MoS2 Quantum Dots as New Electrochemiluminescence Emitters for

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MoS2 Quantum Dots as New Electrochemiluminescence Emitters for Ultrasensitive Bioanalysis of Lipopolysaccharide Min Zhao, Anyi Chen, Dan Huang, Yaqin Chai, Ying Zhuo, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01558 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 13, 2017

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Analytical Chemistry

MoS2 Quantum Dots as New Electrochemiluminescence Emitters for Ultrasensitive Bioanalysis of Lipopolysaccharide Min Zhao, An-Yi Chen, Dan Huang, Ya-Qin Chai, Ying Zhuo,∗ Ruo Yuan∗ Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China

ABSTRACT

Cd-based semiconductor quantum dots (QDs) with size-tunable luminescence and high quantum yield have become the most promising electrochemiluminescence (ECL) emitters. However, their unavoidable biotoxicity limited their applications in bioassays. Here, the nontoxic and economical MoS2 QDs prepared by chemical exfoliation from the bulk MoS2 were first investigated as new ECL emitters, and then the possible luminescence mechanism of MoS2 QDs was studied using ECL-potential curves and differential pulse voltammetry (DPV) methods in detail. With MoS2 QDs as the ECL emitters and triethylamine (TEA) as the efficient coreactant, a practical and label-free aptasensor for lipopolysaccharide (LPS) detection was constructed based on aptamer recognition-driven target-cycling synchronized rolling circle amplification. Comparing to conventional stepwise reactions, this target-cycling

∗

Corresponding authors at: Tel.: +86 23 68252277, fax: +86 23 68253172.

E-mail addresses: [email protected] (Y. Zhuo), [email protected] (R. Yuan). 1

ACS Paragon Plus Environment


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synchronized rolling circle amplification achieved more efficient signal amplification and simpler operation. The developed assay for LPS detection demonstrated a wide linear range of 0.1 fg/mL to 50 ng/mL with limit of detection down to 0.07 fg/mL. It is worth mentioning that MoS2 QDs with stable ECL emission exhibited a great application potential in ECL bioanalysis and imaging as a new type of excellent emitter candidates. KEY WORDS: MoS2 QDs; electrochemiluminescence; rolling circle amplification; target-cycling; lipopolysaccharide

INTRODUCTION

Electrochemiluminescence (ECL) methodology, as its unique characteristics of excellent controllability, high sensitivity and low-cost instruments,1 has been widely used as a powerful tool in many analytical chemistry related areas of food analysis2, environmental monitoring3 and clinical diagnosis4. Over the last decade, Cd-based quantum dots (QDs) have attracted increasing interests in ECL assays as emitters due to their size-tunable luminescence, high quantum yield, easy functionalization.5-7 However, increasing evidences indicate that most Cd-based QDs exhibit unavoidable biotoxicity due to the leakage of toxic metal ions, which drives researchers to seek more superior species to satisfy the requirement of biological applications. The emerging MoS2 QDs, a class of unique graphene-analogous transition-metal dichalcogenide nanomaterial, possess the above excellent properties of Cd-based QDs, even more important, they present preeminent biocompatibility due to their 2


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heavy-metal-free

characteristics.8.9

For

example,

Dong

et

al.

synthesized

green-emission MoS2 QDs with quantum yield as high as 19% for bioimaging.10 Dai and coworkers developed size-tunable MoS2 QDs as photoluminescent probes for intracellular microRNA detection and multiphoton bioimaging.11 Shaijumon’s group12 and Wu’s group8 successively reported that MoS2 QDs exhibited high efficiency of electro-catalytic activity in hydrogen evolution reaction. However, these works mainly focused on the photoluminescent and electro-catalytic properties, the ECL behavior of MoS2 QDs has never been explored before. Here, we first investigated the ECL emission of MoS2 QDs as new ECL emitters with the coreactant of triethylamine (TEA) and further introduced MoS2 QDs/TEA system into biosensing application. Lipopolysaccharide (LPS, also referring to bacterial endotoxins) is the primary constituent in the outer cell wall of Gram-negative bacteria and possesses extremely toxic activity to the host.13,

