JP4811247B2 - Microchip and analytical device using the same - Google Patents

Description

  The present invention relates to a microchip for electrochemically or optically analyzing a biological fluid and an analysis device using the microchip.

  Conventionally, there is a method of electrochemically or optically analyzing a biological fluid using a microchip in which a microchannel is formed. As a method for electrochemical analysis, as a biosensor for analyzing a specific component in a sample solution, for example, a current value obtained by a reaction between glucose in blood and a reagent such as glucose oxidase supported in the sensor is obtained. Some measure blood glucose levels by measuring them.

  In the analysis method using a microchip, the fluid can be controlled using a rotating device having a horizontal axis, and the sample liquid is measured, the cytoplasmic material is separated and separated using a centrifugal force. Therefore, various biochemical analyzes can be performed.

  As a conventional sample solution collecting method for introducing a sample into a microchip, there is an electrochemical biosensor shown in FIG. 7, and the sample solution is introduced into the cavity 212 from the suction port 208 by capillary action, and the working electrode 201, It is guided to a position where the counter electrode 202 and the reagent layer 210 are located. At this time, the sample is quantitatively collected with the volume of the cavity 212. The current value generated by the reaction between the sample solution and the reagent at the working electrode 201 and the counter electrode 202 is read by connecting to an external measuring device (not shown) through leads 203 and 204 (Patent Document 1).

  Further, in the centrifugal transfer biosensor shown in FIG. 8, the sample liquid is quantitatively collected from the inlet 313 into the first capillary cavity 312 by capillary action, and then the centrifugal force is applied to the sample liquid in the capillary cavity 312. The liquid is transferred to the receiving cavity 317 via the first flow path 314, and the reagent that has reacted with the reagent in the receiving cavity 317 is centrifuged, and only the solution component is collected in the second cavity 316 by capillary force, and optically collected. Can read the reaction state (Patent Document 2).

Further, in the centrifugal transfer type biosensor shown in FIG. 9, the sample is transferred from the inlet port 409 to the outlet port 410 by capillary force, and each capillary cavity 404a-f is filled with the sample solution, and then generated by rotation of the biosensor. The sample liquid in each capillary cavity is distributed at the position of each vent hole 406a-g by centrifugal force and transferred to the next processing chamber (not shown) through each connected microconduit 407a-f (patented) Reference 3).
JP 2001-159618 A Japanese National Publication No. 4-504758 JP-T-2004-529333

  However, in the conventional configuration, when the sample liquid disappears before filling the capillary cavity, or when the sample liquid is separated from the inlet during sampling, bubbles due to the influence of the surface tension near the inlet are mixed, If the orientation of the device is changed during handling, the sample in the capillary cavity may move and bubbles may be mixed in. If bubbles are mixed in, the missing sample solution will be added later and aspirated. Even if it tries to make it, since the bubble in a capillary cavity cannot escape outside, it had the subject that a bubble part could not be filled and a fixed amount of sample liquid could not be extract | collected.

  The present invention solves the conventional problems by making the cross-sectional shape of the capillary cavity connecting the injection port of the microchip and the holding chamber rectangular, and making the width of the outlet side of the capillary cavity wider than the width of the injection port. Therefore, an object of the present invention is to provide a microchip that can prevent air bubbles from being mixed into the capillary cavity and can collect a sample solution as many times as necessary until the capillary cavity is filled.

In order to solve the above-described conventional problems, an analysis device of the present invention includes an inlet for collecting fluid by capillary action, a capillary cavity connected to a holding chamber for holding the fluid collected through the inlet, the provided, the depth of the bristles Hososuga stream is formed by the cavity path shallower than the depth of the holding chamber, and analysis with a microchip formed by wider configuration than the inlet area to the outlet area of the capillary cavity A device for transferring the fluid injected into the inlet of the microchip to the holding chamber that temporarily holds a predetermined amount of the fluid through the capillary cavity of the microchip, and the holding chamber is required Until the amount of fluid is reached, it can be spotted multiple times without generating bubbles and accumulated in the holding chamber, and the holding cha The fluid that has accumulated from the bar is obtained by, characterized in that in transferring the measurement chamber for measuring the fluid optically through a capillary channel to measure the fluid analysis.

