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Paper-based Fluorogenic Device for Detection of Copper Ions in Biological System Meirong Wu, Fengtai Suo, Jia Zhou, Qiuyu Gong, Lei Bai, Buxiang Chen, Qiong Wu, Cheng-wu Zhang, Hai-Dong Yu, Xiao Huang, Lin Li, and Wei Huang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00435 • Publication Date (Web): 27 Sep 2018 Downloaded from http://pubs.acs.org on September 27, 2018
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Paper-based Fluorogenic Device for Detection of Copper Ions in Biological System Meirong Wu†, Fengtai Suo†, Jia Zhou†, Qiuyu Gong†, Lei Bai†, Buxiang Chen†, Qiong Wu*†, Chengwu Zhang†, Haidong Yu†, Xiao Huang†, Lin Li*† and Wei Huang†, ‡ †
Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing, 211816, P.R. China ‡
Shanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU), 127 West Youyi Road, Xi'an 710072, P.R. China Supporting information
ABSTRACT: A new paper-based device is fabricated for detection of copper ions in biological system, which is much inexpensive, fast, and environmental friendly. Compared with the advanced diagnostics which have been used widely in wellequipped laboratories, the paper-based devices are affordable in resource-limited settings because basic infrastructure and trained operators are not prerequisites for using such devices. This work presents a novel fluorogenic device for rapid, highly accurate, sensitive and selective detection of copper ions (Cu2+) based on fluorescence “turn on” of the probe which is consisted of fluorescein moieties and oxyalkyl chain on the microfluidic paper-based analytical devices. The probe exhibits emissions at 520 nm, under the excitation wavelength of 460 nm. The fluorescence can be selectively “turned on” upon exposure to Cu2+, and the signal will be enlarged due to the higher concentration. The lowest detection limit of this fluorogenic device is 0.41 pM. In addition, this device can be employed to effectively detect Cu2+ in the biological system as urine, serum, and cytochylema. In the presence of Cu2+, the device would undergo a distinct fluorescence intensity increasing, which can be readily observed in the trace of Cu2+ concentrations. Compared with references on Cu2+ detection, our test paper presents lower detection limit and more portable fabrication. The paper-based fluorogenic sensor is cost-effective, high sensitivity, good selectivity, user-friendly and easy-to-use, indicating its great application promise for comprehensive sample and point-of-care detection. KEYWORDS: paper-based devices, high selectivity, fluorogenic, copper ion
INTRODUCTION 2+
Copper ion (Cu ) is one of the essential trace elements and heavy metal ions for many living organisms, which are closely related to human health. It plays a significant role in bone formation, cellular respiration and other physiological processes. It also serves as an important catalytic cofactor on synthesizing of hemoglobin, elastin and collagen.1,2 The imbalance of Cu2+ may cause many diseases such as anemia, pancytopenia, Wilson's disease, and Alzheimer's disease.3-5 Nevertheless, an increasing in Cu2+ is not only deleterious to living organisms, liver, kidney and the central nervous system, but may also lead to serious environmental pollution. The U.S. Environmental Protection Agency (EPA) has explicitly stipulated that the level of Cu2+ in drinking water should be no more than 20 µM.6 Consequently, it is crucial to monitor and control the amount of Cu2+ to the associated health concerns and environmental monitoring. Many proven techniques that were developed to detect Cu2+ including atomic absorption spectroscopy/emission spectroscopy (AAS), inductively coupled plasma mass spectroscopy (ICP-MS), inductively coupled plasma optical emission spectrometer (ICP-OES), voltammetry and potentiometry.7-9 However, these techniques are usually expensive and should be operated by professionals. Hence, they cannot be widely used in developing countries or remote areas due to limited laboratory resources and professionals, it’s rather difficult to widely use them in developing countries.10,11 The paper-based device is an emerging high efficiency analytical method for meeting the rapid and cost-effective detection.12 The paper-based devices are simplest, low-cost and portable analytical platforms. They are either semi-quantitative or quantitative for single analyte or multiple analytes. The main
