One-Pot Hydrothermal Synthesis of mSiO2-N-CDs with High Solid-State Photoluminescence as a Fluorescent Probe for Detecting Dopamine

An effective fluorescent probe (mSiO2-N-CDs) was prepared by embedding N-CDs into mesoporous silica via a simple one-pot hydrothermal reaction and applied to the detection of dopamine (DA). Mesoporous silica not only provided a skeleton to prevent the aggregation of N-CDs but also a medium for the centrifugal collection of N-CDs, avoiding the need for dialysis and freeze-drying. The formation process, phase composition, morphology, and luminescence properties of the composite were studied in detail. The synthesized mSiO2-N-CDs possessed spherical morphology, a smooth surface, and a diameter of approximately 150 nm. The fluorescence results indicated that mSiO2-N-CDs emitted intense blue color fluorescence at 465 nm under the optimal excitation of 370 nm. Because the mesoporous silica effectively inhibited the self-quenching caused by the aggregation of N-CDs, the quantum yield of solid mSiO2-N-CDs powder reached 32.5%. Furthermore, the emission intensity of the solid mSiO2-N-CDs remained constant for 28 days. The good sensitivity and selectivity of mSiO2-N-CDs for DA enabled the establishment of a rapid, simple, and sensitive DA detection method. The linear range was 0–50 µM and the limit of detection was calculated to be 107 nM. This method was used for the determination of DA in urine, with recovery rates ranging between 98% and 100.8%. In addition, the sensing mechanism was characterized by fluorescence lifetime decay and UV–VIS spectral analysis.


Introduction
As a key neurotransmitter in the human hypothalamus and pituitary gland, dopamine (DA) plays a vital role in regulating the nervous system, cardiovascular system, kidney, hormone secretion system, etc. DA imbalances can lead to psychiatric disorders (such as Parkinson's disease, epilepsy, depression, anxiety disorders, [1][2][3][4] etc.).Therefore, it is very important to detect DA and its precursors (such as tyrosine and phenylalanine) accurately and sensitively for the diagnosis of nervous system diseases.
Technologies for the rapid and accurate detection of DA and its precursors are becoming increasingly important in the medical field.To date, various analytical methods for detecting DA and its precursors-such as high-performance liquid chromatography (HPLC) analysis [5,6], electrochemical sensors [7][8][9][10][11][12][13][14][15][16] and fluorescence sensors [17]-have been developed.Although HPLC is accurate, efficient, and sensitive, the need for expensive instruments and specialized operators has limited its application in the field of rapid DA detection.In recent years, electrochemical technologies have gained widespread interest because of their good sensitivity and low cost.However, the selectivity of electrochemical sensors is easily interfered with by other biological molecules, which limits their application

Preparation of Mesoporous Silica Spheres (mSiO 2 )
The preparation of silica was slightly modified according to previous reports [43,47].Triethanolamine (30 mg) was mixed with water (15 mL) under stirring at 80 • C. Subsequently, cetyltrimethylammonium bromide (CTAB, 190 mg) and sodium salicylate (NaSal, 84 mg) were added to the solution.After stirring for 1 h, tetraethyl orthosilicate (TEOS, 2 mL) was injected drop by drop into the above solution.After continuous stirring for 2 h, the reaction solution was separated by centrifugation for 20 min (10,000 rpm) to yield a white precipitate.Eventually, the resulting precipitate was ultrasonically dispersed in an HCl/CH 3 OH solution (1:10 v/v, 100 mL), and the organic template was removed by continuous reflux at 80 • C for 6 h.The template removal process was repeated once.The mixture was centrifuged (10,000 rpm) for 20 min and the precipitate was cleansed repeatedly with deionized water and ethanol, then dried at 60 • C in an oven.The obtained mSiO 2 was characterized before further use.

Preparation of mSiO 2 -N-CDs
Citric acid (CA, 2.1 g) and urea (UR, 1.8 g) were added to deionized water (50 mL) and the mixture was stirred until a transparent solution formed.Then, the obtained mSiO 2 (358 mg) was ultrasonically dispersed in this solution and the mixture was transferred to a hydrothermal reactor and kept at 160 • C in an oven for 12 h.After cooling, the mixture was centrifuged (10,000 rpm) for 20 min and the precipitate was cleansed repeatedly with deionized water and ethanol.It was then placed in a vacuum drying chamber for 24 h at 40 • C to generate mSiO 2 -N-CDs.The obtained product was stored at 4 • C for further applications.

Preparation of N-CDs
N-CDs were prepared by the one-pot hydrothermal method: CA (2.1 g) and UR (1.8 g) were added to deionized water (50 mL), then transferred into a 100 mL autoclave and maintained at 160 • C for 12 h.After cooling down, the mixture was separated by centrifugation (10,000 rpm, 10 min).The supernatant was collected and purified through a dialysis process (10,000 MWCO, 48 h), then further freeze dried to obtain solid N-CDs.

