DOI: 10.1002/chem.201500909

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& Synthesis Design

Luminescence-Functionalized Metal–Organic Frameworks Based on a Ruthenium(II) Complex: A Signal Amplification Strategy for Electrogenerated Chemiluminescence Immunosensors Cheng-Yi Xiong, Hai-Jun Wang, Wen-Bin Liang, Ya-Li Yuan, Ruo Yuan,* and Ya-Qin Chai*[a] Abstract: Novel luminescence-functionalized metal–organic frameworks (MOFs) with superior electrogenerated chemiluminescence (ECL) properties were synthesized based on zinc ions as the central ions and tris(4,4’-dicarboxylicacid2,2’-bipyridyl)ruthenium(II) dichloride ([Ru(dcbpy)3]2 + ) as the ligands. For potential applications, the synthesized MOFs were used to fabricate a “signal-on” ECL immunosensor for the detection of N-terminal pro-B-type natriuretic peptide (NT-proBNP). As expected, enhanced ECL signals were obtained through a simple fabrication strategy because

Introduction Metal–organic frameworks (MOFs) are novel microporous materials, which have many superior properties, such as large internal surface areas, tunable size, and easy modification, synthesized by assembling metal ions with organic ligands in appropriate solvents.[1–3] These excellent characteristics allow potential applications in gas storage,[4, 5] chemical separation,[6, 7] fluorescent sensors,[8–10] catalysis,[11–13] and drug delivery.[14–16] In addition, electrogenerated chemiluminescence (ECL) technology has received much attention and become a powerful detection method due to its high sensitivity, low background signal, and rapid response.[17–23] However, the application of MOFs in the ECL system has rarely been reported because small organic molecules as traditional ligands of MOFs lack good redox activity, favorable ability of electron transfer, and good ECL activity; these properties are necessary for ECL. Thus, the use of a metallic complex with good luminescence performance as a ligand has become a target in the synthesis of MOFs.[24–26] Moreover, the use of a metallic complex as a ligand may endow MOFs with high electron-transfer capacities, good charge selectivity, and satisfactory electrochemical stability.[27–29]

[a] C.-Y. Xiong, Dr. H.-J. Wang, W.-B. Liang, Y.-L. Yuan, Prof. Dr. R. Yuan, Prof. Dr. Y.-Q. Chai Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education College of Chemistry and Chemical Engineering Southwest University, Chongqing 400715 (P.R. China) E-mail: [email protected] [email protected] Chem. Eur. J. 2015, 21, 9825 – 9832

luminescence-functionalized MOFs not only effectively increased the loading of [Ru(dcbpy)3]2 + , but also served as a loading platform in the ECL immunosensor. Furthermore, the proposed ECL immunosensor had a wide linear range from 5 pg mL¢1 to 25 ng mL¢1 and a relatively low detection limit of 1.67 pg mL¢1 (signal/noise = 3). The results indicated that luminescence-functionalized MOFs provided a novel amplification strategy in the construction of ECL immunosensors and might have great prospects for application in bioanalysis.

Tris(4,4’-dicarboxylicacid-2,2’-bipyridyl)ruthenium(II) dichloride ([Ru(dcbpy)3]2 + ), which is known for its excellent ECL activity, has six carboxyl groups in its molecule structure that can coordinate to Zn2 + ions; thus it is a promising ligand of Zn2 + for the synthesis of luminescence-functionalized MOFs.[30] The unique structure of the MOFs increased the loading of [Ru(dcbpy)3]2 + effectively and provided many active sites, so that the proposed MOFs were easy to modify and functionalize; thus it might have a broad range of applications in advanced scientific and technological fields. The coreactants, which are important intermediates, are of positive significance in ECL signal amplification.[31–35] However, traditional coreactants of the ruthenium complex have volatile properties and biological toxicity, such as triethylamine and tripropylamine, are often added to the detection solution, which may increase measurement errors. According to our previous reports, polyethylenimine (PEI), which is a superior coreactant of a ruthenium complex, can decrease measurement error while it is immobilized on a luminophore.[36–38] Thus, PEI was modified on the surface of proposed luminescence-functionalized MOFs to enhance the ECL signal. Inspired by all of these perspectives, novel luminescencefunctionalized MOFs were synthesized based on [Ru(dcbpy)3]2 + as a ligand and Zn2 + as the central ion (Scheme 1) and applied to the construction of an immunosensor for the sensitive detection of N-terminal pro-B-type natriuretic peptide (NTproBNP), which is a diagnostic serum marker for heart failure.[39–41] First, Nafion was modified on the surface of MOFs. Through electrostatic adsorption with Nafion, PEI was modified on the MOFs. The AuNPs were decorated onto PEI through Au¢N bonds to label the detection antibodies (Ab2) for the preparation of Ab2 bioconjugates (Ab2/AuNPs-PEI/Nafion/lumi-