    The present invention solves the conventional problems by making the cross-sectional area of the outlet side of the capillary cavity connecting the inlet of the microchip and the holding chamber larger than the cross-sectional area of the inlet side, and into the capillary cavity. It is an object of the present invention to provide a microchip that can prevent air bubbles from being mixed and can collect a sample solution any number of times until the capillary cavity is filled, and can reliably collect a predetermined amount of sample solution.

Hereinafter, embodiments of the microchip of the present invention will be described in detail with reference to the drawings.
(Embodiment 1)
FIG. 1 shows a cross-sectional shape example of the capillary cavity 3 and the cavity in the microchip 200 according to the first embodiment of the present invention. In the first embodiment, a case where the cross-sectional shape is rectangular is shown as an example. FIG. 1A shows a front view, a top view, and a rear view of the microchip 200 in order from the top, and FIG. FIG. 1C shows a lower substrate of the analysis device 100 including the microchip 200. FIG. Although the structure of the analysis device 100 will be described with reference to FIG. 6, the microchip 200 is formed by being integrally formed with the lower substrate of the analysis device 100. Here, FIGS. 1A and 1B are enlarged views of the capillary cavity 3 that forms a part of the flow path shown in the analysis device 100. FIG. The microchip 200 is integrally formed when the flow path of the analysis device 100 is formed, and the flow paths are connected and formed. The microchip 200 shown in the first embodiment includes a capillary cavity 3, an inlet 1, and an outlet 2. In this embodiment, the cross-sectional shape of the capillary cavity 3 of the microchip 200 is rectangular, and the inlet width L ₁ of the capillary cavity 3 is set smaller than the outlet width L ₂ of the capillary cavity 3. This is because the width L _{2 of the} outlet 2 is expanded from the inlet 1 side in order to obtain a capillary force that allows the sample liquid to be held in the inlet 1 even when the microchip 200 is directed in the direction of gravity. Is.

As an example, in the present invention, the inlet width L ₁ of the capillary cavity 3 is 2 mm, the outlet width L ₂ of the capillary cavity 3 is 11 mm enlarged from the inlet 1 side, the length h of the capillary cavity 3 is 6 mm, and the capillary tube The thickness t of the cavity 3 is set to 0.02 mm. Further, the external dimensions of the microchip 200 were 11 mm × 6 mm as shown in FIG. 1, and the capillary cavity 3 had a thickness t of 0.02 mm. The size of the microchip 200 can be changed as appropriate in order to obtain an appropriate size for the sample liquid collecting portion of the analysis device 100. The thickness of the lower substrate 10 in which the microchip 200 is integrated is 3 mm, the thickness of the upper substrate 9 is 5 mm, and the external dimensions of the analysis device 100 including the cap are 10 mm in thickness and are approximately 65 mm square. The thickness of the capillary cavity 3 forming the flow path of the sample solution, that is, the depth of the flow path is 0.02 mm. On the other hand, the depth of the holding chamber 4 connected to the capillary cavities 3 and formed on the lower substrate of the analysis device 100 is 0.3 mm to 0.5 mm and the thickness of the capillary cavities 3 (that is, the depth of the flow path). ) Form deeper. By setting in this way, the sample liquid injected into the capillary cavity 3 does not proceed to the holding chamber 4 only by the capillary force, and utilizes the centrifugal force obtained by rotating the analysis device 100, thereby using the sample liquid. It is for transporting.
Of course, the same effect can be obtained with any shape such as a circle or an ellipse as long as the capillary cavity 3 has a cross-sectional shape other than a rectangular shape as long as the capillary force acts.

  Further, the wall of the capillary cavity 3 is formed of a hydrophilic material as shown in FIG. 2 so that the contact angle with the wall surface of the capillary cavity 3 is less than a predetermined angle. Specifically, the contact angle with the wall surface of the capillary cavity 3 is provided so that the inside of the capillary cavity 3 is less than 90 degrees, which is generally said to be hydrophilic. Naturally, the smaller the contact angle, the easier it is for the sample liquid to travel in the flow path, which means that the capillary force works strongly.

  Next, the relationship between capillary force and gravity will be described.

Here, as parameters defining the capillary force, the width of the inlet 1 is L ₁ , the width of the outlet 2 is L ₂ , the surface tension of the sample liquid is γ, the contact angle is θ, the cross-sectional area of the inlet 1 is S ₁ , Assuming that the cross-sectional area of the outlet 2 is S ₂ , the force A to move the liquid by the capillary force is generally (γ × cos θ × L ₁ / S ₁ ) − (γ × cos θ × L ₂ / S ₂ ). Can be defined. On the other hand, when the injection port 1 is directed upward, the force B that tries to move downward due to gravity is ρgh. Here, the density is ρ, the acceleration of gravity is g, and the length of the capillary cavity 3 is h. And by setting A> B, even when the inlet 1 is turned upward, the liquid is held in the inlet 1 by the capillary force that the liquid tends to face upward, and it is possible to prevent the entry of bubbles. .

