The bonding interaction amongst Influenza A and the peptide “PeB,” which selectively binds the viral surface protein hemagglutinin, has been investigated using electrically controlled deoxyribonuclic acid (DNA) nanolevers in the journal Advanced Materials.

PeB is conjugated to DNA strands that are bonded to complementary anchors and fixed on the electrode surface of a “switchSENSE” biochip. A fluorophore is attached to the surface-tethered DNA strand, whereas the complementary strand has a multivalent configuration containing up to three PeB peptides. A negative voltage is used to keep the nanolevers erect (static).

As the current epidemic of the SARS-CoV-2 virus has demonstrated, viral pandemics represent a tremendous threat to humanity. A similar pandemic, known as the Spanish flu, was induced by an influenza A virus and led to millions of fatalities around the world. Annual outbreaks of different intensity are still caused by influenza A viruses. The globe was recently confronted with the swine flu in 2009.

Influenza A is an Orthomyxoviridae virus that is encased in a lipid bilayer that contains three cell membranes: hemagglutinin (HA), neuraminidase (NA), and the M2 proton channels. Neuraminidase is a glycoside cleaver that is essential for the discharge of viral particles from infected cells.

Subsequent to viral fusing, hemagglutinin binds to the host cell’s sialic acid (SA)-containing cellular receptors. Understanding viral internalization and infection requires kinetic analysis of affinity and fervency coefficients of various receptor binding to hemagglutinin.

a) Top view of a switchSENSE microfluidic biochip with four flow channels. Each channel comprises six gold electrodes. The more detailed view shows the six independent measurement electrodes in a row within the microfluidic channel. b) Schematic overview of a single stranded DNA nanolever (NL-B48) immobilized on a gold electrode via thiol coupling. The nanolever carries a fluorophore at the lateral end. c) The DNA monolayer is functionalized with the ligand of interest by hybridization of the complementary DNA strand carrying a receptor molecule at the distal end. d) Dynamic measurement mode: The double stranded DNA nanolever is actuated to a switching motion by applying an alternating potential. The fluorescence signal is gradually quenched by energy transfer upon approaching the gold surface. Read-out of this mode is the switching speed of DNA nanolevers. The switching speed is slowed down upon binding of an analyte to the ligand molecule adding friction to the nanolever. e) Static measurement mode: DNA nanolevers are kept at an upright position. Read-out of this mode is the change in fluorescence intensity upon binding of an analyte due to changes in the local environment of the dye. Drawings are not to scale.

a) Top view of a switchSENSE microfluidic biochip with four flow channels. Each channel comprises six gold electrodes. The more detailed view shows the six independent measurement electrodes in a row within the microfluidic channel. b) Schematic overview of a single stranded DNA nanolever (NL-B48) immobilized on a gold electrode via thiol coupling. The nanolever carries a fluorophore at the lateral end. c) The DNA monolayer is functionalized with the ligand of interest by hybridization of the complementary DNA strand carrying a receptor molecule at the distal end. d) Dynamic measurement mode: The double stranded DNA nanolever is actuated to a switching motion by applying an alternating potential. The fluorescence signal is gradually quenched by energy transfer upon approaching the gold surface. Read-out of this mode is the switching speed of DNA nanolevers. The switching speed is slowed down upon binding of an analyte to the ligand molecule adding friction to the nanolever. e) Static measurement mode: DNA nanolevers are kept at an upright position. Read-out of this mode is the change in fluorescence intensity upon binding of an analyte due to changes in the local environment of the dye. Drawings are not to scale. Image Credit: Kruse M et al., Advanced Material

The ability to calculate not just the dissociation constant but also rate constants is one of the key features of the switchSENSE technique. These kinetic metrics are becoming increasingly important in determining the effectiveness of candidates in medication development.

Other methods for determining dissociation constants exist, but they lack the capacity to resolve bonding kinetic parameters. Radioligand binding tests, microscale thermophoresis (MST), and isothermal calorimetry are some examples of these.

Surface plasmon resonance is one technology that allows for real-time monitoring of kinetic parameters (SPR). SPR measures and switchSENSE technologies are similar in that they both have benefits and drawbacks. As a result, a similar naming system is used.

The quantifiable metrics of kinetic parameters in a timely manner, label-free detection, minimal sample expenditure, and high sensitivity of the technology are all benefits of SPR and switchSENSE. The disadvantages of both procedures are that one of the participants must be immobilized, which may have an impact on binding behavior, and that mass transfer is limited….

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