English

• Baicalein (5, 6, 7-trihydroxyflavone), one of the natural polyphenols, refers to the main ingredients of the famous Chinese herbal medicine scutellaria baicalensis georgi (Huangqin). A great number of studies have indicated that baicalein possesses various pharmacological effects including antioxidant^[1], anti- inflammatory^[2], anticancer^[3], antiviral^[4], etc. However, the overdose of baicalein may cause serious side effects such as dizziness, hyperspasmia, and coma^[5]. Thus, it is quite meaningful to monitor the concentration of baicalein in biological samples. So far, some detection methods for baicalein have been developed, including thin‑layer chromatography^[6], high‑ performance liquid chromatography^[7], voltammetry^[8-9], capillary electrophoresis^[10-11], high-performance liquid chromatography/tandem mass spectrometry^[12] and UV-Vis spectrophotometry^[13]. Although these methods can detect baicalein, most have low sensitivity, complicated sample pretreatment, and poor biological compatibility. Fluorescent sensors have received widespread attention because of their high sensitivity, good selectivity, simple operation, and in vivo/vitro detection.

  Carbon dots (CDs), as a novel type of zero‑ dimensional luminescent nanomaterials, have attracted much attention due to their unique properties including superior photo‑stability, favorable biocompatibility, excellent fluorescence properties, photobleaching resistance, low toxicity, easy synthesis from diverse carbon sources and good water solubility^[14-15]. These unprecedented properties endow CDs with various applications such as biological imaging^[16], catalysis^[17], photoelectric devices^[18], anti-counterfeiting^[19], and fluorescent sensors^[20]. It is crucial to improve active functional groups on the surface of CDs by doping heteroatoms (such as nitrogen, sulfur, and boron) for specific detection of target analytes. So far, there have been limited reports on baicalein detection based on carbon dots doped with heteroatoms. Hu et al. synthesized nitrogen and sulfur dual-doped CDs using citric acid and N‑acetyl‑L- cysteine, and discovered it could sensitively detect baicalein (the detection limit is 0.21 μmol·L^-1) and temperature^[21].

  To construct a more sensitive and environment-friendly detection system for baicalein, in the present work, we utilized p-phenylenediamine and L-cysteine as precursors to prepare N, S-CDs with low toxicity and water solubility via a one‑pot solvothermal method. Scheme 1 illustrated the synthesis route for N, S-CDs, and the turn-off response to baicalein. The blue-green fluorescent N, S-CDs can detect baicalein with high sensitivity and selectivity, and the detection limit of baicalein is 85 nmol·L^-1. In addition, the sensor platform can detect baicalein in serum and urine with a high recovery rate, providing potential possibilities for practical applications.

  Scheme1

  

  Scheme1.  Schematic illustration of the synthesis of N, S-CDs and detection of baicalein

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  1.   Experimental

  1.1   Material and measurement

  L-cysteine (99%), p-phenylenediamine (97%), 2- (4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (HEPES, 99%), and baicalein (98%) were purchased from Aladdin company and used directly without further purification treatment. Fetal bovine serum was obtained from Zhejiang Tianhang Biotechnology Co., Ltd. and morning urine was collected from a healthy volunteer in the laboratory. Ultra-water (18.2 MΩ·cm at 25 ℃) was prepared using a Milli-Q reference system and all the reagents were analytically pure.

  Transmission electron microscope (TEM) images were performed on a Tecnai F20 field emission microscope (America). X‑ray diffraction (XRD) measurements were recorded using a D8 Advance Powder X-ray diffractometer (Germany) operated at 40 kV and 40 mA, utilizing Cu Kα radiation (λ=0.154 06 nm) and a scanning range of 10°-80°. Fourier transform infrared (FTIR) spectra were conducted on an FTIR-8400S spectrometer (Japan). X-ray photoelectron spectroscopy (XPS) was carried out on the ESCALAB 250Xi (America) instrument. Steady-state fluorescence spectra were recorded by an F-7000 FL spectrofluorometer (Hitachi, Japan). UV-Vis absorption spectra were measured on a Specord S600 spectrometer (Germany). Time-resolved fluorescence decays were performed on an FLS920 spectrometer (Edinburgh) using a laser (343.4 nm) as the light source.

