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Target-Triggered Switching On and Off the Luminescence of Lanthanide Coordination Polymer Nanoparticles for Selective and Sensitive Sensing of Copper Ions in Rat Brain Pengcheng Huang, Fang-Ying Wu, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01155 • Publication Date (Web): 01 Jun 2015 Downloaded from http://pubs.acs.org on June 3, 2015

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Analytical Chemistry

Target-Triggered Switching On and Off the Luminescence of Lanthanide Coordination Polymer Nanoparticles for Selective and Sensitive Sensing of Copper Ions in Rat Brain

Pengcheng Huang,1* Fangying Wu,1* Lanqun Mao2

1

College of Chemistry, Nanchang University, Nanchang 330031, China

2

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for

Living Biosystems, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100190, China

1

ACS Paragon Plus Environment


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ABSTRACT Copper ions (Cu2+) in the central nervous system play a crucial role in the physiological and pathological events, so simple, selective, and sensitive detection of cerebral Cu2+ is of great importance. In this work, we report a facile yet effective fluorescent method for sensing of Cu2+ in rat brain using one kind of lanthanide coordination polymer nanoparticles, AMP-Tb, as the sensing platform. Initially, a cofactor ligand, 5-sulfosalicylic acid (SSA), as the sensitizer, was introduced into the non-luminescent AMP-Tb suspension, resulting in switching on the luminescence of AMP-Tb by the removal of coordinating water molecules and concomitant energy transfer from SSA to Tb3+. The subsequent addition of Cu2+ into the resulting SSA/AMP-Tb can strongly quench the fluorescence because the specific coordination interaction between SSA and Cu2+ rendered energy transfer from SSA to Tb3+ inefficient. The decrease ratio of the fluorescence intensities of SSA/AMP-Tb at 550 nm show a linear relationship for Cu2+ within the concentration range from 1.5 to 24 µM with a detection limit of 300 nM. The method demonstrated here is highly selective and is free from the interference of metal ions, amino acids, and the biological species commonly existing in the brain such as dopamine, lactate, and glucose. Eventually, by combining the microdialysis technique, the present method has been successfully applied in the detection of cerebral Cu2+ in rat brain with the basal dialysate level of 1.91 ± 0.40 µM (n = 3). This method is very promising to be used for investigating the physiological and pathological events that cerebral Cu2+ participates in.

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INTRODUCTION As very important endogenous messengers present in organs and bodies, metal ions play essential roles in many fundamental physiological and pathological events, especially those related to brain function activity.1,2 Among of them, copper ion (Cu2+) is one kind of the most abundant transition metal elements and has been demonstrated to substantially participate in the cellular metabolism, growth, and immune system development.3,4 On one hand, Cu2+ is identified as an important catalytic cofactor for many enzymatic reactions in which enzymes like cytochrome c oxidase, superoxide dismutase, and tyrosinase are involved.5-7 On the other hand, the dysregulation in the cellular homeostasis of Cu2+ may cause cell death and serious neurological diseases, such as Wilson disease, Alzheimer’s disease, and prion disease.8-11 In this regard, it is crucial to develop a simple, selective, and sensitive method for the detection of Cu2+ in the central nervous system to understand its physiological and pathological functions. To date, several methods have been established for the detection of Cu2+, including atomic absorption spectrometry (AAS),12 inductively coupled plasma mass spectroscopy (ICP-MS),13 inductively coupled plasma-atomic emission spectrometry (ICP-AES),14 electrochemical sensors,15 colorimetric assays,16 and fluorescence methods.17 Among these analytical methods, fluorescence methods have gained considerable attention mainly due to high sensitivity, simplicity, and fast response.17,18 Although recently some works about cell imaging of Cu2+ using fluorescent sensors have been reported,17a,17c-e there still remains a big challenge for the fluorescence detection of Cu2+ in the central nervous system, for example rat brain, because the intrinsic chemical complexity essentially necessitates the proposed methods with high analytical performance particularly in sensitivity and selectivity.15c,15d,15e,19 Lanthanide-based fluorescent sensors have some advantageous spectral characteristics over traditional fluorescent sensors, including long fluorescence lifetime, large Stokes shifts, and sharp line-like emission bands, which make them very suitable for time-resolved luminescence detection by readily distinguishing from the background luminescence.20-22 As a newly-emerging fascinating sensing platform in lanthanide-based fluorescent sensors, lanthanide coordination polymer nanoparticles (Ln-CPN), self-assembled by lanthanide ions and organic linkers via coordination bonds, have activated growing interests because of their structural tailorability and adaptive capability of guest encapsulation besides such characteristics mentioned above analogous 3



