NO161945B - PROCEDURE AND APPARATUS FOR DETERMINATION OF SUBSTANCES IN SOLUTION WITH AN OPTICAL CHAIRMAN. - Google Patents

Description

The present invention relates to the use of a waveguide for determining the concentration of a substance (or analyte) in solution in a liquid by measuring the degree (concentration-dependent) of its binding with or dissociation from a particular reactant, e.g. a conjugated part in a complexation reaction. More particularly, the invention relates to a method for determining an analyte in solution, where a layer of an analyte-reactant product is built up on the surface of a light waveguide core that transmits a light wave signal that is reflected several times in total, where this layer formation has the effect of changing its optical properties to thus modify the wave signal and where the modification is measured and used for said determination. Thus, the invention is applicable to a large number of chemical and biological systems and is particularly good for the determination of bioactive molecules in low concentration, in reactions of the immunoassay type, i.e. reactions based on the formation of complexes by the addition of antibody- (AB) to antigen- (AG) molecules or vice versa.

There are already many methods in this area

for achieving the above-mentioned determination based on classical techniques from biochemistry. E.g. chemical reactions can be used to measure a given analyte in a number of different ways. Classical systems involve titration or reaction with a special reagent that gives a colored product or pre-precipitate. The requirement for this measurement system is that the reactant is in equivalence or in excess, so that the product can be measured by ordinary photometry, turbidimetry, colorometry, etc. The measurement system is chosen in accordance with the size of the signal to be measured. At very low analyte concentrations, measurement will be difficult and greater discrimination can be achieved, e.g. by concentrating the reaction product locally, e.g. by solvent extraction, centrifugation, etc. which can be time-consuming and expensive. However, the above disadvantage was greatly reduced when a practical system for measuring biochemical analytes in extremely low concentrations became available in 1960. This microanalytical system (radioimmunoassay) took advantage of

the properties of biological systems for molecular recognition (antigen-antibody reactions) and the extreme sensitivity to radioactive measurements (radioactive isotope labelling). A significant feature of this breakthrough was the method of limited reactant analysis with tracer labeling used for measuring the distribution of analyte to be measured between reaction-bound and free parts (see e.g.: Review Paper "The theoretical aspects of saturation analysis", R.P. Ekins in "In vitro procedures with radioisotopes in medicine", International Atomic Energy Agency, June 1970). Although immunoassays were first described as reagent-limited assays, similar practical systems were later described for reagent-excess methods (see Miles et al., Biochem. J. 108, 611 (1968)).

In addition to volumetric and gravimetric analyses, the present methods will thus include highly sensitive

in

with methods such as colorometry, spectroscopy and radioactive measurements. However, many such techniques have now become obsolete as they are time-consuming, require relatively large amounts of analyte to be accurate, are based on difficult-to-prepare and difficult-to-store reagents or require expensive and cumbersome equipment and highly skilled operators. Thus, there is now a tendency to develop more applicable methods that require less reagent and which can be carried out safely, quickly and accurately by moderately skilled personnel. Among such methods1 which have been developed in recent times, some include the use of optical waveguides which include reactant. For analysis, the waveguide is brought into contact with the analyte in solution whereby a reaction with the reactant on the waveguide occurs with the result that the optical properties of the latter are modified. Measurements of such modification then provide the required data for analyte determination. According to the teachings of some later references (e.g. US-PS 4,050-895 (Hardy) and WO 81 100 912 (Buckles)) the conductors may consist (Buckles) of a porous light-transmitting core which is impregnated with reactant into which the analyte will diffuse during the reaction. Or (Buckles

or Hardy) the waveguide may consist of a non-porous light-transmitting core (eg glass) coated with a porous or permeable cladding which is impregnated with reactant and into which the analyte will diffuse. Furthermore, in a particular case applied to immunoassay (Hardy, example 3), a rod-shaped waveguide is coated with an antibody layer bound by diphenyldimethoxysilane and reacted with polystyrene latex beads treated with an antigen. The antigen-treated beads will then attach to the conductor and modify the light signal output from the latter, which variation is used for analytical determination.

The above techniques have their value, but they will not work on certain types of analysis that involve reaction kinetics. It is certainly well known that degrees can provide important analytical data, especially in the case of automated test systems, and since the reactions occur within permeable or porous bodies will always also entail a preliminary diffusion of the analyte into the substance, and since the diffusion process in general is much slower than the chemical reactions, the degree of the latter cannot be measured directly, whereby in such cases only equilibrium data can be obtained. Also, in the known prior art, designs involving the use of a transparent core coated with a reactant sheath with a refractive index less than that of the core are avoided for the reason that it must be expected that a low sensitivity will be the expected result . In such a case, of course, most of the light signal introduced at the entrance to the conductor will move inside the core by a total reflection process and in such a case, as is commonly expected, the interaction of the signal with the reaction products located in the sheath, i.e. .on the outside of the core, could only be minimal. Consequently, it was taken into account in the prior art that the refractive index of the cladding n£ (where the reaction takes place) is always greater than that (n^) • of the core to allow light injected into the core at the entrance to be refracted into the sheath and from this point will continue to move in the sheath all the way to the exit from the conductor. Now, contrary to the content of some of the prior art, the output signal (the result of light that has been modified by passing through the reaction products: reactant + analyte in the envelope) will not easily return to the core and reach the rear end of this (this behavior is due to the elementary optical principles to be discussed later) and, for measurements, the output light detector must be placed very close to the sample probe (ie at the rear end of the envelope). Such a device is not always practically possible to construct, namely when the conductor (plus the sheath) is immersed in a liquid for measuring an analyte in solution.

The present invention avoids these disadvantages as it does not include any porous core or sheath and no diffusion through a matrix structure (sheath or core). Also with the present invention, the light signal that moves inside the waveguide by total reflection will neither be transmitted nor be carried within the reactant-analyte product; instead, only the short wave component of the signal input (ie the part of the wave extending into the region outside the core in the case of total reflection) will be included. Thus, as the area for the effect of the short-lived wave is only a fraction of the wavelength (A), the amount of product required (reactant + analyte) will be extremely small and a very high sensitivity

(with regard to the total amount of substance to be analysed)

can be achieved.

Thus, an aim of the present invention is to provide a method for rapid and accurate determination, with an optical waveguide, of the concentration of chemical substances or analyte in solution in a liquid. Another purpose of the invention is to provide an analytical method for determining biological analytes with great specificity and sensitivity. Another purpose is to provide an analytical method which can be easily used by moderately skilled persons and which requires only a small amount of analytical solution. Furthermore, it is a purpose of the invention to provide a versatile and automatic measuring device intended for carrying out the above-mentioned method.

These objectives (and further further objectives will appear in the course of this description) are suitably fulfilled by the present invention which includes the use of a waveguide core, whose refractive index (n-^) is chosen to be higher than that (^J for said analyte solution and that a ratio n^/n2 is provided such that the depth of penetration into the solution of the electromagnetic field associated with said light signal traveling in the conductor practically matches or exceeds the thickness of said layer of analyte-reactant product. Thus, including the method, e.g. contacting a section of a light (non-porous) waveguide core which is coated with a thin film of a reactant substance on an analyte with a solution of said analyte to thus enable said analyte to react with said reactant in the film and form a reactant-analyte product layer, that the corresponding optical changes are observed in relation to the time used for the light signal to be through the core at the exit of the core as a result of said product layer formation and comparison of the obtained data with standard reference data obtained in a similar manner from calibration samples of the analyte, where the refractive index (n^) in said core is greater than (n2) for the solution and the thickness of the film is only a fraction of the signal wavelength.

It will be appropriate at this stage to state certain general information about the transmission of light in a core by so-called total reflection. This can be better done with the help of part of the attached drawings, where: Fig. 1 schematically illustrates the total reflection process for an incident beam N at the boundary between a dense (n^) and a thin (n,>) medium at an angle t) greater than 9 , the critical total reflection angle. In this figure, Eq is the first quantity for the electric field component of the light at zero depth in the thinner medium, Z is the depth of the penetration axis and d^ is defined in the below-

end discussion. R is the reflected ray.

Fig. 2 illustrates the fractional penetration depth

for the electromagnetic field in a thinner mass medium for total internal reflection against the angle of incidence for a number of interfaces. The penetration depth is unlimited at the critical angle and is approx. 1/10 of the wavelength at stray incidence for relatively high medium index." A^ = A/n^ is the wavelength in the denser medium. (Taken from N.Y. Harrick, International Reflection Spectroscopy, Wiley 1967).

Fig. 3 illustrates the interaction between an evanescent wave and a layer of reaction product.

Physical insight into the interaction mechanism at a reflective surface can be obtained from a more fundamental approach using the Maxwell equations. In the present case, i.e. reflection in a dense medium at the boundary with a thinner medium, the following question must be answered: what is the electromagnetic field distribution in the thinner medium outside the reflection boundary surface for total internal reflection?

In this case, a wave function exists

in the thinner medium which progresses parallel to the interface. Its electric field amplitude falls exponentially with distance from the surface (see Fig. 1); hence it is called an evanescent wave. In the ideal case and if the thinner medium has no absorbing property itself at the wavelength in question, there is no net flow of energy into the non-absorbing thinner medium, as the time-averaged energy described by the Poynting vector (see, e.g., M. Born & W. Wolf, "Principles of Optics", Pergamon Press (1959)) is zero. Mathematically, the electric field can be described by the exponential function:

The depth of penetration d , defined as the distance required for the electric field amplitude to drop to e<-1 >of its value at the surface, is given by where A^ = A/n^ is the wavelength in the denser medium and n2_^ = n2 ^ nl is f°r^1°l<^et nie 11 about the ref raks ion index for the thinner medium divided by that for the denser medium. But-one with these conditions is illustrated by fig. 2 in which the penetration depth divided by the incident and reflected wavelength A^ is plotted against the angle of incidence 0 for a number of interfaces (ie for different ratios of n-j/n^). It should be stated that the penetration depth is approx. 1/10 of the wavelength in cases where the difference between the refractive indices is large, i.e. when n2/n^ is small, and this is close to the straying angle (A = 90°). This penetration becomes unreasonably large when A approaches X. At a fixed angle, the penetration depth is greater in the case of a small index difference (ie when n?_^ approaches 1). The penetration depth is also proportional to the wavelength and consequently greater at longer wavelengths. As an example, if the dense medium is glass (n-^ = 1.5) and the thinner medium is an aqueous analyte (n2 s 1.3), n2/n^ = 0.867 which corresponds approximately to curve 11 in Fig. 2. In this case, the penetration will be approx. 1/3 of the wavelength at stray angle, theoretically untuned at critical angle (60°), but already below 1 at 65°.

