Building a Three-Dimensional NanoâBio Interface for Aptasensing: An

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Building Three Dimensional Nano-Bio Interface for Aptasensing: a Novel Analytical Methodology Based on Steric Hindrance Initiated Signal Amplification Effect Xiaojiao Du, Ding Jiang, Nan Hao, Jing Qian, Liming Dai, Lei Zhou, Jianping Hu, and Kun Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02368 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 2016

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Analytical Chemistry

Building Three Dimensional Nano−Bio Interface for Aptasensing: a Novel Analytical Methodology Based on Steric Hindrance Initiated Signal Amplification Effect Xiaojiao Du,† Ding Jiang,‡ Nan Hao,† Jing Qian,† Liming Dai,† Lei Zhou,† Jianping Hu,† and Kun Wang*,† † Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China ‡ School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China

ABSTRACT:

The

development

of

novel

detection

methodologies

in

electrochemiluminescence (ECL) aptasensor fields with simplicity and ultrasensitivity, is essential for constructing biosensing architectures. Herein, a facile, specific and sensitive methodology was developed unprecedentedly for quantitative detection of microcystin-LR (MC-LR) based on three dimensional boron and nitrogen co-doped graphene hydrogels (BN-GHs) assisted steric hindrance amplifying effect between the aptamer and target analytes. The recognition reaction was monitored by quartz crystal microbalance (QCM) to validate the possible steric hindrance effect. Firstly, the BN-GHs were synthesized via self-assembled hydrothermal method and then applied as the Ru(bpy)32+ immobilization platform for further loading the biomolecule aptamers due to their nano-porous structure and large specific surface area.

ACS Paragon Plus Environment


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Interestingly, we discovered for the first time that, without the aid of conventional double-stranded DNA configuration, such three dimensional nanomaterials can directly amplify steric hindrance effect between the aptamer and target analytes to a detectable level and this facile methodology could be for an exquisite assay. With the MC-LR as a model, this novel ECL biosensor showed a high sensitivity and a wide linear range. This strategy supplies a simple and versatile platform for specific and sensitive determination of a wide range of aptamer-related targets, implying that three dimensional nanomaterials would play a crucial role in engineering and developing novel detection methodologies for ECL aptasensing fields.


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■ Introduction The establishment of rapid, facile, ultrasensitive, and highly selective detection strategies, for identifying and quantifying a trace amount of target molecules has continuously been a core of pushing the boundary on analytical chemistry,1 notably in electrochemiluminescence (ECL) fields. ECL is chemiluminescence triggered by electrochemical technique,2 possessing merits of high sensitivity, low background noise and instrument simplicity,3,4 which has been extensively used as a powerful tool in many analytical chemistry related areas.5 In the past few decades, various primary methodologies have been developed for ECL assays, such as inhibiting

6,7

and enhancing effects

8-10

on recorded ECL signals directly

caused by ECL reaction among target analytes, the luminophor and co-reactant.11 However, such methodologies are poor in selectivity, preventing them from being widely employed. Recently, ‘‘signal-off’’ and ‘‘signal-on’’ ECL aptasensors based on the coupling of resonance energy transfer (RET) and DNA aptamers have been a hot topic due to their high specificity and binding affinity.12 Although the ECL resonance energy transfer (ECL-RET) aptasensors exhibit excellent performances in terms of sensitivity and selectivity, their extensive applications are limited because of the difficulty in search of a suitable donor/acceptor pair and operational complexity of detecting process.13 Therefore, it is highly essential to develop a novel methodology with excellent performances and a relatively simple process. Immunoassays, particularly the electrochemical immunosensors, involving the



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antibody-hapten reaction have been ideally suited for meeting the assay requirements over the past two decades.14 As is known to all, two primary methodologies have been studied for immunoassays: steric hindrance resulting from the formation of an immunocomplex to hinder the transfer of a coreactant; the coreactant consumption or generation with enzymatic reaction, which constitutes a simple signal-off typed assaying protocol.15 To our best knowledge, aptamers are a kind of well-known antibody mimetics with high specificity and high affinity to specific targets, which have some pivotal merits over antibodies, such as comparatively smaller size, low-cost synthesis, an in-vitro selection procedure, and reversible denaturation.16,17 Inspired by the above features of the detection mechanism in immunosensing areas, it can be drawn an analogy and envisaged that whether the facile signal-off typed detection mechanism caused by steric hindrance could also be applied in ECL aptasensors. For signal-off assays, it is a challenging task to improve the sensitivity of assays.18 Generally, when evaluating the performances of a method for quantitative analysis, sensitivity is one of the most important parameters in the biosensor construction.19 Therefore, considerable efforts have been dedicated to exploiting new detection methodologies and signal amplification strategies to enhance the sensitivity in ECL sensing so far,20-24 such as ECL biosensor based on aptamer and hyper-branched rolling

circle

amplification

and

exonuclease-catalyzed

target

recycling

amplification.25,26 In spite of the outstanding sensitivity of these amplification strategies has been achieved, they may suffer from the high cost of enzyme and DNA


