Performance of a Flow-Through Enzyme Reactor Prepared from a Silica Monolith and an α-Poly(D-Lysine)-Enzyme Conjugate.

Horseradish peroxidase (HRP) is covalently bound in aqueous solution to polycationic α-poly(D-lysine) chains of ≈1000 repeating units length, PDL, via a bis-aryl hydrazone bond (BAH). Under the experimental conditions used, about 15 HRP molecules are bound along the PDL chain. The purified PDL-BAH-HRP conjugate is very stable when stored at micromolar HRP concentration in a pH 7.2 phosphate buffer solution at 4 °C. When a defined volume of such a conjugate solution of desired HRP concentration (i.e., HRP activity) is added to a macro- and mesoporous silica monolith with pore sizes of 20-30 µm as well as below 30 nm, quantitative and stable noncovalent conjugate immobilization is achieved. The HRP-containing monolith can be used as flow-through enzyme reactor for bioanalytical applications at neutral or slightly alkaline pH, as demonstrated for the determination of hydrogen peroxide in diluted honey. The conjugate can be detached from the monolith by simple enzyme reactor washing with an aqueous solution of pH 5.0, enabling reloading with fresh conjugate solution at pH 7.2. Compared to previously investigated polycationic dendronized polymer-enzyme conjugates with approximately the same average polymer chain length, the PDL-BAH-HRP conjugate appears to be equally suitable for HRP immobilization on silica surfaces.

Biocompatible chemical network of α-cellulose-ESBO (epoxidized soybean oil) scaffold for tissue engineering application.

Despite many desirable properties, the use of α-cellulose in biomedical applications is limited because of its poor processability. Here we demonstrate that the chemical network of α-cellulose and epoxidized soybean oil (ESBO) can be adequately processed into biocompatible, self-standing, highly-porous scaffolds for tissue engineering applications. First, α-cellulose was dissolved in N-Methylmorpholine N-oxide monohydrate (NMMO.MH) and chemically crosslinked by ESBO. Then, the porous scaffolds of α-cellulose-ESBO were fabricated by solvent exchange and freeze-drying techniques. The scaffolds were evaluated for morphology, thermal and mechanical stability, and in vitro cell attachment and cell viability. Scanning electron microscopy images and Brunauer-Emmett-Teller results suggested that porous scaffolds provide a good surface and internal structure for cell adhesion and growth. Specifically, the α-cellulose-ESBO scaffolds support the homogeneous attachment and proliferation of MG63 cells. Overall, our results suggest that α-cellulose-ESBO chemically crosslinked networks are biocompatible and demonstrate a remarkable capacity for the development of tissue engineering platforms.

A two-enzyme cascade reaction consisting of two reaction pathways. Studies in bulk solution for understanding the performance of a flow-through device with immobilised enzymes.

Enzyme-catalysed cascade reactions in flow-through systems with immobilised enzymes currently are of great interest for exploring their potential for biosynthetic and bioanalytical applications. Basic studies in this field often aim at understanding the stability of the immobilised enzymes and their catalytic performance, for example, in terms of yield of a desired reaction product, analyte detection limit, enzyme stability or reaction reproducibility. In the work presented, a cascade reaction involving the two enzymes bovine carbonic anhydrase (BCA) and horseradish peroxidase (HRP) - with hydrogen peroxide (H2O2) as HRP "activator" - was first investigated in great detail in bulk solution at pH = 7.2. The reaction studied is the hydrolysis and oxidation of 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) to 2',7'-dichlorofluorescein (DCF), which was found to proceed along two reaction pathways. This two-enzyme cascade reaction was then applied for analysing the performance of BCA and HRP immobilised in glass fiber filters which were placed inside a filter holder device through which a DCFH2-DA/H2O2 substrate solution was pumped. Comparison was made between (i) co-immobilised and (ii) sequentially immobilised enzymes (BCA first, HRP second). Significant differences for the two arrangements in terms of measured product yield (DCF) could be explained based on quantitative UV/vis absorption measurements carried out in bulk solution. We found that the lower DCF yield observed for sequentially immobilised enzymes originates from a change in one of the two possible reaction pathways due to enzyme separation, which was not the case for enzymes that were co-immobilised (or simultaneously present in the bulk solution experiments). The higher DCF yield observed for co-immobilised enzymes did not originate from a molecular proximity effect (no increased oxidation compared to sequential immobilisation).