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

Abstract

We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti-PSA in solution and immobilized Fab anti-PSA cross-reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.

UV-Light Exposure of Insulin: Pharmaceutical Implications upon Covalent Insulin Dityrosine Dimerization and Disulphide Bond Photolysis

“…Reduction of SS upon UV excitation of aromatic residues has been shown for proteins such as cutinase and lysozyme [1], [3], [27], bovine serum albumin [28], [29], prostate specific antigen [30], alpha-lactalbumin [4] and antibody Fab fragments [31].…”

Photo-synthesis of protein-based nanoparticles and the application in drug delivery

“…The formation of intermolecular disulfide bonds can further enhance the assembly of lysozyme molecules to form oligomers or even larger aggregates [24]. As mentioned above, the photo-induced unfolding of protein bovine serum albumin [28,38], prostate specific antigen [39], β-lactoglobulin [33], and antibody fab fragments [40,41] have also been investigated previously (see Table 1). ) - …”

Polymer-antibody fragment conjugates for biomedical applications

“…Alternatively, recombinantlyproducted Fab fragments, which typically lack free thiols,can be functionalizedwithshort, cysteine-containing peptide tags that facilitate their detection and purification in the recombinant expression platformin order to allow a similartargeting couplingonactivated surfaces [57].Light-assisted molecular immobilization (LAMI) is an alternative approach that has beenused to exploit thiol chemistry in Fab conjugation, where irradiation with UV led to the generation of free thiols from intramolecular disulfide bridges in an anti-prostate specific antigen Fab. The thiols were then used to covalently orient the Fab on thiol reactive surfaces [75] butthe obvious concern is how modification of native disulfides that are often critical for structural stability might affect the confirmation and activity of irradiated proteins.…”