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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Early life stress induces irritable bowel syndrome from childhood to adulthood in mice

Background Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorder. Traditionally, early life stress (ELS) is predisposed to IBS in adult. However, whether ELS induces IBS in early life remains unclear. Methods Separated cohort studies were conducted in neonatal male pups of C57BL/6 mice by maternal separation (MS) model. MS and non-separation mice were scheduled to be evaluated for prime IBS-phenotypes, including visceral hypersensitivity, intestinal motility, intestinal permeability, and anxiety-like behavior. Ileal contents and fecal samples were collected and analyzed by 16S rRNA gene sequencing and bacterial community analyses. Subcellular structures of intestinal epithelial, such as epithelial tight junctions and mitochondria, were observed under transmission electron microscopy. Results MS induced visceral hypersensitivity and decreased total intestinal transit time from childhood to adulthood. In addition, MS induced intestinal hyperpermeability and anxiety-like behavior from adolescence to adulthood. Besides, MS affected intestinal microbial composition from childhood to adulthood. Moreover, MS disrupted intestinal mitochondrial structure from childhood to adulthood. Conclusion The study showed for the first time that MS induced IBS from early life to adulthood in mice. The disrupted intestinal mitochondrial structure and the significant dysbiosis of intestinal microbiota in early life may contribute to the initiation and progress of IBS from early life to adulthood.

Tao, Enfu↗
DOE OSTI2018

Proteogenomic Analysis of Burkholderia Species Strains 25 and 46 Isolated from Uraniferous Soils Reveals Multiple Mechanisms to Cope with Uranium Stress

Two Burkholderia spp. (strains SRS-25 and SRS-46) were isolated from high concentrations of uranium (U) from the U.S. Department of Energy (DOE)-managed Savannah River Site (SRS). SRS contains soil gradients that remain co-contaminated by heavy metals from previous nuclear weapons production activities. Uranium (U) is one of the dominant contaminants within the SRS impacted soils, which can be microbially transformed into less toxic forms. We established microcosms containing strains SRS-25 and SRS-46 spiked with U and evaluated the microbially-mediated depletion with concomitant genomic and proteomic analysis. Both strains showed a rapid depletion of U; draft genome sequences revealed SRS-25 genome to be of approximately 8,152,324 bp, a G + C content of 66.5, containing a total 7604 coding sequences with 77 total RNA genes. Similarly, strain SRS-46 contained a genome size of 8,587,429 bp with a G + C content of 67.1, 7895 coding sequences, with 73 total RNA genes, respectively. An in-depth, genome-wide comparisons between strains 25, 46 and a previously isolated strain from our research (Burkholderia sp. strain SRS-W-2-2016), revealed a common pool of 3128 genes; many were found to be homologues to previously characterized metal resistance genes (e.g., for cadmium, cobalt, and zinc), as well as for transporter, stress/detoxification, cytochromes, and drug resistance functions. Furthermore, proteomic analysis of strains with or without U stress, revealed the increased expression of 34 proteins from strain SRS-25 and 52 proteins from strain SRS-46; similar to the genomic analyses, many of these proteins have previously been shown to function in stress response, DNA repair, protein biosynthesis and metabolism. Overall, this comparative proteogenomics study confirms the repertoire of metabolic and stress response functions likely rendering the ecological competitiveness to the isolated strains for colonization and survival in the heavy metals contaminated SRS soil habitat.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Bioactive diterpenoids impact the composition of the root-associated microbiome in maize (Zea mays)

Abstract Plants deploy both primary and species-specific, specialized metabolites to communicate with other organisms and adapt to environmental challenges, including interactions with soil-dwelling microbial communities. However, the role of specialized metabolites in modulating plant-microbiome interactions often remains elusive. In this study, we report that maize ( Zea mays ) diterpenoid metabolites with known antifungal bioactivities also influence rhizosphere bacterial communities. Metabolite profiling showed that dolabralexins, antibiotic diterpenoids that are highly abundant in roots of some maize varieties, can be exuded from the roots. Comparative 16S rRNA gene sequencing determined the bacterial community composition of the maize mutant Zman2 ( anther ear 2 ), which is deficient in dolabralexins and closely related bioactive kauralexin diterpenoids. The Zman2 rhizosphere microbiome differed significantly from the wild-type sibling with the most significant changes observed for Alphaproteobacteria of the order Sphingomonadales. Metabolomics analyses support that these differences are attributed to the diterpenoid deficiency of the Zman2 mutant, rather than other large-scale metabolome alterations. Together, these findings support physiological functions of maize diterpenoids beyond known chemical defenses, including the assembly of the rhizosphere microbiome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Use of Split‐Intein Proteins to Design a Small Molecule Biosensor in Plants

