JPH11271216A - Method and device for fixing material to surface of chip to be measured - Google Patents

Abstract

PROBLEM TO BE SOLVED: To positively fix material such as physiological active material to the surface of a chip to be measured. SOLUTION: The device includes liquid holders 41, 42 capable of holding a solution S containing material to be fixed in the condition of being swollen upward by surface tension and a chip-to-be-measured holder 45 capable of holding a chip A to be measured with the surface positioned downward. The chip-to- be-measured holder 4 is put near to the solution S held at the end of the liquid holder 42 to adhere the solution S to the chip A to be measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for immobilizing a substance such as a physiologically active substance on the surface of a measurement chip, and a method for measuring the same.

[0002]

2. Description of the Related Art It is common practice to drop a sample liquid or the like on the surface of a measurement chip and observe and analyze the sample liquid or the like by an appropriate measuring means. For example, a sample liquid is placed on a light-transmitting substrate, irradiated with light, and the sample is analyzed based on variables such as the refractive index and the absorptance of reflected light and transmitted light. Optical analysis methods that can detect changes in biologically active substances with high sensitivity in various test methods using immune reactions in clinical tests, etc., for example, optical analysis methods using surface plasmon resonance (SPR) Is mentioned.

In the case of the above-mentioned optical analysis method using surface plasmon resonance, a measuring chip used in an optical analyzer generally corresponds to a light-transmitting substrate, a metal thin film and an object to be analyzed from below. A measurement chip comprising an immobilized membrane on which a physiologically active substance is immobilized, and having such a configuration,
It is set on the prism of the optical analyzer so that the transparent substrate side faces. The sample liquid is continuously fed to the immobilized membrane surface on which the physiologically active substance is immobilized using a liquid supply pump, or the liquid surface of the cell containing the sample liquid is brought into contact with the immobilized membrane, An interaction between the physiologically active substance and the analyte is caused (for example, see Japanese Patent Publication No. 5-2181, Japanese Patent Application Laid-Open No. 63-75542, etc.). Also,
In order to immobilize the physiologically active substance on the immobilization film, a method in which the substrate provided with the immobilization layer is immersed in a concentrated solution of the physiologically active substance or dropped using a microdispenser has been adopted.

[0004]

As described above, in the case of the optical analysis method using surface plasmon resonance, the optical analysis of the incident light irradiating the back surface of the translucent substrate and the reflected light from the metal thin film is performed. Required information from the quantitative analysis, the physiologically active substance on the metal film on the surface of the translucent substrate constituting the measurement chip may have a very small area, and may be immobilized on the immobilization film. The purpose can be sufficiently achieved if a very small amount of the sample liquid is present on the physiologically active substance. However, a method of immobilizing a physiologically active substance by immersing a substrate provided with an immobilization layer in a concentrated solution of a physiologically active substance requires a large amount of a concentrated solution. In addition, even when the interaction between the physiologically active substance and the analyte is caused by the method described in the above publication, preparation of a large amount of sample solution is required, and time and cost are required for sample preparation. There is a loss.

The present inventors have used a conventionally known microdispenser as a means for supplying a trace amount of a concentrated solution or a sample solution of a physiologically active substance onto the surface of a measurement chip, ie, an immobilization film on the surface of a light-transmitting substrate. An attempt was made to drop a droplet on the surface of the measurement chip. However, depending on the hydrophilicity / hydrophobicity of the measurement chip surface and the nature of the solution to be dropped, for example, as shown in FIGS.
The droplet P that falls from the tip of the needle 11 does not stay at a predetermined position on the substrate 20 that is below the tip of the needle 11, but is displaced. Experience that it is difficult to keep the droplets in place. In particular, when the surface of the measurement chip had a hydrophobic surface, and the protein solution was dropped on the surface, the above phenomenon was likely to occur. The required analysis can be performed by dropping an amount that can cover the desired site even if the position is slightly displaced.However, protein solutions are generally expensive. You must avoid as much as possible.