application of these kinds of devices is to let people living in the developing world have access to affordable and environmentally friendly disease diagnosis. Different kinds of detection methods were proposed to detect the response signal from paper-based diagnostics, such as electrochemistry, colorimetry, chemiluminescence, electrochemiluminescence and fluorescence.13-15 Up to now, colorimetric detection has been the most commonly assay method used on microfluidic paper-based analytical devices (µPADs). And the fluorescence assay, another kind of optical method, inherently possesses much higher sensitivity than colorimetric method. Fluorescent methods for detecting the copper ions have been widely applied owing to their great sensitivity, good selectivity and rapid response. So the fluorescent dyes, noble metal nanoparticles, quantum dots, upconversion nanoparticals and carbon dots have been attracted by their advantages which conclude of small size, high fluorescence intensity, good biocompatibility for fluorescent indicators or labels on the paper-based devices.16-18 Generally, the fluorescent dyes have been widely used as fluorescent indicators or labels in the paper-based devices.19-21 Organic fluorophores with rigid structure and electron-donor group are inclined to have higher fluorescent density. Meanwhile, the polarity of solvent and size of fluorophores affect the luminescent behavior of fluorophores. So they are characterized with small size, high fluorescence intensity, good biocompatibility, and easy surface modifications for covalent conjugations. The fluorescent dyes in labeling compound show high fluorescence quantum yield, good light stability and low temperature coefficient.22-23 In this study, a highly sensitive and selective fluorogenic paper-based device for Cu2+ detection was developed based on
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RESULTS AND DISCUSSION
As shown in scheme 1, the PP2 and PP3 can be respectively synthesized by coupling 1-bromo-2-(2-(2-methoxyethoxy) ethane and 1-bromododecane with the PP1. All compounds were prepared according to procedures described in a previous literature.25 The structures of the PP2 and PP3 were fully characterized by 1H NMR, 13C NMR analysis (Figure S1). The synthetic background and original spectrum of all compounds were described in detail in Supporting Information. The three probes showed low fluorescence, almost close to zero. The composition of the probe was fluorescein hydrazide, which can identify copper ion. The difference between the PP2 and PP3 was the long chain structure. The PP2 had an oxyalkyl chain and PP3 had an alkyl chain. The PP1, PP2 and PP3 all responded to the copper ion. And the UV-vis spectrum showed absorption peak centered at 490 nm. The fluorescence spectrum of the three probes exhibited an emission peak at 520 nm with an excitation of 460 nm (Figure S2).
To investigate the sensitivity of the three synthesized probes, we increased the concentration of Cu2+ to detect the fluorescence intensity of the probes. As could be seen in Figure 2, the fluorescence intensity increased with the copper ion concentration under UV lamp, and exhibited a good linear
(A) FL.Intensity (a.u.)
fluorescence of probes (10 µM) had no obvious change in the pH range 3-11. However, when Cu2+ were added, the fluorescence of probes increased evidently as the pH value increased and then decreased when reaching the peak. All these probes had the same feature that when pH was 10, the probe had the best response. It indicated that we should use this pH to detect the performance of paper-based devices.
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fluorescence “turn on” of fluorescein on µPADs. The µPADs was easy to fabricate and can be finished fast in 1 min by the wax-printer and the heater. The probe is consisted of fluorescein moieties and oxyalkyl chain, which increased the affinity of the probe and the paper. When the sensor was exposed to Cu2+, it would “turn on” fluorescence, and the fluorescence intensity would be enhanced as the Cu2+ concentration increased. The fluorogenic paper-based detection limit of this highly sensitive probe was as low as 0.41 ppm. In addition, this device was effectively employed to detect Cu2+ in the biological samples such as urine, serum, and cytochylema. Hence, the paper-based fluorogenic sensor is cost-effective, high sensitivity, good selectivity, disposable and equipment-free, indicating its potential application incomprehensive sample and point-of-care detection.