As E decreases exponentially, the region outside the interface in which the amount of energy for interaction with the product is still substantially consistent with the depth Z where the electric field magnitude is still a substantial fraction of EQ, e.g. a value of at least 0.1 EQ, better, the range in which E is between EQ and EQ/3. Thus, for optimum interaction efficiency the thickness of the reaction-product film should be approx. adapted to the depth for this area. This is illustrated in fig. 3 showing an incident (N) and a reflected beam (R), the zero depth vector EQ for traveling wave and a film of product reactant plus analyte (A) whose thickness approximately matches the penetration depth d^ where E is approx. EQ/3. In fig. 3, the influence of the refractive index of the thin film A is not considered significant because the thickness of this film does not exceed the depth of penetration of the traveling wave. However, change in refractive index for the thin medium due to growth of analyte-reactant film in the reaction area will be so small that a corresponding change in the value for the critical angle of reflection is practically insignificant, with the exception of reflective conditions very close to the angle of reflection. Support for this point of view, which constitutes an unexpected advantage of the invention in relation to prior art, can be found e.g.

in the aforementioned N.Y. Harrick reference, page 51.

Another point that should be highlighted for comparison with prior art relates to the efficiency of interaction between the light signal and the reaction product. In classic spectrometric systems, a light signal is passed through a transparent holder that contains the analyte (beaker or cuvette) and part of the energy is absorbed by the sample leading to a certain degree of absorption that is measured. Nevertheless, this method is not particularly effective as the amount of analyte should be relatively large to provide significant interaction with the light signal under normal conditions. In contrast, in the present invention where there is interaction between a traveling wave and a film whose thickness is approximately adapted to the penetration of the wave, the efficiency will be significantly higher as there is a strong light amplification effect in the interacting area. However, as has been shown e.g. in the previously mentioned Harrick reference, the field strength of the traveling wave within its interacting region with the analyte-reactant layer will be much stronger than that of the incoming signal. This is indeed the case due to the simultaneous presence of both incoming and outgoing beam field amplitudes.

The above-discussed optical foundations which are appropriate for the understanding of the operating principles of the invention refer to the use of unpolarized light. In practice, it is important to note that the first magnitude of the electric field at zero depth (EQ) is dependent on the state of polarization of the incident light wave. Thus, in some cases, polarized light will be advantageous instead of ordinary light, when used in carrying out the invention (it will be seen in the following that in the case of measurement of signal change by ellipsometry, polarized light is essential)

and, in such cases, the various optical parameters can be controlled and optimized for maximum response and sensitivity; e.g. a choice of a suitable incident polarization angle (eg polarization parallel or perpendicular to the plane of incidence) can be made to maximize EQ.

From the above considerations, the following advantages of the invention in relation to prior art can be fully understood. Thus, in the case of a sample containing a particularly specific reactant for an analyte, the thickness of the product layer will usually be determined by the respective size of the product molecules. E.g. in a typical immunoassay, the product layer may be formed by a first film of an antibody and a second film of an antigen. The thickness of this can depend on the types of molecules from a few Angstroms to several hundred Angstroms or more. Now based on the thickness of the layer, the refractive index for the core can be chosen and also in some cases the wavelength so that the above-mentioned parameters will be adapted as much as possible. To give an illustrative example, if the layer involved is relatively thin, high refractive index cores will be chosen (eg sapphire, n = 1.8; silicon, n = 3.4) and if compatible with the used optical methods (e.g. absorption, scattering, fluorescence, etc), shorter wavelengths will also be chosen. This will allow minimization of the interaction of traveling waves with areas of analyte solution deeper than the thickness of the room where the analytical reaction takes place and thus minimize the influence of unwanted external factors (background noise, the presence of impurities in the solution, etc.). Obviously, none of the methods from the prior art can provide such possibilities. It must also be stated when assessing the differences between the invention and prior art where the thickness of the covering extends well beyond the penetration of the evanescent wave (whereby the refractive index (n2) of the covering becomes decisive in contrast to what happens with the invention)

and where n2 is made greater than the index (n^) of the core,

the light signal will first be introduced into the core and refracted into the sheath and will not easily re-enter the core and be present at the exit thereof as is obviously assumed by some (see e.g. WO 81 100 912). Admittedly, when a light signal moves inside a thinner medium that is surrounded by a denser medium, refraction of the light signal into the envelope will occur.

Thereby, the refracted wave will be totally reflected by the outer side boundary of the sheath and will strike back towards the core.

Now, since the index n^ of the core is less than that of the envelope, the wave will do one of two things: if the angle of incidence is greater than the critical angle, the wave will again be reflected and will rest completely in the envelope.

If the angle of incidence is less than the critical angle, the wave will pass through the core and penetrate to the other side into the envelope, etc. Thus, in no case will a wave originally introduced into the core and refracted inside the envelope return only to the core and be present therein if it is not still surrounded by the enclosure. This is perfectly illustrated in fig. 3B of US-PS 4,050,895. No defect of this type exists in the invention in which the key light signal only moves in the core and not in the outer layer containing reactant and analyte.

The optical changes included in the method according to the invention can relate to different types of phenomena. E.g. the following phenomenon may be included: absorption of light moving in the nucleus; change in scattering of the light signal of the reaction product; change in fluorescence of the reaction product upon excitation of the light signal in the nucleus. Furthermore, the excitation signal in the core can be polarized and ellipticity polarization factors can be subjected to modification of the analytical reaction and be measured. Each of these possibilities-

is described in more detail in the following description.

The types of analytical measurements that can be carried out with the present invention are so numerous that it is practically impossible to specify them all. However, a few examples will be given in the following by way of illustration. However, before proceeding in this direction, it is appropriate to develop something fundamental regarding the application of the invention to "limited reagents" and "excess reagents" in assays which are desirably used in biological and diagnostic assays. For the purpose of such a discussion, one should conventionally call the analyte "antigen" AG and the reactant "antibody" AB. It should not be necessary to say that the reverse condition and is valid.

"Excess reactant" analysis relates to cases in which an excess of a reactant in relation to the analyte is used. "Limited reagent" essentially includes the use of a system in which the test substance or analyte (containing the antigen to be measured) is treated with a limited amount of a particular reagent (the antibody) to give an analyte-reactant product (e.g. eg an AG.AB complex) plus certain amounts of residual analyte. When the reaction is allowed to go to completion, i.e. the analysis progresses to equilibrium ("saturation analysis"), i.e. limitation of conjugate reactant

(AB) saturated with analyte, it is necessary to add, before

.the reaction (or in subsequent analyses, at some time

before final equilibrium is reached, i.e. before measurement), a fixed amount of a labeled form of analyte (AG<*>) to the reaction mixture under test. E.g. for an antigen to be analyzed with an antibody reagent, the ratio of labeled antigen (AG<*>) to unlabeled (unknown) will remain the same

in said residual analysis as it was at the beginning. Since the known amount of AB used will bind a known amount of AG + AG<*->mixture, it is sufficient to determine the residue AG<* >or AG<*> bound to AB (using its label) to calculate the amount of AG originally present in the sample.

To give a simplified example, it can be assumed that the sample contains x equivalents of an enzyme (AG) to be measured using a known quantity g of an enzyme conjugate (AB) which forms an AG-AB complex (with e.g. a 1:1 molecular ratio of both components). Then the reaction a equivalents of the same enzyme to be measured, but in labeled form (AG<*>) will be added to the sample. Thereby, during the reaction, part of g equivalents of antigen (AG + AG<*>) will be consumed by g equivalents of antibody. Now, after removing the complex from the mixture, the residue AG<*> will be found by conventional means. It is found by subtracting the value measured from the remaining AG<*>, that the amount actually used was b equivalents, it becomes clear as AG and AG<*> are chemically identical and consumed at the same rate that the ratio of consumed AG<*> to consumed AG, i.e. b/g - b should be equal to the original ratio a/x, from which x = a(g - b)/b can be calculated.

This type of approach is quite attractive although in the previously known applications, it suffers from certain disadvantages, one of which is the general requirement that the complex (the mixture of AG<*>.AB + AG.AB) must be separated from the reaction medium, which of and to is troublesome and a possible source of error. Now, when applied to the present invention, this disadvantage is not present because the complex formed automatically removes the analyte from solution as it is deposited on the waveguide. To illustrate the application of the present method to the "saturation type of analyses", one must again start with a waveguide, i.e. an optical fiber which is coated with a special antibody AB which is immersed in a buffer solution and allowed to enter equilibrium with it. The unknown amount of complementary antigen AG to be determined is added as before, but at the same time with a known small amount of the same antigen labeled with a molecule that has special optical properties (AG<*>), e.g. optical absorption, fluorescence etc.) 1 which can be detected by the interaction of the evanescent wave at the surface of the coated fiber using suitable optical devices which will be described later. Now, since both labeled and unlabeled AG are essentially identical in reactivity to AB-coated fiber optics, but only the labeled substance can be detected via its labeling, the change in optical property measured through the fiber (e.g., drop in absorbance if an absorbent label is used) be inversely proportional to the unknown concentration of AG and can be determined by reference to a suitable series of known standards. This type of application is illustrated in one of the following examples.

