Nitrogen-Doped Graphene Quantum Dots@SiO2 Nanoparticles as Electrochemiluminescence and Fluorescence Signal Indicators for Magnetically Controlled Aptasensor with Dual Detection Channels.

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Nitrogen-Doped Graphene Quantum Dots@SiO2 Nanoparticles as Electrochemiluminescence and Fluorescence Signal Indicators for Magnetically-Controlled Aptasensor with Dual Detection Channels Chengquan Wang, Jing Qian, Kun Wang, Mengjuan Hua, Qian Liu, Nan Hao, Tianyan You, and Xingyi Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09300 • Publication Date (Web): 02 Nov 2015 Downloaded from http://pubs.acs.org on November 3, 2015

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ACS Applied Materials & Interfaces

Nitrogen-Doped Graphene Quantum Dots@SiO2 Nanoparticles as Electrochemiluminescence and Fluorescence Signal Indicators for Magnetically-Controlled Aptasensor with Dual Detection Channels Chengquan Wang,†,§ Jing Qian,‡,§ Kun Wang,*,‡ Mengjuan Hua,‡ Qian Liu,‡ Nan Hao,‡ Tianyan You,‡ Xingyi Huang*,† †

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, P.

R. China ‡

Key Laboratory of Modern Agriculture Equipment and Technology, School of

Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, P. R. China

1

ACS Paragon Plus Environment


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ABSTRACT: We proposed a facile method to prepare the nitrogen-doped graphene quantum dots (NGQDs) doped silica (NGQDs@SiO2) nanoparticles (NPs). The NGQDs@SiO2 NPs were further explored as versatile signal indicator for ochratoxin A (OTA) aptasensing by combination with electrochemiluminescence (ECL) and fluorescence (FL) detection. In this strategy, the core-shell Fe3O4@Au magnetic beads (MBs) acted as a nanocarrier to immobilize the thiolated aptamer specific for OTA and the amino-modified capture DNA (cDNA) was efficiently tagged with NGQDs@SiO2 NPs. The multifunctional aptasensor was thus fabricated by assembly of the NGQDs@SiO2 NPs onto the surface of Fe3O4@Au MBs through the high specific DNA hybridization. Upon OTA incubation, the aptamer linked with Fe3O4@Au MBs preferred to form aptamer-OTA complex, which resulted in the partial release of the preloaded NGQDs@SiO2 NPs. The more OTA molecules in the detection system, the more NGQDs@SiO2 NPs were released into the bulk solution and the less preloaded NGQDs@SiO2 NPs were accumulated on the magnetic electrode surface. This provided a dual channel for OTA detection by combination with the enriched solid-state ECL and homogeneous FL detection. The FL assay exhibits a wide dynamic range and is more reproducible due to the homogeneous detection while the ECL assay possesses a lower detection limit and is preferable by using cheaper instrument. One can obtain a preliminary screen from FL assay and a more accurate result from ECL assay. Integrating the virtues of dual analytical modality, this aptasensing strategy well balanced the rapidity, sensitivity, and dynamic range, making it promising to other targets with aptamer sequences.

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KEYWORDS:

nitrogen-doped

graphene

quantum

dots,

silica,

aptasensor,

electrochemiluminescence, fluorescence, ochratoxin A 

INTRODUCTION

Graphene quantum dots (GQDs), single or few-layered graphene sheets with sizes smaller than 10 nm, are a novel type of zero-dimensional carbon nanomaterials.1,2 Compared with the traditional QDs containing cytotoxic heavy metal constituents, GQDs have attracted tremendous attention due to their robust biological and chemical inertness, low cytotoxicity and good biocompatibility, and numerous possible applications.3–5 Both the theoretical and experimental studies have demonstrated that the doping of heteroatoms can offer more active sites and effectively tune their optical property.6–8 Therefore, the preparation, properties, and advanced applications of nitrogen-doped GQDs (NGQDs) have become one of the headline-grabbing topics.9 In line with these intensive studys, NGQDs with favorable fluorescence (FL) properties for biolabeling and bioimaging,10,11 peroxidase-like catalytic activity for biosensing,12 and electrocatalytic activity for light-emitting diodes13 were reported. Recently, the studys concerning the detection of targets using the FL property of NGQDs have attracted increasing interest due to the high sensitivity and selectivity of the FL technique.14–18 Meanwhile, some NGQDs-based electrochemiluminescence (ECL) sensors have been developed for ascorbic acid19 and proteins20,21 detection because ECL analysis has many advantages over FL due to the absence of background from unselective photoexcitation.22,23 However, some disadvantages associated with the pristine NGQDs just like poor chemical stabilities in harsh chemical environments

