Construction of Highly Efficient Resonance Energy Transfer Platform

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Construction of Highly Efficient Resonance Energy Transfer Platform inside a Nanosphere for Ultrasensitive Electrochemiluminescence Detection Miao-Miao Chen, Ying Wang, Shi-Bo Cheng, Wei Wen, Xiuhua Zhang, Shengfu Wang, and Wei-Hua Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05074 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

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Analytical Chemistry

Construction of Highly Efficient Resonance Energy Transfer Platform inside a Nanosphere for Ultrasensitive Electrochemiluminescence Detection Miao-Miao Chen, † Ying Wang, # Shi-Bo Cheng, † Wei Wen, # Xiuhua Zhang,*, # Shengfu Wang, # and Wei-Hua Huang,*, † † Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China # Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules＆College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, China * E-mail: [email protected] * E-mail: [email protected]

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ABSTRACT Electrochemiluminescence (ECL) detection has attracted increasing attention as a promising analytical approach. A considerable number of studies showed that ECL intensity can be definitely improved by resonance energy transfer (RET), while the RET efficiency is strongly dependent on the distance between exited donors and acceptors. Herein we disclose for the first time a highly enhanced RET strategy to promote

the

energy

transfer

efficiency

by

co-encapsulating

the

donor

([Ru(bpy)3]2+)/acceptor (CdTe quantum dots, CdTe QDs) pairs into a silica nanosphere. Plenty of [Ru(bpy)3]2+ and CdTe QDs closed packed inside a single nanosphere greatly shortens the electron-transfer path and increases the RET probability, therefore significantly enhancing the luminous efficiency. Further combining with molecularly imprinting technique, we develop a novel ECL sensor for ultrasensitive and highly selective detection of target molecules. Proof of concept experiments showed that extremely low detection limits of sub-fg/mL (S/N=3) with broad linear ranges (fg/mL to ng/mL) could be obtained for detection of two kinds of mycotoxins (α-ergocryptine and ochratoxin A) that are recognized as potential health hazards at very low concentrations. This strategy combining enhanced RET system and molecularly imprinting technique, represents a versatile ECL platform toward low-cost, rapid, ultrasensitive and highly selective detection of target molecules in diverse applications.

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INTRODUCTION Electrochemiluminescence (ECL) is a light-emission progress generated from the excited species sourced by exergonic electron transfer reactions.1-3 By utilizing potential as the excitation source rather than an external light, ECL is performed at a low background signal compared to photoluminescence, with advantages of high sensitivity, facile controllability and simplified optical equipment. So far, ECL has become a widely applicable technique from fundamental studies to practical use in biological and chemical analysis.4-9 For ultrasensitively sensing trace amounts of target molecules, many efforts have been made to enhance the emitted light intensity of ECL sensors. Erenow, a variety of signal amplification strategies are rapidly established and continuously reinvented for ECL assays. Due to the fact that the ECL intensity relies heavily on the electron transfer efficiency between the luminophore and coreactant, intramolecular self-enhanced ECL emitters have been manifested by covalently attaching coreactants onto ECL active luminophore. Compared with intermolecular reaction, intramolecular reaction greatly shortens the electron transfer path and reduces the complication of mass transport between the reactants during the lifetime of radical intermediates, therefore leading to the reinforced sensitivity for ECL sensing.10-15 To increase the excitation rate of luminophores, surface plasmon-coupled ECL has also been extensively investigated for signal enhancement.16-18 The excited luminophores can motivate noble metal, such as a thin gold film or Au nanoparticles (NPs)19, 20 to engender localized surface plasmon resonance (LSPR) field. The distance dependent 3