  1.2   Synthesis of N, S-CDs

  The N, S-CDs were prepared via a simple and low-cost solvothermal method. A solution of p-phenylenediamine (0.50 g) and L-cysteine (0.50 g) in 25 mL ethanol was added to a 50 mL Teflon autoclave and heated at 180 ℃ in an electric blast drying oven for 10 h. After cooling to room temperature, the suspension was filtered by a 0.22 μm membrane, and the solvent was evaporated. Then, the residue was dissolved in water, centrifuged at 8 000 r·min^-1 for 10 min, and purified with a dialysis membrane with MWCO of about 1 000 Da for 48 h. The dialysis bag solution was lyophilized to obtain N, S-CDs powders.

  1.3   Sample preparation

  The stock buffer solution of HEPES (100 mmol·L^-1) was prepared by dissolving HEPES into neat water and using NaOH solution (1 mol·L^-1) to adjust pH to 7.4. HEPES buffer solution (10 mmol·L^-1, pH 7.4) was obtained by diluting the above solution with water. The stock solution of N, S-CDs (0.5 mg·mL^-1) was prepared in water and the stock solution of baicalein (2.5 mmol·L^-1) was prepared in DMF.

  1.4   Fluorescence measurement assays

  A N, S-CDs solution (0.5 mg·mL^-1, 500 μL) was added to a 25 mL volumetric flask, and diluted with 10 mmol·L^-1 HEPES to the mark to obtain the sensor platform. For each fluorescence test, 2.5 mL above solution was placed in a quartz cuvette (1 cm×1 cm×3.5 cm) and the corresponding spectra were recorded. Then, different volumes of baicalein (2.5 mmol·L^-1) were added cumulatively through pipettes and stirred by capillary tubes, and the resulting spectra were scanned. The excitation wavelength was set at 360 nm and the excitation and emission slit widths were both 5 nm.

  1.5   Detection of baicalein in serum and urine

  Serum (obtained from Zhejiang Tianhang Biotechnology Co., Ltd.) was diluted with HEPES buffer solution (10 mmol·L^-1, pH 7.4) to a volume fraction of 1% and used directly. Human urine (obtained from a healthy volunteer) was centrifuged for 10 min (8 000 r·min^-1) to remove the insoluble matrix and the supernatant was diluted with 10 mmol·L^-1 HEPES (pH 7.4) to the corresponding percentage in volume (10%). Then, the sensor platform was built using 1% serum and 10% urine, respectively.

  1.6   Determination of quantum yield

  The determination of quantum yield of the N, S-CDs was determined by using quinine sulfate (in 0.1 mol·L^-1 H₂SO₄ aqueous solution, Φ_R=0.56) as the reference, and calculated using the following equation 1^[22]:

  $ \Phi_{\mathrm{F}}=\Phi_{\mathrm{R}} \frac{\int I_{\mathrm{F}}}{\int I_{\mathrm{R}}} \frac{A_{\mathrm{R}}}{A_{\mathrm{F}}} \frac{n_{\mathrm{F}}^2}{n_{\mathrm{R}}^2} $

  (1)

  where the subscripts F and R represent the N, S-CDs, and reference compounds, respectively. Φ represents the fluorescence quantum yield and ∫I represents the integrated fluorescence intensity. A is the absorbance at 360 nm (less than 0.05), and n is the refractive index of the solvent.

