Simultaneous Detection of Adenosine Triphosphate and Glucose Based on the Cu-Fenton Reaction

Both adenosine triphosphate (ATP) and glucose are important to human health, and their abnormal levels are closely related to angiocardiopathy and hypoglycaemia. Therefore, the simultaneous determination of ATP and glucose with a single test mode is highly desirable for disease diagnostics and early recognition. Herein, a new fluorescence on/off switch sensing platform is developed by carbon nanodots (CNDs) to detect ATP and glucose simultaneously. The fluorescence of CNDs can be quenched by Cu2+ and hydrogen peroxide (H2O2), due to the formation of hydroxyl radicals (·OH) produced in the Cu-Fenton reaction. Based on the high affinity of Cu2+ with ATP, the fluorescence of CNDs will recover effectively after adding ATP. Additionally, glucose can be efficiently catalyzed by glucose oxidase (GOx) to generate H2O2, so the platform can also be utilized to analyze glucose. Under optimum conditions, this sensing platform displays excellent sensitivity and the linear ranges are from 0.1 to 7 μM for ATP with a limit of detection (LOD) of 30.2 nM, and from 0.1 to 7 mM for glucose with a LOD 39.8 μM, respectively. Benefiting from the high sensitivity and selectivity, this sensing platform is successfully applied for simultaneous detection of ATP and glucose in human serum samples with satisfactory recoveries.


Introduction
As is well known, both adenosine triphosphate (ATP) and glucose are sources of energy and important for energy metabolism. ATP supplies energy for metabolic processes and glucose is a metabolic intermediate in biological systems. Furthermore, the ATP and glucose levels in blood are indicators of human health conditions. For example, the excessive production of ATP by creatine kinase is the pathogenesis of angiocardiopathy [1] and the aberrant concentration of ATP will cause energy disturbance, resulting in hypoglycaemia, ischemia, and Parkinsons' disease [2]. On the other hand, the high glucose levels will produce diabetes mellitus, which damages the eyes, kidneys, feet, and heart, and also causes a series of angiocardiopathy [3], while hypoglycaemia is usually due to the low concentration of glucose. This means that both the levels of ATP and glucose are related to angiocardiopathy and hypoglycaemia by virtue of the coexistence of ATP and glucose in biological samples [4][5][6][7], thus, the simultaneous determination of ATP and glucose with a single test mode is highly desirable for disease diagnostics and early recognition.
A comparison with the traditional methods, such as chromatography and electrochemical techniques [8,9], fluorescence analysis receives a great deal of attention due to its simplicity, rapid response,

Instruments
The fluorescence spectra were recorded with a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan). Transmission electron microscopy (TEM) images were recorded on a JEM-2100PLUS (JEOL, Japan). The Fourier transform infrared (FT-IR) spectra of the samples were analyzed using Thermo Nicolet Nexus 470 FT-IR ESP spectrometer (Nicolet, WI, USA). The ultraviolet-visible (UV-vis) absorption spectra were obtained on a Cary 300 Bio UV-vis spectrophotometer (Varian, Palo Alto, CA, USA) and the pH values of solutions were measured using a pH meter (Mettler Toledo FE20, Zurich, Switzerland).

Synthesis of CNDs
Typically, 0.8 g AA was dissolved in 20 mL water. Then the above solution was stirred thoroughly and then transferred into a 50 mL autoclave reactor. The reactor was heated to 160 • C in a constant temperature drying oven for 70 min. After cooling to room temperature, a clear yellow aqueous dispersion containing CNDs was gained. Subsequently, the resulting yellow solution (20 mL) was dried in a drying oven at 60 • C to constant weight (for about 14 h). Then, 39 mg powder of carbon nanodots was weighed and diluted in 1 mL water to obtain CNDs solution (39 mg/mL). The prepared solution of CNDs was diluted 100 times before use, and the concentration was 0.39 mg/mL.

