DE102023204533B4 - Procedures for conducting biochemical tests on samples - Google Patents

Abstract

Method for carrying out biochemical investigations on samples containing biological molecules as analytes and also receptors with high binding affinity for these biological molecules, to each of which at least one molecule which, as a result of an enzyme, emits electromagnetic radiation in the form of luminescence within a first wavelength interval is bound after an incubation period,
in which several cavities (3) are arranged discretely to one another on a surface of a substrate (1), and each of the cavities (3) contains a sample, and
above the openings of the cavities (3) a plurality of detectors (5) with which the detection of electromagnetic radiation as a result of luminescence of luminescent molecules contained in the samples, which are arranged in an array arrangement within at least a first and a second wavelength interval of the emitted electromagnetic radiation, takes place, and
an electronic evaluation unit is connected or connectable to the detectors (5), with which the proportions of electromagnetic radiation of the first and second wavelength intervals emitted as a result of luminescence detected by the detectors (5) are recorded and compared, so that the proportion of receptor-bound biological molecules in the individual samples is determined relative to one another, and
an optical transmission element (7) is arranged between the openings of the cavities (3) and the detectors (5), in which channel-shaped openings (7.1) are formed which are associated with the openings of the cavities (3), through which electromagnetic waves emitted as a result of luminescence Radiation from the samples is directed onto the detectors (5).

Description

The invention relates to a method for conducting biochemical analyses on samples. The samples contain biological molecules as analytes and also receptors with a high binding affinity for these biological molecules. At least one molecule is bound to each receptor, emitting electromagnetic radiation within a specific first wavelength interval. This can exploit the fact that molecules suitable for luminescence, which emit electromagnetic radiation in a first wavelength interval, emit electromagnetic radiation in a second wavelength interval, which differs from the first wavelength interval, upon binding to a receptor contained in the sample, as a result of luminescence. For example, radiation in the first wavelength interval, for example from the wavelength range of blue light, that is only emitted from a sample as a result of luminescence can be detected. After an incubation period, during which receptors with luminescent molecules bind to analytes, electromagnetic radiation from the second wavelength interval, which may be in the red light range, for example, can also be detected, depending on the number of biological molecules bound in this way. The detected proportions of electromagnetic radiation from the different wavelength intervals can be used to draw conclusions about the presence of analytes in the respective sample, for example, the presence of certain antibodies, and this can even be quantified. In this case, an analyte can be firmly bound to a receptor, so that this bond can be maintained even if a molecule luminescent in the first wavelength interval has bound to an analyte with the receptor.

The invention is particularly suitable for biomedical applications, i.e., detection based on antigen-antibody reactions. Such detection methods can be used in human medical diagnostics, veterinary medicine, food analysis, biotechnology, and much more. The potential areas of application likely extend even further. Technically, the goal is to build such a system with a dedicated image sensor. The measurement results can be read and evaluated via USB using a PC or smartphone, for example.

• + Die jeweilige zu untersuchende Probe wird mit den Nachweisreagenzien gemischt, die Messung erfolgt dann nach einer Zeitspanne („Inkubationsszeit“) von wenigen Minuten.
• + Dabei wird in der Lösung durch ein Enzym Lumineszenz erzeugt. Die Farbe/Wellenlänge(n) der Lumineszenz ist - aufgrund des speziellen Designs der zum Nachweis verwendeten Sensorproteine - von der
• Konzentration der jeweiligen Analyt-Moleküle in der Probe abhängig. + Demzufolge kann bei der Messung einzig die Farbe der emittierten elektromagnetischen Strahlung in mindestens zwei vorgegebenen Wellenlängenbereichen des Lumineszenz-Lichts gemessen werden. Dabei erfolgt eine Prüfung dahingehend, dass das Verhältnis der Intensitäten in beiden Wellenlängenbereichen vor und nach einer Bindung von Analyt an Rezeptoren ausgewertet wird.
  • + Diese Messungen können in Laborgeräten (Spektrometern oder Photometern) durchgeführt werden, die das von der Probe emittierte Spektrum an infolge Lumineszenz emittierter elektromagnetischer Strahlung mit guter Wellenlängenauflösung detektieren können. Dieser Ansatz ist nicht auf Anwendungen vor Ort (am s.g. Point-of-Care) übertragbar.
  • + Nach dem Stand der Technik wurden die zu untersuchenden Proben auf ein Filterpapier pipettiert, um die infolge Lumineszenz emittierte elektromagnetische Strahlung mittels einer SpiegelreflexKamera zu erkennen. Die Auswertung erfolgt dabei durch nachträgliche Analyse der Farbinformation in den Aufnahmen der Kamera. Daraus kann geschlussfolgert werden, dass eine Farbbestimmung mittels eines kommerziellen rgb-Bildsensors möglich und ausreichend ist.
  • + Es ist auch bekannt, ein Smartphone als Detektor zu nutzen, und die Kamera dieses Smartphones durch eine Zusatzlinse mit „Blackbox“ (Abschirmung von Umgebungslicht) auszustatten. Dort wird bisher jedoch nur die Intensität der infolge Lumineszenz emittierten elektromagnetischen Strahlung nicht deren Wellenlängen genutzt.
  • + Anstelle der Detektion der Biolumineszenz als Intensitätssignal können derartige Nachweise z.B. mit nahe an der Probe angeordneten Detektoren (wie z.B. Fotodioden, Avalanche-Fotodioden, Fotomultiplier) erfolgen. Dazu können zwei Fotomultiplier als empfindliche Detektoren in Verbindung mit zwei unterschiedlichen Spektralfiltern angewendet werden. Dadurch ist auch die Detektion eines Farbverhältnisses (unabhängig von der Helligkeit der Lumineszenz) als Messsignal möglich.