electromagnetic field in LSPR further increases both the excitation rate and the emission factor of luminophores, consequently boosting the ECL emission. Recently, increasing attentions have been paid on ECL resonance energy transfer (ECL-RET) by leveraging of overlapped spectra of donors emission and acceptors absorption in close proximity.21-23 In this circumstance, the acceptor is excited via energy transferred nonradiatively from an excited donor. For example, ruthenium (II) complexes, the most extensively studied luminophore, can be served as a versatile energy acceptor of multiple ECL donors, such as CdS quantum dots (QDs)23, g-C3N4 nanosheet22, luminol24 to construct ratiometric ECL sensors for accurate quantification of analyte. Notably, when ruthenium (II) complexes are acted as luminous donors coupling to an acceptor with higher quantum yield (e.g. CdTe QDs), the ECL intensities can be enhanced by transferring the energy from excited ruthenium(II) complexes to CdTe QDs and inducing emission of the acceptor.25, 26 But considering that the RET efficiency is strongly dependent on the distance between donors and acceptors,27-29 RET is severely hampered when donors and acceptors are isolated dispersed at the electrode or in aqueous medium26. To address this issue, a recent study showed that covalently linking the donor and acceptor to form a single nanostructure could obviously enhance the ECL intensity.29 This offer a promising approach to improve ECL-RET efficiency, while developing highly efficient ECL-RET nanostructure for enforcing ultrasensitive, highly reproducible and convenient detection still remains a big challenge. In this work, we developed a novel ECL-RET strategy to improve RET efficiency 4


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by co-encapsulating the donor ([Ru(bpy)3]2+) and acceptor (CdTe QDs) into a silica nanosphere with a facile one step sol-gel technique. The ECL-RET can occur inside single nanospheres through this design, and plenty of [Ru(bpy)3]2+ and CdTe QDs distributed inside nanospheres efficiently decrease the distance from the excited donor to acceptor. This greatly increase the RET probability and improve the RET efficiency, therefore endowing the nanosphere with excellent ECL performance (Scheme 1a). By immobilizing such RET efficiency-enhanced nanospheres on the electrode and further coating a molecularly imprinted polymer (MIP) film thereon, we obtained a solid-state MIP-ECL sensor with high sensitivity and selectivity (Scheme 1b). Here, the performances of as prepared sensor were evaluated by highly sensitive and selective detection of mycotoxins. Mycotoxins including such as ergot alkaloid, ochratoxin, fumonisins are the fungal secondary metabolites and produced in the improper storage of food supplies. These natural toxic contaminants even at very low concentrations can pose a serious threat on human and animal health, ranging from skin irritation to immunosuppression, neurotoxicity and carcinogenicity.30-34 Using this senor in detection of two kinds of mycotoxins (α-ergocryptine and ochratoxin A), an extremely low detection limit of sub-fg/mL (S/N=3) with a broad linear range (fg/mL to ng/mL) was obtained. This work demonstrates a versatile and robust ECL sensing platform by introducing the nanospheres with self-enhanced RET efficiency, indicating it promising potential toward ultrasensitive detection of target analytes in diverse applications.

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Scheme 1. Schematic illustration of the synthesis of CdTe-Ru@SiO2 nanospheres (a) and fabrication of the MIP-ECL platform for mycotoxin detection (b). Each CdTe QD is surrounded by three [Ru(bpy)3]2+ for simplicity.

EXPERIMENTAL SECTION Reagents and Apparatus. Tris (2, 2-bipyridyl) dichlororuthenium (II) hexahydrate (Ru(bpy)3Cl2·6H2O) was purchased from Green kaemmer (Beijing, China). Tripropylamine (TPrA), cadmium chloride hemipentahydrate (CdCl2.2.5H2O) and sodium tellurite (IV) (Na2TeO3), methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA), and azodiisobutyronitrile (AIBN) were obtained from Aladdin Chemistry Co., Ltd. Ochratoxin A (OTA), α-ergocryptine，ergocristine， ergocornine，glucose，fructose, ascorbic acid, and pure starch sample were all supplied by Cayman Chemical Company. All other chemicals were of analytic grade from Sigma-Aldrich used as received. Phosphate buffer solution (0.1 M KH2PO4-Na2HPO4; PBS; pH 7.0) was assigned to be a base solution. Millipore ultrapure water was used 6


