JP2007222072A - Parallel microanalysis of chemical substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To screen the influence of an environmental chemical contaminant, etc., on an in vivo enzyme reaction by using a microchip. <P>SOLUTION: Equivalent flow channels 40 of μm order are formed on both the face side and the back side of a silicon substrate 10 and a heat generation means constituted on an ITO film is arranged at a reaction part in the middle of the flow channels. Each flow channel 40 independently has an independent chemical substance supply port 50 connected to the flow channel and a common chemical substance supply port 60 branched and connected to the flow channel 40. For example, a reaction inhibitor is supplied from the independent chemical substance supply port 50 and an enzyme and a substrate corresponding to the enzyme are supplied from the common chemical substance supply port 60, a reaction is performed in the flow channels 40 to screen a plurality of reaction inhibitors. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a chemical substance analysis method using a microchip, and can be effectively applied particularly to screening the effects of various environmental pollutants on in vivo enzyme reactions.

  The technology described below has been studied by the present inventors in completing the present invention, and the outline thereof is as follows.

  In recent years, a micrometer-order channel with a width of 1 mm or less is formed on a substrate, and a chemical substance is allowed to flow through the channel. Therefore, a technique for efficiently performing in a short time has been proposed.

  Among such techniques, a technique has also been proposed in which a plurality of substrates on which a flow path for passing a chemical substance is stacked and the flow paths of each layer are connected to expand the flow path in a three-dimensional manner. . Non-Patent Document 1 shows that the yield of the chemical reactant generated in the flow path can be easily increased by adopting a laminated structure of a plurality of sheets. Further, Non-Patent Document 2 also describes a liquid feeding port when a chemical substance is caused to flow through a flow path.

  Naturally, temperature control is also required in the reaction using such a microchip. Conventional general temperature control is performed by leaving the microchip in the thermostat for a required time, and the temperature of the microchip itself is equal to the temperature in the thermostat. This was a method of waiting for the set temperature to reach equilibrium. However, in such a method, it takes time and effort to control the reaction temperature, and temperature control in which the temperature is appropriately changed according to the reaction situation and the situation is confirmed is almost impossible.

Regarding temperature control, Patent Document 1 proposes a technique in which a heating means is formed by patterning an ITO film, and the ITO film is separately patterned into a meandering line to constitute a temperature sensor.
Y.Kikutani, "Pile-up glass microreactor" Lab Chip, 2002, 2, p193-196 Y.Kikutani, T.Hotiuchi, K.Uchiyama, H.Hisamoto, M.Tokeshi, T.kitamori, `` Glass Microchip with Three-Dimensional Micrchannel Network for 2 × 2 Parallel Synthesis '', Lab on a Chip, 2002, 2, p188-192 Japanese Patent Application Laid-Open No. 2002-85961

  Elucidation of sugar chain function is one of the important fields of biotechnology research. Sugar chains are said to function as codes for information transmission in recognition and interaction between cells. It is expected to elucidate the occurrence mechanism of diseases related to sugar chains and sugar chain-related enzymes, and it is expected to lead to the treatment method, and it is a big problem in future sugar chain research.

  On the other hand, the effects on human bodies such as diseases caused by environmental chemical substances have not been fully elucidated. It is extremely important to elucidate how and what chemical substances are involved in the in vivo enzyme reaction and the adverse effects. Screening work is indispensable for such elucidation.

  However, the conventional screening methods use many reagents and require a long time and a large amount of cost. In particular, the use of many reagents leads to re-environmental contamination of the human body by environmental chemical substances. For this reason, attempts have been made to screen using as little reagent as possible, but no effective method has been developed yet.

  Therefore, the present inventor considered that the amount of reagent to be used can be suppressed to the necessary minimum by using a microchip that has been attracting attention in recent years. Furthermore, if such a microchip is used, the amount of waste liquid processed after the test can be remarkably reduced, and this should lead to a reduction in environmental burden.

  However, since such a microchip is extremely small, it is difficult to increase its processing accuracy. For example, it may be difficult to carry out the manufacturing process in which one piece is equivalent to one piece, and it may possibly affect the reproducibility of the test.

  In chemical analysis using a microchip, it is necessary to dilute the sample in several steps. In particular, in the quantitative analysis, drops on the vessel wall also have a very serious effect.

