JPH11271307A - Measuring chip for optical analyzing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve an initial analysis target sufficiently with an extremely small amount of a sample solution, by providing at least one measuring part of a substance to be detected and also at least one reference, and correcting part for a translucent substrate in which a physiologically active substance layer is formed on its one side. SOLUTION: A metal thin film is arranged on a translucent substrate 1, and a fixing film fixing a physiologically active substance is formed on it. At the time of analysis, a few drops of a sample solution (d) are dribbled to the introducing opening S1 of the void space S of a measuring chip A1. The sample solution (d) enters the inside of the void space S, which is a sample chamber, and reaches a discharging opening S2. As a result, the surface of an analysis region 10 is filled with the sample solution (d), and the physiologically active substance and an object to be analyzed on the fixing film act with each other. Next, the measuring chip A1 is transferred into an optical diagnosing device to perform analysis using surface plasmon resonance. By this, it is possible to simplify the pretreatment of analysis and to eliminate wastage of a sample solution to establish low-cost optical analyzing operation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring chip for an optical analyzer, and more particularly, to a method for irradiating a physiologically active substance layer provided on a light-transmitting substrate with light toward a sample solution, thereby forming a sample on the substrate. For optical analysis, it is intended to compensate for the effects of non-specific binding, sample physical properties, and temperature, and to be used effectively when observing a detected substance accurately over time. .

[0002]

2. Description of the Related Art It is common practice to dispose a sample liquid on a light-transmitting substrate, irradiate the sample with light, and analyze the sample based on variables such as the refractive index and the absorptance of reflected light and transmitted light. . For example, in various test methods using an immune reaction in a clinical test or the like, an optical analysis method capable of detecting a change in a physiologically active substance with high sensitivity, for example, surface plasmon resonance (S
For example, an optical analysis method using (PR) is used.

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 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 interaction between the physiologically active substance and the analyte is performed. (See, for example, Japanese Patent Publication No. 5-2181, Japanese Patent Application Laid-Open No. 63-75542).

As described above, most of the supply of a sample liquid to a measurement chip in a conventional optical analyzer uses a liquid supply pump or a sample liquid storage cell. In the case of the optical analysis method used, necessary information is obtained from optical analysis of incident light irradiating the back surface of the light-transmitting substrate and light reflected from the metal thin film, and the surface of the light-transmitting substrate is obtained. If a very small amount of sample liquid is present on the metal thin film, the purpose can be sufficiently achieved. However, in actuality, since a very small amount of sample is used, simultaneous measurement for compensation and reference is required to obtain accurate measurement results.

[0005] As a conventional technique relating to this, there are a sensing part having two or more detection surfaces, each having a functional group (Japanese Patent Application Laid-Open No. 4-501606), and a sensing part having a sensing substance fixed on a metal thin film. Having a non-fixed reference portion (Japanese Patent Laid-Open No. 5-288672);
A metal thin film having a detection part and a reference part, further comprising a coating layer of a certain material covering the outer surface of the reference part (Japanese Patent Application Laid-Open No. 9-257699), and one having a sub-zone (Japanese Patent No. 2504877) There is. However, each of them has a configuration in which the function of the reference / correction unit cannot be sufficiently performed.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned inconvenience of a conventional measuring chip for an optical analyzer by supplying a very small amount of sample liquid to the measuring chip. The object of the present invention is to provide a measurement chip for an optical analysis device which can sufficiently achieve the initial analysis purpose only by performing the preparation, measurement, and judgment work in a short time and at low cost. .

[0007]

Means for Solving the Problems As a result of intensive studies in view of the above problems, the present inventors have found that by providing the reference / compensation unit on the same substrate as the measurement unit, a sufficient amount of sample liquid for analysis purposes can be obtained. The inventors have found that the sample is introduced into the sample liquid chamber on the substrate, and have completed the present invention. That is, the present invention comprises (1) a light-transmitting substrate, and a physiologically active substance layer formed on one surface of the light-transmitting substrate. (2) a measurement chip for an optical analysis device, wherein the correction unit has at least one or more of each at the same time.
The measurement chip for an optical analyzer according to claim 1, wherein the translucent substrate has no physiologically active substance layer in the reference / correction unit.