14

There is a strong desire for rapid and sensitive

monitoring of LPS due to its high biological activity and toxicity even at a low concentration of picomole per milliliter range. In 2012, a high affinity aptamer of LPS was first selected by Choe’s group15, which bridged the gap between the needs of highly sensitive detection and the high specificity with controllable modification. Recently, the development of nucleic acid amplification technologies has become a powerful method in sensitivity improvement of bioassays.16-19 Especially, isothermal nucleic acid amplification technologies20, 21, for example, rolling circle amplification (RCA)22, 23, hybridization chain reaction (HCR)24 and catalyzed hairpin assembly (CHA)25 et al., have widely used in quantitative assessment of nucleic acids, proteins 3



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and small molecules due to their excellent specificity and high amplification efficiency. Zhang and coworkers proposed a RCA-reliant sensitive biosensor for microRNA detection based on the microRNA transforming to target-related DNA as a primer and then triggering RCA.22 Huck’s group developed a RCA-based visual fluorescence method for gene expression evaluation by the reverse transcription of mRNA to DNA as a primer for further RCA initiation.23 Summarized by the above-mentioned cases, RCA was usually executed through two steps-dependent reactions of (1) target transformation from target RNA to DNA as a primer and (2) the initiation of RCA by the primer. In our previous work, protein transformation to DNA for triggering CHA was designed to construct a sensitive aptasensor, which not only expanded the application of nucleic acid amplification into the protein detection, but also introduced the target-cycling strategy for signal amplification.26 However, the two steps-dependent reactions still remained some defects of the high errors risk, the complicated operation and the prolonged process. Thus, it is a fierce challenge to develop efficient target transformation-free dual amplification of target-cycling and RCA for aptasensor construction. Here, a multifunctional circular recognition probe (CRP), as LPS recognition element and RCA template, was designed to realize aptamer recognition-driven target-cycling synchronized RCA for more efficient amplification and simpler operation. In this work, a label-free “on-off” MoS2 QDs-realized ECL aptasensor with aptamer recognition-driven target-cycling synchronized RCA was developed for sensitive detection of LPS. Initially, MoS2 QDs encapsulated Pd-Au convex 4


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hexoctahedrons composites (abbreviation as MoS2@Pd-Au, Scheme 1A) were immobilized onto the working electrode surface for further assembling the primer to fabricate the biosensing platform to obtain the strong ECL signal as “on” state with the use of TEA as the coreactant. Interestingly, Pd-Au convex hexoctahedrons with large surface area and excellent electrical conductivity could not only immobilize massive MoS2 QDs but also improve electron transfer of the biosensing interface to amplify the ECL signals. Next, a multifunctional circular recognition probe (CRP), which was made up of LPS aptamer (I), complementary sequence of primer (c-primer) domain (II) and C-rich domain (III), was designed. As expect, in the presence of target LPS, LPS specifically bond to the aptamer (I) to form the LPS-aptamer complex. Meanwhile, the c-primer domain (II) was released and further combined with the primer immobilized onto the surface of the electrode to launch target-cycling and RCA via one-step-dependent reaction. Then, the generated RCA product with numerous G-rich sequence embedded hemin to form hemin/G-quadruplex. As a result, mass hemin/G-quadruplex effectively quenched the ECL emission of MoS2 QDs/TEA system to obtain the weak ECL signal as “off” state, making the ECL changes increase with the increasing LPS concentrations. Conversely, in the absence of target LPS, c-primer domain (II) was blocked by LPS aptamer (I) to forbid the start of RCA, which could not embed hemin for quenching the ECL signal of MoS2 QDs/TEA system. On account of the high ECL emission of the MoS2 QDs and the efficient quenching system, this proposed aptasensor exerted highly sensitive detection of LPS. Significantly, MoS2 QDs provided promising ECL emitters candidates to develop 5



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high-performamce ECL systems for the applications in the bioassays.

Scheme 1. Schematic Illustration of the Ultrasensitive “on-off” ECL Aptasensor for LPS Detection based on Aptamer Recognition-driven Target-cycling Synchronized RCA. (A) The Preparation Process of MoS2@Pd-Au.

EXPERIMENTAL METHODS

Materials and Reagents.

