Enzyme-free Target Recycling and Double-Output Amplification

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Biological and Medical Applications of Materials and Interfaces

Enzyme-free target recycling and double-output amplification system for electrochemiluminescent assay of mucin 1 with MoS2 nanoflowers as co-reaction accelerator Shengkai Li, Zhiting Liu, Jiyang Li, Anyi Chen, Yaqin Chai, Ruo Yuan, and Ying Zhuo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02262 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

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ACS Applied Materials & Interfaces

Enzyme-free target recycling and double-output amplification system for electrochemiluminescent assay of mucin 1 with MoS2 nanoflowers as co-reaction accelerator Sheng-Kai Li, Zhi-Ting Liu, Ji-Yang Li, An-Yi Chen, Ya-Qin Chai, Ruo Yuan,* and Ying Zhuo* Key Laboratory of Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People’s Republic of China ABSRTACT: In this work, a sensitive electrochemiluminescent assay of mucin 1 (MUC1) was developed with the advantages of target recycling amplification strategy and effective MoS2 nanoflowers (MoS2 NFs)-based signal probe. Briefly, the target MUC1 triggered enzyme-free recycling and double-output amplification process was executed to acquire masses of single-stranded DNA (ssDNA) as mimic target, which further participated the catalytic hairpin assembly (CHA) process for signal amplification. Meanwhile, MoS2 NFs were prepared as an effective co-reaction accelerator, which not only possessed excellent catalytic performance for H2O2 decomposion

to

largely

enhance

the

luminous

intensity

of

N-(aminobutyl)-N-(ethylisoluminol) (ABEI)-H2O2 ECL system, but also offered a desirable platform for ABEI functionalized Ag nanoparticles (ABEI-Ag complexes) loading via Ag-S binding. The experimental results showed the proposed aptasensor

∗

Corresponding authors at: Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected] (R. Yuan), [email protected] (Y. Zhuo). 1

ACS Paragon Plus Environment

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had a good linear relationship in the range of 1 fg/mL to 10 ng/mL for MUC1 detection, and the limit of detection was 0.58 fg/mL (S/N = 3). In addition, the aptasensor had nice stability, selectivity and huge potential to be applied in clinical research. KEYWORDS:

electrochemiluminescence,

double-output

amplification,

MoS2

nanoflowers, co-reaction accelerator, aptasensor

1. Introduction In recent years, target recycling amplification strategies have aroused wide interest in bioassay due to their excellent efficiency and superb selectivity, which could transfer target molecule into output DNA mimic target through elaborate sequences design.1-3 However, most of the target recycling amplification strategies were carried out with the aid of at least a kind of enzyme.4-6 Expensive enzymes need harsh storage conditions and even suffer from instability, which could influence the amplification efficiency.7 Thus, it is particularly necessary to establish an enzyme-free target recycling amplification strategy for sensitive target molecules determination. For example, Carlo et al.8 transferred target into single-stranded DNA (ssDNA) as mimic target through strand displacement amplification to realize the detection of microRNA. Hou’s group9 reported an enzyme-free amplification strategy on the basis of catalytic hairpin assembly (CHA) to obtain plenty of double-stranded DNA (dsDNA) as mimic target for protein assay. The amplification strategies above could obtain one mimic target in one circle of target recycling, resulting in the limited amplification efficiency. To achieve higher amplification efficiency, a target triggered