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as well as the complex manipulations of the biosensor construction process. Therefore, it is preferable to construct a novel ECL signal-off sensing system with high sensitivity, low cost and, especially, handleability. In the recent years, graphene-based nanomaterials have been conceived as ideal candidates in the design of biosensing systems with advanced functions because they can facilitate signal transduction; namely, the signal of recognition events can be amplified by several orders of magnitude, causing the extremely high sensitivity.27,28 Compared with two-dimensional graphene, three-dimensional graphene hydrogels (3D GHs), representing a new class of ultralight and porous carbon based materials and associated with higher strength-to-weight and surface-area-to-volume ratios, have aroused extensive attention recently.29,30 They can supply multidimensional electron transport pathways and provide great convenience to assemble more ECL luminophore molecules such as Ru(bpy)32+ on its surface for enhancing the ECL intensity of luminophores and further boosting the immobilization amount of biomolecules.18,31

Such

a

solid-state

ECL

sensor

is

better

and

more

environment-friendly than Ru(bpy)32+ ECL system in solution phase due to reducing the consumption of expensive ECL reagent Ru(bpy)32+. Besides, very recently, both theoretical and experimental studies have showed that chemical co-doping with two elements, nitrogen (N) and boron (B), was an effective method to tailor the its chemical and physical properties for more widespread applications.32-34 As a result, it is feasible to develop simple, ultrasensitive, highly selective and cost-effective biosensing platforms coupling with 3D GHs nanomaterials co-doped with nitrogen



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(N) and boron (B) in ECL fields. Inspired by the peculiar property of 3D GHs nanomaterial, this work developed a simple and pragmatic ECL biosensing strategy for ultrasensitive and specific detection directly by integrating target-initiated steric hindrance effect with the signal amplification of 3D BN-GHs nanomaterial in single analysis system. The quartz crystal microbalance (QCM) technique was performed to provide evidence for it. The microcystin-LR, the most hepatotoxic congener among all microcystins and potential threat on animals and human health, was exploited as a model target to verify the proposed strategy. Besides, we have demonstrated that the mechanism of the proposed detection methodology in detail. Since both pricy labeling and sophisticated probe immobilization processes are avoided, this method exhibits outstanding advantages of simplicity and low cost. This strategy may become an alternative method for simple, sensitive, and selective aptamer-related targets and have great potential to be applied in more extensive researches. ■ EXPERIMENTAL SECTION Materials and Reagents. Graphite was purchased from Qingdao Tianhe Graphite Co., Ltd. Ammonium pentaborate (NH4B5O8), ethylenediamine tetraacetic acid (EDTA) and tripropylamine (TPrA) were acquired from Aladdin (Shanghai, China). Ru(bpy)3Cl2•6H2O and Nafion (5 wt%) was purchased from Sigma Aldrich. Microcystin-LA (MC-LA), microcystin-YR (MC-YR) and MC-LR were obtained from J&K Chemical Ltd. (Shanghai). Graphite oxide (GO) was prepared from natural graphite by Hummers' method.35 The oligonucleotides were synthesized and


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HPLC-purified by Sangon Biotech Co., Ltd. (Shanghai). Single-stranded DNA (ssDNA) aptamer for MC-LR used in this work was selected as the following sequences:36 5′-GGC GCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC-3′). Standard microcystin-LR samples had been dissolved in binding buffer (50 mM Tris−HCl, pH 7.4, containing 0.1 M NaCl, 5 mM MgCl2, 0.2 M KCl, 1.0 mM EDTA) to obtain various concentrations of microcystin-LR. The DNA solution was prepared by dissolving DNA into binding buffer. Phosphate-buffered saline (PBS, Na2HPO4–NaH2PO4, 0.1 M) was prepared in the laboratory. All other reagents were of analytical purity and used as received. Ultrapure water (18.4 MΩ) purified by a milli-QTM system (Millipore) was utilized throughout. The contaminated environmental water samples were collected from Yudai River (in the city of Zhenjiang, China) and kept in a beaker which were filtered using a 0.45 µm Millipore cellulose membrane immediately and finally stored at 4 oC prior to analysis. Tap water samples were obtained in our laboratory and the human blood serum samples were acquired from Jiang Bin Hospital and all the experiments were implemented on the day of blood collection. Apparatus. The scanning electron microscopy (SEM) images were recorded with a field-emission scanning electron microscope (JEOL JSM-7001F) equipped with an energy-dispersive X-ray spectroscope (EDS) operated at an acceleration voltage of 10 kV. The X-ray photoelectron spectroscopy (XPS) data were carried out on an