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and conjugate aggregation, would limit their effectiveness for such applications. To solve the problem, NGQDs have been covalently bound on carbon nanotubes which were used as promising ECL indicator for immunoassay.24 Coating NGQDs with inert materials such as the nontoxic silica might be a preferable choice for solving above problems in application. A silica coating can not only provide both chemical and physical shielding from the external environment, but also prevent flocculation of particles and species from adsorbing onto the surface, thereby improving the chemical and optical stabilities.25 The silica shell can also enable versatile possibilities for further surface functionalization, and has high water dispersibility and good biocompatibility,25,26 which leads to the luminophor doped silica being widely used in biosensors.27 Encapsulating organic dyes28,29 or inorganic nanocrystals30–32 with silica has received intensive investigations and the silica coating can well retain the optical properties of the doped luminophor.28,33 However, the preparation of luminescent NGQDs doped silica (NGQDs@SiO2) nanoparticles (NPs) with a biocompatible and stable silica shell has not been reported in literatures. Food contamination is one of the most important worldwide issues and leads to 2 million deaths annually,34 while the pollution caused by mycotoxins is one important branch.35 Ochratoxin A (OTA), one of the most toxic mycotoxins, frequently presents in various agricultural commodities and known to be neurotoxic, genotoxic, teratogenic, myelotoxic, carcinogenic to mammalian species.35,36 The intensifying legislative framework worldwide as well as the increasing awareness about OTA has aroused the need for efficient analytical methods to prevent the risk of OTA

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consumption.37 The emergences of aptamers make the aptasensor attractive due to their obvious advantages over antibodies.38 Since the first select of the aptamer specific for OTA in 2008,39 different versions of aptasensor towards OTA have been reported combined with FL,40,41 ECL,42 electrochemical,37,43,44 conductometric,45 and colorimetric46 transducers. However, studies devoted on the combination of two or more signal transducers for use in one aptasensor is scarce but it is highly desirable due to the diverse aims of detection, i.e., preliminary screens and accurate determination.47 To integrate the virtues of different analytical modalities, novel NGQDs@SiO2 NPs have been prepared and an aptasensor based on target-induced structure switching of aptamers was developed using NGQDs@SiO2 NPs as versatile indicator. The Fe3O4@Au magnetic beads (MBs) acted as a nanocarrier to immobilize the aptamer and the NGQDs@SiO2 NPs served as the tag to efficiently label the capture DNA (cDNA). The aptasensor was fabricated by bringing the NGQDs@SiO2 NPs onto MBs surface through DNA hybridization between aptamer and cDNA. Upon OTA addition, the aptamer preferred to form an aptamer-OTA complex, leading to the partial release of the preloaded NGQDs@SiO2 NPs from the MBs surface into the bulk solution. After magnetic separation, the released and unreleased NGQDs@SiO2 NPs provided a dual channel for OTA detection by combination with the enriched solid-state ECL and homogeneous FL analysis. 

EXPERIMENTAL SECTION Reagents. Tetraethylorthosilicate (TEOS), tris (hydroxymethyl) aminomethane

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(Tris), and glutaraldehyde, were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). (3-aminopropyl) triethoxysilane (APTS), tris (2-chloroethyl) phosphate (TCEP), 6-mercapto-1-hexanol (MCH), OTA, fumonisin B1 (FB1), and aflatoxins B1 (AFB1) were obtained from Sigma-Aldrich. cDNA: 5’–CCT TTA CGC CAC CCA CAC CCG ATC–NH2–3’ and aptamer: 5’–GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA–SH–3’ were purchased from Sangon Biotech Co., Ltd (China). DNA oligonucleotide stock solutions were prepared with 10 mM Tris-HCl buffer (pH 7.4, containing 10 mM NaCl and 5 mM MgCl2) and kept refrigerated in dark. The NGQDs were synthesized by a hydrothermal treatment14 (Supporting Information) and the Fe3O4@Au MBs were obtained according to our previous reports.48 Double-distilled water was used throughout the study. Apparatus. Samples’s morphology and size were checked by transmission electron microscopy (TEM) technique (JEOL 2100, JEOL, Japan) or atomic force microscopy (AFM) (Bruker Dimension Icon, Germany). UV-vis absorption spectra were measured on UV-2450 spectrophotometer (Shimadzu, Japan). Fourier transform infrared (FTIR) spectrum was received on a flourier transform spectrometer (Tensor 27, Bruker, Germany). FL spectra were recorded on a Hitachi F-4500 FL spectrophotometer (Tokyo, Japan). X-ray photoelectron spectroscopy (XPS) was collected on ESCALAB 250 multitechnique surface analysis system (Thermo Electron Co., America). The ECL emission was detected using a MPI-A ECL analyzer (Xi’an Remex Analysis Instrument Co. Ltd., China) with the PMT of 800 V. Three-electrode system was employed with a modified magnetic glassy carbon

