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WO2025176605A1 - Procédé permettant d'analyser les interactions entre biomolécules - Google Patents

Procédé permettant d'analyser les interactions entre biomolécules

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Publication number
WO2025176605A1
WO2025176605A1 PCT/EP2025/054203 EP2025054203W WO2025176605A1 WO 2025176605 A1 WO2025176605 A1 WO 2025176605A1 EP 2025054203 W EP2025054203 W EP 2025054203W WO 2025176605 A1 WO2025176605 A1 WO 2025176605A1
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WO
WIPO (PCT)
Prior art keywords
biomolecule
carrier
type
cells
biomolecules
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/054203
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English (en)
Inventor
Paolo Romele
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Hexagonfab Ltd
Original Assignee
Hexagonfab Ltd
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Filing date
Publication date
Application filed by Hexagonfab Ltd filed Critical Hexagonfab Ltd
Publication of WO2025176605A1 publication Critical patent/WO2025176605A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to the field of measuring molecular interactions between biomolecules.
  • it relates to the measurement of interactions between membrane proteins in a cell and further biomolecules.
  • the present invention relates to the use of such measurement methods in binding assays, affinity measurements, and kinetics measurements.
  • determining a binding state means to evaluate if a specific drug molecule, such as an antibody, binds to a specific membrane protein.
  • determining binding affinity and binding kinetics means to evaluate how quickly and strongly a specific drug molecule, such as an antibody, binds to a specific membrane protein and how quickly this binding is released again.
  • BLI Bio-Layer Interferometry
  • SPR Surface Plasmon Resonance
  • ELISA ELISA assays
  • a capture molecule e.g., antigen
  • the biosensor is then brought into contact with the sample (e.g., antibody), either by moving the biosensor into the sample or by flowing the sample over the biosensor.
  • the measurement is performed by means of interferometry, whereas SPR measures changes in the properties of a surface plasmon.
  • a capture molecule e.g., antigen
  • the sample e.g., antibody
  • an enzyme is bound to the sample, often by using a detection antibody.
  • the measurement is performed by analysing a colour change caused by a reaction between the enzyme and a substrate.
  • BLI and SPR are surface sensitive, i.e. , only interactions within a distance of around 200 nm from the surface can be measured.
  • ELISA does not suffer from the same distance limitations, but does not provide time resolved binding information.
  • ELISA methods have been developed, wherein the enzymesubstrate reaction is not measured by colorimetric or fluorescent detection but by electrochemical detection.
  • cell-based ELISA assays are known, where the capture molecule is not isolated, but entire cells are fixed in the wells of a mictrotiter plate.
  • Microfluidic cytometry is another commonly used technique for biological assays.
  • screening and analysis in small volumes of fluids containing cells is performed in microscale fluidic devices.
  • the specific fluorescent probe- labelled cells flow in suspension through a laser beam of the measuring device which measures light scattering and fluorescence. While being a useful tool for measuring multiple structural and functional parameters in cells, performance of time dependent binding assays, affinity measurements or kinetic measurements using microfluidic cytometry is complicated.
  • the cells to be investigated are contained in solution or suspension, which requires a labour-intensive and impractical repeated charging and discharging of sample and buffer solutions/suspensions into and out of the device.
  • SPR systems For measuring binding affinities and kinetics, most commonly a SPR system is used today. With a standard SPR method the behaviour of the surface plasmon is read out with a coherent light source.
  • SPR systems have the disadvantage that they are incompatible with entire cells due to limitations of the technology. In particular, SPR cannot be used for measurements using a whole cell because cells are too large for this surface sensitive technology, because cells move, and because cells absorb and release salts. This results in the occurrence of interfering signals and additionally, leads to the problem that liquid flow channels of the apparatus, which are very thin, are blocked by the cells. Therefore, SPR systems usually work with isolated molecules.
  • the present invention provides a method for analysing interactions between biomolecules, wherein the measurement can be performed without isolating molecules from a cell.
  • the method of the invention is described in the claims. Preferred embodiments are described in the dependent claims.
