Artificial transmembrane proteins for detecting intracellular or intravesicular biomolecular interactions

Abstract

Disclosed herein is an artificial transmembrane protein for use in a biomolecule detection device for detecting intracellular or intravesicular biomolecule interactions, the artificial transmembrane protein comprising an extracellular or extravesicular binding structure, a hydrophobic transmembrane domain, and an intracellular or intravesicular domain having an intracellular or intravesicular receptor structure, wherein the receptor structure is configured to interact with an intracellular or intravesicular component of a biomolecule interaction to be detected, and wherein the extracellular or extravesicular binding structure is configured to bind to a membrane recognition element arranged along a plurality of predetermined lines of the biomolecule detection device.

Description

TECHNICAL FIELD

[0001] The present invention is in the field of artificial transmembrane proteins, in particular for detecting intracellular or intravesicular biomolecular interactions. The invention further relates to a biomolecular detection device for analyzing a cell, a vesicle or a component of a cell or a vesicle comprising an artificial transmembrane protein, and to a method for detecting intracellular or intravesicular biomolecular interactions in a cell, a component of a cell or a vesicle or a component of a vesicle. BACKGROUND

[0002] Detection devices are used as biosensors, for example in a wide variety of applications. One particular application is the detection or monitoring of binding affinities or processes. For example, with the aid of such biosensors, various assays can be performed to detect the binding of target samples to binding sites. Typically, a large number of such assays are performed on a biosensor at points arranged in a two-dimensional microarray on the surface of the biosensor. The use of microarrays provides a tool for simultaneously detecting the binding affinities or processes of different target samples in high-throughput screening. In order to detect the affinity of a target sample to bind to a particular binding site, a large number of capture molecules of a predetermined type are immobilized at individual points on the outer surface of the biosensor (for example by inkjet spotting or photolithography). Each point forms a separate measurement zone for the predetermined type of capture molecule. The binding of a target molecule to a particular type of capture molecule is detected and used to provide information about the binding affinity of the target molecule with respect to the particular capture molecule.

[0003] A known technique for detecting the binding affinity of a target sample makes use of fluorescent labels. Fluorescent labels are capable of emitting fluorescence upon excitation. The emitted fluorescence has a characteristic emission spectrum which identifies the current fluorescent label at a particular point. The identified fluorescent label indicates that a labeled target molecule has bound to a particular type of binding site present at the point.

[0004] A sensor for detecting a labeled target sample is described in the article "Zeptosens' protein microarrays: A novel high performance microarray platform for low abundance protein analysis", Proteomics 2002, 2, S. 383-393, Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany. The sensor described therein comprises a planar waveguide arranged on a substrate. The planar waveguide has an outer surface on which a plurality of binding sites can be attached. Furthermore, the planar waveguide has a plurality of in-coupling lines for coupling a coherent light beam into the planar waveguide in such a way that the coherent light beam propagates along the planar waveguide. The coherent light propagates through the planar waveguide under total internal reflection, wherein an evanescent field of the coherent light propagates along the outer surface of the planar waveguide. The depth at which the evanescent field penetrates into the optically thinner medium at the outer surface of the planar waveguide is of the order of a fraction of the wavelength of the coherent light propagating through the planar waveguide. The evanescent field excites fluorescent labels of a labeled target sample bound to the binding sites arranged on the surface of the planar waveguide. Since the depth at which the evanescent field penetrates into the optically thinner medium at the outer surface of the planar waveguide is very small, only the labeled sample bound to the binding sites immobilized on the outer surface of the planar waveguide is excited. The fluorescence emitted by these labels is then detected with the help of a CCD camera.

[0005] While it is in principle possible to detect binding affinities using fluorescent labels, the disadvantage of this technique is that the signal detected is generated by the fluorescent label and not by the binding partner itself. Furthermore, labeled target samples require additional preparation steps. In addition, labeled target samples are relatively expensive. A further disadvantage is a distortion of the results due to steric hindrance of the fluorescent label on the target sample, which can interfere with the binding of the target sample to the capture molecule. A further disadvantage is a distortion of the results due to photobleaching or quenching effects of the label. Furthermore, fluorescent labels can significantly influence the chemical, biological, pharmacological and physical properties of the compounds of interest. Thus, measurements relying solely on fluorescent labels can be distorted by the presence of such labels. Furthermore, fluorescence spectroscopy requires labeling of any compound or cellular component of interest. Thus, only interactions with the specific compound labeled can be observed, while any further interactions cannot be observed. SUMMARY

[0006] A key requirement for the analysis of biological samples is to distinguish between specific and non-specific binding of a compound or moiety of interest to a binding site. Known strategies to address this issue, such as surface plasmon resonance (SPR) or Mach Zehnder interferometry, strongly rely on reference measurements and are only applicable to measurements under static conditions. Thus, such techniques are generally not suitable for measurements in complex environments, such as detecting biomolecular interactions within living cells. SPR measures the change in refractive index when a receptor-ligand binding occurs near the sensor surface. However, a drawback of this technique is that it is susceptible to any change in refractive index near the sensor surface. Thus, non-specific binding remains a major issue. In particular, refractometric sensors cannot distinguish between molecules that actually bind to the target of interest and the mere presence of other compounds and additional effects that influence the refractive index.

[0007] Since G-protein coupled receptors (GPCRs) are associated with the development and progression of many diseases, such as pain, asthma, inflammation, obesity and cancer, they have developed into particularly prominent targets for candidate drugs, and detailed analysis of these receptors in living cells is highly desirable. Whole-cell assays for determining GPCR activity traditionally rely on the detection of different intracellular second messengers (cAMP, Ca 2+ ), relocation of fluorescently labeled proteins (inhibition of protein recruitment to the receptor or receptor internalization), or expression of reporter genes under the control of a GPCR-activated signaling cascade. However, as mentioned above, fluorescent labeling requires extensive biomolecular modifications, such as overexpression of proteins or introduction of fluorescent labels, which are not always possible or desirable, as these would alter cell physiology or drug pharmacology. Furthermore, GPCRs often modulate more than one effector, which can lead to cross-sensitivity in these assays.

[0008] Label-free cell assays, such as resonant waveguide grating biosensors, do not require any molecular labeling (see Paulsen et al., Photonics Nanostruct. Fundam. Appl. 2017, 26, 69). Typically, such refractometric sensors monitor the refractive index above the sensor chip by means of a propagating evanescent wave that defines the sensing volume by its penetration depth. Redistribution of cellular content within this sensing volume leads to an overall change in refractive index, resulting in the much appreciated holistic picture of dynamic mass redistribution (DMR). In turn, DMR is inherently cross-sensitive, meaning that different GPCR-mediated signaling pathways cannot be spatiotemporally deconvoluted by the sensor.

[0009] As an alternative to optical label-free whole cell assays such as SPR, dynamic mass redistribution based on resonant waveguide gratings (RWG), symmetric waveguide sensors and quantitative phase imaging have been used to study morphological changes and phenotypic cellular responses in drug discovery. It is worth noting, however, that these methods can only provide information about cytosolic mass, solute concentration and volume changes, but not about intracellular or intravesicular processes.

[0010] It is therefore a general object of the present application to improve the state of the art with respect to detecting intracellular or intravesicular interactions, preferably completely or partially avoiding the drawbacks of the state of the art.

[0011] In an advantageous embodiment, an artificial transmembrane protein is provided which can be specifically engineered such that it is configured for interacting with a specific intracellular or intravesicular component and at the same time is capable of monitoring direct or indirect interaction with said intravesicular or intracellular component.

[0012] In a further advantageous embodiment, an artificial transmembrane protein is provided which is capable of single cell measurements.

[0013] The general object is generally achieved by the subject matter of the independent claims. Further advantageous and exemplary embodiments result from the description and the drawings.

