To find out the optimal temperature for hybridization between aptamer and cDNA on MBs, we mixed FAM-labelled aptamer with MBs-cDNA at different temperatures. After magnetic separation, we measured the fluorescence intensity of the supernatant. The reduction of fluorescence intensity was used to evaluate the efficiency of the hybridization (Fig. ). Normally, a temperature suitable for DNA hybridization is considered to be about 20-25 °C lower than the melting temperature (Tm). In our present system, the Tm of aptamer and cDNA is 80 °C and 40 °C, respectively. Therefore, we tested the hybridization at three different temperatures: 20 °C, 30 °C and 40 °C. As shown in Fig. , hybridization at 20 °C resulted in the highest uptake of FAM-aptamer by the cDNA on the MBs. Due to that the RCA time determines the length of the product DNA, it affects directly the intensity of fluorescence after adding the F-Q probe. We therefore studied the effect of carrying out the RCA reaction for 30 min, 60 min, 90 min, 120 min and 180 min. The RCA reaction was stopped by heating at 65 °C for 10 min. After adding the F-Q probe and incubation, we measured the fluorescence intensity of the mixture. As shown in Fig. , the fluorescence intensity increased when the reaction time increased from 30 min to 120 min. After 120 min the fluorescence intensity did not increase further. Therefore, we selected 120 min as the optimal RCA reaction time.