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Escalab-MKII spectrometer with Mo Kα X-rays as the excitation source. X-ray diffraction (XRD) and Raman spectra were obtained using Cu target with a Panalytical high resolution XRD-1, PW 3040/60 unit and a RM 2000 microscopic confocal Raman spectrometer, respectively. The

ECL

measurements

were

acquired

using

a

model

MPI-A

electrochemiluminescence analyzer (Xi’ an Remex Analysis Instrument Co. Ltd. Xi’ an, China) with 800 V photomultiplier tube voltage with the potential 0.2 V to 1.2 V. A three-electrode system was composed of a modified glassy carbon electrode (GCE, ϕ = 3 mm) as the working electrode, a Pt wire as the counter electrode and an Ag/AgCl as the reference electrode and all cyclic voltammograms (CVs) were scanned from 0.2 V to 1.2 V with scan rate of 100 mV/s. Electrochemical impedance spectroscopy (EIS) was recorded in a 0.1 M KCl solution containing 5 mM Fe(CN)63−/4− at 200 mV with a frequency range from 0.1 Hz to 10 kHz, and the amplitude of the applied sine wave potential in each case was 5 mV, which was obtained with a ZENNIUM electrochemical workstation (Zahner Instruments, Germany). CHI 400C electrochemical workstation, crystals and QCM cell system were purchased from Chenghua Instruments Co. (Shang-hai, China), in which the QCM cell consisted of a detection cell and Ag/AgCl as reference electrode and Pt wire as counter electrode. Preparation of the Boron and Nitrogen Co-doped Graphene Hydrogels (BN-GHs). The weighed NH4B5O8 (100 mg) and 3 mL GO (3 mg mL−1) mixture was initially under sonication treatment for 20 min. The mixed stable suspension was


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transferred into a Telfon-lined autoclave, hydrothermally treated at 180 oC for 12 h. In the whole process, the raw material NH4B5O8 not only functioned as the source of boron but also the source of nitrogen synchronously. Then, the autoclave was naturally cooled to room temperature and the product was taken out (denoted as BN-GHs). Finally, the monolith was washed repeatedly with distilled water, and freeze-dried into an aerogel for further use. As a control, the U-GHs, N-GHs and B-GHs were also prepared using the same procedure with the addition of the appropriate dopants as needs (Figure S1). For instance, ammonia (NH3•H2O) and boric acid (H3BO3) were utilized for N-GHs and B-GHs, respectively. In addition, the boron and nitrogen co-doped graphene (BNG) were also prepared using the same experiment condition. Fabrication of the ECL Aptasensor. The fabrication procedure of the aptasensor was shown in Scheme 1A. Prior to the modification, the GCE was polished to a mirror-like with 0.3 and 0.05 µm alumina slurry sequentially. After a short period sonication in ethanol and water, the GCE was dried at ambient temperature. 2 mg mL−1 BN-GHs suspension was prepared by dispersing 1.0 mg BN-GHs in 0.5 mL ultrapure water with ultrasonic agitation for about 30 min. Then, the as-prepared BN-GHs suspension and 30 µL of 5 % Nafion were mixed with ultrasonic agitation for 5 min. 6 µL of mentioned suspension was cast on the surface of GCE, and allowed to dry. After that, the Nafion/BN-GHs/GCE was immersed into 0.1 mM Ru(bpy)3Cl2 solution for 3 h to adsorb Ru(bpy)32+, denoted as Ru(bpy)32+/Nafion/BN-GHs/GCE. For comparison, Ru(bpy)32+/Nafion/U-GHs/GCE, Ru(bpy)32+/Nafion/N-GHs/GCE