[0014] According to a first aspect of the present application, the general object is achieved by an artificial transmembrane protein in a biomolecule detection device for detecting intracellular or intravesicular biomolecule interactions. The artificial transmembrane protein comprises an extracellular or extravesicular binding structure, a hydrophobic transmembrane domain and an intracellular or intravesicular domain comprising an intracellular or intravesicular receptor structure. The receptor structure is configured to interact with an intracellular or intravesicular component of a biomolecule to be detected, and wherein the extracellular or extravesicular binding structure is configured to bind to a membrane recognition element arranged along a plurality of predetermined lines of the biomolecule detection device. It is to be understood that the interaction of the receptor structure with the intracellular or intravesicular component of a biomolecule to be detected refers to an interaction in the sense of biomolecules. Thus, such an interaction can for example be the binding of a compound to a receptor. The intracellular or intravesicular receptor structure can be specifically designed, for example by genetic engineering, to interact with any given intracellular or intravesicular component of interest. Thus, the artificial transmembrane protein is a biomimetic membrane receptor designed for a specific purpose by existing biotechnological procedures.

[0015] Preferably, the biomolecule detection device that can be combined with the artificial transmembrane protein according to any embodiment described herein for detecting biomolecular interactions of interest comprises an evanescent illuminator having an optical coupling unit configured for generating an evanescent field on a first surface of the evanescent illuminator from coherent light having a predetermined wavelength. The first surface of the evanescent illuminator comprises a template nano-pattern comprising a coherent arrangement of a plurality of predetermined lines along which binding structures of a membrane recognition element for a cell, a vesicle or a cellular or vesicular component of a transmembrane protein, preferably a laterally diffusible artificial transmembrane protein, are arranged. A laterally diffusible transmembrane protein is a transmembrane protein that can diffuse within the membrane of a cell or a vesicle. The membrane recognition element is configured to bind to the binding structure of the laterally diffusible artificial transmembrane protein for forming a transmembrane nano-pattern within the cell, the vesicle or the cellular or vesicular component based on the template nano-pattern of the evanescent illuminator by locally immobilizing the artificial transmembrane protein such that light of the evanescent field is scattered by the cell, the vesicle or the cellular or vesicular component bound to the membrane recognition element. As understood by the skilled person, an evanescent illuminator is an element capable of generating an evanescent field from coherent light of a light source. The predetermined lines on the evanescent illuminator are arranged such that light scattered by the cell, the vesicle or the cellular or vesicular component bound to the membrane recognition element constructively interferes at predetermined detection sites, wherein the difference in optical path length is an integer multiple of the predetermined wavelength of the coherent light. As understood by the skilled person, the optical path length refers to the product of the geometrical length of the path followed by light through a given system and the refractive index of the medium through which it propagates. Since the extracellular or extravesicular binding structure of the artificial transmembrane protein is configured to bind to the membrane recognition element arranged along the plurality of predetermined lines of the biomolecule detection device, the template nano-pattern of the biomolecule detection device is transferred into a living cell by selective binding of the laterally diffusible transmembrane protein within the cell, thereby generating a transmembrane nano-pattern in the cell itself. Typically, the membrane recognition elements of a plurality of different predetermined lines bind to the same cell, i.e. more than one membrane recognition element binds or is configured to bind to a single cell, vesicle or cellular or vesicular component.

[0016] Because the extracellular or extravesicular binding structure is configured to bind to the membrane recognition elements arranged along the plurality of predetermined lines of the biomolecule detection device, and the receptor structure can be specifically designed to interact with any given intracellular or intravesicular component of interest by, for example, genetic engineering, the artificial transmembrane protein provides a detection window specific to the detection of biomolecules of interest. The template nano-pattern of the biomolecule detection device allows the transfer or replication of the template nano-pattern of the biomolecule detection device into the membrane of a living cell by selective binding of the artificial transmembrane protein that can diffuse laterally within the cell. As a result, any biomolecular interaction involving the interaction of the artificial transmembrane protein, even intercellular processes, can be selectively detected. After binding between the membrane recognition element and the binding structure of the artificial transmembrane protein, evanescent light is scattered and constructively interfered at the predetermined detection site. The constructive interference of scattered light is related to all bound transmembrane proteins and produces a quadratic scale of the measured intensity with respect to the number of transmembrane proteins. Importantly, the random scattering of background molecules that are not bound to the molecular recognition element has the same probability of being constructive and destructive. As a result, cross-sensitivity is significantly reduced. Surprisingly, even though living cells and likewise vesicles contain highly uneven surfaces on the nanoscale, almost no cross-sensitivity is observed due to any cellular scattering, in particular membrane scattering. While the measurement of living cells can be easily disturbed by signals that suppress the light scattered at the bound membrane recognition elements, no significant loss of sensitivity is observed and only a slight distortion of the focused diffraction signal is observed.

[0017] In general, the coherent light has a predetermined wavelength and is preferably monochromatic, in particular at a single wavelength. In general, visible or near-infrared light can be used. A part of the evanescent field is coherently scattered by scattering centers consisting of biomolecules from the cell, vesicle or cell or vesicle components, which bind to the membrane recognition elements arranged on different predetermined lines. The scattered electric field at any location can be determined by adding the contributions of each scattering center and then calculating the intensity by squaring the resulting phasor. The maximum of the scattering intensity is located at the predetermined detection position because the predetermined lines are arranged such that at the predetermined detection position the optical path lengths of the light scattered by different scattering centers differ by an integer multiple of the wavelength of the light. Any scattered light that increases the detectable signal at the detection position satisfies the requirement of constructive interference. The intensity pattern at the predetermined detection position preferentially forms, but is not limited to, a diffraction-limited Airy disk. Essentially, any shape accessible to Fourier optics is possible. For any shape, the signal can be best recovered using a matched filter.

[0018] Isolated membrane recognition element - scattering of a transmembrane complex with a refractive index n R embedded in a medium with a refractive index n0in plane polarized light is:

[0019]

[0020] wherein

[0021] r is the distance of the compound of interest to the detection site

[0022] λ is the vacuum wavelength

[0023] I R is the intensity of the plane polarized field incident on the artificial transmembrane complex

[0024] V R is the volume of the artificial transmembrane complex;

[0025] and wherein

[0026]

[0027] M R is the molar mass of the artificial transmembrane complex,

[0028] N A is the Avogadro constant,

[0029] is the refractive index increment of the compound of interest.

[0030] the total mass density Γ tot and by simple operations, also the number of receptors can be calculated by the intensity measured at the Airy disk center at the predetermined detection site:

[0031]

[0032] wherein

[0033] NA is the numerical aperture of the circular template nano-pattern

[0034] is the analyte efficiency of the manufactured template nano-pattern

[0035] D is the diameter of the circular template nano-pattern

[0036] is the average intensity incident on the set of receptors

[0037] is the intensity at the Airy disk center, i.e. at the predetermined detection site.

[0038] Therefore, depending on the experimental design, changes in the intensity of scattered light provide a means to derive the molecular mass of the interacting partner, the total bound mass, or the number of receptors involved during a biomolecular interaction involving an artificial transmembrane protein. For example, if a certain messenger compound binds to the artificial transmembrane protein, the corresponding mass increase can be calculated. For example, an intracellular or intravesicular receptor structure can be configured to interact with a first biomolecular interacting component A of known molecular mass. Upon binding of component A, a change in diffraction intensity is observed. This intensity change can be used to calculate the number of receptors interacting with component A. When a second component B binds to component A or the same receptor structure, a further change in intensity is observed. When components A and / or B subsequently detach from the receptor structure, the mass of the corresponding complex decreases again, and the signal intensity changes. Assuming that B binds to the same number of binding sites as A, the molecular mass of the complex, as well as the molecular mass of B, can be calculated at any given measurement time, thereby allowing real-time monitoring of biomolecular interactions over time. Due to the mass difference, information can be obtained about any compound that is directly or indirectly bound or unbound to the artificial transmembrane protein.

[0039] In some embodiments, the artificial transmembrane protein further comprises a linker domain, which is configured to promote the interaction between the intracellular or intravesicular components of the intracellular or intravesicular receptor structure and the biomolecule to be detected, wherein the linker domain is arranged between the intracellular or intravesicular receptor structure and the hydrophobic transmembrane domain. Preferably, the linker domain does not contain bulky hydrophobic residues that may interfere with protein folding, such as tryptophan. Therefore, the linker domain can be composed of amino acids glycine, serine, alanine, glutamine, proline and phenylalanine or is only composed of glycine. Small amino acids such as glycine can provide the higher flexibility of the linker domain. Polar residues, such as glutamine increase the solubility of the joint in water. The linker domain can generally have a length of 4 to 25 residues, preferably 4 to 20 residues.

