A Combination of Membrane Filtration and Raman-Active DNA Ligand Greatly Enhances Sensitivity of SERS-Based Aptasensors for Influenza A Virus

Abstract

Biosensors combining the ultrahigh sensitivity of surface-enhanced Raman scattering (SERS) and the specificity of nucleic acid aptamers have recently drawn attention in the detection of respiratory viruses. The most sensitive SERS-based aptasensors allow determining as low as 104 virus particles per mL that is 100-fold lower than any antibody-based lateral flow tests but 10–100-times higher than a routine polymerase chain reaction with reversed transcription (RT-PCR). Sensitivity of RT-PCR has not been achieved in SERS-based aptasensors despite the usage of sophisticated SERS-active substrates. Here, we proposed a novel design of a SERS-based aptasensor with the limit of detection of just 103 particles per ml of the influenza A virus that approaches closely to RT-PCR sensitivity. The sensor utilizes silver nanoparticles with the simplest preparation instead of sophisticated SERS-active surfaces. The analytical signal is provided by a unique Raman-active dye that competes with the virus for the binding to the G-quadruplex core of the aptamer. The aptasensor functions even with aliquots of the biological fluids due to separation of the off-target molecules by pre-filtration through a polymeric membrane. The aptasensor detects influenza viruses in the range of 1·103–5·1010 virus particles per ml.

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

“…RHA0385 aptamer was chosen as a recognizing element of the sensor, since it was shown to have high affinity and selectivity towards the influenza A virus ( Zhdanov et al, 2022b ). The dissociation constant of the complex between RHA0385 and influenza A hemagglutinin is 0.6 nM ( Bizyaeva et al, 2021 ); and the dissociation constant of the complex between RHA0385 and influenza A virus is as low as 1.1 pM ( Zhdanov et al, 2022b ).…”

“…Biological media are challenging for SERS-based sensors as non-specific adsorption of biomolecules drastically decreases the signals from the target molecules. For example, blood plasma is to be diluted at least 1,000-times for colloidal SERS-sensors ( Zhdanov et al, 2022a ), whereas the effect from impurities of the allantoic fluid was observed even after 10 6 -times dilution with a buffer ( Zhdanov et al, 2022b ). Membrane filtration makes the task much easier for decreasing the concentration of non-specific compounds.…”

“…Membrane filtration makes the task much easier for decreasing the concentration of non-specific compounds. For example, influenza A virus was detected with 10 5 -times lower limit of detection due to the membrane filtration ( Zhdanov et al, 2022b ). Porous filters that provide SERS and/or SEF effects are high interest ( Kristavchuk et al, 2017 ; Feng et al, 2020 ).…”

“…Porous filters that provide SERS and/or SEF effects are high interest ( Kristavchuk et al, 2017 ; Feng et al, 2020 ). The filters can be used for both prefiltration of the sample ( Laserna et al, 1988 ; Muniz-Miranda et al, 1997 ; Zhdanov et al, 2022b ); and as flow-through chip with simultaneous filtration and signal enhancement capabilities ( Taurozzi and Tarabara, 2007 ; Feng et al, 2020 ; Serebrennikova et al, 2022 ).…”

Lithographic SERS Aptasensor for Ultrasensitive Detection of SARS-CoV-2 in Biological Fluids

“…Recently, aptamer-based analysis methods have been actively developed due to their low cost, high stability, ease of modification, and superior specificity and affinity. Combining the surface enhancement of Raman scattering and luminescence with molecular recognition through aptamers has a promising future as it allows to create effective and inexpensive sensor designs for detecting molecular targets of various nature, such as low-molecular-weight compounds [ 23 ], protein toxins [ 24 ], bacteria [ 25 ], viruses [ 12 , 26 ], and circulating cancer cells [ 27 , 28 ].…”