Abstract
A plasmon-enhanced fluorescence-based antibody-aptamer biosensor — consisting of gold nanoparticles randomly immobilized onto a glass substrate via electrostatic self-assembly — is described for specific detection of proteins in whole blood. Analyte recognition is realized through a sandwich scheme with a capture bioreceptor layer of antibodies — covalently immobilized onto the gold nanoparticle surface in upright orientation and close-packed configuration by photochemical immobilization technique (PIT) — and a top bioreceptor layer of fluorescently labelled aptamers. Such a sandwich configuration warrants not only extremely high specificity, but also an ideal fluorophore-nanostructure distance (approximately 10–15 nm) for achieving strong fluorescence amplification. For a specific application, we tested the biosensor performance in a case study for the detection of malaria-related marker Plasmodium falciparum lactate dehydrogenase (PfLDH). The proposed biosensor can specifically detect PfLDH in spiked whole blood down to 10 pM (0.3 ng/mL) without any sample pretreatment. The combination of simple and scalable fabrication, potentially high-throughput analysis, and excellent sensing performance provides a new approach to biosensing with significant advantages compared to conventional fluorescence immunoassays.Graphical abstract
Highlights
Fluorescence-based techniques are widely employed and rapidly emerging as a leading methodology in biotechnology, biomedicine, and life sciences [1, 2]
Despite the lack of order in the AuNP arrangement entailed a reduction of the fluorescence amplification, the limit of detection (LOD) provided by this biosensor in a complex matrix like human blood lies in the picomolar range
The Ab-analyte-Apt* sandwich used in this work consisted of anti-PLDH as the capture bioreceptor layer that offers effective detection of any malaria biomarkers Plasmodium lactate dehydrogenase (PLDH) [34], whereas malaria Apts* used as the top bioreceptor layer warrants a cost-effective and highly specific targeting of Plasmodium falciparum lactate dehydrogenase (PfLDH) with discrimination from Plasmodium vivax LDH (PvLDH) [35]
Summary
Fluorescence-based techniques are widely employed and rapidly emerging as a leading methodology in biotechnology, biomedicine, and life sciences [1, 2]. Plenty of plasmonic substrates consisting of twodimensional (2D) arrays of metal nanostructures have been developed as PEF-based biosensing platforms [5, 7,8,9]. In the last few years, several milestones have been achieved in terms of FE factor (up to 105-fold) [10] and limit of detection (LOD) (down to fM level) [9]. The complexity of these approaches usually limits the success of PEF-based fluoroassays in point-of-care testing and mass screening [3]. 2D arrays of gold nanoparticles (AuNPs) are attractive candidates as fluorescence enhancers because of cost-effective and scalable fabrication, tunable plasmonic properties, and wide The complexity of these approaches usually limits the success of PEF-based fluoroassays in point-of-care testing and mass screening [3]. 2D arrays of gold nanoparticles (AuNPs) are attractive candidates as fluorescence enhancers because of cost-effective and scalable fabrication, tunable plasmonic properties, and wide
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