JP3897265B2 - Method and apparatus for immobilizing a substance on the surface of a measuring chip - Google Patents

Description

[0001]
BACKGROUND 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 an apparatus measurement for the method.
[0002]
[Prior art]
Usually, a sample solution or the like is dropped on the surface of the measurement chip, and the sample solution or the like is observed and analyzed by an appropriate measurement means. For example, a sample liquid is placed on a light-transmitting substrate and irradiated with light, and the sample is analyzed based on variables such as the refractive index and absorption rate of reflected light and transmitted light. , Optical analysis methods that can detect changes in physiologically active substances with high sensitivity in various inspection methods using immune reactions in clinical tests, for example, optical analysis methods using surface plasmon resonance (SPR), etc. Is mentioned.
[0003]
In the case of the optical analysis method using the surface plasmon resonance described above, the measurement chip used in the optical analyzer is generally a translucent substrate, a metal thin film, and a physiologically active substance corresponding to the analysis object from below. The measurement chip having such a configuration is set on the prism of the optical analyzer so that the translucent substrate side faces. The sample liquid is continuously fed to the immobilized membrane surface on which the physiologically active substance is immobilized using a feed pump, or the liquid surface of the cell containing the sample solution is brought into contact with the immobilized membrane, An interaction between the physiologically active substance and the analyte is caused (see, for example, Japanese Patent Publication No. 5-2181, Japanese Patent Application Laid-Open No. 63-75542). In order to immobilize the physiologically active substance on the immobilization film, a method in which a substrate provided with an immobilization layer is immersed in a concentrated solution of the physiologically active substance or dropped by a microdispenser is used.
[0004]
[Problems to be solved by the invention]
As described above, in the case of an optical analysis method using surface plasmon resonance, necessary information is obtained from optical analysis of incident light irradiated on the back surface of a light-transmitting substrate and reflected light from the metal thin film. 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 a very small amount of the physiologically active substance immobilized on the immobilization film. If the sample liquid is present, the purpose can be sufficiently achieved. However, a method of immobilizing a physiologically active substance by a method in which a substrate provided with an immobilization layer is immersed in a concentrated solution of the physiologically active substance requires a large amount of concentrated solution. Further, even when the interaction between the physiologically active substance and the analyte is caused by the method as described in the above publication, it is necessary to prepare a large amount of sample solution, which requires time and cost for sample preparation. There is a loss.
[0005]
The present inventors have used a conventionally known microdispenser as a means for supplying a concentrated solution or sample solution of a trace amount of physiologically active substance onto the measurement chip surface, that is, a fixed film on the surface of the translucent substrate. Attempts were made to drop droplets on the surface. 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 . 7 a to 7 c, the droplet P falling from the tip of the needle 11 of the microdispenser 10 is below the tip of the needle 11. Experience not only staying at a predetermined position on the substrate 20 but also causing a positional shift, making it difficult to hold the droplets in a predetermined place for the time required for immobilization or reaction. did. In particular, when the surface of the measuring chip has a hydrophobic surface and the protein solution is dropped on the surface, the above phenomenon is likely to occur. Although it is possible to perform the required analysis if the amount that can cover the desired site is dropped even if there is some misalignment, the protein solution is generally expensive, and mass production is Avoid as much as possible.
[0006]
As a countermeasure, as shown in FIG. 8 , a droplet P is formed on the tip of the needle 11 of the microdispenser 10 ( FIG. 8a), and the solution is still ejected even if the solution contacts the substrate surface. After injecting a predetermined amount ( Fig. 8b), an attempt was made to pull up the needle 11 ( Fig. 8c). With this method, the drop position is relatively stable, and the droplet can be held at a predetermined position for the required time, and analysis in a very small amount is possible, but control on the microdispenser side is not easy, and mass production level This method is not effective.
[0007]
An object of the present invention is to eliminate the above-described inconvenience occurring when a substance such as a physiologically active substance is immobilized on the surface of the measurement chip, and a liquid containing the substance to be immobilized is measured on the measurement chip. It is an object of the present invention to provide a method and an apparatus for immobilizing a substance on the surface of a measuring chip that can be reliably immobilized on the surface of the substrate.
