B,N-carbon dots-based ratiometric fluorescent and colorimetric dual-readout sensor for H2O2 and H2O2-involved metabolites detection using ZnFe2O4 magnetic microspheres as peroxidase mimics

Accepted Manuscript Title: B,N-carbon dots-based ratiometric fluorescent and colorimetric dual-readout sensor for H2 O2 and H2 O2 -involved metabolites detection using ZnFe2 O4 magnetic microspheres as peroxidase mimics Authors: Na Xiao, Shi Gang Liu, Shi Mo, Yu Zhu Yang, Lei Han, Yan Jun Ju, Nian Bing Li, Hong Qun Luo PII: DOI: Reference:

S0925-4005(18)31341-8 https://doi.org/10.1016/j.snb.2018.07.097 SNB 25066

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

12-3-2018 3-7-2018 19-7-2018

Please cite this article as: Xiao N, Liu SG, Mo S, Yang YZ, Han L, Ju YJ, Li NB, Luo HQ, B,N-carbon dots-based ratiometric fluorescent and colorimetric dual-readout sensor for H2 O2 and H2 O2 -involved metabolites detection using ZnFe2 O4 magnetic microspheres as peroxidase mimics, Sensors and Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.07.097 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

B,N-carbon dots-based ratiometric fluorescent and colorimetric dual-readout sensor for H2O2 and H2O2involved metabolites detection using ZnFe2O4 magnetic microspheres as peroxidase mimics

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Na Xiaoa, Shi Gang Liua, Shi Mob, Yu Zhu Yanga, Lei Hana, Yan Jun Jua,

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Nian Bing Lia,* and Hong Qun Luoa,*

School of Chemistry and Chemical Engineering, Southwest University, Chongqing

Department of Physics, City University of Hong Kong, Tat Chee Avenue, Kowloon,

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400715, PR China

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Hong Kong, China

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Corresponding Author. Tel: +86 23 68253237; fax: +86 23 68253237; E-mail

Graphical Abstract

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address: [email protected] (NB Li); [email protected] (HQ Luo).

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Research Highlights

 Complex synthesis and specific modifications are needless for ZnFe2O4 and B,N-CDs.

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 Colorimetric and ratiometric fluorescence assay for H2O2, glucose and UA is designed.

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 Dual-readout signal makes the experimental results more convincing.

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 The proposed method is simple, flexible, high specific and sensitive.

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 This method has potential applications in H2O2-involved metabolites detection.

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

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Abstract In this work, a two-dimensional optical sensing platform for H2O2, glucose, and uric acid (UA) detection is developed, which integrates the advantages of colorimetric and

peroxidase-like activity are synthesized to catalyze the oxidation of o-

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ratiometric fluorescent techniques. ZnFe2O4 magnetic microspheres with an intrinsic

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phenylenediamine in the presence of H2O2, producing a typical yellow substance

(oxOPD) with an absorption peak at 420 nm. The oxOPD can significantly quench the fluorescence of boron and nitrogen co-doped CDs (B,N-CDs) at 430 nm through the

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inner filter effect and generate a new fluorescence emission peak at 556 nm. Thus, the

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fluorescence intensity ratio (I556/I430) can be utilized for quantitative analysis of the

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concentrations of H2O2 and H2O2-involved metabolites (glucose and UA). The

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colorimetric “naked-eye” readout based on the color change of solution can also be

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established to determine H2O2, glucose, and UA levels. The detection limit based on colorimetric sensing for H2O2, glucose, and UA are 0.09, 0.9, and 0.9 μM,

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respectively, and 0.1, 8, and 1 μM using ratiometric fluorescent sensing. Furthermore,

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this strategy can detect glucose and UA in human serum with satisfactory results, and provide potential applications in the detection of metabolites related to H2O2 release.

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Keywords: Colorimetry; ratiometric fluorescence; H2O2; H2O2-involved metabolites; ZnFe2O4 magnetic microspheres; B,N-carbon dots

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1. Introduction Hydrogen peroxide (H2O2), as a kind of reactive oxygen species, is a vital metabolite in living system. Also, it is considered to be a signal molecule and biomarker of oxidative stress and physiological activity [1]. Additionally, H2O2

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possesses a substantial risk for biological system and has a significant relationship

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with some central nervous system diseases [2,3]. Numerous metabolites in human

body (glucose, uric acid, cholesterol, lactate, xanthine and choline) can produce H2O2 as a byproduct through the corresponding enzyme catalytic reaction [4,5].

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Accordingly, it is valuable and significant to develop effective detection methods for

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significant diagnostic parameter in serum [6].