Bulk

molybdenum

disulfide

powders,

lipopolysaccharide

6


(LPS)

from

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Escherichia coli 055:B5, gold chloride (HAuCl4), cetylpyridinium chloride monohydrate

(CPC),

cetyltrimethylammonium

bromide

(CTAB),

potassium

tetrachloropalladate (K2PdCl4), chitosan (CS) and 6-mercapto-1-hexanol (MCH) were brought from Sigma-Aldrich Chemical Co. (St. Louis, MO, U.S.A.). Ascorbic acid (AA), triethylamine (TEA), sodium citrate, ethylenediaminetetraacetic acid (EDTA) and N, N-dimethylformamide (DMF) were purchased from Kelong Chemical Inc. (Chengdu, China). T4 DNA ligase and 10×reaction buffer were purchased from Takara Biotechnology Company Ltd. (Dalian, China). The HPLC-purified DNA oligonucleotides, deoxyribonucleoside triphosphate (dNTPs), Phi29 DNA polymerase and 10×reaction buffer were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The synthetic DNA sequences were listed in Table S1 (see Supporting Information). The preparation of the circular recognition probe (CRP) was described in the Supporting Information. The phosphate buffer solution (PBS, pH 8.0) containing 0.5 M Na2HPO4, 0.5 M KH2PO4 and 0.1 M KCl was used as the electrolyte solution. All the solutions were prepared using deionized water (specific resistance of 18.2 MΩ·cm). Pd-Au convex hexoctahedrons (abbreviation as Pd-Au CHs) were prepared according to the previous report27 by Zhang et al..

Apparatus.

The ECL and electrochemical measurements were carried out using a model MPI-A multifunctional analyzer (Xi'An Remax Electronic Science & Technology Co.

7



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Ltd., Xi'An, China) and a CHI660C electrochemistry workstation (Shanghai Chenhua Instruments, China), respectively. A conventional three-electrode system was consisted of a modified glass carbon electrode (working electrode), a Ag/AgCl electrode (reference electrode) and a platinum wire (counter electrode). Gel electrophoresis was executed with BG-verMIDI standard vertical electrophoresis apparatus (Baygene, Beijing, China). The ultraviolet-visible (UV-vis) absorption and photoluminescenece spectra of MoS2 QDs were performed using a UV-2450 UV-Vis spectrophotometer (Shimadzu, Tokyo, Japan) and an F-5700 spectrofluorophotometer (Hitachi, Tokyo, Japan), respectively. The morphologies of different nanomaterials were characterized by Tecnai G2 F20 high-resolution transmission electron microscopy (HRTEM, FEI, U.S.A), S-4800 field emission scanning electron microscopy (FESEM, Hitachi, Tokyo, Japan), Multimode 8 atomic force microscopy (AFM, Bruker, Germany).

Preparation of MoS2 QDs and MoS2@Pd-Au. Initially, MoS2 QDs were synthesized according to the published work8 with minor modification as follows. Briefly, 0.25 g bulk molybdenum disulfide powders were added into 25 mL DMF. Followed by continuous sonication for 5 h, the above resultant mixture was refluxed with gently stirring for 6 h at 140 °C. Next, the resultant mixture was sonicated for another 2 h and then centrifuged at 8000 rpm to collect the light yellow supernatant. Later, the prepared MoS2 QDs were dialyzed against deionized water for more than 48 h to obtain MoS2 QDs aqueous solution.

8


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Subsequently, 1 mL MoS2 QDs aqueous solution was added into 2 mL Pd-Au CHs solution. Then, the above mixture was gently stirred for 8 h and centrifuged at 8000 rpm to obtain MoS2 QDs encapsulated Pd-Au CHs composites (abbreviation as MoS2@Pd-Au) by the Pd/Au-S covalent bond28-30. The prepared MoS2@Pd-Au were stored at 4 °C under a dark environment until further use.

Fabrication of Biosensor.

The glassy carbon electrode (GCE, 4 mm in diameter) was continuously polished with 0.3 µm and 0.05 µm alumina slurries and then ultrasonicated in deionized water to obtain a mirror-like electrode. Next, the prepared MoS2@Pd-Au were dispersed in 1.0 mL of 0.02% CS solution, which were dropped onto GCE and dried at room temperature

to

form

a

uniform

film

(CS-MoS2@Pd-Au/GCE).

Later,

CS-MoS2@Pd-Au/GCE was further treated with 15 µL of 1% glutaraldehyde for 2 h to activate the CS film for cross-linking with the amino-terminated primer and then incubated with 20 µL of primer for another 2 h to obtain a primer-assembled electrode (primer/MoS2@Pd-Au/GCE). Followed by blocking with 1.0 mM MCH for 40 min, the biosensor (MCH/primer/MoS2@Pd-Au/GCE) was fabricated and stored at 4 °C.

ECL Measurement Procedure.