2


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enzyme-free recycling and double-output amplification system based on the acquirement of two mimic targets in one circle of target recycling was proposed to require a large number of ssDNA as mimic target, which further participated the CHA process for signal amplification. Significantly, the application with the combination of enzyme-free target recycling and double-output amplification process has not been attempted in bioanalysis. As a powerful analytical technique, the solid-state electrochemiluminescence (ECL) analytical technique has attracted substantial research efforts due to the prominent virtues of excellent controllability, high sensitivity, low cost and background noise.10-12 The enhancement of ECL intensity was suggested as the most desirable approach to enhance the detection sensitivity. Recently, we found the introduction of co-reaction accelerator into luminophore/co-reactant system could largely amplify the ECL intensity, because it could interact with co-reactant to boost the generation of free radical intermediate for the obtaintion of more excited state of the luminophore.13-15 The latest research16 reported that polyethylene glycol (PEG) functionalized MoS2 nanoflowers (MoS2 NFs) with good affinity could greatly promote the decomposition of H2O2. Based on this, we deduced MoS2 NFs could act as an effective co-reaction accelerator of N-(aminobutyl)-N-(ethylisoluminol) (ABEI)-H2O2 ECL system for the decomposition of H2O2 to generate masses of superoxide radical (O2•−) and hydroxyl radical (OH•) which could react with ABEI to produce the excited state substance for ECL emission.17,18 Moreover, MoS2 NFs possessed the prominent virtues of large specific surface area, good water solubility,

3



biocompatibility, and the S atoms on the surface offered a lot of vacant orbitals to combine with noble metal atoms via coordinate bond.19-21 Hence, ABEI functionalized silver/MoS2 NFs (ABEI-Ag-MoS2 NFs) complexes were prepared as signal tags in this work, in which MoS2 NFs were acted as the efficient co-reaction accelerator of ABEI-H2O2 ECL system and provided a desirable platform for masses of ABEI-Ag complexes loading via Ag-S binding.22 Significantly, the proposed ABEI-Ag-MoS2 NFs tags with luminophore and co-reaction accelerator showed high luminous intensity, which exhibited great potential application in bioanalysis. Mucin 1 (MUC1) was regarded as a significant protein biomarker for mammary cancer,23-25 thus its quantitative detection would exert a profound influence in early clinical diagnosis. In our previous work,26-28 some enzyme-assisted target recycling amplification strategies were applied to fabricate ECL aptasensors for the detection of MUC1. Herein, an ECL aptasensor with enzyme-free target recycling and double-output amplification strategy was fabricated for ultrasensitive MUC1 assay using MoS2 NFs as the co-reaction accelerator for ABEI-H2O2 ECL system. As exhibited in Scheme 1, hairpin probe 2 (HP2) was firstly immobilized on the Au nanoparticles (Au NPs) modified glass carbon electrode (GCE) surface to acquire the electrode interface. In the presence of target MUC1, aptamer-MUC1 complexes were obtained to trigger an enzyme-free target recycling and double-output amplification process (Cycle I). Therefore, a great deal of ssDNA were obtained as mimic target (output S1) based on the acquirement of two mimic targets in one circle of target recycling. Then, a CHA process (Cycle II) was triggered by output S1 to capture

4


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masses of ABEI-Ag-MoS2 NFs labeled signal probe. Hence, the constructed ECL aptasensor exhibited much wider linear range and lower detection limit for trace MUC1 assay.

Scheme 1. Design principle of the fabricated MUC1 aptasensor: (A) preparation of ABEI-Ag-MoS2 NFs/HP3 signal probe, (B) enzyme-free target recycling and double-output amplification process and (C) preparation of the modified electrode and probable mechanism of ECL signal generation.

2. Experimental Section 2.1. Instrumentation, reagents and DNA sequences All instrumentation, reagents and DNA sequences used in this work were listed in Supporting Information (SI) 1.1 and 1.2. The DNA pretreatment procedure was showed in SI 1.3. 2.2. Interfacial modification of Au@Fe3O4

