Label-Free Electrochemiluminescence Aptasensor for 2,4,6

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A label-free electrochemiluminescence aptasensor for 2,4,6-trinitrotoluene based on bilayer structure of luminescence functionalized graphene hybrids Guixin Li, Xiuxia Yu, Danqing Liu, Xiaoying Liu, Fang Li, and Hua Cui Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02913 • Publication Date (Web): 14 Oct 2015 Downloaded from http://pubs.acs.org on October 20, 2015

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Analytical Chemistry

A label-free electrochemiluminescence aptasensor for 2,4,6trinitrotoluene based on bilayer structure of luminescence functionalized graphene hybrids ⊥

⊥

Guixin Li‡† , Xiuxia Yu†§ , Danqing Liu†, Xiaoying Liu†, Fang Li†, 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 ‡ Laboratory for Pollution Monitoring and Control, School of Chemistry and Chemical Engineering, Xinjiang Normal University, Urumqi, Xinjiang 830054, P.R. China § College of Marine Science, Shandong University at Weihai, Weihai 264209, China

*Corresponding author. Prof. H. Cui, Tel: +86-551-63600730 Fax: +86-551-63600730 Email: [email protected]

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ABSTRACT: The electrochemiluminescence (ECL) behavior of N-(aminobutyl)-N-(ethylisoluminol)/hemin dual-functionalized graphene hybrids (A-H-GNs) and luminol functionalized silver/graphene oxide composite (luminol-AgNPs-GO) was investigated under cyclic voltammetry and pulse potential. It was found that A-H-GNs and luminol-AgNPs-GO exhibited excellent ECL activity. On this basis, a label-free ECL aptasensor for 2,4,6-trinitrotoluene (TNT) detection was developed based on bilayer structure of luminescence functionalized graphene hybrids consisting of A-H-GNs and luminol-AgNPs-GO. First, positively charged chitosan coated A-H-GNs were modified on the surface of indium-doped tin oxide electrode by simple dripping and drying in the air; after that, the modified electrode was immersed in negatively charged luminol-AgNPs-GO modified with aptamer (aptabiotin-SA-luminol-AgNPs-GO) to form apta-biotin-SA-luminol-AgNPs-GO/CS-A-H-GNs/ITO electrode (i.e. aptasensor) by electrostatic interaction. In the presence of TNT, a remarkable decrease in ECL signals was observed due to the formation of aptamer–TNT complex. TNT could be detected based on the inhibition effect. The aptasensor exhibits a wide dynamic range from 1.0×10−12–1.0×10−9 g/mL with a low detection limit of 6.3×10−13 g/mL for the determination of TNT, which is superior to most previously reported bioassays for TNT. Moreover, the proposed aptasensor has been successfully applied to the detection of TNT in environmental water. It is sensitive, selective, and simple, avoiding complicated labeling and purification procedures. Due to the wide target recognition range of aptamer, this strategy provides a promising way to develop new aptasensor for other analytes. 2,4,6-trinitrotoluene (TNT) is a dual use compound that has found applications both for peaceful industrial and military/terrorist purposes. It is used as ammunition/explosive and in the manufacturing of dyes, plasticizers, herbicides, etc. Because of its persistence, traces of this harmful chemical can be found in soil and groundwater in the vicinity of manufacturing plants and areas with past or current military activities for a long time. TNT contamination has a serious adverse effect on all life forms of our ecosystem.1-3 In humans, TNT can cause anemia, abnormal liver function, skin irritation and weakened immune system. It has been classified as a potential carcinogen by the U.S. Environmental Protection Agency (EPA).4 Due to its contamination on the environment and the risk of human health as well as the growing homeland security concerns, selective and ultrasensitive detection of TNT has attracted increasing attention.5-9 Over the past years, several techniques have been employed for the detection of TNT at trace amount, including surface plasmon resonance,10,11 fluorescence,12,13 colorimetry,14 electrochemistry,15-18 chemiluminescence (CL)19 and electrochemiluminescence (ECL),20-22 etc. In particular, CL and ECL methods are considered as one of the most promising approaches for TNT detection due to their high sensitivity and ease of automatization. Unfortunately, these methods reported mostly involve complicated labeling and purification procedures, making these methods time-consumed and impeding the wide application of these methods. To overcome these shortcomings, it is highly desired to develop a simple, sensitive and convenient method for the determination of TNT at trace amount. Graphene Oxide (GO) consists of atomically thin sheet of graphite, and has recently attracted much attention due to its unique optical, mechanical, thermal and electrochemical properties.23-27 GO has been proved to be satisfied tunable platforms for sensors because of its good dispersion in water and versatile surface functionalization.28-30 In our previous work, novel N-(aminobutyl)-N-(ethylisoluminol)/hemin dualfunctionalized graphene hybrids (A-H-GNs)31 and luminol functionalized silver/graphene oxide composite (luminolAgNPs-GO)32 were successfully synthesized, which both showed excellent CL activity when reacting with H2O2. Moreover, they have good dispersion and stability in solution. However, the ECL behavior of A-H-GNs and luminol-AgNPsGO and their analytical applications have not been explored. Herein, the ECL behavior of A-H-GNs and luminol-AgNPsGO was investigated under cyclic voltammetry and pulse

