Figure 1.
Diagrammatic representations of compact waveguide-mode sensor system.
Photograph of the handy portable prototype of the sensing system. The spectral readout system is employed in the setup. Xe lamp was used as the light source.
Figure 2.
Preliminary analyses with antibody and immuno-AuNPs.
(a) Dot-blot analyses with immuno-AuNPs. Different concentrations of immuno-AuNPs were spotted on the nitrocellulose membrane. AuNPs with a diameter of 10, 20, or 40 nm were tested. (b) SEM images for the attachment of immuno-AuNPs on the CDI surfaces. The scale bar is shown. Figure inset for enlarged view. (c) Western blot analyses using the antibody generated for A/Udorn/307/1972. Lane 1: A/Udorn/307/1972 virus with uncleaved HA; Lane 2: virus with partially cleaved HA by 0.5 µg/ml TPCK-trypsin treatment at 34°C for 20 min; Lane 3: virus with completely cleaved HA by 2.5 µg/ml TPCK-trypsin treatment at 34°C for 20 min. The hemagglutinin, HA (72 kDa) is cleaved into HA1 (55 kDa) and HA2 (25 kDa). The nucleocapsid protein, NP (56 kDa) is cleaved into a 53-kDa protein. Virus proteins were separated by 18% SDS-PAGE containing 3 M urea. The anti-Udorn antibody strongly react with HA2 and M1, modestly with HA and HA1, and weakly with NP(56) and NP(53). (d) Representation of the attachment of molecules on the sensing plate. The capture antibody was attached on the N,N′-carbonyldiimidazole (CDI) surface, followed by the virus and immuno-AuNPs.
Figure 3.
Spectrum shows the interactions of A/Udorn/307/1972 and immuno-AuNP.
(a) Intact viruses and those treated with different detergents are shown. The detergents were used at a concentration of 0.5%. Vertical arrows on the figures indicate the direction of the spectral changes. Shift only occurs after the final. The green and brown lines represent the reflectivity measured after the attachment of virus and immuno-AuNPs, respectively, on the CDI surface immobilized with the capture antibody and ethanolamine. (b) Graphical representation of intact and detergent-treated viruses. Error bars are shown with averaged values.
Figure 4.
Analyses of Triton X-100 treated A/Udorn/307/1972 with immuno-AuNPs.
(a) Spectrum shows the interactions of A/Udorn/307/1972 and immuno-AuNPs. The virus was treated with different titers of Triton X-100 (0.25, 0.5, 0.75, 1, 2, and 3%). Shift only occurs after the final. The green and brown lines represent the reflectivity measured after the attachment of virus and immuno-AuNPs, respectively, on the CDI surface immobilized with the capture antibody and ethanolamine. Vertical arrows on the figures indicate the direction of the spectral changes. (b) Graphical representation of titrated Triton X-100 with viruses. Error bars are shown with averaged values. (c) Analyses of the signal magnitude of the waveguide sensing system using intact and Triton X-100 treated A/Udorn/307/1972. Error bars are shown with averaged values. (d) Waveguide spectrum with Triton X-100 treated 8 ×104 PFU/ml viruses. The green and brown lines represent the reflectivity measured after the attachment of the virus and immuno-AuNPs, respectively, on the CDI surface immobilized with the capture antibody and ethanolamine. Vertical arrow on the figures indicate the direction of the spectral changes.
Figure 5.
Analyses of pre-mixed A/Udorn/307/1972 with immuno-AuNPs.
(a) Waveguide spectrum of 8×105 PFU/ml viruses that were pre-mixed with immuno-AuNPs; 10% of the immuno-AuNPs were used. Vertical reflectivity changes are considered. Gold nanodots forming a confined ring on the surface of the virus are shown as figure inset. The blue and brown lines represent the reflectivity measured after the attachment of ethanolamine and immuno-AuNP-virus complex, respectively, on the CDI surface immobilized with anti-A/Udorn/307/1972 capture antibody. (b) SEM images shown with “O” ring formed on the viral surface. Different sizes of AuNP particles were used with diameters of 10, 20, and 40 nm. (c) Spectral analyses with the waveguide using different concentrations of immuno-AuNPs as the pre-mixer with the virus. immuno-AuNPs from 10% to 1% were used as the decreasing titer. (d) Waveguide spectrum with 8×105 PFU/ml Triton X-100–treated viruses that were pre-mixed with immuno-AuNPs. Vertical reflectivity changes are considered. Formation of nanodots on the surface of the virus is shown as a figure inset. The blue and brown lines represent the reflectivity measured after the attachment of ethanolamine and immuno-AuNP-virus complex, respectively, to the CDI surface immobilized with anti-A/Udorn/307/1972 capture antibody.
Figure 6.
Interactions of A/Brisbane/10/2007 and immuno-AuNP.
(a) SEM images shown with nanodots formed on the ruptured A/Brisbane/10/2007 surface. Size of immuno-AuNP particles used with diameter of 10 nm. (b) Spectrum shows the interactions of A/Brisbane/10/2007 and immuno-AuNPs. Different dilutions of viruses were used (1∶100, 1∶1000, 1∶10000 and 1∶100,000). These dilutions represent 12,000, 1,200, 120, and 12 pg/µl, respectively. Shift only occurs after the final. The green and brown lines represent the reflectivity measured after the attachment of virus and immuno-AuNPs, respectively, on the CDI surface immobilized with the capture antibody and ethanolamine. Vertical arrows on the figures indicate the direction of the spectral changes. (c) Graphical representation of spectral changes with the interactions of A/Brisbane/10/2007 and immuno-AuNPs. Error bars are shown with averaged values. Values less than 3% was considered to be within the error ranges and the minimum detectable limit is 120 pg/µl of A/Brisbane/10/2007.
Figure 7.
Analyses of pre-mixed A/Udorn/307/1972 with immuno-AuNPs on captured anti-rabbit IgG.
(a) Waveguide spectrum of A/Udorn/307/1972 that was pre-mixed with immuno-AuNPs; Viruses from 8×102 PFU/ml to 8×105 PFU/ml were titrated. 10% of the immuno-AuNPs were used. Vertical reflectivity changes are considered. Gold nanodots forming a confined ring on the surface of the virus are shown as figure inset. The blue and brown lines represent the reflectivity measured after the attachment of ethanolamine and immuno-AuNP-virus complex, respectively, on the CDI surface immobilized with anti-rabbit IgG as the capture antibody. (b) Graphical representation of spectral changes with the above interactions. Error bars are shown with averaged values. Values less than 3% was considered to be within the error ranges and the minimum detectable limit is 8×103 PFU/ml of A/Udorn/307/1972.