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throughout the experiments. ECL-potential profiles were recorded by employing a MPI-A ECL analyzer (Xi’an Remex Analytical Instrument Co., Ltd., China, 185 − 650 nm), consisting of a three-electrode system with a glassy carbon working electrode (GCE, 5 mm in diameter), a Pt counter electrode, and an Ag/AgCl (saturated) reference electrode. High resolution transmission electron microscope (HRTEM) and energy-dispersive X-ray (EDX) images were recorded by 300 KV Titan Probe corrected TEM, Titan G2 60-300. The X-ray photoelectron spectroscopy (XPS) measurements were conducted with a Thermo Scientific, ESCALAB 250Xi. Scanning electron microscopy (SEM, Zeiss Sigma field-emission), UV-vis spectrometer (Shimadzu, UV-3600) and atomic force microscope (AFM, Oxford Instrument, Cypher ES, Asylum Research) were used. Electrochemical impedance spectroscopy (EIS) was performed on a CHI 660E electrochemistry workstation (Shanghai CH Instrument, China). Preparation of CdTe-Ru@SiO2 Nanospheres. Synthesis of CdTe QDs was carried out with Na2TeO3 as the Te source and 3-mercaptopropionic acid (MPA) as stabilizer via a one-step hydrothermal route.35 Typically, CdCl2 ‧2.5H2O (6.25 mM) and MPA (7.5 mM) were dissolved evenly with ultrapure water, forming the cadmium precursor. The pH of the solution was adjusted to 10.00 by addition of 1 M NaOH. With a stream of high-purity nitrogen atmosphere, Na2TeO3 (3.125 mM) and NaBH4 (5 mg) were successively added with vigorous magnetic stirring. Then the reaction proceeded at 100 °C for 2 h and finally the mixture was cooled down to gain the CdTe QDs sample. 7



CdTe-Ru@SiO2 nanospheres were acquired through a water-in-oil (W/O) microemulsion method that has been previously described for preparation of Ru@SiO2 nanospheres.36,

37

Due to the opposite charge, [Ru(bpy)3]2+ complex

preferentially electrostatic attracts the carboxyl group of MPA on the surface of CdTe QDs. The prepared CdTe QDs (170 µL) reacted overnight with [Ru(bpy)3]2+ (80 mM, 170 µL) in a conical flask, and then cyclohexane (7.5 mL), TX-100 (1.77 mL), and n-hexanol (1.8 mL) were injected into the mixture with constant agitation for 30 min. Following the rapid injection of the precursor TEOS (100 µL), the polymerization reaction was initiated by 60 µL NH3·H2O. After reaction protected from light in the sealed container for 24 hours, the resultant products were isolated with acetone, centrifuged (10000 rpm, 8 min) and purified with ethanol, water, ethanol in succession for 3 times. The obtained CdTe-Ru@SiO2 nanospheres were dispersed in 600 µL PBS, which was stored at 4 °C for further use. Fabrication of α-ergocryptine Imprinting ECL Sensor. The fabrication process was displayed in schematic illustration. Glassy carbon electrode (GCE) was pretreated with 0.3 and 0.05 µm aluminum slurry for about 15 min. After being ultrasonically washed with ethanol/water solution, the fresh GCE was dried naturally. The as-prepared CdTe-Ru@SiO2 nanospheres blended well with 1 wt% chitosan (CS) in the position of rapid agitate strength for 2 h. Then 5 µL mixture was vertically cast onto the electrode surface, dried in air and denoted as CdTe-Ru@SiO2/GCE. Subsequently, 2 µL of crosslinking solution containing α-ergocryptine as template molecules mingled with the functional monomer of MAA, cross-linker of EDMA and 8


