Preparation of bright fluorescent polydopamine-glutathione nanoparticles and their application for sensing of hydrogen peroxide and glucose

Sensors and Actuators B 259 (2018) 467–474

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Preparation of bright fluorescent polydopamine-glutathione nanoparticles and their application for sensing of hydrogen peroxide and glucose Li Tang, Shi Mo, Shi Gang Liu, Na Li, Yu Ling, Nian Bing Li ∗ , Hong Qun Luo ∗ Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

a r t i c l e

i n f o

Article history: Received 16 July 2017 Received in revised form 11 December 2017 Accepted 12 December 2017 Available online 13 December 2017 Keywords: Polydopamine-glutathione nanoparticles Glutathione Fluorescent sensor Hydrogen peroxide Glucose

a b s t r a c t A novel water-soluble fluorescent polydopamine derivative, polydopamine-glutathione nanoparticles (PDA-G (-S-)NPs), was synthesized by the Michael addition reaction between dopamine (DA) and reduced glutathione (GSH). Compared to a direct polymerization of dopamine, the as-prepared PDA-G (-S-)NPs have stronger fluorescence emission intensity. Also, the synthesis does not need any hazardous organic solvents and the process is simple. Additionally, the roles of GSH and hydrogen peroxide (H2 O2 ) in enhancing the fluorescence intensity are discussed in detail. The thioether in PDA-G (-S-)NPs is easily oxidized by hydrogen peroxide to sulfoxide and sulfone groups, accompanied by a decrease in fluorescence intensity. Therefore, the PDA-G (-S-)NPs can be applied to construct a fluorescent sensor for the sensitive detection of hydrogen peroxide. Based on the transformation of glucose into gluconic acid and H2 O2 in the presence of glucose oxidase, the PDA-G (-S-)NPs system was further utilized to sensing glucose. The linear ranges and detection limits of hydrogen peroxide and glucose are (0.5–6 ␮M, 2–130 ␮M) and (0.15 ␮M, 0.6 ␮M), respectively. © 2017 Elsevier B.V. All rights reserved.

1. Introduction In recent years, the thiol-X reaction has gained extensive attention in polymer modification and organic synthesis [1–5] due to its various advantages, such as mild reaction conditions, fewer by-products, higher functional group compatibility, and higher conversion [6–9]. Since Allen et al. initially proposed the thiol-Michael addition reaction in the 1960s [10], it rapidly became indispensable to organic synthesis and essential issues on fundamental analysis and practical implementation in polymer chemistry [11,12]. Dopamine [13–15] is inclined to be oxidized and polymerized to form polydopamine (PDA) under aerobic and alkaline conditions [16–18]. With excellent biocompatibility and many other prominent properties, polydopamine nanoparticles are widely investigated in the field of optics, electricity, or magnetism [19–22]. The particles were always generated by the oxidation and polymerization of dopamine. However, it is an intrinsic fluorescent polymer with weak fluorescence intensity. Therefore, enhanc-

∗ Corresponding authors. E-mail addresses: [email protected] (N.B. Li), [email protected] (H.Q. Luo). https://doi.org/10.1016/j.snb.2017.12.071 0925-4005/© 2017 Elsevier B.V. All rights reserved.

ing the fluorescence intensity of PDA has become a meaningful research topic. In 2015, Tseng et al. utilized hydroxyl radicals in H2 O2 to degrade large particles of PDA into small polydopamine dots (PDs), leading to the great enhancement of the fluorescence intensity [23]. In addition, Zuo et al. prepared a novel watersoluble polydopamine-polyethyleneimine (PDA-PEI) copolymer by the Michael addition reaction between PEI and PDA. As a result, the hydrogen bonds formed between PEI and PDA twisted the plane structure of PDA, which decreased the intro- or intermolecular coupling and enhanced the fluorescence intensity of PDA-PEI [24]. Inspired by the above-mentioned studies, we synthesized polydopamine derivatives by a thiol-Michael addition reaction between thiols and dopamine. This work can identify the sulfhydryl compounds by a colorimetric method and establish a fluorescent sensor for the detection of H2 O2 and glucose based on the interaction between thioether of PDA-G (-S-)NPs and H2 O2 . The preparation procedures of fluorescent nanoparticles were described as follows: Firstly, dopaquinone (the oxidation product of dopamine (DA)) reacted with GSH to form a dopamine derivative by the Michael addition reaction under alkaline conditions, and then its cyclization and polymerization took place with the addition of H2 O2 , resulting in the formation of polydopamine-glutathione nanoparticles (PDA-G (-S-)NPs). Interestingly, H2 O2 can oxidize the structure of

