A Ratiometric Fluorescence Universal Platform Based on N, Cu

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

A Ratiometric Fluorescence Universal Platform Based on N, Cu Codoped Carbon Dots to Detect Metabolites Participating in H2O2-generation Reactions Yunsu Ma, Yao Cen, Muhammad Sohail, Guanhong Xu, Fangdi Wei, Menglan Shi, Xiaoman Xu, Yueyue Song, Yujie Ma, and Qin Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10548 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

A Ratiometric Fluorescence Universal Platform Based on N, Cu Codoped Carbon Dots to Detect Metabolites Participating in H2O2-generation Reactions Yunsu Ma, Yao Cen, Muhammad Sohail, Guanhong Xu, Fangdi Wei, Menglan Shi, Xiaoman Xu, Yueyue Song, Yujie Ma, Qin Hu∗ School of pharmacy, Nanjing medical university, Nanjing, Jiangsu 211166, PR China

Keywords: N, Cu codoped carbon dots, ratiometric fluorescence, H2O2, cholesterol, xanthine

Abstract In this work, a new kind of N, Cu codoped carbon dots (N/Cu-CDs) was prepared via a facile one-pot hydrothermal method by using citric acid monohydrate, copper acetate monohydrate and diethylenetriamine. The prepared N/Cu-CDs with a high quantum yield (50.1 %) showed excitation-independent emission at 460 nm. The structure and fluorescence properties of N/Cu-CDs were characterized by high-resolution transmission electron microscopy, fluorescence spectrofluorometer, FT-IR spectrometer, UV-Visible spectrophotometer and X-ray photoelectron spectroscopy. N/Cu-CDs were applied to establishing a ratiometric fluorescence probe toward H2O2 based on the inner filter effect (IFE) between N/Cu-CDs and DAP (2, 3-diaminophenazine, the oxidative product of o-phenylenediamine), and provided a ratiometric fluorescence universal platform for detection of the metabolites participating in H2O2-generation reactions (cholesterol and xanthine). The proposed

∗

Corresponding author: Tel. / fax: +86-2586868468; E-mail: [email protected] 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

method was demonstrated to be ultrasensitive and highly selective for cholesterol and xanthine assay with detection limits of 0.03 and 0.10 µM, respectively. The fluorescence probe built was applied to the determination of cholesterol and xanthine in human serum with satisfactory results.

1. INTRODUCTION Carbon dots (CDs) are a new member of the functional nanocarbon family1. Owning to their unique optical properties, multiple functional groups, excellent biocompatibility, chemical and photostability, CDs have attracted a large amount of concerns for applications in the field of bioimaging2, 3, sensing4, 5, optoelectronic conversion6, 7and nanomedicine8. To improve the structures and photophysical properties of CDs, several physical or chemical synthetic methods of CDs have been developed. These methods can be classified into two types, namely top-down9-12 and bottom-up strategies13,

14

. On account of the complex and extreme synthetic

conditions of the top-down strategies, the bottom-up strategies have been received growing attention due to their easy handle and high productivity. To improve the photophysical performance and enlarge application scope of CDs, there are two kinds of functional strategies including surface functionalization and heteroatom doping 15. However, surface functionalization using polymers or small organic molecules has the drawbacks of occupying the position functionalized for specific analytical15, 16. Thus, heteroatom-doping into CDs has been becoming a more powerful approach to improve the fluorescence properties of CDs owing to its facile and easy manipulating feature11,

17, 18

. For example, nitrogen-doped CDs prepared by using citric acid 2


Page 2 of 26

Page 3 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


monohydrated and ammonia show a high quantum yield (QY) of 40.5%19. Additionally, nitrogen and sulfur codoped CDs generating from [C4mim][Cys] and sulfuric acid show great performance on Cu2+ detection20. As reported in the references21-23, metal ions like Fe3+, Cu2+, and Hg2+ usually quenched the fluorescence of CDs by interacting with the phenolic hydroxyl groups on the surface. It is a promising way to reduce the phenolic hydroxyl groups of CDs by metal doping in production, and then the metal ions insensitive CDs may be used as biosensors, avoiding the interference of metal ions that normally exist in sensing systems. Unlike the nonmetal elements doping, the research of metal doping are in the infancy

24

.

Copper element was selected to dope in CDs due to its safety and easy interaction with chemical groups on the surface of CDs. Nitrogen element was selected to codope in CDs to increase the QY of CDs. To date, there have been only two references about the synthesis of N, Cu codoped CDs and their applications as photocatalytic materials24, 25. There is no report about N, Cu codoped CDs used as fluorescence materials in biosensing. It is necessary to produce fine N, Cu codoped CDs with high QY, and explore their fluorescence properties and application in biosensing. H2O2, as one type of reactive oxygen species, is an inevitable byproduct of cell metabolism and a common marker and signal molecule of physiological activity and oxidative stress26. It is well-known that a large variety of metabolites in human body (cholesterol, xanthine, glucose, lactate, choline, glutamate, alcohol) are catalyzed by enzymes to produce H2O227. It is meaningful and valuable to develop efficient methods for monitoring H2O2. Many fluorescence materials including organic 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 26

molecule, dye, semiconductor quantum dots were used to develop fluorescence probe for H2O2 detection28-31. The ratiometric fluorescence measurement, as one of fluorescence measurement can afford simultaneous recording of two measurable signals at one excitation wavelength, which overcome the drawbacks that single fluorescence measurement is easily influenced by detection conditions and probe concentrations. However, CDs were rarely used in ratiometric fluorescence probe for H2O2 determination32, and there was no report about the heteroatom doped CDs used in this probe. Due to the excellent properties of the heteroatom doped CDs, it is essential to build a ratiometric fluorescence universal platform for detection of H2O2 and metabolites participating in H2O2-generation reactions with easy operation, high sensitivity and good selectivity. In this work, citric acid monohydrate, together with copper acetate monohydrate and diethylenetriamine were used to synthesize N/Cu-CDs for the first time. The structure and outstanding optical properties of N/Cu-CDs were characterized by high-resolution transmission electron microscopy (TEM), spectrofluoromete, FT-IR spectrometer, UV-visible spectrophotometer and X-ray photoelectron spectroscopy (XPS). A ratiometric fluorescence universal platform based on the metal insensitive N/Cu-CDs to detect the metabolites participating in H2O2-generation reactions was developed.

