Confocal microscopy study of polymer microcapsules for enzyme immobilisation in paper substrates

Abstract

The goal of this research is to develop the technology platform required for the production of bioactive paper based on enzymes as bioactive agents. The immobilization platform described here is based on microencapsulation, which consists in the entrapment of biomolecules in the core of hollow spheres made by a semipermeable membrane. The capsules containing the enzymes can be either deposited on paper or mixed with paper pulp to prepare a bioactive paper. The activity of encapsulated laccase was compared with that of free enzyme using its reaction with the o-phenylenediamine (OPD) substrate. Confocal Laser Scanning Microscopy (CLSM) is used to study the location of protein in microcapsules and provides explanations for differences in activity of encapsulated laccase. The location of protein in microcapsules was determined using BSA modified with the fluorescent tag sulforhodamine. Polyethyleneimine microcapsules were modified with fluorescein isothiocyanate allowing the simultaneous identification of capsule walls and of encapsulated proteins. From CLSM analysis, proteins were found to favor the wall of the capsules because of strong ionic attraction with the charged polymer. BSA was found to some extent in the core of the capsules and encapsulation of higher loadings increased the proportion of core proteins. We will also present our results on the incorporation of microcapsules in a paper substrate. CLSM was used in this section to determine the distribution and density of tagged microcapsules in the paper substrate. The response of immobilized laccase to a common substrate will also be described.

Characterisation and applications of microcapsules obtained by interfacial polycondensation

“…Bioactive papers prepared by the immobilisation of PEI microcapsules presented a higher retention of the enzyme on the paper sheets due to the size of the capsules and favourable electrostatic interactions between paper and microcapsules (Kouisni and Rochefort, 2009). The enzymatic activity was also maintained for a longer period of time in the paper compared with free laccase due to the protection from bacteria and proteases offered by the semipermeable membrane (Guerrero et al, 2011).…”

“…The physical processes of obtaining microencapsulation are the emulsion and encapsulator method for the same chemical process, namely interfacial polycondensation. PEI microcapsules were prepared by interfacial polycondensation due to the benefits of this method, as mentioned in the introduction (Hébert and Rochefort, 2008;Rochefort et al, 2008;Kouisni and Rochefort, 2009;Rochefort, 2010b, 2011b;Savolainen et al, 2011). Figure 6 illustrates the zeta potential of empty and laccase microcapsules prepared by the emulsion method at different pHs of the buffer solution.…”

Advancements in Assessments of Bio-Tissue Engineering and Viable Cell Delivery Matrices Using Bile Acid-Based Pharmacological Biotechnologies

“…This method of analysis is used in cell encapsulation research as it can illustrate the homogenous or inhomogeneous nature of polymer and/or co-polymer distribution within the microcapsules. In addition, such a technique can provide an indication of any polymer-excipient interactions, as well as potential permeability of the surrounding membrane [ 12 , 13 ]. In terms of traditional CLSM on live cells, the technique does have a significant disadvantage, it causes phototoxicity.…”

A colorimetric paper sensor for lactate assay using a cellulose-Binding recombinant enzyme

“…The adsorbed enzymes are also largely vulnerable to harsh external conditions, leading to concerns on sensor reliability. Other methods such as chemical cross-linking [12], thin film entrapment [13], and microencapsulation (capsules deposited or mixed with the pulp) [14] could improve the efficiency of enzyme immobilization. However, those are generally achieved mostly in a manner with very limited accessibility of the enzymes for assay reactions, and generally with big compromises in the complexity and cost of sensor preparation.…”