Triethanolamine-Modified Gold Nanoparticles Synthesized by a One

Feb 7, 2018 - Triethanolamine-Modified Gold Nanoparticles Synthesized by a One-Pot Method and Their Application in Electrochemiluminescent ...
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Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Triethanolamine-Modified Gold Nanoparticles Synthesized by a OnePot Method and Their Application in Electrochemiluminescent Immunoassy Xiaoli Qin,† Chaoyue Gu,† Minghan Wang,† Yifan Dong,† Xin Nie,† Meixian Li,† Zhiwei Zhu,† Di Yang,‡ and Yuanhua Shao*,† †

Beijing National Research Center for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China ‡ Institute of Cardiovascular Disease, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China S Supporting Information *

ABSTRACT: In many electrochemiluminescent (ECL) systems, coreactants play crucial roles in the redox-induced light emission process at the electrode surface. In this work, a novel and environment-friendly nanoplatform for ECL immunosensing enabled by triethanolamine (TEOA)-modified gold nanoparticles (TEOA@AuNPs) is reported. The monodisperse TEOA@AuNPs are fabricated by one-pot synthesis using TEOA as both reducing and stabilizing agent. Then the TEOA@AuNPsmodified electrode not only acted as coreactant for Ru(bpy)32+ ECL system but also provided a carrier for antibody 1 to form label-free immunosensor through an interaction between antigen and antibody. The unique structure of the TEOA@AuNPs loaded a large amount of coreactant of Ru(bpy)32+, which shortened the electron-transfer distance from the AuNPs surface to the appended TEOA molecules, thereby greatly enhancing the ECL efficiency and amplifying the ECL signal. In addition, Ru(bpy)32+-doped silica (RuSiO2) nanoparticles and antibody 2 were combined to form a composite for labels and a sandwichtype ECL immunosensor has been constructed. The possible mechanism of those ECL systems have also been proposed and confirmed by the EC-MS hyphenated technique. The human cardiopathy biomarker, cardiac troponin I (cTnI), was detected in a wide linear concentration range and the limit of detection (LOD) was 34 or 5.5 fg mL−1 by using the proposed label-free or labeling ECL immunoassay method.

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system is one of the most popular ECL systems and the basis of commercial ECL immunoassay and DNA analysis devices.4,14 Despite its great efficiency in generating ECL signal, the TPA has some disadvantages such as toxicity, volatility, poor solubility, and low electrochemical oxidation rate. At the moment, to maintain a more general analytical applicability, a series of tertiary amine compounds have been developed as alternative ECL coreactants, such as triethanolamine (TEOA), 3 , 1 5 , 1 6 trimethylamine (TEA), 1 7 , 1 8 and 2(dibutylamino)ethanol (DBAE).19 Usually, tertiary amines are more effective than secondary amines, primary amines, and other kinds of coreactants. To develop more efficient and environment-friendly ECL assay, TEOA might be a promising coreactant because it is much less toxic, more soluble, and cheaper than other popular coreactants in aqueous solution. Yuan and co-workers employed graphite-like carbon nitride (gC3N4) as a luminophore and TEOA as a coreactant to

lectrochemiluminescence (ECL) is a useful technique for qualitative and quantitative analysis of various samples in multiple applications, such as food safety,1,2 clinic testing,3,4 and environmental monitoring5,6 because it has simple setup and high sensitivity due to its absence of background optical signal.7,8 At present, three major ECL luminophores, metal complexes, luminol, and nanomaterials, have all been employed for practical applications. For example, the tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy)32+) and the luminol-based ECL biosensors have been widely used in immunoassay,9,10 DNA analysis,11−13 and cancer cell detection.14,15 Compared with the molecular luminophores, nanoluminophores, such as metal nanoclusters, nanocrystals, and carbon nanomaterials, have received a great deal of attention in recent years.7,8 In the approach involving coreactant, selection of a proper coreactant for ECL systems is the key. In such ECL systems, the exited state is usually generated through the reaction between the luminophore and the coreactant, whose electrochemical oxidation or reduction is first carried out. In the past few decades, tri-n-propylamine (TPA) has been exclusively used as the coreactant for ECL systems. The Ru(bpy)32+/TPA © XXXX American Chemical Society

