Highly sensitive detection of human cardiac myoglobin using a reverse sandwich immunoassay with a gold nanoparticle-enhanced surface plasmon resonance biosensor

• Published in: Analytica chimica acta Vol. 759; pp. 105 - 109
• Main Authors: Gnedenko, Oksana V., Mezentsev, Yury V., Molnar, Andrey A., Lisitsa, Andrey V., Ivanov, Alexis S., Archakov, Alexander I.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 08-01-2013; Elsevier
• Subjects: Antibodies, Immobilized - immunology; Antibodies, Monoclonal - immunology; Cardiac myoglobin; Gold - chemistry; Gold nanoparticles; Humans; Immunoassay - methods; Limit of Detection; Myocardium - chemistry; Myoglobin - analysis; Myoglobin - blood; Myoglobin - immunology; Nanoparticles - chemistry; Reverse sandwich immunoassay; Signal amplification; Surface plasmon resonance; Surface Plasmon Resonance - methods; Surface plasmon resonance; Reverse sandwich immunoassay; Cardiac myoglobin; Gold nanoparticles; Signal amplification
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2012.10.053

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► A highly sensitive immunoassay for detection of cardiac myoglobin was designed. ► A gold nanoparticles were used for SPR signal amplification. ► The limit of detection of cMb in a serum sample was found to be as low as 10pM.
A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10pM (approx. 0.18ngmL−1), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment.