Highly efficient electrogenerated chemiluminescence quenching of PEI enhanced Ru(bpy)32+ nanocomposite by hemin and Au@CeO2 nanoparticles

Biosensors and Bioelectronics 63 (2015) 392–398

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Highly efficient electrogenerated chemiluminescence quenching of PEI enhanced Ru(bpy)32 þ nanocomposite by hemin and Au@CeO2 nanoparticles Lin-Ru Hong, Ya-Qin Chai, Min Zhao, Ni Liao, Ruo Yuan n, Ying Zhuo n Key Laboratory of Luminescence and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China

art ic l e i nf o

a b s t r a c t

Article history: Received 16 April 2014 Received in revised form 13 July 2014 Accepted 22 July 2014 Available online 1 August 2014

In this work, a new signal amplified strategy based on the quenching effect of hemin and Au nanoparticles decorated CeO2 nanoparticles (Au@CeO2 NPs) for ultrasensitive detection of thrombin (TB) is reported for the first time. Herein, the poly(ethylenimine) (PEI) enhanced Ru(bpy)32 þ nanocomposite was implemented by direct chemical polymerization, which could provide the desirable enhanced initial ECL signal. Furthermore, the detection aptamer of thrombin (TBA 2) was immobilized on Au@CeO2 NPs to form TBA 2/Au@CeO2 conjugates. Then, the G-rich DNA of TBA 2 sequence could fold into a G-quadruplex structure to embed hemin to obtain the quenching probe of hemin/TBA 2/Au@CeO2 conjugates. In the presence of target TB, the sandwiched structure could be formed between capture aptamer (TBA 1), TB and hemin/TBA 2/Au@CeO2 conjugates, thereby resulting in a proportional quenching in ECL response with TB, due to the quenching of both hemin and Au@CeO2 NPs. As a result, the signal-off aptasensor showed a wider linear range response from 10  13 to 10  8 M with lower detection limit of 0.03 pM. & 2014 Elsevier B.V. All rights reserved.

Keywords: Electrochemiluminescence Aptasensor Hemin Au@CeO2 nanoparticles Thrombin Quenching

1. Introduction Electrochemiluminescence (ECL) is superior to photoluminescence in terms of its inexpensive reagent, low background noise, high detection sensitivity, and a wide dynamic range (Richter, 2004; Miao, 2008; Bertoncello and Forster, 2011). Usually, the ECL signals can be obtained from the luminophores such as Ru (bpy)32 þ (Kim and Kim, 2014), luminal (Li and Cui, 2013), semiconductor nanocrystals (Maity et al., 2013; Bertoncello and Forster, 2009) and so on. Due to the excellent stability, high luminescence, wide application range of pH and electrochemical reversibility of Ru(bpy)32 þ and its derivatives, there are many reports focused on the enhanced ECL based on the Ru(bpy)32 þ system using co-reactants (Chen et al., 2013; Crespo et al., 2012; Wu et al., 2013). However, the investigation of ECL quenching has been relatively rare. Present studies have implied that there are four kinds of efficient ECL quenchers of Ru(bpy)32 þ (Huang et al., 2013). First one is the metal complex, the most typical kind is ferrocene (Fc) and its derivatives (Wang et al., 2009; Li et al., 2014). n

Corresponding authors. Tel.: þ 86 23 68252277; fax: þ 86 23 68253172. E-mail addresses: [email protected] (R. Yuan), [email protected] (Y. Zhuo). http://dx.doi.org/10.1016/j.bios.2014.07.065 0956-5663/& 2014 Elsevier B.V. All rights reserved.