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to those of conventional lanthanide-based counterparts; 23 but unfortunately, they usually show quite weak fluorescence due to quenching effect of water molecules or low chemical stability in aqueous solution, 23c,23e,24 and even worse, in most cases, relatively poor selectivity to analytes would be encountered owing to the facts that commonly-incorporated aromatic ligands lack target-specific binding sites and that complicated experimental procedures render difficulties in post-modification for Ln-CPN to improve their selective recognition to analytes.25 Herein, we develop a new strategy for simple, selective, and sensitive sensing of Cu2+ in rat brain using a Ln-CPN-based “OFF-ON-OFF” fluorescent sensor combined with in vivo microdialysis for dialysate sampling (Scheme 1). In this study, one kind of water-stable Ln-CPN, constructed from adenosine monophosphate (AMP) and terbium ion (Tb3+), i. e. AMP-Tb, was exploited as the sensing platform. Although AMP-Tb is non-luminescent as reported previously,23e,24b by rationally selecting a cofactor ligand, 5-sulfosalicylic acid (SSA), as the sensitizer to introduce the as-formed AMP-Tb network, the luminescence of AMP-Tb can be greatly enhanced via energy transfer from SSA to Tb3+ (the so-called antenna effect).21,24 And very interestingly, the enhanced luminescence could specifically be weakened by the addition of Cu2+ since strong coordination interaction between SSA and Cu2+ efficiently suppressed such antenna effect. It should be noted that the network of AMP-Tb is very favorable for full contact between SSA and external Cu2+ due to the nanoscaled particle size, thus leading to good sensitivity. Additionally, SSA plays indispensable roles in both modulating the “ON/OFF” luminescent state of AMP-Tb by antenna effect or sequentially coordinating to Cu2+ and, improving the selectivity towards Cu2+ using AMP-Tb as the sensing platform. Therefore, our strategy demonstrated here is very simple yet effective for fluorometric sensing of cerebral Cu2+. As far as we know, this is the first report on the direct fluorescent detection of Cu2+ in rat brain, which holds great promise for further research on physiological and pathological events associated with Cu2+.

EXPERIMENTAL SECTION Chemicals and Reagents. Adenosine monophosphate (AMP), Tb(NO3)3·5H2O, and copper-zinc superoxide dismutase (Cu-Zn SOD) from bovine erythrocytes were purchased from aladin-reagent Co. Ltd. (Shanghai, China). Dopamine (DA), ascorbate acid (AA), lactate, 4


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5-hydroxytryptamine (5-HT), 3, 4-dihydroxyphenylacetic acid (DOPAC), glucose, and tyrosinase from

mushroom

were

all

purchased

from

Sigma-Aldrich

(Shanghai,

China).

2-[4-(hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 5-Sulfosalicylic acid dihydrate (SSA), and other chemicals of at least analytical reagents (e. g., amino acids and metallic salts) were obtained from Beijing Chemical Corporation (Beijing, China) and used without further purification. Artificial cerebrospinal fluid (aCSF) was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM) into doubly distilled water, and the solution pH was adjusted to 7.4. All aqueous solutions were prepared with doubly distilled water. Apparatus and Measurements. Fluorescence spectra were performed on a F-4600 fluorescence spectrometer (Hitachi, Japan) equipped with a xenon lamp source and a 1.0 cm quartz cell. The excitation wavelength was set at 328 nm, and emission spectra were recorded by observing fluorescence intensity of Tb3+ at ca. 550 nm. The slit widths were both 5 nm for excitation and emission. The photomultiplier tube (PMT) voltage was set at 400 V. UV-vis absorption spectra were conducted at room temperature on a UV-2550 spectrophotometer (Shimadzu, Japan). Fourier transform infrared (FT-IR) spectra were recorded with KBr pellets on a Nicolet 5700 FTIR Spectrometer (Nicolet, USA). Scanning electron microscopy (SEM) images were collected on a scanning electron microscope S-4800 (Hitachi, Japan) at 15.0 kV. All experiments were carried out at room temperature. Preparation of AMP-Tb. AMP-Tb was prepared according to the previous report.23e Briefly, 4.5 mL of Tb(NO3)3 aqueous solution (10 mM) was added into 4.5 mL of HEPES buffer (0.1 M, pH 7.4) containing AMP (10 mM) to form a white precipitate under stirring at room temperature. The obtained precipitate was purified by centrifugation and washed several times with water, and finally, was re-dispersed in 9 mL of HEPES buffer (0.1 M, pH 7.4) to form AMP-Tb suspension for use. Switching on the Luminescence of AMP-Tb by the Cofactor Ligand SSA. To switch on the luminescence in AMP-Tb, the stock solution of SSA (1 mM) was successively added into AMP-Tb suspension (500 µL) until the fluorescence intensity of Tb3+ at ca. 550 nm reached to its saturated value. For control experiments, we studied several analogues of SSA, phenol, benzoic acid, benzenesulfonic acid, and salicylic acid, about antenna effect on the luminescence. 5