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H2O2. For many metabolic-related diseases, such as diabetes, glucose level is a

Uric acid (UA) is another important marker molecule of many diseases related to

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the change of UA level in human urine and serum. Ordinarily, the normal level of UA

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should be 0.12-0.46 mM in serum [7]. It has been demonstrated that an excessive concentration of UA in serum (hyperuricemia) is closely associated with numerous

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metabolic disorders [7,8]. In addition, an extreme low concentration of UA results in oxidative stress or multiple sclerosis [9,10]. Hence, quantitative detection the

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metabolites level seems particularly necessary in the diagnosis of related diseases. Until now, various analytical techniques for detecting H2O2, glucose, and UA

have been reported, mainly including liquid chromatography [11,12], electroanalysis [6,13], and optical methods [14-16]. Among these methods, optical techniques like 4

fluorescence (FL) and colorimetric/UV–vis absorption methods have attracted considerable attention due to their rapid response, high sensitivity, practicality, simplicity and low cost [17,18]. Because of the easy identification of color changes with the naked eye, colorimetric biosensors can be used for on-site analysis and point-

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of-care test without using expensive or sophisticated instruments, suggesting that it is significantly valuable for real-time measurement.

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Horseradish peroxidase (HRP) plays an important role in biomolecular

colorimetric analysis [19]. In particular, o-phenylenediamine (OPD) is chosen as the

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peroxidase substrate for HRP since colorless OPD can be catalytically oxidized by

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HRP/H2O2 to form the yellow product (oxOPD), which has a strong fluorescence

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emission when excited at 430 nm [20,21]. Nevertheless, due to the inherent

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shortcomings of natural enzymes, for example, the high cost of extraction and

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separation, the strict conditions of the catalytic reaction, and the poor stability [22], the widespread application of natural enzymes is greatly restricted. To solve this problem,

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the peroxidase mimicking with good stability has been developed. And several

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nanomaterials with intrinsic peroxidase-like property have been reported, including nanoparticles and nanocomposites [23-31], nanoclusters [32-34], nanosheets [35], and

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graphene oxide [36]. These enzyme-like nanomaterials show many intrinsic advantages over natural enzymes, such as simple synthesis, less consumption, high catalytic activity, low cost, and good stability. Su and co-workers reported that ZnFe2O4 magnetic microspheres had the several advantages over HRP and other enzyme-like 5

nanomaterials, such as monodispersion, high catalytic efficiency, easy and rapid separation and good stability [37]. However, they only used ZnFe2O4 magnetic microspheres to build colorimetric sensors for measuring the glucose level. Therefore, it is meaningful to designing a dual readout signal for colorimetric and ratio

Until now, the application of gold nanoparticles [2,7], upconversion

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fluorescence determination of analytes using ZnFe2O4 as a substitute for HRP.

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nanoparticles [10,38], CdS quantum dots [39]), carbon nanodots [40,41], and CdTe nanoparticles [42] as fluorescent probes to detect metabolites have also attracted

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considerable attention. Unfortunately, most of those reported fluorescent sensing

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platforms are based on single fluorescence measurement, which is easily affected by

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the instrumental efficiency, probe concentrations, and environmental changes. These

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factors may interfere with signal output. In contrast, ratiometric sensing can

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effectively normalize the variation and obtain more accurate readouts by its selfcalibration of two different emission bands [43-45]. Carbon dots (CDs) have been

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developed as a new promising fluorescent probes due to their chemical stability,

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excellent fluorescence properties, low toxicity, and biocompatibility [46]. Particularly, CDs can be applied as a substitute for the semiconductor quantum dots, which usually consist of toxic heavy metals [47]. Although the development of biosensing platforms

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have made great progress based on fluorescent CDs, heteroatom-doped CDs are rarely used as ratio fluorescent probes to determine H2O2 [48]. Due to the excellent properties of heteroatom-doped CDs, it is particularly necessary to construct a good 6

selective, highly sensitive, and easily-operated multifunctional nanoplatform for detecting H2O2 and H2O2-generation reaction involved metabolites. As far as we know, the combination of heteroatom-doped CDs and ZnFe2O4 magnetic microspheres to construct the ratiometric fluorescence detection of H2O2 has not been

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reported.

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In this study, we synthesized B,N-CDs and ZnFe2O4 magnetic microspheres, the B,N-CDs have a fluorescence emission peak at 430 nm upon excitation at 365 nm. With an intrinsic peroxidase-like activity, the prepared ZnFe2O4 magnetic

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microspheres can be combined with the B,N-CDs to develop a colorimetric and

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ratiometric fluorescent sensing strategy to detect H2O2, glucose, and UA. The

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mentioned ZnFe2O4 magnetic microspheres can catalyze the oxidation of OPD in the presence of H2O2, glucose/glucose oxidase (GOx), and UA/uricase to form yellow

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oxOPD, which emits a yellow light emission at 556 nm. The absorption band of

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oxOPD at 420 nm can overlap with the emission peak of B,N-CDs at 430 nm, resulting in fluorescence quenching of B,N-CDs owing to the inner filter effect (IFE).

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The results showed that the fluorescence intensity at 430 nm decreased gradually and the fluorescence intensity at 556 nm increased continuously. Therefore, the

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fluorescence intensity ratio of oxOPD to B,N-CDs (I556/I430) can be applied to quantitatively analyze H2O2, glucose, and UA. Furthermore, the absorption peak of oxOPD at 420 nm increased gradually and the color of the solution became deep with the increase of H2O2, glucose, and UA concentrations. Thus, a colorimetric sensing 7

platform was established to determine the concentrations of H2O2, glucose, and UA based on the color change. In the end, B,N-carbon dots-based ratiometric fluorescent and colorimetric dual-readout sensor for sensitive and selective detection of H2O2, glucose, and UA using ZnFe2O4 magnetic microspheres as peroxidase mimics was

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constructed.

