Synthesis of o-phenylenediamine-based Zn-doped carbon dots with excitation-independent emission for the qualitative detection of ClO⁻

2.1. Materials and apparatus

Aniline, o-phenylenediamine, zinc sulfate heptahydrate, sodium hypochlorite (>10% active chlorine), hydrogen peroxide (AR, 30 wt. % in H₂O), ABTS [2,2’-azinobis (3-ethylbenzothiazoline-6-sulphonic acid ammonium salt)], sodium nitroferricyanide dihydrate, and tert-butyl hydroperoxide were obtained from Aladdin Reagent Co. Ltd. (Shanghai, China).

The morphology and composition of Zn-CDs were characterized using TEM, XPS, FTIR, Raman, fluorescence spectra, and UV-vis absorption spectra.

2.2. Preparation of Zn-CDs

1 mL aniline, 1 g o-phenylenediamine, and 0.575 g zinc sulfate heptahydrate were dissolved in 70 mL deionized water, which was subsequently transferred to an autoclave with a capacity of 100 mL. The solution was heated at 180°C and maintained for 12 h and allowed to cool to room temperature naturally. Then, the solution was extracted using a centrifuge at 8000 rpm for 10 min, filtered thrice using micro-filtration membranes (φ = 0.22 µm), and dialyzed for 1 day using dialysis bags (MWCO 1000 Da). The resulting solutions were concentrated into brown solids using a rotary evaporator. The obtained solids were then dissolved in deionized water to prepare Zn-CDs aqueous solutions at a concentration of 1 g·L⁻¹ for future use.

2.3. Effect of pH on Zn-CDs

The aqueous solutions with varying pH values of 2.11, 2.73, 3.70, 4.93, 6.03, 6.93, 7.55, 8.35, 9.07, 10.05, 11.02, and 11.89 were prepared using NaOH and H₂SO₄. Then, 12 aliquots of 100 μL Zn-CDs (1 g·L^-1) solutions were added to each of the 12 different 2 mL aqueous solutions with corresponding pH values. Reversibility of pH effect on fluorescence of Zn-CDs was assessed by adjusting pH values between 2.73 and 11.02 through the addition of NaOH or H₂SO₄.

2.4. Effect of metal ions on Zn-CDs

Different metal ion solutions (K⁺, Ca²⁺, Na⁺, Mg²⁺, Pb²⁺, Ba²⁺, Cd²⁺, Co²⁺, Mn²⁺, NH₄⁺, Ni²⁺) were prepared using corresponding salts at a concentration of 10^-2 mol/L, respectively. 2 mL of each metal ion solutions were separately added into multiple portions of 100 μL Zn-CDs solutions (1 g·L^-1).

2.5. Scavenging effect of Zn-CDs on ABTS^{• +}

ABTS is an abbreviation for 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), which is used to generate 2,2-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^•+) under the oxidation of potassium persulfate, as shown in Scheme S1(a). ABTS^•+ is a synthesized and water-soluble substance, which is applied to the measurement of the total antioxidant activity of pure substances, foods, and drinks in aqueous solutions [22]. ABTS^•+ has maximum absorption at wavelengths of 415, 645, 734, and 815 nm. The absorption peak at 734 was selected as the characteristic absorption peak in Scheme S1(b). The scavenging effect of Zn-CDs on ABTS^•+ was investigated to assess the antioxidant properties of Zn-CDs. ABTS^•+ was synthesized by using ABTS and K₂S₂O₈ as reactants. Specifically, 20 mg of ABTS and 3.4 mg of K₂S₂O₈ were mixed in 5.21 mL of deionized water, which was kept for 12 h in the dark to yield a blue-green ABTS^•+ aqueous solution. Concentration of ABTS^•+ was measured through characteristic absorbance at 734 nm.

The concentrations of Zn-CDs were diluted in 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg·L^-1, respectively. Eleven parts of ABTS^•+ aqueous solutions (2 mL) were separately added to 11 different concentrations of Zn-CDs solutions (1 mL). After 3 min, UV-vis absorption spectra were recorded using a spectrophotometer. EC₅₀ (50% effective concentration) value was calculated to provide further insight into the antioxidant properties of Zn-CDs based on the absorbance at 734 nm [23].

