Bimetallic Metal–Organic Framework Fe/Co-MIL-88(NH₂) Exhibiting High Peroxidase-like Activity and Its Application in Detection of Extracellular Vesicles

Abstract

Metal–organic frameworks (MOFs) have many attractive features, including tunable composition, rigid structure, controllable pore size, and large specific surface area, and thus are highly applicable in molecular analysis. Depending on the MOF structure, a high number of unsaturated metal sites can be exposed to catalyze chemical reactions. In the present work, we report that using both Co­(II) and Fe­(III) to prepare the MIL-88­(NH2) MOF, we can produce the bimetallic MOF that can catalyze the conversion of 3,3′,5,5″-tetramethylbenzidine (TMB) to a color product through a reaction with H2O2 at a higher reaction rate than the monometallic Fe-MIL-88­(NH2). The Michaelis constants (K m) of the catalytic reaction for TMB and H2O2 are 3–5 times smaller, and the catalytic constants (k cat) are 5–10 times higher than those of the horseradish peroxidase (HRP), supporting ultrahigh peroxidase-like activity. These values are also much more superior to those of the HRP-mimicking MOFs reported previously. Interestingly, the bimetallic MOF can be coupled with glucose oxidase (GOx) to trigger the cascade enzymatic reaction for highly sensitive detection of extracellular vesicles (EVs), a family of important biomarkers. Through conjugation to the aptamer that recognizes the marker protein on EV surface, the MOF can help isolate the EVs from biological matrices, which are subsequently labeled by GOx via antibody recognition. The cascade enzymatic reaction between MOF and GOx enables the detection of EVs at a concentration as low as 7.8 × 104 particles/mL. The assay can be applied to monitor EV secretion by cultured cells and also can successfully detect the different EV quantities in the sera samples collected from cancer patients and healthy controls. Overall, we prove that the bimetallic Fe/Co-MIL-88­(NH2) MOF, with its high peroxidase activity and high biocompatibility, is a valuable tool deployable in clinical assays to facilitate disease diagnosis and prognosis.

Highly Stable Fe/Co-TPY-MIL-88(NH2) Metal–Organic Framework (MOF) in Enzymatic Cascade Reactions for Chemiluminescence-Based Detection of Extracellular Vesicles

“…We compared the K m and k cat values with those of Fe/Co-MIL-88(NH 2 ) acquired in our previous work. 17 As shown in Figure 2c, a K m of 0.697 mM to H 2 O 2 was found for Fe/Co-TPY-MIL-88(NH 2 ), close to that of Fe/Co-MIL-88(NH 2 ); but the K m of Fe/Co-TPY-MIL-88(NH 2 ) to TMB was about 4 times larger. This comparison indicates that blocking the open metal sites by ligand did not affect the affinity of H 2 O 2 to MOF, but reduced the affinity of TMB.…”

“…The K m and k cat against TMB, was also measured (Figure S7). We compared the K m and k cat values with those of Fe/Co-MIL-88­(NH 2 ) acquired in our previous work . As shown in Figure c, a K m of 0.697 mM to H 2 O 2 was found for Fe/Co-TPY-MIL-88­(NH 2 ), close to that of Fe/Co-MIL-88­(NH 2 ); but the K m of Fe/Co-TPY-MIL-88­(NH 2 ) to TMB was about 4 times larger.…”

“…Many MOFs have been found to possess high peroxidase-mimicking activity and are capable of generating high signals to enable sensing of low-abundant biomarkers, as what we reported previously for the Fe/Co-MIL-88(NH 2 ) MOF. 17 However, one big hurdle in applying MOFs in biomedical fields, including in biosensing, is that they contain unsaturated, open metal sites, which could be easily attacked by electron-rich ligands such as water molecule, hydroxide anion and biomolecules in clinical samples, reducing the MOF stability in such environments. It has been reported that, with the insertion of a second ligand, more metal−ligand bonds could be formed to block the open metal sites and improve the stability of MOF.…”

“…In our previous work, 17 we developed the bimetallic MOF of Fe/Co-MIL-88(NH 2 ) and demonstrated that the addition of Co(II) can greatly enhance the peroxidase-like activity of the MOF compared to the monometallic Fe-MIL-88(NH 2 ), owing to the synergistic action of Co(II) and Fe(III) in the Fentonlike reactions. The peroxidase-mimicking MOF can be coupled with glucose oxidase (GOx) to form the cascade reaction system, and be applied for colorimetric detection of a promising biomarker family, extracellular vesicles (EVs) 18 with a limit of detection (LOD) ∼ 1 × 10 5 EV particles/mL.…”

“…MOF crystals were washed twice with ethanol at 60 °C or room temperature and then dried by vacuum to remove the residual solvents that may impede the subsequent enzyme activity measurement and sensing performance. Fe/Co-MIL-88(NH 2 ) was prepared as literature described (condition 1) 17 to be compared with the newly designed Fe/Co-TPY-MIL-88(NH 2 ) for stability, activity, and sensing performance. Fe/Co-MIL-88(NH 2 ) used for N 2 77 K adsorption test was prepared by condition 2, which was slightly different from condition 1 (see Figure S4 caption text).…”