JP5116864B2 - Micro analysis chip - Google Patents

Description

  The present invention relates to a micro-analysis chip used for trace chemical analysis of biological substances and substances in the natural environment. More specifically, the present invention can smoothly transfer a plurality of test solutions having different viscosities and interfacial tensions. The present invention relates to a micro analysis chip that can be used.

  Immunoassays such as ELISA (enzyme immunoassay), CLEIA (chemiluminescence enzyme immunoassay), RIA (radioimmunoassay), and LTIA (latex immunoturbidimetric assay) are widely used as protein measurement methods. .

  The immunoassay is known as an important analysis / measurement method in the medical field, biochemical field, measurement field such as allergen and the like. However, the conventional immunoassay has a problem that the operation is complicated and it takes more than a day for the analysis.

  Under such circumstances, a micro analysis chip has been proposed in which a micro-order channel is formed on a substrate and an antibody or the like is immobilized in the micro-channel.

  When performing analysis using a microanalysis chip, a solution is introduced into the detection part or reaction part of the microanalysis chip from the introduction port or introduction channel, the solution is reacted in the microanalysis chip, and the discharge port or discharge is performed. It is necessary to perform a series of steps of discharging the solution from the flow path to the outside of the micro analysis chip. Conventionally, the transfer of the solution in the microanalysis chip has been performed using an external power source such as a pump or a valve. However, since the pump and the valve are larger than the micro analysis chip, there is a problem that it is difficult to reduce the size of the entire apparatus. A method of arranging relatively small micropumps and microvalves inside and outside the microanalysis chip has also been proposed, but this method requires a complicated microfabrication technique and thus lacks practicality.

  On the other hand, as a simple method for transferring a solution in a chip, a technique using the capillary force of a hydrophilic flow path has been proposed (see, for example, Patent Document 1). FIG. 12 shows an example of a micro analysis chip using a capillary force. In such a microanalysis chip, when a solution is dropped at the inlet 401, the solution moves through the flow path 402 by capillary force, and the solution can be discharged without requiring an external force such as a pump. As a method for discharging the solution filled in the flow path, for example, as shown in Patent Document 4, there is a method in which an absorber that absorbs the solution is installed at the outlet, and the absorber is sucked to discharge the solution. Proposed.

  As a simple solution transfer switching (solution flow switching) method, Patent Document 1 proposes a micro valve (electro-wetting valve) using electro-wetting. FIG. 13 shows an example of a micro analysis chip using an electrowetting valve. An electrowetting valve including a working electrode 405 and a reference electrode 406 is provided in the flow path 402 of the micro analysis chip. The surface of the working electrode 405 is hydrophobic when no voltage is applied, and becomes hydrophilic when a voltage is applied. For this reason, it is possible to switch between stopping and moving the solution (opening and closing the solution flow) by applying a voltage.

  FIG. 14 shows the operating principle of the electrowetting valve. FIG. 14A shows a state in which no voltage is applied, and FIG. 14B shows a state in which a voltage is applied between the working electrode 405 and the reference electrode 406. In the state where no voltage is applied, the hydrophobic film 407 is formed on the surface of the working electrode 405, so that the solution 408 that has moved in the flow path by capillary force stops when it reaches the working electrode 405. To do. By applying a voltage, the surface of the working electrode 405 is hydrophilized by the effect of electrowetting, and the stopped solution 408 passes through the working electrode 405 and moves in the flow path.

  Incidentally, as one of the applications of the micro analysis chip, there is a determination chip for metabolic syndrome.

  Metabolic syndrome refers to a disease state in which the accumulation of fat in the viscera weakens the effect of insulin, which is a hormone that lowers blood sugar, resulting in abnormal sugar and lipid metabolism. In metabolic syndrome, visceral fat obesity, hypertension, diabetes, and hyperlipidemia, which are risk factors for arteriosclerotic diseases, often occur in duplicate, and the risk of arteriosclerotic disease is due to duplication. It rises further.

  As diagnostic criteria for metabolic syndrome, for example, in Japan, in 2005, the diagnostic criteria were presented by the Japanese Society of Internal Medicine. According to the diagnostic criteria, the waist is not less than a prescribed value (85 cm for men and 90 cm for women), and (a) the maximum blood pressure is 130 mmHg or more, the minimum blood pressure is 85 mmHg or more, (b) the fasting blood glucose level Is diagnosed as metabolic syndrome when two or more of the three items are 110 mg / dL or more, (c) neutral fat is 150 mg / dL or more, or HDL-cholesterol is less than 40 mg / dL.

  The above diagnostic criteria are only for diagnosing metabolic syndrome after onset. However, metabolic syndrome is a disease that can be prevented from occurring by improving lifestyle habits. Therefore, it is extremely important to detect metabolic syndrome early or in advance, early treatment and improvement of life, and development of a method for easily predicting and predicting the onset and progression of metabolic syndrome is strongly desired.

  Under such circumstances, as a method for determining metabolic syndrome, a method for detecting a specific protein such as adiponectin involved in the development of metabolic syndrome has been proposed (Patent Document 2). Adiponectin is an insulin-sensitive hormone that is secreted specifically from adipocytes, and is present in blood in a relatively high concentration (5 to 10 μg / mL). Adiponectin is an adipocyte-specific secreted protein, but has a significantly low blood concentration in obese individuals. For example, adiponectin concentration is decreased in coronary artery disease and type 2 diabetes. Adiponectin can be regarded as a molecule involved in both insulin resistance and arteriosclerosis.

  When measuring the amount of adiponectin in blood by immunoassay, it is necessary to perform pretreatment such as removal (separation) of red blood cells, monomerization (decomposition) of multimers, dilution, etc. on the blood sample. As a separation method, a method such as centrifugation or separation using a filter is used. As a decomposition method, methods such as boiling treatment, decomposition by the action of a reducing agent, a surfactant, a protease and the like are used (for example, see Patent Document 3).

JP 2006-220606 A WO2005 / 038457 WO2005 / 038458 JP 2000-297761 A JP 2003-149252 A

  If a specific protein such as adiponectin involved in the development of metabolic syndrome can be detected using a micro-analysis chip, it becomes possible to diagnose metabolic syndrome and determine its risk level easily in a short time.

  However, the conventional immunoassay as described above is complicated and requires a lot of time. Therefore, when a protein contained in blood such as adiponectin is measured by an immunoassay method such as ELISA using a microanalysis chip, an enzyme-labeled antibody is added to the collected and pretreated blood sample. It is necessary to send a plurality of liquids having different viscosities and interfacial tensions such as a solution, a cleaning liquid, and a substrate solution. Therefore, a liquid feeding control technique for these liquids is required.

  In particular, in a micro-analysis chip using a capillary force as a driving force, when a plurality of liquids having different viscosities and interfacial tensions are fed, it is difficult to control the liquid feeding because the capillary force acting on each liquid is different. In addition, when a microvalve using electrowetting is used for each of a plurality of liquids, it is difficult to sequentially control a plurality of liquids having different viscosities and interfacial tensions because the valves are mutually affected.