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Full Paper signal. In this fabrication, PEI as an excellent coreactant, could greatly increase the ECL intensity of the proposed MOFs and immobilize AuNPs to label Ab2. Furthermore, AuNPs could promote electron transport, which might enhance the ECL signal. In particular, the porous structure of MOFs with a large specific area increases the loading of luminophore and coreactant, which can further effectively enhance the ECL response of the proposed immunosensor. Therefore, herein, we provide a feasible method for the use of a metallic complex instead of small organic molecules in the synthesis of MOFs and extend their application to bioanalysis.

Results and Discussion Characteristics of luminescence-functionalized MOFs

Scheme 1. A) and B) The coordination modes of [Ru(dcbpy)3]2 + and Zn2 + . C) The potential 3D structure of the proposed luminescence-functionalized MOFs.

nescence-functionalized MOFs; Scheme 2). At the same time, platinum nanoparticles (PtNPs) were electrodeposited on the glass carbon electrode (GCE) for the abundant modification of capture antibodies to recognize NT-proBNP specifically (Ab1). Finally, through sandwiched immunoreactions with NT-proBNP, the obtained Ab2 bioconjugates could be modified on the electrode surface and provide a significantly enhanced ECL

The morphology of luminescence-functionalized MOFs was characterized by SEM. As shown in Figure 1, the luminescencefunctionalized MOFs were hexangular nanoplates with a diameter of 150 to 250 nm. To characterize the ECL properties of the luminescence-functionalized MOFs, the ECL spectrum was compared with that of pure [Ru(dcbpy)3]2 + . The ECL spectrum was obtained by collecting the maximum ECL intensity during the cyclic potential sweep with a series of optical filters by using a model MPI-E II electrochemiluminescence analyzer. The potential range of luminescence-functionalized MOFs was from 0.2 to 1.5 V and the potential range of [Ru(bpy)3]2 + (bpy = 2,2’-bipyridine) was from 0.2 to 1.25 V at a scan rate of 0.1 V s¢1. As shown in Figure 2 A, the ECL emission peak and half-peak width of the luminescence-functionalized MOFs were l = 681 and 131 nm, respectively, whereas the ECL emission peak and half-peak width of pure [Ru(dcbpy)3]2 + were l = 638 and 125 nm. Moreover, the intensity– wavelength curve was based on Equation (1).



Nh lAt

ð1Þ

in which h, l, A, and t represent the Planck constant, wavelength, unit area, and unit time, respectively, for N photons arriving in A within time t. Through integral calculations for the intensity– wavelength curve from l = 550 800 P to 800 nm, I=Itotal of Rul¼550

MOFs was 90.40 %, whereas 825 P I=Itotal of pure [Ru(dcbpy)3]2 +

l¼550

Scheme 2. Illustration of A) the preparation of Ab2 bioconjugate and B) the fabrication of the proposed ECL immunosensor. Chem. Eur. J. 2015, 21, 9825 – 9832

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was 90.38 % through integral calculations for the intensity–wave-

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Figure 1. SEM image of luminescence-functionalized MOFs.