  Therefore, it is preferable that a plurality of analyzes can be performed using the same reagent without causing individual differences in instruments, devices, and the like. For example, if the test target can be analyzed under the same conditions as much as possible by forming a plurality of equivalent flow channels on the microchip and using the same reagents as much as possible, compare each other. More preferable.

  However, regarding the equivalence of a plurality of flow paths in a microchip, there are many technical problems, and it has not been fully achieved yet. At present, parallel analysis cannot be performed sufficiently for quantitative analysis under the same conditions using a plurality of flow paths.

  In addition, in the microchip, in addition to the equivalence of the flow paths, the accuracy in feeding a reagent or the like as described above is also a problem. Since the amount is very small and diluted several times, even a small amount of liquid leakage has a serious effect and is fatal to the test.

  An object of the present invention is to improve equivalence of analysis environments of a plurality of flow paths in parallel analysis using a microchip.

  An object of the present invention is to provide a technique necessary for analysis or the like for screening the influence of an environmental pollutant chemical substance on an in vivo enzyme reaction or the like using a microchip.

  The above and other objects and novel features of the present invention will be apparent from the description of this specification and the accompanying drawings.

  Of the inventions disclosed in the present application, the outline of typical ones will be briefly described as follows.

  The present invention is a parallel microanalysis method of a chemical substance using a microchip, and the microchip has a plurality of μm-order flow paths that penetrate in the thickness direction and communicate with both front and back surfaces, Each of the channels is formed as an equivalent channel having a recovery rate of at least 97% of the initial supply amount to each of the plurality of channels, and the plurality of channels are independent of each of the plurality of channels. The plurality of flow paths have a common reaction substance supply port that is branched and connected to each of the plurality of flow paths, and is supplied from the independent reaction substance supply port. The reacted reactant and the reactant supplied from the common reactant supply port are reacted in each of the plurality of flow paths, the reaction product after the reaction is recovered, and the reaction product in each of the plurality of flow paths is recovered. Analyze reaction status by analytical means It is characterized in.

  In such a configuration, the initial supply amount when the recovery rate of each of the plurality of flow paths is 97% or more is 150 μl or more and 310 μl or less. In the above configuration, the plurality of flow paths are formed with a processing accuracy within 1% with respect to the order of 100 μm. In any one of the configurations described above, the reactants that react with each other are supplied from the common reactant supply port, and the reaction-inhibitory substance that may inhibit the reaction is supplied from the independent reactant supply port. A parallel microanalysis method for chemical substances. In this configuration, the reaction inhibiting substance is an environmental pollutant.

  In the parallel microanalysis method of the chemical substance, the reactant supplied from the common reactant supply port is an enzyme and a substrate that reacts with the enzyme. The enzyme is a glycolytic enzyme, and the substrate is a polysaccharide having an atomic group separated by the glycolytic enzyme. The sugar chain degrading enzyme is chitinase, and the polysaccharide is a structural polysaccharide.

  Among the inventions disclosed in the present application, effects obtained by typical ones will be briefly described as follows.

  According to the present invention, it is possible to perform parallel analysis with good accuracy in a short time using an extremely small amount of reagent, for example, the influence of an environmental pollutant on the in vivo enzyme reaction and the like.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that components having the same function are denoted by the same reference symbols throughout the drawings for describing the embodiment, and the repetitive description thereof may be omitted.

  The present invention is a parallel microanalysis technique for chemical substances that enables screening of a plurality of chemical species using a microchip having a plurality of flow paths. In such parallel microanalysis, particular consideration was given to the equivalence of a plurality of flow paths and the liquid feed equivalence of reagents and the like to each flow path.

  The flow path formed in the microchip is both on the order of μm in width and depth. For example, the width is 400 μm and the depth is 100 μm. The plurality of channels in the microchip for performing the reaction are formed equivalently. The equivalence of the flow path itself is suppressed within a processing accuracy of 1% with respect to the processing of a flow path width of the order of 100 μm, for example.

  In addition, with respect to the initial supply amount of 150 μl or more and 310 μl or less of the same substance to a plurality of flow paths, the recovery rate in each flow path is 97% or more, and an equal volume is sent to each flow path It is. In screening using such a microchip, the amount of a chemical substance such as a reagent used can be suppressed to, for example, μl order.

