A reduced graphene oxide-based fluorescence resonance energy transfer sensor for highly sensitive detection of matrix metalloproteinase 2

Abstract

A novel fluorescence nanoprobe (reduced nano-graphene oxide [nrGO]/fluorescein isothiocyanate-labeled peptide [Pep-FITC]) for ultrasensitive detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the Pep-FITC comprising the specific MMP2 substrate domain (PLGVR) onto the surface of nrGO particles through non-covalent linkage. The nrGO was obtained by water bathing nano-graphene oxide under 90°C for 4 hours. After mixing the nrGO and Pep-FITC for 30 seconds, the fluorescence from Pep-FITC was almost completely quenched due to the fluorescence resonance energy transfer between fluorescein isothiocyanate (FITC) and nrGO. Upon cleavage of the amide bond between Leu and Gly in the Pep-FITC by protease-MMP2, the FITC bound to nrGO was separated from nrGO surface, disrupting the fluorescence resonance energy transfer process and resulting in fluorescence recovery of FITC. Under optimal conditions, the fluorescence recovery of nrGO/Pep-FITC was found to be directly proportional to the concentration of MMP2 within 0.02–0.1 nM. The detection limit of the nrGO/Pep-FITC was determined to be 3 pM, which is approximately tenfold lower than that of the unreduced carboxylated nano-graphene oxide/Pep-FITC probe.

Covalent linking peptide to hydrothermally reduced graphene oxide for ultrasensitive detection of matrix metalloproteinase 9

“…in vivo (Akers et al 2012) and in vitro (Li et al 2015). Based on the specific cleavage site of MMP2 between the Leu and Gly of Pro-Leu-Gly-Val-Arg, some nanoprobes via engineering a dye-labeled peptide (Pep-dye) containing the MMP2-specific cleavage substrate onto the surface of nanoparticle have been fabricated for sensitive MMP2 detection Li et al 2011;Feng et al 2011;Song et al 2013;Xi et al 2016). Wang and coworkers (2014) used poly(m-phenylenediamine) (PMPD) as an acceptor to develop PMPD-Pep-FITC probe showing 32 pM of detection limit for MMP2 .…”

“…Using the good optical absorptivity of graphene oxide (GO), Song and coworkers developed a GO-Pep-FITC probe that could detect MMP2 at concentration as low as 35 pM (Song et al 2013). Considering the higher optical absorptivity of reduced graphene oxide (rGO) over GO, we recently developed a non-covalent rGO-based FRET nanoprobe (nrGO/Pep-FITC) with 3 pM of detection limit for MMP2 detection (Xi et al 2016). Similarly, Nguyen and coworkers noncovalently bound fluorescent gold nanocluster-labeled peptide containing the specific cleavage site of MMP9 to GO for MMP9 detection, and the detection limit of this probe was 0.15 nM (Nguyen et al 2017).…”

“…Pep-FITC (NH 2 -Lys-Gly-Pro-Arg-Ser-Leu-Ser-GlyLys-FITC) was synthesized by Wuhan Fine Peptide Co. Ltd. Graphene oxide (GO) flake, latent proMMP9, 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4-aminophenylmercuric acetate (APMA), dimethyl sulfoxide (DMSO), other reagents employed, and all buffers have been described in our previous publication (Xi et al 2016). …”

“…GO was dissolved in tri-distilled water and sonicated just as described in our previous publication (Xi et al 2016). NaOH and ClCH 2 COOH were added to nGO suspension and then sonicated for 1 h. The reaction liquid was divided into four parts, and then stirred for 0, 1, 2, and 3 h, respectively.…”

“…Characterizations of materials, including atomic force microscopic imaging (AFM), f-potentials and polydispersity index (PdI) analysis, ultraviolet-visible (UV-vis) spectra, fourier transfer infrared (FTIR) spectra, and Raman spectra, have been described in our previous publication (Xi et al 2016). The fluorescence intensity at 518 nm of FITC with 480 nm excitation was measured using Luminescence spectrometer (LS 55; PerkinElmer) for the concentration-dependent cleavage of nrGO-Pep-FITC by MMP9 or microplate reader (Infinite M200; Tecan) for other experiments.…”