Initially, the aptamer recognition-driven target-cycling synchronized RCA process was implemented with the prepared biosensor (MCH/primer/MoS2@Pd-Au/GCE) as follows. 15 µL of 1× reaction buffer containing 1 µM as-prepared CRP, 100 U/mL phi29 DNA polymerase, 1.0 mM dNTPs, and various concentrations LPS was 9



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dropped onto the surface of biosensing platform and reacted for 2 h at 37 °C. Followed by gently rinsing with PBS (pH 8.0) to remove the unbound reagents, RCA/MCH/primer/MoS2@Pd-Au/GCE was further incubated with 15 µL of 0.1 mM hemin stock solution for 1 h to form hemin/G-quarduplex. The ECL measurement was executed with the MPI-A ECL analyzer by scanning the potential from -1.0 V to 1.3 V with a scan rate of 0.5 V/s in 0.1 M PBS (pH 8.0) containing 20 mM TEA. The changes of ECL intensity (∆ECL), defined as ∆I = I0 – I (where I stood for the ECL intensity with samples and I0 stood for the ECL intensity with blank), were positively related to the LPS concentrations.

RESULTS AND DISCUSSION

Morphology Characterization of MoS2 QDs

Typical HRTEM images of the MoS2 QDs were depicted with different scales in Figure 1A and Figure 1B. Figure 1A exhibited uniform and monodisperse MoS2 QDs in narrow distribution of 4.2 ± 0.1 nm diameter. The highly paralleled and ordered lattice fringe with the d-spacing of 0.23 nm was observed (Figure 1B and 1C), which was corresponding to the (103) faces of MoS2 well-crystals31,

32

. Moreover, the

apparent aureole appeared in the selected area electron diffraction (SAED) patterns (Figure 1D), revealing the polycrystalline of MoS2 QDs. The thickness of the MoS2 QDs was also surveyed by AFM (Figure 1E). A line profile painted on the AFM image displayed a height difference of ~1.7 nm (Figure 1F), validating the mono-layered or bi-layered MoS2 QDs in agreement with the previous work8, 33. 10


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Figure 1. HRTEM images of the MoS2 QDs with 20 nm scale (A) and 5 nm scale (B). The inset of (A): the size distribution of MoS2 QDs. The amplified HRTEM image (C) and the relevant SAED patterns (D) of MoS2 QDs. AFM image (E) of MoS2 QD and the line profile (F) along the white line in the AFM image.

TEM and EDX Elemental Mapping Characterization of MoS2@Pd-Au

The formation of MoS2@Pd-Au was confirmed using TEM. Figure 2A was a typical TEM image of Pd-Au CHs, which showed angular polyhedral shape with multiple facets and the uniform size with a diameter of 90 ± 10 nm. The TEM image of MoS2@Pd-Au composites (Figure 2B) revealed that the Pd-Au nanoparticles (dark region) were coated with thin substances (grey region) formed by some small distorted granules, which might be the MoS2 QDs.

11



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Figure 2. TEM images of Pd-Au CHs (A) and MoS2@Pd-Au (B).

Further insight into the element distribution of MoS2@Pd-Au was verified by energy dispersive X-ray spectroscopy (EDX) elemental mapping with a field emission scanning electron microscope (FESEM). As shown in Figure 3B and 3C, mass Au(0) formed from Au3+ reduction was grew up with the seeds of Pd nanoparticles. Since the surfactant CPC was used during the synthesis process of Pd-Au CHs, the carbon and oxygen elements were observed (Figure 3D and 3E). As expected, MoS2 QDs with the main elements of Mo (blue region, Figure 3F) and S (purple region, Figure 3G) were homogeneously distributed on the surface of Pd-Au CHs. It confirmed that MoS2 QDs were decorated onto the surface of Pd-Au CHs based on the Pd/Au-S bond28-30. Additionally, the composition proportion of MoS2@Pd-Au was investigated by FESEM-EDX spectrum in Figure S1 (Supporting Information).

12


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Figure 3. FESEM image of MoS2@Pd-Au (A). EDX elemental mappings of Au (B), Pd (C), C (D), O (E), Mo (F) and S (G).

Optical Properties of MoS2 QDs.