At this point, it is appropriate to make a clear statement with regard to the labeling used to

measure the reaction that takes place. Usually, in immunoassays, it is not possible to measure the analyte directly because the concentrations of analytes and reagents are extremely low. Since the equilibrium mixture in limited reagent analyzes essentially only contains an excess of analyte and a fixed amount of bound complex, when the latter cannot be measured directly and when the latter is a fixed amount, no quantitative assessment of the original analyte concentration can be obtained even after separation of the complex and excess of antigen. Added labeled tracer (a small amount of the labeled analyte) is necessary to allow measurement via labeling according to its distribution between bound and free fraction.

However, if the analyte has an intrinsic property

which can be detected or measured when concentrated locally (ie on-site separation and on-site concentration), e.g. on the surface of the fiber optic zone, then the addition of a labeled analyte track will no longer be necessary. Thus,

in the present invention, the on-site concentration of analyte-reactant complex would allow a measurement of the analyte without having to resort to a labeled trace fraction. However, local concentration of analyte-reactant complex will

(as the amount of the reactant is fixed) do not allow the quantitative determination of the original analyte concentration if the reaction between analyte and reactant is not measured kinetically or if the total amount of analyte is not

is less than the number of reactant binding sites. Thus, using on-site separation and concentration of the complex binding analyte in connection with a sensitive detection system in kinetic form which in the present invention will allow quantitative measurement of the analyte in the limited reactant system without having to resort to a labeled tracer.

With regard to labeled systems, a distinction can be drawn between tracer systems in which a labeled version of the analyte is added in trace amounts to the reaction and the labeled reagent systems in which a label is attached to the particular reagent. The first trace reagent is usually used in limited reagent assays (e.g. standard radioimmunoassays), while the latter is usually used in excess reagent assays (e.g. standard layer assays) and can be used in certain forms of kinetic reaction assays (e.g. Kronick et al, US-PS 3,939,350); labeled analyte and labeled reagent systems are suitable for the present invention if the label used is capable of being measured in an optical wavelength system (e.g. absorbing or fluorescent labelling).

For the next part of the discussion, reference must be made to another part of the attached drawings, where

fig. 4 is a diagram illustrating a first type of analysis called the "direct type of analysis".

Fig. 5 is a diagram illustrating a competing "limited reactant" type of assay. Fig. 6 is a diagram illustrating an intrinsically competitive "limited reactant" type of assay. Fig. 7 is a diagram illustrating a serial "saturation" type of analysis. Fig. 8 is a diagram illustrating a "layer" (sandwich) type of analysis.

The most straightforward case of analysis to which the invention can be applied is schematically shown in fig. 4. This is called the "direct" type analysis. In this analysis, a waveguide core 1 of which only a part (with refractive index n1) is shown is equipped with a film of antibody AB and this core is immersed in an analyte solution with a refractive index n2 (n^ > n^) containing a antigen AG? to be determined. The antigen will attach to the AB molecules in a proportion proportional to the antigen concentration (AG?) (since the amount of AB film on the core is a fixed unit) and once this proportion is determined, it can be brought into accordance with standard proportions which is obtained from calibration of AG solutions and (AG?) can be determined. Thus, the amount of AB available will be "limited" or it may be in excess and the reaction may go to an equilibrium stage. For measuring the AB-AG complex formation by means of optical changes that occur in the nucleus, the various aforementioned techniques can be used (i.e. extinction of the signal by absorption, scattering and fluorescence phenomena, etc.), provided that the formation of AB .AG forms the required optical changes. Thus, the sample will work particularly well on large molecules that are able to scatter light or have observable properties at certain wavelengths or emit fluorescence during excitation at certain wavelengths. If such properties are lacking, then the samples will be of little value and the "limited reagent" type sample will be advantageously used with an amount of 1 of the labeled AG (AG<*>) added to the analyte. This is illustrated in fig. 5 in which the characters (letters and numbers) used correspond to those in fig. 4. For this test, the amount of AG<*> does not need to be known, provided that it is standardized (ie always constant for a set of calibration curves and 1 sample), since the proportions of analytical reactions with different amounts of AG? will always be directly proportional to the AG<*>/AG? ratio (assuming, of course, that observable optical changes in the waveguide are due only to labeled reaction product AG<*>.AB).

In another, very convenient variation of the "limited reagent" type of assay, the waveguide core is coated with a known or fixed amount of the same antigen

(preferably in pure form) to be determined in the analytical sample, after which the nucleus is brought into contact with the sample and at the same time a fixed amount of freely detectable antibody is added. This is illustrated in fig. 6, where the references are the same as used before. It appears from the figure that the reference amount of antibody AB will simultaneously react with the reference AG on the core and with AG? to be measured. Thus will the observed proportion (as indicated by the optical changes occurring in the nucleus) be brought into relation to AB/AG? the mole ratio and brought into context with the standard reference proportion curve which will give the desired result. Obviously, in this case AB must have observable properties in the optical method used for the sample, i.e. AB can have absorption at suitable A values or be adapted to scatter light or the like. Alternatively, if AB is not directly observable on the conductor by reaction with AG, it can be felt, e.g. coupled with a fluorescent agent or other optically measurable label.

A further system includes the so-called serial testing as illustrated in fig. 7. In this test, a core is arranged with a fixed amount of AB, this amount is in excess of the corresponding equilibrium which is necessary to bind AG? to be determined. Thus, in a first step, the coated core is brought into contact with the sample whereby available AG? is bound and thus removed from the sample. Then the core is brought into contact with the mark AG<* >which will fill the cavities in the antibody layer on the core. Measurement after or during the reaction of the optical changes due to the labeling of AG<*> will provide the necessary data for calculating the initial amount of AG?.

Finally, the invention can also be applied to the case of analysis which is termed "layer" analysis or "sandwich" analysis regarding the determination of antigen with more than one binding site for antibodies, i.e. the antigen has sites numbered (1), (2). .. (n) which is able to bind with 1, 2 or n different antibodies. This is shown schematically in fig. 8 where the first antibody is designated as AB1, the second antibody as AB2 and a two-site antigen as <2)AG(1). In this case, it is assumed that the antigen is not measurable optically by itself while AB2 is measurable after reaction at the appropriate second site of AG. Thus, in this case where (2)AG(1)? is to be determined, a core with a first reference coating of a first antibody (AB1) will be used

and contact is formed with the antigen solution. The latter will thus bind to the nucleus at its first binding site, after which the second antibody AB2 in a reference quantity is added and its proportion of binding at site 2 of antigen is measured based on optical changes that occur in the nucleus as a result of this binding. Of course, in this procedure the first reaction AB1 + (1)AG(2) can be allowed to go to equilibrium before the addition of the reference amount of AB2; or, alternatively, a simultaneous type test can be carried out, i.e. AB2 can be added to the solution at the same time as the AB1 coated conductor core. This is particularly applicable where AB^ and AB^ are different monoclonal antibodies (see, for example, Uotila et al., Journal of Immunological Methods, 42 (1981), 11 - 15).. Also any variations of the aforementioned analysis systems can be adapted by the person skilled in the art without deviating from the framework of the invention, whereby the operating parameters for any of such variations are determined with reference to the calibration of dissolution samples of the analyte.

Examples of devices for carrying out the various embodiments of the method according to the invention in the area of optical absorption, fluorescence and scattering as well as ellipsometric measurements will now be produced with reference to the following part of the attached drawings, where: Fig. 9 is a schematic diagram of an apparatus for on-site measurement of changes in the light signal traveling through an optical fiber caused by unprecipitation of a complex film on the optical fiber,

fig. 10 schematically on an exaggerated scale, a fiber optic zone that can be used in the apparatus of fig. 9; fig. 10a is a partial top view of said probe and fig. 10 b is a side section thereof along the line B-B in fig. 10a,

fig. 11 is a schematic view, on an enlarged scale, of another probe design particularly intended for light variation measurements, while fig. 1a is a front view and fig. 11b a side view,

fig. 12 is a schematic diagram of the light signals at the detector of an apparatus corresponding to that in fig. 9, but used for fluorescence measurements, whereby fig. 12a relates to the situation before the reaction is started and fig. 12b relates to the situation some time after the start of the test,

fig. 13a schematically shows (top view) an apparatus for measuring optical changes caused in a waveguide by light scattering,

fig. 13b is a schematic partial side view of the apparatus in fig. 13a,

fig. 14 is a schematic view on an exaggerated scale of part of a modification of the embodiment of FIG. 13a and 13b,

fig. 15 schematically shows part of another embodiment,

fig. 16 schematically shows an apparatus for measuring optical changes caused, in a waveguide, by the fluorescence phenomenon,

fig. 17 schematically shows, on a greatly exaggerated scale, part of the apparatus in fig. 16 to show the reflection angles for incident and fluorescent light,

fig. 18a shows schematically (top view) an apparatus for measuring ellipsometric optical changes in an optical fiber caused by the formation of a complex film on the fiber, and

fig. 18b is a cross-sectional view of a part of such an apparatus, taken along the line A-A in fig. 18 a.

