# Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds
## 2 Biosensors Based on Bivalent Binding Modes
### Biosensors Based on Proximity-Induced Hybridization  
The concept of bivalent biosensors based on proximity-induced nucleic acid hybridization was first reported by the [PERSON] group in 2002 [19]. This strategy is based on the DNA ligation of two adjacent probe oligonucleotides via bivalent binding; it is called proximity ligation assay (PLA). Two oligonucleotides containing an aptamer for the cytokine platelet-derived growth factor B-chain (PDGF-B) were ligated to each other using T4 DNA ligase and connector oligonucleotides in the presence of only a homodimeric form of PDGF-B (PDGF-BB) (Figure 2). The ligated product was amplified by real-time polymerase chain reaction (qPCR) and analyzed using a 5\({}^{\prime}\)-nuclease assay, affording a fluorescence signal. This PLA using bivalent interactions allowed for the detection of a target protein, PDGF, on a zeptomole scale (40 \(\times\) 10\({}^{-21}\) mol). The concept of proximity ligation has been further applied to protein-specific glycosylation [20]; the quantification of certain \(O\)-GlcNAc proteins [21]; the detection of tumor-derived exosomal PD-L1 [22], cytokines [23], and other proteins [24; 25; 26; 27; 28; 29; 30; 31]; as well as COVID-19 diagnosis [32]. Moreover, this concept has been combined with an enzymatic amplification system to afford an ultrasensitive protein sensor [33]. Nine years later, in 2011, a modified PLA called the proximity extension assay (PEA) was suggested by the [PERSON] group to overcome the limitations of PLA; notably, it showed moderate efficacy in human plasma [29]. PEAs utilize two antibody-conjugated oligonucleotide probes to detect specific antigens with bivalent interactions. In the presence of an antigen, the two probes cooperatively bind to the antigen and hybridize with each other using short overhangs. Unlike the PLA method, PEAs then employ DNA polymerase to extend the hybridizing oligonucleotides; this produces fully extended double-stranded DNA templates. The obtained template is then quantified by qPCR (Figure 3). The PEA method has shown high sensitivity and specificity and has thus been able to detect femtomolar levels of interleukin-8 (IL-8) and glial-derived neurotrophic factor (GDNF) proteins, even in human plasma. Recently, PEA strategies have also been utilized for measuring the levels of single-cell proteins [34], detecting extracellular vesicle (EV) proteins [35; 36], C-reactive protein, cardiac troponin I [37], or plasma proteins [38], monitoring drug-target interactions, [39] or profiling proteins in plasma samples of COVID-19 patients [31; 40]. Figure 2: Structure and conceptual scheme of proximity ligation assay (PLA) for the detection of PDGF-BB. Reprinted by permission from Springer Nature Ltd.: _Nat. Biotechnol._ 2002, 20, 473–477, doi:10.1038/nbt0502-473. Copyright (2002) [19]. In addition to PLAs and PEAs, relatively simpler designs have been adopted for biosensors that afford signals arising directly from proximity-induced hybridization. In this design, signaling molecules are directly linked to DNA scaffolds. A recent study reported by the [PERSON] group in 2021 [41] described the development of a detection system, proximity-DNA point accumulation in nanoscale topology (pPAINT), that relies on the spatial proximity between split docking sites. Namely, two rationally designed DNA molecules comprising docking, stem, and leash parts afford the hybridization of the stem and a complete docking strand when the two targets are in close proximity. Moreover, the transient binding of the imager strand to the docking strand allowed for the collection of fluorescent signals at the docking site, and a super-resolution image of the protein-protein interaction was reconstructed (Figure 4). This method enables the visualization of \(\alpha\)- and \(\beta\)-tubulin in close proximity with high sensitivity and low false-positive rates. A similar biosensor design was reported in the same year by [PERSON] et al. [42].