PMC:4996384 / 11046-21595 JSONTXT

{"project":"2_test","denotations":[{"id":"27600212-17999554-69481644","span":{"begin":449,"end":451},"obj":"17999554"},{"id":"27600212-22950078-69481645","span":{"begin":578,"end":580},"obj":"22950078"},{"id":"27600212-22863116-69481646","span":{"begin":984,"end":986},"obj":"22863116"},{"id":"27600212-24069412-69481647","span":{"begin":987,"end":989},"obj":"24069412"}],"text":"3. Results and Discussion\n\n3.1. Functionalization of Gold Surfaces\nIn view of the detection of small molecules by SPRi detection, we aimed at an amplification of the SPRi signal by the use of gold nanoparticles (AuNPs). Those nanoparticles have two main effects: besides an amplification phenomenon due to their mass, we may also expect a coupling between the localized surface plasmon of the AuNPs and the surface plasmons of the biosensor itself [30]. The latter effect is present as soon as the AuNPs are sufficiently close to the sensor surface (distance lower than 10 nm) [31,32]. However, this amplification requires successful functionalization on both gold surfaces with anti-fouling molecules in order to avoid non-specific adsorption. \nPEG molecules are commonly used to decrease protein spontaneous adsorptions on surfaces and thus increase signal to noise ratios. This functionalization approach was successfully used for the detection of proteins by aptamer microarrays [21,22]. We adapted this method to the case of sandwich assays with split-aptamers grafted to the gold surface of SPRi prisms and nanoparticles. Two different lengths of thiolated PEG molecules have been tested for self-assembling monolayer (SAM) formation on gold: short and long ones of respectively 300 Da (PEG300) and 2 kDa (PEG2000). As can be seen from Figure 1, the longer PEG co-grafted with the split-aptamer sequences gave impressive results. While injecting gold nanoparticles grafted with Split-APT and PEG2000, no signal was observed on the gold surface of the SPRi prisms (Au curve in yellow). Furthermore, no signal was observed also on spots grafted with negative control sequences CN8 and PEG2000 (CN8 curve in grey). The lack of non‑specific interactions and SPRi signal on both cases insures that any SPRi signal observed on other spots effectively corresponds to specific interactions.\nFigure 1 Reflectivity shifts observed upon injection of gold nanoparticles grafted with Split-APT sequences on various spots of the Surface Plasmon Resonance imaging (SPRi) biosensor surface without the target adenosine present in the solution. Signal increase is observed only on Split-APT8 spots through hybridization of the hairpin stems of the split‑aptamers. APT8 and APT4 spots do not present signal shift due to a folding of the complete aptamer on the surface while Split-APT4 sequences present stems (four bases) too short to hybridize with the Split-APT sequences grafted on the gold nanoparticles. Lack of non-specific signal is also confirmed on control spots with sequences CN8 and Split-APT or on pure gold. The same experiments were realized with PEG300 on one or both gold surfaces (prisms or AuNPs). These trials were always leading to increased non-specific signals compared to PEG2000 grafting. Thus, the grafting with PEG300 was not considered for the following experiments, only PEG2000 co‑grafting of the spotsand the AuNPs were considered. In conclusion, it seems that the hydrophilic behavior brought by the long PEG chains avoid any non-specific interactions and justifies the use of this original grafting strategy for sandwich assays with AuNP SPRi signal amplification.\n\n3.2. Hybridization of Split-APT AuNPs without Adenosine \nThe 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.\nFigure 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).\nFigure 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.\n\n3.3. Adenosine Detection with Split-APT8 Sequence \nIn this section, the detection limit of the sandwich assay with the APT8 splitting sequences were explored. AuNPs were grafted with Split-APT and the biosensor with Split-APT8 sequence. We have already seen that a specific signal (signal ON) is present without adenosine. A series of different adenosine concentrations ranging from 20 to 100 μM in presence of 200 pM of AuNPs were injected on the microarray in the same conditions as the injection of AuNPs alone. The injection of AuNPs was stopped after 24 min without reaching equilibrium. All the experiments present a leveling-off after the arrest of nanoparticles injection. The stability observed for the complexes between AuNPs functionalized with Split-APT and the Split-APT8 probe surface is high enough to avoid any desorption of the AuNPs. The stabilization of the complex formed by the target and the split-aptamers leads to an increased SPRi reflectivity shift for an increasing concentration of adenosine (Figure 4). A detection limit of 50 μM may be deduced from the range of adenosine concentration considered. The selectivity of the biosensor assay was validated by an injection of 100 μM of guanosine (G curve) whose reflectivity shift is comparable to the injection of AuNPs alone.\nFigure 4 A series of different adenosine (A) concentrations have been incubated in presence of Split-APT coated gold nanoparticles. Higher SPRi signals on Split-APT8 spots are observed upon increased concentrations allowing a detection limit of 50 μM. The injection of 100 μM of guanosine (G) does not significantly modify the SPRi signal compared to the injection of gold nanoparticles (AuNPs) alone implying a good selectivity of the biosensor.\n\n3.4. Adenosine Detection with Split-APT4 Sequence\nThe case of the biosensor grafted with Split-APT4 sequence differs from the previous one with Split-APT8 since no specific signal (signal OFF) is observed without adenosine. We injected a series of different adenosine solutions (from 1 nM to 1 μM) in presence of 200 pM of AuNPs in the same conditions than the injection of AuNPs alone. The injections of AuNPs with or without adenosine were longer and stopped after 40 min due to the reduced SPR signal observed compared to the Split-APT8 probes. The stabilization of the complex formed by the target and the split-aptamers leads to a specific signal (signal ON) with an increased SPRi reflectivity shift. This increased signal is dependent of the concentration of adenosine (Figure 5) with a limit of detection (LOD) of 50 nM. This LOD is three order of magnitude lower than the one obtained with Split-APT8 biosensor. Two main reasons may explain this huge effect. First of all, it is known that signal OFF—signal ON biosensors are generally more efficient than the ones with an increased signal ON in presence of the targets. This is principally due to the fact that it is easier to detect an increased signal form the background noise than from an important reference signal. However, this requires a limited non-specific signal to reduce the noise which is precisely the case with our grafting procedure. Furthermore, the sequence of the split‑aptamers may also explain a large part of the LOD improvement. Since the number of hybridization bases in the stem is reduced for Split-APT4 than for Split-APT8 the stabilizing effect of the target on the complex formation is relatively more important. Finally, the selectivity of the biosensor assay was also validated by an injection of 1 μM of guanosine (G curve) whose reflectivity shift is weak and comparable to the injection of AuNPs alone.\nFigure 5 A range of adenosine (A) concentrations have been incubated in presence of Split-APT coated gold nanoparticles. Higher SPRi signals on Split-APT4 spots are observed upon increased concentrations allowing a detection limit of 50 nM. The injection of 1 μM of guanosine (G) does not significantly modify the SPRi signal compared to the injection of gold nanoparticles (AuNPs) alone implying a good selectivity of the biosensor.\n\n4."}