Second one is the organic molecule of phenols and catechols (McCall et al., 1999). In addition, the quantum dots (Wu et al., 2011) and pristine carbon nanotube (Tang et al., 2013) also exhibit the quenching effect on ECL of Ru(bpy)32 þ . Hemin, a well-known natural porphyrinatoiron complex, has the redox pair of Fe(III)/Fe (II) similar to metal complex of Fc which is reported to quench quantum dots previously (Deng et al., 2013). CeO2 nanoparticles (CeO2 NPs) are as well as a promising material for the fabrication of H2O2 biosensors with the interesting properties like electrocatalytic, non-toxicity, biocompatibility, chemical stability and high electron transfer capability. To our knowledge, the quenching effect of hemin or CeO2 NPs on ECL of Ru(bpy)32 þ is never reported before. In quenching ECL, it is important to achieve the enhanced background response by immobilizing the luminophores. Thus, many Ru(bpy)32 þ immobilization approaches have been proposed for biosensor construction, including Langmuir–Blodgett technique (Capone et al., 2014), layer-by-layer self-assembly technique (Yang et al., 2010), and sol gel membrane technique (Huang and Qiu, 2014). In this work, a novel method for immobilizing Ru (bpy)32 þ is implemented by direct chemical polymerization to obtain ECL enhanced Ru(bpy)32 þ nanocomposite, which realized the simultaneous immobilization of Ru(bpy)32 þ and the co-reactant to obtain strong luminous signal. The polymer monomer

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complexes of the Ru(bpy)32 þ nanocomposite were formed between a polymer with amino groups and an organic monomer with a carboxylic group. Herein, the PEI is chosen as the polymer with amino groups, which not only acts as the matrix of Ru (bpy)32 þ immobilization but also serves as an ideal co-reactant of Ru(bpy)32 þ to enhance the ECL emission intensity. The acrylic acid (AA) is chosen as an organic monomer with a carboxylic group. Then the Ru(bpy)32 þ is doped in the aqueous solution with the two precursors, such polymer–monomer complexes can form micelles through electrostatic interactions. We use potassium persulfate (K2S2O8) for initiation of the polymerization of AA, followed by cross-linking of the complexes with glutaraldehyde (GA) to end polymerization led to the formation of dense film of ECL enhanced Ru–PEI–PAA composite, resulting in a number of luminescent molecules of Ru(bpy)32 þ irreversibly embedding into the inner compact film. We found that this strategy can be very versatile for the preparation of stable and efficient luminescent composite. Herein, based on the ECL enhanced Ru–PEI–PAA composite, we designed an ultrasensitive aptasensor for thrombin detection by the dual quenching of hemin and Au@CeO2 NPs. Au@CeO2 NPs were first prepared and used to immobilize detection aptamer of thrombin (TBA 2). Then, the quenching probe of hemin/TBA 2/ Au@CeO2 conjugates was obtained by adding hemin to react with TBA 2/Au@CeO2 conjugates since the G-rich DNA of TBA 2 sequence could fold into a G-quadruplex structure to embed hemin. For aptasensor construction, Ru–PEI–PAA composite was prepared and coated onto the bare electrode to achieve the enhanced ECL signal. Then nano-Au was dropped onto Ru–PEI–PAA composite. Subsequently, SH-TBA 1 was anchored on the nano-Au/Ru–PEI–PAA modified electrode. Following that, the resultant electrode was blocked with bovine serum albumin (BSA) to avoid the nonspecific adsorption. Finally, the proposed aptasensor was incubated with thrombin (TB) standard solution and hemin/TBA 2/Au@CeO2 conjugates. The quantitative detection was based on the change in the ECL response before and after sandwich reaction. With the efficient quenching probe, the proposed aptasensor obtained a wide linear range and a relatively low detection limit for TB.

2. Experiment 2.1. Reagents and materials Ru(bpy)3Cl2  6H2O, branched polyethylenimine (PEI), thrombin (TB), CeO2 NPs, hemin and hemoglobin (Hb) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gold chloride (HAuCl4) and bovine serum albumin (BSA, 96–99%) were purchased from Shanghai Medpep Co. Ltd. (Shanghai, China). K2S2O8 was purchased from Shanghai Chemical Reagent Company (Shanghai, China). Nano-Au of 16 nm diameter was received by reducing gold chloride tetrahydrate with citric acid at 100 °C for half an hour (Zhuo et al., 2006). Sodium borohydride (NaBH4) was obtained from Kelong Chemical Inc. (Chengdu, China). The sequence of capture thrombin aptamer (TBA 1, 5 μL) is as follows: 5′-SH(CH2)6-GGTTGGTGTGGTTGG-3′, the sequence of detection thrombin aptamer (TBA 2, 5 μL): 5′-NH2-(CH2)6-GGTTGGTGTGGTTGG-3′, which were obtained from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Phosphate buffer solution (PBS) (pH 7.4) was prepared by using 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl. All chemicals and solvents used were of analytical grade. Double distilled water was used throughout this study.