[0040] In further embodiments, the extracellular or extravesicular binding structure is configured to establish a covalent bond with a membrane recognition element arranged along a plurality of predetermined lines of a biomolecule detection device. For example, the extracellular or extravesicular binding structure may comprise a chemical moiety capable of forming a covalent bond, such as an electrophile, a nucleophile, a dienophile, a diene, a 1,3-dipole, or a dipolephile.

[0041] In certain embodiments, the extracellular or extravesicular binding structure comprises a nucleophile, preferably a thiol or thiolate.

[0042] In further embodiments, the extracellular or extravesicular binding structure comprises or consists of a SNAP tag or a CLIP tag. The SNAP tag is a 182 residue polypeptide and accepts 6- a benzyl cytosine derivative as a substrate, instead of O 2 - a benzyl cytosine derivative as a substrate, instead of O 6 - a benzyl cytosine derivative as a substrate, instead of O

[0043] In some embodiments, the artificial membrane protein is a one-way transmembrane protein.

[0044] In further embodiments, the artificial transmembrane protein is

[0045] (a) Type I, wherein the intracellular or intravesicular domain is arranged adjacent to the C-terminus and the extracellular or extravesicular domain is arranged adjacent to the N-terminus; or

[0046] (b) Type II, wherein the intracellular or intravesicular domain is arranged adjacent to the N-terminus and the extracellular or extravesicular domain is arranged adjacent to the C-terminus.

[0047] In Type I artificial transmembrane proteins, the N-terminus is configured to face the outside of the cell, while the C-terminus is configured to remain in the cytosol. Conversely, in Type II artificial transmembrane proteins, the N-terminus is configured to remain in the cytosol, while the C-terminus is configured to face the outside of the cell.

[0048] In more specific embodiments of Type I, the order of domains from N-terminus to C-terminus can be: an extracellular or extravesicular binding structure, preferably with a SNAP or CLIP tag, followed by a hydrophobic transmembrane domain, optionally followed by a linker domain, followed by an intracellular or intravesicular domain with an intracellular or intravesicular receptor structure. Preferably, positively charged amino acids are arranged in front of the linker domain. Optionally, the extracellular or extravesicular binding structure comprises a cleavable signal peptide, which can preferably be arranged in front of the SNAP or CLIP tag, i.e. closest to the N-terminus.

[0049] In a more specific embodiment of type II, the order of domains from N- to C- terminus can be: an intracellular or intravesicular domain with an intracellular or intravesicular receptor structure, optionally followed by a linker domain, then a hydrophobic transmembrane domain, then an extracellular or extravesicular binding structure, preferably with a SNAP or CLIP tag. Optionally, the intracellular or intravesicular domain can comprise a cleavable signal peptide, which can preferably be arranged in front of the receptor structure.

[0050] In some embodiments, the artificial transmembrane protein is of type I or type II, and the intracellular or intravesicular domain comprises a higher amount of positively charged amino acid residues than the extracellular or extravesicular domain. In particular, the positively charged amino acids can be selected from lysine, arginine or histidine, preferably lysine. In particular, the frequency of occurrence of these amino acids in the intracellular or intravesicular domain can be 3 to 4 times higher than the rest of the artificial transmembrane protein. The higher amount of positively charged amino acids enables a more efficient orientation of the artificial transmembrane helix within the membrane.

[0051] In further embodiments, the intracellular or intravesicular receptor structure is configured to interact with the beta subunit of a protein kinase in the cAMP pathway or to interact with a receptor tyrosine kinase (RTK). If the intracellular or intravesicular receptor structure is configured to interact with an RTK, the receptor structure can comprise a Grb2 protein.

[0052] In some embodiments, the intracellular or intravesicular receptor structure can comprise a fluorescent protein, such as eYFP.

[0053] In some embodiments, the intracellular or intravesicular receptor structure is a designed receptor, an artificial binder or other functional molecule, such as an antibody, an antibody fragment, a nanobody, an affimer, etc.

[0054] In further embodiments, the artificial transmembrane protein comprises a cleavable signal peptide adjacent to the N-terminus of the artificial transmembrane protein for interacting with a protein transport system and controlling the translocation of the artificial transmembrane protein.

[0055] Generally, the cleavable signal peptide consists of 18-26 amino acids, which can form a positively charged N-terminal n-region, a central hydrophobic h-region and a polar C-terminal c-region. Preferably, the cleavage site is comprised in the c-region and is configured to be recognized by a signal peptidase.

[0056] In some embodiments, the extracellular or extracapsular binding structure comprises an affinity tag configured to interact, preferably selectively interact, with a membrane recognition element of the biomolecule detection device. For example, the affinity tag can be a HA (human influenza hemagglutinin), FLAG or 6His affinity tag. The use of an affinity tag is beneficial because the protein conformation can be altered by molecular interactions, which can limit or prevent the recognition of the extracellular or extracapsular binding structure of the artificial transmembrane protein by an antibody acting as a membrane recognition element. This can be avoided by the affinity tag in the extracellular or extracapsular binding structure.

[0057] In some embodiments, the artificial transmembrane protein comprises an intracellular or intracapsular fluorescent protein or a protein configured to interact with other intracellular elements, such as biomolecule tags. For example, for intracellular sortase-mediated protein immobilization. Preferably, the protein is configured to specifically interact with other intracellular elements.

[0058] In some embodiments, the transmembrane protein is label-free, in particular fluorescence label-free. Preferably, the transmembrane protein does not contain an artificial marker moiety that can be excited upon irradiation. As used herein, a fluorescence label-free transmembrane protein is a transmembrane protein that does not contain a fluorescent small molecule, i.e. a molecule with a molecular weight below 900 Da.

[0059] According to another aspect, the present application relates to a cell, vesicle or cell or vesicle component comprising an artificial transmembrane protein according to any embodiment described herein or a nucleic acid sequence encoding an artificial transmembrane protein according to any embodiment described herein. The nucleic acid sequence can be introduced, preferably in vitro, into a cell and expressed therein for use in a biomolecule detection device. According to another aspect, the present application relates to a recombinant nucleic acid molecule comprising at least one nucleic acid sequence encoding an artificial transmembrane protein according to any embodiment described herein.

[0060] According to another aspect, the present application relates to a vector, preferably a plasmid vector, comprising a recombinant nucleic acid molecule as described in any embodiment herein.

[0061] According to another aspect, the present application relates to the use of a vector according to any embodiment described herein for the in vitro expression of an artificial transmembrane protein, comprising:

[0062] providing a cell and introducing into the cell a vector according to any embodiment described herein and expressing the artificial transmembrane protein.

[0063] In some embodiments, the use further comprises the step of cleaving the signal peptide from the artificial transmembrane protein.

[0064] According to another aspect, the present invention relates to a biomolecule detection device for analyzing cells, vesicles, or cell or vesicle components, comprising an artificial transmembrane protein according to any embodiment described herein, wherein the biomolecule detection device comprises an evanescent illuminator having an optical coupling unit, wherein the optical coupling unit is configured to generate an evanescent field from coherent light having a predetermined wavelength on a first surface of the evanescent illuminator, the first surface of the evanescent illuminator comprising a template nanopattern comprising a coherent arrangement of a plurality of predetermined lines, along which are arranged optical fibers for the artificial transmembrane protein, cell, vesicle, or cell. A membrane recognition element having a binding structure for a cell or vesicle component, wherein the membrane recognition element is configured to bind to a binding structure of an artificial transmembrane protein, and is used to form a transmembrane nanopattern within the cell, vesicle, or cell or vesicle component based on a template nanopattern of an evanescent illuminator, so that light of the evanescent field is scattered by the cell, vesicle, or cell or vesicle component bound to the membrane recognition element, and wherein the predetermined lines are arranged so that light scattered by the cell, vesicle, or cell or vesicle component bound to the membrane recognition element constructively interferes at a predetermined detection site, wherein the difference in optical path length is an integer multiple of a predetermined wavelength of the coherent light.

