WO2002025245A1

WO2002025245A1 - Method and device for detecting an analyte in a sample holder

Info

Publication number: WO2002025245A1
Application number: PCT/EP2001/010389
Authority: WO
Inventors: Michael Müller; Herwig Brunner; Frank Vitzthum; Jürgen Bernhagen
Original assignee: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority date: 2000-09-22
Filing date: 2001-09-08
Publication date: 2002-03-28
Also published as: DE10047007A1; DE10047007C2; AU2002212218A1

Abstract

The invention relates to a method and device for detecting an analyte in a sample holder (PT). According to the invention, at least one liquid is provided in the sample holder, and the analyte is contained in and/or on top of a liquid, gaseous or solid uptake medium. To this end, the geometric configuration of an interface of the liquid is used for detecting the analyte in the sample holder by optically measuring a measured value that is dependent on the shape of the interface.

Description

_VE R _FA H N _RE _AND VORR _I AUTION FOR THE DETECTION OF AN ANALYTE IN A SAMPLE CARRIER

Besehreibung

The invention relates to a method and an apparatus for detecting an analyte in a sample carrier, said Anal ^'yt optionally in and / or on a liquid or gaseous or solid recording medium is contained under the existence of at least one liquid.

Spectrophotometric and luminometric methods for the determination of analytes on the basis of absorption and luminometric properties of the analytes are already known. Furthermore, detection reactions are used in a known manner in order to achieve spectrophotometric or luminometric effects, with the aid of which the analytes can be detected indirectly. These known primary detection methods are relatively complicated, complex and expensive.

The object of the invention is to provide a method and a device for carrying out the same, which should enable an alternative detection of an analyte in a sample carrier, with the spectrophotometry and / or luminometry possibly only being used secondarily.

To solve the problem, a method is proposed with the features of claim 1 characterized in that the geometric configuration of an interface of the liquid is used to detect the analyte in the sample carrier by measuring a measurement value dependent on the shape of the interface.

The substances to be analyzed, analytes, are not only pure gases, liquids or solids. It is also possible to determine qualitatively and / or quantitatively substances that are in gases, liquids, at solids or interfaces. The method not only offers the possibility to record the properties of static systems in equilibrium, but also to track the kinetics of dynamic systems. For example, the diffusion of surface-active components or their formation or change in reactions can be recorded. Furthermore, the binding and / or reaction of components of gases and / or liquids with surface-bound partners can be detected. In addition, reactions, in particular enzymatically catalyzed reactions that take place in liquids, at surfaces or interfaces, can be recorded. It is also conceivable to use two or more immiscible liquids with different densities. If necessary, a gas can also be present in the multi-component system. Interactions at the interfaces, liquid-liquid, liquid-solid, liquid-gaseous and solid-gaseous, can thus be investigated. For example, it is conceivable to investigate surface-active enzymes, such as lipases. It is also possible to erase interfacial to track stations. The determination is carried out by optical systems so that, for example, spectrophotometric and / or luminometric techniques can be used in addition.

In contrast to the prior art, the invention does not primarily utilize the spectrophotometric and / or lu-geometric properties of the analytes or the detection reactions to determine the properties of gases, liquids and / or surfaces. Instead, it uses the geometrical configuration (for example in the form of a meniscus formation) of an interface of a liquid which is located in a sample carrier.

Menisci are formed depending on the physico-chemical properties of the surfaces of the sample carrier system used as well as the liquid (s) and / or gases contained therein. These menisci lead to local changes in the height of the liquid column (s) and act as optical lenses. Using suitable devices, the method according to the invention can be used to determine material properties, in particular polarity, of liquids and / or surfaces and thus also for the detection of analytes. The properties and / or the composition of gases can also be determined.

Conventional, relatively complex detection reactions are advantageously not required. If necessary, it can be based on expensive, analytical specific, human-toxic and environmentally hazardous reagents. Since time-consuming and material-intensive detection reactions do not have to be carried out, the detection is accelerated, so that costs can be reduced further. In addition, the method is extremely flexible and can be used universally. The sensitivity is preferably very high by selecting the dimensions of the sample carriers to be small, so that the greatest possible meniscus formation occurs. Accordingly, small sample volumes are preferably used. In addition, the measurement effects are most evident at very low analyte concentrations. Parallelization and automation is also possible. The invention is particularly attractive for the bio-chip or lab-on-a-chip area. The new method can therefore not only be used successfully for a so-called "low sample throughput", but in particular also for a "high sample throughput".

