Biology subjects

Boghossian, A. A.

Publications and source records attributed to Boghossian, A. A..

8 recordsLinked to original sources

bioRxiv2023

Directed evolution of nanosensors for the detection of mycotoxins

In this study, we develop and apply a directed evolution approach to engineer the optical sensing properties of DNA-wrapped single-walled carbon nanotubes (DNA-SWCNTs) towards mycotoxins, a class of molecules critical to detect in the food industry. We successfully demonstrate the creation of sensors for the detection of both the aflatoxin B1 (AFB1) and fumonisin B1 (FB1) mycotoxins based on the specific response of the (9,4) and (7,5) SWCNT chirality fluorescence peaks, respectively. The resulting chirality-specific responsivity was used to demonstrate the multimodal detection of both mycotoxins at different wavelengths of light in the presence of complex food medium. Moreover, we show that directed evolution can be used not only to improve the chiral-dependent selectivity of our sensors to the mycotoxins, but also the sensor sensitivity and fluorescence intensity through multiple rounds of evolution. The approach demonstrated in this study is versatile and could be generalized to other SWCNT sensors as well as other nanosensors comprising a biological element.

biophysics↗

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

A simple micropreparative gel electrophoresis technique for purification of proteins, nucleic acids, and bioconjugates

The biochemical and biomedical fields hinge on the ability to effectively separate and purify biological macromolecules. Though this need is largely addressed with a variety of chromatographic and electrophoretic purification techniques, such techniques are usually laborious, time-consuming, and often require complex and costly instalments that are inaccessible to most laboratories. In this work, we introduce a simple micro-preparative (MP) method based on polyacrylamide gel electrophoresis (PAGE) to purify biological samples containing proteins, nucleic acids, and complex bioconjugates. Using a conventional vertical slab system, we demonstrate the extraction of purified DNA, proteins, and DNA-protein bioconjugates from their respective mixtures using MP-PAGE. We apply this system to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using specialized commercial devices. We also demonstrate the purification of folded enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, comparable to purities achieved using a two-step size exclusion and immobilized metal-ion affinity chromatography purification procedure. Finally, we demonstrate the successful isolation of an EYFP-DNA bioconjugate sample that otherwise could not be processed using the two-step chromatography procedure. MP-PAGE thus offers a rapid and versatile means of purifying a variety of biomolecules without the need for specialized equipment.

biochemistry↗

bioRxiv2021

Modulating the properties of DNA-SWCNT sensors using chemically modified DNA

Properties of SWCNT-based sensors such as brightness and detection capabilities strongly depend on the characteristics of the wrapping used to suspend the nanotubes. In this study, we explore ways to modify the properties of DNA-SWCNT sensors by using chemically modified DNA sequences, with the aim of creating sensors more suitable for use in in vivo and in vitro applications. We show that both the fluorescence intensity and sensor reactivity are strongly impacted not only by the chemical modification of the DNA but also by the method of preparation. In the absence of modifications, the sensors prepared using MeOH-assisted surfactant exchange exhibited higher overall fluorescence compared to those prepared by direct sonication. However, we demonstrate that the incorporation of chemical modifications in the DNA sequence could be used to enhance the fluorescence intensity of sonicated samples. We attribute these improvements to both a change in dispersion efficiency as well as to a change in SWCNT chirality distribution. Furthermore, despite their higher intensities, the response capabilities of sensors prepared by MeOH-assisted surfactant exchange were shown to be significantly reduced compared to their sonicated counterparts. Sonicated sensors exhibited a globally higher turn-on response towards dopamine compared to the exchanged samples, with modified samples retaining their relative intensity enhancement. As the increases in fluorescence intensity were achieved without needing to alter the base sequence of the DNA wrapping or to add any exogenous compounds, these modifications can - in theory - be applied to nearly any DNA sequence to increase the brightness and penetration depths of a variety of DNA-SWCNT sensors without affecting biocompatibility or reducing the near-limitless sequence space available. This makes these sensors an attractive alternative for dopamine sensing in vitro and in vivo by enabling significantly higher penetration depths and shorter laser exposure times.

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↗