The subject of this invention is also the detection of analytes or "analyte molecules" in a preferably aqueous sample for medical/diagnostic sensor technology. Certain molecules contained in the sample (usually proteins or nicotinamide adenine dinucleide or NADPH/NADP+ or nicotinamide adenine dinucleide in phosphorylated form NADH/NAD+) are to be detected by their binding to a corresponding antibody. Antibodies can be special synthetic proteins that fulfill the functions described below. The basic types of detection reactions as well as the necessary synthetic sensor proteins are known and, for example, Q. Yu, L. Xu et al., “Semisynthetic sensor proteins enable metabolic assays at the point of care,” Science 361, (2018), pp 1122-1126 Published. Reference is made to the contents of this publication. The following facts are important:

• + The sample to be examined is mixed with the detection reagents, and the measurement is then carried out after a period (“incubation time”) of a few minutes.
• + An enzyme generates luminescence in the solution. The color/wavelength(s) of the luminescence depends on the
• The concentration of the respective analyte molecules in the sample depends on this. Therefore, the measurement can only measure the color of the emitted electromagnetic radiation in at least two specified wavelength ranges of luminescence light. This test involves evaluating the ratio of the intensities in both wavelength ranges before and after the analyte binds to receptors.
  • + These measurements can be performed using laboratory instruments (spectrometers or photometers) that can detect the spectrum of electromagnetic radiation emitted by the sample due to luminescence with good wavelength resolution. This approach is not transferable to on-site applications (at the so-called point-of-care).
  • + According to the state of the art, the samples to be examined were pipetted onto filter paper to detect the electromagnetic radiation emitted by luminescence using a single-lens reflex camera. The evaluation is performed by subsequent analysis of the color information in the camera images. From this, it can be concluded that color determination using a commercial RGB image sensor is possible and sufficient.
  • + It is also known to use a smartphone as a detector and to replace the camera of this smartphone with an additional lens with a “black box” (shielding from ambient light) from However, so far only the intensity of the electromagnetic radiation emitted as a result of luminescence has been used, not its wavelengths.
  • + Instead of detecting bioluminescence as an intensity signal, such detection can be performed using detectors positioned close to the sample (e.g., photodiodes, avalanche photodiodes, photomultipliers). Two photomultipliers can be used as sensitive detectors in conjunction with two different spectral filters. This also allows the detection of a color ratio (independent of the brightness of the luminescence) as a measurement signal.

So are WO 01/350079 A1 Fluoremeters with light sources that generate low heat are known.

The object of the invention is to provide possibilities for a wavelength-resolved determination for detection on samples, with which bioluminescence can be detected quickly and with sufficiently high measurement accuracy using a very simple, small hardware system and can be evaluated simply and in a short time, whereby the losses of electromagnetic radiation emitted as a result of luminescence, which emanates from samples and which cannot be detected with suitable detectors, are to be minimized.

In the method according to the invention, several cavities are arranged discretely on the surface of a substrate. Each cavity contains a sample. The samples can be identical or different.

