Direct optical detection of viral nucleoprotein binding to an anti-influenza aptamer

Abstract

We have demonstrated label-free optical detection of viral nucleoprotein binding to a polyvalent anti-influenza aptamer by monitoring the surface-enhanced Raman (SERS) spectra of the aptamer-nucleoprotein complex. The SERS spectra demonstrated that selective binding of the aptamer-nucleoprotein complex could be differentiated from that of the aptamer alone based solely on the direct spectral signature for the aptamer-nucleoprotein complex. Multivariate statistical methods, including principal components analysis, hierarchical clustering, and partial least squares, were used to confirm statistically significant differences between the spectra of the aptamer-nucleoprotein complex and the spectra of the unbound aptamer. Two separate negative controls were used to evaluate the specificity of binding of the viral nucleoproteins to this aptamer. In both cases, no spectral changes were observed that showed protein binding to the control surfaces, indicating a high degree of specificity for the binding of influenza viral nucleoproteins only to the influenza-specific aptamer. Statistical analysis of the spectra supports this interpretation. AFM images demonstrate morphological changes consistent with formation of the influenza aptamer-nucleoprotein complex. These results provide the first evidence for the use of aptamer-modified SERS substrates as diagnostic tools for influenza virus detection in a complex biological matrix.

Figure 1

(a) SERS spectrum of the anti-influenza aptamer (1000 nM) - PEG spacer (100 nM) complex on a Ag nanorod substrate, (b) anti-influenza aptamer complex and binding buffer blank control, (c) anti-influenza aptamer complex and RSV (10⁵ PFU/mL) negative control, (d) anti-influenza aptamer complex and nucleoproteins from A/Uruguay, (e) anti-influenza aptamer complex and nucleoproteins from A/Brisbane, and (f) anti-influenza aptamer complex and nucleoproteins from B/Brisbane. Virus concentrations in (d) – (f) were adjusted to 1μg/mL (relative to HA content). Each spectrum shown is an average of 10 individual spectra for each particular sample. The dashed vertical lines in (a), (b), and (c) indicate the characteristic oligonucleotide bands for the influenza aptamer and its controls. The dashed vertical lines in (d), (e), and (f) indicate the oligonucleotide bands that changed after binding of the nucleoproteins. Asterisks indicate the presence of new bands in the aptamer complex corresponding to binding of the protein target.

Figure 2

Log-log plot of the absolute SERS intensity of the 731 cm⁻¹ band of the anti-influenza aptamer as a function of concentration in the range from 0.1 to 5000 nM.

Figure 3

Dendrogram produced by hierarchical cluster analysis (HCA) of the SERS spectra shown in Fig. 1 using Ward’s method and label-coded as follows: (A) anti-influenza aptamer, blank buffer, and RSV negative control, (B) anti-influenza aptamer incubated with influenza nucleoproteins from A/Uruguay, A/Brisbane, and B/Brisbane. A total of 240 spectra were used to generate this dendrogram, corresponding to 40 spectra in each of the 6 sample categories.

Figure 4

SERS spectra of the anti-vasopressin aptamer, controls, vasopressin, and influenza nucleoproteins on a Ag nanorod substrate after incubation at 37 °C for 8 hours. (a) anti-vasopressin aptamer (1000 nM) - spacer (100 nM), (b) vasopressin aptamer with blank binding buffer control, (c) vasopressin aptamer with vasopressin (1μg/mL), (d) vasopressin aptamer with nucleoproteins from A/Uruguay, (e) vasopressin aptamer with nucleoproteins from A/Brisbane, and (f) vasopressin aptamer with nucleoproteins from B/Brisbane. Virus concentrations in (d) – (f) are 1μg/mL (relative to HA content). Each spectrum shown is an average of 10 individual spectra for each particular sample. The dashed vertical lines in (a), (b), and (c) indicate the characteristic oligonucleotide bands for the anti-vasporessin aptamer. Asterisks indicate the presence of new bands in the aptamer complex corresponding to binding of the protein target.

Figure 5

Dendrogram produced by hierarchical cluster analysis (HCA) of the SERS spectra shown in Fig. 4 calculated using Ward’s method and label-coded as follows: (A) anti-vasopressin aptamer complex and influenza nucleoproteins from A/Uruguay, A/Brisbane, B/Brisbane, (B) anti-vasopressin aptamer (1000 nM) - spacer (100 nM) complex, along with blank buffer control, and (C) anti-vasopressin aptamer complex and vasopressin (1μg/mL). A total of 240 spectra were used to generate this dendrogram, corresponding to 40 spectra in each of the 6 sample categories.

Figure 6

(Top) 500 nm × 500 nm 2-D AFM images of (a) bare Ag nanofilm substrate, (b) Ag substrate after injection of the disulfide anti-influenza aptamer at a concentration of 100 pM for 3 hours, (c) Ag substrate after injection of (1-Mercaptoundec-11-y)tetra(ethyleneglycol) at a concentration of 10 pM for 3 hours, (d) Ag substrate after injection of A/Uruguay diluted in the binding buffer to a concentration of 1 pg/mL (HA) content at 37°C for 3 hours. (Middle) The plots show the surface roughness line scans corresponding to the green line on each of the 2-D AFM images immediately above it in Panels (a), (b), (c) and (d). (Bottom) Panels (e), (f), (g), and (h) are the representative 3-D AFM images of the 2-D images in (a), (b), (c) and (d), respectively.