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At first, Au@Fe3O4 were obtained by combining amino modified Fe3O4 magnetic beads (Fe3O4 MB) with 16 nm Au NPs via Au-N binding.29 Next, 500 µL doubled-stranded DNA (dsDNA, 2 µM) and 1 mL Au@Fe3O4 were mixed fully in a centrifuge tube, then the mixture was shaken using a shaking table at 4 °C overnight to gain dsDNA-Au@Fe3O4 solution via Au-S binding.30 Finally, 100 µL 0.25% bovine serum albumin (BSA) was added into the mixture above for 1 h to block remaining nonspecific binding sites. 2.3. Preparation of functionalized MoS2 NFs Functionalized MoS2 nanoflowers (MoS2 NFs) were prepared according to the literature16 with a little alteration. Concretely, 0.5 g polyethylene glycol (PEG, Mw = 20, 000) and 0.1766 g H24Mo7N6O24·4H2O were dissolved in 20 mL deionized water in 50 mL beaker. Then, 10 mL thiourea (TU, 2 mM) aqueous solution was added into the above solution drop by drop in 20 min under continuous stirring. Afterwards, the mixture was put into a Teflon-lined stainless steel autoclave and kept at 180 °C for 12 h. Finally, the products were collected by washing with ethanol and deionized water for three times, then dried at 60 °C for future use. In addition, MoS2 NFs without the functionalization of PEG were obtained according to the same preparation method of MoS2 NFs just without the addition of PEG. 2.4. Synthesis of ABEI-Ag-MoS2 NFs nanocomposites The ABEI-Ag-MoS2 NFs nanocomposites were obtained by employing a one-step method. At first, 0.02 g dried MoS2 NFs were dissolved in 5 mL deionized water.

Then,

5

mL

ethanol,

1

mL

AgNO3

6


(10

mM)

and

2

mL

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N-(Aminobutyl)-N-(ethylisoluminol) (ABEI, 0.02 M) aqueous solution were added. Next, the mixed solution was ultrasonicated for 10 min and stirred for 12 h in the dark at ambient temperature. After the reaction, the nanocomposites were acquired by centrifugation and washing with deionized water, then dispersed in 1 mL phosphated buffered solution (PBS, 0.1 M, pH 7.4) for further application. 2.5. Preparation of signal probe The obtained ABEI-Ag-MoS2 NFs solution was ultrasonicated for 10 min, then 200 µL hairpin probe 3 (HP3, 2 µM) was put into the above solution and stirred for 12 h in ice bath. After that, the precipitate was obtained through centrifugation and washing with deionized water. Lastly, the ABEI-Ag-MoS2 NFs/HP3 signal probe (20 mg/mL) was dispersed in 1 mL PBS (0.1 M, pH 7.4) and stored at 4 °C for use. Other different signal probes were prepared with the similar method above and the detailed operation steps were described in SI 1.5 and 1.6. 2.6. Construction of sensing interface First of all, the glassy carbon electrode (GCE) was polished with 0.3 and 0.05 µm alumina slurry and then washed thoroughly to acquire a mirror-like surface. After that, the Au NPs/GCE was gained through immersing the air-dried GCE in 2 mL HAuCl4 solution (1%) for electrostatic deposition at -0.2 V for 30 s. Whereafter, 10 µL hairpin probe 2 (HP2, 2 µM) was immobilized on the Au NPs/GCE surface via Au-S binding, then blocked with 0.25% BSA for about 40 min. 2.7. Assay procedure Above all, 100 µL dsDNA-Au@Fe3O4 solution in centrifuge tube was performed

7



by magnetic separation and then remove the supernatant. Hairpin probe 1 (HP1, 2 µM), S2 (2 µM) and different concentration of MUC1 were added into the centrifuge tube and kept shaking at room temperature for 2 h. Next, the supernatant (referred to as sample solution) was collected by magnetic separation. After that, sample solution and ABEI-Ag-MoS2 NFs/HP3 signal probe solution (volume ratio 1:1) were mixed together, then 10 µL the mixture above was incubated on the resultant electrode surface for 2 h. Ultimately, ECL measurements were carried out in 2 mL PBS (0.1 M, pH 7.4) containing 3 mM H2O2 with the potential from 0 to 0.55 V, the photomultiplier tube of 800 V and scan rate of 100 mV/s. 3. Results and discussion 3.1. Morphology characterization of the prepared nanomaterials SEM characterization was used to examine the surface morphologies of MoS2 NFs and ABEI-Ag-MoS2 NFs. As illustrated in Fig. 1A and 1B, the MoS2 NFs showed flower-like structure and uniformed shape with average particle size about 400 nm. However, we observed masses of small and bright nanoparticles evenly covered the surface of MoS2 NFs (Fig. 1C and 1D), indicating the generated Ag nanoparticles (Ag NPs) were combinded with MoS2 NFs via Ag-S binding. Besides, SEM image of Au NPs and transmission electron microscope (TEM) image of Au@Fe3O4 were showed in Fig. S1 (SI 1.7).

