Biomolecules were spotted on top of a nanoporous alumina membrane using a CFM (continuous flow microfluidic spotter) system. On their own, CFM spots are 3 ~ 4 orders of magnitude more sensitive than conventional printing methods, such as pin and fempto-jet printing. The addition of a three-dimensional substrate takes greater advantage of the 3 dimensional-flow and addressing afforded by the CFM printer, further increasing spot sensitivity and efficiency. The CFM printed spots on the nanoporous alumina substrates showed high signal to background ratios, were highly saturated, and still produced a measureable signal at very low immobilization concentrations. In short, this technique produces highly concentrated biomolecular spots from dilute samples, all without any microfabrication process.

The microarray technique has proven to be a rapid and effective tool for genomics and proteomics research. Nonetheless, the conventional microarray system has limitations that include expensive instrumentation, low active spot concentrations, and the need for highly concentrated samples. To overcome these drawbacks, the CFM system was developed and tested with proteins and lipids. The spot fluorescence intensity was increased by 300% using the CFM system compared to micropipette spots. For CFM system, GAP (gamma amino-propyl-silane) coated glass slides usually used for protein immobilization, but, the binding capacity is limited. To improve each microspot’s sensitivity and efficiency, the three-dimensional microarray has been developed and tested with nanoporous membranes without micro/nanofabrication process.

The nanoporous alumina membranes were attached to glass slides using a thin layer of spin coated, partially-cured PDMS. Alexafluor 554 Protein A conjugate (Invitrogen, USA) was chosen for the initial benchmark study, and diluted 2 fold serially in 1X PBS buffer from a starting concentration of 100 ug/mL down to 20 ng/mL. The PDMS print-head was coated with TWEEN 20 (Biorad), and rinsed with Millipore water. The deposition profile is as follows: the slide was docked to print-head, each of the 48 wells of the print-head was loaded with 80 uL of sample, and then the flow through the print-head was reversed for 15 min at 60 ul/min. Following deposition, the sample was recovered so that the spots could be rinsed with 0.1X PBS buffer for 2 minutes. After rinsing, the slides were undocked and imaged at 554 nm excitation using an automated Olympus IX81 TIRFM fluorescence microscope.

Using identical spotting procedures, the protein A spot density on nanoporous AOM was higher than on the surface modified glass substrate This is because the nanoporous AOM substrate has a large surface area, which increases the binding opportunities for biomolecules. Also, the biomolecules remain on top of the nanoporous substrate due to capillary forces. These forces are generated from vertical nanopores structures within the membrane, and they keep the bio-molecules on the surface of the membrane while water is drawn through. This is a unique combination of technologies, namely printing via flow in a 3-dimensional microfluidic system onto a similarly 3D surface. Nanoporous AOM, when used as a substrate for the microfluidic spotter system, increased the efficiency of the system and improved the quality of microspots.