One-step synthesis of enzyme-stabilized gold nanoclusters for fluorescent ratiometric detection of hydrogen peroxide, glucose and uric acid

Accepted Manuscript One-step synthesis of enzyme-stabilized gold nanoclusters for fluorescent ratiometric detection of hydrogen peroxide, glucose and uric acid

Feifei Meng, Huaqin Yin, Yong Li, Shiyue Zheng, Feng Gan, Gang Ye PII: DOI: Reference:

S0026-265X(18)30518-6 doi:10.1016/j.microc.2018.06.006 MICROC 3206

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

29 April 2018 3 June 2018 5 June 2018

Please cite this article as: Feifei Meng, Huaqin Yin, Yong Li, Shiyue Zheng, Feng Gan, Gang Ye , One-step synthesis of enzyme-stabilized gold nanoclusters for fluorescent ratiometric detection of hydrogen peroxide, glucose and uric acid. Microc (2017), doi:10.1016/j.microc.2018.06.006

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ACCEPTED MANUSCRIPT One–step synthesis of enzyme-stabilized Gold Nanoclusters for fluorescent ratiometric detection of hydrogen peroxide, glucose and uric acid Feifei Meng1, Huaqin Yin1, Yong Li1, Shiyue Zheng1, Feng Gan1*, Gang Ye2* 1. School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, P.R. China. 2. Department of Gastroenterology, the First Affiliated Hospital of Jinan University, Guangzhou 510630,

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P.R. China

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* E-mail: [email protected]

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Abstract: This paper reports a novel enzyme-functionalized gold nanocluster with dual emission characteristics. We applied the reductivity of tyrosine residues in catalase to reduce

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gold ions so as to fabricate catalase conjugated gold nanoclusters (CAT-GNCs) under strong alkaline conditions. The whole synthetic process only took 15 min. We observed the

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fluorescence of CAT-GNCs can be rapidly quenched by hydrogen peroxide, which indicates that the activity of catalase was not severely affected although it was used as reductant in the synthesis of the CAT-GNCs. The CAT-GNCs were used as a ratiometric fluorescence sensor

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to detect H2O2, and the linear range was 10-80 µM (R2 = 0.9939). The detection limit was as

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low as 25 nM. The CAT-GNCs were also used to determine the glucose and uric acid, with the linear ranges of 5-500 µM (R2 = 0.9938) and 10-200 µM (R2 = 0.9910), respectively; and the

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detection limits of 3 µM and 40 nM, respectively. Finally, the CAT-GNCs were used in real serum samples, the recovery rates of the glucose and UA were more than 86.37% and 86.90%,

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respectively. The results demonstrated that the CAT-GNCs have the potential for use. Keywords: Catalase, GNCs, hydrogen peroxide, glucose, Uric acid

1. Introduction

Enzymes are special proteins with high selectivity and catalytic activity, which makes them to play an outstanding role in biological metabolic systems and important reagents for qualitative or quantitative analysis

[1]

. In recent years, researchers have carried out a new 1

ACCEPTED MANUSCRIPT exploration of combining the excellent substrate selectivity of enzymes with the superior optical properties of gold clusters to prepare new kinds of biosensors. The new biosensors have the specificity of enzymes, the biocompatibility and convenience detection of the nanomaterials in the meantime. For example, early scientific research has successively used DNase [2], lysozyme [3], trypsin [4], and pepsin [5] to synthesize gold nanoclusters with different colors and effects, and these gold nanoclusters have been widely used in environmental [6-8]

, biomolecular detection

[9-12]

[13-14]

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monitoring

, and cell imaging

. However, these gold

then further to biconjugate with antibodies

[15]

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clusters only use the enzyme as templates to complete the synthesis of gold nanoclusters, and or other targeted biomolecules

[16]

to endow

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them specific identification functions and biocompatibility. The advantage of the enzymes’ special binding on the target material have not been utilized. One important reason might be

et al.