  1.7   Determination of detection limit

  The detection limit (DL) of baicalein based on 3σ IUPAC was calculated by using the following equations^[23]:

  $ S_{\mathrm{b}}=\sqrt{\frac{\sum\limits_{i=1}^n\left(x_i-\bar{x}\right)}{n-1}} $

  (2)

  $ S=\frac{\Delta I}{\Delta c} $

  (3)

  $ \mathrm{DL}=\frac{3 S_{\mathrm{b}}}{S} $

  (4)

  where S_b represents the standard deviation of fluorescence intensity of N, S-CDs for 11 blank tests. ΔI and Δc represent the change in fluorescence intensity and baicalein concentration, respectively. Therefore, S is the slope of the linear change of fluorescence intensity with baicalein concentration. The detection limit of N, S-CDs for baicalein was calculated by equation 4.

  2.   Results and discussion

  2.1   Characterization of N, S-CDs

  Using TEM to investigate the structure of the synthesized N, S-CDs, as shown in Fig. 1a, it was found that the CDs were approximately spherical objects with good dispersion and the diameters of the particles were mainly distributed in the range of 1.6-5.1 nm with an average diameter of ca. 3.5 nm (Fig. 1b). The XRD spectrum of the N, S-CDs showed there were strong peaks near 17.8° (Fig. 1c), indicating good crystallinity, which was a typical characteristic of CDs synthesized from phenylenediamine^[24-25]. The functional groups on the surface of N, S-CDs were also investigated using FTIR. As depicted in Fig. 1d, the broad peaks at 3 340 and 3 216 cm^-1 are ascribed to the stretching vibrations of N—H and O—H, illustrating the existence of amino and hydroxyl groups, which gives N, S-CDs excellent water solubility^[26]. The located peaks at 2 927 and 2 629 cm^-1 demonstrate the presence of C—H and S—H stretching vibrations, respectively^[27]. The characteristic absorption peaks at 1 621, 1 514, 1 435, 1 294, and 1 085 cm^-1 are assigned to the stretching vibrations of C=O, S=O, C—N, C—S, and C—O—C, respectively^[28].

  Figure 1

  

  Figure 1.  TEM image (a), particle size distribution diagram (b), XRD pattern (c), and FTIR spectrum (d) of N, S-CDs

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  In addition, the chemical composition of N, S-CDs was also analyzed by XPS. From the XPS full spectrum of the CDs (Fig. 2a), it can be found that there are four binding energy peaks located at 285.0, 400.0, 531.0, and 168.0 eV which belong to C1s, N1s, O1s and S2p, and the element contents of C, N, O, and S are 67.0%, 14.3%, 13.8%, and 4.9%, respectively. The C1s spectrum of N, S-CDs can be resolved into three peaks at 284.4, 285.1 and 287.7 eV corresponding to the C—C, C—N, and C=O bond (Fig. 2b). The N1s band showed two peaks, relating to N—H (398.9 eV) and C—N (400.2 eV) (Fig. 2c). The O1s spectrum depicted two peaks at 530.9 and 531.75 eV belonging to C=O and C—OH/C—O—C bond, separately (Fig. 2d). The high-resolution spectrum of S2p included four peaks with binding energy of 163.3, 164.55, 167.45, and 168.45 eV, respectively (Fig. 2e). The binding energy of 163.3 (2p_3/2) and 164.55 eV (2p_1/2) corresponds to C—S bond and the binding energy of 167.45 (2p_3/2) and 168.45 eV (2p_1/2) corresponds to S=O bond^[26-28]. The area ratio of C—S (2p_3/2) to C—S (2p_1/2) or S=O (2p_3/2) to S=O (2p_1/2) is approximately 2∶1. Besides, the relative align="center"ontents of various functional groups were shown in Table 1. The above results illustrate that N and S have successfully co-doped in the water-soluble CDs.