  The present invention has been made to solve the above-described problems, and provides a microanalysis chip capable of sequentially feeding a plurality of liquids having different viscosities and interfacial tensions, and thereby providing a microanalysis chip in blood. It aims at measuring a specific protein simply and accurately.

The present invention for solving the above problems includes a main flow path including a detection unit and / or a reaction unit,
A first introduction channel and a second introduction channel for introducing a liquid into the main channel from the upstream side of any of the detection unit and the reaction unit, and the first introduction channel and the second introduction flow A first valve and a second valve provided on each of the passages for opening and closing the liquid flow; an air hole connected to the upstream side of the main flow path; and preventing the liquid from flowing into the air hole. A micro-analysis chip that includes an inflow prevention unit and a discharge unit connected to the downstream side of the main flow path and performs liquid feeding using a capillary force as a driving force.

  In this configuration, an air hole is connected to the upstream side of the main flow path, and liquid feeding using a capillary force can be stabilized by the air hole. In addition, by providing the inflow prevention portion, it is possible to prevent the liquid from flowing into the air holes and to reliably perform the function of the air holes.

  The said structure WHEREIN: The said main flow path and the said air hole can be set as the structure connected through the air hole path. In addition, the inflow prevention portion may be formed of a hydrophobic portion and provided near the boundary between the air hole passage and the main flow passage.

  In the above configuration, the main channel groove, the first introduction channel groove, the second introduction channel groove, and the third introduction channel groove are formed. One substrate and a second substrate that covers the first substrate may be overlaid.

  By adopting this configuration, the configuration of the microanalysis chip can be simplified.

  In the above configuration, if each of the first valve and the second valve is an electrowetting valve, the microchip can be reduced in size.

  The electrowetting valve preferably has at least a working electrode and a reference electrode, and may further include a counter electrode. As a working electrode of such a valve, a configuration composed of a metal thin film or a configuration composed of a metal thin film and a thin film provided thereon can be adopted.

  The thin film on the metal thin film preferably has a thickness of 100 nm or less in order to operate well. Moreover, it is preferable that the contact angle of the said thin film with respect to the pure water whose specific resistance is 18 Mohm * cm at 25 degreeC is 80 degree | times or more. Moreover, it is preferable that a thin film consists of a substance containing a fluorine-containing substance or a thiol group.

  In the introduction flow path including the electrowetting valve, the downstream groove width is preferably larger than the upstream groove width so that the stopped liquid can easily flow into the main flow path.

  The operating potential of each electrowetting valve is preferably 3 V or less.

  The said structure WHEREIN: It is preferable to set it as the structure by which the said main flow path and the said discharge part are connected via the discharge flow path.

  Further, when an absorber is connected to the downstream end of the discharge channel, a stable capillary force can be obtained by the absorber. Further, since the absorber absorbs the liquid, the liquid does not pollute the external environment.

  If the discharge channel is provided with a valve for opening and closing the liquid flow, the liquid can be stopped in the main channel located on the upstream side thereof, so that detection and reaction efficiency can be improved.

  The microanalysis chip of the present invention is suitable for detecting proteins, particularly adiponectin.

  As described above, according to the present invention, it is possible to stably control a plurality of liquids having different characteristics in a micro analysis chip without requiring external power, and measurement of a specific protein can be performed. It is possible to realize a micro analysis chip that can be simply and highly accurately performed.

FIG. 1 is a plan view and a cross-sectional view of the microanalysis chip according to the first embodiment. FIG. 2 is a structural diagram of the first substrate and the second substrate of the micro-analysis chip according to the first embodiment. FIG. 3 is a plan view of the microanalysis chip according to the first embodiment. FIG. 4 is a structural diagram of the first substrate and the second substrate of the microanalysis chip according to the first embodiment. FIG. 5 shows the measurement results of the adiponectin concentration by the microanalysis chip according to Example 1. FIG. 6 is a structural diagram of the introduction flow path of the working electrode portion of the micro analysis chip according to the third example. FIG. 7 is a plan view of the microanalysis chip according to the second embodiment. FIG. 8 is a plan view and a cross-sectional view of the pretreatment unit of the micro analysis chip according to the second embodiment. FIG. 9 is a plan view of the microanalysis chip according to the fourth embodiment. FIG. 10 is a plan view and a cross-sectional view of the pretreatment unit of the microanalysis chip according to the fourth embodiment. FIG. 11 is an operation explanatory diagram of the preprocessing unit of the microanalysis chip according to the fourth embodiment. FIG. 12 shows a conventional example of a micro analysis chip using capillary force. FIG. 13 is a conventional example of a micro analysis chip using an electrowetting valve. FIG. 14 shows the operating principle of the electrowetting valve. (A) is a state where no voltage is applied, and (b) is a state where a voltage is applied between the working electrode and the reference electrode.

  EMBODIMENT OF THE INVENTION Below, the form for implementing this invention is demonstrated in detail using drawing.

(Embodiment 1)
As shown in FIG. 1A, the microanalysis chip according to the present embodiment includes a main flow path 14 including a detection unit 13, and open holes 1, 2, and 3 for introducing liquid into the chip. First to third introduction channels 4, 5, 6 that introduce liquid into the main channel 14 from the upstream side of the detection unit 13, a discharge channel 9 provided on the downstream side of the main channel, and a discharge A discharge portion 7 provided at the end of the flow path 9 and an air hole path 12 connected to the air hole 11 are provided. The first to third introduction flow paths 4, 5, and 6 are provided with first to third valves that open and close the liquid flow, respectively.

  FIG. 1B shows an XY cross-sectional view of FIG. The micro-analysis chip according to the present embodiment is configured by bonding the first substrate 15 in which the grooves for the respective channels are formed and the second substrate 16 that covers the first substrate.

FIG. 2 shows the structure of the first substrate 15 and the second substrate 16 of the microanalysis chip according to the present embodiment.
As shown in FIG. 2A, the first substrate 15 includes a groove for the main flow path 14, a groove for the first to third introduction flow paths 4, 5, and 6, a groove for the discharge flow path 9, A hole for the discharge part 7, a groove for the air hole path 12, a through hole for the air hole 11, and a through hole for the open holes 1 to 3 are formed.

  Here, the groove widths of the first to third introduction channels 4 to 6, the discharge channel 9, the air hole channel 12, and the main channel 14 do not change in width in the flow direction. Moreover, the groove width of the main flow path 14 is the largest, and the groove width of the 1st introduction flow path 4 is the widest among the 1st-3rd introduction flow paths 4-6. Further, the groove width of the air hole path 12 is larger than the first introduction flow path 4 and smaller than the main flow path 14.

  As shown in FIG. 2B, the second substrate 16 is a substrate that seals grooves and through holes formed in the first substrate 15 from below. The second substrate includes detection electrodes 17, 18, and 19, electrowetting valve operating electrodes 20, 21, 22, 23, and 24, and electrowetting valve reference electrodes 26, 27, and 28 that constitute a detection unit. 29, the electrode pad 30, the extraction electrode 34, and the hydrophobic part 31 are formed. Moreover, the absorber 32 is mounted in the discharge part so that it may contact the downstream end of a discharge flow path.

