Detection of Cobalamin and In Vitro Cell Imaging Based on Nitrogen-Doped Yellow Fluorescent Carbon Dots with Nano Architectonics

As novel fluorescent nanomaterials, carbon dots have attracted increasing research attention because of their simple synthesis, robust fluorescence, low toxicity, and easy functionalisation. Previous research was focused on preparing carbon dots from biomass and chemical materials; however, most of these carbon dots exhibited blue fluorescence. Moreover, the fluorescence quantum yield was generally low, significantly limiting their application in biological imaging. To broaden the application scope of carbon dots, this study prepared long-wavelength emitting nano-carbon dots that exhibited increased quantum yield. Novel N-doped yellow fluorescent nano-carbon dots (Y-CDs) were synthesised via a hydrothermal method using L-tartaric acid and urea as the precursors. The Y-CDs had a high quantum yield (15.9%) and demonstrated photostability at various pHs, temperatures, and ionic strengths. The Y-CDs could detect cobalamin effectively and selectively, showing a linear relationship between fluorescence intensity and cobalamin concentration. The related coefficient was 0.997, and the detection limit was 2.101 μmol/L. In addition, the Y-CDs were successfully used as an imaging probe for MDA-MB-231 cells. Therefore, the Y-CDs developed in this study can be used for cobalamin detection and cell imaging.


Introduction
Carbon dots (CDs) are a type of fluorescent nanomaterial that have a size of <10 nm. CDs were first synthesised in 2004. In that year, Professor Xu conducted cataphoresis to purify single-walled carbon nanotube fragments. In the process, the nanoparticles with fluorescence characteristics were separated. In 2006, Professor Sun used graphite and black charcoal as a raw material for the synthesis of fluorescent nanoparticles [1][2][3][4]. These nanoparticles exhibited improved fluorescence. It was the first time CDs were nominated officially. Compared with organic dyestuff and quantum dots, CDs possess good luminescence properties, have an easy synthesis route, and exhibit low consumption and high biocompatibility and fluorescence without bleaching [5][6][7]. Therefore, the research on CDs has attracted widescale attention. At present, there are two main methods for CD synthesis, namely, the top-to-bottom approach based on the bulk material and the bottom-totop approach based on the small molecule. During early research, graphite and carbon fibre were mainly used as raw materials. The preparation methods included arc discharge, laser corrosion, or electrochemistry. The CDs synthesised by the abovementioned two methods mainly emit blue short-wavelength light, and their quantum efficiency is low [8,9]. Upon further research, CD synthesis was conducted using raw materials such as biomass, e.g., strawberries, lime juice, and silkworm chrysalises. Moreover, carbon-based molecules such as citric acid, urea, and ethylenediamine (EDA) were used [10,11]. The synthesis methods involved microwave and ultrasonic irradiation and the hydrothermal method because they are rapid and convenient. The current research focuses on the synthesis and  In this manuscript, the MDA-MB-231 cells were cultivated by professor Xinguo Zhang. The place these cells were cultured was the school of pharmaceutical sciences, Southern Medical University.

Preparation of N-Doped Y-CDs
The one-step hydrothermal method was conducted to synthesise N-doped Y-CDs with L-tartaric acid, urea, and DMF as the raw materials [22][23][24]. First, 2.0 g of L-tartaric acid and 1.0 g of urea were dissolved in 10 mL of DMF. After ultrasonication for 5 min, the mixture was transferred to a 40 mL polytetrafluoroethylene (PTEF)-lined vessel. Next, the vessel was placed in a stainless steel hydrothermal reactor and heated at 180 • C for 4 h in an electric blast drying oven. After the reaction, 20 mL of absolute alcohol was added to dilute the solution in order to remove macromolecular impurities. Then, the mixture was subjected to centrifugation at 8000 rpm for 5 min. The supernatant was filtered using a 0.22 µm Millipore filter. After vacuum distillation, the mixture was freeze-dried for 24 h to obtain the Y-CDs.

