Polymer Microstructures Fabricated via Laser Ablation Used for Multianalyte Protein Microassay

Abstract

This paper describes a novel technique for the fabrication of a multianalyte protein microassay on the basis of the creation of microwells that locally enhance the adsorption of proteins. The microwells are fabricated via a localized laser ablation of a protein-blocked thin gold layer (∼50 nm) deposited on a poly(methyl methacrylate) film. The microablation of gold induces local chemical and physical changes in the top surface of the polymer as well as a higher specific surface, which cooperate to achieve a higher and more reproducible surface concentration of proteins in microwells. The fabrication platform consists of a computer-controlled laser ablation system, comprising a research-grade inverted optical microscope, a pulsed nitrogen laser emitting at 337 nm, a programmable XYZ stage, and a picoliter pipet mounted on the XYZ stage. The microwells with diameters of 5−20 μm, 1−5 μm, and submicron widths are readily achieved by focusing through a 20× dry objective, a 40× dry objective, or a 100× oil immersion lens, respectively. One variant of the method uses a sequence of local ablation and “flood” coverage with protein solution. The second variant uses the microablation of the whole microassay followed by the “spatially addressable” deposition of different protein solution with a picoliter pipet mounted on the same fabrication platform. The analytical performance of the device required only a 2−7 μL volume of sample and a single dilution step. The results indicate that antibody arrays can be used to identify different proteins, yielding results within a few minutes of sample addition with acceptable assay repeatability. It was observed that the microassays comprising line-shaped microstructures offer a higher reproducibility as well as offering the opportunity to encode the information (e.g. type of antibody, concentration) through a combination of vertical lines in a “bar code”, “informationally addressable” mode and not in a 2D, spatially addressable mode like in the classical microarrays.