Dual-stabilizer-capped CdSe quantum dots for “Off–On” electrochemiluminescence biosensing of thrombin by target-triggered multiple amplification

Abstract

In this work, new dual-stabilizer-capped CdSe quantum dots (QDs) with unique electrochemiluminescence (ECL) were prepared, and used to design a promising “off–on” ECL biosensor for highly sensitive assay of thrombin by target-triggered multiple amplification for the first time. New poly(diallyldimethylammonium chloride) (PDDA)–graphene oxide (GO) nanocomposites with good electroconductibility and much surface area was used to assemble a large number of CdSe QDs on the electrode, showing excellent ECL and stability. The ECL of CdSe QDs was efficiently quenched by gold nanoparticles (off state), the presence of target thrombin resulted in the generation of numerous DNA sequences (t1) by using multiple DNA cycle amplification techniques. Subsequently, the specific hybridization of abundant t1 to the quencher probe caused significantly amplified ECL enhancement (on state), which was used for highly sensitive detection of thrombin. More importantly, the biosensor with the dual-stabilizer capped CdSe QDs demonstrated highly intense ECL emission, and the DNA devices by the cleavage reaction were cycled for multiple rounds, which greatly amplified the ECL signal and much improved the detection sensitivity. The developed assay protocol also shows high selectivity for the target and can be performed in homogeneous solutions. These distinct advantages make it a potential platform for detecting various types of biomolecules. This flexible biosensing system exhibits excellent performance in real human serum assay, which has wider potential applications than other ECL reagent-based system.

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Introduction

Electrochemiluminescence (ECL) has remarkable features such as simplicity, rapidity, high sensitivity and easy controllability. Since Bard's group reported stable ECL from Si QDs in 2002,¹ QDs have rapidly become one of the most popular ECL species,² and biomolecular determinations based on QDs ECL have been widely reported.^3–5 Compared with conventional ECL-emitting species such as luminol or Ru(bpy)₃²⁺, QDs ECL possesses many advantages, such as neutral detection conditions and easy realizability in both cathodic and anodic processes with diverse co-reactants, leading to multifarious biosensing strategies.^6,7 However, the limited ECL efficiency of QDs is one barrier for developing QD-based ECL sensors, as the ECL intensity of QDs cannot come to that of conventional ECL reagents, such as Ru(bpy)₃²⁺.⁸ Recently, it was found that the novel CdSe QDs with dual-stabilizers (mercaptopropionic acid and sodium hexametaphosphate) display highly intense and eye-visible ECL emission,⁹ and was used to the ECL detection of dopamine.¹⁰

In addition, quantitative detection of specific proteins plays an increasingly important role in the biomedical field, especially in disease diagnosis.^9,11 However, protein biomarkers are present in ultralow concentrations in biological samples of patients in the early stage of disease progression. Therefore, the development of higher sensitive ECL methods for protein identification and determination is highly demanded. To improve the sensitivity of ECL detection, a variety of DNA signal amplification strategies have been reported to enhance the detection signal.^12,13 In general, signal amplification can be achieved by nanomaterials, enzyme catalytic reaction, and target recycling reaction. While the signal amplification approaches based on biobarcodes,¹⁴ nanomaterial composites¹⁵ and enzyme¹⁶ labels for aptamer-based protein detection require extensive label preparation and conjugation processes. Very recently, DNA cycle amplification devices have now become powerful tools due to the specificity of molecular recognition capability and their robust physicochemical properties. A new class of signal amplification strategies based on nuclease (exonuclease or endonuclease)-assisted target recycling has been reported.^17,18 The nuclease with sequence specific activity can degrade the probe DNA of the probe/target dsDNA strands and release the target DNA to achieve recycling amplification. This signal amplification strategy has been demonstrated to be useful in detecting trace levels of targets.

Aptamers, artificial oligonucleotides originating from an in vitro strategy termed systematic evolution of ligands by exponential enrichment (SELEX),¹⁹ can bind a number of specific molecular targets, such as small chemicals, proteins, and even cells, with high affinity and specificity comparable to that of antibodies.²⁰ Aptamers have shown overwhelming advantages over antibodies as recognition probes for protein detection, owing to their high affinity and specificity, target versatility, easy synthesis and regeneration. Among various aptamer-based protein sensing strategies such as fluorescence,²¹ colorimetry,²² surface plasmon resonance (SPR),²³ electrochemistry²⁴ and electrochemiluminescence (ECL),^25,26 the ECL approach has attracted special attention due to its high sensitivity.