Scanning Electron Microscopy (SEM)
SEM was conducted to observe CD sample morphology and size. For sample preparation, the CD samples were first mixed with absolute alcohol. Upon ultrasound irradiation for 10 min, the homogeneous suspension was formed. Next, a conductive resin was stuck to a copper platform. A total of 1-2 drops homogeneous suspension were dropped on the conductive resin by a capillary, which avoided the contact between the capillary and conductive resin. After air drying, the samples were subjected to gold sputtering. The JSM-7001F system was used for SEM, and the accelerating voltage was set to 15 kV.

Transmission Electron Microscopy (TEM)
In order to acquire the information about the internal structure and morphology of the CD samples, TEM was conducted. For sample preparation, the CD samples were first dispersed in absolute alcohol. After ultrasound irradiation for 5 min, a homogeneous suspension was formed. A few drops of the homogeneous suspension were dropped on a copper mesh by a capillary. After air drying, the JEM-2100F system was used for TEM (FEI company, Columbia, SC, USA).

X-ray Diffraction (XRD) Analysis
The crystalline and crystalline phase purity of the samples were analysed by XRD (Shimadzu corporation, Kyoto, Japan). The Cu target was used as the radiant. The testing voltage was set to 40 kV. The testing current was set to 26 mA. The scanning range was 10 • -80 • . The scanning speed was 10 • /min.

Infrared (IR) Spectroscopy
IR spectroscopy was conducted to gain information about the functional groups of the CD samples. The IR spectrum of KBr pellets was considered as the blank control. Moderate KBr was weighed and mixed with the CD samples uniformly (1:200). The mixture was ground to powder. This powder was placed under an IR lamp for drying, which avoided water interference. After tablet compression, the samples were placed into an infrared spectrometer (Thermo Fisher Scientific technology company, Waltham, MA, USA). The wavenumber range was 400-4000 cm −1 . The resolution rate was 4 cm −1 .

Determination of Y-CD Fluorescence
Absolute alcohol was used as the solvent to prepare a Y-CD solution (2.5 mg/mL). Its emission spectrum was collected from 460 to 800 nm on an FLS980 fluorescence analyser in transient and steady states. The excitation wavelength was 450 nm. The slit width was 2.0 nm, and the detention time was 0.1 s. The illuminant used for emission spectroscopy and the photostability test was a xenon lamp (450 W). A 320 nm laser was used for the fluorescence lifetime test.

Interference Factor of Y-CDs Fluorescence
(1) Absolute alcohol, distilled water, carbinol, acetone, and PBS buffer solution (pH 7.40) were used as solvents to repair Y-CD solutions (2.5 mg/mL). From each solution, 3 mL was extracted for fluorescence test. (2) Absolute alcohol was used as a solvent to prepare the Y-CD solution (2.5 mg/mL).
The excitation wavelength was varied from 400 nm to 500 nm to obtain excitationdependent spectra by using 3 mL of the solution. (3) Absolute alcohol was used as a solvent to prepare the Y-CD solutions (2.5, 5, 15, 45, and 150 mg/mL). The solution (3 mL) was excited using 450 nm-wavelength light to obtain concentration-dependent spectra. (4) NaOH solution (2 mol/L) and HCl solution (1 mol/L) were added to a PBS buffer to adjust its pH to 2-12. The PBS solutions with different pHs were used as solvents to prepare the Y-CD solutions (2.5 mg/mL). The fluorescence spectra of the Y-CDs (3 mL) at various pHs were collected. (5) Absolute alcohol was used as a solvent to prepare the Y-CD solution (2.5 mg/mL).
The excitation wavelength was 450 nm. Using 3 mL of the solution, the fluorescence emission spectra (460-800 nm) were scanned from 20 to 65 • C at 5 • C intervals. In the thermostat, the temperature was held for 5 min with every increase of 5 • C. (6) NaCl solutions (0.4-1.2 mol/L) were used as solvents to prepare the Y-CD solutions (2.5 mg/mL). Next, 3 mL of these solutions were scanned by a fluorescence spectrometer to investigate the effect of ionic strength.

Detection of QYs of Y-CDs
A comparison measurement method was employed in this experiment. Rhodamine 6 g (r6g) with a QE of 98% was used as a standard. The emission and UV-vis spectra of r6g and Y-CDs were collected, and the QY of Y-CDs (x) can be calculated by Equation (1).
where I is the integral intensity of the fluorescence emission spectrum, η is the solvent refractive index, and A is the absorbance.