Received: November 29, 2017 Accepted: January 24, 2018

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DOI: 10.1021/acs.analchem.7b04952 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry determine rutin.3 The electron-withdrawing hydroxyethyl groups of TEOA exhibit positive effect on ECL intensity. Also, a series of ruthenium(II) tris-bipyridyl complexes covalently linked with TEOA for an ECL system were studied by Kehr and co-workers.15 Although there have been several reports regarding TEOA as the coreactant for the ECL system, studies on its other roles and effect on the ECL performance are still lacking. To date, with the development of nanotechnology, many nanomaterials, such as silicon nanoparticles, quantum dots, carbon nanomaterials, and gold nanoparticles (AuNPs),7,8 have been employed in the ECL to improve analytical performance. Among them, AuNPs have attracted much attention because of their unique properties, such as excellent conductivity, good biocompatibility, fascinating electrocatalytic activity, and large surface area.20 For example, AuNPs-functionalized g-C3N4 nanohybrids were used to construct an ECL immunosensor for detection of carcinoembryonic antigen, which was reported by Chi and co-workers.21 You and co-workers developed an ECL immunosensor for the analysis of HIV-1 p24 antigen, which combined Ru(bpy)32+-doped silica (RuSiO2) with AuNPs-functionalized graphene (P-RGO@Au) to form a PRGO@Au@RuSiO2 and were used as a label.22 Also, gold nanoclusters can be used as luminescent probes to the ECL system; for instance, the near-infrared ECL emission of Au25+ clusters was observed in the annihilation of electrogenerated Au252+ and Au252− species and enhanced in the path of a coreactant system with benzoyl peroxide for the first time by Ding and co-workers. 23 Furthermore, Cui et al. have demonstrated for the first time that luminol and HAuCl4 could directly react in aqueous solution to synthesize AuNPs, and the luminol could be immobilized as a capping reagent of AuNPs assembled on the electrode for developing a H2O2 ECL sensor.24 Although there have been many investigations of AuNPs in ECL systems, in fact no study about it attaching with coreactants to amplify the ECL signals has been reported. In this work, first of all, TEOA and HAuCl4 have been tested to see whether they can be used to synthesize monodisperse, stable, and useful AuNPs. The experimental results demonstrate that the monodisperse TEOA@AuNPs are indeed fabricated by one-pot synthesis using TEOA as both reducing and stabilizing agents. Then we concentrate on the TEOA@ AuNPs for the Ru(bpy)32+ ECL system, its mechanism involved, and the effects of related experimental conditions. Finally, with use of TEOA@AuNPs, the label-free or labeling ECL immunosensor has been constructed for the analysis of human cardiopathy biomarker (cardiac troponin I, cTnI), and a new detection approach is proposed based on TEOA@AuNPs as the coreactant in the field of ECL sensing.