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evocating agent of AIBN was put on the nanospheres-modified layer simultaneously. Under ultraviolet radiation of 365 nm for 1 min, the α-ergocryptine imprinting ECL sensor denominated as MIP/CdTe-Ru@SiO2/GCE was obtained. As a control, nonmolecularly imprinted polymers (NIP) were prepared under identical conditions but omitting the template in the reaction system. ECL Assay. The ECL signal generated by performing the potential step program was measured with a photomultiplier tube (PMT, high-voltage 600 V, magnitude 3), at a constant distance, under the cell and inside a homemade dark box. The MIP/CdTe-Ru@SiO2/GCE was eluted in ethanol for some time, after which it was rinsed with double distilled water to remove unbound reagents thoroughly. Immerged in a serious of α-ergocryptine solutions having known concentrations, the sensors were further evaluated in the subsequent analytical performance. In each immobilization step, ECL emission signals were measured in 3 mL 0.1 M PBS (pH 7.0) containing 2 mM TPrA as an oxidative coreactant with a scan range from 0 to 1.1 V and a scan rate of 100 mV/s via cyclic voltammetry.

Results and Discussion Characterization of the CdTe-Ru@SiO2 Nanospheres CdTe QDs with a uniform diameter about 4 nm (Figure S1a) and fluorescent emission peak at 627 nm (Figure S1b) were firstly prepared, which was then coencapsulated into SiO2 nanospheres together with [Ru(bpy)3]2+. As illustrated in Figure 1a, the as-prepared CdTe-Ru@SiO2 display a well-defined and uniform 9



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assembly of closely packed nanospheres. Comparing with Ru@SiO2 in Figure S1c as well in our previous work,

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the adulteration of QDs has no significant effect on the

shape of nano-structure during the building-up reaction. The Zeta potential of CdTe-Ru@SiO2 was measure to be -28.7 mV (Figure S1d), indicating the nanospheres were negatively charged. This is beneficial to stabilize positively charged [Ru(bpy)3]2+ by electrostatic interaction. HRTEM imaging was carried out to characterize the CdTe-Ru@SiO2 nanospheres, and

the

nanospheres

display

a

nearly

spherical

morphology

and

good

monodispersibility (Figure 1b) with an average diameter of 52.8 ± 8.1 nm (mean ± SD, n = 276, Figure 1c). Notably, HRTEM images clearly show that plenty of CdTe QDs are well dispersed inside the silica nanospheres (Figure 1b), and elemental mapping using energy dispersive X-ray spectroscopy (EDX) indicates the coexistence of Ru, Si, Cd, O, Te elements in the nanospheres (Figure 1d). X-ray photoelectron spectroscopy (XPS) was used to further confirm the element composition (Figure S2, and Figure 1e, f). The appearance of the characterized XPS peaks including Ru 3d5/2 at 280.8 eV, Cd 3d5/2 at 405.1 eV and Te 3d5/2 at 572.3 eV confirms the existence of ruthenium, cadmium, and tellurium species in the nanospheres. Additionally, the trace of Si 2p, O 1s signals are also observed.38-40 Taken together, these combined results indicate that the co-encapsulation of [Ru(bpy)3]2+ and CdTe QDs by SiO2 nanospheres occurred successfully. This provides a facile and efficient approach to improve ECL-RET efficiency by reducing the distance between closely co-existed donor and acceptor. Figure 1b shows that the 10


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distance between each QDs is no more than 20 nm. Given the fact that the negatively charged QDs are surrounded by a mass of [Ru(bpy)3]2+, we can conclude that the distance between [Ru(bpy)3]2+ and CdTe QDs should be less than 10 nm in a silica nanosphere, which is exactly located within the range of RET path length.4

Figure 1. Characterization of the CdTe-Ru@SiO2 nanospheres. (a) SEM image; (b) HRTEM image. The inset shows the representative enlarged morphology image of individual, and the QDs are marked by arrows for clarity; (c) Size distribution of the nanospheres; (d) TEM image of several nanospheres and corresponding EDX elemental mapping images; (e, f) X-ray photoelectron spectra of solid-state nanospheres, (e) Ru 3d and C1s signals, (f) Cd 3d signals.

