Miniaturized electrospraying as a technique for the production of microarrays of reproducible micrometer-sized protein spots

Electrospraying in a stable cone-jet mode; at < 400 mum above a substrate is shown to be a powerful technique to produce arrays of identical micrometer-sized spots consisting of biologically active substances. Aqueous solutions with a surface tension of 0.04 N m(-1) and conductivities ranging from 0.04 to 2.2 S m-l were sprayed at ultralow now rates ranging from 100 to 300 pL s(-1). The charged jet that emanates from the cone tip breaks up into a spray of charged droplets that are deposited in the form of a uniform spot of 130-350 mum in diameter by spraying during 0.5-3 s at 220-400 mum above a substrate, respectively. After a spot was deposited, spraying was stopped instantaneously by increasing the distance between the capillary-tip and the substrate by an additional 100 mum using a computer-controlled x-y-z table. This was immediately followed by a rapid shift of the substrate 400 mum sideways and 100 mum upward, thus causing spraying to resume instantaneously because of the increased electric field strength, which resulted in the deposition of the next spot. It is shown here that spraying of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6P-DH), and pyruvate kinase (PK) on a liquid layer resulted in the complete preservation of their activities despite the high solution conductivity of 3.3 S m-l and high currents ranging from 300 to 500 nA. LDH and PK activities were fully preserved after spraying onto dry aluminum by adding 0.05 M buffer and 0.5 and 1 wt % of trehalose, respectively, to the spray solutions. Electrospraying allows for accurate dispensing of liquid volumes as small as 50 pL. Enzymatic activities of LDH and PK are fully preserved after spraying.