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Scheme 1. Schematic illustration of the sensing mechanism of PDA-G (-S-)NPs for H2 O2 and glucose.

thioether in the product to sulfoxide and sulfone groups, with a decrease of the fluorescence signal. Therefore, using these fluorescent PDA-G (-S-)NPs, we can establish a sensor to detect H2 O2 . The level of glucose in the blood is usually closely linked with various diseases including diabetes and hypoglycemia [25–28]. Therefore, the determination of glucose content plays an important role in the diagnosis of diabetes and clinical trials. Because the glucose oxidase (GOx)-catalyzed oxidation of glucose by molecular oxygen yields gluconic acid and H2 O2 , the constructed fluorescent sensor can also be used to detect glucose. The preparation and sensing mechanism of PDA-G (-S-)NPs are shown in Scheme 1. 2. Experimental section 2.1. Reagents and materials 3-Hydroxytyramine hydrochloride (dopamine) was purchased from Adamas Reagent Co., Ltd. (Shanghai, China). Tris(hydroxymethyl) aminomethane (Tris), glutathione (GSH, reduced), cysteine (Cys), N-acetyl-cysteine (N-Cys), phenylalanine (Phe), glutamic (Glu), arginine (Arg), lysine (Lyb), tyrosine (Tyr), tryptophan (Try), histidine (His), alanine (Ala), and threonine (Thr) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Hydrogen peroxide (H2 O2 , 30 wt%) was ordered from Chengdu Kong Chemical Reagents Factory, China. GOx was received from Songon Biotech Co., Ltd. (Shanghai, China). Glucose, fructose, sucrose, lactose, mannose, and maltose were purchased from Sigma-Aldrich (USA). NaH2 PO4 -Na2 HPO4 (200 mM, pH 6.0-7.0) and Tris-HCl (50 mM, pH 7.0–10.0) buffers were needed. Ultrapure water with a resistivity 18.2 M cm was used in all the experiments. Human serum samples were provided by the local hospital and diluted to one hundred fold with ultrapure water after centrifugation at 1000 rpm for 5 min. 2.2. Instruments Excitation and emission spectra of the systems were measured on a Hitachi F-2700 spectrofluorometer (Tokyo, Japan). Slit widths of all spectra were fixed at 10 nm with a PMT voltage of 400 V. A UV–vis 2450 spectrophotometer (Shimadzu, Japan) was utilized to record UV–vis absorption spectra. A Bruker AVANCE III (600 MHz) (Bruker, Germany) was used to measure nuclear magnetic res-