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electrode (mGCE), an Ag/AgCl (saturated KCl), a Pt wire as the working, reference, and the counter electrode, respectively. Preparation of the NGQDs@SiO2 NPs. NGQDs@SiO2 NPs were prepared according to our reported work.49 Briefly, 40 mL of NGQDs solution was added into 100 mL of ethanol under vigorous stirring, followed by adding 1 mL of APTS solution. After stirring for 6 h, 1 mL of ammonia solution was added under vigorous stirring, and 20 mL of 19:1 (v/v) ethanol/TEOS was added dropwise in four equal shares every 30 min. The mixture was further stirred for 24 h and the NGQDs@SiO2 NPs were then obtained after centrifugation and washing. Conjugation of cDNA on NGQDs@SiO2 NPs. The resultant NGQDs@SiO2 NPs was dispersed in 40 mL of ethanol, followed by adding 1 mL of APTS. After stirring for 6 h, the amino-terminated NGQDs@SiO2 NPs were collected which were then reacted with 10 mL of glutaraldehyde for 2 h under gentle shaking at 37 oC. After separation and washing, the glutaraldehyde-functionalized NGQDs@SiO2 NPs were redispersed in 40 mL of 10 mM Tris-HCl buffer and 200 μL cDNA (100 μM) was added to react with the aldehyde groups. After incubation for another 2 h with gentle shaking at 37 oC, the cDNA modified NGQDs@SiO2 NPs (cDNA-NGQDs@SiO2) were obtained. After centrifuging and washing steps, they were redispersed in 40 mL of 10 mM Tris-HCl buffer for later use. Aptasensor Fabrication. Aptamer coupled Fe3O4@Au MBs (MB-aptamer) were prepared according to our previous work (Supporting Information).48 40 mL of the cDNA-NGQDs@SiO2 suspension was added to the MB-aptamer resultants and

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the hybridization reaction between cDNA and aptamer was allowed to proceed for 2 h at 37 °C under gentle shaking (Scheme 1A). After rinsing with Tris-HCl buffer for several

times,

the

MB-aptamer/cDNA-NGQDs@SiO2

magnetically-controlled

bioconjugations were collected under a magnetic field and then redispersed in 40 mL Tris-HCl buffer for further study. Scheme 1. Schematic Illustration of the Aptasensor Fabrication (A) and Its Working Principle for OTA Detection by Using Two Detection Channels (B).

Detection Procedure. 100 μL of OTA containing solution was added to 400 μL of the magnetically-controlled bioconjugations. After incubation at 37 °C for 50 min, the released NGQDs@SiO2 NPs were then collected after magnetic separation and the magnetically-controlled bioconjugations was redispersed in 1 mL of Tris-HCl buffer. Then the pretreated mGCE was immersed shallowly into the suspension and all of the bioconjugations were modified on the surface of mGCE in 2 min. The modified mGCE was washed with Tris-HCl buffer and put into electrolyte solution to record its

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ECL response. Then the collected solution containing NGQDs@SiO2 NPs was diluted to a final volume of 2 mL to record its FL intensity. 

RESULTS AND DISCUSSION Characterization of the NGQDs. The resulting NGQDs are relatively uniform

in size and exhibit a relatively narrow size distribution between 1.1 and 4.9 nm with an average diameter of 3.1 nm (Figure 1A). The corresponding AFM image (Figure 1B) reveals a typical topographic height of 1.0–3.8 nm, suggesting that the resultant NGQDs consist of a fewer layers of C–N sheets.50 Figure 1C shows the FTIR spectrum of the as-obtained NGQDs. The O-H stretching vibration was appeared at 3420 cm–1.51 The peak located at 1590 cm–1 with high intensity corresponded to the asymmetric stretching vibrations of the carboxylate anions.50 Additionally, the C-O and O-H deformation peak was also observed at 1406 and 1290 cm–1, respectively.51,52 N-H stretching vibration was observed at 3220 cm–1 which expressed from –NH3+ structure, revealing that the types of nitrogen existed on the surface of NGQDs.51 The wide scan XPS spectrum (Figure 1D) demonstrated that the NGQDs contained carbon (C), nitrogen (N), and oxygen (O) elements. In contrast, no N peak was detected in pure GQDs resultants (Figure S1). To further evaluate the nitrogen configuration in NGQDs, the C1s XPS has been deconvoluted into four peaks at 284.6, 285.2, 286.1, and 288.2 eV (Figure 1E), attributing to C-C,15 C-N,50 C-O,51 and O-C=O53 bond, respectively. The narrow scan N1s XPS of NGQDs (Figure 1F) demonstrates two predominant peaks at 399.2 and 400.5 eV,51 which reveal the existence of both pyrrolic-like and amino N atoms, respectively.51 These results