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To investigate the effect of pH on the fluorescence enhancement, AMP-Tb suspension in the absence or in the presence of 4 µM SSA was added into HEPES buffer at different pH (3.0-11). For the effect of reaction time, the fluorescence intensities of Tb3+ at ca. 550 nm were recorded every one-minute interval after SSA (150 µL, 1 mM) was added into AMP-Tb suspension. Switching off the Luminescence of SSA/AMP-Tb for Cu2+ Sensing. For fluorometric sensing of Cu2+, initially, 150 µL of an aqueous solution of SSA (1 mM) was added into AMP-Tb suspension (500 µL), and the resulting mixture was shaken well and allowed to stand by for 12 min at room temperature. After that, different volumes of Cu2+ (1 mM) were continuously added into the above mixture and the fluorescence intensities of Tb3+ were monitored accordingly. In Vivo Microdialysis. Animal surgery and in vivo microdialysis were carried out as reported previously.15c,15d,15e,19 Adult male Sprague-Dawley rats (350-400 g) were purchased from Center of Health Science, Peking University and housed on a 12: 12 h light-dark scheduler with food and water ad libitum. The animals were anaesthetized with chloral hydrate (345 mg/kg, i.p.) and positioned onto a stereotaxic frame. The microdialysis guide cannula (BAS/MD-2250, BAS) was implanted into the striatum using standard stereotaxic procedures. Throughout the surgery, the body temperature of the animals was maintained at 37 °C with a heating pad. After the rats were allowed to recover for at least 24 h, a microdialysis probe was first implanted into the rat striatum and then perfused with aCSF at 2.0 µL/min. After continuously perfusing the probe for at least 90 min for equilibration, the microdialysate was collected for fluorometric sensing of cerebral Cu2+.

RESULTS AND DISCUSSION Switching on the Luminescence of AMP-Tb by SSA via Antenna Effect. To accomplish direct fluorescent detection of Cu2+ in rat brain, we chose AMP-Tb as the lanthanide-based sensing platform. Nucleotide-lanthanide coordination polymers are a novel subclass of coordination polymer nanoparticles recently and possess some excellent physico-chemical properties, especially extremely good water-stability even in a very high concentration of salt, for example rat brain microdialysates in this study, which renders them superior to a number of nanomaterials in biological applications.26 AMP-Tb was prepared by self-assembly of AMP and Tb3+ in aqueous solution. As shown in Figure S1-A, the resulting AMP-Tb nanoparticles were colloidal spheres with diameters at about 40 nm, which is almost consistent with the previous report.23e From Figure 6


