Dehydroabietic acid (99.06%) was purchased from Jusheng Technology Co., Ltd. (Hubei, China). Ethylenediamine, Al(NO₃)₃·9H₂O, NaH₂PO₂·H₂O, Ca(NO₃)₂·4H₂O,Cd(NO₃)₂·4H₂O, Cu(NO₃)₂·3H₂O, Fe(NO₃)₃·9H₂O, AgNO₃ and NaNO₃ were purchased from Bodi Chemical Industry Co., Ltd. (Tianjin, China). Ni(NO₃)₂·6H₂O, Pb(NO₃)₂, Sr(NO₃)₂, Ba(NO₃)₂, C₂H₂ClO₂Na, CH₃COONa and NaNO₂ were purchased from Fuyu Fine Chemical Co., Ltd. (Tianjin, China). EDTA-2Na, B₄O₇Na₂, C₂O₄Na₂, Na₂HPO₄, NaCl, NaF, NaHCO₃, Co(NO₃)₂·6H₂O, FeCl₂, Na₂SO₃, Na₂SO₄, Na₂SiO₃, Na₂S₂O₃ and NaH₂PO₄ were purchased from Yongda Chemical Reagent Co., Ltd. (Tianjin, China). All chemicals were of analytical purity and were used as received. Deionized water was prepared using a smart-q30 UT ultrapure water system (Zhiang Instrument Co., Ltd., Shanghai, China). The cell counting kit-8 (CCK-8) assay was purchased from Dojindo Laboratories (Kumamoto, Japan), Normocin was purchased from InvivoGen (San Diego, CA, United States). Dulbecco’s modified Eagle’s medium/Ham’s F12 medium (DMEM/F12), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were all Gibco brand and were purchased from Thermo Fisher Scientific Inc., Waltham, MA, United States). HUVECs was purchased from the Chinese Academy of Sciences (Shanghai, China).

The morphology, structure and atomic composition of the Bio-CDs were determined using a JEM-2100 transmission electron microscope (JEOL Ltd., Tokyo, Japan), a Fourier Transform infrared (FTIR) spectrometer (Perkin Elmer Inc., Waltham, MA, United States) and an ESCALAB 250Xi X-ray photoelectron spectrometer (Thermo Fisher Scientific, New York, NY, United States), respectively. The ultraviolet-visible (UV-vis) absorption spectrum was recorded using a TU-1950 spectrofluorometer (Persee General Instrument Co., Ltd., Beijing, China). Fluorescence was measured using an LS55 fluorescence spectrometer (Perkin Elmer Inc., Waltham, MA, United States). Time-resolved fluorescence spectra were recorded using a DeltaFlex modular fluorescence lifetime instrument (Horiba Jobin Yvon IBH, Ltd., Glasgow, United Kingdom). Fluorescence images were captured using a DMI4000 B inverted fluorescence microscope (Leica Microsystems Inc., Wetzlar, Germany).

Ethylenediamine (1 ml) was added to a mixture of dehydroabietic acid (0.4 g) and deionized water (60 ml) in a 100 ml Teflon-lined stainless autoclave and the mixture was stirred thoroughly using a glass rod. The mixture was heated to 200°C for 5 h and then allowed to cool to room temperature. The resulting solution was filtered through a 0.22 μm ultrafiltration membrane and then freeze-dried to provide the crude product. The crude product was ground to a powder, dissolved it with absolute ethanol, and filtered to separate insoluble matter. The insoluble matter was dried under vacuum at 50°C to provide Bio-CDs.

