JPH11271185A - Droplet-immobilizing device for measurement chip and measurement chip preparation device with it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a droplet immobilization device for a measurement chip that can effectively immobilize a droplet with a low affinity with the surface of a measurement chip onto the surface. SOLUTION: In a droplet immobilization device to a measurement chip A, a dropped droplet P needs to be retained on a surface for a long time. A device 30 is provided with machine frames 34 and 35 and a stick 33 that is inclined and extended from the machine frame to the lower part, and the stick 33 can be dipped into the droplet P where the tip part is dropped when the droplet P is dropped into the measurement chip. The stick 33 is preferably constituted by a material that has a higher affinity with the droplet P than that between the droplet P and the surface of the measurement chip A.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a droplet fixing device for a measurement chip which can effectively fix a droplet having low affinity to the surface of the measurement chip, and a measurement chip having the fixing device. It relates to a preparation device.

[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 a fixing film on which a physiologically active substance is fixed, and having such a configuration, is set on a prism of an optical analyzer so that the light-transmitting substrate side faces. The sample liquid is continuously fed to the fixed membrane surface on which the biologically active substance is fixed 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 the biologically active substance is And an analyte (see, for example, Japanese Patent Publication No. 5-2181 and Japanese Patent Application Laid-Open No. 63-75542). Further, 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]

In the case of the optical analysis method utilizing surface plasmon resonance as described above, the optical analysis of incident light irradiating the back surface of the light-transmitting substrate and light reflected from the metal thin film are performed. The bioactive substance on the metal film on the surface of the light-transmitting substrate that constitutes the measurement chip may have a very small area, and the bioactive substance immobilized on the immobilization film If a very small amount of sample liquid is present on the substance, the purpose can be sufficiently achieved. 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 providing a trace amount of a concentrated solution or sample solution of a physiologically active substance on the surface of a measurement chip, ie, on a fixed 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, FIG.
a to 8c, as shown in FIG.
The droplet P falling from the tip of the needle 11 of the needle 1
(1) It does not stay at a predetermined position on the substrate 20 which is a position below the front end, and causes a positional shift, and it is difficult to hold the droplet at a predetermined place for a time required for fixing or reaction. Experienced that. 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.
Is formed at the tip of the needle 11 of the microdispenser 10 (FIG. 9a), the solution is still ejected even when the solution comes into contact with the substrate surface, and after ejecting a predetermined amount (FIG. 9b), the needle 11 is pulled up. (FIG. 9c) The method was tried. 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.

An object of the present invention is to provide a measuring chip which requires a droplet such as a concentrated solution or a sample solution of a physiologically active substance to be dropped on the surface of the measuring chip and kept at the place for a predetermined time. In order to solve the above-mentioned inconvenience caused in the above, for example, it is possible to reliably hold and fix the droplet dropped by an appropriate droplet dropping means such as a micro dispenser on the surface of the measurement chip, It is another object of the present invention to provide a measuring chip droplet fixing device and a measuring chip preparation device having the fixing device, which can surely perform a required analysis with a minimum required amount of sample liquid.

[0008]

According to the present invention, there is provided a measuring chip drop fixing apparatus for solving the above-mentioned problems, which requires a dropped chip to be held on a surface for a predetermined time. A liquid drop fixing device, comprising a machine frame and a stick extending downwardly from the machine frame, wherein the stick is a liquid whose tip is dropped when a droplet is dropped on a measurement chip. It is characterized in that it can enter into the droplet.

In use, the drop fixing device for the measuring chip is set so that the tip of the stick is close to the drop drop position on the surface of the measuring chip, and the droplet dropping device and the surface of the measuring chip are connected. Set in between. When a droplet is dropped from the droplet dropping device in that state, the tip of the stick is positioned in the droplet dropped on the surface of the measurement chip, and the movement of the droplet is restricted. In that state, for example,
Wait for the liquid droplets (concentrated solution of biologically active substance or sample liquid) to react with the material on the surface of the measurement chip, and after a predetermined time, remove the droplet fixing device for the measurement chip, and attach the measurement chip to the analyzer. And perform the desired analysis work.

As described above, by using the droplet fixing device for a measurement chip according to the present invention, even if the affinity between the surface of the measurement chip and the sample liquid (droplet) is extremely low, a predetermined time can be obtained. , It is possible to reliably hold the droplet at a predetermined surface position of the measurement chip, and it is possible to easily prepare a measurement chip that can reliably perform the required analysis with the minimum necessary liquid volume Becomes The stick is tilted downward instead of vertically so as not to hinder the drop of the liquid droplet from above, and the tilt angle is arbitrary.

