RAPID DIAGNOSTICS USING PHASE COUPLING OF ANTIGENS
20230400463 · 2023-12-14
Inventors
Cpc classification
International classification
Abstract
A method is provided with which biomolecules (5) such as viruses, for example, are quantitatively determined and/or separated very rapidly. Antigens or receptors (2) are directly or indirectly fixed so closely together on a substrate (1) that they couple together in phase. A dynamic charge displacement field is significantly increased compared to a single antigen or receptor (2) by means of the phase coupling, so that biomolecules (5) which are located in the range of the larger charge displacement field move immediately in the direction of the phase-coupled antigens or receptors (2). The binding kinetics are significantly faster than in the case of conventional methods. The invention enables, for example, the construction of a rapid test for coronavirus which can deliver a result within seconds. The sensitivity which can be obtained is that of one virus. The method can be applied to liquids (6) or aerosols (7).
Claims
1. A method for the quantitative determination and/or separation of biomolecules, the method comprising the step of fixing a plurality of antigens or receptors, each with at least one alpha helix or a beta sheet, directly or indirectly on a substrate so closely together that vibrations of the plurality of antigens or receptors couple together in phase via electric charges or mechanically, wherein an electric charge displacement field of the plurality of antigens or receptors which are coupled in phase is formed which is larger compared to an electric charge displacement field of an individual antigen or receptor, wherein the biomolecules in the electric charge displacement field of the plurality of antigens or receptors which are coupled in phase couple to the antigens or receptors specifically and rapidly.
2. The method as claimed in claim 1, wherein the biomolecules each contain at least two alpha helices or two beta sheets.
3. The method as claimed in claim 1, wherein antibodies which specifically bind to the biomolecules are used as the antigens or receptors.
4. The method as claimed in claim 1, wherein a liquid layer containing the biomolecules is formed above the substrate with the plurality of antigens or receptors which are coupled in phase, wherein the liquid layer has a layer thickness up to a maximum layer thickness at which all or the predominant fraction of the biomolecules in the liquid couple to the antigens or receptors.
5. The method as claimed in claim 1, wherein biomolecules are present in an ambient air or respiratory air aerosol, wherein the ambient air or respiratory air aerosol is guided over the substrate with the plurality of antigens or receptors which are coupled in phase, and wherein the temperature of the substrate with the plurality of antigens or receptors which are coupled in phase is controlled such that condensation of the ambient air or respiratory air aerosol occurs on the substrate.
6. The method as claimed in claim 1, wherein method is used to quantitatively determine and/or to separate the biomolecules, comprising neurotransmitters, interleukins, chemokines, enzymes, antibodies, viruses, bacteria and/or cells, from circulating blood, from a cerebrospinal fluid or from respiratory air from a human being or animal in vitro.
7. The method as claimed in claim 1, wherein the biomolecules are in a liquid or in an ambient air or respiratory air aerosol which is guided in a defined continuous flow process or throughflow process over the substrate with the plurality of antigens or receptors which are coupled in phase.
8. The method as claimed in claim 1, wherein an oxidizing material comprising silicon or aluminum is used as the substrate and blocking of regions of the substrate which are not occupied by antigens or receptors is carried out by means of oxidation or by means of other organic or inorganic materials.
9. The method as claimed in claim 1, wherein regions of the substrate which are not occupied by antigens or receptors are oxidized and an oxide layer which is formed thereby is rendered hydrophobic by a natural or synthetic coating.
10. The method as claimed in claim 1, wherein a linear or elliptically polarized laser beam is directed onto the substrate with the plurality of antigens or receptors which are coupled in phase with the coupled biomolecules and a laser beam which is reflected therefrom is detected in a detector.
11. The method as claimed in claim 10, wherein a camera is used as the detector.
12. The method as claimed in claim 10, wherein scattered light produced from the biomolecules coupled to the antigens or receptors is detected and analyzed for the quantitative determination of the number of coupled and therefore separated biomolecules.
