FI96800C - Device for performing an analysis - Google Patents

Description

96800

Device for performing the analysis

The invention relates to an apparatus for performing an analysis of the type set out in the preamble of appended claim 1.

Immunological assays generally use assay wells, which may be in a special microtiter plate and in which the immunological reaction is allowed to proceed. The result is measured by the physical quantity obtained from the well. Examples of this type of method are competitive binding methods using a fluorescence label in which excitation energy is applied to an analyte in a well in the form of electromagnetic radiation of a suitable wavelength and the fluorescence of the well is measured. Methods are described, for example, in U.S. Patents 4,374,120, 4,808,541 and 5,124,268. For the structure and measurement arrangement of a plate used in various photometric measurements 20, reference is made, for example, to U.S. Patent 4,648,250, which discloses a light source below the well and a detector above the well to detect a change in light due to a substance in the well.

A disadvantage of immunoassay methods is that special substances that cause this phenomenon have to be used to induce fluorescence. 1. labels. Attaching these substances to the analyte requires an additional step.

It is known to use the SPR phenomenon to determine the biochemical components 35 present in a cuvette-like structure, e.g. as disclosed in EP-A-286195 and 517930.

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The object of the invention is to provide a new type of device for performing an analysis in which the SPR phenomenon can be exploited without special reagents, without, however, decisively changing the structure of the analysis wells 5 and the dosing of the substances to be analyzed. To achieve this object, the device according to the invention is mainly characterized by what is set forth in the characterizing part of the appended claim 1. The analysis wells then form 10 well plates with several wells. The measurement based on the SPR phenomenon is performed through suitable prism structures bounding from the bottom direction of the well plate to the bottom, the cross-sections of which remain constant in the direction of the well rows. The detectors are then also located on the bottom side of the well plate and not on the mouth side of the wells, as in conventional measuring devices using a transmitted light beam, e.g. in microtiter plates.

For other preferred embodiments of the invention, reference is made to the appended dependent claims.

The invention will now be described in more detail with reference to the accompanying drawings, in which Figures 1a-e show examples of different well structures, Figures 2a-c show different alternatives of the measurement principle with a well according to the invention, Figure 3 shows a combination of wells used, Figures 4a and b show measurements With the combination of wells according to Fig. 3, Fig. 5 shows the effect of changes in concentration on the measured values, and Fig. 6 shows the effect of biomolecules on the measured values.

Figures 1a-1e show an assay well 1 for immunological assays, which is included in a well plate according to the invention. The result of the immunological reaction in the well can be read optically based on the Surface Plasmon Resonance (SPR) technique known from e.g. FI patents 85766 and 84940. The well consists of the actual reaction space 2 and the prism 3. Both are made of 15 transparent plastics. , for example polystyrene, by compression into a single unit. Attached to the inner surface of the reaction space 2, which is at the same time one interface of the prism 3 connected to the well, is a layer 4 of SPR-producing material. This may be a thin gold well vaporized or sputtered on the surface 20. The outer surface of the layer 4 may be formed by a thin additional layer of dielectric material (e.g. polystyrene, glass or diamond) to protect the actual material of the layer 4 or to improve the adhesion of the biomolecule.

Attached to the outer surface of the material layer 4 is a biomolecule, such as an antibody, antigen or peptide, to which the analyte molecule to be measured in the sample specifically binds. With this coating, the pit can be stored for a long time in a dry place.

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The measurement takes place by injecting into the well. sample and reacting it with the immobilized molecular layer on the surface of layer 4. Through the prism 3 forming the bottom of the well, a 35 p-polarized light beam or light beam is applied to the material layer 4 at the interface between the prism 3 and the reaction space 2. At the interface, light is totally reflected back to the prism i 'at prism values greater than a certain cut-off value. than the refractive index of the liquid in reaction space 2. At a certain wavelength of light and the value of the incident angle in the total reflected light, the so-called

5 surface plasmon resonance, in which case the reflected light disappears. In this case, all the energy of light is transferred to the electromagnetic wave propagating in the material layer 4, such as in the plasma formed by the free electrons in gold. The resonance phenomenon confirms the so-called total reflection occurring in the reaction space 2 10. evanescent electric field. This field extends only a few hundred nanometers from the gold surface to reaction space 2. The Evanescent field sees a surface reaction, e.g., the formation of a specific complex between the antibody bound to the surface of the material layer 15 and the antigen in the sample, because it represents a certain refractive index change at the layer surface. The binding is measurable from the reflected light because the resonance (loss of light 20) shifts to another value of the incident angle.

