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.

Recent Advances in the Application Peptide and Peptoid in Diagnosis Biomarkers of Alzheimer’s Disease in Blood

“…The fluorescence sensing platform can be used to monitor BACE1 with a detection limit of 66 pM and has been proven to be suitable for screening BACE1 inhibitors. Kim S. et al (2020) constructed a magnetic graphite oxide (MGO) alkenyl FRET biosensor, on which they synthesized an N-terminal FITC-labeled peptide specific to BACE1 (EVNLDA). The resonance energy transfer from FITC to MGO results in the quenching of the fluorescence generated from FITC.…”

Sensing of Digestive Enzymes—Diagnosis and Monitoring of Pancreatitis

“…As can be reflected in the above introduction, the fluorescence detection method of digestive enzymes often uses nutrients as the material connecting the fluorescent substance to the quenched substance (Figure 6) [81]. Fluorescence is released in the presence of digestive enzymes.…”