EP1129336A1

EP1129336A1 - Device and method for rapid evaluation of bonding reactions in structured interferometric microtitre plates

Info

Publication number: EP1129336A1
Application number: EP98966390A
Authority: EP
Inventors: Gunnar Brink; Joachim RÄDLER; Erich Sackmann
Original assignee: Jandratek GmbH
Current assignee: Jandratek GmbH
Priority date: 1997-12-23
Filing date: 1998-12-21
Publication date: 2001-09-05
Also published as: WO1999034196A1; DE19757706C2; DE19757706A1; AU2275299A

Abstract

The invention relates to an interferometric method which makes it possible to determine a modification in the optical density of an adsorbate layer in microtitre plates. The method is based on the integration of laterally structured interference layers into the base of a biofunctionialized microtitre plate. The optical reflection-interference of the plate is imaged and evaluated by means of image processing. The average quadratic difference of standardized intensities is used to determine a modification in the optical density of the adsorbate. The method is highly sensitive, quantitative, economical and robust with respect to external perturbations. One of the substantial advantages is that the ligands do not need to be marked.

Description

Device and method for the rapid evaluation of binding reactions in structured interferometrisehen

Micriter plates

The present invention relates to a device and a method for rapidly evaluating binding reactions in structured interferometric microtiter plates, in particular a device and a method for determining the change in the optical thickness of a layer by means of light interference.

A number of immunological tests require that either the antigen or the antibody be coupled to a solid surface. Solid supports such as latex balls, plastic tubes etc. are used for this. By far the most successful and most widespread carrier is the microtiter plate. Techniques such as the ELISA (enzyme-linked i munosorbent assay) test or radiological immune tests are carried out as standard in disposable microtiter plates. Microtiter plates are plastic trays made of transparent plastics (polystyrene, polypropylene etc.) with a number (typically 96) of small reaction pots. Proteins, such as antibodies, can be bound in the hydrophobic containers by physisorption. The addition of the solutions to be tested and the subsequent washing cycles are often carried out by automated pipetting machines. The reading of the plates can also be automated. For In ELISA tests, the color reaction is measured based on the optical absorption of the solution, in radiological tests the amount of radioactive substances. However, for this type of tests, firstly fluorescence or radioactive labels are necessary and secondly, in particular ELISA tests are not always quantitative, ie only detect the presence but not the exact amount of a substance.

In contrast to this are biosensors, which deliver exact values, for example the optical layer thickness of an adsorbate or the mass coverage of a surface. Biosensors are reversible and continuously working sensors for the detection of proteins, nucleic acids or poly sugars. Such highly sensitive transducers play a major role if, in addition to the presence of a ligand, its concentration is also to be determined. Quantitative sensors can be used in particular to determine the binding constant of a ligand. In addition to the development of suitable substrates and chemical coupling processes, the automation of the quantitative determination of the receptor-ligand binding must still be optimized.

The best known quantitative surface techniques that determine optical layer thicknesses include surface plasmon resonance (Liedberg, et al. 1983; Surface Plasmon Resonance for Gas Detection and Biosensing, Sensors and Actuators, 4: 299-304), which reflecto Electric interference spectroscopy (Brecht et al. 1993; Interferometric immunoasay in a FIA system: a sensitive and rapid approach in label-free immunosensing. Biosensors & Bioelectronics. 8: 387-392), as well as quartz scales. The first two techniques measure optical layer thicknesses with a resolution in the Angstrom range, while the Quartz scale measures mass loadings with a detection limit in the μg range.

In R dler, et al. 1993, Imaging optical thicknesses and Separation distances of phospholipid vesicles at solid surfaces, J. Physics II France 3: 727-748, shows that reflection-interference-contrast-microscopy (RICM) shows quantitatively evaluable interference intensities for simple layer systems. In particular, planar systems of evaporated magnesium fluorite with adhering vesicles, spread membranes or lipid monolayers transferred by Langmuir-Blodgett technique are imaged. It is shown that the change in intensity (shown for example in FIG. 1b) in the case of protein adsorption to an interference layer of constant thickness D (in this case 48 nm) can be measured or can be imaged on a CCD camera using RICM. When measuring changes in layer thickness, however, a planar interference layer only provides a signal which is subject to numerous interferences.

In contrast, the object of the invention is to provide a device, a method and a method for producing the device which allow (s) to determine the change in the optical thickness of a layer, preferably an adsorbate layer in microtiter plates. This object is achieved with the features of the claims.

