JP2005257603A - Method for manufacturing reactor, reactor, analysis method, and immobilization method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing a reactor to easily immobilize biochemical substances at desired positions not only on a flat plate but also even in a micro-flow path without using solid particles as solid phase, a reactor obtained by this method, an analysis method, and an immobilization method. <P>SOLUTION: Light is irradiated to prescribed positions on a substrate w1 with a light absorption layer M put thereon, thereby removing the absorption layer M at relevant positions. Then, a biochemical substance T1 is position-selectively adsorbed to the substrate w1 at a position from which the absorption layer M has been removed by thereinto introducing a solution of the biochemical substance T1. Then, light is irradiated to another prescribed position to remove the absorption layer M at a relevant position. A biochemical substance T2 is position-selectively adsorbed to the substrate w1 at a position from which the absorption layer M has been removed by thereinto introducing a solution of the biochemical substance T2. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a reactor in which a biochemical substance is immobilized, a manufacturing method thereof, an analysis apparatus using the reactor, and a fixing method. In particular, even when the inner wall of the microchannel is a solid phase, a fixing method and a reactor manufacturing method capable of fixing a biochemical substance at a predetermined position of the solid phase, and a reactor and an analyzer obtained by this manufacturing method About.

  In recent years, in the field of analytical chemistry, research on micro technology (Micro Electro-Mechanical System, hereinafter referred to as MEMS) has progressed rapidly, and micro-measurement of an analyzer (μ-TAS; The movement of Micro Total Analytical System or Lab on a chip) has spread to analysis fields such as proteins, genes, biochemistry, etc., and research is being carried out.

As a bio-MEMS analyzer that performs an immune reaction, an enzyme reaction, and the like, for example, an attempt to analyze using a microchip with solid fine particles as a reaction solid phase has been reported (for example, see Non-Patent Document 1). According to this method, by introducing solid particles such as beads as the reaction solid phase, the surface area on which the antibody is immobilized can be increased, and analysis with higher sensitivity is possible.
In addition, a method has been reported in which a solution of an active substance having a chemically reactive site with respect to the inner wall of the channel is introduced into the capillary channel and the active substance is immobilized on the inner wall by a chemical reaction (see, for example, Non-Patent Document 2). .)
Furthermore, a method of immobilizing an enzyme at a predetermined position by reacting a photocrosslinking agent on a flat resin surface by ultraviolet lithography and further reacting with the enzyme has been proposed (see Non-Patent Document 3). ).
"Development of a dioxin measurement system in a flue using BioMEMS, report on the results (first year)", New Energy and Industrial Technology Development Organization, March 2002 J. et al. P. Cutler, S. C. Jacobson, N.M. Matsubara, J.H. M.M. Ramsey (JP Putler, SC Jacobson, N. Matsubara, J. R. Ramsey), Analytical Chemistry (Anal-Chem), 1998, 70, 3291. Journal of American Chemical Society, (USA), 1993, Vol. 115, page 814. Journal of American Chemical Society.

However, in the bio-MEMS analyzer using solid particles described in Non-Patent Document 1, it is necessary to produce a precise fine structure for retaining the solid particles in the analyzer, and it is necessary to control the particle size of the solid particles. There was a problem that the production cost was high. In addition, since it takes time to insert and discharge solid particles, it is difficult to perform quick measurement.
Further, in the case of Non-Patent Document 2, although solid particles are not used, there is a problem that the active substance cannot be immobilized only at a desired position because the entire inner wall of the flow channel is chemically modified.

Moreover, in the method of nonpatent literature 3, it cannot fix unless it is on the flat plate of an open state. Therefore, when trying to form a microchannel, it is necessary to fix a lid on an open flat plate after the enzyme is immobilized. However, practical fixing means inactivate biochemical substances such as enzymes in both plasma irradiation and thermal bonding.
Therefore, according to the method of Non-Patent Document 3, it is possible to immobilize a biochemical substance at a desired position on an open flat plate, but the biochemical substance is placed at a desired position in the microchannel. It was difficult to fix.

  The present invention has been made in view of the problems of the prior art, and does not use solid particles as a solid phase, but also puts biochemical substances at desired positions not only on a flat plate but also in a microchannel. It is an object of the present invention to provide a method for producing a reactor that can be easily fixed, a reactor and an analyzer obtained by this method, and a method for fixing.

