Natural Peptide Probe Screened for High-Performance Fluorescent

Jan 10, 2019 - By virtue of rich coordination sites (amidogen and carboxyl) and fluorescence property of aromatic amino acids that which are also esse...
0 downloads 0 Views 2MB Size
Download PDF
This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes.

Article Cite This: ACS Omega 2019, 4, 793−800

http://pubs.acs.org/journal/acsodf

Natural Peptide Probe Screened for High-Performance Fluorescent Sensing of Copper Ion: Especially Sensitivity, Rapidity, and Environment-Friendliness Xiaoxuan Li,† Zhihe Qing,*,†,‡ Younan Li,† Zhen Zou,†,‡ Sheng Yang,† and Ronghua Yang*,†,‡ †

School of Chemistry and Food Engineering, Changsha University of Science and Technology, Changsha 410114, P. R. China State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, P. R. China

‡

ACS Omega 2019.4:793-800. Downloaded from pubs.acs.org by 91.243.91.218 on 01/10/19. For personal use only.

S Supporting Information *

ABSTRACT: Copper, one of the most important metal elements, has been a new favorite in research areas. However, the Cu2+ detection strategy with high-efficient and biocompatibility maintains importance. Here, we made effort to develop a new sensor with above advantages for Cu2+ detection. By virtue of rich coordination sites (amidogen and carboxyl) and fluorescence property of aromatic amino acids that which are also essential for living organism, monomers and combinations of them are designed to interact with Cu2+, attractively, the short peptide of tryptophane−phenylalanine (Trp−Phe) held stronger fluorescence emission and displayed much more significant response to Cu2+ than other molecules; thus, Trp−Phe was screened as a new fluorescent sensor for Cu2+ detection. It had a rapid response to the target ion via a coordination-mediated PET effect, the signal change reached the maximum within 10 s; cell proliferation experiments verified that the screened peptide had excellent biocompatibility, implying that it had great environment-friendliness and convenience for practical application in Cu2+ detection; high selectivity and a wide linear detection range from 10 to 1500 nM were achieved, with a lowest detectable concentration of 10 nM, which are superior to conventional optical strategies; through analysis of real water samples, good recoveries and consistencies with classical inductively coupled plasma mass spectrometry indicated that this strategy had promising potential for practical application, especially significant for Cu2+ detection in drinking water; and in addition, Cu2+ antidotes identification was successfully carried out, implying useful contribution to medical therapy and sewage treatment.

■

INTRODUCTION In living organisms, copper at low concentration levels plays an important role as a micronutrient, through cofactor-dependent activation of many protein enzymes,1−4 and is the third transition metal essential to human health following zinc and iron.5−7 However, on the opposite side, copper is becoming one of the major heavy-metal environmental pollutants, with the increasing of industrial pollution and mineral corrosion. Especially in drinking water, its toxicity has been deemed to be second only to mercury ion.8−10 Over-ingestion of copper ion (Cu2+) can result in severe damages in gastrointestinal, kidney, and liver by facilitating the generation of reactive oxygen species that can disorder the cellular metabolism balance.11−13 Some associations have been discovered between the risk and a few of serious diseases, including amyotrophic lateral sclerosis,14,15 Menkes syndrome,16,17 Alzheimer’s disease,18,19 and Wilson’s disease.20−22 The safety limit of copper ion in drinking water has been, respectively, set at 1.3 mg/L (ac. 20 μM) and 2.0 mg/L (ac. 30 μM) by the U.S. Environmental Protection Agency (EPA) and the World Health Organization (WHO).23 Thus, the analysis and monitoring of copper ion in environment and food samples have become increasingly significant. © 2019 American Chemical Society

Fluorescent analysis strategy has attracted great interest by virtue of the high sensitivity, simple operation, and fast detection speed of fluorescence technique. In the past years, with the development of molecular engineering and nanotechnology, multifarious fluorescent materials have been designed and synthesized, such as inorganic fluorescent metal nanoparticles24 and organic fluorophores.25 These fluorescent materials have provided tools for constructing probes in analytical chemistry. Undoubtedly, distinct advance for biochemical sensing have been made by these developed fluorescent methods. However, tedious synthesis, purification, and/or complicated modification are generally required for the preparation of these fluorescent probes;26−29 in particular, most fluorescent materials are not out of the limit which originates from their low-biocompatibility and even biotoxicity.30,31 The low-biocompatibility and biotoxicity of these materials not only result in inconvenience in practical operation but also lead to subsequent pollution after being discharged into the environment. Thus, it is of longing and Received: September 25, 2018 Accepted: December 24, 2018 Published: January 10, 2019 793

DOI: 10.1021/acsomega.8b02513 ACS Omega 2019, 4, 793−800

ACS Omega

Article

challenging to construct “green” probes for biochemical sensing based on more biocompatible materials, with environment-friendliness, rapidity, and simplicity.32 In this work, inspired by the fact that Cu2+ holds strong coordination ability toward amidogen and carboxyl,33 the molecular mechanism that the coordination of the Lewis basic sites on fluorophores to metal ions can cause fluorescence change through photoinduced electron transfer (PET)34 and the fact that aromatic amino acids hold both coordination sites and florescent property, we first made effort to develop a “green” probe for Cu2+ detection based on its regulation on the emission of natural amino acids-based materials, via coordination-mediated PET effect. Aromatic amino acids and their short combinations (peptides) were used as fluorescent molecule library, the fluorescence properties of them were measured, and their optical interaction with Cu2+ was investigated. Attractively, compared with others, the short peptide of tryptophane−phenylalanine (Trp−Phe) held stronger fluorescence emission and displayed much distincter response to Cu2+. Thus, Trp−Phe dipeptide was screened as a fluorescent sensor for copper ion detection. As a result, highselectivity, sensitivity, and rapid-response were achieved. In addition, because the peptide is composed of natural amino acids which are essential for living organism including plants and animals, one can conclude that a natural peptide-based fluorescent probe is highly biocompatible and environmentfriendly.