Recently, graphene oxide (GO) has been extensively studied due to its unique and excellent electronic, thermal, and mechanical properties.^27,28 Specifically, the formation of GO-based nanocomposites is greatly advantageous to the design of versatile material for high-performance biosensors, owing to their good water dispersibility, large accessible surface area, and good biocompatibility.^29,30

In this study, inspired by the unique ECL of a novel dual-stabilizers-capped CdSe QDs and advantages of multiple amplification strategy, we prepared the CdSe QDs with excellent ECL, and reported a promising “off–on” ECL biosensor for highly sensitive detection of thrombin by target-triggered multiple amplification technique. A large number of CdSe QDs were assembled on the electrode by using the PDDA–GO nanocomposites, showing highly intense ECL and well stability. Taking advantages of the high amplification efficiency of enzyme-aided multiple recycling amplification technique and efficient quenching of gold nanoparticles to ECL of CdSe QDs, the target thrombin triggers the generation of many DNA sequences (t1), and thereafter the specific binding of t1 to MB release abundant quencher probe. Using the significantly enhanced ECL signal, we demonstrate the specific and sensitive detection of thrombin. The highly sensitive ECL detection of protein macromolecules is achieved with a new quadratic signal amplification approach by using the DNAzyme probes, which can be performed in homogeneous solution. This flexible biosensing system also exhibits excellent performance in real human serum thrombin assay. These advantages make our approach a potential platform for highly sensitive ECL detection of various biomarkers for early disease diagnostic applications.

Results and discussion

Characterization of the dual-stabilizers-capped (DSC) CdSe QDs

The transmission electron microscopy (TEM) image of the DSC CdSe QDs was shown in Fig. 1A, the nanoparticles possessed uniform morphology and size, and the average diameter was about 5 nm. The photoluminescence (PL) spectra of the CdSe QDs were presented in Fig. 1B, the PL emission peaks of the QDs were at 510 nm and 630 nm, which may be attributed to the individual and aggregated CdSe QDs respectively.³¹ The PL intensity was high, indicating that the CdSe QDs possess good luminescent property.

Fig. 1 (A) TEM image and (B) photoluminescence (PL) of the dual-stabilizers-capped CdSe QDs.

ECL behavior of the dual-stabilizers-capped CdSe QDs

Fig. S1† showed the ECL–potential curve and cyclic voltammogram (CV, inset) of the dual-stabilizers-capped CdSe QDs. The CdSe QDs show highly intense ECL. The reason may be as follows: the surface cadmium atoms of CdSe QDs to the dual stabilizers can effectively remove the nonradiative surface states and deep surface traps for enhanced ECL efficiency; the other is that the CdSe QDs are favorable for the electrochemical involved electron and hole injection processes.³² A cathodic peak at −0.93 V is observed in the CV response, corresponding to the reduction of S₂O₈²⁻. There is an ECL peak at −1.49 V in the ECL–potential curve, which results from the reaction of the CdSe QDs with S₂O₈²⁻. The QDs are reduced to nanocrystalline species (QD⁻˙), while the S₂O₈²⁻ is reduced to strong oxidant SO₄⁻˙. Then SO₄⁻˙ reacts with QD⁻˙ to form the excited state (QD*) that could emit light. The possible ECL mechanisms are as follows.³³

QD + e⁻ → QD⁻˙ (1)

S₂O₈²⁻ + e⁻ → SO₄²⁻ + SO₄⁻˙ (2)

QD⁻˙ + SO₄⁻˙ → QD* + SO₄²⁻ (3)

QD* → QD + hν (4)

ECL detection of thrombin based on the dual-stabilizers-capped CdSe QDs by target-triggered multiple amplification strategy

