Signal-Switchable Electrochemiluminescence System Coupled with

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Signal-Switchable Electrochemiluminescence System Coupled with Target Recycling Amplification Strategy for Sensitive Mercury Ion and Mucin 1 Assay Xinya Jiang, Huijun Wang, Haijun Wang, Ruo Yuan, and Yaqin Chai Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02501 • Publication Date (Web): 16 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

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Analytical Chemistry

Signal-Switchable Electrochemiluminescence System Coupled with Target Recycling Amplification Strategy for Sensitive Mercury Ion and Mucin 1 Assay Xinya Jiang, Huijun Wang, Haijun Wang, Ruo Yuan∗, Yaqin Chai∗

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR of China

∗

Corresponding author. Tel.: +86-23-68252277; Fax: +86-23-68253172.

E-mail address: [email protected] (R. Yuan), [email protected] (Y. Q. Chai)

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Abstract: In the present work, we first found that mercury ion (Hg2+) has an efficient quenching effect on the electrochemiluminescence (ECL) of N-(aminobutyl)-N-(ethylisoluminol) (ABEI). Be inspired by this discovery, an aptamer-based ECL sensor was fabricated based on Hg2+ triggered signal switch coupled with exonuclease I (Exo I)-stimulated target recycling amplification strategy for ultrasensitive determination of Hg2+ and mucin 1 (MUC1). Concretely, the ECL intensity of ABEI-functionalized silver nanoparticles decorated graphene oxide nanocomposite (GO-AgNPs-ABEI) was initially enhanced by ferrocene labeled ssDNA (Fc-S1) (first signal switch “on” state) in the existence of H2O2. With the aid of aptamer, assistant ssDNA (S2) and full thymine (T) bases ssDNA (S3) modified Au nanoparticles (AuNPs-S2-S3) were immobilized on the sensing surface through the hybridization reaction. Then, via the strong and stable T-Hg2+-T interaction, abundance of Hg2+ were successfully captured on the AuNPs-S2-S3 and effectively inhibited the ECL reaction of ABEI (signal switch “off” state). Finally, the signal switch “on” state was executed by utilizing MUC1 as an aptamer-specific target to bind aptamer, leading to the large decrease of the captured Hg2+. To further improve the sensitivity of the aptasensor, Exo I was implemented to digest the binded aptamer, which resulted in the release of MUC1 for achieving target recycling with strong detectable ECL signal even in low level of MUC1. By integrating the quenching effect of Hg2+ to reduce the background signal and target recycling for signal amplification, this proposed ECL aptasensor was successfully used to detect Hg2+ and MUC1 sensitively with a wide linear response.

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INTRODUCTION Recently, sensor based on electrochemiluminescence (ECL) has been regarded as a sensitive, simple, rapid, and reliable technique for small molecule, protein, DNA and cell assay.1-4 In particular, aptamer-based ECL sensor has become a hotspot and widely been applied in sensing field on account of the excellent stability, the high binding affinity and specificity of the aptamer as well as its structural switching property.5,6 So far, one main method to ameliorate the sensitivity of the ECL sensor is increasing the ECL signal through various amplification strategies, such as using nanomaterials with excellent conductivity to promote electron transfer,7,8 improving the luminescence efficiency of luminohores,9,10 increasing the loading number of signal molecules, 11 , 12 and applying target recycling amplification strategy. 13 , 14 Among these strategies, target recycling amplification strategy triggered by nuclease including exonuclease, 15 , 16 polymerase, 17 , 18 and endonuclease 19 , 20 has attracted increasing interest in sensor fabrication for the reason that it can not only yield a apparent detectable signal in trace level of target, but also need no any further modification step. Exonuclease I (Exo I) with digestion function can stimulate the nucleotides of single-stranded DNA to remove in the direction of 3' to 5', and it has been successfully implemented to digest the aptamer in the protein-aptamer complex.16,

21,

Therefore, Exo I is expected to be applied in the construction of

aptamer-based ECL sensor to accomplish the cyclical usage of target protein with desirable amplified ECL signal. Previous studies indicated that “on-off-on” signal switch strategy has been regarded as a promising method to improve the sensitivity of the sensor for it not only largely 3



