# Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds
## 4 Conclusions and Outlook  
In this review, the design strategies and applications of biosensors based on the bivalent and multivalent interactions of nucleic acid scaffolds were explored. These scaffolds  
Figure 16: Principle of an ion-responsive logic sensor based on tetrahedral DNA nanostructures. Forster resonance energy transfer (FRET) signals between Cy3 and Cy5 are observed only in the presence of both K\({}^{+}\) and H\({}^{+}\) ions. Adapted with permission from _Anal. Chem._ 2020, 92, 9273–9280, doi:10.1021/acs.analchem.0c01612. Copyright (2020) American Chemical Society [121]. Figure 17: **(top)** A star motif found in spatial patterns of epitopes of the dengue virus (DENV). **(bottom)** Design strategy of a DENV-targeting sensor based on a star-shaped DNA nanoarchitecture. Reprinted by permission from Springer Nature Ltd.: _Nat. Chem._ 2020, 12, 26–35, doi:10.1038/s41557-019-0369-8. Copyright (2020) [129]. exhibit several advantageous features, such as rigidity, structural diversity, and an ability to easily conjugate ligands at specific positions and precisely controlled distances; consequently, biosensors with enhanced specificity and sensitivity have been designed to detect and visualize diverse biologically relevant components, mainly proteins and cells. Since this review primarily described strategies for transducing binding events into observable signals, the biosensors described herein were categorized by the manner in which their nucleic acid scaffolds respond structurally to bivalent or multivalent interactions; these include proximity-induced hybridization or DNA strand displacement for bivalent interactions and chain elongation or nanostructure formation for multivalent interactions. Based on the examples presented here, we have proposed general strategies for designing bi- or multivalent biosensors. In the case of biosensors based on bivalent interactions, many examples have been reported owing to the simplicity of their design. The central principle for producing signals from bivalent interactions is changing the state of the nucleic acids involved, namely their structures. For example, the distance between the ligands on the scaffold can be made to be either longer or shorter than that between the two binding sites. In the former case, bivalent interactions bring the nucleic acids in close proximity to generate or release a new activation strand; these generated or released strands serve as starting points for proximity-induced hybridization or strand displacement strategies, respectively (Figure 18A, top). In contrast, steric-strain-induced structural changes are applicable in the latter case (Figure 18A, bottom). This is shown in the work of [PERSON]'s group [97]. The practical design of biosensors is performed by simply adding signaling molecules to specific positions of nucleic acids. Moreover, the activation strand produced by bivalent recognition would be able to conjugate with any kind of DNA nanodevice or enzymatic amplification system to devise ultrasensitive biosensors with significantly low detection thresholds. Multivalent interactions are commonly observed among proteins, viruses, and cell-to-cell interactions. Since the binding sites of such biomolecules are usually spread throughout 3D space, not 2D space, the construction of a multiple-ligand display that compliments the size, pattern, and shape of a receptor array is an essential task for the design of biosensors. To this end, two predominant strategies can be utilized to achieve multivalent recognition via nucleic acid scaffolds: one is chain elongation, and the other is the use of DNA nanostructures. The chain elongation strategy is adopted for the recognition of randomly distributed and fluid biomolecule receptors.