interact with Cu2+. Typically, 2.5 μM Trp−Phe was added into 300 μL 10 mM MOPS buffer solution (pH = 7.0, 150 mM NaCl), and the fluorescence peak intensity (F0) was measured with an excitation wavelength of 280 nm. Cu2+ (2.5 μM) was added into the above solution and shaked for a while. Subsequently, the resulted fluorescence peak intensity (F) was measured. The signal change ratio ((F0 − F)/F0) was calculated by the fluorescence intensities of the probes in the absence and presence of Cu2+. Afterward, the job’s plot measure was used to make an investigation of the binding ratio between the probe and Cu2+. The proportion between probe and Cu2+ was changed with inherent total concentration of 5.0 μM, and the molar concentration ratio of Trp−Phe was adjusted from 0 to 1. The fluorescence spectra were detected in 300 μL MOPS buffer solution (10 mM MOPS, pH = 7.0, 150 mM NaCl). In addition, the photostability of the screened peptide was tested by a time-base fluorescence measure because the photostability of a fluorescent sensor which influences the repeatability of detection and its application is very important. The probe in different concentrations from 0.5 to 5.0 μM in 300 μL MOPS was continuously excited with 280 nm, and the fluorescence intensity was recorded. Results indicated that Trp−Phe held good photostability and promising potential for applications. Biocompatibility of the Screened Peptide Probe. The effect of the screened peptide probe on cell’s proliferation and morphology was first investigated by confocal microscopy imaging. Hela cell (150 μL) of index period was added into 1.5 mL DEME medium (10% bovine serum albumin, 1% penicillin−streptomycin solution) in four laser confocal dishes (a, b, c, and d), respectively. The laser confocal dishes were placed into cell culture incubator for 48 h under 37 °C and 4.5% CO2. Subsequently, a 2.5 μM probe was added into a, b, and c, while d was set as the control group without addition of the probe and incubated for another 6 h. Then, the cell imaging experiments were implemented at different times [(a) 0; (b) 24; (c) 48; (d) 48 h] by a FV500+ix70 laser scanning confocal microscope (Olympus, Japan) with a 40× objective lens. Then, the effect of the screened peptide probe on cell proliferation was statistically evaluated by the MTT cytotoxicity assay. Hela cell (100 μL) of index period was cultivated in a well of 96-well plate for 24 h, and then the probe of different concentrations was added. After 48 h, 10 μL of MTT (5 mg/ mL) was added and then incubated for 4 h. Finally, 150 μL of dimethyl sulfoxide was introduced into the resulted cell solution, and the absorbance was measured by the microplate reader. All experiments were repeated three times. The cell viability percentage of the control group without the addition of the probe was set as 100%, cell viability percentages of other groups were calculated by comparison with that of the control group. Cu2+ Detection. After confirming that the screened short Trp−Phe could be exploited for Cu 2+ sensing with biocompatibility, its detection performance was further investigated. First, response-rapidity of the probe to Cu2+ was tested by fluorescence real-time monitoring of Trp−Phe (2.5 μM) in 300 μL of 10 mM MOPS buffer solution (pH = 7.0, 150 mM NaCl) before and after addition of Cu2+. The fluorescence spectra after addition of Cu2+ were also detected at different times from 0 to 50 s. Then, to prove selectivity of this probe, different ions, including Li+, K+, Ag+, Hg2+, Mg2+,

■

EXPERIMENTAL SECTION Chemicals and Apparatus. Amino acid reagents and other salts with a purity of at least analytical grade were purchased from Dingguo Biological Technology Co., Ltd. (Beijing, China) and used without further treatment. Short peptides were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) purified by high-performance liquid chromatography. 3-(N-Morpholino) propanesulfonic acid (MOPS) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). The stock solutions of amino acids (500 μM) were prepared by dissolving them in double distilled water. The stock solutions of short peptides (500 μM) were prepared by dissolving it in isopropanol. The MOPS buffer containing 150 mM NaCl was prepared in double-distilled water whose pH was then adjusted to 7.0. The double-distilled water used in this work was prepared with the Milli-Q system (Millipore, USA). All pH measurements were carried out by a model 868 pH meter (Orion, USA). The fluorescence spectra were measured on the PTI QM40 fluorospectrophotometer systems (Photo Technology International, USA). Ex and Em slits of the spectrophotometer were both set at 8.00 nm with a response time of 2 s, fluorescence data were then recorded with a 1.0 cm × 1.0 cm quartz cuvette containing 300 μL solution, and all measurements were carried out at room temperature. The 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were carried out by Microplate reader (Tecan, Swiss) to test the biocompatibility of the probe. The experiments of cell imaging were obtained from a FV500+ix70 laser scanning confocal microscope (Olympus, Japan). Screening the Cu2+-Responsive Peptide Probe. First, to screen an ideal Cu2+-responsive biocompatible probe, various amino acids and short peptides, including tryptophan, phenylalanine, tyrosine, Trp−Phe dipeptide, Tyr−Trp dipeptide, and Phe−Tyr dipeptide, were designed and used to 794

DOI: 10.1021/acsomega.8b02513 ACS Omega 2019, 4, 793−800

ACS Omega

Article

Figure 1. Screening Cu2+-responsive fluorescent peptide probe. (A−C) Fluorescence spectra change of fluorescent short peptides as a function of the addition of Cu2+. The insets in (A−C) show the structures of corresponding peptides. (D) Fluorescence peak intensity of different fluorescent short peptides without (red bar) and with Cu2+ (black bar). (E) Photostability investigation of Trp−Phe with different concentrations by timescanning spectroscopy. (F) Left: Real-time fluorescence monitoring of Trp−Phe in the absence (red) and presence (black) of Cu2+; Right: fluorescent spectra of Trp−Phe after the addition of Cu2+ at different times from 0 to 50 s.

