Intramolecular charge transfer-induced fluorescence enhancement of carbon dots for rapid detection of sugammadex sodium and clinical injection sample analysis

2.1. Materials and instrumentations

Further details can be found in the Supplementary Material.

2.2. Preparation of CDs

CDs were acquired according to a reported work [20]. 0.1000 g PEI and 0.0100 g BPB were mixed and dissolved into 20 mL and placed in a 50 mL hydrothermal reactor after being mixed well. Then, the mixture was heated at 180°C for 12 h to obtain yellow solution. The product was dialyzed against to water through dialysis (molecular weight cut off of 3500 Da) for 24 h. The resulting product was stored at 4°C until further use.

2.3. Detection procedure of SUG

The fluorescent assay of SUG was carried out in a PBS system (pH 7.0). Firstly, 10 μL of CDs solutions were mixed with 170 μL of the above PBS solutions. Then, 20 μL of SUG solutions with a certain concentration was added. After 5 min, the fluorescence spectrum of the mixture was collected and relevant intensity at 460 nm was screened. The instrument conditions were set as 365 nm for excitation, 5 nm for both excitation and emission slit. All the tests were carried out in three independent replicates.

2.4. Clinical sample assay

The pharmaceutical SUG injection as the real sample was selected for the practical application. The pharmaceutical was diluted for 100 times before processing. 20 μL of real sample solutions or sample with standard SUG solutions (0.15, 0.25, 0.30 g/L) were added into PBS solutions (pH 7.0) containing 10 μL of CDs. The fluorescence spectrum of the system was collected and relevant intensity at 460 nm was screened. The content of SUG in sample solutions and in the SUG injection as well as the detection recoveries were calculated accordingly.

3.1. Characterization of CDs

In order to establish SUG-sensitive fluorescence response, nitrogen-doped CDs were synthesized from polyethyleneimine (PEI) and bromophenol blue (BPB). Figure 1(a) showed the TEM pattern of CDs in which nanodots with diameters below 5 nm were observed. Figure 1(b) recorded the size distribution of CDs and the diameter ranging from 1.74 to 4.85 nm and the average size of 2.48 nm were concluded. Figure 1(c) displayed the X-ray diffraction (XRD) profile of CDs, in which a wide peak ranging from 20° to 30° was observed. The characteristic diffraction represented the (002) plane of graphite with a certain degree of deviation due to the doping of heteroatoms [26]. Figure 1(d) recorded the FT-IR spectrum of CDs to specify the functional groups. The broad peak centered at 3420 cm^-1 was from the O−H. The sharp absorption at 1630 cm^-1 represented the C=O or C−Br. The absorption at 1515 and 1175 cm^-1 came from the C=C and C−O/C−N, respectively. Furthermore, the chemical compositions of CDs were tested through XPS in Figure 2. As shown in the XPS spectrum (Figure 2a), elements C, O, N, S, and Br were detected. The high-resolution spectra of C 1s, O 1s, N 1s, S 2p, and Br 3d of CDs have been listed in Figures 2(b-f), respectively, and the corresponding binding forms have been exhibited in Table S1 in detail. The results demonstrate the morphology, structure, and composition of CDs.

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Figure 1.

The morphology and structural characterization of CDs. (a) TEM pattern; (b) size distribution histogram; (c) XRD profile; (d) the FT-IR spectrum.

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Figure 2.

The chemical constitution of CDs. The XPS (a) full scan spectrum, and partial spectra from (b) C 1s, (c) O 1s, (d) N 1s, (e) S 2p and (f) Br 3d of CDs.

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3.2. Spectral response of SUG

Figure 3(a) compared the fluorescence emission spectra of CDs before and after adding SUG. The CDs displayed a maximal emission at 460 nm (black line) and it was proved that the emission intensity at 460 nm was assuredly improved by SUG (blue line). The insets showed the practical photos of CDs and CDs+SUG under UV light illumination. The blue fluorescence of CDs turned brighter after adding SUG, visually proving the fluorescence enhancement. The ultraviolet visible (UV-Vis) absorption variations of CDs before and after introducing SUG were presented in Figure 3(b). The results showed that the absorbance of CDs before 250 nm increased in the present of SUG at a certain degree. It was reported that interaction between substances enhanced the rigidity of the molecular skeleton [27] and therefore, increased the spectral absorbance [28], which proving an interaction between CDs and SUG.

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Figure 3.

The fluorescence and UV-Vis spectral response to SUG. (a) The fluorescence emission spectra of CDs with or without SUG. Insets are the photos of (i) CDs and (ii) CDs + SUG under UV light, respectively. (b) The UV-Vis absorption spectra of CDs with or without SUG.

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3.3. Fluorescence sensing mechanism

To reveal the specific form of action between CDs and SUG, molecules representing the partial structure of SUG were proposed to compare their effect on the fluorescence of CDs. As shown in Figure 4(a), ethylene diamine tetraacetic acid (EDTA, standing for carboxyl), γ-cyclodextrin (standing for the main molecular skeleton), 2-thiapropane (standing for thioether), and glucose (standing for hydroxy) with equivalent molar quantities to the representative structures in SUG were carried out. It turned out that only EDTA (yellow line) showed the same fluorescence enhancement behavior as SUG. The result demonstrated that carboxyl played a key role during the fluorescence improvement of CDs.

