Biology subjects

Reggente, M.

Publications and source records attributed to Reggente, M..

6 recordsLinked to original sources

bioRxiv2024

Covalent conjugation of glucose oxidase on single-walled carbon nanotubes for glucose sensing

Glucose sensing and monitoring are crucial for biological and medical applications. Compared to existing methods, real-time detection and long-term monitoring are still required. Single-walled carbon nanotubes (SWCNTs) have excellent optical properties for sensing applications, which provide the possibility for designing a new generation of glucose sensors. In this study, we describe a method for covalently conjugate glucose oxidase (GOx) on SWCNTs as an optical glucose sensor. The functional groups are introduced by a photocatalytic reaction which acts as the handle for protein loading on SWCNTs. In this sp3 defect reaction, the optical properties of SWCNTs can be maintained. With a convenient bioconjugation reaction, the GOx could be covalently linked with SWCNTs. Compared to the non-covalent immobilization conjugates, the covalent conjugate sensor exhibits a much stronger optical response toward glucose, and the stability of the biosensor also increases in harsh conditions. At the same time, we also report the changing ratio of the original E11 and defected E11* peak during the bioconjugation reaction, which is also inspiring for reaction monitoring on SWCNTs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/618143v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@e1ef8org.highwire.dtl.DTLVardef@11354d7org.highwire.dtl.DTLVardef@2717cforg.highwire.dtl.DTLVardef@8c57bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Photoluminescence brightening of single-walled carbon nanotubes through conjugation with graphene quantum dots

Spanning the tissue transparency window, the near-infrared (NIR) photoluminescence (PL) of single-walled carbon nanotubes (SWCNTs) can optically penetrate biological tissue for deep-tissue imaging and optical sensing. SWCNTs are often functionalized with single-stranded DNA (ssDNA) to yield biocompatible, responsive, and selective sensors. However, the low brightness of these ssDNA-wrapped SWCNTs sensors restricts the depth at which such sensors can be implanted in the tissue. This work demonstrates the PL enhancement of ssDNA-wrapped SWCNTs by incorporating biocompatible graphene quantum dots (GQDs). Two kinds of GQDs, pristine (PGQDs) and nitrogen-doped (NGQDs), were fabricated and characterized. Thermodynamically, both GQDs were shown to significantly increase the fluorescence efficiency of ssDNA-SWCNTs with the same degree of PL enhancement after 3 h. Furthermore, a correlation between the diameter of the SWCNTs and the PL enhancement factor was found; the larger the SWCNT diameter, the higher the PL increase upon adding GQDs. For instance, a 30-fold enhancement was achieved for (8,6) chirality while it was only 2-fold for the (6,5) chirality. Our experiments showed that adding GQDs increases the surface coverage of SWCNTs suspended by ssDNA, limiting water molecules access to the nanotube surface, thus increasing the fluorescence efficiency. Kinetically, NGQDs brightened SWCNTs much faster than PGQDs. The PL intensity reached a plateau in 2 min following the addition of NGQDs, while it was still increasing even after 1 h upon the addition of PGQDs. We show that NGQDs can act as reducing agents to decrease the amount of dissolved oxygen, which quenches the SWCNTs PL. This advancement provides a promising tool for engineering the brightness of NIR sensors for biomedical applications such as single-molecule imaging of individual SWCNTs using NIR confocal microscopy and deep tissue sensing.

biophysics↗

bioRxiv2023

Living Photovoltaics based on Recombinant Expression of MtrA Decaheme in Photosynthetic Bacteria

At the center of microbial bioelectricity applications lies the critical need to express foreign heme proteins that are capable of redirecting the electron flux of the cells metabolism. This study presents bioengineered Synechocystis sp. PCC 6803 cells capable of increased electrogenicity through the introduction of a genetic construct for cytochrome expression. We could demonstrate the functional expression of the periplasmic MtrA decaheme c-type cytochrome from Shewanella oneidensis, a dissimilatory metal-reducing exoelectrogen, inside Synechocystis. Protein expression was verified through western-blotting and immunostaining, and oxygen evolution, optical density, and absorption measurements confirm sustained cell activity and viability under the tested expression conditions. Furthermore, the bioengineered cells show enhanced mediated exoelectrogenicity, as confirmed through a colorimetric iron assay and electrochemical measurements. Compared to wildtype cells on graphite electrodes, the bioengineered cells show a 2-fold increase in light-dependent, extracellular electron transfer, achieving photocurrent densities of 4 A/cm2 under white light illumination of [~]500 mol m-2s-1. The increased capacitance obtained under illumination and suppressed photocurrents in the presence of the photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suggest increased extraction of photosynthetically derived electrons from the recombinant cells. The improved bioelectricity transport across the outer membranes, as achieved through the heterologous heme expression inside cyanobacteria, presents new opportunities for re-wiring the metabolisms of light-harvesting microbes.