Fluorescence Responses of Cu 2+
The fluorescence detection of Cu 2+ was performed as follows: briefly, 35 µL CNDs (0.39 mg/mL), 50 µL H 2 O 2 (0.1 M), different concentrations of Cu 2+ , 200 µL HCl-CH 3 COONa buffers (pH = 5) and water were mixed together, and the final volume was 1 mL. After incubation for 10 min at room temperature, the fluorescence of the mixture was measured at 380 nm with an excitation of 315 nm. With increasing the concentration of Cu 2+ , the fluorescence of CNDs decreased linearly and the fluorescence differences were expressed as F 0 -F, in which F 0 and F represented the fluorescence intensities of CNDs-H 2 O 2 in the absence and presence of Cu 2+ , respectively.
Additionally, the LOD is the limit of detection, which is defined as the concentration of analyte that corresponds to three times the signal-to-noise ratio (S/N = 3) [18]. LOD is calculated according to the expression LOD = 3σ/K, where σ is the standard deviation for the blank solution (n = 10), and K is the slope of the calibration curve.

Fluorescent H 2 O 2 Assay
A typical H 2 O 2 detection procedure was conducted as follows: Firstly, 35 µL CNDs (0.39 mg/mL), 60 µL Cu 2+ (0.01 mM), 200 µL HCl-CH 3 COONa buffers (pH = 5), various concentrations of H 2 O 2 and water were mixed thoroughly. The final volume of the mixture was 1 mL. After incubation for 10 min at ambient temperature, the concentration of H 2 O 2 linearly responded to the fluorescence differences of F 1 -F 2 (F 1 and F 2 represented the fluorescence intensity of CNDs-Cu 2+ in the absence and presence of H 2 O 2 ).

Fluorescence Detection of ATP
The assay was carried out as follows: (i) 200 µL HCl-CH 3 COONa buffers (pH = 5), 60 µL Cu 2+ (0.01 mM) and ATP with different concentrations were added to a certain amount of water and reacted for 10 min at room temperature; (ii) 35 µL CNDs (0.39 mg/mL) and 50 µL H 2 O 2 (0.1 M) were injected to the mixture and the volume of final solution was 1 mL. After the mixture reacted for 10 min at room temperature, a linear correlation was found between the concentration of ATP and the fluorescence differences of F 4 − F 3 , in which F 3 was the fluorescence intensity of CNDs-Cu 2+ -H 2 O 2 system and F 4 was the fluorescence intensity of the system with the addition of various concentrations of ATP.

Fluorescence Measurement of Glucose
Firstly, the mixture containing 13 µL GOx (765 U/mL) and different concentrations of glucose was incubated at 37 • C for 60 min, and then 200 µL HCl-CH 3 COONa buffers (pH = 5), 60 µL Cu 2+ (0.01 mM) and 35 µL CNDs (0.39 mg/mL) were added to the mixture. Ten minutes later, their fluorescence spectra were recorded. The concentration of glucose linearly responded to the fluorescence differences of F 5 − F 6 , where F 5 and F 6 were the fluorescence of CNDs-Cu 2+ -GOx system in the absence and presence of glucose.

Real Sample Detection
The real sample detection in our assay was carried out as follows: Typically, a 1.5 mL blood sample was kept at 37 • C in a water bath for about 0.5 h. Then, 3.0 mL acetonitrile was added and followed by centrifugation at 4000 rpm for 20 min to remove large molecules and proteins. Subsequently, the supernatant was removed and kept in a water bath (60 • C) for about 1 h to evaporate acetonitrile. The supernatant of the serum sample was diluted 10 times and measured by the present sensing system to detect ATP and glucose. Then, the standard ATP and glucose solutions with different concentrations were spiked into the human serum samples, respectively, to investigate the reliability of this assay.

Validation Test of TMB
The validation of the Cu-Fenton activity was performed as follows: the experiments were performed in 200 µL HCl-CH 3 COONa buffers (pH = 5) containing 5 mM TMB, 500 mM H 2 O 2 and differing concentrations of Cu 2+ followed by the addition of water to a final volume of 1 mL. After reaction for 10 min, the colour was present in these samples.