length curve from l = 550 to 825 nm. These results indicated that the redshift of the ECL spectrum was caused by the changing energy of some photons; this demonstrated that [Ru(dcbpy)3]2 + was the ligand distributed inside of the MOFs. To further investigate the ECL properties of luminescencefunctionalized MOFs, the real-time transformation of wavelength and ECL intensity was obtained by changing the excitation potential from 0.2 to 2.5 V. As shown in Figure 2 B, there was no clear ECL emission when the potential was from 0.2 to 1.2 V. A strong ECL emission occurred when the potential changed to 1.3 V and an ECL emission peak was observed when the potential was 1.5 V. Furthermore, the ECL intensity decreased with increasing potential from 1.5 to 2.5 V. Meanwhile, the ECL emission maximum wavelength was l … 680 nm and also appeared at 1.5 V, which was larger than the maximum excitation potential of pure [Ru(dcbpy)3]2 + (1.2 V).[30] These results indicated that most of the [Ru(dcbpy)3]2 + ligand was distributed inside of the MOFs and the ECL emission of luminescence-functionalized MOFs was not caused by single molecules of [Ru(dcbpy)3]2 + , but by [Ru(dcbpy)3]2 + –MOFs, which might need more energy to turn into high-energy states. To confirm that the changing energy of some photons caused the redshift in the ECL spectrum, FL spectra of luminescence-functionalized MOFs and [Ru(dcbpy)3]2 + were recorded (Figure 2 C and D). As the results revealed, the maximum excited wavelength and maximum emission wavelength of pure [Ru(dcbpy)3]2 + were at l = 550 and 609 nm, whereas the maximum excited wavelength and maximum emission wavelength of the Ru-MOFs were at l = 565 and 620 nm. The redshift in the FL spectrum demonstrated that the energy of some photons really changed and [Ru(dcbpy)3]2 + was the ligand distributed inside of the MOFs. To investigate the structure of luminescence-functionalized MOFs, XRD was used. As shown in Figure 3 A, the XRD patterns of luminescence-functionalized MOFs were similar to MOFs reported previously.[28] The XRD peaks at 12 and 208 shifted very little, whereas the three XRD peaks from 15 to 208 shifted to a greater extent than that reported in the literature. Moreover, some known peaks were missing and some unknown peaks appeared in the proposed XRD patterns. These experimental results implied that luminescence-functionalized MOFs had Chem. Eur. J. 2015, 21, 9825 – 9832

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Figure 2. A) ECL spectra of luminescence-functionalized MOFs and [Ru(dcbpy)3]2 + . B) Intensity–wavelength–potential curve of luminescencefunctionalized MOFs. C) Fluorescence (FL) spectrum of luminescencefunctionalized MOFs. D) FL spectrum of [Ru(bpy)3]2 + .

complicated crystal structures that might be different from known materials and were very difficult to determine by crystallographic structural analysis with existing technology. Simultaneously, selected-area electron diffraction (SAED) was performed to investigate the structure of luminescence-functionalized MOFs. The results in Figure 3 B also demonstrated that the

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Figure 3. A) XRD patterns and B) electron diffraction patterns of luminescence-functionalized MOFs.

luminescence-functionalized MOFs had complicated crystal structures that were hard to analyze with existing technology. To the best of our knowledge, the RuII ion formed d2sp3hybrid orbitals that constructed a regular octahedral structure in [Ru(dcbpy)3]2 + . Therefore, every carboxylate group was at each corner of the regular octahedron in space and could coordinate to one zinc ion. The zinc center adopts a tetrahedral geometry by coordinating to four oxygen atoms of four carboxylate groups belonging to four different [Ru(dcbpy)3]2 + molecules. These units repeated in space to form a potential 3D structure of the proposed luminescence-functionalized MOFs (Scheme 1 A–C). Elemental analysis of AuNPs-PEI/Nafion/luminescencefunctionalized MOFs To prove the successful synthesis of the AuNPs-PEI/Nafion/ luminescence-functionalized MOFs, X-ray photoelectron spectroscopy (XPS) was utilized for elemental analysis. As anticipated, the characteristic peaks for Zn 2 p, O 1 s, N 1 s, C 1 s, and Ru 3 d could be directly observed in Figure 4 A. The double peaks at 1045.1 and 1022 eV represented the XPS signature of Zn 2 p for the resulting Zn2 + ion (Figure 4 B). The peaks at 288.6, 281.2, and 84.2 eV were the XPS signatures of Ru 3 d and Au 4 f. (Figure 4 C and D). All of these results implied that the AuNPs-PEI/Nafion/luminescence-functionalized MOFs was synthesized.

