Immobilization of fish chromatophores for use as a micro-biosensor for biological toxins

Abstract

Chromatophores isolated from the Siamese fighting fish, Betta splendens represent a class of living cells that provide a vivid color response to microbial pathogens and environmental toxins. The selection of the most appropriate microcarrier and the development of the optimal technique for the chromatophore immobilization in order to enable directed transport of the sensor cells throughout microchannels of the biosensor, as well to preserve the cell survival and its functionality was studied. Microcarriers derived from glass, polystyrene and gelatin (collagen) were tested as substrates for chromatophore attachement. Gelatin microcarriers were found to be the most suitable, due to high attachment efficiency (95% of attached cells), preservation of the cell viability and enhanced cell sensitivity. The optimum conditions for fish cell immobilization on collagen microcarriers were determined based on the cell-to-microcarrier bead ratio and the pH of the solution. The rate of cell attachment to the gelatin microcarrier followed first-order kinetics. Pretreatment of the gelatin beads with fibronectin, known as a cell attachment-promoting agent, resulted in a 10% higher attachment rate constant (k).

Hromatofore izolovane iz ribe Sijamski borac vrste Betta splendens predstavljaju vrstu živih ćelija koja reaguje vidljivom promenom boje i oblika na dejstvo mikrobnih toksina i zagađivača okoline. U radu je ispitivan izbor najpogodnijeg nosača i razvoj optimalne tehnike za imobilizaciju hromatofora u cilju omogućavanja usmerenog transporta senzorskih ćelija kroz predviđenu strukturu mikrokanala biosenzora. Odgovarajuća imobilizaciona tehnika je potrebno da ostvari dobru vijabilnost i funkcionalnost ćelija. Testirani su mikronosači na bazi stakla polistirena i želatina (kolagena). Želatinski mikronosač se pokazao najpogodnijim u pogledu efikasnosti vezivanja (95 % ćelija je vezano) i očuvanja vijabilnosti i senzitivnosti ćelija na toksine. Optimalni uslovi za imobilizaciju ćelija na želatinski nosač su utvrđeni i to: odnos broja ćelija i broja mikronosača, i pH imobilizacije. Takođe je praćena kinetika vezivanja ćelija na želatinski nosač, koja je bila kinetika prvog reda. Pre tretman želatinskih nosača sa fibronektinom, koji je poznat kao agens koji pomaže vezivanje ćelija, uticao je na povećanje kinetičke konstante prvog reda za 10%.