Construction of ultrasensitive label-free aptasensor for thrombin detection using palladium nanocones boosted electrochemiluminescence system

Accepted Manuscript Construction of ultrasensitive label-free aptasensor for thrombin detection using palladium nanocones boosted electrochemiluminescence system Hui-Min Wang, Yan Fang, Pei-Xin Yuan, Ai-Jun Wang, Xi-Liang Luo, Jiu-Ju Feng PII:

S0013-4686(19)30779-0

DOI:

https://doi.org/10.1016/j.electacta.2019.04.093

Reference:

EA 34041

To appear in:

Electrochimica Acta

Received Date: 25 March 2019 Accepted Date: 14 April 2019

Please cite this article as: H.-M. Wang, Y. Fang, P.-X. Yuan, A.-J. Wang, X.-L. Luo, J.-J. Feng, Construction of ultrasensitive label-free aptasensor for thrombin detection using palladium nanocones boosted electrochemiluminescence system, Electrochimica Acta (2019), doi: https://doi.org/10.1016/ j.electacta.2019.04.093. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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Construction of ultrasensitive label-free aptasensor for thrombin detection using palladium nanocones boosted

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electrochemiluminescence system

Hui-Min Wang,a Yan Fang,a Pei-Xin Yuan,a* Ai-Jun Wang,a Xi-Liang Luo,b Jiu-Ju Feng a*

College of Geography and Environmental Sciences, College of Chemistry and Life

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a

b

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Sciences, Zhejiang Normal University, Jinhua 321004, China

Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College

of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China

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*Corresponding author: [email protected] (P.X. Yuan); [email protected] (J.J. Feng).

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Abstract Aptamer-based bioassay of biomarker has broad applications in clinical research and disease diagnosis. In this work, a label-free aptamer biosensor for ultrasensitive (TB)

detection

was

constructed

based

on

highly

enhanced

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thrombin

electrochemiluminescence (ECL) of functional palladium nanocones (Pd NCs) with tripropylamine (TPA) system. Herein, well-defined Pd NCs were initially

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functionalized by mercaptoethanol (MCH) as an ECL emitter and immobilized onto

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the glassy carbon electrode (GCE) initially grafted with amine-terminated polyamidoamine (PAMAM) via electro-oxidation reaction. Under optimal conditions, the ECL intensity of the Pd NCs/TPA system was about 2.5 times enhancement when compared to that of bare GCE. The ECL and cyclic voltammetry (CV) were

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synchronously employed to illustrate the enhanced ECL mechanism. By virtue of the linkage of the modified Pd NCs with specified TB aptamer (TBA) containing sulfydryl group, a label-free ECL detection platform was designed. After full coverage

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of the nonspecific sites with bovine serum albumin (BSA), an ECL biosensor for TB

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detection was constructed by taking the advantage of the steric hindrance effects, showing the wider linear range, lower detection limit of TB (even down to 6.76 fM), superb selectivity and stability. The current strategy provides a highly sensitive platform for detection of various biomolecules in bioanalysis.

Keywords:

Electrochemiluminescence;

Palladium nanocones; Thrombin 2

Amine-terminated

polyamidoamine;

ACCEPTED MANUSCRIPT 1. Introduction Thrombin (TB), as a kind of specific serine protease and a biomarker in blood and human tissues, is the fibrous matrix of blood clots in the bloodstream [1, 2],

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which correlates with many diseases (e.g. lung neoplasm, cardiovascular diseases and thromboembolic disease) [3, 4]. Therefore, various methods for TB detection have been widely applied in clinical research and disease diagnosis [5] such as

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enzyme-linked immunosorbent assay [6], direct electrochemical detection [5] and

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specific sequence aptamer [7]. As we all know, aptamer has the stronger binding to TB, lower steric hindrance effects, superior stability and selectivity alternative to antibody, which extensively explored as excellent probe in biosensing devices [8, 9]. Now, multiple detection methods have been developed for aptamer bioassay,

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including fluorescence [10], amperometry [11], chemiluminescence [12] and electrochemiluminescence (ECL) [13, 14]. Among them, ECL has particularly gained extensive attention in bioassay, where ruthenium complexes [15] or semiconductor

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quantum dots (QDs) usually serve as emitters [16, 17]. Lately, the nanocrystal-based

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ECL emitters offer novel route [18]. However, the high-toxicity of heavy metals and insufficient ECL emission are the main bottlenecks to expand their practical applications.