Optical properties of the MoS2 QDs were characterized by UV-vis absorption, photoluminescence (PL) and ECL spectra. As exhibited in Figure 4A, the UV-vis absorption was observed with distinct absorption peaks at 230 nm and 301 nm (green dashed line), which was assigned to the excitonic features of MoS2 QDs34. The PL spectra of MoS2 QDs were investigated by setting up different excitation wavelengths from 350 nm to 410 nm (Figure 4A). The strongest peak at 456 nm was observed at the excitation wavelength of 370 nm. In addition, the inset of Figure 4A presented the photographs of the MoS2 QDs, showing bright blue luminescence under the radiation of 365 nm UV light. Figure 4B demonstrated the ECL emission spectrum (blue line) of the MoS2 QDs with the maximum emission wavelength at 625 nm. A large red-shifted (169 nm) of ECL emission spectrum was observed comparing to the PL spectrum of MoS2 QDs with PL emission maximum at 456 nm. It could be attributed 13



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to the electronic injection into a surface trap within the band gap of QDs35.

Figure 4. UV-vis absorption spectrum and the PL spectra of MoS2 QDs (A). The inset of A: photographs of MoS2 QDs under the radiation of natural light (left) and 365 nm UV light (right). Normalized PL excitation spectrum, PL emission spectrum and ECL emission spectrum of MoS2 QDs (B).

Mechanism Investigation of the MoS2 QDs ECL System.

ECL and DPV measurements were implemented with bare GCE in the MoS2 QDs solution to validate the possible luminescent mechanism of MoS2 QDs-based ECL system. As curve a displayed in Figure 5A, a slight ECL signal (about 520 a.u.) was observed (curve a, green line) in the absence of TEA. Meanwhile, the DPV included the oxidation curve a' and reduction curve a'' (green lines in Figure 5B). A significant oxidation peak at 1.112 V was observed from the oxidation curve a', which was ascribed to the oxidation of MoS2 QDs (equation 1). In addition, a negligible reduction peak at -0.392 V was observed from the reduction curve a'', which was ascribed to the reduction of dissolved oxygen36. However, when the TEA coreactant was supplied into the working buffer, the ECL signal dramatically enhanced to ~9050 14


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a.u. (curve b in Figure 5A). Moreover, the oxidation and reduction currents within a range of 1.0 to 1.3 V were remarkably increased (curve b' and b'' in Figure 5B). It was attributed to the electronic losing of TEA to form strong reductant TEA• (equation 2) which further reduced MoS2 QDs(h)+ to generate the excited state MoS2 QDs* (equation 3). Subsequently, MoS2 QDs* relaxed to the ground state and emitted light simultaneously (equation 4). The possible ECL mechanisms were outlined as following equations: MoS2 QDs ‒ e‒ → MoS2 QDs(h)+

.

(1)

TEA ‒ e‒ → TEA•+ → TEA• + H+

(2)

MoS2 QDs(h)+ + TEA• → MoS2 QDs* + products MoS2 QDs* → MoS2 QDs + hν

.

(3) (4)

The ECL quenching mechanism was explored by adding hemin into the mixture of MoS2 QDs and TEA. As shown in Figure 5A, when hemin was added into the above solution, the ECL intensity exhibited a notable decrease from 9050 a.u. to 3425 a.u. (Figure 5A, curve c), manifesting the quenching of hemin toward MoS2 QDs/TEA ECL system. Meanwhile, the oxidation peak current of MoS2 QDs significantly decreased (Figure 5B, curve c'), revealing that hemin reduced the MoS2 QDs(h)+ (equation 1). Considering the mechanism of MoS2 QDs/TEA ECL system, this ECL quenching and current decrease were most likely speculated as follows: hemin(FeII) was oxidized to hemin(FeIII) on the surface of the working electrode with the positive scan of -1.0 to 1.3 V (equation 5). Then hemin(FeIII) could compete with MoS2 QDs (h)+ to react with TEA•, which decreased the produce of MoS2 QDs* (equation 6). 15



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Moreover, typical electron transfer might also occurred between MoS2 QDs* and hemin(FeIII) to directly produce MoS2 QDs (h)+ (equation 7). The ECL emission would be reduced via either of the two routes and the possible ECL mechanisms were outlined as following equations: Hemin(FeII) ‒ e‒ → Hemin(FeIII)

(5)

Hemin(FeIII) + TEA• → Hemin(FeII) + products

(6)

Hemin(FeIII) + MoS2 QDs*→ Hemin(FeII) + MoS2 QDs(h)+

(7)

Figure 5. ECL intensity-potential profiles (A) and DPVs (B) of bare GCE detecting in PBS containing different substances: (a) MoS2 QDs, (b) MoS2 QDs + 20 mM TEA, and (c) MoS2 QDs + 20 mM TEA + 0.05 mM hemin.