Above the openings of the cavities are several detectors arranged in an array to detect electromagnetic radiation resulting from luminescence from the luminescent molecules contained in the samples within at least a first and a second wavelength interval of the emitted electromagnetic radiation. Preferably, they can form an array in a row and column arrangement. The substrate should be made of a material that scatters at least 90% of the electromagnetic radiation emitted as a result of luminescence from the molecules contained in the respective samples, so that the majority of the emitted electromagnetic radiation cannot penetrate the substrate and is not lost for detection.

However, the walls of the cavities can also be coated with a coating that reflects the electromagnetic radiation emitted as a result of luminescence in order to achieve the same effect.

An electronic evaluation unit is connected or connectable to the detectors, which is designed to detect the proportions of electromagnetic radiation of the first and second wavelength intervals emitted as a result of luminescence and to relate them to one another, so that the proportion of biological molecules bound to a receptor in the individual samples can be determined.

An optical transmission element is arranged between the cavity openings and the detectors. Channel-shaped openings are formed in the openings, corresponding to the cavity openings, through which electromagnetic radiation emitted by luminescence from the samples impinges on the detectors. The openings form hollow channels through which the emitted electromagnetic radiation from the respective samples can pass toward the detectors.

An immersion layer, preferably with a liquid or gel, can be formed between the optical transmission element or a cover, behind which the detectors are arranged in the radiation direction of the emitted electromagnetic radiation, and a lid with which the openings of the cavities are closed.

It is also advantageous if the wall of the channel-shaped openings is provided with a coating that reflects the emitted electromagnetic radiation or if the optical transmission element is made of a material that reflects at least 50% of the emitted electromagnetic radiation.

The free cross-section of the channel-shaped openings, through which the emitted electromagnetic radiation is directed toward the detectors, should be larger in the area facing the detectors relative to the area facing the opening of the respective cavity. For this purpose, channel-shaped openings can be shaped like a cone or truncated pyramid. Channel-shaped openings can also have curved or arched inner walls, for example, with a concave or convex shape.

The inner walls of the channel-shaped recesses should be aligned at an angle or curved (e.g., concave or convex) so that total reflection, or at least near total reflection, can be achieved for the emitted electromagnetic radiation. At least 80% of this electromagnetic radiation can be reflected and then directed onto the detectors.

To prevent total internal reflection, a transparent element can be arranged at or in the area of the openings of the cavities or at the inlet openings for emitted electromagnetic radiation of the channel-shaped openings of the optical transmission element. The transparent element has a surface facing the detectors, where the emitted electromagnetic radiation is refracted. This prevents back reflections from samples of emitted electromagnetic radiation. For this purpose, the surface of the transparent element facing the detectors can be hemispherical.

The transparent element can be integrated into the lid that closes the openings of the cavities.

On the surface of the substrate where the cavity openings are located, there may be at least one surface area from which detectors are used to detect the dark spot. For this purpose, a sufficiently large distance may be maintained between at least two cavity openings.

In 1 A simple design of a device for conducting biochemical analyses on samples containing biological molecules that emit electromagnetic radiation within a first specific wavelength interval due to luminescence, and also a receptor with high binding affinity for these luminescent molecules, for example the luciferase NanoLuc, is shown. When luminescent molecules bind with receptor molecules to biological molecules of the respective analyte, electromagnetic radiation is emitted within a second wavelength interval due to luminescence. The wavelengths of the first and second wavelength intervals differ. In contrast, electromagnetic radiation emitted by molecules not bound to a receptor as a result of luminescence has wavelengths of a first wavelength interval. Both different electromagnetic radiations can be detected spatially resolved using the detectors.

The challenge with such a system is the fact that only a low intensity of electromagnetic radiation emitted as a result of luminescence is available, although this can be detected with high efficiency by color-sensitive image sensors as detectors. Secondly, it can be made possible to perform multiple determinations on different samples simultaneously (e.g., to achieve negative control, positive control, and sample detection), so that a single detector (such as a color-sensitive photodiode or a spectrometer) is not sufficient. Commercially available, inexpensive image sensors can be used in the invention. This allows adaptation of the luminescence spectra to the color filters of the respective detector (or, conversely, adaptation of the filter spectra on the image sensor to the luminescence wavelengths).

The invention makes it possible to almost completely detect the electromagnetic radiation emitted by the samples due to luminescence with the detectors, as losses due to radiation in other directions can be avoided and significantly reduced. In particular, the design of a transmission element also prevents significant losses due to excessively large angles of incidence of the electromagnetic radiation emitted by the respective samples upon reaching the detectors.