In addition, as an aspect of immobilizing a substance on the surface of the measurement chip, an aspect in which a substance such as a physiologically active substance is immobilized on an immobilization film formed on the surface of the chip, and the substance thus immobilized In contrast, there are two modes in which the analyte in the sample solution interacts, but the “immobilization method” and “immobilization apparatus” in the present invention are used as including both modes.
[0008]
[Means for Solving the Problems]
Substance immobilizing method for measuring chip surface by inventions of the present application to solve the aforementioned problem, leave the state that bulges upward by surface tension liquid containing a to be immobilized material bulging out liquid On the other hand, the measurement chip is separated from the liquid after the surface of the measurement chip is brought into close contact and allowed to stand for a predetermined time .
[0009]
Te above Symbol onset bright odor, the free movement of the liquid containing the substance to be immobilized is regulated to a desired surface region of the measuring chip to be immobilized to the substance for a predetermined time, i.e., contained in the liquid The liquid can be reliably held for the time until the reaction between the substance and the substance on the surface of the measuring chip proceeds and is immobilized. Therefore, a required substance can be reliably fixed 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 measuring chip is a hydrophobic substrate that immobilizes the translucent substrate and the bioactive substance formed on one surface of the translucent substrate. This is particularly effective in the case of a measurement chip for an optical analyzer having an immobilization film.
[0010]
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 in a state where the liquid is swollen upward by surface tension, and a surface of the measuring chip below. At least a measurement chip holder capable of holding the measurement chip in a posture directed toward the head, and a moving mechanism that operates to move the measurement chip holder toward and away from the tip of the liquid holder. An apparatus for immobilizing a substance on the surface of a measuring chip is also disclosed.
The liquid holder may be connected to a container containing a liquid containing a substance to be immobilized via a conduit, and the solution in the container may be supplied to the liquid holder via the conduit. A liquid containing a substance to be immobilized may be dropped into the liquid holder using a micro dispenser.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
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 using surface plasmon resonance as an optical analyzer will be described as an example. Not only this but the measurement chip used for other arbitrary analysis devices is also included.
[0012]
The surface plasmon resonance phenomenon is due to the fact that the intensity of monochromatic light reflected from the boundary between an optically transparent substance such as glass and the metal thin film layer depends on the refractive index of the sample on the metal exit side. Yes, so the sample can be analyzed by measuring the intensity of the reflected monochromatic light. The measuring chip used in the analyzer is basically a translucent substrate, a metal thin film formed on one surface of the substrate, and an immobilized film for immobilizing a physiologically active substance formed on the metal thin film. It consists of.
FIG. 1 illustrates the measurement chip. A metal thin film 2 is disposed on a translucent substrate 1, and an immobilization film 3 for immobilizing a physiologically active substance 4 is formed thereon. The translucent 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.
[0013]
The metal thin film 2 only needs to be such that surface plasmon resonance can occur. Examples of the metal include gold, silver, platinum, copper, and aluminum, and these can be used alone or in combination. it can. In consideration of adhesion to the translucent substrate 1, an intervening layer made of chromium or the like may be provided between the translucent substrate 1 and a layer made of gold, silver, or the like. The thickness of the metal thin film 2 is preferably 100 to 2000 mm, and is usually about 100 to 500 mm.
[0014]
The physiologically active substance 4 is not particularly limited as long as it can interact with an analyte (for example, an antigen or the like), and examples thereof include immune proteins, enzymes, microorganisms, and bacteria. 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, that is, IgG, IgM, IgA, IgE, and IgD can be used. Specifically, when the analyte is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. When using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, use antibodies such as anti-atrazine antibodies, anti-kanamycin antibodies, and anti-methamphetamine antibodies. be able to.