  The thickness of the first substrate 15 is about 0.1 mm to 10 mm, and the thickness of the second substrate 16 is about 0.01 mm to 10 mm. The open holes 1, 2, 3 and the air hole 11 may be through holes having a diameter of 10 μm or more.

  When optical detection is performed by the detection unit of the micro analysis chip, one or both of the materials used for the first substrate 15 and the second substrate 16 are transparent as proposed in, for example, Patent Document 5. Alternatively, it is desirable to use a translucent material. This is because it is necessary to irradiate the test liquid flowing in the main flow path 14 with excitation light and detect the fluorescence generated by the excitation light to measure the amount of the target substance, and therefore use a material that hinders the detection of fluorescence. This is because they cannot. Examples of such a transparent or translucent material include glass, quartz, thermosetting resin, thermoplastic resin, and film. Of these, silicon resins, acrylic resins, and styrene resins are preferable from the viewpoints of transparency and moldability. In addition, when performing electrochemical detection, there are no such restrictions on materials.

  On the other hand, in order to perform electrical control and electrical measurement at the detection unit of the micro analysis chip, it is necessary to form electrodes on the substrate or the surface of the substrate. For this reason, it is preferable that one or both of the substrate and the substrate is a material capable of forming an electrode. As a material capable of forming an electrode, glass, quartz, silicon and the like are preferable from the viewpoint of productivity and reproducibility. Note that, with the current technology, it is difficult to form an electrode on an uneven portion, so it is preferable to form an electrode on a flat substrate (second substrate).

  For forming the flow path, for example, a method of directly processing the substrate, a method by machining, a method by laser processing, a method by etching with chemicals or gas, an injection molding using a mold, a press molding, a method by casting, etc. There is. Among them, a method using a mold and a method using etching are preferable because of high reproducibility of the shape dimensions.

  The width and height of the main flow path 14 are not particularly limited, but are set to dimensions that allow the solution to penetrate due to the wetness of the solution and the capillary force. The height is preferably set to about 1 μm to 5 mm. The width is preferably set to about 1 μm to 5 mm.

  The width and height of the grooves for the first to third introduction flow paths 4, 5, and 6 are adjusted according to the properties of the solution flowing through the flow paths, such as viscosity and interfacial tension, by the wetness of the solution and the capillary force. Each is set to a size that can penetrate. The height is preferably set to about 1 μm to 5 mm. The width is preferably set to about 1 μm to 1 mm.

  The number of introduction flow paths is not limited to three, and is set according to the number of liquids necessary for detection. When measuring by immunoassay, it is preferable to provide three or more introduction channels. Moreover, the structure which introduce | transduces a liquid into a main channel from a downstream rather than a detection part may be sufficient as a part of introduction channel.

  In the first to third introduction flow paths 4, 5 and 6, electrowetting valves each having at least a reference electrode and a working electrode are formed as valves for opening and closing the liquid flow.

  The first to third introduction flow paths 4, 5, 6 are provided with working electrodes 20, 21, 22 for electrowetting valves, respectively, directly below the open holes 1, 2, 3 (the working electrode On the upstream side), reference electrodes 26, 27, and 28 for electrowetting valves are provided, respectively.

  Each of the working electrode and the reference electrode is wired to the electrode pad 30 by the lead electrode 34, and the applied voltage is controlled by an external device (not shown) connected to the electrode pad 30 to operate the valve.

  The introduction channel on the working electrode is preferably hydrophobic when no voltage is applied in order to stop the liquid reliably. Therefore, it is preferable to make part or the whole surface of the substrate 15 hydrophobic by using a hydrophobic material for the substrate 15 itself or forming a hydrophobic film.

  The working electrodes 20, 21, and 22 are formed of a gold thin film. Carbon or bismuth may be used in addition to gold. These materials have the advantage that, when a voltage is applied to the working electrode, the generation of hydrogen and the like is small and the electrode is unlikely to deteriorate.

  A thin film having a contact angle of 80 degrees or more with respect to pure water having a specific resistance of 18 MΩ · cm can be provided on the surface of the working electrodes 20, 21, and 22. By adopting this configuration, the liquid can be reliably stopped in a state where no voltage is applied, and the valve can be stably operated.

  As this thin film, a fluorine-containing substance or a substance containing a thiol group is suitable. By using these substances, the contact angle on the working electrode can be made larger than 90 degrees, and it is easy to stop the liquid with the valve without applying a voltage, so that the valve operation is performed more stably. Can do. The thin film is not limited to the above substances, and may be any film having a surface contact angle larger than that of the gold thin film.

  The thickness of the thin film on the gold thin film is preferably 100 nm or less. According to this configuration, the voltage required for the operation of the valve can be reduced, and the system can be downsized.

  Moreover, the working electrodes 20, 21, and 22 can be configured to form only a metal thin film.

  When the metal surface is exposed to natural air, a thin film (contact angle 60 degrees to 85 degrees) made of carbon deposits or the like is formed on the surface. This film is a very thin film having a contact angle of less than 90 degrees with respect to the pure water but a low hydrophilicity with a contact angle with respect to the pure water of 60 to 85 degrees and 1 nm or less. Therefore, it functions sufficiently as an operating electrode of the electrowetting valve. Further, there is an advantage that the applied voltage required for the valve operation can be reduced as compared with the case where the thin film as described above is formed.

  It is preferable to narrow the groove width of the working electrode portion of the introduction channel. According to this configuration, the liquid can be easily stopped on the working electrode without applying a voltage, and the valve operation can be performed more stably. Moreover, it is preferable to set the groove width of the introduction flow path in accordance with the characteristics of the liquid flowing in each introduction flow path. In this case, it is possible to adjust the capillary force of the valve portion in accordance with the characteristics of the liquid, and there is an advantage that the valve operation can be stably performed for a plurality of different liquids. The width of the working electrode portion is preferably about 1 μm to 500 μm.

  Further, the groove width of the introduction flow path is not constant, and may be larger downstream than the upstream of the introduction flow path. In this case, the liquid can be easily stopped by the valve, and the liquid can easily flow at the connection portion between the introduction flow path and the main flow path, so that liquid feeding can be performed more stably.

  The reference electrodes 26, 27, 28 for the electrowetting valve are made of silver / silver chloride. The formation of silver / silver chloride has the advantage that there is little change in potential when a current is passed through the electrode. In addition to silver / silver chloride, gold, carbon, or bismuth may be used.

  The voltage applied between the working electrode and the reference electrode varies depending on the configuration of the working electrode, but is preferably 3 V or less. In particular, when the working electrode is composed of a gold thin film and a thin film formed by exposing the surface of the gold thin film to air, the operation can be performed at an applied voltage of 1 V or less. By reducing the applied voltage, the system can be miniaturized and applied to portable devices.

  In this embodiment, the electrowetting valve is a two-electrode system using a working electrode and a reference electrode, but a three-electrode system with a counter electrode may be used. By using three electrodes, the number of electrodes increases and the structure becomes complicated, but variations in operating voltage can be suppressed.

  In this embodiment, an electrowetting valve is used as a microvalve, but the present invention is not limited to this. A diaphragm type valve or the like that can stop or start inflow of liquid can be used.