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2. Experimental section 2.1. Materials

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3-Aminophenylboronic acid monohydrate (3-APBA⋅H2O), UA, glutathione

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(GSH), dopamine (DA), lysine (Lys), and leucine (Leu) were obtained from Aladdin

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Reagent Co., Ltd. (Shanghai, China). NaCl, urea, ascorbic acid (AA), KCl, H2O2,

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(30%), OPD, FeCl3.6H2O, ZnCl2, polyethylene glycol, ethylene glycol, CH3COONa

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(NaAc) and CH3COOH (HAc) were acquired from Kelong Chemical Reagent Co., Ltd. (Chengdu, China). Glucose, maltose, fructose, and lactose were purchased from

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Sigm-Aldrich (Shanghai, China). Cysteine (Cys), GOx, and uricase were ordered

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from Sangon Biotech Co. Ltd. (Shanghai, China). All the reagents used in this work were analytical grade and ultrapure water (18.2 MΩ cm) was used throughout the

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study.

2.2. Apparatus The fluorescence was measured on an F-2700 spectrofluorophotometer (Hitachi Ltd., Japan). A UV-2450 spectrophotometer (Shimadzu Instrument Co., Ltd., China) 8

was utilized to collect the UV-vis absorption spectra. The transmission electron microscopy (TEM) and scanning electron microscope (SEM) images were collected using the JEM-2100 (JEOL Ltd., Japan) and Hitachi-4800 (Hitachi Co., Ltd., Japan), respectively. Fourier transform infrared (FTIR) spectrum was collected using a

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Bruker IFS 113v spectrometer (Bruker, Germany). X-ray photoelectron spectroscopy (XPS) was measured with an ESCALab 250Xi (Thermo Fisher Scientific, USA). An

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X-ray diffraction (XRD) pattern was conducted on a Bruker-D8 diffractometer

(BrukerAXS Co., Ltd., Germany). Fluorescence lifetime decays were determined

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using a FLSP920 fluorescence spectrometer (Edinburgh, UK). ζ potentials were

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measured on a NanoBrook Omni (Brookhaven Instrument Co., Ltd., USA). A

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2.3. Preparation of B,N-CDs

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monitor the pH of the solutions.

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METTLER TOLEDO-FE28 pH meter (Mettler Toledo, China) was applied to

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The B,N-CDs were synthesized through a hydrothermal method using a sole precursor 3-APBA⋅H2O as the nitrogen, carbon, and boron source. Briefly, 3-

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APBA⋅H2O (5.0 mg) was dissolved into ultrapure water (5.0 mL). Then, the pH value of solution was adjusted to 10.0 with NaOH (1.0 M) under the constant agitation. The

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resulting solution was transferred to the Teflon-lined autoclave and kept at 160 °C for 8 h. After the reactor cooled down to room temperature, the resulting yellowishbrown solution was centrifuged at 10, 000 rpm for 20 min to remove large aggregates.

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Finally, the obtained suspension of B,N-CDs were stored at 4 °C for further applications. 2.4. Synthesis of ZnFe2O4 magnetic microspheres

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ZnFe2O4 magnetic microspheres were synthesized via a hydrothermal method according to the previously reported synthesis procedure [49]. Briefly, 1.35 g of

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FeCl3.6H2O and 0.34 g of ZnCl2 were dissolved into 40 mL of ethylene glycol to form a yellow clear solution. Subsequently, 1.0 g of polyethylene glycol 4000 and 3.6 g of NaAc were added to the above solution. The mixture was vigorously stirred for 30

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min, then transferred into a 100 mL Teflon-lined autoclave and heated at 200 °C for 8

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h. After cooled down to room temperature, the black products were rinsed with

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2.5. Procedures for sensing H2O2

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absolute ethanol for four times and dried under vacuum.

Briefly, 5 mM OPD, different volumes of H2O2, HAc-NaAc buffer solution (0.1

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M, pH 6.15) and 0.6 mg/mL ZnFe2O4 were mixed (the final volume is 600 μL) and

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incubated at 60 °C for 30 min. Then, ZnFe2O4 magnetic microspheres were separated from the mixture solution by a magnet (time < 1 min). After that, 4 μL of B,N-CDs (1

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mg/mL) was added to 500 μL of the above mixture and incubated at room temperature for 5 min. The fluorescence emission spectra under 365 nm excitation and absorption spectra at 420 nm were recorded, respectively. 2.6. Procedures for sensing glucose and UA 10

For the detection of glucose, 15 μL of glucose with various concentrations were firstly mixed with 3 μL of 2 mg/mL GOx and 12 μL of PBS buffer (1/15 M, pH 6.15) and incubated at 37 °C for 30 min to produce H2O2, the following operations were consistent with Section 2.5. For the detection of UA, various concentrations of

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standard UA were firstly mixed with 100 μg/mL uricase and incubated at 40 °C for 30 min to produce H2O2. The following procedures were performed according to Section

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2.5.

