Conductive AFM patterning on oligo(ethylene glycol)-terminated alkyl monolayers on silicon substrates: proposed mechanism and fabrication of avidin patterns

Abstract

Micro- and nanopatterns of biomolecules on inert, ultrathin platforms on nonoxidized silicon are ideal interfaces between silicon-based microelectronics and biological systems. We report here the local oxidation nanolithography with conductive atomic force microscopy (cAFM) on highly protein-resistant, oligo(ethylene glycol) (OEG)-terminated alkyl monolayers on nonoxidized silicon substrates. We propose a mechanism for this process, suggesting that it is possible to oxidize only the top ethylene glycol units to generate carboxylic acid and aldehyde groups on the film surface. We show that avidin molecules can be attached selectively to the oxidized pattern and the density can be varied by altering the bias voltage during cAFM patterning. Biotinylated molecules and nanoparticles are selectively immobilized on the resultant avidin patterns. Since one of the most established methods for immobilization of biomolecules is based on avidin-biotin binding and a wide variety of biotinylated biomolecules are available, this approach represents a versatile means for prototyping any nanostructures presenting these biomolecules on silicon substrates.

Figure 1

Contact mode AFM height (a, 3 × 3 μm², 30 nm contrast) and friction (b, 1.0 V contrast) images of the patterns (lines) generated by cAFM using the same tip on an OEG-alkyl film with an applied bias voltage of 4, 5, 6, 7, 8, 9, and 10 V (corresponding to the lines from left to right) and a tip velocity of 9 μm/s. The sample was then derivatized with PAMAM dendrimers, followed by treatment with gold nanoparticles, and the topography of the same area imaged with contact mode AFM (c, 3 × 3 μm², 30 nm contrast). (d) and (e) are line profiles designated by the white lines in (a) and (c), respectively.

Figure 2

(a) Topographic and (b) the corresponding friction contact mode AFM images of the lithographic patterns on OEG films generated with a bias voltage of 15, 14, and 13 V (left to right) and an identical tip velocity of 85 μm/s; (c) topographic contact mode AFM images of the same regions after treatment with an avidin solution; (d) line profiles of image (a) and (c) designated by the white lines; scanning size, 12 × 12 μm²; z range, 10 nm, 0.2 V.

Figure 3

Fluorescence images of four 10 × 10 μm² squares on OEG films (ellipsometric thickness ~36 Å) generated by cAFM followed by treatment with EDC and fluorescent FITC-avidin (a); with EDC and nonfluorescent avidin, then biotinylated BSA, followed by FITC-avidin that binds the biotinylated BSA (b); with EDC and avidin, then BSA (without biotin label), followed by FITC-avidin with little binding due to the lack of biotin on the pattern (c); and with EDC and avidin, followed by fluorescently labeled biotin (d).

Figure 4

Topographic AFM images obtained in contact mode of the lithographic patterns generated on OEG films with a tip velocity of 85 μm/s under bias voltage of 10 V at a relative humidity of 37% (a), and the topographic images of the same pattern obtained in tapping mode in water upon treatment with avidin in the presence of EDC (b and g), followed by binding with biotinylated Qdot (c, h) with line profiles (d, e, and f) designated by the white lines in (a), (b) and (c), respectively. Scanning size: 6 × 6 μm² for (a–c) and 2.5 × 2.5 μm² for (g) and (h); z range: 10 nm for (a–c) and 5 nm for (g) and (h).

Figure 5

Illustration of the cAFM system and two pathways (a and b) for the oxidation of the OEG-alkyl monolayers. Pathway a: oxidation near the silicon interface (anode), initiated by anodic oxidation of the HO^– (that arrives near the alkyl layer) via hole tunneling from the interface to form HO^•, followed by hydride abstraction and formation of hemiacetals and esters, finally upon hydrolysis generating a hole presenting alcohols, aldehydes, and carboxylic acids in A. Pathway b: oxidation on the film surface initiated by formation of HO^• and H^• at the AFM tip (cathode) through eqs (1)–(4), followed by a series of reactions forming hydroperoxides and peroxy radicals that decompose to form alcohols, aldehydes, and carboxylic acids in B without substantial degradation of the OEG film. A short duration of bias voltage and high packing density of the film can block the Pathway a and favor the Pathway b.