Dual‐Luminophore‐Labeled Gold Nanoparticles with Completely Resolved Emission for the Simultaneous Imaging of MMP‐2 and MMP‐7 in Living Cells under Single Wavelength Excitation

Abstract

Although considerable effort has been devoted to the design of various nanoprobes for the fluorescent detection of multiple biomarkers in a single assay, they often suffer from emission-overlapping, owing to small Stokes shifts and wide emission spectra, which results in cross-talk and inaccurate quantification. Herein, we report the design and synthesis of a new nanoprobe for multienzyme detection with completely resolved emission peaks under single-wavelength excitation. The probe was assembled by attaching a cleavable peptide spacer, which was comprised from a matrix metalloproteinase-2 (MMP-2) substrate and a MMP-7 substrate, onto the surface of gold nanoparticles (AuNPs) through cysteine residues. A lanthanide complex, BCTOT-Eu(III) (BCTOT=1,10-bis(5'-chlorosulfo-thiophene-2'-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone), and 7-amino-4-methylcoumarin (AMC) were attached to the N terminus and the C terminus of the peptide, respectively. In the presence of one or both targeting enzymes, the substrate was cleaved and fluorescence resonance energy transfer (FRET) between the dyes and AuNPs was prohibited, thereby resulting in the dramatic fluorescence emission of dyes. Importantly, there was no emission cross-talk between the two dyes, thereby ensuring accurate detection of each enzyme. Based on this, the simultaneous fluorescence image of MMP-2 and MMP-7 was accomplished in living cells under single wavelength excitation. The apparent differences in the fluorescence imaging indicated distinct differences between the expression levels of MMPs between the human normal liver cells and the human hepatoma cells.

Persistent Chemiluminescent Glow of Phenoxy‐dioxetane Luminophore Enables Unique CRET‐Based Detection of Proteases

“…For this reason, turn-on fluorescent probesf or MMPs alwaysr ely on aF RET-based approach. [45][46][47][48][49][50][51][52] The insights gained from fluorescent probes forM MPs led us to the realization that chemiluminescent probes for MMPs must dependo naC RET donor-acceptor pair and not on the classic design of dioxetane-based probes, describedi nF igure 1A.W e, therefore, soughtt oe valuatet he feasibility of our Glow-CRET design ( Figure 1B), by preparing as uitable Glow-CRET probe for MMPs ( Figure 4A). The probe consistso ft he glow-luminophore 7-HC-CL as the chemiluminescence donor and a4 -(4-nitrophenylazo)aniline quencher as the CRET acceptor.…”

Noninvasive Detection and Imaging of Matrix Metalloproteinases for Cancer Diagnosis

“…NIR dyes (excitation 650-900 nm), with deeper tissue penetration ability, low tissue damage, and less nonspecific tissue autofluorescence, are more popular in small animal imaging [23]. The reported quenchers included nanoparticles (NPs) [21,24,25], small molecule quenching agents [26], or triple-helical peptide [29,30].…”

“…Gold nanoparticles (GNPs) have been widely used to develop ''turn-on'' fluorescent nanoprobes based on FRET [21,24,25]. The fluorophore-labeled peptides are usually attached onto the surface of GNPs through covalent linkage with cysteine residue.…”

“…It could be a promising probe for the diagnosis of human liver tumor. Moreover, a dual-luminophore-labeled gold nanoprobe composed of two dyes, AMC and BCTOTEu III , has been synthesized to achieve the same excitation wavelength (350 nm) induced completely resolved emission wavelengths, 449 and 613 nm, respectively [25]. These two dyes were attached onto the N terminus and C terminus of a peptide, which was comprised of MMP-2-and MMP-7-sensitive substrate, corresponded to the activation of MMP-2 (449 nm) and MMP-7 (613 nm), respectively.…”

“…To date, more than 20 human MMPs have been characterized, including collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -10, -11), matrilysins (MMP-7), metalloelastases (MMP-12), membrane-type MMPs (MT-MMPs: MMP-14-17, MMP- [24][25], and other MMPs (MMP-19-23, MMP-26-28) [3]. MMPs share similar general structural features, typically consisting of four distinct domains: a pro-domain, containing a thiol group (-SH) and a furin cleavage site, which keeps MMPs inactivated until it is removed; a catalytic domain, containing the zinc binding site, in which the substrate cleavage takes place; a linker region of variable length; and a hemopexin-like domain [4].…”