The work considers a combination of three enhancing approaches for immunochromatographic assay (ICA) and the integration of their impacts into changes of the limit of detection (LOD). Human fatty acid binding protein (FABP), an early biomarker of acute myocardial infarction, was the target analyte. Starting from the common ICA protocol with an LOD equal to 11.2 ng/mL, three approaches were realized: (1) replacement of spherical gold nanoparticles with gold nanoflowers having a branched surface (20-fold lowering the LOD); (2) enhanced labeling of immune complexes via nanoparticle aggregates (15-fold lowering); (3) in-situ growth of bound nanoparticles by reduction of gold salts (3-fold lowering). Single and combined implementations of these approaches have been studied. It has been shown that the LOD decrease for combined approaches is close to the multiplied contribution of each of them. The final LOD for FABP was 0.05 ng/mL, which is 220 times lower than the LOD for the common ICA protocol. The efficiency of the enhanced ICA with three combined approaches was confirmed by testing human serum samples for FABP presence and content. The development presents a new efficient technique for rapid sensitive detection of FABP for medical diagnostics. Moreover, the demonstrated multiple enhancements could be applied for various demanded analytes.
Keywords: cardiomarker; fatty acid binding protein; gold nanoparticles; immunochromatography; sensitivity enhancement.