Figure 4. XPS analysis for A) the full region of XPS for AuNPs-PEI/Nafion/ luminescence-functionalized MOFs, B) the Zn 2 p region, C) the Ru 3 d region, and D) Au 4 f region.

Characteristics of immunosensor fabrication The fabrication process of the proposed immunosensor was characterized by cyclic voltammetry measurements in 0.1 m phosphate buffer solution (PBS; pH 7.4) containing 5.0 mm [Fe(CN)6]3¢/4¢ (acting as a redox probe) and 0.1 m KCl. As shown in Figure 5 A, the bare GCE showed a pair of well-defined redox peaks of [Fe(CN)6]3¢/4¢ (curve a). When PtNPs were deposited on the GCE, the redox peak currents increased remarkably (Figure 5 A, curve b). This might be due to PtNPs having superior conductivity and a large specific area, which can promote electron transfer. Then, decreases in redox current were observed after the addition of Ab1, BSA, and NTproBNP (Figure 5 A, curves c, d, and e). One possible reason might be that the Ab1, BSA, and NT-proBNP protein layers on Chem. Eur. J. 2015, 21, 9825 – 9832

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the electrode hindered electron transfer. All of these observations confirmed that the proposed immunosensor was fabricated successfully. Furthermore, EIS was also performed for different modified electrodes in 5 mm [Fe(CN)6]3¢/4¢ to evaluate interfacial changes in the fabrication process of the ECL immunosensor. As shown in Figure 5 B, the bare GCE showed a semicircle and Ret = 46.69 W (curve a). When the PtNPs were deposited on the GCE, the Ret decreased significantly (Figure 5 B, curve b, Ret = 15.66 W). This might be due to superior conductivity and a large specific area of the PtNPs. Then, clear increases in Ret were observed when Ab1, BSA, and NT-proBNP were incubated

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Figure 6. ECL profiles of the proposed ECL immunosensor in different concentrations of NT-proBNP: a) 0.005, b) 0.01, c) 0.05, d) 0.1, e) 0.5, f) 1, g) 13.4, and h) 25 ng mL¢1. The inset shows the logarithmic calibration curve for NT-proBNP in a 10 % aqueous solution of ethanol containing 0.1 m Zn(NO3)2.

Stability, selectivity, and reproducibility of the immunosensor

Figure 5. A) Cyclic voltammograms at a) bare GCE, b) PtNPs/GCE, c) Ab1/ PtNPs/GCE, d) bovine serum albumin (BSA)/Ab1/AuNPs/GCE, and e) NTproBNP/BSA/Ab1/PtNPs/GCE in 5.0 mm [Fe(CN)6]3¢/4¢ containing 0.1 m KCl by scanning the potential from ¢0.2 to 0.6 V at a scan rate of 0.1 V s¢1. B) The electrochemical impedance spectroscopy (EIS) images for a) bare GCE, b) PtNPs/GCE, c) Ab1/PtNPs/GCE, d) BSA/Ab1/AuNPs/GCE, and e) NT-proBNP/ BSA/Ab1/PtNPs/GCE in 0.1 m KCl containing 5 mm (1:1) [Fe(CN)6]3¢/4¢.

