Various microanalytical devices have been proposed for rapid measurements of proteins and peptides in blood and urine. This is because an enzymatic reaction or immunoreaction occurs effectively in a microchannel due to the extremely large surface-to-volume ratio compared with conventional measurements. However, it is difficult to obtain sufficient sensitivity in the microchannel using the conventional detection method. We have therefore developed a novel detection principle based on a surface reaction which is suitable for microchannels, and applied it to micro-immunodevices.
Thiols are known to be adsorbed on a gold surface, and form a dense self-assembled monolayer. We employed this characteristic for a highly sensitive immunoassay by obtaining the surface preconcentration of thiol molecules (thiocholine) formed by the enzyme (acetylcholinesterase) reaction. Figure 1 shows a schematic of our immunoassay by electrogenerated chemiluminescence determination. Thiocholine is produced from acetylthiocholine by the labeled acetylcholinesterase. The thiocholine molecules are collected and accumulated on the gold surface by gold-thiol binding. Finally, the luminescence intensity is measured when the gold is oxidized with ruthenium complex. Since the luminescence intensity becomes greater as the amount of accumulated thiocholine increases, the analyte concentration can be estimated from the luminescence intensity.
Figure 2 shows portable surface plasmon resonance equipment and a micro-immunodevice with the above thiocholine system. This equipment makes it possible to measure trace peptides around the pg/ml level within 30 min because the reactions (immunoreaction, enzyme reaction and thiocholine accumulation) take place to a sufficient degree in the microchannel.
We have developed a novel immunoassay method that is suitable for microdevices. Unlike conventional absorption and emission measurements, the sensitivity of our method is independent of the optical length.Therefore, the sensitivity is not decreased by downsizing. Instead, our method makes it possible to perform measurements with high sensitivity in a microchannel because of its high collection efficiency and high linear flow rate.