Digital diffraction detection of protein markers for avian influenza

Abstract

Rapid pathogen testing is expected to play a critical role in infection control and in limiting epidemics. Smartphones equipped with state-of-the-art computing and imaging technologies have emerged as new point-of-use (POU) sensing platforms. We herein report a new assay format for fast, sensitive and portable detection of avian influenza-associated antibodies.

Fig. 1. Molecular detection with digital diffraction diagnostic (D3) system

(a) The detection of target molecules is based on the transmittance changes of silica beads (Si-MBs) measured by the holographic imaging system. The assay consists of two steps. First, target analytes (e.g. proteins, antibodies) are captured on Si-MBs via affinity ligands, and subsequently labeled with Au nanoparticles (AuNPs). Second, Ag shells are overgrown on the beads using AuNPs as seeds. The transmittance of the Si-MBs decreases as the shell grows. (b) Transmission electron micrographs of bare, AuNP-coated, and Ag-coated Si-MBs (from left to right). Scale bars: 500 nm (left); 50 nm (middle and right). (c) Microscope images of Si-MBs before and after Ag-shell growth were shown. (d) An imaging module was attached on a smartphone, and a bead sample was imaged.

Fig. 2. Diffraction characterization of silica beads with and without Ag shells

(a) Diffraction patterns and reconstructed images of a bare, a control, and an Ag-coated Si-MBs (diameter, 7 μm). The control was prepared by mixing avidin-coated Si-MBs with pegylated AuNPs. The Ag-coated Si-MB could be distinguished from other Si-MB types. Scale bars: 20 μm. (b – d) Cross-sectional profiles across the bead diameter (x-axis) with and without Ag coating: (b) diffraction intensity; (c) transmittances; (d) phase.

Fig. 3. Assay optimization

(a) The transmittance changed during the Ag-coating, reaching a saturation in 5 min after the reaction started. (b) Optimization of the light source. Among the wavelengths tested (420, 470, 530 and 590 nm), the transmittance difference between bare and Ag-coated Si-MBs was the largest with 470-nm illumination. (c) Transmittance histograms of bare (reference), control, and Ag-coated Si-MBs. The optical transmittance cutoff (0.45) was determined to differentiate Ag-coated beads from non-coated ones. (d) Samples were prepared by mixing Ag-coated and non-coated Si-MBs at different ratios. From the reconstructed images, the counting algorithm identified Ag-coated beads according to the transmittance cutoff. The measured ratios agreed well with the expected values (R² = 0.98). Experiments were performed in duplicate, and the graphs are displayed as mean ± s.d in (a), (b) and (d).

Fig. 4. Detection of avian influenza antibody

(a) Anti-hemagglutinin antibody (HA-Ab) was detected using Si-MBs coated with HA Tag peptide. Transmittance (left) and phase (right) changes are shown. (b) Correlation between D3 and ELISA measurements. (c) Titration experiments. Samples were prepared by spiking different amounts of HA-Ab into chicken sera. The D3 assay showed about 10-times better sensitivity than conventional enzyme-linked immunosorbent assay (ELISA). All experiments were performed in duplicate, and the graphs are displayed as mean ± s.d.