Due to their relative synthetic and chemical simplicity compared to antibodies, aptamers afford enhanced stability and functionality for the detection of environmental contaminants and for use in environmental monitoring. Furthermore, nucleic acid aptamers can be selected for toxic targets which may prove difficult for antibody development. Of particular relevance, aptamers have been selected and used to develop biosensors for environmental contaminants such as heavy metals, small-molecule agricultural toxins, and water-borne bacterial pathogens. This review will focus on recent aptamer-based developments for the detection of diverse environmental contaminants. Within this domain, aptamers have been combined with other technologies to develop biosensors with various signal outputs. The goal of much of this work is to develop cost-effective, user-friendly detection methods that can complement or replace traditional environmental monitoring strategies. This review will highlight recent examples in this area. Additionally, with innovative developments such as wearable devices, sentinel materials, and lab-on-a-chip designs, there exists significant potential for the development of multifunctional aptamer-based biosensors for environmental monitoring. Examples of these technologies will also be highlighted. Finally, a critical perspective on the field, and thoughts on future research directions will be offered.

/pdf/aptamer-based-biosensors-for-environmental-monitoring-196jl1ch67.pdf

Aptamer-Based Biosensors for Environmental Monitoring.

This article provides a comprehensive review of biosensing with DNAzymes, providing an overview of different sensing applications while highlighting major progress and seminal contributions to the field of portable biosensor devices and point-of-care diagnostics. Specifically, the field of functional nucleic acids is introduced, with a specific focus on DNAzymes. The incorporation of DNAzymes into bioassays is then described, followed by a detailed overview of recent advances in the development of in vivo sensing platforms and portable sensors incorporating DNAzymes for molecular recognition. Finally, a critical perspective on the field, and a summary of where DNAzyme-based devices may make the biggest impact are provided.

Biosensing with DNAzymes

Catalytic Nucleic Acids: Biochemistry, Chemical Biology, Biosensors, and Nanotechnology

Due to the considerable environmental impact and the controversial animal welfare associated with industrial meat production, combined with the ever-increasing global population and demand for meat products, sustainable production alternatives are indispensable. In 2013, the world's first laboratory grown hamburger made from cultured muscle cells was developed. However, coming at a price of $300.000, and being produced manually, substantial effort is still required to reach sustainable large-scale production. One of the main challenges is scalability. Microcarriers (MCs), offering a large surface/volume ratio, are the most promising candidates for upscaling muscle cell culture. However, although many MCs have been developed for cell lines and stem cells typically used in the medical field, none have been specifically developed for muscle stem cells and meat production. This paper aims to discuss the MCs' design criteria for skeletal muscle cell proliferation and subsequently for meat production based on three scenarios: (1) MCs are serving only as a temporary substrate for cell attachment and proliferation and therefore they need to be separated from the cells at some stage of the bioprocess, (2) MCs serve as a temporary substrate for cell proliferation but are degraded or dissolved during the bioprocess, and (3) MCs are embedded in the final product and therefore need to be edible. The particularities of each of these three bioprocesses will be discussed from the perspective of MCs as well as the feasibility of a one-step bioprocess. Each scenario presents advantages and drawbacks, which are discussed in detail, nevertheless the third scenario appears to be the most promising one for a production process. Indeed, using an edible material can limit or completely eliminate dissociation/degradation/separation steps and even promote organoleptic qualities when embedded in the final product. Edible microcarriers could also be used as a temporary substrate similarly to scenarios 1 and 2, which would limit the risk of non-edible residues.

/pdf/microcarriers-for-upscaling-cultured-meat-production-59vbr0du5n.pdf

Microcarriers for Upscaling Cultured Meat Production.

Although rational genetic engineering is nowadays the favored method for microbial strain improvement, random mutagenesis is still in many cases the only option. Atmospheric and room-temperature plasma (ARTP) is a newly developed whole-cell mutagenesis tool based on radio-frequency atmospheric-pressure glow discharge plasma which features higher mutation rates than UV radiation or chemical mutagens while maintaining low treatment temperatures. It has been successfully applied on at least 24 bacterial and 14 fungal species, but also on plants, dinoflagellates, and other microbial communities for the improvement of tolerance to medium components, to increase cellular growth and production of cellular biomass, to enhance enzyme activity, and to increase the production of various chemicals. Achievements like 385.7% of acetic acid production enhancement in Acetobacter pasteurianus give this new mutagenesis tool a promising future. However, certain questions remain regarding optimal operational conditions, the effects at subcellular levels, and standard operation procedures, which need to be addressed to facilitate applications of ARTP in microbial breeding and other fields such as evolution of enzymes.

