Development of microplate-based biosensors for high-throughput measurements

Abstract

Nowadays, the increasing number of samples in environmental analysis, biomedical studies and drug screening requires the chemists to analyse a large number of samples within a short time without raising the cost of analysis of each sample. The standard microtiter plate (or microplate) is a 96-well container with small operating volume (total volume: 360ul for each well) and the flat clear bottom is suitable for optical measurement. The microtiter plate is a convenient platform to be modified as high-throughput biosensors for small sample analysis. In this project, microplate-based biosensors for dissolved oxygen measurement, toxicity tests, biochemical oxygen demand (BOD) determination and carbohydrate-protein interaction studies have been developed. ORMOSIL (organically modified silicate) optical oxygen sensors on microtiter plates have been developed via a simple, convenient and low-cost method. The ORMOSIL high-throughput oxygen sensors prepared from tetramethoxysilane (TMOS) and dimethoxy dimethylsilane (DiMe-DMOS) with tris(4,7-diphenyl-l,10-phenanthroline)ruthenium(II), [Ru(dpp)3]2+, as the sensing dye were coated onto the well bottom surface of a 96-well polystyrene microtiter plate to give a high-throughput system for dissolved oxygen measurement. The ORMOSIL high-throughput oxygen sensors adhere on the well bottom surface strongly and give linear Stern-Volmer calibration plots with high sensitivity and long term stability. The applicability of this ORMOSIL high-throughput oxygen sensor platform for cell-based toxicity assays has been studied. The toxicity of two antimalarial drugs chloroquine and quinacrine on a yeast model {Saccharomyces cerevisiae) for studying drug resistance was evaluated by measuring their effect on the respiration rate (dissolved oxygen consumption rate) of yeasts which was monitored by the ORMOSIL oxygen sensor. The ICso values (concentration of a drug that exhibits 50% respiration inhibition) of the drugs obtained by ORMOSIL oxygen sensor were found to be in good agreement with those determined by conventional optical density (OD595) method. In addition, ORMOSIL high-throughput oxygen sensors in combination with aerobic assimilatory microorganisms can also serve as biosensors for BOD determination. The BOD biosensor was constructed by coating a microbial film on top of the ORMOSIL oxygen sensing film. The microbial film was fabricated by immobilizing a bacterial species, Stenotrophomonas maltophilia, in sol-gel-poly(vinyl alcohol) matrix. The results obtained with the BOD biosensor were in agreement with those of the conventional BODs method for different wastewater samples. A simple, convenient, low-cost and high-throughput microplate-based carbohydrate sensor has been constructed for carbohydrate-protein interaction study. The high-throughput carbohydrate sensor was constructed by covalent immobilization of aminated carbohydrate onto TESPI-ORMOSIL film (TESPI = 3-(triethoxysilyl)propyl isocyanate) on a microtiter plate. The TESPI-ORMOSIL film was prepared by coating a layer of 3-(triethoxysilyl)propyl isocyanate (TESPI) onto a transparent ORMOSIL film. The application of the high-throughput carbohydrate sensors in studying carbohydrate-protein interaction has been demonstrated by the well established carbohydrate-lectin system. The results show that this microplate-based carbohydrate microarray sensor is practical for high-throughput analysis of carbohydrate-protein interactions.