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Figure 4.

The fluorescence sensing mechanism study. (a) The fluorescence discrepancy of CDs with molecules representing the partial structure of SUG. (b) The time-resolved fluorescence decay of CDs before and after reacting with SUG.

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To further explore the essence of the fluorescence enhancement, the Zeta potential of CDs before and after introducing SUG was tested in Figure S1. It was proved that CDs displayed a positive potential of +3.85 mV because of the protonation of N atoms in CDs. However, SUG containing abundant carboxyl and hydroxy (whose structure was listed in Figure S2) bonded with CDs through electrostatic interaction, which led to a sharp drop in Zeta potential (-11.60 mV after adding SUG). Figure 4(b) recorded the fluorescence decay spectra at 460 nm of CDs with or without SUG. The relevant fitting data have been shown in Table S2. The fluorescence lifetime of CDs and CDs+SUG was computed to be 5.60 and 6.12 ns, respectively. The stable lifetimes suggested a static effect for the interaction between CDs and SUG. Combining with the UV-Vis spectra, functional groups identification, Zeta potential, and fluorescence lifetime results (Figure 3b, Figures 4(a,b), and Figure S1), -NH₂ in CDs interacted with -COOH in SUG was proved. Further, an abundant amount of literature has reported that the intermolecular charge transfer (ICT) occurred with the interaction between the amino and carboxyl groups, which improved the fluorescence [25,29-32]. Therefore, ICT activated by -NH₂ in CDs and -COOH in SUG took responsibility for the fluorescence enhancement. This was consistent with the conclusion that carboxyl-containing EDTA can enhance the fluorescence of CDs.

3.4. Optimization of analytical parameters

As an established fluorescent assay for SUG, the optimal detection parameters, like pH and time, were investigated. Figure S3 investigated SUG-triggered fluorescence change of CDs in PBS under various pH conditions. Figure 5(a) presents the fluorescence enhancement efficiency (F/F₀) of the CDs upon reacting with SUG, where F₀ and F were defined as the initial fluorescence intensity of the mixture and the one after reacting with SUG, respectively. The results showed the homogeneous fluorescence enhancement efficiencies at pH from 4 to 10, proving the universality of this method. Furthermore, a comparison of the fluorescence of CDs with and without SUG over time has been provided in Figure S4, and their fluorescence enhancement efficiency has been summarized in Figure 5(b). Time-stable fluorescence enhancement phenomenon was obtained, providing a handy operation for detection. For convenience, further detection was performed under PBS with pH 7.0 after mixing CDs with SUG for 5 min.

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Figure 5.

The optimization of analytical parameters. (a) The fluorescence enhancement efficiency under different pH values. (b) The fluorescence enhancement efficiency at different time intervals.

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3.5. Fluorescent detection of SUG

Under the above detection conditions, the calibration curve was constructed by plotting the fluorescence enhancement efficiency versus the concentration of SUG. Figure 6(a) illustrates the evolution of the fluorescence spectra of the CDs upon the addition of increasing concentrations of SUG, in which the gradually improved spectra were observed. Figure 6(b) displayed two linear ranges of 0.001-0.07 and 0.07-0.5 g/L with equations of F/F₀=1.0538+4.8116C (R²=0.9907) and F/F₀=1.3455+0.7941C (R²=0.9988), respectively. The limit of detection (LOD) was determined to be 0.44 mg/L (3σ). Besides, the effect of possible coexisting interferences on fluorescence response was investigated in Figure 6(c). Except for SUG, other species, including Na+, Zn2+, NH4+, proline, aspartic acid, threonine, leucine, glycine, lysine, methionine, tryptophan, glutamic acid, arginine, serine, cysteine, alanine, histidine, uric acid, urea, BSA, glucose, and dopamine, showed no response to CDs. The corresponding spectra have been recorded in Figure 6(d). The phenomenon demonstrating fluorescence enhancement of CDs by only SUGs was observed. The results confirmed that the CDs exhibited satisfactory selectivity for SUG detection. The comparison of analytical performances of this work with other reported methods have been detailed in Table S3. It was concluded that this method possessed a satisfactory linear range and LOD. Moreover, the fluorescent assay with short response time and CDs with preparation simplicity and low cost in this work made it a promising method for SUG detection.

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Figure 6.

The fluorescent detection of SUG based on CDs. (a) The evolution of the fluorescence spectra of the CDs upon increasing concentrations of SUG. (b) The linear calibration curve for SUG detection through fluorescent enhancement. (c) Selectivity of CDs for SUG detection via fluorescence enhancement and (d) the corresponding spectra of CDs system with coexisting compounds equivalent with SUG (species number 1-23 stood for SUG, Na⁺, Zn²⁺, NH₄⁺, proline, aspartic acid, threonine, leucine, glycine, lysine, methionine, tryptophan, glutamic acid, arginine, serine, cysteine, alanine, histidine, uric acid, urea, BSA, glucose, and dopamine, respectively.)

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The SUG injection for practical analysis was carried out. The detection results have been displayed in Table 1. Quantitative analysis showed that the amount of SUG in the target was 0.1055 g/L, and the recovery covered 94.95-100.35%. According to the dilution ratio, the content in the original pharmaceutical was 105 g/L, which was close to the marked content (200 mg/2mL). These results demonstrated the high accuracy and reliability of the proposed method.