Figure 18.

Electrochemical platform (right) and scheme (left) for the detection of human methyltransferase activity from crude cell lysates. (right) The electrochemical detection platform contains two electrode arrays, each with 15 electrodes (1 mm diameter each) in a 5 × 3 array. Multiple DNAs are patterned covalently to the substrate electrode by an electrochemically activated click reaction initiated with the patterning electrode array. Once a DNA array is established on the substrate electrode platform, electrocatalytic detection is then performed from the top patterning/detection electrode. (left) Overview of electrochemical detection scheme at each electrode of the 5 × 3 array. DNA, patterned onto the bottom electrode using the copper-activated click chemistry, is electrocatalytically detected from the top electrode using methylene blue (MB+) as the electrocatalyst and ferricyanide for amplification. Crude cell lysate is then added to the surface containing the patterned DNA. If methyltransferase (green) is present (blue arrows), the hemimethylated DNA on the electrode is methylated (green dot) by the methyltransferase to a fully methylated duplex; if methyltransferase is not present (red arrows), the hemimethylated DNA is not further methylated. A methylation-specific restriction enzyme, BssHII (purple), is then added. If the DNA is fully methylated (blue arrows), the electrochemical signal remains protected, and the DNA is not cleaved. However, if the DNA remains hemimethylated (red arrows), it is cut by the restriction enzyme, and the electrocatalytic signal associated with MB+ binding to DNA is diminished significantly. Adapted with permission from ref (479). Copyright 2014 Proceedings of the National Academy of Sciences of the United States of America.