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1 and the inset in Figure S1-A, it can be seen that AMP-Tb is non-luminescent. As described in many literatures about lanthanide complexes, this could be attributed to quenching effect of water molecules occupied the residual electron orbitals except those AMP coordinated to in the coordination sphere of Tb3+.23c,23e,24 However, in the presence of SSA, the luminescence of AMP-Tb was significantly enhanced (Inset in Figure S1-B). The four strong emission peaks at 494, 550, 590, and 625 nm are the characteristic peaks of Tb3+,24b while the excitation peak at 328 nm resulted from the absorption of SSA. These results indicate that the luminescence of AMP-Tb was efficiently sensitized by the introduction of SSA into the coordination networks. We also investigated the dependence of the fluorescence intensity of AMP-Tb on the concentration of SSA, the pH of the reaction media, and the reaction time (Figure S2-A, B, C). It was found that the fluorescence intensity increased with the increase of SSA concentration, whereas it leveled off when the concentration of SSA reached 0.23 mM. For pH effect, the fluorescence intensity reached the maximum value at neutral media upon the addition of SSA, which is very good for subsequent sensing of cerebral Cu2+. The kinetic luminescence increase of AMP-Tb toward SSA (0.23 mM) was monitored, and it can be completed within 12 min. Thus, for the following fluorescent detection of Cu2+, the optimal concentration of SSA was fixed at 0.23 mM and the equilibrium time after adding SSA was set at 12 min. Figure 2 displayed FT-IR spectra of AMP-Tb in the absence and presence of SSA. For AMP, peaks at 1482, 1109 and 978 cm-1 were assigned to the N7-C8 stretching (νN7-C8) band in the adenine subunit of AMP, the phosphate antisymmetric (νasPO3) and symmetric (νsPO3) vibration bands, respectively.23e For AMP-Tb, the wavenumber of AMP was shifted to 1484, 1113 and 989 cm-1, respectively, suggesting that both the adenine moieties and phosphate groups of AMP coordinated to Tb3+. For SSA/AMP-Tb, relative to those of AMP-Tb, the wavenumbers of the corresponding peaks slightly shifted (1483, 1110 and 987 cm-1), on the other hand, there did not exist the characteristic peaks of SSA (e. g., the stretching bands of the sulfonic groups (SO3) and the C=O stretching band of the carboxylic group);27 this demonstrates that the binding of SSA to form the ternary complex had little effect on the structural integrity of host assemblies consisting of AMP and Tb3+, which is also supported by the SEM image (Figure S1-B) judged from the morphology and size. The absorption spectra of SSA, SSA-Tb, AMP-Tb, and SSA/AMP-Tb were depicted in Figure 7



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3. SSA exhibited a strong absorption peak at 297 nm,28 and SSA-Tb formed by mixing SSA with Tb3+ led to a red shift to 307 nm, reflecting the complexation between SSA and Tb3+. In the case of SSA/AMP-Tb, besides the absorption peak of the maximum absorption of nucleobases at 260 nm,29 a small shoulder component around 300 nm was observed, which indicates the complexation between SSA and Tb3+ could also take place by the addition of SSA into AMP-Tb, and thus greatly sensitize the luminescence of AMP-Tb. In general, such a sensitization that the introduction of an appropriate cofactor ligand into the lanthanide complexes leads to enhanced luminescence is also called as antenna effect, in which the antenna ligand replaces the coordinating water molecules and at the same time, upon UV light irradiation, causes energy transfer from it to lanthanide ions.24 To more clearly understand antenna effect between SSA and AMP-Tb, we directly investigated the fluorescence and absorption spectra of the complex SSA-Tb in the absence of AMP. As shown in Figure S3, Tb3+ is non-luminescent, while SSA gives a distinct characteristic emission peak at 405 nm.28 Whereas, upon the addition of SSA into Tb3+, the fluorescence intensities of the four characteristic emission peaks of Tb3+ dramatically increased, accompanied by an obvious decrease of the fluorescence intensity at 405 nm corresponding to SSA; meanwhile, a large red shift was also observed in the absorption spectra for SSA when it coordinated to Tb3+, as demonstrated above. Undoubtedly, both of them could explain the process about energy transfer from SSA by strongly absorbing UV light to the emissive 5D4 state of Tb3+ and the same one also existing in the ternary complex SSA/AMP-Tb (Scheme 1A). To gain more insight into the binding fashion between SSA and AMP-Tb, we compared a series of analogues of SSA, phenol, benzoic acid, benzenesulfonic acid, and salicylic acid, about antenna effect on the luminescence of AMP-Tb with SSA. As seen in Figure S2-D, phenol, benzoic acid, and benzenesulfonic acid could not sensitize the luminescence, whereas salicylic acid could. We also found that, because of slower response time and weaker maximal fluorescence intensity, the antenna effect of salicylic acid was not strong as SSA; therefore, we speculate that the adjacent carboxylate group and hydroxyl group of benzene ring in SSA coordinated to Tb3+, and that there existed electrostatic interaction between the sulfonic group and Tb3+;23e the former played a main role in the antenna effect in this study and to some extent, the latter also promoted such effect because it could facilitate energy transfer by further strengthening the binding between 8


