Quenching Electrochemiluminescence Immunosensor Based on

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Biological and Medical Applications of Materials and Interfaces

Quenching electrochemiluminescence immunosensor based on resonance energy transfer between ruthenium (II) complex incorporated in UiO-67 metal-organic framework and gold nanoparticles for insulin detection Guanhui Zhao, Yaoguang Wang, Xiaojian Li, Xue Dong, Huan Wang, Bin Du, Wei Cao, and Qin Wei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04786 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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ACS Applied Materials & Interfaces

Quenching electrochemiluminescence immunosensor based on resonance energy transfer between ruthenium (II) complex incorporated in UiO-67 metal-organic framework and gold nanoparticles for insulin detection

Guanhui Zhao, Yaoguang Wang, Xiaojian Li, Xue Dong, Huan Wang, Bin Du, Wei Cao*, Qin Wei*

Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China

* Corresponding author E-mail: [email protected] (W. Cao). E-mail: [email protected] (Q. Wei). Tel.: +86 53182767890 Fax: +86 53187161600

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ABSTRACT This work describes a sandwich-type electrochemiluminescence (ECL) strategy for insulin detection by using Ru(bpy)32+ as luminophore which was encapsulated in UiO-67 metal-organic framework (UiO-67/Ru(bpy)32+). Since UiO-67 possesses the characteristics of large specific surface area and porosity, more Ru(bpy)32+ could be loaded onto its surface and holes, thus greatly improving the electrochemiluminescence efficiency. Furthermore, the ECL resonance energy transfer (ECL-RET) could occur between UiO-67/Ru(bpy)32+ (ECL donor) and Au@SiO2 nanoparticles (ECL acceptor), resulting in conspicuously decreased of ECL response. The ECL spectrum of UiO67/Ru(bpy)32+ which exhibited strong ECL intensity has suitable spectral overlap with absorption spectrum of Au@SiO2, which further proved the occurrence of the ECLRET action. The ECL intensity decreased with the increase of the concentration of insulin. In addition, the sandwich-type ECL immunosensor was applied to insulin detection and the ECL decrease efficiency was found to be logarithmically related to the concentration of the insulin antigen in the range from 0.0025 ng mL-1 to 50 ng mL1

with the limit of detection of 0.001 ng mL-1. Meanwhile, this work provides an

important reference for the application of MOFs in ECL and ECL-RET study, and also exhibits potential capability in the detection of other hormone.

KEYWORDS: Electrochemiluminescence; Resonance energy transfer; UiO-67 metal-organic framework; Ru(bpy)32+; Gold nanoparticles

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INTRODUCTION Insulin plays a significant role in regulation of carbohydrate and fat metabolism. And the diabetes and related diseases would be triggered when the situation of glucose homeostasis and hormone release are abnormal.1-4 Diabetes also has become one of the most severe threats for human health and the patients verge to be younger.5 Hence, bringing out an ultrasensitive way to determine the level of insulin effectively is increasingly momentous in the diagnosis of diabetes of insulinoma and trauma. Electrochemiluminescence (ECL) has attracted wide attention and has been applied in multiple domains including bio-detecting and bio-sensing technology.6-11 However, finding suitable and stable luminescent materials is a major research problem in construction of ECL sensors.12 Among numerous illuminants, Ru(bpy)32+ is a prevalent luminophor because of its admirable ECL efficiency and favourable stability in aqueous solution and non-aqueous solvents.13 These properties generate preponderances such as demanding minimal amounts of costly or poisonous reagents and simplified experimental design considerations especially in the solid-phase immobilized applications.14 However, the ruthenium cations are water-soluble and it is necessary to search for excellent materials for the fixation of Ru(bpy)32+. For the past few years, nanoscale metal-organic frameworks (NMOFs) have drawn greatly attention as supporter of ECL luminophores due to their exceptional high porosity and well crystallinity.15-16 Therefore, the exceptionally stable Zr-based NMOFs of UiO-67 with the average diameter of 90 nm was investigated in this work, which was assembly by the organic 3