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To evaluate the selectivity of the probe for Cu2+, a variety of metal cations, amino acids and anions were investigated in selectivity binding experiments. The concentrations of these possible interfering ions or amino acids were 10 fold higher than that of Cu2+. The fluorescence enhancement of the probe (5 µM) in 1 : 5 (v/v) MeCN/H2O (37 oC) upon addition of various metal cations, amino acids and anions was depicted in Figure 1. Significant fluorescence enhancement of the probe was observed only when Cu2+ was added, other metal cations had a negligible fluorescent intensity and little interference. The insert figures verified that the fluorescence intensity was enhanced when adding the same equiv of Cu2+ to the interference cations solution, the fluorescence intensity was increased the same as the situation when there were only Cu2+ present. This was attributed to the formation of the ringopened fluorescein derivative.25 It was obvious that the three probes had a high selectivity and a good anti-jamming capability for Cu2+ recognition. For practical applicability, the different pH conditions for probes were investigated (Figure S3). From Figure S3 (A) and (B), the probes of PP1 and PP2 had the same feature and exhibited the best response when pH was 10. Also Figure S3 (C) showed that the pH value has less influence on PP3 than the other two probes. Without Cu2+, the
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Figure 1. The fluorescence intensity of the (A) PP1, (B) PP2 and (C) PP3 (5 µM) upon addition of 10 µM of Cu2+ and 100 µM of various metal cations, amino acids and anions in acetonitrile/water solution (1 : 5) at 37 °C. 1, Ni2+; 2, Ag+; 3, Al3+; 4, Mn2+; 5, Co2+; 6, Zn2+; 7, Na+; 8, Cr+; 9, K+; 10, Ba2+; 11, Fe2+; 12, Ca2+; 13, Fe3+; 14, Mg2+; 15, F-; 16, HCO3-; 17, SO42-; 18, Br-; 19, Cl-; 20, I-; 21, HPO42-; 22, CO32-; 23, L-Cys; 24, Met; 25, Pro; 26, Thr; 27, Gly; 28, Try; 29, Glu; 30, His; 31, Tyr; 32, Phe; 33, Orn; 34, Arg; 35, Dia; 36, Ala; 37, Gly; 38, Val; 39, Ser; 40, Asp; 41, Iso; 42, DL-Cys; 43, GSH; 44, Hcy; 45, blank; 46, Cu2+. The insert photos were the fluorescence effect of the corresponding probes with the same concentration on the samples expect 45 and 46.
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Figure 2. Fluorescence emission spectra (λex = 460 nm) of (A) PP1, (C) PP2 and (E) PP3 with concentrations of Cu2+ (0-10 equiv) added increasing in 1:5 (v/v) acetonitrile-water solution at 37 oC for 2 h. Figure (B), (D), (F) showed the calibration curve of fluorescence intensity vs Cu2+ concentration, and the fitted equation was (B) Y = 1.2914X+7.7916, with R2 = 0.9886, (D) Y = 1.2016X+7.2204, with R2 = 0.961 and (F) Y = 1.3953X+8.3575, with R2 = 0.9787.
correlation in the concentration range from 0-10 µM. A linear regression curve was then fitted to the data of the fluorescent intensity and the concentration of the Cu2+, and the point at which this line crossed the axis was served as the limit value. The detection limits of PP1, PP2 and PP3 were defined as 3 times of the standard deviation of background (3σ) and calculated to be 9.25×10-7 M, 9.79 ×10-7 M and 1.02×10-6 M, respectively. Among the three probes, the response of PP1 to Cu2+ was the best. Being one of the most important criteria of an excellent chemosensor, the practical application of the three probes were explored by test paper experiments. The paper device was designed as 96 well plates which can be tested by the Microplate System. When the probe was anchored to the paper, there was no obvious color change in the detection zone until Cu2+ was added, and the fluorescence could be seen under the UV lamp (Figure 3). And the fluorescence intensity tended to increase with the increasing concentration of Cu2+ under the UV lamp. The results exhibited the lowest visibility when the concentration was 10 nM by naked eye. And different Cu2+ concentrations increased were detected on the paper sensors (SI Figure S4). Therefore, Cu2+ can be detected in the paper-based fluorescence sensors by naked eye when the concentration ranging from 10 nM to 1 M. The paper device also suggested that the probe had a high selectivity and a good anti-jamming capability for Cu2+ recognition (Figure S5).