[0011] Preferably, the stick is made of a material such that the affinity between the stick and the dropped droplet is higher than the affinity between the dropped droplet and the surface of the measurement chip. For example, the measurement chip is composed of a translucent substrate, a metal thin film, and a fixing film on which a physiologically active substance corresponding to an analyte is immobilized from below, and a droplet to be dropped is a protein solution (biologically active substance). If so, it is recommended that the stick be made of a material such as stainless steel, carbon, glass, and the like. The diameter of the stick also depends on the amount of liquid to be dropped, but when preparing a measurement chip for performing optical analysis using the surface plasmon resonance (SPR) described above, a stainless steel of about 0.2 mm to 1.2 mm is used. The needle was effective.

In the droplet fixing device for a measurement chip according to the present invention, preferably, a movable machine frame is used as the machine frame. A stick is attached to the movable machine frame, for example, so that the machine frame can be rotated at an angle of 90 ° with one end serving as a fulcrum,
Alternatively, at least the tip of the stick can be moved toward and away from the surface of the measurement chip to which the droplet is to be fixed, such as by being able to selectively take up and down positions. . With this configuration, it is easy to continuously and uniformly hold and fix the droplet on the surface of the measurement chip.

[0013] More preferably, a plurality of sticks are attached to the machine frame so as to be able to operate on a plurality of measurement chips simultaneously. In this case, for example, it is easy to simultaneously and uniformly fix a sample liquid to a measurement chip case containing a large number of measurement chips such as four rows and five stages.

The present invention further comprises at least one or a plurality of measuring chips, the above-described droplet fixing device for measuring chips, and a microdispenser for dropping liquid droplets on the surface of the measuring chips. A measurement chip preparation device is also disclosed. By controlling the movement of the micro-dispenser in three axial directions and the drop control of the droplets using a known control device, it is possible to simultaneously and uniformly prepare a measurement chip in which the droplets are fixed.

[0015]

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 measurement chip suitable for use in an immunosensor using surface plasmon resonance as an optical analyzer will be described as an example.
The present invention is not limited to this, and includes a measurement chip used for any other analysis device.

The surface plasmon resonance phenomenon means that the intensity of monochromatic light reflected from the 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 measurement chip used in the analyzer is basically composed of a translucent substrate, a metal thin film formed on one surface of the substrate, and a fixing film for immobilizing a physiologically active substance formed on the metal thin film. Consists of

FIG. 1 illustrates the measurement chip, in which a metal thin film 2 is disposed on a light-transmitting substrate 1, and a fixing film 3 for fixing 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 0.1.
About 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 a layer on which the physiologically active substance 4 is supported or immobilized. For example, as a porous material, a fabric made of synthetic fiber, natural fiber, inorganic fiber, or the like, A knitted fabric, a nonwoven fabric, a porous inorganic or organic material, or the like is used (Japanese Unexamined Patent Publication No.
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 general, such a method of immobilizing the physiologically active substance 4 on the immobilization membrane 3 may be performed by a conventional method. For example, a method in which a predetermined amount of the physiologically active substance 4 is brought into contact with the immobilization membrane 3 for a predetermined time, It can be immobilized by a method such as impregnation, microdispenser, ink jet, etc., which will be described later. In the case where the droplet fixing device for a measurement chip according to the present invention is used, a means such as a microdispenser capable of dropping a droplet on the surface of the measurement chip from above is used.

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 Although a measurement chip suitable for use in an immunosensor utilizing surface plasmon resonance comprising a physiologically active substance has been proposed, when a physiologically active substance is immobilized on such a hydrophobically bound membrane, The apparatus for fixing a droplet for a measurement chip according to the present invention is particularly effective.

Next, an example of preparing a measurement chip used for an optical analyzer utilizing surface plasmon resonance as described above will be described as an example. A preferred embodiment of the apparatus will be specifically described. FIG. 2 shows an example of a measurement chip. In this measurement chip A for an optical analyzer, the translucent substrate 1 has a side of about 18 mm,
It is a glass plate having a thickness of about 0.1 to 0.5 mm. As a material for the substrate 1, besides, for example, unstretched polyethylene terephthalate, unstretched polycarbonate, triacetate, etc., it has a light-transmitting property, does not show anisotropy with respect to polarized light, and has excellent workability. Resin materials can also be used effectively. In the center of one surface of the translucent substrate 1, an analysis area 5 in which the metal thin film 2 and the fixed film 3 are stacked is described.
Are formed.

As a specific example, for example, a layer made of chromium and then a layer made of gold are 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 translucent substrate 1. 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.