precursor of 2, 2-bipyridine-5, 5-dicarboxylic acid and the metal precursor of zirconium (IV) chloride.17 The prepared UiO-67 nanoparticles owned holes with size of 12 Å and 23 Å respectively on the surface, which were enough to package the luminophore of Ru(bpy)32+ cations (11.5 Å) via a facile solvothermal reaction. The Ru(bpy)32+ cations were both integrated into the pores of the MOF structure and strongly adsorbed on the surface of nanoparticles. After Ru(bpy)32+ were incorporated into the multihole UiO-67 nanoparticles, the luminescence lifetime and quantum yield of Ru(bpy)32+ were much enhanced giving the credit to the space limitation effect of MOF pores, which could greatly improve the luminous efficiency and intensity of the ECL immunosensor.18 Herein, on account of the superior ECL performance of the UiO-67/Ru(bpy)32+, an ultrasensitive sandwich-type immunosensor was prepared for insulin detection, which was based on the resonance energy transfer between luminophore Ru(bpy)32+ embedded inside the MOF NPs and Au nanoparticles loaded on silicon spheres. In this work, Au@SiO2 was a fascinating RET acceptor because of the large surface area, excellent biocompatibility, and the large density of gold nanoparticles loaded onto the silicon spheres which could enhance the quenching efficiency especially.19 In addition, The ECL spectrum of UiO-67/Ru(bpy)32+ which exhibited strong ECL intensity has suitable spectral overlap with absorption spectrum of Au@SiO2,20-21 which could give rise to the quench of donor emission through nonradiative energy dissipation in the acceptor in this RET system.22-23 Therefore, this sensing ECL-RET platform based UiO-67 metal-organic framework may open up a new avenue for selective and ultrasensitive detection of disease biomarkers. 4


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EXPERIMENTAL SECTION Preparation of Ab2-Au@SiO2. 5 mg of Au@SiO2 and 100 μL of 10 μg mL-1 Ab2 solution were mixed in 2 mL of PBS and oscillated for 24 h. The sediment was gathered by centrifugation and rinsed with PBS for once to remove the redundant Ab2 which have not participated in reaction. Afterwards, the sediment was re-dispersed in 1 mL of 1% BSA solution and shaken overnight at 4 °C to seal the non-specific active locus on the surface of Au@SiO2. The prepared Ab2-Au@SiO2 was gathered by centrifugation and rinsed by PBS for once before decentralized in 1 mL of PBS for succeeding usage. Assembled the Sandwich-type Immunosensor. The schematic diagram was shown in Scheme 1. Firstly, the GCE (4 mm) was polished to a mirror successively and washed thoroughly by ultrapure water and ethanol.24-25 6 μL of UiO-67/Ru(bpy)32+ solution was then dropped to the preprocessed GCE surface to act as ECL nanoemitters. Subsequently, 3 μL of miscible liquids of EDC (20 mg) and NHS (10 mg) was dropped to the surface of GCE at room temperature for 1 h to activate the carboxylic acid groups of UiO-67. Then 6 μL of Ab1 solution (1 μg mL-1) was spread on the pre-prepared electrode surface by incubating at 4 °C overnight and blocked the non-specific active sites with 4 μL of 0.5% BSA solution for 2 h. Following that, 6 μL of insulin antigen with different concentrations were modified to the electrode surface. In the end, 5 μL of Ab2- Au@SiO2 solution (3 mg mL-1) was incubated onto the above pretreated electrode for 1.5 h. It should be emphasized that each step should be rinsed with pH 7.5 PBS buffer solution to remove the unbound substance. The sandwich-type ECL immunosensor has been assembled and reserved at 4 °C for the recognition of insulin. 5



Scheme 1. A Schematic diagram for the construction of the ECL immunosensor.

RESULTS AND DISCUSSION Characterization of UiO-67, UiO-67/Ru(bpy)32+, SiO2, and Au@SiO2. The SEM photographs of UiO-67 and UiO-67/Ru(bpy)32+ were shown as Figure S1 and both UiO67 and UiO-67/Ru(bpy)32+ were homogeneous nanoparticles. Figure 1A and B were HRTEM images of UiO-67 composites and UiO-67 NPs before loading Ru(bpy)32+. It could be found that the diameter of UiO-67/Ru(bpy)32+ (~ 100 nm) composites was a bit larger than pure UiO-67 NPs which mean diameter was 90 nm with a wellproportioned size distribution. It was attributed that Ru(bpy)32+ was immobilized onto the surface of UiO-67 via electrostatic adsorption.18 Moreover, the results of HRTEM energy dispersive X-ray analysis (EDAX) mapping (Figure 1A′ and B′) and EDS (Figure S2) showed the uniform distribution of Zr, Ru, C, O, and N elements in the UiO-67/Ru(bpy)32+ composites and UiO-67 NPs clearly, which further confirmed that Ru(bpy)32+ cation was scattered among the surface and holes of UiO-67 NPs. In addition, the ECL behaviors of UiO-67 and UiO-67/Ru(bpy)32+ were also explored and 6


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shown in Figure S3 and it could be found that the individual UiO-67 had almost no ECL response while UiO-67/Ru(bpy)32+ had excellent ECL performance.