8


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Fig. 1. SEM images of (A, B) MoS2 NFs and (C, D) ABEI-Ag-MoS2 NFs under different scales.

3.2. Contact angle experiments and UV-vis, Ramam, XPS analysis The contact angle experiments were performed on a glass slide with a water droplet (5 µL) of MoS2 NFs with and without the functionalization of PEG. The contact angle for MoS2 NFs without the functionalization of PEG was 21.7° (n = 5, Fig. 2A), which was much bigger than that for MoS2 NFs with the functionalization of PEG (10.6°, n = 5, Fig. 2B). It can be seen from the inserts, functionalized MoS2 NFs exhibited homogeneous in aqueous solution while MoS2 NFs without the functionalization of PEG showed poor dispersivity. The results indicated that the PEG functionalized MoS2 NFs used in this work possessed good water solubility and dispersivity. UV-vis analysis were firstly used to characterize ABEI and the prepared nanomatarials. As depicted in Fig. 2C, the UV-vis absorption spectra of ABEI presented an obvious absorption peak at 230 nm approximately, which rooted in the UV absorption of -NH2 group. MoS2 NFs exhibited two different absorption peak at around 208 and 230 nm, which was attributed to the excitonic peaks of MoS2.31 9



However, the UV-vis absorption spectra of ABEI-Ag-MoS2 NFs showed a newly broad absorption band centered at 400-600 nm, indicating the generation of Ag NPs.32 Raman spectroscopy was further performed to examine the compositions and phases of the prepared nanomaterials. The Raman spectroscopy of MoS2 NFs was exhibited in curve a of Fig. 2D. The peaks at 376 cm-1 and 405 cm-1 represented the E2g1 and A1g modes, respectively, which were consistent with that reported in the literature.16 However, the characteristic peaks of ABEI-Ag-MoS2 NFs complexes (curve b of Fig. 2D) had a red shift of the E2g1 mode and a blue shift of the A1g mode because of the combination of MoS2 NFs and metal nanomaterials,33 indicating the generation of ABEI-Ag-MoS2 NFs composites. In addition, XPS analysis were applied to analyze the elemental composition of ABEI-Ag-MoS2 NFs complexes and the results were showed in SI 1.8.

Fig. 2. The shape of a water droplet on the glass slide of MoS2 NFs (A) without and (B) with the functionalization of PEG. The inserts stand for the pictures of the corresponding nanomaterial dispersed in water under visible light. (C) UV-vis absorption spectra of different materials. (D)

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Ramam spectrum of (a) MoS2 NFs and (b) ABEI-Ag-MoS2 NFs complexes with laser excitation at a wavelength of 532 nm.

3.3. Feasibility analysis To evaluate the feasibility of enzyme-free target recycling and double-output amplification process (Cycle I, Part B of Scheme 1), the prepared samples of diverse control tests after magnetic separation were firstly performed by non-denatured polyacrylamide gel electrophoresis (PAGE) analysis. As shown in Fig. 3A, two strips of HP1 and S2 in lane a (sample solution without the addition of MUC1) can be observed because HP1 could not hybridize with S2 spontaneously. A newly band emerged in the bottom of lane b (sample solution without the addition of S2) and lane c (sample solution), which positions were paralleled with that of the band in lane d (2 µM S1, as the DNA marker), indicating the generation of mimic target (output S1). The newly band in lane c was brighter than that in lane b which coincided with the design principle of Cycle I. The results can be explained as follows: In lane b, one target molecule could only transfer to one mimic target and Cycle I was not triggered in the absence of S2. In lane c, Cycle I based on the acquirement of two mimic targets in one circle of target recycling was executed to obtain masses of mimic target in the presence of MUC1 and S2. Besides, PAGE analysis were also performed to validate the execution of catalytic hairpin assembly (CHA, Cycle II, Part C of Scheme 1) process. Lane e (2 µM HP2 + 2 µM HP3) exhibited two separate bands, illustrating HP2 could not hybridize with HP3 spontaneously. Nevertheless, a new strip appeared in the top of lane f (2 µM HP2 + 2 µM HP3 + 2 µM S1), indicating the running of