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and Ru(bpy)32+/Nafion/B-GHs/GCE were prepared using a similar process. Before use, the Ru(bpy)32+ modified electrode was rinsed thoroughly with ultrapure water to remove the excess Ru(bpy)32+. Finally, the resultant electrodes were immersed into 5 µM aptamer solution for 2 h to form the aptasensor through strong electrostatic adsorption. After the incubation, the electrodes were rinsed by binding buffer to remove excess non-adsorbed aptamer. Before the aptasensor was used to detect target MC-LR, 20 µL different concentrations of MC-LR was incubated on the aptamer-modified electrode at room temperature for 35 min and then the electrode was rinsed again with binding buffer and subjected to electrochemical measurements. ■ RESULTS AND DISCUSSION The Novel Detection Methodology. Scheme 1A displayed the schematic representation of the ECL aptasensor construction and the mechanism for the detection of MC-LR. When the target analyte MC-LR was incubated with its aptamer, the ECL signals declined dramatically. The reason for the decrement of the ECL signals when the detection process was carried out can be elaborated as follows. The specific binding of target analyte MC-LR to its aptamer blocked the approaching of the coreactant TPrA to Ru(bpy)32+ of the electrode interface and resulted in a decrease in ECL emission. Furthermore, the ECL intensity decreased gradually with the increase of MC-LR concentration. As a result, the quantitative detection of MC-LR was acquired by monitoring the ECL signal decrease after bounding to MC-LR.


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In order to study whether the constructed ECL aptasensor was the diffusion-controlled

here,

CVs

of

the

prepared

aptamer-based

Ru(bpy)32+/Nafion/BN-GHs/GCE in 0.1 M PBS (pH=7.4) at different scan rates were investigated. Figure 1 showed that the CVs peak currents increased linearly with the increasing square root of scan rates (υ1/2), indicating that the adsorbed Ru(bpy)32+ contained in the hybrids on the electrode surface experienced a diffusion-controlled process within the film.37,38 To further confirm the feasibility of the proposed detection principle, the QCM experiments were implemented. The QCM technique is an ultrasensitive mass-measuring device where the dynamic process of biochemical interaction is monitored utilizing an oscillating crystal.39 As the biomolecules are immobilized on its surface, the mass increases, which is correlated with the specific binding reaction, causing a decrease of the oscillating frequency according to Sauerbrey’s equation.40 The equation is as follows:

∆f = -2f 02

∆m A ρµ

(1)

In which ∆f (Hz) is the measured frequency shift produced as a result of a mass alteration, f0 (Hz) represents the fundamental resonant frequency with no attached mass, ∆m (g) stands for the electrode mass change, A (cm2) denotes the piezoelectrically active area, ρ is the density of the quartz, and µ represents the shear modulus of quartz. Figure 2 displayed the typical frequency response versus time profile of the constructed biosensor recorded before and after incubated with 5 pM MC-LR in 0.1



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M

PBS

(pH=7.4).

As

was

shown

in

Figure

Page 12 of 37

2,

the

modification

of

aptamer/Ru(bpy)32+/Nafion/BN-GHs film onto a quartz crystal reduced the frequency changes

(∆f),

while

MC-LR

molecules

were

then

captured

onto

the

aptamer/Ru(bpy)32+/Nafion/BN-GHs-coated crystal to further decrease ∆f, which reflected the increment of adsorbed mass due to the specific binding between the MC-LR molecules and their corresponding aptamer (Equation 1). The results may provide the valid evidence for the proposed steric hindrance-assisted methodology. Such a detection methodology has been rarely reported in previous reports in ECL biosensing, which might be attributed to that conventional nanomaterial-based electrode film in this analytical model is not enough to provide such a high sensitivity, while BN-GHs in our work provide a hierarchical framework with maximum access to the doping sites within highly exposed graphene sheets and multidimensional electron transport pathways,41 thus leading a dramatic change in ECL intensity. Briefly, as illustrated in Scheme 1B, three dimensional BN-GHs framework functioned as amplifying the ECL decrement (−∆E) to accessible detection range caused by of MC-LR-aptamer complexes increased the steric hindrances and therefore, it is feasible to achieve the intention of quantification rather than complex detection procedure in a simple and efficient analytical model. The explanation could be further confirmed in Figure 3. It can be seen that the 3D BN-GHs based apasensor exhibited the powerful ECL quenching efficiency (56.1 %) with 5 pM MC-LR (Figure 3B), while two dimensional BNG had extremely low ECL quenching efficiency, which was almost not visible and unavailable for constructing valid transducers (Figure 3A). Moreover,


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the composite BN-GHs can also serve as an excellent Ru(bpy)32+ immobilization nanomatrix for ECL sensing utilizing high binding capacity of 3D porous BN-GHs to Ru(bpy)32+. Overall, all these reflect the great significance of introducing 3D networks materials into such an analytical model to constitute a new detection methodology in this work. Mechanism of the ECL Biosensor. Since Leland and Powell reported the ECL of Ru(bpy)32+ with TPrA as a coreactant in 1990,42 the classical Ru(bpy)32+/TPrA system has garnered considerable attention and been exploited for a diverse range of significant applications.43 The generalized excitation schemes for electrochemically induced Ru(bpy)32+ luminescence are elaborated as follows (Equal a to e).44

