Ultra-small gold nanoclusters (AuNCs) have emerged as agile probes for in vivo imaging, as
they exhibit exceptional tumour accumulation and efficient renal clearance properties.
However, their intrinsic catalytic activity, which can enable increased detection sensitivity, has
yet to be explored for in vivo sensing. By exploiting the peroxidase-mimicking activity of AuNCs
and the precise nanometer size filtration of the kidney, we designed multifunctional protease
nanosensors that respond to disease microenvironments to produce a direct colorimetric
urinary readout of disease state in less than 1 h. We monitored the catalytic activity of AuNCs
in collected urine of a mouse model of colorectal cancer where tumour-bearing mice showed
a 13-fold increase in colorimetric signal compared to healthy mice. Nanosensors were
eliminated completely through hepatic and renal excretion within 4 weeks after injection with
no evidence of toxicity. We envision that this modular approach will enable rapid detection of
a diverse range of diseases by exploiting their specific enzymatic signatures.