To realize colorimetric detection for RBD, we selected to use HRP-loaded DNA flower to catalyze the commonly used colorimetric reaction between TMB and H2O2. As shown in Fig. , when one of the essential components of the RCA reaction was missing, it was not possible to obtain a clear absorbance band as a response to RBD. Also, omitting any component of the colorimetric reaction (HRP, TMB, or H2O2) made it impossible to observe the absorbance band at 450 nm. These results confirm that both the RCA-enabled DNA flower and the HRPcatalyzed reaction are indispensable for the colorimetric detection of RBD. Formation of HRP-loaded DNA flower can be achieved using the same RCA reaction as used in the fluorometric detection (Fig. ). The morphology of the DNA flowers (DFs), with and without loaded HRP, were studied by scanning electron microscopy (SEM) (Fig. and). The extended rolling circle amplification generated long DNA strands, which complexed with Mg2PPi to form flower-like microparticles. The uneven, petal-like structure of the DFs provides a larger surface area, making the encapsulated HRP readily accessible to the substrates TMB and H2O2. For colorimetric detection, the signal output depends on the amount of HRP trapped in the DNA flowers, as well as the concentration of TMB and H2O2.