onance (NMR) spectra by dissolving the freeze-dried product in D2 O. Fourier transform infrared (FT-IR) spectra were measured on a Bruker IFS (Germany) 113 v spectrometer. A JEOL-2100 (Tokyo, Japan) system was used to record transmission electron microscopy (TEM) images at 200 kV. Zeta potential and hydrodynamic diameter measurements were performed on a Zetasizer Nano-ZS90 (Malvern Instruments Ltd.). 2.3. Synthesis of the fluorescent polydopamine-glutathione nanoparticles PDA-G (-S-)NPs were synthesized from dopamine and GSH via the Michael addition reaction. Typically, 4 mg of dopamine was firstly dissolved in 2 mL of Tris–HCl buffer solution (pH 8.5) with magnetic stirring for 2 min, and then 4.8 mg of GSH was added. The mixture continuously reacted for 2 h with the color changing from brown to pale yellow. All the above experiments were carried out at room temperature. Subsequently, 200 ␮L of H2 O2 (30 wt%) was injected into the above solution dropwise and incubated via hydrothermal treatment at 50 ◦ C for 7 h. For purification, the as-prepared PDA-G (-S-)NPs solution was dialyzed against ultrapure water through a dialysis membrane (molecular weight cutoff 1000 Da) for 24 h. The purified product was freeze-dried under vacuum and stored in the refrigerator (−20 ◦ C) for long-term preservation and dissolved with ultrapure water if needed (PDA-G (-S-)NPs: 2.5 mg mL−1 ). 2.4. The effect of pH, H2 O2 , and glucose on PDA-G (-S-)NPs The effect of pH on the fluorescence intensity of PDA-G (-S)NPs was investigated. 100 ␮L of PDA-G (-S-)NPs (2.5 mg mL−1 ) was mixed completely with 400 ␮L of Tris-HCl (50 mM) buffer solution with pH ranging from 6.0 to 10.0. Then the fluorescence emission spectra were recorded at a maximum excitation of 380 nm. All measurements were carried out at room temperature. The effect of H2 O2 on the fluorescence intensity of PDA-G (-S)NPs was studied. Firstly, 100 ␮L of PDA-G (-S-)NPs (2.5 mg mL−1 ) was introduced to 380 ␮L of Tris-HCl buffer (50 mM, pH 9.0) with stirring sufficiently. Then, 20 ␮L of H2 O2 with different concentrations (0–25 ␮M) were added and the mixtures were incubated at room temperature for 10 min. The subsequent operations and fluorescence measurements were the same as above.

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Fig. 1. (A) The influences of several compounds containing sulfhydryl groups reacting with DA on the fluorescence spectrum and intensity of the obtained product. (B) The comparison of fluorescence intensity of product by using sulfhydryl-containing GSH and sulfhydryl-free amino acids (Phe, Glu, Arg, Lyb, Tyr, Try, His, Ala, and Thr) as the reaction reagent to react with DA. (C) The appearance changes (up: under the irradiation of visible light, down: under the irradiation of UV light) when various reactants (Cys, N-Cys, GSH, Blank, Phe, Glu, Arg, Lyb, Tyr, Try, His, Ala, and Thr) were added to dopamine solutions without H2 O2 . (D).The appearance changes (up: under the irradiation of visible light, down: under the irradiation of UV light) when various reactants (Cys, N-Cys, GSH, Blank, Phe, Glu, Arg, Lyb, Tyr, Try, His, Ala, and Thr) were added to dopamine solutions with H2 O2 .

The glucose sensing was conducted according to the following steps: GOx solution (10 mg mL−1 , 20 ␮L) was mixed with different concentrations of glucose (20 ␮L) and the mixtures were incubated at 37 ◦ C for 1 h. After cooling to room temperature, PDA-G (-S)NPs solution (2.5 mg mL−1 , 100 ␮L) and 360 ␮L of Tris-HCl buffers (50 mM, pH 9.0) were added to the above solution. After reaction for 10 min, the fluorescence intensity of the mixtures was measured by the fluorescence spectrometer. To detect the glucose in the human serum sample, the glucose solution was replaced with 1% human serum sample (20 ␮L), and other steps are the same as the procedure described above. 3. Results and discussion 3.1. Mechanism of synthesis of fluorescent PDA-G (-S-)NPs and sensing of H2 O2 To study the reaction mechanism of GSH and DA, the optical properties of the synthesized dopamine derivatives using cysteine, N-acetyl-cysteine, and several common amino acids as the reagents were measured. As displayed in Fig. 1A, compared with Nacetyl-cysteine and cysteine, the fluorescence intensity of PDA-G (-S-)NPs is the strongest when GSH was employed as the reactant. The differences in the fluorescence intensity are associated with the nucleophilicity of three thiol compounds as mentioned above. Fig. 1B reveals the fluorescence intensity of PDA derivatives utilizing GSH and several thiol-free amino acids as the reactants, respectively. Evidently, the fluorescence intensity of PDA-G (-S)NPs with thiol-containing GSH is the highest.

