US-20260126425-A1 - NANOBODY-FUNCTIONALIZED BIOLOGICAL NANOPORES AND MEANS AND METHODS RELATED THERETO

Abstract

The invention relates to means and methods for analysis of analytes using nanopore-based sensors, for example, to methods, nanopore systems and devices for the stochastic detection of (label-free) analytes in complex samples, for example the specific detection of a protein biomarker in a bodily sample. Provided is a method for detecting the presence of at least one analyte in a sample using a nanopore system comprising a cis chamber comprising a first conductive liquid medium in liquid communication with a trans chamber comprising a second conductive liquid medium through a modified nanopore, comprising: (a) adding a sample to be analyzed for the presence of an analyte to the cis chamber; (b) optionally applying an electrical potential across the modified nanopore; (c) measuring ionic current passing through the modified nanopore, wherein said modified nanopore is a biological nanopore that is functionalized with a 5 to 50 kDa, preferably 10 to 40 kDa, recognition element (e.g., proteinaceous recognition element) R capable of specifically binding to the analyte.

Inventors

• Giovanni Maglia
• Xialin ZHANG
• Jørgen Kjems

Assignees

• RIJKSUNIVERSITEIT GRONINGEN
• AARHUS UNIVERSITET

Dates

Publication Date

20260507

Application Date

20250530

Priority Date

20221202

Claims (20)

1. 1 .- 138 . (canceled)
2. 139 . A method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluid chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a first side and a second side, wherein the nanopore is coupled to a protein recognition element, wherein the protein recognition element is configured to move between an internal region of the nanopore and an external region of the nanopore; and (b) contacting the protein recognition element with an analyte.
3. 140 . The method of claim 139 , wherein the protein recognition element is coupled to the nanopore via a linker.
4. 141 . The method of claim 139 , wherein the protein recognition element is between about 5 kilodaltons to about 50 kilodaltons
5. 142 . The method of claim 139 , wherein the protein recognition element comprises a nanobody, a Fab fragment, a single-chain variable fragment (scFv), an antibody, a monobody, an affimer, an affibody, an Adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.
6. 143 . The method of claim 139 , wherein the protein recognition element couples to the analyte.
7. 144 . The method of claim 143 , wherein the protein recognition element coupled to the analyte effects movement of the protein recognition element.
8. 145 . The method of claim 144 , wherein effecting the movement of the protein recognition element generates a change in (i) a frequency of the movement of the protein recognition element or (ii) a noise or a magnitude of a current of the nanopore system.
9. 146 . The method of claim 139 , wherein the analyte comprises a protein, a peptide, a small molecule, a protein assembly, a protein/DNA assembly, a protein/RNA assembly, a steroid, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a cell, a dendrimer, a polymer, or any combination thereof.
10. 147 . The method of claim 139 , wherein the protein recognition element is coupled to at least one subunit of the nanopore via a linker.
11. 148 . The method of claim 147 , wherein the at least one subunit of the nanopore comprises a monomer of a pore-forming protein.
12. 149 . The method of claim 148 , wherein the pore-forming protein comprises cytolysin A (ClyA), pleurotolysin (PlyAB), YaxAB, perforin-2, tripartite alpha-pore forming toxin, secretin, Helicobacter pylori OMC, SpoIIIAG, Gasdermin-A3, or any combination thereof.
13. 150 . The method of claim 148 , wherein the pore-forming protein comprises one or more mutations.
14. 151 . The method of claim 139 , wherein the nanopore is coupled to another protein recognition element.
15. 152 . The method of claim 139 , wherein the nanopore system further comprises a pair of electrodes configured to generate an electrical potential across the nanopore.
16. 153 . The method of claim 152 , wherein movement of the protein recognition element between an internal region of the nanopore and an external region of the nanopore effects a change in a current of the nanopore system.
17. 154 . A system comprising: (a) a fluid chamber; and (b) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into (1) a first side and (2) a second side, wherein the nanopore is coupled to a protein recognition element, wherein the protein recognition element is configured to move between an internal region of the nanopore and an external region of the nanopore.
18. 155 . The system of claim 154 , wherein the protein recognition element is coupled to the nanopore via a linker.
19. 156 . The system of claim 154 , wherein the protein recognition element is between about 5 kilodaltons to about 50 kilodaltons.
20. 157 . The system of claim 154 , wherein the protein recognition element comprises a nanobody, a Fab fragment, a single-chain variable fragment (scFv), an antibody, a monobody, an affimer, an affibody, an Adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