The present invention includes the following aspects.
[1] A method for producing a solid phase capable of adsorbing a biochemical substance and a reactor in which the biochemical substance is adsorbed and fixed at a predetermined position of the solid phase, wherein the light absorption is removable on the solid phase by light. A first step of stacking layers; a second step of irradiating the predetermined position with light to remove the light absorption layer at the position; and the biochemical substance on the solid phase at the position where the light absorption layer has been removed. And a third step of adsorbing the reactor.

[2] The method for producing a reactor according to [1], further including a step of forming a flow path having the solid phase on the inner surface side before the second step.
[3] The reactor according to [1] or [2], wherein a plurality of types of biochemical substances are fixed at different predetermined positions by repeating the second step and the third step one or more times. Production method.
[4] A reactor manufactured by the manufacturing method described in any one of [1] to [3].
[5] An analyzer comprising a reactor and a detector for detecting a reaction in the reactor, wherein the reactor is the reactor described in [4].

  [6] A method for adsorbing and fixing a biochemical substance at a predetermined position on a solid phase capable of adsorbing a biochemical substance, the first step of laminating a light absorbing layer removable by light on the solid phase And a second step of irradiating the predetermined position with light to remove the light absorption layer at the position, and a third step of adsorbing the biochemical substance to the solid phase at the position where the light absorption layer has been removed. A method for immobilizing a biochemical substance, comprising:

According to the invention of the aspect of [1], since the solid phase can be exposed at the position irradiated with light, the biochemical substance can be immobilized only at a desired position.
According to the invention of the aspect of [2], the biochemical substance can be fixed at an arbitrary position in the flow channel after the flow channel is formed. In this case, since it is not necessary to perform a bonding process such as plasma irradiation after fixing the biochemical substance, the biochemical substance is not transformed or deactivated. Therefore, the biochemical substance can be easily fixed at a desired position even in the microchannel. According to the invention of the aspect [3], a plurality of types of biochemical substances can be fixed at arbitrary positions. Can be Therefore, a complex reaction system can be formed, and integration of reactions can be realized.

According to the invention of the aspect of [4], a reactor in which a biochemical substance is immobilized at a desired position can be obtained.
According to the invention of the aspect of [5], since a reactor in which a biochemical substance is immobilized at a desired position is used, it is possible to make an analyzer that can perform various analyzes quickly and easily and can be easily downsized. it can. Further, the inside of the flow path can be set to a micro level, and in that case, a very small area also increases as a specific surface area. Therefore, the analysis can be performed by fixing a very small amount of biochemical substance, and is particularly suitable for analysis using an expensive biochemical substance.
According to the invention of the aspect of [6], the biochemical substance can be immobilized at a desired position.

[reactor]
A reactor according to an embodiment of the present invention will be described with reference to FIG. However, the present invention is not limited to the embodiments described below. In the following drawings, for convenience of explanation, the dimensional ratio of each part does not match the actual one.

As shown in FIG. 1, the reactor of this embodiment is configured by bonding a substrate w1 and a substrate w2. A flow path f having an inlet f1 and an outlet f2 is formed between these two substrates.
Although the light absorption layer M is provided on the flow path f side of the substrate w1, a part thereof is removed. Then, biochemical substances T1 and T2 are adsorbed and fixed to the removed portions.

The substrate w1 has a surface on the channel f side that functions as a solid phase in the present invention. Therefore, it is necessary that at least the surface on the channel f side can adsorb the biochemical substance to be fixed.
Moreover, it is preferable that it is transparent with respect to the light used in the manufacturing method mentioned later. This is because when it is opaque, it is necessary to protect it from the irradiation light so that it is not removed together with the light absorption layer M by light.
Moreover, since it uses as a reactor, it is preferable that it has chemical resistance, and it is preferable that it is a material with easy joining with the board | substrate w2.
Examples of the material of the substrate w1 include glass such as quartz, polydimethylsiloxane resin (hereinafter referred to as “PDMS”), polyfluorinated ethylene resin, polyester resin, polymethyl methacrylate resin, polystyrene resin, metal, and the like.
Of these, glass, PDMS, polyester resin, and polymethyl methacrylate resin are preferable because of their high transparency, moldability, and bondability.