Testing and Calculation of Fluorescence Lifetime
For the test, an FLS980 transient and steady fluorescence analyser was equipped with a 450 nm laser. The monitoring wavelength was 550 nm. The fluorescence lifetime of the Y-CDs can be calculated by Equation (2).
where I(t) is the fluorescence intensity of the Y-CDs at 550 nm, I 0 is the initial fluorescence intensity (t = 0), A is the fluorescence lifetime constant, and τ is the fluorescence lifetime of the decaying exponential. The average fluorescence lifetime can be calculated by Equation (3).

Fluorescence-Based Cobalamin Detection
The fluorescence-based detection of cobalamin is a simple and rapid analytical process. First, 100 µL of Y-CD solution (6 mg/mL), 2100 µL of PBS solution (pH 7.400), and 800 µL of VB12 solution were added to a cuvette and mixed thoroughly. The emission spectra under 450 nm excitation were measured. For real sample analysis, cobalamin solutions of different concentrations were added to the veterinary cobalamin injection (0.5 mg/mL). The fluorescence spectra of these samples were detected as mentioned above, and the measurement was conducted in triplicate. The corresponding recycling rate and relative standard deviation (RSD) were calculated.

Selective Research on Cobalamin Detection
Selectivity is important in real sample analysis. In a system of cobalamin, common vitamins were used as disruptors. At the relevant concentrations of disruptors, the fluorescence spectra were collected. According to the effects of the disruptors on the fluorescence of the Y-CDs, the selectivity of Y-CDs for cobalamin detection was verified [25][26][27][28].

Effect of Cobalamin on Y-CD UV Spectrum
A solution of the Y-CDs (50 µg/mL) and a cobalamin solution (200 µM) were mixed, and the UV absorption spectrum of the mixture was scanned from 400 to 750 nm.

Effect of Cobalamin on Y-CD Fluorescence Lifetime
A solution of the Y-CDs (50 µg/mL) and a cobalamin solution (0-200 µM) were mixed. The experimental conditions were the same as those mentioned in Section 2.7. At different cobalamin concentrations, the fluorescence lifetimes of the Y-CDs were tested.

Cytotoxicity Investigation and In Vitro Cell Imaging
MDA-MB-231 human breast cancer cells were cultured in RMPI-1640 media containing 10% Australian foetal bovine serum and 1% penicillin-streptomycin solution. First, the MDA-MB-231 cells were inoculated to a 96-well plate and placed into a 5% CO 2 incubator at 37 • C for 24 h. The culture solution is H 2 O. When the cell density reached about 5 × 10 3 -10 × 10 3 per well, the culture medium in the well was suctioned and the cells were washed with PBS solutions (pH 7.40). Next, 100 µL of the culture medium containing 2, 8, 15, 20, 30, and 150 mg/mL Y-CDs was added. Two groups of the samples were prepared following the abovementioned steps. These samples were placed in a 5% CO 2 incubator at 37 • C for 24 h. The culture solution is H 2 O. After incubation, the Y-CD solution was extracted, and the cells were washed with PBS (pH 7.40). Next, 10 µL of CCK8 solution was added into each well and incubated for 4 h. After incubation, the absorption value at 450 nm was determined using a microplate reader.
MDA-MB-231 cells (1 mL) were added into a 15 mm glass bottom cell culture dish for inoculation, and placed in a 5% CO 2 incubator at 37 • C for 12 h. Next, the culture medium was extracted and replaced by 1 mL of the culture medium containing 20 mg/mL of Y-CDs. After incubating for 1 h and 4 h, the medium was discarded and replaced with 2 mL of 4% paraformaldehyde (POM). The solution was stewed for 20 min to fix the cells. Then, a PBS solution (pH 7.40) was used to wash POM. Finally, an anti-fluorescence quenching mounting medium was added, and the cells were observed under an inverted LSCM.

