Optical Nanoantenna for Single Molecule-Based Detection of Zika Virus Nucleic Acids without Molecular Multiplication

Abstract

Because of the limited signal-to-background ratio, molecular diagnostics requires molecular amplification of the target molecules or molecular signal amplification after target recognition. For direct molecular detection, we demonstrate a purely physical fluorescence enhancement process which can elevate the fluorescence signal of single fluorescent dyes by several orders of magnitude. To this end, DNA origami-based optical antennas with a height of around 125 nm are used, which utilize metallic nanoparticles to create a hotspot where fluorescence signals are enhanced by plasmonic effects. By equipping the hotspot with a molecular beacon-like structure, we combine the plasmonic signal enhancement with a specific signal generation, leading to an enhanced and therefore easy to detect signal only in the presence of the specific target nucleic acid. Exemplified with Zika virus detection, we show the applicability of this approach by detecting Zika-specific artificial DNA and RNA both in buffer and in heat-inactivated human blood serum. We show the sensitivity against small nucleotide variations of this approach, allowing the discrimination of closely related pathogens. Furthermore, we show how the modularity offered by DNA nanotechnology enables multiplexing by incorporating orthogonal fluorescent labels for the simultaneous detection of different sequences. The achieved signal enhancement will allow technically simplified signal detection, paving the way for single molecule-based point-of-care diagnosis.

DNA-Functionalized Dye-Loaded Polymeric Nanoparticles: Ultrabright FRET Platform for Amplified Detection of Nucleic Acids

“…The heating at 40°C is probably necessary in order to increase the probability of collision between DBCO group grafted to oligonucleotide and azide group at the particle surface, in line with relatively slow DBCO/azide reaction kinetics reported recently for other type of polymeric NPs. 28c However, in phosphate buffer containing 12 mM Mg 2+ ions, required for formation of stable duplexes, 25 DNA-NPs conjugates (NP-SurC) showed relatively large size (Figure 3a), probably because of partial aggregation of the obtained NPs.…”

“…6c,20e Very recently, Tinnefeld and co-workers pioneered the amplified detection of NAs based on plasmonics nanoantenna positioned precisely in space with help of DNA origami. 25 The amplification achieved was 7.3 on average, although higher values were observed for some NPs. In this respect, our organic nanoantennas are expected to offer new possibilities.…”

Betanodavirus and VER Disease: A 30-year Research Review

“…Despite all the significant progress achieved in NNV detection over the last few years, recent advances linked to the use of nanotechnology, already reported in human and animal viruses [298][299][300][301][302][303][304][305][306], could provide a substantial improvement in these diagnostics in the near future. Different nanostructures are available at present, such as nanoparticles (NP), carbon nanotubes (CNT), dendrimers, and quantum dots (QDs) among others, and can be applied to the identification of nucleic acids, proteins and viral particles as well as antibodies [307,308].…”

The Growing Development of DNA Nanostructures for Potential Healthcare‐Related Applications

“…Therefore, errors in placement on a sensor configuration can dampen the signal to noise ratio by many orders of magnitude. To this end, DNA‐based “nanoantennas” have been engineered to precisely colocalize plasmonic NPs with fluorescent target capture motifs . In one representative example, a rigid DNA origami hb (125 nm) was modified to attach a spherical silver NP (80 nm) and a fluorescence‐quencher labeled DNA hairpin that was complementary to the target nucleic acid analyte (Figure C).…”