The apparatus shown schematically in fig. 9 essentially comprises the following components: a) A fiber optic 1 whose central part passes through a container or cuvette 2 intended for a liquid analyte to be determined. The coating on the fiber section immersed in the liquid has been removed so that this part can be coated before operation with a thin film of a special complexing reagent of substances dissolved in the liquid and to be determined. The composition of fiber 1

and container 2 forms the sample probe for the apparatus.

b) A splitting disc 3 is turned by a motor 4

and is equipped with two diametrically opposed windows with filters

5 and 6, plus a hole 7.

c) A main light source 8, a collimator lens 9,

an annular opening 10 and a focusing lens 11 for introducing an angularly selected light beam into the probe fiber core. In this particular embodiment, the fiber is of the multitype and the opening 10 is arranged for the passage of particular types according to certain requirements to be discussed below. However, with modifications to be discussed later, the present apparatus can also be used in connection with low-mode fibers, e.g. single fibers. The focusing lens, which is shown schematically in the drawings, is in reality an optical system analogous to a microscope objective, but which can be adjusted, in addition to its displacement along the optical path,

sideways and up and down for accurate placement of the beam in front of the fiber front end. d) The apparatus then comprises a main light detector 12 for converting the output light signal from the core into a

electrical signal, a locking amplifier 13 and a display device 14. A reference signal is also provided by another source 15, and the light from this is pulsed when passing through the hole 7 in the splitting disc and is directed to a detector 16, which detector then sends corresponding electrical pulses to the latching amplifier 13.

An embodiment of an analytical zone usable with the present apparatus is shown in fig. 10 (fig. 10a + 10b) which shows a plastic holding tank 2 where S-shaped grooves or slots 21a and 21b are cut into two opposite sides. A part of the fiber optics 1 with its coating 22 has been inserted and is held firmly by said track. The coating of the middle part of the fiber has been removed, e.g. by etching with a suitable solvent, and then coated with a thin film 23 of a particularly complex conjugate of the substances to be determined, such substances being represented by small squares 24.

The description of the operation of the present invention follows as follows: Filter 5 is first selected to pass through the core a wavelength X^ which is modified by absorption at the internal reflection point due to absorption of part of the evanescent wave by the coating of complex material that will form between the film 23 and the dissolved molecules to be analyzed 24. For illustration purposes, it can be said that 23 shows a very thin layer of an antibody (AB) and 24 represents molecules of a special antigen (AG) to be determined, whereby the analysis is of the "direct type" as illustrated in fig. 4. The filter 6 is selected for passing a wavelength A2 through the core, which is essentially not absorbed by such complex coatings and consequently is not affected by the immunological reaction present. Thus, when the splitter 3 rotates, two signals and A2 are alternatively fed to the probe core, whereby one signal will be progressively absorbed with growth of the coating 23 + 24, and the other signal A2 will serve as a reference signal, i.e. for calibration and compensation purposes ( cell or fiber replacement, etc). The probe is prepared by selecting a suitable length of optical fiber (multimode in the case shown in Fig. 10), inserting it into the grooves 21a and 21 so that the middle part is held horizontally in the cuvette 2. Watertightness is provided by then filling the grooves with a rubber compound or cementing solution and allow it to dry, The coating of the central part is etched away. For a resin coating, this is done with a suitable organic solvent for this coating. For a glass coating fiber, the etching is carried out with dilute hydrofluoric acid. In the latter case, it is not necessary for the coating to be completely etched for reasons explained below. Then the solution or etching solution will have to be removed and the cell carefully cleaned with distilled water, the fiber core coated with a thin film of antibody (AB) by usual means, e.g. by filling the cell with an AB solution so that AB is deposited on the nucleus as a uniform layer

(if for some reason the possibility that the surface of the fiber does not have sufficient affinity for the molecule deposited thereon, binding can be provided in advance by special treatment known in the art, e.g. grafting binding sites, application of a reactive interlayer ,

etc. In this regard, there is a rich literature (as described in European patent application 29,411). After emptying the cell and cleaning, the latter is mounted on the apparatus at the location indicated between the lens 8 and the detector 12 and a suitable buffer solution is introduced therein. The device is then activated and the splitter is rotated at an appropriate speed, e.g. 120 rpm. The signals A^ and A2 are fed to the core of the probe and the detector 12 converts them into approximately square electrical pulses which are fed to the lock-in amplifier 13.

A reference locking signal is also provided at each revolution, via the hole 7, of the source 15 and the detector 16 which enables the amplifier to distinguish (in case of coincidence) between the pulses from and A2- In practice, the control is preferably built up so that said pulses have approximately the same size before the start of the reaction. Then, at time zero, the sample to be analyzed (a solution AG) is added to the cuvette in the probe 2 and mixed rapidly with the buffer (eg with a stirrer or a gas bubbling device_s©m not shown). The signal due to A-^ starts a progressive change at the exit of the core of the probe, as a result of the molecules of the analyte AG binding to the fiber core (for building the AB.AG complex) and said complex absorbing part of the fleeting wave energy to A^, but a speed proportional to the concentration of AG and corresponding variations are provided on the amplifier in the form of corresponding electrical pulses from the detector 12. Thus, the calculation circuit which is connected to the locking amplifier 13 will calculate data which is written from the relationship for A- the ^/A2~ signals and provide the results to the display system 14, e.g. in the form of a proportion curve which is recorded on a diagram (of course another type of display can also be used if desired: digital display or oscilloscope, etc). The proportion data obtained is then compared with standard data obtained from solutions of AG of known concentrations and the unknown concentration is provided by interpolation. Such calculations can be done manually or can be done automatically using a microcomputer, where the reference data is created in the data store. By choosing suitable periods during the time course of the reaction, desired reaction proportion data can be selected and separated from other proportion data due to the reaction for disturbing and unwanted reactions that progress more or less simultaneously, but with different proportions. In addition or alternatively, the equilibrium conditions can be derived by extrapolation and by this the time it takes to carry out each measurement in comparison with normal equilibrium measurements can be reduced. Further considerations on this question can be found in "Kinetic Versus Equilibrium Methods of Analysis" by B.W. Renoe et al., in "Centrifugal Analysis in Clinical Chemistry", Price & Spencer, Praeger (1980) and Analytical Chemistry 50/02, (1978), 1611-1618.

It is appropriate at this point to state that the sensitivity for the measurements can be varied depending on the light mode order supplied to the fiber input, i.e. changing the opening 10 with ring setting and width. This is easy to understand if you remember that the absorption phenomenon leads to a totally reflected beam inside one core and due to a coating on the outside, the core can only affect the volatile wave component of the beam, i.e. the electrical components that penetrate the coating. Thus, total reflection angles for the mode chosen will be sufficiently shallow with respect to the fiber axis to ensure full reflection (even if the refractive index of the aqueous solution is slightly modified by the growth of coatings surrounding the fiber core) and sufficiently deep to provide a sufficient density for the reflection sites along the fiber (of course this is only at such sites where the evanescent wave interacts with the complex coating). Thus, a sample sensitivity optimization can be achieved by correctly setting aperture parameters, whereby this depends on the respective core, sample resolution and complex refractive indices. Such settings can be determined by the expert in the field for each type of measurement and can then be incorporated into the construction for the field operator's special use.

It is also interesting to note that by changing the settings of the opening's 10 parameters, the present device can be brought into operation in a different "class".

E.g. by correctly setting this opening, the device can be designed for operation with light mode close to the critical angle for total reflection Øc for starting the system in question. In the case where, during the test, the difference An = n1 - n» should decrease with the complex formation, the angle of total reflection at the core coating boundary will change so that some of the modes (or all) will be suddenly broken on the outside of the fiber and a sharp cut-off of the signal will occur. Such a device will therefore give a very high sensitivity to very small amounts of analyte molecules. However, it is less suitable for quantitative measurements.

It has been said above that when using a glass coating fiber optic such a coating will not necessarily be completely etched away for the present use. Admittedly, during operation, as the evanescent wave will in reality penetrate the coating a few tenths of a nm, it may be possible to have a residual glass coating around the core with a thickness below that required for the evanescent wave to significantly interact with the reaction materials, and this will provide a less strict control condition for the etching operation. Thick fibers are also less fragile than thinner ones.

A type of fiber holder which will increase the sensitivity of the sample by a large factor is shown schematically in fig. Ila and b. This holder consists of two helically wound flanges made of an inert plastic (e.g. plexiglass or polyester) on which a piece of optical fiber is wound and clamped by means of clamps 33 and 34. Front and the ends of the fiber, resp. 35 and 36, is bent sideways to

be applicable with light introduction lenses 11 and detector 12.

The middle straight portions of the windings 37 of the fiber are uncovered while the remaining curved sections 38 rest in grooves which retain their original protective coating. The etching of the uncovered parts is carried out by first covering the lower flange of the holder with a suitable silicone rubber cementing solution and allowing it to dry. The holder is then immersed in an etching solution down to the lower surface of the upper flange, whereby all intermediate sections of the coating will be removed, while the lower rubber-protected flange remains untouched. When carrying out the tests and after coating the uncovered part of the fiber with AG or AB as before, the holder is correctly positioned by means of a hook 39 in the optical path of the device. Then a cuvette containing the reaction medium is brought in from below, the reaction medium is added and the measurements are carried out as before.

It should be pointed out that the entire above discussion with regard to the present invention relates to the use of fiber optics to be used in an aqueous or organic medium, that the refractive index is close to that of water (i.e. around 1.3) while the index n-^ for the glass core to the fiber is closer to 1.5 and the index n^ of the active coating is intermediate, i.e. <1.5 but >1.3. This condition is essential here because in the case where n0 > n^, the light would be refracted into the coating, then back into the core, etc. As has been seen previously, such a method is not possible for itself, e.g. it is described in Nature 257 (1975) by Hardy et al who worked with mad-rod waveguides and had to use rather thick polymer coatings which contained, embedded, some reactant to be determined, but it will not be practical in the present case as the surrounding solutions constantly will have refractive indices that are smaller than the fiber core n^ and only very thin coatings (on the order of magnitude a few Ångstrøm to a few tenths of a nm) are used. In contrast, Hardy et al worked in air and did not measure rates of the reactions.

