Highly Chemiluminescent Magnetic Beads for Label-Free Sensing of 2

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Highly Chemiluminescent Magnetic Beads for Label-free Sensing of 2, 4, 6-Trinitrotoluene Weijun Kong, Xiaoning Zhao, Qiuju Zhu, Lingfeng Gao, and Hua Cui Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 29 May 2017 Downloaded from http://pubs.acs.org on May 30, 2017

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Analytical Chemistry

Highly Chemiluminescent Magnetic Beads for Label-free Sensing of 2, 4, 6-Trinitrotoluene Weijun Kong,† Xiaoning Zhao,‡ Qiuju Zhu,† Lingfeng Gao,† Hua Cui*† †CAS Key Laboratory of Soft Matter Chemistry, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China ‡Beijing Yunci Technology Co., Ltd. Room 308 Building 2, 8 Life Science Park Road, PKUcare Industrial Park Changping District. Beijing, 102200, P. R. China

*Hua Cui, Fax: +86-551-63600730. Email: [email protected] ABSTRACT: Until now, despite the great success acquired in scientific researches and commercial applications, magnetic beads (MBs) were used no more than a carrier in most cases in bioassays. In this wok, highly chemiluminescent magnetic beads containing N-(4-aminobutyl)-N-ethyl isoluminol (ABEI) and Co2+ (Co2+/ABEI/MBs) were firstly synthesized via a facile strategy. ABEI and Co2+ were grafted onto the surface of carboxylated MBs by virtue of carboxyl group and electrostatic interaction. The asprepared Co2+/ABEI/MBs exhibited good paramagnetic, satisfactory stability, and intense CL emission when reacted with H2O2, which was more than 150 times than that of ABEI functionalized MBs. Furthermore, it was found that 2, 4, 6-trinitrotoluene (TNT) aptamer could attach to the surface of Co2+/ABEI/MBs via electrostatic interaction and coordination interaction between TNT aptamer and Co2+, leading to a decrease in CL intensity due to that catalytic site Co2+ was blocked by the aptamer. In the presence of TNT, TNT would bind strongly with TNT aptamer and detach from the surface of Co2+/ABEI/MBs, resulting in partial restore of CL signal. Accordingly, label-free aptasensor was developed for the determination of TNT in the range of 0.05-25 ng/mL with a detection limit of 17 pg/mL. This work demonstrates that Co2+/ABEI/MBs is easily connected with recognition biomolecules, which is not only a magnetic carrier but also a direct sensing interface with excellent CL activity. It provide a novel CL interface with magnetic property which is easily to separate analytes from sample matrix to construct label-free bioassays.

For past decades, magnetic materials have attracted considerable attention due to their excellent performance in the fields such as bioassays,1-3 drug delivery,4-6 electronics,7,8 magnetic resonance imaging,9-11 and environmental remediation.12-15 As a typical magnetic materials, magnetic beads (MBs) can be concentrated, trapped or used in rapid separation by applying a magnetic field. Owing to its outstanding properties, such as high loading capacity, high magnetic susceptibility and good biocompatible, MBs have extensively used for label-based bioassays such as immunoassays16-19 and DNA assays.20-23 Particularly, label-based chemiluminescence (CL) technology with MBs has been successfully commercialized in immunoassays. However, they still suffer from complicated labeling, purification and washing procedures and need secondary antibody, which makes them labor-intensive, time-consuming and expensive. In addition, the conjugation chemistry involved in the labeling process increases the risk of damaging the activities of the biomolecules such as antibody and enzyme. In recent years, label-free bioassays based on signal change initiated by the specific interaction between recognition element and target have attracted increasing research interests. They are simple, time-saving, and low-cost, avoiding tedious labeling procedures. The label-free bioassays with CL detection is one of most promising method for the determination of small molecules and biomacromolecules since CL detection has advantages of high sensitivity, wide linear range and simple instrumentation. They often need to construct a CL inter-