Intrinsically amplifying detectors, such as photomultipliers or avalanche photodiodes, can be used. At least two detectors should be used per measurement, which increases the effort required for parallel measurements (e.g., for the usual positive and negative controls parallel to the actual measurement).

Furthermore, a high supply voltage (>100 V) is required for this type of detector, and integration with spectral filters must be done separately. This significantly increases the complexity, but since these components are already included in a commercially available color camera (or, in extreme cases, even a webcam) and are inexpensive, they can be used as detectors.

The invention will be explained in more detail below by way of example.

• 1 In schematischer Form ein Beispiel einer Vorrichtung, die bei der Durchführung des erfindungsgemäßen Verfahrens eingesetzt werden kann;
• 2 ein Beispiel mit einem zusätzlichen Übertragungselement;
• 3 ein Beispiel mit zusätzlichen transparenten Elementen;
• 4 ein weiteres Beispiel mit zusätzlichen transparenten Elementen und
• 5 Aufsichten auf eine Oberfläche eines Substrats mit den Öffnungen von Kavitäten.

Showing:

• 1 In schematic form, an example of an apparatus which can be used in carrying out the method according to the invention;
• 2 an example with an additional transmission element;
• 3 an example with additional transparent elements;
• 4 another example with additional transparent elements and
• 5 Top views of a substrate surface with the openings of cavities.

1. a) Emission durch den Deckel 2 (Strahlen 10, 11, 11') in Richtung des Detektors 5
2. b) Nicht detektierte Emission durch das Substrat 1 (Strahl 13), und
3. c) Emission durch das oder auch in das Substrat 1 z.B. nach Totalreflexion (Strahl 12) an der Grenzfläche zur Luft,

so dass eine vollständige Detektion der gesamten emittierten elektromagnetischen Strahlung verhindert wird, da die Detektoren 5 oberhalb der Öffnungen der Kavitäten 3 angeordnet sind. Weiterhin kann angemerkt werden, dass die Detektoren 5, wie der in 1 schematisch dargestellte Bildsensor, immer nur ein begrenztes Winkelspektrum der emittierten elektromagnetischen Strahlung detektieren können. Die Effizienz der Detektion dieser Strahlung nimmt mit steigendem Einfallswinkel ab. Dies begrenzt die Effizienz einer Anordnung, bei der die Detektoren ohne zusätzliche Elemente direkt an den Proben angeordnet sind. 1 shows a basic structure of a device. In a substrate 1 (predominantly made of plastic or glass), cavities 3 are formed in which the different molecules are contained, preferably in an aqueous solution. The individual cavities 3 can contain the same or different samples for detection. The electromagnetic radiation emitted as a result of luminescence can be emitted in all directions (for example, indicated by rays 10, 11, 11', 12, 13), i.e., the entire solid angle of 4 π. This includes

1. a) Emission through the cover 2 (rays 10, 11, 11') towards the detector 5
2. b) Undetected emission through substrate 1 (beam 13), and
3. c) Emission through or into the substrate 1, e.g. after total reflection (ray 12) at the interface to the air,

so that a complete detection of all emitted electromagnetic radiation is prevented, since the detectors 5 are arranged above the openings of the cavities 3. Furthermore, it can be noted that the detectors 5, as in 1 The image sensor shown schematically can only detect a limited angular spectrum of the emitted electromagnetic radiation. The efficiency of detecting this radiation decreases with increasing angle of incidence. This limits the efficiency of an arrangement in which the detectors are arranged directly on the samples without additional elements.

The substrate 1 is made of a material that scatters at least 95% of the electromagnetic radiation emitted by the samples contained in the cavities 3. This prevents the emission of the electromagnetic radiation emitted as a result of luminescence by the substrate 1, and increased detection efficiency can be achieved. The openings of the cavities 3 are covered with a lid 2 that is transparent to the electromagnetic radiation emitted by the samples as a result of luminescence. The detectors 5 are arranged on a carrier 4 and covered and protected by a cover glass 6 in the direction of the openings of the cavities 3.

With a device like the one in 2 As shown, the problem of the reduced proportion of electromagnetic radiation emitted as a result of luminescence, which impinges on the detectors 5 for detection and can be evaluated, can be further counteracted. The procedure is such that the substrate 1 is formed from a material that scatters the electromagnetic radiation emitted as a result of luminescence, or the surfaces of the cavities 3 containing samples are provided with a coating that reflects the electromagnetic radiation emitted as a result of luminescence, so that each beam 10 - 13 of the electromagnetic radiation emitted as a result of luminescence emitted in a cavity 3 can leave the respective cavity 3 in the direction of the detectors 5 after possibly multiple scattering or reflection at the interface between the cavity 3 and the substrate material.