Another important point that should be discussed is the question of monofibres. Of course, with very small modifications which are known in the field and which mainly concern the introduction of light into the fiber, a single type of fiber optics could also be used in the present method. <Such fibers generally have a very thin core (a few microns) which is surrounded by a relatively thick coating and their transmission is strongly wavelength dependent, e.g. a fiber with only 5% attenuation per meters at 850 nm can have an attenuation of approx. 40% at 800 and 900 nm (see T.G. Giallorenzi in Proceedings of the IEEE 66 (19 78), 748). Now, for a monotype conductor in a fiber at wavelength X, the following expression V = 8.9a/n^AnA must have a value below 2.4 (A n = n, 1 - n2„ and a is the fiber radius in microns). Above V = 2.4, the fiber is no longer in single mode. When the value V is small (e.g. below 1), the guided single mode will be more loosely bound, i.e. the field spreads significantly beyond the physical core of the fiber (e.g.

two or three times the core diameter). Thus, the transmission properties of single-mode fibers will be very sensitive to small changes (A-changes) in the refractive index of the coating, i.e. that of the organic coating formed during the test of the present invention. Of course, this index of refraction is the key variable present in the present analysis performed with single mode fibers as the thickness of the complex layer grows during the reaction. While internal reflections in a multimode fiber allow the evanescent wave in the immediate vicinity of the reflecting branch to pass parallel to the core over a very short distance (which can be defined by Maxwell's equations) at each reflection, the single mode fiber will allow the passage of this evanescent wave fraction parallel to the core along the entire effective length of the fiber. Thus, the use of single-mode fibers in the present apparatus will facilitate an increase in sensitivity compared to multi-mode fibers.

Single mode fibers can be used without complete etching of the original coating as the guided light fields extend substantially into the coating and also into the complex layer formed during the reaction. Modifications: which must be made to the apparatus when using single-mode fibers are essentially of an optical nature, ie introduction of light into the fiber and detection of the signal. Such optical variations are not described here as they are known to those skilled in the art and are discussed in the literature (see, for example, the above-mentioned Giallorenzi article and the references therein).

The present apparatus can also be used for carrying out the method according to the invention using fluorescence effects instead of absorption or refractive index changes. To do this, the following modifications should be performed: a) A fluorescently labeled antigen (AG<*>) will be added to the analyte AG. Thus, the method will be operated under the conditions of "limited reagent" immunoassay as discussed above (see Fig. 5). b) Filter 5 will be selected for an A^ which is specific for excitation of fluorescence to be measured and filter 6 will be selected for blocking A^, but passage of fluorescence emitted wavelength A^. c) An additional filter with optical properties identical to filter 6 will be inserted between the fiber rear end and

the detector 12.

Then the probe with buffer medium and its fiber optics 1 coated (as usual) with an AB film will be installed and the apparatus will be set for reaction as shown schematically in fig. 12a. This figure shows a sequence of pulses from the detector 12. The pulses marked R are reference pulses which are produced when the filter 6 is in the beam. The pulses E are the emitted fluorescence pulses which are introduced into the fiber by the fluorescence complex AB.AG<*->molecule which is formed on the fiber core. Thus, at the start the E level (fig. 12a) is approx. zero from no fluorescence or only residual background noise. Then the analyte containing the antigen to be determined plus a known amount of fluorescence labeled AG<*> is added. A fluorescent coating will gradually form at a rate proportional to the AG concentration and an emitted fluorescence signal appears in the fiber and increases progressively with time as shown in fig. 12b. This variation is measured, supplied to the amplification and calculation sections of the apparatus and recorded as the desired ratio. Then visual or calculated comparisons are made with recorded data and standard reference data from which the desired analytical results are obtained using techniques specific to "reagent limited" analyzes as discussed above.

In the present embodiment, the fluorescence formed by the coating on the outside of the fiber core is essentially re-introduced into the fiber and provides the signal for use in the sample. A related phenomenon has recently been described (see The Journal of Cell Biology 89, 141 - 145 (1981)) with respect to a glass block.

When using a fiber optic, advantages can also be derived from a great sensitivity as the usable length of the fiber can be made quite significant even if confined in a very small space and when using very small volumes of solutions. It should also be mentioned that the measure of fluorescence of coatings in immunoassays has already been reported

(see M.N. Kronick et al, Journal of Immunological Methods

(1975), 235-240). In such a case, however, the fluorescence was measured through antibody solution and only one internal reflection site was used, which gave poor sensitivity as only a very limited part of the total fluorescence emission could be processed. In the case of the use of fiber optics as in the present invention, lateral fluorescence absorption can also be measured, e.g. by using a flat-wound piece of fiber and placing the detector axially to the winding and thus collecting a larger part of the fluorescent light emitted by the fiber.

The present embodiment offers many advantages compared to the known test methods and devices. For example: a) The sensitivity is proportional to the length of the submerged fiber part, very sensitive probes can be produced even if they have small dimensions. b) Measurements can be carried out over a wide range of wavelengths in the visible, ultraviolet and infrared bands. c) A wide range of substances (biological or non-biological) can be tested, among which can be mentioned medicines, haptens, enzymes, peptides, proteins, hormones, bacteria, viruses and cells. A more complete list of target-only analytes can be found e.g. in US-PS Nos. 3,817,837 and 4,299,916.

An interesting special case is when testing

of blood samples for transfusion with regard to possible antibodies in the recipient's blood. Thus, optical fiber probes can be produced with a thin film containing blood components from the patient and tested against blood cells of potential donors. In case of a cross-reactivity, the cells will precipitate on the fiber (due to reaction of their own AG centers with AB from the fiber) and this reaction can be easily measured by one of the typical absorption bands for hemoglobin (e.g. at 555 nm) . Another interesting special case is the possibility of using a waveguide sensor in vivo to make qualitative or quantitative measurements of analytes in the body or excreted from the body. E.g. in diagnosis, it will be possible to analyze the quantity of circulating insulin in reaction to a glucose load test and, in the case of treatment with injected hormones, the in vivo sensor can detect circulating hormone concentration. The possibility of using in vivo sensors has now been described by Buckles, but the present invention will have the added advantage of the absence of a label in immunoassay mode (where many labels are highly active compounds with possible toxic or carcinogenic effects) and improved light transmission will thus avoid the need for special transmission fibers for coupling

the signal in and out of the sensor as described by Buckles.

The apparatus shown schematically in fig. 13a and 13b comprise the following key components: An optical probe composed of a waveguide plate with sloping edge 41 is held by brackets (not shown) and by a counter plate 42 held in precisely regulated parallel facing for hold the waveguide 41. The plate 41 can be made of a high-quality float glass that is precisely cut, or of quartz. The plate 4 2 can be a microscope plate. The space 41a between the plates 41 and 42 is of the order of a fraction of a mm. This ensures that an analyte solution can easily be introduced into this space where it will be held by capillary forces. The apparatus further comprises a light source 43 (in this particular embodiment the light source is a He-Ne laser which provides polarized light), a splitting disk 44 which is operated by a motor 4 5 and which is equipped with a hole 4 6 for the generation of a pulsating light signal which is to be sent towards the optical probe through an optical system comprising focusing lenses 47 and 48, a mirror 49, a polarizing plate 50, a cylindrical lens 51 and a diaphragm 52 for minimizing scattered light. The reason why the light signal is pulsed is to provide maximum phase-sensitive amplification of the detected signal.

The apparatus further comprises a photomultiplier detector 53 having a high voltage source 54, the output of which is connected to a latching amplifier 55 via a two-way switch 56. The photomultiplier is arranged to collect light scattered by the product formed on the waveguide plate in space 41a when the result of a chemical reaction that occurs between a reactant and an analyte of the solution fills space 41a. The diffused light is indicated by arrows 57.

The apparatus further comprises a detector 58 for collecting light which comes out at the output of the waveguide core after multiple reflection therein as shown in the drawing. The exit

of this detector can alternatively be fed to the locking amplifier 55 through the switch 56. The reference signals for the device are provided with a semi-transparent mirror 59 which emits a small part of the light from the source to a detector 60, where the latter forms an intensity reference signal which is fed to an analogue divider 61, to compensate possible variations for the source during the measurements. A pulsating locking reference signal is arranged by means of a mirror 61 and a detector 62, while the corresponding electrical signal is fed to the amplifier 55. The apparatus finally comprises an electronic unit 62 which will comprise a display element (in the same way as in the previous embodiments ) and a registration

device for recording output data and possibly a microcomputing unit for calculating the measured data and for carrying out the required comparisons with the stored reference data from the calibration experiment.

In practice, the device works in the following way. The first step is to prepare the optical probe by depositing a film of reactive substances on the surface of the waveguide 41 which defines the space 41a. Details of how this is done are given below. Then the conductor 41 and the plate 42 are mounted on the apparatus and the optical and electrical system is started up. After a few minutes of warm-up, the controls are set for zero reaction from the photomultiplier 52 (or alternatively full transmission from the detector 58). Then the analyte solution (a few pl) is pipetted into the compartment 41a whereby the analyte in solution begins to react with the reactant film on the conductor 41. When the analyte-reactant product reaches the scattering sites (large molecules, agglomerates etc), a signal that has been scattered will now the photomultiplier tube 53, which is amplified, processed and displayed in relation to time by the display included in the unit 62. This results in a proportion curve, slope data is recorded and calculated against standard data obtained from calibration samples and stored in the storage unit of the electronic unit 62 This calculation gives the desired analytical results (e.g. the concentration of unknown substances in the analyte solution) according to common devices. If the output of the detector 58 is not used, the edge 41b; until the conductor 41 could be masked to minimize the formation of backscattered light. Unmasking can be done e.g. with black paper or paint.

