## 3 Results and Discussion
As shown in Figure 3(a), PCR amplification with EMG607 magnetic nanoparticles is restored with increasing Taq polymerase concentrations of 0.4-0.8 \(\upmu\)g/\(\upmu\)L. Accordingly, when the concentration of the Taq polymerase increases, the inhibitory threshold concentration shifts to a higher level, as shown in Figure 3(b), and similar behavior is also observed when EMG707 or EMG1111 particles are used instead of the EMG607 type. From the experimental results obtained, we found that only Taq polymerase of a DNA synthetic enzyme was sacrificed by magnetic nanoparticles, while other PCR components (such as the template DNA, both oligo primers and the deoxyribonucleotide triphosphate (dNTP)) were not sacrificed. The quantity of Taq polymerase used for PCR will decrease with an increase in the number of nanoparticles. Figure 4 also shows that all product yields were linearly enhanced by the addition of extra Taq polymerase at each threshold concentration, and as a result, all PCR amplifications were  
Figure 3: Amplified DNA bands with EMG607 magnetic nanoparticles having different amounts of Taq polymerase observed using agarose gel electrophoresis. Lane 1: control without magnetic nanoparticles; lanes 2–9: 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 \(\upmu\)g/\(\upmu\)L of EMG607 magnetic nanoparticles, respectively. (**a**) The amounts of Taq polymerase used were 0.2–0.8 \(\upmu\)g/\(\upmu\)L; (**b**) Dependence of PCR product yield with EMG607 magnetic nanoparticles on amounts of Taq polymerase. completely restored at a Taq polymerase concentration of 0.6 \(\upmu\)g/uL. The same amounts of Taq polymerase that should have been used in PCR were sacrificed by the magnetic nanoparticles at each threshold concentration. In short, EMG707 and EMG1111 nanoparticles sacrificed the amounts of Taq polymerase more effectively than EMG607, although their threshold concentrations of EMG707 and EMG1111 were substantially lower than that of EMG607. Next, we examined the state of magnetic nanoparticles in PCR solutions during PCR thermal cycles. The distributions of magnetic nanoparticle diameters were measured before and after heat treatment, as shown in Figure 5(a). Figure 4: Dependence of PCR product yield with several types of magnetic nanoparticles at threshold concentration on amounts of Taq polymerase. Figure 5: **(a)** Particle size distribution of EMG607, EMG707 and EMG1111 magnetic nanoparticles before and after heat treatment in PCR thermal cycles; **(b)** TEM image of EMG607, EMG707 and EMG1111 magnetic nanoparticles after heat treatment in PCR thermal cycles. At room temperature without an external magnetic field, the magnetic nanoparticles EMG607, EMG707 and EMG1111 tend to aggregate to form clusters, and the size of clusters depends on surface properties of particles, although it is considered that superparamagnetic particles do not aggregate only by magnetic interactions. After the PCR thermal cycles, the size of the magnetic nanoparticle clusters was increased by heat treatment, regardless of their initial size. The heat treatment denatured the surface properties of the nanoparticles and then reduced their zeta potential, thereby promoting nanoparticle aggregation. Figure 5(b) shows TEM images of EMG607, EMG707 and EMG1111 magnetic nanoparticles after the PCR thermal cycles. It can be seen that they also aggregated to form irregular cluster structures. EMG707 and EMG1111 tended to strongly aggregate, and EMG1111 formed the largest clusters, while a considerable number of EMG607 magnetic nanoparticles remained stably dispersed. These results suggest a correlation between the size and shape of magnetic nanoparticle clusters and the concentration of the magnetic nanoparticles inhibiting PCR amplification. We also analyzed the structure of EMG607 magnetic nanoparticle clusters heat-treated with the addition of Taq polymerase in PCR thermal cycles using TEM and TEMT. Figure 6(a) shows TEM images of EMG607 magnetic nanoparticle clusters heat-treated in these PCR thermal cycles without Taq polymerase.