Figure 3. Fluorescence intensity of PP2 (5 mM) and addition of Cu2+ (10 ppM-1 µM) on the paper-based device by UV by naked eye.
For multiplex detection, the concentrations of PP1, PP2 and PP3 were optimized respectively. With the concentration of Cu2+ increased, the fluorescence intensity enhanced gradually. Figure 4 (A) and 4 (B) showed that the probes detected different concentration gradients of Cu2+. And it demonstrated a good linear correlation in the concentration range from 10 ppm to 100 mM. Figure 4 (C) showed that the detection limit of PP1 was 2.81 nM, and the fluorescence intensity tended to be stable when Cu2+ concentration reached 100 µM; Figure 4 (D) suggested that the detection limit of PP2 was 0.41 pM, and the fluorescence intensity inclined to be stable when Cu2+ concentration of was 100 nM. Meanwhile, PP3 indicated that it wasn’t an appropriate probe for paper device. Observing from Figure 4 (E), the detection limit of PP1 was 0.181 µM, that of PP2 was 6.99 nM, and there was no reaction on PP3. However, from the fluorescence images of Figure S4 (A), we can see that a good linear regression curve was found between the fluorescence intensive and the concentration of Cu2+ from 10 µM to 10 mM (R2 = 0.9903), and the detection limit was measured to be 10.13 nM (SI Figure S4), closing to the above result (Figure 4 (F), 6.99 nM). Reasons responsible for such difference could be as follows: different unit intervals and the sensitivity of the instruments. These data suggested that PP2 was a good probe for detecting Cu2+, while PP1 and PP3 were not suitable for fabricating paper based devices. The fluorescence emission spectra of the probes on the paper were studied as well (Figure S6). It showed the fluorescence intensity improved as the concentration of copper ions increased. Yet, only PP2 exhibited a good linear correlation when the concentration ranging from 1 pM to 100 mM. Besides, PP2 probe could reach the maximum in 1 h, while PP1 and PP3 would use 2 h (Figure S7). Meanwhile, PP2 and PP3 reached the maximum earlier than the PP1.
Figure 4. The fluorescence intensity images of PP1, PP2 and PP3, Cu2+ concentrations added increasing (10 ppM - 100 mM) on the paper (concentrations probe was 5 mM (A) and 0.5 mM (B)) at 37 oC. (C-D) showed the calibration curve of fluorescence intensity vs Cu2+ concentration ((C) 5 mM and 0.5 mM (E) of PP1, (D) 5 mM and 0.5 mM (F) of PP2).
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Simultaneously, the laser confocal images of paper@PP2@Cu2+ were studied in order to prove the ability of PP2 for specific imaging of Cu2+ on the fiber surface. From these pictures, it is clearly observed that the fluorescence enhanced as Cu2+ concentrations increased (1 pM - 10 M) on the paper based device. The curve also proved that lower concentration of Cu2+ could be seen and the fluorescence intensity increased with the concentration increasing (Figure 5). From Figure 5, 1 pM concentration of Cu2+ showed a difference from the negative control. However, the cellulose structures were intertwined, and the focus images were just captured for portion.
Figure 5. Laser confocal images of paper@ PP2@Cu2+ in the presence of increasing concentrations of Cu2+ (1ppM-1µM) on the paper based device. (A), paper; (B), only probe; (C), 1 pM; (D), 10 pM; (E), 100 pM; (F), 1 nM; (G), 10 nM; (H), 100 nM; (I), 1 µM; (J), 10 µM; (K), 100 µM; (L), 1 mM; (M), 10 mM; (N), 100 mM; (O), 1 M; (P), 10 M; (Q), the fluorescence intensity curve of the laser confocal images.