As a countermeasure, as shown in FIG. 10, a droplet P is formed at the tip of the needle 11 of the microdispenser 10 (FIG. 10a), and even when the solution comes into contact with the substrate surface, the solution is still ejected. And after injecting a predetermined amount (FIG. 10B)
Next, a method of raising the needle 11 (FIG. 10C) was attempted. By this method, the drop position is relatively stable, the droplet can be held at a predetermined position for a required time, and the analysis of a small amount is possible, but the control on the microdispenser side is not easy, and the mass production level This method is not effective.

[0007] An object of the present invention is to eliminate the above-mentioned inconvenience caused when a substance such as a physiologically active substance is immobilized on the surface of a measurement chip, and a liquid containing the substance to be immobilized. It is an object of the present invention to provide a method and an apparatus for immobilizing a substance on the surface of a measurement chip, which can surely immobilize the substance on the surface of the measurement chip. The mode of immobilization of the substance on the surface of the measurement chip includes a mode of immobilizing a substance such as a physiologically active substance on an immobilization film formed on the surface of the chip, and a mode of immobilizing the substance thus immobilized. In contrast, there are two modes in which an analyte in a sample solution interacts, but the “immobilization method” and “immobilization apparatus” in the present invention are used as including both modes.

[0008]

In order to solve the above-mentioned problems, a method for immobilizing a substance on the surface of a measurement chip according to the invention of Brother 1 of the present application is to expand a liquid containing a substance to be immobilized upward by surface tension. The measurement chip is separated from the liquid after the surface of the measurement chip is brought into close contact with the swollen liquid and left for a predetermined time. In the method for immobilizing a substance on the surface of a measurement chip according to the invention of Brother 2 of the present application, a liquid containing a substance to be immobilized is stored in a container, and the surface of the measurement chip is applied to the liquid in the container. The measurement chip is separated from the liquid after being left in contact for a predetermined time.

In any of the above inventions, the free movement of the liquid containing the substance to be immobilized is restricted, and the liquid is placed on a desired surface portion of the measurement chip on which the substance is to be immobilized for a predetermined time, that is, in the liquid. Time until the reaction between the substance contained in the sample and the surface substance of the measurement chip progresses and is fixed,
The liquid can be reliably held. Therefore, the required substance can be reliably immobilized with a small amount of liquid. In the immobilization method according to the present invention, the substance to be immobilized is a physiologically active substance, and the measurement chip has a light-transmitting substrate and a hydrophobic substance for immobilizing the physiologically active substance formed on one surface of the light-transmitting substrate. It functions particularly effectively in the case of a measurement chip for an optical analyzer having an immobilized film.

The present invention also provides, as an apparatus for carrying out the above method, a liquid holder capable of holding a liquid containing a substance to be immobilized upward by surface tension, and a measuring chip. It has at least a measuring chip holder capable of holding the measuring chip with its surface facing downward, and a moving mechanism that operates to move the measuring chip holder toward and away from the tip of the liquid holder. Also disclosed is an apparatus for immobilizing a substance on the surface of a measurement chip, which is characterized by the following. The liquid holder may be connected to a container containing a liquid containing a substance to be immobilized via a pipe, and the solution in the container may be supplied to the liquid holder via the pipe. A liquid containing a substance to be immobilized may be dropped into the liquid holder using a microdispenser.

[0011]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail based on preferred embodiments with reference to the accompanying drawings. In the following description, a case where a physiologically active substance is immobilized on the surface of a measurement chip suitable for use in an immunosensor utilizing surface plasmon resonance as an optical analyzer will be described as an example. , But not limited to this
It also includes a measurement chip used for any other analyzer.

The surface plasmon resonance phenomenon means that the intensity of monochromatic light reflected from a boundary between an optically transparent substance such as glass and a metal thin film layer depends on the refractive index of a sample on the exit side of the metal. Therefore, the sample can be analyzed by measuring the intensity of the reflected monochromatic light. The measuring chip used in the analyzer is basically composed of a translucent substrate, a metal thin film formed on one surface of the substrate, and an immobilization film for immobilizing a physiologically active substance formed on the metal thin film. It is composed of FIG. 1 illustrates the measurement chip, in which a metal thin film 2 is disposed on a light-transmitting substrate 1 and an immobilization film 3 for immobilizing a physiologically active substance 4 is formed thereon. The light-transmitting substrate 1 is generally made of glass or a material transparent to laser light, and has a thickness of about 0.1 to 5 mm.