Structure and Morphology Characterisation of Y-CDs
The elemental composition, functional groups, and morphological structure of the Y-CDs were analysed by XPS, FT-IR spectroscopy, XRD, and TEM. Figure 1a shows the full XP spectrum, wherein three binding energy peaks (Cs, Os, and Ns) are detected. The elemental proportions of C, O, and N are 74.39%, 23.07%, and 1.61%, respectively. Figure Figure 2a, the small particles in the Y-CDs disperse evenly. The interpolated image is the grain diameter distribution map. The distribution of particles sizes is between 2 nm and 5 nm, and is largely concentrated at 3.8 nm. From Figure 2b, with the geometric measurement in MATLAB, the cell interval was found to be 0.21 nm, which is similar to that in the graphene layers.

Fluorescence Stability of Y-CDs
From the interpolated map in Figure 3a, the alcohol solution with 2.5 mg/mL Y-CDs was found to be a flaxen, transparent, and clear liquid under natural light. Under 395 nm UV radiation, the solution emits yellowish green fluorescence. From Figure 3b, under 450 nm UV radiation, the solution emits yellow fluorescence. From the UV absorption curve in Figure 3a, the UV absorption area exists at <300 nm. It is caused by the π−π* transition induced by C=C and C=N. The absorption band exists at 350-500 nm. It is caused by the n-π* and π-π* transitions induced by C=O and the aromatic group structure. The band gap transition at 450 nm is caused by the interface excitation of the Y-CDs. From Figure 3a, the optimum excitation wavelength and maximum emission wavelength of the Y-CD alcohol solution are 450 nm and 550 nm, respectively. The chromatic coordinates were as follows: CIE (x, y) = (0.390, 00.525). From Figure 3b, the solution was found to emit yellow fluorescence. According to the test and calculation, the quantum efficiency of the Y-CD alcohol solution and aqueous solution were found to be 16.7% and 4.5%, respectively. In the view of chemistry, the hydrogen bond in alcohol is stronger than that in water. Therefore, the molecular dipole moment affects the surface electronic state further, which increases the quantum efficiency of the Y-CD alcohol solution. In this study, all luminescent property tests of the Y-CDs were carried out in the alcohol solution. There are many luminescence centres in the CDs. Thus, the PL characteristics related to excitation were considered as special characteristics. When the excitation wavelength increases from 400 nm to 500 nm, the emission peaks gradually shift from 510 nm to 575 nm. The emission characteristics of the Y-CDs is related to the excitation wavelength. This may be related to the differences in the luminescence centres or the radiation contribution of the surface states. From Figure 3c, with the change in the excitation wavelength, the emission wavelength of the Y-CD alcohol solution also red shifts and the fluorescence intensity decreases, which is attributed to the Y-CDs possessing multiple luminescence centres. From Figure 3d, when the excitation wavelength is 450 nm, the Y-CD alcohol solution emits fluorescence at 550 nm. The fluorescence intensity reaches the maximum. XP spectrum, wherein three binding energy peaks (Cs, Os, and Ns) are detected. The elemental proportions of C, O, and N are 74.39%, 23.07%, and 1.61%, respectively. Figure 1b shows the peak fitting results of Cs, and the peaks at 284.3 eV, 284.8 eV, and 288.6 eV correspond to C-C/C=C, C=N, and C=O, respectively. Similarly, N-O, C=O, and amino groups are identified in the high-resolution XP spectra of Os and Ns [29][30][31]. These results agree with the FT-IR spectrum in Figure 1e.    Figure 2a, the small particles in the Y-CDs disperse evenly. The interpolated image is the grain diameter distribution map. The distribution of particles sizes is between 2 nm and 5 nm, and is largely concentrated at 3.8 nm. From Figure 2b, with the geometric measurement in MATLAB, the cell interval was found to be 0.21 nm, which is similar to that in the graphene layers.