  The groove width and groove height of the discharge channel 9 are not particularly limited, but are set to dimensions that allow the solution to penetrate due to the wetness of the solution and the capillary force. The height is preferably set to about 1 μm to 5 mm. The width is preferably set to about 1 μm to 5 mm.

  In the present embodiment, one discharge unit is provided, but the present invention is not limited to this. Two or more discharge units may be provided in accordance with the number and amount of liquid to be discharged.

  In the discharge unit 7, the first substrate 15 is open to the atmosphere, and the second substrate 16 is provided with an absorber 32 connected to the downstream end of the discharge channel.

  Absorber is a liquid absorber, and any material that absorbs liquid using capillary force such as polymer absorber, porous material, hydrophilic mesh, sponge body, cotton, filter paper, etc. It does not matter.

  With this configuration, the liquid can be discharged in a short time, and the measurement time can be shortened. Moreover, there exists an advantage that the outflow to the exterior of a liquid can be prevented by absorbing a liquid with an absorber.

  In addition, an electrowetting valve is provided in the discharge channel. A working electrode 23 is formed in the discharge channel 9, and a reference electrode 29 is formed in the vicinity of the discharge channel of the main channel 14.

  By providing a valve in the discharge channel, the discharge of the liquid from the main channel 14 to the discharge unit can be controlled, and the liquid can be stopped in the main channel 14 for a certain period of time. Further, there is an advantage that a certain amount of discharge can be easily performed.

  The groove width and groove height of the air hole path 12 are set so that the influence between the valves is reduced when the liquid is sequentially sent to the main flow path 14 using a plurality of valves. The groove width of the air hole path 12 is preferably narrower than the groove width of the main flow path 14 and wider than any of the minimum groove widths of the first to third introduction flow paths 4, 5, 6.

  The present configuration including the air holes 12 has an advantage that when liquids are sequentially fed, the influence between the valves is reduced, malfunction of the valves is prevented, and the valves can be operated stably. The height is preferably set to about 1 μm to 5 mm. The width is preferably set to about 1 μm to 1 mm.

  Further, a hydrophobic portion 31 is provided in the vicinity of the boundary between the air hole passage 12 and the main passage 14.

  The hydrophobic portion 31 is a portion where the contact angle between the first substrate 15 and the second substrate 16 is 90 degrees or more. For example, a hydrophobic material such as a fluorine-based hydrophobic agent or a negative resist is applied to the second substrate 16. It can be formed by providing a part.

  By providing the hydrophobic portion 31, it is possible to prevent the liquid from flowing into the air hole path 12, to reliably perform the function of the air hole, and to operate a plurality of valves stably. .

  The air hole path 12 and the discharge flow path 9 are opposite to the detection unit 13 provided in the main flow path 14, that is, the air hole path 12 is the upstream side of the main flow path 14, and the discharge flow path 9 is the main flow path. 14 on the downstream side.

  With this configuration, it is possible to reliably discharge the liquid in the main flow path 14, and it is possible to suppress the deterioration in the quality of the detection signal due to the influence of the liquid remaining without being discharged, and perform detection with good reproducibility. There is an advantage that can be.

  The electrode pad 30 and the extraction electrode 34 perform input of electrical control signals, output of detection signals, and the like. When a gold electrode is used, the production process can be used in combination with a detection electrode and the process can be simplified. In addition, you may form using the electroconductive material containing materials, such as platinum, aluminum, copper.

  The micro-analysis chip shown in FIG. 1 can control the introduction and feeding of a plurality of liquids. For example, an antibody or the like is immobilized in the main flow path 14 and an antigen-antibody reaction is caused by flowing a liquid containing an antigen. An antigen-antibody reaction is performed by flowing a solution containing an antibody, an enzyme substrate reaction is further performed by flowing a substrate solution, and the amount of the electrode active substance generated by the enzyme-substrate reaction is detected with a detection electrode, thereby reducing the amount of the antigen. It can be used for antigen measurement using an immunoassay method.

  By performing the following procedure using this microanalysis chip, a specific protein such as adiponectin involved in the development of metabolic syndrome can be measured, so that metabolic syndrome can be determined.

(1) Immobilizing the antibody on the detection electrode 17 (2) From the first introduction channel 4, the pretreatment (separation, dilution, decomposition) blood sample and enzyme-labeled antibody mixture are mixed into the main channel 14 Introduced in, discharged after stopping for a certain time.
(3) The cleaning buffer solution is introduced into the main channel 14 from the second introduction channel 5 and discharged.
(4) The substrate solution is introduced from the third introduction flow path 6 and stopped for a certain time.
(5) The amount of specific protein in the blood sample is measured by electrochemical detection.

  With this configuration, it is possible to perform measurement of a specific protein by an immunoassay simply and in a short time. Further, by measuring the amount of adiponectin involved in the development of metabolic syndrome, metabolic syndrome can be determined easily and accurately. Further, the use of the micro analysis chip has the advantage that the system can be reduced in size and cost and can be easily applied to portable devices.

  In particular, since the minimum diameters (minimum groove widths) of the first to third introduction flow paths are different, the first introduction flow path having a large minimum diameter is used for a highly viscous liquid (blood sample), for example. For the low liquid (cleaning liquid, substrate solution), there is an advantage that the liquid feeding control becomes easy by using the second or third introduction flow path having a small minimum diameter.

  In this embodiment mode, the case where electrochemical detection is performed is shown, but optical detection may be performed. For example, by immobilizing an antibody or the like in the main flow path 14 part, an antigen-containing liquid is allowed to flow from the first introduction flow path to cause an antigen-antibody reaction, and a washing liquid is flowed from the second introduction flow path to be washed. For the optical measurement in which the liquid containing the labeled antibody is flowed from the third introduction channel to cause the antigen-antibody reaction, and the main channel 14 is irradiated with excitation light and the amount of the antigen is measured by the amount of fluorescence. Available.

(Embodiment 2)
Next, a micro analysis chip having a structure different from that of the first embodiment will be described in detail with reference to the drawings.

FIG. 7 shows a microanalysis chip according to the present embodiment.
The micro analysis chip according to the present embodiment is the same as that of the first embodiment except that the introduction flow path 4 includes a pretreatment unit 50 that performs liquid pretreatment (separation of solids, decomposition of multimers, etc.). It is the same. For this reason, only the preprocessing unit 50 will be described in detail, and the other description will be omitted.

  A plan view of the preprocessing unit 50 is shown in FIG. 8A, and a side view thereof is shown in FIG. 8B. The first substrate 15 and the second substrate 16 are formed using the same processing method as in the first embodiment.

  The preprocessing unit 50 includes a separation processing unit 51 that removes red blood cells contained in a blood sample to be measured, and a decomposition processing unit 52 that performs monomerization of multimers. The decomposition processing unit 52 is formed on the flow path 71, and the separation processing unit 51 is formed in the injection hole 72.

  The width and height of the flow path 71 are not particularly limited, but are set to dimensions that allow the solution to penetrate due to the wetness of the solution and the capillary force. The height is preferably set to about 1 μm to 5 mm. The width is preferably set to about 1 μm to 5 mm. The injection hole 72 may be a through hole having a diameter of 10 μm or more.