DA Detection Method
The detection of DA was analyzed in phosphate buffer (PBS, pH = 7.4).The emission spectra of the mixture containing mSiO 2 -N-CDs (0.25 mg/mL) and various concentrations of DA (0-50 µM) were tested under the optimal excitation of 370 nm.All samples were tested in triplicate.
To investigate the selectivity of mSiO 2 -N-CDs for DA detection, fluorescence intensity tests were conducted with the addition of interfering ions such as Ca 2+ , Zn 2+ , Na + , K + , Mg 2+ , and interfering biomolecules such as CA, UA, cysteine, glucose, galactose, fructose, epinephrine, and norepinephrine.The above interfering agents and DA in the same concentration (50 µM) were added to the mSiO 2 -N-CDs solution separately, and their emission intensities were compared with that of the solution without interfering substances.All experiments were performed in triplicate.

Real Sample Detection
In order to verify the feasibility of this method, the urine of healthy volunteers was used as the analysis sample and standard addition experiments were conducted.The urine was centrifuged at 10,000 rpm for 10 min to remove the sediments, and the supernatant was diluted 10-fold with phosphate buffer solution (PBS, pH = 7.4).The urine samples were spiked with DA (0.5, 1, 5, 40 µM), and DA concentration was determined by the proposed method.All experiments were conducted in triplicate.

Formation Process, Morphology and Composition of mSiO 2 -N-CDs
A novel fluorescent probe for DA detection (mSiO 2 -N-CDs) was designed and synthesized using mesoporous SiO 2 as the framework and N-CDs as the fluorescent unit (Scheme 1).First, silica spheres were prepared using TEOS as the silica source, with CTAB and NaSal as the porous templates.After the templates were removed by HCl/CH 3 OH reflux, mesoporous silica spheres were obtained.After hydrothermal treatment at 160 • C for 12 h, UR and CA were carbonized together and N-CDs formed in the mesoporous silica pores.
urine was centrifuged at 10,000 rpm for 10 min to remove the sediments, and the su natant was diluted 10-fold with phosphate buffer solution (PBS, pH = 7.4).The urine s ples were spiked with DA (0.5, 1, 5, 40 μM), and DA concentration was determined by proposed method.All experiments were conducted in triplicate.

Formation Process, Morphology and Composition of mSiO2-N-CDs
A novel fluorescent probe for DA detection (mSiO2-N-CDs) was designed and s thesized using mesoporous SiO2 as the framework and N-CDs as the fluorescent u (Scheme 1).First, silica spheres were prepared using TEOS as the silica source, with CT and NaSal as the porous templates.After the templates were removed by HCl/CH3 reflux, mesoporous silica spheres were obtained.After hydrothermal treatment at 160 for 12 h, UR and CA were carbonized together and N-CDs formed in the mesopor silica pores.1C, it can be seen that the obtai mSiO2-N-CDs possess a regular spherical appearance with a diameter of approxima 150 nm and good dispersion.By comparing the light and dark area of the mSiO2-N-C it could be determined that the nanospheres are not solid structures and contain m stripe-like channels, which are suitable for embedding N-CDs.As shown in the red circ some dark spots appeared in the pore channels, with very similar morphology to tha the spherical N-CDs.The successful embedding of N-CDs in mSiO2's pore channel further confirmed by X-ray photoelectron spectroscopy (XPS), ultraviolet absorption sp troscopy, FTIR spectroscopy, and fluorescence spectroscopy.
The XPS spectrum is presented in   1C), and X-ray photoelectron spectroscopy (XPS) of mSiO 2 -N-CDs (Figure 1D). Figure 1A,B indicates that N-CDs are composed of a large number of black spherical dots with a diameter of approximately 2-4 nm.From Figure 1C, it can be seen that the obtained mSiO 2 -N-CDs possess a regular spherical appearance with a diameter of approximately 150 nm and good dispersion.By comparing the light and dark area of the mSiO 2 -N-CDs, it could be determined that the nanospheres are not solid structures and contain many stripe-like channels, which are suitable for embedding N-CDs.As shown in the red circles, some dark spots appeared in the pore channels, with very similar morphology to that of the spherical N-CDs.The successful embedding of N-CDs in mSiO 2 's pore channels is further confirmed by X-ray photoelectron spectroscopy (XPS), ultraviolet absorption spectroscopy, FTIR spectroscopy, and fluorescence spectroscopy.
The XPS spectrum is presented in Figure 2A shows the UV-VIS spectra of the obtained samples, including mSiO2 curve), NCDs (blue curve), and mSiO2-N-CDs (red curve).There is no obvious abso peak in the black curve, while the absorption peak at 350 nm that appear in the blue can be attributed to the π→π* transition of the N-CDs [48].In the red curve, the abso peaks are consistent with pure N-CDs, thus further confirming that N-CDs were e ded successfully in the channels of the mesoporous silica.Figure 2B shows the FTIR tra of the prepared samples.In the black curve (mSiO2), the absorption band nea cm -1 is determined as the tensile vibration of -OH, the peak at 1625 cm -1 is caused bending vibration of H-O-H, the two strong peaks at 1080 and 796 cm -1 are due asymmetric and symmetric stretching of Si-O-Si, respectively, and the peak at 96 corresponds to the symmetric stretch of Si-OH.Thus, the black curve is consisten pure mesoporous silica [41].The blue curve represents N-CDs, and in the red (mSiO2-N-CDs), as well as the characteristic peaks of mesoporous silica, an abso band corresponding to -NH2 appears near 3210 cm -1 , a strong characteristic peak o shows around 1679 cm -1 , and the deformation vibration peak of -OH in -COOH sh 1386 cm -1 .These results indicate that N-CDs with abundant amino and carboxyl g were successfully embedded in the mesoporous silica.Figure 2A shows the UV-VIS spectra of the obtained samples, including mSiO 2 (black curve), NCDs (blue curve), and mSiO 2 -N-CDs (red curve).There is no obvious absorption peak in the black curve, while the absorption peak at 350 nm that appear in the blue curve can be attributed to the π→π* transition of the N-CDs [48].In the red curve, the absorption peaks are consistent with pure N-CDs, thus further confirming that N-CDs were embedded successfully in the channels of the mesoporous silica.Figure 2B shows the FTIR spectra of the prepared samples.In the black curve (mSiO 2 ), the absorption band near 3420 cm −1 is determined as the tensile vibration of -OH, the peak at 1625 cm −1 is caused by the bending vibration of H-O-H, the two strong peaks at 1080 and 796 cm −1 are due to the asymmetric and symmetric stretching of Si-O-Si, respectively, and the peak at 965 cm −1 corresponds to the symmetric stretch of Si-OH.Thus, the black curve is consistent with pure mesoporous silica [41].The blue curve represents N-CDs, and in the red curve (mSiO 2 -N-CDs), as well as the characteristic peaks of mesoporous silica, an absorption band corresponding to -NH 2 appears near 3210 cm −1 , a strong characteristic peak of C=O shows around 1679 cm −1 , and the deformation vibration peak of -OH in -COOH shows at 1386 cm −1 .These results indicate that N-CDs with abundant amino and carboxyl groups were successfully embedded in the mesoporous silica.