13. The method as claimed in claim 10, wherein the substrate, with the plurality of antigens or receptors which are coupled in phase, is used multiple times for the quantitative determination and/or separation of biomolecules from different liquids or ambient air or respiratory air aerosols until a relationship between the number of biomolecules coupled to the antigens or receptors and the scattered light produced therefrom is no longer linear.
14. The method as claimed in claim 10, wherein for successive quantitative determinations and/or separations of biomolecules using the same substrate and the same plurality of antigens or receptors which are coupled in phase, the variation in the scattered light produced with respect to the previous quantitative determination and/or separation is analyzed analysed for the quantitative determination of the number of coupled and therefore separated biomolecules.
15. The method as claimed in claim 1, wherein the plurality of antigens or receptors is indirectly fixed to the substrate such that the plurality of antigens or receptors is applied to an elastic or rigid membrane comprised of a lipid membrane or a cell membrane or virus membrane with epitopes, and the membrane is floated onto the substrate on a layer of water.
16. The method as claimed in claim 1, further comprising a step for the qualitative assessment of a biomolecule load based on the quantitatively determined number of coupled biomolecules compared to a specified reference value.
17. An analytical means for the quantitative determination and/or separation of biomolecules, the analytical means comprising: a substrate; and a plurality of antigens or receptors each with at least one alpha helix or a beta sheet, wherein the plurality of antigens or receptors is directly or indirectly fixed on the substrate so closely together that vibrations of the plurality of antigens or receptors are coupled together in phase via electric charges or mechanically, wherein a larger electric charge displacement field of the plurality of antigens or receptors which are coupled in phase is formed compared to an electric charge displacement field of a single antigen or receptor, wherein the larger electric charge displacement field of the plurality of antigens or receptors which are coupled in phase can be used to couple biomolecules to the antigens or receptors specifically and rapidly.
18. An analytical means as claimed in claim 17, wherein the antigens or receptors are antibodies which specifically bind to the biomolecules.
19. An analytical means as claimed in claim 17, wherein the temperature of the substrate with the plurality of antigens or receptors which are coupled in phase can be controlled such that an ambient air or respiratory air aerosol containing the biomolecules condenses on the substrate.
20. An analytical means as claimed in claim 17, wherein the substrate comprises an oxidizing material and regions of the substrate which are not occupied by antigens or receptors are occupied by means of oxidation or by another organic or inorganic material.
21. An analytical means as claimed in claim 17, wherein regions of the substrate which are not occupied by antigens or receptors have an oxide layer which is hydrophobic because of a natural or synthetic coating.
22. An analytical means as claimed in claim 17, wherein the plurality of antigens or receptors are indirectly fixed on the substrate such that the plurality of antigens or receptors is applied to an elastic or rigid membrane and the membrane is floated onto the substrate on a layer of water.
23. An analytical device for the quantitative determination and/or separation of biomolecules in a liquid or an ambient air or a respiratory air aerosol, wherein the analytical device comprises: an analytical means as claimed in claim 17, a continuous flow or throughflow device for guiding the liquid or the ambient air or respiratory air aerosol over the analytical means, a laser beam decoupler for decoupling a linear or elliptically polarised laser beam onto the analytical means, and a detector for detecting a laser beam reflected from the analytical means.
24. The analytical device as claimed in claim 23, wherein the analytical means is disposed in the analytical device so that the analytical means can be changed after a single use or after multiple uses.
25. The analytical device as claimed in claim 23, wherein the detector is a camera.
26. The analytical device as claimed in claim 23, further comprising an analysis unit which is equipped and configured to analyze scattered light produced from the biomolecules coupled to the antigens or receptors of the analytical means, which is detected by the detector for the quantitative determination of the number of coupled biomolecules.
27. The analytical device as claimed in claim 26, wherein the analysis unit is further equipped and configured to qualitatively assess a biomolecule load based on the quantitatively determined number of coupled biomolecules in comparison to a specified reference value.
