JPH01502930A - Improved analytical techniques and equipment - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] IMPROVED ANALYTICAL TECHNIQUES AND DEVICE THEREOF FIELD OF INVENTION This invention provides qualitative and and/or analytical techniques by which quantitative detection can be determined. It concerns an apparatus in which the technique may be carried out.

Background of the invention The analytical techniques mentioned depend on the object of analysis that is to be analyzed and the particular type of table. between a receptor substance coated on the surface, e.g. a ligand or a specific binding partner. It is based on the affinity of (a) A surface with a pre-shaped contoured cross-section on a substrate combining at least a predetermined portion of the species to be analyzed. The surface area is coated with a thin film material that can be (b) optically active with respect to radiation at least exceeding the code; The stained surface is brought into contact with the sample and the nuclide is deposited onto the thin film material on the surface. Measure qualitative and/or quantitative changes in optical properties as a result of binding Description of an analytical technique comprising observing the optical properties of said surface portion in order to For details, see international patent publications WO34102578 and WO36101901. Referenced.

As described in these publications, preformed relief sections typically is an optical grating which can be a simple single grating or two or more intersecting gratings. shape, whose ridges have a square, sinusoidal or triangular cross-section, and whose ridges have a square, sinusoidal or triangular cross-section When used in the specification, reference to a grid is intended to include all such grids. It is.

In public WO34102578, the analysis target to be analyzed (e.g. Changes in the optical properties of the lattice as a result of binding (e.g., specific antigens in serum) , essentially (1) the mass or size of the bound analyte and 2) its dielectric properties. caused as a result.

In published WO36101901, the binding result is the color attached to the sensitive surface. It is monitored by changes in the fluorescent properties of the labeled binding partner.

In both of the aforementioned techniques, the grating plane is at least as large as the radiation used for illumination. opaque at the wavelength of the radiation, so the grating can be considered a reflective grating . as a result of analytical techniques, as a result of the deposition of analytes or other substances on the grid; The changes in the properties of the grating that occur are due to the reflection characteristics of the grating (WO84102578) and In the above specification as a change in fluorescence emission characteristics (WO86101901) It appears as described in. However, the positive results of such analytical techniques The operation relies on the ability to illuminate the lattice surface, and therefore specific binding reactions. Any material other than that which binds to the surface through will block the passage of light. Therefore, it is important to consider how such a test can be carried out “in the wet”. It was difficult to reach.

“In a wet manner” means using a liquid in contact with the grating surface, and When using traditional techniques, this can occur if light is absorbed or scattered at the excitation or observation wavelength. This type of analysis is particularly difficult.

It is therefore an object of the present invention to provide an improved method and apparatus, which This allows analytical techniques to be used “in the wet” and to detect light absorption by suspended particles in the liquid or It can even be performed in the presence of scattering.

Summary of the invention Therefore, in its broadest sense, the present invention provides an analysis method for ligands in a sample. This method provides a method for optical structures that can exhibit surface plasmon resonance. The sample is incubated in contact with one surface of the sample, and said surface is directly or indirectly specifically, adsorb onto it a specific binding partner for the ligand desired to be detected. or coupled to another part of the optical structure with radiation of the appropriate wavelength. Irradiate the surface: and the surface plasmon resonance features of the optical structure are unique to the ligand. whether it changes due to the formation of a complex between the target binding partner and, if desired, measuring the degree and/or rate of

Preferably, the optical structure is a diffraction grating of transparent plastic or glass material. and the grid is coated with a thin layer of metal or metal-like; This layer may be used to illuminate or observe the grid, at least for research purposes. partially reflective and partially transparent at the wavelength of radiation that Ru.

Preferably, the ligand to be analyzed will be an antibody or an antigen, and its specific A complementary binding partner may be an antigen or an antibody.

The invention further provides an apparatus for detecting one or more ligands in a sample. The equipment shall contain a reservoir for holding the sample to be analyzed. at least the inner surface portion of the reservoir exhibits surface plasmon resonance. said reservoir, which in use will come into contact with the sample. The surface of the bath directly exposes the specific binding partner for the ligand desired to be detected. or indirectly adsorbed onto or bound to it. .

