DEVELOPMENT OF RAPID IMMUNOASSAY TESTS BY USING A MICRO-ANALYTICAL FLOW SYSTEM COUPLED WITH ELECTROCHEMICAL DETECTION

摘要

In this work a rapid competitive assay for rlgG detection as a fast means for use in routine analysis is presented.This was achieved by using a micro-analytical flow system (ImmuSPEED™) for rapid and quantitative immunochemical analysis. This is an innovative system which combine a special cartridge (ImmuCHIP™), containing eight polymer microchannels, with a computer-controlled instrument for the control of fluidics. The higher surface-to-volume ratio in the channels and the shorter diffusion distances lead to very low detection limits and short reaction times, whereas the presence of micro-electrodes in each channel allows the direct quantification of the affinity reaction by electrochemical measurement within the microchannel.In our approach an indirect competitive assay format was performed, by immobilising rlgG onto the surface of microchannels. Then, a sample solution containing an unknown amount of rlgG and a fixed concentration of anti-rlgG labelled with alkaline phosphatase was introduced in the modified channel in order to have the competition. Finally, the channel is filled with the substrate solution (containing p-aminophenyl phosphate 10 mM), and a real time amperometric evaluation of the enzyme kinetic was performed.All the steps of the immunoassay occurred through hydrodynamic loading of the different solutions through the channels; the speed (μl/min) and duration of the flow, the number of loading step and incubation parameters were optimised. The effectiveness of the system was demonstrated by analysing rlgG concentrations in sample solutions within 5 min, obtaining the same sensitivities as well as in the ELISA tests.