Characterization of CNDs
As shown in the TEM image (Figure 1a), the CNDs exhibit an approximately spherical shape and are well-dispersed. These nanoparticles have a size distribution from 1 to 3.4 nm and the average diameter of CNDs is about 1.8 nm by estimating the size of 100 dots in the TEM image. The structure and composition of CNDs are characterized by FT-IR spectroscopy ( Figure 1b). The CNDs have a main absorption of C-H stretching vibration at 2900 cm −1 , the C=O stretching at 1740 cm −1 , and the peak at 3452 cm −1 is related to the C-OH bond stretching vibrations. It demonstrates the presence of oxygen-containing functional groups such as hydroxyl and carbonyl groups on the surface of particles. Additionally, these nanodots show a strong peak at 260 nm (Figure 1c), which is ascribed to π-π* transition of carbon [19]. Next, the emission is found to be excitation-dependent, including wavelength and intensity in a well-regulated mode within the exciting range of 285-345 nm ( Figure S1). Although the emission peak shifts from 375 to 390 nm with increasing excitation wavelengths, its intensity reaches a maximum at 315 nm excitation (Figure 1c). The excitation-dependent emission spectra have been reported by various works involving CNDs [20,21]. Meanwhile, the CNDs solution under UV light exhibits bright blue fluorescence, whereas it is yellow under daylight. with different concentrations were spiked into the human serum samples, respectively, to investigate the reliability of this assay.

Validation Test of TMB
The validation of the Cu-Fenton activity was performed as follows: the experiments were performed in 200 μL HCl-CH3COONa buffers (pH = 5) containing 5 mM TMB, 500 mM H2O2 and differing concentrations of Cu 2+ followed by the addition of water to a final volume of 1 mL. After reaction for 10 min, the colour was present in these samples.

Characterization of CNDs
As shown in the TEM image (Figure 1a), the CNDs exhibit an approximately spherical shape and are well-dispersed. These nanoparticles have a size distribution from 1 to 3.4 nm and the average diameter of CNDs is about 1.8 nm by estimating the size of 100 dots in the TEM image. The structure and composition of CNDs are characterized by FT-IR spectroscopy ( Figure 1b). The CNDs have a main absorption of C-H stretching vibration at 2900 cm −1 , the C=O stretching at 1740 cm −1 , and the peak at 3452 cm −1 is related to the C-OH bond stretching vibrations. It demonstrates the presence of oxygen-containing functional groups such as hydroxyl and carbonyl groups on the surface of particles. Additionally, these nanodots show a strong peak at 260 nm (Figure 1c), which is ascribed to π-π* transition of carbon [19]. Next, the emission is found to be excitation-dependent, including wavelength and intensity in a well-regulated mode within the exciting range of 285-345 nm ( Figure  S1). Although the emission peak shifts from 375 to 390 nm with increasing excitation wavelengths, its intensity reaches a maximum at 315 nm excitation ( Figure 1c). The excitation-dependent emission spectra have been reported by various works involving CNDs [20,21]. Meanwhile, the CNDs solution under UV light exhibits bright blue fluorescence, whereas it is yellow under daylight.

Fluorescence Quenching of CNDs
According to Figure 2, when only H2O2 or Cu 2+ is mixed with CNDs, the emission at 380 nm changes inconspicuously. However, by both adding H2O2 and Cu 2+ , the fluorescence of CNDs is