(3) The measuring chip for an optical analyzer according to claim 1, wherein the translucent substrate is provided with a coating layer of a certain material covering an outer surface of the reference / correction unit. 4. The measurement for an optical analyzer according to claim 2, wherein the material covering the outer surface of the reference / correction portion of the light-transmitting substrate is a layer which is hydrophilic and has no functional group. Chip, (5) the translucent substrate is used for a surface plasmon resonance measurement device.
A measurement chip for an optical analyzer according to any one of the above-mentioned items.

[0009] The feature of the present invention is to refer to a single chip.
The reference / correction unit and the detection unit may be provided independently. Alternatively, the detection / substance unit may be independent, or the detection substance may be provided on the entire surface and covered with the reference / correction substance.
The reference / correction unit has no functional group, and is characterized by being hydrophilic in order to increase the wettability of the metal surface. As a merit of this hydrophilicity, the sample liquid uniformly touches the chip, and accurate temperature compensation becomes possible.

Examples of the substance having no functional group include inorganic compounds, for example, glass, silica, alumina, ceramic or metal oxide. Sol gel (coating of a dispersion or the like) or the like is used. In the present invention, the optical analyzer refers to any device capable of optically analyzing an object to be analyzed. For example, in addition to the above-described optical analyzer using surface plasmon resonance, an ultraviolet spectroscope , An infrared spectrometer, a visible light spectrometer, a fluorescence spectrometer, a Raman spectrometer, and the like. Further, the measurement chip for an optical analyzer generally refers to a member capable of carrying in and out an object to be analyzed in a light irradiation area in the optical analyzer.

The sample liquid chamber may be disposed directly on the light-transmitting substrate. However, a measurement chip used in an optical analyzer for performing various measurements utilizing an immune reaction in a clinical test or the like. In this case, a fixing film for fixing a physiologically active substance is provided on the surface of the light-transmitting substrate. The fixing film does not need to be provided over the entire surface of the light-transmitting substrate, and may be formed at least in a region irradiated with light for the purpose of analysis. Also,
In the case of a measurement chip used for an optical analyzer utilizing surface plasmon resonance as described above, a metal thin film is provided on at least a part of the surface of a light-transmitting substrate, and the surface of the metal thin film is provided with the physiologically active substance. Is further provided.

[0012]

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 utilizing surface plasmon resonance as an optical analysis device will be described as an example, but the measurement chip for an optical analysis device of the present invention includes: The present invention includes, but is not limited to, a measurement chip used for any other optical analyzer.

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 the sample on the metal emission side. 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 light-transmitting substrate, a metal thin film formed on one surface of the substrate, and a fixing film for fixing a physiologically active substance formed on the metal thin film. Be composed.

FIG. 1 illustrates the measuring 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 only needs to be capable of generating surface plasmon resonance, and examples of the metal include gold, silver, platinum, copper, and aluminum. These may be used alone or in combination. it can. In consideration of the adhesion to the translucent substrate 1,
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. Metal thin film 2
Has a thickness of preferably 100 to 2000 °, and usually about 100 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 the 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.

The method of immobilizing the physiologically active substance 4 on the immobilization membrane 3 may be performed by a conventional method. For example, a method of bringing a predetermined amount of the physiologically active substance 4 into contact with the immobilization membrane 3 for a predetermined time, immersion, and impregnation , Micro dispensing, inkjet, gravure printing, screen printing and the like. Incidentally, the applicant of the present invention, in the earlier application, Japanese Patent Application No. 9-241276, describes a method for immobilizing a physiologically active substance on a fixing membrane by forming a hydrophobic bond or an electrostatic bond instead of a chemical bond (covalent bond). Is disclosed.