FIGS. 3 and 4 show that a concentrated solution of a physiologically active substance is applied on the fixing film 3 of the measuring chip A for an optical analyzer by using the measuring chip droplet fixing apparatus 30 according to the present invention. This figure shows a case where a physiologically active substance is immobilized by dropping. The measuring chip droplet fixing device 30 includes a U-shaped leg 31 large enough to accommodate the measuring chip A, a standing plate 32 erected from the U-shaped leg 31, and a little from the upper edge of the standing plate 32. The stick 33 is made of stainless steel and extends downward while being inclined downward. When the measuring chip droplet fixing device 30 is put into a use state, the length and the inclination angle of the stick 33 are as shown in FIG. The tip of the stick 33 is slightly (may be about 0.1 to 5 mm) away from the surface of the analysis area 5 of the measurement chip A as shown in FIG.

In immobilizing a physiologically active substance, FIG.
As shown in FIG. 4, the microchip dispenser 10, which is an example of a device for dropping a concentrated solution of a physiologically active substance, is positioned so that the analysis region 5 of the measurement chip A is positioned below the tip of the needle 11. And then
The measuring chip droplet fixing device 30 is attached to the stick 3
3 is set so that the tip is located substantially at the center of the analysis area 5.

The microdispenser 10 is operated via a control device (not shown) to drop a concentrated solution droplet P of the physiologically active substance 4. When the droplet P falls and reaches the surface of the analysis area 5, the tip of the stick 33 enters the inside of the droplet P or comes into contact with the upper part of the droplet. In this example, the stick 33 is made of stainless steel, and the stick 33 and the stick 33 have a higher affinity than the fixed film (hydrophobically bonded film such as an organosilicon film) 3 and the droplet P of the concentrated solution of the physiologically active substance 4. The affinity with the droplet P is high. Therefore, the droplet P is held at the position where it is dropped without changing its position.

By leaving this state for a predetermined time (about 2 hours), the reaction between the fixed film 3 and the physiologically active substance 4 progresses, and the biologically active substance 4 is immobilized on the fixed film 3. After the immobilization is completed, the measurement chip A is taken out and the excess solution is washed away, whereby the measurement chip A for an optical analyzer as schematically shown in FIG. 1 can be obtained.

As described above, by using the droplet fixing device 30 for a measurement chip according to the present invention, only one drop of a concentrated solution sample solution of a physiologically active substance is dropped onto the analysis area 5 of the measurement chip A. In addition, it is possible to easily prepare the measurement chip A for an optical analyzer to which the amount of the physiologically active substance required for the analysis is securely immobilized. Therefore, waste of concentrated solution of expensive bioactive substance can be eliminated,
A low-cost optical analysis task is established.

Using the measurement chip A for an optical analyzer prepared as described above to perform a required analysis by, for example, the above-described optical analysis using surface plasmon resonance, as shown in FIG. Is set on the prism 40 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. When it is desired to perform the analysis with a small amount of the sample solution, the sample solution is dropped with the physiologically active substance 4 immobilized thereon using the measuring chip droplet fixing device 30 according to the present invention. A desired reaction may be performed by holding the sample liquid.
The method of using the measurement chip droplet fixing device 30 in this case is the same as that of immobilizing a physiologically active substance.

FIGS. 6 and 7 show another embodiment of the measuring chip droplet fixing device 30 according to the present invention. This measuring chip droplet fixing device 30 has legs 35... Standing at four corners of a square machine frame 34, and horizontal bars 36. They are arranged at intervals. A stick 33 made of a stainless steel material is attached at predetermined intervals to the horizontal rails 36 and the machine frame portion parallel to the horizontal rails 36, as shown in FIGS. In the illustrated example, four sticks 33 are attached to each of a five-stage machine frame or a horizontal rail, and a total of 20 sticks 33 are attached to the machine frame.

In using the measuring chip droplet fixing device 30, preferably, the same number of measuring chips A as the number of the sticks 33 are arranged at the lower position of the tip of each stick 33 in the same posture as described above. You. In the illustrated example, a measurement chip holder 20 is used, and a total of 20 measurement chips A, 4 × 5 stages, are fitted in the holder 20.

Holes 2 are formed at the four corners of the measuring tip holder 20.
1 are formed, and legs 35 provided on the machine casing 34 are inserted into the holes 21. In use, a measurement chip holder 20 in which a required number of measurement chips A are fitted is first prepared, and the measuring chip droplet fixing device 30 is mounted thereon.
Is fixed so that the leg 35 is inserted into the hole 21.
It is placed at a position below the micro-dispenser 10 controlled in three axial directions (X, Y, Z), and the micro-dispenser 10 is operated via a control device (not shown), thereby forming a droplet P of the concentrated solution of the physiologically active substance 4. Is sequentially dropped on the analysis area 5 of each measurement chip A. The droplet P is held as it is at the position where it is dropped, and the immobilization of the physiologically active substance proceeds.