Understanding how plants perceive their environment is fundamental to advancing agricultural productivity and sustainability. Many biological small molecules, including those involved in microbial recognition, act rapidly at the plant cell surface, but the absence of tools to visualise these dynamics has limited our ability to dissect plant–microbe communication. To address this gap, we sought to create a genetically encoded biosensor that couples ligand-induced protein dimerization with the production of a fluorescent reporter. Inteins are peptide regions that excise themselves from precursor proteins and ligate the flanking chains (exteins). When each half of a split intein is fused to one of two dimerizing proteins, ligand binding brings them into proximity, inducing intein splicing and ligation of flanking extein sequences (Kang et al. 2022). Similar to previous studies, we split the yeast vacuolar ATPase subunit 1 (VMA1) intein, creating a protein biosensor that produces eGFP upon protein dimerization after ligand binding (Figure 1A) (Mootz et al. 2003). Specifically, eGFP halves (i.e., non-functional N- and C-terminal GFP fragments) were fused to the intein halves, resulting in two fusion proteins: N-terminal GFP::N-terminal intein and C-terminal intein::C-terminal GFP (Figure 1A).

Boone, Brandon A. [Oak Ridge National Laboratory (↗
DOE OSTI2016

Genome evolution and nitrogen fixation in bacterial ectosymbionts of a protist inhabiting wood-feeding cockroaches

By combining genomics and isotope imaging analysis using high-resolution secondary ion mass spectrometry (NanoSIMS), we examined the function and evolution of Bacteroidales ectosymbionts of the protist Barbulanympha from the hindguts of the wood-eating cockroach Cryptocercus punctulatus . In particular, we investigated the structure of ectosymbiont genomes, which, in contrast to those of endosymbionts, has been little studied to date, and tested the hypothesis that these ectosymbionts fix nitrogen. Unlike with most obligate endosymbionts, genome reduction has not played a major role in the evolution of the Barbulanympha ectosymbionts. Instead, interaction with the external environment has remained important for this symbiont as genes for synthesis of transporters, outer membrane proteins, lipopolysaccharides, and lipoproteins have been retained. The ectosymbiont genome carried two complete operons for nitrogen fixation, a urea transporter, and a urease, indicating the availability of nitrogen as a driving force behind the symbiosis. NanoSIMS analysis of C. punctulatus hindgut symbionts exposed in vivo to 15 N 2 supports the hypothesis that Barbulanympha ectosymbionts are capable of nitrogen fixation. This genomic and in vivo functional investigation of protist ectosymbionts highlights the diversity of evolutionary forces and trajectories that shape symbiotic interactions. The ecological and evolutionary importance of symbioses is increasingly clear, but the overall diversity of symbiotic interactions remains poorly explored. Here in this study, we investigated the evolution and nitrogen fixation capabilities of ectosymbionts attached to the protist Barbulanympha from the hindgut of the wood-eating cockroach Cryptocercus punctulatus . In addressing genome evolution of protist ectosymbionts, our data suggest that the ecological pressures influencing the evolution of extracellular symbionts clearly differ from intracellular symbionts and organelles. Using NanoSIMS analysis, we also obtained direct imaging evidence of a specific hindgut microbe playing a role in nitrogen fixation. These results demonstrate the power of combining NanoSIMS and genomics tools for investigating the biology of uncultivable microbes. This investigation paves the way for a more precise understanding of microbial interactions in the hindguts of wood-eating insects and further exploration of the diversity and ecological significance of symbiosis between microbes.

09 BIOMASS FUELS↗
DOE OSTI2026

Membrane Composition Influences Expression Yield of Plant Cytochrome P450s in E. coli Lysate-Based Cell-Free Systems

Plant cytochrome P450 enzymes are central to natural product biosynthesis, but remain difficult to express in microbial hosts due to their transmembrane nature. Lysate-based, cell-free expression systems allow supplementation with artificial membranes to support the expression and translocation of transmembrane proteins. We developed a framework to systematically test liposomal membrane compositions to enhance the plant P450 expression yield. Adjustments to common phospholipid ratios or the addition of plant galactolipids had minimal impact on expression. In contrast, blended liposomes containing Egg PC, sterol-conjugated phospholipids, and PEGylated lipids produced concentration-dependent increases in expression. Expression of an Escherichia coli mechanosensitive channel and three plant P450s improved more than 2-fold, with some P450s showing up to 14-fold enhancement. Furthermore, these findings highlight membrane composition as a key determinant of the P450 expression yield in cell-free expression systems. While P450 activity was not measured, these findings provide a framework for future workflows toward achieving functional plant transmembrane enzymes for the bioproduction of natural products.

