Journal: Nature Communications

Article Title: Nanopore sensing of protein and peptide conformation for point-of-care applications

doi: 10.1038/s41467-025-58509-8

Figure Lengend Snippet: a Common biomarkers (cells, nucleic acids, protein and peptides, other small molecules such as vitamins (nicotinamide, vitamin B3) found in different biofluids (examples displayed as drops: saliva (blue), sweat (white), blood/serum (red), urine (yellow)). b Schematic representation of a tertiary structure of a protein altered by interaction with a substrate, post-translational modification (PTMs) or mutations, and the role of conformation specificity in receptor or ligand binding. c Examples of methods for biomarker quantification (ELISA, Mass spectrometry) and conformational analysis of proteins (X-ray diffraction, NMR, CryoEM). d Schematic representation of a nanopore experiment. A single channel (aerolysin) is inserted in a lipid bilayer, and a difference in potential (driving force) is applied by two electrodes in the cis and trans compartments. Analytes with different amino acid enantiomers, sequence, and/or PTMs driven interact with the pore producing a characteristic drop in the current. Schematic representation of events showing the open pore current ( I 0 ), blockade current ( I b ) resulting in a blockade level (Δ I b ) over a dwell time (Tt), and σ (sigma) the standard variation of the event, characteristics of the electrical signal detecting the peptide. Extraction of the characteristic parameters for each signal provides information on analyte size, charge, shape, and volume. Created in BioRender. Ratinho, L. (2025) https://BioRender.com/m45s676 .

Article Snippet: Ed., Boersma et al. © 2012 WILEY_VCH Verlag GmbH & Co. KGaA, Weinheim . c Current trace showing the discrimination of two enkephalin peptides that differ by two amino acid enantiomers with a FraC nanopore in 1 M KCl, pH 3.8.

Techniques: Modification, Ligand Binding Assay, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Sequencing, Extraction

Journal: Nature Communications

Article Title: Nanopore sensing of protein and peptide conformation for point-of-care applications

doi: 10.1038/s41467-025-58509-8

Figure Lengend Snippet: a Schematic representations of current blockades that related to relative ratio of analyte size to pore diameter in direct and indirect detection methods. b Direct discrimination of Aβ peptides that differ by one amino acid with unique blockade characteristics using an Aerolysin nanopore in 1 M KCl, 10 mM Tris, 1 mM EDTA, pH 8. Figure adapted from Angew. chem., Xin et al. © 2022 Wiley_VCH GmbH c Direct detection and characterization of a several proteins with different shapes and sizes: Bovine Thrombin (BT: 35 kDa), Streptavidin (SA: 53 kDa), Hemoglobin (HG: 64 kDa) and C-reactive protein (CPR: 125 kDa) with a YaxAB nanopore in 150 mM NaCl, 150 mM Tris, pH 7.5. Figure adapted from ACS nano, Straathof et al. . d Principle of the detection of the viral envelope protein MPVX A29 bound to an antibody and functionalized aptamer with a nanopipette. Figure adapted from Nano letters, Cai et al. . e Schematic representation of a tFhuA nanopore engineered to attach an Adnectin 1 monobody binding EGFR in 5% FBS. Current traces and histogram of the blockade level of the EGFR-pore interaction in 5% FBS. Figure adapted from Nature Com., Ahmad et al. . Created in BioRender. Ratinho, L. (2025) https://BioRender.com/s75n220 .

Article Snippet: Ed., Boersma et al. © 2012 WILEY_VCH Verlag GmbH & Co. KGaA, Weinheim . c Current trace showing the discrimination of two enkephalin peptides that differ by two amino acid enantiomers with a FraC nanopore in 1 M KCl, pH 3.8.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Nanopore sensing of protein and peptide conformation for point-of-care applications

doi: 10.1038/s41467-025-58509-8

Figure Lengend Snippet: a A protein (P) is trapped inside a pore by molecular trapping. Partner binding (B), results in a complex with a different conformation. The change in pore volume occupied by the complex or a change in conformation can be detected by a shift of blockade level (P + B) in real time. b 160 Current trace, in real-time, of the capture of an engineered E. coli dihydrofolate reductase (DHFR tag ) complexed with methotrexate (MTX), free or bound to NADPH or NADP+ inside a ClyA nanopore in 150 mM NaCl, 15 mM Tris HCl, pH 7,5. Reprinted (adapted) with permission from Soskine, M., et al. Single-Molecule Analyte Recognition with ClyA Nanopores Equipped with Internal Protein Adaptors. J. Am. Chem. Soc . 137 , 5793–5797 (2015). Copyright 2015, American Chemical Society . c) Current trace of the capture of SBD1 (substrate binding domain 1) and GBP (glucose binding protein) in a ClyA nanopore unbound, and bound to respectively, Asparagine and Glucose in a 100-fold dilution of sweat in 150 mM NaCl, 15 mM Tris HCl, pH 7,5. Figure adapted from Nature Com. Galenkamp et al. . Created in BioRender. Ratinho, L. (2025) https://BioRender.com/v49u400 .

