment, FIG. 4 (a) is a plan view, and FIG. 4 (b) is a cross-sectional view taken along line BB in FIG. 4 (a). .

  The material of the measurement substrate 206 is glass (BK7). On the upper surface of the measurement substrate 206, a metal thin film 209 is formed at a position corresponding to the flow path 205 formed in the flow path substrate 201, the planar shape of which is the same as the planar shape of the flow path 205. The metal thin film 209 is composed of a gold thin film 207 and a titanium oxide thin film 208.

  The gold thin film 207 serves as an area (sensing portion (SPR observation area)) for observing a change in refractive index that occurs near the interface between the plasma and the coagulation reagent, in an area corresponding to the planar shape of the third channel 205-3. The titanium oxide thin film 208 is formed in a part (on the side where the coagulation reagent flows), and the titanium oxide thin film 208 is formed in all regions except the SPR observation region corresponding to the planar shape of the flow path 205.

  FIG. 5 is a side sectional view showing a state in which the flow path substrate 201 and the measurement substrate 206 are combined. The blood coagulation test chip 200 of the present embodiment is obtained by treating the flow path substrate 201 shown in FIG. 3 and the measurement substrate 206 shown in FIG. 4 with oxygen plasma using a reactive ion etching apparatus. The channel 205 is manufactured by bonding so that the planar shape of the path 205 and the planar shape of the metal thin film 209 match.

  The blood coagulation test chip 200 is used as a main part of the blood coagulation test apparatus, and is installed in the SPR measurement apparatus. A tube having an inner diameter of 1 mm is connected to the outlet port 204, and a suction pump is connected to this tube. In addition, a dispensing device for dispensing plasma or a coagulation reagent to the blood coagulation test chip 200 is provided. The configuration of the blood coagulation test apparatus using the blood coagulation test chip 200 is not shown.

[Blood coagulation test]
When a blood coagulation test is performed using the blood coagulation test chip 200, the suction pump is operated, and then the plasma is supplied to the first inlet 202 using the pipette capable of simultaneously dropping two kinds of solutions. A coagulation reagent is simultaneously dropped into the introduction port 203.

Then, as shown in FIG. 6, the plasma introduced from the first introduction port 202 flows through the first flow path 205-1 and the coagulation reagent introduced from the second introduction port 203 passes through the second flow path. It flows through 205-2 and flows into the third flow path 205-3. The coagulation reagent and plasma that have flowed into the third flow path 205-3 come into contact with each other at the center of the third flow path 205-3 , and the contacted surface serves as an interface, which causes a coagulation reaction. However, it flows toward the outlet 204.

  In FIG. 6, 301 is plasma and 302 is a coagulation reagent. Reference numeral 303 denotes an interface between plasma and a coagulation reagent. Reference numeral 304 denotes a line for observing SPR, and the blood coagulation test chip 200 is mounted such that the line 304 for observing SPR straddles the third flow path 205-3.

  FIG. 7 shows a refractive index for a certain time when plasma and a coagulation reagent are introduced into the flow path 205 of the blood coagulation test chip 200. The horizontal axis in FIG. 7 indicates the SPR observation point, and the vertical axis indicates the refractive index change at each SPR observation point.

The position where the SPR observation point is “0” is a position where an interface between plasma and a coagulation reagent is formed, that is, a position where a coagulation reaction occurs. In FIG. 7, n1 is the refractive index of the low refractive index component generated by the coagulation reaction, and n2 is the refractive index of the coagulation reagent. The refractive index of n1 was obtained when plasma with a clotting activity rate of 86% was used. In plasma with a clotting activity rate of 43%, a refractive index of n3 is obtained. Δn ₁₂ and Δn ₃₂ are obtained by taking the difference from the refractive indexes of n1 and n3 based on the refractive index n2 of the coagulation reagent.

  Thus, when the coagulation activity rate is halved, the refractive index difference is also halved, and this difference changes depending on the height of the coagulation activity rate. Blood coagulation ability can be measured using the difference between the refractive index n1 of the low refractive index component and the refractive index n2 of the coagulation reagent as an index. Moreover, since it is confirmed that the refractive index of plasma is higher than that of the low refractive index component, it can be confirmed that the coagulation reagent does not flow in the SPR observation region. Furthermore, when this measurement was repeated, no change was observed in the refractive index n2 of the coagulation reagent and the refractive index n1 of the low refractive index component. This means that the surface of the SPR observation region (gold thin film 207) is not contaminated.