  • the inventive method is a method for analysing an interaction between at least one type of first biomolecule(s) and at least one type of second biomolecule(s), comprising the following steps:
  • step (S2) before or after step (S2), the following step (S21 ) can be performed.
  • step (S21 ) is performed before step (S2), i.e., in step (S2), a solution or dispersion containing at least one type of second biomolecule(s) linked to a fluorescent marker is provided.
  • Connecting the second biomolecule and the fluorescent marker can be performed by any suitable technique known to the skilled person, e.g., by conjugation chemistry.
  • Step (S21) can also be done in advance, e.g., several hours or days before providing the solution in step (S2).
  • step (S21) binding of a fluorescent marker can be performed later during step (S31 ).
  • Step (S3) Dipping the carrier from step (S1 ) into the solution from step (S2) or step (S21 ).
  • step (S31 ) can be performed after step (S3).
  • step (S21 ) or step (S31 ) is performed.
  • first and second biomolecule(s) are bound together, wherein the first biomolecules are contained on the surface of a cell, i.e. , within the membrane of a cell, and a fluorescent marker is bound to the second biomolecules.
  • Option 1 The binding between first and second biomolecules is investigated, i.e., how strong, how fast (k on ), or how long the first biomolecule binds to the second kind of biomolecule.
  • step (S4) is performed after step (S3)/(S31 ).
  • Option 2 The release of binding between first and second biomolecules is investigated, i.e., how quickly the binding is released (k O ff).
  • step (S5) is performed after step (S3)/(S31 ).
  • step (S4) is performed after step (S3)/(S31 ) and subsequently, step (S5) is performed.
  • Step (S5) comprises the following steps (S51 ), and (S52).
  • Step (S3), step (S4), and optionally step (S31), are repeated until a defined end condition is reached, and/or step (S51 ) and step (S52) are repeated until a defined end condition is reached.
  • step (S4) (Option 1), or after step (S5) (Option 2), or after steps (S4) and (S5) (Option 3) the following step (S6) is performed.
  • step (S6) can also be performed as immediate step whenever fluorescence measurements have been performed, e.g., between steps (S4) and (S5) in Option 3.
  • FIG .1 is a diagram showing the individual steps of a method according to the invention.
  • FIG. 2 is a sketch illustrating the performance of kinetics measurement with the inventive method as described in Example 1 below.
  • method steps (S3) and (S4) are repeated each three times, but this is only exemplary, and any suitable number of repetitions can be employed.
  • FIG. 3 illustrates a time-dependent signal resulting from the measurement shown in FIG. 2.
  • the obtained signal can be fitted to a binding model.
  • the method of the present invention allows to measure binding events between biomolecules, e.g., if a specific antibody binds to membrane proteins in a cell. Further, with the inventive method, binding affinity and binding kinetics of biomolecules can be measured, e.g., how quickly and strongly a specific antibody binds to membrane proteins in a cell.
  • the present invention further relates to the use of the inventive method in a binding assay, an affinity measurement method, or a kinetics measurement method.
  • Biomolecular binding events are commonly described mathematically by a binding or affinity constant Ka [M’ 1 ] and a dissociation constant Kd [M],
  • Ka [M’ 1 ] and Kd [M] The binding reaction is characterized by the on-rate constant k on or association rate (k a ) [M’ 1 s -1 ], and the unbinding reaction is characterized by the off-rate constant k O ff or dissociation rate (kd) [s’ 1 ], respectively.
  • the binding constant Ka and the dissociation constant Kd are defined as follows: where [R], [L] and [RL] represent the concentration of unbound free receptors R, the concentration of unbound free ligand L and the concentration of receptorligand complexes RL for the reaction R + L «- ⁇ RL.
  • step (S3) When investigating a biomolecule which is a potential drug molecule, it is important to know how fast/strong/long it binds to the target, usually a protein. Just as important is the determination of how quickly the binding is released.
  • step (S3) To perform corresponding kinetics and affinity measurements related to the formation of bonds, e.g., determination of k on , step (S3), step (S4), and optionally step (S31 ) are repeated until a defined end condition is reached.