For the analysis of glucose and UA in human serum samples, the serum samples

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were firstly centrifuged at 12,000 rpm for 20 min to remove particulates. Then, the

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serum samples were spiked with different concentrations of standard glucose and UA

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solutions, respectively. The final serum samples were 200-fold and 75-fold diluted for detecting glucose and UA, respectively. The fluorescence emission spectra of the

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diluted serum samples were measured as described in Section 2.6.

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2.7. Reusability of the ZnFe2O4 magnetic microspheres as the peroxidase mimics

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The reusability of ZnFe2O4 magnetic microspheres was measured after five consecutive repeated cycles according to the procedure described in Section 2.5. After reaction for 30 min, the ZnFe2O4 magnetic microspheres were separated from the

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mixture solution by a magnet before measuring the absorbance and fluorescence intensity of oxOPD. The recycled magnetic microspheres were washed three times with ultrapure water and then reused in the next cycle.

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3. Results and discussion 3.1. Characterization of the B,N-CDs and ZnFe2O4 magnetic microspheres The morphology and structure of the B,N-CDs were analyzed by the TEM and

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high-resolution TEM (HRTEM) images. As shown in Fig. S1, the B,N-CDs are of good monodispersity with a narrow size distribution around 2.0-2.5 nm. The HRTEM

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image of B,N-CDs (Fig. 1A) reveals well-resolved lattice fringes with a lattice spacing of 0.22 nm, which corresponds to the graphite 100 [50]. The FT-IR

spectroscopy was applied to characterize the surface structure of B,N-CDs, as shown

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in Fig. S2. The absorption peak at 3385 cm−1 is ascribed to NH stretching vibration.

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The band at 1651 cm−1 is assigned to C=C stretching vibration and 1402 cm−1

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represented CN stretching vibration. Other stretching vibration bonds, BO (1340 cm−1), COC (1267 cm−1), COH (1123 cm−1), CB (1094 cm−1), BOH (1024

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cm−1) [50], and OH (650-930 cm−1) are also presented in the spectrum. The above

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observations indicated that the functionalized groups, such as NH2, C=O, COOH and OH, existed in the B, N co-doped CDs. The XPS spectra further confirmed the

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above results. As shown in Fig. S3A, the B,N-CDs contain B, C, N, and O four dominant elements which correspond to the peaks at 531.6, 399.6, 285.6, and 191.6

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eV, respectively. The high-resolution survey scan spectrum of C 1s (Fig. S3B) reveals four peaks at 288.2, 286.2, 284.8, and 283.9 eV that correspond to C=O, C−O/C−N, C−C/C=C, and C−B bonds [51]. The three peaks at 400.9, 399.6, and 398.9 eV in the N 1s spectrum (Fig. S3C) are assigned to graphitic N, pyrrolic N, and pyridinic N 12

[52], respectively. The B 1s spectrum (Fig. S3D) shows two peaks at 192.7 and 191.8 eV, which are attributed to B−O and B−C bonds. The optical properties of B,N-CDs can be observed in Fig. 1B, the fluorescence peak position at 430 nm was barely changed under different excitation wavelengths (320 to 390 nm). The excitation-

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independent emission characteristic was probably attributed to the less surface defects and narrow size distribution of B,N-CDs [53,54]. B,N-CDs also possessed an

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excellent optical stability, Fig. S4A and S4B show that the normalized fluorescence

intensity of B,N-CDs were almost unchanged when it was incubated 1 h at pH (3.0-

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7.0) and temperature (25-90 °C), respectively. At the same time, the fluorescence

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intensity of prepared B,N-CDs had no significant attenuation after storage for two

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months under 4 °C (Fig. S4C).

The morphology of the ZnFe2O4 magnetic microspheres was observed using

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SEM. As shown in Fig. 1C, ZnFe2O4 magnetic microspheres are spherical in shape

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and dispersive with average diameter of 260 nm. Moreover, the XRD pattern of the as-prepared ZnFe2O4 magnetic microspheres is shown in Fig. 1D. The diffraction

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peaks at 62.5°, 56.8°, 53.2°, 43.0°, 35.4°, and 30.0° are corresponding to the (440), (511), (422), (400), (311), and (220) planes of ZnFe2O4 (JCPDS: 22-1012),

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respectively. The XRD data illustrated that the ZnFe2O4 magnetic microspheres were a well-defined spinel ferrite compounds. (Here Fig. 1) 3.2. Peroxidase mimics activity of ZnFe2O4 magnetic microspheres 13

To evaluate the peroxidase-like activity of ZnFe2O4 magnetic microspheres, the peroxidase substrate OPD was studied in the presence of ZnFe2O4 magnetic microspheres and H2O2. From Fig. S5A, the typical oxOPD absorption peak at 420 nm was observed in the B,N-CDs/ZnFe2O4/H2O2/OPD system, within 30 min reaction

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under 60 °C (curve 5) and the color of the solution became deep yellow (photo 5 inset in Fig. S5A). By comparison, other control experiments displayed that the B,N-CDs,

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B,N-CDs/OPD, B,N-CDs/ZnFe2O4/OPD, and B,N-CDs/H2O2/OPD systems had no obvious absorption peak at 420 nm and no significant color change. These results

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confirmed that ZnFe2O4 magnetic microspheres exhibit peroxidase mimics activity

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and can accelerate the oxidation of OPD in the presence of H2O2 to produce oxOPD.

