Device for conducting biochemical examinations on samples

Abstract

The present invention relates to a device for conducting biochemical examinations on samples which contain biological molecules as analytes and receptors with a high binding affinity for the analytes, to which a luminescent molecule is bound. The device comprises a substrate (1), on the surface of which a plurality of cavities, containing a sample, are arranged discretely relative to one another. Above the openings of the cavities (3), detectors (5) designed to capture luminescence radiation within at least a first and a second wavelength interval from the luminescent molecules contained in the samples are arranged in an array arrangement. An electronic evaluation unit is connected or connectable to the detectors (5) and configured to acquire the proportions of luminescence radiation of the first and of the second wavelength interval detected using the detectors (5) and to relate the said proportions to one another such that it is possible to determine, relative to one another, the proportion of biological molecules bound to receptors in the individual samples. An optical transfer element (7) in which channel-shaped breakthroughs (7.1), which are assigned to the openings of the cavities (3), are formed is arranged between the openings of the cavities and the detectors (5).

Description

[0001] Device for conducting biochemical analyses on samples. The invention relates to a device 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 that emits electromagnetic radiation within a specific first wavelength interval is bound to each receptor. This can exploit the fact that, in the case of molecules suitable for luminescence, which emit electromagnetic radiation in a first wavelength interval, electromagnetic radiation is emitted in a second wavelength interval, which differs from the first wavelength interval, as a result of luminescence after binding to a receptor contained in the sample.For example, radiation emitted from a sample in the first wavelength interval, such as the blue light wavelength range, can be detected as a result of luminescence. After an incubation period, during which receptors with luminescent molecules bind to the respective receptors, 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 possibly even to quantify these.In this case, an analyte can be firmly bound to a receptor, so that this binding can be maintained even if a molecule luminescent in the first wavelength interval has been bound to an analyte with the receptor.

[0002] 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.

[0003] This invention also relates to 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 published, for example, in Q. Yu, L. Xu et al., "Semisynthetic sensor proteins enable metabolic assays at the point of care", Science 361, (2018), pp. 1122-1126. Reference is made to the content 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.

[0004] + An enzyme generates luminescence in the solution. Due to the specific design of the sensor proteins used for detection, the color / wavelength(s) of the luminescence depends on the concentration of the respective analyte molecules in the sample.

[0005] + Consequently, the measurement can only measure the color of the emitted electromagnetic radiation in at least two specified wavelength ranges of luminescence light. This involves evaluating the ratio of the intensities in both wavelength ranges before and after the analyte binds to receptors.

[0006] + 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).

[0007] + 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.

[0008] + It is also known to use a smartphone as a detector and to equip the smartphone's camera with an additional lens with a "black box" (shielding from ambient light). However, this approach currently only uses the intensity of the electromagnetic radiation emitted as a result of luminescence, not its wavelengths. + Instead of detecting bioluminescence as an intensity signal, such detection can be carried out, for example, with detectors arranged close to the sample (such as photodiodes, avalanche photodiodes, photomultipliers). For this purpose, two photomultipliers can be used as sensitive detectors in conjunction with two different spectral filters. This also makes it possible to detect a color ratio (independent of the brightness of the luminescence) as a measurement signal.

[0009] 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.

[0010] In a device according to the invention, several cavities are arranged discretely relative to one another on the surface of a substrate. Each cavity contains a sample. The samples can be identical or different.

[0011] Above the openings of the cavities are several detectors arranged in an array for detecting electromagnetic radiation resulting from luminescence of 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 of 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.

[0012] 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.

[0013] 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.

[0014] Advantageously, an optical transmission element can be arranged between the openings of the cavities and the detectors. Channel-shaped openings are formed in the optical transmission element, corresponding to the openings of the cavities, 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.

[0015] An immersion layer, preferably made of 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.

[0016] 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.

[0017] 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 compared 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.

[0018] The inner walls of the channel-shaped recesses should be aligned at an angle or curved (e.g., concave or convex) to achieve total internal reflection, or at least near total internal reflection, of the emitted electromagnetic radiation. At least 80% of this electromagnetic radiation should be reflected and then directed to the detectors.

