| 초록 |
In biological studies, real-time measurement of chemical and physical parameters at the single cell level has been of great interest to researchers. The PEBBLE (Probes Encapsulated By Biologically Localized Embedding) concept was introduced, specifically using optical nanosensors to make intracellular measurements. Optical nanosensors consist of an inert, biocompatible matrix in which a sensing material and/or an optical reporter are entrapped. Most of the optical nanosensors reported so far have used fluorophores as the sensing material because of their high sensitivity and relative ease of measurement. However, recently there have been efforts to incorporate bioactive molecules such as enzymes and antibodies, which are more complex sensing materials, in order to expand the scope of analytes that can be detected with this technology. Therefore, encapsulation of enzymes and other biomolecules as sensing agents in these nanosensors is an important challenge in this field. Here, we describe the fabrication and characterization of poly(ethylene glycol) (PEG) hydrogel spheres containing the enzyme, horseradish peroxidase (HRP), for potential use as optical micro- and nanosensors. HRP was encapsulated in the PEG hydrogel spheres by reverse emulsion photopolymerization, yielding spheres from 500 nm to 20 µm. After sphere fabrication and HRP encapsulation, enzyme activity within the spheres and sensitivity to hydrogen peroxide of the spheres was investigated. The fluorescence emission response of HRP-loaded PEG hydrogel spheres changed as a function of H2O2 concentration in the presence of 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), with nanomolar concentrations of hydrogen peroxide. The results indicate that after polymerization the enzyme activity of HRP was still maintained and that the HRP-loaded PEG hydrogel spheres could be potentially used as optical sensors for intracellular analysis, drug screening or in micro-total analytical systems (µ-TAS). |
