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Proceedings Paper

Novel microfabrication stage allowing for one-photon and multi-photon light assisted molecular immobilization and for multi-photon microscope
Author(s): Odete Gonçalves; Scott Snider; Ruben Zadoyan; Quoc-Thang Nguyen; Henrik Vorum M.D.; Steffen B. Petersen; Maria Teresa Neves-Petersen
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Paper Abstract

Light Assisted Molecular Immobilization (LAMI) results in spatially oriented and localized covalent coupling of biomolecules onto thiol reactive surfaces. LAMI is possible due to the conserved spatial proximity between aromatic residues and disulfide bridges in proteins. When aromatic residues are excited with UV light (275-295nm), disulphide bridges are disrupted and the formed thiol groups covalently bind to surfaces. Immobilization hereby reported is achieved in a microfabrication stage coupled to a fs-laser, through one- or multi-photon excitation. The fundamental 840nm output is tripled to 280nm and focused onto the sample, leading to one-photon excitation and molecular immobilization. The sample rests on a xyz-stage with micrometer step resolution and is illuminated according to a pattern uploaded to the software controlling the stage and the shutter. Molecules are immobilized according to such pattern, with micrometer spatial resolution. Spatial masks inserted in the light path lead to light diffraction patterns used to immobilize biomolecules with submicrometer spatial resolution. Light diffraction patterns are imaged by an inbuilt microscope. Two-photon microscopy and imaging of the fluorescent microbeads is shown. Immobilization of proteins, e.g. C-reactive protein, and of an engineered molecular beacon has been successfully achieved. The beacon was coupled to a peptide containing a disulfide bridge neighboring a tryptophan residue, being this way possible to immobilize the beacon on a surface using one-photon LAMI. This technology is being implemented in the creation of point-of-care biosensors aiming at the detection of cancer and cardiovascular disease markers.

Paper Details

Date Published: 21 February 2017
PDF: 17 pages
Proc. SPIE 10079, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications IX, 100790F (21 February 2017); doi: 10.1117/12.2250567
Show Author Affiliations
Odete Gonçalves, Aalborg Univ. (Denmark)
Scott Snider, Newport Corp. (United States)
Ruben Zadoyan, Newport Corp. (United States)
Quoc-Thang Nguyen, NeurAccel Biosciences (United States)
Henrik Vorum M.D., Aalborg Univ. (Denmark)
Steffen B. Petersen, Aalborg Univ. (Denmark)
Maria Teresa Neves-Petersen, Aalborg Univ. (Denmark)

Published in SPIE Proceedings Vol. 10079:
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications IX
Samuel Achilefu; Ramesh Raghavachari, Editor(s)

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