[0015]
The enzyme is not particularly limited as long as it shows activity against the analyte or a substance metabolized from the analyte, and various enzymes such as oxidoreductase, hydrolase, isomerase, A desorption enzyme, a synthetic enzyme, etc. can be used. Specifically, glucose oxidase can be used if the analyte is glucose, and cholesterol oxidase can be used if the analyte is cholesterol. In addition, when analyzing pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc., the substances to be metabolized from these substances show a specific reaction, for example, acetylcholinesterase Enzymes such as catecholamine esterase, noradrenaline esterase, and dopamine esterase can be used.
[0016]
The microorganisms and bacteria are not particularly limited, and various microorganisms and bacteria including E. coli can be used. Further, the physiologically active substance 4 may be a DNA base chain, and can specifically bind a complementary base chain.
The immobilization film 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, as a porous material, synthetic fiber, natural fiber, inorganic fiber, or the like is used. A woven fabric, a knitted fabric, a non-woven fabric, a porous inorganic or organic material, or the like 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.
[0017]
In order to immobilize the physiologically active substance 4 on such an immobilized membrane 3, it is necessary to bring a predetermined amount of the physiologically active substance 4 into contact with the immobilized membrane 3 for a predetermined time by some means. The method and apparatus for immobilizing a substance on the surface of a measuring chip according to the present invention is extremely effective for use in immobilizing a substance in such a case. In the following, using the method and apparatus according to the present invention, the physiologically active substance 4 is applied to the immobilized film 3 formed on the surface of a measurement chip suitable for use in an immunosensor using surface plasmon resonance as an optical analyzer. An example of immobilization will be specifically described.
[0018]
In addition, in Japanese Patent Application No. Hei 9-241276, the present applicant has disclosed a transparent substrate, a metal film disposed on the transparent substrate, and a hydrophobic film such as an organic silicon film disposed on the metal film. A measurement chip suitable for use in an immunosensor using surface plasmon resonance comprising a bound or electrostatically bound membrane and a physiologically active substance disposed on the membrane has been proposed. The immobilization method and apparatus according to the present invention are particularly effective when immobilizing a physiologically active substance on a bound membrane.
[0019]
FIG. 2 shows an example of a measuring chip. In the measuring chip A for an optical analyzer, the translucent substrate 1 is a glass plate having a side of about 18 mm and a thickness of about 0.1 to 0.5 mm. It is. Other materials for the substrate 1, such as unstretched polyethylene terephthalate, unstretched polycarbonate, triacetate, etc., are transparent, exhibit no anisotropy with respect to polarized light, and have excellent workability. The resin material possessed can also be used effectively. An analysis region 5 in which the metal thin film 2 and the fixed film 3 are laminated is formed at the center of one surface of the translucent substrate 1.
[0020]
As a specific example, for example, a layer made of chromium and then a layer made of gold were formed by sputtering on a blue plate glass having a size of 13 mm × 18 mm and a thickness of 0.3 mm as the light-transmitting substrate 1. Sputtering was performed at 100 W for 30 seconds for chromium and 100 W for 150 seconds for gold. The resulting chromium layer was 32.2 mm thick and the gold layer was 474 mm thick. The translucent substrate having the above metal layer was immersed in an undecanethiol 1 mM ethanol solution for 24 hours to form an organic thin film layer (hydrophobically bonded film such as an organic silicon film) corresponding to the fixed film 3 in FIG. .
[0023]
As described above, according to the present invention, a small amount of a concentrated solution S of a physiologically active substance can be reliably brought into close contact with the portion of the analysis region 5 of the measurement chip A, and the physiologically active substance is placed at a required position. It is possible to easily prepare the measurement chip A for an optical analyzer that is securely fixed. Therefore, the waste of the concentrated solution of expensive physiologically active substance can be eliminated, and the optical analysis work at low cost is established.
[0024]
FIG. 3 shows an example of an apparatus for carrying out the method for immobilizing a substance on the surface of a measuring chip according to the present invention . In this example, a physiologically active substance can be immobilized simultaneously on a plurality of measuring chips A. I am doing so. The apparatus 40 has a liquid holder 41 that can hold the solution S containing the substance to be immobilized in a state where it bulges upward due to surface tension, and the surface of the measurement chip A (surface of the reaction region 5) downward. The measuring chip holder 45 is configured to hold the measuring chip A in the oriented posture. The liquid holder 41 has a plurality of cylindrical bodies 42 standing upright, and a first pipe line 43 for communicating the lower ends of the respective cylindrical bodies 42, and the first pipe line 43 is connected to the inside of the container 60 containing the solution S via the second pipe 44 and the liquid supply pump Pa.