Fluorescence Quenching of CNDs
According to Figure 2, when only H 2 O 2 or Cu 2+ is mixed with CNDs, the emission at 380 nm changes inconspicuously. However, by both adding H 2 O 2 and Cu 2+ , the fluorescence of CNDs is quenched effectively. While the amount of H 2 O 2 is fixed, the emission of CNDs sensitively responds to the concentration of Cu 2+ in the range from 1 nM to 2 µM with LOD 0.65 nM ( Figure S2), which is lower than most works [22,23]. Additionally, it should be noted that when the concentration of Cu 2+ is higher than 4 µM ( Figure S3), it can directly quench the fluorescence of CNDs without the addition of H 2 O 2 , which is consistent with previous literature [24], but Cu 2+ ions with a low amount (especially below 2 µM) is invalid. Additionally, with the unchanged amount of Cu 2+ , the fluorescence of these nanoparticles also decreases linearly when the concentration of H 2 O 2 increases from 25 µM to 5 mM with LOD 7.8 µM ( Figure S4). Like the Fenton reaction between Fe 2+ and H 2 O 2 , Cu 2+ and H 2 O 2 can also produce the reactive ·OH. In this process, Cu 2+ oxidizes H 2 O 2 to generate O 2 •and Cu + , and Cu + is able to react with excess H 2 O 2 to form ·OH [25][26][27][28], which can catalyse the oxidation of TMB to produce the blue colour reaction ( Figure S5). TMB oxidation-induced colour development is positively correlated with Cu 2+ concentrations. Therefore, the ·OH is believed to quench the fluorescence of CNDs, and the quenching efficiency reaches the maximum at pH 5 ( Figure S6). Cu-Fenton reaction is different from the Fe-Fenton reaction, and it usually requires a small amount of Cu 2+ and a large amount of H 2 O 2 , which just correspond to the low concentration of ATP and high concentration of glucose in human serum. Based on the binding of ATP to Cu 2+ and arising H 2 O 2 in the reaction between glucose and GOx, ATP, and glucose can be simultaneously detected with a single test mode (Scheme 1). quenched effectively. While the amount of H2O2 is fixed, the emission of CNDs sensitively responds to the concentration of Cu 2+ in the range from 1 nM to 2 μM with LOD 0.65 nM ( Figure S2), which is lower than most works [22,23]. Additionally, it should be noted that when the concentration of Cu 2+ is higher than 4 μM ( Figure S3), it can directly quench the fluorescence of CNDs without the addition of H2O2, which is consistent with previous literature [24], but Cu 2+ ions with a low amount (especially below 2 μM) is invalid. Additionally, with the unchanged amount of Cu 2+ , the fluorescence of these nanoparticles also decreases linearly when the concentration of H2O2 increases from 25 μM to 5 mM with LOD 7.8 μM ( Figure S4). Like the Fenton reaction between Fe 2+ and H2O2, Cu 2+ and H2O2 can also produce the reactive •OH. In this process, Cu 2+ oxidizes H2O2 to generate O2 •-and Cu + , and Cu + is able to react with excess H2O2 to form •OH [25][26][27][28], which can catalyse the oxidation of TMB to produce the blue colour reaction ( Figure S5). TMB oxidation-induced colour development is positively correlated with Cu 2+ concentrations. Therefore, the •OH is believed to quench the fluorescence of CNDs, and the quenching efficiency reaches the maximum at pH 5 ( Figure S6). Cu-Fenton reaction is different from the Fe-Fenton reaction, and it usually requires a small amount of Cu 2+ and a large amount of H2O2, which just correspond to the low concentration of ATP and high concentration of glucose in human serum. Based on the binding of ATP to Cu 2+ and arising H2O2 in the reaction between glucose and GOx, ATP, and glucose can be simultaneously detected with a single test mode (Scheme 1).  quenched effectively. While the amount of H2O2 is fixed, the emission of CNDs sensitively responds to the concentration of Cu 2+ in the range from 1 nM to 2 μM with LOD 0.65 nM ( Figure S2), which is lower than most works [22,23]. Additionally, it should be noted that when the concentration of Cu 2+ is higher than 4 μM ( Figure S3), it can directly quench the fluorescence of CNDs without the addition of H2O2, which is consistent with previous literature [24], but Cu 2+ ions with a low amount (especially below 2 μM) is invalid. Additionally, with the unchanged amount of Cu 2+ , the fluorescence of these nanoparticles also decreases linearly when the concentration of H2O2 increases from 25 μM to 5 mM with LOD 7.8 μM ( Figure S4). Like the Fenton reaction between Fe 2+ and H2O2, Cu 2+ and H2O2 can also produce the reactive •OH. In this process, Cu 2+ oxidizes H2O2 to generate O2 •-and Cu + , and Cu + is able to react with excess H2O2 to form •OH [25][26][27][28], which can catalyse the oxidation of TMB to produce the blue colour reaction ( Figure S5). TMB oxidation-induced colour development is positively correlated with Cu 2+ concentrations. Therefore, the •OH is believed to quench the fluorescence of CNDs, and the quenching efficiency reaches the maximum at pH 5 ( Figure S6). Cu-Fenton reaction is different from the Fe-Fenton reaction, and it usually requires a small amount of Cu 2+ and a large amount of H2O2, which just correspond to the low concentration of ATP and high concentration of glucose in human serum. Based on the binding of ATP to Cu 2+ and arising H2O2 in the reaction between glucose and GOx, ATP, and glucose can be simultaneously detected with a single test mode (Scheme 1).