In order to cause a hydrophobic bond, it is necessary to have a hydrophobic surface in the outermost layer. Therefore, a gold compound containing a saturated alkyl chain terminal or a fluorine atom or a mixture of gold and fluorine is used. In a hydrophobic bond, in order to form a gold compound containing a fluorine atom or a mixture of gold and fluorine, a conventional thin film forming technique such as sputtering, vacuum deposition, ion plating, CVD, plasma polymerization, Dry etching, spin coating, etc. can be used.

As for the electrostatic bonding, a sulfur compound can be used between the two in order to immobilize a physiologically active substance on the gold surface as a physical adsorption method, and among them, alkanethiol is preferable. With this method, detachment of the physiologically active substance from the metal film was reduced.

Next, a preferred embodiment of a measuring chip used for an optical analyzer utilizing the above surface plasmon resonance will be specifically described. 2 is an example, FIG. 2A is an exploded perspective view, and FIG. 2B is a perspective view after assembly. In the measurement chip A1 for an optical analyzer, the above-mentioned translucent substrate 1 has one side of 18
It is a glass plate having a thickness of about 0.1 mm and a thickness of about 0.1 to 0.2 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.

The material of the side plate 12 and the top plate 13 is not limited to a Teflon sheet, and materials such as industrial plastic and glass may be used. The size of the space S is also arbitrary as long as the sample liquid can enter from the inlet S1 to the vicinity of the outlet S2 by capillary action as described later. 1 and side plate 12, top plate 13
May be determined by calculation or experimentally according to the material of the material. In general, in the case of capillary action, in liquid (sample liquid)
The height H of the liquid column raised from a capillary vertically attached to is given by the following equation when the tube wall is completely wetted by the liquid: γ = (１ ／) × r × ρ × g × H (r: tube , Ρ: density difference between liquid and gas in contact with it, g: gravitational acceleration), which may be set as a basic formula and set on a case-by-case basis.

In FIG. 2b, reference numeral 20 denotes a liquid-absorbing pad, which is a superabsorbent polymer such as cloth, nonwoven fabric, absorbent cotton, filter paper, or a polyacrylic acid, polyvinyl alcohol, or polyethylene oxide superabsorbent gel. Made of material. 3 and 4 show the measurement chip A for this optical analyzer.
1 shows an operation procedure for performing an optical analysis using surface plasmon resonance. As shown in FIG. 3A, the operator brings the tip of the pipette 30 containing the sample solution close to the inlet S1 of the cavity S of the measuring tip A1, and drops one or several drops. The dropped sample liquid d penetrates into the space S, which is the sample chamber, by the capillary phenomenon, and the discharge port S
2 (FIG. 3b). As a result, the analysis region 1 exposing at least a part of its surface to the space S.
0 is filled with the sample solution d, and the fixed film 3
Interaction between the physiologically active substance 4 immobilized on the sample and the analyte in the sample liquid d occurs.

The thus-prepared measuring chip A1 is carried into the optical analyzer, and as shown in FIG. 4, its back side is arranged so as to be in contact with the prism 31 of the apparatus. Optical analysis using resonance is performed. That is, a buffer solution or a reference solution is dropped on a measurement chip, a baseline is measured, the buffer solution is removed using a water absorbing pad or the like, and then a target surface plasmon resonance measurement is performed by dropping a sample solution. Can be. When the above-mentioned liquid absorbing pad 20 is used, the liquid absorbing pad 20 is brought into contact with the discharge port S2 side of the measuring chip A1 at that position or in a state where it has been taken out of the optical analyzer to absorb the sample liquid d. (See FIG. 3c). When the liquid absorbing pad 20 is used in a state where the liquid absorbing pad 20 is attached to the optical analyzer, the next sample liquid can be dropped simultaneously with the liquid absorption.

As described above, 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 several sample liquids each time. In addition to simplifying the pre-analysis work, waste of the sample liquid can be eliminated, and a low-cost optical analysis work is established. Further, the sample liquid once entering the sample liquid chamber S does not easily leak out and may touch the operator's hand, so that the handling at the time of loading and unloading to and from the analyzer is extremely rapid. This has the advantage of being easy and improving the analysis accuracy.