  Figure 2

  

  Figure 2.  XPS survey spectrum (a) (Inset: pie chart of the element contents of C, N, O, and S) and high-resolution spectra of C1s (b), N1s (c), O1s (d), and S2p (e) of N, S-CDs

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  Table 1

  

  Table 1.  Area proportion of chemical binds in N, S-CDs XPS spectra

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  C1s				N1s			O1s			S2p
  C—C	C—N	C=O		N—H	C—N		C=O	C—OH/C—O—C		C—S (2p3/2)	C—S (2p1/2)	S=O (2p3/2)	S=O (2p1/2)
  48.5%	48.7%	2.8%		59.6%	40.4%		60.7%	39.3%		31.1%	15.4%	35.6%	17.9%

  2.2   Basic photophysical properties of N, S-CDs

  Considering that the N, S-CDs will be used for detection in biological fluids, we investigated the basic photophysical properties of N, S-CDs in buffer solution (10 mmol·L^-1 HEPES, pH 7.4). As shown in Fig. 3a, the weak absorption peaks around 290 and 350 nm may be caused by π-π* and n-π* transitions of C=C and C=O bonds^[29]. The N, S-CDs solution used for testing is almost colorless under natural light, but emits blue-green fluorescence under a 365 nm UV lamp (Inset of Fig. 3a). Fig. 3b illustrates the emission spectra of N, S-CDs at different excitation wavelengths from 320 to 410 nm, in which the maximum fluorescence intensity was obtained when the excitation wavelength was located at 350 or 360 nm. However, the maximum emission peak at 480 nm did not shift, indicating the excitation-independent behavior, uniform particle size, and surface states of N, S-CDs^[30]. In addition, the relative fluorescence quantum yield of the present N, S-CDs is 16.8% using quinine sulfate as standard.

  Figure 3

  

  Figure 3.  (a) UV-Vis absorption, excitation, and emission spectra of N, S-CDs in 10 mmol·L^-1 HEPES buffer solution (Inset: photographs of N, S-CDs under natural light and 365 nm UV light); (b) Emission spectra of N, S-CDs aqueous solution at different excitation wavelengths

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  2.3   Photostability of N, S-CDs

  To achieve optimal experimental conditions, an investigation was conducted into the potential effects of pH, ionic strength, radiation time, and storage time. As displayed in Fig. 4a, as the pH increased from 2 to 12, the fluorescence intensity reached its maximum with no change significantly between 4-9, this illustrates it can maintain good fluorescence performance in environments ranging from weak acid to weak base. The decrease in fluorescence intensity under strong acid (pH < 4) or alkaline conditions (pH > 9) may be due to the protonation or deprotonation process of the surface functional groups of N, S-CDs^[31]. The fluorescence intensity of N, S-CDs remains almost unchanged with the concentration of NaCl rising from 0 to 1.0 mol·L^-1, suggesting that N, S‑CDs have good salt tolerance (Fig. 4b). In addition, even after continuous UV irradiation (365 nm, 16 W) for 60 min or storage at 4 ℃ for three months (Fig. 4c and 4d), there was no significant change in fluorescence intensity, which indicates that the N, S-CDs have excellent anti-photobleaching effect and long-term stable fluorescence performance.

  Figure 4

  

  Figure 4.  Effect of (a) pH, (b) ion strength by various concentrations of NaCl, (c) radiation time under continuous UV light irradiation (365 nm), and (d) storage time on the fluorescence intensity of N, S-CDs

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  2.4   Fluorescence detection of baicalein with N, S-CDs

  We systematically tested the sensing behavior of N, S-CDs to different concentrations of baicalein. As displayed in Fig. 5a, the fluorescence intensity of N, S-CDs gradually decreased upon the baicalein addition, showing a quenching response. The fluorescence intensity ratio (I₀/I) increased with the increase of baicalein concentration and exhibited a good linear fitting over the low concentration range (0-30 μmol·L^-1) (Fig. 5b). Moreover, the concentration‑dependent quenching behavior follows the Stern-Volmer equation 5:

  Figure 5

  

  Figure 5.  (a) Fluorescence spectra of N, S-CDs upon titration of various concentrations of baicalein; (b) Relationship between I₀ /I and baicalein concentration (Inset: the linear relationship between I₀/I and c_Baicalein)

  (b) Each value represents the average of three parallel measurements.