In this sensing platform, o-phenylenediamine (OPD) was oxidized by

H2O2 in the

presence

of horseradish peroxidase (HRP) to produce

2,

3-diaminophenazine (DAP) which exhibited a yellow light emission at 572 nm. DAP then quenched the fluorescence of N/Cu-CDs due to inner-filter effect (IFE). A 4


Page 5 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ratiometric fluorescence biosensor toward H2O2 was established by measuring the fluorescence signals of DAP and N/Cu-CDs. Furthermore, the ratiometric fluorescence biosensor was demonstrated ultrasensitive and highly selective for detection of the metabolites (cholesterol and xanthine) involved in H2O2-generation reaction.

2. EXPERIMENTAL SECTION 2.1. Chemical and reagents Horse radish peroxidase (HRP, ≥ 300 U mg-1), xanthine (98 %) and o-phenylenediamine (OPD) were purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Hydrogen peroxide (H2O2, 30 %), citric acid monohydrate, diethylenetriamine, and copper acetate monohydrate were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Cholesterol (99 %) and cholesterol esterase (> 15 U mg-1) were purchased from Aladdin Industrial Corporation (Shanghai, China). Cholesterol oxidase (> 20 U mg-1) was purchased from TOYOBO Biotechnology Co., Ltd (Shanghai, China). Xanthine oxidase (> 10 U mg-1) was purchased from Shanghai Yuanye Biological Technology co., LTD (Shanghai, China). The purified water used in the study was prepared using Direct-Q water purification system (Millipore, USA). All other chemicals used in this work were of analytical grade and used without further purification. The healthy human serum samples were friendly supplied by First Affiliated Hospital–Nanjing Medical University (Jiangsu, China). 2.2. Measurement and apparatus 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The

fluorescence

emission

spectra

were

measured

Page 6 of 26

on

an

F4600

spectrofluorometer (Hitachi, Japan) with 380 nm excitation. Fluorescence decay curves were performed with a steady state and transient state fluorescence spectrometer (FM-4P-TCSPC, Horiba Jobin Yvon, France). FT-IR spectrum of N/Cu-CDs was recorded by using a Tensor-27 FT-IR spectrometer (Bruker, Germany). UV-vis absorption spectra were obtained on a UV-Visible spectrophotometer (UV-2450, Hitachi, Japan). The morphology of N/Cu-CDs was analyzed with a Transmission electron microscopy (TEM, JEM-1010, Hitachi, Japan). Zeta potential and nano-particle size were measured by dynamic light scattering (DLS, ZS90, Malvern, U.K.). The powder X-ray photoelectron spectroscopy (XPS) patterns were collected using an X’TRA diffractometer (ARL, Switzerland). 2.3. Synthesis of N/Cu-CDs N/Cu-CDs were prepared using one-pot hydrothermal method. Citric acid monohydrate (CA, 1.2 g) and copper acetate monohydrate (0.15 g) were dissolved in 20 mL of purified water. Then, the resulting solution was added with diethylenetriamine (DETA, 0.15 mL). The mixture was dissolved by an ultrasonic method for 10 min, then added into a 30 mL Teflon-lined stainless steel autoclave and heated at 230 °C for 12 h. After cooled to room temperature, the final products were collected and dialyzed through dialysis-membrane (500 MWCO) over 72 h to remove the unreacted materials. The resulting hazel solution was distilled by reduced pressure distillation to remove extra water. Brown powder of N/Cu-CDs was obtained by vacuum drying method. 6


Page 7 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


As the reference materials, N-CDs was prepared via the same method of N/Cu-CDs except the addition of copper acetate monohydrate, and CDs was prepared via the same method of N -CDs except the addition of DETA. 2.4. H2O2 sensing For the detection of H2O2, 190 µL of 14 mM OPD (10 mM phosphate buffer (PB), pH = 6.6), 10 µL of 1 mg mL-1 HRP (10 mM PB, pH = 6.6) and 10 µL variable concentrations of H2O2 were mixed. The resulting solution was incubated at 37 °C in dark for 30 min. After that, 20 µL of 200 µg mL-1 N/Cu-CDs solution (10 mM PB, pH = 6.6) was added into the mixture and incubated at 25 °C in dark for 1 min. The fluorescence spectra were measured under 380 nm excitation for quantitative analysis of H2O2. 2.5. Cholesterol sensing For the detection of cholesterol, 200 µL variable concentrations of cholesterol and 10 µL of 0.6 mg mL-1 ChOX were mixed with 190 µL of 14 mM OPD (10 mM PB, pH = 6.6) and 10 µL of 1 mg mL-1 HRP (10 mM PB, pH = 6.6). The resulting solution was incubated at 37 °C in dark for 30 min. The following operation was carried out according to section 2.4. For the detection of cholesterol in human serum, the serum (1 mL) was added with cholesterol esterase (5µL, 5 mg/mL), and then diluted by a factor of 200 using PB. The diluted serum was measured as described above. 2.6. Xanthine sensing For the detection of xanthine, 200 µL variable concentrations of xanthine with 10 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

µL of 0.2 mg mL-1 XOD were mixed with 190 µL of 14 mM OPD (10 mM PB, pH = 6.6) and 10 µL of 1 mg mL-1 HRP (10 mM PB, pH = 7.0). The resulting solution was incubated at 37 °C in dark for 30 min. The following operation was carried out according to section 2.4. For detection of xanthine in human serum, the serum was diluted by a factor of 10 using PB, and then measured as described above.