face to produce analytical signal. Earlier work demonstrated that CL functionalized nanomaterials such as CL reagent/metal ion (catalyst) bifunctionalized gold nanoparticles,24,25 graphene oxide26 and multiwalled carbon nanotubes27 are good interface for label-free bioassays. However, in most of cases, such CL functionalized nanomaterials need to be further immobilized on a carrier such as microplate and electrode to construct a CL interface for label-free bioassays so that the separation can be carried out after they react with recognition biomolecules and analytes in samples. In this work, we report for the first time that MBs are employed as immobilized substrate for CL reagent and catalyst metal ions, which makes them not only a conventional magnetic carrier but also a novel material with outstanding CL activity. N-(4-aminobutyl)-N-ethyl isoluminol (ABEI) and Co2+ were directly grafted on the surface of carboxylated MBs to form Co2+/ABEI functionalized MBs (Co2+/ABEI/MBs) by virtue of carboxyl group and electrostatic interaction. The asprepared Co2+/ABEI/MBs were characterized by scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS) and inductively coupled plasma atomic emission spectrometry (ICP-AES). The CL behavior of Co2+/ABEI/MBs were studied when reacting with H2O2. The as-prepared Co2+/ABEI/MBs with good paramagnetic property exhibited excellent CL activity. Furthermore, it was found that 2, 4, 6trinitrotoluene (TNT) aptamer (a small peptide) could attach

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Scheme 1. Schematic illustration for fabrication of Co2+/ABEI/MBs

to the surface of Co2+/ABEI/MBs, leading to a decrease in CL intensity. The interaction mechanism between small peptide and Co2+/ABEI/MBs was explored. In the presence of TNT, TNT would bind strongly with TNT aptamer and detach from the surface of Co2+/ABEI/MBs, resulting in partial restore of CL signal. Accordingly, label-free aptasensor was developed for the determination of TNT. The optimized conditions and the analytical performance of the proposed aptasensor for TNT were investigated. Finally, the applicability of the developed aptasensor in real water samples was also studied.

EXPERIMENTAL SECTION Reagents and materials. Carboxylated MBs with average size of ~55 µm was provided by Beijing Yunci Technology Co., Ltd. (China). ABEI was obtained from TCI (Japan), a stock solution of ABEI (4 mM) was prepared by dissolving ABEI in NaOH solution (0.1 M) and was kept at 4 °C. The sequence of the TNT peptide aptamer (TNT-apt) is (N terminus) Trp-His-Trp-Gln-Arg-Pro-Leu-Met-Pro-Val-Ser-Ile-Lys (C terminus), which was purchased from GL Biochem Co. Ltd. (China). 1-(3-Dimethylaminopropyl)-3ethylcarbodiimidehy-drochloride (EDC), Nhydroxysuccinimide (NHS), 2,4,6-trinitrotoluene, nitrobenzene (NB), 2-nitrotoluene (2-NT), 3-nitrotoluene (3-NT), and 2,4-dinitrotoluene (2,4-DNT) were purchased from Aladdin Reagent (China). All other reagents were of analytical grade. Ultrapure water was prepared with a Milli-Q system (Millipore, France) and used throughout. Activation buffer and washing buffer for EDC and NHS was 25 mM 2-(Nmorpholino) ethanesulfonicacid (MES) buffer at pH 5.5. Dilution buffer for TNT-apt and TNT was 0.01 M phosphate buffer saline at pH 7.0 (PBS). All the buffers were sterilized at 121 °C for 20 min in an autoclave sterilizer. Synthesis of Co2+/ABEI/MBs. In a typical synthesis, 6.0 mL of carboxylated MBs was washed thrice with MES buffer after ultrasonicated for 5 min. Subsequently, the suspension was dispersed in 6.0 mL of MES buffer containing 200 mg of EDC and 200 mg of NHS under fiercely stirring at room temperature. Following 30-min activation, the mixture was washed twice with washing buffer, followed by the addition of 200 µL of ABEI (4 mM) stock solution under continuous stirring. After 12-h reaction at room temperature, the resulted ABEI/MBs was washed thrice and resuspended in ultrapure water. 1.0 mL of Co2+ aqueous solution (10 mM, optimized in

