# Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds
## 3 Biosensors Based on Multivalent Binding Modes
### Biosensors Based on DNA Nanostructures  
Owing to their rigidity arising from their double helix structure, DNA nanostructures with specific shapes have emerged as scaffolds for multivalent interactions of biosensors. Various recognition systems have been designed by introducing aptamers [15; 99; 108; 109; 110] or ligands [111; 112] into specific positions of 2D or 3D DNA nanostructures such as DNA origami, tiles, and tetrahedral nanostructures capable of a trivalent binding [7; 113; 82; 114; 115; 116; 117; 118]. For instance, in 2021, [PERSON] et al. developed a cell surface imaging system based on a nanoarchitecture that mimics the behavior of membrane proteins [119]. In this study, a T-cho3 DNA nanoscalefold was designed to interact with cell membranes. This nanoscaffold has a pendant DNA probe and three cholesterol tags and forms a tetrahedral structure through the self-assembly of four DNA oligonucleotides; as such, it binds to cell membranes through trivalent binding and has been applied to stimuli-responsive cell imaging. Without potassium ion stimulation, the pendant DNA probe is blocked by the ATP aptamer, whereas when potassium ions stimulate the cell to secrete ATP, the ATP aptamer is released, and the pendant DNA probe then exists in a free form that initiates the HCR for the fluorescent imaging of cells (Figure 15). Furthermore, such molecular-recognition-initiated DNA assembly can be utilized as a scaffold for multivalent interactions in cell membranes. This allows the design of a system that can observe and manipulate cell-to-cell interactions using stimuli [120]. Figure 14: (**A**) Construction of a multivalent binding scaffold using the rolling circle amplification (RCA), fixed complementary (FC) DNAs, and molecular beacon-type aptamers. (**B**) Detection of B16 cells using the designed multivalent binding scaffolds. Adapted with permission from _ACS Biomater. Sci. Eng._ 2020, 6, 3114–3121, doi:10.1021/acsbiomaterials.0c00288. Copyright (2020) American Chemical Society [104]. In the same year, [PERSON] and co-workers developed an H\({}^{+}\)/K\({}^{+}\) dual-responsive logic sensory system that operates on the surface of cancer cells [121]. The main strategy of this work involves an in situ dimeric framework nucleic acid (FNA) assembly on the cell surface. To this end, two different tetrahedral DNA nanostructures (TDNs) bearing three cholesterols at each of their three vertices were designed for multivalent interactions (Figure 16). These two TDNs also contain an i-motif-forming sequence and a branching strand for further hybridization at their remaining vertex; the branching strand in one of the TDNs is blocked by aptamer AS1411. Once the TDNs are positioned on the cell membrane, as directed by cholesterol molecules, they can be linked to each other under certain conditions to produce signals. Low pH enables TDNs to assemble into dimeric nanostructures via i-motif formation, and K\({}^{+}\) ions promote hybridization between the branching strands of TDNs by inducing a G-quadruplex formation of aptamer AS1411. Thus, only in the presence of both H\({}^{+}\) and K\({}^{+}\) can the two TDNs form stable dimeric assemblies by proximity-induced hybridization, which produces a foster resonance energy transfer (FRET) signal (Figure 16). Using this concept, an ion-responsive logic sensor was developed. This study demonstrates that multivalent binding scaffolds constructed using DNA nanostructures are useful for sensors that probe extracellular environments. Recently, trivalent binding based on 3D nanostructures has also been applied to other systems for imaging cancer cells [122; 123; 124; 125]. For instance, the trivalent binding signals of tetrahedral structures have been amplified by RCA for the clear visualization of T-cells [126]. Additionally, a DNA framework sensor was developed for the pH analysis of cell surfaces [127], and the response of tetrahedral nanoprobes to pH variation has been applied to image exocytosis and retrieve synaptic vesicles [128].