(a) ( Rubpy )3 2+  →( Rubpy )33+ + e (b) ( Rubpy )33+ + TPrA  →( Rubpy )3 2+ + TPrA•

+

+

(c) TPrA•  → TPrA• + H + (d) ( Rubpy )33+ + TPrA•  → *( Rubpy )3 2+ + products (e) * ( Rubpy )3 2+  →( Rubpy )3 2+ + hv Therefore, in this work, initially, the Ru(bpy)32+ is reversibly oxidized to Ru(bpy)33+ at an electrode surface (Equal a), which is capable of oxidizing the amine (TPrA) to form its radical cation TPrA•+ (Equal b) and then TPrA• (Equal c) in the absence of microcystin-LR. The free radical of TPrA• reacts with Ru(bpy)33+ to generate the excited-state *Ru(bpy)32+ (Equal d), suggesting the important role played by the aminium free radical of TPrA• in efficient production of the *Ru(bpy)32+ ECL emission source. Besides, previous work showed that the diffusion amount of TPrA contributed to the variation of relative ECL intensity,45 meaning that more TPrA approaching Ru(bpy)32+ of the electrode interface, stronger ECL emission would be



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observed. As a result, the possible quenching mechanism of ECL emission is speculated subsequently. In the presence of MC-LR, the target analyte MC-LR would specifically bind to its aptamer forming space steric hindrance effect, which greatly limits the approaching of TPrA. This effect blocked the coreactant TPrA having the access to Ru(bpy)32+ of the electrode interface and further impede the production of the free radical of TPrA•, thus resulting in a decrease in ECL emission (Equal d). Characterization of the As-obtained BN-GHs. XPS characterization was carried out to explore the content and configuration of doped nitrogen and boron in the BN-GHs samples. In Figure 4A, the full range XPS analysis of the resultant BN-GHs samples distinctly showed the presence of boron (B), carbon (C), nitrogen (N) and oxygen (O), and the corresponding B 1s, C 1s, N 1s and O 1s peaks centered at 191.3, 284.5, 399.4 and 531.7 eV, respectively.46-48 The chemical composition of the samples determined by XPS method was listed in Supporting Information Table S1. From this, significant contents of N (≈2.95 % atomic percent) and B (≈0.74% atomic percent) were incorporated in BN-GHs. In the high-resolution B 1s spectrum of BN-GHs (Figure 4B), the B 1s spectra were deconvoluted into two peaks with one peak at 191.0 eV for N−B−C moieties and another at 192.1 eV for BCO2 species.46 The N 1s spectra were fitted into three peaks assignable to N−B bonding and pyridinic nitrogen (398.3 eV), pyrrolic nitrogen (399.8 eV) and quaternary nitrogen (401.1 eV).47,48 Besides, three distinguishable peaks (285.1 eV, 289.0 eV and 289.9 eV) in the C 1s spectrum (Figure 4C) corresponded to the chemical environment of carbon atoms bonded to carbon, nitrogen and boron in graphene.49


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Figure 5A presented a digital photo of the resulting monolithic BN-GHs while the inset is a photo of that after lyophilization. The morphology and microstructure of the as-prepared BN-GHs were further investigated by SEM characterization as shown in Figure 5B. It is obvious that the as-obtained BN-GHs exhibited well-defined and interconnected

three

dimensional

(3D)

porous

networks

with

micrometer

interconnected pores. Raman spectra further offered additional evidence of the introduction of B and N atoms into graphene lattice of GHs. Figure 5C displayed the Raman spectra of the U-GHs, N-GHs, B-GHs, and BN-GHs. The spectra exhibited two remarkable peaks at approximately 1352 and 1586 cm−1 corresponding to the well-defined D band and G band, respectively.34 The G band at 1586 cm−1 associated with the bond stretching of all sp2-bonded pairs could be used to explain the degree of graphitization, and the D band at 1352 cm−1 originates from the sp3 defect sites and partially disordered structures of the sp2 domains.34,50 Although there was no significant shift in the position of D and G bands, the intensity ratio of ID/IG increased from GHs (0.97) to BN-GHs (1.13), suggesting that the increasing presence of N or B heteroatoms in the target products brought about a noticeable change in the ordering degree of the hexagonal lattice.51 Figure 5D showed the XRD patterns of GO, GHs and BN-GHs. The feature peak of GO at 11.4o was assigned to the (002) graphite crystal with a c-axis interlayer space of 0.78 nm, implying the complete graphite exfoliation. For BN-GHs, the peak at 11.4o vanished, while a new broad peak at 25.7o appeared with a d-spacing of 0.35 nm corresponding to the (002) plane of graphene, suggesting that the preparation process