Fluorescence Stability of Y-CDs
From the interpolated map in Figure 3a, the alcohol solution with 2.5 mg/mL Y-CDs was found to be a flaxen, transparent, and clear liquid under natural light. Under 395 nm UV radiation, the solution emits yellowish green fluorescence. From Figure 3b, under 450 nm UV radiation, the solution emits yellow fluorescence. From the UV absorption curve in Figure 3a, the UV absorption area exists at <300 nm. It is caused by the π−π* transition induced by C=C and C=N. The absorption band exists at 350-500 nm. It is caused by the

Fluorescence Stability of Y-CDs
Fluorescence stability is an important property governing the application scope of CDs. Figure 4a shows the pH-dependent fluorescence spectra of the Y-CDs. The emission wavelength does not change significantly. In the pH range of 1.5-4, the fluorescence intensity increases rapidly because the functional groups on the Y-CD surface are hydrolysed under the strong acid condition. When the pH value is 4-8, the fluorescence intensity increases and remains stable. The fluorescence intensity is slightly enhanced under an alkaline condition (pH [8][9][10][11][12]. Different solvents were used to dissolve the Y-CDs at the same concentration. As shown in Figure 4b, the fluorescence intensity of the Y-CDs in ethanol is higher than those in carbinol, acetone, distilled water, and the buffer solution. Different solvent polarities cause the rearrangement of the surface electrons of the Y-CDs. The dipole moment changes greatly and the fluorescence intensity of the Y-CDs decreases.

Fluorescence Stability of Y-CDs
Fluorescence stability is an important property governing the application scope of CDs. Figure 4a shows the pH-dependent fluorescence spectra of the Y-CDs. The emission wavelength does not change significantly. In the pH range of 1.5-4, the fluorescence intensity increases rapidly because the functional groups on the Y-CD surface are hydrolysed under the strong acid condition. When the pH value is 4-8, the fluorescence intensity increases and remains stable. The fluorescence intensity is slightly enhanced under an alkaline condition (pH [8][9][10][11][12]. Different solvents were used to dissolve the Y-CDs at the same concentration. As shown in Figure 4b, the fluorescence intensity of the Y-CDs in ethanol is higher than those in carbinol, acetone, distilled water, and the buffer solution. Different solvent polarities cause the rearrangement of the surface electrons of the Y-CDs. The dipole moment changes greatly and the fluorescence intensity of the Y-CDs decreases. In Figure 5, the fluorescence intensity of the Y-CDs (2.5 mg/mL) remains unchanged in 0.50-1.50 M NaCl solutions, confirming that the Y-CDs exhibit good salt tolerance at high ionic strength. Therefore, the Y-CDs demonstrate excellent fluorescence stability at different pHs, temperatures, and ionic strengths. In Figure 5, the fluorescence intensity of the Y-CDs (2.5 mg/mL) remains unchanged in 0.50-1.50 M NaCl solutions, confirming that the Y-CDs exhibit good salt tolerance at high ionic strength. Therefore, the Y-CDs demonstrate excellent fluorescence stability at different pHs, temperatures, and ionic strengths.

Fluorescence-Based Detection of Cobalamin
Considering their excellent stability, the Y-CDs were used for VB12 detection. As shown in Figure 6, the emission intensity at 530 nm quenches in the presence of VB12 at different concentrations (0, 25, 50, 100, 150, 200 μM). With the increasing VB12 concentration, the emission intensity of the Y-CDs decreases gradually. When the concentration is higher than 100 μM, the emission peak is divided into a doublet because of the spectral overlap of the VB12 absorption peak and the Y-CD emission peak. After gaussian decomposition, as shown in Figure 6, the Y-CD emission peak is divided into two peaks (542 nm and 600 nm), suggesting the presence of at least two luminescence centres in the Y-CDs. The absorption peak of VB12 overlaps with the peak at 542 nm. Therefore, the IFE occurs Wavelength(nm)  In Figure 5, the fluorescence intensity of the Y-CDs (2.5 mg/mL) remains unchanged in 0.50-1.50 M NaCl solutions, confirming that the Y-CDs exhibit good salt tolerance at high ionic strength. Therefore, the Y-CDs demonstrate excellent fluorescence stability at different pHs, temperatures, and ionic strengths.

Fluorescence-Based Detection of Cobalamin
Considering their excellent stability, the Y-CDs were used for VB12 detection. As shown in Figure 6, the emission intensity at 530 nm quenches in the presence of VB12 at different concentrations (0, 25, 50, 100, 150, 200 μM). With the increasing VB12 concentration, the emission intensity of the Y-CDs decreases gradually. When the concentration is higher than 100 μM, the emission peak is divided into a doublet because of the spectral overlap of the VB12 absorption peak and the Y-CD emission peak. After gaussian decomposition, as shown in Figure 6, the Y-CD emission peak is divided into two peaks (542 nm and 600 nm), suggesting the presence of at least two luminescence centres in the Y-CDs. The absorption peak of VB12 overlaps with the peak at 542 nm. Therefore, the IFE occurs Wavelength(nm) Figure 5. PL spectra of the Y-CDs in NaCl solutions with different ionic strengths (λ ex = 450 nm).

