Polymethyldopa Nanoparticles-Based Fluorescent Sensor for

Aug 28, 2018 - (18) In 2012, Zhang et al. obtained fluorescent polydopamine organic .... reaction with ethanolamine between methyldopa and metyrosine,...
0 downloads 0 Views 654KB Size
Download PDF
Subscriber access provided by UNIV OF DURHAM

Article

Polymethyldopa Nanoparticles-Based Fluorescent Sensor for Detection of Tyrosinase Activity Guoyong Liu, Jiahui Zhao, Shasha Lu, Shuang Wang, Jian Sun, and Xiurong Yang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00684 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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

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

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

ACS Sensors

Polymethyldopa Nanoparticles-Based Fluorescent Sensor for Detection of Tyrosinase Activity Guoyong Liu†,‡, Jiahui Zhao†,§, Shasha Lu†,‡, Shuang Wang,†,‡, Jian Sun*,†, and Xiurong Yang*,†,‡ †State

Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡University of Science and Technology of China, Hefei, Anhui 230026, China §University of Chinese Academy of Sciences, Beijing 100049, China *Fax: +86 431 85689278. E-mail: [email protected], [email protected]

KEYWORDS: tyrosinase, fluorescent sensor, methyldopa, ethanolamine, polymethyldopa nanoparticles ABSTRACT: Being a typical copper-containing oxidase, tyrosinase plays critical roles in biological activity and its aberrant expression might cause diverse skin diseases. Herein, we, for the first time, found an interesting green fluorogenic reaction between methyldopa and ethanolamine. By combining transmission electron microscopy, UV−visible absorption spectrum, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and MALDI-TOF mass spectrum analysis, we have confirmed that there is a reliable method for preparing the bright green fluorescent polymethyldopa nanoparticles (PMNPs) by simply mixing methyldopa and ethanolamine at room temperature. Inspired by such simple and convenient fluorogenic reaction, a novel polymethyldopa nanoparticles-based fluorescent sensor for detection of tyrosinase activity was developed by using the commercially available metyrosine as a substrate, accompanied by the tyrosinase-catalyzed specific conversion of metyrosine into methyldopa. According to the intrinsic sensitivity/selectivity of fluorescence technology and unambiguous response mechanism, our fluorescent sensor exhibits excellent sensing performance and can be utilized in the determination of the tyrosinase activity in real biological samples and inhibitor screening.

Tyrosinase (EC 1.14.18.1) can be found in plants, bacteria, fungi, and animal tissues. As a copper-containing oxidase, it can convert monophenol to catechol, and then to the corresponding orthoquinone product.1 Tyrosinase plays critical roles in vertebrate pigmentation2 and food browning process.3 The aberrant expression of tyrosinase has been regarded as a reason of the melanoma cancer.4 Many previous studies have found that the disordering of tyrosinase might lead to vitiligo5 and Parkinson disease.6 Given the importance of tyrosinase in pathophysiology, a convenient assay of its activity is important and necessary. To date, various sensing methods have been reported for detection of tyrosinase activity, such as colorimetry,7 electrochemistry,8 fluorometry,9 and radiometry.10 Among them, fluorescent assays have been widely used because of their simple operation, low cost, and high sensitivity. Most of these fluorescent assays are applied to monitor tyrosinase activity by employing multifarious fluorescent probes like near-infrared probe,11 gold/silver nanoclusters,12 inorganic semiconductor quantum dots,13 and carbon quantum dots.14 However, there still exist some shortcomings in these fluorescent probes, for example, the time-consuming synthesis procedure for near-infrared probe, high cost for gold/silver nanoclusters, high toxicity for inorganic semiconductor quantum dots, and complex modification for carbon quantum dots. More significantly, most of the current

fluorescent assays are based on the tyrosinase-catalyzed oxidation of dopamine to o-quinone moiety and take advantage of the fluorescence quenching effect of o-quinone moiety on the specific fluorophore. Such fluorescence turnoff sensing systems suffer from low sensitivity, high background and laborious labeling processes.15,16 Therefore, it is still urgent to establish a straightforward and rapid fluorescence turn-on strategy for tyrosinase activity monitoring. Recently, polydopamine with intrinsic fluorescence properties have drawn extensive attention in the biomedical applications.17 Polydopamine can be obtained by the oxidation and self-polymerization of dopamine in the presence of aerobic and alkaline conditions. The alkaline condition is usually provided by buffer solution (e.g., Tris buffer), which allows the polydopamine to have weak or none fluorescence.18 In 2012, Zhang et al. obtained fluorescent polydopamine organic nanoparticles by using H2O2 as oxidant to oxidize self-polymerized dopamine for 5 h.19 Lin et al. prepared fluorescent polydopamine dots by using NaOH and H2O2 as oxidants to oxidize polydopamine through heating.20 Liu et al. fabricated fluorescent organic nanoparticles by using dopamine and polyethyleneimine through Michael addition and Schiff base reaction.21 However, few articles reported the preparation of fluorescent nanoparticles by