In the solution, the invention is based on the basic idea that due to variations in the spacing between the top and bottom of a layer, for example an adsorbate layer, there are varying interference patterns when irradiated with light if the optical thickness of the layer changes. For this purpose, a biofunctionalized microtiter plate microstructured interference layers introduced or applied.

The optical reflection interference of the plate is imaged and evaluated with the help of image processing. The mean square difference of the nominal intensities of the interference patterns before and after adsorption is used to determine the change in the optical layer thickness of the adsorbate. The device / method according to the invention is based on optical interference, the thickness of the interference-capable coating varying. This is resolved by optical imaging and evaluated by a simple image processing method.

The advantages of the invention are numerous:

1. The ligands to be detected do not have to be pretreated or labeled.

2. The evaluation of a plate with a large number of tests is possible in one step and can be accomplished in seconds with the help of a fast computer.

3. The determination of the change in the optical layer thickness of the adsorbate from the mean square difference of the nominated intensities has a high sensitivity with a layer thickness resolution of one angstrom (1 Å).

4. The method is robust against external fluctuations in the lighting intensity, as well as against irregularities in the exact microstructuring of the plate.

5. Since the described microtitre test is based on an imaging process, obvious malfunctions such as the Precipitation of reagents or the accumulation of a speck of dust can easily be noticed. 6. The imaging process can be easily combined with fluorescence techniques.

The method should therefore be used as an interferometric immunoassay, the advantages being particularly evident in automated systems with a high throughput of microtiter plates. It is therefore also explained below how structured microtiter plates can be produced inexpensively on a plastic basis.

The invention is explained in more detail below with reference to the accompanying drawings. Show it:

La shows a representation of an integrated interference layer with a laterally varying layer thickness D, the adsorbate layer δ carrying receptors and an optical layer thickness change Δδ being measured;

1b shows a simulation of the reflectivity as a function of the location-dependent layer thickness D of the interference layer; the curves correspond to adsorbate layer thicknesses of δ = 10, 20 and 30 Å;

FIG. 1c shows the mean square difference of the normalized intensities S as a function of the optical layer thickness change Δδ;

2a shows a representation of a microtiter plate with 12 reaction droplets;

2b shows an interfering recess integrated in each potty (and filled with the plastic 2); 2c shows a representation of an interference pattern. Of a depression, as can be seen in the imaging reflection interference microscopy;

3 shows a structure for the optical imaging of the reflectivity of the microtiter plate by means of reflection-interference-contrast microscopy; and

4 shows a structure for the optical imaging of the reflectivity of the microtiter plate by means of laser scanning.

The basis of the method according to the invention is to apply a biofunctionalized layer directly to an interference-capable substrate. In the simplest case, a first thin (preferably heterogeneous) layer 2 with a refractive index n2 different from nl is applied to a section of a carrier 1 (for example glass or plastic) with a refractive index nl (for example nl = 1.5) (see FIG. 1a) , with n2 preferably in the range from 1.3 to 1.4. This first layer is provided with a biocompatible adsorbate layer 3 with calculation index n3, preferably a polymer layer, which contains antibodies or receptors. The optical

Layer thickness δ of this biocompatible layer 3 increases when ligands 4 are bound from an aqueous solution 17 with a calculation index /. To detect the increase in layer thickness, the substrate is illuminated from below, ie from the side facing away from the biofunctionalized layer 3, with preferably monochromatic light, and the resulting interference pattern is imaged on a camera. The intensity of the reflected light is determined by the superposition of all reflected partial beams (see: Azza, R. et al. 1975; Ellipsometry and polarized light, Amsterdam, North Holland). Both the thickness D of the interference layer 2 as well as the thickness δ of the adsorbate film 3. For the exact determination of a change in layer thickness Δδ, use is now made of the fact that the thickness D of the interference layer 2 varies laterally, so that a large number of interference maxima and minima are produced.

Fig. La shows a first embodiment. This shows, for example, a triangular depression and thus a linear change in the thickness D corresponding to the oblique course of the triangular surfaces from the location. The ratio of the intensities corresponding interference Irefl ^ O i ^st i ⁿ Fig. Lb. It can be seen here that the position of the maxima shifts with an increase in the thickness δ of the adsorbate layer. The interference images are now processed in such a way that the change in layer thickness Δδ is determined only from the mean square phase shift. This corresponds to the shift in the reflectivity curves shown in FIG. 1b. This processing makes the determination of the change in thickness of the adsorbate layer 3 independent both of fluctuations in the incident light and of the precise shape and quality of the microstructured interference layer. A number of maxima and the same number of minima are preferably used for the evaluation.