Fluorescence Recovery of CNDs-Cu 2+ -H 2 O 2 System by ATP
As shown in Figure 3, when the ATP is introduced in the CNDs-Cu 2+ -H 2 O 2 system, the quenched fluorescence recovers effectively because Cu 2+ ions exhibit a strong binding affinity to ATP with respect to multiple phosphates, while ATP alone will not influence the fluorescence of CNDs, CNDs-Cu 2+ , and CNDs-H 2 O 2 . All of results indicate that ATP indeed binds with Cu 2+ ions, which cannot react with H 2 O 2 to form ·OH. At the same time, the reaction between ATP and Cu 2+ ions can complete within 10 min, suggesting a rapid reaction rate ( Figure S7). The enhanced fluorescence positively correlates with the concentration of ATP in the linear range from 0.1 to 7 µM with LOD 30.2 nM (Figure 4). Comparing with the previous reports (Table 1), our method is not inferior to others [29][30][31][32][33][34][35] and also exhibits relatively wide linear ranges and a low limit of detection of ATP.

Fluorescence Recovery of CNDs-Cu 2+ -H2O2 System by ATP
As shown in Figure 3, when the ATP is introduced in the CNDs-Cu 2+ -H2O2 system, the quenched fluorescence recovers effectively because Cu 2+ ions exhibit a strong binding affinity to ATP with respect to multiple phosphates, while ATP alone will not influence the fluorescence of CNDs, CNDs-Cu 2+ , and CNDs-H2O2. All of results indicate that ATP indeed binds with Cu 2+ ions, which cannot react with H2O2 to form •OH. At the same time, the reaction between ATP and Cu 2+ ions can complete within 10 min, suggesting a rapid reaction rate ( Figure S7). The enhanced fluorescence positively correlates with the concentration of ATP in the linear range from 0.1 to 7 μM with LOD 30.2 nM ( Figure  4). Comparing with the previous reports (Table 1), our method is not inferior to others [29][30][31][32][33][34][35] and also exhibits relatively wide linear ranges and a low limit of detection of ATP.

Detection of Glucose Based on CNDs-Cu 2+ System
H2O2 is the main product of the reaction between glucose and GOx, so CNDs-Cu 2+ -H2O2 system can be utilized to analyse glucose. As illustrated in Figure 5, there is no significant difference when glucose or GOx is added in CNDs and CNDs-Cu 2+ system. When glucose is oxidized by GOx, the fluorescence of CNDs-Cu 2+ platform was quenched effectively. The fluorescence difference was proportional to the concentration of glucose in the range from 0.1 to 7 mM with LOD 39.8 μM ( Figure  6). The fluorescence response of this platform to the glucose ranging from 0 to 1.0 mM is depicted in Figure S8, where the slope of the linear equation is similar to that of glucose with concentration from 0.1 to 7.0 mM, indicating that the glucose with low concentrations can be also sensitively detected. In comparison with the reported studies (Table 2), this new strategy is demonstrated to not be inferior to other works [36][37][38][39][40][41]. Additionally, CNDs involved in this assay, as a novel probe, are easily synthesized, experimentally convenient, and low-cost, and can be used to detect ATP and glucose simultaneously.