The metal thin film 2 is only required to be capable of generating surface plasmon resonance.
Gold, silver, platinum, copper, aluminum and the like can be mentioned, and these can be used alone or in combination. In addition, an intervening layer made of chromium or the like may be provided between the light-transmitting substrate 1 and a layer made of gold, silver, or the like in consideration of the adhesion to the light-transmitting substrate 1. The thickness of the metal thin film 2 is preferably 100 to 2000 °
It is about 00 to 500 °.

The physiologically active substance 4 is a substance to be analyzed (for example,
The protein is not particularly limited as long as it can interact with an antigen or the like, and examples thereof include an immunoprotein, an enzyme, a microorganism, and a bacterium. As the immunity protein, for example, an antibody having an analyte as an antigen can be used. The antibody is not particularly limited, and various immunoglobulins, ie, Ig
G, IgM, IgA, IgE, IgD can be used. Specifically, when the analyte is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks and the like as an antigen, for example, use an antibody such as an anti-atrazine antibody, an anti-kanamycin antibody, an anti-methamphetamine antibody be able to.

The enzyme is not particularly limited as long as it has an activity on an analyte or a substance metabolized from the analyte, and various enzymes such as oxidoreductase, hydrolase, isomerism, etc. Synthase, lyase, synthase and the like can be used. Specifically, when the analyte is glucose, glucose oxidase can be used, and when the analyte is cholesterol, cholesterol oxidase can be used. Also, pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics,
When cocaine, heroin, cracks, and the like are used as the analytes, an enzyme such as acetylcholinesterase, catecholamine esterase, noradrenaline esterase, and dopamine esterase that shows a specific reaction with a substance metabolized from them can be used. .

The microorganisms and bacteria are not particularly limited, and various microorganisms and bacteria including Escherichia coli can be used. The physiologically active substance 4 may be a DNA base chain, and can specifically bind a complementary base chain. The immobilization membrane 3 for immobilizing the physiologically active substance 4 may be any layer on which the physiologically active substance 4 is supported or immobilized. For example, the porous material may be made of synthetic fiber, natural fiber, inorganic fiber, or the like. A woven fabric, knitted fabric, nonwoven fabric, or a porous inorganic or organic material is used (see JP-A-3-164195). Further, it may be a thin film made of a material having a specific functional group based on a chemical or biochemical reaction.

In order to immobilize the physiologically active substance 4 on the immobilization membrane 3, it is necessary to keep a predetermined amount of the physiologically active substance 4 in contact with the immobilization membrane 3 for some time. The method and apparatus for immobilizing a substance on the surface of a measurement chip according to the present invention are extremely effective for use in immobilizing a substance in such a case. Hereinafter, using the method and the apparatus according to the present invention, the physiologically active substance 4 is immobilized on the immobilized film 3 formed on the surface of a measurement chip suitable for use in an immunosensor utilizing surface plasmon resonance as an optical analyzer. An example of immobilization will be specifically described.

The applicant of the present invention has filed Japanese Patent Application No.
No. 241276, a transparent substrate, a metal film disposed on the transparent substrate, a hydrophobic or electrostatically bonded film such as an organosilicon film disposed on the metal film, and disposed on the film A suitable measurement chip for use in an immunosensor utilizing surface plasmon resonance, which comprises a physiologically active substance, has been proposed. In that case, the immobilization method and device according to the invention are particularly effective.

FIG. 2 shows an example of a measuring chip. In this measuring chip A for an optical analyzer, the above-mentioned translucent substrate 1 has a side of about 18 mm and a thickness of 0.1 to 0.5 mm.
It is a glass plate of the degree. Other materials for the substrate 1 include:
There is translucency like unstretched polyethylene terephthalate, unstretched polycarbonate, triacetate, etc.
A resin material that does not show anisotropy with respect to polarized light and has physical properties excellent in processability can also be used effectively. In the center of one surface of the translucent substrate 1, the above-mentioned metal thin film 2 and fixed film 3 are provided.
Are formed to form an analysis region 5 in which are stacked.