The representation in FIG. 1b is based on the following refractive indices: support 1 = glass, n ^ = 1.51; first layer 2 = interference layer, n2 = 1.38; second layer 3 = adsorbate (protein), n3 = 1.55; aqueous solution 17, n4 = 1.33.

In a further embodiment, only the first layer 2 is applied to the carrier 1, so that an attachment to the layer 2 takes place. O

As a further embodiment, the first layer 2 or second layer 3 does not have the receptors to which ligands bind, but conversely the ligands to which the receptors bind. The mutual binding effect of receptors and ligands is used here.

In a further embodiment, instead of a carrier with recesses, a flat or smooth carrier is used, on which the layer 2 is applied in such a way that the distance between the top and bottom varies. In this way it is achieved in this embodiment that the thickness D of the layer 2 varies. Layer 2 is preferably applied in the form of drops or droplets (for example sprayed on or applied with a pipette). These are allowed to dry or polymerize. Alternatively, a flat layer 2 is applied, to which a structure is then printed.

A swellable material is preferably used for the second layer 3, which in the dry state has a thickness of approximately 5 to 200 Å, preferably approximately 10 to 20 Å, while in the swollen state it has thicknesses between 10 and 100,000 Å, preferably 10,000 Å. In this swollen state, this layer preferably has a refractive index n3 of 1.31.

The change in the optical thickness of the layer to be determined can take place, for example, in the following ways. On the one hand, the physical thickness and thus also the optical thickness of the layer change due to deposits, on the other hand, a change in the optical thickness can be based on a change in the refractive index, which results from deposits in the layer. An interference image is recorded before and after (or during) the addition of the ligand and the following operations are carried out over the surface of the interference pattern. (In the case of a multiple test, such as the microtiter plate shown below, these operations are of course carried out separately for each binding surface.) First, the intensities are standardized:

The <> means the averaging over all NxM pixels. Then the difference between the normalized intensities Iγ, N of the images before and after the binding is formed and divided into squares:

wherein the averaging is carried out over all NxM pixels of an image section which is preferably the same size or smaller than the interference pattern at the bottom of a microtiter potty. Averaging is preferably carried out over a square area. S is referred to as the "mean square difference of the normalized intensities" of two interference patterns (I _v , I _j j) before and after the adsorption. The following applies to the value S obtained in this way:

where λ denotes the light wavelength used and n- _j _ the respective refractive indices as defined above. Equation (3) is used in many practical cases where NEN the adsorbate layer is small compared to λ / 4 is sufficient to directly determine the optical layer thickness increase Δδ from the measured value for S. The respective accuracy of equation (3) can be determined by comparison with the numerical values for given refractive indices and layer thicknesses δ. Thus, for small changes in layer thickness, S is proportional to the change in layer thickness Δδ. The exact relationship can be determined numerically and stored in a table. For the borderline case that the adsorbate layer is exactly zero before adsorption, the above relationship (3) can be derived by neglecting multiple reflection. Fig. Lc shows the size S of a simulated sample, which was calculated according to equations (1) and (2) as a function of

Change in layer thickness Δδ. It was assumed that the adsorbate layer δ is homogeneous, that is, it is not dependent on the location.

Manufacture of interferometric microtiter plates

Optically high-quality, laterally structured interference layers can be produced by standard vapor deposition of dielectric layers on glass. In this case, the structuring would take the form of masks which are either shifted or exchanged between the individual vapor deposition cycles.

According to the first embodiment, plastic plate or the carrier substance is structured. This is easy and inexpensive. An optically low or non-birefringent polymer (polycarbonate, cyclic olefir-e) can, for example, be injection molded onto a microtiter plate mold (FIG. 2a). This consists of an arrangement of small plastic pots with a flat bottom. In a second Step, a depression 5 of a few micrometers depth is pressed in each potty with the aid of a stamp (FIG. 2b). For example, the recess is between 5 and 2000 nanometers deep. Several possibilities are conceivable for the exact shape of the recess 5. For example, monotonously increasing, preferably continuous shapes, such as concave, convex, conical or roof-shaped depressions, represent simple solutions. In the last step, a second polymer (layer 2) with a different refractive index is applied from the solution to the depression 5 in the casting process, but can - as shown in Fig. La - extend beyond the recess 5. Since the diameter of the stamp is constant, the layer thickness D can be produced very reproducibly by applying a defined amount of solvent. The second plastic 2 should be soluble in a solvent that does not attack the plastic 1. By drying the layer 2, a meniscus can form on the surface.