The prism 3 forming the bottom of the analysis well 1 can be comprised as a part having an interface 3a through which the light beam enters the prism, an interface from which the light 25 is totally reflected back to the prism, and an interface 3b through which the totally reflected light beam leaves the prism. The prism 3 may be, for example, in the shape of a half-cylinder (Fig. 1a) or an ordinary triangular prism (Fig. Ib), its interfaces 3a and 3b, through which the light beam enters and leaves, converging at the bottom of the pit, forming either a triangular apex or a semicircle. Depending on the value of the angle of incidence the resonance occurs, the prism may also be part of the above, the outer surface of the bottom of the analysis-35 being straight between the above-mentioned interfaces 3a, 3b, i.e. a kind of truncated prism (Figures le and Id). Figure 1e shows a prism structure in which the interfaces 3a, 3b are in the middle of the prism structure, sending each other in the direction of the reaction space 2, i.e. a kind of cavity is formed in the bottom. The incoming light beam refracting at the interface 3a is reflected from the outer surface of the prism towards the layer 4, either due to the total reflection 5 or by means of the mirror reflection provided by the reflective layer 3c attached to the outer surface. The light reflected from the interface bordering the layer 4 is also reflected in one of the above ways also from the second outer surface again to the second interface 3b, where it is refracted from the prism.

Figures 2a-2c show the measurement event in a simplified form. The light source 5 is a laser or a light emitting diode (LED). The measurement of the reflected light 15 can be made with a photodiode acting as a detector 6 if only changes in the intensity of the reflected light at a certain angle are measured (Figure 2a). Figure 2b shows the introduction of light by the optical fiber 13, whereby the beam propagating from the end of the fiber can be paralleled by collimating optics 7. In an analogous manner, the light from the prism can be introduced into the detector along the optical fiber 14. If the entire resonance curve is recorded as a function of angle as shown in Figure 2c, 25 focusing optics 12 are used between the light source 5 and the prism 3 and the total reflected scattered light beam is measured by a multi-detector CCD line detector 15. The beam can be collimated with optic 11 if necessary. relatively far from the prism.

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Although separate optics are shown in Figures 2a-c, it is also possible to connect the optics to the prism 3, whereby the lens structures can be located on the surfaces 3a and 3b. The prism and optics can then be made into a uniform structure, e.g. by casting.

In the structure according to the invention, several analysis wells 1 can be measured simultaneously, whereby they 6 96800 can be arranged either in a row or in a matrix, as shown in Fig. 3. The wells can be arranged successively in a plastic strip 8 with several wells side by side. The strips 8 comprising these several wells 1 5 can further be placed side by side, thus forming a conventional structure of the well plate 9, i.e. a microtiter plate. The bases of the strips can then have the shape of a prism 3 of cross-section shown in Figures 1a-e. The base of the strip 10 thus consists of a continuous prism structure with a constant cross-section, and individual prisms 3 are formed at each well 1.

Two different methods 15 can be used to read the well plate 9, as shown in Figures 4a and b: 1. The light beam from the light source 5 (e.g. laser diode) is divided into parallel light beams on each prism 3 of the well plate 20 9, for example by a holography element 10 and an associated collimator, that the angle of incidence is the same in each case, Fig. 4a. Each reflected beam has its own light detector 6, which measures changes in intensity.

25 2. The scattered light beam from the light source 5 is collimated into a parallel beam by optics 7. The parallel rays hit the entire area of the well plate 9. The total reflected light in which the beams 30 are parallel is collected by a two-dimensional CCD detector 15 with adjacent ones; detectors, 1 for each analysis well.

This creates an image of the plate in which the displacement of the resonance of a particular well is seen as a change in the intensity of the light coming from the well, Figure 4b.

For reading the well plates 9, it is also possible to use the registration of the whole resonance curve li 7 96800 according to Fig. 2c as a function of the angle of incidence for each well 1. The optical arrangement is then the illumination of Köhler known per se.