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Figure S1) was added to 0.5 mL of ABEI/MBs suspension and incubated at room temperature for 20 min under constant shaking. Finally, the resulting suspension was washed twice and dispersed in 2.0 mL water or other medium for further use. Characterization. The morphology of Co2+/ABEI/MBs was characterized by SEM on a JEM-2010 scanning electron microscope (Hitachi, Japan). XPS spectrum was obtained from an X-ray photoelectron spectroscopy consisting of an ESCALABMK II electron spectrograph (VG Scientific, UK) and an Al Ka radiation as the X-ray source. ICP-AES measurement was carried out on an Optima 7300 DV plasma atomic emission spectrometry (PerkinElmer, USA). CL spectra were measured on an F-7000 FL spectrometer (Hitachi, Japan) operated with the lamp off. CL Measurements.CL was measured with a centro LB960 microplate luminometer (Berthold, Germany). In a typical assay, 100 µL of Co2+/ABEI/MBs and control substances were added into each well of 96-well microplate, respectively, and then 100 µL of H2O2 (100 µM) solution dissolving in 0.1 M NaOH (pH 13.0) was injected into each well. While the H2O2 solution was injected into the system, the light emission was collected by the microplate luminometer and recorded as CL kinetic curves. The measurement time was optimized as 30 s with a time interval of 0.1 s. Detection of TNT. First, TNT-apt/Co2+/ABEI/MBs were synthesized by adding 800 µL of Co2+/ABEI/MBs into 2.0 mL of PBS buffer (pH 7.0) containing TNT-apt (1 µg/mL) under constant shaking. After 4-h reaction at room temperature, the suspension was washed twice and dispersed with 1.6 mL of PBS buffer for further use. For the CL detection of TNT, 200 µL of TNT with different concentrations (0.05, 0.1, 0.5, 1, 5, 10, 25 ng/mL) was incubate with 200 µL of the as-prepared TNT-apt/Co2+/ABEI/MBs suspension under gentle shaking at 37 °C for 30 min, followed by washing twice with PBS buffer via magnetic separation and dispersed with water. Afterward, 100 µL of H2O2 in 0.1 M NaOH solution (pH 13.0) was injected into the microwell of 96-well plate with 100 µL aqueous dispersion of the as-prepared TNT-apt/Co2+/ABEI/MBs hybrids after interacted with TNT, and then the CL kinetic curves of each well were recorded.

RESULTS AND DISCUSSION Synthesis and Characterization of Co2+/ABEI/MBs. As shown in Scheme 1, a simple strategy without extra chelators for the preparation of Co2+/ABEI/MBs was developed. To begin with, the mixture of ABEI solution and EDC-activated MBs suspension were violently stirred to prepare ABEI/MBs. Subsequently, the as-prepared ABEI/MBs suspension was mixed with 10 mM Co2+ aqueous solution and incubate for 20 min under constant shaking. After washing, Co2+/ABEI/MBs was obtained. To confirm the successful preparation of Co2+/ABEI/MBs, The as-prepared Co2+/ABEI/MBs was investigated by SEM, XPS, and ICP-AES. The morphology of Co2+/ABEI/MBs was characterized by SEM. As shown in Figure 1A, the carboxylated magnetic beads have an average diameter of ~55 µm. After coating with the ABEI and Co2+, ABEI/MBs (Figure 1B) and Co2+/ABEI/MBs (Figure 1C) did not show much difference from MBs in morphology. And both the as-prepared Co2+/ABEI/MBs and carboxylated magnetic beads showed excellent monodispersion. As shown in Figure 1D, after modi-


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Figure 1. SEM images of (A) MBs (B) ABEI/MBs (C) Co2+/ABEI/MBs (D) Photograph of MBs andCo2+/ABEI/MBs under external magnetic field. -fication, there is no significantly changes of magnetism observed from the Co2+/ABEI/MBs to MBs, the Co2+/ABEI/MBs could be separated quickly under external magnetic field. The XPS results of the as-prepared Co2+/ABEI/MBs are shown in Figure 2A. Compared the XPS survey of ABEI/MBs with that of MBs, there was a new component of N 1s at 398.5eV, indicating the existence of ABEI molecules on the surface of ABEI/MBs composites. In addition, the XPS peaks located at around 780 eV attributed to Co 2p were observed in the XPS survey of Co2+/ABEI/MBs, demonstrating that there was cobalt element in Co2+/ABEI/MBs. Figure 2B, 2C and 2D shows the C 1s, N 1s and Co 2p spectra of Co2+/ABEI/MBs, respectively. The C 1s spectrum of Co2+/ABEI/MBs was curvedfitted into five peaks at 284.7, 285.5, 286.1, 287.3 and 289.3 eV. The peak at 289.3 eV corresponding to the carbon atom in the -COOH group on the surface of MBs was observed. The component at 287.3 eV was due to the CO-NH group which come from the amidation reaction between ABEI and the COOH group on MBs. The component at 286.1 eV was associated with the carbon atom in the C-N group. Similarly, the N 1s spectrum of Co2+/ABEI/MBs was curved-fitted into two components at 399.7 and 401.8 eV. The peak at around 399.7 was attributed to the nitrogen atoms in the N-C while the component at 401.8 eV was due to NH-C=O group. Such peaks were come from the immobilized ABEI. Besides, the Co 2p spectrum of Co2+/ABEI/MBs was curved-fitted into four components at 797.3, 781.6, 802.4 and 786.1 eV. The components around 797.3 and 781.6 eV were due to the cobalt element in the Co-O group, which indicating the coordination interactions between Co2+ and the carboxyl groups on MBs. Meanwhile, the components at 802.4 and 785.1 eV were attributed to the multielectron excitation intense satellite lines of the high spin cobalt (II) core. The evidence above revealed that the Co element on Co2+/ABEI/MBs was divalent cobalt ion. The C 1s, N 1s and the Co 2p spectrum of Co2+/ABEI/MBs indicated that ABEI molecules and Co2+ coexisted on the surface of Co2+/ABEI/MBs. The composition of the as-prepared Co2+/ABEI/MBs hybrids was also investigated by ICP-AES. The results demonstrated that there was no Co element in MBs and ABEI/MBs samples, while Co element existed with a concentration of 551.6 µg/g in Co2+/ABEI/MBs.