393

2.2. Apparatus The ECL measurement was carried out on a model MPI-A electrochemiluminescence analyzer (Xi’an Remax Electronic Science &Technology Co. Ltd., Xin, China) with the voltage of the photo-multiplie tube (PMT) set at 700 V, the potential scan from 0.2 to 1.25 V and the scan rate of 100 mV/s in the process of detection. A CHI 660A electrochemical workstation (Shanghai Chenhua Instrument, China) was used for Electrochemical Impedance Spectroscopy (EIS). The SEM images of CeO2 NPs and Au@CeO2 NPs were recorded by a Hitachi S-4800 scanning electron microscope (Tokyo, Japan) at an acceleration voltage of 20 kV. UV–vis absorption bands were performed on a UV-2450 (Shimadzu, Japan). IR spectra were recorded using a FTIR-650 (Gangdong, Tianjin). All experiments were performed with a conventional three-electrode system, in which the modified glassy carbon electrode (GCE, Ф ¼4 mm) was the working electrode, a platinum wire was the counter-electrode and an Ag/AgCl (sat. KCl) was the reference electrode. 2.3. Preparation of Ru-PEI-PAA composite The Ru–PEI–PAA composite was obtained by direct chemical polymerization method. Firstly, 2 mL AA (about 99.5%) was added into 20 mL double distilled water and then mixed with 2 mL PEI (1–2%). After vigorously stirring for a few minutes, the carboxyl groups of AA combined with the amino of PEI via electrostatic interaction to form homogeneous solution. Then, 40 μL 5 mg/mL Ru(bpy)3Cl2  6H2O was injected into the above solution under vigorous stirring to obtain the homogeneous mixture. Afterwards, the initiator of 24 mL 1 M K2S2O8 was added followed by deaeration with N2 for 30 min at 80 °C. Subsequently, the temperature was reduced to 60 °C with vigorous stirring for 100 min. Finally, the obtained Ru–PEI–PAA composite was cross-linked by 2 mL GA (50%) at 40 °C for 2 h. The Ru–PEI–PAA composite was stored at 4 °C prior to use. 2.4. Preparation of hemin/TBA 2/Au@CeO2 Hemin/TBA 2/Au@CeO2 conjugates as quenching probe for “sandwich” reaction were synthesized by the following procedure. The dispersed CeO2 NPs were first suspended in 2 mL 1% BSA aqueous solution and stirred for 4 h at 4 °C. The product of BSA coated CeO2 NPs was collected through centrifugation, which was put into 1 mL 16 nm nano-Au solution under stirring for 12 h to adsorb nano-Au. Then, the product was subjected to the centrifugation at 12,000 rpm for 15 min and washed 3 times by double distilled water to obtain Au@CeO2 NPs. 15 μL NH2–TBA 2 were added in the dispersed Au@CeO2 NPs solution with stirring for 12 h at 4 °C. When 10 μL 1 mM hemin was introduced, the G-rich DNA of TBA 2 sequence could fold into a G-quadruplex structure to obtain the hemin-G-quadruplex/Au@CeO2 conjugates. The products were purified by centrifugation and washed with double distilled water three times. The resultant hemin/TBA 2/Au@CeO2 conjugates were stored in the refrigerator at 4 °C when not in use. 2.5. The fabrication of the sandwich-type ECL aptasensor A glassy carbon electrode (GCE, Ф ¼4 mm) was firstly polished with 0.3 and 0.05 μm alumina powder to obtain mirror-like surface, followed by washing thoroughly with double distilled water and ethanol, double distilled water, respectively. Finally, the cleaned electrode was allowed to dry at room temperature. The cleaned GCE was coated with 20 μL Ru–PEI–PAA composite and dried in the air. Then 10 μL nano-Au was dropped onto Ru– PEI–PAA composite to form covalent bonds with PEI and dried for