When using the strips 8, the light rays can be directed to a row of prisms 3 forming a continuous structure, for example from a row of several light sources 5. The detectors 6 can be arranged in a row, respectively, and when using the CCD detector 15 it only needs to be a 10-dimensional detector. If it is desired to register the entire resonance curve as a function of the angle of incidence, an arrangement according to Fig. 2c with a row of light sources 5 and a two-dimensional detector of a multi-detector as a CCD detector 15 can be used.

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Compared to, for example, a conventional ELISA or RIA assay in the SPR cuvette and well plate presented here, a faster assay is achieved because no labeling (additional reagents) is required. The only additional step that may be required is to wash the SPR cuvette prior to measurement to remove non-specifically bound material. The simple measurement procedure also enables the development of cheap, portable measuring devices for environmental analytics, for example. 25

The light from the light source must be p-polarized. If the light source does not itself contain a polarizer, this is achieved by a separate polarizer placed between the light source and the prism, which is indicated by reference numeral 16 in Figures 2b, 2c and 4b.

· Multiple sample measurements as well as positive and negative control samples can also be used on the well plate to confirm the result. In addition, the strips 35 or plates may have separate, enclosed control coatings to calibrate the measurement.

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The well strip and well plate can be made so that their external dimensions are consistent with existing wells, strips and plates. In this case, no new dosing and incubation equipment is required.

5 Em. the structures can be made, for example, by injection molding from a plastic of suitable optical quality, after which the bottoms of the wells 1 can be coated with a layer of SPR material 4 by some known technique. Instead of assembling separate strips 8, the well plate 10 can also be made by casting in one piece. in the way of prism structures of constant cross-section parallel to the well rows.

Figure 5 shows the effect of a 300-80 mM NaCl dilution series on the light intensity obtained in the analysis. As the series of experiments shows, the concentrations of the liquid in the reaction space adjacent to the thin gold well clearly affect the light intensity. The measurement 20 is performed in the descending range of the intensity curve as a function of the incident angle, where an increase in the concentration at a certain constant value of the incident angle causing the SPR phenomenon causes the curve to shift to the right and an increase in the intensity values of the transmitted light 25.

Figure 6 shows the effect of synthetic peptide and HIV antibody binding on intensity values. In time interval A (0-750 s), 30 buffer solutions have been added to the well. At step B (750 s), rinsing and addition of peptide (50 μg / ml) have been performed. Between C (1400-1600 s), rinses with buffer solution have been performed, at point D (1600 s), antibody (1/24000 titer) has been added, and between E (3400-3600 s), 35 rinses have been performed. Em. the effect of the interactions between the measures and the biomolecules on the intensity estimates of the SPR measurement is clearly observable, and the figure is a good example of how the reaction in reaction state 2

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9 96800 or more reaction conditions 2 can be monitored continuously in the device according to the invention.

Although the purpose of the invention is to monitor and quantitatively or qualitatively analyze the reactions between biomolecules mentioned above, the invention can be applied to all types of analyzes in which a change in the analyte in the reaction state causes a sufficiently clear change in the light passing through the prism.

Claims (3)

96800 10 An apparatus for performing an analysis having an analysis well (1) for placing an analyte, a light source (5) directed towards the analysis well and a detector (6) for receiving a light beam obtained from the light source (5) directed to the analysis well (1), the reaction space 10 (2) the bottom is coated with a layer (4) of SPR-inducing material, optionally with an additional coating, the bottom portion of the analysis well (1) adjacent to layer (4) is transparent to the light-inducing light with said material of SPR and is shaped for incoming a prism (3) comprising an interface (3a) for light, an interface (3b) for outgoing light and an interface providing total reflection, in addition to which the light source (5) is directed through the prism (3) towards the reaction space (2) 20 and a detector (6) is positioned so as to receive light from the prism (3), characterized in that that the analysis wells (1) form a well plate (9) comprising a plurality of wells, the bottom of which has adjacent prisms (3) at the bottoms of the respective reaction spaces 25 (2), forming rows of wells (1); parallel prism structures with constant cross-sections perpendicular to the longitudinal direction of the rows. Device according to Claim 1, characterized in that the well plate (9) is formed side by side. placed strips (8), the bases of which consist of continuous prism structures of constant cross-section. 35 11 96800 Device according to claim 1, characterized in that focusing light optics (12) are provided between the light source (5) and the prism (3), wherein a 2-dimensional detector (15) comprising several detectors is arranged to receive light at different incident angles. to measure. 12 96800