[0025]
Further, the apparatus 40 includes an appropriate moving mechanism including a screw mechanism that operates to move the measuring chip holder 45 toward and away from the tip of the cylindrical body 42 of the liquid holder 41.
At the time of immobilization, the liquid supply pump Pa is operated by an appropriate control mechanism to send out 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 its surface tension. It is assumed that the tubular body 42 bulges from the tip. In this state, the measurement chip holder 45 is lowered to bring the bulging portion of the concentrated solution S into close contact with the fixed film 3 (reaction region 5) of the measurement chip A, and left for a predetermined time. In the meantime, the reaction between the fixed membrane 3 and the physiologically active substance 4 progresses, and the liquid holder 41 and the measurement chip holder 45 are separated, whereby a measurement chip for an optical analyzer as schematically shown in FIG. A large number (25 in the illustrated example) of A can be obtained simultaneously.
[0026]
4 and 5 are examples of other apparatuses for carrying out the method for immobilizing a substance on the surface of a measuring chip according to the present invention . This apparatus 50 is configured to move a solution S containing a substance to be immobilized upward by surface tension. The liquid holder 51 having a large number of bottomed holes 52 that can be held in a bulged state, and a large number of measurements with the surface of the measuring chip A (the surface of the reaction region 5) facing downward A measurement tip holder 55 ( FIG. 5 ) capable of holding the tip A, and further, the measurement tip holder 55 is moved toward and away from the tip of the bottomed hole 52 of the liquid holder 51. An appropriate moving mechanism that functions in the above is provided. In this example, as shown in FIG. 5 , the moving mechanism includes vertical holes 53 formed at the four corners of the liquid holder 51, through holes 56 formed at the four corners of the measurement chip holder 55, and the vertical holes 53 and the through holes 56. And a pin 54 inserted through the pin.
[0027]
At the time of immobilization, as shown in FIG. 4 , only the liquid holder 51 is placed below the micro dispenser 10 controlled in the three axial directions (X, Y, Z), and the micro dispenser 10 is connected via a control device (not shown). The concentrated solution S of the physiologically active substance 4 is dropped into the bottomed hole 51 of each measurement chip A. At that time, the dropping amount is controlled so that a part of the dropped concentrated solution S bulges upward from the upper end surface of the bottomed hole 51 due to the surface tension.
[0028]
After the concentrated solution S is dropped into all or necessary bottomed holes 51, the liquid holder 51 is moved to the side, and the pins 54 are set up in the vertical holes 53. Next, the measurement chip holder 55 is gradually lowered in such a posture that the surface of each measurement chip A (surface of the reaction region 5) is directed downward and the pin 54 is passed through the through holes 56 formed in the four corners. Let As a result, the liquid holder 51 and the measurement chip holder 55 gradually approach each other, and the bulging portion of the concentrated solution S and the fixed film 3 (reaction area and region 5) of the measurement chip A are brought into close contact with each other. As shown schematically in FIG. 1, the liquid holder 51 and the measuring chip holder 55 are separated after allowing the fixed membrane 3 and the physiologically active substance 4 to proceed by leaving in this state for a predetermined time. A large number (16 in the figure) of measuring chips A for an optical analyzer can be obtained simultaneously.
[0029]
When performing the required analysis by optical analysis using the surface plasmon resonance described above, for example, using the measurement chip A for an optical analyzer prepared as described above, as shown in FIG. It is set on the prism 70 of the optical analyzer so that the translucent substrate 1 side faces. The sample solution to be analyzed is continuously fed to the fixed membrane surface 3 to which the physiologically active substance 4 is fixed using a feed pump, or the liquid surface of the cell containing the sample solution is in contact with the fixed membrane. And causes interaction between the physiologically active substance and the analyte. However, such a method requires a large amount of sample solution. When it is desired to perform analysis with a small amount of sample solution, the analysis region 5 having the physiologically active substance 4 to which the sample solution is immobilized using the method and apparatus for immobilizing a substance on the measurement chip surface according to the present invention. What is necessary is just to stick to. After the sample solution is held at that position for a predetermined time and a desired reaction is performed, the measurement chip A having the analysis target thus prepared on the surface may be set for optical analysis.