  • An example for determining k on is shown in Example 1 and FIG. 2 and 3.
  • step (S51 ) and step (S52) are repeated until a defined end condition is reached.
  • any suitable end condition can be defined, but it is preferred that the defined end condition is reaching a steady-state condition or expiry of a predefined period of time.
  • a certain amount of second biomolecules is contained in solution.
  • the carrier with the attached cells is dipped into the solution with second biomolecules, the second biomolecules start binding to the first biomolecules comprised in the cells on the carrier.
  • an equilibrium condition is reached, such that no further increase in bound second biomolecules can be detected (steady-state as end condition).
  • the measurement can be repeated until a steadystate is reached, or it can be aborted beforehand if it takes too long (expiry of a defined period of time as end condition).
  • the time span between repetition of measurements in step (S4) or step (S52) is not particularly limited. However, typically the formation and release of a binding between biomolecules is a rather slow interaction, such that the measurement of about one data point per 20 seconds is sufficient.
  • a measurement is performed every 120, every 90, every 60, every 40, every 30, every 20, or every 10 seconds, preferably every 60, or every 20 seconds.
  • step (S51 ) When measuring the release of bonds (e.g., determination of k O ff) a pure buffer solution is used in step (S51 ), and during the measurement the signal becomes smaller. It possible to measure the release of bonds (e.g., determination of k O ff) after measuring the formation of bonds, (e.g., determination of k on ) (Option 3), or to measure the release of bonds directly, i.e. , without measuring the formation of bonds beforehand, (Option 2). To do this, the carrier is immersed in the solution in step (S3) until binding has occurred (without measurement), after which the release of bonds is measured. Usually, the measured values of fluorescence measurement performed in step (S4) or step (S52), i.e.
  • the fluorescent changes are proportional to the number of binding events between the at least one type of first biomolecule(s) and the at least one type of second biomolecule(s).
  • the interaction between the at least one type of second biomolecule(s) and the at least one type of first biomolecule(s) calculated in step (S6) is at least one selected from the group consisting of the number of binding events, the association rate (k a ), the dissociation rate (kd), and the binding affinity. Binding kinetics can be quantified.
  • Performing the fluorescence measurement in step (S4) and/or step (S52) may include obtaining a sensor signal from a light-detection sensor.
  • the fluorescence measurement may be an auto-fluorescence measurement or a stimulated fluorescence measurement.
  • a light source may be switched on.
  • the sample may be exposed with a certain light intensity and light dose (light intensity x time) to trigger fluorescence.
  • the acquired signals may each be scaled with the respective light dose of each iteration, to make them comparable.
  • An active control of the light exposure may be used, e.g., using a photodiode.
  • the fluorescence measurement may be based on a sensor signal of a lightdetection sensor such as a photodiode, a photomultiplier, or an avalanche photodetector.
  • a spectral filter may be used to exclude light at wavelengths different from the fluorescence wavelength.
  • the carrier - to which the cells and the first biomolecules and the second biomolecules and the fluorescent marker are attached - may be placed next to an optical detector including, e.g., a collecting lens, the spectral filter, and the sensor. Then, for stimulated fluorescence measurements, the stimulating light source is activated. The light source emits light in a spectral range offset from the fluorescence light emitted by the fluorescent marker. The fluorescent marker is exposed to the stimulating light and the fluorescence light is then picked up by the optical detector. For auto-fluorescence measurements, stimulating light is not required.
  • step (S1 ) the cells can be attached to the carrier by any suitable technique known to the skilled person.
  • step (S1 ) is performed by dipping the carrier into a solution comprising the cells, for a time t5 sufficient for the cells to form a bonding to the surface of the carrier, or by growing the cells onto the surface of the carrier.
  • the carrier may include a carrier surface to which the cells are attached.
  • the carrier surface is bio-functionalized to facilitate the attachment of cells.
  • bio-functionalizing the surface of the carrier such as forming self-assembled monolayers.
  • sulphur in an alkanethiol adheres to the carrier surface while the other end of the molecule can be functionalized with various chemical groups.