Ca2+, Sr2+, Ba2+, Mn2+, Pb2+, Zn2+, Cu2+, Cd2+, Co2+, Ni2+, Sn2+, Cr3+, Al3+, and Fe3+, were added into 300 μL of MOPS buffer solution (10 mM MOPS, pH = 7.0, 150 mM NaCl) with 2.5 μM Trp−Phe. The spectra were recorded in the emission wavelength from 320 to 500 nm with an excitation wavelength of 280 nm. All data were repeated three times. The signal change ratio ((F0 − F)/F0) was then calculated to evaluate the specificity of this probe. In addition, the sensitivity was investigated by fluorescence titration experiments with Cu2+ concentration from 0 to 3000 nM, in 300 μL MOPS buffer solution containing 2.5 μM probe. All detections were repeated three times. The relationship between Cu2+ concentration and signal change ratio ((F0 − F)/F0) was plotted, and the linear detection range and lowest detectable concentration were evaluated. Application of the Screened Probe. On the basis of its ideal performance, the probe was then used to detect Cu2+ in river water. The water sample was obtained from the Xiangjiang River filtered by a 0.22 μm syringe filter to remove large-scale insoluble materials. The analytes were prepared by spiked real water samples without or with Cu2+ of different concentration levels and then detected by our proposed method mentioned above and a classic inductively coupled plasma mass spectrometry (ICPMS) method, and all detections were repeated three times. Recovery percentage, standard deviation, and consistency to ICPMS were calculated and valuated. To further explore the application of this probe, screening of copper antidotes was carried out, subsequently. As well known that because of abundant protein, milk and egg are generally used to relieve heavy metal poisoning, it is necessary to know what ingredients of proteins are acting as antidotes. Here, amino acids, the ingredients of proteins, were used to seize Cu2+ in the Trp−Phe/Cu2+ system, and fluorescence change was measured. Samples were prepared by adding 2.5 μM dipeptide probe and 2.5 μM Cu2+ into 300 μL of MOPS buffer solution (10 mM MOPS, pH = 7.0, 150 mM NaCl), followed by the addition of 2.5 μM amino acids. The antagonism effect

of each antidote on Cu2+ could be directly determined by the recovery of fluorescence intensity, and concentration-dependent antagonism was also investigated by altering the concentration of antidotes.

■

RESULTS AND DISCUSSION

Screening Cu2+-Responsive Natural Peptide Probe. As well known, amino acids are important nutrients for living organisms; thus, the functional materials which are composed of natural amino acids are biocompatible and environmentfriendly; furthermore, aromatic amino acids are fluorescent because of their conjugate structures, providing alternative units for constructing optical probes; in addition, rich functional groups (amidogen and carboxyl) on amino acidbased materials provide interaction sites to surrounding interesting substances. Here, aromatic amino acids and their combinations (short peptides) are designed and synthetized as fluorescent molecules; their fluorescence properties and optical interaction with Cu2+ were investigated. As shown in Figure S1, when the monomer molecules of aromatic amino acids were used as fluorophores, there was low fluorescence emission of the monomers and negligible effect on their fluorescence was detected from the addition of Cu2+. Attractively, when Trp−Phe was used as the reporter, high fluorescence emission intensity was displayed and obvious response to Cu2+ was detected (Figure 1A). In other words, as a function of the addition of Cu2+ into the solution containing Trp−Phe dipeptide, a distinct decrease in fluorescence peak intensity at 350 nm was recorded. Although other two combinations of aromatic amino acids were instead of Trp− Phe, low fluorescence emission and much weaker response to Cu2+ was observed (Figure 1B,C). It’s most likely because the specific element (mainly nitrogen) and structure of Trp−Phe which provides a perfect binding site to Cu2+. The orbital overlap occurs when Cu2+ is close to the N atom, forming a complex, and then the energy transfer occurs from ligand to metal causing the fluorescence of Trp−Phe quenching. In addition, to further illustrate the integrated information on 795