Figure 1. The HRTEM and EDAX mapping images of UiO-67/Ru(bpy)32+ (A), (A′, 1-5) and UiO-67 (B), (B′, 1-4). Since BPDC was used as organic-ligand to synthesize UiO-67 MOFs, the surface of UiO-67 had multitudinous carboxy groups which could link with Ab1. As shown in FT-IR spectrogram (Figure 2A), the absorption peak at 3425 cm-1 was stretching vibration of O-H and the absorption peaks at 1600 cm-1 and 1409 cm-1 were double bond stretching vibration of C=O and single bond vibration of O-H in carboxy groups. Compared with the FT-IR spectrogram of UiO-67 (a), the carboxy groups were still in good condition in UiO-67/Ru(bpy)32+ composites (b). Figure 2B showed the N2 adsorption-desorption isotherms and the pore size distribution (inset) of UiO-67 7



nanoparticles (a) and UiO-67/Ru(bpy)32+ composites (b) and it could be figured out the curves were belonged to type IV.26-27 For UiO-67 and after incorporating Ru(bpy)32+, the average pore sizes were 3.644 nm, 2.779 nm and the BET specific surface areas were 1040.152 m2 g-1, 1301.926 m2 g-1 which were higher than many other materials. It was worth emphasizing that the average pore size of UiO-67 was larger than UiO67/Ru(bpy)32+ while the specific surface areas of UiO-67 was smaller than UiO67/Ru(bpy)32+, which further proved that Ru(bpy)32+ was loaded onto the surface and pores of UiO-67 successfully.

Figure 2. The FT-IR characterization (A), BET adsorption-desorption curves and corresponding hole size schematic curves (inset) (B), XRD spectrum (C), zeta potential (D) of UiO-67 nanoparticles (a) and UiO-67/Ru(bpy)32+ composites (b). Figure 2C showed the XRD spectrogram of UiO-67 and UiO-67/Ru(bpy)32+, 8


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indicating that the crystallinity of UiO-67 was not destroyed after encapsulating Ru(bpy)32+. As Figure 2D shown, the surface potential of UiO-67 changed from -13.3 to -1.2 due to the positive Ru(bpy)32+ neutralized with a number of negative UiO-67 nanoparticles. As the carrier of Au NPs, the smaller size of SiO2 would made the occurrence of RET behavior between Au@SiO2 and Ru(bpy)32+ more beneficial. Hence, the morphology of SiO2 was investigated by TEM characterization. As Figure 3A shown, the morphology of SiO2 was uniformly spherical with an average diameter of 70 ~ 75 nm. Figure 3B showed Au nanoparticles were well distributed on the surface of SiO2 spheres.

Figure 3. The TEM photographs of SiO2 (A) and Au@SiO2 (B). Characterization of This Immunosensor. The establishment route of the proposed immunosensor were convinced by recording ECL signal (Figure 4A) and electrochemical impedance spectrum (EIS, Figure 4B) of different established phases. As ECL-potential responses shown (Figure 4A), the GCE/UiO-67/Ru(bpy)32+ showed a significantly strengthen ECL signal at 1.4 V which was decreased after Ab1 was 9



combined to the modified-GCE. The ECL response further decreased when BSA and insulin antigen were dropped onto the modified-GCE due that Ab1, BSA, and insulin antigen were molecular protein which could depress the contact between Ru(bpy)32+* and TPA•+/TPA•. The ECL intensity was down to minimum when Ab2-Au@SiO2 bedecked to the pretreated GCE, which because in the process of RET partial energy of Ru(bpy)32+* was transferred to the receptor. As EIS behavior shown in Figure 4B, with the construction of each step of the immunosensor, the impedance value increased with varying degrees. That was because each step all contained protein molecules, which would hamper the electrons transfer on the electrode surface.