Figure 2. (A) Survey XPS data of (a) MBs, (b) ABEI/MBs and (c) Co2+/ABEI/MBs. The deconvolution of (B) C1s, (C) N1s and (D) Co2p spectra of Co2+/ABEI/MBs. Assemble mechanism. The assembling mechanism of Co2+/ABEI/MBs was studied. First, ABEI was immobilized on the surface of carboxylated MBs via a typical EDC/NHS coupling reaction. It is obviously that the carboxyl full-covered MBs was negatively charged around pH 7.0. Therefore, positively charged Co2+ could be easily adsorbed on the surface of MBs through electrostatic interaction. Zeta-potential measurements were performed to study the contribution of electrostatic interaction (Table S1). The measured zeta-potentials of MBs, ABEI/MBs and Co2+/ABEI/MBs in aqueous solution were -23.7, -12.5 and -7.5 mV, respectively. Compared with MBs, the zeta-potentials of ABEI/MBs were positive-shifted by 11.2 mV. As we mentioned above, ABEI was immobilized through EDC/NHS coupling reaction. The formation of amide bond will cost equivalent carboxyl group, reducing the electrical negativity of MBs. Moreover, compared with ABEI/MBs, zeta-potentials of Co2+/ABEI/MBs were further positiveshifted by 5.0 mV, suggesting that positively charged Co2+ was assembled on the surface of negatively charged ABEI/MBs by electrostatic interaction. In addition, according to the XPS results shown in Figure 2D, Co2+ could also be attached onto ABEI/MBs through coordination interactions between Co2+ and the carboxyl groups from MBs owing to the Co-O bonding observed in XPS spectra. Therefore, in this case, Co2+ could be adsorbed onto ABEI/MBs by means of not only electrostatic interaction but also coordination interactions. CL Property of Co2+/ABEI/MBs. Co2+ has been intensively used as a catalyst to enhance CL signal in the luminol-H2O2 system. Considering the existence of Co2+ on the Co2+/ABEI/MBs, the as-prepared hybrids which contain ABEI (an analogue of luminol) might have outstanding CL activity. The CL behavior of the as-prepared Co2+/ABEI/MBs was investigated by static injection on a microplate luminometer. As shown in Figure 3, a strong CL emission signal is observed when 100 µL of 0.1 M H2O2 in 0.1 M NaOH was injected to 100 µL of Co2+/ABEI/MBs. In comparison, the CL intensity of Co2+/ABEI/MBs was about 150 times higher than that of ABEI/MBs under the same conditions. In spite of the increment on CL signal, after the addition of free Co2+ (the same amount as Co2+ on the surface of Co2+/ABEI/MBs) to ABEI/MBs, the CL intensity (Figure 3, inset A) was still 6 times weaker than that of Co2+/ABEI/MBs. Thus, Co2+/ABEI/



