Fluorescent EGTA-derived carbon dots for turn on-off-on detection of Fe3+ and ascorbic acid via hydrothermal synthesis and cellular imaging

Abstract

We present a novel, one-step hydrothermal synthesis of carbon dots (CDs) with intense blue fluorescence and a remarkably high quantum yield of 50%, using ethylene glycol tetraacetic acid (EGTA) as a single precursor eliminating the need for additional passivation agents. This streamlined strategy represents a significant advancement in the efficient production of functional CDs. The resulting CDs exhibit dual-mode fluorescence sensing, selectively detecting Fe³⁺ through a fluorescence quenching mechanism, with an ultra-low detection limit of 23 nM. Notably, the quenched fluorescence is fully restored upon the introduction of ascorbic acid (AA), enabling a highly sensitive "off-on" detection system with a detection limit of 21 nM. This approach demonstrates excellent selectivity for AA over dopamine and other amino acids, providing a reliable method for distinguishing AA in complex biological matrices. Furthermore, the CDs-based Fe³⁺/AA sensing system proves highly effective for bioimaging applications, allowing for clear visualization of Fe³⁺ and AA in living cells. Compared to conventional commercial assays, this method is cost-effective, simple, and scalable, offering a powerful tool for next-generation biosensing and bioimaging. The fluorescence-based "off-on" mechanism, combined with the versatility of CDs in real-sample analysis, highlights the innovation and practical value of this approach.

Fig. 1

The CDs as prepared are displayed in representative TEM (a,b) and HRTEM (c) images. Image (c) shows the crystalline lattice. Histogram (d) shows the size distribution of the CDs.

Fig. 2

XRD pattern of CDs.

Fig. 3

The FTIR spectra of (a) pure EGTA, (b) CDs.

Fig. 4

Full XPS spectrum of CDs (a), and High resolution XPS spectra of C 1s (b), (c) N 1s and (d) O 1s for the CDs.

Fig. 5

Fluorescence emission spectra of CDs in HEPES (12 mM) buffer at pH = 7.4 (excitation wavelength (λ_ex) from 280 to 480 nm).

Fig. 6

Fluorescence spectra of CDs in HEPES (12 mM) buffer at pH 7.4 with varying concentrations of Fe³⁺. Top to bottom Fe³⁺ concentration: (0, 0.4, 2, 4, 6, 12, 16, 20, 24, 28, 32, 36, 40, 48, 56, 64, 72, 78, 84, 90, 96, 100, 106, 112,120, 128, 136, 144, 152,160, 170, 180, 190, 200, 220, 240, 280, and 320 µM) (λ_ex = 320 nm).

Fig. 7

CDs’ (120 µg/mL) fluorescence response to various metals (500 µM), Fe³⁺ (80 µM), and HEPES (12 mM) buffer at pH 7.4 (λ_ex = 320 nm).

Fig. 8

Fluorescence spectra of CDs-Fe (III) at pH 7.4 in HEPES (12 mM) buffer containing varying concentrations of AA. AA concentration in ascending order: (0, 2, 4, 6, 8, 10, 12, 14, 16, 24, 32, 40, 52, 64, 76, 88, 100, 120, 140, 160, 180, 200, 260 and 300 µM) (λ_ex = 320 nm).

Fig. 9

CDs-Fe (III) fluorescence response to various anions and amino acids (HEPES (12 mM) buffer at pH 7.4) (λ_ex = 320nm). The anions and amino acids are all concentrated at 500 µM.

Fig. 10

Schematic representation of CDs, how Fe³⁺ and AA bind to CDs, and how Fe³⁺and AA quench on–off-on CDs.

Fig. 11

Live BHK-2 cell confocal fluorescence images. (1) 1a: Fluorescence image of pure BHK-2 cells stimulated by UV light; 1b: Bright field microscopy image of pure BHK-2 cells. 1c: an ultraviolet light-excited layer of pure BHK-2 cells. (2) BHK-2 cells cultured with CDs for one hour at 37 °C were shown in a fluorescence image. (3) Fluorescence picture of BHK-2 cells stained with CDs after 30 min at 80 μM Fe³⁺. (4) Fluorescence picture of CDs- Fe³⁺ stained BHK cells after 30 min at 140 μM AA.