One aspect of the device of the invention is that the device is located outside the reservoir and away from the sample during use. means for irradiating the surface of the wax optical structure; and a surface plate of the optical structure. The characteristics of rasmon resonance are altered by the formation of a complex between the ligand and a specific binding partner. determine whether and, if desired, the degree and/or rate of The method further comprises: means for analyzing the reflected radiation.

Typically, the reservoir comprises a shallow grooved well with a flat bottom; a metal or metal-like thin film formed on the upper surface of the side and applied to the grating surface; It has a diffraction grating that partially reflects and partially transmits the transparent film.

Where it is important to protect the metal from liquids or other substances placed in the storage tank. If the metal film is preferably co-coated with a further film of inert material, This inert material, when activated by light of the appropriate wavelength, forms a diffraction grating. While still allowing the particles to exhibit surface plasmon resonance, the metal and the inside of the reservoir interfere with chemical interactions between reactants.

An advantage of the present invention is that “in the wet” and in the presence of scattered particles in a liquid sample, This analytical technique can be used when using samples.

A further advantage is that reactions can be monitored as they occur and that Therefore, the end point of the analysis can be predicted, thereby reducing the time required to perform the analysis. It is possible to reduce the length of.

An additional advantage is that there is no need to separate the sample from the sensor before measurement. That is.゛ The invention therefore provides a method for preparing whole blood samples or other samples containing light-scattering compounds or particulate matter. Suitable for use with biological samples.

The present invention also allows for wet or dry competitive assays in which the antibodies in the reagents are fluorescently labeled. or in which the secondary antibody is fluorescently labeled. This allows for patch assays to be performed.

The detection means are capable of discriminating between different wavelengths of the received light, and in particular are capable of discriminating between different wavelengths of light received. Light of a wavelength corresponding to that produced by the optical label is present in the light re-emitted from the grating. Fluorescently labeled substances can be used if it is possible to determine whether is possible.

In a typical device, a monochromatic or semi-monochromatic light source is used as the primary light source to illuminate the grating. Although colored light sources are used and typically lasers are used, the light source The choice is not limited to lasers, and the angle of incidence of the light may be changed. It will be understood that Instead, use a multicolored, e.g. white light source In order to keep the angle of incidence constant and detect the surface plasmon resonance effect, The characteristic wavelengths of the emitted light are analyzed.

The invention will now be briefly explained with reference to the attached figures.

FIG. 1 shows the surface plasmon resonance effect due to back illumination of the diffraction grating; and FIG. 2 shows an apparatus and a book by which an analysis may be carried out according to the invention. 1 schematically illustrates embodiments of features of preferred configurations of the invention;

Figure 1 is coated with 50 nm of silver by vacuum evaporation and helium Injection molding irradiated from the back side of the grating with on-laser (λ=633r+a+) The results obtained from the diffraction grating shown in FIG. The intensity of the reflected light is determined by the change in the angle of incidence (θ). was monitored accordingly. Reflectivity of surface plasmon resonance over a range of angles A characteristic dip was observed. Metallizing test without grid When illuminating one area of the device, the intensity of the reflected light changes over the range of angles covered. It didn't make any difference at all.

In FIG. 2, the shallow groove well 10 is shown as a plastic material with a flat bottom 12. formed on the upper inner surface of this flat bottom by a suitable method, such as contact molding or mechanical A diffraction grating 14 is formed by machining or otherwise.

The upper surface of the grating 14 is semi-reflective metal or metal-like and has a metal foil. If an inert intermediate layer is required between the film and the reagents placed in the well, Its surface is coated with a layer of a suitable buffering material, for example silicon oxide.

The grid contains a specific antigen, antibody or other binding material that may be labeled with a fluorescent compound. coated with a thin film of material containing a mating partner, and the wells are analyzed. Add a liquid containing the specific antibody, antigen or complementary binding partner to be used. It is prepared so that

The product containing the wells is assembled into a device shown schematically at 16, which It includes a laser light source 18 from which light is emitted and suitable optical means (not shown). irradiate the bottom of the well at an appropriate angle of incidence using a causes surface plasmon resonance. A wavelength-sensitive photodetector 20 is also included in the housing 1 6 and the output from the detector 20 is installed inside the housing. sent to electrical processing and display devices such as 22, which may or may not include .

Adjustments to detector 20 and processing and display circuitry 22 are made until the well is in place. The first reading occurs when the liquid is irradiated and contains no ligand. It is done as follows.