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Thus, H2O2, glucose, and UA can be detected by monitoring changes in absorbance at

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420 nm. Meanwhile, as shown in Fig. S5B, the fluorescence intensity of the oxOPD at

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556 nm was gradually increased with increasing H2O2 concentration, indicating that the catalytic activity of ZnFe2O4 magnetic microspheres relied on the H2O2

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concentration.

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To further investigate the effect of B,N-CDs on the enzymatic properties of ZnFe2O4 magnetic microspheres, we compared the peroxidase-like activities of B,N-

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CDs and ZnFe2O4 magnetic microspheres. As shown in Fig. S5A (curves 4 and 5), the mimic enzyme activity was only ascribed to ZnFe2O4 magnetic microspheres, excluding B,N-CDs. The absorbance (Fig. 2A) and fluorescence intensity (Fig. S6) of OPD catalyzed by ZnFe2O4 magnetic microspheres were consistent with those in the 14

absence and presence of B,N-CDs, respectively. All the data indicated that B,N-CDs do not affect the peroxidase-like activity of ZnFe2O4 magnetic microspheres, and there was no increase in enzymatic properties of ZnFe2O4 magnetic microspheres when they were mixed together.

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In addition, the stability of ZnFe2O4 magnetic microspheres was also

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investigated after incubation at different pH and temperatures (Fig. S7A and S7B).

The results showed that there was no significant change in the catalytic performance of ZnFe2O4 magnetic microspheres incubated for 2 h at pH (1.0-12.0) and temperature

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(0-80 °C). The catalytic activity of ZnFe2O4 magnetic microspheres remained stable

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even after one month (Fig. S7C), which indicated that it had good stability during

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long-term storage.

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3.3. Reusability of the ZnFe2O4 magnetic microspheres The reusability of ZnFe2O4 magnetic microspheres as the peroxidase mimics was

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examined by cyclic catalytic experiments. The ZnFe2O4 samples were collected after

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each cycle and reused in the next cycle. As shown in Fig. S8A and S8B, the catalytic activity of ZnFe2O4 magnetic microspheres decreased slightly in the fifth cycle, which may be ascribed to the loss of magnetic microspheres in each cycle. However, the

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activity remained above 85% after five consecutive cycles. These results implied that the ZnFe2O4 magnetic microspheres possessed outstanding reusability in the process of catalytic reaction.

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3.4. Colorimetric and ratiometric fluorescent response mechanism for H2O2, glucose, and UA The response mechanism of the colorimetric and ratiometric fluorescent sensing for H2O2, glucose, and UA are illustrated in Scheme 1: (1) H2O2 is produced from

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glucose and UA oxidized by their specific oxidoreductase (GOx and uricase) and O2

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(Scheme 1A); (2) Scheme 1B shows that the OPD is oxidized by H2O2 with ZnFe2O4 magnetic microspheres, producing oxOPD with a yellow light emission at 556 nm (inset 1). Meanwhile, a typical yellow color can be easily observed by naked eye

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(inset 2) with an absorption peak at 420 nm; (3) In Scheme 1A, the ZnFe2O4 magnetic

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microspheres are removed by a magnet and the oxidation products oxOPD can

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effectively quench the fluorescence of B,N-CDs (emission at 430 nm) and generate a new fluorescence emission peak at 556 nm. The absorbance of oxOPD at 420 nm and

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the fluorescence intensity ratio of oxOPD to B,N-CDs (I556/I430) are determined for

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UA.

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quantitative colorimetric and ratiometric fluorescence analysis of H2O2, glucose, and

(Here Scheme 1)

The possible mechanism for B,N-CDs fluorescence quenching was IFE, which

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refers to the good overlap between the excitation or emission spectra of fluorophores and the absorption spectrum of the absorber [55]. As shown in Fig. 2B, the oxOPD had an absorption band at 420 nm, which significantly overlapped with the emission spectrum (430 nm) of B,N-CDs to induce IFE. To further confirm the fluorescence 16

quenching mechanism, the UV–vis absorption spectroscopy, fluorescence lifetime, and zeta potential were measured. The UV–vis absorption peak of oxOPD remained unchanged after adding B,N-CDs in Fig. 2A, which suggested that the ground-state complex was not formed and fluorescence resonance energy transfer (FRET) between

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them was excluded [56]. As shown in Fig. 2C, the fluorescence lifetimes of B,N-CDs before and after adding oxOPD were 3.41 and 3.33 ns, respectively. Almost invariable

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fluorescence lifetime suggested that there was no energy or electron transfer between B,N-CDs and oxOPD [20,57,58]. Moreover, the zeta potentials of oxOPD and B,N-

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CDs dispersed in the HAc-NaAc buffer solution (0.1 M, pH 6.15) were -25.85 and -

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19.77 mV, respectively. Both oxOPD and B,N-CDs possessed negative charge, the

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distance between B,N-CDs and oxOPD is hardly shorter than 10 nm owing to the

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weak electrostatic attraction of them. Therefore, the FRET between oxOPD and B,N-

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CDs cannot occur [59]. Based on the above results, it can be concluded that the

(Here Fig. 2)

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CDs.

