. 2022 Dec 15;2(1):12. doi: 10.1007/s43939-022-00033-3

Table 3.

Comparison of the covalent and non-covalent conjugation strategies in bioconjugation processes and possible interactions between biomolecules and nanoparticle’s surface in bioconjugation process [96, 176, 180–182]

Non-covalent interactions	Advantages	Disadvantages
Spontaneous absorption of biomolecules onto the surface of stabilised NPs	• Convenient and simple • Do not require any additional chemical components • Most useful in understanding physicochemical interactions at the nano–bio interface • Used to reduce non-specific reactions and aggregation • Affinity-based receptor-ligand systems provide strong bonds with high binding affinity to cells and resistance to: o pH; o Temperature variations; o Denaturants	• Random orientation of proteins reducing its activity • Necessity of a high concentration of biomolecules for the preparation of biomolecule–gold particle conjugates • Difficult control over biological response due to the bioconjugate is mainly formed by electrostatic attractions • The binding is greatly influenced by changes in: o pH; o Ionic strength e.g., increasing the electrolyte concentration shields the attractive electrostatic interaction causing the desorption of biomolecules • Possible displacement by other molecules on the NPs surface specially in complex biological samples • Washing buffers can remove loosely bound proteins and reduce non-specific interactions
Electrostatic interactions Positively charged groups in biomolecules are attracted by the negative charged surface of the metal NPs or vice versa
Hydrophobic interactions Attraction between hydrophobic parts of the biomolecule and the metal nanoparticle surface.
Chemisorption Donation of unshared electron pairs from free atoms of the biomolecule to the metal conducting electrons (e.g. sulphur-gold)
Adaptive molecules Affinity-based receptor-ligand systems such as streptavidin–biotin

Non-covalent interactions

Advantages

Disadvantages

Spontaneous absorption of biomolecules onto the surface of stabilised NPs

• Convenient and simple

• Do not require any additional chemical components

• Most useful in understanding physicochemical interactions at the nano–bio interface

• Used to reduce non-specific reactions and aggregation

• Affinity-based receptor-ligand systems provide strong bonds with high binding affinity to cells and resistance to:

o pH;

o Temperature variations;

o Denaturants

• Random orientation of proteins reducing its activity

• Necessity of a high concentration of biomolecules for the preparation of biomolecule–gold particle conjugates

• Difficult control over biological response due to the bioconjugate is mainly formed by electrostatic attractions

• The binding is greatly influenced by changes in:

o pH;

o Ionic strength

e.g., increasing the electrolyte concentration shields the attractive electrostatic interaction causing the desorption of biomolecules

• Possible displacement by other molecules on the NPs surface specially in complex biological samples

• Washing buffers can remove loosely bound proteins and reduce non-specific interactions

Electrostatic interactions

Positively charged groups in biomolecules are attracted by the negative charged surface of the metal NPs or vice versa

Hydrophobic interactions

Attraction between hydrophobic parts of the biomolecule and the metal nanoparticle surface.

Chemisorption

Donation of unshared electron pairs from free atoms of the biomolecule to the metal conducting electrons (e.g. sulphur-gold)

Adaptive molecules

Affinity-based receptor-ligand systems such as streptavidin–biotin

Covalent interactions	Advantages	Disadvantages
Chemical bond between biomolecules and the surface of NPs	• Thermally stable (desirable in applications that require thermo cycling) • Allows 10–40% more protein via covalent • Prevents elution of bound protein increasing the stability • Correct spatial biomolecule orientation can be difficult via physical adsorption, whilst covalent attachment can orient the molecule properly, yielding increased activity and lower reagent consumption	• Effectiveness limitations when release is required as in drug release systems • Attachment of highly active antibodies can be impaired due to pH requirement for successful cross-linking reaction • Depending on the strategy, covalent binding can originate random orientation of proteins reducing its biomolecule activity • Covalent attachment can force the protein to have unfavourable interactions with the NP surface coating ligand, causing unfolding
Conjugation chemistry Exploiting functional groups on both particles and biomolecules such as bifunctional linkers of mediator linkers
Maleimide coupling Diels–Alder cycloadditions with dienes; 1,3-dipolar cycloadditions with nitrones and azides; Michael-type additions with thiols and amines
Click chemistry Refers to a family of reactions that are modular, stereospecific, and high yielding

Covalent interactions

Advantages

Disadvantages

Chemical bond between biomolecules and the surface of NPs

• Thermally stable (desirable in applications that require thermo cycling)

• Allows 10–40% more protein via covalent

• Prevents elution of bound protein increasing the stability

• Correct spatial biomolecule orientation can be difficult via physical adsorption, whilst covalent attachment can orient the molecule properly, yielding increased activity and lower reagent consumption

• Effectiveness limitations when release is required as in drug release systems

• Attachment of highly active antibodies can be impaired due to pH requirement for successful cross-linking reaction

• Depending on the strategy, covalent binding can originate random orientation of proteins reducing its biomolecule activity

• Covalent attachment can force the protein to have unfavourable interactions with the NP surface coating ligand, causing unfolding

Conjugation chemistry

Exploiting functional groups on both particles and biomolecules such as bifunctional linkers of mediator linkers

Maleimide coupling

Diels–Alder cycloadditions with dienes; 1,3-dipolar cycloadditions with nitrones and azides; Michael-type additions with thiols and amines

Click chemistry

Refers to a family of reactions that are modular, stereospecific, and high yielding