[17]

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the loss or degeneration of the enzyme activity during the fabrication processes. In 2011, Wen used horseradish peroxidase as model protein and template to prepare red

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fluorescence gold nanoclusters while the activity of horseradish peroxidase was still kept. They also used the nanoclusters to detect the biological activity of hydrogen peroxide. In

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2013, wang et al. [18] used glucose oxidase to synthesize gold nanoclusters by an etching

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reaction, which still maintains the biological activity of the glucose oxidase. The materials were successfully used for glucose detection. All these works show that enzymes could play an important role in the development of new kinds of biosensors.

synthesis

[19-22]

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In this paper, we use catalase (CAT) to synthesize gold nanoclusters (GNCs) by one step . Catalase is a marker enzyme of peroxisomes that can catalyze the

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decomposition of hydrogen peroxide into oxygen and water. The amino acid residues of the CAT have 20 tyrosine that can be used as a reducing agent under appropriate conditions and 4 cysteine that can act as a polyvalent ligand to stabilize the metal core，so it could be a good template for the synthesis of GNCs. We designed suitable reaction conditions to synthesize CAT coupled GNCs (CAT-GNCs) successfully. More importantly, we found that the biological activity of CAT was still kept. The CAT-GNCs were successfully used for the detection of hydrogen peroxide. Rather good results were obtained. To the best of our knowledge, this is the first time to synthesize of CAT coupled GNCs. 2

ACCEPTED MANUSCRIPT 2. Experimental

2.1. Chemicals and materials

All of the chemicals were of reagent grade and were used without further purification.

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Catalase was purchased from Meilun Biotech (Shanghai) Co., Ltd. HAuCl4•3H2O, lactose, sucrose, fructose, maltose were purchased from Sinopharm Chemical Reagent Co., Ltd

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(Shanghai, China). The serum samples were obtained from Zhongkechenyu (Beijing, China).

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Nitrate salts including Ca(NO3)2, KNO3, Mg(NO3)2 and NaNO3 were of analytical grade and obtained from Guangzhou Chemical Regent Factory (Guangzhou, China). Amino acid

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including lysine, tyrosine, glycine, and proline were purchased from Sigma-Aldrich (St. Louis, MO). Bovine Serum Albumin (BSA) was purchased from Sigma. Catalase, Glucose, glucose

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oxidase, Urease and urea were purchased from Meilun Biotechnology Co., Ltd (Shanghai, China); Twice-distilled water was used throughout all experiments; Disodium phosphate,

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Sodium dihydrogen phosphate and Sodium hydroxide was purchased from Damao Chemical

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(Tianjin, China); Buffer solutions with pH ranging from 3 to 11 were prepared as follows: NaH2PO4/H3PO4: pH 3-4, NaH2PO4/Na2HPO4: pH 5-8, NaH2PO4/NaOH: pH 9-11.

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2.2. Instrumentations

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The absorption spectra of CAT and CAT-GNCs were measured on UV 3150 Spectrophotometer (Shimadzu, Japan). Transmission electron microscope (TEM) images of CAT-GNCs were obtained on a JEM-2010HR (Jeol Ltd, Japan) with an accelerating voltage of 200 kV. The Fourier Transform Infrared (FTIR) spectra of CAT and CAT-GNCs were measured on the Nicolet Avatar FTIR model 330 Spectrometer (Thermo, America) with the range from 400 cm-1 to 4000 cm-1. The exaction and emission spectra were measured on RF-5301 Fluorospectrophotometer (Shimadzu, Japan). The X-ray photoelectron spectroscopy (XPS) of the CAT-GNCs were measured on ESCALab250 (Thermo, America). The fluorescent lifetime of CAT-GNCs was measured on FLSP920 photoluminescence 3

ACCEPTED MANUSCRIPT spectrometer (Edinburgh Instruments Ltd).

2.2. Preparation of the CAT-GNCs and the BSA–GNCs

All glasswares were thoroughly cleaned with aqua regia (HNO3/HCl, volume ratio 1:3)

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and rinsed extensively with double distilled water prior to use. The CAT (25 mg/mL, 2 mL) was rapidly added into a boiling HAuCl4 (5 mM, 2 mL) under stirring at 20 rpm. After 2 min,

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the solution of NaOH (1 M, 180 µL) was added and the reaction was allowed to proceed under stirring at 100 °C for 15 min. During this period, the color of the solution changed from

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dark red to blackish green, and to deep brown at last. The solution was taken away from heating and cooled down to the room temperature. Then, the solution was centrifugated (13,

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000 rpm, 30 min) to remove larger particle sizes. After that, the solution was collected and stored at 4 °C in the dark until further characterization. In the Results and Discussion section,

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we proved that the CAT-GNCs were successfully fabricated. The BSA stabilized Au clusters

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(BSA–GNCs) were also fabricated using the one-step synthetic route as reported before [23].