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reduces the background signal but also eliminates the false positive signal. In this signal switch strategy, the higher initial signal switch “on” state obtained could greatly improve the sensitivity as well as the lower signal switch “off” state obtained. Therefore, investigators devoted noteworthy efforts to enhancing the initial signal switch “on” state as much as possible and quenching the signal switch “off” state to the minimum. For example, Zhao et al. constructed a highly sensitive ECL aptasensor based on Au@nano-C60 nanocomposite and poly-L-histidine as enhancer for the first signal switch “on” state, and hemin/G-quadruplex DNAzymers as effective quencher for the signal switch “off” state.22 Du and co-workers demonstrated an “on-off-on” ECL sensor based on GO-CoPc hybrids adsorb superoxide anions (O2-) as coreactant for signal amplification (first signal switch “on” state), and ethanol consumes the coreactant of O2- for signal quenching (signal switch “off” state).23 To the commonly used luminophore of N-(aminobutyl)-N-(ethylisoluminol) (ABEI), its enhanced ECL signal had been widely studied. 24 , 25 However, the quenching on ABEI ECL signal was relatively studied a little. Expecially, metal ion caused ABEI ECL signal quenching has not been investigated until recently. Mercury (Hg2+) has been proven to be a highly toxic and deleterious heavy metal element to the environment even human for it can cause severe damage in the brain, immune system and other organs.26 Thus, in order to prevent mercury poisoning, it is central to construct sensitive Hg2+ sensors to monitor its content in water or food. Recently, numerous sensors have been developed to determine Hg2+ including electrochemical method, 27 fluorescence method, 28 colorimetric method, 29 and so forth. However, using ECL sensor to detect Hg2+ is relatively rare. Herein, we first found that Hg2+ has 4


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an efficient quenching effect on the ECL emission of ABEI. Thus, Hg2+ was selected as a novel and effective quencher in “on-off-on” signal-switchable ECL sensor fabrication, which could also provide a new determination channel for Hg2+ simultaneously. Mucin 1 (MUC1) is a transmembrane protein that has been identified as cancer biomarker as its highly expression always companies with various cancers such as breast, ovarian, prostate, and pancreatic cancer.

30

Thus, identification and

quantification of MUC1 are quite central to meet the demand in clinical diagnosis. In this study, we constructed a novel signal-switchable ECL aptasensor by using ferrocene (Fc) as enhancer to amplify the signal, Hg2+ as a novel quencher to decrease the signal and MUC1 as model analyte to realize signal recovery for highly sensitive determination of Hg2+ and MUC1, respectively. As demonstrated in Scheme 1, ABEI-functionalized silver nanoparticles decorated graphene oxide nanocomposite (GO-AgNPs-ABEI) was firstly synthesized and coated on the electrode surface. The first signal switch “on” state was achieved by the introduction of ferrocene labeled ssDNA (Fc-S1) on GO-Ag-ABEI modified electrode for the reason that Fc catalyzed ·

H2O2 to product hydroxyl radical (OH ),31 which further catalyzed the ECL reaction of ABEI with enhanced ECL signal. To obtain a high signal switch efficiency of the system, assistant ssDNA (S2) and full thymine (T) bases ssDNA (S3) modified Au nanoparticles (AuNPs-S2-S3) were utilized to capture the quencher of Hg2+ via the T-Hg2+-T pairing interaction.32 Thus, plenty of Hg2+ were successfully captured on the electrode surface, leading to an extremely low signal switch “off” state which caused by the efficient quenching effect of Hg2+. Finally, the aptamer was dissociated 5



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from the matched double-stranded DNA in the presence of MUC1 and digested by Exo I, which led to the release of Hg2+ as well as MUC1. Then the released MUC1 participated in another specific recognition reaction with rest aptamer, achieving the target recycling. Simultaneously, based on this recycling, large amounts of Hg2+ were liberated from the sensing surface, leading to significant signal recovery (signal switch “on” state). In this signal switch strategy, the decrease and recovery of ECL signal depended on the loading amount of Hg2+ and the concentration of target MUC1, respectively. Therefore, the proposed aptasensor was successfully applied to detect Hg2+ and MUC1 sensitively. Further investigation exhibited that the linear range for Hg2+ was 10.0 pM to 1.0 mM with the limit of detection estimated to be 3.1 pM and the linear range for MUC1 was 10 fg mL-1 to 30 ng mL-1 with the limit of detection estimated to be 2.6 fg mL-1.