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SSA and Tb3+. This result is also evidenced by FT-IR spectra of SSA and SSA-Tb (Figure S4). Switching off the Luminescence of SSA/AMP-Tb in the Presence of Cu2+. As depicted in Figure 4 and the inset in Figure S1-C, the further addition of Cu2+ into the resulting SSA/AMP-Tb suspension gave rise to a remarkable fluorescence quenching, and this course finished very rapidly (Figure S5), while no change was obtained for AMP-Tb under identical conditions (Figure S6). This result indicates that SSA/AMP-Tb showed a good response toward Cu2+. As discussed above, the introduction of SSA can facilely switch on the luminescence of AMP-Tb via antenna effect by the replacement of coordinating water molecules and concomitant energy transfer, so we hypothesize that switching off the luminescence of SSA/AMP-Tb could presumably arise from the presence of Cu2+ to form a new complex SSA-Cu, which broke the coordination bond between SSA and Tb3+ through competitive interaction and hence, largely weakened antenna effect due to the inhibition of energy transfer (Scheme 1A). To validate this hypothesis, the absorption spectra were firstly measured (Figure 3). Different from the absorption spectra of SSA/AMP-Tb with a small shoulder component around 300 nm, a new broad shoulder peak at ca. 310 nm emerged after adding Cu2+, which was ascribed to the characteristic absorption peak of SSA-Cu,27 suggesting the formation of SSA-Cu and the breakdown of SSA-Tb. FT-IR spectra (Figure S4) also testified that SSA-Cu could be easily formed estimated by quite an obvious alteration for the corresponding spectrum of SSA in the presence of Cu2+. Furthermore, when Cu2+ was added into the SSA-Tb suspension, the fluorescence spectra revealed an interesting phenomenon (Figure S3): the fluorescence intensities of the four characteristic emission peaks of Tb3+ declined, whereas the fluorescence of SSA (λmax = 405 nm) kept constant. However, in the case of free SSA under the same conditions, the fluorescence of SSA dropped remarkably upon the addition of Cu2+ (Figure S3). These results reflect that Cu2+ can directly quench the fluorescence of free SSA and that, nevertheless, in the presence of Tb3+ to form SSA-Tb, Cu2+ turned to quench the fluorescence of Tb3+, indicating that the negative effect Cu2+ had on the luminescence of SSA-Tb were indeed by inhibiting energy transfer from SSA to Tb3+. So our proposed hypothesis about switching off the luminescence of SSA/AMP-Tb by Cu2+ could substantially be confirmed. Note that, judging by FT-IR spectra (Figure 2) and the SEM image (Figure S1-C), the addition of Cu2+ did not influence the network of SSA/AMP-Tb, which demonstrates that Cu2+ 9



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only changed the coordination microenvironment of SSA within AMP-Tb but not completely separated SSA from the network, implying good stability of AMP-Tb as the host, again. Sensitivity and Selectivity. To evaluate the sensitivity of the assay, different concentrations of Cu2+ were added into the SSA/AMP-Tb suspension. As illustrated in Figure 5A, the fluorescence intensity of SSA/AMP-Tb gradually decreased with the increasing concentration of Cu2+, and the ratio (I0-I)/I0, where I0 and I are the fluorescence intensities of SSA/AMP-Tb at 550 nm before and after adding Cu2+, respectively, showed a linear response toward Cu2+ within a concentration range from 1.5 to 24 µM ((I0-I)/I0 = 0.024 C/µM + 0.135, R = 0.974, Figure 5B). The detection limit was about 300 nM, calculated from S/N = 3. This value is comparable to that of the existing fluorescent sensors for Cu2+, but is lower than the concentration of cerebral Cu2+ in rat brain reported previously,15c,15d,15e so the present strategy could as expected be used for fluorometric sensing of cerebral Cu2+, as will be demonstrated below. As is known that the brain system is very complex, it requires the established methods with high analytical performance not only in sensitivity but also more especially in selectivity. To investigate the selectivity of our method, different kinds of species that may possibly be considered to interfere with the Cu2+ sensing were tested to observe their influences on the fluorescence intensity of SSA/AMP-Tb. First, various metal ions such as abundant cerebral cations (K+, Na+, Mg2+, Ca2+) with 100-fold concentration as that of Cu2+, and trace metal cations may be included in the rat brain (Zn2+, Al3+, Co2+, Fe3+, Fe2+, Ni2+, Ag+, and Cu+) with the same concentration as that of Cu2+, were tested. As shown in Figure 6A, only Cu2+ can result in a significant fluorescence quenching. The competition experiment of Cu2+ with the coexistence of other kinds of metal cations is also performed. Figure 6B displayed that there were little interferences for Cu2+ sensing because in this case, the fluorescence quenching efficiency toward SSA/AMP-Tb were almost the same as that when Cu2+ added only. We next investigated the selectivity against natural amino acids and copper-containing proteins (Figure 6C and D). Similarly, amino acids showed no obvious fluorescence quenching toward SSA/AMP-Tb. For the competition experiment, negligible effects they had on the process that Cu2+ led to the fluorescence quenching were observed. It is noted that although copper-containing proteins like tyrosinase (molecular weight: 32.5 kDa) and Cu-Zn SOD (molecular weight: 133 kDa) caused potential interference toward Cu2+ sensing, such interference could essentially be ruled out since 10