Photoluminescent Properties of mSiO2-N-CDs
The photoluminescent (PL) performance of the obtained mSiO2-N-CDs dispersed water (0.25 mg•mL -1 ) were characterized in detail, as shown in Figure 3A,B.The PL spect show the typical excitation and emission peaks of N-CDs, consisting of a wide excitatio peak and a broad emission peak.The best excitation peak is near 370 nm and the highe emission peak is around 465 nm.As can be seen from Figure 3B, the emission peak d not move with the change of the excitation wavelength (300-380 nm); however, the emi sion intensity varied with the excitation wavelength and the optimal excitation wav length is 370 nm.Unlike many previous reports on CDs [48,49], mSiO2-N-CDs exhibite a stable emission peak showing no shift with the adjustment of excitation wavelengt indicating that the size and surface of the prepared mSiO2 particles are uniform.

Photoluminescent Properties of mSiO 2 -N-CDs
The photoluminescent (PL) performance of the obtained mSiO 2 -N-CDs dispersed in water (0.25 mg•mL −1 ) were characterized in detail, as shown in Figure 3A,B.The PL spectra show the typical excitation and emission peaks of N-CDs, consisting of a wide excitation peak and a broad emission peak.The best excitation peak is near 370 nm and the highest emission peak is around 465 nm.As can be seen from Figure 3B, the emission peak did not move with the change of the excitation wavelength (300-380 nm); however, the emission intensity varied with the excitation wavelength and the optimal excitation wavelength is 370 nm.Unlike many previous reports on CDs [48,49], mSiO 2 -N-CDs exhibited a stable emission peak showing no shift with the adjustment of excitation wavelength, indicating that the size and surface of the prepared mSiO 2 particles are uniform.

Photoluminescent Properties of mSiO2-N-CDs
The photoluminescent (PL) performance of the obtained mSiO2-N-CDs dispersed in water (0.25 mg•mL -1 ) were characterized in detail, as shown in Figure 3A,B.The PL spectra show the typical excitation and emission peaks of N-CDs, consisting of a wide excitation peak and a broad emission peak.The best excitation peak is near 370 nm and the highest emission peak is around 465 nm.As can be seen from Figure 3B, the emission peak did not move with the change of the excitation wavelength (300-380 nm); however, the emission intensity varied with the excitation wavelength and the optimal excitation wavelength is 370 nm.Unlike many previous reports on CDs [48,49], mSiO2-N-CDs exhibited a stable emission peak showing no shift with the adjustment of excitation wavelength, indicating that the size and surface of the prepared mSiO2 particles are uniform.As shown in Figure 3C,D, the solid mSiO 2 -N-CDs produce bright blue fluorescence with a quantum yield (QY) of 32.5% under 365 nm UV irradiation.For further investigation of the fluorescence stability of mSiO 2 -N-CDs, the effect of storage time on the emission intensity was studied.From Figure 3C, it can be seen that the emission intensity of solid mSiO 2 -N-CDs remained almost constant for 28 days (the relative standard deviation was 1.07%).The comparative photos and emission spectra of solid N-CDs and mSiO 2 -N-CDs are presented in Figure S1: mSiO 2 -N-CDs powder emitted strong blue fluorescence under a 365 nm ultraviolet lamp (inset photograph) and solid N-CDs showed much lower fluorescence than that of mSiO 2 -N-CDs due to the ACQ.The stability emission may have been due to the interaction of the amino (-NH 2 ) and carboxyl (-COOH) groups on the surface of the N-CDs with the hydroxyl (-OH) groups of the silica pore channels.This interaction effectively avoided ACQ of the N-CDs.Thus, the prepared mSiO 2 -N-CDs possessed stable emission intensity and would be suitable for practical applications.