FIG. 5 shows another embodiment of the measuring chip for an optical analyzer according to the present invention. The measuring chip A2 is different from the measuring chip A1 in that the width of the top plate 13 is shorter than the width of the side plate 12. In this case, since the discharge port side of the sample liquid chamber (vacant space) S has a portion S3 which is partially opened upward, the work of discharging the sample liquid becomes easy. In the case of the measuring chip A2 of this form, it is effective to use a liquid absorbing pad 20a having a convex portion 21 large enough to enter the open portion S3 on the side surface thereof. Can be faster.

FIG. 6 shows still another embodiment of the measuring chip for an optical analyzer according to the present invention. The measuring chip A3 is different from the measuring chip A1 in that the shape of the sample liquid chamber (vacancy) S is larger in the cross-sectional area of the outlet S2 than in the inlet S1. ing. In this case, there is an advantage that the measurement can be sped up by increasing the discharge speed of the sample liquid.

FIG. 7 shows still another embodiment of the measuring chip for an optical analyzer according to the present invention. In the measuring chip A4, the width of the top plate 13 of the measuring chip A3 is shorter than the width of the side plate 12. Therefore, as in the case of the measurement chip A2, a portion S4 in which the discharge port side of the sample liquid chamber (vacant space) S is partially opened upward is formed, thereby facilitating the discharge operation of the sample liquid. Also in this case, the liquid-absorbing pad 20b has a convex portion 22 (preferably a trapezoidal convex portion 22 as shown in the figure) large enough to enter the open portion S4 on its side surface. That is valid.

FIG. 8 shows a transport holder 40 for facilitating the setting and taking out of the optical analysis device measuring chip A1 from the optical analysis device. The transfer holder 40 is made of a material such as a general-purpose plastic such as polypropylene or polyvinyl chloride. One side is a measurement chip engaging portion 41, and the other side is a holder portion 42 for a worker to carry and carry by hand. ing. The measuring chip engaging portion 41 has concave grooves 43 formed on both sides, and the peripheral edge of the light transmitting substrate 1 of the measuring chip A1 is inserted into the concave grooves 43, 43. Locked to 40. Reference numeral 45 denotes a stop member used as needed, which is attached to the opening side of the measuring chip engaging portion 41 by means of an insertion pin 46 or the like, and the measuring chip A1
To avoid accidentally moving and falling off. Further, the entire holder including the measurement chip may be disposable.

The above description is a description of a preferred embodiment in a case where the measuring chip for an optical analyzer according to the present invention is used as a measuring chip of an optical analyzer utilizing surface plasmon resonance. There exists a modification of. For example, in the case where analysis is performed using light transmitted through the sample solution d instead of reflected light as a variable, such as optical analysis for detecting a color reaction of an enzyme, the metal thin film 2 or the fixed film 3 may be used.
Is unnecessary, and the top plate 13 is made of a translucent material. In addition, this may be used as long as the visible light region is detected. However, in the case where the ultraviolet region is detected, it is recommended to use a configuration using quartz glass.

Example 1 In this example, a measurement chip having the basic layer structure shown in FIG. 1 was prepared. As the transparent substrate, a blue plate glass (manufactured by Matsunami Glass Industry Co., Ltd.) having a size of 13 mm × 18 mm and a thickness of 0.3 mm was used. On this transparent substrate,
A layer made of chromium and then a layer made of gold were formed by sputtering. Sputtering is 100 W for 30 seconds for chromium and 100 W for 150 seconds for gold.
Seconds. The thickness of the obtained chromium layer was 32.2 °, and the thickness of the gold layer was 474 °.