on the electrode (Figure 5 B, curves c, d, and e, Ret = 28.82, 47.75, and 91.18 W, respectively). One potential reason might be that the Ab1, BSA, and NT-proBNP significantly hinder electron transfer. Analytical performance of ECL immunosensors The dynamic linear range of the proposed immunosensor was evaluated by incubating different concentrations of NT-proBNP. The ECL intensity increased with increasing NT-proBNP concentration from 5 pg mL¢1 to 25 ng mL¢1 (Figure 6). The limit of detection (LOD) was 1.67 pg mL¢1 (signal/noise (S/N) = 3). The regression equation was I = 1035.6(œ 13.1)lgcNT-proBNP + 3122.3(œ 21.2) with a correlation coefficient of R2 = 0.9988 (in which I is the ECL intensity and cNT-proBNP is the concentration of NT-proBNP). The results in Table 1 allow those obtained with the proposed immunosensor to be compared with those previously reported. The results indicated that the proposed immunosensor could be an appropriate tool for the detection of NT-proBNP.

The stability was monitored by consecutive cyclic potential scanning for over 1000 s. From the results shown in Figure 7 A, there were no significant changes in the ECL response. The satisfactory stability might contribute to the excellent electrochemical properties of the luminescence-functionalized MOFs. The selectivity and specificity were evaluated with the assistance of some possible interfering substances, such as CEA, AFP, and PSA (Figure 7 B). Compared with the blank sample, there were few changes to the ECL response when the immunosensors were incubated with 5 ng mL¢1 CEA, 5 ng mL¢1 AFP, and 5 ng mL¢1 PSA. Compared with the ECL response obtained from the interfering substances, no clear changes were observed when the proposed immunosensor was incubated with 0.05 ng mL¢1 NT-proBNP. These results showed that the proposed immunosensor had good selectivity for the detection of NT-proBNP. The relative standard deviations (RSDs; of the ECL response) of intra- and interassays were utilized to assess the reproducibility of the proposed immunosensor. Neither of the RSDs were more than 5 %, which illustrated that the proposed immunosensor had acceptable reproducibility. ECL characterization of the amplified strategy

To amplify the effect of PtNPs in the sensing interface modified layer, two different immunosensors were constructed for comparison. First, AuNPs were replaced with PtNPs for electrodeposition on the GCE for the conTable 1. Comparison of our research with other methods for NT-proBNP detection. struction of the sensing interDetection limit [pg mL¢1] Refs. Measurement protocol Linear range [ng mL¢1] face. With the same detection microfluidic immunoassay 0.005–4 3 [39] procedure, the contrast between electrochemical immunoassay 0.02–100 6 [40] the two different immunosenelectrochemiluminescence 0.005–25 1.67 this work sors is revealed in Figure 8 A.

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Full Paper immunosensors with two bioconjugates (Ab2/AuNPs-PEI/ Nafion/luminescence-functionalized MOFs and Ab2/AuNPs-PEI/ Nafion/[Ru(dcbpy)3]2 + ) were revealed Figure 8 B. The ECL signal of the immunosensor with Ab2/AuNPs-PEI/Nafion/luminescence-functionalized MOFs (Figure 8 B, curve c) was higher than that for the immunosensor with Ab2/AuNPs-PEI/Nafion/ [Ru(dcbpy)3]2 + (Figure 8 B, curve d). These results imply that luminescence-functionalized MOFs could increase the loading of [Ru(dcbpy)3]2 + to amplify the ECL intensity in the proposed immunosensor. Analysis of human serum samples Recovery experiments were explored in human serum to assess the feasibility of the proposed ECL immunosensor through the standard addition method. Preliminary results showed that the recovery was from 96.8 to 106.3 % (Table 2). This acceptable recovery implied that the proposed ECL immunosensor could be applied to real detection of NT-proBNP.