3. RESULTS AND DISCUSSIONS 3.1. Characterization of N/Cu-CDs As shown in Fig.1A, N/Cu-CDs presented a spherical shape with a diameter of 1 to 3 nm, evenly dispersing in the aqueous solution. The size of N/Cu-CDs was smaller than that of N-CDs (Fig.S1A). Additionally, the lattice fringe of 0.21 nm was seen in the high-resolution TEM image (insert of Fig.1A), which was also smaller than that of N-CDs (insert of Fig.S1A). The structure of N/Cu-CDs and functional groups on the surface N/Cu-CDs were characterized with FT-IR spectra (Fig.1B). The broad vibration band of N/Cu-CDs around 3400 cm−1 was ascribed to－OH stretching vibration, which was much lower than that of N-CDs, and the absorptions slightly below 3000 cm−1 and at 1380 cm−1 were ascribed to C－H alkyl stretching vibration and bending vibration, respectively. The peak at 1680 cm−1 was ascribed to C＝O stretching vibration. These results indicated that there were－OH，C－H and C＝O on the surface of N/Cu-CDs. Besides, the peaks at 1580 and 1490 cm−1 were attributed to the vibrations of C＝C in benzene ring, indicating that there were aromatic structures in N/Cu-CDs. The vibration bands at 1450 and 1200 cm−1 were assigned to the stretching vibrations of C－N and C－O, respectively33. Unlike 8


Page 8 of 26

Page 9 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


N-CDs, a couple of bonds appeared at metal-sensitive region (1000－920 cm−1 ) in the FT-IR spectrum of N/Cu-CDs, which could be assigned to the N－Cu－N

24, 34

.

The results of FT-IR spectrum indicated that the N and Cu were successfully codoped in N/Cu-CDs. XPS spectroscopy was used for further identification of the functional groups in the N/Cu-CDs. Fig.1C showed the full scan survey of N/Cu-CDs. There were four main peaks at 284, 400, 532, and 933 eV, which represented C 1s, N 1s, O 1s and Cu 2p, respectively. According to the C1s XPS spectra (Fig.1D), the carbon existed in four different states: C＝C/C–C, C–N/C–O, COO– and C＝O (284.3, 286.1, 288.5, and 287.5 eV)17, 35. As shown in the high-resolution N 1s spectra of N/Cu-CDs (Fig.1E), the peak at 399.4 eV confirmed the presence of pyrrolic N, and the peak at 400.3 eV attributed to graphite N36. Considering the N–Cu–N bond appeared in the FT-IR spectrum, the copper and nitrogen were supposed to exist as porphyrin-metal complexes in N/Cu-CDs. The spectrum of Cu 2p (Fig.1F) had two peaks: the peaks at 933.0 and 953.2 eV assigned to Cu 2p3/2 and Cu 2p1/2 spectra of N－Cu－N covalent bound in porphyrin-metal complex. These results from XPS data were in good accordance with FT-IR analysis of N/Cu-CDs. From the above data, it was clear that nitrogen and copper were codoped in N/Cu-CDs. 3.2. Optical properties of N/Cu-CDs The optical properties of N/Cu-CDs were researched in detail based on the analysis of UV−vis absorption and fluorescence spectrum. The UV-vis absorption spectrum of N/Cu-CDs (Fig.2A) showed significant absorption bands at ~240 and 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 26

~350 nm, which resulted from π－π* transition of sp2 carbon and n－π* transition of C＝O/C＝N in N/Cu-CDs, respectively. Compared with the absorption of N-CDs band at 300-400 nm, the UV-vis absorption spectrum of N/Cu-CDs had a broader absorption band at 300-500 nm owing to the transition metal-copper doped37 (Fig.S1B). As shown in Fig.2B, the prepared blue emissions N/Cu-CDs displayed special fluorescence properties. The fluorescence peak position located at 460 nm was almost invariable when the excitation wavelength changed from 300 to 390 nm, which revealed the excitation-independent emission characteristic of N/Cu-CDs. While the CDs prepared using CA showed excitation-dependent emission (Fig.S1C). As previously reported, the blue emission of CDs was assigned to the intrinsic near-band edge recombination of electron−hole pairs localized in sp2 clusters38, 39. Based on the calculated band gap and size-dependent emission of quantum dots, the excitation-independent emission of N/Cu-CDs was probably caused by only a single near-band edge transition mode occurred in the fluorescence process40. The excitation-dependent of CDs were ascribed to the new energy levels in the band gap which were caused by surface trapping states. When CDs were excited at different wavelengths, the multiple transition modes occurred in different probabilities, which leaded to the excitation-dependent emission. The surface traps on the prepared N/Cu-CDs were effectively passivated by functional groups, like –C=O, –C–O, COO–, C–N, and N–Cu–N, and only the radiative transition of sp2 carbon produced fluorescence

emission.

A

single

recombination

mode

leaded

to

the

excitation-independent fluorescence behavior. This is a possible mechanism for the 10


Page 11 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


excitation-independent fluorescence property. N/Cu-CDs also exhibited an excellent optical stability. As the image of stability against photobleaching (Fig.S2A) shown, the fluorescence intensity of N/Cu-CDs in various solutions with different pH levels from 3.6 to 8.0 had no significant attenuation within 7500 seconds of continuous scanning (600 v, excited at 380 nm). Additionally, the fluorescence intensity of N/Cu-CDs also showed no change when stored at room temperature for two weeks (Fig.S2B). Moreover, the QY of N/Cu-CDs in water was as high as 50.1 % using quinine sulfate as a reference. The QY of N/Cu-CDs was much higher than that of Cu-N CDs in previous report (17.3 %)24. The increase of QY might be caused by different carbon source. These properties suggested that the prepared N/Cu-CDs had a promising potential for further application. 3.3. Ratiometric fluorescence determination for H2O2, cholesterol and xanthine. 3.3.1. Principle of the ratiometric fluorescence universal platform based on N/Cu-CDs to detect cholesterol and xanthine In this study, a novel N/Cu-CDs based ratiometric fluorescence probe which responded to H2O2 was developed. It provided a universal platform to detect the substrates which could be oxidized by their specific oxidoreductases to produces H2O2, like cholesterol and xanthine. As illustrated in scheme 1, CA, DETA, and copper acetate monohydrate were used to prepare N/Cu-CDs by one-pot hydrothermal method, and N/Cu-CDs with blue light emission at 460 nm were obtained. H2O2 was produced from cholesterol and xanthine oxidized by their specific oxidoreductases (ChOX and XOD) and O2. Then, OPD was oxidized by H2O2 and HRP and 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

