*2.3. Optimization of OTA Detection Conditions 2.3. Optimization of OTA Detection Conditions*

**Figure 5.** Fluorescence spectra of NMM with different substances: (**a**) H1, H2 and OTA; (**b**) H1 and H2; (**c**) H1 and OTA; (**d**) H2 and OTA. Experimental conditions: [H1] = [H2] = 300 nM, [OTA] = 10 nM. *2.3. Optimization of OTA Detection Conditions*  For the sake of obtaining optimal performance of the proposed detection platform, several For the sake of obtaining optimal performance of the proposed detection platform, several important parameters were optimized on the basis of single-factor experiments. Firstly, the effect of K+ concentration on the changes of fluorescence intensity was studied because the sequences of Grich oligonucleotide can form stable G-quadruplex structures under the action of K+. Furthermore, the K+ can mutual coordinate with carbonyl oxygen atoms of the G-residues and be embedded in the central of two stacked G-tetrads [36]. As shown in Figure 6a, F0 and F represent the measured fluorescence value at 608 nm before and after adding OTA, respectively. With the increase of K+ concentration, the amount of fluorescence intensity change (F-F0) gradually increased and reached to the maximum at 20 mM, indicating that 20 mM of K+ could completely accelerate the folding of Gquadruplex structures. Therefore, 20 mM K+ concentration was chosen for the next experiment. For the sake of obtaining optimal performance of the proposed detection platform, several important parameters were optimized on the basis of single-factor experiments. Firstly, the effect of K<sup>+</sup> concentration on the changes of fluorescence intensity was studied because the sequences of G-rich oligonucleotide can form stable G-quadruplex structures under the action of K+. Furthermore, the K<sup>+</sup> can mutual coordinate with carbonyl oxygen atoms of the G-residues and be embedded in the central of two stacked G-tetrads [36]. As shown in Figure 6a, F<sup>0</sup> and F represent the measured fluorescence value at 608 nm before and after adding OTA, respectively. With the increase of K<sup>+</sup> concentration, the amount of fluorescence intensity change (F-F0) gradually increased and reached to the maximum at 20 mM, indicating that 20 mM of K<sup>+</sup> could completely accelerate the folding of G-quadruplex structures. Therefore, 20 mM K<sup>+</sup> concentration was chosen for the next experiment.

important parameters were optimized on the basis of single-factor experiments. Firstly, the effect of K+ concentration on the changes of fluorescence intensity was studied because the sequences of Grich oligonucleotide can form stable G-quadruplex structures under the action of K+. Furthermore, the K+ can mutual coordinate with carbonyl oxygen atoms of the G-residues and be embedded in the Subsequently, the HCR reaction time for H1 and H2 was also optimized. As the reaction time gradually increased in Figure 6b, the degree of hybridization between H1 and H2 deepened and the Subsequently, the HCR reaction time for H1 and H2 was also optimized. As the reaction time gradually increased in Figure 6b, the degree of hybridization between H1 and H2 deepened and the fluorescence change remained steady after 60 min, manifesting an equilibrium for HCR assembly between H1 and H2. Consequently, the time for HCR was set at 60 min in the subsequent experiments.

central of two stacked G-tetrads [36]. As shown in Figure 6a, F0 and F represent the measured fluorescence value at 608 nm before and after adding OTA, respectively. With the increase of K+ concentration, the amount of fluorescence intensity change (F-F0) gradually increased and reached to the maximum at 20 mM, indicating that 20 mM of K+ could completely accelerate the folding of Gquadruplex structures. Therefore, 20 mM K+ concentration was chosen for the next experiment. Subsequently, the HCR reaction time for H1 and H2 was also optimized. As the reaction time Moreover, the concentration of NMM and its reaction time with the G-quadruplexes produced in the experiment also directly affect the fluorescence intensity of the solution. According to Figure 6c, 1.5 µM of NMM displayed the highest fluorescence change in the detection because the lower concentration of NMM cannot provide sufficient fluorescence intensity for the G-quadruplexes produced in the reaction, and the higher concentration of NMM may engender increased background fluorescence signal. It was worth noting that the incubation time with NMM had little impact on the variation of fluorescence

gradually increased in Figure 6b, the degree of hybridization between H1 and H2 deepened and the

intensity (Figure 6d). Hence, in order to save the general time for the experiment, 10 min was selected as the combination time with NMM. experiment, 10 min was selected as the combination time with NMM.

variation of fluorescence intensity (Figure 6d). Hence, in order to save the general time for the

*Toxins* **2020**, *12*, x FOR PEER REVIEW 7 of 13

fluorescence change remained steady after 60 min, manifesting an equilibrium for HCR assembly between H1 and H2. Consequently, the time for HCR was set at 60 min in the subsequent experiments. Moreover, the concentration of NMM and its reaction time with the G-quadruplexes produced in the experiment also directly affect the fluorescence intensity of the solution. According to Figure 6c, 1.5 µM of NMM displayed the highest fluorescence change in the detection because the lower concentration of NMM cannot provide sufficient fluorescence intensity for the G-quadruplexes produced in the reaction, and the higher concentration of NMM may engender increased background

**Figure 6.** The fluorescence intensity change at 608 nm versus: (**a**) the concentration of K+. [NMM] = 1.5 µM, and the reaction time of HCR and NMM are 60 min and 10 min, respectively; (**b**) the reaction time of HCR. [NMM] = 1.5 µM, [K+] = 20 mM, and the combination time of NMM is 10 min; (**c**) the concentration of NMM. [K+] = 20 mM, and the reaction time of HCR and NMM are 60 min and 10 min, respectively; (**d**) the reaction time of NMM. [NMM] = 1.5 µM, [K+] = 20 mM, and the reaction time of HCR is 60 min. Other conditions: [H1] = [H2] = 300 nM. Error bars, SD, *n* = 3. **Figure 6.** The fluorescence intensity change at 608 nm versus: (**a**) the concentration of K+. [NMM] = 1.5 µM, and the reaction time of HCR and NMM are 60 min and 10 min, respectively; (**b**) the reaction time of HCR. [NMM] = 1.5 µM, [K+] = 20 mM, and the combination time of NMM is 10 min; (**c**) the concentration of NMM. [K+] = 20 mM, and the reaction time of HCR and NMM are 60 min and 10 min, respectively; (**d**) the reaction time of NMM. [NMM] = 1.5 µM, [K+] = 20 mM, and the reaction time of HCR is 60 min. Other conditions: [H1] = [H2] = 300 nM. Error bars, SD, *n* = 3.