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3 h in the air. Subsequently, the nano-Au/Ru–PEI–PAA modified electrode was incubated with 10 μL 2 μM SH-TBA 1 for about 13 h at 4 °C. To reduce nonspecific adsorption, the modified electrode was blocked with 10 μL 0.25% BSA aqueous solution by 40 min treatment. 2.6. Measurement procedure The proposed aptasensor was incubated with 10 μL TB standard solution with different concentrations for 40 min at 37 °C. After that, 10 μL TBA 2 conjugates were dropped onto the obtained electrode for 40 min to form sandwich-type complex. To remove the physically adsorptive species, the resultant electrode was rinsed with double distilled water each step. The obtained aptasensor was investigated with a MPI-A ECL analyzer in PBS (0.1 M, pH 7.4) at room temperature in the process of detection. The ECL intensity of the BSA/TBA 1/nano-Au/Ru–PEI–PAA modified GCE was recorded I0, and after the proposed aptasensor was incubated with different concentrations of TB and 10 μL TBA 2 conjugates, the ECL intensity was recorded I. Finally, the ECL quenching response (ΔI¼ I0  I) was obtained. Our protocol for sensitive ECL detection of TB based on hemin/TBA 2/Au@CeO2 quenching probe is illustrated in Scheme 1.

3. Results and discussion 3.1. The UV–vis and IR spectra of the Ru-PEI-PAA composite UV–vis and IR spectra of the Ru–PEI–PAA composite are shown in Fig. 1. As shown in Fig. 1A, AA (curve a) exhibited maximum absorptions at 205 nm and 233 nm and the PEI (curve b) exhibited a maximum absorption peak at 220 nm. Moreover, Ru(bpy)32 þ (curve c) had three UV absorptions at approximately 228 nm, 286 nm and 453 nm, respectively. While Ru–PEI–PAA composite (curve d) not only displayed a broad absorption at approximately 210 nm, but also contained a peak at 288 nm which corresponded to absorption bands of PAA with large bathochromic shifts (55 nm). In addition, the absorption at 454 nm of the Ru–PEI– PAA (curve d) could attribute to the Ru(bpy)32 þ . IR spectra of Ru– PEI–PAA composite are also shown in Fig. 1B. As shown in Fig. 1B, the Ru–PEI–PAA composite produces the strong absorption at

1812.85 cm  1 (C=O stretching of acylamino), suggesting that the polymerization of AA with PEI. These results clearly confirmed that the Ru–PEI–PAA composite was successfully formed by chemical polymerization reaction. 3.2. The SEM image of Ru-PEI-PAA composite, Au@CeO2 and CeO2 NPs Furthermore, the morphologies of Ru–PEI–PAA composite, CeO2 NPs and Au@CeO2 NPs were investigated by SEM (Fig. 2). From Fig. 2A, the Ru–PEI–PAA composite displayed a hollow core– shell structure with an average size about 200 nm in diameter, which was also well dispersed. Fig. 2B displays a panoramic SEM image of CeO2 NPs. The CeO2 NPs exhibit cubic shape with average size of 80 nm which is consistent with reported work (Wang and Feng, 2003). As shown in Fig. 2C, many tiny spots with homogeneous structure unevenly distributed on the CeO2 NPs surface, indicating that nano-Au particles were successfully adsorbed on CeO2 NPs surface. 3.3. ECL and EIS characterization of the aptasensor fabrication The ECL characterization was an effective method for probing the process of electrode modification. The fabrication process of the aptasensor was characterized by ECL in 0.1 M PBS (pH 7.4). As shown in Fig. 3A, the ECL signal of Ru–PEI–PAA modified electrode was obtained (curve a), because PEI could act not only as matrix to immobilize Ru(bpy)32 þ but also as an ideal and effective co-reactant of Ru(bpy)32 þ to significantly enhance the ECL of Ru(bpy)32þ . Then ECL intensity was obviously enhanced when nano-Au was dropped onto Ru–PEI–PAA composite modified electrode (curve b), because nano-Au made it easier for the electron transfer. However, after incubating with TBA 1, BSA and TB, respectively, the ECL signal decreased successively (curves c–e). The reason was that they could hinder the electron transfer. Finally, TBA 2 conjugates were incubated with the resultant electrode; an obvious decreased ECL response was obtained because Ru(bpy)32þ luminescence was quenched by hemin/ TBA 2/Au@CeO2 conjugates. (Fig. 3A, curve f). EIS was also used to monitor the process of electrode modification. Fig. 3B shows the results of EIS at different modification stages in 5 mM Fe(CN)64  /3  solution. Before modification, EIS of the bare GCE was investigated, which displayed a very small

Scheme 1. Schematic representation of the aptasensor preparation process and response mechanism. (A) Preparation of hemin/TBA 2/Au@CeO2 probe. (B) Preparation of Ru–PEI–PAA composite.