subsequently produced DAP, a yellow light emission material (at 572 nm), which could quench the fluorescence of N/Cu-CDs. The ratio of the fluorescence intensity of DAP to N/Cu-CDs was measured for quantitative analysis The possible mechanism for N/Cu-CDs quenching was probably due to IFE. As shown in Fig.3A and Fig.S3A, N/Cu-CDs showed metal ions insensitivity, and was only quenched by DAP. Obviously, N/Cu-CDs acted as the donator, and DAP, the oxidation product of OPD, was an acceptor, and the other materials (HRP, OPD) in this system showed no effects on the fluorescence of N/Cu-CDs except DAP. Fig.3B also proved that there was a great overlay between the absorbance spectrum of DAP (around 450 nm) and the emission spectrum of N/Cu-CDs excited at 380 nm. In order to confirm if the experimental phenomenon caused by photoinduced electron transferor or Förster-resonance-energy transfer (FRET), the fluorescence lifetimes of N/Cu-CDs before and after the production of DAP were measured41-43. As shown in Fig.3C, the fluorescence lifetimes of N/Cu-CDs had no change before and after the fluorescence quenched by DAP, which indicated that there was no electron or energy transfer between N/Cu-CDs and DAP44. For FRET, the distance between fluorescence donor and acceptor should be less than 10 nm45. However, the zeta potentials of all components were nearly electroneutrality in this system (Fig.S4), the distance between N/Cu-CDs and DAP hardly shorter than 10 nm due to the weak electrostatic attraction of them. Thus, the FRET could not occur in this system. All the results suggested that the mechanism for the quenching of N/Cu-CDs by DAP was due to IFE. 12


Page 12 of 26

Page 13 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.3.2.

H2O2 determination

Under optimized conditions (Fig.S5), the changes of fluorescence intensity of N/Cu-CDs and DAP were recorded after the addition of different concentrations of H2O2 from 0.025 µM to 0.60 mM. As shown in Figure 4A, with the increase of H2O2 concentrations, the fluorescence intensity of N/Cu-CDs decreased gradually while the fluorescence intensity of DAP increased. Under the optimal conditions, Fig.4B showed that a good linear relationship was between the ratiometric fluorescence intensity (I572/I460) and H2O2 concentration in the range of 0.025 to 400 µM (y = 0.01509x + 0.07058, r = 0.9992, n = 3). The limit of detection (LOD) based on 3σ/K (where σ is the standard deviation of blank measurement, and k is the slope of calibration graph) was 0.01 µM, which was comparable or better than most of existing fluorescence probes for H2O228. N-CDs and CDs as the reference materials were also studied this detection system. The effects of metal ions, OPD and HRP on the fluorescence of reference materials were first investigated. Unlike metal ions insensitive N/Cu-CDs (Fig.S3A), the fluorescence of N-CDs and CDs were significantly quenched by adding Fe3+ (Fig.S3B, S3C) since the phenolic hydroxyl groups on the surface of N-CDs and CDs interacted with Fe3+23, 46, 47. Additionally, the fluorescence of the reference materials was also quenched by OPD. The results indicated that N-CDs and CDs cannot be used as probes in this sensing system. These results demonstrated that the prepared ratiometric fluorescence sensing platform had promising potential for highly sensitive detection of H2O2, suggested a universal platform to detect the metabolites which participate in H2O2-generation 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

reactions (e.g, cholesterol48, xanthine49, glucose26, lactate, choline, L-lysine, pyruvate, glutamate50, 51). As the proof-of-concept demonstration, cholesterol and xanthine were measured as below. 3.3.3.

Determination of cholesterol

Cholesterol can be oxidized to choleste-4-en-3-one and H2O2 by ChOX and O252,53. Thus, the probe which sensitively responds to H2O2 can be used to detect cholesterol. Under the optimal conditions (Fig.S6), with the increase of the concentration of cholesterol up to 0.60 mM, the fluorescence intensity of DAP (at 572 nm) was found to rise gradually while the fluorescence intensity of N/Cu-CDs (at 460 nm) decreased (Fig.5A). Fig.5B showed the relationship between the concentration of cholesterol and the ratiometric fluorescence intensity (I572/I460). A good linear relationship (r = 0.9992) was obtained in the concentration range of 0.05 − 100 µM (y = 0.01309x + 0.1155, r = 0.9992, n = 3). The LOD was measured to be 0.03 µM, which is comparable or even superior to those achieved by using other methods52, 54. Additionally, the specificities of the probe were measured. The results (Fig.5D) showed that when the concentration of cholesterol was 0.05 mM and other interferents were 0.50 mM, the interferents (amino acid, carbohydrate, and metal ions) had no influence on the ratiometric fluorescence measurement. The reason should be ascribed to ChOX catalytic specificity for cholesterol. These results indicated that the prepared probe had excellent sensitivity and selectivity for cholesterol determination. The results of precision and accuracy of N/Cu-CDs based ratiometric fluorescence probe are displayed in Table S1. They were studied by assaying 0.10, 14


Page 14 of 26

Page 15 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.50 and 80.00 µM of cholesterol in three separate runs. As illustrated in Table S1, the intra-day and inter-day relative standard deviations (RSDs) were below 6.9 % and 4.3 %, respectively. The accuracies were all in the range of 93.0 – 107.2 %. The results suggested that the prepared probe had a promising potential for detection of cholesterol in human serum. Thus, N/Cu-CDs based ratiometric fluorescence probe was applied to the determination cholesterol in human serum by a standard curve method. Because most of cholesterol bonds with fatty acid to form cholesterol ester in blood, cholesterol esterase was used to hydrolyze cholesterol ester to produce free cholesterol in cholesterol detection. As shown in Table S2, the results of serum cholesterol determination, the recoveries for cholesterol in human serum samples were between 90.3 % and 108.0 %, the RSDs were from 1.9 % to 7.8 %. The detected cholesterol levels in these three serum samples were in accordance with the reported (2.86 – 5.98 mM)54. All these results indicated that N/Cu-CDs based ratiometric fluorescence probe was a promising design in determination of cholesterol in human serum. 3.3.4.