  As described above, in the present invention, a plurality of environmental pollutions are obtained by using an equivalent flow path having a recovery rate of 97% or more with respect to a liquid feeding amount of 150 μl or more and 310 μl or less in a state where the amount of reagent used is suppressed to μl. Screening of the inhibition status of a substance against an in vivo enzyme reaction can be performed in a short time, efficiently and at a low cost.

  As shown in FIG. 1, in the microchip 100, glass plates 20 and 30 are bonded to both surfaces of a silicon substrate 10 by anodic bonding. As the glass plates 20 and 30, for example, Pyrex (registered trademark) can be used.

  As shown in FIGS. 2A and 2B, a channel 40 through which a chemical substance can flow is formed in a groove shape on both surfaces of the silicon substrate 10. For example, four flow paths 40 are formed on both surfaces of the silicon substrate 10, respectively, and are provided meandering as shown in FIGS. 2 (a) and 2 (b). In addition, a part of the flow path 40 forms a reaction part in which chemical substances supplied into the flow path 40 react with each other. In the figure, the reaction part is shown in a frame shape.

  On the front surface 10a side of the silicon substrate 10, independent chemical substance supply ports 50 (50a, 50b, 50c, 50d) are provided so as to be independently connected to the four flow paths 40 on the back surface 10b. The independent chemical substance supply port 50 penetrates in the thickness direction of the silicon substrate 10 and exits to the back surface 10b side, and is independently connected to the four flow paths 40 provided on the back surface 10b side. The independent chemical substance supply ports 50 (50a, 50b, 50c, 50d) are configured in this manner, so that different chemical substances can be supplied to the four flow paths 40 in parallel. It has become.

  Further, on the front surface 10a side of the silicon substrate 10, common chemical substance supply ports 60 (60a, 60b) are provided which are branched and connected to the four flow paths 40 on the back surface 10b. The common chemical substance supply port 60 is divided into two hands on the front surface 10a side as indicated by an arrow, each of the two divided hands penetrates in the thickness direction of the silicon substrate 10 to reach the back surface 10b side, and further on the back surface 10b side. Divided into two hands, each is connected to four channels 40.

  By providing the common chemical substance supply port 60 so that it can be branched and connected to a plurality of flow paths in this way, the same chemical substance with the same concentration can be sent to each flow path. It is possible to secure accuracy in parallel analysis performed on the road.

  As shown in FIGS. 2A and 2B, the common chemical substance supply port 60 having such a configuration is composed of two common chemical substance supply ports 60a and 60b. From the common chemical substance supply ports 60a and 60b, Different chemicals that react with each other can be supplied separately.

  Further, the supply ports 41 formed in the independent chemical substance supply port 50 and the common chemical substance supply port 60 pass through a through passage 42 provided in the glass plate 20 covering the surface side of the silicon substrate 10 as shown in FIG. The glass tube 43 is fixed to the surface side of the glass plate 20. The glass tube 43 is fixed to the surface of the glass plate 20 with an adhesive or the like so that it can be used as the liquid feeding port 44.

  Conventionally, a screw hole is formed in a jig that fixes the microchip at a position that coincides with the supply port of the microchip, and a Teflon (registered trademark) screw is screwed into the screw hole. The mouth provided in was used as a liquid feeding port. In such a configuration, liquid leakage often occurs from a screw hole or the like during liquid feeding.

  If the Teflon (registered trademark) screw is tightened too much, the microchip will be broken, so that moderate tightening is necessary. However, in such a case, if the tightening is weak, liquid leakage occurs from the gaps in the screw holes.

  Furthermore, when there are a plurality of Teflon (registered trademark) screws corresponding to a plurality of flow paths, if each tightening condition is different, the ease of flow of the solution or the like in each flow path is different depending on the tightening method. The difference came out, and as a result, the amount of liquid delivered in each flow path was different.

  However, as described above, by fixing the glass tube 43 matched to the diameter of the supply port 41 to the formation position of the so-called supply port 41 of the independent chemical substance supply port 50 and the common chemical substance supply port 60 as a liquid supply tube. It is no longer necessary to install Teflon (registered trademark) screws that cause liquid leakage or differences in the amount of liquid to be fed, and it is possible to ensure equivalency at the time of liquid feeding without considering the tightening condition of the screws surrounding it. became.

