Graying the self-assembly of gold nanoparticles for improved enzyme activity assays

Despite the simplicity and usability of gold nanoparticle (AuNP)-based colorimetry in a large panel of bioassays, this technique still suffers from a poor detection limit. To overcome this issue, we report an improved AuNP-based colorimetric assay in combination with simple centrifugation and silver enhancement. As a proof-of-concept, two types of enzymes (i.e., protein phosphatase and protease) were constructed based on the self-assembly of thiol-stabilized AuNPs in the presence of peptides and metal ions. When the AuNP solutions were subjected to a short cycle of centrifugation and silver enhancement, the signal-to-background ratio (SBR) was distinctly improved by a factor of 5-10 in both enzyme activity assays, as compared to that of AuNP-based colorimetry. The detection limit achieved by using the silver enhancement was determined to be improved by a factor of 1.0-3.4. Furthermore, grayscale images of the silver enhancement allowed for a rapid and simple enzyme assay that did not require measuring the absorbance. Due to its ability to improve the detection senstivity in a facile way, we anticipate that this approach will be suitable for use in many AuNP colorimetric assays.