Ltd. (Shanghai, China). Monoclonal antibody against cTnI (anti-cTnI), cTnI, and patients’ plasma samples were all provided by the First Affiliated Hospital of Nanjing Medical University. All aqueous solutions were prepared with ultrapure water (≥18 MΩ-cm) and all the reagents were analytical grade or better. Apparatus and Characterizations. The UV absorption spectra were recorded with an Hitachi U-4100 spectrophotometer (Hitachi Co., Japan). Fourier transform infrared (FTIR) spectra were measured from a KBr window on a Nicolet iS50 FT-IR spectrophotometer (Thermo Fisher Scientific Inc., USA) and Spectrum Spotlight 200 FT-IR microscope (PerkinElmer, Inc., USA). X-ray photoelectron spectroscopy (XPS) measurements were performed by an Axis Ultra spectrometer (Kratos Analytical Ltd., Japan) and the binding energy was calibrated by the O 1s peak at 532.4 eV and the C 1s peak at 284.8 eV. Characterizations of transmission electron microscopy (TEM) were carried out on a JEM-2100F electron microscope (JEOL, Japan) under the accelerating voltage of 200 kV. Mass spectrometric (MS) experiments with in situ electrochemical reactions were carried out on an Agilent 6300 series ion trap mass spectrometer (Agilent Technologies, Inc., USA). The main experimental parameters of the MS and the fabrication of the hybrid ultramicroelectrodes were conducted like those in our previous work.26 The ECL measurements were conducted on a model MPIEII electrochemiluminescence analyzer (Xi’An Remax Electronic Science & Technology Co. Ltd., China). Cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS) were performed with a CHI 660C electrochemical workstation (Shanghai Chenhua Instruments, Co., China). A glassy carbon electrode (GCE) with the diameter of 3 mm served as the working electrode (WE) after polishing and sonication cleaning procedures.27 A Pt wire electrode and an Ag/AgCl (3 M KCl) electrode were used as the counter electrode and the reference electrode, respectively. Synthesis of the TEOA@AuNPs. A 17.5 mL aqueous solution was heated to 100 °C; 1 mL of TEOA (0.1 M) and 100 μL of HAuCl4 (42.8 mM) were added and vigorously stirred. Then the mixture was heated for 50 min. The solution rapidly changed from purple to light purple, wine red, indicating the formation of AuNPs. The citrate@AuNPs were synthesized according to a previous report.28 For purification of the TEOA@AuNPs, 10 mL of the AuNPs solution was dialyzed (Mw 3500) against water for over 2 days. Modification of RuSiO2 Nanoparticles with Antibody. According to a previous report,29,30 the modification of antibody on the RuSiO2 nanoparticles was prepared. First, 1 mL of TEOS and 800 μL of Ru(bpy)3Cl2 (0.1 M) solution were premixed with 5 mL of ethanol and vigorously stirred for 10 min. Then, 750 μL of NH3·H2O solution was added to the ethanol solution and continuously stirring for 1 h. After that, the solution was sonicated for 10 min and centrifuged at 5000 rpm for 10 min; the orange precipitation was washed three times with ethanol and dispersed into 6 mL of ethanol. Two milliliters of the above RuSiO2 nanoparticles solution was diluted with ethanol to 6 mL and 200 μL of APTES was added. After being stirred for 30 min, the amino-terminated RuSiO2 nanoparticles were obtained after being centrifuged and washed with water several times to remove the excess APTES. Then 5 mL of GA (2.5%) solution was added to the modified RuSiO2 nanoparticles solution and reacted at 37 °C for 4 h under

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EXPERIMENTAL SECTION Chemicals. The following chemicals were used as received: chloroauric acid (HAuCl4, ≥99.995%), Ru(bpy)3Cl2·6H2O (≥99.95%), 3-aminopropyltriethoxysilane (APTES, ≥98%), and bovine serum albumin (BSA, ≥98%) were purchased from Sigma-Aldrich. The electrochemical reactions solution (0.1 M PBS1, pH 7.0) and washing and blocking buffer solution for immunoassay (0.01 M PBS2, pH 7.4) were prepared by the previously described procedure.25 TEOA (≥98%) was obtained from Xilong Scientific Co., Ltd. (Guangdong, China). Tetraethylorthosilicate (TEOS, ≥98%) was obtained from J&K Scientific Ltd. (Beijing, China). Glutaraldehyde (GA, 50%) was obtained from Sinopharm Chemical Reagent Co. B

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Figure 1. Effects of reaction temperature on the fabrication of the TEOA@AuNPs. From (A) to (E), the reaction temperatures are 30, 60, 80, 90, and 100 °C, respectively. (F) and (G) are the corresponding absorption spectra and photo images for the samples from (A) to (E). All the reaction times were 50 min. According to these data, well-defined monodisperse AuNPs could be obtained at 100 °C, which was then chosen for the fabrication. The 50 nm scale bar in panel (E) also applies for other TEM pictures.