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395

1.0

Transmittance [%]

3 Absorbance

b 2

d

1

a

c

0.8

0.6

0.4

0

300 450 Wavelength/nm

1000

600

1812.85 2000 3000

Wavenumber

4000

cm-1

Fig. 1. UV–vis absorption bands (A) and IR spectra (B) of Ru–PEI–PAA composite (A: (a) AA; (b) PEI; (c) Ru(bpy)32 þ ; (d) Ru–PEI–PAA composite).

Fig. 2. SEM images of Ru–PEI–PAA composite (A), CeO2 nanoparticles (B) and Au@CeO2 (C).

b a

12.0k

-450

9.0k c d e

6.0k 3.0k 0.0 0.6

Z"/ohm

ECL intensity / a.u

-600

-300 -150

a

f

0.8

1.0 1.2 Potential / v

1.4

0 0

b cde 200 400

f 600

800

Z'/ohm

Fig. 3. The ECL (A) and EIS (B) characterization of the different modified electrode: (a) Ru–PEI–PAA/GCE; (b) nano-Au/Ru–PEI–PAA/GCE; (c) TBA 1/nano-Au/Ru–PEI–PAA/GCE; (d) BSA/TBA 1/nano-Au/Ru–PEI–PAA/GCE; (e) the above e modified electrode was incubated with TB (0.1 nm); (f) the above f modified electrode was incubated with hemin/ TBA 2/Au@CeO2 (the inset of red curve in (B) is bare GCE). ECL measured in pH 7.4 PBS (pH 7.4, 3.0 mL) containing 0.1 M KCl. Scan rate: 100 mV/s. EIS tracked in 5 mmol L  1 Fe(CN)64  /3  . The frequency range of EIS is at 1  10  2 to 1  106 Hz. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

semicircle (GCE, the inset of red curve). After coating with Ru–PEI– PAA composite, the semicircle diameter (curve a) was enlarged because the Ru–PEI–PAA composite could perturb the interfacial electron-transfer. When nano-Au was further modified on selfenhanced Ru–PEI–PAA composite modified electrode, the electron transfer resistance decreased obviously (curve b), which might be

that the nano-Au could facilitate electron-transfer. However, the electron transfer resistance increased successively when TBA 1 (curve c) and BSA (curve d) were immobilized onto the electrode attributing to their insulation effects. Following that, the semicircle diameter (curve e) increased again when the resultant electrode was incubated in TB standard solution because of protein

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Fig. 4. Comparison of ECL responses with different TBA 2 conjugates with 0.1 nM TB: (A) TBA 2/Au, (B) TBA 2/Au@CeO2, and (C) hemin /TBA 2/Au@CeO2. In addition, the red ECL potential curve stands for the modified GCE electrode of BSA/TBA 1/nano-Au/Ru–PEI–PAA. (D) ECL responses of the bare GCE in different solutions: (a) Ru(bpy)32 þ /PEI aqueous solution; (b) Ru(bpy)32 þ /PEI aqueous solution with CeO2; (c) Ru(bpy)32 þ /PEI aqueous solution with hemin; (d) Ru(bpy)32 þ /PEI aqueous solution with CeO2 and hemin. Scan rate of 100 mV/s. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