  The four flow paths 40 to which the independent chemical substance supply port 50 and the common chemical substance supply port 60 having such a configuration are connected reach the back surface 10b from the front surface 10a side, meander the back surface 10b side, and then the thickness of the silicon substrate 10 It penetrates in the vertical direction and rises to the surface 10a side, and further meanders to reach the discharge port 70 (70a, 70b, 70c, 70d).

  The discharge ports 70 (70a, 70b, 70c, 70) provided on the road terminal side of the flow channel 40 are not shown in the figure, as in the case of forming the supply port 41, but are not illustrated. Through, the glass plate 20 is connected to a glass tube fixed to the surface of the glass plate 20 with an adhesive or the like, so that the reaction product or the like can be collected quantitatively.

  The flow path 40 having such a configuration is formed on both the front and back surfaces of the silicon substrate 10 as follows. That is, as shown in FIG. 4, an oxide film 101 is formed on the surface of the silicon substrate 10 with a predetermined layer thickness by thermal oxidation. A photoresist 102 of a photosensitive material is applied on the formed oxide film 101 with a predetermined layer thickness. The photoresist 102 is exposed to light of a predetermined wavelength using a mask on which a flow path pattern is formed, developed after exposure, and a flow path forming pattern is formed on the photoresist 102.

The photoresist 102 on which the flow path formation pattern is formed is further used as a mask, and the oxide film 101 is etched to form a flow path formation mask on the silicon substrate 10. Using such a flow path forming mask, the ICP multi-beam processing apparatus is irradiated with plasma gas SF ₆ to dry-etch the silicon substrate 10, thereby forming a groove corresponding to the flow path 40. Thereafter, the mask used for forming the flow path 40, that is, the oxide film 101 and the photoresist 102 is removed.

Thus, by using the ICP multi-beam processing apparatus, the flow path 40 formed by irradiating SF ₆ as the plasma gas could be formed more uniformly than the conventional one. That is, the plurality of flow paths 40 are formed equivalently.

  Since the silicon substrate 10 is selected as the substrate for forming the flow path, and the flow path 40 is formed on the silicon substrate 10 by ICP processing, each equivalent flow path 40 with high accuracy and high aspect ratio is provided. Can be formed. That is, a more equivalent flow path 40 can be formed than when ICP processing is applied to a glass substrate or when wet etching is performed on the silicon substrate 10 with a chemical solution.

  For example, formation with a processing accuracy of 1% or less was possible for a channel width of 100 μm. Therefore, it has become possible to form a plurality of equivalent flow paths 40 required for parallel analysis by reducing variations due to processing accuracy between the flow paths.

  Glass plates 20 and 30 such as Pyrex (registered trademark) are bonded to the front surface 10a and the back surface 10b of the silicon substrate 10 in which the flow path 40 having such a configuration is formed. At the time of bonding, anodic bonding or the like is applied, the flow path 40 is formed, and bonding is reliably performed even if the bonding surface of the silicon substrate 10 is slight. In this way, the upper side of the flow path 40 formed on the front surface 10a and the back surface 10b is formed with the glass plate 20, and the lower surface side of the flow path 40 is formed with the glass plate 30, thereby forming the flow path 40 that does not leak. Yes.

  In the case shown in FIG. 4, the state in which the glass plate 20 is anodically bonded to the surface 10 a is simply shown. For such anodic bonding, for example, the temperature was set to 400 ° C. and the applied voltage was set to 500V.

  Further, from the meandering start portion of the flow path 40 formed on the back surface 10b, the chemical substances supplied from the independent chemical substance supply port 50 and the common chemical substance supply port 60 are mixed and reacted to form a reaction part. ing. However, the reaction part constitutes a part of the flow path 40 and exists only as an extended state of the flow path 40, and the configuration is not particularly different from other flow path 40 portions. It is merely divided as its main role.

  Although not shown, the glass plates 20 and 30 covering the flow path 40 are provided with heat generating means so as to control the heating according to the flow path 40. As such heat generating means, for example, an ITO (Indium Tin Oxide) film is provided on the surface side of the glass plates 20 and 30. Although not shown, an ITO metal is formed on the ITO film, and platinum is formed between the electrode metals.

