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.

AFM analysis of the polymer microstructures used for novel multianalyte protein microassay

“…The similar depths of ca. 20 suggest that the top gold layer was effectively ablated, although the results indicate that some residual and/or redeposited gold was present on the surface after the first laser treatment. As well, region I, being at the focal spot and therefore most affected by the laser beam, did not increase in depth with the second laser treatment, but exhibited an increase in the height of the central hump instead.…”

“…Protein / solid interactions are complex and fabricating a biochip surface which can accommodate different proteins is a complex matter. The variation in the morphology and surface chemistry in the shallow microstructures effectively 'combinatorialize' the surface properties of the microstructures, thus facilitating the patterning of different proteins for used in multi-analyte protein micro-assays 20 . The physical insights into the formation of these microstructures as shown in the present study provide a basis for further optimization of the technology.…”

“…Preparation of gold-coated PMMA film. Details of the method used to prepare the gold-coated PMMA film were described elsewhere 20 . Essentially, a 4 wt% solution of PMMA in 99% propylene glycol methyl ether acetate (PGMEA) was spin-coated at 3000 rpm for 40 s on glass slides or cover slips primed with hexamethyldisilazane.…”

“…These complex interactions can also result in important changes in the protein structure and bioactivity, further complicating the patterning of different proteins on the same surface. We recently describe a novel technique for the fabrication of microstructures on the same solid surface having a range of surface properties that can be used for patterning different proteins and in multianalyte protein micro-assay device 20 . The micro-structures are fabricated via a localized laser ablation of a protein-blocked thin gold layer deposited on a poly(methyl methacrylate) (PMMA) film.…”

Submicro foaming in biopolymers by UV pulsed laser irradiation

“…Laser processing of organics substrates, such as polymers and biopolymers, is of application for the fabrication of artificial biocompatible extracellular matrices or scaffolds for culture and growth of biological tissue [1][2][3][4] . Laser assisted manipulation and destruction of biomaterials can be carried out with unprecedent precision and selectivity.…”