The substrate w2 is positioned on the light incident side in the manufacturing method described later. Therefore, it is necessary to be transparent to the light to be used and is preferably transparent. This is because, when there is no transparency, the light absorption layer cannot be removed by light in the manufacturing method described later.
Moreover, since it uses as a reactor, it is preferable that it has chemical resistance, and it is preferable that it is a material with easy joining with the board | substrate w1, and formation of the flow path f.
Examples of the material of the substrate w2 include glass such as quartz, PDMS, polyester resin, polymethyl methacrylate resin, polystyrene resin, and polyfluorinated ethylene resin.
Of these, glass, PDMS, polyester resin, and polymethyl methacrylate resin are preferable because of their high transparency, moldability, and bondability.
It is particularly preferable that the substrate w1 is glass or PDMS and the substrate w2 is PDMS.

The light absorption layer M is required to be made of a material that has absorptivity to light used in the manufacturing method described later and can be removed by this light.
Moreover, it is preferable that the light absorption layer M has low adsorptivity with respect to the biochemical substance to be fixed. Even if there is some degree of adsorptivity, immobilization of the biochemical substance can be prevented by using a blocking agent, as will be described later in the production method.
The light-absorbing material may be a metal, but chromium is preferable because it efficiently absorbs light having a wide range of wavelengths. Gold is suitable without requiring a blocking agent because protein is hardly adsorbed by the action of molecules having thiols.
Although there is no limitation in particular in the thickness of the light absorption layer M, it is preferable to set it as 10-100 nm. When the thickness is 10 nm or more, the irradiated light is sufficiently absorbed and heated, so that ablation (jetting due to heating) is likely to occur and removal is facilitated. Moreover, the light quantity required for removal does not become excessive by setting it as 100 nm or less.

Examples of the biochemical substances T1 and T2 include proteins, DNA, glycoproteins, glycolipids, cells, viruses, and bacteria.
The biochemical substances T1 and T2 are adsorbed and fixed to the substrate w1. This adsorption may be physical adsorption or chemical adsorption, but is preferably physical adsorption because it is easy to produce.

Next, a method for manufacturing a reactor according to an embodiment of the present invention will be described with reference to FIGS.
As shown in FIG. 2, first, a substrate w1 is prepared. And as shown in FIG. 3, the light absorption layer M is laminated | stacked by vapor deposition etc. on the substantially whole surface of the position corresponding to the flow path f of this board | substrate w1. The light absorption layer M may be laminated slightly wider than the position corresponding to the flow path f, but it is preferable that the light absorption layer M be stacked almost completely coincident with the position corresponding to the flow path f. This is to facilitate the bonding with the substrate w2.

Next, as shown in FIG. 4, a substrate w <b> 2 in which a recess that becomes the flow path f is prepared in advance, and the side on which the recess is formed is attached to the substrate w <b> 1 as an inner surface. Although there is no limitation on the method of forming the concave portion that becomes the flow path f in advance on the substrate w2, it can be formed using, for example, a convex mold. Further, there is no particular limitation on the bonding method between the substrate w1 and the substrate w2, and a known bonding means such as bonding by plasma irradiation, anodic bonding, or thermal bonding may be appropriately employed according to the material of each substrate.
At this stage, the flow path f side of the substrate w1 is covered with the light absorption layer M.

Next, as shown in FIG. 5, the blocking agent B is coated on the inner surface of the flow path f. The blocking agent B is for suppressing the non-specific adsorption of the biochemical substances T1 and T2 on the substrate w2 or the light absorption layer M. If the substrate w2 and the light absorption layer M are not adsorptive with respect to the biochemical substances T1 and T2, the use of the blocking agent B is unnecessary.
Examples of the blocking agent B include bovine serum albumin (BSA), non-fat dry milk, casein, and γ globulin.
In order to coat the blocking agent B, a solution of the blocking agent is introduced from the inlet f1 into the flow path f, retained for a predetermined time, and then discharged from the outlet f2 and washed.

Next, as shown in FIG. 6, light is irradiated to a place (predetermined place) where the biochemical substance T1 is to be fixed. Thereby, the light absorption layer M at the irradiation position absorbs the light and is removed by ablation. At this time, the blocking agent B on the light absorption layer M to be removed is also removed at the same time. As a result, as shown in FIG. 7, the substrate w1 is exposed at the irradiation position.
There is no limitation in particular in the wavelength of the light to irradiate, For example, the light of 200-1500 nm can be used. Light with a shorter wavelength is preferable because the irradiation place can be easily narrowed down. In addition, a laser beam is preferable because a thin parallel beam with sharp directivity can be obtained.