From the viewpoint of ultimate practically, the PP2 probe was immobilized onto the paper to build a platform for detection the Cu2+ (SI Figure S8). And the added three different concentrations (25 nM, 35 nM, 45 nM) of the copper ion standard solution into the samples were also studied. All these samples were then tested on the paper-based devices. The results described that this method realized a good average recovery rates from 89%-124% for Cu2+ detection (Table 1). The results of human serum demonstrated that the real-world applicability of the paper-based device for clinical serum samples from patients whose blood Cu2+ level was obviously higher than that of healthy individuals. The above results suggested that our paper-based fluorogenic device provided a novel platform for detecting the Cu2+ from the serum, urine, cells and the lake water samples. Moreover, it was more significant that the results of paper-based fluorogenic devices were almost unchanged after being stored for two weeks in the dark. Besides, being pollution free in nature, these devices can be burnt after use (SI Figure S8).
Table 2 shows the comparison of paper-based fluorogenic devices for detection of Cu2+ reported in literatures. Owing to their capability detecting Cu2+ with high selectivity and sensitivity, these sensors had received considerable attention. Since the maximum contamination level of Cu2+ in drinking water has been regulated by the U.S. Environmental Protection Agency (EPA) as 20 µM30, our work was applied to biology samples rather than drinking water. The fabrication of the device can be finished in 2 min.
CONCLUSIONS
In summary, the paper-based fluorogenic device has been designed and synthesized in a rather simple way. The
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Table 1 Determination of Cu2+ with spiked concentration in serum samples, urine, HepG-2 cells, and lake water on paper-based devices. Sample
Detected (nM） ）
Added (nM） ）
Founded (nM)
Recovery
Serum (human)
125
Serum (bovine)
52.73
Urine
17.88
HepG-2 (2%)
0.14
Lake water
0.08
25 35 45 25 35 45 25 35 45 25 35 45 25 35 45
160±7.85 175±6.89 196±5.94 79.43±0.85 102.84±7.55 121.47±11.87 38.66±2.11 47.76±2.56 62.3±0.29 29.74±2.23 40.1±2.5 52.97±3.92 29.07±1.99 31.08±1.99 50.98±2.94
112% 113% 118% 102% 117% 124% 90% 90% 99% 118% 114% 117% 116% 89% 113%
fluorogenic paper sensor demonstrated a highly selective and sensitive recognition and determination of Cu2+ over other kinds of interference anions, cations and amino acids in the biological system31-32. The detection limit was as low as 0.41 ppM on the paper devices. The paper-based device for detecting the concentration of Cu2+ could last for 3 months with no obvious change. For longer use, the probe should be separately kept from the paper, and stick to the principle of on-site fabrication for on-site use. This strategy presents promising prospects for Cu2+ monitoring in environmental aqueous samples33. Finally, the introduction of oxyalkyl chain as an affinity group to paper was critical to the detection system of Cu2+, and these results will make the rational design of more excellent chemosensors easier in the future.
EXPERIMENTAL SECTION
Materials and Instruments. All reagents and chemicals were purchased from commercial suppliers and used without further purification, except when specified. All metal salts were derived from their nitrate or chloride salts. PP1 was synthesized according to literature procedures.31 1H and 13C NMR spectra were measured with an Avance AV-300 spectrometer using solvent peak as internal reference at 25 °C. Absorption and fluorescence spectra were recorded on an UVvis spectrometer and on a spectrofluorometric at room temperature. The fluorescence paper images were captured by an OmegaLum W Multicolor fluorescence, chemiluminescence, and visible light gel imaging system (Aplegen, America) and a Nikon Eclipse TE2000U inverted fluorescence microscope equipped with a cooled CCD camera (Nikon, Japan). Scanning electron microscope (SEM) images were obtained with a JSM-7800F SEM system (JEOL, Japan). Synthesis of 2-amino-3’-hydroxy-6’-(2-(2-(2methoxyethoxy)ethoxy)spiro[isoindoline-1,9’-xanthen]-3one (PP2). A solution of PP1 (100 mg, 0.3 mmol) and Cesium carbonate (195.5 mg, 0.6 mmol) were mixed in a 250 mL flask under a dry nitrogen atmosphere. And the solution of 1bromo-2-(2-(2-methoxyethoxy)ethoxy)ethane (54.9 mg, 0.3 mmol) was subsequently added dropwise by syringe over 30 minutes under vigorous stirring. The reaction was then heated to 80 °C and continued at this temperature for 8 hours. Then, the mixture solution was washed by sodium chloride for 3 times, extracted by ethyl acetate and dried by anhydrous sodium sulfate. Finally, the product was purified by flash
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ACS Applied Bio Materials Table 2 Comparison of paper-based fluorogenic devices for detection of Cu2+ reported in literatures.