As the surface plasmon resonance biosensor, SPR-20 manufactured by Electrochemical Instruments was used, and its sensor head and supply / drainage system were modified and used. The transparent substrate having the metal layer was immersed in a 1 mM ethanol solution of undecanethiol for 24 hours to form an organic thin film layer. In this step, it is possible to perform the processing more uniformly and efficiently by immersing the entire substrate than by performing partial processing. A physiologically active substance can be immobilized based on hydrophobic bonds. (See above)

Example 2 The basic layer configuration is the same as that of FIG. 1, but the layout of the substance-to-be-detected and the reference / correction section is arranged as shown in FIG. One or more of the substance to be detected measuring unit and the reference / correction unit are provided on the same chip, respectively, and can correspond to a plurality of inspection items. Immobilization of the physiologically active substance is required in the detection / measurement section, but is not necessarily provided in the reference / correction section. In this embodiment, no physiologically active substance is provided in the reference / correction unit. According to this embodiment, a constant value is always obtained for a signal for reference, and a stable and accurate measurement of a detection signal can be performed. In addition, depending on the physiologically active substance immobilized on the reference / correction site, non-specific adsorption is unavoidable, so that the layer configuration in the present embodiment is more effective.

(Example 3) Only the reference and correction parts were coated on the layer structure of Example 1. As a result, the hydrophilicity of the surface can be improved, the measurement error can be minimized, and stable performance against temperature fluctuation can be exhibited.

(Embodiment 4) With respect to the layer structure of the embodiment 2,
Only the reference and correction sites were coated. If the coating agent is firmly fixed, the same effect as that of the third embodiment can be obtained. In other words, the hydrophilicity of the surface can be improved, the measurement error can be minimized, and stable performance is exhibited even when the temperature changes.

[0036]

By using the measuring chip for an optical analyzer according to the present invention, required optical analysis can be performed with a very small amount, waste of the sample liquid can be eliminated, and the optical cost can be reduced at low cost. Analytical work can be established. In addition, it is expected that an error caused by the skill level of the worker is reduced, and it is possible to provide anyone with an opportunity to carry out the measurement of the inspection very accurately and simply.

[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 according to the present invention, FIG. 2a is an exploded perspective view, and FIG.
b is a perspective view after assembly.

FIG. 3 is a view for explaining a use mode of the measurement chip for an optical analyzer in FIG. 2;

FIG. 4 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. 5 is a perspective view illustrating another example of a measurement chip for an optical analyzer.

FIG. 6 is a perspective view illustrating still another example of the measurement chip for an optical analyzer.

FIG. 7 is a perspective view illustrating still another example of the measurement chip for an optical analyzer.

FIG. 8 is a perspective view illustrating an example of a measurement chip for an optical analyzer having a transport holder.

[Explanation of symbols]

A1 to A4: measuring chip for optical analyzer, 1: translucent substrate, 2: metal thin film, 3: fixed film, 4: bioactive substance,
Reference numeral 10: analysis area, 12: side plate, 13: top plate, S: sample liquid chamber (vacant space), S1: inlet, S2: outlet, 20, 20
a, 20b: Liquid absorption pad.

Claims (5)

[Claims] 1. A light-transmitting substrate comprising: a light-transmitting substrate; and a physiologically active substance layer formed on one surface of the light-transmitting substrate, wherein the light-transmitting substrate has at least a substance-to-be-detected measurement section and a reference / correction section. A measurement chip for an optical analysis device, wherein each of the measurement chips has at least one of them simultaneously. 2. The measuring chip for an optical analyzer according to claim 1, wherein the translucent substrate has no physiologically active substance layer in the reference / correction section. 3. The measuring chip for an optical analyzer according to claim 1, wherein the translucent substrate includes a coating layer of a certain material covering an outer surface of the reference / correction unit. 4. The optical device according to claim 2, wherein the material for covering the outer surface of the reference / correction portion of the light-transmitting substrate is a layer which is hydrophilic and has no functional group. Chip for dynamic analyzer. 5. The measuring chip for an optical analyzer according to claim 1, wherein said translucent substrate is used for a surface plasmon resonance measuring device.