Advantageously, the analyte or the liquid is penetrated with at least one light beam and a light intensity dependent on the geometric configuration of the interface is determined by means of a detector unit for detecting the analyte in the sample carrier. A light beam passing through the analyte or the liquid is particularly suitable for the detection of the analyte, since a change in the light intensity, which can be measured precisely by means of the detector unit, occurs depending on the geometrical configuration of, for example, a meniscus, which draws conclusions at least on the existence of the analyte in the sample. carrier and if necessary allow a qualitative determination and / or quantification of the analyte.

A photometric measurement or a luminometric measurement is advantageously carried out by means of the detector unit. Both a photometric and a luminometric measurement by means of the detector unit permits reproducible and reliable detection of the analyte in a sample carrier, which is interspersed with at least one light beam.

The detector unit is preferably moved while changing the distance to the interface. In this way it is possible to demonstrate whether a meniscus arising at the interface is concave or convex in relation to the detector unit.

According to a preferred embodiment variant, an optical lens system arranged between the meniscus and the detector unit is moved in order to optimize the measurement. The measurement of the light intensity by means of the detector unit can be optimized with regard to the measurement precision and / or the measurement area.

To achieve the object, a device for detecting an analyte in a sample carrier is also proposed, which has the features of claim 9. The device according to the invention comprises a sample holder for holding a liquid, gaseous or solid, if necessary Recording medium contained analytes under the presence of a liquid with an interface, a light source for emitting at least one light beam passing through the analyte or the liquid and a detector unit for determining a light intensity dependent on the geometric configuration of the interface. A device designed in this way is particularly suitable for carrying out the method according to the invention, and advantageously the design of the sample holder, the detector unit and / or the light source can be flexibly adapted to the analytes to be detected in each case in a sample holder. It is possible, for example, to provide the detector unit with a curved detector surface, to provide a lens system that can be moved to optimize the measurement between the sample carrier and the detector unit and / or to design the detector unit such that it has at least one frontal detector section and / or at least with respect to the sample carrier has a side detector section.

Further advantageous embodiments of the invention result from the description.

The invention is explained in more detail below in several exemplary embodiments with the aid of an associated drawing. Show it:

Figure 1 is a schematic representation of a device according to the invention according to a first embodiment; FIGS. 2A, each a schematic representation 2B, 3 of physical effects on a meniscus;

Figures each a schematic representation 4A to 4E development of light beams with different angles of incidence;

FIGS. 5A to 5J each show schematic representations of different embodiments of a device according to the invention or different embodiments of a method according to the invention;

Figures various test results in 6 to 8 diagram form;

FIG. 9 shows the result of a location-dependent signal resolution as a function of a detector curvature in diagram form;

Figure 10 shows an inventive device with a lens system;

Figures each a schematic representation ILA, 11B development of a device according to the invention with a planar and with a curved interface;

Figures each a schematic representation 12A, 12B of different embodiments of the device according to the invention with a scanning unit; FIGS. 13A, 13B each show a schematic representation of the device according to the invention with a plurality of light sources or with mirrors;

Figure 14 is a schematic representation of a

Sample carrier with two different liquids;

FIGS. 15A, 15B each show a schematic representation of a sample carrier with three different liquids and

Figures each show a schematic representation 16A, 16B of a sample holder with inflow and outflow systems.

The basic features of the devices and methods according to the invention are illustrated below using the influence of circular menisci (M) in vertical spectrophotometric measurements of liquids in sample carriers (FIGS. 1 and 4). The arrangement according to the invention of a sample holder (PT) filled with a liquid with respect to a light beam (L) and a detector unit (D) is shown schematically in FIG. 1 (detector elements D _A , D _B , D _et c); (r _D ) radius of the detector unit; (r _G ) limit radius; (r _L ) radius of the light beam, (r _PT ) inner radius of the sample carrier (PT) location-dependent course of the partial light beam, or a photon with the x output coordinate (r _P ); (y _D ) Distance of the detector unit.

The detector unit (D) consists of at least one detector element (D _A ) and is present Example with three detector elements (D _A , D _B D _c ). A plurality of detector elements, each with the smallest possible dimensions, for example "charge coupled device detectors", "diode arrays" and / or light guide systems or "light guide arrays", are preferably to be used. If necessary, detector optics can be located between the liquid and the actual detector unit (D). The detector optics can be constructed from lenses or lens systems and filters, for example interference filters, polarization filters, etc. The inclusion of a grating system for spectral analysis is also conceivable. The planar or concave or convex curved detector unit can be arranged at a preferably variable angle (ε, FIG. 1) to the optionally transparent, preferably planar bottom of the sample carrier. In the following ε = 90 ° (Figure 1). In Figure 1, the sample holder has a cylindrical (φ = 90 °) symmetry. Other measurements are also possible.