DOI: 10.1021/acsomega.8b02513 ACS Omega 2019, 4, 793−800

ACS Omega

Article

their fluorescence emission and response to Cu2+, the fluorescence measurements are summarized in Figure 1D; Trp−Phe displayed the strongest fluorescence emission than aromatic amino acids and other combinations and had a significant decrease of approximately 88% in fluorescence when Cu2+ was added; in contrast, a little or negligible response to Cu2+ was displayed when other molecules were instead of Trp−Phe. Thus, by virtue of its high fluorescence emission and great response to Cu2+, the Trp−Phe dipeptide was screened as a fluorescent sensor for copper ion detection. The photostability of a fluorescent sensor is an important factor which influences the repeatability of detection and its practical application. As shown in Figure 1E, with the continuous irradiation of excitation light from 0 to 200 s, negligible decay was monitored for Trp−Phe of different concentrations, and it is more stable than traditional fluorescent dyes with poor anti-photobleaching capability,35 indicating that the screened Cu2+-responsive Trp−Phe sensor held good photostability and promising potential for applications. In addition, the detection speed is another important parameter for the evaluation of a sensing method, and rapid detection is more desirable in practical application. When Trp−Phe dipeptide was used as a sensor, the real-time monitoring of its response to Cu2+ was investigated. As shown in Figure 1F, with the addition of Cu2+, the fluorescence of Trp−Phe decreased quickly, the signal change reached the maximum with 10 s, which is much more rapid than many conventional optical strategies. Thus, the screened Trp−Phe dipeptide held excellent potential for Cu2+ detection with good photostability and response-rapidity. Interaction Mechanism between the Screened Trp− Phe and Cu2+. It’s well known that the optical property of fluorescent probes is very important. The optical property of Trp−Phe was first been detected (Figure S2). Subsequently, the interaction mechanism between Trp−Phe and its target Cu2+ was investigated. The effect from the electrostatic interaction between Cu2+ and the negatively charged carboxyl groups of peptides was first investigated by titrating sodium chloride (NaCl) in the Trp−Phe-contained buffer. From Figure S3, as there is the increase of NaCl concentration in the buffer solution, no matter whether there was Cu2+ (Figure S3B) or not (Figure S3A), the fluorescence intensity of Trp− Phe did not change obviously, and the degree of signal reduction as a function of Cu2+ remained almost the same (Figure 2A). One can conclude that the fluorescence response of Trp−Phe to Cu2+ could not be attributed to the electrostatic

interaction between Cu2+ and the negatively charged carboxyl groups of peptides. Thus, we inferred that the fluorescence response of Trp−Phe to Cu2+ should be due to the ionselective coordination effect between the Lewis basic sites of Trp−Phe and Cu2+, resulting in fluorescence decrease through PET. Then, the combination ratio between Trp−Phe and Cu2+ was investigated by a Job’s plot method.36,37 As shown in Figure 2B, with the increase of the ratio ([Trp−Phe]/([Trp− Phe] + [Cu2+])), the degree of fluorescence change (ΔF = F0 − F) as a function of the addition of Cu2+, increased first and then decreased, where F0 was the fluorescence intensity of Trp−Phe itself and F was that after the addition of Cu2+. When the ratio ([Trp−Phe]/([Trp−Phe] + [Cu2+])) was 0.5, the ΔF reached the maximal value, indicating that the combination ratio between Trp−Phe and Cu2+ was 1:1. Biocompatibility Investigation. Environment-friendliness and biocompatibility are hot spots of concern in modern scientific research, especially in chemistry, biology, and environmental science. More environment-friendliness and biocompatibility will more facilitate the practical application of new materials and/or methods. Thus, we here investigated the biocompatibility of Trp−Phe through its effect on cell proliferation. The cell proliferation in the presence of Trp− Phe was observed through confocal microscopy imaging (Figure 3A). It could be obviously observed that the amounts of cells increased with the prolongation of the culture time, and the cell morphology almost remained intact, without cell apoptotic fragments. Compared with the control group without the addition of Trp−Phe (Figure 3B), after culture for the same time (48 h), the group with Trp−Phe was coincident in cell amount and morphology. Subsequently, the statistical evaluation was carried out by MTT cytotoxicity assay,38 as shown in Figure 3C, Trp−Phe has no negative influence on cell growth, even its concentration reached 10 μM. These results demonstrated that the screened Trp−Phe has excellent biocompatibility, implying that it had great environment-friendliness and convenience for practical application in Cu2+ sensing. Copper Ion Detection. Because pH is an important factor influencing the coordination reaction, low pH will lead to strong acid effect on the host and high pH will result in obvious oxyhydrate of metal ions; to get better performance for Cu2+ detection by applying Trp−Phe as a sensor, the pH of detection system was first optimized by the manner of fluorescence change ratio ((F0 − F)/F0), where F0 was the fluorescence intensity of Trp−Phe itself at 350 nm and F was that after the addition of Cu2+. As a result, pH 7.0 was chosen as the optimal value, which is approximate to ambient condition and convenient for practical operation (Figure S4). Then, the selectivity for Cu2+ detection was investigated. As shown in Figure S5, an observed depress of fluorescence peak of Trp−Phe was induced by the introduction of Cu2+, while little change was resulted in by other ions. Fluorescence decrease ratios in the presence of different ions were shown in Figure 4A, a distinct ratio displayed for only Cu2+, without influence from other ions at the used concentrations, indicating that there was a good selectivity for Cu2+ in our strategy. Subsequently, Trp−Phe fluorescence emission, responding to different concentrations of Cu2+, was recorded. It can be directly observed in Figure 4B that the fluorescence emission peak of Trp−Phe had a regular decrease as the concentration of Cu2+ increased. To further demonstrate the detection

Figure 2. (A) Evaluation of Cu2+-responsive performance of Trp−Phe probe under different NaCl concentration in buffer. The results are analyzed by a manner of fluorescence change ratio ((F0 − F)/F0), where F0 is the fluorescence intensity of Trp−Phe before addition of Cu2+, and F is that after addition of Cu2+. (B) Job’s plot method for determining the combination ratio between Trp−Phe and Cu2+. 796