With an ideal radiation-scattering material (no absorption, only light scattering), it can be achieved that the rays 12 and 13 are emitted only in the direction of the detectors 5.

Between the substrate 1 with the luminescent samples contained in the cavities 3 and the detectors 5, an optical transmission element 7 can advantageously be arranged, in which channel-shaped openings 7.1 are formed, assigned to a respective cavity 3, so that the electromagnetic radiation emitted as a result of luminescence, emanating from the respective sample, can impinge on detectors 5 through the correspondingly arranged opening 7.1. These reduce the angular spectrum of the electromagnetic radiation impinging on the detectors 5 and increase the illuminated area of the detectors 5. Ideally, these structures operate in an etendue-preserving manner, i.e. $$A\cdot {\left(n\cdot \text{sin}\phi \right)}^2=const.$$

Here A is the luminous area in the medium with the refractive index n, and φ is the maximum illumination angle measured from the normal of the surface A.

Electromagnetic radiation is emitted into a cavity 3 with an area A _cav at the refractive index of the aqueous solution containing the sample, n~1.34, at angles of up to 90°. The radiation exits the substrate 1, limited by total internal reflection. Thus, a maximum efficiency of approximately 56% is achieved upon entering the apertures 7.1 of the transmission element 7 with n~1, if a non-scattering substrate 1 or one not provided with a reflective coating is used.

The transmission element 7 should not be transparent to the electromagnetic radiation emitted as a result of luminescence, and the wall 7.2 of the openings 7.1 should be provided with a coating that reflects the emitted electromagnetic radiation. In the simplest case, the openings 7.1 can be shaped like a truncated cone, but can also be formed with a parabolically curved wall, whereby it is advantageous if the free cross section of the opening gen 7.1 through which the emitted electromagnetic radiation is directed towards detectors 5.

This efficiency in detection can be achieved with a device according to 3 be further improved.

In the example after 3 An immersion layer 9 with a suitable liquid is formed between the cover 2 and the transmission element 7. In addition, 2 and 3 Top views of the respective substrate 1 with the cavities 3 in different arrangements are shown below.

The total reflection on the surface of the lid 2, with which the openings of the cavities 3 can be closed, can be achieved by applying an optical element 8 that is transparent to the electromagnetic radiation emitted as a result of luminescence (see 4 ) with a surface pointing in the direction of the detectors 5, at which the emitted electromagnetic radiation is refracted, which may be hemispherically curved, for example, and/or by means of an immersion layer 9, which is arranged or formed between the optical transmission element 7 and the cover 2, as shown in 3 shown, can be prevented.

This allows the above-mentioned efficiency to theoretically be increased to 100%. It is limited only by the optically sensitive area of the detectors 5 and possible residual losses due to reflections from the cover 2, optical element 8, and optical transmission element 7.

With such a device it is possible to carry out several measurements with the detectors 5. The lower part of the 2 and 3 shows, as an example, the arrangement of five or three such structures in front of a rectangular detector array, viewed from above. The optical transmission element 7 can be fixedly mounted in front of the detectors 5. The substrate 1 can be coupled to this system in an aligned manner using an immersion layer 9.

1. a) Die Kavitäten 3 sind in das die elektromagnetische Strahlung streuende Substrat 1 integriert; das transparente optische Element 8 kann auf dem Deckel 2 angeordnet oder dort ausgebildet sein. Der Deckel 2 kann in Form einer entsprechend ausgebildeten Deckelfolie ausgebildet sein. Dies erfordert eine justierte Montage des Deckels 2 auf dem Substrat 1, um die korrekte Position der transparenten optischen Elemente 8 in Bezug zu den jeweiligen Kavitäten 3 zu gewährleisten.
2. b) Transparente optische Elemente 8 und Kavitäten 3 können von beiden Seiten in eine Platte als Substrat 1 geprägt und diese am Ende durch eine streuende Folie/Platte als Deckel 2 verschlossen werden, wie es beispielhaft aus 4 hervorgeht.