Reading out the reflection interference signal

The reflectivity of the microtiter plate can be imaged with the aid of reflection microscopy under reflected light (see FIG. 3). If monochromatic light is generated with the aid of a narrow-band interference filter, the interference becomes visible in the laterally structured depressions 5 described above. In practice, further the ratio of interference signal to background brightness verbes ^¬ lets fibers by the anti-Flex technology for Pluta (Pluta, M; Advanced Light Microscopy, Elsevier, Amsterdam 1989) uses be ^¬. 3 shows the beam path of such a reflection-interference-contrast microscope according to Pluta. If the sample chamber is placed directly on the immersion oil λ / 4 plate 7 applied, so the first reflective interface is the glass-magnesium fluoride 2 interface (Fig. La).

3 shows a structure for the optical imaging of the reflectivity of the microtiter plate by means of reflection-interference-contrast microscopy. The carrier 1 with the introduced interference layer 2 is arranged over a λ / 4 plate 7 (optional). In between is a medium 6 with the same refractive index as the support

1 and the λ / 4 plate 7 for reflection-free coupling. This medium is preferably an immersion oil or silicone. The light emitted by a mercury vapor lamp 14 is directed onto the layer to be measured via a bandpass filter 13, which generates monochromatic light, a polarizer 12 (optional), a semi-transparent mirror 9 and an objective 8. The reflected light penetrates the semitransparent mirror 9 and reaches an camera 11 via an analyzer 10 (optional). This can be a CCD camera.

A second possible readout method (readout device) consists of scanning the microtiter plate with a laser or a laser diode. The reflected light is recorded with a one-dimensional detector. If the plate is scanned, an interference signal again arises, as shown in FIG. 1b, and the evaluation is carried out analogously to the imaging reflection interference.

Fig. 4 shows an embodiment with laser scanning. Scanning the microtiter plate with a laser 19 is advantageous in applications in which the speed of the reading process is critical. In this In the embodiment, imaging optics are dispensed with and the microtiter plate is scanned by a laser beam, the intensity of the reflected beam being measured. In Fig. 4, the laser beam is deflected by a rotatable mirror 20 so that it sweeps over the sample and is reflected on a one-dimensional detector 21 (preferably CCD). The depressions of the microtiter plate in this embodiment of the scanning are preferably roof-shaped or wedge-shaped depressions, that is to say only varied in a direction corresponding to the scanning direction. In this case, the averages according to formulas (1) and (2) correspond to the averaging along a line. Reference numeral 18 denotes a glass block with an anti-reflective coating.

Claims

Claims:

Substrate with a carrier (1) and with a layer (2) arranged on at least one section of the carrier (1) or with two layers (2, 3) lying one above the other, the layer (2) resting on the carrier (1) in at least a section has a thickness that changes depending on the location, the first or the second layer can bind substances on its free surface and thereby change its optical thickness and the layers are dimensioned such that light interference can be observed on them.

The substrate of claim 1, wherein the variation in thickness is monotonic.

The substrate of claim 1 or 2, wherein the thickness varies linearly or non-linearly.

The substrate of claim 2 or 3, wherein the top and / or bottom of a portion of the first layer (2) has a convex or concave or conical or roof-like shape.

The substrate of claim 1, wherein the carrier (1) has at least one recess (16).

The substrate of claim 1 or 5, wherein the carrier, preferably in the region of the recess (16) has at least one recess (5).

The substrate of claim 6, wherein the first layer (2) at least partially fills the depression (5).

The substrate of claim 6 or 7, wherein the recess (5) has a monotone wall.

9. The substrate of claim 8, wherein the wall has a linear or non-linear shape.

10. The substrate of claim 8 or 9, wherein the wall has a convex or concave or conical or roof-shaped shape.

11. Substrate according to one of claims 1 to 10, wherein the first layer (1) is heterogeneous.

12. Substrate according to one of claims 1 to 11, wherein the first layer (2) has a refractive index n2 which is different from the refractive index nl of the carrier (1).

13. The substrate of claim 12, wherein n2 is less than nl.

14. Substrate according to one of claims 1 to 13, wherein the second layer (3) is arranged on the side of the first layer (2) facing away from the carrier (1).