Based on the above results, the fluorescence enhancement caused by GSH lies in the existence of thiol group rather than carboxyl, amino and peptide groups. Based on the interaction between thiol and DA, a visualization detection of thiol-containing compounds can be established. Cys, N-Cys, GSH, Phe, Glu, Arg, Lyb, Tyr, Try, His, Ala, and Thr were introduced into dopamine solutions, respectively. Fig. 1C shows the color change of the dopamine solution after the addition of the above substances. Once thiol compounds of cysteine, N-acetyl-cysteine, and glutathione were added, separately, a color of solutions changed from brown to light yellow, immediately. However, when other amino acids were added, the colors of those solutions kept nearly unchanged. Furthermore, after introducing H2 O2 , the solutions were treated by hydrothermal treatment at 50 ◦ C for 7 h. Afterward, the solutions corresponding to the compounds containing the thiol group were yellow in the visible light and blue in the UV lamp, whereas the solutions of the thiol-free amino acids solutions were brown in visible light and cyan under UV light as shown in Fig. 1D. Since GSH has the greatest effect on enhancing the fluorescence intensity of polydopamine, in this paper, glutathione and dopamine were used as raw materials to form PDA-G (-S-)NPs. During the synthesis process, dopamine was easily oxidized to dopaquinone in Tris-HCl (100 mM, pH 8.5) buffer solution [29,30]. The electrodeficiency character of generated dopaquinone has the ability to accept electrons from polymerized products under the irradiation of light [31], which can give rise to intramolecular photoinduced electron transfer (PET), leading to a low fluorescence intensity of the final products [32]. The PET effects were destroyed in the presence of GSH, due to the strong electron-donating effects of

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Fig. 2. (A) The UV–vis absorption spectra of different reactants: curve a (DA), curve b (GSH), curve c (DA + GSH), curve d (GSH + H2 O2 ), curve e (DA + H2 O2 ), and curve f (DA + GSH + H2 O2 ); (GSH: 5.2 mM, DA: 10.5 mM, H2 O2 : 200 ␮L). (B) Fluorescence emission spectra of PDA-G (-S-)NPs (2.5 mg mL−1 ) under different excitations from 320 to 440 nm.

thioethers groups. Naturally, the as-prepared PDA-G (-S-)NPs emit a stronger fluorescence after polymerized. Because reduced glutathione is an antioxidant, it is necessary to introduce an oxidant to oxidize the dopamine derivative. In this work, H2 O2 was selected to promote the fluorescence intensity of the system and control the size of nanoparticles [23,33]. The sizes of PDA-G (-S-)NPs gradually increased with increasing dosage of H2 O2 , but excessive H2 O2 can degrade large PDA-G (-S-)NPs into small particles [34]. In addition, a higher pH condition is more conducive to the combination of GS- with dopamine, leading to fluorescence elevation of PDA-G (-S-)NPs. Accordingly, there exists a certain relationship between the fluorescence intensity of PDA-G (-S-)NPs and the pH value. However, the fluorescence intensity decreases, when the pH value is greater than 9, which can be explained by the fact that glutathione is likely to hydrolyze under a stronger alkaline condition [35]. Therefore, in the subsequent sensing of H2 O2 , Tris-HCl buffer of pH 9.0 was selected, with the introduction of different concentrations of H2 O2 , the fluorescence intensity of the as-prepared fluorescent probe decreased gradually and had a good linearity. It is reported that the hydrogen peroxide can oxidize thioether to sulfoxide and sulfone groups [36,37]. Therefore, the reaction mechanism of H2 O2 and PDA-G (-S-)NPs can be expressed as follows: PDA-G(-S-)NPs + H2 O2 → PDA-G(-SO-)NPs

(1)

PDA-G(-SO-)NPs + H2 O2 → PDA-G(-SO2 -)NPs

(2)

The above reactions can produce sulfoxide and sulfone groups, which have great polarity: the [O] with negative charges and the [S] with positive charges. Thence, the conjugated large ␲ system can form among sulfur atom, amino and the phenolic hydroxyl in polymers, which leads to the UV–vis absorption redshift. This phenomenon coincides with the previous report about the solvent effect of sulfoxide and sulfone groups on the UV–vis absorption of some aminoazobenzene dyes [38]. In addition, under the oxidation of hydrogen peroxide, the electron-donating thioethers were changed to sulfoxide groups and sulfone groups with increasing electron-withdrawing ability, which can lead to a decrease in fluorescence intensity. We have investigated the effect of H2 O2 on the UV–vis absorption and fluorescence intensity of PDA-G (-SO2 )NPs. The UV–vis absorption spectra and fluorescence spectra of PDA-GNPs in the absence and presence of H2 O2 were measured. When compared to curve a in Fig. S1, curve b shows a redshift of absorption after the introduction of H2 O2 . And the fluorescence intensity of PDA-G (-S-)NPs in the presence of H2 O2 (curve d) is far below than that of PDA-G (-S-)NPs in the absence of H2 O2 (curve c). Therefore, we infer that the decrease in the fluorescence intensity