In Figure 1, the starting point of the light beam (light source LQ) is placed under the sample holder (PT) and the position of the detector unit (D) above it. Inverse or other systems are also conceivable. The light source (LQ) can provide light beams with different properties through the use of monochromators, for example gratings or corresponding filters as well as polarization filters and lens systems or light guides. If, for example, radially symmetrical cylindrical light beams as well as sample carriers and detector units are used, is for the x coordinate, the use of radii (r) in relation to the axis of symmetry with r = 0 makes sense. The y coordinate includes the relevant areas of the liquid column in the sample holder as well as the distance of the detector unit from the transparent bottom of the sample holder, which is set to zero. Information on the y coordinate that relates to the liquid column is described with the layer thickness (d), otherwise y is used. Line 22 in FIG. 1 shows a planar interface, while line 23 shows a concave meniscus.

There are various effects in the formation of menisci, which are mutually dependent, but are described separately below. On the one hand, the layer thickness of the liquid in a meniscus changes continuously depending on the curvature of the meniscus (FIG. 2), on the other hand, the light is refracted at the curved interface (FIG. 3). This makes up the lens effect of a meniscus. There is also a change in reflection. This increases with increasing meniscus curvature and the diameter of the light beam. Scattering effects are hardly noticeable. Furthermore, the radius of the detector unit (r _D ) or the detector elements (r _D , _A , _B , etc.) and its distance from the meniscus or from the sample carrier bottom play a role (FIG. 4). In order to capture this fact mathematically, a light beam with radius (r _L ) is divided into partial light beams with certain radii (r _P ). Above a certain limit radius' (r _G ) of the partial light beam due to the refraction, it will no longer reach the detector element of the detector unit with radius (r _D ), which is closer to x = 0. The result of this is that the signal of this detector element decreases and the intensity of a refracted light beam (1 ^) measured there is less than that of the refracted light beam (I _B ) or the unbroken light beam (I). If reflection and scatter are neglected, I _B can be set equal to I. I _B and I are intensities of light rays that have already passed through a medium and can therefore be distinguished from the intensities of the light rays that are radiated into the medium (I _D ).

To further describe the invention using the example of the influence of circular menisci, in vertical spectrophotometric measurements of liquids in sample carriers, the determination of the layer thickness is shown separately. The layer thickness can be determined from the description of the curvature of the meniscus. This curvature mainly depends on the properties of the liquids and the sample carrier walls as well as on the gravitation. For example, when a concave meniscus is formed, the adhesive forces are greater than the cohesive forces. In extreme cases, the adhesive forces are so great that the liquid almost completely wets the sample carrier wall and thus hits it approximately at the angle γ = 0. A correspondingly small sample carrier radius (r _{? Τ} ) can therefore enable an almost semicircular meniscus formation (FIG. 2A). The value for the layer thickness (d _(M) ) depending on the meniscus based on r = 0 is calculated according to the Pythagorean theorem for a convex meniscus:

and for a concave too:

! '-' r ² + (r _n -d. _U) ) ² = r? - (2)

Since the binomial formulas are the same, a quadratic equation results, which provides the following solution:

d = r ^w / - <rr _π τr ^x (3)

The layer thickness depends on the radius (d _(r) ) at any point (Z) on a convex meniscus:

d _lr , = d _r - ₀ ~ d ( _M ) '(4)

and with a concave meniscus after:

d (r) = d _r = o + d ( _M ). (5)

Furthermore, the radius-dependent absorption (Abs- _r )) is calculated for spectrophotometric measurements according to Bougner-Lambert-Beers law:

Abs _(r , = ε-c "d _(r) . ^• ( ^β )

Here, c is the concentration of the chromophore and ε its molar, decadal absorption coefficient at a certain wavelength. As The analyte itself, a reaction product, or another absorbent substance, but preferably a liquid, can serve as a chromophore. In aqueous solutions, for example, measurements in the infrared range are advantageous because water absorbs them. In systems with several liquids or also with a gas, the spectrophotometric properties of one or more components can be used. As a rule, however, the angle γ will not be zero. The choice of the sample carrier radius plays a role here. Therefore, not only semicircular menisci are formed, but also circular menisci. In addition, it is conceivable that menisci with location-dependent meniscus radii (R ( _r )) form (FIG. 2B), which can be described as follows:

With the beam path, it must be taken into account here that a focal cloud does not occur, but a focal cloud. However, this can be neglected within certain limits. If, for example, only the area of the meniscus that is struck by the light beam is considered, this local condition can also adequately describe a location-independent meniscus radius.

In order to include the layer thickness independently of the function in the calculation of the absorption, a solution can be used by determining the average layer thickness (d _m ). To calculate the average layer thickness, the mean value of the integral calculation, with which the following results:

The following applies analogously to the consideration of the volume including the angle functions (χ = rcosφ; γ = rsinφ):

1st case; Limit case: r _L = r _PT ; d _m = d _pG ;

2nd case; As a rule: r _L <r _PT ; d _m ≠ d _pG .