28. The analytical device as claimed in claim 23, further comprising a temperature control unit which is equipped and configured to control the temperature of the analytical means so that an ambient air or respiratory air aerosol containing the biomolecules condenses on the analytical means.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] In the drawings:
[0049]
[0050]
[0051]
DESCRIPTION OF PREFERRED EMBODIMENTS
[0052] Exemplary Production of Electrical Phase Coupling with Blocking:
[0053] In the case of electrical phase coupling (
[0054] Oxide-free or oxide-reduced silicon may, for example, be obtained by freeing a silicon wafer with a SiO.sub.2 layer from its oxide layer using hydrofluoric acid. Application of antigens or antibodies in aqueous solution to the oxide-free or oxide-reduced silicon wafer (1) leads to coupling of the antigens or antibodies (2), so that these sit securely on the silicon wafer and are orientated in the same direction.
[0055] In the case of aluminium, aluminium foil may be used as the substrate (1). During the production process, the foil is rolled in the folded state, wherein the inside of the folded foil is substantially free from oxide. Oxidation only begins when the foil is pulled apart. If antigens or antibodies in aqueous solution are applied to the foils at this point in time, then they adhere immediately.
[0056] In order to obtain the phase coupling via charges, the mutual distance between the antigens or antibodies (2) must be so small that the charges of adjacent antigens or antibodies (2) can interact with each other.
[0057] Antigens (2) have positive charges in the form of NH.sub.3.sup.+ ions and negative charges in the form of COO.sup.− ions; the isoelectric point is between the positive and the negative charges. In addition, antigens have at least one beta sheet or alpha helix. It is generally known that both beta sheets as well as alpha helices behave like linear or non-linear springs. It is also generally known that the structures vibrate or can vibrate in the terahertz range. For this, the structures can only be moist or in aqueous solution.
[0058] By applying the mathematical methods of engineering mechanics and using Maxwell's equations, it can be shown that automatic phase coupling of the antigen vibrations occurs when the antigens (2) are sufficiently densely disposed adjacent to each other.
[0059] For an antigen distance of 3 nm, phase coupling occurs via electric charges in all cases. This can readily be demonstrated mathematically using the two adjacent mass-spring-mass system model, wherein both masses are electrically charged. The two mass-spring-mass systems synchronise independently of the starting conditions with time. The only prerequisite is that both mass-spring-mass systems have the same resonance frequency.
[0060] Phase coupling is particularly strong when a flat plane can pass through all of the isoelectric points of the antigens (2). Thus, the substrate (1) should be as smooth as possible. However, phase coupling also occurs when a flat plane cannot be passed through the isoelectric points. An increase in the sensitivity of the method may also be obtained when several antigen layers or other layers are applied. This additional layer is preferably to be introduced between the substrate (1) and the antigen layer (2).
[0061] By overlaying of the individual fields, the antigens (2) which are fixed and which vibrate in phase produce a resultant electric charge displacement field which is larger than the respective charge displacement field of an individual antigen (2). The range of the charge displacement waves increases with the root of the number of coupled antigens (2). This can be calculated with conventional electrical engineering methods. For a rapid test, it is therefore necessary for sufficient antigens (2) to be coupled in phase.
[0062] Experiments have shown: if whole antibodies (2) are used instead of simple antigens, then the burst kinetics of ligands or biomolecules (5) accelerate significantly.
[0063] After producing an antigen or antibody layer which is not necessarily complete, blocking of unoccupied regions of the substrate (1) is carried out by means of molecules (3) or by means of the natural oxidation process (3). Natural blocking by oxidation can be carried out via a natural or synthetic oxidation process. In the case of a natural oxidation process, the natural oxygen of the air is used; in the case of a synthetic oxidation process, additional oxygen is supplied. Unfortunately, SiO.sub.2 is hydrophilic, but it can be made hydrophobic. To this end, the SiO.sub.2 layer can simply be exposed to normal air. After a certain time, a hydrophobic layer (4) is formed on the SiO.sub.2. This process can be shortened by vaporizing oil or fat in the vicinity. An extremely thin hydrophobic film then forms on the SiO.sub.2. This extremely thin hydrophobic film improves blocking of unoccupied regions of the substrate (1).
[0064] As an alternative to SiO.sub.2 blocking, blocking with other substrates may also be carried out. Possible examples in this regard are lipids or TRIS. These substrates have the task of preventing non-specific binding to regions of the substrate (1) which are not occupied by antigens or receptors (2).