potential. It was found that A-H-GNs and luminol-AgNPs-GO exhibited excellent ECL activity. On this basis, a label-free ECL aptasensor was successfully developed for the detection of TNT based on bilayer structure of luminescence functionalized graphene hybrids consisting of A-H-GNs and luminol-AgNPs-GO. A-H-GNs and luminol-AgNPs-GO were for the first time used for the design of label-free sensor. A-HGNs could provide strong ECL signal, but would be difficult to be connected with single strand DNA due to multilayer stacking of ABEI and hemin on the surface of graphene. Luminol-AgNPs-GO could not only further increase ECL intensity but also provide bind sites for the connection of recognition elements aptamers by virtue of AgNPs. Therefore, the assembly of A-H-GNs with luminol-AgNPs-GO to form the bilayer structure of luminescence functionalized graphene hybrids could offer an excellent ECL signal interface with recognition site to construct sensing platform. The process of the assembly of the sensor was characterized by electrochemical impedance spectra (EIS) and ECL. The conditions for the detection of TNT were optimized and the analytical performance of the proposed aptasensor was studied. Finally, the applicability of the proposed aptasensor for determining TNT in real water samples was explored.

EXPERIMENTAL SECTION Chemicals and materials. Standard solution (1.0 mg/mL) of TNT, 2,4-dinitrotoluene (DNT), p-nitrotoluene (NT) and nitrobenzene (NB) was purchased from Aladdin Reagent (Shanghai, China). Bovine serum albumin (BSA) and streptavidin (SA) were purchased from Solarbio (Beijing, China). GO was purchased from XFNANO Materials Tech Co. Ltd. (Nanjing, China). N-(aminobutyl)-N(ethylisoluminol) (ABEI) stock solution was prepared by dissolving ABEI in 0.1 M NaOH and kept at 4 ºC. A stock solution of luminol (0.01 M) was prepared by dissolving luminol (Sigma) in 0.1 M NaOH aqueous solutions. 0.5% chitosan (CS) (from Country medicine group, Shanghai) solution was prepared by dissolving CS in 2% acetic acid solution with magnetic stirring for 2 h. The TNT peptide aptamer was purchased from GL Biochem Co. Ltd. (Shanghai, China) and purified using high performance liquid chromatography. The sequence of the biotinylated aptamer (biotin-apta) is as follows: (N terminus) Trp-His-Trp-Gln-ArgPro-Leu-Met-Pro-Val-Ser-Ile-Lys-Cysteamine (C terminus). This sequence has been reported as a selective peptide aptamer for TNT.33,34 All other reagents were of analytical grade. 2


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Ultrapure water was prepared by a Millipore Milli-Q system and used throughout.

measurements were carried out with the homemade ECL system (Supporting Information section S3).36, 37 The ECL intensity difference ∆I in the absence and presence of TNT under a double-step potential was used for quantification.