bioengineering↗

bioRxiv2023

Implementation of a flavin biosynthesis operon improves extracellular electron transfer in bioengineered Escherichia coli

Bioelectrochemical systems (BES) are promising for energy, sensing, environmental, and synthesis applications. Escherichia coli were previously bioengineered for application in BES by introduction of extracellular electron transfer (EET) pathways. Inspired by the metal-reducing (Mtr) pathway of Shewanella oneidensis MR-1, several of its cytochromes were heterologously expressed in E. coli, leading to increased EET rates and successful application in BES. Besides direct electron transfer, S. oneidensis MR-1 is known to secrete flavins that act as redox mediators and are crucial for high EET rates. Here we co-express the Mtr pathway and a flavin biosynthesis pathway in E. coli, to enhance EET in engineered strains. The secretion of both flavin mononucleotide and riboflavin was increased up to 3-fold in engineered strains. Chronoamperometry revealed an up to ~3.4-fold increase in current over the wild type when co-expressing cytochromes and flavin biosynthesis genes, and a ~2.3-fold increase when expressing flavin biosynthesis genes on their own. Thus, the introduction of flavin biosynthesis genes yields in a distinct, yet complementary EET mechanism, and holds promise for application in BES.

bioengineering↗

bioRxiv2021

Tailored extracellular electron transfer pathways enhance the electroactivity of Escherichia coli

Extracellular electron transfer (EET) engineering in Escherichia coli holds great potential for bioremediation, energy and electrosynthesis applications fueled by readily available organic substrates. Due to its vast metabolic capabilities and availability of synthetic biology tools to adapt strains to specific applications, E. coli is of advantage over native exoelectrogens, but limited in electron transfer rates. We enhanced EET in engineered strains through systematic expression of electron transfer pathways differing in cytochrome composition, localization and origin. While a hybrid pathway harboring components of an E. coli nitrate reductase and the Mtr complex from the exoelectrogen Shewanella oneidensis MR-1 enhanced EET, the highest efficiency was achieved by implementing the complete Mtr pathway from S. oneidensis MR1 in E. coli. We show periplasmic electron shuttling through overexpression of a small tetraheme cytochrome to be central to the electroactivity of this strain, leading to enhanced degradation of the pollutant methyl orange and significantly increased electrical current to graphite electrodes.

bioengineering↗

bioRxiv2021

Distinguishing dopamine and calcium responses using XNA-nanotube sensors for improved neurochemical sensing

To date, the engineering of single-stranded DNA-SWCNT (DNA-SWCNT) optical biosensors have largely focused on creating sensors for new applications with little focus on optimising existing sensors for in vitro and in vivo conditions. Recent studies have shown that nanotube fluorescence can be severely impacted by changes in local cation concentrations. This is particularly problematic for neurotransmitter sensing applications as spatial and temporal fluctuations in the concentration of cations, such as Na+, K+, or Ca2+, play a central role in neuromodulation. This can lead to inaccuracies in the determination of neurotransmitter concentrations using DNA-SWCNT sensors, which limits their use for detecting and treating neurological diseases. Herein, we present new approaches using locked nucleic acid (LNA) to engineer SWCNT sensors with improved stability towards cation-induced fluorescence changes. By incorporating LNA bases into the (GT)15-DNA sequence, we create sensors that are not only more resistant towards undesirable fluorescence modulation in the presence of Ca2+ but that also retain their capabilities for the label-free detection of dopamine. The synthetic biology approach presented in this work therefore serves as a complementary means for enhancing nanotube optoelectronic behavior, unlocking previously unexplored possibilities for developing nano-bioengineered sensors with augmented capabilities.

bioengineering↗