TXTL↗
DOE OSTI2023

The Shr receptor from Streptococcus pyogenes uses a cap and release mechanism to acquire heme–iron from human hemoglobin

Streptococcus pyogenes (group A Streptococcus ) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, S. pyogenes uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a “cap and release” mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr–Hb complex which function to transiently uncap HID1 from the heme bound to Hb’s β subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr’s NEAT domains. These dynamics may maximize the efficiency of heme scavenging by S. pyogenes , enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Yeasts from temperate forests

Yeasts are ubiquitous in temperate forests. While this broad habitat is well-defined, the yeasts inhabiting it and their life cycles, niches, and contributions to ecosystem functioning are less understood. Yeasts are present on nearly all sampled substrates in temperate forests worldwide. They associate with soils, macroorganisms, and other habitats and no doubt contribute to broader ecosystem-wide processes. Researchers have gathered information leading to hypotheses about yeasts' niches and their life cycles based on physiological observations in the laboratory as well as genomic analyses, but the challenge remains to test these hypotheses in the forests themselves. Here, we summarize the habitat and global patterns of yeast diversity, give some information on a handful of well-studied temperate forest yeast genera, discuss the various strategies to isolate forest yeasts, and explain temperate forest yeasts' contributions to biotechnology. Here, we close with a summary of the many future directions and outstanding questions facing researchers in temperate forest yeast ecology. Yeasts present an exciting opportunity to better understand the hidden world of microbial ecology in this threatened and global habitat.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, in this study, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three P. putida strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h -1 on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators gacS, gacA, and turA along with large duplication and intergenic deletion events that impacted the heterologously-expressed tphAB II catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of tphAB II and tpaK displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve P. putida growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Transitions of foliar mycobiota community and transcriptome in response to pathogenic conifer needle interactions

Profiling the host–mycobiota interactions in healthy vs. diseased forest ecosystems helps understand the dynamics of understudied yet increasingly important threats to forest health that are emerging due to climate change. We analyzed the structural and functional changes of the mycobiota and the responses of Pinus contorta in the Lophodermella needle cast pathosystem through metabarcoding and metatranscriptomics. When needles transitioned from asymptomatic to symptomatic, dysbiosis of the mycobiota occurred, but with an enrichment of Lophodermella pathogens. Many pathogenicity-related genes were highly expressed by the mycobiota at the necrotrophic phase, showing an active pathogen response that are absent in asymptomatic needles. This study also revealed that Lophodermella spp. are members of a healthy needle mycobiota that have latent lifestyles suggesting that other pine needle pathogens may have similar biology. Interestingly, Pinus contorta upregulated defense genes in healthy needles, indicating response to fungal recognition, while a variety of biotic and abiotic stresses genes were activated in diseased needles. Further investigation to elucidate the possible antagonistic interplay of other biotic members leading to disease progression and/or suppression is warranted. This study provides insights into microbial interactions in non-model pathosystems and contributes to the development of new forest management strategies against emerging latent pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Support for Next-Generation Ecosystem Experiments (NGEE Arctic) Field Campaign Report

An important challenge for Earth System Models (ESMs) is to represent land surface and subsurface processes and their complex interactions in a warming climate. This is especially important for arctic ecosystems where permafrost extent, topography, hydrology, vegetation, disturbance, and biogeochemistry are inextricably linked. The implications of such linkages include permafrost thaw and deepening of the active layer, microbial decomposition of vulnerable soil organic matter, altered productivity and migration of tall woody shrubs, and watershed-scale changes in surface and groundwater transport and storage. Although ESMs describe some of these interactions for high-latitude ecosystems, their representation requires extensive confrontation with field and laboratory observations to test and improve models, and to use those models to inspire new observations and experiments. The Next-Generation Ecosystem Experiments (NGEE Arctic) is a 10-year project (2012 to 2022) to improve our predictive understanding of carbon-rich arctic system processes and feedbacks to climate. This is achieved through experiments, observations, and synthesis of existing data sets that strategically inform model process representation and parameterization, and that enhance the knowledge base required for model initialization, calibration, and evaluation. One question of special interest to the NGEE Arctic project addresses how above- and below-ground plant functional traits might change across environmental gradients, and what are the consequences for arctic ecosystem C, water, and nutrient fluxes? Arctic plant traits, and their variation in response to changing environmental conditions, will play a key role in the response of tundra ecosystems to warming, permafrost thaw, and the wetter or drier conditions expected in the future. The appropriate representation of these functional traits in models is necessary to accurately represent ecosystem C, water, and nutrient cycling in tundra ecosystems, now and in the future. We characterized the variation in above- and below-ground plant traits in response to varying edaphic and environmental conditions in Utqiaġvik (formerly Barrow), Alaska. These new data, combined with remote-sensing and synthesis activities across the Arctic and the globe, have been used to inform model structure and parameterization of key processes such as photosynthesis, nutrient dynamics, and trait variation across the landscape.