An improvement of the embodiment described above is illustrated in fig. 14. Instead of using a perfectly homogeneous working surface for the waveguide 41, one can first treat this fist to give it discontinuity. E.g. one can modify the inherent adhesion of the surface to reactant in accordance with a certain pattern (achievable, for example, by known photolithographic techniques). In the case shown in fig. 14, the surface 41c of the conductor 41 has been roughened in the area indicated by the number 41d, whereby such areas are parallel strips over a wavelength width and separated by a distance of the same order of magnitude. Such a lattice-like pattern can be obtained by first covering the surface with a photoresist, exposing the resist layer by means of a photographic film with the negative image of the lattice, developing (ie dissolving the unexposed areas in a suitable solvent) and light etching of the uncovered areas, after development, e.g. with HF. After the final removal of the resist, the plate has a lattice pattern or stripes with alternating zones of higher and lower affinity for proteins (antigens or antibodies). Thus, when the plate is brought into contact with a reactant (AB), the latter will mainly stick to the etched areas as indicated in fig. 14 by the letters AB. At this stage, the thickness of the pattern will not be sufficient to provide dispersion. However, in the presence of antigen (which is of the correct light-scattering property), the latter will bind to the bands bearing antigen, i.e. as indicated by AG in the drawing. This discontinuous type of layer will provide definite patterns of diffraction scattered light as opposed to the stray scattering mode of the main embodiment and thus improves the directivity and collection efficiency (of the photomultiplier tube) of the signal that is scattered. In fig. 14, the arrows 65 indicate the zeroth order of diffraction and the arrows 66 indicate the first order of diffraction. When collecting a particular order (e.g. the first) the collection of the signal by means of the photomultiplier tube 54 will be greatly enhanced and the signal-to-noise ratio is increased by this modification compared to the collection of randomly scattered light which is only around 10% of the total scattered light.

For another modification, see fig. 15, a perfectly flat and even surface 41c on a waveguide is sprayed with micro-droplets of antibody (AB) and during the analysis antigen AG attaches only to such preferred areas. Such a construction constitutes a statistical diffuser that spreads light in the form of a cone whose size and geometric parameters depend on the size and distribution of the drops. Thus, a directional effect has also been achieved here, which promotes an increase in the efficiency of signal collection. Another advantage of providing a diffraction light signal is to minimize the importance of accidental scattering such as by dust particles or scratches in the glass at larger sizes (of the next order of magnitude, ie about 10 X or more). In such cases the diffraction angle will be smaller and the resulting diffracted light will not reach the photomultiplier tube, its presence may be omitted.

The apparatus shown in fig. 16 is generally similar to what has already been discussed in connection with fig. 13, but it is intended for fluorescence measurements instead of scattered light measurements. This apparatus essentially consists of an optical system represented by block 70, which is practically identical to the system used in the apparatus of fig. 13. Therefore, the details are not repeated for overview reasons. This system 70 contains a pulsating light source and means for providing a sample signal with correct excitation of wavelength which is correctly directed towards a waveguide 71 and a reference signal to a reference detector 80. In a similar way as in the second embodiment, there is a plate 72 which together with the conductor 71 determines a thin space 71a for the introduction of analyte solution, made of reactant which is coated on the upper surface of the conductor 71. The apparatus also comprises a side detector (the photomultiplier tube 73 and its supply 74), a core-output detector 78 and blocking filters 76 and 77 (these features are missing in the previous embodiment). The electronic components of the apparatus include two integrating amplifiers 81 and 82, a multiplexer 83, an analog-to-digital converter 84, a microprocessor 85 and a display-recording device 86. All these elements are common and their operation is known to those skilled in the art.

The operation of the device in the side recording mode is exactly the same as that described for the spread version. Thus, after the preparation of the waveguide 71 with a layer of reactant on the upper surface and on the plate 72, the analyte solution will be introduced to give a fluorescent reactant-analyte product. The optical unit 70 transmits a test signal of the correct wavelength'^ for emission of fluorescence of wavelength A2- The emitted fluorescent light passes across the screen 76 (which screens all other wavelengths including scattered emission: light) and strikes the photo-multiplier tube 73, where a signal proportional to the fluorescence (and to the extent of the reaction) is produced. This signal, together with the reference signal from the detector 80, is fed to the multiplex amplifier 83 which alternatively feeds them to the remaining electronic elements whereby a treatment similar to that described previously will occur.

Simultaneously or alternatively with the aforementioned operation, the signal that appears at the core output and falls on the detector 78 can also be measured. To understand how this is possible, reference is made to fig. 17. This figure shows part of the conductor 71, the plate 72 and the space between them 71a and schematically a layer of AB.AG product (the product that is the result of the reaction of reactant AB plus an

listen AG of which the proportion is to be measured in the "sample). The figure also shows the different light rays that are included in the excitation of the fluorescence process, i.e. N is the incident ray at A^, R is the reflected ray of wavelength and Rj is the formed backward and forward fluorescent beam (A2)- 9^ is the angle of incidence and 9^c is the critical angle for A,.0_ is the critical angle for

1 f c

A,,. As shown, the radiated light N now hits the inner surface of the wall at an angle 0. greater than the critical angle 0. and it is thus reflected (R). However, part of the transient excitation wave is absorbed by the AB.AG layer and energy is re-emitted with a shorter wavelength A2« Due to the principle of reciprocity (confirmed quantitatively by C.K. Corniglia et al. in J.O.S.A. 62, (4), 1972, 479 - 486 ), want

now excited molecules emit ephemeral photons that behave exactly like the incident ephemeral wave photons. So-

thus, fluorescence emitted by the molecules close to the interface between dense and thin (AB-AG layer) will progress into the dense medium at angles greater than the critical angle Q^c for X^. Currently, peak fluorescence is observed when 0., is close to 0. and at angles close to 0_ (see R.E.

i ic ^ fc

Brenner et al., Fiber Optics, Advances in R s D, Providence, RI, U.S.A.; 19 - 23 June 1978, NY Plenum Press (1979)). Thus, the emitted fluorescence will concentrate the maximum intensity within a relatively narrow angle range when it is guided in the waveguide, the output at several interacting locations is added up to give a higher intensity signal at the output of the core. Another important point is that, due to the refractive indices being different for different wavelengths, the excited signal and the fluorescence signal will follow paths with different angles of reflection in the conductor and will emerge from the output also at different angles from each other. Accordingly, there is an inherent optical separation of the emitted beam from the excitation beam at the exit of the core and the detector 78 may be suitably positioned to be in the path of the fluorescence signal (A^) while avoiding the re-excitation beam even in the absence of filter 77.

The apparatus shown in fig. 18a and 18b are intended for the measurement of optical changes that occur in an optical fiber waveguide that has been transmitted during its formation

of a product on the surface of said fibre, whereby this measurement is carried out by means of ellipsometry. Ellipsometric measurement applications for bioanalysis have been described in detail in the applicant's European application EP 81 810 255.0 and the reader is referred to the contents of this application for general consideration in this area. The main difference in the present application compared to the previous description is the use of an optical fiber instead of generally known flat reflective surfaces. Ellipsometry is based on measurements of changes in the degree of elliptical polarization of light caused by the presence of a film of material deposited on reflective surfaces. Based on the special property

and geometry for optical fibers in comparison with a flat surface on a waveguide, additional features must be taken into account. First, to maintain a beam of light that is linearly polarized in a fiber, one must use a special group of light modes. These modes are defined as HE^m modes, with m = 1, 2, 3... n(n is limited by the size is the refractive index of the fiber in question, see E. Snitzer and H. Osterberg, J. Opt. Soc. Am. 51, 499 (1961)). Secondly, in order to be able to measure the full extent of any polarization modification caused by a reaction occurring on the surface of the fiber, it is important that the signals in any direction of polarization are transmitted equally well along the waveguide. Thus, geometric deviations such as may be caused by the mechanical load on the fiber should be avoided, because in such cases a particular polarization direction could be favored. Accordingly, in the present apparatus the optical probe (of which the key component is the optical fiber) is made short and rigid and immovable to minimize unwanted vibrational influence or other possible disturbances or deviations.

To preferentially emit HE-^ modes in an optical fiber, the radial light intensity should have a Gaussian distribution, e.g. that obtained from a laser radiation in the fundamental mode and the beam should be directed axially and centrally towards the fiber end. This connection can be achieved using a microscope objective. In order to minimize background drift, it is preferred to work with just one mode rather than several modes at the same time. In the present apparatus, the HE^ mode (lowest single mode) was chosen for reasons of simplicity.

In principle, multimode fibers can be used with single-mode signals. This can be made possible (eg the correct Gaussian distribution can be chosen) by letting the incident beam diameter at the fiber entrance face be 0.65 x the diameter of the fiber core. However, this is not in reality satisfactory because other modes with different polarization states are also emitted to a certain extent and a partially depolarized signal is obtained at the probe output. Consequently, it is strongly preferred to use a single-mode fiber in this embodiment as it will essentially only transmit the HE^ mode whereby no higher-order conversion of the modes and thus decrease in the degree of polarization will occur. Consequently, a higher signal-to-noise ratio is achieved than compared to the use of multimode fibers.

In the present embodiment, the fiber length is preferably 10 cm or less for the above reasons. A high-quality single-mode fiber is preferred in terms of depolarization and birefringence properties. Fibers obtained by the CVD technique, as described in H. Aulich et al., Applied Optics 19, 22, 3735 (1980), are preferred.

The apparatus shown in fig. 18a and 18b comprise an optical zone comprising a partially etched piece of optical fiber 101 with a core 101a and a symmetrically removed cladding 101b. The technique for removing the coating is the same as described for other optical fiber applications in this specification. At a very short distance from the uncovered fiber core (part of a mm) on both sides of it, two glass plates 102 are placed which are held rigidly on "U-shaped brackets 103. The clad end parts of the fiber waveguide are clamped by means of clamps not shown fixed to an adjustable stand (not shown) whereby the latter enables the position of the fiber to be accurately controlled laterally and up and down. The space 104 between the fiber core and the plates 102 is the reaction site, and the fluids to be sampled are held by capillary forces between fibers and plates.