Fluorescence-Based Detection of Cobalamin
Considering their excellent stability, the Y-CDs were used for VB12 detection. As shown in Figure 6, the emission intensity at 530 nm quenches in the presence of VB12 at different concentrations (0, 25, 50, 100, 150, 200 µM). With the increasing VB12 concentration, the emission intensity of the Y-CDs decreases gradually. When the concentration is higher than 100 µM, the emission peak is divided into a doublet because of the spectral overlap of the VB12 absorption peak and the Y-CD emission peak. After gaussian decomposition, as shown in Figure 6, the Y-CD emission peak is divided into two peaks (542 nm and 600 nm), suggesting the presence of at least two luminescence centres in the Y-CDs. The absorption peak of VB12 overlaps with the peak at 542 nm. Therefore, the IFE occurs between the VB12 absorption peak and the 544 nm absorption peak, dividing the Y-CD emission peak gradually.
As shown in Figure  between the VB12 absorption peak and the 544 nm absorption peak, dividing the Y-CD emission peak gradually.

Quenching Mechanism for Cobalamin Detection
The systematic fluorescence quenching mechanism includes static quenching, dynamic quenching, Forster resonance energy transfer (FRET), photo-induced electron transfer (PET), and IFE. Different quenching mechanisms occur under different conditions. The fluorescence emission of the Y-CDs and the UV absorption spectra of VB12 are shown in Figure 8. A large overlapping area is found at 430-620 nm, suggesting that VB12 can absorb a part of the fluorescence emitted by the Y-CDs. When the Y-CDs constantly emit fluorescence, the quantity of absorption fluorescence depends on VB12 concentration. Therefore, VB12 can quench the fluorescence of the Y-CDs, showing a linear relationship within a specific concentration range. According to the nature of spectral overlap, it can be deduced that the quenching mechanism may be related to the FRET or IFE. For verification of the quenching mechanism, fluorescence lifetime attenuation must be considered. between the VB12 absorption peak and the 544 nm absorption peak, dividing the Y-CD emission peak gradually.

Quenching Mechanism for Cobalamin Detection
The systematic fluorescence quenching mechanism includes static quenching, dynamic quenching, Forster resonance energy transfer (FRET), photo-induced electron transfer (PET), and IFE. Different quenching mechanisms occur under different conditions. The fluorescence emission of the Y-CDs and the UV absorption spectra of VB12 are shown in Figure 8. A large overlapping area is found at 430-620 nm, suggesting that VB12 can absorb a part of the fluorescence emitted by the Y-CDs. When the Y-CDs constantly emit fluorescence, the quantity of absorption fluorescence depends on VB12 concentration. Therefore, VB12 can quench the fluorescence of the Y-CDs, showing a linear relationship within a specific concentration range. According to the nature of spectral overlap, it can be deduced that the quenching mechanism may be related to the FRET or IFE. For verification of the quenching mechanism, fluorescence lifetime attenuation must be considered.