[0030]
In the above description, the method and apparatus for immobilizing a substance on the surface of a measurement chip according to the present invention is applied to the measurement chip of an optical analyzer using surface plasmon resonance. However, the present invention is not limited to this. Used in various fields, such as preparation of measurement chips when analyzing light that has passed through the sample liquid instead of reflected light as a variable, such as optical analysis that detects the color reaction of enzymes Is possible. Further, as the moving mechanism that operates to move the measuring chip holder toward and away from the liquid holder, the one shown in FIG. 5 is merely an example, and an electric moving means such as a screw driving mechanism or a servo motor. Conventionally known ones such as a drive mechanism can be used as appropriate.
[0031]
【The invention's effect】
By using the method and apparatus for immobilizing a substance on the surface of a measuring chip according to the present invention, it becomes possible to reliably immobilize a required substance on the surface of the measuring chip with a small amount of liquid, and the required analysis can be performed at low cost. And can be performed with high efficiency.
[Brief description of the drawings]
FIG. 1 is a schematic cross-sectional view for explaining 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 view for explaining an example of an apparatus for carrying out the method for immobilizing a substance on the surface of a measuring chip according to the present invention.
FIG. 4 is a view for explaining another example of an apparatus for carrying out the method for immobilizing a substance on the surface of a measuring chip according to the present invention.
FIG. 5 is a diagram for explaining a use state of the apparatus shown in FIG. 4 ;
FIG. 6 is a diagram for explaining an example in which a measurement chip for an optical analyzer is set and used in an optical analyzer.
FIG. 7 is a diagram for explaining the behavior of a dropped liquid droplet.
FIG. 8 is a diagram for explaining the behavior of a dropped liquid droplet.
[Explanation of symbols]
A ... Measuring chip for optical analyzer, 1 ... Translucent substrate, 2 ... Metal thin film, 3 ... Immobilization film, 4 ... Bioactive substance, 5 ... Analysis region, 10 ... Micro dispenser, 30, 35 ... Cylindrical shape Container, 40, 50 ... Device for immobilizing substance on measurement chip surface, 41, 51 ... Liquid holder, 42 ... Cylindrical body, 43 ... Pipe line, 45, 55 ... Measurement chip holder, S ... Liquid (for bioactive substances) Concentrated solution)

Claims (5)

  The liquid containing the substance to be immobilized is swelled upward due to surface tension, and the measurement chip is separated from the liquid after leaving the measurement chip in close contact with the swelled liquid for a predetermined time. A method of immobilizing a substance on the surface of a measuring chip characterized by For an optical analyzer, the substance to be immobilized is a physiologically active substance, and the measurement chip has a translucent substrate and an immobilized film for immobilizing the physiologically active substance formed on one surface of the translucent substrate. 2. The method for immobilizing a substance on the surface of a measurement chip according to claim 1 , wherein the substance is a measurement chip.   A liquid holder that can hold the liquid containing the substance to be immobilized in a state where it bulges upward due to surface tension, and a measurement chip that can hold the measurement chip with the surface of the measurement chip facing downward An apparatus for immobilizing a substance on the surface of a measurement chip, comprising at least a holder and a moving mechanism that operates to move the measurement chip holder toward and away from the tip of the liquid holder. The liquid holder is connected to a container containing a liquid containing a substance to be immobilized via a conduit, and further includes a liquid supply means for supplying the solution in the container to the liquid holder via the conduit. 4. The apparatus for immobilizing a substance on the surface of a measuring chip according to claim 3, further comprising: The apparatus for immobilizing a substance on the surface of a measurement chip according to claim 3 , further comprising a micro dispenser for dropping a liquid containing the substance to be immobilized in the liquid holder.

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