To determine the optimal concentration of the primer, 100 nM circle template (HBA-CT) and different concentrations of RCA primer (HBA-P) (table S1) were annealed in 1× T4 DNA ligase buffer to form template-primer complexes with a nicked DNATFBS. Then, 2 U of T4 DNA ligase was added, and the mixture was incubated at 37°C for 30 min to seal the 5′-phophate and 3′-hydroxyl ends of the nicked circular template. For RCA reaction, 2 μl of Phi29 DNA polymerase buffer, 0.5 μl of 25 mM dNTPs, 0.1 μl of phi29 DNA polymerase (100 U/μl), and 1 μl of Nb.BbVCI (10 U/μl) were introduced into the system at a final volume of 20 μl. The RCA reaction mixture was incubated at 37°C for 2 hours on a PCR thermocycle instrument, and the reaction was terminated by heating at 85°C for 10 min. To determine the optimal HosA concentration, 100 nM HosA-circle probe and 50 pM HosA-PC were used to prepare the template-primer complexes. Then, different concentrations of HosA (0, 1.5, 3, 4.5, and 6 nM) were mixed with the template-primer complexes and incubated at 25°C for 20 min to allow complete interaction between protein and DNA. Subsequently, the ligation and RCA reaction were performed as mentioned above.

To characterize the 4-HBA biosensor with RCA output, different concentrations of the 4-HBA from 0 to 200 nM were added to the aTFs-DNA complex under the above determined conditions and incubated at 37°C for 30 min. The ligation and RCA reactions were performed as above. Then, visible output and real-time fluorescence output were measured. For visible output, the solutions of 3.6 mM ABTS2− and 3.6 mM H2O2 were simultaneously added to the DNAzyme solution and diluted with TE buffer to yield a total volume of 100 μl. Data were obtained by recording the absorbance at 420 nm after 5 min of the reaction, and photographs of the results were taken by the Tanon 1600 Gel Imaging System. For real-time fluorescence output, the RCA reaction containing 5 μM ThT was incubated at 37°C on a Roche LightCycler 480II real-time PCR system, and real-time fluorescence was monitored at intervals of 1 min. For the construction of TC and UA biosensors, the procedures were the same as above for the optimization of the 4-HBA biosensor. For the construction of the UA and TC biosensors, the procedures were the same as the abovementioned optimization of the 4-HBA biosensor.

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