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Development of a New Generation of Plasmonic Nanomaterials as Promising Colorimetric Reporters for Lateral Flow Assay

par Gosselin, Bryan
Président du jury Valkenier, Hennie
Promoteur Bruylants, Gilles
Co-Promoteur Jabin, Ivan
Publication Non publié, 2024-02-29

Thèse de doctorat

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Résumé :

Lateral flow assays (LFAs), such as pregnancy tests or antigen tests, are probably the most widespread point-of-care testing. They combine all their advantages: rapid, low cost, ease of use and straightforward signal. However, LFAs, in biomedical diagnostics, are sometimes considered as not sufficiently reliable due to their poor sensitivity. Indeed, the analyte in extremely low amounts is not detected, increasing the false negative rate. Many strategies were recently developed to enhance the sensitivity of LFAs. In this thesis, investigations were focused on the colorimetric reporters involved in the assay. Indeed, the red-colored signal observed in most of the LFAs is due to the binding of gold nanoparticles (AuNPs), used as colorimetric reporters, at the test line. AuNPs belong to the plasmonic nanomaterials class, which are able to strongly absorb light. However, silver nanoparticles are known to have higher extinction coefficient compared to their gold counterpart, but suffer from a low stability against external stress, limiting their applications. Recently, calix[4]arene-based coating emerged as promising strategy to drastically enhance stability of plasmonic nanoparticles. First, the calix[4]arene-coated AgNPs, with a 20 nm average diameter, were synthesized and used for the detection of Anti-SARS-CoV-2 IgG in clinical samples. Calix[4]arene coating conferred remarkable stability to the resulting AgNPs, as no degradation was observed over several months. In comparison with lateral-flow immunoassays (LFIAs) based on classical gold nanoparticles, the limit of detection for Anti-SARS-CoV-2 IgG was reduced by 1 order of magnitude and similar signals were observed with 10 times fewer particles with our AgNPs-based assay. These results represent one of the first examples of the use of AgNP-based LFA in the case of real clinical samples.Secondly, silver nanoplates (AgNPls) synthesis was developed. In contrast to AgNPs, the absorption spectrum of this anisotropic nanostructure can be easily tuned as a function of their size. For instance, AgNPls with a 35 nm average size present a dark blue color in suspension, leading to a strong contrast with the nitrocellulose (NC) membrane compared to yellow AgNPs. A radical improvement of the lifetime of the material from 1 day for AgNPls coated with citrate to more than 900 days for calix[4]arene-coated AgNPls, as well as the stability in acidic conditions, phosphate saline buffer (PBS) or biofluid, was observed. In comparison to our previous AgNPs-based assay, the limit of detection was lowered by 2 for the detection of Anti-SARS-CoV-2 IgG in human fluid. Additionally, the wide range of colors provided by the AgNPls allowed the design of a multicolor multiplex assay for the simultaneous detection of multiple analytes.Thirdly, using AgNPls as colorimetric reporters, peptide aptamers were investigated as promising alternative to conventional antibodies for use as recognition units in lateral flow assays (LFAs). Indeed, antibodies are highly versatile as they can be synthesized to target specific analytes with a high affinity, however, they suffer from several drawbacks such as high production cost, low stability and batch-to-batch reproducibility issues. The peptide-based assay detected Mdm2, a well-established cancer biomarker, in low nanomolar range even in complex matrixes such as cell lysates. The utilization of peptide aptamers demonstrated superior performance and extended shelf life compared to polyclonal antibodies, underlining their potential as recognition units in LFAs. In addition to their ease of handling, peptide aptamers utilization also offers the prospect of substantial cost reductions compared to conventional antibody-based LFAs.Fourthly, Au@Ag core-shell nanostructures were investigated to further enhance the sensitivity of LFA. In this study, candidates with different Ag thicknesses were selected depending on their optical advantages, such as high extinction coefficient or color suspension. Subsequently, all core-shell NPs were functionalized with a calix[4]arene layer, imparting superior stability against external stresses, such as dispersion in PBS, when compared to NPs functionalized with traditional ligands. Their efficacy as colorimetric reporters was evaluated in a lateral flow assay for Troponin I detection, demonstrating positive signals down to 1 ng/ml, surpassing the detection limit of the parent 37 nm gold NPs (2.5 ng/ml). Notably, the grey color of the core-shell Au@Ag10@Au NPs provided strong contrast against the white NC membrane, facilitating T line visualization even at low intensities. Despite lacking optimization of the LFA parameters, this assay competes with the limit of quantification of most commercial LFAs for Troponin I detection, promising the development of highly sensitive assays.Finally, a convenient and rapid characterization technique to confirm the presence of biomolecules, such as antibodies or nucleic acid, on the NPs was developed. This versatile technique relies on the LFA principle and, therefore, can be applied to any nanoparticles with optical properties in the visible region. In conclusion, this work opens new perspectives to silver nanomaterials for their use in biomedical applications due to the calix[4]arene coating strategy, increasing significantly their resulting stability and facilitating further bioconjugation.