Summary of modifications to biological, solid-state, hybrid, semi-synthetic and de novo nanopores. The purpose, challenges, and future potential of each class of modification are highlighted.

Pore type	Modification	Technological requirements fulfilled	Challenges and limitations	Future perspective	Ref.
Biological	-Introduction of charged or hydrophobic residues via mutagenesis	-Dwell time control	-Changes to pore stability and electro-osmotic flow	-Label-free, real-time detection of proteins and charged molecules -Fingerprinting and detection for proteins	23, 24, 26, 50 and 51
		-Signal differentiation	-Limited by viable mutations
			-Limited by membrane stability
	-Introduction of reactive handle via mutagenesis	-Dwell time control	-Limited by viable mutations and inherent pore instability	-Design of molecular machines, observations of reaction mechanisms, enhanced substrate detection	34, 35, 38–46 and 48
		-Signal differentiation
		-Multiple reads
		-Capture of molecules
		-Observation of reactive intermediates	-The use of unnatural amino acids is underexplored
	-Introduction of reactive handles via chemical modification	-Signal differentiation	-Limited to available solvent exposed reactive handles -Potential lack of selectivity	-Real-time observations of reaction mechanics at the single-molecule level	52 and 53
		-Observation of reactive intermediates
Solid state	-Surface functionalisation with reactive inorganic coating	-Signal differentiation	-Challenging to create a monodisperse layer and achieve consistent pore geometry	-High stability biomimetic channels -Applications for on-site, high-sensitivity measurements	12 and 16
		-Dwell time control
		-Channel selectivity
	-Surface functionalisation lipid bilayer	-Capture of molecules	-Relies on non-specific interactions between lipid and analyte	-Label-free, and real-time detection of native proteins	13 and 62
		-Signal differentiation
	-Surface functionalisation with DNA	-Capture of molecules	-Design of DNA sequences. -Attachment to solid matrixes	-High sensitivity detection of target DNA and RNA sequences for medical applications	56, 57, 61 and 62
		-Channel selectively
		-Dwell time control
Hybrid/semi-synthetic	-Combination of biological and solid-state pores	-Enhanced stability	-Challenging to fabricate	-Extremely high-stability pores with atomically precise geometries	4 and 58
		-Signal differentiation
	-Sequestering of membrane active sections of known proteins	-Capture of molecules	-Less-reliable stability compared to native pores -Difficult to predict structure	-Custom pores with tailored size and functionality	64
	-Combination of existing proteins into a bespoke channel	-Signal differentiation	-Similar issues to native pores with rapid translocation beyond the limit of resolution	-Utilisation of properties from multiple proteins for new sensing and detection applications	65 and 66
		-Capture of molecules
de novo	-Synthesis of synthetic transmembrane nanopore	-Signal differentiation	-Challenges in the design of viable channels -Difficult to predict structure	-Custom-designed bespoke channels	68
		-Capture of molecules