[0065] Template nanopatterns allow the transfer of template nanopatterns of biomolecule detection devices into living cells, generating transmembrane nanopatterns in the cells themselves by selectively binding to transmembrane proteins that can diffuse laterally within the cells. Typically, multiple different predetermined lines of membrane recognition elements are bound to the same cell, i.e., more than one membrane recognition element is bound to or configured to bind to a single cell, vesicle, or cell or vesicle component. As a result, any biomolecular interaction involving transmembrane protein interactions, even intercellular or intracellular processes, can be selectively detected. After binding between the membrane recognition element and the binding structure of the transmembrane protein, evanescent light is scattered and constructively interferes at the predetermined detection site. The constructive interference of the scattered light is associated with all bound transmembrane proteins and produces a quadratic scaling of the measured intensity relative to the number of transmembrane proteins. Importantly, random scattering of background molecules that are not bound to the molecular recognition element has the same probability of being constructive and destructive. As a result, cross-sensitivity is effectively suppressed. Surprisingly, even though living cells and vesicles contain highly uneven surfaces at the nanoscale, almost no cross-sensitivity is observed due to any cell scattering, especially membrane scattering. Although measurements of living cells may be easily disturbed by suppressing the signal of light scattered at the bound membrane recognition element, no significant loss of sensitivity is observed. This is surprising because the cells, vesicles, or cell or vesicle components are significantly larger than the distance between the predetermined lines of the nanopattern. Therefore, in the embodiments disclosed herein, multiple membrane recognition elements of different predetermined lines are typically bound to the same cell. Typically, the distance between two directly adjacent predetermined lines is 1 / 2 to 1 / 100, preferably 1 / 30 to 1 / 100, that of a single cell.

[0066] In some embodiments, the evanescent illuminator comprises or is a carrier having a planar waveguide arranged on a carrier surface and a light coupler as a light coupling unit for coupling coherent light of a predetermined wavelength into the waveguide such that the coherent light propagates through the planar waveguide having an evanescent field of the coherent light propagating along a first surface of the planar waveguide and wherein the first surface of the planar waveguide comprises a template nano-pattern.

[0067] In some embodiments, the evanescent illuminator is a total internal reflection system configured to provide a coherent light beam of a predetermined wavelength and a predetermined angle onto a first surface of the evanescent illuminator by an optical coupling unit, in particular by a prism or any other suitable optical element.

[0068] In some embodiments, the plurality of predetermined lines comprises curved lines having a curvature configured such that light of the evanescent field scattered by cells, vesicles or vesicle or cell components or biomolecules thereof bound to the membrane recognition element interferes at the predetermined detection site.

[0069] Preferably, the overall shape of the template nano-pattern can be circular, in particular round.

[0070] In preferred embodiments, the curved lines are arranged with a decreasing distance between adjacent lines in the direction of propagation of the light for focusing the light scattered by the cells, vesicles or cell or vesicle components to the predetermined detection site. Alternatively, the plurality of predetermined lines can comprise straight lines arranged at a predetermined angle to the propagation of the light coupled to the evanescent illuminator. An additional coupler can be used to focus the diffracted light to the predetermined detection site.

[0071] In further embodiments, the plurality of predetermined lines is arranged on the outer surface of the evanescent illuminator in such a way that their positions in the x j ,y j coordinates are geometrically defined by the following equations:

[0072]

[0073] wherein

[0074] λ is the vacuum wavelength of the propagating light;

[0075] N is the effective refractive index of the guided mode in the planar waveguide; N depends on the thickness of the planar waveguide, the refractive index of the carrier, the refractive index of the medium on the first surface of the planar waveguide and the polarization of the guided mode; in the case of a total internal reflection system, N can be represented by the angle of total internal reflection

[0076] n S is the refractive index of the carrier

[0077] f is the focal length of the predetermined detection position; ​

[0078] A0 is an integer, taking a value close to the product refractive index n S and the focal length of the predetermined detection position f of the carrier divided by the wavelength l;

[0079] j is a consecutive integer, representing the index of the respective row.

[0080] The selected integer A0 assigns a negative x value at the center of the lines with negative j values and a positive x value at the center of the lines with positive j values. Or in other words, the integer A0 defines the origin of the x, y coordinate system which is used to position the lines on the outer surface of the evanescent illuminator; the selected A0 value places the detection position at x = 0, y = 0, z = -f.

[0081] In some embodiments, the at least one cell, vesicle or cell or vesicle component is bound to the membrane recognition element by the binding structure of the artificial transmembrane protein.

[0082] According to another aspect, the present application relates to a kit comprising an artificial transmembrane protein according to any embodiment described herein, or a cell according to any embodiment described herein, or a recombinant nucleic acid molecule according to any embodiment described herein, or a vector according to any embodiment described herein, and a biomolecule detection device according to any embodiment described herein.

[0083] In some embodiments, the kit further comprises a protein of interest configured for an intracellular or intravesicular biomolecule interaction, wherein the protein of interest comprises a high mass moiety, in particular a gold nanoparticle. The use of such a high mass moiety is beneficial as the higher molecular weight has a beneficial effect on the signal strength obtained, thus even single cell measurements can be performed. As the skilled person understands, a high mass moiety typically has a significantly larger molecular weight than the artificial transmembrane protein. For example, the high mass moiety can also be a protein or a protein aggregate overexpressed. Preferably, the mass of the high mass moiety can be at least 150 kD.

[0084] According to another aspect, the present application relates to a label-free method for detecting an intracellular or intravesicular biomolecule interaction in a cell, cell component or vesicle or vesicle component using a biomolecule detection device according to any embodiment as described herein, the method comprising the steps of:

[0085] - optionally providing a cell, cell component or vesicle or vesicle component or vesicle component comprising an artificial transmembrane protein according to any embodiment described herein;

[0086] - applying the cell, cell component or vesicle or vesicle component to the membrane recognition element of the biomolecule detection device or providing the biomolecule detection device with the at least one cell, vesicle or cell or vesicle component bound to the membrane recognition element by the binding structure of the artificial transmembrane protein;

[0087] - generating a coherent light beam at a predetermined beam generation position relative to the plurality of predetermined lines, the coherent light beam having a predetermined wavelength and being incident on the membrane recognition element having the bound transmembrane protein in a manner that a diffracted portion of the incident coherent light beam constructively interferes at a predetermined detection site relative to the plurality of predetermined lines, wherein a difference in optical path length is an integer multiple of the predetermined wavelength of the coherent light to provide a signal representative of the membrane recognition element, the predetermined detection site having the artificial transmembrane protein bound thereto of the cell, the vesicle, or a cell or vesicle component;

[0088] - measuring the signal representative of the membrane recognition element, the artificial transmembrane protein bound thereto of the cell, the vesicle, or a cell or vesicle component.

[0089] The label-free method for detecting an intracellular or intravesicular biomolecule interaction in a cell, a cell component, or a vesicle or a vesicle component is a method that does not rely on an interaction between light, in particular coherent light, and a label. Such a label is for example an artificial label moiety that can be excited upon irradiation, for example a fluorescent label. Frequently used fluorescent labels are fluorescent small molecules, i.e. fluorescent molecules having a molecular weight below 900 Da.

[0090] In some embodiments, the method further comprises the step of providing a substance that interacts with the intracellular or intravesicular component that interacts with the artificial transmembrane protein’s receptor structure or with the biomolecule to be detected, for example a drug, a drug candidate, a small molecule, or an antibody.

[0091] In certain embodiments, the cell or cell component comprising the artificial transmembrane protein is provided by in vitro transfection of a cell with a vector according to any of the embodiments described herein, expressing the artificial transmembrane protein in the cell, and optionally removing part of the membrane of the cell for providing the cell component.