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could partially restore the graphitic crystal structure because of the reduction effect of high temperature and co-doping.52 In addition, compared with the GHs, the XRD peak for BN-GHs shifted to a higher degree (ca. 25.7o) obtained from GO under the same conditions (ca. 24.4o), suggesting the smaller interlayer spacing as a result of most presumably by the heterogeneous B-N-dopants.51,53 Characterization of the Fabricated ECL Aptasensor. The ECL behaviors of the aptasensor were recorded and the results were illustrated in Figure 6A. When Ru(bpy)32+ was adsorbed on Nafion/BN-GHs modified electrode in blank PBS solution, an obvious ECL signal appeared (curve a) due to the presence of luminescence reagent Ru(bpy)32+. After adding TPrA as coreactants, a markedly enhanced ECL signal could be observed (curve b), implying that TPrA played a vital role in this ECL system, whereas the ECL signal dropped with the combination of aptamer due to the fact that the film of MC-LR aptamer enhanced the steric hindrance of the electrode interface and impeded the electron transfer (curve c). It was worth noting that the ECL intensity drastically decreased after incubation of 5 pM MC-LR (curve d), based on which a novel detection methodology was proposed in our work for the first time. Meanwhile, the EIS technique, as a tool for evaluating electron transfer resistance,54 was utilized to characterize the corresponding stepwise modification of the electrodes in the 5.0 mM [Fe(CN)6]3−/4− solution (Figure S2). Moreover, the relative standard deviation (R.S.D.) was 5.3 % for successive measurements (n=12) after the aptasensor incubated with 5 pM MC-LR (Figure 6B), suggesting a good repeatability of the proposed aptasensor.


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ECL and Electrochemical Performances of the Modified GCEs. CVs methods were performed to investigate electrochemical behaviors of the different materials modified electrodes (Figure 6C). Of the four electrodes, it was apparent that BN-GHs/GCE based aptasensor (curve d) was the most prominent in the ECL system with highest peak current, the lowest onset reduction potential at 0.95 V, and the lowest onset oxidization potential at 1.18 V for Ru3+/Ru2+ system, which was probably attributed to a synergistic catalysis effect towards Ru(bpy)32+/TPrA system after the co-doping of graphene hydrogel with B and N atoms.55 Correspondingly, following the co-doping with N and B, the highest ECL enhancement was realized via the synergism catalysis between B and N atoms for the reaction between the solid-phase Ru(bpy)32+ and coreactant TPrA (Figure 6D). This was because that not only the isolated N and B atoms served as active sites through charge transfer with neighboring C atoms, but also interaction between adjacent N and B atoms could facilitate the charge transfer with neighboring C atoms.56 Concretely, the BN-GHs based aptasensor exhibited the largest ECL emission efficiency, 2.8 times, 1.41 times and 1.69 times more than that of the U-GHs (curve a), B-GHs (curve b) and N-GHs (curve c), respectively, in the presence of the same TPrA concentration (5 mM). The results demonstrated that the combination doping of B and N atoms in graphene hydrogel exhibited the maximal catalytic activity for Ru(bpy)32+ based ECL enhancement. On the basis of above facts, a robust ECL platform was constructed and could be extensively applied in sensing fields. Optimization of ECL Aptasensor. The Effects of aptamer concentration and



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incubation time with MC-LR molecules on the ECL aptasensor response to 5 pM MC-LR were investigated in 0.1 M PBS (pH=7.4). As depicted in Figure 7A, several ECL aptasensors were prepared with different aptamer concentrations to compare their ECL emission. The ECL response decreased with increasing the aptamer concentration from 1.0 to 5.0 µM, implying that more MC-LR molecules were immobilizated on the electrode surface, while the aptamer concentration further increased to 5 µM and beyond 5 µM, the response kept constant. This meant that the amounts of aptamer immobilized on electrode surface reached saturation point. Therefore, the optimized aptamer concentration for this aptasensor was 5 µM. Moreover, the incubation time between aptamer and target analytes MC-LR is also an important parameter to optimize the performances of the biosensor. Figure 7B showed that ECL intensity of the constructed aptasensor obviously decreased with the increasing incubation time from 10 to 30 min and then reached a plateau in 35 min, suggesting that 35 min was enough and thus selected as the optimal incubation time in the following studies. Analytical Performances. Under the optimum conditions, the developed aptamer/Ru(bpy)32+/Nafion/BN-GHs/GCE electrode was applied in the quantitative detection of MC-LR. The ECL−time curves of the assembled ECL aptasensor clearly indicated the different responses in different concentrations of MC-LR. As presented in Figure 8, the ECL response was found to be linearly decreased with increasing the concentration of MC-LR from 0.1 pM to 1000 pM. The detection limit of MC-LR was calculated to be 0.03 pM (S/N=3). The proposed ECL aptasensor showed much lower