The key to this faultiness is the sluggish kinetics at the electrode surface [19],

and thereby many efforts are focused on surface modification with carbon materials [20, 21] and advanced nanomaterials (e.g. Au, Pt, and SiO2) to enlarge the electrode surface area and facilitate the electronic kinetics [22, 23]. However, the poor stability 3

ACCEPTED MANUSCRIPT at high potential seriously limits the ECL applications in practice, due to the weak binding of the attached materials with electrodes and tremendous background responses [24]. polyamidoamine

(PAMAM)

is

highly

branched

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Amine-terminated

macromolecules with precisely defined star-like structures containing hydrophilic terminal functional amino-groups [25, 26]. It is found that PAMAM can be effectively

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anchored onto glassy carbon electrode (GCE) by direct electro-grafting [27]. The

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grafted PAMAM can create more binding sites easily accessible for subsequent immobilization of advanced materials with remarkable enhancement in ECL as emitters [26, 28].

Herein, mercaptoethanol (MCH) was employed to functionalize palladium

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nanocones (Pd NCs), which were initially prepared by a facile wet-chemical approach (Scheme 1A) with poly-L-lysine (PLL) as a green protecting ligand (Fig. S1, Supplementary Information, SI). The resulting functional Pd NCs had strong

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adsorption onto the enriched amino groups in PAMAM previously electrografted on

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the electrode surface by cyclic voltammetry (CV), defined as Pd NCs/PAMAM/GCE for simplicity. Besides, the ECL and CV measurements were synchronously conducted to illustrate the enhanced ECL mechanism for PAMAM towards Pd NCs/TPA system, followed by the linkage of the specified TB aptamer (TBA) with terminal thiol group. After eliminating the nonspecific sites with bovine serum albumin (BSA), a novel label-free ECL biosensor was obtained for TB detection (Scheme 1B). 4

ACCEPTED MANUSCRIPT 2. Experimental 2.1. Preparation of functional Pd NCs The uniform Pd NCs were prepared through a one-pot hydrothermal reaction

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based on our previous work [29], which were obtained with H2PdCl4 as the precursor, PLL as the green protecting ligand and formaldehyde (HCHO) as the reducing agent. In order to remove the capped PLL, the as-obtained Pd NCs were further

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functionalized with MCH by virtue of the stronger affinity of the thiol group in MCH

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to Pd surface than the amino groups in PLL. In brief, 10 mM MCH solution was put into the Pd NCs suspension, and consecutively stirred for 12 h to obtain a homogeneous Pd NCs suspension. In the controls, hydrogen peroxide (H2O2) and acetic acid (CH3COOH) were also employed as the modifying agents instead of MCH,

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while the other operation parameters remained constant. 2.2. Preparation of Pd NCs/PAMAM/GCE

Before construction of the enhanced ECL electrode (i.e. Pd NCs/PAMAM/GCE),

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the GCE was initially cleaned to obtain a mirror-like surface [30]. Then, the electrode

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was immersed into 0.1 M LiClO4 electrolyte containing 10 µM PAMAM and continuously sweeping for 20 cycles by CV from 0.0 to 1.0 V with the scan rate 10 mV s-1. The resultant electrode was defined as PAMAM/GCE for clarity. After being dried in air, the PAMAM/GCE was sequentially coated with 10 µL

of the functional Pd NCs suspension (1mg mL–1), which was denoted as Pd NCs/PAMAM/GCE for simplicity.

5

ACCEPTED MANUSCRIPT 2.3. Fabrication of label-free ECL biosensor for TB detection The Pd NCs/PAMAM/GCE was primarily coated with 10 µL of TBA (1.0 µM) at 4 °C for 12 h, completely washed with phosphate buffer solution (PBS, pH 7.4) to

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remove the residue, and further incubated with 10 µL of a BSA solution (1.0 wt.%) for 2 h at 37 °C to block the nonspecific binding sites. After washing efficiently with PBS, the modified electrode was incubated with 10 µL of TB solutions with different

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concentrations at 37 °C for 40 min. Eventually, the resulting electrode was washed

and ECL measurements.

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completely with PBS and stored in the refrigerator (4 °C) for further electrochemical

The ECL measurements were performed in the PBS (pH 7.4) including 30 mM TPA with the potential window of 0.0 - 1.4 V at the scan rate of 100 mV s-1.

presented in SI.