DA Detection Performance
The high selectivity of mSiO 2 -N-CDs for the detection of DA was demonstrated by fluorescence intensity tests.Various potentially interfering molecules (including CA, UA, cysteine, glucose, galactose, fructose, epinephrine, and norepinephrine) and ions (Ca 2+ , Zn 2+ , Na + , K + , and Mg 2+ ) were introduced into the mSiO 2 -N-CDs solution (0.25 mg•mL −1 ).The fluorescence intensity of these solutions was then compared with the fluorescence intensity of equivalent solutions with DA added.Here, the concentrations of DA and the interfering analytes were both 50 µM.The results are shown in Figure 4A: Except for epinephrine, norepinephrine, and DA, the fluorescence intensity of mSiO 2 -N-CDs solution did not change significantly.The structures of epinephrine and norepinephrine are similar to that of DA, which resulted in fluorescence quenching.Nevertheless, the fluorescence quenching effect was weaker than that of DA.Additionally, the concentration of epinephrine (<300 pg•mL −1 ) and norepinephrine (<400 pg•mL −1 ) in blood serum is much lower than that of DA [49,50].Therefore, these data indicate the specificity and selectivity of the mSiO 2 -N-CDs nanoprobe for DA detection.As shown in Figure 3C,D, the solid mSiO2-N-CDs produce bright blue fluorescence with a quantum yield (QY) of 32.5% under 365 nm UV irradiation.For further investigation of the fluorescence stability of mSiO2-N-CDs, the effect of storage time on the emission intensity was studied.From Figure 3C, it can be seen that the emission intensity of solid mSiO2-N-CDs remained almost constant for 28 days (the relative standard deviation was 1.07%).The comparative photos and emission spectra of solid N-CDs and mSiO2-N-CDs are presented in Figure S1: mSiO2-N-CDs powder emitted strong blue fluorescence under a 365 nm ultraviolet lamp (inset photograph) and solid N-CDs showed much lower fluorescence than that of mSiO2-N-CDs due to the ACQ.The stability emission may have been due to the interaction of the amino (-NH2) and carboxyl (-COOH) groups on the surface of the N-CDs with the hydroxyl (-OH) groups of the silica pore channels.This interaction effectively avoided ACQ of the N-CDs.Thus, the prepared mSiO2-N-CDs possessed stable emission intensity and would be suitable for practical applications.