ECL behavior of A-H-GNs and luminol-AgNPsGO. A-H-GNs and luminol-AgNPs-GO were synthesized as described previously.31,32 The detailed processes of synthesis are described in Supporting Information section S1. As-prepared A-H-GNs were centrifuged at 12500 rpm for 15 min to remove excess reagents and the precipitates were dispersed with 0.5% chitosan solution. 50 µL of A-H-GNs in chitosan solution was dropped into the reservoir of the ITO electrode, drying naturally in air. The modified ITO electrode was used as working electrode. The ECL behavior of A-HGNs was investigated under cyclic voltammetry. When a potential was scanned on the modified ITO electrode in 0.02 M carbonate buffer containing 1.5 mM H2O2 from 0.0 V to 1.5 V, the profile of ECL intensity vs. potential was recorded. The ECL behavior of A-H-GNs was also studied under a doublestep potential on the ITO electrode. When a double-step potential (30 s pulse period, 0.1 s pulse time, 0.8 V pulse potential) was applied to the electrode in 0.02 M carbonate buffer containing 1.5 mM H2O2, a pulse ECL signal was obtained. The ECL behavior of luminol-AgNPs-GO or luminol was studied using the same method as A-H-GNs.

RESULTS AND DISCUSSION ECL behavior of A-H-GNs and luminol-AgNPsGO. The ECL behavior of A-H-GNs was investigated under cyclic voltammetry in 0.02 M carbonate buffer containing 1.5 mM H2O2. As a control experiment, ABEI instead of A-HGNs was also examined under the same conditions. The curves of ECL intensity versus applied potential and the cyclic voltammogram were recorded simultaneously. The typical profile of current intensity/electrode potential and ECL intensity/electrode potential of A-H-GNs (solid line) and ABEI (dash line) are shown in Figure 1A and Figure 1B. When the potential was swept from 0 to 1.5 V, the oxidation peak of ABEI started at ca. 0.5 V, and reached to maximum at 0.8 V. Meanwhile, the ECL of A-H-GNs also appeared from 0.5 V and reached its peak at 0.8 V, indicating that the ECL emission was from ABEI molecules attached on the surface of A-H-GNs. The ECL generation of A-H-GNs might be due to that ABEI and H2O2 were electro-oxidized to ABEI radical and O2•–, respectively, on ITO electrode, and ABEI radical reacted with O2•– to give rise to light emission.36, 38 And excellent ECL activity of A-H-GNs was due to the catalytic effect of hemin.32 Furthermore, ECL behavior of A-H-GNs was studied under a double-step potential on the ITO electrode in 0.02 M carbonate buffer containing 1.5 mM H2O2, as shown in Figure 2A. When a double-step potential (30 s pulse period, 0.1 s pulse time, 0.9 V pulse potential) was applied to the electrode, a pulse ECL signal was obtained. Figure 2A displayed ECL signals for ten times, which were quite strong and stable. The results imply that such A-H-GNs may be used as selfassembly platform for the construction of sensors.

Preparation of luminol-AgNPs-GO modified with aptamer. 25 µL SA (1.0 mg/mL) was added to 1.0 mL re-prepared luminol-AgNPs-GO. After incubated at room temperature for half an hour, 250 µL of 5.0 % BSA solution was added to the final concentration of 1.0 % BSA, and stirred for 5 min. After that, as-prepared mixture was centrifuged at 12500 rpm for 15 min (Universal 320, Hettich, Germany), and the sediments were dispersed with 300 µL of 0.03 M pH 8.0 Tris-HCl by shortly ultrasonic operation. Subsequently, biotinylated-aptamer was added to SA coated luminol-AgNPsGO solution, followed by incubation at 37 ºC for 30 min. The reacting solution was centrifuged at 12500 rpm for 15 min, and the precipitates were dispersed with 0.03 M pH 8.0 TrisHCl buffer by shortly ultrasonic operation. The luminolAgNPs-GO modified with aptamer (apta-biotin-SA-luminolAgNPs-GO) was ready for the further experiments.