Figure 2. Structural characterizations of the TEOA@AuNPs. (A) High-resolution TEM image of an individual TEOA@AuNP and corresponding fast Fourier transformation diffractogram (B). (C) FT-IR spectra of the TEOA@AuNPs (red curve) and TEOA (black curve). XPS survey spectra (D) and high-resolution spectra of C 1s (E) and O 1s (F) of the TEOA@AuNPs.

was coated on the TEOA@AuNPs/GCE surface and incubated at 4 °C overnight in a 100% moisture-saturated environment. After the excess Ab1 was removed with PBS2, 6 μL of 3% BSA was dropped onto the electrode surface at 4 °C for 1 h to block the nonspecific binding sites and then washed with PBS2 (BSA/Ab1/TEOA@AuNPs/GCE). After that, the modified electrode was exposed to 6 μL of PBS2 containing different concentrations of antigen or serum sample at 37 °C for 1 h to form antigen/BSA/Ab1/TEOA@AuNPs/GCE (immunoelectrode 1) after being washed with PBS2. This electrode was incubated in 6 μL of labeled Ab2 at 37 °C for 1 h to form Ab2-

stirring. The mixture was centrifuged and washed three times with water to remove excess GA. The precipitation was dispersed into 2 mL of 0.1 mg mL−1 anti-cTnI 2 (Ab2) and stirred for 4 h under a 37 °C water bath. After the mixture was centrifuged and washed with PBS2, the Ab2-modified RuSiO2 nanoparticles (Ab2-RuSiO2) were obtained after being redispersed in 1 mL of PBS2 containing 1% BSA and stored at 4 °C before use. Fabrication of Immunoelectrodes. Twenty microliters of TEOA@AuNPs suspension was dropped on the pretreated GCE surface and dried in air. Then 6 μL of anti-cTnI 1 (Ab1) C

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data (Figure 2E,F versus Figure S4 in the Supporting Information), a carboxyl, a new group, appeared in the asprepared TEOA@AuNPs, which agreed with FT-IR characterization. These results indicated that the hydroxyl group in TEOA could reduce Au3+ into Au0, which leads to formation of gold particles, while the hydroxyl groups are oxidized to carboxyl groups (as shown in Scheme 1).31,33

RuSiO2/antigen/BSA/Ab1/TEOA@AuNPs/GCE (immunoelectrode 2). The fabricated electrodes were finally rinsed with PBS2 to carry out further ECL assay. ECL Detection. The ECL signal was recorded in 0.1 M PBS1 containing none or 100 μM Ru(bpy)32+ with the immunoelectrode 1 or immunoelectrode 2 as the working electrode. The cyclic voltammogram was recorded from 0 to 1.35 V with 100 mV s−1.

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Scheme 1. Schematic Diagram of (1) Construction of TEOA@AuNPs and (2) the Immunoassay Systems: (a) Label-Free Approach; (b) Labeling Method