As a specific example, for example, a layer made of chromium and then a layer made of gold are formed on a 13 mm × 18 mm, 0.3 mm thick blue plate glass as the light-transmitting substrate 1 by sputtering to form an analysis region 5. did. Sputtering was performed at 100 W for 30 seconds for chromium and 100 W for 150 seconds for gold. The thickness of the obtained chromium layer was 32.2 °, and the thickness of the gold layer was 474 °. The light-transmitting substrate having the metal layer was immersed in a 1 mM ethanol solution of undecanethiol for 24 hours, and FIG.
An organic thin film layer (hydrophobically bonded film such as an organic silicon film) corresponding to the fixed film 3 was formed.

FIG. 3 shows a method for immobilizing a substance on the surface of a measurement chip according to the present invention, in which a physiologically active substance is coated on the fixing film 3 (reaction region 5) of the measurement chip A for an optical analyzer shown in FIG. This is an explanation of one mode of immobilization. First, a concentrated solution S of a physiologically active substance is placed in an upright bottomed cylindrical container 30, and a conventionally known appropriate liquid such as a microdispenser or a pipette is used. The concentrated solution S is supplied using a supply unit, and a part of the supplied concentrated solution S
0 is swelled upward from the upper end of FIG.
a). From above the cylindrical container 30 in this state, the measurement chip A for an optical analyzer is lowered with the fixed film 3 (reaction region 5) facing downward (or the cylindrical container 30 is raised). The bulging portion of the concentrated solution S and the fixing film 3 of the measurement chip A are brought into close contact with each other (FIG. 3B). If left for a predetermined period of time (about 2 hours) in a state of close contact, the reaction between the fixed film 3 and the physiologically active substance 4 proceeds, and the biologically active substance 4 is immobilized on the fixed film 3. After the immobilization is completed, the measurement chip A is separated from the close contact with the concentrated solution S, whereby the measurement chip A for an optical analyzer as schematically shown in FIG. 1 can be obtained.

FIG. 4 shows another embodiment of the method for immobilizing a substance on the surface of a measurement chip according to the present invention. In this case, a bottomed cylindrical container 35 having an open top is used. The container 35 is sized so that the chip A can enter therein, and a predetermined amount of the concentrated solution S is stored in the container 35 (FIG. 4A). The measuring chip A is accommodated in the container with the fixing film 3 (reaction region 5) facing downward from above, and the concentrated solution S and the fixing film 3 of the measuring chip A are brought into close contact with each other (FIG. 4B). By taking out the measurement chip A from the container 35 after a predetermined time has elapsed, the measurement chip A for an optical analyzer as schematically shown in FIG. 1 can be obtained.

As described above, according to the present invention, a small amount of the concentrated solution S of the physiologically active substance can be securely brought into close contact with the analysis region 5 of the measurement chip A, and It is possible to easily prepare the measurement chip A for an optical analyzer securely fixed at a required position. Therefore, waste of a concentrated solution of an expensive physiologically active substance can be eliminated, and a low-cost optical analysis operation is established.

FIG. 5 shows an example of an apparatus for carrying out the method for immobilizing a substance on the surface of a measurement chip according to the present invention. In this example, a plurality of measurement chips A. It can be fixed. Device 40
The liquid holder 41 capable of holding the solution S containing the substance to be immobilized upward by surface tension, and the surface of the measurement chip A (the surface of the reaction region 5) facing downward. And a measurement chip holder 45 capable of holding the measurement chip A in a posture. The liquid holder 41 has a plurality of cylindrical bodies 42 standing upright and a first pipeline 43 for communicating the lower ends of the tubular bodies 42 with each other. 43 is the pipeline 4 of the younger brother 2
4 and the solution supply pump Pa, it is connected to the container 60 containing the solution S.

