Methods for carrying out biochemical investigations on samples

Abstract

Methods for carrying out biochemical investigations on samples containing biological molecules as analytes and also receptors with high binding affinity for these biological molecules, to which at least one molecule is bound after an incubation period, which emits electromagnetic radiation in the form of luminescence within a first wavelength interval as a result of an enzyme. in which several cavities (3) are discretely arranged relative to each other on the surface of a substrate (1) and each cavity (3) contains a sample, and above the openings of the cavities (3) several detectors (5) with which the detection of electromagnetic radiation resulting from 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, is carried out, and an electronic evaluation unit is connected or can be connected to the detectors (5) with which the proportions of electromagnetic radiation of the first and second wavelength intervals detected by the detectors (5) as a result of luminescence are recorded and compared, so that the proportion of biological molecules bound to receptors in the individual samples is determined relative to each other, 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 corresponding to the openings of the cavities (3), through which electromagnetic radiation emitted from the samples as a result of luminescence is directed towards the detectors (5).

Description

[0001] 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 method 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.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. Specific 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 binding to a corresponding antibody. Antibodies can be special synthetic proteins that fulfill the functions described below. The basic types of detection reactions and the necessary synthetic sensor proteins are known and have been 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 for this purpose: + 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 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, equipping the smartphone's camera with an additional lens that acts as a "black box" (shielding ambient light). However, this approach currently uses only the intensity of the electromagnetic radiation emitted by luminescence, not its wavelengths. + Instead of detecting bioluminescence as an intensity signal, such detection can be performed using detectors arranged 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.

[0004] For example, WO 01 / 350079 A1 discloses fluorometers with light sources that generate low heat.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] An optical transmission element is arranged between the openings of the cavities and the detectors. Channel-shaped openings are formed in the openings, corresponding to the openings of the cavities. Electromagnetic radiation emitted by luminescence from the samples impinges on the detectors through these openings. The openings form hollow channels through which the emitted electromagnetic radiation from the respective samples can pass toward the detectors.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] The inner walls of the channel-shaped recesses should be oriented 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.

[0015] 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, 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 toward the detectors can be hemispherical.

[0016] The transparent element can be integrated into the lid that closes the openings of the cavities.

[0017] 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.

[0018] In Fig. 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.

[0019] 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).

[0020] 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.

[0021] 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).

[0022] 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, for example, a commercially available color camera (or, in extreme cases, even a webcam) and are inexpensive, they can be used as detectors.

[0023] The invention will be explained in more detail below by way of example.

[0024] Showing: Fig. 1 In schematic form, an example of an apparatus which can be used in carrying out the method according to the invention; Fig. 2 an example with an additional transmission element; Fig. 3 an example with additional transparent elements; Fig. 4 another example with additional transparent elements and Fig. 5 Top views of a substrate surface with the openings of cavities.

[0025] Fig. Figure 1 shows a basic structure of a device. Cavities 3 are formed in a substrate 1 (predominantly made of plastic or glass), in which the various molecules are contained, preferably in an aqueous solution. The individual cavities 3 can contain identical or different samples for detection. The electromagnetic radiation emitted as a result of luminescence can be emitted in all directions (for example, indicated by beams 10, 11, 11', 12, 13), i.e., the entire solid angle of 4π. This includes a) Emission through the cover 2 (rays 10, 11, 11') towards the detector 5 b) Undetected emission through substrate 1 (beam 13), and c) Emission through or into the substrate 1, e.g. after total reflection (beam 12) at the interface to the air, so that a complete detection of the entire 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 Fig. The image sensor shown schematically in Figure 1 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.

[0026] 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 by 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.

[0027] With a device like the one in Fig. 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.

[0028] 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.

[0029] 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⋅(n⋅sinφ)2=const.

[0030] 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.

[0031] Electromagnetic radiation is generated in a cavity 3 with area A cav At the refractive index of the aqueous solution containing the sample, n~1.34, 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.

[0032] 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, is enlarged toward the detectors 5.

[0033] This efficiency in detection can be achieved with a device according to Fig. 3 can be further improved.

[0034] In the example after Fig. 3, an immersion layer 9 with a suitable liquid is formed between the cover 2 and the transmission element 7. In addition, Fig. 2 and Fig. 3 shows top views of the respective substrate 1 with the cavities 3 in different arrangements.

[0035] The total reflection on the surface of the cover 2, with which the openings of the cavities 3 can be closed, can be achieved by applying an optical element 8 which is transparent to the electromagnetic radiation emitted as a result of luminescence (see Fig. 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 Fig. 3 can be prevented.

[0036] 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.

[0037] With such a device it is possible to carry out several measurements with the detectors 5. The lower part of the Fig. 2 and Fig. Figure 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.

[0038] 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 ( Fig. 4). There are two ways to do 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 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 relative 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 lid 2, as is shown for example in Fig. 4.

[0039] In the Fig. 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.

[0040] 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.

[0041] 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.

[0042] For this detection unused parts, in Fig. 5 are 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

[1] 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 radiation emitted from the samples as a result of luminescence is directed onto the detectors (5). [2] Method according to claim 1, characterized by that the wall (7.2) of the channel-shaped openings (7.1) is provided with a coating that reflects the emitted electromagnetic radiation or the optical transmission element (7) is formed from a material that reflects at least 50% of the emitted electromagnetic radiation, so that the emitted electromagnetic radiation is directed in the direction of the detectors (5). [3] Method according to one of the preceding claims, characterized bythat the walls (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 by total reflection. [4] Method according to one of the preceding claims, characterized by that the emitted electromagnetic radiation is guided through channel-shaped openings (7.1) whose free cross-section, through which the emitted electromagnetic radiation is directed, is directed in the direction of detectors (5) and is enlarged in the area towards the detectors (5) compared to the area which points in the direction of the opening of the respective cavity. [5] Method according to one of the preceding claims, characterized bythat 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), an element (8) is arranged in each case, which element is transparent to the emitted electromagnetic radiation and 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. [6] Method according to the preceding claim, characterized by that the surface of the element (8) facing towards the detectors (5) is hemispherical, which prevents back reflections of electromagnetic radiation emitted by samples. [7] Method according to one of the preceding claims, characterized bythat 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.

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