11



CHA process with the introduction of S1. Besides, ECL signals of distrinct control tests were executed to further explore the practicability of the aptasensor. Experiments were performed the same as the proposed reaction format, the concentration of MUC1 and H2O2 were 10 pg/mL and 3 mM, respectively. As exhibited in Fig. 3B, a fairly low ECL signal (570 a.u.) was observed on the aptasensor without the incubation of MUC1, the ECL response was originated from background signal caused by physical adsorption of the signal probe on the sensing interface. The ECL intensity of the aptasensor in the absence of S2 (Fig. 3C) increased to 2003 a.u., because in the absence of S2, one target could transfer to only one mimic target (the target was not recycled) for CHA process with an M amplification ratio (M is the recycle times of Cycle II). Thanks to the proposed protocol of target recycling and double-output amplification system, the ECL intensity rose to 6017 a.u. (Fig. 3D), which was almost 3 times than that of Fig. 4C. The obvious enhancement was attributed to the fact that one target could transfer to two mimic targets within one target recycle time, then CHA process was executed by mimic target recycle to capture a larger amount of signal probe, resulting in a 2N × M amplification ratio (N is the recycle times of Cycle I, M is the recycle times of Cycle II). Based on the results above, we confirmed the proposed strategy was feasible for MUC1 detection.

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Fig. 3. (A) PAGE analysis of the samples: lane a, sample solution without the addition of MUC1; lane b, sample solution without the addition of S2; lane c, sample solution; lane d, 2 µM S1; lane e, 2 µM HP2 + 2 µM HP3 and lane f, 2 µM HP2 + 2 µM HP3 + 2 µM S1. ECL curves of the aptasensors incubated with (B) the sample solution without MUC1 (neither Cycle I nor Cycle II was executed), (C) the sample solution without S2 (one target was transferred to only one mimic target for the execution of Cycle II), (D) the sample solution without based on the proposed reaction format (both Cycle I and Cycle II were executed).

3.4. Comparison of the ECL responses with other HP3 signal probes In order to explore the amplification performance of MoS2 with different morphologies of nanosheets (NSs) and NFs, ECL signals of the aptasensors incubated with different signal probes were recorded under the same experimental conditions when MUC1 concentration was 10 pg/mL. The three signal probes were ABEI-Ag labeled HP3 (ABEI-Ag/HP3, named probe A), ABEI-Ag modified MoS2 NSs labeled HP3 (ABEI-Ag@MoS2 NSs/HP3, named probe B), and the proposed signal probe (ABEI-Ag@MoS2 NFs/HP3, named probe C). As exhibited in Fig. 4, the ECL

13



responses of red curves were obtained after aptasensors incubated with probe A, probe B and probe C, respectively, while the blue curves represented the ECL responses of corresponding control line. As shown in Fig. 4A, the ECL response of probe A raised about 2134 a.u. compared to the control line (480 a.u.) because of the capture of ABEI-Ag complexes. It can be found in Fig. 4B that the ECL signals of the biosensor incubated with probe B increased about 2682 a.u. compared with that of control line (854 a.u.), illustrating MoS2 NSs could help the decomposition of H2O2 for ECL signal amplification. However, the probe C (∆I = 5377 a.u., Fig. 4C) could boost ECL response much more obviously than probe B, which was assumably ascribed to the fact that MoS2 NFs as the effective co-reaction accelerator possessed more catalytic active sites for H2O2 decomposition and larger specific surface area for ABEI-Ag complexes loading compared to MoS2 NSs. Significantly, the control line of the biosensor incubated with probe C (546 a.u.) was smaller than that incubated with probe B (854 a.u.) because MoS2 NFs with the functionalization of PEG could help to reduce nonspecific adsorption on the electrode surface. Hence, probe C of ABEI-Ag-MoS2 NFs/HP3 was a good choice for trace MUC1 assay in this work. Besides, mechanism of ECL signal generation and enhancement for the aptasensor was discussed in SI 1.10.