  The separation processing unit 51 is provided with a filter for removing red blood cells. As a material of the filter, glass fiber, metal, nylon, polyester, carbon, or the like can be used. Among them, glass fiber is particularly suitable for removing red blood cells. The number of filters may be one, or two or more. Separation performance can be improved by stacking two or more sheets.

  The separation processing unit 51 is not limited to a separation method using a filter, and may be configured to dampen and separate red blood cells using, for example, a pillar structure as long as the red blood cells can be removed.

  The decomposition processing unit 52 is provided with a heater for making the protein in the blood into a single amount by heat treatment. As the material of the heater, nickel, chromium, tantalum, nickel chromium alloy or the like is used.

  The decomposition processing unit 52 is not limited to the decomposition method by heat treatment, and may be configured to use a method using an action with a reducing agent or a surfactant.

  In the present embodiment, the configuration includes both the separation processing unit and the decomposition processing unit, but only one of them may be provided. In addition, a dilution processing unit for performing dilution may be provided in the preprocessing unit.

  By using this pretreatment unit 50, red blood cells can be separated from the blood injected from the injection hole 72 by a filter, and adiponectin in the blood can be singulated by heat treatment. Therefore, since the adiponectin concentration in the blood can be measured easily and accurately without performing pretreatment outside by the micro analysis chip according to the present embodiment, it is possible to simplify the time and effort involved in the measurement.

(Example)
EXAMPLES Next, although an Example demonstrates this invention, the scope of the present invention is not limited to these Examples.

Example 1
The present example is related to the first embodiment.
3 and 4 show a microanalysis chip according to the present example.
As shown in FIG. 3, the microanalysis chip of this example includes first to third introduction flow paths 4, 5, and 6 each having open holes 1, 2, and 3 for introducing a liquid into the chip, The discharge passages 9 and 10 connected to the two discharge portions 7 and 8 respectively, and the air hole passage 12 connected to the air hole 11 are connected to the main flow passage 14 provided with the detection portion 13.

As shown in FIG. 4, the micro-analysis chip is composed of two substrates. In the first substrate 15, a groove for the main flow path 14, a through hole for the open holes 1, 2, 3, a groove for the first to third introduction flow paths 4, 5, 6, a discharge flow path 9, A groove for 10, a hole for the discharge portions 7 and 8, a groove for the air hole path 12, and a through hole for the air hole 11 are formed.

  The second substrate 16 includes detection electrodes 17, 18, 19, electrowetting valve working electrodes 20, 21, 22, 23, 24, electrowetting valve reference electrodes 26, 27, 28, 29, and electrode pads. 30, the hydrophobic part 31 is formed, and the absorbers 32 and 33 are placed on the discharge part.

Grooves for the main flow path 14 in the first substrate 15, through holes for the open holes 1, 2, and 3, first to third
For the production of the grooves for the inlet channels 4, 5, 6, the grooves for the outlet channels 9, 10, the holes for the outlets 7, 8, the grooves for the air holes 12, and the through holes for the air holes 11 Used a resin molding method using a mold. For the production of the mold, a resist pattern was formed on a silicon substrate by photolithography, and then etching was performed by a dry etching process. The prepared mold form was placed, and silicon rubber (polydimethylsiloxane) (Zill pot 184 manufactured by Toray Dow Corning Co., Ltd.) was poured into the thickness until it became 2 mm, and heated at 100 ° C. for 15 minutes to be cured. After curing, the mold and the cured silicon rubber were separated from the mold, and the silicon rubber was shaped into a length of 15 mm, a width of 30 mm, and a thickness of 2 mm to produce a first substrate.

  In the first substrate 15, open holes 1, 2, and 3 were formed by punching as through holes having a diameter of 2 mm, and air holes 11 were formed as through holes having a diameter of 1 mm. The discharge parts 7 and 8 have a shape penetrating the first substrate 15 as a whole, and are formed by a mold.

  The width of the main flow path 14 was set to 1000 μm, the width of the discharge flow paths 9 and 10 was set to 50 μm, and the width of the air hole path 12 was set to 500 μm. The flow path height was all 50 μm.

  The width of the introduction channel is set in accordance with the characteristics such as the viscosity of the liquid to be flowed. The width of the first introduction channel 4 is 400 μm, the width of the second introduction channel 5 is 300 μm, and the third introduction channel. The width of the channel 6 was 300 μm. (Excluding valve working electrode)

  The second substrate was prepared by cutting a quartz substrate having a thickness of 600 μm into a length of 17 mm and a width of 34 mm with a dicing saw.

  The second substrate 16 is previously provided with detection electrodes 17, 18, 19, electrowetting valve working electrodes 20, 21, 22, 23, 24, electrowetting valve reference electrodes 26, 27, 28, 29, electrodes A pad 30 was produced.

  Electrowetting valve working electrodes 20, 21, 22, 23, and 24 are prepared by patterning a resist by photolithography, forming a titanium layer 50 nm and a gold layer 100 nm by sputtering, and then patterning by lift-off. Formed. The size of the electrowetting valve working electrode was 500 μm in length and 500 μm in width.

  A thin film (contact angle 60 ° to 85 °) made of carbon deposits is formed on the surface of the gold thin film by exposure to natural air.

  For the production of the electrowetting valve reference electrodes 26, 27, 28, 29, after patterning the resist by photolithography, a silver layer of 1 μm was formed by sputtering, and patterned electrodes were formed by lift-off. After the electrode was prepared, the surface of Ag was subjected to chlorination treatment to prepare an electrode having an Ag / AgCl layer. For the chlorination treatment, a voltage of +100 mV for 50 seconds was applied to the electrode in 0.1 M hydrochloric acid. The size of the reference electrode for the electrowetting valve was 1000 μm in length and 1000 μm in width.

  In the present embodiment, the electrowetting valve is a two-electrode system including a working electrode and a reference electrode, but a three-electrode system including a counter electrode may be used. Further, the microvalve may be other than the electrowetting valve and may be any one that can stop or start the flow of liquid, such as a diaphragm type valve.

  The width of the introduction flow path on the working electrode is narrower than that of other portions, and the width is set in accordance with characteristics such as the viscosity of the liquid to be flowed. The width of the first introduction channel 4 on the working electrode was 100 μm, the width of the second introduction channel 5 was 50 μm, and the width of the third introduction channel 6 was 30 μm.

  For the production of the detection electrode 17 (working electrode), the detection electrode 19 (counter electrode) and the electrode pad 30, similarly, after patterning a resist by photolithography, a titanium layer 50 nm and a gold layer 100 nm are formed by sputtering, An electrode patterned by the lift-off method was formed. The size of the detection electrode 17 was 1000 μm in length and 1000 μm in width, and the size of the detection electrode 19 was 1000 μm in length and 1500 μm in width. The electrode pad 30 has a size of each electrode of 1000 μm and a width of 1500 μm, and is formed at an interval of 0.05 inch.