The fabrication principle for ECL assay of thrombin based on the dual-stabilizers-capped CdSe QDs by target-triggered multiple amplification strategy was shown in Scheme 1. The hairpin DNA probe (HP1) contained an aptamer sequence for thrombin and the primer recognition fragment. In the presence of the target thrombin, it binds to the aptamer loop region, leading to the exposure of the primer recognition fragment. Subsequently, the added primer hybridizes with the recognition fragment and proceeds with polymerization under the assistance of Bst-polymerase, which displaces the target and leads to the formation of a partially complementary DNA duplex. The displaced target again binds with other intact HP1 to initiate the recycling amplification cycle (recycling I). Moreover, the polymerization-generated partially complementary DNA duplexes (cDNA) with 5′-phosphorylated termini can be recognized by λ exonuclease, which catalytically digests the DNA duplexes and releases the corresponding complementary strands and blue sequences (s1). Then, the released complementary strands can hybridize with other HP1 to form partially complementary DNA duplexes, and λ exonuclease further cleaves these duplexes to initiate recycling II, which will result in the generation of numerous single-stranded DNA s1. Meanwhile, the gold NPs as the ECL quencher were linked to the thiol-modified hairpin DNA2 (HP2) by Au–S bond. After the dual-stabilizers-capped CdSe QDs as ECL emitters were assembled on the PDDA–GO composites modified-electrode, the HP2 quenching probe was conjugated to the QDs through covalent interactions. In the presence of large number of released DNA s1, it hybridized with HP2 to form the double-stranded recognition region of Nb.BbvCI, leading to scission of HP2 under the effect of Nb.BbvCI, and released DNA fragments with quencher and s1. Then DNA s1 can hybridize to other HP2 to continue the strand-scission cycle III, which result in the massive release of quencher from HP2. After the quencher was washed off the electrode, much enhanced ECL signal by multiple amplification was obtained, which was used to detect the target thrombin. Importantly, due to the quadratic amplification nature of the proposed strategy, the presence of low levels of target is expected to generate a large number DNA s1, which can potentially result in a significant change of ECL signal for detection of thrombin.

Scheme 1 Schematic representation strategy for ECL detection of thrombin based on the dual-stabilizers-capped CdSe QDs by target-triggered multiple amplification strategy.

SEM characterization of the ECL biosensor

Fig. 2 shows the field-emission scanning electron microscopy (FE-SEM) images of the modified electrodes. The PDDA–GO nanocomposites with good electroconductibility and many amino groups were firstly immobilized on the electrode, then the CdSe QDs were assembled on the nanocomposites. It was observed that the PDDA–GO with bulk shape and many small nanoparticles of CdSe QDs were formed on the electrode (Fig. 2A), and the surface was very smooth. By comparison, when the DNA-gold nanoparticles were further conjugated to the CdSe QDs on the electrode, the surface morphology became obviously different, a thick film with many nanoparticles spread over the surface uniformly (Fig. 2B). The results demonstrate that the ECL biosensor based on CdSe QDs and DNA-gold nanoparticles quencher was successfully fabricated.

Fig. 2 SEM images of (A) PDDA–GO/CdSe QDs, (B) PDDA–GO/CdSe QDs/DNA-gold NPs on the electrode.

ECL detection of thrombin based on DSC CdSe QDs by enzyme-aided multiple DNA cycle amplification strategy

The strategy feasibility for thrombin detection based on ECL quenching of the DSC QDs by enzyme-aided multiple DNA cycle amplification strategy was investigated. Fig. 3 showed that the background ECL on the bare electrode was very low (curve a), while the DSC CdSe QDs modified electrode displayed very high ECL signal (curve b). When the MB2-gold NPs as ECL quencher was linked to the CdSe QDs on the electrode, the ECL signal obviously decreased (curve c), indicating that ECL of the CdSe QDs was efficiently quenched by the gold NPs. In the presence of target thrombin together with the mixture containing primer, DNA probe, Bst-polymerase, λ exonuclease and endonuclease, the ECL signal obviously enhanced (curve d), indicating that the strategy based on ECL quenching of the CdSe QDs by multiple amplification could be used for thrombin detection.

Fig. 3 ECL–time curves for (a) bare Au electrode, (b) DSC CdSe QDs modified electrode, (c) ECL quenching by gold NPs (d) enhanced ECL in the presence of target thrombin by multiple cycle amplification strategy.

PAGE characterization of reaction products by enzyme-aided multiple DNA cycle amplification

To demonstrate the feasibility of the proposed enzyme-aided multiple DNA cycle amplification method for the assay of target thrombin, we performed nondenaturating PAGE analysis (Fig. S2†). Only a weak DNA band is observed in the presence of hairpin DNA and primer DNA, (Fig. S2,† lane 1). In addition, when Bst DNA polymerase and dNTPs were also added to the mixture of hairpin DNA and primer DNA, no obvious change of DNA band was observed (lane 2), indicating that only Bst DNA polymerase and dNTPs could not result in the polymerization. In contrast, when target thrombin was further added to the same mixture of lane 2, a distinct and strong DNA band was observed (lane 3), demonstrating that the binding of thrombin to its aptamer loop region lead to the exposure of the primer recognition fragment and subsequent polymerization under the assistance of Bst-polymerase, so the polymerization product was obtained. In the presence of hairpin DNA, primer DNA, Bst-polymerase, target thrombin dNTPs and lambda exonuclease, the strong DNA band of digestion reaction products were observed, suggesting that the enzyme-aided multiple DNA cycle amplification can be initiated by further adding lambda exonuclease (lane 4).