Figure 4. The ECL behavior (A) and electrochemical impedance spectroscopy (B) of bare GCE (a), GCE/UiO-67/Ru(bpy)32+ (b), GCE/UiO-67/Ru(bpy)32+/Ab1 (c), GCE/UiO-67/Ru(bpy)32+/Ab1/BSA (d), GCE/UiO-67/Ru(bpy)32+/Ab1/BSA/antigeninsulin (50 ng mL-1) (e), GCE/UiO-67/Ru(bpy)32+/Ab1/BSA/insulin-antigen/Ab2Au@SiO2 (f). Mechanism of This Immunosensor. In this work, Ru(bpy)32+ luminophores were anodic electrochemiluminescence with the presence of tripropylamine as co-reaction reagent. There were two typical hypothetical mechanisms of Ru(bpy)32+-tripropylamine 10


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system.28 One supposed theory of producing Ru(bpy)32+* was shown in Route 1: at lower potential, TPA was directly oxidized to TPA•+ which to converse to TPA• by deprotonation. Then, the Ru(bpy)32+ cation was reduced to produce Ru(bpy)3+ by TPA• radical species and then Ru(bpy)3+ reacted with TPA•+ to generate Ru(bpy)32+*. Another course was shown as Route 2: at higher potential, Ru(bpy)32+ was translated to Ru(bpy)33+ by oxidization and then reacted with TPA• radical to produce Ru(bpy)32+*.

Figure 5. The characterization of UV-vis of Au@SiO2 nanoparticles (a) and The characterization of ECL emission of UiO-67/Ru(bpy)32+ composites (b). To investigate the descend of the ECL intensity caused by the Au@SiO2 nanoparticles on the GCE, the ECL emission spectrum of UiO-67/Ru(bpy)32+ (Figure 5) and the ultraviolet absorption spectrum of Au@SiO2 nanoparticles were explored and found that there was suitable overlaps between them. Therefore, the resonance energy transfer maybe occur between UiO-67/Ru(bpy)32+ and Au@SiO2 nanoparticles, resulting in the decline of the ECL signal. Route 1 TPA – e

TPA•+

(1) 11



TPA•+

TPA• + H+

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(2)

TPA• + Ru(bpy)32+

Ru(bpy)3+ + product

(3)

Ru(bpy)3+ + TPA•+

Ru(bpy)32+* + product

(4)

Route 2 Ru(bpy)32+ – e TPA – e TPA•+

Ru(bpy)33+

(1)

TPA•+

(2)

TPA• + H+

(3)

Ru(bpy)33+ + TPA•

Ru(bpy)32+* + product

(4)

Optimization of the Detection Conditions. The value of the pH in this measurement environment was vital to the accuracy of test results.29-32 Thus, the different pH effects were studied and pH 7.5 was chose as the optimal pH at which the ECL luminescence efficiency of UiO-67 metal-organic frameworks was greatest (Figure 6A). Since TPA acted as the co-reactant of Ru(bpy)32+, the anodic ECL response depended on the quantity of TPA. The results of the experiment were shown in Figure 6B, 10 mmol L-1 TPA was selected in the whole experiment process.33 Moreover, the concentratrtion of UiO-67/Ru(bpy)32+ composites would affect the ECL luminescence intensity and then influence the sensitivity of the immunosensor. Obtained from Figure 6C, the ECL intensity increased with the increasing concentration of UiO-67/Ru(bpy)32+ (until 1.5 mg mL-1). Hence, the concentration of 1.5 mg mL-1 of UiO-67/Ru(bpy)32+ was applied in the system. Because Au@SiO2 nanoparticles were the energy recipient in this ECL-RET system, the amount of Au@SiO2 nanoparticles could effect the degree of energy transmit. It could be obsered from Figure 6D that the 12


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ECL-RET efficiency tended to be gentle after 2.5 mg mL-1. Therefore, 2.5 mg mL-1 Au@SiO2 nanoparticles was chosen to incubate with Ab2.

Figure 6. The effects of pH (A), quantity of tripropylamine (B), concentration of UiO-67/Ru(bpy)32+ (C), concentration of Au@SiO2 (D) (the concentration of insulinantigen was 50 ng mL-1) on ECL intensity. Performance of the ECL Immunosensor., The ECL immunosensor analysis for insulin detection was investigated under optimal conditions. As shown in Figure 7A, the ECL response declined with the increase quantity of insulin incubated onto the GCE, and a satisfying linearly dependent between ECL signal and the logarithmic of the quantity of insulin (Figure 7B) in a broad detection scope of 0.0025 - 50 ng mL-1. The corrected linear equation was I = 65522.84 – 1407.53 lg c with the correlation index of 0.988, and the minimum detection limit was 0.001 ng mL-1. The results revealed that the suggested ECL immunosensor was excellently sensitiveness and held a meaningful 13