RESULTS AND DISCUSSION Characterization of the TEOA@AuNPs. The TEM images and UV−vis absorption spectra were used to characterize the AuNPs synthesized by the TEOA with different reaction temperatures. As shown in Figure 1A−E, the as-synthesized AuNPs are nearly spherical and the diameters of AuNPs decrease with the increasing reaction temperature. The size of AuNPs at 100 °C is 19 ± 1.6 nm in diameter from the TEM (Figure S1 of the Supporting Information, SI). The TEM images also show the diameters of AuNPs decrease with the increasing reaction times (Figure S2). When HAuCl4 was added into TEOA solution, the color of the mixture solution changed from purple to wine red at 100 °C (Figure S2J). UV− vis spectra recorded at different reaction times show that the absorption peak of AuNPs gradually blue-shifts, and the adsorption intensity increased with increasing reaction time. According to these data, the system became stable after 40 min of reaction and an absorption peak of AuNPs appeared at 526 nm, which is the typical plasmonic resonance absorption peak of AuNPs.31 For better stability of the products, a further 10 min of reaction was conducted. As a result, 50 min reaction time was employed for the fabrication of the TEOA@AuNPs. Also, using different TEOA/HAuCl4 ratios to synthesize the AuNPs was considered (Figure S3). The results indicated that the best monodisperse AuNPs were obtained as the molar ratio equal to 20:1, which was employed throughout in this work. From the above experiments, it indicates that AuNPs could be facially fabricated using TEOA as both stabilizer and reducing agent in mild conditions. After the mixing of TEOA with HAuCl4, the solution was heated to 100 °C for 50 min; AuNPs colloidal solution was reliably obtained and it can be kept for more than 4 months. To know the effects of TEOA in the AuNPs fabrication, more investigation and the corresponding products have been studied. Figures 2A,B confirm the good crystallinity of the TEOA@ AuNPs due to its distinct lattice fringes. Several techniques were employed to characterize the surface chemistry of the products. First, the FT-IR spectra of TEOA and TEOA@ AuNPs were detected. As shown in Figure 2C, the similar profiles of the two curves mean that the major feature groups of TEOA molecules are retained in the obtained products: such as, we can observe the obvious peaks at 3313 and 1067 cm−1, which were assigned to the hydroxyl groups of TEOA.31 Compared with the original TEOA, as the relative intensity of hydroxyl groups (3313 cm−1) decreased, the peak of carbonyl groups (1748 and 1633 cm−1) appeared and the intensity significantly increased after reaction with HAuCl4. Second, the XPS was used to further provide the structure information (Figure 2D). The C 1s XPS spectrum (Figure 2E) exhibits four peaks at 284.8, 286.2, 287.2, and 288.8 eV, resulting from C−C, C−OH, C−N, and O−CO bonds, respectively,32 while for the O 1s spectrum (Figure 2F) three peaks at 532.3 and 533.3 eV, which are attributed to C−O and O−CO bonds, respectively, were observed.33 According to the contrast of XPS

In almost all previous reports for ECL systems, TEOAs were used as coreactants in the solution. However, a few used TEOA on the AuNPs surface as scaffolds for luminous molecules or proteins, and the formed composites are promising for the employment of ECL-based biosensors, which indeed limit the applications of this molecule. Therefore, to demonstrate the potential applications, the interactions of the TEOA@AuNPs and Ru(bpy)32+ were first studied. As shown in Figure 3A, the intensity of ECL is gradually enhanced with the added TEOA@ AuNPs produced by increase of temperature. Because of the smaller and monodisperse size/structure with more functional molecules, the more luminescent probes can enter into the AuNPs surface and react with them. We have chosen commonly citrate@AuNPs instead of TEOA@AuNPs in Ru(bpy)32+ aqueous solution and tried to see whether they will work. Low ECL peak is obtained at the bare GCE. At the citrate@AuNPs-modified electrode, the ECL signal increased because the AuNPs not only enlarged the specific surface area but also improved the conductivity and accelerated the electron-transfer rate. In addition, the ECL signal further increased significantly with the TEOA@AuNPs-modified electrode, revealing that TEOA@AuNPs can react with Ru(bpy)32+ to generate strong ECL and as a nanocarrier for the ECL bioassay. Mechanism of This ECL System. Ru(bpy)32+ and tri-npropylamine (TPrA) are widely used as ECL reagents, and the mechanisms have been reported.26,34 The analogous reaction D

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Figure 3. ECL intensity−potential curves of 20 μL of TEOA-Au with different reaction temperatures (A) and different nanoparticles (B) dried on GCE in in 0.1 M PBS (pH 7.0) containing 100 μM Ru(bpy)32+. Scan rate: 100 mV s−1. Scan potential: 0−1.35 V. PMT = 800 V.