Scheme 1. Fabrication of the signal-switchable ECL aptasensor for Hg2+ and MUC1.

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EXPERIMENTAL SECTION Reagents and materials. Ultrapure water was used throughout this experimental process. Graphene oxide (GO) was purchased from Nanjing Xianfeng Nano Co. (Nanjing, China). AgNO3, HgCl2, ethanol, H2O2 and sodium citrate were purchased from Chemical Reagent Co. Ltd. (Chongqin, China). N-(4-aminobutyl)-Nethylisoluminol (ABEI), bovine serum albumin (BSA, 96-99%), and gold chloride (HAuCl4) were obtained from Sigma (St. Louis, MO, USA). Human Mucin (MUC1) was purchased from North Connaught Biotechnology (Shanghai, China). Human serum was obtained from Xinqiao Hospital (Chongqin, China). Oligonucleotides utilized in the present work (see Table 1) were provided by Sangon Biotech Co. Ltd. (Shanghai, China). Exonuclease I (Exo I) and 10 × Exo I buffer (67 mM Glycine-KOH (pH 9.5 at 25 oC), 6.7 mM MgCl2, 10 mM 2-mercaptoethanol, 37 oC incubation) were obtained from the New England Biolabs (U.S.A.). In addition, the DNA hybridization buffer (pH 7.4) was prepared by 10 mM Tris-HCl, 1.0 mM EDTA and 1.0 M MgCl2. The detection phosphate buffer solution (PBS, 0.1 M, pH 8.0) was prepared by Na2HPO4 (0.1 M), KH2PO4 (0.1 M) and KCl (0.1 M). Table 1. The used oligonucleotides in our present work. Sequences (from 3' to 5')

Oligonucleotides ferrocene labeled ssDNA (Fc-S1):

HS-(CH2)6-TTC GTC GTC AAC TAG GA-Fc

aptamer:

GTG GTC CCA TAG GTT TCC TAG TTG ACG ACG

assistant ssDNA (S2):

AAC CTA TGG GAC CAC-(CH2)6-SH

full thymine (T) bases ssDNA (S3):

HS-(CH2)6-TTT TTT TTT TTT TTT TTT TT

Instruments. Electrochemical impedance spectroscopy (EIS) experiments and ECL experiments were monitored respectively with a CHI 660C electrochemical workstation (Shanghai, China) and a MPI-A ECL analyzer (Xi’an, China) using a 7



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conventional three-electrode system. It contains a modified glassy carbon electrode (GCE), Ag/AgCl electrode and platinum wire. For characterization of different materials, scanning electron microscopy (SEM, Hitachi, Tokyo, Japan), Raman microscope (Renishaw, UK) and UV-vis spectrophotometer (Shimadzu, Tokyo, Japan) were utilized. Synthesis of GO-Ag-ABEI nanocomposite. The GO-Ag-ABEI nanocomposite was synthesized using a one-step method according to the literature 33 with some modification. 2 mL AgNO3 (10 mM) and 0.4 mL GO (2 mg mL-1) were added into a mixing solution containing 9 mL ethanol and 5 mL ultrapure water. Then, 1 mL ABEI (0.02 M) was quickly added and reacted over night with stirring in darkness at room temperature. The GO-Ag-ABEI nanocomposite was obtained by centrifugation and washing with ultrapure water. Synthesis of AuNPs-S2-S3. Firstly, 16 nm AuNPs were prepared according to the literature.34 Then, a total of 100 µL S2 (2.5 µM) was added into 1 mL AuNPs with slight stirring for two hours. After that, 400 µL S3 (2.5 µM) was added to the mixing above with stirring for over night. Finally, for the purpose of blocking the possible remaining sites of AuNPs, 150 µL BSA (1%) was added and reacted for 1 h. The synthesis process was carried out under 4 oC. The AuNPs-S2-S3 precipitate was centrifuged and dispersed in 0.1 M PBS and conserved at 4 oC for further use and characterization. Fabrication of the signal-switchable ECL aptasensor. As shown in Scheme 1, 8 µL GO-Ag-ABEI nanocomposite was first spreaded on a cleaned GCE surface to form a