1

ACS Paragon Plus Environment

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

using dopamine analogues,22 especially based on the oxidation of dopamine analogues by ethanolamine (a strong alkaline). Inspired by the aforementioned self-polymerization, Michael addition and Schiff base reaction, we develop a novel method for preparing the bright green fluorescent polymethyldopa nanoparticles (PMNPs) by simply mixing methyldopa (a dopamine analogue) and ethanolamine at room temperature for 2 h. A series of characterization techniques are applied to study and confirm the formation mechanism of fluorescent PMNPs. Compared to the fluorescent polydopamine nanoparticles, the preparation of fluorescent PMNPs does not need longer time and higher temperature, which is advantageous for designing the biosensing system. Furthermore, we notice that methyldopa can be obtained by hydroxylation of metyrosine with the help of tyrosinase, and metyrosine cannot react with ethanolamine. Such difference in the reaction with ethanolamine between methyldopa and metyrosine ensures that metyrosine can be used as a substrate to determine the tyrosinase activity. Tyrosinase-incubated metyrosine solution shows bright green fluorescence under the action of ethanolamine, and the fluorescence intensities associate with the tyrosinase concentrations. Thus, under the unequivocal sensing principle, a novel fluorescent sensor is developed and demonstrated for tyrosinase activity detection based on in situ synthesis of fluorescent PMNPs. The proposed sensor has a good sensitivity and selectivity toward tyrosinase in both aqueous solution and real serum sample. This sensor is also utilized to screen inhibitor of tyrosinase by using kojic acid as a model. To our knowledge, this is the first report on the synthesis and application of polymethyldopa nanoparticles-based fluorescent sensor. Therefore, our study may introduce a new prospect to sense tyrosinase activity and screen its potential inhibitors.

EXPERIMENTAL SECTION Chemicals. Metyrosine was obtained from Aladdin Industrial Corporation (Shanghai, China). Methyldopa was acquired from Shanghai Sangon Biotechnology Co., Ltd. (China). Tyrosinase from mushroom, lysozyme, trypsin, human serum albumin (HSA), bovine serum albumin (BSA), EcoR I, glucose oxidase (GOx), alkaline phosphatase (ALP), ethanolamine, sodium hypochlorite, sodium borohydride and kojic acid were acquired from Sigma-Aldrich Corporation (Shanghai, China). Clinical fetal bovine serum samples were kindly gifted from the Second Hospital of Jilin University (Changchun, China). All of the other chemicals bought from Beijing Chemical Reagent Co. (China) were analytical grade and used as received. Ultrapure water produced by a Millipore system (USA) was utilized in all aqueous solution. Instruments. UV–visible spectra were measured on a CARY 500 UV−Vis−NIR Varian spectrophotometer (CA, USA). Fluorescence spectra were surveyed on a Hitachi F4600 spectrofluorometer (Tokyo, Japan). Transmission electron microscopy (TEM) measurement was obtained by using a FEI Tecnai G2 F20 S-TWIN (OR). Fourier transform infrared spectroscopy (FT-IR) spectra were taken on a Bruker Optics VERTEX 70 spectrometer (Ettlingen, Germany) in the transmission mode. X-ray photoelectron spectroscopy (XPS) was carried out on a Thermo ESCALAB VG

Page 2 of 8

Scientific 250 (West Sussex, UK). The MALDI-TOF mass spectrum was performed on an autoflex III smart beam MALDITOF/TOF Mass spectrometer (Bruker, Germany). Preparation and Purification of Fluorescent PMNPs. In a typical preparation, 108 μL of ethanolamine was added into 20 mL of 10 mM methyldopa solutions. The mixture was kept for 2 h at room temperature, followed by adding 1 mL of 6 M HCl. After that, 1000 Da cutoff dialysis bag was utilized to purify the resulting solution against ultrapure water for 8 h, during which the ultrapure water was replaced every 0.5 h. The solid-state PMNPs were obtained by freeze-drying for further characterization. Detection of Tyrosinase Activity by in Situ Synthesis of Fluorescent PMNPs. For detection of tyrosinase activity, 450 μL of 3 mM metyrosine and 4.5 μL of tyrosinase with different activities varying from 0 to 9 U/mL were injected into 445.5 μL of phosphate buffer (10 mM, pH 6.0). After incubation for 50 min at 37 °C and equilibrium for 5 min at room temperature, 100 μL of 900 mM ethanolamine was injected into the resulting solutions. Then the above mixture solution was incubated for another 60 min at room temperature. The fluorescence spectrum was carried out when excited at 380 nm. To evaluate the selectivity of the proposed fluorescent sensor for tyrosinase activity, other nonspecific enzymes/proteins, biological metal ions, and reactive oxygen species (ROS) instead of tyrosinase were utilized, including HSA, trypsin, lysozyme, EcoR I, GOx, ALP, BSA, Mg2+, Ca2+, K+, H2O2, and ClO-. Diluted fetal bovine serum (1%) was used in this assay to monitor the tyrosinase activity in real sample. Inhibitor Screening. To assess the efficiency of inhibitor, 4.5 μL of tyrosinase (1000 U/mL) and 9 μL of kojic acid solutions with different concentrations were injected into 436.5 μL of phosphate buffer (10 mM, pH 6.0) and then kept for 10 min at room temperature. 450 μL of 3 mM metyrosine was injected into the above mixture solution. The resulting solutions were incubated for 50 min at 37 °C and then for 5 min at room temperature. Finally, 100 μL of 900 mM ethanolamine was added into the solutions. The fluorescence spectra were obtained when excited at 380 nm after incubation for another 60 min at room temperature.