  • biomolecules can be directly adsorbed onto the surface, using the affinity between metal and certain functional chemical groups.
  • a layer-by-layer-assembly is possible where oppositely charged polyelectrolytes, proteins, or nanoparticles form a multi-layer structure.
  • biomolecules can be covalently bonded to the surface of carriers via bioconjugation.
  • a reactive group of the biomolecule can be used or a linker molecule can be relied upon that links the carrier surface and the biomolecules.
  • Another option is electrochemical grafting where certain organic molecules can be grafted onto the surface of the carrier using electrochemical process. Once a linker layer is established, additional biomolecules can be added to the carrier surface to facilitate adhesion of cells.
  • the carrier is biofunctionalized before performing step (S1 ).
  • a preferred method of biofunctionalizing the carrier is as follows: The surface of the carrier is made of a suitable material, such as gold.
  • the first step is to add a linker layer onto the surface, e.g., a suitable linker is a short polymer with a sulphur group on one end and a carboxylic acid on the other end. Attaching the linker layer onto the surface can be achieved with common techniques known to the skilled person. Once this linker layer is established it is possible to link additional biomolecules, often proteins, to the carboxylic acid group. There are a large number of proteins that adhere to cells, and which are commercially available. Such proteins will be linked to the carboxylic acid group.
  • the carrier surface is functionalized with a linker and proteins capable of being linked to the carboxylic acid groups of the linker and capable of adhering to the cell surface are brought into contact with the functionalized carrier surface to form a linkage between the proteins and the carboxylic acid groups of the linker.
  • the cells are attached to the carrier surface by bringing the cells into contact with the functionalized carrier surface under conditions allowing the proteins to adhere to the cells, such that the proteins bound to the linker adhere to the cells.
  • the cells comprising at least one type of first biomolecule(s) are contained in a well of a mictrotiter plate in suspension and the carrier (which has preferably been bio-functionalized in advance) is dipped into this well to attach the cells to the carrier surface.
  • attaching the cells to the carrier surface can be achieved by growing the cells onto the surface of the carrier. That means, the cells are grown directly on the carrier ahead of the experiment.
  • Step (S11 ) is performed, wherein the number of cells attached to the carrier surface is determined. This determination of surface coverage is important for standardization, as the signal strength depends on the number of cells.
  • Step (S11 ) is preferably performed by electrochemical impedance spectroscopy (EIS), changes in light adsorption induced by the added layer of cells on the carrier or image analysis of light microscopy images.
  • EIS electrochemical impedance spectroscopy
  • a further advantage of the present invention is the possibility to use the method in an easy set-up.
  • a common measurement system for performing fluorescent measurements of assays in wells of a multi-well mictrotiter plate can be employed.
  • a suitable measurement system is disclosed in DE 10 2024 101 875, which can be equipped with a fluorescent detector.
  • the solutions in step (S2), and/or step (S21), and/or step (S3), and/or step (S31 ), and/or step (S51 ) are contained in wells of a mictrotiter plate. More preferably, the solutions of all steps, i.e.
  • step (S2), step (S3), step (S21 ) or step (S31 ) and optionally step (S51 ) are contained in wells of a mictrotiter plate.
  • the carrier can be dipped, for instance by a robotic actuator, into the individual wells of a mictrotiter plate.
  • the method is performed with a computer-controlled robotic system, and the dipping into the wells is performed by a robotic actuator.
  • steps (S2) to (S4) or steps (S2) to (S5) can be repeated 1 to 100 times without replacing the solution(s) in step (S3), and/or step (S31 ) and/or step (S51 ).
  • the carrier can be regenerated and used for the next measurement.
  • the robotic actuator can, in particular, place the carrier in a measurement position next to an optical detector for a fluorescence measurement.
  • the measurement position may be defined by a carrier holder fixedly coupled to the optical detector, the carrier holder engaging the carrier at a well-defined pose with respect to the optical detector. This ensures that the fraction of the fluorescence light picked up by the optical detector is the same for multiple iterations of the fluorescence measurement and not affected by placement errors of the robotic actuator.