In practical applications, the use of immersion liquids (in the current case, even water could be used) is difficult. This can be circumvented by integrating the optical function of the transparent element 8 with the substrate 1 or its cover 2 ( 4 ). There are two ways to do this:

1. a) The cavities 3 are integrated into the substrate 1 that scatters the electromagnetic radiation; the transparent optical element 8 can be arranged on the cover 2 or formed there. The cover 2 can be designed in the form of a correspondingly shaped cover film. This requires an adjusted mounting of the cover 2 on the substrate 1 to ensure the correct position of the transparent optical elements 8 in relation to the respective cavities 3.
2. b) Transparent optical elements 8 and cavities 3 can be embossed from both sides into a plate as substrate 1 and this can be closed at the end by a scattering film/plate as lid 2, as is shown for example in 4 emerges.

In the 1 to 4 In the examples shown, the measurement signals recorded by the detectors 5 can be fed to an electronic evaluation unit (not shown), in which the evaluation for the individual samples can be carried out.

As already explained above, the entire area with the detectors for detecting electromagnetic radiation emitted by the samples as a result of luminescence does not have to be used to detect this radiation.

In order to be able to reliably detect the low intensities of the electromagnetic radiation emitted as a result of luminescence using detectors 5, measured values should be recorded over longer periods of time, for example in the range (100 ms ... 10 s), and taken into account in the evaluation.

For this detection unused parts, in 5 shown hatched, are protected from external light by the optical transmission element 7 and can be used as a dark signal for a dark adjustment for correction during data processing of the measurement signals recorded by the detectors 5.

Claims (7)

Method for carrying out biochemical investigations on samples containing biological molecules as analytes and also receptors with a high binding affinity for these biological molecules, to each of which at least one molecule is bound which, as a result of an enzyme, emits electromagnetic radiation in the form of luminescence within a first wavelength interval after an incubation period, in which a plurality of cavities (3) are arranged discretely to one another on a surface of a substrate (1), and each of the cavities (3) contains a sample, and above the openings of the cavities (3) a plurality of detectors (5) are arranged, with which the detection of electromagnetic radiation as a result of luminescence of luminescent molecules contained in the samples Molecules that are arranged in an array arrangement within at least a first and a second wavelength interval of the emitted electromagnetic radiation, and an electronic evaluation unit is connected or can be connected to the detectors (5), with which electronic evaluation unit the proportions of electromagnetic radiation emitted as a result of luminescence, detected by the detectors (5), of the first and second wavelength intervals are recorded and put into relation so that the proportion of biological molecules bound to receptors in the individual samples is determined relative to one another, and an optical transmission element (7) is arranged between the openings of the cavities (3) and the detectors (5), in which channel-shaped openings (7.1) are formed that are associated with the openings of the cavities (3), through which electromagnetic radiation emitted as a result of luminescence from the samples is directed onto the detectors (5). Procedure according to Claim 1 , characterized in that the wall (7.2) of the channel-shaped openings (7.1) is provided with a coating reflecting the emitted electromagnetic radiation or the optical transmission element (7) is formed from a material reflecting at least 50% of the emitted electromagnetic radiation, so that the emitted electromagnetic radiation is directed in the direction of the detectors (5). Method according to one of the preceding claims, characterized in that the wall (7.2) of the channel-shaped depressions are aligned or curved in such a way that the emitted electromagnetic radiation is completely or at least almost completely reflected there with total reflection. Method according to one of the preceding claims, characterized in that the emitted electromagnetic radiation is guided through channel-shaped openings (7.1), the free cross section of which, through which the emitted electromagnetic radiation is directed, is directed in the direction of detectors (5) and is enlarged in the region in the direction of detectors (5) compared to the region which points in the direction of the opening of the respective cavity. Method according to one of the preceding claims, characterized in that at or in the region of the openings of the cavities (3) or at inlet openings for emitted electromagnetic radiation of the channel-shaped openings (7.1) of the optical transmission element (7) there is arranged in each case an element (8) which is transparent to the emitted electromagnetic radiation and which is provided with a surface facing in the direction of the detectors (5) at which the emitted electromagnetic radiation is refracted, and with which total reflection is avoided. Method according to the preceding claim, characterized in that the surface of the element (8) facing in the direction of the detectors (5) is hemispherical, with which back reflections of electromagnetic radiation emitted by samples are avoided. Method according to one of the preceding claims, characterized in that on the surface of the substrate (1) on which the openings of the cavities (3) are arranged, detection is carried out on at least one surface area with detectors (5) to carry out a dark calibration.

Images