Direct Detection of Protein Biomarkers in Human Fluids Using Site-Specific Antibody Immobilization Strategies

Abstract

Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

Recent advances in nanoplasmonic biosensors: applications and lab-on-a-chip integration

“…In general terms, although some publications have dealt with this controversy [7,8,11], there is still a lack of convincing studies that confirm whether nanoplasmonics is competitive enough with SPR in terms of surface sensing performance. However, a few recent works demonstrate that sensitivity levels are in the same order of magnitude, although the sensitivity seems to improve and be higher at low analyte concentration in the case of LSPR, both in a competitive assay [12] and in direct approaches [13,14]. This could be partially due to the strong LSPR field confinement of the nanostructures compared to SPR, which becomes more evident at low target concentrations, especially in direct assays.…”

“…A white light source and a small spectrophotometer are used, which somehow hinder complete integration, although both components can be substituted by LEDs and by commercially available miniaturized spectrophotometers, respectively. With a simple microfluidic and LabView-developed software, bioanalytical applications relevant in the clinical field have been developed reaching excellent levels of sensitivity, even when analyzing real clinical samples [13,14]. Expanding the microfluidics to a multiplexed format would place this kind of platform closer to the LOC concept.…”

“…The strategy was applied to the direct detection of protein biomarkers such as the human chorionic gonadotropin (hCG), C-reactive protein (CRP), and focal adhesion kinase (FAK) protein [14]. Direct detection in urine was demonstrated, keeping unaltered the LODs compared to buffer conditions, although for the analysis in serum it was necessary a 1:10 dilution factor.…”

Surface Plasmon Resonance: Advances of Label-Free Approaches in The Analysis of Biological Samples

“…Unfortunately the biotinylation of the bioreceptor might impair its properties and structure. More recently, Soler et al [44] demonstrated the use of a ProLinker TM molecule for site-specific antibody immobilization and its application on novel nanoplasmonic biosensors resulting in promising advantages for its application in clinical and biomedical diagnostics. Comprehensive literature about site-specific immobilization techniques can be found elsewhere [45,46].…”

Nanoplasmonic Biosensors for Label-free Deciphering of Cellular Pathways

“…Moreover, real applications as clinical or environmental diagnosis require well-defined surfaces, minimizing non-specific adsorptions from complex samples. We have developed several techniques to ensure a proper functionalization of the plasmonics sensors for evaluating real samples [2,3].…”