RESULTS AND DISCUSSION Synthesis and Characterization of Fluorescent PMNPs. Similar to the formation of polydopamine, we speculate that methyldopa will also self-polymerize to produce the PMNPs under aerobic and alkaline conditions. Thus, ethanolamine (a strong alkaline primary amine) is chosen to oxidize methyldopa. By simply mixing methyldopa and ethanolamine, the mixture solution exhibits yellow under visible light while appearing intense green emission under 365 nm ultraviolet light (inset of Figure 1A). Meanwhile, no fluorescence can be observed under 365 nm ultraviolet light in either methyldopa or ethanolamine solution. As shown in Figure 1A, ethanolamine has no obvious absorption, and the absorption peak of methyldopa at 280 nm is attributed to ππ* transition of C=C.23 Apart from the peak at 280 nm, a new absorption peak at 380 nm appears in the UV–visible absorption spectra of the fluorescent PMNPs, which can be ascribed to n-π* transitions of C=O/C=N.24 The fluorescent PMNPs show maximum emission wavelength at 525 nm

2

ACS Paragon Plus Environment

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

ACS Sensors with a maximum excitation wavelength at 380 nm. The emission peaks of fluorescent PMNPs are fixed at 525 nm with excitation wavelengths ranging from 340 to 420 nm (Figure 1B). The relative fluorescence quantum yield (QY) is 2.09% with quinine sulfate (QY = 54%) as the reference in 0.05 M H2SO4 (Figure S1).25 The morphology of fluorescent PMNPs was imaged by TEM. As shown in Figure 1C and the inset of Figure 1C, the fluorescent PMNPs display spherical nanoparticles with diameters from 1.5 nm to 11.5 nm. Compared with FT-IR spectra of methyldopa (Figure 1D red line), a new absorbance peak at 1043 cm-1 emerges in fluorescent PMNPs (Figure 1D black line), which can be ascribed to the C−N asymmetric stretching vibration,26 implying the possible Michael addition reaction between aromatic rings and amine groups.27 Other characteristic functional groups can be proved by stretching vibration mode, such as the O−H/N−H (3260 cm-1),28 C=O/C=N (1602 cm1),29 C=C (1515 cm-1),30 C−N= (1394 cm-1),31 and C−O (1282 cm-1).32

Figure 1. (A) The UV−visible absorption spectra of ethanolamine (a), methyldopa (b), fluorescent PMNPs (c), and the excitation (d), and emission (e) spectra of the fluorescent PMNPs. Inset photos display the fluorescent PMNPs under ambient (left) and 365 nm ultraviolet light (right), respectively. (B) Fluorescence emission spectra of the fluorescent PMNPs at various excitation wavelengths. (C) TEM image of the fluorescent PMNPs. The scale bar is 100 nm. Inset shows the size distribution histogram. (D) FT-IR spectra of methyldopa (red line) and the fluorescent PMNPs (black line).

The elemental analysis and chemical compositions of the fluorescent PMNPs were investigated by XPS. As depicted in Figure S2A, five peaks observed at 198, 268, 285, 401 and 532 eV are ascribed to Cl 2p, Cl 2s, C 1s, N 1s, and O 1s, respectively.33 Trace quantities of chlorine should be produced by HCl because the polymerization reaction was blocked by adding HCl.34 Thus, the full survey of XPS spectrum reveals that the fluorescent PMNPs mainly consist of C, N, and O elements. The high resolution C 1s spectrum demonstrates that the fluorescent PMNPs contain C=C (284.5 eV), C−C (284.6 eV), C−N/C−O (286.2 eV), and C=N/C=O (288.2 eV) bonds (Figure S2B).35 The high resolution N 1s spectrum indicates the existence of C=N (398.6 eV), C−N−C (399.6 eV) and N−H (401.3 eV) species (Figure S2C).36 The high resolution O 1s spectrum implies that the PMNPs have C=O (531.2 eV) and C−O (532.5 eV) bonds (Fig-