Quenching Mechanism for Cobalamin Detection
The systematic fluorescence quenching mechanism includes static quenching, dynamic quenching, Forster resonance energy transfer (FRET), photo-induced electron transfer (PET), and IFE. Different quenching mechanisms occur under different conditions. The fluorescence emission of the Y-CDs and the UV absorption spectra of VB12 are shown in Figure 8. A large overlapping area is found at 430-620 nm, suggesting that VB12 can absorb a part of the fluorescence emitted by the Y-CDs. When the Y-CDs constantly emit fluorescence, the quantity of absorption fluorescence depends on VB12 concentration. Therefore, VB12 can quench the fluorescence of the Y-CDs, showing a linear relationship within a specific concentration range. According to the nature of spectral overlap, it can be deduced that the quenching mechanism may be related to the FRET or IFE. For verification of the quenching mechanism, fluorescence lifetime attenuation must be considered. Figure 9 shows the time-resolved decay of Y-CDs in the presence and absence of VB12. When the VB12 concentration varies from 0 to 200 µM, the fluorescence lifetime does not change. This indicates that no new chemical compound is generated during the quenching process, which is contrary to the FRET mechanism. When the fluorophore coexists with the other absorbing materials, the stimulated luminescence or emission light is absorbed by the fluorophore or other absorbing materials, quenching the fluorescence. This phenomenon is the fluorescence IFE. In this process, no new chemical compound is generated, and the quenching mechanism of the Y-CDs is more likely to be the IFE.  Figure 9 shows the time-resolved decay of Y-CDs in the presence and absence of VB12. When the VB12 concentration varies from 0 to 200 μM, the fluorescence lifetime does not change. This indicates that no new chemical compound is generated during the quenching process, which is contrary to the FRET mechanism. When the fluorophore coexists with the other absorbing materials, the stimulated luminescence or emission light is absorbed by the fluorophore or other absorbing materials, quenching the fluorescence. This phenomenon is the fluorescence IFE. In this process, no new chemical compound is generated, and the quenching mechanism of the Y-CDs is more likely to be the IFE.

Content Assay Via Cobalamin Injection
To explore the practical application of the Y-CDs, veterinary cobalamin injection is applied for content assay. The concentration of cobalamin injection (0.5 mg/mL) is 368.903 μM, exceeding the detection range of the Y-CDs. Therefore, the cobalamin injection is diluted to 92.226 μM. The cobalamin concentration is detected as 92.283 μM. The absolute error value is 0.057 μM. The relative error value is 0.06%. To compare the preliminary fluorescence detection, the organic dyestuff and quantum dots are used as the fluorescent carbon dots. Under the same experimental environment, the cobalamin content assay is carried out. Through comparison and analysis, the error in the Y-CD fluorescence detection is the minimum. Based on the results summarised in Table 3, this method is more suitable for actual cobalamin detection.    Figure 9 shows the time-resolved decay of Y-CDs in the presence and absence of VB12. When the VB12 concentration varies from 0 to 200 μM, the fluorescence lifetime does not change. This indicates that no new chemical compound is generated during the quenching process, which is contrary to the FRET mechanism. When the fluorophore coexists with the other absorbing materials, the stimulated luminescence or emission light is absorbed by the fluorophore or other absorbing materials, quenching the fluorescence. This phenomenon is the fluorescence IFE. In this process, no new chemical compound is generated, and the quenching mechanism of the Y-CDs is more likely to be the IFE.

Content Assay Via Cobalamin Injection
To explore the practical application of the Y-CDs, veterinary cobalamin injection is applied for content assay. The concentration of cobalamin injection (0.5 mg/mL) is 368.903 μM, exceeding the detection range of the Y-CDs. Therefore, the cobalamin injection is diluted to 92.226 μM. The cobalamin concentration is detected as 92.283 μM. The absolute error value is 0.057 μM. The relative error value is 0.06%. To compare the preliminary fluorescence detection, the organic dyestuff and quantum dots are used as the fluorescent carbon dots. Under the same experimental environment, the cobalamin content assay is carried out. Through comparison and analysis, the error in the Y-CD fluorescence detection is the minimum. Based on the results summarised in Table 3, this method is more suitable for actual cobalamin detection.

Content Assay Via Cobalamin Injection
To explore the practical application of the Y-CDs, veterinary cobalamin injection is applied for content assay. The concentration of cobalamin injection (0.5 mg/mL) is 368.903 µM, exceeding the detection range of the Y-CDs. Therefore, the cobalamin injection is diluted to 92.226 µM. The cobalamin concentration is detected as 92.283 µM. The absolute error value is 0.057 µM. The relative error value is 0.06%. To compare the preliminary fluorescence detection, the organic dyestuff and quantum dots are used as the fluorescent carbon dots. Under the same experimental environment, the cobalamin content assay is carried out. Through comparison and analysis, the error in the Y-CD fluorescence detection is the minimum. Based on the results summarised in Table 3, this method is more suitable for actual cobalamin detection.

