# Multivalent Aptamer Approach: Designs, Strategies, and Applications
## 3 Design Strategies
### Heterovalent Aptamers  
Heterovalent aptamer strategies, also known as aptamer cocktails, use two or more types of aptamers that target different moieties of the target. In comparison with single type of aptamer, heterovalent aptamer approaches have been shown to result in the cooperative enhancement of overall aptamer performances. For example, an 18-fold improvement of limit of detection (LOD) was observed using an aptamer cocktail-modified electrode (three different aptamers targeting different moieties on the surface of _E. coli_ cells) compared to an electrode modified with a single-type aptamer [87]. The aptamer-cocktail strategies have shown exciting potentials in CTC detections. Traditionally, CTC detections face issues such as low CTC occurrences in clinical samples and difficulties in targeting appropriate CTC biomarkers, since the presence of these surface markers is known to be non-uniform between individual patients [18]. To overcome these obstacles, one can target different arrays of CTC biomarkers simultaneously to both increase binding avidities and prevent the off-target effects due to the reduced or loss of certain surface biomarker expressions on the CTCs [88]. For example, [PERSON] et al. [18] developed a dual-aptamer-tethered network system that allowed DNA-triggered reversible isolation-and-release of CTCs by tethering Sgc8c and Sgc4f aptamers onto a ssDNA scaffold. This dual-type aptamer network system showed significantly higher capturing efficiency compared to a single-type aptamer network. Similarly, [PERSON] et al. [89] combined the aptamer-cocktail strategy and poly(dimethylsiloxane) (PDMS) microfluidic detection platform to detect CTCs, as shown in Figure 3. The researchers showed that the aptamer cocktail exhibited a synergetic effect in target capturing and that the aptamer-cocktail approach demonstrated a higher overall capturing efficiency when compared with the single-type aptamer approach. Another way of utilizing heterovalent aptamers is multiplex detection, which allows for the simultaneous detection of more than one target. Multiplex detections significantly reduce sample size and runtime required for detection. For example, by combining aptamer-conjugated gold nanoparticles (AuNPs) with magnetic graphene nanosheets, [PERSON] and coworkers [90] developed a sensitive electrochemical platform for detecting CTCs in blood samples. Both Sgc8 and Td05 aptamers were incorporated to target two types of CTCs: CCRF-CEM cells and Ramos cells, respectively. This platform demonstrated successful detection of rare CTCs in clinical blood samples. In another interesting study, [PERSON] et al. [91] developed an optical multiplex system based on a 3D triangular-shaped DNA nanotube (DNANT), as shown in Figure 4. As a proof-of-the-concept, three different aptamers to individually target thrombin, adenosine triphosphate (ATP), and insulin were conjugated onto three edges of the nanotube. This work demonstrated that the aptamer-conjugated DNA nanotube was able to achieve multiple binding activities that also generated discriminative signals for multiple targets in one detection system. The authors further suggested that by simply altering the design of the scaffold from triangular to square, pentagonal, and hexagonal nanotubes, it would be possible to simultaneously detect four, five, or even six targets. Figure 4: Schematic drawing of constructions of 3 aptamer-DNANTs and schematic representation of thrombin-DNANT, ATP-DNANT, and insulin-DNANT [91]. Figure 3: Schematic description of aptamer-cocktail-based microfluidic approach for CTC detection. (**a**) A microfluidic device is composed of an aptamer-conjugated silicon nanowire and a PDMS chaotic mixer. (**b**) When a single type aptamer is incorporated, the capturing affinity of the device is relatively weak due to the lack of synergistic binding. (**c**) By using an aptamer cocktail, the synergistic effects among individual aptamers lead to an enhanced capturing affinity.