The Potential of Nanopore Technologies in Peptide and Protein Sensing for Biomarker Detection
Abstract
1. Introduction
2. Biomarkers: Identification and Detection Methods
3. Clinical Utility and Translational Potential
4. Nanopore Setups and Applications for Protein and Peptide Detection
4.1. Types of Nanopores and Their Limitations
Nanopore | Geometry and Size | Distinct Functional Characteristics | Refs. | |
---|---|---|---|---|
Biological | α-Hemolysin | Mushroom-shaped, transmembrane heptamer. Outer protein vestibule and β-barrel in membrane Vestibule ~3.6 nm diameter and ~5 nm length; β-channel ~2.6 nm diameter and ~5 nm length Constriction zone~1.4 nm diameter | Robust and stable channel in lipid membranes Hydrogen bonds with polar residues inside the channel | [77,78,79] |
MspA (Mycobacterium smegmatis porin A) | Conical shape (funnel shape)—symmetric octamer Diameter of ∼1.2 nm and a length of ∼0.6 nm | Short and very narrow channel (~2–4 residues occupy the narrowest area) leading to higher translocation rates and, potentially, higher resolution (fewer amino acids simultaneously in the pore) | [79,80] | |
Aerolysin | β-barrel Diameter ~1.0 nm, length ~10 nm | The lumen of the pore contains a high density of charged residues, which confer a pronounced electrostatic character to the interior. Very long and narrow pore | [81,82] | |
ClyA (Cytolysin A) | Barrel-shaped Diameter of cis vestibulum ~6 nm; diameter of trans vestibulum ~3 nm; total length ~13 nm | Strong electrostatic interactions with positively charged molecules; hydrogen bonds with polar residues. Large lumen that accepts native folded proteins | [83,84] | |
OmpG (Outer membrane protein G) | Monomer β-barrel with 14 chains Diameter ~2–2.9 nm; length ~3 nm | Naturally exhibits gating behavior due to the flexibility of the L6 loop, which can spontaneously block or unblock the channel | [85] | |
Solid-state | SiN | Cylindrical, thickness is flexible and can be reduced to < 5 nm Pore diameter can be controlled in manufacturing | Robust and chemically inert solid nanopore, compatible with CMOS processes; provides durability and precise pore size control | [79,86] |
Graphene | Two-dimensional sheet of atomic carbon (atomic thickness ~0.34 nm) Diameter~2 nm to 25 nm | Very ‘sticky’ hydrophobic surface, which can adsorb biopolymers and slow translocation | [87] | |
MoS2 (Molybdenum disulphide) | Membrane—monolayer of MoS2 ~0.7 nm thick Pore size has been observed to range from 1 to 2 nm | Detect molecular translocation via ionic signal (as a biological nanopore) Electrically amplifier response (via electronic effects, as a sensor) | [88] | |
Hybrid | α-Hemolysin in solid pore | α-hemolysin protein pore (heptamer ~10 nm total diameter) inserted into a larger solid membrane nanopore (e.g., ~20 nm in SiN) | The biological pore controls interactions with translocated molecules, but the system becomes more mechanically stable due to the solid support | [89] |
Solid nanopores (SiN) functionalized with biomolecular ligand | The basic geometry is still that of the solid pore. The functionalized molecule may reduce the effective pore size (e.g., a 10 nm diameter SiN pore with a DNA aptamer attached may have a smaller functional area) | Specific interactions are brought about by the attached biomolecular ligand, in addition to non-specific electrostatic interactions with the solid pore walls | [90] | |
DNA Origami Nanopores | Customizable 2D and 3D structures Diameter between 2 and 20 nm | Charge distribution can be modified by nanopore design | [91,92,93,94] |
4.2. Technological Considerations and Control Strategies for Protein Translocation
4.2.1. Challenges and Emerging Strategies in Protein Translocation and Unfolding
4.2.2. Analytical Strategies in Biological and Solid-State Nanopores
Protein Analysis with Biological Nanopores
Protein Analysis with Solid-State Nanopore
Protein Analysis with Hybrid Nanopores