Table 2. Preliminary analysis results of real samples. Figure 7. A) The ECL response of the proposed ECL immunosensor incubated with 0.1 ng mL¢1 NT-proBNP under continuous cyclic potential scans for over 1000 s. B) The ECL response of the proposed ECL immunosensor incubated with a) blank; b) carcinoembryonic antigen (CEA; 5 ng mL¢1); c) a-fetoprotein (AFP; 5 ng mL¢1); d) prostate specific antigen (PSA; 5 ng mL¢1); e) NT-proBNP (0.05 ng mL¢1); f) a mixture containing CEA (5 ng mL¢1) and NT-proBNP (0.05 ng mL¢1); g) a mixture containing CEA (5 ng mL¢1), AFP (5 ng mL¢1), PSA (5 ng mL¢1), and NT-proBNP (0.05 ng mL¢1). Scanning potential from 0.2 to 1.5 V at a scan rate of 100 mV s¢1.

The ECL signal of the immunosensor with PtNPs (Figure 8 A, curve a) was significantly higher than that of the immunosensor with AuNPs (Figure 8 A, curve b). This result indicated that PtNPs had a greater amplification effect than AuNPs. Furthermore, the amplification effect of luminescence-functionalized MOFs on the ECL intensity was also investigated. Thus, [Ru(dcbpy)3]2 + was replaced with luminescence-functionalized MOFs to prepare the Ab2 bioconjugate with the immunosensors BSA/Ab1/PtNPs/GCE. After performing sandwiched immunoreactions, the contrasting results from the two www.chemeurj.org

Added [ng mL¢1]

Found [ng mL¢1]

Recovery [%]

1 2 3 4

13.4 1.0 0.5 0.1

12.97 1.06 0.49 0.103

96.8 106.3 99.2 103.0

Conclusion

Figure 8. A) ECL profiles of immunosensors electrodeposited with PtNPs (a) and AuNPs (b). B) ECL profiles of immunosensors with c) Ab2/AuNPs-PEI/ Nafion/luminescence-functionalized MOFs, and d) Ab2/AuNPs-PEI/Nafion/ [Ru(dcbpy)3]2 + . The immunosensors were all incubated with 0.5 ng mL¢1 NT-proBNP.

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Sample number

A novel luminescence-functionalized MOFs with excellent ECL performance was prepared by using [Ru(dcbpy)3]2 + as a ligand and Zn2 + as the central ion. The luminescence-functionalized MOFs were utilized to construct a “signal on” ECL immunosensor for the detection of NT-proBNP. The results implied that the proposed ECL immunosensor had good selectivity, satisfactory stability, and a wide linear range for the detection of NTproBNP. Thus, luminescence-functionalized MOFs were demonstrated to have a high specific surface area, superior redox activity, good electrochemical stability, and strong ECL emission. Moreover, adopting a metallic complex instead of a small organic molecule as the ligand proved to be a promising way to synthesize functionalized MOFs. In addition, luminescencefunctionalized MOFs as a combination of ECL and MOFs provided a novel signal amplification strategy in ECL technology, and thus, might have wide applications in ECL immunoassays, photoelectrochemistry, surface-enhanced Raman scattering, biological imaging, and so on.

Experimental Section Reagents and materials NT-proBNP antibodies and antigens were obtained from Southwest hospital (Chongqing, P.R. China). Standard solutions of AFP, PSA, and CEA were bought from Biocell (Zhengzhou, P.R. China).

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Full Paper [Ru(dcbpy)3]2 + was from Suna Tech Inc. (Suzhou, P.R. China). The reagents PEI (50 %), HAuCl4·4 H2O, H2PtCl6·6 H2O, Nafion (5 %), BSA (96–99 %), and sodium citrate were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Zn(NO3)2, EtOH, and n-propanol (NPA) were purchased from Chongqing Chuandong Chemical (Group) Co., Ltd. PBS at pH 7.4 was prepared by mixing standard stock solutions of 0.1 m K2HPO4, 0.1 m NaH2PO4, and 0.1 m KCl and adjusting the pH with 0.1 m HCl or NaOH, then diluting it with ultrapure water. AuNPs were prepared by reducing gold chloride with citric acid according to a procedure previously reported in the literature.[42] The average diameter of the AuNPs was about 15–20 nm, as characterized by SEM (data not shown). All chemicals were of analytical grade and used without further purification. All solutions were prepared with ultrapure water and stored in the refrigerator (4 8C).