ure S2D).37 The newly formed C=N bonds indicate the occurrence of Schiff base reaction. To explore the possible chemical structures of the fluorescent PMNPs, MALDI-TOF mass spectrum measurement was performed. The fragment ion peaks at m/z 416, 461 and 483 in Figure S3A might be corresponding to the possible chemical structures of a, b and c (Figure S3B), respectively, implying the incorporation of ethanolamine into the fluorescent PMNPs through selfpolymerization, Schiff base and Michael addition reaction.38 Assuming that there is no relative molecular mass of ethanolamine, we cannot get the structures of b and c, which further confirm the reaction of ethanolamine with methyldopa. Control experiments were further performed to reveal the reaction mechanism. A sufficient quantity of ascorbic acid (a common reducing reagent) was injected into methyldopa solution before adding ethanolamine. Meanwhile, NaOH, instead of ethanolamine, was used to oxidize the methyldopa. As shown in Figure S4A line 1 and line 2, no fluorescence signal could be observed in the control experiments, indicating that ascorbic acid could reduce quinone derivative to phenol derivative to block the in situ synthesis of fluorescent PMNPs,39 and only in alkaline environment, methyldopa could not produce fluorescent PMNPs.40 In addition, the as-prepared fluorescent PMNPs were treated with a sufficient quantity of NaBH4 (a special reducing reagent that can reduce C=N group). As shown in Figure S4B, C, after reduction by NaBH4, the absorption peak at 380 nm and fluorescence intensity at 525 nm decrease, accompanied by a blue shift of the emission position. These results are consistent with previous study, indicating that the C=N group in fluorescent PMNPs is reduced.33 We speculate that the C=N group in fluorescent PMNPs might play an important role in luminescent center.40 Thus, by combining FT-IR spectrum, XPS spectrum, MALDI-TOF mass spectrum, and control experiments analysis, the formation mechanism might be as follows. In the presence of ethanolamine, methyldopa is first oxidized by strong base into quinone derivative. And then the unstable quinone derivative reacts with ethanolamine to produce fluorescent PMNPs via selfpolymerization, Michael addition and Schiff base reaction. Establishment and Optimization of Tyrosinase Sensing System.

Figure 2. (A) Fluorescence emission spectra at increasing concentrations of methyldopa. (B) Fluorescence intensities versus different concentrations of methyldopa in the presence of 90 mM ethanolamine.

The synthesis conditions of the fluorescent PMNPs were optimized by monitoring the fluorescence intensities and UV−visible absorption spectra changes over time. Stable fluorescence intensities can be observed at 2 h in the presence of 90 mM ethanolamine (Figure S5A). The absorbance of the mixture of methyldopa (100 μM) and ethanolamine (90 mM)

3

ACS Paragon Plus Environment

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

no longer increases in 2 h (Figure S5B). Therefore, the favorable experimental conditions of the concentration of ethanolamine and the reaction time were 90 mM and 2 h, respectively. To obtain the response at low concentration, we investigated the effect of methyldopa concentration within 100 μM on the fluorescent PMNPs in detail. With the concentration of methyldopa varying from 0 to 100 μM, the fluorescence intensities at 525 nm of fluorescent PMNPs increased gradually (Figure 2A). A good linear relationship (R2 = 0.993) was acquired as the concentrations of methyldopa ranging from 0 to 80 μM (Figure 2B), implying good conditions for detection of methyldopa-related targets.41 We notice that methyldopa is a product of hydroxylation of metyrosine with the help of tyrosinase. Apart from that, no fluorescence can be observed under 365 nm ultraviolet light in the mixture solution of metyrosine and ethanolamine (Figure S6). Due to the difference in the reaction with ethanolamine between methyldopa and metyrosine, we choose commercially available metyrosine as the substrate of tyrosinase for developing a convenient fluorescence turn-on tyrosinase activity assay.

Page 4 of 8

the presence of oxygen and tyrosinase, metyrosine is oxidized to methyldopa, which further reacts with ethanolamine to produce fluorescent PMNPs (Figure 3A). Consequently, the strategy of tyrosinase-enabled in situ formation of fluorescent PMNPs is proposed, which can be applied for detection of tyrosinase activity.42 To detect tyrosinase activity, we optimized the effect of pH, the concentration of metyrosine, the incubation time of metyrosine with tyrosinase, and the reaction time of tyrosinase-incubated metyrosine with ethanolamine on detection of tyrosinase activity. As shown in Figure S7A, the pH value was first optimized to 6.0. With the increase of metyrosine concentration, the fluorescence intensity at 525 nm of fluorescent PMNPs increased gradually (Figure S7B). Considering the solubility of metyrosine in water, 1.5 mM metyrosine was recommended as the concentration of substrate. The fluorescence intensities reached the maximum at 50 min and then a little decreased with the further increase of incubation time (Figure S7C). Therefore, the favorable incubation time was 50 min. When the reaction time of tyrosinase-incubated metyrosine with ethanolamine was more than 60 min, the fluorescence intensities of three concentrations of tyrosinase almost kept constant (Figure S7D). Hence, the optimum reaction time of tyrosinase-incubated metyrosine with ethanolamine was 60 min. Compared with the reaction time of methyldopa with ethanolamine, the reaction time of tyrosinase-incubated metyrosine with ethanolamine was decreased because tyrosinase could convert methyldopa into methyldopachrome,43 which was favorable for the reaction. Fluorescent Sensor for Tyrosinase Activity Detection.

Figure 3. (A) Principle of tyrosinase-triggered formation of fluorescent PMNPs. (B) Absorption and (C) fluorescence emission spectra of metyrosine (1), mixture of metyrosine and ethanolamine (2), mixture of ethanolamine and tyrosinase (3), mixture of metyrosine and tyrosinase (4), mixture of tyrosinase-incubated metyrosine and ethanolamine (5), and the as-prepared fluorescent PMNPs by mixing methyldopa and ethanolamine at room temperature for 2 h (6), respectively. The concentrations of metyrosine, methyldopa, ethanolamine and tyrosinase used in this are 1.5 mM, 100 μM, 90 mM, and 9.0 U/mL, respectively.