Exemplary Embodiment for Mechanical Phase Coupling:
[0065] As an alternative or in addition to coupling via charges, as described above, mechanical phase coupling of the antigens or antibodies may occur. An example of mechanical phase coupling is illustrated in
[0066] Antigens or antibodies (2) on an elastic or rigid membrane (8) are placed on a substrate (1) with or without an oxide layer (3) and which is as smooth as possible (for example a silicon substrate). The membrane may, for example, be a cell membrane or virus membrane with epitopes. A monolayer of water (9) automatically forms between the lipid membrane (8) and the substrate surface (1); the monolayer enables the membrane to vibrate, and so mechanical phase coupling of the antigens can occur.
[0067] The mechanical phase coupling of metronomes which are positioned on a movable plate is known. This principle also applies to biomolecules.
[0068] In the case of mechanical phase coupling as described above, blocking can be dispensed with, because lipid membranes make suitable blockers. In order to obtain mechanical phase coupling, the distance between the antigens or antibodies (2) may be larger compared to electrical phase coupling, because the antigens or antibodies (2) are mechanically coupled by means of the membrane (8).
Exemplary Embodiment with Electrical or Mechanical Phase Coupling
[0069] The rest of the procedure is largely the same, irrespective of the phase coupling (mechanical or electrical).
[0070] Above the antigens or antibodies (2) is a liquid layer (6) with the biomolecules (5) to be analysed. The liquid layer (6) is, however, only thick enough to allow the electric charge displacement waves to penetrate with sufficient strength. The field strength here is large enough for the biomolecules (5), for example enzymes, antibodies, viruses, bacteria or cells, to be recognised at any location in the liquid layer (6) where antigens or antibodies (2) are found. They will then automatically move in the direction of the antigens or antibodies in order to couple to them. The thinner the layer, the faster will the coupling process occur. If the aforementioned general conditions are satisfied, then all of the biomolecules (5) will couple, i.e. there is a significant deviation from the law of mass action. This has been shown in experiments. The non-linear kinetics of the law of mass action are replaced by a controlled process with linear kinetics. This controlled process is significantly faster (by powers of ten) than the law of mass action and leads to the development of rapid tests.
[0071] Experimentally, a region of approximately 2 mm could be identified in which biomolecules (5) such as viruses recognise their antigens or antibodies (2) and move towards them; in the case of bacteria and cells, something similar can be expected. In theory, this region could be extended to more than 10 mm by optimization of the receptor-layer system.
[0072] It has also been shown experimentally that in this region, the biomolecules move towards the substrate at a constant speed. In experiments with IgE antibodies, a speed of 2 mm/s was determined; with viruses, the speed was 0.1-0.2 mm/s.
[0073] In the case of a column of liquid (6) of 1 mm above the antigen or antibody-coated substrate (2), all biomolecules in the form of IgE antibodies are deposited from the liquid column within 0.5 seconds. In the case of viruses, the time is ca. 5-10 seconds. Instead of a continuous liquid column (6), aerosols (7 in
[0074] In order to detect aerosols (7) metrologically, the substrate (1) should be colder than the aerosol (7) or be below the dew point. This means that the aerosols (7) will condense on the antigen layer or antibody layer (2) and the coupling process will occur.
[0075] The metrological evaluation may, for example, be carried out in accordance with U.S. Pat. No. 6,168,921, but since then, other further developments based on this have been developed which have a significantly higher sensitivity. With the aid of this technology, a variation in layer thickness of 20 femtometres can be detected. The individual biomolecules (5) are in fact larger, but the optical diffraction limit means that mean virtual layer thicknesses which are significantly below one atomic diameter can be measured. If a 160 nm virus with a volume of 2.1*10.sup.−3 μm.sup.3 is coupled to a measurement surface area of 3 mm.sup.2, then this corresponds to a mean virtual layer thickness increase of 0.71 femtometres on the 3 mm.sup.2 substrate surface. In the case of an instrumentational engineering resolution, then the method has a resolution of 28 viruses (5), for example.