[0092] In some aspects, the present application is described by the following clauses:

[0093] Clause 1 : An artificial transmembrane protein for use in a biomolecule detection device for detecting an intracellular or intravesicular biomolecule interaction, the artificial transmembrane protein comprising an extracellular or extravesicular binding structure, a hydrophobic transmembrane domain, and an intracellular or intravesicular domain having an intracellular or intravesicular receptor structure, wherein the receptor structure is configured to interact with an intracellular or intravesicular component that interacts with the biomolecule to be detected, and wherein the extracellular or extravesicular binding structure is configured to bind to a membrane recognition element arranged along a plurality of predetermined lines of the biomolecule detection device.

[0094] Clause 2: The artificial transmembrane protein according to clause 1, further comprising a linker domain configured to facilitate interaction between the intracellular or intravesicular receptor structure and an intracellular or intravesicular component of the biological molecule to be detected, wherein the linker domain is arranged between the intracellular or intravesicular receptor structure and the hydrophobic transmembrane domain.

[0095] Clause 3: The artificial transmembrane protein according to any one of clauses 1 or 2, wherein the extracellular or extravesicular binding structure is configured to establish a covalent bond with the membrane recognition element arranged along a plurality of predetermined lines of the biological molecule detection device.

[0096] Clause 4: The artificial transmembrane protein according to any one of the preceding clauses, wherein the extracellular or extravesicular binding structure comprises a nucleophile, preferably a thiol or thiolate.

[0097] Clause 5: The artificial transmembrane protein according to any one of the preceding clauses, wherein the extracellular or extravesicular binding structure is a SNAP tag or a CLIP tag.

[0098] Clause 6: The artificial transmembrane protein according to any one of the preceding clauses, wherein the artificial transmembrane protein is

[0099] (a) Type I, wherein the intracellular or intravesicular domain is arranged adjacent to the C-terminus and the extracellular or extravesicular domain is arranged adjacent to the N-terminus; or

[0100] (b) Type II, wherein the intracellular or intravesicular domain is arranged adjacent to the N-terminus and the extracellular or extravesicular domain is arranged adjacent to the C-terminus.

[0101] Clause 7: The artificial transmembrane protein according to clause 7, wherein the artificial transmembrane protein is Type II and wherein the intracellular or intravesicular domain comprises a higher amount of positively charged amino acid residues than the extracellular or extravesicular domain.

[0102] Clause 8: The artificial transmembrane protein according to any one of the preceding clauses, wherein the intracellular or intravesicular receptor structure is a designed receptor or other functional molecule, such as an antibody, an antibody fragment, a nanobody or an affimer.

[0103] Clause 9: The artificial transmembrane protein according to any one of the preceding clauses, further comprising a cleavable signal peptide adjacent to the N-terminus of the artificial transmembrane protein for interaction with a protein transport system and for controlling translocation of the artificial transmembrane protein.

[0104] Clause 10: The artificial transmembrane protein according to any one of the preceding clauses, wherein the extracellular or extravesicular binding structure comprises an affinity tag configured for interaction with the membrane recognition element.

[0105] Clause 11: The artificial transmembrane protein according to any one of the preceding clauses, wherein the transmembrane protein is label-free, in particular fluorescence label-free.

[0106] Clause 12: A cell, vesicle or cellular or vesicular component comprising the artificial transmembrane protein according to any one of clauses 1 to 11 or a nucleic acid sequence encoding the artificial transmembrane protein according to any one of clauses 1 to 10.

[0107] Clause 13: A recombinant nucleic acid molecule comprising at least one nucleic acid sequence encoding the artificial transmembrane protein according to any one of clauses 1 to 10.

[0108] Clause 14: A vector, preferably a plasmid vector, comprising the recombinant nucleic acid molecule according to clause 12.

[0109] Clause 15: Use of a vector according to clause 13 for expressing an artificial transmembrane protein in vitro, comprising:

[0110] providing a cell and introducing the vector according to clause 13 into the cell and expressing the artificial transmembrane protein.

[0111] Clause 16: The use according to clause 14, further comprising the step of cleaving the signal peptide from the artificial transmembrane protein.

[0112] Clause 17: A biomolecule detection device for analyzing a cell, vesicle or cellular or vesicular component comprising the artificial transmembrane protein according to any one of clauses 1 to 10, the biomolecule detection device comprising an evanescent illuminator having an optical coupling unit configured to generate an evanescent field from coherent light having a predetermined wavelength on a first surface of the evanescent illuminator, the first surface of the evanescent illuminator comprising a template nano-pattern comprising a plurality of predetermined lines along which membrane recognition elements for a binding structure of the artificial transmembrane protein of the cell, vesicle or cellular or vesicular component are arranged, wherein the membrane recognition elements are configured to bind the binding structure of the artificial transmembrane protein to form a transmembrane nano-pattern within the cell, vesicle or cellular or vesicular component based on the template nano-pattern of the evanescent illuminator such that light of the evanescent field is scattered by the cell, vesicle or cellular or vesicular component bound to the membrane recognition elements, and wherein the predetermined lines are arranged such that light scattered by the cell, vesicle or cellular or vesicular component bound to the membrane recognition elements constructively interferes at a predetermined detection site, wherein the difference in optical path length is an integer multiple of the predetermined wavelength of the coherent light.

[0113] Clause 18: The biomolecule detection device according to clause 17, wherein at least one cell, vesicle or cellular or vesicular component is bound to the membrane recognition elements by the binding structure of the artificial transmembrane protein.

[0114] Clause 19: A kit comprising:

[0115] a. the artificial transmembrane protein according to any one of clauses 1 to 11, or

[0116] the cell according to clause 12, or

[0117] the recombinant nucleic acid molecule according to clause 13, or

[0118] the vector according to clause 14, and

[0119] b. the biomolecule detection device according to clause 16 or 17.

[0120] Clause 20: The kit according to clause 19, further comprising a protein of interest configured for intracellular or intravesicular biomolecule interaction, wherein the protein of interest comprises a high quality moiety, in particular a gold nanoparticle.

[0121] Clause 21 : A label-free method of detecting intracellular or intravesicular biomolecule interaction in a cell, a cellular component or a vesicle or a vesicular component using the biomolecule detection device according to clause 16 or 17, the method comprising the steps of:

[0122] - providing a cell, a cellular component or a vesicle or a vesicular component comprising the artificial transmembrane protein according to any one of clauses 1 to 11;

[0123] - applying the cell, cellular component or vesicle or vesicular component to the membrane recognition element of the biomolecule detection device;

[0124] - generating a coherent light beam at a predetermined beam generation position relative to the plurality of predetermined lines, the coherent light beam having a predetermined wavelength and being incident on the membrane recognition element having the bound transmembrane protein in a manner that a diffracted portion of the incident coherent light beam constructively interferes at a predetermined detection site relative to the plurality of predetermined lines, wherein a difference in optical path length is an integer multiple of the predetermined wavelength of the coherent light to provide a signal representative of the membrane recognition element with the artificial transmembrane protein bound thereto at the predetermined detection site;

[0125] - measuring the signal representative of the membrane recognition element with the artificial transmembrane protein bound thereto with the cell, vesicle or cellular or vesicular component.

[0126] Clause 22: The method according to clause 21, further comprising the step of providing a substance that interacts with the intracellular or intravesicular component that interacts with the receptor structure of the artificial transmembrane protein or with the biomolecule to be detected.

[0127] Clause 23: The method of clause 21 or 22, wherein the cell or cells comprising the artificial transmembrane protein are provided by in vitro transfection of the cells with the vector of clause 14, expression of the artificial transmembrane protein in the cells, and optionally removal of portions of the cell membrane in order to provide the cellular components. BRIEF DESCRIPTION OF DRAWINGS

[0128] Figure 1 A schematic diagram of a biomolecule detection device according to one embodiment of the application is shown.

[0129] Figure 2 A schematic cross-sectional view of a biomolecule detection device according to another embodiment of the application is shown.

[0130] Figure 3a An artificial transmembrane protein for detecting intracellular interactions of the cAMP pathway according to one embodiment of the application is shown.

[0131] Figure 3b Signals expected from wild-type cells during a control experiment to detect the cAMP pathway and using G-protein knockout cells are shown.