[0019] 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 apertures of the optical transmission element. It has a surface facing toward the detectors, at which 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 toward the detectors can be hemispherical.

[0020] The transparent element can be integrated into the lid that closes the openings of the cavities.

[0021] 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 dark calibration. For this purpose, a sufficiently large distance may be maintained between at least two cavity openings.

[0022] Figure 1 shows a simple design of a device for conducting biochemical tests on samples containing biological molecules that emit electromagnetic radiation due to luminescence within a first specific wavelength interval, and also a receptor with high binding affinity for these luminescent molecules, for example the luciferase NanoLuc. When luminescent molecules bind with receptor molecules to biological molecules of the respective analyte, electromagnetic radiation is emitted due to luminescence within a second wavelength interval. The wavelengths of the first and second wavelength intervals differ. In contrast, electromagnetic radiation emitted due to luminescence by molecules not bound to a receptor has wavelengths of a first wavelength interval.Both different electromagnetic radiations can be detected spatially resolved with the detectors.

[0023] 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 carry out as many determinations as possible on different samples simultaneously (e.g. to achieve negative control, positive control and detection of the sample), so that a single detector (such as a color-sensitive photodiode or a spectrometer) is not sufficient. The invention can use commercially available, inexpensive image sensors. In this case, it is possible to adapt the luminescence spectra to the color filters of the respective detector (or conversely, to adapt the filter spectra on the image sensor to the luminescence wavelengths).

[0024] The invention makes it possible to detect almost all of the electromagnetic radiation emitted by the samples as a result of 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.

[0025] 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).

[0026] 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. Since these components are already included in, for example, a commercially available color camera (or, in extreme cases, even a webcam) and are inexpensive, they can be used as detectors.

[0027] The invention will be explained in more detail below by way of example.

[0028] Showing:

[0029] Figure 1 shows in schematic form an example of a device which can be designed according to the invention;

[0030] Figure 2 shows an example with an additional transmission element;

[0031] Figure 3 shows an example with additional transparent elements;

[0032] Figure 4 shows another example with additional transparent elements and

[0033] Figure 5 Top views of a surface of a substrate with the openings of cavities.

[0034] Figure 1 shows a basic structure of a device. In a substrate 1 (mainly 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 n. This includes a) emission through the cover 2 (rays 10, 11, 11') in the direction of the detector 5 b) undetected emission through the substrate 1 (ray 13), and c) emission through or into the substrate 1, e.g.after total internal reflection (beam 12) at the interface with the air, so that 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, such as the image sensor schematically shown in Figure 1, can only ever 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.

[0035] The substrate 1 is formed from 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 through 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.

[0036] With a device as shown in Figure 2, 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. This is done by forming the substrate 1 from a material that scatters the electromagnetic radiation emitted as a result of luminescence, or by providing the surfaces of the cavities 3 containing samples 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 being scattered or reflected multiple times at the interface between the cavity 3 and the substrate material.

[0037] With an ideal radiation-scattering material (no absorption, only light scattering), it is possible to ensure that the beams 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 the 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.

[0038] A • (n • sin <p) 2 = const .

[0039] Here A is the luminous area in the medium with the refractive index n, and <p der maximale Beleuchtungswinkel gemessen von der Normalen der Oberfläche A.

[0040] Electromagnetic radiation is generated in a cavity 3 with area A cav At a refractive index of n~1.34 of the aqueous solution containing the sample, the radiation is emitted at angles of up to 90°; the exit of the radiation from the substrate 1 is limited by total internal reflection. Thus, when entering the apertures 7.1 of the transmission element 7 with n~1, a maximum efficiency of approximately 56% is achieved if a non-scattering substrate 1 or one not provided with a reflective coating is used.

[0041] The transmission element 7 should be opaque to the electromagnetic radiation emitted by luminescence, and the wall 7.2 of the apertures 7.1 should be provided with a coating that reflects the emitted electromagnetic radiation. In the simplest case, the apertures 7.1 can be shaped like a truncated cone, but can also be formed with a parabolically curved wall. It is advantageous if the free cross-section of the apertures 7.1, through which the emitted electromagnetic radiation is directed, increases toward the detectors 5. This detection efficiency can be further improved with a device according to Figure 3.