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  $ I_0 / I=1+K_{\mathrm{sv}} c_{\text {Baicalein }} $

  (5)

  where I₀ and I are the fluorescence intensity of N, S-CDs at 480 nm in the absence or presence of various quencher concentrations (i.e. baicalein), and c_Baicalein is the concentration of baicalein. K_SV represents the Stern-Volmer constant, which can be extracted from the relationship plot between I₀/I and c_Baicalein. According to the linear regression equation y=1.000+0.026x (R²=0.999), K_SV is 2.6×10⁴ mol·L^-1, indicating a strong quenching ability. Meanwhile, the response time was fast for each concentration of baicalein, and the spectra were very stable after adding baicalein and stirring for about 30 s. The detection limit of the present sensor for baicalein was determined to be 85 nmol·L^-1 via the 3σ method. Compared with the reported detection methods for baicalein, although the detection limit of this method is not the lowest, it is simple, fast, and sensitive (Table 2).

  Table 2

  

  Table 2.  Comparison of various reported baicalein detection methods

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  Method	Linear range / (μmol·L-1)	Detection limit / (μmol·L-1)	Reference
  High performance liquid chromatography	1.85-1480	1.480	[7]
  Voltammetry	0.08-8.0	0.050	[9]
  Capillary electrophoresis	0.5-1000	0.224	[10]
  LC-MS	0.074-1.85	0.015	[12]
  P-g-C3N4 nanosheets	2.0-30	0.053	[5]
  N, S-CDs	0.69-70	0.210	[21]
  N, S-CDs	0-30	0.085	This work

  2.5   Selectivity and interference experiments of N, S-CDs to baicalein

  To investigate the selectivity, the fluorescence emission spectra of the sensor platform were measured in the presence of a series of analytes including common metal ions, anions, and biological molecules at 150 μmol·L^-1. As shown in Fig. 6a, except for baicalein, no other interfering substances exhibited a notable quenching response. In addition, when exposed to 365 nm UV light, the quenching of the blue-green emission of the sensor solution could only be visualized by adding baicalein, suggesting the present sensor has an excellent selectivity to baicalein, and can be visualized under 365 nm UV light (Inset of Fig. 6a). Then, competitive experiments were conducted to investigate the influence of different possible interferents. As displayed in Fig. 6b, the quenching response to baicalein and 10‑equivalent interferent or interferent mixture (blue bars) was quite similar to the quenching response to baicalein alone and much greater than that to interferent alone (grey bars). It turns out that the present sensor platform has excellent selectivity and strong anti-interference capability for baicalein detection.

  Figure 6

  

  Figure 6.  (a) Fluorescence spectra of N, S-CDs upon titration of different analytes at 150 µmol·L^-1 (Inset: photos of N, S-CDs in the absence and presence of 150 µmol·L^-1 analytes under 365 nm UV light); (b) I₀/I of N, S-CDs to baicalein (150 µmol·L^-1) in the absence and presence of various interferents (1.5 mmol·L^-1)

  (b) 1: Baicalein alone, 2: Na⁺, 3: K⁺, 4: Ca²⁺, 5: Mg²⁺, 6: Zn²⁺, 7: Cl^-, 8: Br^-, 9: I^-, 10: NO^3-, 11: S^2-, 12: Glucose, 13: Cys, 14: Hcy, 15: GSH, 16: Mixture; Each value is the average of three parallel measurements.

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  2.6   Sensing mechanism study of N, S‑CDs to baicalein

