Carbon nanotube-based multicolor fluorescent peptide probes for highly sensitive multiplex detection of cancer-related proteases

Abstract

In this work, a novel carbon nanotube (CNT)-based multicolor fluorescent peptide nanoprobe is developed for rapid, sensitive, and multiplex detection of cancer-related proteases in homogeneous solution. To prepare the nanoprobe, three peptide substrates, each labeled at the C-terminal with a fluorescent dye (i.e., fluorescein isothiocyanate (FITC), cyanine dye Cy3, cyanine dye Cy5), that respond to one of three different proteases are co-conjugated to the surface of CNTs. This conjugation brings the dyes into the proximity of the CNT surface, which leads to significantly quenched fluorescence due to highly efficient long-range energy transfer from the dyes to CNTs. However, upon incubation with the targeted proteases, specific peptide cleavage occurs and releases the dyes from the CNT surface, which results in the fluorescence recovery that provides the basis for a quantitative measurement of protease activity. With the use of three cancer-related proteases, matrix metalloproteinase-7 (MMP-7), matrix metalloproteinase-2 (MMP-2), and urokinase-type plasminogen activator (uPA), as the proof-of-concept analytes, the nanoprobe could simultaneously detect these proteases with high sensitivity and specificity. The limits of detection for this method are obtained in the range 0.5 pg mL⁻¹ to 500 pg mL⁻¹, which are two orders of magnitude lower than many previously reported methods. Moreover, the suitability of this CNT-based sensing platform for complex biological sample analysis has also been demonstrated. This approach holds great promise as a routine tool for the high-throughput screening of proteases in proteomics and clinical diagnostics.