Ink-Jet Printing for drug srreening on microarrays: from covalent approaches to in-liquid-droplets assays

Drug screening is the complex process of retrieving chemical compounds able to modulate the activity of biological targets which are of interest for certain diseases. Conventional miniaturized drug screening technologies are based on robotic dispensers coupled with microwell arrays. However these devices require time and reagent consuming (micro-, nanoliter scale) instrumental tools, liquid handling robotics and complex detectors. Here we show a low-cost and efficient drug screening methodology based on inkjet printing for delivering molecular systems in picoliter volumes coupled with easily-implemented detection tools for probing target-drug interaction. We firstly show up a screening platform for a model enzyme/substrate couple and we extend this approach to systems of clear interest for medicinal chemistry. The approach was initially proved with a model enzyme system like Glucose Oxidase substrate covalently linked to a functionalized silicon oxide support. On this support an enzymatic substrate (D-glucose)/inhibitor (D-glucal) couple was accurately dispensed. A simple colorimetric detection method based on the production of a red quinoneimine dye in a reaction catalyzed by Horseradish Peroxidase proved the screening capability of the microarray at the single spot level. Occurrence of competitive inhibition was verified at the solid-liquid interface with a similar behavior occurring for such system in a solution phase. Afterwards, this methodology has been extended to CYP450 enzymes like CYP3A4, one of the main targets for the phase I drug metabolism via a droplet microreactors arrays containing CYP3A4 enzyme mixed with model inhibitors (i.e. ketoconazole and erythromycin) and enzymatic chemiluminescent substrates (Luciferin- Isopropylacetate). Enzymatic activity in picoliter liquid spots was detected by using a low cost optical method. Accordingly, bioluminescence given by D-luciferin upon reaction with oxygen in a reaction catalyzed by Luciferase enzyme leads to a production of photons that increase spot brightness which can be quantified by Charge-coupled device camera.