In comparison to a planar interface and its layer thickness (d _pG ), light rays with radii that are smaller than the sample carrier radius result in a change in the average layer thickness, provided that reflection phenomena on the sample carrier can be neglected. According to the Bougner-Lambert-Beer law:

Abs = ε-c "d _m , (10)

is the measurement signal, for example the absorption, of samples at the same volume with a planar interface unlike that with a meniscus. With concave menisci, the measurement signal is lowered, with convex it increases. It must be taken into account that a spherical meniscus, be it a semicircular one or not, only arises in the limit case or under ideal conditions. It is therefore only possible to speak of a focal point under ideal conditions. If there are deviations, it is a focus cloud. For the sake of simplicity, a defined focus was assumed in the representations and calculations. Approximately, the calculations also apply to a focus cloud.

It is also conceivable that only the area of the meniscus that is struck by the light beam is considered. Approximately, if the light beam diameter is not too large, a constant meniscus radius can be defined locally so that a focal point can be calculated.

In addition to the change in layer thickness, the refraction of the light can also be used analytically. This is determined by Snellius' law of refraction: sinör ⁿ 2 [Lu _f t)

= n,

^• R ¹ ' ² ' (11)

described (Figure 3). When a light beam passes from medium 1 (liquid A) to medium 2 (air, other gases or a liquid B that is not miscible with liquid A), the quotient of the sine values of angle of incidence (α) and angle of refraction (ß) is equal to that inverse ratio of the absolute refractive indices (n) of both ^media . As a rule, the refractive index (nι _{# 2} ) of substances compared to air is given. n _1/2 is not depends only on the medium itself, but also on the wavelength of the light in question. This so-called dispersion is determined empirically. At a defined wavelength, the angle of refraction can therefore be calculated using the angle of incidence. Since the angle of incidence is dependent on the tangent slope at the meniscus, the refraction of the light depending on the meniscus can be determined by the dependence of the tangent slope on the meniscus (m.τM (r)) according to:

be determined.

The intensity (I ^D ) measured at the detector unit results from the signal generated there, for example the voltage (U), which depends on the intensity of the light, i.e. on the number of incident photons and their energy ( Figure 4). To describe this fact, the x coordinate (r _PD ) of a partial light beam that strikes the detector unit is compared with the x output coordinate of the partial light beam (r _P ), the distance between the detector unit and the meniscus surface (y _D - d ₍ M)) and the angle of incidence and refraction are determined as follows (FIG. 3):

r ^• p, _Dir - = r _P + (y _D - d _m ) tan (ß - a); (13)

Case: τ _PDirp) ≤ r _DA , the partial light _beam hits the detector unit or the detector element D _A (FIGS. 4B and C),

The intensity of a refracted light beam (1 °) measured at the detector element D _A thus corresponds to the intensity of a refracted light beam

(I _B ) and that of an unbroken light beam

(I): i ι ^D _B = ι _B = i;

2nd case: r _{PD {rp)} > r _DA , the partial light beam does not hit the detector element D _A (FIGS. 4D and E),

± _B <IB = I; the intensity at an adjacent detector element increases accordingly.

In order to examine the dependence of the intensity of a refracted light beam measured on the detector unit or the detector elements more precisely, the photon density (Pd) is introduced. The photon density of a light beam is expressed in photons per area (A) and time (t):

Pd = -. (14)

A - t

Since the time interval of the measurement is predetermined by the device according to the invention and is not influenced by the refraction, this equation is based on: ^{Pd =} A - ⁽ 15 ⁾

reduced. Assuming that in the case of a refracted light beam the photon density decreases qμadratically with the distance, for example starting from a virtual or real focal point (B _v , B _r ) (FIGS. 4D and E), the following relationship results (FIG. 4A):

Therefore, the following applies to the photon _density at the height y _{D of} the detector unit (P _yD / Ay _D ) based on the photon density of the light beam emerging from the liquid (P ^ / A _m ) with the average layer thickness (d:

With the photon density of the light beam P ₀ / Ao entering the medium, the following results for P _yD / A _yD :

Since the photon density at y _{D is} the same for the light beam cone and the detector unit (P ° / A ° _D ), the following applies:

A ^D pD. -_ p - -H-. _{(1 9]}

™ -ya

Accordingly, the following applies to the intensity of a refracted light beam at the detector unit or a detector element (7 °) in the case of radial symmetry:

This results in the detector element D _A (FIG. 4) with:.