As shown in Figure 3B, C, metyrosine (line 1), mixture of metyrosine and ethanolamine (line 2), and mixture of ethanolamine and tyrosinase (line 3) have no absorption at 380 nm and fluorescence at 525 nm. The results show that metyrosine has no fluorescence signal at 525 nm and ethanolamine cannot react with metyrosine or tyrosinase. Tyrosinase-incubated metyrosine solution generates wide absorption band varying from 360 to 700 nm, while no fluorescence signal could be observed (line 4 in Figure 3B, C). However, in the presence of ethanolamine, tyrosinase-incubated metyrosine solution shows a new absorption peak at 380 nm and strong fluorescence signal at 525 nm (line 5 in Figure 3B, C), which is consistent with the as-prepared fluorescent PMNPs by mixing methyldopa and ethanolamine (line 6 in Figure 3B, C), implying the formation of the fluorescent PMNPs. The results suggest that the reaction can be triggered by tyrosinase-catalyzed hydroxylation of metyrosine specifically. Until now, we have demonstrated that in

Figure 4. (A) Fluorescence emission spectra of fluorescent PMNPs toward various concentrations of tyrosinase. (B) The fluorescence intensity against the concentration of tyrosinase. (C) Corresponding photos under 365 nm ultraviolet light. The final concentrations of tyrosinase are 0, 0.1, 0.4, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 U/mL, respectively. (D) Selectivity investigation of the sensing system for tyrosinase activity. The concentrations of tyrosinase, enzymes/proteins, biological metal ions, and ROS are 5 U/mL, 10 μg/mL, 250 μM, and 10 μM, respectively.

Tyrosinase activity measurements were carried out under optimal experimental conditions. With the concentrations of tyrosinase varying from 0 to 9 U/mL, the fluorescence intensities at 525 nm of fluorescent PMNPs increased

4

ACS Paragon Plus Environment

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

ACS Sensors gradually (Figure 4A). As shown in Figure 4B, the regression equation could be expressed as F = - 6.14 + 28.26 Ctyrosinase (R2 = 0.994) with a linear range from 0.40 to 7.0 U/mL, where F was the fluorescence intensity at 525 nm of fluorescent PMNPs. The detection limit was 0.10 U/mL, which was comparable to some previously reported assays for tyrosinase (Table S1).7,9,44-50 Gradually increased green fluorescence could be observed under 365 nm ultraviolet light with the increase of tyrosinase activity (Figure 4C). To validate the specificity of the sensing system, the interfering substances, including trypsin, lysozyme, EcoR I, GOx, ALP, HSA, BSA, Mg2+, Ca2+, K+ H2O2 and ClO-, with and without tyrosinase were investigated in this assay under the same conditions. As shown in Figure 4D, the interfering substances have a negligible effect on the sensing system. Above results indicate that the proposed sensing system has good sensitivity and selectivity toward tyrosinase. Accordingly, we have developed a novel fluorescent sensor for tyrosinase activity monitoring based on in situ synthesis of fluorescent PMNPs. To illustrate the practicability of the proposed sensor in real sample, tyrosinase activity was measured in 1% fetal bovine serum samples. As shown in Table S2, a satisfactory recovery varying from 94.0% to 106.3% was acquired with relative standard deviation ranging from 2.56% to 5.74%, implying the proposed sensor could be utilized in biological matrices for tyrosinase activity monitoring. Investigation of Tyrosinase Inhibitor. Tyrosinase associates with the browning process and melanin formation, whereas tyrosinase inhibitors can effectively inhibit these processes. The screening of tyrosinase inhibitors is of great significance in food browning prevention and skin disease treatment. We choose kojic acid (a common tyrosinase inhibitor) as a model to evaluate the utility of our proposed sensor for screening tyrosinase inhibitor. As shown in Figure 5A, the fluorescence intensity of our test solution decreases gradually with the concentrations of kojic acid varying from 1 to 100 μM. We acquired a representative sigmoidal curve by means of plotting the fluorescence intensity against the logarithm of the concentration of kojic acid (Figure 5B). The IC50 value (concentrations of inhibitor when tyrosinase activity is inhibited by 50%) was calculated to be 13 μM. Our result and previously reported result have the same order of magnitude,51 indicating the feasibility of this sensor for potential inhibitor of tyrosinase screening.

CONCLUSION In summary, we have found a fluorogenic reaction by easily mixing methyldopa and ethanolamine at room temperature for the first time. The reaction mechanism is studied by FTIR, XPS, MALDI-TOF mass, etc. We infer that methyldopa is first oxidized by strong base into quinone derivative, followed by the reaction with ethanolamine to produce fluorescent PMNPs. Due to the difference in the reaction with ethanolamine between methyldopa and metyrosine, a novel fluorescent sensor is successfully developed based on the in situ synthesis of fluorescent PMNPs for detection of tyrosinase activity. Our sensor not only has good sensitivity and selectivity toward tyrosinase but also exhibits good performances in real serum sample analysis and screening its related inhibitor. We believe that fluorescent PMNPs hold great potential in biosensing applications.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. Fluorescence quantum yield measurements, XPS spectra, MALDI-TOF MS spectrum, additional fluorescence and absorption analysis data, photograph of control experiment, parameter optimized data, data comparisons, and tyrosinase activity detection in real serum sample.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel.: +86 431 85262056; Fax: +86 431 85689278. *E-mail: [email protected]. Tel: +86 431 85262063. Fax: +86 431 85689278.