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More detailed information of the Reagents and materials, and Apparatus were

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3. Results and discussion

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3.1. Materials characterization The morphology and structure of the metal nanocrystals were critically

investigated by transmission electron microscopy (TEM) images [31]. As the lowand medium-magnification TEM images show (Fig. 1A, B), the well-defined Pd nanocrystals with triangular cone-like architectures almost remain after the treatment with MCH, with an average side length of 60 nm similar to those initially prepared with PLL [29]. Moreover, the photographs of the Pd NCs were taken after further 6

ACCEPTED MANUSCRIPT functionalization with MCH, H2O2 and CH3COOH under the same conditions (Fig. S2A, B, C). As expected, only the treatment with MCH produces a black homogeneous suspension. It means the highly improved stability of the Pd NCs after

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the ligand change between MCH and PLL. The high-resolution TEM (HRTEM) images (Fig. 1C, D) reveal the well-resolved lattice fringes of the functional Pd NCs with the interplanar spacing

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distances of 0.224 nm and 0.225 nm correlated with the (111) planes of the face

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centered cubic (fcc) Pd (0.224 nm) [32]. These observations demonstrate that the Pd NCs are enclosed by four Pd (111) facets [33].

X-ray photoelectron spectroscopy (XPS) measurements were performed to investigate the surface features of the modified Pd NCs. As described in Fig. 1E, S, C,

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N, O and Pd elements show up in the survey XPS spectrum of Pd NCs, revealing the successful functionalization of Pd NCs [34, 35]. In addition, Fig. 1F shows the predominant metallic Pd0 in the high-resolution Pd 3d XPS segment, showing the

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efficient reduction of the Pd precursor in this work [36].

3.2. Characterization of the enhanced ECL electrode To establish the enhanced ECL platform, the PAMAM was initially

electro-grafted onto the GCE by continuously scanning within the potential range of 0.0 - 1.0 V. As Fig. 2A depicts, a large irreversible oxidation peak appears at 0.8 V in the first cycle (curve b), unlike that in the absence of PAMAM (curve a), showing the efficient polymerization of PAMAM on the electrode surface via the C-N bond. 7

ACCEPTED MANUSCRIPT Furthermore, the oxidation peak currents decline steeply in the sequential cycles, indicating the formation of the stable C-N bond between PAMAM and the electrode [27].

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Then, the electrochemical behaviors of PAMAM/GCE was further investigated by CV in 0.1 M KCl electrolyte containing 5 mM [Fe(CN)6]4-/[Fe(CN)6]3- (Fig. 2B). There is a pair of reversible redox peaks of Fe3+/Fe2+ detected at 0.24/0.12 V with

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slight enlarged difference in the peak potentials and the declined peak currents (curve

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b) in comparison to bare GCE, accompanied with a pair of strong redox peaks emerged at 0.22/0.14 V (curve a). These scenarios reveal the efficient adherence of PAMAM onto the GCE as a functional layer.

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3.3. ECL enhancement mechanism of PAMAM to Pd NCs/TPA system The Pd NCs were assembled on the electrode surface via strong interactions between amino/imino groups of PAMAM and Pd NCs [37, 38]. The ECL and CV

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measurements were conducted synchronously to illustrate the enhancement

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mechanism of PAMAM to Pd NCs/TPA system. As seen in Fig. 3A, an obvious ECL emission peak emerges at 1.2 V on Pd NCs/GCE with the peak intensity of 2580 a.u. in the presence of 30 mM TPA (curve a), and an obvious oxidation peak emerge at 0.99 V with the peak current of 0.21 mA (Fig. 3B, curve a). Most impressively, the ECL emission peak current is roughly 2.5 times enhancement for the Pd NCs/PAMAM/GCE (Fig. 3A, curve b), accompanied by the roughly 2.5-time enlargement in the oxidation peak current of Pd NCs/PAMAM/GCE (Fig. 3B, curve b) 8

ACCEPTED MANUSCRIPT relative to that of Pd NCs/GCE (Fig. 3B, curve a). Fig. S3 shows the CV plots of Pd NCs (curve a) and Pd NCs-PAMAM (curve b) in the absence of 30 mM TPA, in which an obvious oxidation peak emerges at 0.81 V

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for Pd NCs/GCE at a slow scan rate of 10 mV s−1, while a homologous peak with slightly decreased current is detected at Pd NCs-PAMAM (curve b). It indicates that PAMAM has negligible impacts on the oxidation rate of Pd NCs. Besides, there is a