of PDA-G (-S-)NPs after the addition of H2 O2 is mainly because of the transform of the thioether to sulfoxide and sulfone groups. It is noteworthy that in the synthesis of PDA-G (-S-)NPs, the main role of H2 O2 is as oxygen resource to promote the production of PDA-G (-S-)NPs. In the preparation of PDA-G (-S-)NPs, the amount of hydrogen peroxide (200 ␮L) was controlled, which was not enough to oxidize the thioether of PDA-G (-S-)NPs as revealed in Fig. S2A. This result can be explained as follows: Due to the removal of active oxygen by GSH, H2 O2 played a significant role in the regulation of the particle sizes of PDA-G (-S-)NPs. Moreover, the temperatures are different in both the synthesis and sensing procedures. 3.2. Characterization of polydopamine-glutathione nanoparticles The PDA-G (-S-)NPs were characterized by fluorescence spectroscopy, UV–vis absorption spectroscopy, nuclear magnetic resonance (NMR) spectra, Fourier transform infrared (FT-IR) spectra, zeta potential, hydrodynamic diameter, and transmission electron microscopy (TEM) image. Fig. 2A shows the corresponding UV–vis absorption spectra of different reactants. From the UV–vis absorption spectra of curves e (DA + H2 O2 ) and f (DA + GSH + H2 O2 ), it can be seen that the addition of GSH enhanced the intensity of the absorption peak at 400 nm. From curves b (GSH), d (GSH + H2 O2 ), and f (DA + GSH + H2 O2 ), we can exclude the possibility that the absorption peak at 400 nm was caused by GSH or the interaction of GSH and H2 O2 . According to the above phenomena, we assume that the fluorescence enhancement of the PDA-G (-S-)NPs may be induced by the reaction product of DA with GSH under the effect of H2 O2 . In order to investigate the role of H2 O2 in the synthesis progress of PDA-G (-S-)NPs, the fluorescence spectra and UV–vis absorption spectra of the products with different dosages of H2 O2 were measured. As shown in Fig. S2A, the fluorescence intensity increased dramatically with the gradual addition of H2 O2 from 50 to 200 ␮L, which can be interpreted that hydroxyl groups from H2 O2 can contribute to the addition reaction between dopamine and glutathione to form fluorescent PDA-G (-S-)NPs. As the amount of H2 O2 continued to increase, the fluorescence intensity remained relatively stable, and finally even appeared a decreasing trend. This is because the strong oxidizing agent, H2 O2 , in alkaline conditions can chemically degrade PDA, leading to the decrease in fluorescence intensity [39]. In addition, the UV–vis absorption spectra of the PDA-G (-S)NPs synthesized with different amounts of H2 O2 were measured.

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Fig. 3. (A) FT-IR spectra of (a) DA, (b) GSH, and (c) PDA-G (-S-)NPs (2.5 mg mL−1 ). (B) 1 H NMR spectrum of PDA-G (-S-)NPs (2.5 mg mL−1 ). (C) 13 C NMR spectrum of DA, GSH, and PDA-G (-S-)NPs (2.5 mg mL−1 ). (D) TEM image of PDA-G (-S-)NPs (2.5 mg mL−1 ).