DOI: 10.1021/acsomega.8b02513 ACS Omega 2019, 4, 793−800

ACS Omega

Article

Figure 3. Biocompatibility and environment-friendliness investigation by cell proliferation experiments. (A) Confocal microscopy imaging of cells after different incubation time (0, 24, and 48 h) in the presence of 2.5 μM Trp−Phe. (B) Confocal microscopy imaging of cells after incubation for 48 h in the absence of Trp−Phe. (C) MTT assay of cells treated with Trp−Phe of different concentrations. The concentrations of Trp−Phe from (a−j): 0, 0.025, 0.05, 0.1, 0.2, 0.5, 1, 10, 50, 100, and 500 μM.

deviation, and the consistency of these methods. All of these results are shown in Table 1, good recovery percents and Table 1. Monitoring Copper Pollution in Real Water Samples by Our Strategy and Classic ICPMS piked (nM) 0 100 200 350 500 750 1000 1500

detected (nM) (meana ± SDb)

ICPMS (nM)

recovery (%)c

consistency (%)d

± ± ± ± ± ± ± ±

16.68 112.02 212.16 367.50 469.68 735.00 998.43 1425.00

89.64 108.11 108.57 95.45 97.67 104.61 97.44

107.61 96.05 110.37 108.28 105.44 102.1 106.56 103.83

17.95 107.59 234.16 397.94 495.22 750.50 1063.95 1479.52

0.0216 0.0388 0.0133 0.0177 0.0201 0.0385 0.0086 0.0142

a

Mean value of three-repeated detections. bStandard deviation. Recovery percent which was calculated by the formula ((Cd − C0)/Cs) × 100%, where C0 is the concentration of the no-spiked sample measured by our approach, Cs was the spiked concentration, and Cd was the concentration in each spiked sample measured by our approach. dConsistency percent which was calculated by the formula (Cd/CICPMS) × 100%, where Cd is the concentration measured by our approach, CICPMS is that measured by ICPMS. c

Figure 4. (A) Selectivity investigation of Trp−Phe probe by the manner of fluorescence decrease ratio (black bars: in the presence of single ion as marked on the abscissa; gray bars: adding Cu2+ following the former). (B) Fluorescence spectra of Trp−Phe after the addition of Cu2+ of different concentrations. (C) Relationship between Cu2+ concentration and the fluorescence decrease ratio ((F0 − F)/F0), where F0 is the fluorescence intensity of Trp−Phe probe before addition of Cu2+, and F is that after addition of Cu2+. The inset in (C) shows the linear detection range from 10 to 1500 nM.

acceptable consistencies were obtained, and these results indicated the reliability for Cu2+ monitoring in real water samples, especially important for Cu2+ analysis in drink water. Cu2+ Antidotes Identification. As well known, by virtue of their protein of high abundance, milk and egg are generally used to relieve heavy metal poisoning, it is necessary to know what ingredients of proteins are acting as antidotes. Here, amino acids, the ingredients of proteins, were used to seize Cu2+ in the Trp−Phe/Cu2+ system, and fluorescence change was measured. As shown in Figure 5A, obvious recovery of fluorescence intensity can be observed when cysteine (Cys) and histidine (His) were used, whereas other amino acids (glycine, alanine, valine, arginine, threonine, lysine, proline, and isoleucine) had negligible effect on Trp−Phe/Cu2+ system, implying obvious detoxification effect from cysteine and histidine against Cu2+, this should be due to the strong binding between Cu2+ and sulfhydryl on Cys or imidazole on His. Then, the concentration-dependent detoxification was further investigated (Figures S6 and 5B); with increase of the concentration of the selected amino acids, negligible change of

performance of the screened Trp−Phe, the relationship between fluorescence change ratio ((F0 − F)/F0) and Cu2+ concentration was revealed in Figure 4C. A wide linear detection range (R2 = 0.9935) from 10 to 1500 nM was obtained with a detectable lowest concentration of 10 nM, which is much lower than the safety limit of copper ion in drinking water set by EPA (20 μM) and WHO (30 μM), indicating a good detection performance and displaying great potential for practical application. To further investigate the practical application for monitoring copper pollution, the water samples from Xiangjiang River (Changsha Section, Hunan Province) spiked with different Cu2+ concentrations, were detected by both our approach and a classic method (ICPMS). The detection performance was evaluated by recovery percentage, standard 797

DOI: 10.1021/acsomega.8b02513 ACS Omega 2019, 4, 793−800

ACS Omega

Article

via a coordination-mediated PET effect and the signal change reached the maximum within 10 s; (3) the screened natural peptide probe has excellent biocompatibility, implying that it had great environment-friendliness and convenience for practical application in Cu2+ detection; (4) promising capability for Cu2+ detection was displayed, including highselectivity, wide linear detection range (10−1500 nM), low detection limit (10 nM), and practical application. Table 2 summarized different Cu2+ detection methods, it is not difficult to find that our strategy has obvious advantages in multipoints in comparison with the developed optical methods. Finally, toward Cu2+-related sewage treatment and emergency treatment, antidote identification was successfully verified by its unique advantages in operation and performance.

Figure 5. Screening copper antidotes by the proposed strategy. (A) Fluorescence spectra of Trp−Phe/Cu2+ system (10 mM MOPS with 150 mM NaCl, 2.5 μM Trp−Phe, 2.5 μM Cu2+) after introduction of different antidotes (cysteine, histidine, glycine, alanine, valine, arginine, threonine, lysine, proline, and isoleucine). (B) Relationship between antidote concentrations and fluorescence intensity.