ECL detection of thrombin

Fig. 4 shows the ECL signal responses upon different concentrations of target thrombin. In the absence of thrombin, the ECL signal was very low (curve a), indicating that little unspecific binding occurs. However, in the presence of target thrombin by enzyme-aided multiple DNA cycle amplification strategy, the ECL peak signal gradually increased with increasing thrombin concentrations (curve b to j), indicating that the thrombin concentration could be quantitatively measured by the ECL signal.

Fig. 4 ECL signal responses for detection of different concentrations of thrombin. The concentrations of thrombin (nM): (a) 0; (b) 1.0 × 10⁻⁵; (c) 1.0 × 10⁻⁴; (d) 1.0 × 10⁻³; (e) 1.0 × 10⁻²; (f) 1.0 × 10⁻¹; (g) 1.0; (h) 1.0 × 10¹; (i) 1.0 × 10²; (j) 1.0 × 10³.

The relationship between ΔI and thrombin concentrations was shown in Fig. 5, the value of ΔI increased with thrombin concentrations in the range from 0.01 pM to 1000 nM. Inset showed that ΔI was linear to the logarithm of thrombin concentrations in the range from 0.1 pM to 100 nM (R = 0.994), the detection limit was 0.04 pM at 3σ, which is lower than the previously reported detection of thrombin.³ A series of three duplicate measusrements of 0.1 nM were used for estimating the precision, and the relative standard deviation (RSD) was 4.8%, suggesting that the ECL method displayed good reproducibility.

Fig. 5 Relationship between ΔI and thrombin concentration, inset: the logarithmic calibration curve for thrombin detection.

Specificity of the ECL-based strategy for thrombin detection

The specificity of the ECL-based strategy for thrombin detection was studied, the influences of some other proteins such as bovine serum albumin (BSA) and lysozyme were examined under the same experimental conditions. Fig. S3† shows that none of these proteins at concentration of 0.1 nM caused obvious ECL change, while only 0.01 nM thrombin resulted in significant ECL enhancement, indicating that those proteins did not interfere with the thrombin assay. The results demonstrated that the proposed amplification strategy exhibited good specificity for thrombin detection.

Analysis of thrombin in real sample

We investigated the applicability of the proposed ECL strategy by detecting thrombin in a human serum sample. The thrombin was spiked into 10-fold-diluted serum samples to examine the applicability of this ECL method. The serum samples were spiked with 6.0, 20.0, 100.0, and 50.0 pM, and then measured by the proposed ECL method. The recoveries were obtained by comparing the measured amounts to that of added human thrombin, which varied from 95.0 to 105.2% (Table 1). These results suggest that the ECL method could be used to the detection of thrombin samples.

Table 1 Recovery of thrombin assay at different concentrations spiked into human serum samples^a

Sample^b	Sample	Spiked	Found	Recovery	RSD
a Each sample was repeated for three times and averaged to obtain the recovery and RSD values. All human serum samples were diluted 10-fold prior to assay.b Samples 1–3 were from healthy people, and 4–5 were from patients.c “—” represents not detectable.
1	—^c	6.0	5.7	95.0	3.6
2	—^c	20.0	20.8	104.0	2.8
3	—^c	100.0	105.0	105.0	3.4
4	7.5	50.0	60.5	105.2	3.6
5	9.5	100.0	114.5	104.6	3.2

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Experimental

Materials and apparatus

Graphite powder, NaNO₃, H₂SO₄, KMnO₄, H₂O₂, polyvinylpyrrolidone (PVP), poly(diallyldimethylammonium chloride) (PDDA, 20%, w/w, in water, MW = 200 000–350 000), CdCl₂·2.5H₂O (98%) were purchased from aladdin-reagent.com. (Shanghai). Sodium hexametaphosphate (HMP), sodium sulfite (AR), selenium (99.9%, powder), mercaptopropionic acid (MPA, 99.8%), and hydrazine hydrate (98%) were purchased from Aldrich. The Bst-DNA polymerase (large fragment), λ exonuclease, endonuclease (Nb.BbvCI, 10 000 U mL⁻¹) was purchased from the NEW ENGLAND Biolabs (NEB). The recognition site of Nb.BbvCI was: 5,…CCTCA|GC…3, 3,…GGAGT|CG…5.