9



suggested the presence of oxygen-rich and nitrogen-containing groups in the NGQDs.52 C

10 1

C 1s

N 1s

O 1s

D

2 3 4 Size / nm

200 400 600 Binding energy / eV

5

2 0 0

40

80

d / nm

120

F C 1s C=C

C-O

O-C=O

C-N

800 279 282 285 288 291 Binding energy / eV

1085 1290

3420 3220

4000 3200 1600 800 Wavenumber / cm-1

160

E

1590 1406

4

Intensity / a.u.

20

h / nm

30

Intensity / a.u.

Fraction / %

40

0

0

Transmitance / %

B

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

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N 1s Pyridinic N NH2

392 396 400 404 408 Binding energy / eV

Figure 1. (A) TEM image of the NGQDs. Inset: size distribution histogram. (B) AFM image of NGQDs deposited on the mica substrate and the height profile along the white line in the AFM image. FTIR (C) and wide scan XPS (D) spectra of the NGQDs. High resolution XPS spectra of C 1s (E) and N 1s (F) of NGQDs. The aqueous dispersion of NGQDs displays a characteristic absorption band at 335 nm and it has optimal maximum excitation and emission wavelengths at 340 nm and 440 nm, respectively (Figure S2). The FL emission peak located at 440 nm remained to be unchanged upon different excitation wavelengths in the range of 300–420 nm, meanwhile, the strongest emission peak corresponded to the excitation wavelength of 340 nm (Figure 2A). The well-defined absorption band and the excitation-independent emission were related to the uniformity of size as well as high crystallinity of the

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as-prepared NGQDs.54 Besides, the aqueous dispersed NGQDs was transparent and clear under daylight while they exhibited strong blue luminescence using 365 nm UV excitation (inset of Figure 2A). Compared to NGQDs, the pure GQDs showed considerably weaker FL emission at about 420 nm (Figure S3), which was an obvious blue shift by 20 nm with respect to that of NGQDs. This result demonstrates that doping by N atoms can effectively modulate the FL properties of NGQDs. Characterization of the NGQDs@SiO2 NPs. NGQDs@SiO2 NPs have been prepared through a typical Stöber method.49 NGQDs were firstly linked with an amino-containing silane agent APTS, and then a silica alkoxide precursor TEOS was added to hydrolyze and co-condense in the ethanol/NH3·H2O mixture, which resulted in NGQDs@SiO2 NPs. The maximum emission peak of NGQDs@SiO2 NPs was observed at about 440 nm at the excitation wavelength of 340 nm (Figure 2B), very similar to that of the free NGQDs at the same excitation wavelength (curve c, Figure 2A). In comparison with the aqueous dispersion of NGQDs, NGQDs@SiO2 suspension exhibited a brown color but with the similar blue luminescence under 365 nm UV illumination (inset of Figure 2B). The results indicated that NGQDs was successfully encapsulated inside of SiO2 and the entrapped NGQDs could well retain its FL properties.28 The XPS survey spectrum (Figure 2C) showed the resultant NGQDs@SiO2 NPs contained C, N, O, and Si elements, in accordance with the constituent elements of NGQDs and SiO2. The successful encapsulation of NGQDs into SiO2 NPs can prevent the pristine NGQDs from conjugate aggregation, a nuisance that commonly occurs when using ultra-small particles as biological labels.55

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Besides, the NGQDs@SiO2 NPs doped with numerous NGQDs would be hugely beneficial to enlarge the detection signal. The TEM image showed that the NGQDs@SiO2 NPs had a chemically clean and homogenized structure with an average diameter of 110 nm (Figure 2D). A1000

B 800

c d

FL Intensity / a.u.