1st case: r „ _. <r _v „ _, ; 7 = 7 _B = 7;

2nd case: r _yo > _yoλ ' _B <- ^1' «, the intensity at an adjacent detector element increases accordingly.

For the absorption, this means an increase for the detector element (D _A ) in the second case after:

Abs = log ₁₀ - ^ <lθg ₁₀ - ^ = log ₁₀ -. (21

B B

Due to the quadratic connection, this will be very noticeable. The occurrence of reflection also leads to a decrease in the measurement signal with increasing curvature of the meniscus and increasing light beam diameter. From this point of view, changes in the layer thickness can only be detected precisely with small light beam diameters. The effects of scattering will hardly be noticeable in this context.

Another way to mathematically understand the facts of the effects of refraction. lies in the calculation of the area of the light beam _cone (A _yD V-

A, _o =]] dxdy (22) and the photon density of a refracted light beam (Pd) measured on the detector unit or the detector elements, using:

Since these are circular areas, the following also applies here:

2π ^r yo

^A _yD ⁼ l \ ^r _L dr _L dφ ≡ πr

0 r _vl (24)

Pd _s = - - - ⁽²⁵⁾

It can thus be shown that the method according to the invention and the device according to the invention necessary therefor are suitable for determining the properties of liquids and / or surfaces. Analytes that are in the liquid and change their properties can thus also be detected.

FIG. 5 shows simplified designs of the devices according to the invention and the effects of meniscus formation in the case of light rays with small radii (a dotted line, FIGS. 5A, C and F) and large radii (two dotted lines, FIGS. 5B, D, E, G, H, I and J) on the beam path and the measurement signal (S) depending on location (r). The location-dependent representation of the measurement signals is shown here only schematically and in a greatly simplified manner for illustration purposes.

The devices according to the invention are shown schematically on the basis of detector units 1, sample carriers 2 with side walls 3, which are covered by corresponding surfaces. chene properties, a transparent bottom 4 in these cases and a light source 5 not shown. The light rays emanating from the light source 5 pass through the liquids 7, which all have the same volume, but are either planar. Form interfaces or menisci.

FIGS. 5A and B show the situation in measurements with a planar interface. There are locally defined signals with a corresponding extent or intensity depending on the light beam used. If a concave meniscus is formed (FIGS. 5C, D and E), a centered light beam with a small radius (FIG. 5C) is ideally not refracted. The layer thickness of the liquid in the center decreases in the case of a concave meniscus, so that in the case of this photometric measurement the signal, that is to say the intensity, increases in comparison with a planar interface. According to Bougner-Lambert-Beer law, this means a decrease in absorption (see also Figure β).

If, on the other hand, a light beam with a large radius is used, the light beam is mainly refracted (FIG. 5D). This reduces the signal, but extends over a wider area. If only one detector unit is considered, the decrease in the signal in photometric measurements leads to a decrease in intensity, which results in an increase in absorption (see also FIG. 7). The situation can be similar with luminometric measurements (FIG. 8). In order to prevent reflection on the sample carrier, it can be useful to design the sample carrier accordingly. For example, a conical geometry can be useful (FIG. 5E). It is also conceivable to use planar or approximately planar sample carriers (FIG. 5F). It can also be advantageous to use materials that minimize reflections. Furthermore, by choosing the appropriate liquid volumes, a fill level can be achieved which reduces the risk of reflection.

If convex menisci are formed, there is an increase in the layer thickness in the case of light rays with small radii compared to a planar interface, thus a reduction in the signal and finally an increase in absorption in the case of photometric measurements (FIG. 5G). Accordingly, a distinction can be made between concave and convex menisci.

When using light rays with large radii, this can be done by changing the distance of the detector unit from the sample carrier or from the surface of the liquid (Figures 5G, H and I). Concave menisci lead to a different dependence of the signal identity distribution on the distance than convex menisci.

FIG. 6 shows the result of tests in which aqueous solutions with different bovine hemoglobin concentrations of the same volumes were also used in conical sample carriers made of non-polar material identical dimensions and a diameter at the height of the meniscus formation of about 6 mm. The solutions were measured photometrically at a wavelength of 977 nm. At this wavelength only water absorbs, not bovine hemoglobin. Light rays with radii in the micrometer range were irradiated in the center of the samples, so that refraction processes could be neglected. A detector element with a constant distance from the sample carrier was used for detection.

The concave meniscus formation, which increased to a maximum with the bovine hemoglobin concentration, was not to be expected in this way. Surprisingly, bovine hemoglobin obviously deposited on the non-polar surface of the sample carrier and thus increased the polarity of the surface of the sample carrier, which increased the adhesive forces. The very significant decrease in layer thickness, which was reflected in a significant reduction in absorption, was also unexpected (see also FIG. 5C). Samples that were shaken vigorously shortly before the measurement (o) surprisingly provided different data than samples that were incubated at room temperature for a while after shaking (Q). For comparison, samples were measured in cuvettes (Δ) that suppress the formation of menisci.