All the DNA sequences were synthesized and purified by SBS Genetech Co. Ltd. (China), and the DNA sequences used in this work are listed in Table 2. Human thrombin was purchased from Shanghai Bio Technology Co., Ltd. 0.1 M PBS buffer (pH 7.4) was prepared according to the standard methods. Chloroauric acid (HAuCl₄) and trisodium citrate were obtained from Shanghai Reagent Company (Shanghai, China). All other reagents were of analytical grade. Double distilled water was used for all experiments.

Table 2 The sequences of the DNA

DNA	Sequence
Hairpin RNA	5′-NH2-TTTTTTTTTTTTTTTCCGGTTGCCCATCCGCTGAGGCAACAACCGG-SH-3′
Primer DNA	5′-TCAAGTCC-3′
Hairpin probe DNA	5′-Phosphate-TAATTCCCAATCAAGTCCGTGGTAGGGCAGGTTGGGGTGACTGCATATAATCGAGGGGACTTGATTGCCTCAGCGGATGGG-3′

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Transmission electron microscopy (TEM) images were recorded using a JEM-2000EXinstrument (Hitachi). Field-emission scanning electron microscopy (FE-SEM) was carried out on a JEOL JSM-6700F instrument. Photoluminescence (PL) spectra were obtained on a F-4500 spectrophotometer (Shimadzu). Absorption measurements were carried out using a Varian Cary 500 UV-vis spectrophotometer. ECL measurements were carried out on a MPI-A ECL analyzer (Xi'An Remax Electronic Science & Technology, Xi'An, China) using a three-electrode system.

Synthesis of PDDA-functionalized graphene oxide

The PDDA-functionalized graphene oxide was synthesized based on a previous work of our group.³³ First, the graphene oxide was prepared from natural graphite powder by the modified Hummers method.³⁴ Then the graphene oxides obtained were exfoliated by ultrasonication in water for more than 1 h. At last, water solution of homogeneous graphene oxide (1.0 mg mL⁻¹) was obtained.

PVP-capped graphene oxide was prepared according to the reported work.³⁰ In a typical procedure, 80 mg of PVP was added to 20 mL of 0.25 mg mL⁻¹ graphene oxide solution, followed by stirring for 30 min. The resulting dispersion was washed, centrifuged three times and dissolved in 5 mL of water.

To obtain PDDA-functionalized graphene oxide (PDDA/GO), 0.1 mL of 20 wt% PDDA was mixed with 16.8 mL of 0.625 M KCl, followed by injecting 4.2 mL of PVP-capped graphene oxide, and the resulting solution was sonicated for 1.5 h. The products were washed and centrifuged three times. Finally, the PDDA/GO was redispersed in 4 mL of water.

Preparation of Na₂SeO₃

Na₂SO₃ (1.7 g) was added to a 100 mL flask, then 50 mL of ultrapure water was added. The solution was stirred, degassed with nitrogen for 10 min and heated to the temperature of 90 °C. Then 0.6 g of selenium powder was quickly injected into the reaction flask, and refluxed for 5 h with nitrogen. After the selenium powder disappeared completely, the light yellow and clear Na₂SeSO₃ solution was obtained.

Syntheses of dual-stabilizers-capped CdSe QDs

CdSe QDs were synthesized according to the literature.^31,35 Briefly, CdCl₂ solution (73 mg), HMP (72.5 mg), and MPA (34.6 μL) were dissolved in 50 mL of H₂O successively. After pH was adjusted to 9.0, 0.80 mL of Na₂SeSO₃ solution (20.0 mM) was added to the mixture. Then, the above mixture was refluxed at 100 °C for 10 min, and 3.67 mL of N₂H₄·H₂O was added and refluxed at 100 °C for another 10 h. The resultant was purified three times with isopropyl alcohol by centrifugation at 10 000 rpm, and stored in the dark at 4 °C. The concentration of the dual-stabilizers-capped CdSe QDs stock solution was estimated to be 7.10 μM with an empirical equation.^33,36