Assembly of label-free ECL aptasensor. An ITO glass slide was used as working electrode and pre-treated according to previous literature35. First, 10 µL of A-H-GNs dispersed with CS was introduced into the reservoir of the ITO electrode and dried in the air to form a CS-A-H-GNs layer on the ITO electrode surface. Following that, 50 µL apta-biotinSA-luminol-AgNPs-GO solution was dropped on the CS-A-HGNs/ITO electrode for 4 h at 4 ºC and rinsed by washing buffer (1.0×10-2 M pH 7.4 Tris–HCl containing 0.1 M NaCl). The apta-biotin-SA-luminol-AgNPs-GO/CS-A-H-GNs/ITO electrode was ready for further experiments. The EIS results of the modified electrodes during different stages indicated the electrodes were modified as expected (Supporting Information section S2). TNT detection. 50 µL aliquots of TNT solution with various concentrations or 30 mM pH 7.4 phosphate buffer containing 20 mM NaCl (blank) were dropped on the aptabiotin-SA-luminol-AgNPs-GO/CS-A-H-GNs/ITO electrode at 37 ºC for 30 min, followed by a thorough washing with the same buffer in order to remove any unbound TNT. ECL

Figure 1. A: CV curves of blank (dot line), ABEI (dash line) and A-H-GNs (solid line); B: IECL/E curves Curves of blank (dot line), ABEI (dash line) and A-H-GNs (solid line) obtained under cyclic voltammertry condition; C: CV curves of blank (dot line), luminol (dash line) and luminol-AgNPs-GO (solid line); D: IECL/E curves 3



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of blank (dot line), luminol (dash line) and luminol-AgNPs-GO (solid line). Potential range from 0 V to 1.5 V; scan rate, 50 Vs-1, pH 10.0, H2O2: 1.5×10-3 M. ECL intensities were recorded with CHI electrochemical workstation.

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luminescence functionalized graphene hybrids with TNT recognition site consisting of A-H-GNs and luminol-AgNPsGO on the surface of ITO electrode by electrical adsorption. The modified apta-biotin-SA-luminol-AgNPs-GO/CS-A-HGNs/ITO electrode was ready for the detection of TNT after rinsing with washing buffer. In the absence of TNT (blank), a strong ECL signal was observed and recorded as I0. In the presence of TNT, a remarkable decrease in ECL signals was observed due to the formation of aptamer–TNT complex and ECL signal I1 was detected. In this way, ∆ I （ ∆ I= I0 - I1） of two events was obtained and can be used to quantify TNT. The bilayer structure of luminescence functionalized graphene hybrids could provide not only an excellent ECL signal interface but also recognition site for TNT.

Figure 2. ECL signals of A-H-GNs (A) and luminol-AgNPs-GO (B) modified ITO electrode obtained under pulse potential. Initial potential 0 V; pulse period 30 s; pulse time 0.1 s; pulse potential 0.9 V, H2O2: 1.5×10-3 M. ECL intensities were recorded with luminometer.

The ECL behavior of luminol-AgNPs-GO was investigated in 0.02 M carbonate buffer containing 1.5 mM H2O2 under the same conditions as the A-H-GNs. The typical profile of current intensity/electrode potential and ECL intensity/ electrode potential of luminol-AgNPs-GO is shown in Figure 1C and Figure 1D. As can be seen, the CV of luminol modified ITO electrode showed that the oxidation peak started at ca. 0.5 V, and reached to maximum at approximately 0.95 V. The ECL of luminol-AgNPs-GO modified ITO electrode also appeared from the 0.5 V and reached its peak at 0.95 V, corresponding to the oxidation of luminol. The ECL generation of luminol-AgNPs-GO might be due to that luminol and H2O2 were electro-oxidized to luminol radical and O2•–, respectively, on ITO electrode, and luminol radical reacted with O2•– to give rise to light emission.36 The ECL behavior of luminol-AgNPs-GO modified on ITO electrode in 0.02 M carbonate buffer containing 1.5 mM H2O2 was also studied under a double-step potential on the ITO electrode. Figure 2B displayed ECL signals for ten times, which were quite stable. The results indicated that luminolAgNPs-GO also possessed excellent ECL performance.