Author Contributions The manuscript was written through contributions of all authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the financial supports by the National Key Research and Development Program of China (2016YFA0201301), the National Natural Science Foundation of China (No. 21435005, 21627808, 21605139), Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SLH019), and the Youth Innovation Promotion Association, CAS (No. 2018258).

REFERENCES

Figure 5. (A) Fluorescence emission spectra of fluorescent PMNPs at increasing concentrations of kojic acid. (B) Kinetic plots of the fluorescence intensities versus the logarithm of kojic acid concentrations. The concentration of tyrosinase is 5 U/mL. The concentrations of kojic acid vary from 1 μM to 100 μM.

(1) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E., Multicopper oxidases and oxygenases. Chem. Rev. 1996, 96, 2563-2606. (2) Slominski, A.; Tobin, D. J.; Shibahara, S.; Wortsman, J., Melanin pigmentation in mammalian skin and its hormonal regulation. Physio. Rev. 2004, 84, 1155-1228. (3) Washington, C.; Maxwell, J.; Stevenson, J.; Malone, G.; Lowe, E. W., Jr.; Zhang, Q.; Wang, G.; McIntyre, N. R., Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one. Arch. Biochem. Biophys. 2015, 577-578, 24-34. (4) Mikami, M.; Sonoki, T.; Ito, M.; Funasaka, Y.; Suzuki, T.; Katagata, Y., Glycosylation of tyrosinase is a determinant of melanin production in cultured melanoma cells. Mol. Med. Rep. 2013, 8, 818-822.

5

ACS Paragon Plus Environment

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

(5) Song, Y. H.; Connor, E.; Li, Y.; Zorovich, B.; Balducci, P.; Maclaren, N., The role of tyrosinase in autoimmune vitiligo. Lancet 1994, 344, 1049-1052. (6) Tessari, I.; Bisaglia, M.; Valle, F.; Samori, B.; Bergantino, E.; Mammi, S.; Bubacco, L., The reaction of alpha-synuclein with tyrosinase: possible implications for Parkinson disease. J. Biol. Chem. 2008, 283, 16808-16817. (7) Xu, Q.; Yoon, J., Visual detection of dopamine and monitoring tyrosinase activity using a pyrocatechol violet-Sn4+ complex. Chem. Commun. 2011, 47, 12497-12499. (8) Yildiz, H. B.; Freeman, R.; Gill, R.; Willner, I., Electrochemical, photoelectrochemical, and piezoelectric analysis of tyrosinase activity by functionalized nanoparticles. Anal. Chem. 2008, 80, 28112816. (9) Wu, X.; Li, X.; Li, H.; Shi, W.; Ma, H., A highly sensitive and selective fluorescence off-on probe for the detection of intracellular endogenous tyrosinase activity. Chem. Commun. 2017, 53, 24432446. (10) Chen, Y. M.; Chavin, W., Radiometric assay of tyrosinase and theoretical considerations of melanin formation. Anal. Biochem. 1965, 13, 234-258. (11) Li, X.; Shi, W.; Chen, S.; Jia, J.; Ma, H.; Wolfbeis, O. S., A nearinfrared fluorescent probe for monitoring tyrosinase activity. Chem. Commun. 2010, 46, 2560-2562. (12) Ao, H.; Qian, Z.; Zhu, Y.; Zhao, M.; Tang, C.; Huang, Y.; Feng, H.; Wang, A., A fluorometric biosensor based on functional Au/Ag nanoclusters for real-time monitoring of tyrosinase activity. Biosens. Bioelectron. 2016, 86, 542-547. (13) Yan, X.; Li, H.; Zheng, W.; Su, X., Visual and fluorescent detection of tyrosinase activity by using a dual-emission ratiometric fluorescence probe. Anal. Chem. 2015, 87, 8904-8909. (14) Chai, L.; Zhou, J.; Feng, H.; Tang, C.; Huang, Y.; Qian, Z., Functionalized carbon quantum dots with dopamine for tyrosinase activity monitoring and inhibitor screening: in vitro and intracellular investigation. ACS Appl. Mater. Interfaces 2015, 7, 23564-23574. (15) Hu, J. J.; Bai, X. L.; Liu, Y. M.; Liao, X., Functionalized carbon quantum dots with dopamine for tyrosinase activity analysis. Anal. Chim. Acta 2017, 995, 99-105. (16) Zhang, F.; Xu, L.; Zhao, Q.; Sun, Y.; Wang, X.; Ma, P.; Song, D., Dopamine-modified Mn-doped ZnS quantum dots fluorescence probe for the sensitive detection of tyrosinase in serum samples and living cells imaging. Sens Actuators, B 2018, 256, 1069-1077. (17) Liu, M.; Zeng, G.; Wang, K.; Wan, Q.; Tao, L.; Zhang, X.; Wei, Y., Recent developments in polydopamine: an emerging soft matter for surface modification and biomedical applications. Nanoscale 2016, 8, 16819-16840. (18) d'Ischia, M.; Napolitano, A.; Ball, V.; Chen, C. T.; Buehler, M. J., Polydopamine and eumelanin: from structure-property relationships to a unified tailoring strategy. Acc. Chem. Res. 2014, 47, 35413550. (19) Zhang, X.; Wang, S.; Xu, L.; Feng, L.; Ji, Y.; Tao, L.; Li, S.; Wei, Y., Biocompatible polydopamine fluorescent organic nanoparticles: facile preparation and cell imaging. Nanoscale 2012, 4, 5581-5584. (20) Lin, J. H.; Yu, C. J.; Yang, Y. C.; Tseng, W. L., Formation of fluorescent polydopamine dots from hydroxyl radical-induced degradation of polydopamine nanoparticles. Phys. Chem. Chem. Phys. 2015, 17, 15124-15130. (21) Liu, M.; Ji, J.; Zhang, X.; Zhang, X.; Yang, B.; Deng, F.; Li, Z.; Wang, K.; Yang, Y.; Wei, Y., Self-polymerization of dopamine and polyethyleneimine: novel fluorescent organic nanoprobes for biological imaging applications. J. Mater. Chem. B 2015, 3, 3476-3482. (22) Gao, Z. F.; Li, T. T.; Xu, X. L.; Liu, Y. Y.; Luo, H. Q.; Li, N. B., Green light-emitting polyepinephrine-based fluorescent organic dots and its application in intracellular metal ions sensing. Biosens. Bioelectron. 2016, 83, 134-141. (23) Jiang, Y.; Wang, Z.; Dai, Z., Preparation of silicon-carbonbased dots@dopamine and its application in intracellular Ag(+) detection and cell imaging. ACS Appl. Mater. Interfaces 2016, 8, 36443650.