North Slope of Alaska, arctic processes (meteorolo↗
DOE OSTI2026

Depth-resolved sagebrush root metabolomics, rhizosphere microbial communities, and geochemistry at the East River Watershed

This data set consists of results from soil nutrient profile, untargeted metabolomics, mass spec imaging, and amplicon sequencing. Data for soil nutrient profile includes common cations (Ca, Mg, Na, and K etc.) extracted from 3 digesting steps – ammonia acetate (for exchangeable cations), nitric acid (for acid dissolved fraction), and hydrofluoric acid/perchloric acid (HF/HClO4) for whole soil digestion. It also includes concentration of organic carbon, inorganic nitrogen (ammonia and nitrate) and phosphorus (Bray-1 P and nitric acid extract), and total nitrogen and phosphorus. Data for untargeted metabolomics includes metabolomic profile for root exudate/tissues and soil extracts from depths at surface soil to saprolite, that were measured using gas chromatography – mass spectrometry (GC-MS), and liquid chromatography – tandem mass spectrometry (LC-MS/MS). Data for mass spec imaging includes spatial distribution of metabolites that were detected and annotated with Fourier transformation ion cyclotron resonance mass spectrometer (FTICR-MS). Data for amplicon sequencing includes the base paired 16S and ITS ribosomal RNA sequences from Miseq Illumina sequencing. All samples were collected from 2 sampling campaign October 2022 and June 2023. Collectively, these datasets enable a mechanistic evaluation of how nutrient acquisition, especially nitrogen and phosphorus, differs between shallow roots operating in soil and deep roots functioning within the fractured bedrock zone. All files are provided as comma-separated values (CSV) fies (.csv) and (GZIP) file (.gz). The compressed .gz FASTQ files can be read directly in R using the dada2 package as part of the amplicon sequence analysis workflow. This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231. This research was performed on a project award 60563 (https://dx.doi.org/10.46936/expl.proj.2022.60563/60008727) from the Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility sponsored by the Biological and Environmental Research program under Contract No. DE-AC05-76RL01830.

EARTH SCIENCE > AGRICULTURE > SOILS > CARBON↗
DOE OSTI2017

Consultancy on Large-Scale Submerged Aerobic Cultivation Process Design - Final Technical Report: February 1, 2016 -- June 30, 2016

NREL is developing an advanced aerobic bubble column model using Aspen Custom Modeler (ACM). The objective of this work is to integrate the new fermentor model with existing techno-economic models in Aspen Plus and Excel to establish a new methodology for guiding process design. To assist this effort, NREL has contracted Genomatica to critique and make recommendations for improving NREL's bioreactor model and large scale aerobic bioreactor design for biologically producing lipids at commercial scale. Genomatica has highlighted a few areas for improving the functionality and effectiveness of the model. Genomatica recommends using a compartment model approach with an integrated black-box kinetic model of the production microbe. We also suggest including calculations for stirred tank reactors to extend the models functionality and adaptability for future process designs. Genomatica also suggests making several modifications to NREL's large-scale lipid production process design. The recommended process modifications are based on Genomatica's internal techno-economic assessment experience and are focused primarily on minimizing capital and operating costs. These recommendations include selecting/engineering a thermotolerant yeast strain with lipid excretion; using bubble column fermentors; increasing the size of production fermentors; reducing the number of vessels; employing semi-continuous operation; and recycling cell mass.

09 BIOMASS FUELS↗
DOE OSTI2016

Current understanding of the correlation of lignin structure with biomass recalcitrance

Lignin, a complex aromatic polymer in terrestrial plants, contributes significantly to biomass recalcitrance to microbial and/or enzymatic deconstruction. To reduce biomass recalcitrance, substantial endeavors have been exerted on pretreatment and lignin engineering in the past few decades. Lignin removal and/or alteration of lignin structure have been shown to result in reduced biomass recalcitrance with improved cell wall digestibility. While high lignin content is usually a barrier to a cost-efficient application of bioresources to biofuels, the direct correlation of lignin structure and its concomitant properties with biomass remains unclear due to the complexity of cell wall and lignin structure. Advancement in application of biorefinery to production of biofuels, chemicals, and bio-derived materials necessitates a fundamental understanding of the relationship of lignin structure and biomass recalcitrance. In this mini-review, we focus on recent investigations on the influence of lignin chemical properties on bioprocessability—pretreatment and enzymatic hydrolysis of biomass. Furthermore, lignin-enzyme interactions and the effects of lignin compositional units, hydroxycinnamates, and lignin functional groups on biomass recalcitrance have been highlighted, which will be useful not only in addressing biomass recalcitrance but also in deploying renewable lignocelluloses efficiently.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