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well-dispersed nanofilm. In this case, the GO-Ag-ABEI nanofilm not only exhibited an ECL signal, but also was employed to immobilize large amounts of Fc-S1 due to its enhanced effective area. After 10 µL Fc-S1 (2.5 µM) was added to the GCE surface and incubated for 12 h at 4 oC, 15 µL BSA (1%) was used to block the nonspecific binding for 1 h. Then, 10 µL aptamer solution and 10 µL the previously prepared AuNPs-S2-S3 solution were successively incubated for 2 h on the GCE surface at 37 oC. After each fabrication step, ultrapure water was utilized to wash the modified electrode and finally the obtained electrode was kept at 4 oC for further use. Analytical procedure. For Hg2+ detection, 20 µL Hg2+ solution with different concentrations was incubated for 90 min on the prepared aptasensor. In a typical test for MUC1, after the prepared aptasensor was first incubated with 20 µL of Hg2+ (1 mM) for 90 min, 20 µL of MUC1 solution with different concentrations containing 30 U Exo I was incubated on the sensing surface for 1 h at 37 oC. Finally, after thorough washing, the obtained aptasensor was put in PBS (2 mL, pH 8.0, 1 mM H2O2) to detect signal from 0.2 to 0.8 V with a scan rate of 200 mV s-1. RESULTS AND DISCUSSION Characterization of GO-Ag-ABEI, AuNPs and AuNPs-S2-S3. Scanning electron microscopy (SEM), Raman spectra and UV-vis absorption spectra were utilized to characterize

the

as-synthesized

GO-Ag-ABEI,

AuNPs

and

AuNPs-S2-S3

nanomaterials. As shown in Figure 1A, large amounts of Ag nanoparticles reduced by ABEI were embedded uniformly on the surface of graphene with a diameter of about 50 nm. Figure 1B showed the UV-vis absorption spectra of pure ABEI and

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GO-Ag-ABEI. We found that the UV-vis absorption spectra of GO-Ag-ABEI (Figure 1B, curve b) presented a broad absorption band in the region of 400-500 nm, indicating the successful synthesis of Ag nanoparticles. In addition, it exhibited the characteristic absorbance of ABEI at 267 nm and 306 nm with a blue-shifted compared with pure ABEI (Figure 1B, curve a). Furthermore, in order to prove the existence of GO, Raman spectra of GO and GO-Ag-ABEI were tested. From the inset of Figure 2B, GO (curve a) and GO-Ag-ABEI (curve b) all displayed the typical D-band and G-band of GO at about 1350 and 1601 cm-1. These results suggested the successful preparation of GO-Ag-ABEI nanocomposite. Figure 1C showed the SEM images of AuNPs and AuNPs-S2-S3 nanocomposite. AuNPs (inset of Figure 1C) showed a uniform spherical structure with a diameter of about 13 nm, which was further confirmed by UV-vis absorption spectra (Figure 1D, curve a). Moreover, after assembling S2 and S3 on the surface of AuNPs, the obtained AuNPs-S2-S3 also showed a spherical structure but with a little blurry (Figure 1C). Figure 1D showed the UV-vis absorption spectra of AuNPs and AuNPs-S2-S3. AuNPs exhibited an absorption peak at 519 nm (Figure 1D, curve a), while the UV-vis absorption spectra of AuNPs-S2-S3 red-shifted to 525 nm with a new absorption peak occurred at 260 nm (Figure 1D, curve b), which was corresponding to the typical DNA absorption demonstrating the successful loading of S2 and S3 on AuNPs.