The present apparatus further comprises two microscope objectives 105 and 106 for coupling the input incident beam with the fiber and the outgoing elliptically polarized signal with the detection system, respectively. The incident beam is formed by a light source 107 (in this embodiment a He-Ne laser) whose output light passes over a polarizer plate 108 and a rotating splitting disc 109 driven by a motor 110 and has a hole 111 for splitting the signal. Next, the detection system of the apparatus comprises a quarter-wave plate 112, a polarization analyzer 113 and a photodetector 114, where these elements correspond to corresponding elements described in the related application EP 81 810 255.0 and are operated in a corresponding manner. Finally, the device comprises a latching amplifier 115 and associated components, i.e. a microcomputer unit 116 and recording device 117, whereby the functions of these electronic components are essentially the same as for the corresponding elements already described earlier in this description. It should also be stated that the split reference signal used for reference purposes is shown in the drawing as coming from the splitting motor 110 and is transmitted to the amplifier 115 by a line 118.

The operation of the apparatus is very similar in essence to that described in the previous embodiments combined with the operation of the apparatus described in said application No. EP 81 810 255.0. Thus, to perform a test, the uncovered portion of the fiber is first coated with a reactant (e.g., antibody) as shown by reference number 120 in the drawing to fill the space with the correct solution as shown in the drawing with dashed patterns. Then, after the usual cleaning and drying step, zeroing of the instrument in the presence of a blank buffer by correct alternative rotation of the polarization ion elements 112 and 113 (see the description in application no.

EP 81 810 255.0), and removal of the blank solution, after which the solution to be tested is introduced to initiate the reaction between antigen analyte and antibody coating which results in growth of reactant-analyte layer on the waveguide core and consequent modification of the elliptically polarized output. The corresponding change from photodetector 114 is amplified and measured in the electrically associated components 116 and 117 and provides the desired data exactly as in the previous embodiments.

For the immobility of a film of antibody on

a waveguide core (or of course of antigen if this is the antibody that must be determined) many methods are known that can be used as already mentioned earlier. When implementing the present invention under reference

for immunoassay testing, it is generally preferred to take advantage of the fact that most types of glass will provide a strong adsorption of proteins via their hydrophobic or hydrophilic regions. Thus, in such a case, the etching fiber will first be cleaned in an aqueous washing solution (e.g. a 2% aqueous washing or cleaning agent solution). It is then cleaned under running water and immersed overnight in concentrated I^SO^. It is then purified with distilled I^O and dried in hot air. It will then easily bind proteins from the solutions, e.g. human IgG in buffers.

In another preferred method, the active part is

of the conductor after cleaning with H^SO^ and cleaning as mentioned above immersed for 1 hour in a 15% (w/v) of TiCl^ in an anhydrous organic solution such as acetone or ethanol. It is then washed with distilled water and 0.1 M phosphate buffer (pH 7) and then brought into contact with a human IgG solution (2 g/l in 0.9% NaCl solution or 0.1 M phosphate buffer, pH 7 ). The fiber (or rather the probe) which has thus been prepared for testing against AG is stored moist in a suitable buffer (or also dry for shorter periods).

The examples that follow illustrate the practical features of the invention.

Example 1

An optical zone including a waveguide of the type described with reference to fig. 13a and 13b and a glass slide was prepared to carry out immuno-chemical analyzes in the following way: glass articles made from the following types of glass (n = 1,523): "Farloses Brillenroh glass B-260, DESAG", were first washed with a warm cleaning -solution, cleaned in distilled water and air-dried. They were then immersed for 5 minutes in concentrated U^SO^ at 95°C, cleaned in distilled water and dried with a clean cloth. The subsequent manipulation was carried out with the utmost care whereby the clean glass surface was not touched and the probe was handled via the edges. The plates were mounted on the apparatus as shown in fig. 13a and 13b, with a gap of 0.3 mm between them. For coating purposes, an approximate (but accurately weighed)

1 g/l antigen solution prepared by dissolving the required amount of solid human immunoglobulin (Serva Biochemicals, Heidelberg, Germany) in 0.9% NaCl solution. About. 0.2 ml

of such a solution was placed between the glass elements 41 and 42 of the probe by means of a syringe and was allowed to stand there for 2 hours at room temperature. During this time, approx. 1 - 10% of the AG that was available in the sample attached to the surface of the waveguide (about 2-20 yg). Then the cell was emptied by absorbing the liquid with an absorbent material and the cell was cleaned with distilled water and the cleaning water was removed as before. Then a 0.1M phosphate buffer (pH 7) containing 2 g/l of bovine serum albumin and 0.5 ml/l of liquid detergent ("Tween 20") was placed in the cell and left there for 1 hour. The serum albumin would fill the "holes" left empty on the slide after coating with antigen (ie, since the antigen does not usually attach to all available areas on the slide and since the antibody to be tested also has affinity for the uncovered slide, the presence of uncoated areas, "the holes"

on the waveguide, consequently introducing errors in the measurements). As the antibody has no affinity for bovine serum albumin, this treatment will simply render unused, uncovered parts of the glass harmless. The cleaning agent will support such an effect.

After again washing and cleaning, the cell was allowed to dry in a stream of clean warm air. It was then ready for the experiment. All analyzes were carried out at room temperature. The various optical and electronic components with the exception of the photomultiplier tube were switched on and allowed to equilibrate and a sample of antibody solution (2 ml) was introduced into the cell using the same device. The antibody chosen was rabbit anti-human immunoglobulin from DAKA IMMUNOCHEMICALS, Copenhagen, Denmark, in 0.9% NaCl. The first solution received was a 10 g/l protein solution with a titer of 900/ml, i.e. this figure (given by the manufacturer) means that each ml of the antibody solution really neutralizes 900 µg of antigen (neutralization here means that when such reciprocal amounts of both ingredients have reacted, the solution will not contain any significant amounts of AG or AB). For calibration purposes, known antibody solutions were thus suitably diluted where they were used for carrying out the test (see table I). As soon as the cell was filled with calibration solution (n = about 1.33), the room was darkened to avoid saturation of the photomultiplier tube and the latter was switched on. The proportion curve formed by the scattered light signal captured by the photomultiplier began its development after a few seconds of equilibration (probably due to the time required to properly wet the protein in the coating). The reaction was allowed to proceed for approx. 2-5 minutes and the slope of the share curve (close to linear) was averaged over the period.

The results are summarized in table I below, which gives the values for the dilution of the samples of antiserum and corresponding calculated titer values as well as the slope of the corresponding proportion curves as measured with the apparatus that was used:

For the analysis of an unknown sample, the same procedure was followed, proportion data recorded and then compared with standard data in Table I. The desired analytical data was obtained by plotting the analytical proportion value obtained against the corresponding titer as given by the data in Table I.

Example 2

The reverse of the experiment described in Example 1 was carried out with the same apparatus and under the same conditions. In this case, the waveguide in the optical probe was coated with a thin film of antibody (rabbit anti-human immunoglobulin) using 0.2 ml of the starting solution with a titer of 900/ml (see example 1). All other operations were the same as described in Example 1. The calibration samples were prepared from known solutions of the antigen solution as given in Table II which also summarizes the slopes of the proportion curves that were recorded.

For analyzing unknown samples of AG it was followed the procedure already described in example 1, i.e. the slope of the corresponding proportion curve under the sample was measured and the value obtained was brought into accordance with the corresponding antigen concentration by comparison with the curve produced from the values summarized in table II.

Example 3

In this example it was signal that was obtained

from light that came out from the rear end of the waveguide and which falls on the detector 58 used for forming the proportion curve. The optical probe was essentially the same as that used in Example 2 (with a coating of AB, rabbit anti-human immunoglobulin). Samples of antigen solutions (similar to example 2) were used whereby the concentrations are shown in Table III below. The rest of the operation was carried out exactly as explained in examples 1 and 2 with the difference that the photomultiplier 53 was not used and that the signal really decreased as the reaction progressed (this is because

the proportion of light that does not reach the output of the waveguide increases as the reaction progresses). The slope of the proportion curves, recorded as a function of the amount of AG present in the sample, is given in Table III.

Example 4

The following example illustrates a competitive analysis method of the type mentioned in connection with fig. 5. For this, a fluorescence measuring device type of the type described with reference to fig. 16. The optical probe was produced by coating the corresponding waveguide 71 with a film of human immunoglobulin (AG) following the same procedure as described in example 1. Antibody AB<*> that was used was labeled with fluorescein isothiocyanate (FITC ) and specific to the antigen tested (it was obtained from DAKO IMMUNOGLOBULINS, Copenhagen). The unlabeled corresponding antibody AB was also obtained from the same source. In the following experiment, the exit surface 71 of the waveguide was coated with black paint to minimize scattering of incident light.

In each experiment, a fixed amount of anti-spor substance AB<*> was added. This amount was 100 µl of Dako product diluted 1/200 in 0.1M phosphate buffer at pH 7 and it was mixed with 100 µl of the sample solution of unlabeled AB at dilutions given in Table IV. The operation was carried out as in the previous examples, the mixed 200 µl portions were introduced between plates 71 and 72. The results are given in the form of slopes for corresponding proportion curves in table IV.

For measurement of the unknown solutions of antibody, the same technique was used (addition of 100 µl of the labeled antibody) and the results were obtained with reference to the standard data given above.

Example 5

The preceding example was repeated, but this time the output from the detector 78 at the rear end of the waveguide was measured. Using the same sample concentrations as given in Table IV, the corresponding proportion results (in increasing order) were obtained (mvolt/min.): 2.9; 4.8; 6.1; 6.6.