4.2.3. Strategies for Nanopore Engineering and Optimization
4.3. Analysis of Protein–Protein and Protein–Drug Interactions
5. Signal Analysis and Bioinformatics Approach
6. Comparative Advantages over Traditional Methods
7. Challenges and Opportunities
8. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ONT | Oxford Nanopore Technologies |
MRI | Magnetic Resonance Imaging |
CA-125 | Cancer Antigen 125 |
ELISA | Enzyme-Linked Immunosorbent Assay |
SDS | Sodium Dodecyl Sulfate |
HD | Huntington’s Disease |
CRP | C-Reactive Protein |
PSA | Prostate-Specific Antigen |
NMR | Nuclear Magnetic Resonance |
RF | Random Forest |
NNet | Neural Network |
MspA | Mycobacterium smegmatis porin A |
ATP | Adenosine Triphosphate |
α-HL | Alpha-hemolysin |
ClyA | Cytolysin A |
OmpG | Outer membrane protein G |
DNA | Deoxyribonucleic acid |
CR | Congo Red |
LRG-1 | Leucine-Rich alpha-2-Glycoprotein 1 |
NNet | Neural Networks |
PTMs | Post-Translational Modifications |
CART | Classification And Regression Trees |
RNNs | Recurrent Neural Networks |
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Method | Principle of the Method | Advantages | Disadvantages | Refs. |
---|---|---|---|---|
X-ray crystallography | Using a mobile and stationary phase system separate molecules according to their physico-chemical properties | Allows precise protein separations | The process can be complex and time-consuming | [30,31,32,33] |
Colorimetric tests | The change in color of a solution when a specific molecule is present, measured using a spectrophotometer | Rapid and easy to use | Requires specific reagents for each target molecule | [34,35] |
ELISA (Enzyme-Linked Immunosorbent Assay) and Western blot | Specific recognition between an antibody and an antigen, followed by detection of the signal generated by an enzyme bound to a secondary antibody | Speed, ease of use, and high sensitivity | Need for specific antibodies | [36,37,38] |
Mass spectrometry | Involves analysis of the mass of protein molecules | Precise identification, analysis of post-translational modifications | Complex interpretation, limitations related to protein size | [39,40] |
Electrophoresis | Separation of proteins according to their electrical charge and size | Efficient separation, versatility | Lower resolution, depending on the references used for sizing | [41,42] |
Nuclear magnetic resonance (NMR) | Uses the magnetic properties of atomic nuclei to obtain structural and dynamic information about molecules | Atomic-level resolution, ability to study dynamics | Low sensitivity, data interpretation complexity, size limitations | [43,44] |
Nanopores | Proteins can change the electrical conductivity of the system, which can be measured and interpreted | Single-molecule analysis, real-time monitoring | Improving sensitivity, complex interpretation | [45,46] |
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Șoldănescu, I.; Lobiuc, A.; Caliman-Sturdza, O.A.; Covasa, M.; Mangul, S.; Dimian, M. The Potential of Nanopore Technologies in Peptide and Protein Sensing for Biomarker Detection. Biosensors 2025, 15, 540. https://doi.org/10.3390/bios15080540
Șoldănescu I, Lobiuc A, Caliman-Sturdza OA, Covasa M, Mangul S, Dimian M. The Potential of Nanopore Technologies in Peptide and Protein Sensing for Biomarker Detection. Biosensors. 2025; 15(8):540. https://doi.org/10.3390/bios15080540
Chicago/Turabian StyleȘoldănescu, Iuliana, Andrei Lobiuc, Olga Adriana Caliman-Sturdza, Mihai Covasa, Serghei Mangul, and Mihai Dimian. 2025. "The Potential of Nanopore Technologies in Peptide and Protein Sensing for Biomarker Detection" Biosensors 15, no. 8: 540. https://doi.org/10.3390/bios15080540
APA StyleȘoldănescu, I., Lobiuc, A., Caliman-Sturdza, O. A., Covasa, M., Mangul, S., & Dimian, M. (2025). The Potential of Nanopore Technologies in Peptide and Protein Sensing for Biomarker Detection. Biosensors, 15(8), 540. https://doi.org/10.3390/bios15080540