  Luminescence of CDs and doped CDs may originate from typical quantum confinement effects and surface defects, and surface defects induced by dopants could modulate the emission wavelength by generating new energy levels^[32]. According to the characterization data, the surface of N, S-CDs contains abundant chemical bonds (such as N—H, C=O, S=O, etc.), which leads to the formation of surface defects and provides possible interactions between N, S-CDs, and analytes^[33]. In general, fluorescence resonance energy transfer (FRET), inner filter effect (IFE), dynamic quenching, and static quenching can induce the fluorescence quenching of fluorescent sensors^[34]. To elucidate the fluorescence quenching mechanism of N, S-CDs by baicalein, the UV-Vis absorption spectrum of baicalein and fluorescence excitation and emission spectra of N, S-CDs were measured in 10 mmol·L^-1 HEPES solution (pH 7.4). As depicted in Fig. 7a, it was obvious that the absorption spectrum of baicalein overlapped with both the fluorescence excitation and emission spectra of N, S-CDs to a considerable extent, indicating that FRET or IFE may induce the quenching response. While the former can shorten the fluorescence lifetime, the latter cannot^[35-36]. Therefore, time‑resolved fluorescence decays of N, S-CDs before and after adding baicalein were tested at 480 nm and the results are presented in Fig. 7b. The average lifetime of N, S-CDs remains almost unchanged in the absence (16.11 ns) and presence (16.45 ns) of baicalein, suggesting that the quenching mechanism is not FRET. Meanwhile, the quenching mechanism is not dynamic quenching, because the lifetime of the sensor platform would change in the presence of a quencher^[34]. In addition, UV-Vis absorption spectra of N, S‑CDs, baicalein, and N, S‑CDs+baicalein were tested. As shown in Fig. 7c, the experimental and theoretical spectra (the absorption spectra of N, S-CDs and baicalein are mathematically superimposed) of N, S-CDs+baicalein almost overlapped, indicating the absence of non-fluorescent ground-state complexes and no static quenching between N, S-CDs and baicalein. Based on the above results and discussion, IFE is considered to be the possible main reason for the fluorescence quenching of N, S-CDs by baicalein in this system.

  Figure 7

  

  Figure 7.  (a) Normalized UV-Vis absorption spectrum of baicalein (100 µmol·L^-1) in 10 mmol·L^-1 HEPES solution and normalized excitation and emission spectra of N, S-CDs sensor platform; (b) Time-resolved fluorescence decays of N, S-CDs in the absence and presence of baicalein (50 µmol·L^-1); (c) UV-Vis absorption spectra of N, S-CDs, baicalein, and N, S-CDs+baicalein

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  2.7   Detection of baicalein in real samples

  Based on the excellent selectivity and sensitivity of the N, S-CDs in detecting baicalein, the sensor platform was further applied to the quantitative determination of baicalein in serum and urine. The measurement results of adding three different concentrations of baicalein (5, 10, 30 μmol·L^-1) were summarized in Table 3. All measurements were taken from the average consequences of three repeated assessments. The recovery rates of baicalein in serum and urine were 98.60%-100.67% (relative standard deviation (RSD), less than 1.03%) and 99.60%-103.00% (RSD, less than 1.05%), respectively. All the results indicate that the present sensor has the potential to detect baicalein in biofluids.

  Table 3

  

  Table 3.  Results for the detection of baicalein in serum and urine (n=3)

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  Sample	Standard added / (μmol·L-1)	Total found / (μmol·L-1)	Recovery / %	RSD / %
  Serum	5	4.93	98.60	0.85
  	10	9.98	99.80	1.03
  	30	30.20	100.67	0.99
  Urine	5	5.15	103.00	1.02
  	10	9.96	99.60	0.98
  	30	30.10	100.33	1.05

  3.   Conclusions

  In this work, we have successfully synthesized blue‑green emitting fluorescent N, S‑CDs using p‑ phenylenediamine and L-cysteine as raw materials via a rapid one-step solvothermal method. The N, S-CDs could be well dispersed in water and have excellent salt tolerance and photo‑stability. The fluorescence sensing experiments showed that the present CD sensor could exhibit high sensitivity and good selectivity detection of baicalein with a detection limit of 85 nmol·L^-1 in the linear range of 0-30 μmol·L^-1. The corresponding sensing mechanism could be attributed to the inner filter effect. Meanwhile, the sensor platform can detect baicalein in serum and urine with recovery rates of 98.60%-100.67% and 99.60%-103.00%, respectively. This method has the advantages of simple operation, low cost, good reproducibility, and high sensitivity, providing a promising approach for constructing fluorescent sensors for detecting other analytes.