The layer thickness is constant here and thus also the absorption from the bovine hemoglobin concentration. The clarity of the effect, especially with extremely low bovine hemoglobin concentration NEN, illustrates the effectiveness of the method according to the invention.

In the experiments according to FIG. 7, the procedure was as in the experiment shown in FIG. 6. However, a human hemoglobin determination according to Drabkin (Drabkin and Austin, J. Biol. Chem., 98, 719, 1932) was carried out here. A light beam with a wavelength of 540 µm and a radius of about 2 mm was used for the measurement.

The absorption increased more or less linearly with increasing human hemoglobin concentration when using cuvettes (B). In contrast, the use of the innovative system (•) provided a dihyperbolic curve with significantly increased sensitivity.

From a certain hemoglobin concentration, the slopes of the two curves were comparable, so that the contribution of the chromophore can be determined taking into account the change in the layer thickness.

According to FIG. 8, another analog test arrangement showed that the meniscus formation, which is dependent on the analyte concentration, can also be evaluated by means of a luminometric measurement. For this, the fluorescence of Schweineherzdiaphorase at 365 n was ^"excited.

With the system (o) according to the invention, there was an approximately dihyperbolic course, in contrast to the experiments in which cuvettes (CD) were used. A linear relationship was shown here. The relative fluorescence resulted from the integration over a defined circular area of the sample carrier of approximately 4 mm.

In Figure 9 it is clear that it can be advantageous to use curved detectors (Dι, ₅ ), for example a "Charge Coupled Device", a "diode array" or preferably a "light guide array". The location-dependent resolution of the signals with a curved detector geometry differ from a planar detector (D ₃ ). This can be used to advantage. It is also conceivable to use a planar detector by a certain angle ε

(D _2/4 ) (see also Figure 1) to then rotate it. Accordingly, it is also conceivable to use a detector, the detector elements of which are not strung together but only in a line. As a consequence, a single detector element can also be used in a corresponding, spatial scanning operation.

However, the detection can also be carried out in an advantageous manner by means of corresponding optical lens systems, as shown by way of example in FIG. 10. An optical lens or a lens system 9 can influence the signal by the movement according to double arrow 10 in space, possibly by changing refractive properties. Depending on the meniscus and the change in the lens system, there are defined signals that can be used to detect or determine an analyte. The possibility of varying the light beam radius by means of diaphragms, optical systems or “light guide arrays” has already been discussed in the description of FIG. 5. FIG. 11 shows an example of how optical lenses or lens systems 11 can be used in a likewise advantageous manner when generating light beams. In the figures, attention was drawn to the representation of the refraction on planar surfaces. If the position of the focal point is varied in a system (double arrow 12; dotted and dashed lines), different refractive properties result with a planar interface (FIG. 11A) than with a curved interface (FIG. 11B). The signals are correspondingly different, so that an analyte can also be detected in this way.

The possibility of a scanning operation is shown in FIGS. 12A and 12B. This can be done from below (Figure 12A). A light beam with the smallest possible radius is preferably used here. With regard to a centered position (dotted line, FIG. 12A), a translocation (double arrow 13, line with long lines, FIG. 12A) and / or the variation of the angle δ (line with short lines, FIG. 12A) provides certain signals depending on the Meniscus. If the sample carrier is enclosed by detector systems 1, the signal acquisition can be optimized. For example, the proportion of the reflection (thin dashed lines, FIG. 12A) can be recorded. If necessary, the detection of the scatter is also possible. If the light beam is irradiated from the side (FIG. 12B), the total reflection can be used in particular (dotted line). Furthermore, a measurement with a defined layer thickness (dashed line) can be carried out, so that the concentration of a chromophore can be determined in a conventional manner. The proportion of meniscus formation in the measurement effect can thus be recorded independently of the absorption of the chromophore.

In addition to the possibilities for determining the contribution of the chromophores to the measurement effect described with reference to FIGS. 7, 8 and 12B, there are further options. For example, the determination can also be carried out by additionally carrying out the samples in cuvettes with defined layer thicknesses. In addition, there is the possibility of rotating the light source 5 and the detector unit 1 in accordance with FIG. 13A such that, in addition to a vertical measurement, a horizontal measurement can also be carried out. It is of course also conceivable to provide a second light source 5 'and a second detector unit 1'. Furthermore, a system of mirrors 14, as shown in FIG. 13B, can be used. Optical fiber systems can also be used instead of the mirror system.