Further, the apparatus 40 includes the liquid holder 41
Of the measuring tip holder 4 toward the tip of the cylindrical body 42 of FIG.
5 is provided with an appropriate moving mechanism such as a screw mechanism which operates to make the 5 approach and separate. At the time of immobilization, the liquid supply pump Pa is operated by an appropriate control mechanism to send the concentrated solution S of the physiologically active substance in the container 60 toward the liquid holder 41, and a part of the concentrated solution S is caused by the surface tension. The cylindrical body 42 is in a state of swelling from the tip. In this state, the measurement chip holder 45 is lowered, and the swelling portion of the concentrated solution S and the fixed film 3 of the measurement chip A are removed.
(Reaction region 5) and left for a predetermined time. In the meantime, the reaction between the immobilizing membrane 3 and the physiologically active substance 4 proceeds, and the liquid holder 41 and the measurement chip holder 45 are separated from each other, whereby the measurement chip for an optical analyzer as schematically shown in FIG. A many (25 in the illustrated),
Can be obtained at the same time.

FIGS. 6 and 7 show another example of an apparatus for carrying out the method for immobilizing a substance on the surface of a measurement chip according to the present invention. This apparatus 50 comprises a solution S containing a substance to be immobilized.
Liquid holder 5 having a large number of bottomed holes 52 capable of holding the swelling upward by surface tension.
1 and a measurement chip holder 55 (FIG. 7) capable of holding a large number of measurement chips A in a posture in which the surface of the measurement chip A (the surface of the reaction region 5) faces downward.
An appropriate moving mechanism that functions to move the measuring chip holder 55 closer to or away from the tip of the bottomed hole 52 of the liquid holder 51 is provided. In this example, the moving mechanism is a liquid holder 51 as shown in FIG.
Vertical holes 53 formed in the four corners of the measuring chip holder 5
5 includes four through holes 56 formed at four corners, and pins 54 inserted into the vertical holes 53 and the through holes 56.

At the time of immobilization, as shown in FIG. 6, only the liquid holder 51 is placed below the microdispenser 10 which is controlled in three axial directions (X, Y, Z), and is controlled via a control device (not shown). Micro dispenser 10
, The concentrated solution S of the physiologically active substance 4 is dropped into the bottomed hole 51 of each measurement chip A. At this time, the amount of dripping is controlled so that a part of the concentrated solution S dropped swells above the upper end surface of the bottomed hole 51 due to its surface tension.

All or required bottomed holes 51
After the concentrated solution S is dropped, the liquid holder 51 is moved to the side, and the pin 54 is set up in the vertical hole 53. Next, the measurement chip holder 55 is gradually lowered in a posture in which the surface of each measurement chip A (the surface of the reaction region 5) faces downward, and a posture in which the pins 54 pass through through holes 56 formed in four corners. Let it. As a result, the liquid holder 51 and the measurement chip holder 55 gradually approach each other, and the swelling portion of the concentrated solution S and the fixing film 3 (reaction area and region 5) of the measurement chip A come into close contact with each other. In this state, the reaction is allowed to proceed for a predetermined period of time to allow the reaction between the immobilizing film 3 and the physiologically active substance 4 to proceed. Then, the liquid holder 51 and the measurement chip holder 55 are separated from each other, as schematically shown in FIG. A large number (16 in the drawing) of the measurement chips A for the optical analysis device can be obtained simultaneously.

When a required analysis is performed by using the measurement chip A for an optical analyzer prepared as described above by, for example, the above-described optical analysis using surface plasmon resonance, the measurement is performed as shown in FIG. Is set on the prism 70 of the optical analyzer so that the transparent substrate 1 side faces. The sample liquid to be analyzed is continuously fed to the fixed membrane surface 3 on which the physiologically active substance 4 is fixed by using a liquid supply pump, or the liquid surface of the cell containing the sample liquid is brought into contact with the fixed membrane. And causes an interaction between the physiologically active substance and the analyte. However, such a method requires a large amount of sample liquid. If it is desired to perform the analysis using a small amount of sample solution, the analysis region 5 having the bioactive substance 4 having the sample solution immobilized thereon, using the method and apparatus for immobilizing the sample solution on the measurement chip surface according to the present invention. It is sufficient to make it adhere to. After holding the sample liquid at that position for a predetermined time to cause a desired reaction, the measuring chip A having the surface of the analyte thus prepared may be set for optical analysis.