potential application in insulin detection. In Table S1, by contrast with many platforms which used to determinate insulin, the UiO-67/Ru(bpy)32+-Au@SiO2 ECL system has a relatively wide detection range and low detection limit, which reflected its favourable potential application value. Superior stability was crucial for real clinical application of an ECL immunosensors.34-35 Figure 7C showed the ECL response situation of this proposed strategy when detecting different concentration of insulin under continuous scanning 3 cycles. It could be observed that an extremely stable ECL signal has been obtained in every last insulin concentration, which ensured the reliability of this suggested immunosensor. To investigate the reproducibility of the sandwich-type immunosensor, ten immunosensors were established through the same steps and measured under the identical condition.36-37 As shown in Figure 7D, the relative standard deviation of the ECL signals was 1.83 % illustrating the excellent reproducibility of this constructed immunosensor. In addition, the binding specificty of the proposed immunosensor was also investigated (Figure S4). When the determinands contained insulin, proatate specific antigen (PSA), alpha fetal protein (AFP), insulin with the interference of PSA, and insulin with the interference of AFP repectively (the concentrations of insulin and interferents were 50 ng mL-1 and 500 ng mL-1), the results showed that only the ECL intenssity of samples included insulin has decreased indicating that the immunosensor has favourable specificty for insulin detecction. The outstanding stability, reproducibility, and specificty all indicated the analytical performance of the suggested strategy was practicable in clinic diagnoses. 14


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Figure 7. ECL intensity (A) and calibration cure (B) of this immunosensor for different quantities of insulin-antigen (0.0025, 0.005, 0.01, 0.05, 0.5, 1, 10, 25, and 50 ng mL-1). Stability (C) (concentration of insulin was selected as 0.0025, 0.01, 0.05, 1, 25, and 50 ng mL-1) and reproducibility (D) of the ECL immunosensor. (Concentration of insulin was 50 ng mL-1) Detection of Insulin in Real Samples. The suggested sandwich-type immunosensor was used to determine insulin in human serum samples (obtained from the hospital of University of Jinan) with standard addition method. The results as Table S2 demonstrated, the insulin levels in one human serum sample was detected to be 26.13 ng mL-1. After the human serum sample was spiked with 0.01, 0.5, and 10 ng mL-1 insulin, the recoveries were 98%, 98%, and 100% respectively, exhibiting the appropriately certainty of measurement.

CONCLUSIONS 15



A sandwich-type electrochemiluminescence immunosensor was successfully constructed based on Ru(bpy)32+ as luminophore which embedded into UiO-67 organicmetal frameworks for insulin detection with preferable sensitivity, specificity, and stability. Since large specific surface area and poriness of UiO-67 organic-metal frameworks, more luminophores were loaded onto the surface and holes and superior anodic ECL response of Ru(bpy)32+ were acquired on electrode. Besides, ECL intensity decreased when Ab2-Au@SiO2 were incubated onto the modified-GCE because of the RET between UiO-67/Ru(bpy)32+ donors and Au@SiO2 acceptors. The feasibility of this proposed immunosensor has been demonstrated by detecting insulin in human serum samples with good reproducibility. Moreover, utilizing the preponderances of MOFs to load ECL luminophores provides a new avenue in the establishment of ECL immunosensors.

ASSOCIATED CONTENT Supporting Information Apparatus used in all experiments; the SEM images of SiO2 and Au@SiO2; the EDS of UiO-67/Ru(bpy)32+; the ECL behaviors of UiO-67/Ru(bpy)32+ and UiO-67; the specificity of the suggested immunosensor; comparison of the performance of the proposed and other sensors for insulin detection; the results of insulin determination in human serum.

AUTHOR INFORMATION Corresponding Authors *Tel: +86 53182767890. Fax: +86 53187161600. E-mail: [email protected] (W. 16


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Cao). *Tel: +86 53182767890. Fax: +86 53187161600. E-mail: [email protected] (Q. Wei). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was supported by the Natural Science Foundation of Shandong Province (No. ZR2016BM20), National Natural Science Foundation of China (Nos. 21575050, 21505051, and 21777056), National Key Scientific Instrument and Equipment Development Project of China (No.21627809), and Qin Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province (No. ts20130937) and UJN.

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MWCNTs-Nafion composite film. Electrochim. Acta 2015, 164, 211-217. (14) Hosseini, M.; Mrk, P.; Norouzi, P.; Moghaddam, M.; Ganjali, M. An enhanced electrochemiluminescence

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Quenching Electrochemiluminescence Immunosensor Based on

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