Plasmonic ELISA for Biomarker Detection: A Review of Mechanisms, Functionalization Strategies, and Emerging Modalities

Abstract

Plasmonic enzyme-linked immunosorbent assay (ELISA) effectively integrates noble metal nanostructures with traditional immunoassays, facilitating rapid, ultrasensitive, and multiplexed biomarker detection. By leveraging localized surface plasmon resonance modulations instigated by biocatalytic reactions and analyte binding, these assays achieve signal amplification through growth, etching, and aggregation mechanisms. Such methodologies significantly enhance detection limits by factors ranging from 10- to over 1000-fold, attaining sensitivity at the subpicogram per milliliter level. Robust surface functionalization methods, including electrostatic adsorption, covalent coupling, and affinity binding, ensure stable immobilization of antibodies while preserving the activity of the nanozymes. Incorporating advanced two-dimensional nanomaterials, such as graphene derivatives and MXenes, further augments the sensitivity (up to ∼200-fold), assay stability, and potential for miniaturization. Emerging modalities, including electrochemical techniques, microfluidics, photothermal methods, surface-enhanced infrared absorption (SEIRA), surface-enhanced Raman scattering, and CRISPR-enabled ELISA, extend the analytical versatility, multiplexing capabilities, and operational speed. Clinical trials, alongside real-world studies, substantiate the efficacy of plasmonic ELISA platforms in early cancer detection, diagnostic evaluation of infectious diseases, and monitoring cardiovascular biomarkers, demonstrating performance comparable to or exceeding that of traditional methodologies. Despite significant advancements, challenges persist with regard to assay standardization, multiplex integration, and large-scale manufacturing. This review presents a comprehensive overview of recent developments, identifies critical knowledge gaps, and outlines future perspectives to expedite the clinical translation of plasmonic ELISA technologies for precision medicine and global health applications.

Keywords: biomarker detection; clinical diagnostics; multiplexed biosensing; nanoparticle surface functionalization; plasmonic ELISA.

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(a) Comparison of conventional ELISA and plasmonic-integrated ELISA, emphasizing enhanced sensitivity and multiplexing via plasmonic nanomaterials through LSPR and nanozyme catalysis. (b) Plasmonic ELISA modalities: (A) plasmonic ELISA achieving ultrasensitive colorimetric detection via noble metal nanoparticles. Reproduced with permission. Copyright 2016, Royal Society of Chemistry. (B) Nanozyme-linked immunosorbent assay (NLISA) using enzyme-mimicking nanomaterials for signal amplification. Reproduced with permission. Copyright 2011, Elsevier. (C) Surface-enhanced Raman scattering (SERS)-based ELISA offering highly specific biomarker detection with spectral resolution. Reproduced with permission. Copyright 2018, Frontiers.

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Unique properties of plasmonic nanostructures: (A) localized SPR. Reproduced with permission. Copyright 2022, MDPI. (B) SERS via local EF enhancement. Reproduced with permission. Copyright 2014, Wiley. (C) MEF via increased radiative rate (Γm) in the presence of nearby AuNR in comparison to that (Γ) in the absence of the AuNR. Reproduced with permission. Copyright 2023, American Chemical Society. (D) SEIRA. Reproduced with permission. Copyright 2017, American Chemical Society. (E) Photothermal effect. Reproduced with permission. Copyright 2019, Royal Society of Chemistry. (F) Plasmon-enhanced photocatalysis. Reproduced with permission. Copyright 2013, American Chemical Society.

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Illustration of the growth-based Plasmonic-ELISA for multicolor visual detection of HER2 ECD. The assay employs NADH-assisted and AA-mediated growth of gold nanobipyramids (Au NBPs). The subsequent size-dependent optical shift facilitates colorimetric detection that corresponds to the HER2 ECD concentration through antibody recognition. Reproduced with permission. Copyright 2020, American Chemical Society.

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Illustration of the etching-based plasmonic enzyme-linked immunosorbent assay (ELISA) for the detection of NT-proBNP utilizing TMB²⁺-mediated alteration of the shape of gold nanorods. The immunoassay initiates the HRP-driven oxidation of TMB, resulting in the production of TMB²⁺, which etches the AuNRs and induces a visible color change that is proportional to the concentration of the biomarker. Reproduced with permission. Copyright 2023, Royal Society of Chemistry.

