# Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds
## 2 Biosensors Based on Bivalent Binding Modes
### Biosensors Based on Proximity-Induced Hybridization
This study is also based on the spatial proximity of DNA strands arising from the presence of a target. The system consisted of gold nanoparticles (AuNPs) bearing fluorophore-labeled DNA and a pair of DNA-antibody conjugates, each having a split fragment of a DNAzyme. In the presence of the target viruses, enteroviruses EV71 and CVB3, the antibody on each conjugate binds to the virus, and, consequently, the activity of the DNAzyme is restored. Then, the fluorophore-labeled DNA, the target of the DNAzyme, is cleaved to release the fluorophore from the AuNPs, which produces a fluorescence signal (Figure 5). This system demonstrates that simple biosensors based on proximity-induced hybridization are capable of detecting viruses in clinical samples. Figure 4: Biosensing strategy of proximity-DNA point accumulation in nanoscale topology (pPAINT) for direct signaling from proximity-induced hybridization induced by bivalent interactions [41]. Figure 3: Conceptual scheme of proximity extension assays (PEA) for the detection of biomolecules such as antigens. Reproduced by permission of Oxford University Press: _Nucleic Acids Res._ 2011, 39, e102, doi:10.1093/nar/gkr424. Copyright (2011) [29]. In 2018, [PERSON] et al. designed the simplest bivalent biosensor for antibodies based on proximity-induced hybridization [43]. While other proximity-induced hybridization methods require an additional strand for signaling, this method requires only a pair of split green fluorescent protein (GFP)-mimic RNA aptamers modified with an antigen as a signaling component. The presence of an antibody gives rise to the assembly of aptamers that afford a binding site for 3,5-difluoro-4-hyroxybenzylidene imidazoline (DFHBI), which yields a fluorescent signal upon binding (Figure 6). Through this method, different types of antibodies were selectively detected. Furthermore, despite its simplicity, the design showed high binding affinity, and target specificity originating from bivalent binding and worked well even in crude cellular extracts. Recently, other, similar approaches have also used proximity-induced hybridization to produce advanced bivalent biosensors; these include an electrochemical proximity assay for the femtomolar detection of insulin [44], a biosensor employing nicking endonuclease-assisted isothermal fluorescence signal amplification for the detection of streptavidin or prostate-specific antigen (PSA) [45], a proximity hybridization-regulated DNA biogate for PSA detection [46], a DNAzyme-based DNA nanomotor for detection of thrombin [47], a  
Figure 5: Principle operation of a biosensor for virus detection. The method is enabled by proximity-induced DNAzyme formation and fluorophore-labeled gold nanoparticles (AuNPs). Adapted with permission from _Anal. Chem._ 2021, 93, 5606–5611, doi:10.1021/acs.analchem.1c00335. Copyright (2021) American Chemical Society [42]. Figure 6: Conceptual scheme for a fluorogenic antibody sensor based on split green fluorescent protein (GFP)-mimic RNA aptamers and 3,5-difluoro-4-hyroxybenzylidene imidazoline (DFHBI). Adapted with permission from _Anal. Chem._ 2018, 90, 1049–1053, doi:10.1021/acs.analchem.7b02102. Copyright (2018) American Chemical Society [43]. cytosensor for the detection of circulating tumor cells [48], a scattering proximity immunoassay based on the dimerization of AuNPs for detecting carcinoembryonic antigen [49], and other enzymatic methods [50; 51]. Moreover, proximity-induced hybridization has been combined with templated reaction techniques to identify antibodies [52]. These recent studies imply that proximity-induced hybridization is a simple yet potent strategy for building biosensors based on bivalent binding.