Figure 3. A schematic for proposed label-free ECL aptasensor for 2,4,6-trinitrotoluene detection based on an assembly strategy of A-H-GNs and luminol-AgNPs-GO.

ECL characterization of the modified electrode. The ECL behavior of the aptasensor was studied with a double-step pulse potential in 0.02 M pH 10.0 carbonate buffer solution (CBS) containing 1.5×10-3 M H2O2. The ECL signal of the modified electrodes during different stages was examined to gain a clear idea of the ECL signal generation, as shown in Figure 4. No ECL responses were observed on a bare ITO electrode (curve a). When CS-A-HGNs were modified on the surface of ITO electrode, a strong ECL signal was observed as shown in curve b of Figure 4, which caused by the reaction of ABEI radicals electrooxidized by ABEI on the surface of A-H-GNs with O2•– electro-oxidized by H2O2. When the apta-biotin-SA-luminolAgNPs-GO was further modified on the electrode, the ECL intensity was increased obviously (curve c). Because the luminol coated on the surface of luminol-AgNPs-GO could be also electro-oxidized to luminol radicals, which could react with O2•– to generate ECL signal, the bilayer structure of luminescence functionalized graphene hybrids could effectively increase the ECL intensity of the system. In the presence of TNT (1.0 × 10-10 g/mL), a decrease in ECL intensity (curve d) was observed, revealing the binding of aptamer with TNT. The decrease in ECL intensity was a comprehensive result. Firstly, the EIS results of the modified electrode showed that the presence of TNT would lead to an increase in electron transfer resistance (Supporting Information S2), indicating that TNT could hinder the electron

Strategy for label-free ECL aptasensor. Figure 3 depicted schematically label-free ECL aptasensor for TNT detection based on A-H-GNs and luminol-AgNPs-GO. First, A-H-GNs was dispersed in CS solution in order to assemble A-H-GNs on the surface of an ITO electrode. LuminolAgNPs-GO was modified with SA, which was then connected with biotinylated TNT aptamer by virtue of biotin-SA interaction to form apta-biotin-SA-luminol-AgNPs-GO in order to get recognition site on the surface of GO. Zeta potential was measured for both of CS-A-H-GNs and aptabiotin-SA-luminol-AgNPs-GO to explore electric charge on the surfaces of the two kinds of nanomaterials. The results showed that +55.3 mV zeta potential was observed for the CSA-H-GNs and -22.7 mV zeta potential for apta-biotin-SAluminol-AgNPs-GO, indicating that they were positively and negatively charged, respectively. Second, positively charged CS-A-H-GNs were modified on the surface of ITO electrode by simple dripping and drying in the air. Third, the modified electrode was immersed in negatively charged apta-biotin-SAluminol-AgNPs-GO for 4 h at 4 ºC to form bilayer structure of 4


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transfer and influenced the electro-oxidation reaction of luminol, leading to a decrease in ECL intensity. In addition, according to the literatures,39, 40 TNT could also interact with the luminophor excited-state N-(aminobutyl)-N(ethylphthalate) and 3-aminophthalateanions from the ECL reactions by virtue of donor-acceptor interaction between primary amines and TNT, leading to an energy loss and quenching of the ECL emission. The stability of the ECL signals was studied. The result showed that the ECL intensity in the fifty periods only decreased 2.4%, indicating that the aptasensor was highly stable.

