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. 2023 Jan 10:10:1076749.
doi: 10.3389/fbioe.2022.1076749. eCollection 2022.

Aptamer-coated track-etched membranes with a nanostructured silver layer for single virus detection in biological fluids

Vladimir Kukushkin  1 , Olga Kristavchuk  2 , Evgeny Andreev  2 , Nadezda Meshcheryakova  3 , Olga Zaborova  3 , Alexandra Gambaryan  4 , Alexander Nechaev  2 , Elena Zavyalova  3
Affiliations

Aptamer-coated track-etched membranes with a nanostructured silver layer for single virus detection in biological fluids

Vladimir Kukushkin et al. Front Bioeng Biotechnol. .

Abstract

Aptasensors based on surface-enhanced Raman spectroscopy (SERS) are of high interest due to the superior specificity and low limit of detection. It is possible to produce stable and cheap SERS-active substrates and portable equipment meeting the requirements of point-of-care devices. Here we combine the membrane filtration and SERS-active substrate in the one pot. This approach allows efficient adsorption of the viruses from the solution onto aptamer-covered silver nanoparticles. Specific determination of the viruses was provided by the aptamer to influenza A virus labeled with the Raman-active label. The SERS-signal from the label was decreased with a descending concentration of the target virus. Even several virus particles in the sample provided an increase in SERS-spectra intensity, requiring only a few minutes for the interaction between the aptamer and the virus. The limit of detection of the aptasensor was as low as 10 viral particles per mL (VP/mL) of influenza A virus or 2 VP/mL per probe. This value overcomes the limit of detection of PCR techniques (∼103 VP/mL). The proposed biosensor is very convenient for point-of-care applications.

Keywords: SERS; aptamer; aptasensor; influenza; membrane; virus.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The nanostructured surface of the tracked-etched membrane. (A) Scanning electron microscopy of the surface. (B) The distribution diagram of a nanoparticle size on the track-etched membrane.
FIGURE 2
FIGURE 2
Surface-enhanced fluorescence (A) and surface-enhanced Raman (B) spectra of Cyanine-3 reporter obtained from the track-etched membrane functionalized with the aptamer before and after the filtration of phosphate-buffered saline.
FIGURE 3
FIGURE 3
Proposed operating principle of the aptasensor. (A) Random orientation of the aptamer with the reporter toward the surface; (B) PBS filtration modified the surface providing a negatively charged surface with increased distance between the reporter and the surface; (C) the filtration of the virus provided specific interactions between aptamers and viral particles orienting the reporter in the common manner near the surface.
FIGURE 4
FIGURE 4
Surface-enhanced fluorescence (A) and surface-enhanced Raman (B) spectra of Cyanine-3 reporter obtained from the track-etched membranes functionalized with the aptamer after the filtration of the probes with different concentrations of influenza A virus.
FIGURE 5
FIGURE 5
Scanning electron microscopy of the track-etched membranes functionalized with the aptamer after the filtration of phosphate-buffered saline (A), 3·108 VP/mL of influenza A virus (B), 2·106 VP/mL of influenza A virus (C), and 30 VP/mL of influenza A virus (D).
FIGURE 6
FIGURE 6
A nanoparticle tracking analysis of the influenza A virus sample. The experimental size distribution of the nanoparticles and its approximation.
FIGURE 7
FIGURE 7
A concentration dependence of a normalized surface-enhanced Raman spectrum intensity of signal from Cyanine-3 reporter on the concentration of influenza A virus and influenza B virus (A). The part of the dependence with a monotonous increase of the signal was used to calculate the limit of detection that is indicated by an arrow (B).
FIGURE 8
FIGURE 8
A concentration dependence of a normalized surface-enhanced fluorescence spectrum intensity of signal from Cyanine-3 reporter on the concentration of influenza A virus and influenza B virus (A). The part of the dependence with monotonous increase of the signal was used to calculate the limit of detection that is indicated by an arrow (B).
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