1 3
Topics in Current Chemistry (2020) 378:12
RAFT polymerization. The encapsulated enzyme showed high enzymatic activity
and stability in a mixture of toluene/t-BuOH, in which the free enzyme undergoes
denaturation [171].
5.4 Chemical Conjugation
Chemical bioconjugation allows the immobilization of biomolecules at the surfaces
of nanoparticles by forming covalent bonds. This method guarantees stable anchoring between the support and the biomolecule. Several works report that supramolecular conjugation, encapsulation or adsorption, are simpler and more economical
methods to immobilize enzymes onto NPs, but show long-term loss of activity since
the enzyme-support binding is weak compared to covalent methods [172, 173].
Covalent immobilization ensures that the number of immobilized molecules
remains constant after conjugation with a small probability of losing the biomolecule during operation of the nanobioconjugate. For conjugates obtained by covalent immobilization of enzymes, greater resistance to deactivation due to the effects
of temperature, organic solvents or pH has been reported [174, 175]. Although the
formation of covalent bonds can affect the active conformation of enzymes, several
reports describe the covalent coupling of these biocatalysts onto metal oxide NPs
as a method for ensuring the increase in the enzymatic activity and stability of the
immobilized biomolecule [176, 177].
The coupling protocol is based on reactions involving amino (–NH 2 ) [178], carboxyl (–COOH) [179], hydroxyl (–OH) [180], azide (N 3
−
) [181] or thiols (–SH)
[182]—functional groups present on the surface of the support as well as in the side
chains of the biomolecule. Of the 20 amino acids, the ones used most often for the
formation of covalent bonds with the support are those that present ionizable groups
in their side chain of biomolecules, such as lysine [183], cysteine [184], tyrosine
[185], histidine [186], arginine [187], and aspartic [188] and glutamic acids [189].
One of the most widely used protocols for the chemical immobilization is known
as the Steglich reaction, or the carbodiimide method, which is based on peptide coupling between a carboxyl group and a primary amine [107, 190]. During the chemical reaction, the carboxylic acid is activated in the presence of a coupling agent
such as 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), forming an activated
ester. Frequently, additives such as N-hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (NHS) are added to increase yields and decrease side reactions. Finally,
the amine reacts with the activated ester to form an amide, with excellent yields
(> 90%) (Scheme 1) [191]. The described methodology should be optimized carefully to avoid any irreversible damage to the biological function of the immobilized
component. If the covalent immobilization is properly designed, the spatial disposition of the biomolecule can be controlled [192].
Alternatively, it is possible to use the high reactivity and selectivity of the isothiocyanate group towards primary amines to form thioureas [193]. This protocol has been
used in biochemistry for the conjugation of antibodies [194], as well as in the labeling of nanoparticles coated with amino groups using fluorescent isothiocyanates for
109
Reprinted from the journal
Topics in Current Chemistry (2020) 378:12
RAFT polymerization. The encapsulated enzyme showed high enzymatic activity
and stability in a mixture of toluene/t-BuOH, in which the free enzyme undergoes
denaturation [171].
5.4 Chemical Conjugation
Chemical bioconjugation allows the immobilization of biomolecules at the surfaces
of nanoparticles by forming covalent bonds. This method guarantees stable anchoring between the support and the biomolecule. Several works report that supramolecular conjugation, encapsulation or adsorption, are simpler and more economical
methods to immobilize enzymes onto NPs, but show long-term loss of activity since
the enzyme-support binding is weak compared to covalent methods [172, 173].
Covalent immobilization ensures that the number of immobilized molecules
remains constant after conjugation with a small probability of losing the biomolecule during operation of the nanobioconjugate. For conjugates obtained by covalent immobilization of enzymes, greater resistance to deactivation due to the effects
of temperature, organic solvents or pH has been reported [174, 175]. Although the
formation of covalent bonds can affect the active conformation of enzymes, several
reports describe the covalent coupling of these biocatalysts onto metal oxide NPs
as a method for ensuring the increase in the enzymatic activity and stability of the
immobilized biomolecule [176, 177].
The coupling protocol is based on reactions involving amino (–NH 2 ) [178], carboxyl (–COOH) [179], hydroxyl (–OH) [180], azide (N 3
−
) [181] or thiols (–SH)
[182]—functional groups present on the surface of the support as well as in the side
chains of the biomolecule. Of the 20 amino acids, the ones used most often for the
formation of covalent bonds with the support are those that present ionizable groups
in their side chain of biomolecules, such as lysine [183], cysteine [184], tyrosine
[185], histidine [186], arginine [187], and aspartic [188] and glutamic acids [189].
One of the most widely used protocols for the chemical immobilization is known
as the Steglich reaction, or the carbodiimide method, which is based on peptide coupling between a carboxyl group and a primary amine [107, 190]. During the chemical reaction, the carboxylic acid is activated in the presence of a coupling agent
such as 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), forming an activated
ester. Frequently, additives such as N-hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (NHS) are added to increase yields and decrease side reactions. Finally,
the amine reacts with the activated ester to form an amide, with excellent yields
(> 90%) (Scheme 1) [191]. The described methodology should be optimized carefully to avoid any irreversible damage to the biological function of the immobilized
component. If the covalent immobilization is properly designed, the spatial disposition of the biomolecule can be controlled [192].
Alternatively, it is possible to use the high reactivity and selectivity of the isothiocyanate group towards primary amines to form thioureas [193]. This protocol has been
used in biochemistry for the conjugation of antibodies [194], as well as in the labeling of nanoparticles coated with amino groups using fluorescent isothiocyanates for
109
Reprinted from the journal