Feasibility Analysis of the Aptasensor for LPS Detection

In order to confirm the feasibility analysis of the aptasensor for LPS detection based on aptamer recognition-driven target-cycling synchronized RCA, we have carried out a control experiment to contrast the ECL responses in the absence/presence

target.

Specifically,

the

developed

LPS

aptasensor

(MCH/primer/MoS2@Pd-Au/GCE) was reacted with the reaction buffer containing 16


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CRP, phi29 DNA polymerase, dNTPs and in the absence/presence of 0.1 ng/mL LPS standard solution, and then incubated with 0.1 mM hemin. As shown in Figure 6, when the MCH/primer/MoS2@Pd-Au/GCEs (curve a) were incubated with blank (test, curve b) and with LPS (curve c), respectively, two obvious different ECL changes of 457 a.u. (∆I1) and 9568 a.u.(∆I2) were observed. Such preliminary results affirmed that the target LPS initiated target-cycling synchronized RCA and then the RCA product with mass G-rich sequences incubated with hemin to form the hemin/G-quadruplex which could quench the ECL signal of MoS2 QDs/TEA system, as expected.

Figure 6. ECL intensity-potential curves of the MCH/primer/MoS2@Pd-Au/GCEs (curve a) incubated with blank solution (curve b) and LPS solution (curve c), respectively. The different modified electrodes were detected in 3 mL PBS containing 20 mM TEA under the potential scanning of -1.0 ~ 1.3V with a scan rate of 0.5 V/s.

Native Polyacrylamide Gel Electrophoresis Characterization of Aptamer Recognition-Driven Target-Cycling Synchronized RCA

In order to confirm the aptamer recognition-driven target-cycling synchronized 17



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RCA, the gel electrophoresis was employed to characterize the different samples. As shown in Figure 7, the CRP, the primer and the CRP/primer mixture in lane 1, lane 2 and lane 3, respectively, displayed clear and non-interacted bands. Lane 4 loaded the mixture of primer, CRP and LPS, while it exhibited little evolution to lane 3. It was attributed that the concentration of LPS was lower than one-tenth of the concentrations of primer and CRP, which caused the little formation of LPS-CRP-primer complex. Lane 5 represented the mixture of primer, CRP, phi29 polymerase and dNTPs in reaction buffer. It showed two bands identical to lane 3, suggesting that RCA wasn’t triggered in the absence of LPS. However, when LPS was added into the above mixture, the primer band was invisible and a distinct band with extremely low migration was observed (lane 6), indicating that aptamer recognition-driven target-cycling synchronized RCA was executed, as expected.

Figure 7. Polyacrylamide gel electrophoresis analysis of different samples. Lane 1, 1 µM CRP; lane 2, 1 µM primer; lane 3, 1 µM primer + 1 µM CRP; lane 4, 1 primer µM + 1 µM CRP + 100 18


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µg/mL LPS; lane 5, 1 µM primer + 1 µM CRP + 100 U/mL phi29 polymerase + 1 mM dNTPs; lane 6, 1 µM primer + 1 µM CRP + 100 U/mL phi29 polymerase + 1 mM dNTPs + 1 µg/mL LPS.

ECL Responses of the Aptasensors toward LPS.

In order to sensitively and efficiently determine of LPS with this developed aptasensor, two key parameters, including the reaction time of RCA and the concentration of hemin, were optimized in Figure S4 of Supporting Information. The results indicated that the optimal reaction time of RCA and the concentration of hemin were 2 h and 0.1 mM hemin, respectively. Under the optimal reaction conditions, the designed aptasensors were incubated with different LPS concentrations. As exhibited in Figure 8, the elevated LPS concentrations from 0.1 fg/mL to 50 ng/mL led to a gradual ECL intensity decrease. The inset of Figure 8 exhibited the calibration plots of ∆ECL (∆I = I0 - I) vs the logarithm of LPS concentration with a linear regression equation of ∆I = 1009.9 lg c + 10077.4 (where ∆I was the change of ECL intensity and c was the LPS concentration) and a correlation coefficient of R = 0.9977. Moreover, the limit of detection (LOD) was calculated to be 0.07 fg/mL according to 3SB/m (where SB is the standard deviation of the blank, and m is the slope of the corresponding calibration curve)37. This proposed method for LPS analysis was compared with other reported methods for LPS detection (Table S2 of Supporting Information). It showed this proposed method for LPS analysis with better sensitivity, which was attributed to the powerful amplification of aptamer recognition-driven target-cycling synchronized RCA and the efficient quenching of hemin toward MoS2

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QDs/TEA system.

