## 3 Results and Discussion
### Effectiveness of ssDNA Adsorption on GNPs Depends on Incubation Temperature  
All the experiments described above were conducted under standardized conditions, that is, the adsorption of oligonucleotides on GNPs was studied at 25 \({}^{\circ}\)C and with a low-ionic-strength solution (4 mM sodium citrate, pH 5.8). The temperature at which the interaction is carried out is one of the decisive factors determining the effectiveness of DNA binding to GNPs. We have previously found that the noncovalent interaction of ssDNA with GNPs proceeds faster at elevated temperatures [4]. Considering this and the fact that the extension of an ssDNA strand raises the probability of formation of its complicated structures, we next investigated in detail the influence of the incubation temperature of the reaction mixture (25, 37, or 45 \({}^{\circ}\)C) on the efficiency of formation of noncovalent ssDNA-GNP associates. One can see that the sequence of ssDNA significantly affects the nature of the dependence of its affinity for GNPs on temperature (Figure 3A-C).  
In the case of binding of 20-mer strands R and F with GNPs, there is a tendency for adsorption capacity to increase with temperature, as expected for ssDNA having a heterosequence that is not prone to the formation of secondary structures (Figure 3A).  
A significant temperature-dependent increase in the capacity of GNPs for ssDNA of the FLn series was observed only for FL (length: 26 nucleotides) (Figure 3B, red solid bars). In the other cases (FLn at \(n>1\)), we did not notice an increase in the capacity with increasing temperature of the mixture incubation (Figure 3B,C). This finding validates the notion of a balance of opposing factors: formation of intra- and/or intermolecular structures versus the enhanced anchoring ability of the L region.  
Our data are suggestive of two possible modes of adsorption of ssDNA on GNPs. For instance, the initial oligonucleotides R and F, which have similar values of affinity for GNPs, and some FTn oligonucleotides (FT2 and FT4) at 25 \({}^{\circ}\)C probably form a network of equivalent contacts between their sequence regions and GNPs, without an obvious adsorption advantage for any of these regions. Raising the incubation temperature increases the degrees of freedom of ssDNA, thereby facilitating the interaction of ssDNA with the nanoparticles. This effect enables a greater number of ssDNA molecules to bind to the GNP surface (Figure 3D, left). In the presence of the \"anchor\" L motif in FL, we observed a pronounced enlargement of the binding capacity with increasing temperature, and the maximum capacity of 98 molecules per GNP was reached at 45 \({}^{\circ}\)C (Figure 3D, right).  
Oligonucleotides FL2 and FL4 (they have an extended \"anchor\") each occupy a greater contact area on the surface of a GNP and at the same time have an elevated propensity for intra-and/or intermolecular structures. These circumstances (taking into account greater electrostatic repulsion with the extension of ssDNA strands) do not allow for a significant increase in the efficiency of this interaction with increasing temperature. Nevertheless, the very presence of gold-binding sequences in FLn (at \(n>1\)) during the formation of the respective noncovalent associates with GNPs enables the mode of predominantly \"anchor\" adsorption (Figure 3D, right), thereby minimizing electrostatic repulsion during the adsorption of such a large number of molecules. For FTn oligonucleotides, there is probably no clear advantage of one mode, as in the case of F and R, as explained below in the affinity-rating analysis section.