Although the method and apparatus for immobilizing a substance on the surface of a measurement chip according to the present invention are applied to a measurement chip of an optical analyzer utilizing surface plasmon resonance, the present invention has been described above. Not limited to, for example, such as optical analysis to detect the color reaction of the enzyme, such as preparation of a measurement chip in the case of analysis using light transmitted through the sample solution instead of reflected light as a variable, It can be used in the field. Further, as a moving mechanism that operates to move the measuring chip holder toward and away from the liquid holder, the one shown in FIG. 7 is merely an example, and an electric moving means such as a screw driving mechanism or a servomotor is used. A conventionally known mechanism such as a driving mechanism according to the above can be used as appropriate.

[0031]

By using the method and apparatus for immobilizing a substance on the surface of a measurement chip according to the present invention, it is possible to reliably immobilize a required substance on the surface of the measurement chip with a small amount of liquid. The analysis can be performed at low cost and with high efficiency.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view illustrating a main part of a measurement chip suitable for use in an optical analyzer utilizing surface plasmon resonance.

FIG. 2 is a perspective view illustrating an example of a measurement chip for an optical analyzer.

FIG. 3 is a diagram illustrating one embodiment of a method for immobilizing a substance on the surface of a measurement chip according to the present invention.

FIG. 4 is a diagram illustrating another embodiment of the method for immobilizing a substance on the surface of a measurement chip according to the present invention.

FIG. 5 is a view for explaining an example of an apparatus for carrying out the method for immobilizing a substance on the surface of a measurement chip according to the present invention.

FIG. 6 is a view for explaining another example of an apparatus for carrying out the method for immobilizing a substance on the surface of a measurement chip according to the present invention.

FIG. 7 is a diagram illustrating a use state of the device illustrated in FIG. 6;

FIG. 8 is a diagram illustrating an example of a case where a measurement chip for an optical analyzer is set and used in the optical analyzer.

FIG. 9 is a diagram illustrating the behavior of a dropped droplet.

FIG. 10 is a diagram illustrating the behavior of a dropped droplet.

[Explanation of symbols]

A: measuring chip for optical analyzer, 1: translucent substrate, 2
... Metal thin film, 3 ... Immobilized film, 4 ... Bioactive substance, 5 ... Analysis area, 10 ... Micro dispenser, 30, 35 ...
Cylindrical containers, 40, 50: Device for immobilizing substances on the surface of the measurement chip, 41, 51: Liquid holder, 42: Cylindrical body, 43
... pipe line, 45, 55 ... measuring chip holder, S ... liquid (concentrated solution of physiologically active substance)

Claims (6)

[Claims] A liquid containing a substance to be immobilized is swelled upward by surface tension, and the surface of the measurement chip is brought into close contact with the swelled liquid and left for a predetermined time. A method for immobilizing a substance on the surface of a measurement chip, wherein the substance is separated from a liquid. 2. A liquid containing a substance to be immobilized is housed in a container, and the surface of the measurement chip is brought into close contact with the liquid in the container and left for a predetermined time, and then the measurement chip is separated from the liquid. A method for immobilizing a substance on the surface of a measurement chip, characterized in that: 3. The substance to be immobilized is a physiologically active substance, and the measurement chip has a translucent substrate and an immobilization film formed on one surface of the translucent substrate for immobilizing the physiologically active substance. 3. The method for immobilizing a substance on the surface of a measurement chip according to claim 1, wherein the method is a measurement chip for an optical analyzer. 4. A liquid holder capable of holding a liquid containing a substance to be immobilized upward by surface tension in a state of being swollen upward, and holding the measurement chip in a posture in which the surface of the measurement chip faces downward. A device for immobilizing a substance on a surface of a measurement chip, comprising at least a measurement chip holder capable of moving the measurement chip holder toward and away from the tip of the liquid holder. . 5. The liquid holder is connected via a conduit to a container containing a liquid containing a substance to be immobilized,
The apparatus for immobilizing a substance on a surface of a measurement chip according to claim 4, further comprising liquid supply means for supplying the solution in the container to the liquid holder through the conduit. 6. The apparatus according to claim 4, further comprising a microdispenser for dropping a liquid containing a substance to be fixed in the liquid holder.