Transmission electron microscopy (TEM) showed that the Bio-CDs were nanoparticles (Figure 1A), with a particle size distribution of 3.9–6.3 nm and an average diameter of 5.07 nm (Figure 1B). The lattice fringe spacing of the Bio-CDs, observed in high-resolution TEM (HRTEM) images, was 0.21 nm (Figure 1A, inset), which corresponds to the graphene (100) plane (Chen et al., 2016; Zheng et al., 2021). The FT-IR spectrum (Figure 1C) of the Bio-CDs showed an absorption peak at 3,305 cm⁻¹, which is characteristic of -NH stretching vibrations (Li et al., 2019a; Shi et al., 2017). The absorption peaks at 1,665 cm⁻¹ and 1,637 cm⁻¹ correspond to the stretching vibration of C=O/C=N groups (Chen et al., 2017; Miao et al., 2018; Shi et al., 2017). A strong peak at 1,566 cm⁻¹ was attributed to N−H bending vibrations (Guo et al., 2019; Shi et al., 2017; Zhu et al., 2013). The FT-IR spectrum thus confirmed the presence of functional groups such as amino and carbonyl groups on the surface of the Bio-CDs. The full X-ray photoelectron spectroscopy (XPS) spectrum (Figure 1D) showed peaks at 285 eV, 399.8 and 530.9 eV, corresponding to C 1s (69.05%), N 1s (12.56%) and O 1s (18.39%), respectively. The high-resolution C 1s spectrum showed a peak at 284.5 eV, attributed to C−C/C=C (Bi et al., 2018; Li et al., 2019a), a peak at 285.8 eV, attributed to C−O/C−N (Nie et al., 2014; Lee et al., 2021) and a peak at 288.2 eV, attributed to C=O/C=N (Bi et al., 2018; Li et al., 2019a) (Supplementary Figure S1A). The high-resolution O1s spectrum showed two components, corresponding to O=C (530.9 eV) (Miao et al., 2018) and C−OH/C−O−C (532.3 eV) (Nie et al., 2014), repectively (Supplementary Figure S1B). The high-resolution N 1s spectrum showed peaks at 399.2 and 400.9 eV (Supplementary Figure S1C), corresponding to amino N (Jiao et al., 2020; Ge et al., 2021b) and pyrrole N (Sheng et al., 2011; Chen et al., 2017), respectively. All of these results indicate that the Bio-CDs have a large number of functional groups. The rich surface functionalization means that the Bio-CDs are highly soluble in water, which is very important for practical applications.

Having established the physical characteristics of the Bio-CDs, we next systematically investigated their optical properties, including UV-vis absorption, photoluminescence (PL) excitation, PL emission, PL decay and PL stability (Figure 2). The UV absorption spectrum of an aqueous solution of Bio-CDs showed a strong absorption peak at ∼300 nm and a shoulder peak at 345 nm (Figure 2A), which were attributed to π-π∗ transitions of C=C and n-π∗ transitions of C=O/C=N bonds, respectively (Jiang et al., 2020; Jiao et al., 2020; Wei et al., 2020). In the PL spectrum of an aqueous solution of Bio-CDs (Figure 2B), the emission wavelength gradually shifted from 427 to 491 nm as the excitation wavelength was increased from 330 to 430 nm. An aqueous solution of Bio-CDs thus show excitation-dependent emission, which is common among previously reported Bio-CDs (Ge et al., 2021c; Zhou et al., 2020). The optimal excitation wavelength of an aqueous solution of Bio-CDs is 370 nm and the maximum emission wavelength is 442 nm; both the optimal excitation and maximum emission wavelengths of solutions of Bio-CDs are essentially unaffected by changes in the concentration of the solution (Supplementary Figure S2). The PL excitation spectrum of an aqueous solution of Bio-CDs is consistent with the PL emission spectrum (Figure 2A). The excitation spectrum coincides with the UV absorption at 345 nm (Figure 2A), indicating that the emission of an aqueous solution of Bio-CDs is associated with the surface state (Liu et al., 2021a). The time-resolved fluorescence spectrum of an aqueous solution of Bio-CDs (Figure 2C) shows that the average fluorescence lifetime is 1.64 ns, fitted by the double exponential decay function. We also recorded the change in fluorescence intensity of an aqueous solution of Bio-CDs (λ_ex = 370 nm) at different temperatures. The emission intensity weakened with increasing temperature (Figure 2D and Supplementary Figure S3), and the fluorescence intensity remained essentially unchanged over several temperature cycles (Supplementary Figure S4). The phenomenon of decreasing emission intensity with increasing temperature is associated with non-radiative relaxation (Ge et al., 2021b; Nie et al., 2014). The fluorescence intensity did not change when an aqueous solution of Bio-CDs was continuously excited with 365 nm UV light for 50 min (Figure 2E). The emission intensity of an aqueous solution of Bio-CDs was also essentially unchanged when the pH of the solution was adjusted between 3.91 and 8.69 (Figure 2F and Supplementary Figure S5). This is the pH range of the intracellular microenvironment, indicating that the Bio-CDs are suitable for cell imaging (Cosnier et al., 2014).