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Figure 1. (A) SEM image of GO-Ag-ABEI. (B) UV-vis spectra of ABEI (curve a) and GO-Ag-ABEI (curve b). Inset is the Ramam spectrum of GO (curve a) and GO-AgABEI (curve b). (C) SEM image of AuNPs–S2-S3. Inset is the SEM image of AuNPs. (D) UV-vis spectra of AuNPs (curve a) and AuNPs–S2-S3 (curve b). Investigation the quenching mechanism of Hg2+ on the ECL of ABEI. To investigate the quenching mechanism of Hg2+ on the ECL of ABEI, ECL and CV responses of pure ABEI and the mixture of ABEI with Hg2+ were first measured without H2O2. As displayed in Figure 2A, the ABEI solution (0.2 mM) had a strong ECL emission (about 1100 a.u.). The ECL signal of ABEI began to emit at around 0.45 V and reached its maximum ECL emission at potential of 0.63 V (Figure 2A, curve a). However, a weak ECL emission (about 200 a.u.) was obtained after adding 0.2 mM Hg2+. In addition, the ECL emission of ABEI shifted to 0.62 V and the maximum ECL emission occured at around 0.73 V (Figure 2A, curve b). From the

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results, we may conclude that the formation of the excited-state ABEI became difficult after adding Hg2+ and the amount of the formed excited-state ABEI was relatively small. The similar phenomena was also observed from their corresponding CV curves. From the figure, we found that ABEI exhibited a noticeable oxidation peak at about 0.57 V (Figure 2B, curve a). After adding 0.2 mM Hg2+, the oxidation peak shifted to 0.69 V (Figure 2B, curve b) and the current value became lower than that of pure ABEI. This result suggested that the oxidation process of ABEI was inhibited after the adding of Hg2+. In addition, in the presence of coreactant H2O2, the ECL and CV responses of pure ABEI and the mixture of ABEI with Hg2+ presented the similar results (Figure 2C and 2D). In conclusion, the reason for the ECL quenching of ABEI by Hg2+ may be that Hg2+ inhibited the oxidation of ABEI, resulting in a small amount of excited-state ABEI formed with a low ECL signal.

Figure 2. (A) ECL responses and (B) CVs of 0.2 mM ABEI (curve a) and 0.2 mM ABEI containing 0.2 mM Hg2+ (curve b). (C) ECL responses and (D) CVs of 0.06

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mM ABEI with 0.03 mM H2O2 (curve a) and 0.06 mM ABEI with 0.03 mM H2O2 containing 0.15 mM Hg2+ (curve b). The experiment was measured in 2 mL PBS (0.1 M, pH 8.0). Characterization of the proposed signal-switchable ECL aptasensor. ECL analysis technique was firstly utilized to characterize the sensing interface of the ECL aptasensor. As shown in Figure 3A, it depicted that bare GCE had almost no ECL signal (curve a), while the GO-Ag-ABEI modified GCE had an obvious ECL emission (curve b) in pH 8.0 PBS with 1 mM H2O2 as coreactant. After Fc-S1 was captured on GCE, the ECL intensity largely enhanced (curve c) due to the reason that ·

Fc could catalyze H2O2 to generate reactive hydroxyl radical (OH ) which play an essential role in the ECL reaction of ABEI. However, the ECL signal decreased gradually after the successive immobilization of aptamer (curve d), BSA (curve e) and AuNPs-S2-S3 (curve f), which were ascribed to the formed biological macromolecule hampering the electron transfer. Evidently, the ECL intensity decreased heavily down to about 700 a.u. (curve g) after the conjugation of Hg2+ due to its efficient quenching effect. Interesting, when target MUC1 with 30 U Exo I was incubated on the sensing surface above, the ECL intensity recovered (curve h). This fact was owing to that MUC1 binded with its aptamer, leading to the release of Hg2+ from the electrode surface. Figure 3B showed the EIS Nyquist plots obtained from the stepwise modification process of ECL aptasensor. From the figure, bare GCE exhibited a minor semicircle 13