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observed fluorescence quenching was attributed to IFE between oxOPD and B,N-

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3.5. Optimum conditions for the detection To acquire a better sensing performance toward H2O2, glucose, and UA, several

important parameters such as the concentrations of OPD and ZnFe2O4, pH value, incubation time, GOx and uricase levels were optimized. The fluorescence of B,NCDs was barely quenched by OPD when the concentration of OPD was less than or 17

equal to 7 mM (Fig. S9). The effect of OPD and ZnFe2O4 concentrations were studied by recording the fluorescence intensity ratio of oxOPD to B,N-CDs (I556/I420). As shown in Fig. S10A and S10B, with increasing OPD and ZnFe2O4 concentrations, the I556/I420 increased rapidly and reached balance when the concentrations of OPD and

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ZnFe2O4 were 5 mM and 0.6 mg/mL, respectively. Therefore, 5 mM OPD and 0.6 mg/mL ZnFe2O4 were selected as the optimum concentrations. The pH effect was

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investigated in the pH range from 3.5 to 7.0. Fig. S10C shows that the fluorescence intensity ratio of I556/I420 reached a maximum value in pH 6.15 HAc-NaAc buffer,

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thus pH 6.15 was selected as the optimal pH. The I556/I420 changed slightly 5 min later

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when B,N-CDs was added to oxOPD (Fig. S10D), thus, 5 min was selected as the

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appropriate incubation time for H2O2, glucose, and UA detection. For glucose and UA

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detection, the effects of GOx and uricase amounts were investigated (Fig. S10E and

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S10F). When the concentrations of GOx and uricase increased to 200 and 100 μg/mL, respectively, the I556/I420 barely changed and then kept constant. Hence, 200 μg/mL

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GOx and 100 μg/mL uricase were chosen in this experiment for glucose and UA

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detection, respectively.

3.6. Colorimetric and ratiometric fluorescent detection of H2O2

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Under optimal conditions, the linear response of H2O2 was evaluated. As

illustrated in Fig. 3A, the absorption peak of oxOPD at 420 nm increased gradually with increasing H2O2 concentration, accompanied with the easy and rapid visible change by the naked eye from colorless to yellow color: a noticeable color change can 18

be observed for 10 μM H2O2 (inset in Fig. 3A). Fig. 3B exhibits a linear response between the absorbance and H2O2 concentration in the range of 0.1-300 μΜ (A420 = 0.0053CH2O2 + 0.1543), with a good linear correlation (R2 = 0.9950). Meanwhile, the fluorescence intensity at 430 nm of B,N-CDs decreased gradually, while the

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fluorescence intensity at 556 nm of oxOPD increased with increasing concentration of H2O2 (Fig. 3C). The linear response in the range of 0.1-200 μM with a regression

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equation: I556/I430 = 0.0210CH2O2 + 0.0503 (R2 = 0.9926) (Fig. 3D). The detection limits (LOD) for H2O2 using the colorimetry and ratiometric fluorescence method based on

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3σ/k were calculated to be 90 nΜ and 0.1 μM, respectively. As summarized in Table

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or superior to the previously reported methods.

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S1, sensitive and convenient H2O2 detection made this sensing platform comparable

(Here Fig. 3)

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3.7. Colorimetric and ratiometric fluorescent detection of glucose and UA

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In view of the importance of monitoring serum glucose and UA levels in serum

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during disease diagnosis, we further applied the platform to detect glucose and UA. As can be seen from Fig. 4A and 4B, the absorption (A420) increased gradually with increasing concentration of glucose and UA, accompanied with a noticeable color

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change observed at 100 μM glucose and 20 μM UA, respectively (inset in Fig. 4A and 4B). And the concentration of UA (100-400 μM) had a remarkable color change from other concentrations (inset in Fig. 4B).The linear response for the detection of glucose and UA ranged from 1-1000 and 1-600 μM, with a regression equation: A420 = 19

0.0013Cglucose + 0.0837 (R2 = 0.9956) and A420 = 0.0017CUA + 0.1209 (R2 = 0.9970), respectively (Fig. 4C and 4D). Moreover, with the increase of glucose and UA concentrations, the fluorescence intensity ratio of I556/I430 also went up gradually (Fig. 5A and 5B). Fig. 5C and 5D shows the relationship between the I556/I430 and the

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concentration of glucose and UA. A good linear relationship was obtained in the glucose and UA concentration range of 20-1000 μM (I556/I430 = 0.0022Cglucose +

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0.1038, R2 = 0.9938) and 1-500 μM (I556/I430 = 0.0041CUA + 0.1397, R2 = 0.9962),

respectively. The LOD for glucose and UA using the colorimetric and ratiometric

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fluorescent methods were calculated to be 0.9 and 8 μΜ, 0.9 and 1 μΜ, respectively.