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proteins cannot diffuse across the semipermeable membrane in microdialysis sampling, as mentioned in the following practical application of direct sensing of Cu2+ in rat brain.19 In addition, the interference from other biomolecules that commonly coexist in the cerebral system such as DA, DOPAC, AA, 5-HT, H2O2, lactate and glucose was examined (Figure 6E and F). It was also found that these bioactive species did not cause interferences for sensing of Cu2+ either. All these results substantially demonstrate that the present method have good selectivity toward Cu2+ against other metal ions, amino acids, and biological species. In view of such good selectivity, we consider it to be attributed to the following two aspects, on one hand, upon the addition of Cu2+ into the SSA/AMP-Tb suspension, the specific and strong coordination interaction would take place between SSA and Cu2+;27,30 on the other hand, like in some reports about lanthanide coordination polymer nanoparticles, many ultrasmall pores are distributed within the network of SSA/AMP-Tb,23h which may exclusively allow Cu2+ to pass through to react with SSA. Apparently, compared with the existing fluorescent sensors for Cu2+, our proposed fluorescent sensor also possesses other excellent properties such as simple fabrication and low technical demands because complicated and time-consuming synthetic procedures and chemical modifications, and accurate control on reaction conditions could be avoided. These properties, along with good sensitivity and selectivity, make the present method show great potential for sensing of Cu2+ in rat brain. Fluorometric Sensing of Cu2+ in Rat Brain. To demonstrate the validity of our established method for direct sensing of Cu2+ in rat brain, SSA (150 µL, 1 mM) was firstly added into the aCSF suspension of AMP-Tb (500 µL) to sensitize the fluorescence; it should be pointed out that the fluorescence of SSA/AMP-Tb is very stable in pure aCSF since AMP-Tb is highly salt-tolerant as mentioned above. The brain microdialysate (5 µL) was then added into the resulting SSA/AMP-Tb suspension (Scheme 1B). As shown in Figure 7, a clear decrease for the fluorescence intensity of SSA/AMP-Tb was observed, suggesting the presence of Cu2+ in rat brain. According to the calibration curve described above (inset in Figure 5B), the value of the basal level of Cu2+ in rat brain microdialysates was determined to be 1.91 ± 0.40 µM (n = 3) (Table S1), which is almost in accordance with the reported values.15c,15d,15e These properties substantially demonstrate that the fluororimetric assay developed in this study by switching on and off the luminescence of AMP-Tb by introducing SSA as the sensitizer and subsequent addition of Cu2+ 11



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would offer a straightforward and reliable approach to direct sensing of Cu2+ in the cerebral system.