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diameter of EIS Nyquist plot (curve a), indicating a low electron-transfer resistance (Ret) value of the Fe(CN)63-/4- redox system. The Ret value of GO-Ag-ABEI nanocomposite modified GCE was increased (curve b) due to the negetively charged aminophthalate ion analogue formed on the surface of AgNPs 35 hindering the electron transfer (the zeta potential was calculated to be -27.6 mV). After S1, BSA, aptamer and AuNPs-S2-S3 were successive immobilized on GCE, the Ret values were gradually increased in order (curve c, d, e and f). The phenomena was ascribed to that the assembled biological macromolecule layers blocked the electron transfer. The results obtained from the EIS were consistent with the ECL results, indicating the successful fabrication of the signal-switchable ECL aptasensor.

Figure 3. ECL characterization (A) and EIS characterization (B) of the signal-switchable ECL aptasensor: (a) bare GCE, (b) GO-Ag-ABEI/GCE, (c) Fc-S1/GO-Ag-ABEI/GCE, (d) BSA/Fc-S1/GO-Ag-ABEI/GCE, (e) aptamer/BSA/FcS1/GO-Ag-ABEI/GCE, (f) AuNPs-S2-S3/aptamer/BSA/Fc-S1/GO-Ag-ABEI/GCE, (g) Hg2+/AuNPs-S2-S3/aptamer/BSA/S1-Fc/GO-Ag-ABEI/GCE, (h) Hg2+/AuNPs-S2-S3/ aptamer/Fc-S1/GO- Ag-ABEI/GCE in the presence of MUC1 with 30 U Exo I. The 14


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ECL signals were detected in PBS (0.1 M, pH 8.0, 1 mM H2O2) and the EIS curves were detected in [Fe(CN)6]3-/4- (5 mM, 0.1 M KCl). Optimization of the experimental conditions. In the first signal switch “on” state, the ECL intensity mainly depended on the incubation time of Fc-S1. When the incubation time of Fc-S1 increased, more Fc were captured on the GO-Ag-ABEI ·

modified GCE, thus a high level of OH was produced from H2O2 catalyzed by Fc. As revealed in Figure 4A, the ECL intensity increased with increasing incubation time and obtained the maximum value at 12 h. Therefore, 12 h was selected as the optimal incubation time for the Fc-S1. The incubation time of Hg2+ affected the sensitivity of the proposed ECL sensor. In order to reach the maximum quenching efficiency, the incubation time of Hg2+ was optimized with 1 µM Hg2+. When incubation time of Hg2+ increased, the ECL quenching efficiency drastically increased (Figure 4B) owing to that large amounts of Hg2+ were captured on the electrode surface via T-Hg2+-T bond. The quenching efficiency arrived at the maximum value at 90 min. Therefore, 90 min was chosen for Hg2+ incubation in this experiment.

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Figure 4. The effects of incubation time of Fc-S1 (A) and Hg2+ (B) (1 µM Hg2+) on ECL response of the signal-switchable aptasensor. Working buffer was 2 mL PBS (0.1 M, pH 8.0, 1 mM H2O2). Analytical performance of the signal-switchable ECL aptasensor. The proposed signal-switchable ECL aptasensor was first used to detect Hg2+. The ECL intensity decreased gradually from 10.0 pM to 1.0 mM (Figure 5A) because more Hg2+ were captured on the sensing surface under the high concentration. The ECL intensity quenching degree linearly responds to the logarithmic value of Hg2+ concentration with a correlation coefficient of 0.9954 (Figure 5B). The regression equation was expressed as the following: IECL = – 2176.6 – 975.7 lg c. The limit of detection was evaluated to be 3.1 pM.36 Compared with some previous reported methods (Table 2), the proposed ECL aptasensor showed a wider detection range as well as a lower detection limit for Hg2+ detection (Table 2).