[0076] Using a camera as the detector (12) means that the resolution can be increased still further. Even if the signal noise of the camera (12) is a factor of 10000 higher than in the case of the method without a camera and the layer thickness resolution of each pixel is only 0.2 nm, in the case of a diffraction limit (lateral resolution of the camera) of 500 nm, then even an individual virus (5) can be identified really well. The resolution limit of the method is then approximately 1/10 viruses.
[0077] If the substrate (1) is very smooth, then the antigen or antibody layer (2) on the substrate (1) does not produce any significant scattered light. If larger biomolecules (5) such as viruses, bacteria or cells are coupled to the antigens or antibodies (2), then this produces scattered light (13). Even biomolecules (5) which are smaller than the diffraction limit produce scattered light (13) which can be seen with the naked eye or with optical aids (for example imaging optics with a camera).
[0078] Experiments have shown that even 40 nm biomolecules (5) can be seen with the naked eye. This is possible because the scattered light (13) has a diverging beam path.
[0079] As an alternative and independently, the present invention may also be defined as follows:
[0080] A method for detecting, removing or filtering out molecules is provided, characterized in that antigens or receptors with at least one molecular spring (alpha helix or beta sheet) are fixed on a substrate, the vibrations being coupled in phase via electric charges or mechanically (for example lipid membranes) in order to produce a larger electric charge displacement field, wherein the larger range of the field is used to bring ligands to their receptors in a specific and rapid manner.
[0081] Optionally, the ligands may have at least two springs (alpha helices or two beta sheets).
[0082] Optionally, antibodies may be used as the antigens or receptors.
[0083] Optionally, the thickness of the liquid layer above the substrate with the phase-coupled antigens may be selected in a manner such that all or the predominant fraction of the ligands couple to the antigens in the liquid.
[0084] Optionally, ligands in ambient air or respiratory air aerosols may be determined, removed or filtered out, wherein the temperature of the substrate with the receptors is controlled in a manner such that condensation of the aerosol on the substrate occurs.
[0085] Optionally, the method may be used to remove biomolecules (neurotransmitters, interleukins, chemokines, enzymes, antibodies, viruses, bacteria or cells) from circulating blood, from cerebrospinal fluid or from respiratory air.
[0086] Optionally, the liquids or aerosol-containing gases (for example ambient air) may involve a continuous flow or throughflow method.
[0087] Optionally, oxidising materials (for example silicon or aluminium) may be used as the substrate and blocking may be carried out by oxidation or using other organic or inorganic materials.
[0088] Optionally, the oxide layers may be rendered hydrophobic by natural contamination or synthetic coating.
[0089] Optionally, the measurement may be carried out optically or mechanically with the aid of a cantilever.
[0090] Optionally, a camera may be used for detection in order to increase the resolution of the method.
[0091] Optionally, the scattered light may be detected with a camera.
[0092] While specific embodiments of the invention have been shown and described in detail to illustrate the application of the principles of the invention, it will be understood that the invention may be embodied otherwise without departing from such principles.
REFERENCES TO LITERATURE
[0093] 1. Deutsches Arzteblatt [German Medical Journal] 24/2020, issue A, page 1194 ff. [0094] 2. American Association for the Advancement of Science (AAAS), News Release 27 May 2020, “One minute electro-optical coronavirus test developed at Ben-Gurion University”, https://www.eurekalertorg/news-releases/497554 [0095] 3. Riβ, Udo, (2011/06/30), “Theory of long distance interaction between antibodies and antigens”, European Biophysics Journal: EBJ. 40. 987-1005. 10.1007/s00249-011-0718-z. [0096] 4. Alexandre Rothen, “IMMUNOLOGICAL REACTIONS BETWEEN FILMS OF ANTIGEN AND ANTIBODY MOLECULES”, Journal of Biological Chemistry, Volume 168, Issue 1, 1947, Pages 75-97, ISSN 0021-9258, https://doi.org/10.1016/S0021-9258(17)35094-9. (https://www.sciencedirect.com/science/article/pii/S0021925817350949)