Abstract
Functional microorganisms play a vital role in removing environmental pollutants because of their diverse metabolic capability. Herein, a function-oriented fluorescence resonance energy transfer (FRET)-based graphene quantum dots (GQDs-M) probe was developed for the specific identification and accurate sorting of azo-degrading functional bacteria in the original location of environmental samples for large-scale culturing. First, nitrogen-doped GQDs (GQDs-N) were synthesized using a bottom-up strategy. Then, a GQDs-M probe was synthesized based on bonding FRET-based GQDs-N to an azo dye, methyl red, and the quenched fluorescence was recovered upon cleavage of the azo bond. Bioimaging confirmed the specific recognition capability of GQDs-M upon incubation with the target bacteria or environmental samples. It is suggested that the estimation of environmental functional microbial populations based on bioimaging will be a new method for rapid preliminary assessment of environmental pollution levels. In combination with a visual single-cell sorter, the target bacteria in the environmental samples could be intuitively screened at the single-cell level in 17 bacterial strains, including the positive control Shewanella decolorationis S12, and were isolated from environmental samples. All of these showed an azo degradation function, indicating the high accuracy of the single-cell sorting strategy using the GQDs-M. Furthermore, among the bacteria isolated, two strains of Bacillus pacificus and Bacillus wiedmannii showed double and triple degradation efficiency for methyl red compared to the positive control (strain S12). This strategy will have good application prospects for finding new species or high-activity species of specific functional bacteria.
Highlights
MATERIALS AND METHODSIdentification, isolation, and domestication of particular functional microorganisms from the environment are necessary for the construction of bioresource reservoirs and environmental bioremediation (Berry et al, 2015)
Upon combining the single-cell sorting platform with the function-oriented graphene quantum dot (GQD)-M probe, azo-respiration functional bacteria can be accurately sorted from environmental samples based on fluorescence labeling, and do not require any pretreatment
The precursor has four positively charged sites of NO2 groups, which allow nucleophilic substitution reactions to occur with several alkaline species such as NH3 and NH2NH2 that are added to the hydrothermal media, implying that the precursor can be fused with graphitized carbon materials at a low temperature (200◦C) (Wang et al, 2014)
Summary
Identification, isolation, and domestication of particular functional microorganisms from the environment are necessary for the construction of bioresource reservoirs and environmental bioremediation (Berry et al, 2015). To increase the detection efficiency, some new methods have been developed in recent years, including Raman-stable isotope probing (Song et al, 2017), immunomagnetic separation (Wen et al, 2018), droplet microfluidic technologies (Watterson et al, 2020), and fluorescence in situ hybridization in combination with fluorescence-activated cell sorting (Grieb et al, 2020) These methods do not allow the detection and screening of target bacteria from complex environmental samples based on their degradation functions. We synthesized a function-oriented probe graphene quantum dots (GQDs-M) based on FRET which showed high sensitivity and selectivity for azo-respiration functional bacteria. Upon combining the single-cell sorting platform with the function-oriented GQDs-M probe, azo-respiration functional bacteria can be accurately sorted from environmental samples based on fluorescence labeling, and do not require any pretreatment. Laser scanning confocal microscopy (LSCM, LSM 700 Zeiss) was used to monitor the distribution of fluorescence in the bacteria
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