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Figure 5. ECL responses of the proposed signal-switchable ECL aptasensor with different concentrations of Hg2+ (A) and MUC1 (C). The calibration plots for Hg2+ (B) and MUC1 (D) detection . As expected, when detection MUC1, the ECL intensity obviously increased with increasing MUC1 concentration (Figure 5C). The ECL intensity increasing degree linearly responds to the logarithmic value of MUC1 concentration in the ranging of 10 fg mL-1 to 30 ng mL-1 with a correlation coefficient of 0.9968 (Figure 5D). The regression equation was expressed as the following: IECL = 6639.8 + 1018.6 lg c. The limit of detection was evaluated to be 2.8 fg mL-1.36 Compared with some previous reported methods (Table 2), the proposed ECL aptasensor exhibited broader linear range as well as a relatively low detection limit for MUC1 determination. The high sensitivity of this aptasensor may be ascribed to the target recycling for signal amplification and low background signal caused by the efficient quenching effect of Hg2+. Table 2. Different methods for Hg2+ ion and MUC1 detection. Method

Hg2+

Fluorescence

0 - 1 nM

0.3 nM

37

Fluorescence

---

1.7 nM

38

Resonance Rayleigh scattering

50 pM - 500 pM

20 pM

39

Electrochemiluminescence

10 pM - 1 mM

3.1 pM

This work

Fluorescence

0.04 µM - 10 µM

28 nM

40

Electrochemical

10 pM - 1 µM

4 pM

41

Fluorescence

0.8 -39.7 µM

250 nM

42

Electrochemiluminescence

10 fg mL-1 -30 ng mL-1

2.8 fg mL-1

This work

MUC 1

Detection range

Detection limit

Analyte

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References


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To investigate the stability of the aptasensor, the ECL emission upon cyclic potential scan for fifteen cycles were tested in the absence and presence of 1 pg mL-1 MUC1 containing 30 U Exo I. As shown in Figure 6A, the sensor presented a relatively stable ECL signal with a relative standard deviations (RSD) of 2.27% after quenching by Hg2+ (curve a). Furthermore, the sensor also presented an excellent stability with a RSD of 0.77% after incubation of 1 pg mL-1 MUC1 containing 30 U Exo I (curve b). In addition, five same prepared aptasensors were used to detect the same concentration of 1 µM Hg2+ or 1 ng mL-1 MUC1 respectively to investigate the reproducibility of the proposed ECL aptasensor. The result implied that these five aptasensors exhibited almost the similar ECL intensity. The RSD for Hg2+ detection was estimated to be 5.2% as well as 3.7% for MUC1 detection, indicating a good reproducibility of the proposed aptasensor. Selectivity, another important performance of the aptasensor, was estimated using other compounds such as CEA and LN as interfering agents to replace target MUC1. According to Figure 6B, the ECL intensity of the aptasensor incubated with pure 10 ng mL-1 CEA or LN was almost the same as that of blank, even if the concentrations of interfering agents were 10-fold higher than that of MUC1. Furthermore, the ECL intensity obtained from the mixture (MUC1 (1 ng mL-1) , LN and CEA (10 ng mL-1) ) showed no obvious change when compared with that obtained from pure target MUC1 (1 ng mL-1). These investigations suggested that the proposed signalswitchable ECL aptasensor possessed excellent stability and specificity.

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Figure 6. (A) ECL responses of the signal-switchable ECL aptasensor under consecutive cyclic potential scans for 15 cycles in the absence (curve a) and presence (curve b) of 1 pg mL-1 MUC1 containing 30 U Exo I. (B) The signal-switchable ECL aptasensor with different interfering targets. Application of the signal-switchable ECL aptasensor in human serum. To investigate the applicability of the proposed signal-switchable ECL aptasensor, four different concentrations of MUC1 samples were firstly prepared using human serum as real sample. Prior to use, the used human serum was initially diluted 50 times with PBS (pH 7.4). Then, the MUC1 samples were prepared with the diluted human serum via a standard addition method. Finally, a recovery study was carried out on these MUC1 samples using the proposed signal-switchable ECL aptasensor. The obtained recoveries of the serum samples were 101.9%, 103.3%, 97.13% and 95.26%, respectively (Table 3). The relative standard deviation (RSD) was between 0.63% and 2.1%, indicating the potential application of the prepared ECL aptasensor for MUC1 in clinical determination.