  For the production of the detection electrode 18 (reference electrode), a resist was patterned by a photolithography method, a silver layer was formed by 1 μm by a sputtering method, and a patterned electrode was formed by a lift-off method. After the electrode was prepared, the surface of Ag was subjected to chlorination treatment to prepare an electrode having an Ag / AgCl layer. For the chlorination treatment, a voltage of +100 mV for 50 seconds was applied to the electrode in 0.1 M hydrochloric acid.

  In this embodiment, two discharge portions 7 and 8 having a length of 2000 μm and a width of 6000 μm are provided. The discharge units 7 and 8 are open to the atmosphere on the first substrate 15 side, and the absorbent bodies 32 and 33 made of cotton are placed on the second substrate 16.

  The discharge channels 9 and 10 are provided with electrowetting valves. Valve discharge electrodes 23 and 24 are formed in the discharge channels 9 and 10, and a valve reference electrode 29 is formed in the vicinity of the discharge channel of the main channel 14.

Further, a hydrophobic portion 31 is provided in the vicinity of the boundary between the air hole passage 12 and the main passage 14. The hydrophobic portion 31 is 500 μm in length and 200 μm in width, and is formed by providing a fluorine-based hydrophobic agent (Durasurf manufactured by Harves Co.) on the second substrate 16.

  The first substrate 15 and the second substrate 16 are subjected to oxygen plasma treatment under conditions of 100 W, an oxygen flow rate of 30 sccm, and 60 seconds to increase the hydrophilicity of the substrate surface, and then the first substrate 15 and the second substrate 16 are self-adsorbed. The microanalysis chip concerning Example 1 was produced.

  Using the micro analysis chip according to Example 1, a test for flowing a liquid was performed.

  A pretreated liquid sample and enzyme-labeled antibody mixture from the open hole 1 (first liquid), a wash buffer solution (second liquid) from the open hole 2, and a substrate solution (third liquid) from the open hole 3, 2 μL each was injected.

  The injected liquid moved through each introduction channel by capillary action and stopped when it reached the valve working electrode in each introduction channel. The first to third liquids have different properties such as viscosity, but by setting the width of the introduction flow path according to the characteristics of the flowing liquid, all the liquids have their respective introduction flow paths. It was able to move smoothly.

  Next, by applying a voltage between the working electrode 20 and the reference electrode 26, the valve in the first introduction flow path 4 was turned on, and the first liquid was introduced into the main flow path 14. The applied voltage was 1V. The first liquid introduced into the main channel 14 moved through the main channel due to capillary action and stopped when it reached the valve working electrode in the discharge channel.

  Next, by applying a voltage of 1 V between the working electrode 23 and the reference electrode 29, the valve in the discharge channel 9 was turned on, and the first liquid was discharged to the discharge unit 7. The discharged first liquid was absorbed by the absorber 32 in the discharge portion 7 and was discharged from the main flow path.

  When the air hole path 12 connected to the air hole 11 is not provided, when the first liquid is discharged, the other liquid in the second to third introduction flow paths 5 and 6 moves, and enters the main flow path. Although it was sometimes introduced by mistake, by providing the air hole 12, such a malfunction could be prevented and the liquid could be discharged stably.

  Next, by applying a voltage of 1 V between the working electrode 21 and the reference electrode 27, the valve in the second introduction flow path 5 was turned on, and the second liquid was introduced into the main flow path 14. The second liquid introduced into the main flow path 14 moved through the main flow path by capillary action and stopped when it reached the valve working electrode in the discharge flow path.

  Next, by applying a voltage of 1 V between the working electrode 24 and the reference electrode 29, the valve in the discharge channel 10 was turned on, and the second liquid was discharged to the discharge unit 8. The discharged second liquid was absorbed by the absorber 33 in the discharge unit 8, and was discharged entirely from the main flow path without malfunction of other valves.

  Finally, by applying a voltage of 1 V between the working electrode 22 and the reference electrode 28, the valve in the introduction flow path 6 is turned on, the third liquid is introduced into the main flow path 14, and the discharge flow path 6 It stopped when it reached the valve working electrode.

  Therefore, according to the present invention, it can be seen that a plurality of liquids having different characteristics can be sent and discharged stably without an external power source.

  Next, the specific protein was measured by immunoassay using the microanalysis chip according to Example 1.

  The concentration of adiponectin involved in the development of metabolic syndrome as a specific protein was measured.

  The antibody (R & D MAB10651) was solidified on the detection electrode 17 in the main channel 14 in advance. The antibody was immobilized by physisorption and incubation at 37 ° C. for 10 minutes.

  Instead of the blood sample, a sample solution having a different concentration of adiponectin (R & D 1065AP) was prepared, and measurement was performed according to the following procedure.

(1) 2 μL of a mixture of adiponectin (0, 10, 100, 500 ng / mL) and enzyme (ALP) labeled antibody (2.5 μg / mL) is introduced into the main channel 14 from the first introduction channel 4 Then, after stopping for 5 minutes, discharge.
(2) 2 μL of a washing tris buffer solution (THAM (tris hydroxylmethylamine): 10 mM, NaCl: 137 mM, MgCl: 1 mM, PH 9.0) is introduced from the second introduction channel 5 into the main channel 14. , Discharge.
(3) 2 μL of a substrate (pAPP (p-Aminophenyl phosphate)) solution (1 mM) is introduced into the main channel 14 from the third introduction channel 6 and stopped.
(4) After 5 minutes, pAP (p-aminophenol) produced by the reaction between the enzyme and the substrate is detected electrochemically (cyclic voltammetry) with the detector electrode, and the peak current value depends on the adiponectin concentration Sex was measured.

  The measurement results are shown in FIG. A calibration curve is obtained when the adiponectin concentration is in the range of 10 to 500 ng / mL, and it can be seen that according to the present invention, the measurement of a specific protein by an immunoassay can be performed simply and in a short time.

(Example 2)
Next, another example according to the first embodiment will be described.
The micro-analysis chip according to this example has the same structure as that of Example 1 shown in FIGS. 3 and 4 except for the shape of the introduction channel of the valve working electrode and the valve working electrode part.

  Similarly to Example 1, the width of the main flow path 14 was set to 1000 μm, the width of the discharge flow paths 9 and 10 was set to 50 μm, and the width of the air hole path 12 was set to 500 μm. The flow path height was all 50 μm.

  The widths of the respective introduction channels are set in accordance with characteristics such as the viscosity of the flowing liquid, the width of the first introduction channel 4 is 400 μm, the width of the second introduction channel 5 is 300 μm, and the third The width of the introduction flow path 6 was 300 μm.

  The second substrate 16 is previously provided with detection electrodes 17, 18, 19, electrowetting valve working electrodes 20, 21, 22, 23, 24, electrowetting valve reference electrodes 26, 27, 28, 29, electrodes A pad 30 was produced.

  Electrowetting valve working electrodes 20, 21, 22, 23, and 24 are prepared by patterning a resist by photolithography, forming a titanium layer 50 nm and a gold layer 100 nm by sputtering, and then patterning by lift-off. Formed. The size of the valve working electrode was 500 μm in length and 500 μm in width.