2.6. Effect of ClO⁻ on Zn-CDs

Effects of ClO⁻, H₂O₂, NO, ROO^•, on Zn-CDs were further investigated. Concentration of ClO⁻, H₂O₂ was determined based on the absorbance at 292, 240 nm [24]. Concentration of NO was obtained by measuring the concentration of sodium nitroferricyanide dihydrate (SNP). Concentration of ROO^• was determined based on the concentration of tert-butyl hydroperoxide (t-BuOOH). 2.5 mL of water, ClO⁻, H₂O₂, NO, and ROO^• (10^-3 M) were added to five aliquots of 100 μL Zn-CDs solutions (0.01 g·L^-1), respectively. The fluorescence spectra were continuously measured 25 times.

3.1. Characterizations of Zn-CDs

The morphology and composition of Zn-CDs were characterized using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared (FTIR). As shown in Figure 1(a), the TEM image of Zn-CDs showed that the synthesized Zn-CDs were evenly dispersed in deionized water, with particle sizes roughly between 3 and 7 nm in Figure S1(a). Additionally, a lattice spacing of 0.29 nm, associated with a crystalline CD (denoted as ‘I’ Zn-CDs), corresponded to the (002) planes of graphene [25]. As illustrated in Figure 1(b), two deconvoluted peaks observed at 284.6, 285.7 eV in the XPS spectrum of C1s, were corresponded to C=C, C-N. [19,21]. The three deconvoluted peaks at 399.0, 400.6, and 401.9 eV in the N1s spectrum were put down to pyrrolic N, pyridinic N, and graphitic N, respectively, as in Figure 1(c) [26]. In Figure 1(d), two peaks at 1046, 1023 eV in the Zn2p XPS spectrum were owed to Zn2p1/2, Zn2p3/2 [19]. It indicated that Zn²⁺ was doped into Zn-CDs because of the formation of a coordination bond between Zn²⁺ and the amino group [27,28]. It could be seen from the FTIR spectrum of Zn-CDs that a peak at 1637 cm^-1 was due to the stretching vibration of C=C, while the N-H stretching vibration was located at 3300 cm^-1, as in Figure 1(e). Therefore, the synthesized Zn-CDs contained a C=C bond and an amino group might be conducive to their fluorescence properties and antioxidant capabilities. In addition, the structure of Zn-CDs was further characterized by the Raman spectrum. It could be seen from Figure S1(b), there were two peaks at 1230 and 1550 cm^-1, which were attributed to the vibration of C-C and the stretching vibration of C=C, respectively. The Raman peak at 1350 cm^-1 corresponded to the D band. The D band was attributed to structural defects or disordered carbon, reflecting the degree of disorder in the material [29]. The Raman peak at 1550 cm^-1 was related to the G band. The G band originated from the in-plane vibration of sp² hybridized carbon atoms in graphite crystals, representing the highly ordered graphitized regions in the material [29]. I_D/I_G was the intensity ratio of the D band to the G band. The larger I_D/I_G showed more defects in the material. The smaller I_D/I_G indicated a higher degree of graphitization in the material [29]. The ratio of I_D/I_G was 0.40, implying that there was an ordered graphite lattice structure and structural defects in Zn-CDs, which were consistent with the results of TEM.