Page 6 of 8

(24) Miao, X.; Qu, D.; Yang, D.; Nie, B.; Zhao, Y.; Fan, H.; Sun, Z., Synthesis of carbon dots with multiple color emission by controlled graphitization and surface functionalization. Adv. Mater. 2018, 30 1704740. (25) Zhang, J.; Yu, S. H., Highly photoluminescent silicon nanocrystals for rapid, label-free and recyclable detection of mercuric ions. Nanoscale 2014, 6, 4096-4101. (26) Sun, B.; Zhao, B.; Wang, D.; Wang, Y.; Tang, Q.; Zhu, S.; Yang, B.; Sun, H., Fluorescent non-conjugated polymer dots for targeted cell imaging. Nanoscale 2016, 8, 9837-9841. (27) Zhao, C.; Zuo, F.; Liao, Z.; Qin, Z.; Du, S.; Zhao, Z., Mussel-inspired one-pot synthesis of a fluorescent and water-soluble polydopamine-polyethyleneimine copolymer. Macromol. Rapid Commun. 2015, 36, 909-915. (28) Gao, T.; Wang, X.; Yang, L. Y.; He, H.; Ba, X. X.; Zhao, J.; Jiang, F. L.; Liu, Y., Red, yellow, and blue luminescence by graphene quantum dots: syntheses, mechanism, and cellular imaging. ACS Appl. Mater. Interfaces 2017, 9, 24846-24856. (29) Gong, X.; Li, Z.; Hu, Q.; Zhou, R.; Shuang, S.; Dong, C., N, S, P co-doped carbon nanodot fabricated from waste microorganism and its application for label-free recognition of manganese(VII) and l-ascorbic acid and AND logic gate operation. ACS Appl. Mater. Interfaces 2017, 9, 38761-38772. (30) Hua, X.-W.; Bao, Y.-W.; Wu, F.-G., Fluorescent carbon quantum dots with intrinsic nucleolus-targeting capability for nucleolus imaging and enhanced cytosolic and nuclear drug delivery. ACS Appl. Mater. Interfaces 2018, 10, 10664-10677. (31) Ding, H.; Yu, S. B.; Wei, J. S.; Xiong, H. M., Full-color lightemitting carbon dots with a surface-state-controlled luminescence mechanism. ACS Nano 2016, 10, 484-491. (32) Wang, T. Y.; Chen, C. Y.; Wang, C. M.; Tan, Y. Z.; Liao, W. S., Multicolor functional carbon dots via one-step refluxing synthesis. ACS Sens. 2017, 2, 354-363. (33) Nie, H.; Li, M.; Li, Q.; Liang, S.; Tan, Y.; Sheng, L.; Shi, W.; Zhang, S. X.-A., Carbon dots with continuously tunable full-color emission and their application in ratiometric pH sensing. Chem. Mater. 2014, 26, 3104-3112. (34) Wang, D.; Chen, C.; Ke, X.; Kang, N.; Shen, Y.; Liu, Y.; Zhou, X.; Wang, H.; Chen, C.; Ren, L., Bioinspired near-infrared-excited sensing platform for in vitro antioxidant capacity assay based on upconversion nanoparticles and a dopamine-melanin hybrid system. ACS Appl. Mater. Interfaces 2015, 7, 3030-3040. (35) Bhattacharya, S.; Sarkar, R.; Chakraborty, B.; Porgador, A.; Jelinek, R., Nitric oxide sensing through azo-dye formation on carbon dots. ACS Sens. 2017, 2, 1215-1224. (36) Lu, S.; Sui, L.; Liu, J.; Zhu, S.; Chen, A.; Jin, M.; Yang, B., Nearinfrared photoluminescent polymer-carbon nanodots with twophoton fluorescence. Adv. Mater. 2017, 29, 1603443. (37) Liu, S.; Tian, J.; Wang, L.; Zhang, Y.; Qin, X.; Luo, Y.; Asiri, A. M.; Al-Youbi, A. O.; Sun, X., Hydrothermal treatment of grass: a lowcost, green route to nitrogen-doped, carbon-rich, photoluminescent polymer nanodots as an effective fluorescent sensing platform for label-free detection of Cu(II) ions. Adv. Mater. 2012, 24, 20372041. (38) Yang, Y.; Qi, P.; Ding, Y.; Maitz, M. F.; Yang, Z.; Tu, Q.; Xiong, K.; Leng, Y.; Huang, N., A biocompatible and functional adhesive amine-rich coating based on dopamine polymerization. J. Mater. Chem. B 2015, 3, 72-81. (39) Du, X.; Li, L.; Behboodi-Sadabad, F.; Welle, A.; Li, J.; Heissler, S.; Zhang, H.; Plumeré, N.; Levkin, P. A., Bio-inspired strategy for controlled dopamine polymerization in basic solutions. Polym. Chem. 2017, 8, 2145-2151. (40) Han, L.; Liu, S. G.; Dong, J. X.; Liang, J. Y.; Li, L. J.; Li, N. B.; Luo, H. Q., Facile synthesis of multicolor photoluminescent polymer carbon dots with surface-state energy gap-controlled emission. J. Mater. Chem. C 2017, 5, 10785-10793. (41) Zhao, J.; Bao, X.; Wang, S.; Lu, S.; Sun, J.; Yang, X., In situ fluorogenic and chromogenic reactions for the sensitive dual-readout assay of tyrosinase activity. Anal. Chem. 2017, 89, 10529-10536.

