Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I

Abstract

Influenza A viruses (IAV) are classified based on their surface proteins hemagglutinin (HA) and neuraminidase (NA). Both pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses pose a significant threat to public health. Effective methods to simultaneously distinguish H1N1 and H5N1 are thus of great clinical value. In this study, a protocol for detection of HA proteins of both H1N1 and H5N1 was established. Specifically, we designed an aptasensor for HA using fluorescence resonance energy transfer (FRET) strategy combined with DNase I-assisted cyclic enzymatic signal amplification. HA aptamers of H1N1 and H5N1 IAVs labeled with various fluorescent dyes were used as probes. Graphene oxide (GO) acted as a FRET acceptor for quenching the fluorescence signal and protected aptamers from DNase I cleavage. The fluorescence signal was recovered owing to aptamer release from GO with HA protein. DNase I-digested free aptamers and HA proteins were able to further interact with more fluorescent aptamer probes, resulting in increased signal amplification. The limits of detection (LOD) of H5N1 HA and H1N1 HA were 0.73 and 0.43 ng/ml, respectively, which were 19 and 27 times higher than LOD values obtained with the DNase I-free system. The recovery rate of HA protein in human serum samples ranged from 88.23 to 117.86%, supporting the accuracy and stability of this method in a complex detection environment. Our rapid, sensitive, and cost-effective novel approach could be expanded to other subtypes of IAVs other than H1N1 and H5N1.

Keywords: DNase I; FRET; H1N1; H5N1; hemagglutinin.

Figure 1

Schematic diagram of dual detection of HA protein from influenza viruses H5N1 and H1N1 based on FRET combined with DNase I.

Figure 2

Optimization of GO reaction conditions. (A) Fluorescence emission spectral signals of the H5N1-FAM probe. (B) H1N1-ROX probe in the presence of different concentrations of GO (0, 10, 20, 30, 40, 50, 60, 80, and 100 μg/ml). (C) Changes in trends of fluorescence signals of H5N1-FAM and H1N1-ROX probes in the presence of different concentrations of GO (0, 10, 20, 30, 40, 50, 60, 80, and 100 μg/ml). (D) Changes in trends of fluorescence signals at different time points (0, 1, 2, 3, 5, 10, 15, and 20 min) in the presence of GO (50 μg/ml).

Figure 3

Selection and optimization of blocking agents. (A) Fluorescence recovery effects of different types of blocking agents. (B) Effects of PEG800 concentration on fluorescence quenching (black bar) without HA and fluorescence recovery (red bar) with HA. (C) Effects of PEG800 concentration on fluorescence signals.

Figure 4

Examination of cross-reactions by monitoring fluorescence spectra of the two probes. (A) Different concentrations of HA of H5N1 in the detection system. (B) Different concentrations of HA of H1N1 in the detection system. A range of HA protein concentrations (0, 200, 500, and 1,000 ng/ml) was examined at a fixed GO concentration of 50 μg/ml.

Figure 5

Feasibility of GO quenching and DNase I cleavage in the reaction system.

Figure 6

Effects of different doses of DNase I on fluorescence recovery for (A) H5N1-FAM and (B) H1N1-ROX probes in the detection system. Effects of incubation times of DNase I on fluorescence recovery for (C) H5N1-FAM and (D) H1N1-ROX probes.

Figure 7

Effects of DNase I on the sensitivity of detection of H5N1 HA. (A) Fluorescence spectra of a series of concentrations of H5N1 HA (0, 0.5, 1, 1.5, 2, 3, 5, 7, 10, 12, and 15 ng/ml) in the presence of DNase I. (B) (F – F0)/F0(fluorescence change) vs. concentration of H5N1 HA in the presence of DNase I. (C) Fluorescence intensities of a series of concentrations of H5N1 HA (0, 10, 20, 30, 50, 70, 100, 120, 150, and 200 ng/ml) in the absence of DNase I. (D) (F – F0)/F0 (fluorescence change) vs. concentration of H5N1 HA in the absence of DNase I. The insets show a linear relationship between (F – F0)/F0 and H5N1 HA concentration in the presence of DNase I (B) and the absence of DNase I (D).

Figure 8

Effects of DNase I on the sensitivity of detection of H1N1 HA. (A) Fluorescence spectra of a series of concentrations of H5N1 HA (0, 0.5, 1, 1.5, 2, 3, 4, 5, 7, 10, 15, and 20 ng/ml) in the presence of DNase I. (B) (F – F0)/F0 (fluorescence change) vs. concentration of H1N1 HA in the presence of DNase I. (C) Fluorescence intensities of a series of concentrations of H1N1 HA (0, 10, 20, 30, 50, 70, 100, 120, 150, and 200 ng/ml) in the absence of DNase I. (D) (F – F0)/F0 (fluorescence change) vs. concentration of H1N1 HA in the absence of DNase I. The insets depict a linear relationship between (F – F0)/F0 and the concentration of HA H1N1 in the presence of DNase I (B) and the absence of DNase I (D).

Figure 9

Specificity analysis of the aptamer sensor. HA proteins from H1N1 and H5N1 act as counterpoint controls for each other. Background control protein BSA and human IgG are at a concentration of 20 ng/ml.