ECL-RET Performance of the CdTe-Ru@SiO2 Nanospheres CdTe-Ru@SiO2 nanospheres were immobilized on a glassy carbon electrode (GCE) for ECL analysis. A nearly straight line of EIS scan for the bare GCE (Figure S3, 11



curve 1) indicates that no substance hinders electronic transfer on the electrode surface. After modification with CdTe-Ru@SiO2, the negatively charged nanospheres prevent [Fe(CN)6]3-/4- from reaching the electrode surface, hence a small semicircle at high frequencies is observed (Figure S3, curve 2). Furthermore, AFM results (Figure 2a) clearly indicate that CdTe-Ru@SiO2 nanospheres were faithfully transferred onto the electrode with Ra value of 12.3 nm.

Figure 2. (a, b) AFM images of CdTe-Ru@SiO2 nanospheres on the GCE surface; (c) Schematic diagram showing the RET progress in a nanosphere; (d) UV–vis absorption spectrum of CdTe QDs (black) and ECL spectrum (blue) of Ru@SiO2/GCE; (e) Normalized ECL spectra of CdTe-Ru@SiO2/GCE with different ratio of Ru(II) to CdTe, here the content of [Ru(bpy)3]2+ was fixed at 170 µL (80 mM) and CdTe QDs with different amounts (curve 1: 30 µL, curve 2: 170 µL, curve 3: 310 µL) were used; (f) ECL responses of CdTe@SiO2/GCE (curve 1), Ru@SiO2/GCE (curve 2), Ru@SiO2/GCE with 5 µL CdTe in solution (curve 3), and CdTe-Ru@SiO2/GCE (curve 4). The UV–vis spectrum was obtained in 0.1 M PBS (pH 7.0), and the ECL spectra were gained through a series of optical filters: 525 nm, 550 nm, 575 nm, 600 nm, 610 nm, 625nm, 650 nm, 675 nm, 700 nm, and measured in 0.1 M PBS (pH 7.0) containing 2 mM TPrA. 12


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The enhanced ECL of CdTe-Ru@SiO2 nanospheres relies on efficient RET from excited [Ru(bpy)3]2+ to CdTe QDs (Figure 2c), which is based on the perfect overlap between ECL spectrum of [Ru(bpy)3]2+ (Figure 2d, blue curve) and UV-vis absorption spectrum of CdTe QDs (Figure 2d, black curve). Figure 2e displays the ECL spectra of CdTe-Ru@SiO2/GCE with different ratio of Ru(II) to CdTe inside the nanospheres. The results show that the ECL-RET leads to a remarkable decline in ECL emission of [Ru(bpy)3]2+ centered at 599 nm as the increase of CdTe QDs, accompanying by a significant increase in CdTe QDs emission at 627 nm (Figure 2e, note in this ECL conditions, CdTe QDs alone do not show any ECL signals which is further indicated in Figure 2f). The CdTe QDs emission reached a constant value when 170 µL or more mount of QDs were used (Figure 2e), which is in agreement with the ECL intensity measurement (Figure S4) and further demonstrates the ECL-RET from [Ru(bpy)3]2+* to CdTe QDs. As illustrated in Figure 2f, the ECL performance of different materials has been investigated using TPrA as the coreactant. Almost no ECL response was appeared when CdTe@SiO2/GCE was used as the control (Figure 2f, curve 1), and ECL emission was generated at a voltage of 1.10 V on Ru@SiO2/GCE with the peak intensity about 1482 a.u. (Figure 2f, curve 2). This is assigned to a single oxidative step including TPrA and Ru complex according to well-established mechanism.41, 42 Followed by adding CdTe QDs in the solution, Ru@SiO2/GCE showed a 2-fold increase in ECL intensity (Figure 2f, curve 3, 3011 a.u.), which is in good agreement with our previous results.26 This suggests the occurrence of RET between the 13