■

ASSOCIATED CONTENT

S Supporting Information *

fluorescence intensity could be detected when glycine was used, while gradual increase of fluorescence intensity could be observed when cysteine and histidine were used, which could be a persuasive certification of the concentration-dependent detoxification. In a word, all these results have sufficiently proved that our strategy not only can be used for highperformance detection of Cu2+ but also has the potential ability to distinguish the efficiency of copper antidotes.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02513. Fluorescence measurement of aromatic amino acids and their response to Cu2+, optical characterization data of Trp−Phe, evaluation of Cu2+-responsive performance of Trp−Phe probe under different NaCl concentration in buffer, optimization of buffer pH, selectivity investigation by fluorescence spectra measurement of Trp− Phe after addition different ions, and fluorescence spectra of Trp−Phe/Cu2+ system in the presence of antidotes of different concentrations (PDF)

■

CONCLUSION In summary, inspired by the fact that Cu2+ can cause fluorescence change through PET after coordination binding with the Lewis basic sites on fluorophores and that natural aromatic amino acids hold great biochemical properties including rich coordination sites, florescent emission, and absolute biocompatibility, we made effort to develop a highperformance and environment-friendly sensing strategy for copper ion detection. From their optical interaction between Cu2+ and aromatic compound, including aromatic amino acids and their short peptides, Trp−Phe was screened as a fluorescent sensor for Cu2+ detection by virtue of its stronger fluorescence emission and much distincter response to Cu2+. This sensor had multifaceted advantages: (1) as an add-andrecord manner, it was of low-cost and simple in its operation without any requirement for complex synthesis and modification; (2) there was a high-rapid response to the target ion

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.Q). *E-mail: [email protected]. Fax: +86-731-88822523 (R.Y.). ORCID

Zhihe Qing: 0000-0002-6268-225X Sheng Yang: 0000-0003-0867-6438 Ronghua Yang: 0000-0001-7873-6892 Notes

The authors declare no competing financial interest.

Table 2. Comparison of Our Proposed Method with Reported Cu2+ Detection Methods method

tool

fluorescence colorimetry fluorescence

beacon modified with fluorophore and quencher AuNPs modified with alkyne or azide functional groups DNA-templated copper/silver nanoclusters quenched by 3-mercaptopropionic acid (MPA) DNA-stabilized silver nanoclusters azide-functionalized magnetic silica nanoparticles clicked on acetylene-functionalized multiwalled-carbon nanotubes AuNPs functionalized with alkylthiol-modified oligonucleotide strands azide- and alkyne-functionalized polydiacetylene vesicles a hybrid system composed of carbon and CdSe/ZnS quantum dots (QDs) integrating N-(2-aminoethyl)-N,N,N′-tris(pyridin-2-ylmethyl)ethane-1,2-diamine (AE-TPEA) a hydrogel functionated by poly T DNA sequence DNA-templated copper nanoparticles and auxiliary reductive reagents direct detection by a natural peptide

fluorescence absorbance colorimetry colorimetry fluorescence fluorescence fluorescence fluorescence