Apparatus The ECL intensity was monitored by using a model MPI-A or MPI-E II electrochemiluminescence analyzer (Xi’An Remax Electronic Science & Technology Co. Ltd., Xi’An, P.R. China). The voltage of the photomultiplier tube (PMT) was set at 800 V. The ECL spectrum was obtained by collecting the maximum ECL intensity during the cyclic potential sweep with a series of optical filters and a spectrometer (Avantes Inc., USA). The scan rate was 0.1 V s¢1 for ECL detection. Cyclic voltammetry measurements were performed on a CHI 660D electrochemistry workstation (Shanghai CH Instruments, P.R. China). The fluorescence measurements were performed on a RF-5301PC spectrophotometer (Shimadzu, Tokyo, Japan) at room temperature with a 150 W xenon lamp (Ushio Inc., Japan) as the excitation source. EIS measurements were performed with a Model IM6ex spectrometer (ZAHNER Elektrick Co., Germany). XRD was performed with an X-ray diffractometer (Ultima IV, Rigaku, Japan). A three-electrode electrochemical cell was composed of a modified GCE (F = 4 mm) as the working electrode, Ag/ AgCl (sat. KCl) as the reference electrode, and a platinum wire as the auxiliary electrode. The GCE, platinum wire, and the AgCl electrode were obtained from Tianjin Aidahengsheng Technology Co. Ltd., P.R. China. The morphologies of different nanomaterials were characterized by SEM (SEM, S-4800, Hitachi, Tokyo, Japan) at an acceleration voltage of 5.0 kV. XPS characterization was carried out by using a VG Scientific ESCALAB 250 spectrometer (Thermoelectricity Instruments, USA) with AlKa X-rays (1486.6 eV) as the light source.

Synthesis of luminescence-functionalized MOFs The luminescence-functionalized MOFs were synthesized by using the following method. First, [Ru(dcbpy)3]2 + (18 mg) was dissolved in NPA/H2O (10 mL; 3:1 v/v). Then, Zn(NO3)2 (54 mg) was dissolved in the prepared solution and sonicated for 1 h. Subsequently, the prepared solution was allowed to react at room temperature for 24 h without stirring. The resulting complex was obtained by centrifugation (8000 rpm, 4 8C, 5 min) and was washed three times with ultrapure water. Finally, the product was dispersed in ultrapure water (2 mL).

Preparation of Ab2/AuNPs-PEI/Nafion/luminescence-functionalized MOFs (Ab2 bioconjugates) The Ab2/AuNPs-PEI/Nafion/luminescence-functionalized MOFs were synthesized as follows (Scheme 2 A). First, Nafion solution (200 mL; 5 %, w/w) was added to the as-prepared luminescence-functionalized MOFs solution (1 mL) and stirred for 2 h. Then, PEI (200 mL; Chem. Eur. J. 2015, 21, 9825 – 9832

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1 %, w/w) was added to the solution, which was stirred for about 6 h. Subsequently, the solution of AuNPs (400 mL) was added to the mixture and stirred for another 12 h. Next, Ab2 (200 mL) was added to the as-prepared AuNPs-PEI/Nafion/luminescence-functionalized MOFs solution and incubated at 4 8C overnight. BSA solution (100 mL; 1 %, w/w) was then added to the Ab2/AuNPs-PEI/ Nafion/luminescence-functionalized MOFs solution to block nonspecific sites. The complex was obtained by centrifugation (8000 rpm, 4 8C, 10 min) and was stored at 4 8C before use.