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Figure 3. A comparison of CL kinetic curves of Co2+/ABEI/MBs (curve a), ABEI/MBs (curve b) with free Co2+, ABEI/MBs (curve c), MBs (curve d). The inset A is the magnification of ABEI/MBs. The inset B is the CL spectra of Co2+/ABEI/MBs (black line) and ABEI (orange line). Reaction condition: 0.1 M H2O2 in 0.1 M NaOH solution. MBs demonstrated excellent CL activity. As displayed in Figure 3, inset B, the CL spectra of Co2+/ABEI/MBs showed a peak centered around 450 nm, which was in accordance with that of the characteristic emission wavelength of ABEI CL reaction. The result indicated that the CL emission above resulted from the reaction of ABEI molecules on the surface of Co2+/ABEI/MBs with H2O2. Effect of Size of Magnetic Beads and pH of H2O2 on CL Activity. The effect of the size of magnetic beads (200 µm, 50µm and 500 nm) on the CL activity was examined as shown in Figure S2. 50 µm MBs showed better CL activity than 200 µm and 500 nm magnetic beads. Therefore, 50µm MBs were selected for further experiments. Besides, the pH value was a crucial factor influencing the CL intensity. In order to obtain the optimal pH for CL emission of Co2+/ABEI/MBs, the effect of the pH of H2O2 on the CL intensity of Co2+/ABEI/MBs was studied. In this study, 0.1 M H2O2 with different pH values were obtained by diluting H2O2 with Britton-Robinson buffer (pH 6.1-11.2) and NaOH solution (pH 12.0-13.0). As seen in Figure S3, the CL intensity rises sharply with increment of the pH and reached the maximum when the pH value was 13.0. This may be due to that appropriate high pH could promote the dissociation of H2O2 to form more intermediate radicals, leading to an increase in CL intensity. Effect of Metal Ions. Earlier studies demonstrated that various metal ions including Co2+, Cu2+, Cr3+, Fe2+, Ni2+, Pb2+ and Ce2+ could catalyze the CL reactions of luminol and its analogues with H2O2.In this study, Co2+ was chosen as a model of metal ion to synthesize metal ion/ABEI/MBs. The effect of other metal ions on the CL efficiency of functionalized MBs was also investigated. A series of functionalized MBs were synthesized with different transition metal ions, such as Co2+, Cu2+, Cr3+, Fe2+, Ni2+, Pb2+ and Ce2+. The CL behaviors of various as-synthesized functionalized MBs were studied as shown in Figure 4. Functionalized MBs synthesized with Co2+ and Cu2+ exhibited outstanding CL activity, showing over 2 orders of magnitude higher CL intensity than that of ABEI/MBs. The CL intensity follows the order: Co2+> Cu2+> Cr3+> Fe2+> Ni2+> Pb2+> Ce2+. Co2+/ABEI/MBs synthesized with Co2+ exhibited strongest CL intensity among all the tested

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Figure 4. CL kinetic curves for reaction of various metal ion/ABEI/MBs with H2O2. Reaction conditions: 100 µL 0.1 M H2O2 in 0.1 M NaOH (pH13.0) was injected into 100 µL Co2+/ABEI/MBs water dispersion in a microwell. metal ions. This may be owing to that Co2+ has the strongest catalytic effect on ABEI CL reactions. Proposed CL Mechanism. The outstanding CL efficiency of Co2+/ABEI/MBs on the ABEI-H2O2 CL system might be owing to the powerful catalytic ability of immobilized Co2+ and the effect of the carboxylated MBs. Our previous work demonstrated that CL catalysis and enhancement of the ABEIH2O2 system related to the generation of oxygen-related radicals including HO●, O2●− and other radical derivatives. Therefore, in order to study the CL mechanism and identify the radical species involved in the CL reaction process, the effects of the radical scavengers and O2 were investigated. As seen in Figure S4A and S4B, both CL intensities significantly decreased with increasing the concentrations of thiourea and superoxide dismutase (SOD), respectively, demonstrating that HO● and O2●− were involved in the reaction. Furthermore, as the Figure S2C shown, the CL intensity decreased in a nitrogen-saturated solution, while increased in an oxygen-saturated solution, indicating that the dissolved oxygen (O2) also play an important part in this CL system. Overall, according to previous studies on Co2+ catalyzed ABEI-H2O2 system,24 it is suggested that heterogeneous catalysis of Co2+ immobilized on the MBs facilitated the formation of HO● radicals, which reacted with ABEI also immobilized on the surface of MBs and the dissolved O2 to yield ABEI●− and O2●−. Finally, ABEI●− reacted with O2●− to produce strong CL emission. Both immobilization of Co2+ and ABEI highly concentrated Co2+ and ABEI molecules on the surface of MBs and the surface of MBs stimulated electron transfer, which also contributed to strong light emission. The reason for that Co2+ functionalized MBs exhibit much better CL activity than those of MBs functionalized with other metal ions in the CL system could be as follows. The CL reaction occurred under highly alkaline conditions and hydroxide ions could react with metal ions to yield precipitates, which may inhibit the catalytic decomposition of hydrogen peroxide.28 Among these metal ions, Co2+ readily complex with HO2− to form a stable complex (Co2+-HO2−) compared with other metal ions, subsequently leading to the formation of OH●. Thus, Co2+ functionalized MBs exhibited the best CL activity. TNT detection. The interaction of Co2+/ABEI/MBs with small peptides was explored by taking TNT-apt as a model peptide. It was found that TNT-apt can be attached onto