  
  
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  Scheme1  Schematic illustration of the synthesis of N, S-CDs and detection of baicalein

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  Figure 1  TEM image (a), particle size distribution diagram (b), XRD pattern (c), and FTIR spectrum (d) of N, S-CDs

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  Figure 2  XPS survey spectrum (a) (Inset: pie chart of the element contents of C, N, O, and S) and high-resolution spectra of C1s (b), N1s (c), O1s (d), and S2p (e) of N, S-CDs

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  Figure 3  (a) UV-Vis absorption, excitation, and emission spectra of N, S-CDs in 10 mmol·L^-1 HEPES buffer solution (Inset: photographs of N, S-CDs under natural light and 365 nm UV light); (b) Emission spectra of N, S-CDs aqueous solution at different excitation wavelengths

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  Figure 4  Effect of (a) pH, (b) ion strength by various concentrations of NaCl, (c) radiation time under continuous UV light irradiation (365 nm), and (d) storage time on the fluorescence intensity of N, S-CDs

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  Figure 5  (a) Fluorescence spectra of N, S-CDs upon titration of various concentrations of baicalein; (b) Relationship between I₀ /I and baicalein concentration (Inset: the linear relationship between I₀/I and c_Baicalein)

  (b) Each value represents the average of three parallel measurements.

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  Figure 6  (a) Fluorescence spectra of N, S-CDs upon titration of different analytes at 150 µmol·L^-1 (Inset: photos of N, S-CDs in the absence and presence of 150 µmol·L^-1 analytes under 365 nm UV light); (b) I₀/I of N, S-CDs to baicalein (150 µmol·L^-1) in the absence and presence of various interferents (1.5 mmol·L^-1)

  (b) 1: Baicalein alone, 2: Na⁺, 3: K⁺, 4: Ca²⁺, 5: Mg²⁺, 6: Zn²⁺, 7: Cl^-, 8: Br^-, 9: I^-, 10: NO^3-, 11: S^2-, 12: Glucose, 13: Cys, 14: Hcy, 15: GSH, 16: Mixture; Each value is the average of three parallel measurements.

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  Figure 7  (a) Normalized UV-Vis absorption spectrum of baicalein (100 µmol·L^-1) in 10 mmol·L^-1 HEPES solution and normalized excitation and emission spectra of N, S-CDs sensor platform; (b) Time-resolved fluorescence decays of N, S-CDs in the absence and presence of baicalein (50 µmol·L^-1); (c) UV-Vis absorption spectra of N, S-CDs, baicalein, and N, S-CDs+baicalein

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  Table 1.  Area proportion of chemical binds in N, S-CDs XPS spectra

  C1s				N1s			O1s			S2p
  C—C	C—N	C=O		N—H	C—N		C=O	C—OH/C—O—C		C—S (2p3/2)	C—S (2p1/2)	S=O (2p3/2)	S=O (2p1/2)
  48.5%	48.7%	2.8%		59.6%	40.4%		60.7%	39.3%		31.1%	15.4%	35.6%	17.9%

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  Table 2.  Comparison of various reported baicalein detection methods

  Method	Linear range / (μmol·L-1)	Detection limit / (μmol·L-1)	Reference
  High performance liquid chromatography	1.85-1480	1.480	[7]
  Voltammetry	0.08-8.0	0.050	[9]
  Capillary electrophoresis	0.5-1000	0.224	[10]
  LC-MS	0.074-1.85	0.015	[12]
  P-g-C3N4 nanosheets	2.0-30	0.053	[5]
  N, S-CDs	0.69-70	0.210	[21]
  N, S-CDs	0-30	0.085	This work

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  Table 3.  Results for the detection of baicalein in serum and urine (n=3)

  Sample	Standard added / (μmol·L-1)	Total found / (μmol·L-1)	Recovery / %	RSD / %
  Serum	5	4.93	98.60	0.85
  	10	9.98	99.80	1.03
  	30	30.20	100.67	0.99
  Urine	5	5.15	103.00	1.02
  	10	9.96	99.60	0.98
  	30	30.10	100.33	1.05

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