Freeform 3D Bioprinting Involving Ink Gelation by Cascade Reaction of Oxidase and Peroxidase: A Feasibility Study Using Hyaluronic Acid-Based Ink

Freeform bioprinting, realized by extruding ink-containing cells into supporting materials to provide physical support during printing, has fostered significant advances toward the fabrication of cell-laden soft hydrogel constructs with desired spatial control. For further advancement of freeform bioprinting, we aimed to propose a method in which the ink embedded in supporting materials gelate through a cytocompatible and rapid cascade reaction between oxidase and peroxidase. To demonstrate the feasibility of the proposed method, we extruded ink containing choline, horseradish peroxidase (HRP), and a hyaluronic acid derivative, cross-linkable by HRP-catalyzed reaction, into a supporting material containing choline oxidase and successfully obtained three-dimensional hyaluronic acid-based hydrogel constructs with good shape fidelity to blueprints. Cytocompatibility of the bioprinting method was confirmed by the comparable growth of mouse fibroblast cells, released from the printed hydrogels through degradation on cell culture dishes, with those not exposed to the printing process, and considering more than 85% viability of the enclosed cells during 10 days of culture. Owing to the presence of derivatives of the various biocompatible polymers that are cross-linkable through HRP-mediated cross-linking, our results demonstrate that the novel 3D bioprinting method has great potential in tissue engineering applications.

LuxAB-Based Microbial Cell Factories for the Sensing, Manufacturing and Transformation of Industrial Aldehydes

The application of genetically encoded biosensors enables the detection of small molecules in living cells and has facilitated the characterization of enzymes, their directed evolution and the engineering of (natural) metabolic pathways. In this work, the LuxAB biosensor system from Photorhabdus luminescens was implemented in Escherichia coli to monitor the enzymatic production of aldehydes from primary alcohols and carboxylic acid substrates. A simple high-throughput assay utilized the bacterial luciferase—previously reported to only accept aliphatic long-chain aldehydes—to detect structurally diverse aldehydes, including aromatic and monoterpene aldehydes. LuxAB was used to screen the substrate scopes of three prokaryotic oxidoreductases: an alcohol dehydrogenase (Pseudomonas putida), a choline oxidase variant (Arthrobacter chlorophenolicus) and a carboxylic acid reductase (Mycobacterium marinum). Consequently, high-value aldehydes such as cinnamaldehyde, citral and citronellal could be produced in vivo in up to 80% yield. Furthermore, the dual role of LuxAB as sensor and monooxygenase, emitting bioluminescence through the oxidation of aldehydes to the corresponding carboxylates, promises implementation in artificial enzyme cascades for the synthesis of carboxylic acids. These findings advance the bio-based detection, preparation and transformation of industrially important aldehydes in living cells.

A Multi-Catalytic Sensing for Hydrogen Peroxide, Glucose, and Organophosphorus Pesticides Based on Carbon Dots

In this work, a facile one-pot hydrothermal route was employed to synthesize a series of fluorescent carbon dots (CDs) by using 20 natural amino acids, respectively, as the starting materials. It was found that the CDs synthesized using phenylalanine could possess the intrinsic peroxidase-like activity that could effectively catalyze a traditional peroxidase substrate like 3, 3’, 5, 5’- tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue solution; thereby, a catalytic sensing system for H2O2 has been developed. On the basis of this catalytic reaction, together with the fact that glucose oxidase (GOx) can catalyze the hydrolysis of glucose to generate H2O2, a sensitive catalytic sensing system for glucose could be further established. Furthermore, based on this catalytic reaction, taken together with the two enzymatic catalytic systems of acetylcholinesterase (AChE) and choline oxidase (CHO), a highly sensitive multi-catalytic sensing system could be successfully developed for organophosphorus (OPs) pesticides such as dimethoate, DDVP, and parathion-methyl. Limit of detections (LODs) of H2O2 and glucose were estimated to be 6.5 and 0.84 μM, respectively. The limit of detection of the sub-nM level could be obtained for tested dimethoate, DDVP, and parathion-methyl OPs pesticides. The established sensing systems can exhibit good practical application performance in serum and several fruit samples.

Morpho-Physiological Testing of NaCl Sensitivity of Tobacco Plants Overexpressing Choline Oxidase Gene

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.

A Crosstalk- and Interferent-Free Dual Electrode Amperometric Biosensor for the Simultaneous Determination of Choline and Phosphocholine

Choline (Ch) and phosphocholine (PCh) levels in tissues are associated to tissue growth and so to carcinogenesis. Till now, only highly sophisticated and expensive techniques like those based on NMR spectroscopy or GC/LC- high resolution mass spectrometry permitted Ch and PCh analysis but very few of them were capable of a simultaneous determination of these analytes. Thus, a never reported before amperometric biosensor for PCh analysis based on choline oxidase and alkaline phosphatase co-immobilized onto a Pt electrode by co-crosslinking has been developed. Coupling the developed biosensor with a parallel sensor but specific to Ch, a crosstalk-free dual electrode biosensor was also developed, permitting the simultaneous determination of Ch and PCh in flow injection analysis. This novel sensing device performed remarkably in terms of sensitivity, linear range, and limit of detection so to exceed in most cases the more complex analytical instrumentations. Further, electrode modification by overoxidized polypyrrole permitted the development of a fouling- and interferent-free dual electrode biosensor which appeared promising for the simultaneous determination of Ch and PCh in a real sample.

Highly Sensitive Fluorescent Detection of Acetylcholine Based on the Enhanced Peroxidase-Like Activity of Histidine Coated Magnetic Nanoparticles

Inspired by the active site structure of natural horseradish peroxidase having iron as a pivotal element with coordinated histidine residues, we have developed histidine coated magnetic nanoparticles (His@MNPs) with relatively uniform and small sizes (less than 10 nm) through one-pot heat treatment. In comparison to pristine MNPs and other amino acid coated MNPs, His@MNPs exhibited a considerably enhanced peroxidase-imitating activity, approaching 10-fold higher in catalytic reactions. With the high activity, His@MNPs then were exploited to detect the important neurotransmitter acetylcholine. By coupling choline oxidase and acetylcholine esterase with His@MNPs as peroxidase mimics, target choline and acetylcholine were successfully detected via fluorescent mode with high specificity and sensitivity with the limits of detection down to 200 and 100 nM, respectively. The diagnostic capability of the method is demonstrated by analyzing acetylcholine in human blood serum. This study thus demonstrates the potential of utilizing His@MNPs as peroxidase-mimicking nanozymes for detecting important biological and clinical targets with high sensitivity and reliability.

Phasic oxygen dynamics confounds fast choline-sensitive biosensor signals in the brain of behaving rodents

Cholinergic fast time-scale modulation of cortical physiology is critical for cognition, but direct local measurement of neuromodulators in vivo is challenging. Choline oxidase (ChOx)-based electrochemical biosensors have been used to capture fast cholinergic signals in behaving animals. However, these transients might be biased by local field potential and O2-evoked enzymatic responses. Using a novel Tetrode-based Amperometric ChOx (TACO) sensor, we performed highly sensitive and selective simultaneous measurement of ChOx activity (COA) and O2. In vitro and in vivo experiments, supported by mathematical modeling, revealed that non-steady-state enzyme responses to O2 give rise to phasic COA dynamics. This mechanism accounts for most of COA transients in the hippocampus, including those following locomotion bouts and sharp-wave/ripples. Our results suggest that it is unfeasible to probe phasic cholinergic signals under most behavioral paradigms with current ChOx biosensors. This confound is generalizable to any oxidase-based biosensor, entailing rigorous controls and new biosensor designs.