  Contamination on the surface of the gold thin film 207 is considered to be due to adsorption of components contained in plasma and adhesion of coagulum, but plasma flows over the SPR observation region where the gold thin film 207 is formed as in this embodiment. By adopting such a liquid feeding method, contamination of the gold thin film 207 can be suppressed and blood coagulation ability can be repeatedly measured.

  Instead of forming the titanium oxide thin film 208 in the region excluding the gold thin film 207 on the bottom surface of the channel 205, a highly biocompatible polymer capable of suppressing adsorption of fibrinogen or the like is modified on the gold thin film 207. It may be.

  In addition, it is conceivable that the titanium oxide thin film 208 is not formed in the region excluding the gold thin film 207 on the bottom surface of the flow path 205 but is left as a glass substrate, but the titanium oxide thin film 208 is formed in the region excluding the gold thin film 205. By making the biomolecules difficult to adsorb positively, a greater effect can be obtained.

  In the above-described embodiment, the test solution is plasma and the blood coagulation ability is measured as blood coagulation ability. However, the test solution is not limited to plasma.

[Extension of the embodiment]
The present invention has been described above with reference to the embodiment. However, the present invention is not limited to the above embodiment. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the technical idea of the present invention.

  DESCRIPTION OF SYMBOLS 1 ... Chip, 2 ... 1st inlet, 3 ... 2nd inlet, 4 ... Outlet, 5 ... Channel, 5-1 ... 1st channel, 5-2 ... 2nd channel, 5-3 ... 3rd flow path, 10 ... Plasma, 11 ... Coagulation reagent, 12 ... Interface, 13 ... Sensing part, 200 ... Blood coagulation test chip, 201 ... Flow path substrate, 202 ... 1st introduction port, 203 ... second inlet, 204 ... outlet, 205 ... channel, 205-1 ... first channel, 205-2 ... second channel, 205-3 ... third channel, 206 ... measurement 207 ... Gold thin film (sensing part (SPR observation region)), 208 ... Titanium oxide thin film, 209 ... Metal thin film, 301 ... Plasma, 302 ... Coagulation reagent, 303 ... Interface.

Claims (3)

A first inlet through which a solution to be inspected is introduced;
A second inlet through which the coagulation activating solution is introduced;
A first flow path through which the solution to be inspected introduced from the first introduction port flows;
A second flow path through which the coagulation activation solution introduced from the second introduction port flows;
The test solution flowing through the first channel and the coagulation activating solution flowing through the second channel flow in, and the inspected solution and the coagulation activating solution flowing in contact with each other along the flow direction. A third flow channel flowing;
A solution to be inspected flowing through the third flow path and a lead-out port through which the coagulation activating solution is led out ,
The solution to be inspected introduced from the first inlet and the coagulation activation solution introduced from the second inlet flow into the third flow path by being sucked from the outlet.
The solution to be inspected and the coagulation activating solution that have flowed into the third flow path are in contact with each other at the center of the third flow path, and the contacted surface serves as an interface. Flowing through the third channel toward the outlet,
A gold thin film is formed on a part of the bottom surface of the third flow path as a region for observing a change in refractive index generated near the interface,
A titanium oxide thin film is formed as a thin film of a material that hardly absorbs biomolecules at least in a region other than the region where the change in refractive index is observed on the bottom surface of the first channel and the bottom surface of the third channel. blood coagulation test chip, characterized in that there. The blood coagulation test chip according to claim 1,
A blood coagulation test chip characterized in that a polymer having high biocompatibility capable of suppressing the adsorption of biomolecules is modified on the region where the change in refractive index is observed . In the blood coagulation test chip according to claim 1 or 2 ,
The blood coagulation test chip is characterized in that the region for observing the change in refractive index is formed on a surface of the bottom surface of the third flow channel on the side where the coagulation activation solution flows .

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