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Compared with many reported methods for glucose and UA detection (Table S2 and

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S3), our dual signal sensor is comparable or even superior to other methods. Glucose is normally present at 3.5 to 5.3 mM in serum for a healthy person [60]

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and ≥7 mM in diabetes patients [61]. UA is normally present at 120 to 460 μM in

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serum [7]. Thus, as a colorimetric and ratiometric fluorescent sensor to identify glucose and UA, the B,N-CDs/ZnFe2O4/OPD/GOx is promising and attractive to meet

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the demands of clinical application, owing to their low cost, reliability, and simplicity. Although the simple colorimetric sensing based on ZnFe2O4 magnetic

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microspheres provided better analytical performance (Tables S1, S2, and S3), the B,N-CDs had no mimic enzyme activity and cannot catalyze the oxidation of OPD in the presence of H2O2, as shown in Fig. S5A (curve 4). This means that H2O2 cannot be detected directly by colorimetry using B,N-CDs as the probe. Nevertheless, a 20

ratiometric fluorescence method based on ZnFe2O4 magnetic microspheres with the peroxidase-like activity and B,N-CDs can be utilized for quantitative analysis of the concentrations of H2O2 and H2O2-involved metabolites. Moreover, as far as we know, the combination of heteroatom-doped CDs and ZnFe2O4 magnetic microspheres to

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construct the ratiometric fluorescence detection of H2O2 has not been reported. We further performed the ratiometric fluorescence analysis of H2O2 and H2O2-involved

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metabolites. Thus, an alternative approach is provided to extend the application of fluorescent B,N-CDs in biological analysis.

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The repeatability test of the proposed method for detecting H2O2, glucose, and

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UA has been performed and the results are shown in Fig. S11. It can be seen from Fig.

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S11A, S11B, and S11C that the (I556/I430) and (A420) of the five parallel samples under the same conditions were similar, respectively. The relative standard deviations

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(RSD) of every five parallel data were all below 5%, indicating that the proposed

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method has a good repeatability.

(Here Fig. 4) (Here Fig. 5)

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3.8. Selectivity of the B,N-CDs/ZnFe2O4/H2O2/OPD for glucose and UA assay Selectivity is a key parameter for evaluating the performance of newly developed

sensors. Thus, some potential interfering chemicals in serum samples including K+, Na+, AA, Leu, DA, glucose, maltose, fructose, lactose, UA, Cys, GSH, urea, and Lys 21

were investigated for detecting glucose and UA. As shown in Fig. 6A and 6B, only glucose and UA caused an obvious increased in fluorescence intensity ratio (I556/I420) compared with other interferences. The high selectivity should be ascribed to GOx and uricase catalytic specificity for GOx and UA, respectively. All these results

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and UA detection even the foreign substances at high concentrations.

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indicated that pronounced selectivity and credible of our sensing system for glucose

(Here Fig. 6)

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3.9. Real samples detection

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To evaluate the feasibility of this method, we detected glucose and UA in diluted

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human serum samples. The corresponding results were obtained as shown in Tables

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S4 and S5. The spiked concentrations of glucose and UA in serum samples were

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consistent with those of added glucose and UA based on the standard addition method. The recoveries of glucose and UA ranged from 90.12% to 96.28% and

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95.90% to 96.32%, respectively, and the RSD was all below 4%, indicating

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satisfactory precision and accuracy in the processes. These results demonstrated that the proposed sensing platform was applicable for glucose and UA monitoring in real

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samples.

4. Conclusions In conclusion, B,N-carbon dots-based ratiometric fluorescent and colorimetric method for detection of H2O2, glucose, and UA using ZnFe2O4 magnetic microspheres 22

as peroxidase mimics has been developed. This dual-readout assay platform offers several obvious advantages: (1) complex synthesis processes and specific modifications are not required for the ZnFe2O4 magnetic microspheres and B,N-CDs; (2) the sensing assay can display colorimetric and ratiometric fluorescent dual-readout

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signal, making the experimental results more convincing; (3) the proposed method is simple, flexible, and has a higher specificity and sensitivity. Furthermore, the sensing

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system has been successfully applied in the detection of glucose and UA levels in

human serum with satisfactory results. Thus, we believe that the applicability of this

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generating reaction in complex clinical biomatrices.

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method can be further expanded to probe various metabolites involved in H2O2-

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Acknowledgments

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This work was financially supported by the National Natural Science Foundation of China (No. 21675131) and the Natural Science Foundation of Chongqing (No.

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CSTC-2015jcyjB50001).