3.2. Fluorescence properties of Zn-CDs

At varying excitation wavelengths, including 375, 400, 425, 450, and 475 nm, fluorescence emission spectra of Zn-CDs were measured and normalized. In Figure S2(a,b), as the fluorescence excitation wavelengths moved from 375-450 nm, fluorescence emission intensity was quite high at 400 nm (Figure S3). Through the ‌utilization of quinine sulfate as a reference, the fluorescence quantum yield of Zn-CDs was measured as 5.21% [28]. Additionally, there was almost no change in normalized fluorescence emission spectra and fluorescence emission peaks at 553 nm, as shown in Figure 2(a). Unlike many traditional CDs, which exhibited excitation-dependent fluorescence emission, while fluorescence emission positions of Zn-CDs did not exhibit blue or red shifts with the change of excitation wavelengths. This observation indicated that the synthesized o-phenylenediamine-based Zn-CDs exhibited excitation-independent fluorescence emission behavior, along with the reported literature [18,30-32]. The photoluminescence mechanism of o-phenylenediamine-based Zn-CDs might be attributed to the molecular state fluorophores [33,34]. The fluorescence excitation spectra and UV-vis absorption spectra were further utilized to investigate the luminescent center of Zn-CDs. Normalized fluorescence excitation spectra of Zn-CDs at fluorescence emission wavelengths (550, 575, and 600 nm) have been shown in Figure 2(b). As fluorescence emission wavelength transitioned from 550 to 600 nm, two peaks at 258 and 405 nm remained consistent. As depicted in Figure 2(c), there were three absorption peaks located at 230, 275, and 440 nm, ‌stemming primarily from n-σ* transition of C-NH, π-π* transition of C=C, conjugated structure in -NH₂ group, and phenazine ring structure [30]. It was inferred that the fluorescence of Zn-CDs was primarily derived from two absorption peaks located at 275 and 440 nm by combining with the fluorescence excitation spectra. Therefore, excitation-independent fluorescence emission of Zn-CDs was beneficial for sensing and imaging applications.

Close

Figure 2.

(a) Normalized fluorescence emission spectra of Zn-CDs at different excitation wavelengths (375, 400, 425, 450, and 475 nm). (b) Normalized fluorescence excitation spectra of Zn-CDs at different emission wavelengths (550, 575, and 600 nm). (c) The UV-vis absorption spectrum of Zn-CDs.

Export to PPT

3.3. Effects of pH and metal ions on the fluorescence of Zn-CDs

Fluorescence emission spectra of Zn-CDs in aqueous solutions with varying pH values have been measured in Figure S4(a). The fluorescence of Zn-CDs exhibited minimal changes under neutral and alkaline conditions, as shown in Figure 3(a). In contrast, when Zn-CDs were in the acidic aqueous solutions, the fluorescence intensities decreased with the decrease of pH. As the pH dropped below 2.73, the fluorescence of Zn-CDs exhibited a quenching phenomenon. It indicated that Zn-CDs could be applied to detect solution acidity. To study the recoverability of Zn-CDs, fluorescence emission spectra and the fluorescence intensities probed at 553 nm were further measured upon the cyclic switching of Zn-CDs under alternating pH conditions of 2.73 and 11.02. The fluorescence of Zn-CDs was sequentially quenched or restored with the alternating change in pH value, as illustrated in Figure S4(b-c). By characterizing the structure of Zn-CDs, it was found that there was only one acid-base functional group, -NH₂, in Zn-CDs. The behaviors of fluorescence quenching and recovery were attributed to protonation and deprotonation of -NH₂ under different acid-base conditions [35]. Therefore, the fluorescence quenching phenomenon of Zn-CDs at pH below 2.73 could be attributed to the protonation of -NH₂. The effect of metal ions on Zn-CDs was further investigated. Various metal ions aqueous solutions (K⁺, Ca²⁺, Na⁺, Mg²⁺, Pb²⁺, Ba²⁺, Cd²⁺, Co²⁺, Mn²⁺, NH₄⁺, Ni²⁺) were individually added into Zn-CDs solutions. As these metal ion solutions were introduced, fluorescence emission spectra and fluorescence intensities of the Zn-CDs remained nearly unchanged, as in Figure S5 and Figure 3(b). It indicated that the fluorescence properties of Zn-CDs were minimally influenced by metal ions. Additionally, the synthesized Zn-CDs exhibited excellent stability in the company of metal ions.

Close

Figure 3.

(a) Fluorescence intensities of Zn-CDs at 553 nm in aqueous solutions with varying pH values. (b) Fluorescence intensities of Zn-CDs at 553 nm mixed with metal ions.