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detection limit compared with previous aptamer-based methods for MC-LR determination.57,58 Interference Study and Sample Analysis. In order to evaluate the selectivity of prepared ECL aptasensor (Figure 9), the ECL responses of the assay to the common potential coexisting interferents, such as two similar structure molecules (MC-LA and MC-YR) and a control DNA were investigated. In this process, −∆E is the decrement of ECL responses of the aptasensor after incubation in 5 pM MC-LR and 100-fold (500 pM) concentration of another interfering substances, which is used as a parameter to measure the selectivity. The relative ECL intensity ratio of aptasensor to MC-LR was set to be 100 %. As was presented in Figure 9, distinct ECL variation was only observed in the presence of MC-LR and the relative ECL intensity ratio of MC-LA and MC-YR were only 4.6 % and 9.2 %, respectively. Besides, to rule out the possibility of the nonspecific adsorption of MCs on the aptasensor, a control experiment was also carried out using non specific DNA sequence via the same precept and incubated with 5 pM MC-LR, but no any significant change was observed (4.7 %). The result indicated that these substances would not interfere with the detection of MC-LR. The super selectivity of the MC-LR sensor can be mainly due to the specific recognition ability of the aptamer and thus the proposed aptasensor only capture the target molecule MC-LR. Furthermore, the robustness of the ECL biosensor was estimated by standard adding MC-LR to tap water, contaminated water samples and human serum samples (Table S2). The recovery was studied by adding four different amounts of MC-LR



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into all the three samples, and the recovery was calculated as 91.0~104.0 %. The concentration of the MC-LR measured by the ECL system was a little higher than the added standard values, which was likely on account of the existing interferents in river water, inducing a positive deviation.59 A decreased recovery percentage was obtained for the spiked tap water sample. We attribute this to the presence of uncontrolled amount of cations which can affect the binding of the aptamer with MC-LR.57 Moreover, the decrease of the MC-LR recovery could be owing to the decomposition of part of the MC-LR molecules by chlorine which usually added as a disinfectant in the drinking water.60 The satisfying results demonstrated that nonsignificant variations of RSD were obtained in all these different sample matrixes, implying the reliability and potential applicability of the biosensor for monitoring trace MC-LR in environmental and clinical samples. ■ CONCLUSIONS In summary, we demonstrated a novel ECL aptasensor based on the “signal-off” switch system towards the Ru(bpy)32+−TPrA system, in which a three dimensional nanomaterial BNGHs were designed to amplify steric hindrance effect between the aptamer and target analytes to a visible level without the aid of double-stranded DNA. In addition to good sensitivity for aptamer-based assay, this proposed detecting methodology avoided expensive labeling and sophisticated probe immobilization processes, providing a proof-of-concept of “signal-off” ECL strategy for reliable detection of a wide spectrum of aptamer relevant analytes in practice.


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■ ASSOCIATED CONTENT

Supporting Information Digital images of the as-prepared graphene hydrogel, the EIS behaviors of modified electrodes, elemental content of BNGHs, data for real samples. This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author * Address: Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu

212013,

P.R.

China.

Tel.: +86

511

88791800;

E-mail

address:

[email protected] Author Contributions The manuscript was written through the contributions of all the authors. All the authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21175061, 21375050 and 21405063), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (No. PAPD-2014-37), Qing Lan Project and Key Laboratory of Modern Agriculture Equipment and Technology (No. NZ201109). ■ REFERENCES



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Scheme 1.

Scheme 1. The schematic representation of the fabrication process and detection mechanism for ECL aptasensor (A); the methodology of the proposed analytical model (B).



Figure 1.

140

50

A

B Current (µ A)

70 Current (µ A)

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

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0 -70

-140

10 mV/s

0.3

-50 -100 -150

500 mV/s 0.0

0

0.6 0.9 Potential (V)

1.2

0

5

10 1/2 15 1/2 Scan rate (mV )

20

Figure 1. (A) Scan rate dependency for thin films of the prepared aptamer-based Ru(bpy)32+/Nafion/BN-GHs/GCE in 0.1 M PBS (pH=7.4) at a scan rate 10 < v < 500 mV/s over the potential range from 0.2 V to 1.2 V vs. Ag/AgCl. (B) The relationship between the anodic and cathodic peak current and scan rates for this data. Error data stand for triplicate results.