CONCLUSIONS In summary, we have developed a novel strategy for direct, selective, and sensitive sensing of Cu2+ in rat brain based on an “OFF-ON-OFF” fluorescent sensor combined with in vivo microdialysis. In our strategy, one kind of lanthanide coordination polymer nanoparticles (Ln-CPN), AMP-Tb, was used as the fluorescent sensing platform. By the introduction of SSA as the sensitizer and the chelator toward Cu2+, we have successfully modulated the “ON” or “OFF” state of the fluorescence of AMP-Tb for Cu2+ sensing because SSA can switch on the fluorescence by antenna effect while based on the specific molecular recognition with SSA, Cu2+ can efficiently suppress such effect to switch off the fluorescence. To the best of our knowledge, this is the first report on the direct fluorescent detection of Cu2+ in rat brain, which is advantageous in simple fabrication, low technical demands, and good sensitivity and selectivity compared with the existing fluorescent sensors for Cu2+ and could thus be beneficial to simple monitoring of Cu2+ and its relevant physiological and pathological events in brain. Considering the compositional diversity and structural flexibility, Ln-CPN-based fluorescent sensors as demonstrated in this study could potentially be further employed for the detection of other kinds of metal ions in the central nervous system, and could thereby open up the pathway to a new sensing platform to understand brain chemistry.

ASSOCIATED CONTENT Supporting Information Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mails: Pengcheng Huang, [email protected]; Fangying Wu, [email protected]. Notes 12


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The authors declare no competing financial interest.

ACKNOWLEDGMENTS We acknowledge the financial support of Natural Science Foundation of China (No. 201365014), Jiangxi Province Science and Technology University Ground Plan project (KJLD No. 14007), Jiangxi Province Natural Science Foundation (JXNSF No. 20132BAB203011), and Doctoral Start-up Funding of Nanchang University. We also greatly appreciate Qin Jiang for preparing microdialysate.

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Scheme 1. Schematic Illustration of (A) the Mechanism of Switching On and OFF the Luminescence of AMP-Tb and (B) Its Application in the Fluorometric Sensing of Cerebral Cu2+ in Rat Brain Microdialysates

A

NH2 N N

O O

P

O

O

N

Tb3+

SSA

N

O

OH

OH

AMP OFF

ON

Cu2+

OFF

SSA

B Pump

Microdialysis

aCSF

Probe

17



a b c d

I (a. u.)

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

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200

300

400

500

600

λ / nm

Figure 1. Excitation (left: a and c) and emission (right: b and d) spectra of AMP-Tb suspension in the absence and in the presence of SSA in HEPES buffer (0.1 M, pH 7.4).

18


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a

νas(PO3) νs(PO3)

ν(N7-C8)

b c d e

1800

1500

1200

Wavenumber / cm

900

-1

Figure 2. FT-IR spectra for (a) AMP, (b) AMP-Tb, (c) SSA/AMP-Tb, (d) SSA/AMP-Tb+Cu2+ , and (e) SSA. The y axis is presented in transmittance.

19



SSA AMP-Tb SSA/AMP-Tb SSA/AMP-Tb+Cu2+ SSA+Cu2+ SSA+Tb3+

Abs. (a. u.)

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

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250

300

350

Wavelength / nm Figure 3. UV-vis absorption spectra of the mixtures prepared by separate addition of SSA (black curve), SSA+Tb3+ (cyan curve), SSA+Cu2+ (green curve) in HEPES buffer (0.1 M, pH 7.4), and the suspension of AMP-Tb (red curve), SSA/AMP-Tb (blue curve), and SSA/AMP-Tb+Cu2+ (purple curve) in HEPES buffer (0.1 M, pH 7.4), respectively.

20


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40 SSA/AMP-Tb SSA/AMP-Tb+Cu2+

30 I (a. u.)

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


20

10

0

500

550

600

λ / nm

Figure 4. Emission spectra (λex = 328 nm) of SSA/AMP-Tb suspension and SSA/AMP-Tb suspension by adding Cu2+ (30 µM) in HEPES buffer (0.1 M, pH 7.4).

21



35

A

B

0

30

0.6

25

24 µM

20

(I0-I)/I0

I (a. u.)

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

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15

0.4

0.2

10 5 0 450

0.0 500

550

600

650

0

5

λ / nm

10

15

20

25

CCu2+ / µM

Figure 5. (A) Fluorescence responses of the aCSF suspension of SSA/AMP-Tb upon the addition of Cu2+ with the various concentrations from 0 to 24 µM. λex = 328 nm. (B) Plot of the ratio of the decrease value to the initial value for the fluorescence intensities of SSA/AMP-Tb at 550 nm ((I0-I)/I0) against the concentrations of Cu2+ ranging from 0 to 24 µM.