Export to PPT

3.4. Scavenging effect of Zn-CDs on ABTS^{• +}

Antioxidant property of Zn-CDs was evaluated through scavenging ABTS^•+. In Figure 4(a), the characteristic absorption peak of ABTS^•+ at 734 nm decreased in pace with Zn-CDs. Concurrently, the blue-green aqueous solution of ABTS^•+ was turned into colorless ABTS, indicating that ABTS^•+ could be effectively scavenged by Zn-CDs through the oxidation-reduction reaction. A negative zeta potential of Zn-CDs determined as -5.23 mV facilitated the interaction between Zn-CD and positively charged ABTS^•+. Correspondingly, the fluorescence of Zn-CDs was quenched by adding ABTS^•+, as in Figure 4(b). It could be observed from Figure 4(c) that the characteristic absorbances of ABTS^•+ at 734 nm progressively decreased with increasing concentrations of Zn-CDs. The EC₅₀ value of Zn-CDs for scavenging ABTS^•+ was calculated to be 4.0887 × 10^-3 g/L in Figure 4(d). Therefore, the scavenging activity demonstrated that Zn-CDs could function as a fluorescent probe with antioxidant capabilities. 

Close

Figure 4.

(a) UV-vis absorption spectra of Zn-CDs, ABTS•+, Zn-CDs + ABTS•+. (b) Fluorescence emission spectra of Zn-CDs, ABTS•+, Zn-CDs + ABTS•+, λEx = 400 nm. (c) UV-vis absorption spectra of ABTS•+ with varying concentrations of Zn-CDs. (d) Linear relationship between concentrations of Zn-CDs and absorbances at 734 nm.

Export to PPT

3.5. Effect of ClO⁻ on Zn-CDs

Antioxidant characteristics of Zn-CDs were further evaluated by assessing the effects of ClO⁻, H₂O₂, NO, and ROO^• on Zn-CDs. Fluorescence emission spectra of a ClO⁻ and Zn-CDs mixed solution were continuously recorded 25 times over 350 s. In Figure 5(a), the fixed fluorescence emission peak at 553 nm progressively faded; meanwhile, a new fluorescence emission peak at 490 nm emerged, signifying a blue shift compared to that of Zn-CDs. Moreover, fluorescence of Zn-CDs at 553 nm could be well-fitted by a single-exponential decay, as shown in Figure S6. The chemical reaction rate constant, derived from fluorescence intensities at 553 nm during the reaction between ClO⁻ and Zn-CDs, was calculated as 0.0077. In Figure 5(b), the changes in the reaction between Zn-CDs and ClO⁻ were further elucidated by UV-vis absorption spectra. Two absorption peaks of Zn-CDs that contribute to the fluorescence emission disappeared, concurrently, a new absorption peak appeared around 400 nm, indicating that Zn-CDs with antioxidant capacity could react with ClO⁻ to form a new luminescent species. The effects of water (blank), ClO⁻, H₂O₂, NO, and ROO^• on the fluorescence of Zn-CDs were further studied. It could be seen from Figure 5(c) that H₂O₂, NO, and ROO^• had no effect on the fluorescence of Zn-CDs compared with the blank sample, while there was an oxidation-reduction reaction between ClO⁻ and Zn-CDs. This indicated that Zn-CDs, as fluorescent probes with antioxidant properties, exhibited good selectivity towards ClO⁻. Therefore, Zn-CDs could be color-changing fluorescent probes to achieve qualitative detection of ClO⁻ (Figures S7-S9).

Close

Figure 5.

(a) Fluorescence emission spectra of Zn-CDs with ClO⁻ continuously measured 25 times within 350 s, λ_Ex = 400 nm. (b) UV-vis absorption spectra of Zn-CDs, ClO⁻, and Zn-CDs + ClO⁻. (c) Fluorescence emission spectra of the effects of water (Blank), H₂O₂, NO, ROO^•, ClO⁻ on Zn-CDs, respectively.

Export to PPT