Description

INCORPORATION BY REFERENCE This application is a continuation of International Patent Application No. PCT/NL2023/050633, filed Dec. 1, 2023, which claims benefit of European Application No. EP22211193.2, filed Dec. 2, 2022, each of which is incorporated herein in their entirety and by this reference thereto. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Aug. 18, 2025, is named 64828-711_301_SL.xml, and is 4,804 bytes in size. BACKGROUND Determining analytes (e.g., target analytes) in a sample is an important aspect of scientific studies. The presence or absence of analytes in a sample can be important for clinical aspects. SUMMARY In an aspect, the present disclosure provides a method for detecting the presence of at least one target analyte in a sample using a nanopore system comprising a cis chamber comprising a first conductive liquid medium in liquid communication with a trans chamber comprising a second conductive liquid medium through a modified nanopore, comprising (a) adding a sample to be analyzed for the presence of a target analyte to the cis chamber, (b) optionally applying an electrical potential across the modified nanopore, and (c) measuring ionic current passing through the modified nanopore, wherein said modified nanopore is a biological nanopore that is functionalized with a 5 to 50 kDa, for example 10 to 40 kDa, proteinaceous recognition element R capable of specifically binding to the target analyte and wherein R dynamically moves in and out of the nanopore to provoke transient current blockage events, and wherein binding of R to the target analyte modulates its dynamic movement, thereby inducing a change in the frequency and/or magnitude of the current blockage events, and wherein the change in the frequency and/or magnitude of current blockage events indicates the presence of the target analyte in the sample. In some embodiments, the modified nanopore is an oligomeric assembly comprising or consisting of monomers of the general formula N-L-R, wherein N is a monomer of a pore-forming toxin having a largest internal diameter (e.g., internal lumen diameter) of 5 nm to 20 nm, and L is a flexible linker attached to the cis entrance of the pore. In some embodiments, binding of R to the target analyte increases the time of R staying outside of the pore, thereby decreasing the frequency and/or magnitude of the current blockage events. In some embodiments of any one of the preceding embodiments, the biological nanopore is functionalized with at least two different proteinaceous recognition elements R′ and R″, for example wherein R′ and R″ bind to distinct sites of the target analyte. In some embodiments of any one of the preceding embodiments, the target analyte is a protein, protein assembly, protein/DNA assembly, protein/RNA assembly, steroid, lipid, lipid membrane, lipid particle, bacterium, virus capsid, virus particle, cell, dendrimer, polymer, or any combination thereof, wherein the target analyte is a protein, when the protein is selected from the group consisting of a folded/native protein, a clinically relevant protein, biomarker, pathogenic protein, cell surface protein. In some embodiments, the target analyte is a protein, preferably selected from the group consisting of a folded/native protein, a clinically relevant protein, biomarker, pathogenic protein, or cell surface protein. In some embodiments of any one of the preceding embodiments, the sample is a complex sample comprising a mixture of proteins, wherein the sample comprises a clinical sample, such as a bodily fluid, such as whole blood, plasma, urine, feces, saliva, cerebrospinal fluid, breast milk and sputum. In an aspect, the present disclosure provides a modified proteinaceous nanopore having a minimal pore diameter of 5 nm that is functionalized via a flexible linker with a 5 to 50 kDa, preferably 10 to 40 kDa, proteinaceous recognition element R that is specifically reactive with a target analyte, preferably a target protein. In preferred embodiments, R can move in and out of the pore to provoke a blocking current. In an aspect, the present disclosure provides a sensor system for protein analysis, comprising a fluid-filled compartment separated by a membrane into a first chamber and a second chamber, electrodes capable of applying a potential across the membrane, and at least one biological nanopore that is functionalized with a 5 to 50 kDa, preferably 10 to 40 kDa, proteinaceous recognition element R capable of specifically binding to a target analyte, and wherein R is positioned via a flexible linker atop of the nanopore to allow for moving in and out of the nanopore to provoke transient current blockage events. In an aspect, the present disclosure provides a nanopore sensor system comprising a cis chamber comprising a first conductive liquid medium in liq