Natural Leukocyte Membrane-Masked Microelectrodes with an Enhanced Antifouling Ability and Biocompatibility for In Vivo Electrochemical Sensing.

Probing chemical information in the central nervous system is essential for understanding the molecular mechanism of brain function. Electrochemistry with tissue-implantable carbon fiber electrodes (CFEs) provides a powerful tool for monitoring the dynamics of neurochemicals in a subsecond time scale; however, the implantation of CFEs into brain tissue immediately causes the nonspecific adsorption of proteins on electrode surfaces. This process can dramatically impact the performance of the electrochemical method in terms of reduced sensitivity and accuracy. Herein, we report a strategy to minimize the electrode biofouling by masking CFEs with leukocyte membranes (LMs). We find that the LM masking endows CFEs with a highly hydrophilic surface that gains a high resistance to nonspecific protein adsorption. The electrode reactivity to target molecules decreases by a small degree due to the membrane coating, but the sensitivity loss of the LM-masked CFEs is greatly lessened even after in vivo implantation for 8 h. This study offers a new method of microelectrode modification by natural cell membranes for sustained sensing performance during long-term in vivo analysis.

Nanoscale ATP-Responsive Zeolitic Imidazole Framework-90 as a General Platform for Cytosolic Protein Delivery and Genome Editing.

Metal-organic frameworks (MOFs) are an emerging class of nanocarriers for drug delivery, owing to their tunable chemical functionality. Here we report ATP-responsive zeolitic imidazole framework-90 (ZIF-90) as a general platform for cytosolic protein delivery and CRISPR/Cas9 genome editing. The self-assembly of imidazole-2-carboxaldehyde and Zn2+ with protein forms ZIF-90/protein nanoparticles and efficiently encapsulates protein. It was found that, in the presence of ATP, the ZIF-90/protein nanoparticles are degraded to release protein due to the competitive coordination between ATP and the Zn2+ of ZIF-90. Intracellular delivery studies showed that the ZIF-90/protein nanoparticle can deliver a large variety of proteins into the cytosol, regardless of protein size and molecular weight. The delivery of cytotoxic RNase A efficiently prohibits tumor cell growth, while the effective delivery of genome-editing protein Cas9 knocks out the green fluorescent protein (GFP) expression of HeLa cells with efficiency up to 35%. Given the fact that ATP is upregulated in disease cells, it is envisaged that the ATP-responsive protein delivery will open up new opportunities for an advanced protein delivery and CRISPR/Cas9 genome editing for targeted disease treatment.

Identification of Flavin Mononucleotide as a Cell-Active Artificial N6 -Methyladenosine RNA Demethylase.

N6 -Methyladenosine (m6 A) represents a common and highly dynamic modification in eukaryotic RNA that affects various cellular pathways. Natural dioxygenases such as FTO and ALKBH5 are enzymes that demethylate m6 A residues in mRNA. Herein, the first identification of a small-molecule modulator that functions as an artificial m6 A demethylase is reported. Flavin mononucleotide (FMN), the metabolite produced by riboflavin kinase, mediates substantial photochemical demethylation of m6 A residues of RNA in live cells. This study provides a new perspective to the understanding of demethylation of m6 A residues in mRNA and sheds light on the development of powerful small molecules as RNA demethylases and new probes for use in RNA biology.

Exploring Ferredoxin-Dependent Glutamate Synthase as an Enzymatic Bioelectrocatalyst.

Ferredoxin-dependent glutamate synthase (Fd-GltS) is reported as an enzymatic bioelectrocatalyst for the first time. By configuring mediated electrochemical interfaces with mediators of different redox potentials, we realize bioelectrosynthesis or bioelectrooxidation of glutamate with recombinant Fd-GltS from cyanobacteria. Particularly, bioelectrocatalytic oxidation of glutamate by Fd-GltS is demonstrated to be oxygen independent. This study reinforces a new catalytic option for developing enzymatic bioelectronic devices for powering, sensing or synthesis.

A versatile artificial metalloenzyme scaffold enabling direct bioelectrocatalysis in solution.

Artificial metalloenzymes (ArMs) are commonly designed with protein scaffolds containing buried coordination pockets to achieve substrate specificity and product selectivity for homogeneous reactions. However, their reactivities toward heterogeneous transformations are limited because interfacial electron transfers are hampered by the backbone shells. Here, we introduce bacterial small laccase (SLAC) as a new protein scaffold for constructing ArMs to directly catalyze electrochemical transformations. We use molecular dynamics simulation, x-ray crystallography, spectroscopy, and computation to illustrate the scaffold-directed assembly of an oxo-bridged dicobalt motif on protein surface. The resulting ArM in aqueous phase catalyzes electrochemical water oxidation without mediators or electrode modifications. Mechanistic investigation reveals the role of SLAC scaffold in defining the four-electron transfer pathway from water to oxygen. Furthermore, we demonstrate that SLAC-based ArMs implemented with Ni2+, Mn2+, Ru3+, Pd2+, or Ir3+ also enable direct bioelectrocatalysis of water electrolysis. Our study provides a versatile and generalizable route to complement heterogeneous repertoire of ArMs for expanded applications.

Role of rare-earth elements in enhancing bioelectrocatalysis for biosensing with NAD+-dependent glutamate dehydrogenase.

Dehydrogenases (DHs) are widely explored bioelectrocatalysts in the development of enzymatic bioelectronics like biosensors and biofuel cells. However, the relatively low intrinsic reaction rates of DHs which mostly depend on diffusional coenzymes (e.g., NAD+) have limited their bioelectrocatalytic performance in applications such as biosensors with a high sensitivity. In this study, we find that rare-earth elements (REEs) can enhance the activity of NAD+-dependent glutamate dehydrogenase (GDH) toward highly sensitive electrochemical biosensing of glutamate in vivo. Electrochemical studies show that the sensitivity of the GDH-based glutamate biosensor is remarkably enhanced in the presence of REE cations (i.e., Yb3+, La3+ or Eu3+) in solution, of which Yb3+ yields the highest sensitivity increase (ca. 95%). With the potential effect of REE cations on NAD+ electrochemistry being ruled out, homogeneous kinetic assays by steady-state and stopped-flow spectroscopy reveal a two-fold enhancement in the intrinsic reaction rate of GDH by introducing Yb3+, mainly through accelerating the rate-determining NADH releasing step during the catalytic cycle. In-depth structural investigations using small angle X-ray scattering and infrared spectroscopy indicate that Yb3+ induces the backbone compaction of GDH and subtle ß-sheet transitions in the active site, which may reduce the energetic barrier to NADH dissociation from the binding pocket as further suggested by molecular dynamics simulation. This study not only unmasks the mechanism of REE-promoted GDH kinetics but also paves a new way to highly sensitive biosensing of glutamate in vivo.

A single-atom Fe-N4 catalytic site mimicking bifunctional antioxidative enzymes for oxidative stress cytoprotection.

Atomically dispersed Fe-N4 sites anchored on N-doped porous carbon materials (Fe-SAs/NC) can mimic two antioxidative enzymes of catalase (CAT) and superoxide dismutase (SOD), and therefore serves as a bifunctional single-atom-based enzyme (SAzyme) for scavenging reactive oxygen species (ROS) to remove excess ROS generated during oxidative stress in cells.