The invention also includes the use of systems with multiple phases or liquids. For example, FIG. 14 shows a system consisting of two liquids A and B which are immiscible with one another. In this case, liquid B has a lower density than liquid A. The formation of the interface liquid-gaseous liquid B can be planar, concave or convex depending on the test conditions. With a suitable combination, this can advantageously lead to a reduction or strengthening of the refraction, reflection and scattering effects and change in layer thickness.

A planar interface always occurs when the sample carrier is filled with liquid B exactly to the edge. If this limit is exceeded, a convex meniscus always forms until the fluid overflows.

The liquid B preferably has a correspondingly high boiling point, so that it can be used as an evaporation protection for the liquid A. Accordingly, measurements can also be carried out at high temperatures, which may even be above the boiling point of liquid A.

In addition to the use of two liquids, the use of further liquids is also conceivable. In a special embodiment, this can be used in a special way. This is shown in FIG. 15. In addition to the use of a third liquid C, the formation of the phase boundary between liquid A and B can optionally also be used. In particular, it is conceivable to use substances which have spectrophotometric and / or luminometric properties and are preferably in the range of the phase boundary. In this embodiment, the devices are preferably with light guides or To provide "light guide arrays" which can be both light source 5 and detector 1. If a planar phase boundary is formed (FIG. 15A), a light beam 6, starting from a specific light guide 16, can shine through the phase boundary or liquid C in order to then strike the opposite light guide element. If a weak meniscus forms, the light beam can in principle still reach the light guide opposite through total reflection within the phase boundary or within the liquid C. If the curvature of the meniscus increases, this is no longer possible. First there is refraction, then there is the total reflection shown in FIG. 15B. In order to determine this, it can be useful to also use detectors at the top and bottom.

The use of detectors at these locations will be particularly important for luminometric measurements. Depending on the shape, planar, concave or convex, the phase boundary or liquid C, the distribution of the luminescence changes. This can be detected particularly well by detectors mounted above or below (see also FIG. 8).

If necessary, it may be sufficient to use an optical fiber or an “optical fiber array” only on one side, since the luminescence or the reflection can differ in the case of photometric determinations in the region of the liquid A, B and the phase boundary or liquid C. Through the use of substances The efficiency of the measurement can be improved with corresponding spectrophotometric and / or luminometric properties that are specifically in the phase boundary or in the liquids. It is also conceivable to use substances that act as fluorescence quencher in order to obtain optimal measurement signals.

Furthermore, the light beams of the respective light guide elements can be irradiated simultaneously or in succession and a scan can be carried out. It is also conceivable to replace at least one liquid with a gel or some other type of matrix.

FIG. 16 shows chamber systems which can be equipped with inflow and outflow systems 17, 18 for the liquid or the gas B and inflow and outflow systems 19, 20 for the liquid or the gas A. Constructions are also conceivable in which only one supply line is used as the inflow and outflow for the respective liquid or even for both. With the systems it is conceivable, for example, to vary volume ratios. For example, a method according to the invention can be helpful in which a liquid is initially wetted and then displaced from certain surface areas. Only through this procedure do menisci form in a certain way. The same can be done by shaking the sample carrier (FIG. 6). It may also make sense to adhere to certain incubation times or temperature conditions. It is also conceivable, for example, to transfer an analyte in a first liquid to a sample carrier whose surface has been functionalized in such a way that it is highly affine for the analyte. If the binding of the analyte does not yet form a sufficient meniscus, the first liquid can be replaced by a second liquid, which forms a characteristic meniscus on the sample carrier surface in the presence of the analyte.

The "attaching" of the analytes to an optionally functionalized sample carrier surface can be carried out non-specifically via van der Waals and ionic interactions, but also via hydrogen bonds, coordinative or covalent bonds.

Another effect that can be exploited analytically is the concentration of analytes in the area of the minimum distance of a concave surface with respect to the bottom of the sample carrier. A functionalization of the surface that is tailored to the analyte or a variation of the surface properties of the sample carrier are also conceivable here. The variation of the liquid volume and thus the height of the liquid column can also be advantageously evaluated here.

The local concentration of charged analytes can also be done, for example, by electric fields. In addition, the properties of the sample carrier surfaces, the liquids, gases and / or the analytes can be changed by using electrical fields be changed so that advantageous effects are achieved for measurements. For example, with the aid of electrodes, it is possible to generate polar surfaces by applying different electrical potentials to a sample carrier with initially non-polar surfaces. The use of time-dependent electrical potentials can be useful for the investigation of magnetic analytes or particles. In addition, the use of spark discharges enables quick reactions to be investigated. Accordingly, this can also be made possible by rapid pressure changes using the inflow and outflow systems.