Cytotoxicity Test and In Vitro Cell Imaging of Y-CDs
Before the cell imaging experiment, the cytotoxicity of the Y-CDs to MDA-MB-231 cells was evaluated using the CCK-8 method. The cell survival rate shown in Figure 10a was determined after the cells were co-cultured with different concentrations of the Y-CDs for 4 h. When the Y-CD concentration reaches 20 mg/mL, the cell survival rate is higher than 90%. When the concentration is higher than 20 mg/mL, the cell survival rate decreases gradually. Combining the effect of the cell survival rate and Y-CD concentration on the fluorescence intensity, 20 mg/mL of Y-CDs was chosen for the cell imaging experiment.
on the fluorescence intensity, 20 mg/mL of Y-CDs was chosen for the cell imaging experiment.
The blue fluorescence emitted from a DAPI dye under 441 nm laser excitation is shown on the left in Figure 10b,c. The cell locations can be marked because the DAPI dye enters the nucleus. After the Y-CDs and cells are incubated for 1 h, a few Y-CDs entered the nucleus. Under the excitation of a 490 nm laser, the Y-CDs exhibit faint yellow fluorescence in the nucleus. After incubation for 4 h, the yellow fluorescence enhances, and the cell contour can be observed, indicating that the Y-CDs entered the nucleus after 4 h. These observations confirm that the Y-CDs have good biocompatibility and can be used in cell imaging.

Conclusions
In this study, tartaric acid and urea were used as raw materials to prepare Y-CDs via a green and simple solvothermal method. The mean size of Y-CDs was 6 nm, and the size distribution was narrow. The Y-CDs exhibited strong excitation-dependent fluorescence at 530 nm with a high QY of 15.9%. The excellent fluorescence stability of the Y-CDs was verified at different pHs, temperatures, and ionic strengths. The Y-CDs could detect cobalamin sensitively and selectively through the IFE mechanism. The emission intensity of the Y-CDs and cobalamin concentration showed a linear correlation at 0-200 μM. The related coefficient was 0.997, and the detection limit was 2.101 μmol/L. The feasibility of this quenching method was demonstrated by detecting cobalamin in real samples. Moreover, the MDA-MB-231 cells co-cultured with the Y-CDs showed insignificant cytotoxicity and exhibited yellow fluorescence under 490 nm excitation. The result indicated that the Y-CDs possess imaging depth because they are small enough to enter the nucleus. Therefore, the Y-CDs can be applied in cobalamin detection and as a multifunctional fluorescent probe for cell imaging. The blue fluorescence emitted from a DAPI dye under 441 nm laser excitation is shown on the left in Figure 10b,c. The cell locations can be marked because the DAPI dye enters the nucleus. After the Y-CDs and cells are incubated for 1 h, a few Y-CDs entered the nucleus. Under the excitation of a 490 nm laser, the Y-CDs exhibit faint yellow fluorescence in the nucleus. After incubation for 4 h, the yellow fluorescence enhances, and the cell contour can be observed, indicating that the Y-CDs entered the nucleus after 4 h. These observations confirm that the Y-CDs have good biocompatibility and can be used in cell imaging.

Conclusions
In this study, tartaric acid and urea were used as raw materials to prepare Y-CDs via a green and simple solvothermal method. The mean size of Y-CDs was 6 nm, and the size distribution was narrow. The Y-CDs exhibited strong excitation-dependent fluorescence at 530 nm with a high QY of 15.9%. The excellent fluorescence stability of the Y-CDs was verified at different pHs, temperatures, and ionic strengths. The Y-CDs could detect cobalamin sensitively and selectively through the IFE mechanism. The emission intensity of the Y-CDs and cobalamin concentration showed a linear correlation at 0-200 µM. The related coefficient was 0.997, and the detection limit was 2.101 µmol/L. The feasibility of this quenching method was demonstrated by detecting cobalamin in real samples. Moreover, the MDA-MB-231 cells co-cultured with the Y-CDs showed insignificant cytotoxicity and exhibited yellow fluorescence under 490 nm excitation. The result indicated that the Y-CDs possess imaging depth because they are small enough to enter the nucleus. Therefore, the Y-CDs can be applied in cobalamin detection and as a multifunctional fluorescent probe for cell imaging.