15. The substrate of claim 14, wherein the second layer (3) has a binding property for a substance.

16. The substrate according to any one of claims 1 to 15, wherein the first layer (2) has a binding property for a substance.

17. The substrate according to claim 15 or 16, wherein the binding property is a binding action between the receptor and ligand.

18. The substrate according to any one of claims 1 to 17, wherein the change in the optical thickness by a change in the physical thickness of the second layer

.he follows. lo

19. The substrate according to any one of claims 1 to 18, wherein the change in the optical thickness is effected by a change in the refractive index of the second layer.

20. Substrate according to one of claims 14 to 19, wherein the second layer (3) has a refractive index n3 different from n2, preferably n2 being less than n3.

21. The substrate according to any one of claims 14 to 20, wherein the second layer (3) is a swellable layer.

22. Substrate according to one of claims 14 to 21, wherein the second layer (3) has a thickness of 5 to 200 Å in the dry state.

23. The substrate according to any one of claims 14 to 22, wherein the second layer (3) in the swollen state has a thickness of 10 to 100,000 Å.

24. The substrate according to any one of claims 6 to 10, wherein the recess (5) is between 1 and 10000 nanometers deep.

25. The substrate of claim 24, wherein the recess (5) is between 5 and 2000 nanometers deep.

26. The substrate of claim 25, wherein the recess (5) is 600 nanometers deep.

27. Substrate according to one of claims 5 to 10, wherein the recess (5) has a depth corresponding to an integer multiple of half the light wavelength in the first layer (2).

28. Sensor for determining the change in the optical thickness of a layer by means of light interference with a substrate according to one of claims 1 to 27.

29. Device for determining the change in the optical thickness of a layer by means of light interference a substrate according to one of claims 1 to 27 and a light source (14), a semi-transparent mirror (9), a lens (8) and a camera.

30. The apparatus of claim 29, further comprising a polarizer (12).

31. The apparatus of claim 29 or 30, further comprising an analyzer (10).

32. The apparatus of claim 29, 30 or 31, further comprising a λ / 4 plate (7).

33. Method for determining the change in the optical thickness of a layer, which changes its optical thickness as a result of the binding of a substance, by means of light interference, in which a) the layer (2) is applied to a carrier (1) and in at least one section one has a thickness that changes depending on the location, b) a reflection interference image of the layer is recorded before the substance is bound to determine reference values and after the substance is bound to determine measured values, c) the determined intensities I in accordance with

normalized, d) the mean square intensity difference

is determined, and e) the optical layer thickness change Δδ according to

is determined.

34. Method for determining the change in the optical thickness of a layer, which changes its optical thickness as a result of the binding of a substance, by means of light interference, in which a) the layer as a second layer (3) lies over a first layer (2), which in turn is applied to a carrier (1) and has a location-dependent thickness in at least one section, b) a reflection interference image of the layer is recorded before the substance is bound to determine reference values and after the substance is bound to determine measured values, c) the determined intensities I according to

normalized, d) the mean square intensity difference

is determined, and e) the optical layer thickness change Δδ according to

is determined.

35. The method of claim 33 or 34, wherein the phase shift is determined from the mean square difference of the no ized intensities of the interference images before and after the addition of the layer.

36. The method of claim 33, 34 or 35, wherein the top and / or top of a portion of the first layer (2) has a convex or concave or conical or roof-like shape.

37. The method according to any one of claims 33 to 36, wherein the second layer (3) is biofunctional.

38. The method according to any one of claims 33 to 37, wherein monochromatic light is used.

39. The method according to any one of claims 33 to 38, wherein the interference patterns are determined before, during and after the addition of the substance.

40. A method for determining the change in the optical thickness of a layer by means of light interference, characterized in that the determination is carried out by changing the light interference in a substrate with a carrier of the layer according to one of claims 1 to 27.

41. The method according to any one of claims 33 to 40, wherein the determination is carried out in a carrier substrate which is produced from two optically transparent plastics with a different refractive index and with printed, sprayed, pressed or cast interference-capable structures is provided.

42. The method according to any one of claims 33 to 41, wherein the structured support of the sensitive surface is imaged by means of reflection-interference-contrast microscopy.

43. The method according to one of the claims 33 to 41, wherein the structured support of the sensitive surface is scanned by means of laser light and photodetectors.

44. The method according to any one of claims 33 to 43, wherein a binding effect between the receptor and ligand is used to determine the change in the optical thickness of the layer.

45. Use of the substrate according to one of claims 1 to 27 in the method according to one of claims 33 to 44.