Fabrication of the ECL immunosensor As shown in Scheme 2 B, the ECL immunosensor was fabricated according to the following steps. First, the GCE was polished with 0.3 and 0.05 mm alumina slurry. After being washed with ultrapure water, the electrode was sonicated in ethanol and ultrapure water. Then, it was dried with nitrogen at room temperature. Subsequently, the GCE was immersed in a 1 % solution of H2PtCl4 (2 mL) for electrochemical deposition. The deposition kept for 30 s under a constant potential of ¢0.05 V to obtain PtNP-film modified electrode. After these steps, capture antibodies (Ab1; 16 mL) were incubated on the modified GCE at 4 8C overnight. Then, the GCE was put in BSA solution (16 mL; 1 %, w/w) for 40 min to block the nonspecific binding sites. NT-proBNP was dropped onto the electrode and incubated for 1 h at room temperature. Finally, the obtained electrode was incubated with Ab2/AuNPs-PEI/Nafion/luminescencefunctionalized MOFs (16 mL) for 2 h. Ultimately, the obtained ECL immunosensor was stored at 4 8C before use. After every modified step, ultrapure water was utilized to wash the GCE to remove excess reagents.

Acknowledgements This work was financially supported by the NNSF of China (51473136, 21275119), the China Postdoctoral Science Foundation (2014M550454), and the Fundamental Research Funds for the Central Universities (XDJK2014C138). Keywords: electrochemistry · luminescence · metal–organic frameworks · sensors · synthesis design [1] M. Eddaoudi, J. Kim, N. Rosi, D. Vodak, J. Wachter, M. O’Keeffe, O. M. Yaghi, Science 2002, 295, 469 – 471. [2] S. Li, F. Huo, Small 2014, 10, 4371 – 4378. [3] K. C. Wang, D. W. Feng, T. F. Liu, J. Su, S, Yuan, Y. P. Chen, M. Bosch, X. D. Zou, H. C. Zhou, J. Am. Chem. Soc. 2014, 136, 13983 – 13986. [4] J. G. Vitillo, L. Regli, S. Chavan, G. Ricchiardi, G. Spoto, P. D. C. Dietzel, S. Bordiga, A. Zecchina, J. Am. Chem. Soc. 2008, 130, 8386 – 8396. [5] B. B. Panella, M. Hirscher, H. Pìtter, U. Mìller, Adv. Funct. Mater. 2006, 16, 520 – 524. [6] A. Centrone, E. E. Santiso, T. A. Hatton, Small 2011, 7, 2356 – 2364. [7] J. W. Jun, M. M. Tong, B. K. Jung, Z. Hasan, C. L. Zhong, Chem. Eur. J. 2015, 21, 347 – 354. [8] X. M. Lin, G. M. Gao, L. Y. Zheng, Y. W. Chi, G. N. Chen, Anal. Chem. 2014, 86, 1223 – 1228. [9] B. Gole, A. K. Bar, P. S. Mukherjee, Chem. Eur. J. 2014, 20, 13321 – 13336. [10] B. Gole, A. K. Bar, P. S. Mukherjee, Chem. Eur. J. 2014, 20, 2276 – 2291. [11] Y. K. Hwang, D. Y. Hong, J. S. Chang, S. H. Jhung, Y. K. Seo, J. Kim, A. Vimont, M. Daturi, C. Serre, G. F¦rry, Angew. Chem. Int. Ed. 2008, 47, 4144 – 4148; Angew. Chem. 2008, 120, 4212 – 4216. [12] C. L. Cui, Y. Y. Liu, H. B. Xu, S. Z. Li, W. N. Zhang, P. Cui, F. W. Huo, Small 2014, 10, 3672 – 3676. [13] C. Huang, R. Ding, C. J. Song, J. J. Lu, L. Liu, X. Han, J. Wu, H. W. Hou, Y. T. Fan, Chem. Eur. J. 2014, 20, 16156 – 16163.

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Received: March 6, 2015 Published online on May 27, 2015

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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Luminescence-Functionalized Metal-Organic Frameworks Based on a Ruthenium(II) Complex: A Signal Amplification Strategy for Electrogenerated Chemiluminescence Immunosensors.

Novel luminescence-functionalized metal-organic frameworks (MOFs) with superior electrogenerated chemiluminescence (ECL) properties were synthesized b...
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