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Figure 5. (A) Schematic illustration of proposed label-free aptasensor for TNT. (B) Comparison of CL kinetic curves of Co2+/ABEI/MBs (curve a), TNT-apt/Co2+/ABEI/MBs in the presence of TNT (curve b), TNT-apt/Co2+/ABEI/MBs in the absence of TNT (curve c). Reaction condition: 0.1 M H2O2 in 0.1 M NaOH solution. Co2+/ABEI/MBs and significantly quenching CL signal (Figure 5B). Zeta-potential was positive-shifted from -7.5 mV to 2.8 mV after positive-charged TNT-apt was assembled on the surface of negatively Co2+/ABEI/MBs (Table S1). The results demonstrated that TNT-apt was connected to the surface of Co2+/ABEI/MBs via electrostatic interaction. On the other hands, TNT-apt is a small peptide containing several carboxyl and amino group. It was reported that amino acid was readily coordinate with Co2+.29 In this case, TNT-apt may coordinate with Co2+ to form a complex, which also contributed the interaction between TNT-apt and Co2+/ABEI/MBs. The CL quenching effect by TNT-apt may be due to catalytic site Co2+ was blocked by the small peptide TNT-apt, leading to a decrease in CL intensity. It is known that the binding between aptamers and its corresponding ligands are usually more stable than nonspecific bindings. Thus, TNT-apt may be detached from the surface of Co2+/ABEI/MBs in the presence of TNT due to stronger binding force between TNT-apt and TNT than that of TNT-apt and Co2+/ABEI/MBs. As seen in Figure 5B, a noticeable increase in CL response of Co2+/ABEI/MBs was observed in the presence of TNT, indicating that TNT-apt was partially removed from the surface of Co2+/ABEI/MBs. This was confirmed by negative-shifted zeta-potential from 2.8 to 2.5 after adding TNT (Table S1). As illustrated in Figure 5A, a simple CL label-free aptasensor for the sensitive and selective detection of TNT was developed based on the competitive binding of TNT with Co2+/ABEI/MBs for TNT-apt. Firstly, TNT-apt was adsorbed on the surface of Co2+/ABEI/MBs via electrostatic and coordinate interaction. Then, the attached TNT-apt was released to liquid phase in the presence of TNT, which could specifically bind with TNT-apt. As the number of TNT-apt on Co2+/ABEI/MBs decreased, the CL signal restored partially. In addition, it was found that small amount of Co2+ with TNT-apt/ composites also detached from magnetic beads, which may also contribute to the partial restore of CL signal (Table S2). Under the optimized experimental conditions, the working curve of the proposed aptasensor was established using different concentrations of TNT. As shown in Figure 6, the CL intensity increased linearly when the concentrations of TNT increased from 0.05 to 25 ng/mL. The linear regression equation was ∆I = 11242 log C + 16426, and the correlation coefficients (R2) was 0.9805. The limit of detection for TNT was 17 pg/mL at a signal to noise ratio of 3. Moreover, the relative standard derivations (RSDs) of five replicate determinations of TNT at 0.1 ng/mL, 1.0 ng/mL and 10 ng/mL were all less than 8.5 %. The results indicate that the proposed aptasensor has good repeatability for TNT detection.

A comparison of analytical performance between the proposed method and other previously reported label-free bioassays for TNT is made. The results listed in Table S3 show that the sensitivity of the present aptasensor is better than that of most earlier reported methods except for two ECL sensors with ECL-functionalized graphene hybrids. However, the complicated multi-step purification processes involved in the two ECL sensors were labor-intensive and time-consuming. With the aid of the facile magnetic separation process, the proposed label-free aptasensor shows high sensitivity for TNT without complicated purification procedures. Specificity. The CL aptasensor was assumed to be highly selective towards TNT relied on TNT-aptamer recognition. To examine the specificity, NB, 2-NT, 3-NT and 2,4-DNT at 5.0 ng/ml instead of TNT were assayed with the proposed method, and the obtained CL signals were compared with TNT at the same concentration. The mixture composed of the four interfering compounds at 5.0 ng/ml was also assayed under the same conditions. As seen in Figure 6, inset, only TNT exhibited strong ∆I, while the four interfering compounds (TNT analogs) showed weak ∆I. Moreover, the mixture showed a similar ∆I with that of TNT. The results above revealed satisfactory specificity of the proposed aptasensor for TNT detection. TNT determination in real samples. To evaluate the applicability of the proposed method in practical samples, eight

Figure 6. Linear relationship between CL response and TNT concentration. The inset shows CL responses of TNT and different interfering species (5.0ng/ml).Reaction conditions: 100 µL 0.1 M H2O2 or various interferents in 5.0ng/ml 0.1 M NaOH solution (pH 13.0) was injected into 100 µL Co2+/ABEI/MBs water dispersion.