FL Intensity / a.u

750 b

500 e

250

a gf

0 320

400 480 560 Wavelength / nm

600 400 200 0 360 450 540 630 720 Wavelength / nm

640 D

Si (O) Si 2s

0

N 1s

C 1s

O 1s

C

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

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200 400 600 Binding energy / eV

800

Figure 2. FL spectra of the NGQDs (A) at various excitation wavelengths (from a to g: 300, 320, 340, 360, 380, 400, 420 nm) and NGQDs@SiO2 NPs (B). Inset: photographs of the aqueous dispersed NGQDs solution (A) and NGQDs@SiO2 NPs (B) under daylight (left) and 365 nm UV irradiation (right). Wide scan XPS spectrum (C) and TEM image (D) of the NGQDs@SiO2 NPs. In addition, the amount of NGQDs used in the synthesis of NGQDs@SiO2 NPs was excessive, as evidenced by the observation of the blue emission from the supernatant fluid under UV light. It should be pointed that no blue luminescence or emission peak

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was monitered from the supernatant fluid after the NGQDs@SiO2 NPs being dispersed in water for two weeks, indicating that the entrapped NGQDs was stable and could not leak out from the silica shell. The additional silica shell can effectively improve the photo and chemical stabilities of NGQDs and also prevents their direct contact with the external analytes, thus providing a reliable signal indicator.7 These advantages make the NGQDs@SiO2 NPs have potential applications in many fields, especially in biosensing and bioimaging. Fabrication and Characterization of the Aptasensor. Fe3O4@Au MBs with an average diameter of 450 nm were successfully obtained by growing a layer of Au on Fe3O4 microspheres (Figure S4). The stepwise fabrication process of the MB-aptamer/cDNA-NGQDs@SiO2 bioconjugations was monitored by EIS exploiting the equimolar [Fe(CN)6]3–/4– as a redox probe (Figure 3A). The most adequate fit for the EIS data was given by a Randles equivalent circuit where Rs, Zw, Ret, and Q corresponded to the resistance of the electrolyte solution, Warburg-diffusion impedance, electron transfer resistance, and the constant phase element, respectively (inset of Figure 3A). We focused on the change of Ret recorded after any further step in the aptasensor fabrication and its value can be adjusted by the semicircle diameter in the Nyquist plots. After the magnetically-controlled absorption of Fe3O4@Au MBs on mGCE, the interfacial Ret increased dramatically from 320 to 1650 Ω due to hindrance of the electron transfer process of redox probe at the electrode surface after modification. When the thiolated aptamer bound to the Fe3O4@Au MBs through the famous Au–S linkage and then treated with MCH, the polyanionic phosphate

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backbone of the aptamer repelled the negatively charged redox couple thus the Ret value was increased to be 2150 Ω.52,56 Another factor that contributed to the Ret increment was the formation of the MCH film on the Fe3O4@Au MBs surface with poor conductivity.52 Afterward, the MB-aptamer was then hybridized with the cDNA to bring the NGQDs@SiO2 NPs on the Fe3O4@Au MBs surface and a further enhancement of Ret was observed, which demonstrated the successful fabrication of the MB-aptamer/cDNA-NGQDs@SiO2 bioconjugations.57 The corresponding cyclic voltammograms (CVs) showed the decrease in peak current and the increase in peak-to-peak along with the aptasensor fabrication process (Figure 3B), which is in good agreement with the EIS studies. The

successful

formation

of

the

MB-aptamer/cDNA-NGQDs@SiO2

bioconjugations was also supported by the ECL measurements in the air-saturated 0.1M pH 7.4 Tris-HCl buffer containing 0.1 M KCl and 0.1 M K2S2O8 (Figure 3C). A weak ECL emission at –1.8 V was recorded from the MB-aptamer modified mGCE whereas

a

large

increase

in

ECL

emission

was

observed

from

MB-aptamer/cDNA-NGQDs@SiO2 modified mGCE at the same position. These results suggested that this ECL emission could be greatly enhanced when NGQDs@SiO2

NPs

were

attached

on

the

electrode

surface.

The

MB-aptamer/cDNA-NGQDs@SiO2 bioconjugations was suitably used as the ECL aptasensor due to the extremely stable ECL signals under continuous potential scanning (Figure 3D). No obvious ECL emission was observed when the same electrode was measured in the air-saturated Tris-HCl buffer containing 0.1 M KCl in

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the absence of K2S2O8 (Figure S5), indicating that S2O82– was essential in the cathodic ECL process.58

A

B Current / uA

900 600 300

4500

b

0.0

-0.3

3000

a

b

-0.6

1500 0 a -2.0

-2.0 -1.5 -1.0 -0.5 0.0 Potential / V

-1.5 -1.0 -0.5 Potential / V

b

c d

0

0.6

D 6000

ECL Intensity / a.u.