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Appendix A. Supplementary data

Author Biographies

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Na Xiao is an MS candidate in School of Chemistry and Chemical Engineering, Southwest University, China. Her major research interest is spectrum analysis. Shi Gang Liu is a doctoral student in School of Chemistry and Chemical Engineering, Southwest University, China. His major research interest is spectrum analysis. 23

Shi Mo is a doctoral student in Department of Physics, City University of Hong Kong, Hong Kong. His research interests include nanoparticles and biomaterials. Yu Zhu Yang is a doctoral student in School of Chemistry and Chemical Engineering, Southwest University, China. Her major research interest is spectrum analysis.

University, China. His major research interest is spectrum analysis.

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Lei Han is an MS candidate in School of Chemistry and Chemical Engineering, Southwest

Yan Jun Ju is an MS candidate in School of Chemistry and Chemical Engineering,

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Southwest University, China. Her major research interest is spectrum analysis.

Nian Bing Li is a professor of chemistry in School of Chemistry and Chemical Engineering,

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Southwest University, China. He received his MS degree in physical chemistry in 1997 and PhD

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degree in material science in 2000 from Chongqing University. During 2000–2002, he was a postdoctoral research fellow in Fuzhou University, China. Since 2006–2007, he was a

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postdoctoral research fellow in Korea Advanced Institute of Science and Technology (KAIST),

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chemical sensors and biosensors.

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Korea. His research interests are the developments of electrochemical devices such as

Hong Qun Luo is a professor of chemistry in School of Chemistry and Chemical

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Engineering, Southwest University, China. She received her MS degree in environmental chemistry from Sichuan University in 1991 and PhD degree in analytical chemistry from

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Southwest China Normal University in 2002. During 2006–2007, she was a visiting scholar in Tohoku University, Japan. Her research is focused on molecular spectroscopy and electrochemical sensor. Supplementary data associated with this article can be found, in the online version, at

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http://dx.doi.org/10.1016/j.snb.201 X.XX.XXX.

24

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Figure captions: Fig. 1. (A) HRTEM image of B,N-CDs. (B) Fluorescence spectra of B,N-CDs excited from 320 to 390 nm. (C) SEM image and (D) XRD pattern of ZnFe2O4 magnetic

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microspheres. Scheme 1. Schematic illustration of colorimetric and ratiometric fluorescent detection

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of H2O2, glucose, and UA.

Fig. 2. (A) UV-vis absorption spectra of oxOPD solution in the absence (solid line)

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and presence (dotted line) of B,N-CDs. (B) Fluorescence emission spectrum of B,N-

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CDs and UV−vis absorption spectra of oxOPD and OPD. (C) The fluorescence

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lifetime spectra of B,N-CDs in the absence and presence of oxOPD. Fig. 3. (A) UV–vis absorption spectra of B,N-CDs/OPD/ZnFe2O4 system upon adding

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different concentrations of H2O2 (from bottom to top: 0, 0.1, 1, 5, 10, 20, 40, 60, 80,

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100, 130, 150, 180, 200, 250, 300, 400, and 500 μM). Inset: the visual picture of the system upon adding different concentrations of H2O2. (B) The linear plot of

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absorbance measured at 420 nm as a function of the H2O2 concentration. (C) Fluorescence spectra of B,N-CDs/OPD/ZnFe2O4 system-based ratiometric probe

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containing various concentrations of H2O2 (0, 0.1, 1, 10, 20, 40, 60, 80, 100, 130, 150, 180, 200, and 250 μM). (D) The linear response range for H2O2.

Fig. 4. (A) UV–vis absorption spectra of B,N-CDs/OPD/ZnFe2O4 system upon adding different concentrations of glucose (from bottom to top: 0, 1, 5, 10, 20, 50, 100, 200, 35

400, 600, 800, and 1000 μM). Inset: the visual picture of the system upon adding different concentrations of glucose. (B) UV–vis absorption spectra of B,NCDs/OPD/ZnFe2O4 system upon adding different concentrations of UA (from bottom to top: 0, 1, 10, 20, 50, 100, 200, 300, 400, 500 and 600 μM). Inset: the visual picture

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of the system upon adding different concentrations of UA. The linear plots of absorbance measured at 420 nm as a function of the glucose (C) and UA (D)

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concentration, respectively.

Fig. 5. (A) Fluorescence spectra of B,N-CDs/OPD/ZnFe2O4 system-based ratiometric

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probe containing various concentrations of glucose (0, 10, 20, 100, 200, 400, 600,

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800, and 1000 μM). (B) Fluorescence spectra of B,N-CDs/OPD/ZnFe2O4 system-

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based ratiometric probe containing various concentrations of UA (0, 1, 10, 50, 100,

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(D), respectively.

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200, 300, 400, 500, and 600 μM). The linear response ranges for glucose (C) and UA

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Fig. 6. Selectivity of glucose and UA detection. (A) The concentrations of glucose and UA were 800 M in glucose sensing, and all other substances were tested at 2.5

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mM. (B) The concentration of UA and other substances were 400 M and 2 mM,

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respectively.

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IP T

SC R

U

N

A

M

Figr-6

42