on the electrode could hinder the electron transfer. Finally, TBA 2 conjugates were dropped onto the obtained electrode; the semicircle diameter (curve f) increased evidently because TBA 2 conjugates may weaken the electron transfer. 3.4. The comparison of different TBA 2 labeled probes In order to explore the effect of CeO2 NPs and hemin for ECL quenching, different TBA 2 labeled probes were prepared, including TBA 2/Au (Probe A), TBA 2/Au@CeO2 conjugates (Probe B) and hemin/TBA 2/Au@CeO2 conjugates (Probe C). The modified GCE electrodes of BSA/TBA 1/nano-Au/Ru–PEI–PAA with the same batch were incubated with 0.1 nm TB standard solution, and then different TBA 2 conjugates were incubated with the above electrodes by sandwich reaction. As illustrated in Fig. 4A, the ECL responses of the aptasensor with TBA 2/Au decreased to a lower value than that of the BSA/TBA 1/nano-Au/Ru–PEI–PAA/GCE. The decrease might be attributed to the loading of TB, which could hinder the electron transfer. When the aptasensor was incubated with TBA 2/Au@CeO2 conjugates, the signal decreased about 1264 a.u., demonstrating the CeO2 NPs could quench Ru(bpy)32 þ luminescence (Fig. 4B). Moreover, when the aptasensor was incubated with the as-prepared probe of hemin/TBA 2/Au@CeO2, the ECL emission was obviously decreased about 3246 a.u., which achieved the desirable ECL quenching efficiency of hemin/TBA 2/ Au@CeO2 conjugates (Fig. 4C). Thus, the results adequately indicated that the hemin and Au@CeO2 NPs in TBA 2 bioconjugates

could quench Ru(bpy)32 þ luminescence signal synergistically for sensitive detection of TB. To investigate the quenching mechanism, ECL response of the bare GCE in different solutions was shown in Fig. 4D. Curve a shows the ECL peak intensity (about 10,000 a.u.) of the bare GCE in Ru(bpy)32 þ /PEI solution, proving the PEI could serve as an ideal co-reactant to enhance the ECL of Ru(bpy)32 þ for signal amplification, which is consistent with our previously reported results (Zhuo et al., 2014). When 5.0 mg/mL CeO2 NPs solution was added into Ru(bpy)32 þ /PEI solution, an obvious quenching signal was observed (curve b). The similar phenomenon was obtained after putting 40 μL 5 mM hemin into Ru(bpy)32 þ /PEI solution (curve c). Thus, the results suggest that both hemin and CeO2 NPs could quench ECL of the Ru(bpy)32 þ /PEI system. Furthermore, the ECL was tested in the Ru(bpy)32 þ /PEI solution containing CeO2 NPs and hemin with the same concentration of curves b and c. The lowest ECL was achieved in curve d, demonstrating that CeO2 NPs and hemin could quench Ru(bpy)32 þ luminescence signal synergistically. Then the quenching effect of the two co-existed quenchers (hemin and CeO2 NPs) was further evaluated by the quenching rate constant (Kq) via Stern–Volmer equation (Xia et al., 1995) (The detailed experimental steps, see Supplementary material). The results indicated that the sum of Khemin and KCeO2 was approximate equal to the K hemin þ CeO2, which was consistent with the Stern–Volmer equation. Thus, we suppose that the possible mechanism might be energy and electron transfer in which the excited state Ru(bpy)32 þ * was oxidized to Ru(bpy)33 þ by hemin

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Fig. 5. (A) The ECL potential curves of the aptasensor with different concentrations of TB in pH 7.4 PBS. TB concentration: (a) 10  3 nM, (b) 10  2 nM, (c) 0.1 nM, (d) 1 nM, (e) 10 nM, and (f) 100 nM. The curve g is BSA/TBA 1/nano-Au /Ru–PEI–PAA/GCE. (Inset: the relationship between the change of ECL intensity and the concentration of TB.) (B) The stability of the proposed ECL aptasensor incubated with 1 nM TB under consecutive cyclic potential scans for 10 cycles. (C) The selectivity of the proposed ECL aptasensor: (a) blank, (b) Hb (100 nM), (c) BSA (0.25%), (d) TB (10 nM), and (e) a mixture containing Hb, BSA, and TB.

and CeO2 NPs as both of them have a redox pair of metal ions. The possible ECL quenching mechanisms could be inferred as follows:

Ru(bpy)32 + − e− → Ru(bpy)33 +

electrode oxidation

(1a)