C 6000

a

20

-20

d a b c 0 1000 2000 3000 4000 Z' / ohm Current / mA

-Z" / ohm

1200


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


0.4 0.2 0.0 Potential / V

-0.2

250

1000

4500 3000 1500

0.0

0 0

500 750 Time / s

Figure 3. EIS (A) and CVs (B) of bare (a), MBs (b), MB-aptamer (c), and MB-aptamer/cDNA-NGQDs@SiO2 (d) modified mGCE in 5.0 mM Fe(CN)63−/4− containing 0.1 M KCl at 100 mV s−1. Inset of (A): the equivalent circuit. (C) ECL-potential curves and corresponding CVs (inset) of MB-aptamer (a) and MB-aptamer/cDNA-NGQDs@SiO2 (b) modified mGCE in 10 mM Tris-HCl buffer containing 0.1 M KCl and 0.1 M K2S2O8. (D) Stability of ECL intensities from the MB-aptamer/cDNA-NGQDs@SiO2 modified mGCE. From the CVs corresponding to the ECL curves (inset of Figure 3C), only one cathodic peak at –1.1 V was observed from MB-aptamer modified mGCE. Meanwhile, two

cathodic

peaks

at

–1.72

and

–0.90

V

were

monitored

for 15



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MB-aptamer/cDNA-NGQDs@SiO2 modified mGCE. This was in good agreement with the observations for pure GQDs bound directly with DNA as label.52 The peak around –1.0 V for both electrodes was attributed to the reduction of S2O82– to anion sulfate

radical

SO4–•.59

The

reduction

peak

potential

of

S2O82–

at

MB-aptamer/cDNA-NGQDs@SiO2 modified mGCE was more positive for ~210 mV and exhibited higher reduction peak current than that of MB-aptamer modified mGCE, attributing to the excellent electrocatalytic activity of NGQDs with doped nitrogen.54,60 An additional peak located at –1.72 V could be assigned to the electrochemical reduction of attached NGQDs on the electrode surface,52,59 which produced negatively charged radicals of NGQDs–•. The strongly oxidizing SO4–• resultants, were then reacted with NGQDs–• via electron-transfer annihilation to generate an excited state (NGQDs*) that finally produce an ECL emission.52,59 When the dissolved oxygen was removed from the electrolyte solution, the ECL intensity was a bit lower than that measured in the air-saturated solution (Figure S5). That is because the dissolved oxygen can be reduced to OOH– upon the potential scan with an initial negative direction and the resulting OOH– could react with NGQDs–• to produce light emitting species NGQDs*.60 Hence, the ECL emission of NGQDs@SiO2 in an air-saturated buffer containing S2O82–, can be expressed as follows: S2O82– + e– NGQDs + e– O2 + H2O +2e–

SO42– + SO4–•

(1)

NGQDs–•

(2)

OOH– + OH–

(3)

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SO4–• + NGQDs–•

NGQDs* + SO42–

2NGQDs–• + OOH– + H2O NGQDs*

3OH– + 2NGQDs*

NGQDs + hv

(4) (5) (6)

Aptasensing Principle and Optimization of Key Parameters. The working principle of the versatile aptasensor is indicated as Scheme 1B. When OTA is present, the aptamer specific for OTA would perfer to complex with the toxin molecule, leading to release of the preloaded NGQDs@SiO2 NPs from MB-aptamer into the bulk solution. The more OTA molecules in the detection system, the more NGQDs@SiO2 NPs were released into the solution and the less NGQDs@SiO2 NPs were accumulated on the mGCE surface. After a one-step incubation procedure and followed a simple magnetic separation and electrode modification, this aptasensor can be used for dual channel detection combined the enriched solid-stated ECL and homogeneous FL analysis. Before OTA detection, some important parameters involved in the detection process should be investigated to maximize the efficiency and sensitivity of the aptasensor. The incubation time of the OTA with its aptamer is a very important parameter on the aptasensor performance. At the early stage of the incubation, the ECL response signal decreased accordingly with the increase of the time and changed very little after 50 min (Figure 4A). To generate a rapid-response method, the time of 50 min was adopted as the incubation time for subsequent detection. The temperature of incubation solution is another important parameter relevant to the aptasensor performance. Figure 4B indicated that the ECL signal reached the minimum intensity

17



when the reaction temperature was 37 °C, which was then selected as the optimal temperature for the following research.

B


A 6000 ECL Intensity / a.u.

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4800 3600 2400 1200 0 20 40 60 80 Incubation Time / min

3150 2700 2250 1800 1350 4

37 o 55 25 Temperature / C

Figure 4. Dependence of the incubation time (A) and temperature (B) on the aptasensor performance. Analytical Performance. Under the optimized detection conditions, ECL measurements were used to quantitatively assess the response range and detection limit of the aptasensor for OTA. In the presence of OTA, decreased NGQDs@SiO2 NPs were remained on the MB-aptamer surface and ultimately resulted in a decreased ECL signal as the concentration of OTA increased (Figure 5A). By analyzing the ECL intensity (IE) with the concentrations of OTA, it is found that the IE displays a good linear negative relationship with the logarithm of OTA concentration in the range from 1 pg mL–1 to 10 ng mL–1 (Figure 5B). The corresponding regression equation can be expressed as IE = 4976.6–919.4 log (cOTA/ng mL–1) with the correlation coefficient of R2 = 0.9932. The limit of detection (LOD) was estimated to be 0.5 pg mL–1 based on S/N=3.