Example 6

A probe cell was prepared by taking a piece of plastic coating silicon oxide fiber from "Quartz & Silice SA", type "PCS Fibrosil WSF-UV" series (diameter 380 ym, core 200 ym, loss 0.14 db/m at 350 nm, 97% transmission) and it was inserted with its ends into the slots of a PVC cell as indicated in fig. 10a and 10b. For sealing, a solution of silicone rubber cementing agent in acetone was used. The coating was etched away by filling the cell with concentrated H 2 SO 4 and leaving it for 1 hour (non-coated part approx. 10 cm long). Then the fiber was made active by the first preferred method described in the last paragraph before the examples.

A film of human immunoglobulin G (IgG), from "Serva Biochemicals", Heidelberg, Germany, was then deposited on the fiber as follows: the phosphate buffer in the cell was removed and replaced by a 2 g/l solution of IgG (10 ml)

in a 0.9% NaCl solution. After 2 hours, the antibody solution was removed and the fiber was cleaned with buffer solution

after which a 2 g/l solution of bovine serum albumin containing 0.5 ml of "Tween 20" (a detergent) in 0.1 M phosphate buffer was introduced and left there for 1 hour. Then the cell was again cleaned with buffer and filled with fresh buffer (10 ml). The cell was installed on the optical bench in the apparatus of fig. 9, filter 5 was selected for X = 280 nm (protein absorption) and filter 6 for guiding >>2 = 340 nm (no protein absorption), after which the adjustment was made to center the beam, have the correct response to the electronic units and allow the system to reach equilibrium with minimum noise. Then 50 µl of various dilutions of antiserum (anti-IgG raised in rabbits and purchased from Dako Immunochemicals, Copenhagen) were added and carefully mixed together using bubbling air. The recording device was then started at time zero and absorbance changes at λ = 280 nm were monitored for approx. 10 minutes. The average rate of change in this absorption signal was recorded and the slope was recorded in arbitrary units. The results are shown in Table V as a function of AG concentration.

Example 7

The procedure was essentially as described

in the above example, but the rabbit IgG was replaced with a correspondingly labeled antiserum. The labeling compound was fluorescein isothiocyanate "isomer-1" (FITC) and the label

antiserum was obtained from "Dako". This material absorbs strongly at 492 nm (this wavelength is actually also the one that excites fluorescein). In this experiment, filter 5

chosen for an X^ = 492 nm and filter 6 for an X^ = 600 nm where no absorption occurs. The tests were performed as already described with different concentrations of FITC-labeled IgG and also using, in another set of measurements, active fiber sections of only 5 cm. The results are shown in Table VI.

Example 8

It appears from the above example that labeling with a fluorescein derivative could be used to modify the absorption spectrum of a protein and consequently increase the sensitivity of the sample in the present invention with respect to unlabeled substances. Consequently, this system can also be used to measure unknown concentrations of unlabeled human IgG (competing measurement types) by using, in addition, a fixed concentration of the label AB, where the ratio of this in relation to labeled substances will then be the determining factor in accordance with the scheme shown in fig. 6. In experiments, the results of which are given in Table VII, the fixed concentration of labeled IgG was 1 µg/ml. The values given in the first column are those for unlabeled IgG.

Example 9

The apparatus of fig. 9 was operated with fluorescence measurement conditions, i.e. the filter 5 for excitation light was for X = 492 nm and the value for filter 6 and for the additional excitation light-blocking filter after the fiber rear end was for A2 = 518 nm.

In practice, the experiment was carried out exactly as in example 8, but with the measurement of an increase in signal (emitted fluorescence) instead of a decrease as in the absorption phenomenon. As in Example 8, the analyte contained 1 µg/ml of fluorescent IgG + various concentrations of unlabeled human IgG as shown in the left column of the table.

It should be clear to the reader that the results described in Examples 1 - 9 have been used as standards for comparison purposes with corresponding samples of unknown concentrations. Comparisons and calculations can be made, as usual, visually or by electronic processing in microcomputing units that can be connected to the devices described.

Claims (20)

1. Method for determining an analyte in solution, where a ]ag (23) of an analyte-reactant product is built up on the surface of a light waveguide core (l) which transmits a light wave signal that is reflected several times in total, where this layer formation has the effect that it changes its optical properties in order to thus modify the wave signal and where the modification is measured and used for said determination, characterized in that a core with a refractive index (n-^) higher than (n2) for the analyte solution is used as a waveguide and with a ratio n^/n^ with such a value that the penetration depth in the solution of the light component associated with the wave signal in the core practically matches or exceeds the thickness of said analyte-reactant product. 2. Method according to claim 1, characterized in that a part of a non-porous light waveguide core (1) which is coated with a thin film of reactant which is specific for an analyte, is brought into contact with a solution of the analyte where red it is enabled that the analyte can react with said reactant in the film and form a reactant-analyte product layer, that the corresponding optical changes with time that appear on the light wave signal traveling through the core at the exit of the core as a result of the formation of said product layer are observed and that proportion data obtained in this way are correlated with corresponding standard reference proportion data obtained in a similar way from calibration samples for the analyte. 3. Method according to claim 1, characterized in that a section of a non-porous waveguide core (1) is coated with a reference amount of analyte to be determined, that a light wave signal is sent into an input in the section, that the light section is brought into contact with the analyte solution while a reference is added beforehand or at the same time amount of reactant to cause the latter to react in competition with the analyte in solution and the reference amount on the core, that the corresponding optical changes with time that appear on the wave signal at an output of the core section as a result of said reaction between reactant and the reference quantity are observed and comparison of ratio data obtained in this way with standard reference ratio data obtained in a similar way from calibration samples of dissolution of the analyte. 4. Method according to claim 2, characterized in that a trace amount of pure analyte in labeled form has been added to the analyte solution, that the presence of said labeling in the reactant-analyte product layer on the waveguide core causes said optical changes to the light wave signal that moves through the core, the size of the change is in direct relation to the ratio of labeled analyte to analyte in solution. 5. Method according to claim 2, characterized in that a core is used with a coating of a reactant in excess of what is stoichiometrically necessary for binding of the analyte in the solution to be determined, that the coated core is brought into contact with analyte top the solution for total binding of the analyte to be determined, that the analyte solution is added to a reference amount of analyte in pure labeled form in order to react with the still intact excess of reactant, that the presence of said labeling in the reactant-analyte product causes the aforementioned optical changes that appear in the wave signal, the size of the change being in direct proportion to the ratio of labeled analyte to analyte that was originally in the solution. 6. Method according to claim 2, where the analyte has more than one binding site for specific binding of more than one type of reactant, characterized in that the core which is coated with a first reactant is brought into contact with said analyte solution, that a reference amount of another is added reactant to bring the second reactant to bind to a second binding site in the analyte, that said optical change is the result of the binding of said second reactant on said second binding site of the analyte which is bound by its first binding site on said first reactant of the core. 7. Method according to claim 2, characterized in that proportion data is interpreted in the form of the slope i on proportion curves relating to the reaction under test or the proportion data were extrapolated to determine equilibrium conditions. 8. Method according to claims 1-7, characterized in that the change in optical properties of the conductor core indicates absorption of the wave signal, which modification leads to a reduction with time of the light signal measured at an output of the core. 9. Method according to claims 1-6, characterized in that the change in optical properties of the conductor core includes a change in a fluorescent light signal, where the signal increases with time and is measured at an output of the core or on its side. 10. Method according to claims 1-6, characterized in that the change in optical properties of the conductor core includes scattering of the light wave signal, and that the extent of such scattering increases with time and is measurable partly in the lateral direction of the scattering area and partly at an exit of the core. 11. Method according to claims 1-6, characterized in that a polarized light signal is used and that said change in optical properties of the conductor concerns elliptical polarization parameters for said signal, which parameters are measured at an output of the core. 12. Method according to claims 1 - 10, characterized in that the core is chosen to be formed of a light-transparent plate, rod or fiber-like means with a refractive index of at least 1.4. 13. Method according to claim 12, characterized in that a multimode fiber is used and that it includes the use of modes that are sufficiently shallow with respect to the fiber axis, to ensure total reflection and sufficient steepness to ensure a high linear density for light signal coverage. the reaction sites. 14. Apparatus for measuring kinetic parameters in a reaction between an analyte with refractive index n2 and a specific reactant, which reaction occurs on the surface of a waveguide (1) with refractive index ni and causes detectable changes in its optical properties, comprising a light source (8 ), devices (9,5,11) for introducing a signal from the source into the entrance to the waveguide (1), detection devices (12) for detecting the light signal which has been subjected to changes during the movement through it and which comes out from there and conversion of the signal into an electrical signal, and devices (13,14) for further processing of the signal into usable data with regard to the reaction, characterized in that the ratio n^/n2 is of such a value that the penetration depth in the solution of the light component which is associated with the wave signal in the core practically matches or exceeds the thickness of said analyte-reactant product. 15. Apparatus according to claim 14; characterized in that the waveguide has the form of a plate or a fiber with a refractive index within the range between approx. 1.4 and approx. 3.5. 16. Apparatus according to claim 14, characterized in that the modified light signal is emitted as a scattered, fluorescent or partially absorbed light signal. 17. Apparatus according to claim 14, characterized in that the waveguide has the form of a spirally wound optical fiber. 18. Apparatus according to claim 15, characterized in that it comprises a counter plate arranged in a non-touching close parallel relationship to the waveguide plate, and that the reaction takes place in the space provided between said plates, the reaction medium being held in place by capillary forces. 19. Apparatus according to claim 14, characterized in that the core (1) is a single-mode or multi-mode optical fiber core. 20. Apparatus according to claim 14, characterized in that the core is a multimode fiber and where the modes are selected with a first reflection angle close to the critical angle, whereby during the reaction a slight change in the refractive index n2 for the thin medium occurs due to growth of analyte -reactant product will result in a partial or total refraction of light on the outside of the core.