## 3 Results and Discussion
EMG607 magnetic nanoparticles formed tiny clusters with extremely small inter-particle spaces. Figure 6(b) shows TEM images of EMG607 magnetic nanoparticle clusters heat-treated in the PCR thermal cycles with 0.6 \(\upmu\)L of Taq polymerase. It can be seen that polymerase stained using the negative staining method was present in the spaces among the nanoparticles. These results suggest that a certain amount of Taq polymerase in the PCR solution was captured in the inter-particle spaces among magnetic nanoparticle formation clusters in the PCR thermal cycles. A TEMT 3D reconstruction image of EMG607 magnetic nanoparticle clusters heat-treated in the PCR thermal cycles with 0.6 \(\upmu\)L of Taq polymerase is shown in Figure 7. Taq polymerase was then stained using the negative staining method with uranyl acetate, in order to scatter the electron beam. It can be seen that the surface of magnetic nanoparticles clusters were decorated with heavily stained Taq polymerase. Figure 7 illustrates that the Taq polymerase was also captured in the spaces among magnetic nanoparticles, although more was collected on the complex surfaces of magnetic nanoparticle clusters. We consider that heating-related thermal convection caused these  
Figure 6: TEM images of EMG607 magnetic nanoparticle clusters heat-treated with the addition of Taq polymerase in PCR thermal cycles. **(a)** without Taq polymerase; **(b)** with 0.6 \(\upmu\)L of Taq polymerase. clusters to move, and that pristine Taq polymerase in the PCR solution was then collected by the effect of their motion _in vitro_. The aggregation and the motion effect of magnetic nanoparticles were therefore induced by heat treatment in the PCR thermal cycles, and as a result efficiently enhanced the capture of Taq polymerase by magnetic nanoparticle clusters in the PCR solution. These results indicate that magnetic nanoparticles with higher cohesion capture more Taq polymerase than those with good dispersibility, and that they have a stronger inhibitory effect on PCR amplification. These outcomes show that some amount of Taq polymerase is captured by magnetic nanoparticle clusters, and consequently, the amount of Taq polymerase that should have been used in PCR is reduced in the PCR solution. There are several reports on the effect of various nanoparticles on PCR amplification [2, 3, 4, 5, 6, 7, 8, 9, 10]. However, their reports did not focus on interaction between the particle clusters and Taq polymerase. Although the negative or positive effect of various nanoparticles on PCR amplification has been reported, the mechanism which causes these effects has never clarified. [PERSON] _et al._[10] concluded that the surfaces of individual gold nanoparticles and Taq polymerase bind to each other, and as a result, PCR amplification is inhibited, due to a reduction in the concentration of the free Taq polymerase in the PCR solution. They also suggested that larger gold nanoparticles have a stronger inhibitory effect on PCR amplification than smaller ones. In this way, the inhibitory effects of gold nanoparticles on PCR amplification are induced by interaction between individual gold nanoparticles and Taq polymerase. In contrast, we focused on the interaction between PCR reagents and magnetic nanoparticle clusters rather than individual magnetic nanoparticles. It is the first report clarifying the effect of the magnetic nanoparticle on PCR amplification, and also clarifying the mechanism behind the effects of superparamagnetic particle clusters on PCR efficiency from estimation of their structures in PCR thermal cycles. We revealed that magnetic nanoparticles formed cluster structures in PCR thermal cycles. The inhibitory effects of superparamagnetic nanoparticles on PCR amplification  
Figure 7: TEMT 3D reconstruction image of EMG607 magnetic nanoparticle clusters heat-treated in PCR thermal cycles with 0.6 \(\upmu\)L of Taq polymerase. strongly depend on their cluster sizes and structures.