A novel reverse colorimetric immunoassay (RCIA) strategy was for the first time designed and utilized for sensitive detection of low-abundance protein (prostate-specific antigen, PSA, used in this case) in biological fluids by coupling highly catalytic efficient catalase with magnetic bead-based peroxidase mimics. To construct such a RCIA system, two nanostructures including magnetic beads and gold nanoparticles were first synthesized and functionalized with anti-PSA capture antibody and catalase/anti-PSA detection antibody, respectively. Thereafter, a specific sandwich-type immunoassay format was employed for determination of PSA by using functional gold nanoparticles as enzymatic bioreactors and anti-PSA-conjugated magnetic beads as a colorimetric developer. The carried catalase, followed by the sandwiched immunocomplex, partially consumed the added hydrogen peroxide in the detection solution, which slowed down the catalytic efficiency of magnetic bead-based peroxidase mimics toward TMB/H2O2, thereby weakening the visible color and decreasing the colorimetric density. Different from conventional colorimetric immunoassay, the RCIA method determined the residual hydrogen peroxide in the substrate after consumption. Under the optimal conditions, the developed RCIA exhibited a wide dynamic range of 0.05-20 ng mL(-1) toward PSA with a detection limit of 0.03 ng mL(-1) at the 3Sblank level. Intra- and interassay coefficients of variation were below 6.1% and 9.3%, respectively. Additionally, the methodology was further validated for the analysis of 12 PSA clinical serum specimens, giving results in good accordance with those obtained by the commercially available enzyme-linked immunosorbent assay (ELISA) method.
A label-free nanozyme MIL-53(Fe) with the dual-function of catalyzing and emitting fluorescence was utilized for turn-on fluorescence detection of hydrogen peroxide and glucose. The proposed strategy provides a cost-effective, safe and sensitive method for the design and development of multiple enzyme cascade assays for various biomolecules.
A new impedimetric immunoassay protocol based on enzyme-triggered formation of tyramine-enzyme repeats on gold nanoparticle (AuNP) was designed for highly sensitive detection of carcinoembryonic antigen (CEA, as a model) by virtue of utilizing enzymatic biocatalytic precipitation toward 4-chloro-1-naphthol (4-CN) on anti-CEA antibody (Ab1)-modified immunosensor. Initially, AuNP was functionalized with horseradish peroxidase and detection antibody (HRP-AuNP-Ab2), and then HRP-tyramine conjugate was utilized for the formation of tyramine-HRP repeats through the triggering of the immobilized HRP on the AuNP with the aid of H2O2. In the presence of target CEA, the carried HRP-tyramine repeats accompanying the sandwiched immunocomplex catalyzed the 4-CN oxidation to produce an insoluble precipitation on the immunosensor, thus causing a local alteration of the conductivity. Three signal-transduction tags including HRP-Ab2, HRP-AuNP-Ab2, and HRP-AuNP-Ab2 with HRP-tyramine repeats were employed for target CEA evaluation, and improved analytical properties were achieved by HRP-AuNP-Ab2 with HRP-tyramine repeats. Using the unique signal-transduction tag, the analytical performance of the impedimetric immunoassay was studied in detail. Under the optimal conditions, the impedimetric immunosensor displayed a wide dynamic working range of between 0.5 pg mL(-1) and 40 ng mL(-1) with a detection limit (LOD) of 0.38 pg mL(-1) relative to target CEA. The coefficients of variation (CVs) were ≤9.3% and 13.3% for the intra-assay and interassay, respectively. The levels of CEA in eight clinical serum specimens were measured by using the developed impedimetric immunosensor. The obtained results correlated well with those from the electrochemiluminescent (ECL)-based immunoassay with a correlation coefficient of 0.998.
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