  • first and second biomolecules are bound together, wherein the first biomolecules are contained within the membrane of a cell and a fluorescent marker is bound to the second biomolecules.
  • Binding a fluorescent marker to the second biomolecules in step (S21 ) or step (S31 ) can be achieved by any suitable method known to the skilled person.
  • the fluorescent marker can be bound directly or via a linker to the at least one type of second biomolecule(s).
  • the fluorescent marker is bound to the at least one type of second biomolecule(s) via a linker, wherein the linker is capable of forming a bond to the at least one type of second biomolecule(s).
  • a preferred example for the linker is a secondary antibody (detection antibody).
  • Binding the fluorescent marker to the at least one type of second biomolecule(s) is preferably performed by dipping the carrier from step (S3) into a solution containing the linker tagged with the fluorescent marker. Preferably, dipping is performed for a time t4 sufficient for the linker to bind to the at least one type of second biomolecule(s).
  • Suitable fluorescent markers are known to the skilled person and commercially available. The skilled person knows how to select a suitable fluorescent marker compatible with the second biomolecules and the type of measurement system.
  • fluorescence markers are GFP (Green Fluorescent Protein), RFP (Red Fluorescent Protein), YFP (Yellow Fluorescent Protein), CFP (Cyan Fluorescent Protein), mCherry, EGFP (Enhanced Green Fluorescent Protein), BFP (Blue Fluorescent Protein), DsRed, mOrange, mPlum, mCerulean, Alexa Fluor series, DyLight Fluors, FITC (Fluorescein Isothiocyanate), TRITC (Tetramethylrhodamine Isothiocyanate), PE (Phycoerythrin), APC (Allophycocyanin), Atto dyes (e.g., Atto 488, Atto 532), Cy dyes (e.g., Cy3, Cy5).
  • GFP Green Fluorescent Protein
  • step (S3) is performed for a time t1 sufficient for at least part of the second biomolecules to be captured by at least part of the first biomolecules
  • step (S51 ) is performed for a time t3 sufficient for at least part of the second biomolecules to be released from at least part of the first biomolecules.
  • Preferred ranges for the duration of individual method steps are a range of 0.1 sec to 24 h for time t1 , preferably 1 sec to 1 h, more preferably 10 sec to 30 min, more preferably 10 sec to 5 min; and a range of 0.1 sec to 24 h for t4, preferably 10 sec to 12 h, more preferably 10 sec to 2h, more preferably 10 sec to 15 min.
  • t1 can be 10 sec
  • t4 can be 30 sec, see also the Examples below.
  • the duration of step (S51 ), time t3, is not particularly limited and depends on the specific types of first and second biomolecules. A skilled person knows how to determine a suitable duration t3 for dipping the carrier into a buffer solution to allow the release of second biomolecules.
  • An exemplarily range is from 0.1 sec to 2 h, preferably 10 sec to 30 min, more preferably 10 sec to 15 min.
  • the carrier in the individual wells can be dipped into a well containing a buffer solution for washing, as required.
  • the buffer solution is not particularly limited, and the skilled person knows how to choose an appropriate buffer solution for particular biomolecules.
  • the method of the present invention is suitable to probe any molecular interaction between biomolecules.
  • the first and second biomolecule is each a protein.
  • the first biomolecule is a surface membrane protein.
  • Exemplary surface membrane proteins are protein A or ionchannel proteins, such as GPCR (G protein-coupled receptor).
  • GPCR G protein-coupled receptor
  • An example for an interaction according to the present invention is an interaction between an antibody and a membrane protein.
  • the at least one type of second biomolecule(s) is an antibody, antigen binding region or fragment thereof
  • the at least one type of first biomolecule(s) is an antibody binding membrane protein.
  • Other interactions can also be measured, for example interactions between cells.
  • the at least one type of second biomolecule(s) contained in the solution or dispersion of step (S2) are contained on the surface of a cell.
