In recent years, inkjet printing, as a new method to fabricate microdroplet microarrays, has been increasingly applied in the field of biochemical diagnostics. To further improve the general applicability of the inkjet printing technology in fabricating biochemical chips, in this work, we introduce a model to describe the multiple injection procedure implemented by the inkjet printing approach, with experimental verification. The multiple injection model demonstrates a new sequential inkjet printing method that generates picoliter-scale multicomponent droplet-in-oil arrays via multistep printing on uniform planar substrates. Based on our previous work on double-inkjet printing, this technique adapts the piezoelectric inkjet printing technology to fabricate an oil droplet array, into which multiple precise injections of secondary droplets with different compositions and volumes can be automatically printed in the required sequence, simultaneously addressing the evaporation issues associated with printing picoliter droplets without external assistance. In this paper, we first describe the theory and characterize the model, which account for the basic principles of sequential inkjet printing, as well as validate the design in terms of multiple injections, droplet fusion, and rapid mixing. The feasibility and effectiveness of the method are also demonstrated in a dual fluorescence assay and a beta-galactosidase enzyme inhibition assay. We believe that applying the sequential inkjet printing methodology in existing inkjet printing devices will enhance their use as universal diagnostic tools as well as accelerate the adoption of inkjet printing in multistep screening experiments.
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