Samples, which may or may not contain ligands, can be prepared in any convenient manner. well and monitor the output of detector 20 as displayed by device 22. monitor and measure any changes.

If fluorescence analysis techniques are used, the detector detects any of the fluorescent labels in the analysis. can be set to determine whether light of a certain wavelength can be detected.

If the liquid sample marked by reference number 24 in the figure contains light scattering particles, these interferes with the transmission of light through the liquid and across the grating, and the liquid It would be expected that this would make any observation of the grating through the diffraction grating impractical. deer However, the surface plasmon resonance effect as measured by the detector 20 is is unaffected by the presence of light-scattering particles, and the analytical technique is “wet”. It has been demonstrated that it can be implemented.

Although the illustrated example shows only one well, the light source 18 can of course include multiple wells. may be used to irradiate wells simultaneously, and corresponding multiple detectors may be used. or analysis of a large number of different samples can be done relatively quickly. One detector is used, set to scan each well in succession. You may be

each can have a different binding partner on it, and these can have a number of different In order to simultaneously measure the analyte, non-specific binding can be detected by comparison with a “control area”. It can be used either way to provide the criteria. And analyzed for example is not itself specific for any ligand that may be present in the sample. It may also carry a binding partner.

international search report mA#”, PCT/GB 8B100176

Claims (14)

[Claims] 1. A method for analyzing ligands in a sample, which expresses surface plasmon resonance. The sample is incubated in contact with one surface of an optical structure that can be The surface may directly contain a specific binding partner for the ligand desired to be detected. or indirectly adsorbed on or bonded to the optical structure; irradiating another surface with radiation of a suitable wavelength; and The resonance characteristics change due to the formation of a complex between the ligand and the specific binding partner. to determine whether and, if desired, the extent and/or the speed of the A method comprising: analyzing reflected radiation. 2. 2. The method of claim 1, wherein the optical structure is a diffraction grating. 3. 3. The method according to claim 1 or 2, wherein the specific binding partner is an antigen or an antibody. Law. 4. the optical structure is partially reflective at the wavelength of the radiation used; and A method according to any one of the preceding claims, wherein the method is partially transparent. 5.A device for detecting one or more ligands, the device being used for testing a storage tank for holding a sample to be sampled, and at least an inner surface of said storage tank. The surface portion comprises an optical structure capable of exhibiting surface plasmon resonance, and the use of That surface of said structure which would then come into contact with said sample is desired to be detected. a specific binding partner for the ligand either directly or indirectly A device that is adsorbed or associated with it. 6. From outside the reservoir, view the surface of the optical structure that will be away from the sample during use. means for irradiating and forming a complex between a ligand and a specific binding partner; whether the surface plasmon resonance characteristics of said optical structure change and as desired. analyze the reflected radiation to determine its extent and/or speed. 6. The apparatus according to claim 5, further comprising means for controlling. 7. comprising a storage tank having a plurality of divided regions, each divided region being as defined in claim 5. each segment that will come into contact with the sample during use. The surface of the region has a different specific binding partner adsorbed on it or binds to it. 6. The apparatus of claim 5 for detecting a plurality of ligands in a sample. 8. 6. A device according to claim 5, comprising a plurality of reservoirs as defined in claim 5. 9. From outside the reservoir, a table of the optical structure that will be separated from the sample in use. means for irradiating the surface; and of the complex between the ligand and the specific binding partner. whether the formation changes the surface plasmon resonance characteristics of the optical structure; and reflected radiation, optionally to measure its extent and/or its speed. Multiple ways to analyze lines, or scan each segment or reservoir in succession. Additionally, there are multiple means for analyzing reflected radiation that can be scanned. 9. A device according to claim 7 or claim 8, comprising: 10. Apparatus according to any one of claims 5 to 9, wherein the optical structure is a diffraction grating. Place. 11. Any of claims 5 to 10, wherein the specific binding partner is an antigen or an antibody. Apparatus according to paragraph 1. 12. the optical structure is partially reflective at the wavelength of the radiation used; and coated with a thin layer of metal or metal-like material that is partially transparent. The device according to any one of Items 5 to 11. 13. Any of claims 5 to 12, wherein the irradiation means is a monochromatic or semi-monochromatic light source. The device described in item (1) above. 14. 14. The apparatus of claim 13, wherein the light source is a laser.