  • the second biomolecule is a surface membrane protein. Performing a measurement for interactions between first and second biomolecules which are each contained on the surface of a cell, i.e. , within the membrane of a cell, is for instance valuable in the field of T-cell therapy, where the binding of T-cells to cancer cells needs to be evaluated.
  • first and second biomolecules that can be measured with the inventive method are interactions between: first biomolecule second biomolecule sugar group on a cell surface protein surface receptor on a cell surface RNA fragment membrane protein on a cell surface small molecule small molecule on a cell surface protein
  • first biomolecule second biomolecule sugar group on a cell surface protein surface receptor on a cell surface RNA fragment membrane protein on a cell surface small molecule small molecule on a cell surface protein
  • Her2 a surface membrane protein
  • the method according to the invention allows for an easy and accurate performance of binding assays, affinity measurements or kinetics measurements. Because the carrier is dipped into and removed from the several wells, no elaborate and/or slow charging and discharging of liquids as in microfluidic cytometry is required. A particularly easy measurement can be performed when the inventive method is performed with an automatic system, and the dipping into the wells is performed by a robotic actuator. A further advantage of the inventive method in comparison to microfluidic cytometry arises from the attachment of cells onto the carrier surface. Thus, after fluorescence measurement, the cells are not lost but still present on the carrier and can be subjected to further investigations. For instance, the condition of the cells can be investigated microscopically by staining.
  • the kind of carrier is not particularly limited and any suitable carrier, e.g., a glass plate or an acrylic plate, can be used.
  • the carrier can be chosen by the skilled person according to the specific experiment. For instance, if step (S7) is included in the method, the carrier should include an electrode to apply the electrical field. If step (S11 ) is included in the method and is performed by EIS, the carrier should include an electrode, in particular, an integrated electrode. If step (S11 ) is included in the method and is performed by light microscopy, i.e. , light microscopy image and image processing, or by light absorption measurement, the carrier does not need to include an electrode.
  • the carrier may include an electrode.
  • the electrode may be adjacent to the carrier surface to which the cells are attached.
  • the electrode may be separated by an insulating layer from the carrier surface and extend along the carrier surface. It would also be possible that the electrode forms the carrier surface to which the cells are attached.
  • a gold electrode may be used. Such gold electrode may be bio-functionalized.
  • the electrode By means of the electrode, it is possible to apply an electric field.
  • the electric field is present at the position of the cells and, more specifically, the fluorescent marker.
  • the carrier may include one or more counterelectrodes so that a well-defined direction of the electric field is achieved.
  • the inventive method further comprises a step (S7), which is performed simultaneously with step (S4) and/or step (S52).
  • step (S7) an electric field is applied to the electrode of the carrier. Applying an electrical field in step (S7) allows to quench fluorescence of impurities bound to the carrier surface.
  • impurities can also lead to fluorescent signals disturbing the signals of the fluorescent markers. Whereas the impurities are bound directly onto the carrier surface, the fluorescent markers are bound via the first and second biomolecules and thus, are further away from the carrier surface.
  • the fluorescence of the fluorescent markers bound to the second biomolecules is not or only weakly quenched by the electric field applied to the carrier, and the fluorescence of impurities bound directly to the carrier surface is quenched completely or to a higher degree.
  • the measurement accuracy can be improved.
  • a common measurement system for performing fluorescent measurements of assays in wells of a multi-well mictrotiter plate is used, wherein the wells are accessible for the fluorescent microscope.
  • Cells comprising a surface membrane protein as first biomolecule, such as breast cells comprising Her2 are attached to the surface of a carrier containing electrodes (step (S1 ) in FIG. 2).
  • the number of cells attached to the carrier surface is determined by electrochemical impedance spectroscopy (EIS) or any other suitable method (step (S11 ), not shown in FIG. 2).
  • the carrier is subjected to fluorescence measurement to measure the fluorescence of the fluorescent markers conjugated to the antibodies that are bound to the surface membrane proteins (step (S4) in FIG. 2).
  • a robotic actuator may engage the carrier and place the carrier in a carrier holder.
  • the carrier holder may be arranged next to an optical detector and optionally a light source.
