Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces

Abstract

Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol-reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/ esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody against PSA [prostate specific antigen]), Major Histocompability Complex class I protein, pepsin, and trypsin. The reaction mechanism behind the reported new technology involves "photonic activation of disulfide bridges," i.e., light-induced breakage of disulfide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol-reactive surfaces (see Fig. 1). Interestingly, the spatial proximity of aromatic residues and disulfide bridges in proteins has been preserved throughout molecular evolution. The new photonic-induced method for immobilization of proteins preserves the native structural and functional properties of the immobilized protein, avoiding the use of one or more chemical/thermal steps. This technology allows for the creation of spatially oriented as well as spatially defined multiprotein/DNA high-density sensor arrays with spot size of 1 mm or less, and has clear potential for biomedical, bioelectronic, nanotechnology, and therapeutic applications.Keywords: biosensors; protein sensor arrays; protein immobilization; spatially oriented arrays; lightinduced protein immobilization Molecules can be immobilized on a carrier or a solid surface either passively through hydrophobic or ionic interactions, or covalently by attachment to activated surface groups. In response to the enormous importance of immobilization for solid phase chemistry, biological screening, and nanotechnology applications, the analytical uses of the technology have been widely explored. Immobilization technology has found broad application in many different areas of biotechnology, e.g., diagnostics, biosensors, affinity chromatography, immobilization of molecules in ELISA assays, and immobilization of molecules onto nanoparticles for drug/gene delivery. The value of immobilization techniques is demonstrated by the recent boost in the development of DNA and protein microarrays' technologies, and is needed for the development of drug/gene delivery technologies for therapeutical purposes (e.g., cancer therapy, gene therapy). Common for most of the described immobilization methods is their use of one or more thermochemical/

Arraying prostate specific antigen PSA and Fab anti‐PSA using light‐assisted molecular immobilization technology

“…This can be obtained by introducing a site-specific group, such as a thiol group, on the Fab fragments which can be done by genetic engineering 26 or using LAMI technology because UV excitation of aromatic residues induces disruption of nearby disulphide bridges, leading to free thiol groups in the molecule. 17,27 Immobilization yield is a function of protein concentration and illumination time. Increasing illumination time allows for arraying proteins using less concentrated protein stock solutions.…”

“…We have previously shown that one can immobilize proteins onto a thiol reactive surface using UV light-assisted molecular immobilization (LAMI). 17,18 Our bioinformatic studies reveal that both PSA and Fab fragments have aromatic residues in close spatial proximity of disulphide bridges, making both proteins putative good candidates for LAMI technology. In this article, we demonstrate that light-assisted immobilization of free PSA and Fab anti-PSA onto thiol-functionalized optical flat slides leads to functional protein arrays.…”

Trends and Perspectives in Immunosensors

“…One simple oriented immobilisation strategy involves chemically coupling the Ab directly onto the transducer surface, e.g. a non-coated gold surface and available thiol (-SH) groups on the probe [18,19]. However, these methods require chemical treatments of an Ab prior to immobilisation, such as carbohydrate oxidation and disulfide bond reduction, which may detrimentally alter it.…”

Molecular Printing Using UV-Assisted Immobilization of Biomolecules

“…It has previously been shown that UV-light absorbed by aromatic residues in biomolecules can induce disruption of nearby disulfide bridges (Prompers et al 1999;Neves-Petersen et al 2002;Vanhooren et al 2002;Neves-Petersen et al 2006). Upon breakage of the disulfide bridge the molecule is left with free, reactive thiol groups that can enter a vast range of chemical reactions.…”

“…By exploiting this effect, biomolecules can be immobilized onto thiol-derivatized surfaces in a well controlled manner. This can be used, for example, in the constrution of sensor microarrays (Neves-Petersen et al 2004;Neves-Petersen et al 2005;Neves-Petersen et al 2006;Duroux et al 2007;www.bionanophotonics.dk). The UV-induced immobilization effect has already been observed with a large range of proteins including hydrolytic enzymes, proteases, alkaline phosphatase, immunoglobulins (Fab fragment), and Major Histocompability Complex Class I protein (Duroux et al 2007;Neves-Petersen et al 2006;Snabe et al 2006).…”