Fig. S2B reveals that the corresponding absorbance at 400 nm, the characteristic absorption peak of PDA-G (-S-)NPs, declined with increasing amount of H2 O2 , which may be due to the different particle sizes of as-prepared nanoparticles with the different dosage of H2 O2 . To further investigate the influence of H2 O2 on the nanoparticle size, hydrodynamic diameter and zeta potential of PDA-G (-S-)NPs were conducted. Fig. S3 is the chart of the hydrodynamic diameter distribution: the mean sizes are 3.765, 10.576, 7.647, and 0.882 nm when the volume of H2 O2 is 0, 50, 200, and 400 ␮L, respectively. Obviously, the amount of H2 O2 played an important role in the change of the size of PDA-G (-S-)NPs, which initially assisted the growth of nanoparticles, and then degraded the large PDAG (-S-)NPs into small particles if it exceeded a critical value [34]. When the volume of H2 O2 was 200 ␮L, the absolute value of zeta potential of PDA-G (-S-)NPs is relatively higher (close to 30), indicating that the nanoparticles are more stable. Although the zeta potential of PDA-G (-S-)NPs is higher when H2 O2 was absent, the fluorescence intensity of nanoparticles is very weak (Table S1). Therefore, the volume of H2 O2 , 200 ␮L, was selected to synthesize PDA-G (-S-)NPs in the following investigation. Fig. 2B shows that the maximum emission wavelength is located at 450 nm and the fluorescence intensity reached to 2000 a.u. when excited at 380 nm. A slight excitation-dependence can also be observed with increase in excitation wavelength from 320 to 440 nm. To examine the change of groups on PDA-G (-S-)NPs, the FT-IR spectra measurement was carried out. The FT-IR spectra are shown in Fig. 3A, in which the feature absorption at 1616 cm−1 in curve a (DA) is the stretching vibration of C C double bonds in an aromatic ring [40]. And the absorption at 1650 cm−1 still exists in curve c (PDA-G (-S-)NPs) which means that the products contain the aromatic ring. In addition, the disappearance of thiol group stretching

vibration in curve c located at 2525 cm−1 indicates that the thiol groups reacted with PDA [41]. The characteristic strong absorption located at 3398 cm−1 in curve c represents the stretching vibration of O H and N H in phenolic hydroxyl [42]. Fig. 3B is the 1 H NMR spectrum of PDA-G (-S-)NPs. The peaks at 6.748-6.560 and 2.011 ppm represent the H in the aromatic rings of grafted catechol moieties and the H in the methylene of GSH, respectively, further demonstrating the successful conjugation of GSH to PDA. And the values of the above results are consistent with those of the previously reported 1 H NMR spectrum for benzene ring and GSH [43,44]. Then, the PDA-G (-S-)NPs were characterized using 13 C NMR in Fig. 3C, the peaks between 100 and 150 ppm that were seen in the spectrum of DA disappeared in the 13 C NMR spectrum of PDA-G (-S-)NPs. This phenomenon can be explained by the fact that the addition of GSH can result in redistribution of electron density in benzene ring and then result in chemical shift moving to low field (150–200) [45]. Fig. 3D is the TEM image of the PDA-G (-S-)NPs with an average size (7–8 nm).

3.3. Optimization of sensing conditions To maximize the fluorescence intensity of PDA-G (-S-)NPs, some parameters including the ratio of DA to GSH, the volume of H2 O2 , and the pH of buffer solutions were optimized. As demonstrated in Fig. S4, the fluorescence intensity of PDA-G (-S-)NPs was the strongest when the ratio of DA to GSH was 1:0.75. Nevertheless, the fluorescence intensity of PDA-G (-S-)NPs became lower when the value of ratio changed. As a result, 1:0.75 was chosen as the optimal ratio of DA to GSH. It can be seen from Fig. S2A, with increasing concentration of H2 O2 from 50 to 200 ␮M, the fluorescence intensity of PDA-G (-S-)NPs increased sharply. And when the amount

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Fig. 4. (A) Fluorescence emission spectra of PDA-G (-S-)NPs (2.5 mg mL−1 ) in pH 9.0 Tris-HCl buffer upon addition of different concentrations of H2 O2 (from 0.5 to 50 ␮M). (B) The linear calibration plot between the concentration of H2 O2 (0.5–6 ␮M) and the variation of fluorescence intensity (F0 − F), respectively. (C) Fluorescence emission spectra of PDA-G (-S-)NPs (2.5 mg mL−1 ) with glucose (from 0 to 1000 ␮M) in the presence of GOx (10 mg mL−1 ) under the optimum excitation wavelength of 380 nm. (D) The linear calibration plot between the concentration of glucose (2.0–130 ␮M) and the variation of fluorescence intensity (F0 − F), respectively.