  • a fluorescence signal may be measured for a suitable time depending on the measurement setup and the concentration.
  • a preferred range is 0.1 to 10 seconds, more preferably 0.2 to 5 seconds, more preferably 0.5 to 1 seconds.
  • an electric field may be applied to the carrier to quench fluorescence of impurities bound to the carrier surface (step (S7), not shown in FIG. 2).
  • a signal intensity p1 dependent on the number of antibodies bound to the surface membrane protein is obtained.
  • step (S3) The carrier is dipped again for 10 seconds into the well with the solution containing the antibody (repetition of step (S3)); and fluorescence measurement is performed (repetition of step (S4)). A signal intensity p2 is obtained. Then, the procedure is repeated again, and a signal intensity p3 is obtained.
  • quenching may be activated and deactivated to obtain two fluorescence signals, i.e., p1 a, p1 b, as well as p2a, p2b as well as p3a, p3b.
  • This may enable to further increase the accuracy of the measurement, because the noise I interference can be quantified by comparing the signal levels of each signal of the fluorescence signal pairs, e.g., by determining p1 a-p1 b.
  • the carrier in the individual wells can be dipped into a well containing a buffer solution for washing, as required.
  • S(t) signal intensity at time t t: total duration of dipping the carrier into the solution containing the antibody.
  • Example 2 is performed in the same way as Example 1. However, step (S31 ) is performed instead of providing a solution containing an antibody which is conjugated with a fluorescent marker (step (S21 )).
  • Cells comprising a surface membrane protein as first biomolecule are attached to the surface of a carrier comprising electrodes (step (S1 )).
  • the number of cells attached to the carrier surface is determined by electrochemical impedance spectroscopy (EIS) or another suited method (step (S11 )).
  • a solution containing an antibody such as Trastuzumab, not conjugated with a fluorescent marker, as second biomolecule is provided in a well of the mictrotiter plate (step (S2)).
  • the carrier is dipped into this solution for 10 seconds. During this time, some of the antibody molecules bind to the surface membrane proteins (step (S3)).
  • the carrier is dipped into a well containing a detection antibody (i.e., a linker) tagged with a fluorescent marker, such as Atto 532, for 30 seconds.
  • a detection antibody i.e., a linker
  • a fluorescent marker such as Atto 532

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Abstract

L'invention concerne un procédé permettant d'analyser les interactions entre des biomolécules. Le procédé est approprié pour mesurer des interactions de biomolécules à l'intérieur de la membrane d'une cellule et peut être utilisé avantageusement dans des dosages de liaison, des procédés de mesure d'affinité et des procédés de mesure de cinétique.
PCT/EP2025/054203 2024-02-23 2025-02-17 Procédé permettant d'analyser les interactions entre biomolécules Pending WO2025176605A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000073777A1 (fr) * 1999-05-28 2000-12-07 Bioarray Solutions, Llc Cytometrie de jeux ordonnes d'echantillons
DE102024101875A1 (de) 2023-09-01 2025-03-06 Hexagonfab Limited Eintauchsensor für elektrochemische messungen und verfahren zur herstellung eines eintauchsensors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000073777A1 (fr) * 1999-05-28 2000-12-07 Bioarray Solutions, Llc Cytometrie de jeux ordonnes d'echantillons
DE102024101875A1 (de) 2023-09-01 2025-03-06 Hexagonfab Limited Eintauchsensor für elektrochemische messungen und verfahren zur herstellung eines eintauchsensors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DANIIL D STUPIN ET AL: "Bio-Impedance Spectroscopy: Basics and Applications", ARXIV.ORG, CORNELL UNIVERSITY LIBRARY, 201 OLIN LIBRARY CORNELL UNIVERSITY ITHACA, NY 14853, 7 May 2020 (2020-05-07), XP081670118 *
SINA BONDZA ET AL: "Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells", FRONTIERS IN IMMUNOLOGY, vol. 8, 24 April 2017 (2017-04-24), XP055644152, DOI: 10.3389/fimmu.2017.00455 *

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