Xantine sensing

Xanthine also can be oxidized by XOD and O2 to produce uric acid and H2O250, 55

. Similarly, in the presence of XOD, N/Cu-CDs based ratiometric fluorescence

sensor could detect xanthine in a range of 0.25 – 75.00 µM with a LOD of 0.1 µM (Fig.6A and Fig.6B) under optimal condition (Fig.S7). A good liner relationship between the concentration of xanthine and the ratiometric fluorescence intensity (I572/I460) was displayed in Fig.6C (y = 0.0063 x + 0.1033，r = 0.9998, n = 3). The 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

selectivity of xantine detection was also tested. As shown in Fig.6D, the interferents had no influence on I572/I460, similar to blank sample. Precision and accuracy of xanthine determination were also evaluated. As shown in Table S3, the intra-day and inter-day RSDs were below 8.8 % and 9.5 %, respectively. The accuracies were all in the range of 90.1 – 98.0 %. In addition, as shown in the results of serum cholesterol determination (Table S4), the recoveries for detection of xanthine in serum were between 91.6 % and 101.2 %. The RSDs were from 2.9 % to 8.6 %. All of the results proved that the sensitivity and selectivity of N/Cu-CDs based ratiometric fluorescence sensor and the probe could be used as a universal platform to detect the metabolites which could be oxidized to produce H2O2.

4. CONCLUSIONS In this work, N, Cu codoped carbon dots were successfully synthesized via a simple one-pot hydrothermal method. By introduced with N and Cu, the prepared N/Cu-CDs showed special fluorescence properties, such as metal ions insensitivity, high QY (50.1 %) and good stability. The surface traps on N/Cu-CDs were effectively passivated by C–O, C=O, COO–, C–N, and N–Cu–N, possibly leaded to the excitation-independent fluorescence behavior. Based on the IFE between N/Cu-CDs and DAP, a ratiometric fluorescence universal platform for the determination of metabolites participating in H2O2-generation reactions was developed. The proposed method was used to detect cholesterol and xanthine as probative experiments with LOD of 0.03 and 0.10 µM, respectively. Furthermore, this sensitive and selective probe was successfully used to detect cholesterol and xanthine in human serum, 16


Page 16 of 26

Page 17 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


indicating its promising potential application in clinical diagnosis.

AUTHOR INFORMATION *Corresponding Author Prof. Qin Hu Tel. / fax: +86-2586868468; E-mail: [email protected] ORCID Qin Hu: 0000-0002-4077-8760 Notes The authors declare no competing financial interest.

ASSOCIATED CONTENT Supporting Information TEM image, Uv-vis, FT-IR spectra of N/Cu-CDs and N-CDs, fluorescence spectra of CDs, zeta potential of materials in the probe, the optimized conditions of detection, precision and accuracy of determination of cholesterol and xanthine.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 61775099 and No. 81173016).