Next, as shown in FIG. 8, a solution of the biochemical substance T1 is introduced from the inlet f1 into the flow path f, and is retained for a predetermined time, and then discharged from the outlet f2. The residence time may be appropriately determined in consideration of the adsorptivity of the biochemical substance T1 to the substrate w1, the concentration of the solution to be introduced, and the like. Thereby, as shown in FIG. 9, the biochemical substance T1 is adsorbed on the substrate w1 exposed by light irradiation.
In addition, after discharging | emitting the solution of biochemical substance T1, it is preferable to wash | clean by introduce | transducing a washing | cleaning liquid. At that time, if the blocking agent B is removed from the light absorbing layer M and the substrate w2, it is preferable to coat the blocking agent B again.

Next, as shown in FIG. 10, light is irradiated to a place (predetermined place) where the biochemical substance T2 is to be fixed. Thereby, the light absorption layer M at the irradiation position absorbs the light and is removed by ablation (spouting by heating). At this time, the blocking agent B on the light absorption layer M to be removed is also removed at the same time. As a result, as shown in FIG. 11, the substrate w1 is exposed at the irradiation position.
As this irradiation light, the same light as the light irradiated to the place which should fix biochemical substance T1 can be used.

Next, as shown in FIG. 12, a solution of the biochemical substance T1 is introduced into the flow path f from the inlet f1, and is retained for a predetermined time, and then discharged from the outlet f2. The residence time may be appropriately determined in consideration of the adsorptivity of the biochemical substance T2 to the substrate w1, the concentration of the solution to be introduced, and the like. Thereby, as shown in FIG. 13, the biochemical substance T2 is adsorbed on the substrate w1 exposed by light irradiation.
And after discharging | emitting the solution of biochemical substance T1, blocking agent B is removed by introduce | transducing a washing | cleaning liquid and fully wash | cleaning, and the reactor of FIG. 1 can be obtained.

In the present embodiment, a reactor in which two types of biochemical substances are fixed is used, but the type name of the reactor to be fixed may be one or three or more. In the case of three or more types, the reactor can be manufactured by repeating the steps from FIG. 6 to FIG.
Each biochemical substance may be immobilized at a plurality of locations. There is no particular limitation on the fixed amount and fixed area per place.

In the present embodiment, the concave portion serving as the flow channel f is formed on the substrate w2 side. However, the concave portion constituting the flow channel f may be formed on either side of the substrate w1 and the substrate w2. There is no limitation on the cross-sectional shape, area, and length of the flow path f. The arrangement of the flow path f is not limited to a straight line, and various changes such as a curved flow path and a branched flow path can be made.
In the present embodiment, the light absorption layer M is laminated on the substrate w1 before the flow path is formed. However, the light absorption layer M may be laminated by plating or the like after the flow path is formed. In this case, since the light absorbing layer M is also laminated on the substrate w2, the surface of the substrate w2 on the flow path f side can function as a solid phase in the present invention, similarly to the substrate w1. However, in that case, when irradiating light to the light absorption layer M on the substrate w2, it is necessary to devise such as filling the flow path with colored water so that the light does not reach the substrate w1 side. .

[Analysis equipment]
The analyzer of the present invention comprises the reactor of the present invention and a detector for detecting the reaction in this reactor. As the detector, an optical detector such as a fluorescence detector and an absorptiometric detector, an electrochemical detector such as a pH electrode, an H ₂ O ₂ electrode, and a conductivity electrode, and various other known detectors can be used as appropriate. .

The reaction in the reactor is performed as follows, for example.
(Reaction example 1)
The first antibody is immobilized on the reactor, and the antigen and the second antibody are sequentially bound using the antigen-antibody reaction. Thereafter, a phosphor is added to the second antibody. By detecting the fluorescence of the phosphor, it is possible to analyze any one of the first antibody concentration, the antigen concentration, and the second antibody concentration.

(Reaction example 2)
Glucose oxidase is immobilized on the upstream side of the reactor, and horseradish peroxidase is immobilized on the downstream side. When a glucose solution to which Amplex Red is added is flowed, glucose is first decomposed by glucose oxidase to generate hydrogen peroxide. Next, horseradish peroxidase produces resorufin from this hydrogen peroxide and Amplex Red. By detecting the fluorescence of resorufin, it is possible to analyze the glucose concentration.