  A lead wire is connected to each electrode metal, and when an electric current is passed through the ITO film via the lead wire and the electrode, the ITO film generates heat. As described above, the ITO film formed on the glass plates 20 and 30 generates heat corresponding to the flow path 40, whereby the reaction portion of the flow path 40 is heated to a predetermined temperature.

  The heat generation state of the ITO film is grasped in real time by a temperature sensor composed of an electrode and platinum. For example, when the heat generation state becomes higher than a predetermined temperature, the current flow is controlled to suppress the heat generation state. Can be controlled.

  With respect to the microchip 100 having such a configuration, the equivalence of the plurality of flow paths 40 formed was evaluated. First, as shown in FIG. 5 (a), only water is supplied to the independent chemical substance supply port 50 and the common chemical substance supply port 60 through the liquid supply port 44, respectively, and as shown in FIG. The recovery rates were compared, and the equivalence of each flow path 40 was evaluated.

  That is, 0.25 μl / min of water is supplied from the four independent chemical substance supply ports 50a, 50b, 50c, and 50d, and 1 μl / min of water is supplied from the common chemical substance supply port 60a. From 60b, 2 μl / min of water was supplied. The recovered amount in each flow path 40 is as shown in FIG. Theoretically, 1 μl / min of water is allowed to flow through each flow path 40 and 100% of the water should be recovered. However, as shown in FIG. The recovery rate was as follows. However, such a recovery rate is within the range of accuracy of measurement error, and it has been confirmed that each flow path 40 is sufficiently formed equivalently.

Further, as shown in FIG. 6 (a), an enzyme (chitinase) having a concentration of 0.15 mg / ml is supplied from the independent chemical substance supply ports 50a, 50b, 50c, 50d with a syringe pump at a flow rate of 0.5 μl / min. Supplied. In addition, an enzyme having the same concentration as described above is supplied from the common chemical substance supply port 60a at a flow rate of 2 μl / min. The side (p-nitrophenyl tri-N-acetyl-β-chitotrioside) having a concentration of 0.5 mM was supplied at a flow rate of 4 μl / min to verify the equivalence of each flow path 40. The reaction solution was recovered at a flow rate of 2 μl / min in an eppen containing 300 μl of an aqueous Na ₂ CO ₃ solution as a reaction stop solution. The amount of liquid fed to each channel was 300 μl.

  In this way, the absorbance of the recovered reaction product para-nitrophenol (p-nitrophenol) was examined at a wavelength of 405 nm. As a result, as shown in FIG. 6B, the absorbances of p-nitrophenol collected in the Eppen at the outlets 70 (70a, 70b, 70c, 70d) corresponding to the four channels are almost the same. there were.

  Further, when the reactants are supplied to the flow channel 40 of this configuration in the range of 150 μl to 310 μl at a flow rate of 2 μl / min in the same manner as described above, the discharge ports 70 (70a, 70b, 70c are passed through the respective flow channels 40. 70d), the recovery amount recovered in the Eppen was a recovery rate of 97% or more as shown in FIG. 7, and it was confirmed that each reaction flow channel was equivalent within this range.

  The four flow paths 40 configured as described above are equivalently formed, and the chemical substances supplied from the common chemical substance supply port 60 can be supplied to the four flow paths 40 in equal amounts. I understood. The recovery amount with the Eppen corresponding to each flow path was also 97% or more, and it was confirmed that almost the same amount of reactant was flowing in each flow path 40.

  Further, as shown in FIG. 8 (a), 0.25 μl / min of water is supplied from each of the independent chemical substance supply ports 50a, 50b, 50c and 50d, and the enzyme is supplied at 1 μl / min from the common chemical substance supply port 60a. Min (concentration 0.6 mg / ml) was supplied, and the substrate was supplied at 2 μl / min (concentration 0.4 mM) from the common chemical substance supply port 60b. The absorbance in each flow path 40 was almost the same as shown in FIG. From these results, the equivalence of each flow path was confirmed.

  Furthermore, as shown in FIG. 9 (a), 0.25 μl / min (concentration: 0.1 mM) of allosamidin as an enzyme reaction inhibitor from the independent chemical substance supply port 50a, and from the independent chemical substance supply ports 50b, 50c, and 50d, Water is supplied at 0.25 μl / min, the enzyme is supplied at 1 μl / min (concentration 0.6 mg / ml) from the common chemical substance supply port 60a, and the substrate is supplied at 2 μl / min (at a common chemical substance supply port 60b). Concentration 0.4 mM).