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

REFERENCES （1）Xu, X.; Ray, R.; Gu, Y.; Ploehn, H. J.; Gearheart, L.; Raker, K.; Scrivens, W. A., Electrophoretic Analysis and Purification of Fluorescent Single-Walled Carbon Nanotube Fragments. J. Am. Chem. Soc. 2004, 126, 12736-12737. （2）Liu, C.; Zhang, P.; Zhai, X.; Tian, F.; Li, W.; Yang, J.; Liu, Y.; Wang, H.; Wang, W.; Liu, W., Nano-Carrier for Gene Delivery and Bioimaging Based on Carbon Dots with PEI-Passivation Enhanced Fluorescence. Biomaterials 2012, 33, 3604-3611. （3）Zhang, J.; Yu, S. H., Carbon Dots: Large-Scale Synthesis, Sensing and Bioimaging. Mater. Today 2016, 19, 382-388. （4）Esteves da Silva, J. C. G.; Gonçalves, H. M. R., Analytical and Bioanalytical Applications of Carbon Dots. Trac-Trend. Anal. Chem. 2011, 30, 1327-1331. （5）Goncalves, H. M. R.; Duarte, A. J.; Esteves da Silva, J. C., Optical Fiber Sensor for Hg (II) Based on Carbon Dots. Biosens. Bioelectron. 2010, 26, 1302-1314. （6）Guo, X.; Wang, C. F.; Yu, Z. Y.; Chen, L.; Chen, S., Facile Access to Versatile Fluorescent Carbon Dots Toward Light-Emitting Diodes. Chem. Comm. 2012, 48, 2692-2698. （7）Li, Y.; Hu, Y.; Zhao, Y.; Shi, G.; Deng, L.; Hou, Y.; Qu, L., An Electrochemical Avenue to Green-Luminescent Graphene Quantum Dots as Potential Electron-Acceptors for Photovoltaics. Adv. Mater. 2011, 23, 776-783. （8）Huang, P.; Lin, J.; Wang, X.; Wang, Z.; Zhang, C.; He, M.; Wang, K.; Chen, F.; Li, Z.; Shen, G.; Cui, D.; Chen, X., Light-Triggered Theranostics Based on Photosensitizer-Conjugated Carbon Dots for Simultaneous Enhanced-Fluorescence Imaging and Photodynamic Therapy. Adv. Mater. 2012, 24, 5104-5114. （9）Ramanan, V.; Thiyagarajan, S. K.; Raji, K.; Suresh, R.; Sekar, R.; Ramamurthy, P., Outright Green Synthesis of Fluorescent Carbon Dots from Eutrophic Algal Blooms for In Vitro Imaging. ACS Sustainable Chem. Eng. 2016, 4, 4724-4731. （10）Rochat, T.; Boudebbouze, S.; Gratadoux, J.-J.; Blugeon, S.; Gaudu, P.; Langella, P.; Maguin, E., Proteomic Analysis of Spontaneous Mutants of Lactococcus Lactis: Involvement of GAPDH and Arginine Deiminase Pathway in H2O2 Resistance. Proteomics 2012, 12, 1792-1805. （11）Barman, M. K.; Jana, B.; Bhattacharyya, S.; Patra, A., Photophysical Properties of Doped Carbon Dots (N, P, and B) and Their Influence on Electron/Hole Transfer in Carbon Dots–Nickel (II) Phthalocyanine Conjugates. J. Phys. Chem. C 2014, 118, 20034-20041. （ 12 ） Qi, B.-P.; Bao, L.; Zhang, Z.-L.; Pang, D.-W., Electrochemical Methods to Study Photoluminescent Carbon Nanodots: Preparation, Photoluminescence Mechanism and Sensing. Acs Appl. Mater. Interface 2016, 8, 28372-28382. （ 13 ） Wang, C.-I.; Wu, W.-C.; Periasamy, A. P.; Chang, H.-T., Electrochemical Synthesis of Photoluminescent Carbon Nanodots from Glycine for Highly Sensitive Detection of Hemoglobin. Green Chem. 2014, 16, 2509. （14）Martindale, B. C. M.; Hutton, G. A. M.; Caputo, C. A.; Reisner, E., Solar Hydrogen Production Using Carbon Quantum Dots and a Molecular Nickel Catalyst. J. Am. Chem. Soc. 2015, 137, 6018-6025. （15）Qian, Z.; Shan, X.; Chai, L.; Ma, J.; Chen, J.; Feng, H., Si-Doped Carbon Quantum Dots: A Facile and General Preparation Strategy, Bioimaging Application, and Multifunctional Sensor. Acs Appl. Mater. Interface 2014, 6, 6797-6805. 18