DA Detection Performance
The high selectivity of mSiO2-N-CDs for the detection of DA was demonstrated by fluorescence intensity tests.Various potentially interfering molecules (including CA, UA, cysteine, glucose, galactose, fructose, epinephrine, and norepinephrine) and ions (Ca 2+ , Zn 2+ , Na + , K + , and Mg 2+ ) were introduced into the mSiO2-N-CDs solution (0.25 mg•mL -1 ).The fluorescence intensity of these solutions was then compared with the fluorescence intensity of equivalent solutions with DA added.Here, the concentrations of DA and the interfering analytes were both 50 μM.The results are shown in Figure 4A: Except for epinephrine, norepinephrine, and DA, the fluorescence intensity of mSiO2-N-CDs solution did not change significantly.The structures of epinephrine and norepinephrine are similar to that of DA, which resulted in fluorescence quenching.Nevertheless, the fluorescence quenching effect was weaker than that of DA.Additionally, the concentration of epinephrine (<300 pg•mL -1 ) and norepinephrine (<400 pg•mL −1 ) in blood serum is much lower than that of DA [49,50].Therefore, these data indicate the specificity and selectivity of the mSiO2-N-CDs nanoprobe for DA detection.After verifying the high selectivity and specificity of the probe for DA detection, the experimental conditions, including environmental pH value and reaction time, were optimized.Figure 4B shows the change in the emission intensity of mSiO2-N-CDs with DA (DA concentration 50 μM) at different pH values (pH 4-9).The results showed that the fluorescence quenching rate was optimal at pH 7.4.
Consequently, pH 7.4 and room temperature were chosen as the conditions for the detection of DA and the effect of reaction duration on the detection performance was explored.Figure 4C shows that the emission intensity of the original mSiO2-N-CDs solution was relatively high, then decreased significantly within 5 min after the addition of DA.After verifying the high selectivity and specificity of the probe for DA detection, the experimental conditions, including environmental pH value and reaction time, were optimized.Figure 4B shows the change in the emission intensity of mSiO 2 -N-CDs with DA (DA concentration 50 µM) at different pH values (pH 4-9).The results showed that the fluorescence quenching rate was optimal at pH 7.4.
Consequently, pH 7.4 and room temperature were chosen as the conditions for the detection of DA and the effect of reaction duration on the detection performance was explored.Figure 4C shows that the emission intensity of the original mSiO 2 -N-CDs solution was relatively high, then decreased significantly within 5 min after the addition of DA.This meant that the interaction between mSiO 2 -N-CDs and DA was relatively fast.Furthermore, the emission intensity remained stable when the incubation time was extended to 120 min.In subsequent experiments, a reaction time of 5 min was selected.Therefore, a rapid and simple method for detecting DA was established.
Under the optimal conditions, the performance of the mSiO 2 -N-CDs probe for DA analysis was determined by adding DA of different concentrations.The corresponding fluorescence spectrum is shown in Figure 5A.As the DA concentration increased, the blue fluorescence of the solution gradually weakened.From Figure 5B, a good linear relationship between F/F 0 and the concentration of DA was obtained: F/F 0 = 0.03743c + 0.99974 (R 2 = 0.99074, with detection limit of 107 nM and linear range of 0-50 µM).The detection limit was calculated based on the equation of D = 3σ/K, in which σ is the standard deviation and K is the slope of the calibration line.The inset in Figure 5B shows photographs of the mSiO 2 -N-CDs solution with (50 µM) and without DA under a 365 nm ultraviolet lamp.Moreover, the repeatability of DA detection by this probe was studied.The mSiO 2 -N-CDs solutions (0.25 mg/mL) were prepared three times to measure a DA solution of 10 µM, and the relative standard deviation (RSD) was 1.55%, indicating that the probe had good repeatability.Table 1 lists the detection range and detection limit of several DA detection sensors, and it can be seen that the obtained mSiO 2 -N-CDs had considerable sensitivity compared with the previously reported sensors.
This meant that the interaction between mSiO2-N-CDs and DA was relatively fast.Furthermore, the emission intensity remained stable when the incubation time was extended to 120 min.In subsequent experiments, a reaction time of 5 min was selected.Therefore, a rapid and simple method for detecting DA was established.
Under the optimal conditions, the performance of the mSiO2-N-CDs probe for DA analysis was determined by adding DA of different concentrations.The corresponding fluorescence spectrum is shown in Figure 5A.As the DA concentration increased, the blue fluorescence of the solution gradually weakened.From Figure 5B, a good linear relationship between F/F0 and the concentration of DA was obtained: F/F0 = 0.03743c + 0.99974 (R 2 = 0.99074, with detection limit of 107 nM and linear range of 0-50 μM).The detection limit was calculated based on the equation of D = 3σ/K, in which σ is the standard deviation and K is the slope of the calibration line.The inset in Figure 5B shows photographs of the mSiO2-N-CDs solution with (50 μM) and without DA under a 365 nm ultraviolet lamp.Moreover, the repeatability of DA detection by this probe was studied.The mSiO2-N-CDs solutions (0.25 mg/mL) were prepared three times to measure a DA solution of 10 μM, and the relative standard deviation (RSD) was 1.55%, indicating that the probe had good repeatability.Table 1 lists the detection range and detection limit of several DA detection sensors, and it can be seen that the obtained mSiO2-N-CDs had considerable sensitivity compared with the previously reported sensors.

Real Sample Detection
The urine DA content of healthy volunteers was detected by adding standard DA, as shown in Table 2.The experimental detection results indicate that the recovery rates of different samples range from 98.0% to 100.8%.The relative standard deviation (RSD) was between 0.71% and 2.0%.These satisfactory results show that this method could be feasible for the analysis of actual samples.

Real Sample Detection
The urine DA content of healthy volunteers was detected by adding standard DA, as shown in Table 2.The experimental detection results indicate that the recovery rates of different samples range from 98.0% to 100.8%.The relative standard deviation (RSD) was between 0.71% and 2.0%.These satisfactory results show that this method could be feasible for the analysis of actual samples.

Possible Mechanism
Usually, the fluorescence quenching process includes two types: dynamic quenching and static quenching [53].The possible quenching mechanism of mSiO 2 -N-CDs on DA was analyzed using fluorescence lifetime decay analysis and UV-VIS spectra.Fluorescence lifetime decay analysis was performed on mSiO 2 -N-CDs with and without DA.As shown in Figure 6A, the average lifetime of the mSiO 2 -N-CDs sensor (τ 0 ) was 6.43 ns.After the addition of DA (10 µM), the lifetime (τ 1 ) was 6.45 ns; that is, the ratio value of τ 0 /τ 1 was about equal to 1.According to previous reports [54,55], if τ 0 /τ 1 = 1, static quenching occurs.This indicates that the fluorescence quenching mechanism may be static quenching.