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Presentation of an aggregation-based plasmonic enzyme-linked immunosorbent assay (ELISA) designed for the detection of oxLDL utilizing aptamer-functionalized AuNPs. In this procedure, the addition of sodium chloride (NaCl) facilitates aggregation, leading to a noticeable color change from red (dispersed) to purple (aggregated) upon the presence of oxLDL. Reproduced with permission. Copyright 2023, Elsevier.

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Noncovalent adsorption of antibodies onto HPNMSNs. Antigens adsorb via electrostatic and hydrophobic interactions, preserving the nanozyme catalytic activity and enhancing immunoassay sensitivity. Reproduced with permission. Copyright 2022, American Chemical Society.

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Covalent conjugation in plasmonic ELISA using mesoporous silica-encapsulated platinum nanoparticles for enhanced peroxidase-like activity in colorimetric immunoassays. Reproduced with permission. Copyright 2015, Elsevier.

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Affinity-based specific interactions in plasmonic ELISA utilizing AuNP@IgG-bio probes for the dual-modal detection of ATC through colorimetric and fluorescent sensing mechanisms. Reproduced with permission. Copyright 2021, American Chemical Society.

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Electrochemical ELISA for IL-6 detection using thiol-modified AuNPs and anti-IL-6 antibodies. AuNPs are electrodeposited on a glassy carbon electrode, followed by antibody immobilization and BSA blocking. IL-6 binding results in a concentration-dependent decrease in electrochemical current, as shown in the current (I) vs potential (E) curves. The calibration plot shows a linear detection range from 10 pg/mL to 100 ng/mL, indicating high sensitivity and specificity. Reproduced with permission. Copyright 2023, Frontiers.

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Microfluidic enzyme-linked immunosorbent assay (ELISA) on a paper-based device enables multiplexed colorimetric detection of allergens. Serum samples enter the sample pad (S) and pass through the conjugate (C), test (T), and absorbent (A) pads. AuNPs with anti-IgE antibodies (AuNP@anti-IgE) on the conjugate pad bind to allergen-specific immunoglobulin E (IgE) in the sample. Various allergens on the test pads capture these complexes, resulting in color changes that are proportional to the concentration of the allergens. Reproduced with permission. Copyright 2023, Elsevier.

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Schematic illustrating the photothermal Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of ochratoxin A (OTA). HRP catalyzes the formation of AA, which subsequently reduces Au­(III) ions to Au(0) on gold seeds, facilitating the generation of gold nanostars (AuNSs). Near-infrared (NIR) laser irradiation induces AuNSs to emit a photothermal signal, which is manifested by an increase in solution temperature. OTA acts as an inhibitor of antibody binding, resulting in a decrease in AuNS formation and a reduction in temperature rise, thus enabling the ultrasensitive detection of OTA. Reproduced with permission. Copyright 2023, Elsevier.

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A Schematic representation of SEIRA-ELISA provided for the detection of DNA hybridization, utilizing a gold (Au) nanoparticle film deposited on a zinc selenide (ZnSe) prism within the framework of the attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS) platform. The employment of the plasmonic substrate enhances infrared absorption, thereby facilitating sensitive spectral monitoring of DNA hybridization within the fingerprint region. Reproduced with permission. Copyright 2018, American Chemical Society.

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Schematic of SERS-ELISA for the detection of the SARS-CoV-2 nucleocapsid protein. (a) Conventional ELISA was performed on a 96-well plastic plate. (b) ELISA was conducted on a 384-well glass plate for increased throughput. (c) SERS-ELISA setup featuring SERS nanotags on a 384-well plate, enhancing detection sensitivity. (d) Comparative evaluation of SERS nanotags CS@SiO₂, HAuNP@SiO₂, and AuNP@SiO₂, demonstrating a superior signal from CS@SiO₂ in their Raman spectra. Reproduced with permission. Copyright 2023, Elsevier.