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2.4. Sample preparation

(a) Preparation of probe: phosphate-buffered saline (PBS) (pH 7.45, 20 mM, 500 µL) was

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added into the CAT-GNCs (50 µL) solution, and then stored at 4 °C in the dark for further use; (b) Sensing of H2O2: the PBS solution (500 µL) was added into the CAT-GNCs solutions

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(each of 50 µL). The H2O2 solutions of different concentrations (10-80 μM, 500 µL) were added into the CAT-GNCs solutions and incubated at room temperature for 15 min before spectral analysis; (c) Sensing of glucose: Glucose and glucose oxidase (GOD) were prepared in PBS solution. The solutions of GOD (0.5 mg/mL, 50 µL) were added to the soulutions of different concentrations of glucose (5-500 µM, 200 µL) and then be incubated at room temperature for 35 min. The prepared CAT-GNCs solutions (each of 200 µL) were added into above GOD and glucose solutions and incubated at room temperature for 15 min before spectral analysis. (d) Sensing of UA: urate oxidase (UOx) and UA were prepared in PBS solution. The solutions of UOx (0.3 mg/mL, 25 µL) were added to the solution of different 4

ACCEPTED MANUSCRIPT concentrations of UA (5-500 µM, 200 µL) and then be incubated at room temperature for 50 min. The prepared CAT-GNCs solutions (each of 200 µL) were added into above UOx and UA solutions and incubated at room temperature for 15 min before spectral analysis. The prepared solutions were vibrated for 30 s after two solutions were mixed.

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2.5. preparation of blood samples

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The blood samples were first centrifuged at 4000 rpm for 5 min. Then, the supernatant serum was directly diluted 10-fold with a 20 mM phosphate buffer. The serum samples were

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incubated with GOD for the degradation of glucose, and then were incubated with UOx for

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the degradation of UA, respectively.

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3. Results and discussion

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3.1. Characterization of the CAT-GNCs

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Fig.1. (A) shows the UV/Vis spectra of CAT (a) and CAT-GNCs (b). It shows that the maximum absorption peaks of the materials are at 276 nm, which come from the feature of aromatic amino acids of protein [23, 24]. At the same time, the disappearance of absorption at

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400 nm of Fig.1 (b) indicated that the conformation of CAT might be changed after the formation of CAT-GNCs. These results are in accord with previous reports that proteins

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containing a tyrosine residue can reduce Au3+ in HAuCl4 by adjusting the reaction pH higher than its pKa (-10) value

[23]

. As CAT contains 527 amino acids, including 20 tyrosine and 4

cysteine, it did act as reducing and capping agent at the same time in our experiments. Fig.1B shows the morphology of the CAT-GNCs through transmission electron microscopy (TEM). We found that most of the CAT-GNCs were well dispersed and the average particle diameter was about 2.0 nm. The quantum yield (QY) of the CAT-GNCs was 0.59%, using Rhodamine B as the calibration standard. Fig.1 C shows infrared spectra of CAT (a) and CAT-GNCs (b). It can be seen that after the formation of the CAT-GNCs, the IR absorption peaks at 3300 cm−1 (amide A, NH stretching) 5

ACCEPTED MANUSCRIPT and 1487cm−1 (amide II, CN stretching and NH bending) [25] were different from that of CAT. The broad spectral band of the CAT in the range of 900–1200 cm−1 is due to C–O, C–C stretches and C–O–H, C–O–C deformation of carbohydrates [26]. Fig.1D shows the in-depth chemical state of the CAT-GNCs through the X-ray photoelectron spectroscopy (XPS). The Au core level showed principal Au 4f7/2 and Au 4f5/2

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components at 83.48 and 87.18 eV binding energy, respectively. The spectrum of Au 4f7/2 was deconvoluted into two distinct components (green and purple curves) at binding energies of

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83.34 eV and 83.78 eV, suggesting that both Au (0) and Au (I) exist in the CAT-GNCs. The best fit of the data indicated that there was a certain amount of Au (I) (40%) on the surface of

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Au core, which benefits the stabilization of CAT-GNCs [27].