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Table 1. Determination of TNT in Water Samples Using the Proposed Method. Water samples

TNT found (ng/mL)

TNT added (ng/mL)

Total TNT detected (ng/mL)

Recovery (%)

1 2 3 4 5 6 7 8

4.8 5.4 5.1 4.5

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

5.3 ± 0.8 4.6 ± 1.6 5.0 ± 1.2 4.7 ± 0.5 10.1 ± 1.0 10.8 ± 1.4 9.6 ± 2.1 9.6 ± 0.6

106.0 92.0 100.0 94.0 106.0 108.0 90.0 102.0

The water samples obtained from Ying River were diluted 120× by water. (n=5) water samples were spiked with TNT standard solutions at 5.0 ng/ml and determined with the proposed method. The water samples were detected after repeated centrifugation to remove the sediment. Sample 1-4 were collected at four locations of the Swan Lake, and sample 5-8 were collected at four locations of the Ying River near a TNT factory. The water samples obtained from Ying River were diluted 120 times by water prior to assay. The results listed in Table 1 showed acceptable recoveries ranging from 90% to 108%, demonstrating its reliability and application potential in real samples.

CONCLUSION Highly chemiluminescent Co2+/ABEI/MBs with good magnetic property have been successfully synthesized via a facile strategy. ABEI was immobilized on the surface of carboxylated MBs via a typical EDC/NHS coupling reaction and Co2+ was directly grafted onto the surface of ABEI/MBs through coordination reaction and electrostatic interaction. The asprepared Co2+/ABEI/MBs exhibited good paramagnetic, satisfactory stability, and intense CL emission. The CL intensity of as-prepared Co2+/ABEI/MBs was about 150 times higher than that of ABEI/MBs when reacted with H2O2 in alkaline solution. Moreover, it was found that TNT-apt could be attached onto Co2+/ABEI/MBs via electrostatic and coordination interaction, resulting in quench of CL signal. The CL quenching effect by TNT-apt may be due to catalytic site Co2+ was blocked by the small peptide TNT-apt, leading to a decrease in CL intensity. In the presence of TNT, TNT-apt detached from the surface of Co2+/ABEI/MBs due to strong specific interaction between TNT-apt and TNT. On this basis, by virtue of Co2+/ABEI/MBs as a sensing platform, a sensitive and selective label-free aptasensor for TNT was established based on the competitive binding of TNT with Co2+/ABEI/MBs for TNT-apt. The aptasensor was also successfully applied in real samples, revealing the great potential of Co2+/ABEI/MBs in bioassays. This work demonstrates that Co2+/ABEI/MBs is easily connected with recognition biomolecules, which is not only a magnetic carrier but also a direct sensing interface with excellent CL activity. It provide a novel CL interface with magnetic property which is easily to separate analytes from sample matrix to construct label-free bioassays.

ASSOCIATED CONTENT Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website. Additional information as noted in text, including: effect of Co2+ concentration on CL response; measurement of zetapotentials; effect of size of magnetic beads on CL activity; effect of pH on CL response; effects of radical scavengers, N2 and O2 on CL intensity; discussion of sensing process; a comparison of TNT aptasensor with previous methods.

AUTHOR INFORMATION Corresponding Author *Tel: +86-551-63600730 Fax: +86-551-63600730 Email: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The support of this research by the National Key Research and Development Program of China (Grant No. 2016YFA0201300) and the National Natural Science Foundation of China (Grant Nos. 21527807 and 21475120) are gratefully acknowledged.