[0132] Figure 3c Signals obtained from wild-type cells during a control experiment to detect the cAMP pathway and using G-protein knockout cells are shown.

[0133] Figure 4a Two different artificial transmembrane proteins for detecting intracellular interactions of the ERK pathway according to other embodiments of the application are shown.

[0134] Figure 4b Signals associated with the artificial transmembrane proteins obtained during detection of the ERK pathway when the ERK pathway is activated by growth factors are shown.

[0135] Figure 4c Signals associated with the artificial transmembrane proteins obtained during detection of the ERK pathway after inhibition of a kinase downstream of the ERK pathway are shown.

[0136] Figure 5 Figures 5a and 5b show a schematic diagram of a biomolecule detection device according to another embodiment of the application.

[0137] Figure 6 Figures 6a and 6b show a schematic diagram of a biomolecule detection device according to another embodiment of the application. DETAILED DESCRIPTION

[0138] Figure 1The present invention shows a biomolecule detection device 1 according to an embodiment of the present invention. The biomolecule detection device 1 includes a carrier 2 having a surface on which a planar waveguide 3 is arranged. The detection device further includes an optical coupler 4 for coupling coherent light L of a predetermined wavelength into the planar waveguide 3, so that the coherent light propagates through the planar waveguide, wherein the evanescent field of the coherent light propagates along the first surface of the planar waveguide 3. The first surface of the planar waveguide is the surface facing away from the carrier 2, i.e., Figure 1 In addition to the optical coupler 4, the first surface of the waveguide 3 also includes a template nano-pattern 5, which includes a plurality of predetermined lines along which the film recognition elements are arranged ( Figure 1 Typically, the light source 6 is used to provide an incident beam of coherent light L toward the optical coupler 4. The optical coupler 4 couples the coherent light L into the planar waveguide 3, where the coherent light L travels along the optical coupler 4. Figure 1 The direction of the arrow shown is propagation toward the template nanopattern 5. If the membrane recognition element is bound to a cell, vesicle, or cell or vesicle component via an artificial transmembrane protein according to any embodiment described herein, light is scattered, and due to the predetermined line, it is arranged so that the light scattered by the cell, vesicle, or cell or vesicle component bound to the membrane recognition element constructively interferes at the predetermined detection site 7. In the specific embodiment shown, the predetermined line is a curve having a curvature configured so that the light of the evanescent field scattered by the cell, vesicle, or cell or vesicle component bound to the membrane recognition element interferes at the predetermined detection site 7. The distance between each membrane recognition element of the template nanopattern 5 and the detection site 7 is called the optical path length.

[0139] Figure 2 A schematic cross-section of a biomolecule detection device 1 is shown, along the propagation direction of light L through a planar waveguide 3, through a template nanopattern 5. The device 1 comprises a carrier 2, on whose surface a waveguide 3 is arranged. Arranged on the first surface of the planar waveguide 3 are living cells 8. Furthermore, the template nanopattern 5 comprises ridges 51 and grooves 52. Ridges 51 are regions along which membrane recognition elements are arranged. Grooves 52 are regions that do not contain any membrane recognition elements. Therefore, the binding structures of the transmembrane proteins of the cell 8 can only bind to the nanopattern 5 at the corresponding ridges. Generally speaking, artificial transmembrane proteins as described herein are laterally diffusible within a cell membrane or vesicle membrane. Therefore, the artificial transmembrane proteins diffuse laterally through the membrane until they are in close proximity to the membrane recognition elements, where they can form covalent bonds to establish a nanopattern within the cell, vesicle, or cell or vesicle component. It should be noted that the widths of the cells, nanopatterns, waveguides, and carriers do not provide any indication of their actual widths or width ratios.

[0140] Attachment Figure 3aA schematic of the cyclic AMP (cAMP) pathway is shown, which is monitored with an artificial transmembrane protein according to one embodiment of the application. The cAMP pathway is a G protein-coupled receptor-triggered signaling cascade that plays a fundamental role in cellular responses. In a resting cell, G proteins are bound to the intracellular site of the GPCR. Upon activation of the GPCR by an external ligand, the alpha subunit of the G protein leaves the GPCR complex and activates adenylyl cyclase (AC), a membrane-bound enzyme with an intracellular active site. In turn, AC converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP), one of the most common second messengers of the cell. Furthermore, AC plays a role in amplifying the signal, as the attack of an external ligand requires the synthesis of thousands of seconds of messenger molecules within a few seconds. The increase in cAMP concentration stimulates cAMP-dependent protein kinase (PKA). PKA is a tetramer composed of two regulatory subunits (type I and type II), which bind two cAMP molecules for each subunit and two catalytic subunits (alpha and beta). Binding of the cAMP molecules leads to phosphorylation of the two catalytic subunits and, thus, to their dissociation from the two regulatory dimers. These phosphorylated subunits activate a wide range of targets, which can be other signaling proteins as well as effector proteins. The last step of the cAMP pathway was chosen as a proof of concept for the working principle of the artificial transmembrane protein according to the application. To detect the dissociation of the catalytic B subunit upon GPCR triggering, an artificial transmembrane protein was constructed with an intracellular or cisternal domain, in which the intracellular or cisternal receptor structure presents a PKA type II-beta regulatory subunit (R2) at its cytosolic site. As can be seen from Figure 3b the expected signal intensity decreases, which is associated with the decrease in mass of the complex upon dissociation of the catalytic beta subunit (dotted line). At the same time, no signal change should be detected in G protein knock-out cells, as the absence of G proteins leads to no activation of AC activity and, thus, no dissociation of the catalytic beta subunit. Figure 3c The signals obtained from HER293 cells (wild type) and G protein knock-out cells are shown. While the signal of the knock-out cells does not provide any signal change, the wild type shows a decrease in signal intensity upon dissociation of the catalytic beta subunit.

[0141] Figure 4aA schematic of the ERK signaling pathway is shown, which has been monitored by two different artificial transmembrane proteins. The ERK pathway is initiated by an external ligand binding to a tyrosine kinase receptor (RTK), which is one of the most common types of enzyme-linked surface receptors. Typically, the inactive RTK receptor consists of two single-pass monomers, which are activated and dimerized once the ligand binds to the extracellular domain. Dimerization involves trans autophosphorylation on specific tyrosine residues (one monomer phosphorylates the other, and vice versa). The phosphorylated tyrosine increases the kinase activity of the RTK and acts as a binding site for specific intracellular proteins. The binding event occurs because these proteins are able to recognize the phosphorylated tyrosine and its surrounding RTK conformation. The RTK-associated protein in turn typically associates with the adaptor protein Grb2 through an SH2 domain. Grb2 has two other SH3 domains for interacting with other proteins. One of the SH3 domains often interacts with Sos, a protein that facilitates GDP exchange with GTP binding to Ras. When Sos activates Ras, the monomeric GTPase, which is bound to GDP when inactive, replaces GDP with GTP, and in turn, Ras further activates Sos in a positive feedback circuit. Ras activates Raf, and Raf in turn activates through a cascade mechanism by Mek the extracellular signal-regulated kinase (commonly referred to as the ERK module). The ERK proteins are in an activated or inactivated state, which corresponds to their phosphorylated and unphosphorylated forms, respectively. The phosphorylated ERK translocates into the nucleus and acts on gene regulatory proteins. The ERK module is typically turned off by ERK itself, which inactivates Raf through negative feedback. To monitor the early cascade effects of growth factors along the ERK pathway, the first artificial transmembrane protein is provided with the Grb2 protein as a receptor structure. In addition, the downstream effects are monitored by the second artificial transmembrane protein, which comprises an artificial binder as a receptor structure for the phosphorylated ERK component. Figure 4b The expected signals of the first artificial transmembrane protein (dashed line) and the second artificial transmembrane protein (solid line) are shown when the ERK cascade is activated by growth factor stimulation. Figure 4c The expected signals of the first artificial transmembrane protein (Grb2, dashed line) and the second artificial transmembrane protein (artificial binder, solid line) are shown after inhibition of the downstream kinases, which will result in a reduction of the signal obtained from the second artificial transmembrane protein only.