Soil organic matter availability and climate drive latitudinal patterns in bacterial diversity from tropical to cold temperate forests

Bacteria are one of the most abundant and diverse groups of micro-organisms and mediate many critical terrestrial ecosystem processes. Despite the crucial ecological role of bacteria, our understanding of their large-scale biogeography patterns across forests, and the processes that determine these patterns lags significantly behind that of macroorganisms. Here, we evaluated the geographic distributions of bacterial diversity and their driving factors across nine latitudinal forests along a 3,700-km north–south transect in eastern China, using high-throughput 16S rRNA gene sequencing. Four of 32 phyla detected were dominant: Acidobacteria , Actinobacteria , Alphaproteobacteria and Chloroflexi (relative abundance > 5%). Significant increases in bacterial richness and phylogenetic diversity were observed for temperate forests compared with subtropical or tropical forests. The soil organic matter (SOM) mineralisation rate (SOM min , an index of SOM availability) explained the largest significant variations in bacterial richness. Variation partition analysis revealed that the bacterial community structure was closely correlated with environmental variables and geographic distance, which together explained 80.5% of community variation. Among all environmental factors, climatic features (MAT and MAP) were the best predictors of the bacterial community structure, whereas soil pH and SOM min emerged as the most important edaphic drivers of the bacterial community structure. Plant functional traits (community weighted means of litter N content) and diversity resulted in weak but significant correlations with the bacterial community structure. Our findings provide new evidence of bacterial biogeography patterns from tropical to cold temperate forests. Additionally, the results indicated a close linkage among soil bacterial diversity, climate and SOM decomposition, which is critical for predicting continental-scale responses under future climate change scenarios and promoting sustainable forest ecosystem services.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Live-Cell Quantum Dot-Based Tracking of Plant & Microbial Extracellular Vesicles

All cellular organisms examined to date release various types of small vesicles called extracellular vesicles (EVs). They were originally thought to be unimportant, but research into EVs as carriers of key messages for communication within large, multicellular organisms or between interacting organisms has gained interest in many different scientific areas of study. Recently, it was discovered that both plants and their pathogenic fungi generate EVs and they are important vehicles for sending plant signals to thwart invasion by fungal pathogens, and in response, fungi use EVs to help evade plant defenses. This project aimed to develop new ways to visualize EVs and their potential nucleic acid cargo isolated from the crop plant, sorghum. We adapted a click chemistry-based method for making quantum dot-based RNA sensors, which will be a useful tool for others studying small RNAs. These sensors were not used detect RNA in sorghum EVs because unlike EVs from Arabidopsis, sorghum EVs were not enriched for specific small RNAs. This interesting difference shifted the project in an exciting direction, which focused on comparing EV proteomes. Our study discovered that greater than 60% of Arabidopsis EV proteins were also found in the sorghum EV proteome, revealing a partial conservation between a eudicot and monocot plant. These studies have raised new, fundamental questions on the conserved function of EVs in plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Biocompatible Water Soluble Polyacrylic Acid Coated CdSe/Cu Quantum Dot Conjugates for Biomolecule Detection

Biocompatible polyacrylic acid functionalized CdSe/Cu quantum dot conjugates were synthesized to be used for biomolecules detection. The study results demonstrate the conjugation of the 2.5-3 nm QD with gram negative bacteria with a low detection limit of 28 cfu/ml. The photoluminescence (PL) intensity was correlated to bacterial count, cellular proteins and exopolysaccharides in the tested samples. Confocal Scanning Laser Microscopy (CSLM) images showed significant QD uptake within the cells, both cytoplasm and DNA were the predominant targeted biomolecules, higher fluorescent uptake was shown in gram negative bacteria than that observed for gram positive bacteria. Moreover, PL showed that there was a distinction between live and dead cells as well as gram negative and gram positive cells. Cell viability was not affected even after 6 days (100% viability) rendering it a non-toxic QD. The method is simple and is performed in a single step within approximately 10 min as compared to multi-step protocols for classical microbial count or fluorescent dye staining. All the above results indicate that the CdSe/Cu-PAA QDs are suitable for biomolecule detection, bio-labeling and bioimaging applications.

77 NANOSCIENCE AND NANOTECHNOLOGY↗