FIG. 3 is a diagram showing the relationship between the width L ₁ of the inlet ₁ and the enlargement ratio of the width of the outlet 2. The horizontal axis shows the change in the width L _{1 of the} inlet 1, and the vertical axis shows the enlargement ratio of the width of the outlet 2. The parameter indicates the length h of the capillary cavity 3 and h = 5 mm to h = 13 mm. Each solid line in the figure shows a change in the length h of the capillary cavity 3, and a shape in which the force of the liquid to advance by the capillary force satisfies A> B is calculated, and the width L _{1 of the} inlet 1 is calculated. Is set to 0.02 mm or more that allows red blood cells to pass through at least, and it is appropriate that the maximum is 2.5 mm or less as an inlet for collecting blood from a human fingertip. The length h of the capillary cavity 3 is preferably about 5 mm to 13 mm in view of the occupied area when the analysis device 100 is designed. Of course, other values can be taken depending on the size of the analysis device 100. In the present embodiment, the size of the analysis device 100 is 65 mm × 54 mm × 10 mm, and the length h of the microchip 200 is suitably about 5 to 13 mm.

As a preferable shape shown in the present embodiment, the width L ₁ of the injection port 1 is set to 2 mm, and the length h of the capillary cavity 3 is set to 6 mm. That is, when the dimension of the inlet width L ₁ is 2 mm, the length h may be about 5 mm to 6 mm. When h = 5 mm, the magnification is 3.13 and the outlet width is L ₂ = 2 mm. When x3.13 = 6.26 mm and h = 6 mm, the magnification is 5.45, and the exit width is L ₂ = 2 mm × 5.45 = 10.9 mm.

Therefore, when the length h of the capillary cavity 3 is set to 6 mm, the width L ₂ of the outlet ₂ is 5.45 times the width L ₁ of the inlet 1, that is, 10.9 mm, and the width L of the inlet 1 _{When 1} is set to 1.5 mm, the length h of the capillary cavity 3 is 2.58, and the width L _{2 of the} outlet 2 is 1.5 mm × 2.58 = 3.87 mm. In FIG. 3, when L ₁ = 2 mm is set, in order to obtain a state in which no bubbles are generated, the length of the capillary cavity 3 is limited to h = 5 mm and h = 6 mm. That is, when the length h of the capillary cavity 3 is set to 7 mm or more (in the case of h = 7 to 13), the gravity toward the outlet 2 side is superior to the capillary force toward the inlet 1 side. There is a possibility that bubbles are generated when the retained sample liquid is transferred to the outlet 2 side and applied twice or three times.

Also, as shown in FIG. 4, when the width L _{1 of the} inlet 1 is set to the minimum 0.02 mm, the width L _{2 of the} outlet 2 is set wider than the width L _{1 of the} inlet 1 so that the capillary force It can be seen that the force of the liquid to advance is A> B. Here, in order to satisfy all h = 5 to 13 mm, which is an appropriate length for installation in the analysis device 100, it is preferable to set the width L2 of the outlet ₂ to 2% or more. When the width L _{1 of the} inlet 1 is set to ₁ mm, the length h of the capillary cavity can correspond to the length h = 5 to 13 mm if the enlargement ratio of the width L ₂ of the outlet 2 is 9 times from FIG. I understand.

On the other hand, by designing the width L _{2 of the} outlet 2 with the enlargement ratio shown in FIG. 3 corresponding to the width L ₁ of the inlet, the inlet 1 can obtain a higher force, and the sample liquid deposited is spotted. Even when the capillary cavity 3 is insufficient during the filling of the capillary cavity 3 or the sample liquid is separated from the inlet 1 in the middle of the filling, the sample liquid is spotted from the inlet 1 again, so that the sample liquid is contained in the capillary cavity 3. It comes into contact with the sample liquid held at the inlet 1 and no bubbles are generated, so that it can be spotted repeatedly until each capillary cavity 3 can hold a predetermined amount of sample liquid.