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Fig. 4. Comparison of the ECL responses with different signal probes, (A) ABEI-Ag/HP3 probe, (B) ABEI-Ag@MoS2 NSs/HP3 probe and (C) ABEI-Ag-MoS2 NFs/HP3 probe, on the basis of control line (blue curve) and 10 pg/mL MUC1 in a standard experimental format (red curve). ∆I is is the ECL intensity of the aptasensor minus the control line.

3.6. ECL response and calibration curves Under optimal experimental conditions (SI 1.11), ECL responses of the aptasensor incubated with different MUC1 concentrations were recorded in 2 mL PBS (0.1 M, pH 7.4) containing 3 mM H2O2 to evaluate the sensing range for MUC1 detection. It can be found from Fig. 6A that the ECL intensities augmented with the increment of MUC1 concentrations from 1 fg/mL to 10 ng/mL. Corresponding linearity curve between ECL intensities (I) and the logarithm of MUC1 concentrations (c) was shown in Fig. 6B. The linear equation was I = 1035.60lgc + 7838.59 with a squared correlation coefficient of 0.996, the limit of detection (S/N = 3) was estimated to be 0.58 fg/mL. Beyond that, this work were compared with some relative study for MUC1 determination. As exhibited in Table 1, we found the proposed aptasensor had much wider detection range and lower detection limit.

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Fig. 5. (A) ECL intensity-time curves of the aptasensor when the MUC1 concentration of curve a-g were 1 fg/mL, 10 fg/mL, 100 fg/mL, 1 pg/mL, 10 pg/mL, 100 pg/mL, 1 ng/mL and 10 ng/mL, respectively. (B) Calibration curve between the ECL response and the logarithm of MUC1 concentrations. Error bars, standard deviation (SD), n = 3. Table 1. Comparison with other works for MUC1 detection Test method

Sensing range

Detection limit

Reference

Fluorescence

0.8-39.7 µM

250 nM

34

EC

10-3-1 µM

0.827 nM

35

ECL

10-2-104 pg/mL

4.5 fg/mL

36

ECL

10-3-103 pg/mL

0.62 fg/mL

37

ECL

10-3-104 pg/mL

0.58 fg/mL

Current research

3.6. Specificity, stability and reproducibility of the aptasensor Undeniably, sustained and stable ECL signals were important for MUC1 determination. Under optimal experimental conditions, the aptasensor incubated with 10 ng/mL MUC1 concentration was scanned for 20 cycles to examine the stability. Obviously, the results in Fig. 6A illustrated nice stability of the aptasensor and the relative standard deviation (RSD, S/N = 3) was 1.09%. Specificity of the aptasensor was studied using four proteins (alpha fetal protein (AFP), carcino-embryonic antigen (CEA), cardiac troponin I (cTnI) and cardiac troponin T (cTnT) ) with concentration of 10 pg/mL as probable interferents and the results were shown in Fig. 6B. Compared with the control line (column a, 567 a.u.), there were no obvious difference in ECL response in assay of AFP (column b, 662 a.u.), CEA (column c, 825 a.u.), cTnI (column d, 771 a.u.) and cTnT (column e, 483 16


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a.u.). Nevertheless, much higher ECL intensity was observed when the aptasensor incubated with 100 fg/mL MUC1 (column f, 3701 a.u.), and it showed no significant difference compared with the aptasensor incubated with the mixture above (column g, 3845 a.u.). In brief, the results fully explained the aptasensor had superb selectivity for trace MUC1 assay. Moreover, reproducibility of the aptasensor was equally vital for MUC1 assay. Three parallel intra-assays and interassays were carried out to monitor the ECL signals (Fig. 6C), corresponding RSD (S/N = 3) were calculated as 2.06% and 4.86%, respectively. The results showed the MUC1 aptasensor had acceptable repeatability.