798

detection time

linear range

detection limit

2−4 min 24 h 1h

35−500 nM not mentioned 5−200 nM

35 nM 50 μM 2.7 nM

2 39 40

1.5 h 10 min

10−200 nM not mentioned

8 nM 1 μM

41 42

2h 24 h 1 min

20−100 μM not mention 1−100 μM

20 μM 3 μM 1 μM

43 44 45

10 min 1 min 10 s

20 μM to 10 mM 5−35 μM 10−1500 nM

20 μM 5.6 μM 10 nM

10 7 this work

refs

DOI: 10.1021/acsomega.8b02513 ACS Omega 2019, 4, 793−800

ACS Omega

■

Article

(17) Zhao, Y.; Zhang, X.-B.; Han, Z.-X.; Qiao, L.; Li, C.-Y.; Jian, L.X.; Shen, G.-L.; Yu, R.-Q. Highly Sensitive and Selective Colorimetric and Off-On Fluorescent Chemosensor for Cu2+ in Aqueous Solution and Living Cells. Anal. Chem. 2009, 81, 7022−7030. (18) Zong, C.; Ai, K.; Zhang, G.; Li, H.; Lu, L. Dual-Emission Fluorescent Silica Nanoparticle-Based Probe for Ultrasensitive Detection of Cu2+. Anal. Chem. 2011, 83, 3126−3132. (19) Qu, Q.; Zhu, A.; Shao, X.; Shi, G.; Tian, Y. Development of a Carbon Quantum Dots-Based Fluorescent Cu2+ Probe Suitable for Living Cell Imaging. Chem. Commun. 2012, 48, 5473−5475. (20) Helmchen, F.; Denk, W. Deep Tissue Two-Photon Microscopy. Nat. Methods 2005, 2, 932−940. (21) Smith, A. M.; Mancini, M. C.; Nie, S. Second window for in vivo imaging. Nat. Nanotechnol. 2009, 4, 710−711. (22) Selvan, G. T.; Varadaraju, C.; Selvan, R. T.; Enoch, I. V. M. V.; Selvakumar, P. M. On/Off Fluorescent Chemosensor for Selective Detection of Divalent Iron and Copper Ions: Molecular Logic Operation and Protein Binding. ACS Omega 2018, 3, 7985−7992. (23) Zhao, H.; Xue, C.; Nan, T.; Tan, G.; Li, Z.; Li, Q. X.; Zhang, Q.; Wang, B. Detection of Copper Ions Using Microcantilever Immunosensors and Enzyme-Linked Immunosorbent Assay. Anal. Chim. Acta 2010, 676, 81−86. (24) Fan, C.; Lv, X.; Liu, F.; Feng, L.; Liu, M.; Cai, Y.; Liu, H.; Wang, J.; Yang, Y.; Wang, H. Silver Nanoclusters Encapsulated into Metal-Organic Frameworks with Enhanced Fluorescence and Specific Ion Accumulation toward the Microdot Array-Based Fluorimetric Analysis of Copper in Blood. ACS Sens. 2018, 3, 441−450. (25) Wu, W.; Chen, A.; Tong, L.; Qing, Z.; Langone, K. P.; Bernier, W. E.; Jones, W. E., Jr Facile Synthesis of Fluorescent Conjugated Polyelectrolytes Using Polydentate Sulfonate as Highly Selective and Sensitive Copper(II) Sensors. ACS Sens. 2017, 2, 1337−1344. (26) Liu, J.; Lu, Y. Colorimetric Cu2+ Detection with a Ligation DNAzyme and Nanoparticles. Chem. Commun. 2007, 4872−4874. (27) Domaille, D. W.; Zeng, L.; Chang, C. J. Visualizing AscorbateTriggered Release of Labile Copper within Living Cells Using a Ratiometric Fluorescent Sensor. J. Am. Chem. Soc. 2010, 132, 1194− 1195. (28) Zong, C.; Ai, K.; Zhang, G.; Li, H.; Lu, L. Dual-Emission Fluorescent Silica Nanoparticle-Based Probe for Ultrasensitive Detection of Cu2+. Anal. Chem. 2011, 83, 3126−3132. (29) Liu, X.; Ardizzone, A.; Sui, B.; Anzola, M.; Ventosa, N.; Liu, T.; Veciana, J.; Belfield, K. D. Fluorenyl-Loaded Quatsome Nanostructured Fluorescent Probes. ACS Omega 2017, 2, 4112−4122. (30) Liu, Y.; Zhao, Y.; Sun, B.; Chen, C. Understanding the Toxicity of Carbon Nanotubes. Acc. Chem. Res. 2012, 46, 702−713. (31) Li, J.; Chang, X.; Chen, X.; Gu, Z.; Zhao, F.; Chai, Z.; Zhao, Y. Toxicity of Inorganic Nanomaterials in Biomedical Imaging. Biotechnol. Adv. 2014, 32, 727−743. (32) Qing, Z.; Qing, T.; Mao, Z.; He, X.; Wang, K.; Zou, Z.; Shi, H.; He, D. dsDNA-specific Fluorescent Copper Nanoparticles as a “Green” Nano-Dye for Polymerization-Mediated Biochemical Analysis. Chem. Commun. 2014, 50, 12746−12748. (33) Faller, P.; Hureau, C.; La Penna, G. Metal Ions and Intrinsically Disordered Proteins and Peptides: from Cu/Zn Amyloid-β to General Principles. Acc. Chem. Res. 2014, 47, 2252−2259. (34) Daly, B.; Ling, J.; de Silva, A. P. Current Developments in Fluorescent PET (Photoinduced Electron Transfer) Sensors and Switches. Chem. Soc. Rev. 2015, 44, 4203−4211. (35) Fan, Z.; Sun, L.; Huang, Y.; Wang, Y.; Zhang, M. Bioinspired Fluorescent Dipeptide Nanoparticles for Targeted Cancer Cell Imaging and Real-Time Monitoring of Drug Release. Nat. Nanotechnol. 2016, 11, 388−394. (36) MacCarthy, P. Simplified Experimental Route for Obtaining Job’s Curves. Anal. Chem. 1978, 50, 2165. (37) Shyamal, M.; Mazumdar, P.; Maity, S.; Samanta, S.; Sahoo, G. P.; Misra, A. Highly Selective Turn-on Fluorogenic Chemosensor for Robust Quantification of Zn (II) Based on Aggregation Induced Emission Enhancement Feature. ACS Sens. 2016, 1, 739−747.

ACKNOWLEDGMENTS This work was supported by the financial support through the National Natural Science Foundation of China (21605008, 21575018, 21505006, and 21705010) and the Open Fund of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (2015003, 2017018).