of H2 O2 continued to increase, the fluorescence intensity changed slightly or even showed a downward trend. In addition, it can be derived from Fig. S3 and Table S1, a small amount of H2 O2 is helpful to the growth of PDA-G (-S-)NPs, but the degradation of PDA-G (-S-)NPs occurred with a much higher dosage. Hence, a dosage of 200 ␮L of H2 O2 was chosen as the optimal value. Based on the above optimization, the effect of the pH of reaction medium on the fluorescence intensity of PDA-G (-S-)NPs was studied. Fig. S5 shows the fluorescence intensity of the as-developed system is linearly related to the pH value at pH 6.0 − 9.0. When the pH is higher than 9.0, the fluorescence intensity decreases. Therefore, the pH value (9.0) of the Tris-HCl buffer solution was selected as the optimal experimental condition. Consequently, the sensor can be applied to construct pH sensing when the ambient pH is in the range of 6.0–9.0. Finally, the time dependence of H2 O2 to the fluorescence quenching of PDA-G (-S-)NPs (2.5 mg mL−1 ) dissolved in Tris–HCl buffer (50 mM, pH 9.0) was investigated. Fig. S6 reveals that in the presence of 5 ␮M H2 O2 , the fluorescence of PDAG (-S-)NPs (2.5 mg mL−1 ) system decreased to a stable value in the time range of 10–30 min, and then continued to decline by a certain value for a longer time. To establish a fast and sensitive sensor, 10 min was selected as the optimal reaction time. 3.4. H2 O2 and glucose sensing based on the fluorescent PDA-G (-S-)NPs system Under the above-mentioned optimal conditions, the sensing ability of PDA-G (-S-)NPs system to H2 O2 was studied. As indicated in Fig. 4A, the fluorescence peak at 450 nm decreased gradually with increasing concentration of H2 O2 in the range from 0.5 to 50 ␮M. A linear relationship between fluorescence variables

(F0 − F) (F0 and F respectively represent the fluorescence intensity of sensing system in the absence and presence of H2 O2 ) and the concentration of H2 O2 was obtained in Fig. 4B. The equation is: (F0 − F) = 57.0865C − 20.6691. (R2 = 0.999).The linearity interval was ranging from 0.5 to 6 ␮M. And the detection limit was approximately 0.15 ␮M according to the 3␴/s rule. Additionally, glucose can be catalyzed by GOx to generate H2 O2 . So, the fluorescent sensor can be further applied to detection of glucose. Since catalytic reaction time has an impact on the amount of H2 O2 , and excess GOx can increase the background signal and the experimental cost, the reaction time of GOx-mediated enzymatic reaction and the amount of GOx are also studied to gain an optimum performance. Fig. S7 shows that the catalytic activity was positively correlated with the concentration GOx when the concentration of GOx was 1–15 mg mL−1 . Due to the enhancement of background signal caused by the excessive GOx, 10 mg mL−1 GOx was selected as the optimum level. Fig. S8 displays that the fluorescence variables (F0 − F) (F0 − F represents the reduced fluorescence intensity aroused by H2 O2 , which was the oxidation product of glucose) kept constant when the reaction time of GOx and glucose was more than 60 min in 37 ◦ C bath water. As a result, 60 min was used as the incubation time in the subsequent glucose detection experiments. Fig. 4C reveals the relationship between the fluorescence variables (F0 − F) of PDA-G (-S-)NPs and the concentration of glucose changing from 0 to 1000 ␮M. Fig. 4D shows the linear relationship between fluorescence variables (F0 − F) of PDA-G (-S)NPs system and the concentration of glucose (C). The equation is: (F0 − F) = 1.9266C + 24.9907. (R2 = 0.998). The linearity interval was ranging from 2.0 to 130 ␮M and the detection limit was approximately 0.6 ␮M according to the 3␴/s rule, which was comparable to that of the previously reported nanoparticles (Table S2). In addition,

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4. Conclusions

Fig. 5. The variable values of fluorescence intensity (F0 -F) of PDA-G (-S-)NPs (2.5 mg mL−1 ) in the presence of glucose (100 ␮M, 20 ␮L) and other carbohydrates and metal ions (200 ␮M, 20 ␮L).