/pdf/microbial-mutagenesis-by-atmospheric-and-room-temperature-39vdqv56ew.pdf

Microbial mutagenesis by atmospheric and room-temperature plasma (ARTP): the latest development

The atmospheric and room-temperature plasma (ARTP) method on the dextranase activity and structure

This study evaluated the ability of a dextranase from a marine bacterium Catenovulum sp. (Cadex) to impede formation of Streptococcus mutans biofilms, a primary pathogen of dental caries, one of the most common human infectious diseases. Cadex was purified 29.6-fold and had a specific activity of 2309 U/mg protein and molecular weight of 75 kDa. Cadex showed maximum activity at pH 8.0 and 40 °C and was stable at temperatures under 30 °C and at pH ranging from 5.0 to 11.0. A metal ion and chemical dependency study showed that Mn2+ and Sr2+ exerted positive effects on Cadex, whereas Cu2+, Fe3+, Zn2+, Cd2+, Ni2+, and Co2+ functioned as inhibitors. Several teeth rinsing product reagents, including carboxybenzene, ethanol, sodium fluoride, and xylitol were found to have no effects on Cadex activity. A substrate specificity study showed that Cadex specifically cleaved the α-1,6 glycosidic bond. Thin layer chromatogram and high-performance liquid chromatography indicated that the main hydrolysis products were isomaltoogligosaccharides. Crystal violet staining and scanning electron microscopy showed that Cadex impeded the formation of S. mutans biofilm to some extent. In conclusion, Cadex from a marine bacterium was shown to be an alkaline and cold-adapted endo-type dextranase suitable for development of a novel marine agent for the treatment of dental caries.

https://www.mdpi.com/1660-3397/16/2/51/pdf

Purification and Characterization of a Biofilm-Degradable Dextranase from a Marine Bacterium.

Selection of a metal ligand modified DNAzyme for detecting Ni2.

Vibrio anguillarum is a bacterial pathogen that causes serious damage to aquatic fish, and its rapid detection and prevention are critical. DNAzymes are DNA-based catalysts with excellent stability. In this study, in vitro selection of DNAzymes was performed using the crude extracellular matrix (CEM) of V. Anguillarum as the target. Different from previous selections targeting bacterial CEM, this work used an unmodified DNA library, allowing easier adoption of the technology. After seven rounds of selection, a DNAzyme named VAE-2 with high activity and specificity was obtained. It showed the highest activity toward V. Anguillarum among eight types of tested bacterial strains. Polyvalent metal ions are needed for its activity. Protease treatment of the CEM and filtration studies indicated that the target is a protein with a molecular weight between 50 k and 100 k Da. A fluorescent biosensor was designed for V. anguillarum with a detection limit down to 4000 cfu/mL, and detection was demonstrated for real fish tissue and feeding water samples. Being the first work of DNAzyme-based sensing of aquatic bacteria, this study indicates that unmodified DNA can be used for targeting bacterial CEM, and it provides a new framework for developing other RNA-cleaving DNAzymes for rapid detection of pathogenic bacteria and water pollution.

Selection of DNAzymes for Sensing Aquatic Bacteria: Vibrio Anguillarum.

The crystal structure of Dextranase from the marine bacterium Arthrobacter oxidans KQ11 (Aodex) was determined at a resolution of 1.4 A. The crystal structure of the conserved Aodex fragment (Ala52-Thr638) consisted of an N-terminal domain N and a C-terminal domain C. The N-terminal domain N was identified as a β-sandwich, connected to a right-handed parallel β-helix at the C-terminus. Sequence comparisons, cavity regions, and key residues of the catalytic domain analysis all suggested that the Aodex was an inverting enzyme, and the catalytic acid and base were Asp439 and Asp420, respectively. Asp440 was not a general base in the Aodex catalytic domain, and Asp396 in Dex49A may not be a general base in the catalytic domain. The thermostability of the S357F mutant using semirational design based on B-factors was clearly better than that of wild-type Aodex. This process may promote the aromatic-aromatic interactions that increase the thermostability of mutant Phe357.

Crystal Structure of GH49 Dextranase from Arthrobacter oxidans KQ11: Identification of Catalytic Base and Improvement of Thermostability Using Semirational Design Based on B-Factors

Wei Ren

Papers

The atmospheric and room-temperature plasma (ARTP) method on the dextranase activity and structure

Purification and Characterization of a Biofilm-Degradable Dextranase from a Marine Bacterium.

Selection of a metal ligand modified DNAzyme for detecting Ni2.

Selection of DNAzymes for Sensing Aquatic Bacteria: Vibrio Anguillarum.

Crystal Structure of GH49 Dextranase from Arthrobacter oxidans KQ11: Identification of Catalytic Base and Improvement of Thermostability Using Semirational Design Based on B-Factors