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immobilized Ru@SiO2 and the dispersed CdTe QDs in solution. As expected, when the donor and acceptor were co-encapsulated inside a single nanosphere, CdTe-Ru@SiO2/GCE displays the highest ECL signal with nearly 6-fold enhancement in ECL intensity (Figure 2f, curve 4, 17835 a.u.) compared with the system when the acceptor were dispersed in solution. The significant enhancement of the ECL responses of [Ru(bpy)3]2+ could be explained by highly efficient RET occurred inside each nanosphere. According to the previously established mechanisms,43, 44 the whole ECL-RET process could be described as follows: [Ru(bpy)3]2+ - e → [Ru(bpy)3]3+

(1)

TPrA - e → TPrA+•

(2)

TPrA+• → TPrA• + H+

(3)

TPrA• + [Ru(bpy)3]3+ → [Ru(bpy)3]2+* + products [Ru(bpy)3]2+* + CdTe → [Ru(bpy)3]2+ + CdTe*

(5)

CdTe* → CdTe + hv Electrostatically

assembled

CdTe-Ru

(4)

(6) complex

are

three-dimensionally

encapsulated in the porous silica nanospheres immobilized on the electrode. The small molecule TPrA as the coreactant reaches the electrode surface to be oxidized, after deprotonation process, the TPrA radicals penetrate into the porous nanospheres to react with [Ru(bpy)3]2+, generating excited [Ru(bpy)3]2+*. Then the energy is nonradiatively transferred from [Ru(bpy)3]2+* to neighboring CdTe QDs (Figure 2c), inducing the emission of QDs. Here the distance (electron-transfer path) between closed packed [Ru(bpy)3]2+* and CdTe QDs is greatly shortened, and energy loss during electron-transfer is also minimized. This definitely improves the RET 14


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efficiency and induces a significantly enhanced ECL signal, thereby providing a self-enhanced ECL platform for further analysis. MIP-ECL Sensor for Highly Sensitive and Selective Detection of Mycotoxins To endow the self-enhanced ECL sensor with high selectivity, a MIP polymer was coated on the nanospheres by using MAA as the functional monomer, EDMA as the cross-linker and AIBN as the photoinitiator.45 Here, two representative mycotoxins (α-ergocryptine and OTA) were selected as the template molecules for analysis. The SEM cross-sectional structure of the MIP-ECL layer is showed in Figure 3a. The thickness of the CdTe-Ru@SiO2 layer was controlled by the volume of CdTe-Ru@SiO2 nanospheres-chitosan liquid mixture and was optimized to be about 2.5 µm (Figure S5). A MIP layer with an approximate thickness of 27 µm (this thickness was also optimized to obtain a maximum ∆IECL, data not shown) uniformly covered the nanospheres. Moreover, there are many semblable wrinkles on the surface of both MIP (Figure 3b) and NIP (Figure 3c), indicating that small molecule templates have little influence on the morphology of the polymer.

Figure 3. SEM micrographs of the MIP-ECL sensor. (a) Cross-sectional morphologies of MIP-ECL film; (b, c) Surface appearance of MIP (b) and NIP (c) layer.

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Figure 4. (a) EIS plots in 0.5 mM [Fe(CN)6]3-/4- (in 0.1 M KNO3) of curve 1: MIP/ CdTe-Ru@SiO2/GCE, curve 2: MIP/CdTe-Ru@SiO2/GCE after template removal and curve 3: MIP/CdTe-Ru@SiO2/GCE after rebinding; (b) ECL signals in 0.1 M PBS (pH 7.0) containing 2 mM TPrA for MIP/CdTe-Ru@SiO2/GCE (the insert for NIP/CdTe-Ru@SiO2/GCE) during before (curve 1) and after elution (curve 2), then rebinding (curve 3) process; (c) ECL signals to different concentrations of α-ergocryptine, from top to bottom: 10-6, 10-5, 10-4, 10-3, 10-2, 10-1, 1, 5, 100, 300 ng/mL; (d) Calibration curve for α-ergocryptine determination. Error bars represent standard deviations, n ≥ 3.