  The absorbance in each channel 40 was as shown in FIG. That is, in the flow path in which allosamidin is involved in the reaction, the absorbance is significantly lower than in other cases. However, the other three flow paths show almost the same high absorbance. It was verified that each channel configuration was independent.

  In the above experiment, it was verified whether or not the reagent may flow backward at the branch point provided in the flow path 40. If the enzyme inhibitor of allosamidine is flowing backward at the branch point, a plurality of flow paths may be used. The enzyme reaction is inhibited, and a result different from that shown in FIG. 9B should be obtained. From these results, it was confirmed that the four flow paths 40 were independent as described above, and no back flow occurred at the branch point.

  Such an equivalent flow path 40 is possible, for example, when the silicon substrate 10 is formed by ICP processing within a width of 400 μm and a depth of 100 μm. In other cases such as etching, an equivalent flow path is not formed, and the equivalence of each of the four is inferior to that of the present invention.

  Thus, screening of chemical substances can be performed using a plurality of equivalent flow paths. An example is shown in FIGS. For example, as shown in FIG. 10, from the independent chemical substance supply ports 50a, 50b, 50c, and 50d, the inhibitor A, B, Supply as C and D. An enzyme is supplied from the common chemical substance supply port 60a, and a substrate that reacts with the enzyme is supplied from the common chemical substance supply port 60b.

  In this way, by using four independent equivalent flow paths 40, reaction products can be produced according to the enzyme reaction inhibitory properties of the four types of chemical substances supplied from the independent chemical substance supply port 50. is there. By analyzing the reaction product, it is possible to know the effects of the four inhibitors. In the reaction part, the temperature was controlled at 37 ° C. using the ITO film.

  As shown in FIG. 10, the reaction products of the enzyme and the substrate in which the inhibitors A, B, C, and D are involved are collected from the discharge ports 70a, 70b, 70c, and 70d, respectively. For example, by supplying chitinase as an enzyme and supplying a p-nitrophenyl group bonded to a polysaccharide as a substrate, the release situation of p-nitrophenol by chitinase when inhibitors A, B, C and D are involved Different.

If such p-nitrophenol is colored with a Na ₂ Co ₃ alkaline solution and the amount of liberation is analyzed with a spectrophotometer, etc., the amount of reagent can be easily, accurately and quickly reduced to, for example, μl. Multiple samples can be analyzed.

  In FIG. 11, as described above, a chitinase acting on a structural polysaccharide in a living body is used as an enzyme, and a polysaccharide having a p-nitrophenyl group joined to a polysaccharide is used as a substrate. By supplying a pollutant, the degree of enzyme activity of chitinase can be analyzed, and the influence of the environmental pollutant on the chitinase activity, that is, the inhibitory property can be examined. In the analysis, the type and concentration of chemical substances can be examined for inhibition.

  In addition, the same chemical substance is supplied from independent chemical substance supply ports at four different concentrations, and the enzyme and substrate are similarly inserted from the common chemical substance supply port to act as an inhibitor (inhibitor). It can also be used to determine the concentration range.

  Alternatively, two types of substances that can be inhibited can be added at two concentrations, and the influences can be examined.

  As mentioned above, the invention made by the present inventor has been specifically described based on the embodiment. However, the present invention is not limited to the embodiment, and various modifications can be made without departing from the scope of the invention. Needless to say.

  In the above description, the analysis according to the present invention has been described by taking the case of the influence of environmental substances in the living body of chitinase as an example. However, the present invention can be applied to reactions of enzymes other than chitinase or in vivo. However, the present invention is not limited to this enzyme reaction, and can also be used for reactions that require precise temperature control.

  The present invention can be effectively used in the field of chemical analysis such as parallel microanalysis of chemical substances related to in vivo enzyme reactions and the like.