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f

3000

g h

1500 0

i

j k l

4800 3600 2400 0

FL Intensity / a.u.

300

0 400

3000 2000 1000

0

1 2 3 4 log(cOTA / ng mL-1)

12 24 36 48 cOTA / ng mL-1

D 600

l

450

150

4000

1200

Time

C 600

5000

a

500 600 700 800 Wavelength / nm

FL Intensity / a.u.

e


B 6000 ECL Intensity / a.u.


A 6000 a bc d 4500

FL 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


450 300 150

600 450 300 150 0 1

0 0

2 3 4 5 log(cOTA / ng mL-1)

50 100 150 cOTA / ng mL-1

200

Figure 5. (A) ECL profiles of the aptasensor corresponding to various OTA concentrations (from a to l: 0, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 50 ng mL–1). (B) The relationship between the ECL intensity and the concentration of OTA. Inset: the calibration curve for the ECL assay. (C) FL spectra of the aptasensor corresponding to various OTA concentrations (from a to l: 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, and 200 ng mL–1). (D) The relationship between the FL intensity and the concentration of OTA. Inset: the calibration curve for the FL assay. Instead of ECL measurements, OTA can be quantitatively detected by observing the FL emission intensity of the NGQDs@SiO2 NPs released in the bulk solution after the magnetic separation. The FL intensity of the collected solution was strongly dependent on the amount of the released NGQDs@SiO2 NPs, which was positive

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correlated to the concentration of OTA (Figure 5C). As expected, the FL intensity (IF) increased as the concentration of OTA increased, while the increased IF is directly proportional to the logarithm of OTA concentration in the range from 20 pg mL–1 to 50 ng mL–1 (Figure 5D). The calibration curve fitted a regression equation of IF = –131.1+138.5 log (cOTA/ng mL–1) with a correlation coefficient of R2=0.9933 and a LOD of 12 pg mL–1 (S/N = 3). It is clear that the enriched solid-stated ECL assay has a lower LOD due to the effective preconcentration of the NGQDs@SiO2 NPs. However, the homogeneous FL assay possessing wide dynamic range can be used as the assisted detection for preliminary screens due to its high-throughput capability and convenient operation. To further confirm the superior performance of proposed aptasensor, characteristics including the linear range and LOD achieved have been compared with those of other aptasensors for OTA reported in literatures (Table S1). As indicated, the present aptasensor exhibited a broader linear range and a lower LOD. Its sensitivity was at least 2 and 10–fold higher than the corresponding valus observed in most other existed aptasensors, using FL or ECL transducers respectively. The selectivity of this biosensor was evaluated by the comparison of the sensing results of FB1, AFB1, OTB and OTA. The response signals to FB1, AFB1 and OTB were neglectable with a concentration of 10 ng mL–1 each, while an obvious increase in response was observed for OTA with a concentration of 5 ng mL–1 (Figure S6), attributing to the specific recognition force of aptamer to OTA. The reproducibility of the aptasensor via dual detction channel was investigated at the OTA concentration of 5 ng mL–1. The relative standard deviation (RSD) for five measurements was 6.31%

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and 4.79% for ECL and FL assay, respectively. Significantly, the RSD value for the FL assay was a bit smaller than that obtained by ECL assay, which indicates the method by measuring the FL intensity is more reproducible by using the homogeneous detection mode. However, the detection channel with ECL is preferable by using cheaper instrument but with sufficient sensitivity. Application in Real Samples. This aptasensor was applied to peanut (without shell) from the local supermarket to investigate its potential practical applications. The preparation of the real testing samples was according to our reported descriptions.48 The peanut samples were spiked with 0.05, 0.5, and 5 ng mL–1 of OTA, then detected by the proposed aptasensor using ECL analysis. The recoveries of the spiked samples ranged from 94.8 to 99.4% with RSD lower than 8.3% (Table S2), demonstrating that the present aptasensor can be applied for the detection of OTA in complex real samples. 