Over recent years, carbon dots have been widely studied for use as ion probes (Li et al., 2020; Liu et al., 2019; Zhou et al., 2020). The fluorescence response of Bio-CDs to various anions and cations was measured by adding the test ion (50 μM) to an aqueous solution of Bio-CDs (20 μg ml⁻¹) and measuring the fluorescence intensity of the solution before (F₀) and after (F) addition of the test ion. Anions had no effect on the fluorescence intensity of the solution of Bio-CDs (Figure 3A). Among the tested cations, Fe³⁺ ions significantly quenched the fluorescence intensity and Pb²⁺ ions significantly enhanced the fluorescence intensity of Bio-CDs ( Figure 3B), whereas the other cations had no effect. The selective quenching of the fluorescence of Bio-CDs by Fe³⁺ ions is attributable to coordination of the Fe³⁺ ions with amino groups on the surface of the Bio-CDs, which disrupts radiative transition and leads to quenching of fluorescence (Atchudan et al., 2018; Gao et al., 2019). We next explored the feasibility of using Bio-CDs to detect Fe³⁺. The fluorescence intensity of the Bio-CDs decreased significantly on addition of Fe³⁺ ions (Figure 3C and Supplementary Figure S6) and, over the range 0–160 μM, the ratio of Fe³⁺ concentration to fluorescence intensity (F ₀ /F) fitted well to the linear equation F/F ₀ = −0.0025c + 1.0006, where c is the concentration of Fe³⁺ (Figure 3D). The LOD was 2.33 μM, based on a signal-to-noise ratio of 3 (3σ/m, where σ is the standard deviation of the blank signal (over three tests) and m is the slope of the linear fit). Because lead is a soft metal, it can covalently or synergistically interact with N-containing groups, such as the many amino groups on the surface of the Bio-CDs, which provide high affinity binding sites for Pb²⁺ ions. A large number of amino groups on the surface of the Bio-CDs can coordinate with Pb²⁺, thus enhancing the fluorescence intensity of the Bio-CDs (Li et al., 2020). We next explored the feasibility of using Bio-CDs to detect Pb²⁺ ions (Li et al., 2020). The intensity of the fluorescence emission of the Bio-CDs (at 437 and 500 nm) was significantly enhanced by the addition of Pb²⁺ (Figure 3E, Supplementary Figure S7 and Supplementary Figure S8A), with the emission intensity at 437 nm increasing significantly faster than that at 500 nm (Supplementary Figure S7). Over the range 0–160 μM, the emission intensity of Bio-CDs at 437 and 500 nm and the ratio of Pb²⁺ concentration to fluorescence intensity (F ₀ /F) fitted well to the linear equations F/F ₀ = 0.0081c + 0.979 and F/F ₀ = 0.0134c + 1.0624, respectively (Figure 3F and Supplementary Figure S8B). The LOD values were 0.27 and 0.33 μM, respectively, showing that the Bio-CDs are well suited to the detection of trace amounts of Pb²⁺.

It has been widely reported that Bio-CDs can be used for cell imaging (Atchudan et al., 2016; Demir et al., 2018; Khairol Anuar et al., 2021; Wang et al., 2021b). Here, we used a standard cell counting kit-8 (CCK-8) to determine the cytotoxicity of Bio-CDs in HUVECs (Zhou et al., 2020). Bio-CDs were found to have good biocompatibility and not to show cytotoxicity when incubated with HUVECs for 24 h, at concentrations as high as 800 μg ml⁻¹ (Figure 4A). We next investigated the potential of Bio-CDs for cell imaging by incubating them with HUVECs for 10 h (Zhou et al., 2020). Cells stained with DAPI, a commonly used nuclear stain, and with Bio-CDs are shown in Figure 4B and Figure 4C, respectively. The two images overlap well (Figure 4D), demonstrating that Bio-CDs can also be used as an effective nuclear stain.