In addition, analytes can be brought into the chamber in the flow using the inflow and outflow systems. The volume of the liquids and / or gases can be kept constant. If the concentration of the analyte in a flowing liquid is kept constant, the analyte is bound to the sample carrier surface until a dynamic equilibrium is reached. The association rate constant can be determined on the basis of the associated meniscus formation and signal change. If the liquid is rinsed without analyte, the time-dependent dissociation of the analyte from the sample carrier surface takes place. The dissociation rate constant can be determined accordingly. This enables the equilibrium constants of the association and dissociation to be calculated. Of course, the use of different analyte concentrations is also conceivable here. bar. These can then be determined based on the extent of the signal.

In addition to inflows and outflows directly into the. Chamber, the use of inflows and outflows 21, 22 in the sample holder, which in this case has cavities, is also conceivable. Thereby thermostatting can take place. In addition, a material exchange that can be measured can take place via permeable, in particular semipermeable membrane properties of the sample carrier surfaces of the side walls 3 and the bottom 4.

The above-described embodiments with regard to the detector unit, light source or light beam and examples with regard to the comparative measurements, phase or liquids can in principle be combined with one another as desired.

The sample container preferably has a radius of 2 mm to 3 mm, but reliable results can also be achieved with a radius of approximately 6 mm.

An analyte can be determined by adding a small volume or a small amount of analyte to an already existing solution (FIGS. 5A or B), which then makes it concave (FIGS. 5C, D or E) or convex (FIGS. 5F) to J) Form menisci.

Because of the high sensitivity of the system

(Figures 7, 8 and 9) the addition of small amounts of analyte is possible. A change in shift the thickness can therefore be neglected. The use of references (with the same volume), such as solutions with known polar properties or additives, is also conceivable. In the case of kinetics, the change in the signal would be tracked over time in relation to the start and end point of a reaction or bond.

Deviations due to absorption must be taken into account. However, meniscus formation provides a characteristic signal distribution with respect to the radius (FIG. 5). This does not apply to absorption in this way. In addition, the aim is preferably not to use the wavelengths at which the analyte absorbs, but rather another, inert substance or, preferably, the solvent (IR range for water). It is conceivable for an absorption measurement to be carried out separately or instead of horizontally in the same sample holder to take account of absorptions. The change in absorption can thus be recorded independently of the meniscus formation.

The decisive factor is the occurrence of effects that give signals that enable the detection or determination of analytes. For example, quantifications can be made using calibration series.

With the method according to the invention it is possible to determine the properties of liquids, in particular their surface tension and refractive indices

Properties of solids or surface surfaces, in particular to determine their interface properties, for example the polarity and roughness as well as the properties of gases.

Claims

Expectations

1. A method for the detection of an analyte in a sample carrier, the analyte optionally being contained in and / or on a liquid or gaseous or solid recording medium in the presence of at least one liquid, characterized in that the geometric configuration of an interface of the liquid for the detection of the analyte in the sample holder by measuring a measurement value dependent on the shape of the interface.

2. The method according to claim 1, characterized in that the analyte or the liquid is penetrated with at least one light beam and a light intensity dependent on the geometric configuration of the interface is determined by means of a detector unit for detecting the analyte in the sample carrier.

3. The method according to any one of the preceding claims, characterized in that a photometric measurement or a luminometric measurement is carried out by means of the detector unit.

4. The method according to any one of the preceding claims, characterized in that the detector unit is moved while changing the distance to the interface.

5. The method according to any one of the preceding claims, characterized in that an optical lens system arranged between the interface and the detector unit is moved for measurement optimization.

6. The method according to any one of the preceding claims, characterized in that a qualitative and / or quantitative detection of the analyte is carried out using suitable calibration series.

7. The method according to any one of the preceding claims, characterized in that a light source emitting the light beam is moved to increase the sensitivity.

8. The method as claimed in one of the preceding claims, characterized in that an electrical, magnetic or electromagnetic field is applied to the sample carrier and / or the liquid in order to optimize the measurement.

9. The device, in particular for carrying out a method according to one of claims 1 to 5, comprising a sample carrier (PT) for receiving an analyte optionally contained in a liquid, gaseous or solid recording medium in the presence of a liquid with an interface, a light source (LQ) for emitting at least one light beam passing through the analyte or the liquid and a detector unit (D) for determining a light intensity which is dependent on the geometric configuration of the interface.

10. Device according to one of the preceding claims, characterized in that the detector unit (D) has a curved detector surface.

11. Device according to one of the preceding claims, characterized in that a lens system (11) is provided between the sample carrier (PT) and the detector unit (D), which is movable for measurement optimization.

12. Device according to one of the preceding claims, characterized in that the detector unit (D) has at least one frontal detector section and / or at least one lateral detector section with respect to the sample carrier (PT).