Robust Magnetized Graphene Oxide Platform for In Situ Peptide Synthesis and FRET-Based Protease Detection

Abstract

Graphene oxide (GO)/peptide complexes as a promising disease biomarker analysis platform have been used to detect proteolytic activity by observing the turn-on signal of the quenched fluorescence upon the release of peptide fragments. However, the purification steps are often cumbersome during surface modification of nano-/micro-sized GO. In addition, it is still challenging to incorporate the specific peptides into GO with proper orientation using conventional immobilization methods based on pre-synthesized peptides. Here, we demonstrate a robust magnetic GO (MGO) fluorescence resonance energy transfer (FRET) platform based on in situ sequence-specific peptide synthesis of MGO. The magnetization of GO was achieved by co-precipitation of an iron precursor solution. Magnetic purification/isolation enabled efficient incorporation of amino-polyethylene glycol spacers and subsequent solid-phase peptide synthesis of MGO to ensure the oriented immobilization of the peptide, which was evaluated by mass spectrometry after photocleavage. The FRET peptide MGO responded to proteases such as trypsin, thrombin, and β-secretase in a concentration-dependent manner. Particularly, β-secretase, as an important Alzheimer's disease marker, was assayed down to 0.125 ng/mL. Overall, the MGO platform is applicable to the detection of other proteases by using various peptide substrates, with a potential to be used in an automated synthesis system operating in a high throughput configuration.

Keywords: biological assays; fluorescence resonance energy transfer (FRET); in situ peptide synthesis; magnetic graphene oxide (MGO); proteases.

Figure 1

Preparation and characterization of graphene oxide (GO) and magnetic graphene oxide (MGO). (A) Schematic of MGO synthesis procedure. (B) Optical microscopy image of MGO. (C) Magnetic hysteresis loop of MGO. (D) UV/Vis absorption spectra of GO and MGO. (E) FT-IR spectra of GO and MGO.

Figure 2

Coupling of polyethylene glycol (PEG) diamine and photocleavable linker (PCL) to MGO.

Figure 3

In situ peptide synthesis on MGO and mass spectrometry of the released peptides. (A) Schematic diagram of synthesized peptide on MGO and photocleavage reaction of β-secretase (EVNLDA) peptide on MGO. (B,C) MALDI-TOF analyses of H-thrombin(LVPRGS)-NH₂ and H-EVNLDA-NH₂ released from the MGO upon UV irradiation (365 nm).

Figure 4

Illustration of MGO fluorescence resonance energy transfer (FRET) sensor based on fluorescence recovery induced by enzymatic cleavage.

Figure 5

Fluorescence signals from fluorescein 5-isothiocyanate FITC-KKK PEGx-MGO biosensors upon tryptic peptide cleavage. (A) Fluorescence emission spectra of FITC-KKK-PEG₈₀₀-MGO FRET biosensors in the presence of trypsin at concentrations of 0 to 160 μg/mL for 30 min. (B) Fluorescence intensity recovery (F/F₀) at 514.5 nm depending on the PEG length in FITC-KKK-PEGx-MGO in response to different concentrations of trypsin. (C) Temporal fluorescence emission spectra of FITC-KKK-PEG₈₀₀-MGO FRET biosensors from 0 to 100 min with 20 µg/mL of trypsin. (D) Time-dependent fluorescence intensity recovery (F/F₀) at 514.5 nm depending on PEG length in FITC-KKK-PEGx-MGO with 20 µg/mL of trypsin.

Figure 6

Fluorescence signals from FITC-LVPRGS-PEG₈₀₀-MGO biosensors upon peptide cleavage by thrombin. (A) Fluorescence emission spectra of FITC-LVPRGS-PEG₈₀₀-MGO FRET biosensors in the presence of thrombin at concentration of 0 to 8 μg/mL for 30 min. (B) Fluorescence intensity recovery (F/F₀) at 514.5 nm from FITC-LVPRGS-PEG₈₀₀-MGO in response to different concentrations of thrombin when compared with BSA. (C) Temporal fluorescence emission spectra of FITC-LVPRGS-PEG₈₀₀-MGO FRET biosensors from 0 to 100 min at 2 μg/mL of thrombin. (D) Time-dependent fluorescence intensity recovery (F/F₀) at 514.5 nm in FITC-LVPRGS-PEG₈₀₀-MGO at 2 µg/mL of thrombin when compared to BSA (2 µg/mL).

Figure 7

Fluorescence signals from FITC-EVNLDA-PEG₈₀₀-MGO biosensors upon peptide cleavage by β-secretase. (A) Fluorescence emission spectra of FITC-EVNLDA-PEG₈₀₀-MGO FRET biosensors in the presence of β-secretase at concentrations of 0 to 8 μg/mL for 30 min. (B) Fluorescence intensity recovery (F/F₀) at 514.5 nm from FITC-EVNLDA-PEG₈₀₀-MGO in response to different concentrations of β-secretase when compared to BSA. (C) Temporal fluorescence emission spectra of FITC-EVNLDA-PEG₈₀₀-MGO FRET biosensors from 0 to 60 min at 2 μg/mL of β-secretase. (D) FRET activities of FITC-EVNLDA-PEG₈₀₀-MGO for various proteins at a protein concentration of 0.1 μg/mL.