Article Snippet: Ed., Boersma et al. © 2012 WILEY_VCH Verlag GmbH & Co. KGaA, Weinheim . c Current trace showing the discrimination of two enkephalin peptides that differ by two amino acid enantiomers with a FraC nanopore in 1 M KCl, pH 3.8.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Nanopore sensing of protein and peptide conformation for point-of-care applications

doi: 10.1038/s41467-025-58509-8

Figure Lengend Snippet: a Schematic representation of the characterization of peptides with different types of amino acid mutations such as deletion, addition, or substitution by a nanopore. b Identification and discrimination of sequential C-terminal deletion of angiotensin by the enzyme ACE and/or ACE2 (angiotensin-converting enzyme) with an aerolysin nanopore, as defined by the distribution of dwell time (ms) against the mean blockade level ( I / I 0 ) in 1 M KCl, 10 mM Tris, 1 mM EDTA, pH 8. Figure adapted from Nature Chem., Jiang et al. . c Current traces showing the discrimination between Endothelin 1 and 2 in a mixture with a FraC nanopore in 1 M KCl, 0,1 M citric acid, 180 mM Tris, pH 4,5. Figure adapted from Nature Com., Huang et al. . d Discrimination of Bradykinin (BK) and Des-Arginine bradykinin (Des-Arg BK) and identification of different conformations in serum (2%) identified with Principal Component Analysis and machine learning with an aerolysin nanopore in 4 M KCl, 25 mM Tris, pH 7,5. Reprinted (adapted) with permission from Greive, et al. Identification of Conformational Variants for Bradykinin Biomarker Peptides from a Biofluid Using a Nanopore and Machine Learning. ACS Nano 18 , 539–550 (2024). Copyright 2024, American Chemical Society . Created in BioRender. Ratinho, L. (2025) https://BioRender.com/t96d726 .

Article Snippet: Ed., Boersma et al. © 2012 WILEY_VCH Verlag GmbH & Co. KGaA, Weinheim . c Current trace showing the discrimination of two enkephalin peptides that differ by two amino acid enantiomers with a FraC nanopore in 1 M KCl, pH 3.8.

Techniques: Biomarker Discovery

Journal: Nature Communications

Article Title: Nanopore sensing of protein and peptide conformation for point-of-care applications

doi: 10.1038/s41467-025-58509-8

Figure Lengend Snippet: a Left, sketch representing the most common PTMs. Right, the overall principle of PTM identification using nanopore technology. b1 Characterization and discrimination of Tau peptides according to different phosphorylation patterns in an engineered aerolysin nanopore (T232K/K238Q) in 1 M KCl, 10 mM Tris, 1 mM EDTA, pH8. Figure adapted from Small Methods, Li et al. © 2020 WILEY_VCH Verlag GmbH & Co. KGaA, Weinheim b2 Current traces showing the identification of different post-translational modifications of α-synuclein peptides with an aerolysin nanopore in 1 M KCl, 10 mM Tris, 1 mM EDTA, pH 7,4. Figure adapted from ACS nano, Cao et al. . c Discrimination between unphosphorylated and phosphorylated biological peptides (FPA and FPA-P) with an Aerolysin nanopore. Different blockade levels are observed for each peptide, enabling their identification in the mixture. In addition, due to the high sensitivity of the aerolysin nanopore, two conformations were identified for the phosphorylated peptide. Figure adapted from ACS Central Science, Stierlen et al. . Created in BioRender. Ratinho, L. (2025) https://BioRender.com/j25c673 .

Article Snippet: Ed., Boersma et al. © 2012 WILEY_VCH Verlag GmbH & Co. KGaA, Weinheim . c Current trace showing the discrimination of two enkephalin peptides that differ by two amino acid enantiomers with a FraC nanopore in 1 M KCl, pH 3.8.

Techniques: Phospho-proteomics

Journal: Nature Communications

Article Title: Nanopore sensing of protein and peptide conformation for point-of-care applications

doi: 10.1038/s41467-025-58509-8

Figure Lengend Snippet: a Schematic representation of different forms amino acids as potentially studied by nanopore: structural isomers, enantiomers, rotamers, conformers, and cis/trans isomers. b Current traces and Current blockade histograms showing the discrimination of dextrogyre and levogyre tyrosine (Tyr) and Phenylalanine (Phe) amino acids with an engineered α-hemolysin nanopore in 1 M KCl, 10 mM MOPS, pH 7.5. Figure adapted from Angew. Chem. Int. Ed., Boersma et al. © 2012 WILEY_VCH Verlag GmbH & Co. KGaA, Weinheim . c Current trace showing the discrimination of two enkephalin peptides that differ by two amino acid enantiomers with a FraC nanopore in 1 M KCl, pH 3.8. Figure adapted from JACS, Versloot et al. . d Discrimination of Aβ 1-7 peptides that differ by one amino acid enantiomer with an OmpF nanopore . e Current traces of L-Arginine and D-Arginine Vasopressin (L- and D-AVP) and the resulting histogram of blockade levels showing the characterization and discrimination of the two peptides in an equimolar mixture with an aerolysin nanopore in 4 M KCl, 25 mM Tris, pH 7.5. The histogram of the blockade levels shows multiple populations representative of a change in conformation or orientation of the peptides—figure adapted from ACS Cent. Sci.; Ratinho et al. . Created in BioRender. Ratinho, L. (2025) https://BioRender.com/k79m010 .

Article Snippet: Ed., Boersma et al. © 2012 WILEY_VCH Verlag GmbH & Co. KGaA, Weinheim . c Current trace showing the discrimination of two enkephalin peptides that differ by two amino acid enantiomers with a FraC nanopore in 1 M KCl, pH 3.8.

Techniques: