Biotin
Previous work has shown that DNAzyme-directed assembly of gold nanoparticles can be utilized to make effective colorimetric biosensors. However, the method is restricted to analytes that are directly involved in phosphodiester cleavage. To expand the methodology to a broader range of analytes, a colorimetric adenosine biosensor based on the aptazyme-directed assembly of gold nanoparticles is reported here. The aptazyme is based on the 8-17 DNAzyme with an adenosine aptamer motif that can modulate the DNAzyme activity through allosteric interactions depending on the presence of adenosine. In the absence of adenosine, the aptazyme is inactive and the substrate strands can serve as linkers to assemble DNA-functionalized 13-nm-diameter gold nanoparticles, resulting in a blue color. However, the presence of adenosine activates the aptazyme, which cleaves the substrate strand, disrupting the formation of nanoparticle aggregates. A red color of separated gold nanoparticles is observed. Concentrations of adenosine of up to 1 mM can be measured semiquantitatively by the degree of blue to red color changes or quantitatively by the extinction ratio at 520 and 700 nm. Under the same conditions, 5 mM guanosine, cytidine, or uridine resulted in a blue color only, indicating good selectivity of the sensor. The color difference can be clearly observed by the naked eye by spotting the resulting sensor solution onto an alumina TLC plate. Since aptamers that can target many classes of important analytes have already been selected, they can be adapted into aptazyme systems through rational design or further selection. Thus, colorimetric biosensors for many analytes of interest can be designed using the method presented here, regardless of whether the analytes are directly involved in the cleavage reaction or not.