Possible Mechanism
Usually, the fluorescence quenching process includes two types: dynamic quenching and static quenching [53].The possible quenching mechanism of mSiO2-N-CDs on DA was analyzed using fluorescence lifetime decay analysis and UV-VIS spectra.Fluorescence lifetime decay analysis was performed on mSiO2-N-CDs with and without DA.As shown in Figure 6A, the average lifetime of the mSiO2-N-CDs sensor (τ0) was 6.43 ns.After the addition of DA (10 μM), the lifetime (τ1) was 6.45 ns; that is, the ratio value of τ0/τ1 was about equal to 1.According to previous reports [54,55], if τ0/τ1 = 1, static quenching occurs.This indicates that the fluorescence quenching mechanism may be static quenching The quenching rate constant Kq can be calculated through the Stern-Volmer equation (F0/F = 1 + KSV[Q] = 1 + Kqτ0[Q]).Here, F0 represents the emission intensity at 465 nm without DA, F represents the emission intensity with the addition of DA, KSV is the Stern-Volmer quenching constant, τ0 is the lifespan without DA, and [Q] is the concentration of DA.From the calculation, it was concluded that the value of kq was 3.8 × 10 12 M -1 •s -1 , much greater than 2.0 × 10 10 M -1 •s -1 .This result further implied the existence of static quenching [55].
Figure 6B presents the UV-VIS spectra of mSiO2-N-CDs, DA, and mSiO2-N-CDs with DA.The absorption peak of mSiO2-N-CDs, appearing around 350 nm, was assigned to the n-π* electron transition of the C=O and C-N groups of the CDs [48].The typical absorption of π-orbitals in aromatic rings resulted in a sharp peak at 280 nm for DA [48].When DA was added into the mSiO2-N-CDs solution, the absorption band around 350 nm was significantly weaker and a new wide absorption band (450-600 nm) was generated, implying The quenching rate constant K q can be calculated through the Stern-Volmer equation (F 0 /F = 1 + K SV [Q] = 1 + K q τ 0 [Q]).Here, F 0 represents the emission intensity at 465 nm without DA, F represents the emission intensity with the addition of DA, K SV is the Stern-Volmer quenching constant, τ 0 is the lifespan without DA, and [Q] is the concentration of DA.From the calculation, it was concluded that the value of k q was 3.8 × 10 12 M −1 •s −1 , much greater than 2.0 × 10 10 M −1 •s −1 .This result further implied the existence of static quenching [55].
Figure 6B presents the UV-VIS spectra of mSiO 2 -N-CDs, DA, and mSiO 2 -N-CDs with DA.The absorption peak of mSiO 2 -N-CDs, appearing around 350 nm, was assigned to the n-π* electron transition of the C=O and C-N groups of the CDs [48].The typical absorption of π-orbitals in aromatic rings resulted in a sharp peak at 280 nm for DA [48].When DA was added into the mSiO 2 -N-CDs solution, the absorption band around 350 nm was significantly weaker and a new wide absorption band (450-600 nm) was generated, implying the formation of a new complex (mSiO 2 -N-CDs-DA) between mSiO 2 -N-CDs and DA [56,57].This might be attributed to the forces between mSiO 2 -N-CDs and DA, such as the hydrogen bonding and the π-π conjugation between them, due to the abundant amino (-NH 2 ) and carboxyl (-COOH) groups on the surface of mSiO 2 -N-CDs, and the amino (-NH 2 ) and hydroxyl (-OH) groups on the DA molecule.This result supports the static quenching mechanism (as depicted in Figure 6C) we proposed.

Scheme 1 .
Scheme 1. Synthesis diagram of mSiO2-N-CDs and their application in DA detection.

Figure 1
Figure 1 presents the TEM view of the N-CDs (Figure 1A,B), mSiO2-N-CDs (Fig 1C), and X-ray photoelectron spectroscopy (XPS) of mSiO2-N-CDs (Figure 1D).Fig 1A,B indicates that N-CDs are composed of a large number of black spherical dots wi diameter of approximately 2-4 nm.From Figure1C, it can be seen that the obtai mSiO2-N-CDs possess a regular spherical appearance with a diameter of approxima 150 nm and good dispersion.By comparing the light and dark area of the mSiO2-N-C it could be determined that the nanospheres are not solid structures and contain m stripe-like channels, which are suitable for embedding N-CDs.As shown in the red circ some dark spots appeared in the pore channels, with very similar morphology to tha the spherical N-CDs.The successful embedding of N-CDs in mSiO2's pore channel further confirmed by X-ray photoelectron spectroscopy (XPS), ultraviolet absorption sp troscopy, FTIR spectroscopy, and fluorescence spectroscopy.The XPS spectrum is presented in Figure 1D.It contains four peaks at 104.3, 15 282.5, 399.8, and 531.7 eV, corresponding to Si 2p, Si 1s, C1s, N1s, and O1s, respectiv This indicates that the probe is composed of C, N, O, and Si, further proving the succes embedding of N-CDs in mSiO2.The high-resolution XPS peaks of C1s, N1s and O1s all presented in Figure S2A-C.The C1s' spectrum is divided into three peaks at C-C/C (281.8 eV), C-N/C-O (283.4 eV), and C=N/C=O (288.3 eV), as shown in Figure S2A.Fr Figure S2B, it can be seen that the N1s are separated into C-N-C (398.67 eV), O=C-N (39 eV), and N-H (401.67 eV).The presence of O=C (530.48 eV) and C-O-C/C-OH (532.62 can be confirmed by Figure S2C.