schemes of Ru(bpy)32+ and TEOA are postulated in Figure 4A, and we tried to apply the EC-MS hyphenated technique to detect key intermediates to confirm the proposed mechanism.26 The mixed solution of 5 mM Ru(bpy)32+ and 100 mM TEOA (10 mM NH4Cl as the supporting electrolyte) was used in the experiment. As shown in Figure 4B, when a high potential of 1.3 V was applied, Ru(bpy)33+ could be detected with the absence of Ru(bpy)3+. When 0.8 V was applied (Figure 4B and Figure S5), the intermediate ions of Ru(bpy) 3 + , [(CH2CH2OH)2N = CHCH2OH]+ and [H2N(CH2CH2OH)2]+, could be successfully detected and Ru(bpy)33+ was absent, whereas if no potential was applied, all of these intermediate ions were absent in the MS spectra. These results indicate that the mechanism of the ECL has two routes. Under a high potential (1.3 V), Ru(bpy)33+ is a key intermediate, which will be reduced to Ru(bpy)32+* by a coreactant to generate luminescence. Under low potential (0.8 V) conditions, only TEOA is oxidized on the electrode and no Ru(bpy)33+ is produced, and a Ru(bpy)3+ is a key intermediate. These conclusions are consistent with what Bard et al. have proposed.32 Electrochemical Characterization of the ECL Sensor. As shown in Figure S6, the assembly process of the ECL sensor was characterized by CV and EIS. Compared with the bare GCE, the peak current gradually decreases after the modification of TEOA@AuNPs, anti-cTnI, BSA, and cTnI. And the peak potential difference between the anodic and the cathodic peaks increases because of the low conductivity of the composites and proteins. At last, the capture of the Ab2-RuSiO2 leads to the great decrease of the peak current and increases of the peak-to-peak separation of anodic and cathodic peak

Figure 4. (A) Mechanistic route of Ru(bpy)32+/TEOA electrochemiluminescence when a voltage of 1.3 or 0.8 V is applied. (B) Detection of the Ru(bpy)33+ ion at 1.3 V and the Ru(bpy)3+ ion at 0.8 V. The inset is the isotopic distributions of Ru(bpy)3+ (detected in red, theoretical in green).

potentials due to the nonconducting nanoparticles reducing the electrode activity. The results of EIS are consistent with the CV results as shown in Figure S6B. ECL Detection of cTnI by the Label-Free Method. The cTnI is considered as the “gold standard” marker for acute myocardial infarction (AMI).35 Here, it was chosen as a model and was investigated by the label-free ECL method and shown in Figure 5. According to the strategy listed in Scheme 1, when the immersion concentration of cTnI in the ECL sensor increased, more specific recognition sites of the binding of cTnI to anti-cTnI can be decreased in the molecular recognition progress, leading to the decrease of ECL signal. As shown in Figure 5, the ECL signals decrease gradually with increasing the concentration of cTnI ranging from 350 fg mL−1 to 350 ng mL−1. A good linear response between ECL intensity and the common logarithm of cTnI concentration was obtained, and E

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Figure 5. ECL response (A) and calibration curve of a decreased value of ECL intensity (ΔI) to logarithmic cTnI concentration (B) in 0.1 M PBS (pH 7.0) containing 100 μM Ru(bpy)32+. Scan rate: 100 mV s −1. Scan potential: 0−1.35 V. PMT = 800 V.