In summary, a novel method was applied to prepare an intrinsic fluorescent polydopamine derivative, PDA-G (-S-)NPs. Due to the production of electron-donating thioether group, the PET effect was weakened and a bright fluorescence intensity of PDA-G (-S)NPs was obtained. The fluorescent nanoparticles were further used to construct the fluorescent sensor for the detection of H2 O2 and glucose. The GOx-catalyzed oxidation of glucose generated H2 O2 , which can oxidize the thioether groups of PDA-G (-S-)NPs to sulfoxide and sulfone groups, leading to the decrease of the fluorescence intensity. Therefore, it is feasible to detect the concentration of H2 O2 and glucose according to the fluorescence signal decrease of PDA-G (-S-)NPs. Also, the sensor has good sensitivity and selectivity over other potential interferences existing in the human body. Consequently, the as-established fluorescent sensor can be used for the detection of glucose in human blood samples with satisfactory results. Acknowledgments

Table 1 Analytical results of glucose in the human serum samples (n = 3). Samples

Added (mM)

Detected (mean, mM)

RSD (n = 3, %)

1 2 3 4

0 1 2 3

4.76 5.78 6.75 7.91

2.8 4.3 3.6 3.1

Recovery (%)

102.0 99.5 105.0

Mean value of three determinations. The detection concentration is 4.86 mM by the local hospital.

we have investigated the time-dependent stability of the proposed method. Fig. S9 shows that the fluorescence intensity of the reaction system is stable in the range from 10 to 90 min. After 90 min, the fluorescence intensity decreased slightly.

3.5. Selectivity of the fluorescent PDA-G (-S-)NPs system for glucose sensing To examine the specificity to glucose sensing, the selectivity of PDA-G (-S-)NPs sensing system was evaluated, respectively. The influence of other substances including several common carbohydrates (sucrose, lactose, maltose, fructose, and mannose), vitamin C, and metal ions (Na+ , K+ , Ca2+ , Mg2+ , and Fe3+ ) on the fluorescence intensity of PDA-G (-S-)NPs system was investigated. As revealed in Fig. 5, the as-constructed PDA-G (-S-)NPs sensor displayed a striking variable value of fluorescence intensity (F0 -F) in the presence of glucose. However, other species have no interferences or only a little difference in fluorescence intensity (F0 -F).

3.6. Glucose sensing in human serum samples The practical application of this sensor is the detection of glucose in human serum samples. The glucose levels of human serum samples were detected by our proposed sensor and the experimental values were compared with local hospital information. The measured results of glucose in human serum samples are shown in Table 1. It is clear that the data obtained using the PDA-G (-S-)NPs sensor were in agreement with the information provided by the hospital and the recoveries of human serum samples were 102.0%, 99.5%, and 105.0%, respectively. Therefore, the constructed sensors have a promising practical application.

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Biographies Li Tang is an MS candidate in School of Chemistry and Chemical Engineering. Southwest University, China. Her major research interests include photochemistry and photoanalytical chemistry. Shi Mo is an MS candidate in School of Chemistry and Chemical Engineering. Southwest University, China. His major research interests include electrochemical corrosion and protection of metal and multifunctional material. Shi Gang Liu is a PhD candidate in School of Chemistry and Chemical Engineering. Southwest University, China. His major research interests include photochemistry and photoanalytical chemistry. Na Li is a PhD candidate in School of Chemistry and Chemical Engineering. Southwest University, China. Her major research interests include photochemistry and photoanalytical chemistry. Yu Ling is a postdoctoral fellow in School of Chemistry and Chemical Engineering, 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. Southwest University, China. He received his MS degree in physical chemistry in 1997 and PhD 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 postdoctoral research fellow in Korea Advanced Institute of Science and Technology (KAIST), Korea. His research interests are the developments of electrochemical devices such as chemical sensors and biosensors. Hong Qun Luo is a professor of chemistry in School of Chemistry and Chemical Engineering, Southwest University, China. She received her MS degree in environmental chemistry from Sichuan University in 1991 and PhD degree in analytical chemistry from Southwest China Normal University in 2002. During 2006–2007, she was a visiting scholar at Tohoku University, Japan. Her research is focused on molecular spectroscopy and electrochemical sensor.