Electrochemical impedance spectroscopy (EIS) was performed to investigate the resistance changes during whole detection process, and the corresponding ECL signals were recorded. MIP-coated ECL sensor exhibits a remarkable resistance (Figure 4a, curve 1), with almost no ECL response (Figure 4b, curve 1). This is ascribed to the insulated MIP polymer layer that hinders electron transfer at the electrode surface. After being eluted with ethanol, the tailor-made cavities as binding sites with memory of the shape size and functional groups of the template molecules were created in the MIP polymer. These cavities could be served as channels to facilitate electron transfer, therefore inducing a significant decrease in resistance 16


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(Figure 4a, curve 2) and drastic increase in ECL signal (Figure 4b, curve 2). Subsequently, the MIP modified electrode was submerged into solution of target molecules, allowing templates to rebind into the film, the resistance increased again (Figure 4a, curve 3) and the ECL intensity declined rapidly (Figure 4b, curve 3). As a comparison, the ECL behaviors of NIP/CdTe-Ru@SiO2/GCE were carried out using the same process. Only very weak signal fluctuations were observed (Figure 4b, insert), which is mainly attributed to the nonspecific absorption between NIPs and the analyte. We optimized the elution and rebinding time to achieve the best ECL performance for detection. The results show that the optimum ECL performance were obtained with the elution time of 5 min (Figure S6a) and the rebinding time of 5 min (Figure S6b). Under the optimal conditions, the sensitivity of the MIP-ECL sensor was evaluated by recording the ECL intensity in a series of α-ergocryptine standard samples with different concentrations. Figure 4c demonstrates that the ECL intensity decreases in response to the increasing concentration. This could be explained by the fact that more cavities were occupied by α-ergocryptine with higher concentration, hindering electron transport to a greater extent and lowering ECL emission. The ∆IECL depends linearly on the logarithm value of the α-ergocryptine concentrations varied from 1 fg/mL to 300 ng/mL with a correlation coefficient of 0.994 (Figure 4d). The regression equation is expressed as ∆IECL = 959.94 lg c + 6878.48 with a detection limit of 0.18 fg/ml (S/N=3), which is 5-6 orders of magnitude lower than that previous reported.46-48 17



Using this self-enhanced MIP-ECL platform, detection of another mycotoxin OTA with the linear range from 1 fg/mL to 200 ng/mL and a detection limit of 0.25 fg/mL was also achieved (Figure S7). It is worth noting that in our previous work26 by transferring energy from excited [Ru(bpy)3]2+ (immobilized on the electrode) to CdTe QDs (dispersed in solution with a high concentration of 2.65 mM), the detection limit was 3.0 fg/mL-1, which is 12 times higher than that in the current work. These results clearly show that using much smaller amount of CdTe QDs we still achieve superior detection performance, further demonstrating the significantly enhanced RET between [Ru(bpy)3]2+ and CdTe QDs inside the single nanospheres. Selectivity and Stability of the MIP-ECL Sensing Platform. To assess the selectivity of this method, six kinds of mycotoxins (ergocristine， ergocornine ， OTA, patulin, zearalenone, and fumonisins B1) and some other ingredients (glucose, fructose, ascorbic acid and the mixture of Fe3+, Cu2+, Ca2+, Zn2+, K+) commonly existed in foodstuffs were subjected as the potentially interfering substances in detection of α-ergocryptine. Excess ten-folds concentration of each interferent was severally added in α-ergocryptine solution (10-4 ng/mL), and there were no significant differences in ∆IECL between standard α-ergocryptine sample and interferent added ones (Figure 5a). The result suggests that the ECL-MIP sensor manifested good anti-interference for selective detection. Stability of the MIP-ECL sensor was also evaluated. As shown in Figure 5b, little obvious fluctuation is observed with the ECL peak intensity during continuous three cycles scanning on each concentration of α-ergocryptine, indicating favorable stability 18


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of the proposed ECL sensor. The electrode-to-electrode reproducibility was also determined with nine independent electrodes, showing an acceptable reproducibility with a RSD of 3.9% (Figure 5c).