Detection of Glucose Based on CNDs-Cu 2+ System
H 2 O 2 is the main product of the reaction between glucose and GOx, so CNDs-Cu 2+ -H 2 O 2 system can be utilized to analyse glucose. As illustrated in Figure 5, there is no significant difference when glucose or GOx is added in CNDs and CNDs-Cu 2+ system. When glucose is oxidized by GOx, the fluorescence of CNDs-Cu 2+ platform was quenched effectively. The fluorescence difference was proportional to the concentration of glucose in the range from 0.1 to 7 mM with LOD 39.8 µM (Figure 6). The fluorescence response of this platform to the glucose ranging from 0 to 1.0 mM is depicted in Figure S8, where the slope of the linear equation is similar to that of glucose with concentration from 0.1 to 7.0 mM, indicating that the glucose with low concentrations can be also sensitively detected. In comparison with the reported studies (Table 2), this new strategy is demonstrated to not be inferior to other works [36][37][38][39][40][41]. Additionally, CNDs involved in this assay, as a novel probe, are easily synthesized, experimentally convenient, and low-cost, and can be used to detect ATP and glucose simultaneously.

Detection of Glucose Based on CNDs-Cu 2+ System
H2O2 is the main product of the reaction between glucose and GOx, so CNDs-Cu 2+ -H2O2 system can be utilized to analyse glucose. As illustrated in Figure 5, there is no significant difference when glucose or GOx is added in CNDs and CNDs-Cu 2+ system. When glucose is oxidized by GOx, the fluorescence of CNDs-Cu 2+ platform was quenched effectively. The fluorescence difference was proportional to the concentration of glucose in the range from 0.1 to 7 mM with LOD 39.8 μM ( Figure  6). The fluorescence response of this platform to the glucose ranging from 0 to 1.0 mM is depicted in Figure S8, where the slope of the linear equation is similar to that of glucose with concentration from 0.1 to 7.0 mM, indicating that the glucose with low concentrations can be also sensitively detected. In comparison with the reported studies (Table 2), this new strategy is demonstrated to not be inferior to other works [36][37][38][39][40][41]. Additionally, CNDs involved in this assay, as a novel probe, are easily synthesized, experimentally convenient, and low-cost, and can be used to detect ATP and glucose simultaneously.

Selectivity for ATP and Glucose
To examine the selectivity of this CNDs-Cu 2+ -H2O2 system for ATP, other analogous molecules, including CTP, ADP, UTP, and GTP are investigated under the optimized conditions. As shown in Figure 7a, only the addition of ATP produces an obvious fluorescence recovery, suggesting the highly selectivity for ATP. Additionally, some interference substances, such as galactose, fructose, sucrose, mannose, lactose, sodium citrate, AA, and KCl, are evaluated. Due to the high specificity of GOx for glucose, other compounds cannot induce the effective fluorescence quenching of CNDs, indicating a high selectivity of our proposed sensing system for glucose.

Detection of ATP and Glucose in Human Serum Samples
To evaluate the applicability of this sensing system, the proposed strategy is used to determine ATP and glucose in human serum samples. The results are shown in Table 3, the concentration of

Selectivity for ATP and Glucose
To examine the selectivity of this CNDs-Cu 2+ -H 2 O 2 system for ATP, other analogous molecules, including CTP, ADP, UTP, and GTP are investigated under the optimized conditions. As shown in Figure 7a, only the addition of ATP produces an obvious fluorescence recovery, suggesting the highly selectivity for ATP. Additionally, some interference substances, such as galactose, fructose, sucrose, mannose, lactose, sodium citrate, AA, and KCl, are evaluated. Due to the high specificity of GOx for glucose, other compounds cannot induce the effective fluorescence quenching of CNDs, indicating a high selectivity of our proposed sensing system for glucose.