Figure 6. ECL response (A) and calibration curve of ECL intensity to logarithmic cTnI concentration (B) in 0.1 M PBS (pH 7.0). Scan rate: 100 mV s −1. Scan potential: 0−1.35 V. PMT = 800 V.

promising for the proposed sensing platform. Then the proposed labeling immunosensor was incubated with 3.5 pg mL−1 cTnI and cyclic scanned in 0.1 M PBS solution to monitor its stability. As shown in Figure 7, the 20 measure-

the detection limit is determined as 34 fg mL−1, which is rather sensitive compared with other reports (Table S1). ECL Detection of cTnI by the Sandwich-Type Immunosensor. The possibility of this strategy for assay sandwich-type ECL immunosensor in response to cTnI was also investigated. Recently, RuSiO2 nanoparticles have attracted much attention due to the fact that they contained large amounts of Ru(bpy)32+ to promote ECL detection.22 In this work, RuSiO2 is chosen as a model label to construct sandwichtype immunosensor in the ECL detection of cTnI. As shown in Figure 6, the ECL intensities increase gradually with the concentration of cTnI ranging from 35 fg mL−1 to 350 ng mL−1 and the detection limits down to 5.5 fg mL−1. The results indicate that the proposed strategy can effectively detect cTnI, either label-free or labeling method, with great sensitivity. Furthermore, in light of the importance of the blood cTnI assay, the specificity and selectivity of this method were investigated. Several interfering substances including BSA, IgG, glucose oxidase (GOx), human heart-type fatty-acid-binding protein (FABP), L-cysteine, and dopamine (DA) have been explored. As shown in Figure S7, only cTnI caused a significant ECL signal, revealing the high specificity of the immunosensor. The reproducibility of the proposed strategy was evaluated using cTnI at 350 pg mL−1 and 3.5 ng mL−1 through five replicative ECL measurements, and the variation coefficient was 5.9% and 6.2%, showing its good repeatability. Finally, to assess the practical application of the proposed strategy, the human blood serums have been used to study. As shown in Table S2, the results agreed well with the hospital results. Therefore, this strategy demonstrates a new ECL way to determine cTnI by label-free or labeling method, which is

Figure 7. Stability of the immunosensor toward 3.5 pg mL−1 cTnI based on continuous cyclic scanning in 0.1 M PBS solution.

ments of the electrode exhibited a coincident ECL response and the relative standard deviation (RSD) was less than 2.0%, revealing its satisfying reversibility and reliability as a sensing signal.

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CONCLUSIONS In summary, we present an eco-friendly method for one-step fabrication of coreactants-functionalized AuNPs using TEOA molecules as both stabilizing and reducing agent. Because the AuNPs possess coreactant molecules on the surface of particles, F