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Fluorescent materials
Production cost
Production time
LOD
Application
Ref
Silver nanoclusters
Fiber paper strips
Few hours
20 µM
Drinking/lake water
Liu at al25
AIE-based fluorophore
Microfluidic paper-based
--
160 nM
--
Song at al27
Carbon Dots
Fiber paper strips
>3 hours
25 nM
Tap/lake water
Liu at al28
CdTe QDs
Glass fiber paper
>12 hours
1.2 µg/L
Lake water
Wang at al29
Fluorescein probe
Microfluidic paper-based
2 min
0.41 ppm
Urine, serum, cytochylema
This work
column chromatography (silica gel, DCM/MeOH = 10/1) afforded PP2 as an off-white crystal. 1H NMR (300 MHz, CDCl3): δ 7.93 (dd, J1 = 6 Hz, J2 = 3 Hz, 1 H), 7.44 (dd, J1 = 9 Hz, J2 = 3 Hz, 2 H), 7.02 (dd, J1 = 9 Hz, J2 = 6 Hz,1H), 6.72 (t, J = 7.5 Hz, 2 H), 6.54 (m, 4 H), 4.09 (t, J = 6 Hz, 2 H), 3.80 (d, J = 3 Hz, 2 H), 3.66 (d, J = 9 Hz, 6 H), 3.54 (d, J = 8 Hz, 2 H), 3.33 (s, 3 H). 13C NMR (75 MHz, CDCl3): δ 166.67, 159.83, 158.26, 153.18, 153.14, 151.1, 133.05, 129.79, 128.69, 128.1, 123.79, 123.26, 112.62, 111.9, 110.32, 109.11, 103.45, 101.94, 77.51, 77.09, 76.67, 70.57, 70.45, 69.51, 67.59, 65.87, 60.46, 58.92. Synthesis of 2-amino-3’-(dodecyloxy)-6’hydroxyspiro[isoindoline-1,9’-xanthen]-3-one (PP3) was synthesized according to the similar procedure described above in PP2 by using 1-bromododecane. 1H NMR (300 MHz, CDCl3): δ 7.96 (dd, J1 = 6 Hz, J2 = 3 Hz, 1 H), 7.48 (m, 2 H), 7.06 (dd, J1 = 6 Hz, J2 = 3 Hz, 1H), 6.77 (s, 1 H), 6.70 (s, 1 H),6.56 (m, 4 H), 3.96 (t, J = 7.5 Hz, 2 H), 3.68 (s, 2 H), 1.79 (dd, J1 = 15 Hz, J2 = 9 Hz, 2 H), 0.90 (t, J = 7.5 Hz,3 H). 13C NMR (75 MHz, CDCl3): δ 166.97, 160.48, 158.30, 153.38, 151.23, 133.26, 128.13, 123.96, 123.43, 112.66, 112.17, 109.66, 109.19, 103.68, 103.81, 103.81, 68.44, 66.23, 32.00, 29.66, 26.11, 22.76, 14.19. Fabrication of the Paper-based Fluorogenic Device. A paper-based device contained 8 rows × 12 columns test zones was fabricated on the Whatman chromatography paper 3#. This device consisted only one layer with the detection areas. The paper’s hydrophobization detection area was designed by the wax. This wax patterned paper contained 96 working zones (1.2 ± 0.2 mm in diameter) independently. The patterns of hydrophobic barriers on a white background were designed with probe for detecting Cu2+. The wax patterns were printed on a filter paper by the wax printer (FUJI XEROX Phaser 8580 DN, America). Then the filter paper with the surface wax sheet was baked at 150 oC for 0.5 min. the wax was penetrated through the paper and formed the hydrophobic patterns. The patterned paper could be cut into slice for user after it was cooled down to room temperature.24 Sensitivity and Selectivity Measurements. A stock solution of Cu2+ (1 M) was prepared by dissolving an appropriate