CONCLUSIONS

A modified Stöber method to prepare the NGQDs@SiO2 NPs was proposed for the first time. Using NGQDs@SiO2 NPs as an indicator for both ECL and FL signal generation, a versatile aptasensor based on OTA-induced structure switching of aptamers was thus developed by combination with the enriched solid-state ECL analysis and homogeneous FL detection. The enriched solid-stated ECL assay has a lower LOD can be used to get accurate results while the homogeneous FL assay possessing wide dynamic range can be used as the assisted detection for preliminary screens due to its high-throughput capability and convenient operation. One can get a

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preliminary screen or more accurate result by simply tuned the detection model. This strategy may provide a bridge between a highly sensitive ECL assay and a rapid FL assay, which can be easily extend to other targets with the accessibility of the aptamer sequences. 

ASSOCIATED CONTENT

Supporting Information Synthesis of the NGQDs, preparation of the MB-aptamer, Comparison of the present aptasensor with others, real sample detection, XPS and FL spectra of the pure GQDs, UV-vis absorption and the FL excitation and emission spectra of NGQDs, TEM image and XRD patterns of the MBs, the results of the control experiments, and the selectivity of the aptasensor. This material is available free of charge via the Internet at http://pubs.acs.org. 

AUTHOR INFORMATION

Corresponding Authors *Tel.: +86 511 88791800; Fax: +86 511 88791708. E–mail: [email protected] (K. Wang), [email protected] (X. Y. Huang). Notes The authors declare no competing financial interest. §

These authors contributed equally to this work.



ACKNOWLEDGMENTS

This work was partially sponsored by the National Natural Science Foundation of China (Nos. 21405063, 21375050, 31071549, and 21175061), the Natural Science

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Foundation of Jiangsu province (No. BK20130481), China Postdoctoral Science Foundation funded project (No. 2015T80517), 2015 fourth "333 high level talent project" of Jiangsu province (No. BRA2015320), and Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 14KJA550001). 

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A novel "dual-potential" electrochemiluminescence aptasensor array using CdS quantum dots and luminol-gold nanoparticles as labels for simultaneous detection of malachite green and chloramphenicol.

Electrochemiluminescence resonance energy transfer between graphene quantum dots and gold nanoparticles for DNA damage detection.

Signal-on dual-potential electrochemiluminescence based on luminol-gold bifunctional nanoparticles for telomerase detection.

Functionalized graphene as sensitive electrochemical label in target-dependent linkage of split aptasensor for dual detection.

Biobar-coded gold nanoparticles and DNAzyme-based dual signal amplification strategy for ultrasensitive detection of protein by electrochemiluminescence.

Fabrication of graphene oxide decorated with nitrogen-doped graphene quantum dots and its enhanced electrochemiluminescence for ultrasensitive detection of pentachlorophenol.

Dual-Mode SERS-Fluorescence Immunoassay Using Graphene Quantum Dot Labeling on One-Dimensional Aligned Magnetoplasmonic Nanoparticles.

A monochromatic electrochemiluminescence sensing strategy for dopamine with dual-stabilizers-capped CdSe quantum dots as emitters.

A novel electrochemiluminescence aptasensor based on in situ generated proline and matrix polyamidoamine dendrimers as coreactants for signal amplication.

Sensitive electrochemical aptasensor for thrombin detection based on graphene served as platform and graphene oxide as enhancer.

Multi-positively charged dendrimeric nanoparticles induced fluorescence quenching of graphene quantum dots for heparin and chondroitin sulfate detection.

Nicking enzyme and graphene oxide-based dual signal amplification for ultrasensitive aptamer-based fluorescence polarization assays.

High-performance flexible graphene aptasensor for mercury detection in mussels.

Magnetically triggered dual functional nanoparticles for resistance-free apoptotic hyperthermia.

An efficient signal-on aptamer-based biosensor for adenosine triphosphate detection using graphene oxide both as an electrochemical and electrochemiluminescence signal indicator.

On-chip graphene oxide aptasensor for multiple protein detection.

Colorimetric aptasensor using unmodified gold nanoparticles for homogeneous multiplex detection.

Electrochemiluminescence recovery-based aptasensor for sensitive Ochratoxin A detection via exonuclease-catalyzed target recycling amplification.

Bare magnetic nanoparticles as fluorescence quenchers for detection of thrombin.

A simple and sensitive impedimetric aptasensor for the detection of tumor markers based on gold nanoparticles signal amplification.

Gold Nanoparticles dotted Reduction Graphene Oxide Nanocomposite Based Electrochemical Aptasensor for Selective, Rapid, Sensitive and Congener-Specific PCB77 Detection.

A universal fluorescence sensing strategy based on biocompatible graphene quantum dots and graphene oxide for the detection of DNA.

A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene-molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification.

Label-free electrochemical lead (II) aptasensor using thionine as the signaling molecule and graphene as signal-enhancing platform.