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(11) Zhang, Y. Y.; Feng, Q. M.; Xu, J. J.; Chen, H. Y. ACS Appl. Mater. Interfaces 2015, 7, 26307−26314. (12) Zhao, M.; Liao, N.; Zhuo, Y.; Chai, Y. Q.; Wang, J. P.; Yuan, R. Anal. Chem. 2015, 87, 7602−7609. (13) Zhang, P.; Lin, Z.; Zhuo, Y.; Yuan, R.; Chai, Y. Anal. Chem. 2017, 89, 1338−1345. (14) Hu, L.; Xu, G. Chem. Soc. Rev. 2010, 39, 3275−3304. (15) Sun, S.; Yang, Y.; Liu, F.; Fan, J.; Peng, X.; Kehr, J.; Sun, L. Dalton Trans. 2009, 7969. (16) Zhou, Z.; Shang, Q.; Shen, Y.; Zhang, L.; Zhang, Y.; Lv, Y.; Li, Y.; Liu, S.; Zhang, Y. Anal. Chem. 2016, 88, 6004−6010. (17) Collinson, M. M.; Novak, B.; Martin, S. A.; Taussig, J. S. Anal. Chem. 2000, 72, 2914−2918. (18) Wightman, R. M.; Forry, S. P.; Maus, R.; Badocco, D.; Pastore, P. J. Phys. Chem. B 2004, 108, 19119−19125. (19) Liu, X.; Shi, L.; Niu, W.; Li, H.; Xu, G. Angew. Chem., Int. Ed. 2007, 46, 421−424. (20) Zhou, W.; Gao, X.; Liu, D.; Chen, X. Chem. Rev. 2015, 115, 10575−10636. (21) Chen, L.; Zeng, X.; Si, P.; Chen, Y.; Chi, Y.; Kim, D. H.; Chen, G. Anal. Chem. 2014, 86, 4188−4195. (22) Zhou, L.; Huang, J.; Yu, B.; Liu, Y.; You, T. ACS Appl. Mater. Interfaces 2015, 7, 24438−24445. (23) Hesari, M.; Workentin, M. S.; Ding, Z. Chem. Sci. 2014, 5, 3814. (24) Cui, H.; Wang, W.; Duan, C. F.; Dong, Y. P.; Guo, J. Z. Chem. Eur. J. 2007, 13, 6975−6984. (25) Qin, X.; Liu, L.; Xu, A.; Wang, L.; Tan, Y.; Chen, C.; Xie, Q. J. Phys. Chem. C 2016, 120, 2855−2865. (26) Qiu, R.; Zhang, X.; Luo, H.; Shao, Y. Chem. Sci. 2016, 7, 6684− 6688. (27) Qin, X.; Xu, A.; Liu, L.; Deng, W.; Chen, C.; Tan, Y.; Fu, Y.; Xie, Q.; Yao, S. Chem. Commun. 2015, 51, 8540−8543. (28) Qin, X.; Xu, A.; Wang, L.; Liu, L.; Chao, L.; He, F.; Tan, Y.; Chen, C.; Xie, Q. Biosens. Bioelectron. 2016, 79, 914−921. (29) Qian, L.; Yang, X. R. Adv. Funct. Mater. 2007, 17, 1353−1358. (30) Dong, Y. P.; Chen, G.; Zhou, Y.; Zhu, J. J. Anal. Chem. 2016, 88, 1922−1929. (31) Lu, Q.; Deng, J.; Hou, Y.; Wang, H.; Li, H.; Zhang, Y.; Yao, S. Chem. Commun. 2015, 51, 7164−7167. (32) Lin, Z.; Waller, G.; Liu, Y.; Liu, M.; Wong, C. P. Adv. Energy Mater. 2012, 2, 884−888. (33) Zhao, Y.; Huang, Y.; Zhu, H.; Zhu, Q.; Xia, Y. J. Am. Chem. Soc. 2016, 138, 16645−16654. (34) Miao, W.; Choi, J. P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 14478−14485. (35) Nandhikonda, P.; Heagy, M. D. J. Am. Chem. Soc. 2011, 133, 14972−14974.

they can substantially increase ECL intensities. Such properties probably result from the specific hydroxyl structures of TEOA and their unique electron-transfer effects with the appended Au surface. Furthermore, according to conventional antibody− antigen interaction, the TEOA@AuNPs can achieve unique ECL immunoassay: the TEOA@AuNPs were used for the construction of label-free immunosensor or TEOA@AuNPs/ RuSiO2 for the labeled one. The possible mechanism of those ECL reactions has also been confirmed by the EC-MS hyphenated technique. In brief, the nontoxic, inexpensive, and cost-effective coreactant-functionalized AuNPs might provide an alternative for traditional ECL analytical strategy and open new avenues for the promising application of coreactant in the field of ECL sensing for cTnI and other biomacromolecules.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b04952. Figures S1−S7 and Tables S1−S2 with brief explanations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-10-62759394. Fax: +86-10-62751708. ORCID

Meixian Li: 0000-0001-8620-4191 Yuanhua Shao: 0000-0003-3922-6229 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support for this work from the National Key Research and Development Program of China (No. 2016YFA0201300), National Natural Science Foundation of China (21335001 and 21575006), and China Postdoctoral Science Foundation (2016M600846) are gratefully acknowledged.

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DOI: 10.1021/acs.analchem.7b04952 Anal. Chem. XXXX, XXX, XXX−XXX