Reduction-responsive immobilised and protected enzymes

Abstract

We report a synthetic strategy to produce nano-immobilised and organosilica-shielded enzymes of which the biocatalytic activity is, by design, chemically enhanced under reductive conditions. The enzymes were immobilised onto silica nanoparticles through a reduction-responsive crosslinker and further shielded in an organosilica layer of controlled thickness. Under reducing conditions, disulphide bonds linking the protein to the carrier material were reduced, triggering enzyme activation. The organosilica shield prevents the enzymes from leaching from the nanobiocatalysts and preserves their integrity.

Fig. 1. The schematic figure illustrates the breaking of the crosslinker DSP and the subsequent release of the enzyme, which is shielded within the organosilica layer and activated upon redox stimuli triggering.

Fig. 2. Synthetic route to reduction-responsive immobilised and shielded enzymes. The main synthetic steps are as follows: (i) amino-modification using APTES, (ii) cross linker [dithiobis(succinimidyl propionate), DSP] reaction with amino functions introduced at the surface of the SNPs. The low concentration of SNPs and electrostatic repulsion of SNPs prevent particle cross-linking; (iii) protein surface anchoring via the reaction with N-hydroxysuccinimide-activated surface and organosilica shield production, and (iv) disulphide bond reduction (using DTT or glutathione) releasing the enzyme within the organosilica shield.

Fig. 3. SEM micrographs of SNPs (a) and SNP–DSP–βgal–OS after 80 min of OS layer growth reaction (b). Size distribution (c) and layer growth kinetics (d) measured on SEM micrographs. Every point is the average of at least 100 particles. All scale bars represent 200 nm. Standard error is calculated as where n is the number of measured particles.

Fig. 4. Enzymatic activity of SNP–DSP–βgal–OS with and without DTT treatment. Error bars represent standard deviation measured on triplicates.

Fig. 5. Kinetics of layer growth (a) and enzymatic activity of SNP–DSP–ASNase–AT with and without DTT treatment corresponding to increasing layer thickness (2.8, 4, and 6.5 nm) after 30, 45, and 60 min of reaction, respectively (b). Enzymatic activity of SNPs–DSP–ASNase–AT and SNPs–Glu–ASNase–AT with and without glutathione (GSH) treatment (c). Cytotoxicity of SNPs–DSP–ASNase–AT and SNPs–Glu–ASNase–AT on HepG2 cells (d). Error bars represent standard deviation measured on triplicates.