[0142] First, it was tested whether the SH3 binding domain of the Grb2 protein as a receptor structure of the first artificial transmembrane protein is functional. In this case, cells comprising the artificial transmembrane protein with Grb2 as a receptor structure and cells comprising the artificial transmembrane protein with eYFP (enhanced yellow fluorescent protein) were treated with a protein specifically targeting Grb2. As shown from the Figure 4dIt can be seen that the signal remains within the baseline for about 20 minutes after injection of the peptide. Thereafter, an increase in the response of the cells with artificial transmembrane proteins containing Grb2 as the receptor structure is observed, while the signal of the cells with artificial transmembrane proteins containing eYFP as the receptor structure remains constant at baseline. The lag between injection and change in response can be due to the time required for the peptide to diffuse through the cell membrane and bind to the Grb2 SH3 domain. Thus, this method allows real-time monitoring of intracellular biomolecular interactions within living cells. Furthermore, these results show that the Grb2 targeting peptide specifically binds to the Grb2 receptor structure within living cells, while it does not bind to a non-specific receptor structure, such as eYFP.

[0143] Figure 5 Figures 5a and 5b show a biomolecule detection device 1'according to embodiments of the application. The device 1'comprises an evanescent illuminator 2' with a light coupling unit 4' configured to generate an evanescent field 9' on a first surface of the evanescent illuminator 2' from coherent light of a predetermined wavelength with a light source 6'. The first surface 5 of the evanescent illuminator 2' comprises a template nano-pattern 5' containing a coherent arrangement of a plurality of predetermined lines along which membrane recognition elements for binding structures of a transmembrane protein 81 of a cell 8' are arranged. As can be seen, by establishing a chemical bond between the membrane recognition elements and the laterally diffusible transmembrane protein 81, the nano-pattern 5' of the evanescent illuminator 2' is transposed into the cell as a transmembrane nano-pattern. In Figure 5 In the embodiment of figure 5a, the light source 6' and the detection unit 7' are physically separate components, while in the embodiment shown in figure 5b, they are an integral part of the evanescent illuminator 2'. Figure 5

[0144] Figure 6 Figures 6a and 6b show alternative embodiments of a biomolecule detection device 1'' according to the application. The biomolecule detection device 1 comprises an evanescent illuminator, which in these particular embodiments is a total internal reflection system configured to provide a coherent light beam at a predetermined wavelength from a light source 6'' onto a first surface of the evanescent illuminator at a predetermined angle by means of a light coupling unit 4''. The light coupling unit in these embodiments is a prism. In Figure 6 In the embodiment shown in figure 6a, the evanescent illuminator comprises a refractive index matching medium 11'' such as a refractive index matching oil, DMSO, glycerol, a water mixture, a hydrogel, etc. and a slide 12'' containing a template nano-pattern 5''. Alternatively, as shown in figure 6b, the evanescent illuminator can be free of a refractive index matching medium 11'' and a slide 12''. In this case, the nano-pattern 5'' is directly provided on the first surface of the light coupling unit 4'', i.e. the prism. Figure 6

[0145] ​​Methods and Materials

[0146] DNA plasmids encoding different artificial transmembrane proteins were purchased from the Invitrogen GeneArt Gene Synthesis service by Thermo Fisher Scientific. All synthetic genes were assembled from synthetic oligonucleotides and / or PCR products and inserted into the pcDNA3.1 (+) vector backbone. Plasmid DNA was purified from transformed bacteria, the concentration was determined by UV spectroscopy, and the final constructs were verified by sequencing by the manufacturer. The sequence identity within the insertion site was 100%. Plasmids were delivered in TE buffer at a concentration of about 1 mg / ml and stored at -80 °C in working aliquots.

[0147] The three signal peptides tested are listed in Table 3.1, while other amino acid sequences can be found in Table 1.

[0148]

[0149] Table 2 shows the structural features of the plasmid vectors encoding some of the artificial transmembrane proteins tested:

[0150] .

[0151] Cell culture and transfection

[0152] HEK293 wild-type and G-protein knockout cells were cultured in complete medium (DMEM medium containing 10% fetal bovine serum) at 37 °C in a cell incubator with 5% CO2. To generate artificial transmembrane protein expressing cells, cells were transfected using Lipofectamine 3000 transfection reagent according to the manufacturer’s protocol.

[0153] To establish stable cell lines, transiently transfected cells were grown in complete medium supplemented with 1 mg / ml G418 for approximately 20 days. After that, neomycin resistant cells were stained using SNAP-Surface 649 dye and selected by flow cytometry.

[0154] For fluorescence imaging, cells were seeded at 50% confluency on 24 glass bottom well plates and transfected after 24 hours as previously described. Transfection media was replaced with complete media 12 hours later. Cells were imaged at 12, 24, 36, and 48 hours post-transfection using an Olympus FluoView FV3000 confocal laser scanning microscope. Prior to imaging, cells were incubated with SNAP-Surface 649 dye for 30 minutes and then washed with warm PBS 3 times. During imaging, cells were kept at 37°C with 5% C02. eYFP and SNAP-Surface 649 channels were acquired simultaneously with a 20x objective, using 514 nm excitation / 527 nm emission wavelengths for the green channel and 651 nm excitation / 667 nm emission wavelengths for the red channel.

[0155] Biomolecule detection device

[0156] Zeptosens thin film optical waveguides were processed using standard procedures (Gatterdam et al., Nature Nanotechnology, 12(11): 1089-1095, September 2017) to coat them with grafted PAA-g-PEG polymers. The amine groups of the polymer were protected with the photosensitive PhSNPPOC group in order to allow further processing. Afterwards, a molecular map pattern was formed on the optical waveguide using the reactive immersion lithography process described previously. Briefly, the polymer-coated waveguide chip was mounted on a custom-made holder that allowed the alignment of the phase mask. After placing the phase mask onto the holder, the polymer was exposed to 405 nm wavelength at 2000 mJ / cm2. The exposed areas were then developed in a 1:1 mixture of water and ethanol for 1 minute. The waveguide chip was then incubated with the BG-GLA-NHS or BC-GLA-NHS substrate to conjugate the SNAP-tag or CLIP-tag, respectively, to the activated amine sites. Afterwards, full-field illumination under UV light was performed to remove the remaining photosensitive groups from the grooves and surroundings. The resulting amine groups were functionalized with the GRGDSPGSC peptide. 2 The dose illuminated the chip to cleave the photosensitive groups from the ridges of the nano-pattern. The activated amine sites were incubated with the BG-GLA-NHS or BC-GLA-NHS substrate to conjugate the SNAP-tag or CLIP-tag, respectively. Afterwards, full-field illumination under UV light was performed to remove the remaining photosensitive groups from the grooves and surroundings. The resulting amine groups were functionalized with the GRGDSPGSC peptide.

[0157] Measurement

[0158] Cells were seeded to 100% confluency on the planar waveguide and allowed to attach for 2-3 hours in complete media while the planar waveguide was kept inside a cell incubator. Afterwards, the media was exchanged for HEPES-buffered complete media or HEPES-buffered HBSS, adjusted to pH 7.4. Measurements were performed on a F3000 ZeptoReader, kept at 35°C with 5% C02. Images were acquired every 15 seconds using a 635 nm laser with an exposure time between 0.1 and 1 second. After a 10-minute baseline was established (30 images), pharmacological operations were performed on the chip.

