Primary Amine Functionalized Carbon Dots for Dead and Alive Bacterial Imaging

Abstract

Small molecular dyes are commonly used for bacterial imaging, but they still meet a bottleneck of biological toxicity and fluorescence photobleaching. Carbon dots have shown high potential for bio-imaging due to their low cost and negligible toxicity and anti-photobleaching. However, there is still large space to enhance the quantum yield of the carbon quantum dots and to clarify their mechanisms of bacterial imaging. Using carbon dots for dyeing alive bacteria is difficult because of the thick density and complicated structure of bacterial cell walls. In this work, both dead or alive bacterial cell imaging can be achieved using the primary amine functionalized carbon dots based on their small size, excellent quantum yield and primary amine functional groups. Four types of carbon quantum dots were prepared and estimated for the bacterial imaging. It was found that the spermine as one of precursors can obviously enhance the quantum yield of carbon dots, which showed a high quantum yield of 66.46% and high fluorescence bleaching-resistance (70% can be maintained upon 3-h-irradiation). Furthermore, a mild modifying method was employed to bound ethylenediamine on the surface of the spermine-carbon dots, which is favorable for staining not only the dead bacterial cells but also the alive ones. Investigations of physical structure and chemical groups indicated the existence of primary amine groups on the surface of spermine-carbon quantum dots (which own a much higher quantum yield) which can stain alive bacterial cells visibly. The imaging mechanism was studied in detail, which provides a preliminary reference for exploring efficient and environment-friendly carbon dots for bacterial imaging.

Keywords: bacteria; carbon dots; imaging.

Scheme 1

Preparation of nitrogen-doped carbon dots and fabrication of primary amine functionalized carbon dots.

Figure 1

(a) Fluorescent emission map and ex–em spectrum of spCD, (b) Relative fluorescent intensity of CD, spCD, NH₂–CD and NH₂–spCD in a constant ultraviolet irradiation of 365 nm for 3 h, (c) CIE 1931 chromaticity chart of spCD at excitation of 300~450 nm (step = 5 nm), (d) Fluorescent lifetime of CD, spCD, NH₂–CD and NH₂–spCD, (e) Relative intensity changes of CD, spCD, NH₂–CD, NH₂–spCD and two dyes for a constant detection.

Figure 2

(a) XRD spectra of CD, spCD, NH₂–CD, NH₂–spCD and the PDF standard card, (b) Diameters and QY values for other works and this work [13,15,16,22,26,27,28,40,41,53,54,55,56,57,58,59,60,61,62,63] (c) TEM and HRTEM of CD and (d) spCD, (e) AFM of NH₂–spCDs (left: planar graph of NH₂–spCDs; The picture was a 5 × 5 μm section of the sample while the color bar at the right represents the height value among the section. middle: particle height marked at the left graph in red circle, right: 3D image corresponding to the left graph).

Figure 3

(a) UV–Vis spectra and (b) FTIR spectra of CD, spCD, NH₂–CD and NH₂–spCD, (c) Changes of zeta potentials after cultured with 3% BZ (BZ represent bacteria that cultured by benzalkonium bromide and QD represent bacterial cultured by corresponding carbon dots after cultured in benzalkonium bromide), (d) Zeta potentials of spCD, NH₂–CD and NH₂–spCD, (e) MTT results for CD, spCD, NH₂–CD and NH₂–spCD culturing with L929 cells. Unite of concentration was mg/mL.

Figure 4

XPS spectra of (a) CD, (b) spCD, (c) NH₂–CD and (d) NH₂–spCD; High-resolution XPS spectra of C1s for (e) CD, (f) spCD, (g) NH₂–CD and (h) NH₂–spCD; High-resolution XPS spectra of N 1s for (i) CD, (j) spCD, (k) NH₂–CD and (l) NH₂–spCD; High-resolution XPS spectra of O 1s for (m) CD, (n) spCD, (o) NH₂–CD and (p) NH₂–spCD.

Figure 5

Imaging of E.coli and S.aureus by CDs and spCDs (bacterial was treated with 60 °C water bath for 1 h).

Figure 6

Imaging of alive E.coli and S.aureus by CD, spCD and NH₂–spCD.