3.2. Hybridization of Split-APT AuNPs without Adenosine 
The main advantage of SPRi detection compared to solution phase assays is the possibility to analyze multiple probes at the same time thanks to the microarray format. We used this opportunity to test various detection strategies and more particularly different aptamer sequences. First of all, two complete adenosine aptamers were considered and grafted to the microarrays. APT4 and APT8 only differ in the number of bases hybridizing in the stem domain close to the recognition site of the adenosine target. This variation of hybridizing bases affects the folding thermodynamics of the apamer and potentially its recognition affinity towards the targets. Besides those complete aptamers, the two corresponding split-aptamers were considered. Split-APT4 and Split-APT8 respectively correspond to the first part of the splitting of APT4 and APT8 whereas Split-APT is the second part common to both APT4 and APT8 (see Figure 2 for the sequence engineering). This last sequence was grafted to the AuNPs and used as a reference due to its possible interactions (in presence or not of adenosine) with the four considered sequences (APT4, APT8, Split-APT4 and Split-APT8). As a control sequence Split-APT was also grafted on the microarrays. A negative control CN8, completely independent from the adenosine aptamer was also considered on the microarrays.
Figure 2  Aptamer sequence engineering into split-aptamers. Split-APT corresponds to the common part of both APT4 and APT8 split sequences whereas Split-APT4 and Split-APT8 are their respective counterparts.   First of all, we tested the interactions of the AuNPs grafted with Split-APT sequences on the microarray (Figure 1). Only spots grafted with Split-APT8 presented a shift in SPR reflectivity upon injection of 200 pM of AuNPs for 45 min at room temperature. This indicates that the eight complementary bases between Split-APT and Split-APT8 are sufficient to lead to specific hybridization (Figure 3B). Strangely enough, APT8, which also presents those eight bases complementary to Split‑APT, does not yielded specific signal (Figure 3A). This may be explained by the folded structure of APT8 at room temperature even without the presence of the target adenosine. This also confirms the difficulty to drive the folding of the aptamer by the presence of small targets since the folding is already present without the target. Finally, the spots Split-APT4 do not present specific signal suggesting that the four complementary bases do not hybridize at room temperature (Figure 3C). As we will see in the following sections, the presence of the adenosine targets stabilizes the complex formed between the split-aptamers sequences and allows for a specific signal on Split-APT4 spots (Figure 3D).
Figure 3  Split-APT gold nanoparticles interacting modes with the aptamer microarray: (A) Split-APT gold nanoparticles do not interact with APT8 spots due to the folding of the complete aptamer; (B) Split-APT gold nanoparticles interacts with Split-APT8 spots through hybridization even without adenosine; (C) Split-APT gold nanoparticles do not interact with Split-APT4 spots without adenosine, but (D) interacts with Split-APT4 spots in presence of adenosine.

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