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blue ECL emission (λm= 466 nm) for the Pd NCs/TPA system (Fig. S4). Based on the

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above experimental data, the ECL reaction mechanism of Pd NCs/TPA is roughly described by the following steps [29] :

Pd NCs - e- → Pd NCs+ (+0.81 V)

(1)

TPA - e- → TPA•+

(2)

(+0.99 V)

(3)

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TPA•+ - H+ → TPA•

Pd NCs+ + TPA• → Pd NCs * + P1

(4)

Pd NCs * → Pd NCs + hv (λm= 466 nm)

(5)

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For this ECL process, equation (2) is the key step and thereby the electrocatalytic

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oxidation rate of TPA is positively correlated with the ECL intensity as expected. It demonstrates that PAMAM can significant accelerate the oxidation rate of TPA (step 2) rather than Pd NCs (step 1). The enhancement of TPA oxidation rate by PAMAM was further demonstrated

by the CV test. As Fig. 4B depicts, the oxidation peak currents of TPA at PAMAM/GCE steeply increase with the scan rates from 5 to 100 mV s−1, while the oxidation potential remains constant. Furthermore, the peak current (ip) shows linear 9

ACCEPTED MANUSCRIPT relationship with the square root of scan rate (v1/2) at the PAMAM/GCE (Fig. 4C, curve b), indicating that TPA oxidation at PAMAM/GCE is a typical “diffusion-controlled” process [39]. Also, the electron transfer from TPA to the

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PAMAM/GCE is the key step in the ECL process. These observations correlate well with those observed at bare GCE (Fig. 4A), while the slope of PAMAM/GCE is 2.24-time greater than that at bare GCE (Fig. 4C, curve a), which strongly verifies that

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PAMAM has the significant ECL enhancement for the rapid TPA oxidation.

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We further discussed the greatly accelerated TPA oxidation by the aid of PAMAM, which is poor conductive without the ability to promote the catalytic oxidation rate of Pd NCs on the electrode surface as discussed before (Fig. S3) [40]. Meanwhile, the polymer cannot enhance the electron transfer efficiency of the

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electroactive probe (i.e. [Fe(CN)6]4-/[Fe(CN)6]3- ) as illuminated above (Fig. 2B). Taken together, the quick elimination of TPA• + for the ECL reaction is mainly responsible for the rapid oxidation rates of TPA (Step 3).

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As we known, the nitrogen atom in -NH2 has a lone pair of electrons to form a

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coordination bond (-NH3+) with hydrogen ions in the system. Thus, the enriched amino groups in the PAMAM would effectively bind protons to enhance the deprotonation rate of TPA•+ [41]. To this regard, the PAMAM/GCE would work as excellent proton collector and Pd NCs as feasible ECL emitter to efficiently promote TPA oxidation, ultimately showing the dramatic amplification of the ECL responses [42]. Moreover, the electro-grafting cycle numbers were further optimized to prepare 10

ACCEPTED MANUSCRIPT the PAMAM/GCE by CV. As Fig. S5 displays, the ECL intensities of the Pd NCs/TPA system continuously enlarge by increasing the cycles for the enhanced PAMAM amount on the electrode surface, and reach a plateau roughly by sweeping above 20

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cycles. Thus, 20 cycles were selected as the optimal in this study.

3.4. Fabrication of the ECL biosensor

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The stepwise construction for the ECL biosensor was comprehensively

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characterized by electrochemical impedance spectroscopy (EIS) to provide electrical information of the modified electrode surfaces [16]. As Fig. 5A illustrates, an almost straight line is detected for bare GCE (curve a), which is characteristic of the diffusion-controlled electrochemical processes, indicating the superb electrical

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conductivity of the electrode itself [43]. After the linkage of PAMAM, the electron transfer resistance (Ret) remarkably increases (curve b), owing to the poor conductivity of the as-formed polymer film [44]. After the attachment of Pd NCs, the

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Ret adversely decreases due to the excellent electronic conductivity of the Pd

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nanostructures (curve c). Subsequently, the Ret values gradually increase via the covalent linkage of Pd NCs with TBA (curve d) and the sequential adsorption of BSA (curve e). It is attributed to the fact that non-conductive biomacromolecular structures of TBA and BSA severely hinder the interfacial electron transfer [45]. After the TBA specifically binds with TB, the Ret enlarges obviously (curve f), indicating the feasibility of the as-constructed ECL aptamer biosensor. The successful fabrication of the ECL biosensor was further identified by the 11