  Next, the optimum configuration range of the length h of the capillary cavity 3 is shown in FIG. The horizontal axis x indicates the length of the capillary cavity 3, and the vertical axis y indicates the width of the inlet 1 of the capillary cavity 3.

The curve of FIG. 5 shows the relationship between the inlet L ₁ and the outlet side enlargement ratio (that is, the outlet side width L ₂ is determined) in FIG. 3 with the length h of the capillary cavity 3 as a parameter. In other words, in order to obtain a state in which bubbles are not generated, the condition is obtained from the condition that the capillary force and gravity are equal.

As shown in FIG. 3, the relationship between the expansion ratio of the width of the inlet 1 and the width of the outlet 2 of the capillary cavity 3 can hold the sample solution in the inlet 1 even if it is affected by gravity. Based on the difference in capillary force between the inlet 1 and the outlet 2, the applicable range of the length of the capillary cavity 3 that allows the inlet 1 to always maintain a positive state is calculated. Within the scope 0.02~2.5mm width L ₁ of the inlet in the figure, magnification of the width of each outlet is plotted when the maximum (when the length of the capillary cavity is the longest). For example, when the inlet width L ₁ is 2 mm, the maximum enlargement ratio of the outlet width is 5.45 times, and the length h of the capillary cavity 3 is 6 mm. The result is substituted into a formula y = ax ⁿ of iterated curve, a, when determining the coefficients of n, it can be expressed by y = 12.196x ^-0.9698. By setting it to fall within the area below this curve, the inlet 1 maintains a higher capillary force than the outlet 2, so that it is possible to hold the sample solution at the inlet 1 and apply twice. It is possible to spot without attaching bubbles when attaching. Further, even when the outlet 2 of the microchip 100 is directed in the direction of gravity, it is possible to reduce the gravity by designing with the width of the inlet 1 and the length of the capillary cavity 3 that can maintain the capillary force of the inlet 1 high. It is possible to hold the sample solution at the inlet 1 without the sample solution moving to the outlet 2 under the influence of the above. Further, from the tendency of FIG. 3, it is necessary to set the length of the capillary cavity 3 longer as the width of the inlet 1 becomes narrower.
(Embodiment 2)
FIG. 6 shows the analysis device 100 to which the microchip 200 is attached.

  6A shows the upper substrate 9 of the analysis device 100, and FIG. 6B shows the lower substrate 10 of the analysis device 100.

  In FIG. 6B, the analysis device 100 temporarily puts a sample solution such as blood injected into the injection port 1 of the microchip 200 shown in Embodiment 1 into the holding chamber 4 by a predetermined amount via the capillary cavity 3. Hold on. The holding chamber 4 carries an analysis reagent (not shown). Then, the sample solution and the analysis reagent are mixed, and the mixed solution is transferred to the measurement chamber 5 via the capillary channel 6. The measurement chamber 5 communicates with a capillary channel 7 having an air opening hole 8. The mixture of the sample liquid and the analysis reagent transferred to the measurement chamber 5 is analyzed by measuring predetermined items by an optical method.

  In the measurement of the sample liquid, the measurement chamber 5 is irradiated with light, and the reaction state between the liquid sample to be examined and the analysis reagent is optically analyzed. Since the absorbance changes depending on the rate of reaction between the sample solution and the analysis reagent, predetermined items are measured by measuring the absorbance of the irradiated light, and the reaction state can be analyzed.

  Here, in the present invention, the capillary cavity 3, the flow path 6, and the flow path 7 are formed with a depth of 0.02 mm to less than 0.3 mm. However, if the sample liquid flows by capillary force, this dimension is used. It is not limited to. In general, since liquid such as blood is measured and analyzed, 0.02 mm to less than 0.3 mm is desirable.

  The holding chamber 4 and the measurement chamber 5 are formed with a depth of 0.3 mm to 0.5 mm, which is the amount of sample solution and the conditions for measuring the absorbance (optical path length, measurement wavelength). The reaction concentration of the sample solution, the type of reagent, etc.). Then, the sample liquid transferred to the measurement chamber 5 is optically measured.

  In the present embodiment, the sample solution is held in the holding chamber 4 through the capillary cavity 3 that communicates with the inlet 1 of the microchip 200 by using the capillary force. The wall surface of the flow path 6 is subjected to a hydrophilic treatment. Examples of the hydrophilic treatment method include a surface treatment method using an active gas such as plasma, corona, ozone, and fluorine, and a surface treatment using a surfactant or a hydrophilic polymer. It is done. Here, hydrophilicity means that the contact angle with water is less than 90 degrees.

