Function-Oriented Graphene Quantum Dots Probe for Single Cell in situ Sorting of Active Microorganisms in Environmental Samples

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.

Keywords: bioimaging; environmental samples; functional bacteria; graphene quantum dots; single-cell in situ sorting.

SCHEME 1

Strategy for using graphene quantum dot (GQD) probes for single-cell sorting.

FIGURE 1

Micromorphology and properties of graphene quantum dots (GQDs). (a,b) Transmission electron microscopy (TEM) and high-resolution (HR)-TEM images of GQDs-N [inset: particle size distribution in the TEM image and fast Fourier transform (FFT) pattern in the HR-TEM image]. (c,d) TEM and HR-TEM data of GQDs-M (inset: particle size distribution in the TEM image and FFT pattern in the HR-TEM image). (e) Part of the honeycomb structure of the GQDs. (f) Atomic force microscopy height sensor image of GQDs-N (inset: height profile along the white line). High-resolution N 1s XPS data of GQDs-N in panel (g) and GQDs-M in panel (h); the concentrations of the corresponding functional groups are included in Supplementary Table 2.

FIGURE 2

Optical properties of functionalized GQDs. Fluorescent spectra of GQDs-N in panel (A), GQDs-M in panel (B). UV-visible absorption, emission, and excitation spectra of GQDs-N in panel (C), and GQDs-M in panel (D). Time-resolved fluorescence spectrum of GQDs-N in panel (E). Changes in the fluorescence intensity I/I₀ at 520 nm as a function of time of GQDs-M1 incubation with strain S12 (1–10 h). I represents the fluorescence intensity of GQDs-M and I₀ indicates the fluorescence intensity of GQDs-N in panel (F).

FIGURE 3

Bioimaging of pure culture of microorganism and real samples with GQDs-M (1 mg/mL) including fluorescent channel, bright-field, and merged channel. (a) S12-bright field; (b) S12-GQDs-M channel; (C) S12-merged channel; (d) BI-42-bright field; (e) BI-42-GQDs-M channel; (f) BI-42-merged channel; (g) artificial microbial community (S12, Escherichia coli, C1)-GQDs-M channel; (h) artificial microbial community-DAPI channel; (i) artificial microbial community-merged channel; (j) environmental sample (Baihua Chong)-bright field; (k) environmental sample-GQDs-M channel; and (l) environmental sample-merged channel.

FIGURE 4

Single-cell sorting of environmental samples. (a–f) Single-cell sorting; (a) (#31) and (d) (RG) are fluorescent images of the sorting chip, (b,e) indicate sorting chip images before sorting, and (c,f) represent sorting chip images after sorting. (g–j) Fluorescence verification; (g,h) are bright field and fluorescent images of sorted strain S1A1, and (i,j) correspond to the sorted strain RCA.

FIGURE 5

Verification of azo decolorization function of sorted bacteria.