ACCEPTED MANUSCRIPT ECL measurements. As Fig. 5B exhibits, nearly no ECL signal is found at bare GCE (curve a) and the PAMAM/GCE (curve b) in the PBS containing 30 mM TPA. After the attachment of Pd NCs onto the PAMAM/GCE, the largest ECL emission emerges

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(curve c), owing to the huge oxidation rate of TPA onto the electrode surface. Further immobilization of TBA (curve d) and BSA (curve e) cause the slight decrease of the ECL intensities, owing to the non-conductive property of the biological substances.

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By virtue of the specific linkage between TBA and TB, the ECL intensity dramatically

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declines (curve f). As a result, the specific aptamer-protein interactions induce the remarkable ECL quenching alternative to the other modification steps, which is the foundation for this detection strategy.

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3.5. ECL detection of TB

The incubation time at 37 °C for linking TBA with TB was of great significance to detect TB in the current system, which was first optimized in the controlled

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experiments (Fig. S6). In the presence of 1.0 pM TB, the variation in ECL intensities

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(∆ECL) displays the great increase with the incubation time up to 40 min and almost reach a platform by further extending the time, demonstrating the saturated binding sites in the current detection. Hence, 40 min was selected as the optimized time for the subsequent experiments. Besides, to obtain the maximal detection signals, sufficient aptamer exists (i.e. 1.0 µM) in this analysis. Under the optimal conditions, the ECL signals are responsive to different concentrations of TB and attenuate gradually with the increase of the TB 12

ACCEPTED MANUSCRIPT concentrations (Fig. 6A). As Fig. 6B shows, the ECL intensity shows a linear relationship with the logarithm of the TB concentration, with a linear range from 0.01 to 100 pM and a low detection limit of 6.76 fM (using 3σ method) [46]. The

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calibration curve is I = -1383lg(c/M) -13455 (R2 = 0.995), where I is the ECL intensity and c is the TB concentration. More notably, the as-fabricated ECL aptasensor exhibits a wider linear range and lower detection limit in the detection of TB, which

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are comparable or even superior to those previously reported (Table S1).

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The stability was further examined to evaluate the current analytical performance by consecutive cyclic scanning in the PBS (pH 7.4) containing 1pM TB. Fig. 6C shows the consecutive intensity-constant ECL peaks with the relative standard deviation (RSD) of 1.45% under continuous potential scans, indicating the improved

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stability and reliability of the ECL system.

The fabricated ECL aptasensor also showed dramatically high selectivity for TB detection. Specially, the selectivity was critically checked by using 100 pM of human

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serum albumin (HSA), hemoglobin (Hb), alpha fetoprotein (AFP), and insulin with

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the concentrations of roughly 100-fold increase alternative to that of the target (i.e. 1 pM TB) (Fig. 6D). Clearly, the ECL intensities of the potential interfering substances show no obvious variation even at the much higher concentrations (e.g. 100 pM) when compared to the standard assay of TB. These scenarios demonstrate the highest selectivity of the developed aptasensor to the target. Moreover, the reproducibility of the fabricated ECL aptasensor was tested by five parallel trials (Fig. S7). Briefly, five aptasensor were prepared individually for 1 pM 13

ACCEPTED MANUSCRIPT TB detection with the RSD of 2.23% under the same conditions, reflecting the desirable reproducibility and accuracy of the aptasensor.

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3.6. Determination of TB in human serum samples The accuracy of the as-fabricated ECL aptasensor was evaluated by testing the TB concentrations in real serum samples with standard addition method [47]. As

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shown in Table S2, different concentrations of TB are spiked into 100-fold diluted

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human serum samples and the recoveries show up between 94.0%-104.1%, reflecting the acceptable accuracy in this analysis. These results confirm that the current sensing method would open a new avenue for determination of TB in clinical diagnosis.