[0159] amino acid sequence

[0160]

[0161]

[0162]

Claims

1. A label-free method for detecting intracellular or intravesicular biomolecular interactions in cells, cell components, vesicles, or vesicular components using a biomolecule detection device, the biomolecule detection device comprising an evanescent illuminator having a light coupling unit, the light coupling unit being configured to generate an evanescent field from coherent light (L) having a predetermined wavelength on a first surface of the evanescent illuminator, the first surface of the evanescent illuminator comprising a template nanopattern (5), the template nanopattern comprising a coherent arrangement of a plurality of predetermined lines, membrane recognition elements for binding structures of artificial transmembrane proteins for the cells, vesicles, or cell components or vesicular components (8) being arranged along the predetermined lines, wherein the membrane recognition elements are configured to bind to the binding structures of the artificial transmembrane proteins to form a transmembrane nanopattern within the cells, vesicles, or cell components or vesicular components (8) based on the template nanopattern (5) of the evanescent illuminator, so that light of the evanescent field is scattered by the cells, vesicles, or cell components or vesicular components (8) bound to the membrane recognition elements, and wherein The predetermined lines are arranged so that light scattered by cells, vesicles or cell components or vesicle components (8) bound to the membrane recognition element undergoes coherent interference at a predetermined detection site (7), wherein the difference in optical path length is an integer multiple of a predetermined wavelength of the coherent light (L); The method comprises the following steps: - providing a cell, a cell component, a vesicle or a vesicle component comprising an artificial transmembrane protein, wherein the artificial transmembrane protein comprises an extracellular or extravesicular binding structure, a hydrophobic transmembrane domain and an intracellular or intravesicular domain with an intracellular or intravesicular receptor structure, wherein the receptor structure is configured to interact with an intracellular or intravesicular component that interacts with a biomolecule to be detected, and wherein the extracellular or extravesicular binding structure is configured to bind to membrane recognition elements arranged along a plurality of predetermined lines of the biomolecule detection device, and wherein the transmembrane protein is free of fluorescent labels; - applying the cell, cell component, vesicle or vesicle component to the membrane recognition element of the biomolecule detection device, thereby binding the artificial transmembrane protein to one of the membrane recognition elements; - generating a coherent light beam at a predetermined light beam generation position relative to the plurality of predetermined lines, the light beam having a predetermined wavelength and being incident on the membrane recognition element with the bound transmembrane protein in such a manner that diffracted portions of the incident coherent light beam constructively interfere at a predetermined detection site relative to the plurality of predetermined lines, wherein the difference in optical path length is an integer multiple of the predetermined wavelength of the coherent light, so as to provide a signal representative of the membrane recognition element with the artificial transmembrane protein having bound thereto a cell, a vesicle or a cellular component or a vesicle component at the predetermined detection site; - measuring a signal representative of the membrane recognition element having the artificial transmembrane protein of the cell, vesicle or cellular component or vesicle component bound thereto.

2. The method according to claim 1, wherein the artificial transmembrane protein further comprises a linker domain configured to facilitate the interaction between the intracellular or intravesicular receptor structure and the intracellular or intravesicular component that interacts with the biological molecule to be detected, wherein the linker domain is arranged between the intracellular or intravesicular receptor structure and the hydrophobic transmembrane domain. 3 . The method according to claim 1 , wherein the extracellular or extravesicular binding structure establishes a covalent bond with the membrane recognition elements arranged along a plurality of predetermined lines of the biomolecule detection device.

4. The method according to any one of claims 1 or 2, wherein the extracellular or extracellular binding structure of the artificial transmembrane protein comprises a nucleophile.

5. The method according to any one of claims 1 or 2, wherein the extracellular or extracellular binding structure of the artificial transmembrane protein is a SNAP tag or a CLIP tag.

6. according to the method described in any one in claim 1 or 2, wherein said artificial transmembrane protein is (a) Type I, wherein the intracellular or intravesicular domain is positioned adjacent to the C-terminus of the artificial transmembrane protein, and the extracellular or extravesicular domain is positioned adjacent to the N-terminus of the artificial transmembrane protein; or (b) Type II, wherein the intracellular or intravesicular domain is positioned adjacent to the N-terminus of the artificial transmembrane protein, and the extracellular or extravesicular domain is positioned adjacent to the C-terminus of the artificial transmembrane protein.

7. The method according to claim 6, wherein the intracellular or intravesicular domain of the artificial transmembrane protein comprises a higher amount of positively charged amino acid residues than the extracellular or extravesicular domain of the artificial transmembrane protein.

8. The method according to any one of claims 1 or 2, wherein the intracellular or intravesicular receptor structure of the artificial transmembrane protein is a designed receptor.

9. according to the method described in any one in claim 1 or 2, wherein said artificial membrane-spanning protein further comprises a cleavable signal peptide adjacent to the N-terminus of said artificial membrane-spanning protein for interacting with a protein transport system and for controlling the translocation of said artificial membrane-spanning protein.

10. The method according to any one of claims 1 or 2, wherein the extracellular or extracellular binding structure of the artificial transmembrane protein comprises an affinity tag configured to interact with the membrane recognition element.

11. The method according to any one of claims 1 or 2, wherein The signal intensity I representing the membrane recognition element of the artificial transmembrane protein having cells, vesicles or cell components or vesicle components bound thereto scat (r) at each membrane recognition element is determined by: in r is the distance from the compound of interest to the detection site, λ is the vacuum wavelength, I R is the intensity of the plane polarization field incident on the artificial transmembrane complex, M R is the molar mass of the artificial transmembrane complex, N A is Avogadro's constant, dn / dc is the refractive index increment; and where n R is the refractive index of the artificial transmembrane protein embedded in a medium with a refractive index of n0.

12. The method according to any one of claims 1 or 2, wherein the cell, cell component, or vesicle or vesicle component comprises more than one artificial transmembrane protein, and wherein when the cell, cell component, or vesicle or vesicle component is applied to the membrane recognition element, a plurality of different predetermined lines of membrane recognition elements bind to the artificial transmembrane proteins of the cell, cell component, or vesicle or vesicle component, thereby forming a transmembrane nanopattern within the cell, vesicle, or cell component or vesicle component based on the template nanopattern.

13. The method of claim 4, wherein the nucleophile is a thiol or a thiolate.

14. The method of claim 8, wherein the designed receptor is an antibody.

15. The method of claim 8, wherein the designed receptor is an antibody fragment.

16. The method of claim 8, wherein the designed receptor is a nanobody.

17. The method of claim 8, wherein the designed receptor is an affibody.

18. A kit comprising: a. An artificial transmembrane protein, comprising an extracellular or extravesicular binding structure, a hydrophobic transmembrane domain, and an intracellular or intravesicular domain having an intracellular or intravesicular receptor structure, wherein the receptor structure is configured to interact with an intracellular or intravesicular component that interacts with a biomolecule to be detected, and wherein the extracellular or extravesicular binding structure is configured to bind to membrane recognition elements arranged along a plurality of predetermined lines of the biomolecule detection device, and wherein the transmembrane protein is not fluorescently labeled; or A cell comprising the artificial transmembrane protein; or A recombinant nucleic acid molecule comprising at least one nucleic acid sequence encoding the artificial transmembrane protein; or A vector comprising a recombinant nucleic acid molecule encoding the artificial transmembrane protein; as well as b. A biomolecule detection device, comprising an evanescent illuminator having an optical coupling unit, wherein the optical coupling unit is configured to generate an evanescent field from coherent light (L) having a predetermined wavelength on a first surface of the evanescent illuminator, wherein the first surface of the evanescent illuminator comprises a template nanopattern (5), wherein the template nanopattern comprises a coherent arrangement of a plurality of predetermined lines, and wherein membrane recognition elements having a binding structure for an artificial transmembrane protein of the cell, vesicle, or cell component or vesicle component (8) are arranged along the predetermined lines, wherein the membrane recognition elements are configured to bind to the human The invention relates to a binding structure of a transmembrane protein to form a transmembrane nanopattern in a cell, a vesicle or the cell component or vesicle component (8) based on the template nanopattern (5) of the evanescent illuminator, so that the light of the evanescent field is scattered by the cell, the vesicle or the cell component or vesicle component (8) bound to the membrane recognition element, and wherein the predetermined line is arranged so that the light scattered by the cell, the vesicle or the cell component or vesicle component (8) bound to the membrane recognition element undergoes coherent interference at a predetermined detection site (7), wherein the difference in optical path length is an integer multiple of the predetermined wavelength of the coherent light (L).

19. The kit according to claim 18, further comprising: c. Configuring a protein of interest for intracellular or intravesicular biomolecular interaction, wherein the protein of interest comprises a high-mass fraction.

20. The kit of claim 19, wherein the high-mass fraction is gold nanoparticles.

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