[0042] In the example shown in Figure 3, an immersion layer 9 containing a suitable liquid is formed between the cover 2 and the transfer element 7. Furthermore, Figures 2 and 3 each show plan views of the respective substrate 1 with the cavities 3 in various arrangements.

[0043] The total reflection on the surface of the cover 2, with which the openings of the cavities 3 can be closed, can be prevented by applying an optical element 8 (see Figure 4) which is transparent to the electromagnetic radiation emitted as a result of luminescence and has a surface facing in the direction of the detectors 5, at which the emitted electromagnetic radiation is refracted, which surface can 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 Figure 3.

[0044] This allows the aforementioned efficiency to be theoretically increased to 100%. It is limited only by the optically sensitive area of ​​the detectors 5 and possible residual losses due to reflections from cover 2, optical element 8, and optical transmission element 7.

[0045] With a device designed in this way, it is possible to perform multiple measurements with the detectors 5. The lower part of Figures 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 permanently mounted in front of the detectors 5. The substrate 1 can be coupled to this system in an adjusted manner using an immersion layer 9.

[0046] In practical applications, the use of immersion liquids (in the current case, even water could be used) is difficult. This can be avoided by integrating the optical function of the transparent element 8 with the substrate 1 or its cover 2 (Figure 4). There are two possibilities for this: 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 in the form of a correspondingly designed 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.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 cover 2, as shown by way of example in Figure 4.

[0047] In the examples shown in Figures 1 to 4, the measurement signals detected 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.

[0048] 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.

[0049] 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.

[0050] Parts not used for this detection, shown hatched in Figure 5, 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 detected by the detectors 5.

Claims

Patent claims 1. Device 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 is bound which emits electromagnetic radiation within a first wavelength interval as a result of luminescence, in which a plurality of cavities (3) are arranged discretely to one another on a surface of a substrate (1), and in each of the cavities (3) a sample is contained, and above the openings of the cavities (3) a plurality of detectors (5),which are arranged in an array arrangement for detecting electromagnetic radiation emitted as a result of luminescence from the luminescent molecules contained in the samples within at least a first and a second wavelength interval of the emitted electromagnetic radiation, and an electronic evaluation unit is connected or connectable to the detectors (5), which is designed to detect and relate the proportions of electromagnetic radiation emitted as a result of luminescence by the detectors (5) of the first and second wavelength intervals so that the proportion of biological molecules bound to receptors in the individual samples can be 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 the electromagnetic radiation emitted from the samples as a result of luminescence hitting the detectors (5).

2. Device according to claim 1, characterized in that the substrate (1) is formed from a material which scatters at least 90% of the electromagnetic radiation emitted as a result of luminescence of the molecules contained in the respective samples, or the wall of the cavities (3) is coated with a coating which reflects the electromagnetic radiation emitted as a result of luminescence.

3. Device according to one of the preceding claims, 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.

4. Device 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.

5. Device according to one of the preceding claims, characterized in that the free cross section of the channel-shaped openings (7.1) through which the emitted electromagnetic radiation is directed in the direction of the detectors (5) is enlarged in the region facing the detectors (5) in relation to the region facing the opening of the respective cavity.

6. Device 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) in order to avoid total reflection, a respective one is provided with a surface facing in the direction of the detectors (5) at which the emitted electromagnetic radiation is refracted. a transparent element (8) is arranged for the emitted electromagnetic radiation.

7. Device according to one of the preceding claims, characterized in that an immersion layer, preferably with a liquid or a gel, is formed between the optical transmission element (7) or a cover (6), behind which the detectors (5) are arranged in the radiation direction of the emitted electromagnetic radiation, and a lid (2) with which the openings of the cavities (3) are closed.

8. Device according to the preceding claim, characterized in that the surface of the element (8) facing towards the detectors (5) is hemispherical.

9. Device according to one of the two preceding claims, characterized in that the transparent element (8) is integrated in the lid (2) with which the openings of the cavities (3) are closed.

10. Device 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, there is at least one surface area from which detection is carried out by detectors (5) in order to carry out a dark calibration.

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