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

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.

Site-Specific Covalent Immobilization of SMA-Stabilized ACE2 for SARS-CoV-2 Recognition and Drug Screening

“…The calibration curve shows that the modified method identifies virus concentrations as low as 80 VP μL –1 (Figure Dc), and the limit of detection (LOD) is estimated to be 3.8 × 10 3 VP mL –1 , defined as 3 × SD of the blank signals. The LOD of this method is 5 orders of magnitude lower than the antibody-based test strip (5.0 × 10 8 VP mL –1 ) and slightly higher than RT-PCR (1 × 10 2 VP mL –1 ) . However, this method takes less than 15 min in a single test, which is faster than RT-PCR (2–3 h), and antigen-based detection can detect the presence of infectious viral entities, which makes up for the lack of a positive nucleic acid test that cannot accurately determine the presence of active and infectious viruses in the tested sample.…”

“…The LOD of this method is 5 orders of magnitude lower than the antibody-based test strip (5.0 × 10 8 VP mL −1 ) and slightly higher than RT-PCR (1 × 10 2 VP mL −1 ). 44 However, this method takes less than 15 min in a single test, which is faster than RT-PCR (2−3 h), and antigenbased detection can detect the presence of infectious viral entities, which makes up for the lack of a positive nucleic acid test that cannot accurately determine the presence of active and infectious viruses in the tested sample. This method has a rapid response to virus aerosols and acceptable sensitivity in the case of SARS-CoV-2 spike pseudovirus simulation, which provides a research basis for the detection of virus particles in the environment such as aerosols and water.…”

Aptamers Targeting Membrane Proteins for Sensor and Diagnostic Applications

“…The rapid and ultrasensitive technique has recently gained considerable momentum with regard to the detection of respiratory viruses for the purpose of point-of-care testing, especially after the COVID-19 pandemic. When combined with the specificity and versatility (here, Raman-active labeling) of nucleic acid aptamers, the technique was able to reach low limits of detection (that could challenge the limits of PCR techniques) as shown for SARS-CoV-2 [163,164] as well as the influenza A virus [165,166]. SERS was also applied recently for the sensitive detection of cancer biomarkers, such as carcinoembryonic antigen (CEA) [167].…”

Nanoisland SERS-Substrates for Specific Detection and Quantification of Influenza A Virus

“…Here we discuss the SERS-based aptasensors with LoD values compared to or lower than the LoD of polymerase chain reaction with reverse transcription (≤10 3 VP/mL) with a time of analysis below 20 min. Interestingly, antibody-based SERS sensors concede to aptasensors in LoD values and time of analysis [21,22]; the possible reason is a large size of proteins as compared with aptamers, as well as the SERS decrease during protein adsorption, demanding a complex multilayer architecture of the sensors.…”

“…So, a Raman label was conjugated to the aptamer; the reorientation of the label during virus binding provided an analytical signal. The LoD of the aptasensor was as low as 10 VP/mL, with a time of analysis of 15 min [22]. Interestingly, the sandwich-like approach with two aptamers for catching and labeling the virus was also realized using polymeric membranes with silver nanoisland coatings with a much higher LoD of 2 × 10 4 VP/mL [23].…”