Redox-activating dip-pen nanolithography (RA-DPN)

Abstract

Dip pen nanolithography (DPN) involves the direct transfer of an ink from a coated atomic force microscope (AFM) tip to a substrate of interest and uses as many as 55,000 pens to form arbitrary patterns of alkanethiols, oligonucleotides, proteins, and viruses. Two limitations of DPN are the difficulty in transporting high molecular weight inks and the need to optimize individually the transport rates and tip inking methods of each molecule. As an alternative strategy that circumvents these two challenges, a method termed redox activating DPN (RA-DPN) is reported. In this strategy, an electrochemically active, quinone functionalized surface is toggled from the reduced hydroquinone form to the oxidized benzoquinone form by the delivery of an oxidant by DPN. While the benzoquinone form is susceptible to nucleophilic attack in Michael-type additions, hydroquinone is not and acts as a passivating agent. Because both forms of the quinone are kinetically stable, the patterned surface can be immersed in a solution of a target containing any strong nucleophile, which will react only where the benzoquinone form persists on the surface. For proof-of-concept demonstrations, quinone surfaces were patterned by the delivery of the oxidant cerric ammonium nitrate and were immersed in solutions of AF549 labeled cholera toxin beta subunit or oligonucleotides modified at the 5' end with an amine and the 3' end with a fluorophore. Fluorescent patterns of both the proteins and oligonucleotides were observed by epifluorescence microscopy. Additionally, RA-DPN maintains the advantageous ability of DPN to control feature size by varying the dwell time of the tip on the surface, and variation of this parameter has resulted in feature sizes as small as 165 nm. With this resolution, patterns of 50,000 spots could be made in a 100 x 100 microm(2) grid.

Figure 1

Fluorescence image of 16 × 21 pattern of (A) Cy3-labelled oligonucleotide and (B) AF549 labeled protein dot features assembled on an Si/SiO₂ surface, prepared by the RA-DPN method with a 26 pen array.

Figure 2

(A) AFM topographical image of a series of oligonucleotide dots of increasing size created by varying dwell times (0.01, 0.05, 0.1, 0.5, 1, 5, 10 s, left to right) results in increasing spot size (350, 450, 500, 650, 740, 800, 830 nm). (B) Plot of Dwell time vs. Feature Area.

Scheme 1