Fig.1. (A) The UV/Vis absorption spectra of aqueous CAT (a) and CAT-GNCs (b); (B) The TEM image of the CAT-GNCs (scale bar = 20 nm); (C) The Fourier Transform Infrared Spectroscopy (FTIR) spectra of CAT (a) and CAT-GNCs (b); (D) The X-ray photoelectron spectroscopy (XPS) of CAT-GNCs

Fig.2A shows the fluorescence spectra of the CAT-GNCs. One can see that the dual-emission peaks exhibited by the CAT-GNCs are at 490 nm and 650 nm, and the intensity 6

ACCEPTED MANUSCRIPT of the later is much stronger. The fluorescence peak around 650 nm is attributed to the CAT-GNCs and it can be seen by naked eyes under 365 nm UV irradiation, which is also recorded in the inset photograph of Fig. 2A. The blue fluorescence at 490 nm might come from the amino acid residues of the CAT (tryptophan, tyrosine and phenylalanine) [23]. Fig.2B shows the lifetime of CAT-GNCs. Time components contain three parts：τ1=3.1010

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(1.84%), τ2=28.0988 (2.05%), τ3=594.116 (96.11%). The results show that the CAT-GNCs have a long lifetime that could facilitate their use in vitro and in vivo fluorescence lifetime

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imaging. The long lifetime of the CAT–GNCs (> 100 ns) are the characteristic of Au (I)–thiol complexes, which is attributed to the ligand–metal charge transfers and Au (I)–Au (I)

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interactions [28].

Fig.2. (A) The emission spectra of the CAT-GNCs (λex = 365 nm) and the inset shows a photograph of

CAT-GNCs.

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CAT-GNCs under room light and UV (365 nm) lamp, respectively. (B) Fluorescence lifetime of the

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3.2. Effect of pH on the fluorescence of the CAT-GNCs To explore the fluorescent response of the CAT-GNCs at different pH values, we tested the fluorescence intensities of the CAT-GNCs in the absence and presence of H2O2 (1 mM) upon the addition of different pH buffer solutions into the CAT-GNCs solutions. As displayed in Fig.3, we found that the fluorescence responses in the absence (a) and presence (b) of H2O2 changed slightly when the pH value of CAT-GNCs solutions varied in the range of 3–11. As the CAT-GNCs will be applied in biological samples, we chose pH 7.4 as the experimental conditions. 7

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Fig.3. The pH dependence response of CAT-GNCs in the absence (a) and presence (b) of H2O2 (1 mM)

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3.3. Detection of hydrogen peroxide

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Fig.4 (A) shows the fluorescent spectra of the CAT-GNCs in the presence of different concentrations of H2O2 solutions. It can be seen that with the increase of the concentration of

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H2O2 added, the first peak (453 nm) of the spectrum increases and the second peak (610 nm) decreases gradually. In addition, the entire emission wavelength exhibited a blue-shift (37 nm),

molecule on GNCs surface

[29]

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which can be attributed to the breakage of Au–S bond that alters the conformation of CAT . Fig.4 (B) shows a linear range from 10 µM to 80 µM with a

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correlation coefficient of 0.9939. The detection limit value was 25 nM by 3σ (σ was the standard deviation of the blank measurements), which was the lowest compared with that of

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previous reports for H2O2 detection utilizing fluorescence method (Table 1).

Fig.4. (A) The fluorescent responses of the solutions of the CAT-GNCs with different concentrations of the H2O2. (B) The linear plot of the fluorescence ratios I610/I453 of the CAT-GNCs sensor versus the concentrations of the H2O2. The error bars represent standard deviations based on three independent 8

ACCEPTED MANUSCRIPT measurements.

As previous reported [29], there were clear evidences that Au-S bond can be degraded in the presence of H2O2 as an oxidant. The RS-Au (R=CH3(CH2)n) bonds were oxidized into RS-SR and even RSO3H by H2O2 [29-31]. Low concentration of H2 O2 : 2RS − Au → RSSR + 2Au

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High concentration of H2 O2 : RS − Au → RSO3 H + HO − Au

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These reactions may lead to the rapid changes of the structure of gold nanoclusters, and the effective quenching fluorescence of the CAT-GNCs. However, this kind of quenching effect is

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meaningless and only limited sensitivity could be provided. In order to demonstrate that the CAT had a certain effect on H2O2 quenching, we used BSA-GNCs as a control experiment [17]

. Each assay was carried out by following the

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which is similar with previous reports

procedure in Section 2.4. The relative fluorescence intensity (I610/I453) of BSA-GNCs (I600/I450)

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decreases about 64.45% when H2O2 (80 µM) was added. However, the relative fluorescence intensity of the CAT-GNCs decreases about 91.61% when H2O2 was added. The results

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proved that CAT still had a certain activity after being used to synthesize of the CAT-GNCs.