REFERENCES (1) Pal, M.; Lee, S.; Kwon, D.; Hwang, J.; Lee, H.; Hwang, S.; Jeon, S. Anal. Chim. Acta 2017, 952, 81-87. (2) Liu, F.; Kc, P.; Zhang, G.; Zhe, J. Anal. Chem. 2016, 88, 711-717. (3) Piovesana, S.; Capriotti, A. L.; Cavaliere, C.; Ferraris, F.; Iglesias, D.; Marchesan, S.; Lagana, A. Anal. Chem. 2016, 88, 12043-12050. (4) Guo, Y. S.; Li, S.; Wang, Y. J.; Zhang, S. S. Anal. Chem. 2017, 89, 2267-2274. (5) Mura, S.; Nicolas, J.; Couvreur, P. Nat. Mater. 2013, 12, 991-1003. (6) Torchilin, V. P. Nature reviews. Drug discovery 2014, 13, 813-827. (7) Tartaj, P.; Morales, M. D.; Veintemillas-Verdaguer, S.; Gonzalez-Carreno, T.; Serna, C. J. J. Phys. D: Appl. Phys. 2003, 36, R182-R197. (8) Gan, W. T.; Gao, L. K.; Xiao, S. L.; Zhang, W. B.; Zhan, X. X.; Li, J. J. Mater. Sci. 2016, 52, 3321-3329. (9) Carrouee, A.; Allard-Vannier, E.; Meme, S.; Szeremeta, F.; Beloeil, J. C.; Chourpa, I. Anal. Chem. 2015, 87, 1123311241. (10) Eto, H.; Hyodo, F.; Nakano, K.; Utsumi, H. Anal. Chem. 2016, 88, 2021-2027. (11) Huang, P.; Qian, X. Q.; Chen, Y.; Yu, L. D.; Lin, H.; Wang, L. Y.; Zhu, Y. F.; Shi, J. L.J. Am. Chem. Soc. 2017, 139, 1275-1284. (12) Naous, M.; Garcia-Gomez, D.; Lopez-Jimenez, F. J.; Bouanani, F.; Lunar, M. L.; Rubio, S. Anal. Chem. 2017, 89, 1353-1361. (13) Linley, S.; Leshuk, T.; Gu, F. X. ACS Appl. Mater. Interfaces 2013, 5, 2540-2548. (14) Tucek, J.; Kemp, K. C.; Kim, K. S.; Zboril, R. Acs Nano 2014, 8, 7571-7612.


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Page 7 of 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(15) Chalasani, R.; Vasudevan, S. Acs Nano 2013, 7, 40934104. (16) Lai, W. Q.; Wei, Q. H.; Xu, M. D.; Zhuang, J. Y.; Tang, D. P. Biosens. Bioelectron. 2017, 89, 645-651. (17) Chang, H. H.; Lv, J.; Zhang, H. C.; Zhang, B.; Wei, W. L.; Qiao, Y. Biosens. Bioelectron. 2017, 87, 579-586. (18) Otieno, B. A.; Krause, C. E.; Jones, A. L.; Kremer, R. B.; Rusling, J. F. Anal. Chem. 2016, 88, 9269-9275. (19) Lin, X. C.; Wang, X. N.; Liu, L.; Wen, Q.; Yu, R. Q.; Jiang, J. H. Anal. Chem. 2016, 9881-9884. (20) Ngo, H. T.; Gandra, N.; Fales, A. M.; Taylor, S. M.; Vo-Dinh, T. Biosens. Bioelectron. 2016, 81, 8-14. (21) Kurt, H.; Yuce, M.; Hussain, B.; Budak, H. Biosens. Bioelectron. 2016, 81, 280-286. (22) Oishi, M.; Sugiyama, S. Small 2016, 12, 5153-5158.3 (23) Jin, M. L.; Liu, X.; van den Berg, A.; Zhou, G. F.; Shui, L. L. Nanotechnology 2016, 27, 335102. (24) Liu, M. X.; Zhang, H. L.; Shu, J. N.; Liu, X. Y.; Li, F.; Cui, H. Anal. Chem. 2014, 86, 2857-2861. (25) Li, F.; Liu, Y. T.; Zhuang, M.; Zhang, H. L.; Liu, X. Y.; Cui, H. Appl. Mater. Interfaces 2014, 6, 18104-18111. (26) Liu, X. Y.; Han, Z. L.; Li, F.; Gao, L. F.; Liang, G. L.; Cui, H. Appl. Mater. Interfaces 2015, 7, 18283-18291. (27) Zhang, H. L.; Cui, H. Nanoscale 2014, 6, 2563-2566. (28) Li, Q.; Li, F.; Shen, W.; Liu, X. Y.; Cui, H. J. Mater. Chem. C 2016, 4, 3477-3484. (29) Sharma, R.; Nagar, M.; Agarwal, M.; Sharma, H. J. Enzyme Inhib. Med. Chem. 2009, 24, 197-2.


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For TOC only


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Figure 5. (A) Schematic illustration of proposed label-free aptasensor for TNT. (B) Comparison of CL kinetic curves of Co2+/ABEI/MBs (curve a), TNT-apt/Co2+/ABEI/MBs in the presence of TNT (curve b), TNTapt/Co2+/ABEI/MBs in the absence of TNT (curve c). Reaction condition: 0.1 M H2O2 in 0.1 M NaOH solution. 286x82mm (300 x 300 DPI)



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TOC 64x47mm (300 x 300 DPI)


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Highly Chemiluminescent Magnetic Beads for Label-Free Sensing of 2

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