1 is a perspective view of a microchip according to the present invention. (A) is a top view which shows the flow-path formation pattern of the surface side of the silicon substrate which comprises a laminated chip, (b) is a top view which shows the condition of the back surface side. It is sectional drawing which showed partially the formation part of a liquid feeding port. It is explanatory drawing which shows typically the procedure of the flow-path formation to a silicon substrate. (A) is explanatory drawing which showed typically the supply condition to the independent chemical substance supply port in the evaluation of the equivalence of a flow path, and a common chemical substance supply port, (b) is the recovery condition in each flow path. It is explanatory drawing shown. (A) is explanatory drawing which showed typically the supply condition to the independent chemical substance supply port and common chemical substance supply port in evaluation of the equivalence of a flow path, (b) is the condition of the light absorbency in each flow path It is explanatory drawing which showed. It is explanatory drawing which showed the collection | recovery amount in each flow path in the experiment of the structure shown to Fig.6 (a). (A) is explanatory drawing which showed typically the supply condition to the independent chemical substance supply port and common chemical substance supply port in evaluation of the equivalence of a flow path, (b) is the condition of the light absorbency in each flow path It is explanatory drawing which showed. (A) is explanatory drawing which showed typically the supply condition to the independent chemical substance supply port and common chemical substance supply port in evaluation of the equivalence of a flow path, (b) is the condition of the light absorbency in each flow path It is explanatory drawing which showed. It is explanatory drawing which shows the outline | summary of the parallel microanalysis using an inhibitor, an enzyme, and a substrate. It is explanatory drawing which shows the enzyme function of chitinase typically.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Silicon substrate 10a Front surface 10b Back surface 20 Glass plate 30 Glass plate 40 Flow path 41 Supply port 42 Through passage 43 Glass tube 44 Liquid supply port 50 Independent chemical substance supply port 50a Independent chemical substance supply port 50b Independent chemical substance supply port 50c Independent chemistry Substance supply port 50d Independent chemical substance supply port 60 Common chemical substance supply port 60a Common chemical substance supply port 60b Common chemical substance supply port 70 Discharge port 70a Discharge port 70b Discharge port 70c Discharge port 70d Discharge port 100 Microchip 101 Oxide film 102 Photo Resist

Claims (8)

A parallel microanalysis method for chemical substances using a microchip,
The microchip has a plurality of channels in the order of μm that penetrate in the thickness direction and connect both the front and back surfaces,
Each of the plurality of channels is formed into an equivalent channel having a recovery rate of 97% or more of the initial supply amount to the plurality of channels.
The plurality of flow paths have independent reactant supply ports connected independently to each of the plurality of flow paths,
The plurality of channels have a common reactant supply port branched and connected to each of the plurality of channels.
The reactant supplied from the independent reactant supply port and the reactant supplied from the common reactant supply port are reacted in each of the plurality of flow paths, and the reaction product after the reaction is recovered to A parallel microanalysis method for chemical substances, characterized by analyzing the reaction state in each of a plurality of flow paths by an analysis means. In the parallel microanalysis method of the chemical substance according to claim 1,
The parallel microanalysis method for a chemical substance, wherein the initial supply amount when the recovery rate of each of the plurality of flow paths is 97% or more is 150 μl or more and 310 μl or less. In the parallel microanalysis method of the chemical substance according to claim 1 or 2,
The parallel microanalysis method for a chemical substance, wherein the plurality of flow paths are formed with a processing accuracy within 1% with respect to the order of 100 μm. In the parallel microanalysis method of the chemical substance according to any one of claims 1 to 3,
From the common reactant supply port, reactants that react with each other are supplied,
A parallel microanalysis method for chemical substances, characterized in that a reaction-inhibiting substance that may inhibit the reaction is supplied from the independent reactant supply port. In the parallel microanalysis method of the chemical substance according to claim 4,
The method for parallel microanalysis of chemical substances, wherein the reaction inhibiting substance is an environmental pollutant. In the parallel microanalysis method of the chemical substance according to claim 4 or 5,
The method for parallel microanalysis of chemical substances, wherein the reactants supplied from the common reactant supply port are an enzyme and a substrate that reacts with the enzyme. In the parallel microanalysis method of the chemical substance according to claim 6,
The enzyme is a sugar chain degrading enzyme,
The method for parallel microanalysis of chemical substances, wherein the substrate is a polysaccharide having an atomic group separated by the sugar chain degrading enzyme. In the parallel microanalysis method of the chemical substance according to claim 7,
The sugar chain degrading enzyme is chitinase,
The method for parallel microanalysis of chemical substances, wherein the polysaccharide is a structural polysaccharide.

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