When the fixation is completed, the machine frame 34 is moved by hand or by an appropriate moving means, and the measuring chip droplet fixing device 30 is lifted from the measuring chip holder 20 and moved to the side. Let it. Thereafter, the excess solution is washed out, and each measurement chip A is taken out from the measurement chip holder 20 to obtain a plurality of measurement chips A for an optical analyzer as schematically shown in FIG. 1 as a simultaneous and continuous operation. be able to.

Although not shown, the measuring chip drop fixing device 30 of the form shown in FIGS.
It is also possible to pivotally attach to one end of the “0” using a hinge so as to be rotatable. In this case, when dropping the droplet,
As shown in the figure, the machine frame 34 is positioned such that the tip of the stick 33 approaches the surface of the measurement chip holder 20, and rotates 90 ° or 180 ° when the measurement chip A is inserted into and removed from the measurement chip holder 20. Position.

In the above description, the droplet fixing device for a measurement chip according to the present invention and the device for fixing a droplet to a measurement chip of an optical analyzer utilizing surface plasmon resonance are described. This is an explanation in the case of using as a measurement chip preparation device having a fixing device, and the fixing device and the preparation device of the present invention are not limited to this, for example, reflection analysis such as optical analysis for detecting a color reaction of an enzyme. It can be used in various fields such as preparation of a measurement chip in a case where analysis is performed using light transmitted through a sample solution instead of light as a variable.

[0038]

By using the measuring chip for an optical analyzer according to the present invention, it is possible to perform required optical analysis simply by dropping one or a few drops of a sample solution each time, and to perform a pre-analysis operation. Not only the post-processing can be simplified, but also the waste of the sample liquid can be eliminated, and the optical analysis operation can be established at low cost. In addition, it is expected that an error caused by the skill level of the worker is reduced, and it is possible to provide an opportunity for anyone to easily and accurately perform the inspection measurement.

[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 side view for explaining a use state of the measuring chip droplet fixing device according to the present invention.

FIG. 4 is a perspective view from above illustrating a use state of the measuring chip droplet fixing device shown in FIG. 3;

FIG. 5 is a diagram illustrating an example of a case where the measurement chip for an optical analyzer of FIG. 2 is used by being set on the optical analyzer.

FIG. 6 is a top perspective view illustrating the use state of another embodiment of the droplet fixing device for a measurement chip according to the present invention.

FIG. 7 is a cross-sectional view illustrating a use state of the measuring chip droplet fixing device shown in FIG. 6;

FIG. 8 is a perspective view from above illustrating a use state of another embodiment of the droplet fixing device for a measurement chip according to the present invention.

FIG. 9 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 ... Fixing film, 4 ... Biologically active substance, 5 ... Analysis area, 10 ... Micro dispenser, 30 ... Drop fixation device for measuring chip, 33 ... Stick, 34 ... Machine frame, 3
5 leg, P droplet

Claims (6)

[Claims] 1. A device for fixing a droplet to a measuring chip, which needs to hold a dropped droplet on a surface for a predetermined period of time, the device comprising a machine frame and a tilt downward from the machine frame. And a stick extending therefrom, the stick being in a state of being able to penetrate into the dropped droplet when the droplet is dropped onto the measuring chip, wherein Drop fixing device. 2. The stick, wherein the stick is made of a material having a higher affinity for a droplet to be dropped than for the droplet to be dropped and a surface of a measurement chip. Item 2. A drop fixing device for a measurement chip according to Item 1. 3. The machine frame is a movable machine frame, so that at least the tip of the stick can approach and separate from the surface of the measurement chip on which the liquid droplet is to be fixed. 3. The droplet fixing device for a measurement chip according to claim 1 or 2, wherein: 4. The machine frame according to claim 1, wherein a plurality of sticks are mounted on the machine frame so that the plurality of sticks can be simultaneously operated on a plurality of measurement chips. Droplet fixing device for measuring chip. 5. A measuring chip having at least one or more measuring chips, a droplet fixing device for measuring chips according to any one of claims 1 to 4, and a microdispenser for dropping liquid droplets on the surface of the measuring chip. A measurement chip preparation device characterized by the above-mentioned. 6. A measurement chip for an optical analysis device having a light-transmitting substrate and a fixing film formed on one surface of the light-transmitting substrate for fixing a physiologically active substance. The measurement chip preparation device according to claim 5, wherein