6

ACS Paragon Plus Environment

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

ACS Sensors (42) Sun, J.; Hu, T.; Chen, C.; Zhao, D.; Yang, F.; Yang, X., Fluorescence immunoassay system via enzyme-enabled in situ synthesis of fluorescent silicon nanoparticles. Anal. Chem. 2016, 88, 97899795. (43) Jiménez, M.; Garcia-Canovas, F.; Garcia-Carmona, F.; Tudela, J.; Iborra, J. L., Study of alpha-methyldopa oxidation by tyrosinase. Int. J. Biochem. 1986, 18, 39-47. (44) Qu, Z.; Na, W.; Liu, X.; Liu, H.; Su, X., A novel fluorescence biosensor for sensitivity detection of tyrosinase and acid phosphatase based on nitrogen-doped graphene quantum dots. Anal. Chim. Acta 2018, 997, 52-59. (45) Yang, X.; Luo, Y.; Zhuo, Y.; Feng, Y.; Zhu, S., Novel synthesis of gold nanoclusters templated with l-tyrosine for selective analyzing tyrosinase. Anal. Chim. Acta 2014, 840, 87-92. (46) Wang, C.; Yan, S.; Huang, R.; Feng, S.; Fu, B.; Weng, X.; Zhou, X., A turn-on fluorescent probe for detection of tyrosinase activity. Analyst 2013, 138, 2825-2828. (47) Wu, X.; Li, L.; Shi, W.; Gong, Q.; Ma, H., Near-infrared fluorescent probe with new recognition moiety for specific detection of tyrosinase activity: design, synthesis, and application in living cells and zebrafish. Angew. Chem., Int. Ed. 2016, 55, 14728-14732. (48) Li, H.; Liu, W.; Zhang, F.; Zhu, X.; Huang, L.; Zhang, H., Highly selective fluorescent probe based on hydroxylation of phenylboronic acid pinacol ester for detection of tyrosinase in cells. Anal. Chem. 2018, 90, 855-858. (49) Liu, B.-W.; Huang, P.-C.; Li, J.-F.; Wu, F.-Y., Colorimetric detection of tyrosinase during the synthesis of kojic acid/silver nanoparticles under illumination. Sens. Actuators, B 2017, 251, 836-841. (50) Baron, R.; Zayats, M.; Willner, I., Dopamine-, l-dopa-, adrenaline-, and noradrenaline-induced growth of Au nanoparticles: assays for the detection of neurotransmitters and of tyrosinase activity. Anal. Chem. 2005, 77, 1566-1571. (51) Kim, T. I.; Park, J.; Park, S.; Choi, Y.; Kim, Y., Visualization of tyrosinase activity in melanoma cells by a BODIPY-based fluorescent probe. Chem. Commun. 2011, 47, 12640-12642.

7

ACS Paragon Plus Environment

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

Page 8 of 8

For TOC only

ACS Paragon Plus Environment

8