In order to prove that the bilayer structure of luminescence functionalized graphene hybrids can effectively improve the ECL intensity of the system and the sensitivity of the aptasensor, we studied the aptasensor without A-H-GNs, via layer by layer assembly of CS and apta-biotin-SA-luminolAgNPs-GO on an ITO electrode. The dynamic range could only achieve the range of 1.0×10-10-1.0×10-9 g/mL with a detection limit of 5.6×10-11 g/mL (Supporting Information S5). The results indicated that the aptasensor without A-H-GNs exhibited lower ECL signals than the proposed aptasensor. The bilayer structure of luminescence functionalized graphene hybrids could effectively increase the ECL intensity of the system and improve linear range and sensitivity of the aptasensor.

Figure 4. ECL signals under pulse potential. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V. ECL signals were obtained a) on a bare ITO electrode, b) on a CS-A-HGNs/ITO electrode, c) on an apta-biotin-SA-luminol-AgNPsGO/CS-A-H-GNs/ITO electrode, d) on a TNT/ apta-biotin-SAluminol-AgNPs-GO/CS-A-H-GNs/ITO electrode. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.5×10-3 M H2O2.

Figure 5. Linear relationship between ECL response and logarithm of TNT concentration. The inset shows ECL responses of TNT and different interfering species. a: 1.0×10-9 g/mL NB; b: 1.0×10-9 g/mL DNT; c: 1.0×10-9 g/mL NT; d: 1.0×10-10 g/mL TNT; e: 1.0×10-10 g/mL TNT with 1.0×10-9 g/mL NB, DNT and NT. Initial potential, 0 V; pulse period, 30 s; pulse time, 0.1 s; pulse potential, 0.9 V. All ECL signals were measured in 0.02 M CBS (pH 10.0) solution containing 1.5×10-3 M H2O2.

Analytical performance of the label-free ECL aptasensor. Under the optimized conditions (pH 10.0, 1.5×10-3 M H2O2, pulse potential of 0.9 V, initial potential of 0 V, pulse time of 0.1s and pulse period of 30 s; Supporting Information section S4), the quantitative behavior of the fabricated ECL aptasensor for TNT was assessed by measuring the dependence of ∆ I upon the concentration of TNT. The calibration curve for the determination of TNT is shown in Figure5. It is obvious that the ECL intensity decreased with an increase of the concentration of TNT. ∆ I was linear with the logarithm of concentration of TNT over the range of 1.0×10-12–1.0×10-9 g/mL. The regression equation was ∆I=30396+2547 × log C (unit of C is g/mL) with a correlation coefficient of 0.9926. ∆ I was the relative ECL intensity calculated by I0 - I1, where I0 and I1 are the ECL intensity without and with TNT, respectively. The detection limit for TNT at a signal-to-noise ratio of 3 (S/N = 3) was estimated to be 6.3×10-13 g/mL. The precisions of the aptasensor are investigated. The relative standard deviation of seven replicate determinations of 1.0×10-10 g/mL TNT with different electrodes was 3.25% (n=7), showing good reproducibility.

A comparison between the proposed label-free ECL aptasensor and other previously reported bioassays for TNT is made (Supporting Information section S6). The results show that the sensitivity of the present ECL aptasensor is superior to most previously reported bioassays for TNT except for one ECL sensor with labeling techenology.20 However, multi-steps of labeling and separation were involved in the ECL sensor with labeling technology, which made the ECL sensor much more complicated for the determination of TNT.20 The proposed label-free ECL aptasensor do not need complicated labeling and separation procedures but exhibit high sensitivity for the determination of TNT.