3.4. Determination of glucose and uric acid

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Sensing of glucose: when the solution of GOD is added to the glucose solution, the glucose

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is converted to gluconic acid and hydrogen peroxide as followings

Glucose+ O2 + H2O

GOD

Gluconic acid + H2O2

Fig.5 (A) shows fluorescence spectra of the CAT-GNCs in the presence of 0.5 mg/mL GOD and 5-500 µM glucose. The inset of the fig.5A shows the fluorescence intensities of CAT-GNCs at 610 nm versus the concentrations of glucose. Fig.5 (B) shows a linear range of 5-500 µM of the relative activity with the concentrations of glucose. The correlation coefficient was 0.9961 and the detection limit was 3 µM by 3σ. 9

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Fig.5. (A) Fluorescence spectra of CAT-GNCs in the presence of 0.5 mg/mL GOD and 5-500 µM glucose.

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The inset shows the fluorescence value of CAT-GNCs at 610 nm versus the concentration of glucose. (B) Plot of the value of I610/I453 versus the concentration of glucose. The error bars represent standard

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deviations based on three independent measurements.

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The reaction of UA detection was as followings

UOx

Allantoin + H2O2 + CO2

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Uric acid + H2O + O2

Fig.6 (A) shows fluorescence spectra of CAT-GNCs in the presence of 0.3 mg/mL UOx and 10-200 µM UA. The inset of the fig.6A shows the fluorescence intensities of CAT-GNCs at

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610 nm versus the concentration of UA. Fig.6 (B) shows a linear range of 10-200 µM of the relative activity with the concentrations of UA. The correlation coefficient was 0.9910 and the

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detection limit was 40 nM by 3σ. The limit of detection for UA is much lower than most reported methods (Table 1).

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Fig.6. (A) Fluorescence spectra of CAT-GNCs in the presence of 0.3 mg/mL UOx and 10-200 µM UA. The

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inset shows the fluorescence value of CAT-GNCs at 610 nm versus the concentration of UA. (B) Plot of the value of I610/I453 versus the concentration of UA. The error bars represent standard deviations based on

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three independent measurements.

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Table1 shows the comparison of the efficiency of the CAT-GNCs with other reported fluorescent sensors. The detection limit of the CAT-GNCs towards H2O2, glucose and UA

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exhibited better results than that of the reported methods. The relative good results could be

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attributed to both the activity of the CAT and dual-emission fluorescence characteristics of the CAT-GNCs, which has the inherent ability of self-calibration to decrease or eliminate the

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errors such as the fluctuation, microenvironment and testing conditions [24, 7].

Table1 Comparison of the linear ranges and the detection limits of different sensors for the determination of

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H2O2, glucose and uric acid Method

determination of H2O2 Fluorescence (FL) Electrochemical assay(EA) Fluorescence (FL) Fluorescence (FL) colorimetric detection Fluorescence (FL) Fluorescence (FL)

Materials

Linear range

Detection limit

CS-AuNCs Ag-Co3O4-rmGO BSA-GNCs GOx-AuNCs Fe3O4@SiO2@Au MNPs CQDs CAT-GNCs

1-100 µM 0.5 ~ 7000 µM 0.1–25 µM 0.5-10 µM 1 ~ 40 µM

0.3 µM 0.3 µM 40 nM 0.23 µM 0.6 µM

32 33 30 34 35

10-150 µM 10-80 µM

3.8µM 25 nM

36 This

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Ref.

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FITC/BSA-AuNCs CS-AuNCs BSA-AuNCs EPPGE 3DGH-AuNPs PANIS/Au CAT-GNCs

13 µM 8.2 µM 3.5 µM

37 38 35

100 nM –1 mM 0.1 μM-5 mM

100 nM 0.1 µM

39 40

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determination of uric acid Fluorescence (FL) Fluorescence (FL) Fluorescence (FL) Electrochemical assay(EA) Electrochemical assay(EA) Electrochemical assay(EA) Fluorescence (FL)

10-150 µM 5-500 µM

3.5 µM 3 µM

36 This work

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0.2–12 mM 5-100 µM 0.7–80 µM 0.1–25 µM 1-60 M 2–1000 mM 10-200μM