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Figure 2.

0 ∆ f (Hz)

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


Without MC-LR

-1k

-2k With MC-LR

-3k 0

100

200 300 Time (s)

400

Figure 2. Frequency responses of the aptamer-based QCM biosensor without and with incubated with 5 pM MC-LR.



Figure 3.

A 2.7 %

5k 3k 2k 0

ECL Intensity (a.u.)

6k ECL Intensity (a.u.)

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

Page 30 of 37

12k

B 56.1 %

8k 4k 0

Time

Time

Figure 3. The different ECL response curves at BNG (A) and BN-GHs (B) based aptasensor in the absence (black line) and presence (red line) of 5 pM MC-LR in 0.10 M PBS (pH=7.4) containing 5.0 mM TPrA, respectively.


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Figure 4.

400 800 1200 Binding Energy (eV)

N 1s

Intensity (a.u.)

O 1s N 1s

B 1s

0

C

B 1s

Intensity (a.u.)

B

C 1s

A Intensity (a.u.)

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


186 189 192 195 Binding Energy (eV)

390

396 402 408 414 Binding Energy (eV)

Figure 4. (A) XPS survey scan spectra of BN-GHs; (B) and (C), high-resolution XPS spectra at B 1s, and N 1s state energies, respectively.



Figure 5.

A

B

C

D a b c d

700

1400 2100 -1 2800 Raman shift (cm )

a

Intensity (a.u.)

Intensity (a.u.)

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

Page 32 of 37

b c

20

40 60 2 Theta (degree)

80

Figure 5. (A) The digital images of the BN-GHs obtained before and after freeze-drying (seeing inset). (B) SEM images of the as-prepared BN-GHs. (C) Raman spectra of GHs (a), NGHs (b), BGHs (c), and BN-GHs (d). (D) The XRD patterns of GO (a), GHs (b), and BN-GHs (c).


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A


16k

b

12k

5k

c d

4k

2k

a

0 0 30

5

10 15 Time (s)

C

B

4k

8k

0 0

20 12k

d

0

a bc

-30 -60



Figure 6.

Current (µ A)

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


50 100 150 200 Time (s)

D

d b c

8k 4k

a 0

-90 0.2

0.4

0.6 0.8 1.0 Potential (V)

0.2

1.2

0.4

0.6 0.8 1.0 Potential (V)

1.2

Figure 6. (A) The ECL behaviors of Ru(bpy)32+/Nafion/BN-GHs/GCE in the absence (a) and presence (b) 5 mM TPrA, (c) after aptamer immobilization, and (d) incubation with 5 pM MC-LR. (B) ECL intensity of aptamer/Ru(bpy)32+/Nafion/BN-GHs/GCE modified electrode in PBS (pH=7.4) containing 5 mM TPA under continuous cyclic voltammetry

for

twelve

cycles.

(C)

CVs

and

ECL

profiles

of

aptamer/Ru(bpy)32+/Nafion/GHs/GCE (a), aptamer/Ru(bpy)32+/Nafion/BGHs/GCE (b), aptamer/Ru(bpy)32+/Nafion/NGHs/GCE

(c)

and

aptamer/Ru(bpy)32+/Nafion/BN-GHs/GCE (d) in 0.1 M PBS solution containing 5 mM TPrA.



Figure 7.

A

12k 8k 4k 0

B



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

Page 34 of 37

1

2

5 8 Captamer (µM)

10

8k

6k

4k

0

10

20 30 40 Binding time (min)

50

Figure 7. Influences of (A) concentrations of aptamer and (B) incubation time with target analytes MC-LR (5 pM).


Page 35 of 37

Figure 8.

12k

A a

8k

4k

j



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


9k

B

6k 3k 0

0

9

Figure

8.

(A)

ECL

responses

of

10

11 12 -logC [M]

the

prepared

13

aptamer-based

Ru(bpy)32+/Nafion/BN-GHs/GCE in 0.1 mol L−1 PBS (pH= 7.4) after incubated with 0, 0.1, 0.5, 1, 5, 10, 50, 100, 500 and 1000 pmol L−1 (from a to j) MC-LR and (B) the corresponding calibration curve for MC-LR on the ECL biosensor. Error bars were derived from the standard deviation of five measurements.



Figure 9.

8000

M C

−∆ E

6000

R -L

4000

C R

D ol

-Y C

N A

A -L

0

tr on

M C

2000

M

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

Page 36 of 37

Figure 9. The selectivity of the proposed aptasensor to the same concentration of MC-LR (5 pM), MC-LA (500 pM), MC-YR (500 pM) and control DNA (5 µM).


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