Selectivity of the label-free ECL aptasensor. The proposed label-free ECL aptasensor was assumed to be highly selective towards TNT as the sensing was relied on TNT–aptamer recognition. Therefore, control experiments were conducted to investigate the specificity of the TNT aptasensor. Some TNT analogs such as DNT, NT and NB were used instead of TNT in the sensing protocol. The results 5



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Table 1. Determination of TNT in environmental water samples using proposed ECL aptasensor Water samples 1 2 3 4 5 6

TNT found 9.2 ng/mL 9.8 ng/mL 10.2 ng/mL

TNT added 10.0 pg/mL 10.0 pg/mL 10.0 pg/mL 10.0 ng/mL 10.0 ng/mL 10.0 ng/mL

showed that only TNT exhibited strong ∆ I (Figure 5, inset). The cross sensitivity of the label-free ECL aptasensor in a mixture of three different TNT analogs including DNT, NT and NB was also examined. ∆ I had little difference with that in pure TNT solution. These results indicate that the developed label-free ECL aptasensor is selective for the detection of TNT.

Total TNT detected 10.5 ± 2.1 pg/mL 9.2 ± 3.7 pg/mL 9.1 ± 5.3 pg/mL 19.5 ± 2.8 ng/mL 19.3 ± 4.2 ng/mL 20.9 ± 6.1 ng/mL

Recovery % 105.0 92.0 91.0 103.0 95.0 107.0

ASSOCIATED CONTENT Supporting Information Additional information as noted in text (S1. Preparation of A-H-GNs and luminol-AgNP-GO, S2. EIS of the modified electrode, S3. Apparatus, S4. Optimization of experimental conditions, S5. Working curve for TNT with apta-biotin-SAluminol-AgNPs-GO modified ITO electrode, S6. A comparison of bioassays for TNT detection). This material is available free of charge via the Internet at http://pubs.acs.org.

Determination of TNT in environmental water samples using the ECL aptasensor. To test the validation of the label-free ECL aptasensor in the real sample matrix, analysis of TNT were performed by determining TNT in water samples. Samples 1~3 were collected at three locations of the Yanjing Lake, and sample 4~6 were collected at three locations of the Ying River near a TNT factory. The water samples were detected after repeated centrifugation to remove the sediment. The water samples obtained from Ying River were diluted 100 times by water to yield testing sample solutions. Table1 shows the results obtained by the proposed aptasensor. The results demonstrated good recoveries (91.0– 107%) and relative standard deviation (2.1–6.1%, n=3), as shown in Table 1, indicating that such sample matrix do not affect the determination and the present aptasensor could be used for the determination of TNT in environmental water samples.

AUTHOR INFORMATION Corresponding Author *Phone: +86-551-63600730. Fax: +86-551-63600730. E-mail: [email protected].

Author Contributions ⊥G.X. Li and X.X. Yu are cofirst authors and 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. Notes The authors declare no competing financial interest.

CONCLUSIONS

ACKNOWLEDGMENT

In summary, it was found that A-H-GNs and luminolAgNPs-GO exhibited excellent ECL activity. On this basis, a novel label-free ECL aptasensor has been proposed for TNT detection based on bilayer structure of luminescence functionalized graphene hybrids consisting of A-H-GNs and luminol-AgNPs-GO. The present ECL aptasensor exhibits a wide dynamic range from 1.0×10-12 g/mL to 1.0×10-9 g/mL with a low detection limit of 6.3×10-13 g/mL, which is superior to most previously reported bioassays for TNT. It has been successfully applied to the detection of TNT in real water samples. Compared with the reported TNT sensors, the present aptasensor has the advantages such as avoiding complicated labeling and purification procedure, which could be an easier and simpler sensing approach to automation in environmental monitor. A-H-GNs and luminol-AgNPs-GO synthesized by our group are for the first time used for the design of sensor, demonstrating that the bilayer structure of luminescence functionalized graphene hybrids consisting of A-H-GNs and luminol-AgNPs-GO is an ideal nanointerface for the construction of label-free biosensors. Due to the wide target recognition range of aptamer, this strategy provides a promising way to develop new aptasensor for other analytes.

The support of this research by the National Natural Science Foundation of PR China (Grant nos. 21475120 and 21173201), the Opening Fund of State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, CAS (Grant no. SKLEAC201408), are gratefully acknowledged.

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For TOC only

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Label-Free Electrochemiluminescence Aptasensor for 2,4,6

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