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4. Conclusions

In summary, a novel label-free aptasensor for TB detection is constructed based on Pd NCs/PAMAM/GCE as largely enhanced ECL platform coupled with TPA

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system. For this protocol, PAMAM was efficiently immobilized onto the electrode via

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electro-grafting by CV for 20 cycles. At the same time, the freshly-prepared Pd NCs were initially functionalized with MCH by replacing PLL and then attached onto the PAMAM/GCE as desired ECL emitter. Both the ECL and CV experiments synchronously confirmed the remarkable ECL enhancement of TPA by the presence of PAMAM. The EIS and ECL plots approved the efficient adhesion of the TBA on the functional Pd NCs and the sequential linkage with TB, as well as the obvious ECL quenching. Under the optimized circumstance, a label-free biosensor towards TB 14

ACCEPTED MANUSCRIPT determination was harvested, showing the higher sensitivity, wider linear range and lower detection limit even down to 6.76 fM. This strategy can be readily expanded for detection of other biomolecules, which shows great potential for practical applications

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in bioanalysis.

Acknowledgment

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This research was supported by National Natural Science Foundation of China

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(No.21475118 and 21675093), Basic Public Welfare Research Project of Zhejiang Province (No. LGG18E010001 and GG19B050003), National Natural Science Foundation of Zhejiang Province (No. Q19B050016), Foundation of Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, Qingdao University of

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Science and Technology (No. SATM201703, STAM201804),the Open Research Fund of Key Laboratory of the Ministry of Education for Advanced Catalysis Materials (Zhejiang Normal University), and PhD Fund of ZJNU for Young Teachers (No.

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zz323205020517002104).

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Captions Scheme 1. Construction processes of the Pd NCs/TPA-based ECL biosensor for TB

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detection.

Fig. 1. Typical low- (A), medium- (B) and high-resolution (C, D) TEM images of Pd

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NCs. The respective survey (E) and high-resolution Pd 3d (F) XPS spectra.

Fig. 2. (A) CV plots obtained in 0.1 M LiClO4 electrolyte without (curve a) and with 10 µM PAMAM (curve b) at 10 mV s-1. (B) CV plots of bare (curve a) and PAMAM-immobilized (curve b) GCE in 0.1 M KCl solution containing 5 mM

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[Fe(CN)6]4-/[Fe(CN)6]3- at 100 mV s-1.

Fig. 3. (A) The ECL-potential curves and (B) CV plots of Pd NCs (curve a) and Pd

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NCs-PAMAM (curve b) coated electrodes in the PBS containing 30 mM TPA.

Fig. 4. (A) CV plots of bare (B) and PAMAM-immobilized GCE in the PBS containing 30 mM TPA at scan rates from 5 to 100 mV s−1. (C) The plot of the anodic peak current versus the square root of scan rate.

Fig. 5. (A) EIS of (a) bare GCE, (b) PAMAM/GCE, (c) Pd NCs/PAMAM/GCE, (d) TBA/Pd NCs/PAMAM/GCE, (e) BSA/TBA/Pd NCs/PAMAM/GCE, and (f) TB/ 24

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Fig. 6. (A) ECL responses by varying the TB concentrations from 0.001 to 1000 pM (curves a-g). (B) The calibration curve between the ECL intensities and the logarithm of the TB concentrations. (C) The consecutive scans of the as-developed Pd NCs/TPA

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system. (D) ECL responses of the aptasensor towards different interferences.

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Figures

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Scheme 1

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Fig. 1 (B)

(A)

(C) 0.225 nm <111>

(D)

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1 nm

60 nm

0.224 nm

100 nm

20 nm

1 nm (F)

N 1s

Intensity / a.u.

O 1s

Pd 3d

S 2p

Pd 3d5/2

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Intensity / a.u.

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200 400 600 800 Binding Energy / eV

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<111>

Pd 3d3/2

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Pd

350

0

Pd

345 340 335 Binding Energy / eV

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Fig. 2

(A)

th

20

5

a

PAMAM-free

0

80 0 -80

-160

0.0

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rd

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Current / µA

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Fig. 3

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Fig. 4

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100 mV s

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60 30

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100 mV s

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ip = 6.45v + 38.68

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Peak current / µ A

(C) 100

0.4 0.8 Potential / V

b

80

a

60

1/2

40

ip = 2.88v 2

+ 33.55

4 6 8 10 -1 1/2 Scan rate (mVs )

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Fig. 5

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400 200

e f

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ECL intensity / *10

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Research Highlights

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► Pd NCs were efficiently functionalized with MCH as a superb ECL emitter.

3

► PAMAM dramatically improved the electron-transfer rates and amplify the ECL

5

intensity of the Pd NCs/TPA system.

► A label-free aptamer biosensor was developed for ultrasensitive TB detection.

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