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0.8

0.5

B

A 0.4 0.3

I/I0

(I0-I)/I0

0.6

0.4

0.2 0.1

0.2 0.0

1

2

3

4

5

6

7

8

9 10 11 12 13

0.0

-0.1

0.5

1

2

3

0.8

C

4

5

5

6

6

7

8

9 10 11 12 13

D

0.4 0.3

I/I0

(I0-I)/I0

0.6

0.2 0.1 0.0

0.4

0.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0.0

-0.1

1

2

3

4

7

8

9 10 11 12 13 14 15 16

0.8

0.5

F

E 0.4

0.6

0.3

I/I0

(I0-I)/I0

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


0.4

0.2

0.2

0.1 0.0

1

2

3

4

5

6

7

0.0

8

1

2

3

4

5

6

7

Figure 6. (A, C, E) Selectivity and (B, D, F) competition experiments for SSA/AMP-Tb toward different physiological interferences. (A) Selectivity of metal ions against Cu2+ : 1 Cu2+, 2 K+, 3 Na+, 4 Mg2+, 5 Ca2+, 6 Zn2+, 7 Al3+, 8 Co2+, 9 Fe3+, 10 Fe2+, 11 Ni2+, 12 Ag+, and 13 Cu+. Concentration of metal ions is 30 µM expect of K+, Na+, Mg2+, Ca2+ which are 3 mM. (B) Competition experiments upon the addition of Cu2+ (30 µM) with the coexistence of the interferences: 1 none, 2 K+, 3 Na+, 4 Mg2+, 5 Ca2+, 6 Zn2+, 7 Al3+, 8 Co2+, 9 Fe3+, 10 Fe2+, 11 Ni2+, 23


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

12 Ag+, and 13 Cu+. Concentration of metal ions is 30 µM expect of K+, Na+, Mg2+, Ca2+ which are 1.5 mM. (C) Selectivity of amino acids against Cu2+: 1 Cu2+, 2 Glycine, 3 Alanine, 4 Phenylalanine, 5 Methionine, 6 Valine, 7 Histidine, 8 Isoleucine, 9 Arginine, 10 Aspartic acid, 11 Proline, 12 Lysine, 13 Serine, and 14 Leucine, 15 Tyrosinase, and 16 Cu-Zn SOD. All the concentration of amino acids is 45 µM, and those of tyrosinase and Cu-Zn SOD are both 30 µg/mL. (D) Competition experiment upon the addition of Cu2+ (30 µM) with the coexistence of the interferences: 1 none, 2 Glycine, 3 Alanine, 4 Phenylalanine, 5 Methionine, 6 Valine, 7 Histidine, 8 Isoleucine, 9 Arginine, 10 Aspartic acid, 11 Proline, 12 Lysine, 13 Serine, 14 Leucine, 15 Tyrosinase, and 16 Cu-Zn SOD. All the concentration of amino acids is 45 µM except of histidine which is 22.5 µM, and those of tyrosinase and Cu-Zn SOD are both 30 µg/mL. (E) Selectivity of biological species against Cu2+: 1 Cu2+ (30 µM), 2 DA (30 µM), 3 glucose (15 mM), 4 AA (30 µM), 5 DOPAC (45 µM), 6 H2O2 (0.5 µM), 7 Lactate (1.5 mM), and 8 5-HT (45 µM). (F) Competition experiment upon the addition of Cu2+ (30 µM) with the coexistence of the interferences: 1 none, 2 DA (30 µM), 3 glucose (15 mM), 4 AA (30 µM), 5 DOPAC (22.5 µM), 6 H2O2 (0.5 µM), 7 Lactate (0.75 mM), and 8 5-HT (22.5 µM). All the concentrations are the final concentrations in the resulting mixtures.

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50 AMP-Tb SSA/AMP-Tb SSA/AMP-Tb+microdialysates

40

I (a. u.)

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


30 20 10 0

500

550

600

λ / nm Figure 7. Fluorescence responses of the aCSF suspension of AMP-Tb (black curve), with the addition of SSA (red curve), and with the addition of rat brain microdialysates (blue curve). λex = 328 nm.

25



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For TOC only:

SSA

AMP-Tb

SSA/AMP-Tb

SSA-Cu/AMP-Tb

OFF

ON

OFF

26


Target-Triggered Switching on and off the Luminescence of

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