PEI − e− → PEI⋅+

electrode oxidation (1b)

PEI⋅+ → PEI⋅ + H+

deprotonation

(2)

electron transfer

(3)

luminescence

(4)

Ru(bpy)33 + + PEI⋅ → Ru(bpy)32 +⁎ + PEI fragments Ru(bpy)32 +⁎ → Ru(bpy)32 + + hv Hemin(Fe(III)) + Ru(bpy) 32 + * → Hemin(Fe(II)) + Ru(bpy) 33 +

luminescence

(5a)

Ce(IV) + Ru(bpy)32 +⁎ → Ce(III) + Ru(bpy)33 +

luminescence

(5b)

3.5. ECL response of the aptasensor to TB concentration The analytical performance of the proposed aptasensors was incubated with different concentrations of TB standard solutions

based on sandwiched reaction. As expected, Fig. 5 shows the ECL intensity decreased with the increasing TB concentrations (Fig. 5A, curves a–f) and the linear range for TB was from 10  13 to 10  8 M with a detection limit of 0.03 pM (S/N ¼3). The change of ECL intensity ΔI was proportional to the logarithm of the concentrations of TB with a correlation coefficient of 0.9973 as shown in Fig. 5A. Comparing with the previous work of TB aptasensor (Table S1, see ESI), the present work showed a wider linear range response and lower detection limit. The stability of this proposed aptasensor to 1 nm TB under continuous cyclic potential scans for 10 cycles is shown in Fig. 5B. The relative standard deviation (R.S.D.) was 1.42%. Moreover, the analytical performances did not present an obvious decline, showing that the aptasensor possessed good stability. To further monitor the selectivity, the proposed aptasensors were incubated with different interfering proteins. Fig. 5C shows the quenching ECL signal (ΔI) of the modified GCE electrodes of BSA/TBA 1/nano-Au/Ru–PEI–PAA after incubation with blank (without TB), 10 nm TB, 100 nm hemoglobin (Hb), 0.25% BSA and the mixture containing Hb, BSA and thrombin, respectively. As can be seen, the ΔI values for foreign proteins were much smaller than those for thrombin. The high ECL quenching signal obtained from a mixed sample, indicating an acceptable detection selectivity for this aptasensor.

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Table 1 Determination of TB added in normal human serum with the proposed aptasensor. Serum sample Added thrombin (nM) Found thrombin (nM) Recovery % 1 2 3 4 5 6

1.0  10  3 1.0  10  2 0.1 1.0 10.0 100.0

9.77  10  4 9.55  10  3 9.23  10  2 9.83  10  1 10.3 96.5

97.7 95.5 92.3 98.3 103.0 96.5

Doctoral Program of Higher Education (20110182120010), Ministry of Education of China (Project 708073), Specialized Research Fund for the Doctoral Program of Higher Education (20100182110015), Natural Science Foundation Project of Chongqing City (CSTC-2010BB4121 and CSTC-2009BA1003), and the Fundamental Research Funds for the Central Universities (XDJK2010C062, XDJK2012A004, and XDJK2014A012, XDJK2014C001), China.

Appendix A. Supplementary information 3.6. Detection of thrombin in human serum samples Recovery experiments were used for monitoring the feasibility of the ECL aptasensor which were performed by standard addition methods in human serum. As shown in Table 1, the recovery (between 92.3% and 103.0%) was acceptable, which provided a promising tool for determining TB in real biological samples.

4. Conclusions In summary, a new ECL quenching system between the luminophor of PEI enhanced Ru(bpy)32 þ nanocomposite and the quenchers of hemin and Au@CeO2 NPs is developed for the first time. The obtained Ru–PEI–PAA composite can enhance ECL intensity to provide the desirable initial ECL signal for construction of a quenching aptasensor. More importantly, a sensitive signal-off aptasensor is constructed based on the synergistical quenching effect of both hemin and Au@CeO2 NPs, which offers new opportunities for sensitive detection of TB at low concentrations. Therefore, this method is promising for other biomolecules diagnostics as well as for bioanalysis in general.

Acknowledgments This work is supported by National Natural Science Foundation of China (21275119, 21105081, and 21075100), Research Fund for the

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.065.

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