  Further, as shown in FIG. 6, the analysis device 100 is configured by bonding an upper substrate 9 and a lower substrate 10, and a fine uneven shape is formed on a surface of the lower substrate 10 facing the upper substrate 9. A microchannel structure is formed, and each function works such as transfer of a sample solution and holding a predetermined amount of solution. The injection port 1 has a shape protruding from one side of the main body of the analysis device 100, so that it can be easily spotted with a fingertip or the like. It has the effect of preventing it. When the mixing of the reagent and the sample liquid reaches a predetermined level, the sample liquid in the holding chamber 4 is carried by the capillary force through the flow path 6 to the inlet of the measurement chamber 5, and the analysis device is moved at a predetermined number of rotations. The centrifugal force generated by rotation is transferred to the measurement chamber 5. The transferred sample liquid is optically measured for predetermined items in the measurement chamber 5.

  The microchip 200 according to the present invention prevents the bubbles from being mixed into the capillary cavity 3 for collecting the sample liquid, and can collect the sample liquid any number of times until the capillary cavity 3 is filled. It has the effect of improving the liquid distribution accuracy, and is useful as a sample liquid collection method, a distribution transfer method, etc. in the analytical device 100 used for measuring the components of biological fluids with an electrochemical sensor or an optical sensor. It is.

(A) Top view, front view, and rear view of microchip in Embodiment 1 of the present invention (b) Cross-sectional view of microchip in Embodiment 1 of the present invention (c) Analysis in Embodiment 1 of the present invention Device perspective view Diagram for explaining contact angle of surface tension The figure which shows the relationship between the injection hole width | variety and exit width | variety of the microchip in Embodiment 1 of this invention. The figure which shows the tendency of the capillary force by the capillary cavity length change in the width of the inlet 0.02mm The figure for demonstrating the relationship between the inlet width of the capillary cavity of the microchip in Embodiment 1 of this invention, and the length of a capillary cavity 1 is an exploded perspective view of an analysis device according to Embodiment 1 of the present invention. The figure for demonstrating the structure of the electrochemical type biosensor of a prior art example The figure for demonstrating the structure of the centrifugal transfer type biosensor of a prior art example. The figure for demonstrating the sample liquid distribution structure of the centrifugal transfer type biosensor of a prior art example

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Injection port 2 Outlet 3 Capillary cavity 4 Holding chamber 5 Measurement chamber 6, 7 Flow path 8 Atmospheric release hole 9 Upper substrate 10 Lower substrate 100 Analytical device 200 Microchip 201 Working electrode 202 Counter electrode 203, 204 Lead 205 Insulating substrate 206 Cover 207 Spacer 208 Suction port 209 Air escape hole 210 Reagent layer 212 Cavity 310 Wall 312 First cavity 313 Inlet 314 Flow path 315 Filter material 316 Upper compartment 317 Low compartment 318 Core 401 Continuous microconduit 402, 403 Upper part 404a-f Capillary cavity 405a-e Conduit portion 406a-g Top vent 407a-f Connected microconduit 408a-f Valve function 409 Inlet port 410 Outlet port

Claims (3)

An inlet for collecting fluid by capillary action; and a capillary cavity connected to a holding chamber for holding the fluid collected through the inlet and having a depth of a flow path formed in the capillary cavity was shallower than the depth of the holding chamber over, and, with an analytical device comprising a microchip formed by wider configuration than the inlet area to the outlet area of the capillary cavity, which is injected into the injection port of the microchip The fluid is transferred to the holding chamber that temporarily holds a predetermined amount through the capillary cavity of the microchip, and the holding chamber reaches the inlet without generating bubbles until the required amount of fluid is reached. Accumulated in the holding chamber so that it can be spotted multiple times, and the fluid accumulated from the holding chamber passes through a capillary channel. Analysis device, characterized in that analyzing and measuring the fluid transported to the measuring chamber for measuring the fluid optically Te. The inlet of the microchip, the analysis device according to claim 1, characterized in that it is configured to protrude from a side surface of said analysis device body. The analysis device according to claim 1 , wherein the fluid injected from the microchip inlet is transferred to the holding chamber from the outlet of the microchip by centrifugal force generated by rotating the analysis device.

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