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Table 3. A recovery study carried out in human serum for MUC1 determination. Added MUC1 concentration / ng mL-1

Concentration found / ng mL-1

Recovery / %

RSD / %

0.001

0.001019

101.9

2.1%

0.01

0.01033

103.3

1.1%

0.1

0.09713

97.13

0.99%

1

0.9526

95.26

0.63%

CONCLUSIONS Inspired by the quenching effect of Hg2+ on the ECL of ABEI, we have successfully developed an ECL aptasensor to detect Hg2+ and MUC1. In order to promote the sensitivity of the aptasensor, an “on-off-on” signal switch strategy and target recycling amplification strategy were used in this study. Concretely, Fc was used as enhancer to amplify the first signal switch “on” state, Hg2+ was used as quencher to inhibit the ECL reaction of ABEI and thus obtained the signal switch “off” state. After incubation of MUC1, an increased ECL signal was obtained (signal switch “on” state) for the reason that MUC1 specifically interacted with its aptamer leading to the release of Hg2+. Furthermore, Exo I was applied to digest the MUC1 binded aptamer with the release of MUC1 which specifically recognized the remaining aptamer again, thus achieving the target recycling. With the target recycling, large amounts of Hg2+ were released from the sensing surface, resulting in significant signal recovery. In conclusion, based on the signal-switchable and target recycling strategies, the proposed signal-switchable ECL aptasensor exhibited high sensitivity and excellent analytical performance for the determination of Hg2+ and MUC1. Therefore, we anticipate that this strategy may provide a novel avenue for quantitative detection of

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Hg2+ and other proteins. AUTHOR INFORMATION ∗

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

ACKNOWLEDGEMENTS This work was financially supported by the NNSF of China (5147313，21575116) and the Fundamental Research Funds for the Central Universities (XDJK2015A002), China. REFERENCES (1) Feng, Y. Q.; Dai, C. H.; Lei, J. P,; Ju, H. X.; Cheng, Y. X. Anal. Chem. 2016, 88, 845-850. (2) Liang, W. B.; Zhuo, Y. Xiong, C. Y.; Zheng, Y. N; Chai, Y. Q.; Yuan, R. Anal. Chem. 2015, 87, 12363-12371. (3) Deiss, F.; LaFratta, C. N.; Symer, M.; Blicharz, T. M.; Sojic, N.; Walt, D. R.. J. Am. Chem. Soc. 2009, 131, 6088-6089. (4) Cao, W. D.; Ferrance, J. P.; Demas, J.; Landers, J. P. J. Am. Chem. Soc. 2006, 128, 7572-7578. (5) Dong, Y. P.; Zhou, Y.; Wang, J.; Zhu, J. J. Anal. Chem. 2016, 88, 5469-5475. (6) Wu, D.; Xin, X.; Pang, X. H.; Pietraszkiewicz, M.; Hozyst, R.; Sun, X. G.; Wei, Q. ACS Appl. Mater. interfaces 2015, 7, 12663-12670 (7) Liu, S.; Zhang, J. X.; Tu, W. W.; Bao, J. C.; Dai, Z. H. Nanoscale 2014, 6, 2419 –2425. (8) Wang, H. J.; Yuan, Y. L.; Chai, Y. Q.; Yuan, R. Small 2015, 11, 3703-3709. (9) Jiang, X. Y.; Wang, H. J.; Wang, H. J.; Zhuo, Y.; Yuan, R.; Chai, Y. Q. Nanoscale 2016, 8, 8017-8023. (10) Wang, H. J.; Yuan, Y. L.; Zhuo, Y.; Chai, Y. Q.; Yuan, R. Anal. Chem. 2016, 88, 2258-2265. (11) Wang, D. F.; Li, Y. Y.; Lin, Z. Y.; Qiu, B.; Guo, L. H. Anal. Chem. 2015, 87, 5966-5972. (12) Shao, K.; Wang, J.; Jiang, X. C.; Shao, F.; Li, T. T.; Ye, S. Y.; Chen, L.; Han, H. Y. Anal. Chem. 2014, 86, 5749-5757. 21



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For TOC only

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Signal-Switchable Electrochemiluminescence System Coupled with

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