After forming the electrode, a photolithography method is used to form a pattern inside the working electrode for the electrowetting valve, and then C ₄ F ₈ (cyclobutane octafluoride) gas is introduced in the plasma to deposit a 50 nm fluorocarbon film. It was. An ICP device (MUC-21) manufactured by Sumitomo Precision Industries was used for depositing the fluorocarbon film. After depositing the fluorocarbon film, the resist and the fluorocarbon film formed on the resist were removed by a lift-off method to form a fluorocarbon film in a desired shape. The contact angle of the fluorocarbon film was 110 ° (at room temperature of 25 ° C., pure water (specific resistance: 18 MΩ · cm)).

  The width of the introduction flow path on the working electrode is the same as that of other portions, and the width is set in accordance with characteristics such as the viscosity of the liquid to be flowed. The width of the first introduction flow path 4 on the working electrode was 400 μm, the width of the second introduction flow path 5 was 300 μm, and the width of the third introduction flow path 6 was 300 μm.

  Using the microanalysis chip according to Example 2, a test for flowing a liquid was performed in the same manner as in Example 1.

  A pretreated liquid sample and enzyme-labeled antibody mixture from the open hole 1 (first liquid), a wash buffer solution (second liquid) from the open hole 2, and a substrate solution (third liquid) from the open hole 3, 2 μL each was injected.

  The injected liquid moved through the introduction channel by capillary action, and stopped before the valve working electrode in the introduction channel. Each liquid has different properties such as viscosity, but by setting the width of each introduction channel according to the characteristics of the flowing liquid, all the liquids move smoothly in the introduction channel. I was able to.

  Next, by applying a voltage between the working electrode 20 and the reference electrode 26, the valve in the first introduction flow path 4 was turned on, and the first liquid was introduced into the main flow path 14. The applied voltage was 1.5V. The first liquid introduced into the main channel 14 moved through the main channel due to capillary action and stopped when it reached the valve working electrode in the discharge channel.

  Next, by applying a voltage of 1.5 V between the working electrode 23 and the reference electrode 29, the valve in the discharge channel 9 was turned on, and the first liquid was discharged to the discharge unit 7. The discharged first liquid was absorbed by the absorber 32 in the discharge portion 7 and was discharged from the main flow path.

  Next, by applying a voltage of 1.5 V between the working electrode 21 and the reference electrode 27, the valve in the second introduction flow path 5 was turned on, and the second liquid was introduced into the main flow path 14. . The second liquid introduced into the main flow path 14 moved through the main flow path by capillary action and stopped when it reached the valve working electrode in the discharge flow path.

  Next, by applying a voltage of 1.5 V between the working electrode 24 and the reference electrode 29, the valve in the discharge channel 10 was turned on, and the second liquid was discharged to the discharge unit 8. The discharged second liquid was absorbed by the absorber 33 in the discharge unit 8, and was discharged entirely from the main flow path without malfunction of other valves.

  Finally, by applying a voltage of 1.5 V between the working electrode 22 and the reference electrode 28, the valve in the third introduction flow path 6 is turned on, and the third liquid is introduced into the main flow path 14, It stopped when it reached the valve working electrode in the discharge channel.

  By forming the fluorocarbon film on the working electrode, the contact angle on the working electrode becomes larger than 90 degrees, and it is easy to stop the liquid with the valve without applying voltage, making the valve operation more stable. Could be done.

  Therefore, according to the present invention, it can be seen that a plurality of liquids having different characteristics can be sent and discharged stably without an external power source.

  Using the microanalysis chip according to Example 2, the specific protein was measured by immunoassay in the same manner as in Example 1. As a result, a calibration curve was obtained when the adiponectin concentration ranged from 10 to 500 ng / mL. Therefore, according to the present invention, it can be seen that measurement of a specific protein by immunoassay can be performed easily and in a short time.

(Example 3)
Next, still another example according to the first embodiment will be described.
The microanalysis chip according to this example has the same structure as that of Example 1 shown in FIGS. 3 and 4 except for the shape of the introduction flow path of the valve working electrode part.

  Similarly to Example 1, the width of the main flow path 14 was set to 1000 μm, the width of the discharge flow paths 9 and 10 was set to 50 μm, and the width of the air hole path 12 was set to 500 μm. The flow path height was all 50 μm.

  The width of the introduction channel is set in accordance with the characteristics such as the viscosity of the liquid to be flowed. The width of the first introduction channel 4 is 400 μm, the width of the second introduction channel 5 is 300 μm, and the third introduction channel. The width of the channel 6 was 300 μm. (Excluding valve working electrode)

  As shown in FIG. 6, the width of the introduction channel of the working electrode section is larger downstream than the upstream of the introduction channel, and the width is set according to the characteristics such as the viscosity of the flowing liquid. Yes. The width of the first introduction flow path 4 on the working electrode is 100 μm in the upstream part, 300 μm in the downstream part, the width of the second introduction flow path 5 is 50 μm in the upstream part, 300 μm in the downstream part, and the third introduction flow path The width of 6 was 30 μm in the upstream part and 300 μm in the downstream part.

  Using the microanalysis chip according to Example 3, a test for flowing a liquid was performed in the same manner as in Example 1.

  A pretreated liquid sample and enzyme-labeled antibody mixture from the open hole 1 (first liquid), a wash buffer solution (second liquid) from the open hole 2, and a substrate solution (third liquid) from the open hole 3, 2 μL each was injected.

  The injected liquid moved through each introduction channel by capillary action and stopped when it reached the valve working electrode in each introduction channel. Each liquid has different properties such as viscosity, but by setting the width of the introduction channel according to the characteristics of the flowing liquid, all the liquids can move smoothly in the introduction channel. did it.

  Next, by applying a voltage between the working electrode 20 and the reference electrode 26, the valve in the first introduction flow path 4 was turned on, and the first liquid was introduced into the main flow path 14. The applied voltage was 1V. The first liquid introduced into the main channel 14 moved through the main channel due to capillary action and stopped when it reached the valve working electrode in the discharge channel.

  Next, by applying a voltage of 1 V between the working electrode 23 and the reference electrode 29, the valve in the discharge channel 9 was turned on, and the first liquid was discharged to the discharge unit 7. The discharged first liquid was absorbed by the absorber 32 in the discharge portion 7 and was discharged from the main flow path.

  Next, by applying a voltage of 1 V between the working electrode 21 and the reference electrode 27, the valve in the second introduction flow path 5 was turned on, and the second liquid was introduced into the main flow path 14. The second liquid introduced into the main flow path 14 moved through the main flow path by capillary action and stopped when it reached the valve working electrode in the discharge flow path.

  Next, by applying a voltage of 1 V between the working electrode 24 and the reference electrode 29, the valve in the discharge channel 10 was turned on, and the second liquid was discharged to the discharge unit 8. The discharged second liquid was absorbed by the absorber 33 in the discharge unit 8, and was discharged entirely from the main flow path without malfunction of other valves.

  Finally, by applying a voltage of 1 V between the working electrode 22 and the reference electrode 28, the valve in the third introduction flow path 6 is turned on, the third liquid is introduced into the main flow path 14, and the discharge flow It stopped when it reached the valve working electrode in the road.