Scheme 1 .
Figure 1 presents the TEM view of the N-CDs (Figure 1A,B), mSiO2-N-CDs (Fig 1C), and X-ray photoelectron spectroscopy (XPS) of mSiO2-N-CDs (Figure 1D).Fig 1A,B indicates that N-CDs are composed of a large number of black spherical dots wi diameter of approximately 2-4 nm.From Figure1C, it can be seen that the obtai mSiO2-N-CDs possess a regular spherical appearance with a diameter of approxima 150 nm and good dispersion.By comparing the light and dark area of the mSiO2-N-C it could be determined that the nanospheres are not solid structures and contain m stripe-like channels, which are suitable for embedding N-CDs.As shown in the red circ some dark spots appeared in the pore channels, with very similar morphology to tha the spherical N-CDs.The successful embedding of N-CDs in mSiO2's pore channel further confirmed by X-ray photoelectron spectroscopy (XPS), ultraviolet absorption sp troscopy, FTIR spectroscopy, and fluorescence spectroscopy.The XPS spectrum is presented in Figure 1D.It contains four peaks at 104.3, 15 282.5, 399.8, and 531.7 eV, corresponding to Si 2p, Si 1s, C1s, N1s, and O1s, respectiv This indicates that the probe is composed of C, N, O, and Si, further proving the succes embedding of N-CDs in mSiO2.The high-resolution XPS peaks of C1s, N1s and O1s all presented in Figure S2A-C.The C1s' spectrum is divided into three peaks at C-C/C (281.8 eV), C-N/C-O (283.4 eV), and C=N/C=O (288.3 eV), as shown in Figure S2A.Fr Figure S2B, it can be seen that the N1s are separated into C-N-C (398.67 eV), O=C-N (39 eV), and N-H (401.67 eV).The presence of O=C (530.48 eV) and C-O-C/C-OH (532.62 can be confirmed by Figure S2C.Scheme 1. Synthesis diagram of mSiO 2 -N-CDs and their application in DA detection.

Figure 1
Figure1presents the TEM view of the N-CDs (Figure1A,B), mSiO 2 -N-CDs (Figure1C), and X-ray photoelectron spectroscopy (XPS) of mSiO 2 -N-CDs (Figure1D).Figure1A,B indicates that N-CDs are composed of a large number of black spherical dots with a diameter of approximately 2-4 nm.From Figure1C, it can be seen that the obtained mSiO 2 -N-CDs possess a regular spherical appearance with a diameter of approximately 150 nm and good dispersion.By comparing the light and dark area of the mSiO 2 -N-CDs, it could be determined that the nanospheres are not solid structures and contain many stripe-like channels, which are suitable for embedding N-CDs.As shown in the red circles, some dark spots appeared in the pore channels, with very similar morphology to that of the spherical N-CDs.The successful embedding of N-CDs in mSiO 2 's pore channels is further confirmed by X-ray photoelectron spectroscopy (XPS), ultraviolet absorption spectroscopy, FTIR spectroscopy, and fluorescence spectroscopy.The XPS spectrum is presented in Figure 1D.It contains four peaks at 104.3, 154.7, 282.5, 399.8, and 531.7 eV, corresponding to Si 2p, Si 1s, C1s, N1s, and O1s, respectively.This indicates that the probe is composed of C, N, O, and Si, further proving the successful embedding of N-CDs in mSiO 2 .The high-resolution XPS peaks of C1s, N1s and O1s are all presented in Figure S2A-C.The C1s' spectrum is divided into three peaks at C-C/C=C (281.8 eV), C-N/C-O (283.4 eV), and C=N/C=O (288.3 eV), as shown in Figure S2A.From Figure S2B, it can be seen that the N1s are separated into C-N-C (398.67 eV), O=C-N (399.29 eV), and N-H (401.67 eV).The presence of O=C (530.48 eV) and C-O-C/C-OH (532.62 eV) can be confirmed by Figure S2C.