Selectivity for ATP and Glucose
To examine the selectivity of this CNDs-Cu 2+ -H2O2 system for ATP, other analogous molecules, including CTP, ADP, UTP, and GTP are investigated under the optimized conditions. As shown in Figure 7a, only the addition of ATP produces an obvious fluorescence recovery, suggesting the highly selectivity for ATP. Additionally, some interference substances, such as galactose, fructose, sucrose, mannose, lactose, sodium citrate, AA, and KCl, are evaluated. Due to the high specificity of GOx for glucose, other compounds cannot induce the effective fluorescence quenching of CNDs, indicating a high selectivity of our proposed sensing system for glucose.

Detection of ATP and Glucose in Human Serum Samples
To evaluate the applicability of this sensing system, the proposed strategy is used to determine ATP and glucose in human serum samples. The results are shown in Table 3, the concentration of

Detection of ATP and Glucose in Human Serum Samples
To evaluate the applicability of this sensing system, the proposed strategy is used to determine ATP and glucose in human serum samples. The results are shown in Table 3, the concentration of ATP in human serum sample (without dilution) measured by our method is about 4.3 µM, which is consistent with the value detected by the high-performance liquid chromatograph (HPLC). With a standard addition method, the real samples are spiked with certain amounts of ATP and the satisfactory recoveries of ATP in the range from 102.4 to 106.4% are reached. On the other hand, the concentration of glucose in human serum sample (without dilution) detected by our method is about 3.41 mM, which is similar to the clinic value provided by local hospital (3.78 mM). Next, the recoveries in the ranges from 98.0 to 104.8% are obtained by spiking different concentrations standard glucose solutions into human serum samples (Table 4). All results demonstrate that this proposed method is practicable, reliable, and can be used for simultaneous determination of ATP and glucose in human serum samples.

Conclusions
Herein, a fluorescence sensing platform based on CNDs for the detection of ATP and glucose with superior sensitivity and selectivity has constructed. The sensing mechanism is that Cu 2+ can react with H 2 O 2 to generate ·OH, which quenches the fluorescence of CNDs. Incorporating both ATP and glucose, which respond respectively to Cu 2+ and H 2 O 2 , the measurements of ATP and glucose can be simultaneously achieved in human serum samples with satisfactory recoveries. Due to the abnormal levels of ATP and glucose closely related to some diseases, this assay has been proposed in an effort to find a proper candidate for use in routine clinical practice.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/18/7/2151/s1, Figure S1. Excitation-dependent fluorescence spectra of CNDs. Figure S2. Fluorescence spectra of CNDs upon addition of different concentrations of Cu 2+ (a) in the presence of H 2 O 2 (5 mM) and the corresponding linear ranges of Cu 2+ (b). Figure S3. Fluorescence spectra of CNDs upon addition of different concentrations of Cu 2+ in the absence of H 2 O 2 . Figure S4. Fluorescence spectra of CNDs upon addition of different concentrations of H 2 O 2 (a) in the presence of Cu 2+ (2 µM) and the corresponding linear ranges of H 2 O 2 (b). Figure S5. Photographs of Cu 2+ -H 2 O 2 -TMB system with different concentrations of Cu 2+ under visible light. Figure S6. Influence of pH values on CNDs-Cu 2+ -H 2 O 2 system. Figure S7. Influence of the incubation time between Cu 2+ and ATP. The concentrations of CNDs, H 2 O 2 , Cu 2+ and ATP were 0.014 mg/mL, 5 mM, 600 nM and 1 µM, respectively. Figure S8. Fluorescence spectra of CNDs in the presence of different concentrations of glucose (a) and the corresponding linear range from 0.1 mM to 1 mM (b). The concentrations of CNDs, Cu 2+ , and GOx were 0.014 mg/mL, 600 nM, and 10 U/mL respectively.

Conflicts of Interest:
The authors declare no conflict of interest.