Electrochemiluminescence detection of thrombin based on CdSe QDs by target-triggered multiple amplification strategy

The gold NPs-MB2 as quencher probe was formed as follows. 1 mL of gold colloid was firstly centrifuged at 10 000 rpm for 10 min, then the precipitate was redispersed in 100 μL of doubly distilled water. After adding 50 μL of 1.0 × 10⁻⁵ M thiol-modified MB2 to the purified gold colloid, the solution was shaken gently for 24 h at room temperature, centrifuged at 15 000 rpm for 30 min. The oily precipitate was then washed with 10 mM phosphate buffer two times, recentrifuged, and redispersed in 0.3 M PBS.

For target-triggered multiple amplification, a series of target thrombin in different concentrations, 10 μL of HAP (10 μM), 10 μL of primer (10 μM), 2 μL of dNTPs (10 mM), 16 U of Bst-DNA polymerase, 12 U of λ exonuclease, and 4 μL of dNTPs (0.2 mM) were first mixed to 50 μL of solution, and then incubated at 37 °C for 90 min.

For ECL detection, the Au electrodes were polished with alumina slurries (1, 0.3, 0.05 μm), washed with deionized water, and dried with nitrogen gas. After 8 μL of PDDA/GO solution was dropped on the electrode surface and dried naturally at the room temperature, 7 μL of carboxy groups functionalized QDs solution was dropped on the electrode and dried. Then 10 μL of gold NPs-MB2 solution was dropped on the electrode and reacted at 37 °C for 12 h. Unconjugated gold NPs-MB2 was removed by washing the electrodes with doubly distilled water. The quenched ECL was measured.

Then the electrode was immersed in the above mixture (50 μL of solution containing target thrombin, HAP, primer, Bst-DNA polymerase, λ exonuclease, and dNTPs), and the next endonuclease (Nb.) was added, which was incubated at 37 °C for 2 h, and then washed twice for ECL measurement.

The ECL emission was detected with a model MPI-A electrochemiluminescence analyzer using a three-electrode system at room temperature. The electrodes were a modified Au disk working electrode (4 mm diameter), a saturated calomel reference electrode, and a Pt counter electrode. The modified electrodes above were in contact with 0.1 M PBS (pH 7.4) containing 0.05 M K₂S₂O₈ and 0.1 M KCl and scanned from 0 to −1.5 V. The spectral width of the photomultiplier tube (PMT) was 200–800 nm, and the voltage of the PMT was −800 V in the detection process. ECL signals related to the thrombin concentrations were measured.

Gel electrophoresis

A 10% nondenaturating polyacrylamide gel electrophoresis (PAGE) analysis of reaction products was carried out in 1 × TBE (9 mM Tris–HCl, pH 7.9, 9 mM boric acid, 0.2 mM ethylenediaminetetraacetic acid, EDTA) at a 120 V constant voltage for 35 min at room temperature. The gel was stained by SYBR Green I, and scanned by a Kodak Image Station 4000 MM (Woodbridge, CT, USA).

Conclusions

In summary, we have prepared a novel dual-stabilizers-capped CdSe QDs with excellent ECL, and developed a multiple amplification strategy by integrating several independent recycling cycles for highly sensitive ECL detection of thrombin. Due to the significant quadratic signal amplifications, the presence of thrombin leads to the generation of numerous DNA sequences (t1), which then result in release of abundant quencher probe and significantly amplified change of ECL signal. Besides, the PDDA–GO nanocomposites with good electroconductibility and high stability was used to assemble a large number of CdSe QDs on the electrode, which also much enhanced ECL of QDs. Compared with other traditional antibody-based sandwich immunoassays or aptamer-based assays, this flexible biosensing system has the advantages of high sensitivity, rapid signal readout, and simplicity. Moreover, this developed quadratic signal amplification approach can be a universal ECL sensing platform for a variety of aptamer-based target analytes, and exhibits excellent performance in real human serum assay.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21175078, 21575072), and the Financial Support by Open Research Fund of Top Key Discipline of Chemistry in Zhejiang Provincial Colleges and Key Laboratory of the Ministry of Education for Advanced Catalysis Materials (Zhejiang Normal University) (No. ZJHX201508).

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, UV-vis, TEM and the optimization and selectivity are included in the ESI and additional figures. See DOI: 10.1039/c5ra24811f

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