Triple Enhancement for Sensitive Immunochromatographic Assay: A Case Study for Human Fatty Acid-Binding Protein Detection

Abstract

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.

Figure 1

Scheme of test strip for ICA with triple enhancement: 1—plastic support, 2—sample pad, 3—pad with mixture of GNFs conjugates with biotinylated proteins, 4—pad with sGNP-streptavidin conjugate, 5—working membrane, 6—analytical zone, 7—control zone, 8—absorbent pad.

Figure 2

sGNPs: TEM image (A) and distribution of diameters (B).

Figure 3

Dependence of the intensity of coloration of the analytical zone of the test strip on the nature of detergents (used at 1% concentration) (A) and the detergent concentration (for the case of Tween 20 use) (B) (n = 5).

Figure 4

Common ICA protocol: Appearance of test strips (A), SEM image of the sGNPs bound in the analytical zone (B) and calibration curve for FABP detection in serum (C) (n = 5).

Figure 5

GNFs preparation: TEM images of the nuclei (A) and the final GNFs (B).

Figure 6

Choice of ICA conditions for test systems with GNPs and GNFs. Dependence of cut off values on the concentration of F5/FABP applied in the analytical zone (A) and on the optical density of the used antibody-nanoparticles conjugates solutions (B) (n = 5).

Figure 7

ICA with the use of GNFs: Appearance of test strips (A), SEM image of the GNFs bound in the analytical zone (B) and calibration curve for FABP detection in serum (C) (n = 5).

Figure 8

ICA with the use of GNPs aggregation: Appearance of test strips (A), SEM image of the sGNPs aggregates bound in the analytical zone (B) and calibration curve for FABP detection in serum (C) (n = 5).

Figure 9

ICA with the use of GNFs aggregation: Appearance of test strips (A), SEM image of the sGNFs aggregates bound in the analytical zone (B) and calibration curve for FABP detection in serum (C) (n = 5).

Figure 10

ICA with triple enhancing approaches: appearance of test strips (A), SEM image of the sGNFs aggregates bound in the analytical zone (B) and calibration curve for FABP detection in serum (C) (n = 5).