  By making the introduction channel width of the working electrode portion larger at the downstream side than at the upstream side, it becomes easier to stop the liquid at the valve, and the liquid flows at the connection portion between the introduction channel and the main channel. It became easy to flow, and liquid feeding could be performed more stably.

  Therefore, according to the present invention, it can be seen that a plurality of liquids having different characteristics can be sent and discharged stably without an external power source.

  Using the microanalysis chip according to Example 3, the specific protein was measured by immunoassay in the same manner as in Example 1. As a result, a calibration curve was obtained when the adiponectin concentration ranged from 10 to 500 ng / mL. Therefore, according to the present invention, it can be seen that measurement of a specific protein by immunoassay can be performed easily and in a short time.

Example 4
FIG. 9 shows a microanalysis chip according to this example according to the second embodiment.
The microanalysis chip according to the present embodiment includes a pre-processing unit 50 that performs liquid separation and decomposition processing upstream of the first introduction flow path 4. The configuration other than the preprocessing unit 50 is the same as that of the first embodiment.

  FIG. 10A shows a plan view and FIG. 10B shows a side view of the blood pretreatment unit 50. The first substrate 15 and the second substrate 16 are formed using the same processing method as in the first embodiment.

  The pretreatment unit 50 includes a filter 54 that removes red blood cells from the blood sample, and a heater 55 that performs multimerization. The heater 55 is provided on the flow path 71, and the filter 54 is provided in the injection hole 72.

  The width of the channel 71 was set to 1000 μm and the height was set to 50 μm. The diameter of the injection hole 72 was set to 3 mm, and the filter 54 was disposed inside the injection hole 72. For the filter 54, Whatman glass fiber filter paper 1GF / D was cut to an outer diameter of 3 mm and two sheets were stacked.

  In the flow path 71, a heater 55 is installed for making the protein in the blood into a single amount by heat treatment. As the heater 55, a nickel chrome alloy thin film heater was used, and the heater 55 was heated by being energized.

Using the microanalysis chip according to this example, a pretreatment experiment using a simulated blood sample was performed. The simulated blood used was a polyscience microbead microsphere added at a concentration of 4.5 × 10 ⁶ cells / μL as simulated blood cells in an 80 mg / mL aqueous solution of bovine albumin containing multimeric human adiponectin.

  FIG. 11A shows a plan view and FIG. 11B shows a side view of the pretreatment unit 50 when the simulated blood is injected from the injection hole 72.

  The simulated blood cell was separated from the simulated blood sample injected from the injection hole 72 by the filter 54, and the sample 62 from which the simulated blood cell component was removed was obtained. A sample 63 that has been singulated can be obtained by heat-treating the sample 62 from which the blood cell component has been removed. For adiponectin in the sample 62 from which the blood cell component was removed, heat treatment was performed at 100 ° C. for 5 minutes. With the microanalysis chip according to the present example, it was possible to separate and monomerize blood cells.

  Therefore, the adiponectin concentration in the blood can be easily and accurately measured by the microanalysis chip according to the present embodiment without any external pretreatment.

  As described above, according to the present invention, it is possible to stably control a plurality of liquids having different characteristics in the microanalysis chip without requiring external power, and the specific protein in the blood can be controlled. Measurement can be performed stably and easily. Such a micro-analysis chip can be applied as an analysis system for determining metabolic syndrome, and has great industrial significance.

1, 2, 3 Open hole 4 1st introduction flow path 5 2nd introduction flow path 6 3rd introduction flow path 7, 8 Discharge part 9, 10 Discharge flow path 11 Air hole 12 Air hole path 13 Detection part 14 Main channel 15 First substrate 16 Second substrate 17, 18, 19 Detection electrodes 20, 21, 22, 23, 24 Electrowetting valve working electrodes 26, 27, 28, 29 Electrowetting valve reference electrode 30 Electrode pad 31 Hydrophobic part 32, 33 Absorber 34 Extraction electrode 50: Pre-processing part 51: Separation processing part 52: Decomposition processing part 54: Filter 55: Heater 61: Blood 62: Blood (after blood cell removal)
63: Blood (after monomerization)
71: Channel 72: Injection hole

Claims (16)

A main flow path provided with a detection part and / or a reaction part;
A first introduction channel and a second introduction channel for introducing a liquid into the main channel from the upstream side of any of the detection unit and the reaction unit;
A first valve and a second valve, each of which is provided in each of the first introduction channel and the second introduction channel, for opening and closing a liquid flow;
An air hole connected to the upstream side of the main flow path;
An inflow prevention portion for preventing liquid from flowing into the air holes;
A discharge portion connected to the downstream side of the main flow path,
A micro-analysis chip that delivers liquid with capillary force as driving force. The micro analysis chip according to claim 1,
The main flow path and the air hole are connected via an air hole path,
A microanalysis chip characterized by that. The micro analysis chip according to claim 2,
The inflow prevention part is composed of a hydrophobic part, and is provided in the vicinity of the boundary between the air hole path and the main flow path,
A microanalysis chip characterized by that. In the micro analysis chip according to claim 1, 2, or 3,
The main flow path and the discharge portion are connected via a discharge flow path,
A microanalysis chip characterized by that. The micro analysis chip according to claim 4,
The discharge unit includes an absorber connected to a downstream end of the discharge flow path.
A microanalysis chip characterized by that. The micro analysis chip according to claim 4 or 5,
The discharge flow path includes a valve that opens and closes the flow of liquid.
A microanalysis chip characterized by that. The micro analysis chip according to any one of claims 1 to 6,
Each of the first valve and the second valve is an electrowetting valve,
A microanalysis chip characterized by that. The micro analysis chip according to any one of claims 1 to 7,
A first substrate on which the main channel groove, the first introduction channel groove, and the second introduction channel groove are formed, and a second substrate that covers the first substrate And are superimposed,
A microanalysis chip characterized by that. The micro analysis chip according to claim 7,
The working electrodes of the first valve and the second valve are made of a metal thin film,
A microanalysis chip characterized by that. The micro analysis chip according to claim 7,
The working electrodes of the first valve and the second valve are composed of a metal thin film and a thin film formed on the metal thin film.
A microanalysis chip characterized by that. The micro analysis chip according to claim 10,
The thin film has a thickness of 100 nm or less.
A microanalysis chip characterized by that. The micro analysis chip according to claim 10,
The contact angle of the thin film with respect to pure water having a specific resistance of 18 MΩ · cm at 25 ° C. is 80 degrees or more.
A microanalysis chip characterized by that. The micro analysis chip according to claim 10,
The thin film is made of a fluorine-containing substance or a substance containing a thiol group.
A microanalysis chip characterized by that. The micro analysis chip according to any one of claims 7, 9 to 13,
The operating potentials of the first valve and the second valve are both 3V or less,
A microanalysis chip characterized by that. The micro analysis chip according to any one of claims 1 to 14,
Protein detection is performed by the detection unit.
A microanalysis chip characterized by that. The micro analysis chip according to claim 15,
The protein is adiponectin;
A microanalysis chip characterized by that.