amount of Cu(NO3)2 in water and then various concentrations were obtained by serial dilution of the stock solution. To access the cation selectivity, the following nitrates were studied: Ni2+, Ag+, Al3+, Mn2+, Co2+, Zn2+, Na+, Cr+, K+, Ba2+, Fe2+, Ca2+, Fe3+, Mg2+. Additionally, the anion of sodium salts were used to investigate the possible anion interference: F-, HCO3-, SO42-, Br-, Cl-, I-, HPO42-, CO32-. And the amino acids were also tested: L-cysteine (L-Cys), DLmethionine (Met), L-(-) proline (Pro), L-threonine (Thr), glycine (Gly), L(-)-tryptophan (Try), glutamic (Glu), Lhistidine (His), L-tyrosin (Tyr), L-phenylalanine (Phe), L-
ornithine (Orn), L(+)-arginine (Arg), (s)-2,6-diaminocaproic acid (Dia), L-alanine (Ala), glycine (Gly) , L-valine (Val), Lserine (Ser), L-aspartic acid (Asp), L-isoleucine (Iso), DLcysteine (DL-Cys), glutathione (GSH), DL-homocysteine (Hcy). Preparation of Test Paper and Analytical Application. To demonstrate the feasibility of the method for in vitro detection, our work chose the HepG-2 cells (1×105/mL), urine (mouse), human serum (normal) and bovine serum for quantifying the Cu2+ content. After being treated, these samples were diluted in incubation buffer solution. And then the fluorescent probe (2 µL, respectively) was applied to each paper zone and incubated at room temperature. After that, a series of different doses of samples were dropped into the center zone until dried off. Finally, the fluorescence was measured under excitation at 460 nm.
ASSOCIATION CONTENT
Supporting Information 13
C NMR spectra of PP2 and PP3, fluorescence and UV-Vis spectra for Cu2+ detection.
H and
AUTHOR INFORMATION
Corresponding Authors Email:
[email protected] Email:
[email protected] Notes The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was financially supported by the National Natural Science Foundation of China (81672508, 61601218, 21675085 and 61505076), Jiangsu Provincial Foundation for Distinguished Young Scholars (BK20170041, BK20170042), Key University Science Research Project of Jiangsu Province (Grant 16KJA180004) and China-Sweden Joint Mobility Project (51661145021).
REFERENCES
(1) Chan, Y. H.; Chen, J. X.; Liu, Q. S.; Wark, S. E.; Son, D. H.; Batteas, J. D. Ultrasensitive Copper(II) Detection Using Plasmon-Enhanced and Photo-Brightened Luminescence of CdSe Quantum Dots. Anal. Chem. 2010, 82, 3671-3678. (2) Zhang, Y. H.; Zhang, H. S.; Guo, X. F.; Wang, H. L-Cysteine-Coated CdSe/CdS Core-Shell Quantum Dots as Selective Fluorescence Probe for Copper(II) Determination. Microchem. J. 2008, 89(2), 142-147. (3) Domaille, D. W.; Que, E. L.; Chang, C. J. Synthetic Fluorescent Sensors for Studying the Cell Biology of Metals. Nat. Chem. Biol. 2008, 4(8), 168-175.
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