Figure 5. (a) Performances of the MIP-ECL sensors tested with different interferences from 1 to 11: 10-4 ng/mL α-ergocryptine, 10-4 ng/mL α-ergocryptine + 10-3 ng/mL ergocristine, 10-4 ng/mL α-ergocryptine + 10-3 ng/mL ergocornine, 10-4 ng/mL α-ergocryptine + 10-3 ng/mL OTA, 10-4 ng/mL α-ergocryptine + 10-3 ng/mL patulin, 10-4 ng/mL α-ergocryptine + 10-3 ng/mL zearalenone, 10-4 ng/mL α-ergocryptine + 10-3 ng/mL fumonisins B1, 10-4 ng/mL α-ergocryptine + 10-3 ng/mL glucose, 10-4 ng/mL α-ergocryptine + 10-3 ng/mL fructose, 10-4 ng/mL α-ergocryptine + 10-3 ng/mL ascorbic acid and 10-4 ng/mL α-ergocryptine + 10-3 ng/mL the mixture of Fe3+, Cu2+, Ca2+, Zn2+, K+. (b) Stability test of the sensor with different concentrations of α-ergocryptine by continuous scanning for three cycles apiece; (c) Reproducibility of nine ECL-MIP sensors after elution in 0.1M PBS (pH 7.0) containing 2 mM TPrA.

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Determination of α-ergocryptine in Starch Samples To evaluate the feasibility of the proposed MIP-ECL sensor in detection of real samples, α-ergocryptine with different concentrations (2.2×10-3, 0.10 and 9.9 ng/mL) were added into starch samples via a standard addition method to record the ECL signals. The recovery was individually calculated to be 94.7, 107.8, 105.2 %, with a satisfactory RSD from 2.61% to 5.31% (Table 1). These results suggest that the proposed sensor can work well in the complicated real samples and has potential applications in detection of various analytes at very low and wide range of concentrations. Table 1. Detection of ergocryptine in starch samples by the MIP-ECL sensor.

samples

starch

Added (ng·mL−1)

Detected (ng·mL−1)

Recovery (%)

RSD (n = 3) (%)

2.2×10-3

2.1×10-3

94.7

5.31

0.10

0.11

107.8

4.45

9.90

10.42

105.2

2.61

CONCLUSION In summary, we propose a facile, versatile and robust strategy for in situ self-enhancement of ECL emission, by co-encapsulation of donors and acceptors inside a single nanosphere. The electron-transfer path between closed packed donor and acceptors is greatly shortened inside the nanospheres, and energy loss during electron-transfer is meanwhile minimized, which greatly improves the RET efficiency 20


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and RET probability, therefore inducing a significantly enhanced ECL signal. Further combination of the excellent recognition ability of MIP, ultrasensitive and highly selective detection of target molecules could be readily achieved. The as-proposed MIP-ECL sensor presents an extremely low detection limit of sub-fg/mL with a broad linear range from fg/mL to ng/mL for mycotoxin detection, meanwhile possessing stable performance and anti-interference ability. Its practicability was demonstrated by determination of real samples with the satisfactory recoveries. This work opens a new way in designing high-performance ECL sensing platform, which will have a broad application in biomedical and chemical analysis especially when untrasensitive detection of target molecules with extremely low concentration is definitely needed.

Supporting Information. Additional Figures S1 to S7. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Authors * E-mail: [email protected] or [email protected] Author Contributions M.-M.C. and Y.W. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Nos. 31501568, 11674085, 21725504).

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Construction of Highly Efficient Resonance Energy Transfer Platform

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