Fig. 6. (A) Stability of the aptasensor when MUC1 concentration was 10 ng/mL. (B) Selectivity of the aptasensor toward (a) control line, (b) 10 pg/mL AFP, (c) 10 pg/mL CEA, (d) 10 pg/mL cTnI and (e) 10 pg/mL cTnT, (f) 100 fg/mL MUC1 and (g) a mixed solution of the above samples. (C) Reproducibility of the aptasensor in three parallel intra-assays and interassays when MUC1 concentration was 100 fg/mL.

3.7. Actual samples assay To test the practicability of the proposed strategy for MUC1 detection, a recovery experiment was implemented by standard addition method. Simply, ECL signals were monitored on the basis of the proposed reaction format with different

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MUC1 concentrations diluted by healthy human serum and then calculate the recovery concentrations according to the linear equation obtained in PBS. As exhibited in Table 2, the recovery was between 97.90% and 111.9% and the RSD was ranged from 1.19% to 5.02%, illustrating the aptasensor had superb accuracy. Hence, we were certain that the biosensor possessed huge potential in practical samples analysis and clinical diagnostics. Table 2. The MUC1 biosensor applied in sera samples (n = 3) Order

Added

Found/(mean value )

RSD/%

Recovery/%

1

10 fg/mL

10.24 fg/mL

3.11

102.4

2

100 fg/mL

111.9 fg/mL

3.67

111.9

3

1 pg/mL

1.033 pg/mL

4.31

103.3

4

10 pg/mL

10.07 pg/mL

5.02

100.7

5

100 pg/mL

102.2 pg/mL

1.64

102.2

6

1 ng/mL

0.979 ng/mL

1.19

97.90

Conclusion Herein, an ECL aptasensor with MoS2 NFs as an effective co-reaction accelerator for ABEI-H2O2 system was fabricated for ultrasensitive MUC1 assay based on a target triggered enzyme-free recycling and double-output amplification strategy. The experimental results indicated the aptasensor provided ultrahigh sensitivity for MUC1, the reasons were listed as follows: (1) Two mimic targets was obtained in one circle of target triggered enzyme-free recycling, which further participated in next recycling of the catalytic hairpin assembly (CHA) process to capture a substantial number of signal probe for signal amplification; (2) MoS2 NFs with good affinity and large 18


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specific surface area largely catalyzed H2O2 decomposition for ECL signal boosting. (3) The S atoms originated from MoS2 NFs could provide vacant orbitals to combine with Ag atoms via Ag-S binding for plenty of ABEI-Ag complexes loading. Furthermore, the proposed MUC1 assay strategy opened up new prospects for clinical analysis.

ASSOCIATED CONTENT Supporting Information Instrumentation (SI 1.1), reagents and biochemicals (SI 1.2), pretreatment of DNA sequences (SI 1.3), non-denatured PAGE analysis process (SI 1.4), preparation of ABEI-Ag/HP3 signal probe (SI 1.5), preparation of ABEI-Ag-MoS2 NSs/HP3 signal probe (SI 1.6), characterization of Au NPs and Au@Fe3O4 (Fig. S1, SI 1.7), XPS analysis of ABEI-Ag-MoS2 NFs complexes (Fig. S2, SI 1.8), CV, EIS and ECL characterization of the biosensor (Fig. S3, SI 1.9), mechanism of ECL signal generation and enhancement (Fig. S4, SI 1.10) and optimization of experimental conditions (Fig. S5, SI 1.11) were placed in Supporting Information.

AUTHOR INFORMATION *

Corresponding authors: Tel: +86-23-68252277; Fax: +86-23-68253172. E-mail address: [email protected] (R. Yuan), [email protected] (Y. Zhuo).

Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the NNSF of China (21675130, 21775124, 21675129, 21575116) and the Fundamental Research Funds for the Central Universities (XDJK2015A002), China.

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