Page 18 of 26

Page 19 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


（16）Siraj, N.; El-Zahab, B.; Hamdan, S.; Karam, T. E.; Haber, L. H.; Li, M.; Fakayode, S. O.; Das, S.; Valle, B.; Strongin, R. M.; Patonay, G.; Sintim, H. O.; Baker, G. A.; Powe, A.; Lowry, M.; Karolin, J. O.; Geddes, C. D.; Warner, I. M., Fluorescence, Phosphorescence, and Chemiluminescence. Anal. Chem. 2016, 88, 170-202. （17）Ding, H.; Yu, S.-B.; Wei, J.-S.; Xiong, H.-M., Full-Color Light-Emitting Carbon Dots with a Surface-State-Controlled Luminescence Mechanism. ACS Nano 2016, 10, 484-491. （18）Wu, Z. L.; Liu, Z. X.; Yuan, Y. H., Carbon Dots: Materials, Synthesis, Properties and Approaches to Long-wavelength and Multicolor Emission. J. Mater. Chem. B 2017, 5, 3794-3809. （19）Liu, Y.; Liao, M.; He, X.; Liu, X.; Kou, X.; Xiao, D., One-step Synthesis of Highly Luminescent Nitrogen-doped Carbon Dots for Selective and Sensitive Detection of Mercury(II) Ions and Cellular Imaging. Anal. Sci. 2015, 31, 971-977. （20）Xu, P.; Wang, C.; Sun, D.; Chen, Y.; Zhuo, K., Ionic Liquid as a Precursor to Synthesize Nitrogen- and Sulfur-co-doped Carbon Dots for Detection of Copper(II) Ions. Chem. Res. Chin. Univ. 2015, 31, 730-735. （21）Zong, J.; Yang, X.; Trinchi, A.; Hardin, S.; Cole, I.; Zhu, Y.; Li, C.; Muster, T.; Wei, G., Carbon Dots as Fluorescent Probes for "Off-On" Detection of Cu2+ and L-cysteine in Aqueous Solution. Biosens. Bioelectron. 2014, 51, 330-335. （22）Guo, Y.; Zhang, L.; Zhang, S.; Yang, Y.; Chen, X.; Zhang, M., Fluorescent Carbon Nanoparticles for The Fluorescent Detection of Metal Ions. Biosens. Bioelectron. 2015, 63, 61-71. （23）Song, Y.; Zhu, C.; Song, J.; Li, H.; Du, D.; Lin, Y., Drug-Derived Bright and Color-Tunable N-Doped Carbon Dots for Cell Imaging and Sensitive Detection of Fe3+ in Living Cells. Acs Appl. Mater. Interface 2017, 9, 7399-7405. （24）Wu, W.; Zhan, L.; Fan, W.; Song, J.; Li, X.; Li, Z.; Wang, R.; Zhang, J.; Zheng, J.; Wu, M.; Zeng, H., Cu-N Dopants Boost Electron Transfer and Photooxidation Reactions of Carbon Dots. Angew. Chem. Int. Ed. 2015, 54, 6540-6544. （25）Xue, N.; Kong, X.; Song, B.; Bai, L.; Zhao, Y.; Lu, C.; Shi, W., Cu-Doped Carbon Dots with Highly Ordered Alignment in Anisotropic Nano-Space for Improving the Photocatalytic Performance. Solar RRL 2017, 1, 1700029. （26）Chen, S.; Hai, X.; Chen, X.-W.; Wang, J.-H., In Situ Growth of Silver Nanoparticles on Graphene Quantum Dots for Ultrasensitive Colorimetric Detection of H2O2 and Glucose. Anal. Chem. 2014, 86, 6689-6694. （27）Li, N.; Than, A.; Wang, X.; Xu, S.; Sun, L.; Duan, H.; Xu, C.; Chen, P., Ultrasensitive Profiling of Metabolites Using Tyramine-Functionalized Graphene Quantum Dots. ACS Nano 2016, 10, 3622-3629. （28）Liu, J.-W.; Luo, Y.; Wang, Y.-M.; Duan, L.-Y.; Jiang, J.-H.; Yu, R.-Q., Graphitic Carbon Nitride Nanosheets-Based Ratiometric Fluorescent Probe for Highly Sensitive Detection of H2O2 and Glucose. Acs Appl. Mater. Interface 2016, 8, 33439-33445. （29）Amjadi, M.; Manzoori, J. L.; Hallaj, T., A Novel Chemiluminescence Method for Determination of Bisphenol A Based on The Carbon Dot-Enhanced HCO3--H2O2 System. J. Lumin. 2015, 158, 160-164. （30）Liu, F.; Bing, T.; Shangguan, D.; Zhao, M.; Shao, N., Ratiometric Fluorescent Biosensing of Hydrogen Peroxide and Hydroxyl Radical in Living Cells with Lysozyme–Silver Nanoclusters: Lysozyme as Stabilizing Ligand and Fluorescence Signal Unit. Anal. Chem. 2016, 88, 10631-10638. （31）RA, H.; SH, M., Fluorescent Detection Method Based on Enzyme Activated Conversion of A 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fluorophore Precursor Substrate. U.S.Patent 1991, 5017475. （32）Du, F.; Min, Y.; Zeng, F.; Yu, C.; Wu, S., A Targeted and FRET- Based Ratiometric Fluorescent Nanoprobe for Imaging Mitochondrial Hydrogen Peroxide in Living Cells. Small 2014, 10, 964-972. （ 33） Liu, L.; Chen, L.; Liang, J.; Liu, L.; Han, H., A Novel Ratiometric Probe Based on Nitrogen-Doped Carbon Dots and Rhodamine B Isothiocyanate for Detection of Fe3+ in Aqueous Solution. J. Anal. Methods Chem. 2016, 2016, 4939582-4939587. （34）Xia, J.; Yuan, S.; Wang, Z.; Kirklin, S.; Dorney, B.; Liu, D.-J.; Yu, L., Nanoporous Polyporphyrin as Adsorbent for Hydrogen Storage. Macromolecules 2010, 43, 3325-3330. （35）Lin, Y.; Yao, B.; Huang, T.; Zhang, S.; Cao, X.; Weng, W., Selective Determination of Free Dissolved Chlorine Using Nitrogen-Doped Carbon Dots as A Fluorescent Probe. Microchimica Acta 2016, 183, 2221-2227. （36）Reckmeier, C. J.; Wang, Y.; Zboril, R.; Rogach, A. L., Influence of Doping and Temperature on Solvatochromic Shifts in Optical Spectra of Carbon Dots. J. Phys. Chem. C 2016, 120, 10591-10604. （37）Wang, H.; Cao, G.; Gai, Z.; Hong, K.; Banerjee, P.; Zhou, S., Magnetic/NIR-Responsive Drug Carrier, Multicolor Cell Imaging, And Enhanced Photothermal Therapy of Gold Capped Magnetite-Fluorescent Carbon Hybrid Nanoparticles. Nanoscale 2015, 7, 7885-7895. （38）Li, H.; He, X.; Kang, Z.; Huang, H.; Liu, Y.; Liu, J.; Lian, S.; Tsang, C. H. A.; Yang, X.; Lee, S. T., Water-Soluble Fluorescent Carbon Quantum Dots and Photocatalyst Design. Angew. Chem. Int. Ed. 2010, 49, 4430-4438. （39）Xu, X.; Bao, Z.; Zhou, G.; Zeng, H.; Hu, J., Enriching Photoelectrons via Three Transition Channels in Amino-Conjugated Carbon Quantum Dots to Boost Photocatalytic Hydrogen Generation. Acs Appl. Mater. Interface 2016, 8, 14118-14124. （40）Yang, L.; Park, C. H.; Son, Y. W.; Cohen, M. L.; Louie, S. G., Energy Gaps in Graphene Nanoribbons. Phys. Rev. Lett. 2007, 99, 186801-186807. （41）Fan, L.; Qin, J. C.; Li, T. R.; Wang, B. D.; Yang, Z. Y., A Novel Rhodamine Chromone-based "Off-On" Chemo Sensor for The Differential Detection of Al(III) and Zn(II) in Aqueous Solutions. Sens. Actuator B-Chemical. 2014, 203, 550-556. （42）Sabherwal, P.; Shorie, M.; Pathania, P.; Chaudhary, S.; Bhasin, K. K.; Bhalla, V.; Suri, C. R., Hybrid Aptamer-Antibody Linked Fluorescence Resonance Energy Transfer Based Detection of Trinitrotoluene. Anal. Chem. 2014, 86, 7200-7204. （43）Huang, S.; Wang, L. M.; Huang, C. S.; Su, W.; Xiao, Q., Amino-Functionalized Graphene Quantum Dots Based Ratiometric Fluorescent Nanosensor for Ultrasensitive and Highly Selective Recognition of Horseradish Peroxidase. Sens. Actuator B-Chemical. 2016, 234, 255-263. （44）Liu, H.; Xu, C.; Bai, Y.; Liu, L.; Liao, D.; Liang, J.; Liu, L.; Han, H., Interaction Between Fluorescein Isothiocyanate and Carbon Dots: Inner Filter Effect and Fluorescence Resonance Energy Transfer. Spectroch. Acta A 2017, 171, 311-316. （45）Wang, W.; Ji, X.; Kapur, A.; Zhang, C.; Mattoussi, H., A Multifunctional Polymer Combining the Imidazole and Zwitterion Motifs as a Biocompatible Compact Coating for Quantum Dots. J. Am. Chem. Soc. 2015, 137, 14158-14172. （46）Xu, Q.; Wei, J.; Wang, J.; Liu, Y.; Li, N.; Chen, Y.; Gao, C.; Zhang, W.; Sreeprasede, T. S., Facile Synthesis of Copper Doped Carbon Dots and Their Application as A "Turn-off" Fluorescent Probe in The Detection of Fe3+ Ions. Rsc Adv. 2016, 6, 28745-28750. （ 47 ） Mohapatra, S.; Sahu, S.; Sinha, N.; Bhutia, S. K., Synthesis of A Carbon-Dot-Bbased Photoluminescent Probe for Selective and Ultrasensitive Detection of Hg2+ in Water and Living Cells. 20