Figure 1 .
Figure1presents the TEM view of the N-CDs (Figure1A,B), mSiO 2 -N-CDs (Figure1C), and X-ray photoelectron spectroscopy (XPS) of mSiO 2 -N-CDs (Figure1D).Figure1A,B indicates that N-CDs are composed of a large number of black spherical dots with a diameter of approximately 2-4 nm.From Figure1C, it can be seen that the obtained mSiO 2 -N-CDs possess a regular spherical appearance with a diameter of approximately 150 nm and good dispersion.By comparing the light and dark area of the mSiO 2 -N-CDs, it could be determined that the nanospheres are not solid structures and contain many stripe-like channels, which are suitable for embedding N-CDs.As shown in the red circles, some dark spots appeared in the pore channels, with very similar morphology to that of the spherical N-CDs.The successful embedding of N-CDs in mSiO 2 's pore channels is further confirmed by X-ray photoelectron spectroscopy (XPS), ultraviolet absorption spectroscopy, FTIR spectroscopy, and fluorescence spectroscopy.The XPS spectrum is presented in Figure 1D.It contains four peaks at 104.3, 154.7, 282.5, 399.8, and 531.7 eV, corresponding to Si 2p, Si 1s, C1s, N1s, and O1s, respectively.This indicates that the probe is composed of C, N, O, and Si, further proving the successful embedding of N-CDs in mSiO 2 .The high-resolution XPS peaks of C1s, N1s and O1s are all presented in Figure S2A-C.The C1s' spectrum is divided into three peaks at C-C/C=C (281.8 eV), C-N/C-O (283.4 eV), and C=N/C=O (288.3 eV), as shown in Figure S2A.From Figure S2B, it can be seen that the N1s are separated into C-N-C (398.67 eV), O=C-N (399.29 eV), and N-H (401.67 eV).The presence of O=C (530.48 eV) and C-O-C/C-OH (532.62 eV) can be confirmed by Figure S2C.

Figure 2 .
Figure 2. (A) UV-VIS spectra of the obtained samples; (B) FTIR spectra of the obtained samples.

Figure 3 .
Figure 3. (A) The PL spectra of mSiO2-N-CDs (inset: photographs of mSiO2-N-CDs dispersed water); (B) the emission spectra of mSiO2-N-CDs under different excitation; (C) the emission spect of the solid mSiO2-N-CDs over time, up to 28 days; (D) photographs of the corresponding mSiO N-CDs powder over time, up to 28 days under 365 nm UV irradiation.

Figure 2 .
Figure 2. (A) UV-VIS spectra of the obtained samples; (B) FTIR spectra of the obtained samples.

Figure 3 .
Figure 3. (A) The PL spectra of mSiO2-N-CDs (inset: photographs of mSiO2-N-CDs dispersed in water); (B) the emission spectra of mSiO2-N-CDs under different excitation; (C) the emission spectra of the solid mSiO2-N-CDs over time, up to 28 days; (D) photographs of the corresponding mSiO2-N-CDs powder over time, up to 28 days under 365 nm UV irradiation.

Figure 3 .
Figure 3. (A) The PL spectra of mSiO 2 -N-CDs (inset: photographs of mSiO 2 -N-CDs dispersed in water); (B) the emission spectra of mSiO 2 -N-CDs under different excitation; (C) the emission spectra of the solid mSiO 2 -N-CDs over time, up to 28 days; (D) photographs of the corresponding mSiO 2 -N-CDs powder over time, up to 28 days under 365 nm UV irradiation.

Figure 4 .
Figure 4. (A) Selectivity of mSiO2-N-CDs towards various interfering analytes (50 μM); (B) the fluorescence quenching efficiency of DA on mSiO2-N-CDs at different pH values; (C) the effect of culture time on the fluorescence quenching.

Figure 4 .
Figure 4. (A) Selectivity of mSiO 2 -N-CDs towards various interfering analytes (50 µM); (B) the fluorescence quenching efficiency of DA on mSiO 2 -N-CDs at different pH values; (C) the effect of culture time on the fluorescence quenching.

Figure 5 .
Figure 5. (A) Fluorescence emission spectra of mSiO2-N-CDs probe in PBS buffer solution (pH = 7.4) with different amounts of DA under 370 nm excitation; (B) linear relationship between fluorescence intensity ratio (F0/F) and concentration of DA under 370 nm excitation (Inset: the photographs of mSiO2-N-CDs solution with DA (50 μM) and without DA under 365 nm ultraviolet lamp).

Figure 5 .
Figure 5. (A) Fluorescence emission spectra of mSiO 2 -N-CDs probe in PBS buffer solution (pH = 7.4) with different amounts of DA under 370 nm excitation; (B) linear relationship between fluorescence intensity ratio (F 0 /F) and concentration of DA under 370 nm excitation (Inset: the photographs of mSiO 2 -N-CDs solution with DA (50 µM) and without DA under 365 nm ultraviolet lamp).

Figure 6 .
Figure 6.(A) Representative lifetime decay profiles of mSiO2-N-CDs in the absence (black line) and presence (blue line) of DA; (B) UV-visible absorption spectra of mSiO2-N-CDs, DA, and mSiO2-N-CDs with DA; (C) the possible quenching mechanism diagram.

Figure 6 .
Figure 6.(A) Representative lifetime decay profiles of mSiO 2 -N-CDs in the absence (black line) and presence (blue line) of DA; (B) UV-visible absorption spectra of mSiO 2 -N-CDs, DA, and mSiO 2 -N-CDs with DA; (C) the possible quenching mechanism diagram.

Table 1 .
Comparison of fluorescence sensors for detection of DA.

Table 1 .
Comparison of fluorescence sensors for detection of DA.

Table 2 .
Detection of DA in human urine sample.

Table 2 .
Detection of DA in human urine sample.