■

REFERENCES

(1) Plastino, J.; Green, E. L.; Sanders-Loehr, J.; Klinman, J. P. An Unexpected Role for the Active Site Base in Cofactor Orientation and Flexibility in the Copper Amine Oxidase from Hansenula Polymorpha. Biochemistry 1999, 38, 8204−8216. (2) Liu, J.; Lu, Y. A DNAzyme Catalytic Beacon Sensor for Paramagnetic Cu2+ Ions in Aqueous Solution with High Sensitivity and Selectivity. J. Am. Chem. Soc. 2007, 129, 9838−9839. (3) Koval, I. A.; Gamez, P.; Belle, C.; Selmeczi, K.; Reedijk, J. Synthetic Models of the Active Site of Catechol Oxidase: Mechanistic Studies. Chem. Soc. Rev. 2006, 35, 814−840. (4) Huang, J.; Zheng, Q.; Kim, J.-K.; Li, Z. A Molecular Beacon and Graphene Oxide-Based Fluorescent Biosensor for Cu2+ Detection. Biosens. Bioelectron. 2013, 43, 379−383. (5) Wu, S.-P.; Huang, R.-Y.; Du, K.-J. Colorimetric Sensing of Cu(II) by 2-methyl-3-[(pyridin-2-ylmethyl)-amino]-1,4-naphthoquinone: Cu(II) Induced Deprotonation of NH Responsible for Color Changes. Dalton Trans. 2009, 4735−4740. (6) Ma, Y.-r.; Niu, H.-y.; Zhang, X.-l.; Cai, Y.-q. Colorimetric Detection of Copper Ions in Tap Water during the Synthesis of Silver/Dopamine Nanoparticles. Chem. Commun. 2011, 47, 12643− 12645. (7) Qing, Z.; Zhu, L.; Yang, S.; Cao, Z.; He, X.; Wang, K.; Yang, R. In Situ Formation of Fluorescent Copper Nanoparticles for Ultrafast Zero-Background Cu2+ Detection and Its Toxicides Screening. Biosens. Bioelectron. 2016, 78, 471−476. (8) Liu, C.; Bai, R.; San Ly, Q. Selective Removal of Copper and Lead Ions by Diethylenetriamine-Functionalized Adsorbent: Behaviors and Mechanisms. Water Res. 2008, 42, 1511−1522. (9) Yang, P.; Zhao, Y.; Lu, Y.; Xu, Q.-Z.; Xu, X.-W.; Dong, L.; Yu, S.H. Phenol Formaldehyde Resin Nanoparticles Loaded with CdTe Quantum Dots: A Fluorescence Resonance Energy Transfer Probe for Optical Visual Detection of Copper(II) Ions. ACS Nano 2011, 5, 2147−2154. (10) Qing, Z.; Mao, Z.; Qing, T.; He, X.; Zou, Z.; He, D.; Shi, H.; Huang, J.; Liu, J.; Wang, K. Visual and Portable Strategy for Copper(II) Detection Based on a Striplike Poly(Thymine)-Caged and Microwell-Printed Hydrogel. Anal. Chem. 2014, 86, 11263−11268. (11) Crichton, R. R.; Dexter, D. T.; Ward, R. J. Metal Based Neurodegenerative Diseases-From Molecular Mechanisms to Therapeutic Strategies. Coord. Chem. Rev. 2008, 252, 1189−1199. (12) Taki, M.; Iyoshi, S.; Ojida, A.; Hamachi, I.; Yamamoto, Y. Development of Highly Sensitive Fluorescent Probes for Detection of Intracellular Copper(I) in Living Systems. J. Am. Chem. Soc. 2010, 132, 5938−5939. (13) Chen, P.; Solomon, E. I. Oxygen Activation by the Noncoupled Binuclear Copper Site in Peptidylglycine α-Hydroxylating Monooxygenase. Reaction Mechanism and Role of the Noncoupled Nature of the Active Site. J. Am. Chem. Soc. 2004, 126, 4991−5000. (14) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo. Science 2003, 300, 1434−1436. (15) Jiang, J.; Gu, H.; Shao, H.; Devlin, E.; Papaefthymiou, G. C.; Ying, J. Y. Bifunctional Fe3O4-Ag Heterodimer Nanoparticles for Two-Photon Fluorescence Imaging and Magnetic Manipulation. Adv. Mater. 2008, 20, 4403−4407. (16) Domaille, D. W.; Zeng, L.; Chang, C. J. Visualizing AscorbateTriggered Release of Labile Copper within Living Cells Using a Ratiometric Fluorescent Sensor. J. Am. Chem. Soc. 2010, 132, 1194− 1195. 799

DOI: 10.1021/acsomega.8b02513 ACS Omega 2019, 4, 793−800

ACS Omega

Article

(38) Gerlier, D.; Thomasset, N. Use of MTT Colorimetric Assay to Measure Cell Activation. J. Immunol. Methods 1986, 94, 57−63. (39) Zhou, Y.; Wang, S.; Zhang, K.; Jiang, X. Visual Detection of Copper(II) by Azide- and Alkyne-Functionalized Gold Nanoparticles Using Click Chemistry. Angew. Chem. 2008, 120, 7564−7566. (40) Su, Y.-T.; Lan, G.-Y.; Chen, W.-Y.; Chang, H.-T. Detection of Copper Ions through Recovery of the Fluorescence of DNATemplated Copper/Silver Nanoclusters in the Presence of Mercaptopropionic Acid. Anal. Chem. 2010, 82, 8566−8572. (41) Lan, G.-Y.; Huang, C.-C.; Chang, H.-T. Silver Nanoclusters as Fluorescent Probes for Selective and Sensitive Detection of Copper Ions. Chem. Commun. 2010, 46, 1257−1259. (42) Song, Y.; Qu, K.; Xu, C.; Ren, J.; Qu, X. Visual and Quantitative Detection of Copper Ions Using Magnetic Silica Nanoparticles Clicked on Multiwalled Carbon Nanotubes. Chem. Commun. 2010, 46, 6572−6574. (43) Xu, X.; Daniel, W. L.; Wei, W.; Mirkin, C. A. Colorimetric Cu2+ Detection Using DNA-Modified Gold-Nanoparticle Aggregates as Probes and Click Chemistry. Small 2010, 6, 623−626. (44) Xu, Q.; Lee, K. M.; Wang, F.; Yoon, J. Visual Detection of Copper Ions Based on Azide- and Alkyne- Functionalized Polydiacetylene Vesicles. J. Mater. Chem. 2011, 21, 15214−15217. (45) Zhu, A.; Qu, Q.; Shao, X.; Kong, B.; Tian, Y. Carbon-DotBased Dual-Emission Nanohybrid Produces a Ratiometric Fluorescent Sensor for in vivo Imaging of Cellular Copper Ions. Angew. Chem. 2012, 124, 7297−7301.

800

DOI: 10.1021/acsomega.8b02513 ACS Omega 2019, 4, 793−800