Page 20 of 26

Page 21 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Analyst 2015, 140, 1221-1228. （48）Dong, Y.; Zhang, J.; Jiang, P.; Wang, G.; Wu, X.; Zhao, H.; Zhang, C., Superior Peroxidase Mimetic Activity of Carbon Dots-Pt Nanocomposites Relies on Synergistic Effects. New J. Chem. 2015, 39, 4141-4146. （49）Maaoui, H.; Teodoresu, F.; Wang, Q.; Pan, G.-H.; Addad, A.; Chtourou, R.; Szunerits, S.; Boukherroub, R., Non-Enzymatic Glucose Sensing Using Carbon Quantum Dots Decorated with Copper Oxide Nanoparticles. Sensors 2016, 16, 1720-1729. （50）Dalkıran, B.; Erden, P. E.; Kılıç, E., Construction of an Electrochemical Xanthine Biosensor Based on Graphene/Cobalt Oxide Nanoparticles/Chitosan Composite for Fish Freshness Detection. J. Turk. Chem. Soc., Section A 2016, 3, 23-44. （51）Contreras, F.; Lares, M.; Castro, J.; Velasco, M.; Rojas, J.; Guerra, X.; Chacin, M.; Dowling, V.; Bermudez, V., Determination of Non-HDL Cholesterol in Diabetic and Hypertensive Patients. . Am. J. Ther. 2010, 17, 337-340. （52）Lin, T.; Zhong, L.; Chen, H.; Li, Z.; Song, Z.; Guo, L.; Fu, F., A Sensitive Colorimetric Assay for Cholesterol Based on The Peroxidase-like Activity of MoS2 Nanosheets. Microchim. Acta 2017, 184, 1233-1237. （53）Sun, X.-T.; Zhang, Y.; Zheng, D.-H.; Yue, S.; Yang, C.-G.; Xu, Z.-R., Multitarget Sensing of Glucose and Cholesterol Based on Janus Hydrogel Microparticles. Biosens. Bioelectron. 2017, 92, 81-86. （54）Hu, Y.; Zhang, Z., Determination of Free Cholesterol Based on A Novel Flow-injection Chemiluminescence Method by Immobilizing Enzyme. J. Lumin. 2008, 23, 338-343. （55）Pundir, C. S.; Devi, R., Biosensing Methods for Xanthine Determination: A Review. Enzyme Microb. Technol. 2014, 57, 55-62.

Figures and Tables

Scheme 1. Schematic illustration of N/Cu-CDs synthesis and the principle of the 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ratiometric fluorescence universal platform based on N/Cu-CDs to detect the metabolites (cholesterol and xanthine) participating in H2O2-generation reactions.

22


Page 22 of 26

Page 23 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. The TEM image of N/Cu CDs (A). Insert: high-resolution TEM and particle size distribution images of N/Cu CDs. FT-IR spectra of N/Cu-CDs and N-CDs (B). XPS spectrum of N/Cu-CDs (C), and high resolution XPS spectra of C1s (D), N 1s (E) and Cu 2p (F).

Figure 2. The UV-vis absorption spectrum of N/Cu-CDs (A). The fluorescence spectra of N/Cu-CDs excited from 300 nm to 390 nm (B).

Figure 3. The fluorescence spectra of N/Cu-CDs, N/Cu-CDs + OPD, N/Cu-CDs + HRP, N/Cu-CDs + DAP (A).The UV-vis absorption spectra of OPD and DAP, and the fluorescence spectrum of N/Cu-CDs (B). The fluorescence lifetime spectra of N/Cu-CDs and N/Cu-CDs + DAP (C). DAP was the product of OPD oxidized by HRP and H2O2 in all measurements.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Fluorescence spectra of the ratiometric fluorescence probe in the presence of various concentrations of H2O2 (A). The curve of ratiometric fluorescence intensity versus the concentration of H2O2 in range of 0 − 2.0 mM (B). Insert: calibration curve of H2O2 detection in the range of 0.025 − 400 µM.

Figure 5. Fluorescence spectra of the ratiometric fluorescence probe in the presence 24


Page 24 of 26

Page 25 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of various concentrations of cholesterol (A). The curve of I572/I460 (ratiometric fluorescence intensity) versus the concentration of cholesterol (0 – 0.60 mM) (B). Calibration curve of the cholesterol detection in range of 0.05 − 100 µM (C). Selectivity of cholesterol detection, the concentrations of cholesterol and interferents were 0.05 mM and 0.50 mM, respectivvely (D).

Figure 6. Fluorescence spectra of ratiometric fluorescence probe in the presence of various concentrations of xanthine (A). The curve of I572/I460 (ratiometric fluorescence intensity) versus the concentration of xanthine (0 – 1.0 mM) (B). Calibration curve of xanthine detection in the range of 0.25 – 75.00 µM (C). Selectivity of xanthine detection. The concentrations of cholesterol and interferents were 0.05 mM and 0.50 mM, respectively (D). 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

96 x 96 DPI

26


Page 26 of 26

A Ratiometric Fluorescence Universal Platform Based on N, Cu

Recommend Documents