Abstract
Ultrasensitive detection of biomarkers is highly significant for disease prognosis and public health treatment. Despite wide acceptance in routine laboratory tests, the conventional enzyme-linked immunosorbent assay (ELISA) has been of limited use for early biomarker detection due to insufficient sensitivity and multiple long incubation time. Several nanoprobes have been introduced to circumvent the limitation, however, rapid, simple, and chemical-free nanoprobe synthesis and sensitive detection methods, particularly for ELISA, are still lacking. In this study, we have synthesized a gold nanoprobe, conjugated with multiple 6X-histidine (6X-his) peptide and nickel-horseradish peroxidase (Ni2+-HRP), for enhancing the colorimetric signal in ELISA. The developed nanoprobe has been tested for the detection of immunologically significant C-reactive protein (CRP) in ELISA format. The performance of designed probe is validated by testing standard and serum samples, and the detection limit of 32.0 pg/mL with R2 = 0.98 is confirmed. Furthermore, a comparative analysis of the developed nanoprobe was performed with ELISA developed on conventional guidelines, the proposed immunoassay showed an increase of 12-fold sensitivity for detecting CRP due to the high loading of 6Xhis peptide and binding of multiple Ni2+-HRP on a gold nanoparticle. Additionally, the proposed assay provides a simple, fast, and cost-efficient (not requiring multiple antibodies) detection of CRP with easy nanoprobe synthesis. Moreover, the developed Histag-HRP functionalized nanoconjugate immunoassay is flexible and can be applied to other biomarkers efficiently by using disease specific antibody.
Highlights
C-reactive protein (CRP), an acute-phase protein (23 KDa), is regarded as a significant biomarker for various inflammatory responses
The conventional enzyme-linked immunosorbent assay (ELISA) signals are generated through 1:1 horseradish peroxidase (HRP)-secondary antibody binding ratio, which can cause insufficient signal generation leading to poor sensitivity for early biomarker screening [19,20]
The disproportionate concentration of sample and coating antibody and improper incubation time may result in poor specificity and selectivity, wastage of antibodies, and high background caused by nonspecific binding [34,37]
Summary
C-reactive protein (CRP), an acute-phase protein (23 KDa), is regarded as a significant biomarker for various inflammatory responses. The conventional ELISA signals are generated through 1:1 HRP-secondary antibody binding ratio, which can cause insufficient signal generation leading to poor sensitivity for early biomarker screening [19,20]. Some modifications, such as introduction of streptavidin and biotin-conjugated beads, and metal nanoparticles conjugates have been adopted to improve this binding ratio. Superior affinity and specificity than antibodies are additional advantages that are making them suitable for diagnostics applications [25] Inspired by these properties of peptides, we have synthesized a peptide nanoprobe and designed an immunoassay for the detection of CRP with amplified signals. The performance of the developed nanoprobe is experimentally evaluated and compared with conventional ELISA using standard solution as well as human serum, and the overall time of assay and cost of the proposed device are discussed
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