Examples of the present invention will be described below, but the scope of the present invention is not limited to these examples.
(Formation of flow path)
First, a glass template having a length of 60 mm, a width of 30 mm, and a thickness of 1 mm was prepared. A chromium thin film having a width of 1 mm, a length of 3 cm, and a thickness of about 30 nm was deposited on a substantially central portion of the substrate.
On the other hand, a convex mold is used in advance on a PDMS plate having a length of 60 mm, a width of 30 mm, and a thickness of 3 mm, and a recess having a width of 1 mm, a length of 3 cm, and a depth of 60 μm, and φ1.6 mm at both ends thereof. What formed two holes penetrated in the opposite surface was prepared.
Then, both were joined by making the chromium thin film side of a glass template and the recessed part side of a PDMS board into the inner surface. Bonding was performed by bonding both after plasma irradiation and further heating.

(Immobilization of protein 1)
A 5% aqueous solution of bovine serum albumin (BSA) was introduced into the channel formed as described above and allowed to stay for 1 hour, and then washed with 1 mL of Tris-HCl buffer.
Next, a pulse oscillation YAG laser (532 nm) is irradiated several times to the place (fixing position 1) where protein 1 is to be fixed in a state where the Tris-HCl buffer is filled in the flow path, and the chromium thin film and BSA at the irradiation position are irradiated. And removed.
Next, a 0.005% Tris-HCl buffer solution of fluorescent protein 1 (Avidin Alexafluoro 488) was introduced into the flow path and allowed to stay for 1 hour, and then washed with 1 mL of Tris-HCl buffer.
When the fixing position 1 was irradiated with excitation light (480 nm), Alexafluoro 488 fluorescence at 530 nm was detected, confirming that protein 1 was immobilized. In addition, even if the other part was irradiated with a laser, only very weak fluorescence was detected, and it was confirmed that protein 1 could be immobilized in a position-selective manner.

(Immobilization of protein 2)
A 5% aqueous solution of bovine serum albumin (BSA) was again introduced into the flow channel after immobilizing protein 1 and allowed to stay for 1 hour, and then washed with 1 mL of Tris-HCl buffer.
Next, a pulse oscillation YAG laser (532 nm) was irradiated several times to the place where the protein 2 should be fixed in a state where the Tris-HCl buffer was filled in the flow path, and the chromium thin film and BSA at the irradiation position were removed.
Next, 0.1% Tris-hydrochloric acid buffer solution of fluorescent protein 2 (horseradish peroxidase) was introduced into the flow path and allowed to stay for 30 minutes, and then washed with 1 mL of Tris-hydrochloric acid buffer.
Next, a buffer solution containing Amplex Red and hydrogen peroxide was introduced into the flow path, and resorufin was generated by the action of horseradish peroxidase.
When excitation light (550 nm) was irradiated to the fixed position 2, fluorescence of resorufin at 580 nm was detected, and it was confirmed that protein 2 was immobilized. In addition, even if the other part was irradiated with laser, only very weak fluorescence was detected except for the fixing position 1, and it was confirmed that the protein 2 could be immobilized in a position-selective manner.

It is sectional drawing of the reactor which concerns on embodiment of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention. It is sectional drawing in the manufacturing process of the reactor of this invention.

Explanation of symbols

w1 ... substrate, w2 ... substrate, M ... light absorption layer, B ... blocking agent, T1 ... biochemical substance, T2 ... biochemical substance, f .... Flow path

Claims (6)

A method for producing a solid phase capable of adsorbing a biochemical substance, and a reactor in which the biochemical substance is adsorbed and fixed at a predetermined position of the solid phase,
A first step of laminating a light absorbing layer removable by light on the solid phase;
A second step of irradiating the predetermined position with light to remove the light absorption layer at the position;
A third step of adsorbing the biochemical substance on the solid phase at the position where the light absorption layer is removed;
The manufacturing method of the reactor characterized by having.   The method for producing a reactor according to claim 1, further comprising a step of forming a flow path having the solid phase on the inner surface side before the second step.   The method for producing a reactor according to claim 1 or 2, wherein the second step and the third step are further repeated one or more times to fix a plurality of types of biochemical substances at different predetermined positions.   A reactor manufactured by the manufacturing method according to any one of claims 1 to 3.   An analyzer comprising a reactor and a detector for detecting a reaction in the reactor, wherein the reactor is the reactor according to claim 4. A method of adsorbing and fixing the biochemical substance at a predetermined position on a solid phase capable of adsorbing the biochemical substance,
A first step of laminating a light absorbing layer removable by light on the solid phase;
A second step of irradiating the predetermined position with light to remove the light absorption layer at the position;
A third step of adsorbing the biochemical substance on the solid phase at the position where the light absorption layer is removed;
A method for immobilizing a biochemical substance, characterized by comprising:

Images