0.19 mM 1.7 µM 120 nM 0.03 µM 5 nM 0.15 µM 40 nM

37 32 30 41 42 43 This work

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Electrochemical biosensor Electrochemistry

20–400 µM 0.02–5.7 mM 5 µM–0.35 mM

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Fluorescence (FL) Fluorescence (FL)

FITC/BSA-AuNCs Au NPs Fe3O4@SiO2@Au MNPs CBU–AuNP 3-aminophenylboronic acid functionalized reduced graphene oxide CQDs CAT-GNCs

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determination of glucose Fluorescence (FL) colorimetric detection colorimetric detection

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3.5. Selectivity of the CAT-GNCs for Glucose and UA detection

The selectivity of the CAT-GNCs for glucose and UA was tested by conducting control

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experiments with many nonspecific species, including glucose analogues (lactose, sucrose, fructose, maltose), part of the amino acid (lysine, tyrosine, glycine, proline) and some of the

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common metal ions in serum (Na+, Mg2+, K+, Ca2+). Each assay was carried out by following the procedure in Section 2.4. From the bar diagram Fig.7A-B the better selectivity of CAT-GNCs towards glucose and UA is confirmed. The results indicated that the CAT-GNCs exhibited high selectivity to glucose and UA, which is attributed to the highly specific and effective catalysis of glucose oxidase to glucose and uricase to UA. It should be pointed out that the real concentrations of the interfering substances are much lower than that of UA and glucose in blood [44], so that the accuracy of our assay was guaranteed.

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Fig.7. (A) Effects of potential interferents towards the fluorescent probe for Glucose. GOD, 0.5 mg/mL;

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glucose and potential interferents, 500 µM; pH 7.4, 20 mM PB buffer; t, 50 min (B) Effects of potential interferents towards the fluorescent probe for UA. UOx, 0.3 mg/mL; UA and potential interferents, 250 µM;

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pH 7.4, 20 mM PB buffer; t, 65 min. The error bars represent standard deviations based on three

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independent measurements. (Relative Intensity = (I0(610/453)-I(610/453))/ I0(610/453)）

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3.6. Determination of glucose and UA in serum samples

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In order to validate the applicability, the CAT-GNCs was employed to detect glucose and UA in serum samples. We added different amounts of glucose and UA into the serum samples, measured the fluorescence intensities of the samples, and then calculated the recoveries of

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glucose and UA, respectively. The process was based on the previously obtained spectral information and the regression equation. Table2 shows the recovery rates of glucose and UA

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in the serum samples and the results were satisfactory. The results also suggested that the reliability of the CAT-GNCs for measuring the concentration of glucose and UA in human serum samples.

Table 2 Determination of glucose and UA contents in real samples (n = 3). Serum samples Glucose

Uric acid

Spiked amounta(µM)

Found amount(µM)

Recoveryb(%)

50 250 500 50 100 200

43.19 ± 0.11 224.35 ± 0.62 459.2 ± 0.80 43.45 ± 1.09 98.25 ± 5.9 190.99 ± 8.5

86.37 ± 0.22 89.74 ± 0.25 91.84 ± 0.16 86.90 ± 2.20 98.25 ± 5.90 95.49 ± 4.3

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Concentrations of standards are expressed as the equivalent concentrations added in the final injected

solutions. Each level of standard added was replicated there times b

Recovery=[(amount observed−original amount)/amount added]×100

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4. Conclusion

In this work, we successfully used CAT as a reducing and stabilizing agent to synthesize a

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dual-emission ratio of fluorescent probe. The CAT-GNCs is red under the irradiation of the UV lamp. As we expected, the bound CAT still has its own activity, which makes the

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CAT-GNCs have the dual properties of the gold nanocluster and the enzyme. The dual nature of the CAT-GNCs also performed well when they were applied to the detection of glucose and

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UA in real serum sample. Thus, the CAT-GNCs could be a potential biological probe for

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clinical examination.

Acknowledgement

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This work was supported by the National Natural Science Foundation of China

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(20875106), Guangdong Natural Science Found Committee (9151027501000003) and State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University (4299001), the

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Highlights

1. The enzyme activity of catalase was still kept although it was used as both reducing and protecting agent in the synthesis of gold nanoclusters. 2. The detection limit is the lowest comparing to similar florescence sensors to the detection

of hydrogen peroxide.

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3. The synthesis process only needs 15 min in a one-step method without adding any other

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organic reagent.

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