Figure 8. ECL intensity-potential curves of the proposed aptasensors incubated with LPS concentrations of 0.1 fg/mL, 1.0 fg/mL, 10 fg/mL, 0.1 pg/mL, 1.0 pg/mL, 10 pg/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, and 50 ng/mL (from a to j). The inset was the calibration plot of ∆ECL (∆I = I0 - I) vs the logarithm of LPS concentration. Error bars represent standard deviations of three parallel experiments. The potential scanning was set from -1.0 to 1.3V with a scan rate of 0.5 V/s.

Selectivity and Stability of the LPS Aptasensor.

To evaluate the selectivity of the designed method for LPS detection, the prepared aptasensors were tested with different non-target substances containing human serum albumin

(HSA),

thrombin

(TB),

carcinoembryonic

antigen

(CEA)

and

immunoglobulin G (IgG), it could be seen that no obvious ∆ECL was observed (Figure 9A). Despite the existence of a large excess (10-fold) of interfering matrix, no signiﬁcant influence was noticed comparing the ∆ECL of the pure LPS (1.0 pg/mL) to that of the mixture containing HSA (10 pg/mL), TB (10 pg/mL), CEA (10 pg/mL) and

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IgG (10 pg/mL). It suggested that the proposed method held high selectivity, which was attributed that the target LPS could switch the secondary structure of CRP due to the highly specific binding between LPS and its aptamer domain and then the LPS-CRP complex further hybridized with primer for proceeding RCA. Next, the RCA product containing G-rich sequence embedded hemin to fold into G-quadruplex structure for effectively quenching the ECL signal of the MoS2 QDs/TEA system. Stability, the other important performance parameter of the biosensor, was further discussed using 1.0 pg/mL LPS as a model under consecutive cyclic potential scans. As shown in Figure 9B, the proposed aptasensor has an excellent stability with a relative standard deviation (RSD) of 1.53 % in 15 cycles.

Figure 9. Selectivity (A) of the proposed ECL aptasensor against different non-targets: HSA (10 pg/mL), TB (10 pg/mL), CEA (10 pg/mL), IgG (10 pg/mL) and a mixture containing 10 pg/mL HSA, 10 pg/mL TB, 10 pg/mL CEA, 10 pg/mL IgG, and 1.0 pg/mL LPS. Stability (B) of the proposed aptasensor under consecutive 15 cyclic potential scans. The potential scanning was set from -1.0 to 1.3V with a scan rate of 0.5 V/s.

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CONCLUSIONS

In summary, the ECL emission of MoS2 QDs was first investigated and applied in a label-free aptasensor for LPS quantitative analysis with target-cycling synchronized RCA. Firstly, a novel MoS2 QDs/TEA-based ECL system was constructed, which was effectively quenched by hemin to realize an “on-off” signal response pattern. Secondly, the multifunctional CRP was designed to achieve a simple, rapid and efficient amplification strategy of aptamer recognition-driven target-cycling synchronized RCA. Moreover, this proposed protocol for LPS detection exhibited outstanding sensitivity, selectivity and stability, demonstrating a promising amplification strategy to develop the diverse aptameric systems. It is worth pointing out that the fascinating MoS2 QDs, as non-toxic, economical and superior emitter candidates, exhibit potential applications in ECL methodology. ASSOCIATED CONTENT Supporting Information Preparation of Circular Recognition Probe (CRP), Schematic Diagram of MoS2 QDs, Native Polyacrylamide Gel Electrophoresis, EDX and FESEM of MoS2@Pd-Au, DPVs of TEA, Electrochemical and ECL Characterization of the ECL Aptasensor, Optimal Conditions for the ECL Aptasensor, Table S1, Table S2 and Preliminary Analysis of Real Samples. This material is available free of charge via the Internet at http://pubs.acs.org/. AUTHOR INFORMATION

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*Corresponding authors Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected] (Y. Zhuo), [email protected] (R. Yuan). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was financially supported by the NNSF of China (51473136, 21575116, 21675129, 21675130) and the Fundamental Research Funds for the Central Universities (XDJK2015A002), China.

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MoS2 Quantum Dots as New Electrochemiluminescence Emitters for

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