Optimization of a Pb2+-Directed Gold Nanoparticle/DNAzyme Assembly and Its Application as a Colorimetric Biosensor for Pb2+

We previously communicated a method for directed assembly of gold nanoparticles using a Pb2+-dependent DNAzyme and demonstrated the application of this system as a colorimetric biosensor. The sensor shows high sensitivity and selectivity toward Pb2+ and undergoes a blue-to-red color transition in the presence of Pb2+. To gain a deeper insight into the analyte-directed nanomaterials assembly and sensing processes, a detailed characterization of the system has been performed. First, we found that the presence of gold nanoparticles had no effect on the Pb2+-dependent activity of the DNAzyme and the presence of DNAzyme has little effect on the melting properties of the DNA-functionalized nanoparticle aggregates, suggesting that the performance of the nanoparticle and DNAzyme systems can be optimized independently. Second, the optimal length of the DNAzyme and the alignment of the DNA-functionalized gold nanoparticles for the assembly and sensing processes have been determined to be 9 base pairs on each end for the DNAzyme, and “head-to-tail” alignment for the DNA-functionalized gold nanoparticles. Third, the optimal stoichiometry of the enzyme to the substrate strands of the DNAzyme was shown to be one to one in nanoparticle aggregates. Finally, the most favorable temperature and pH conditions for the system have also been established, with a temperature of 37 °C and pH of 6.4 to 9.2 as the best operating conditions. The study also revealed that, for most efficient assembly of nanoparticles, the DNA backbone should be rigidified by formation of a double helix with other DNA molecules. These findings allow optimization of the processes for directed assembly of nanomaterials and for colorimetric sensing.