A closed bipolar electrochemiluminescence sensing platform based on quantum dots: A practical solution for biochemical analysis and detection

Journal Pre-proof A closed bipolar electrochemiluminescence sensing platform based on quantum dots: A practical solution for biochemical analysis and detection Wenyuan Zhao (Conceptualization) (Data curation) (Formal analysis) (Investigation) (Methodology) (Validation) (Writing original draft), Ying Ma (Project administration) (Conceptualization) (Methodology) (Writing - review and editing), Jianshan Ye (Funding acquisition) (Methodology) (Writing - review and editing), Jiye Jin (Funding acquisition) (Methodology) (Writing - review and editing)

PII:

S0925-4005(20)30278-1

DOI:

https://doi.org/10.1016/j.snb.2020.127930

Reference:

SNB 127930

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

29 December 2019

Revised Date:

23 February 2020

Accepted Date:

27 February 2020

Please cite this article as: Zhao W, Ma Y, Ye J, Jin J, A closed bipolar electrochemiluminescence sensing platform based on quantum dots: A practical solution for biochemical analysis and detection, Sensors and Actuators: B. Chemical (2020), doi: https://doi.org/10.1016/j.snb.2020.127930

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A closed bipolar electrochemiluminescence sensing platform based on quantum dots: A practical solution for biochemical analysis and detection

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Wenyuan Zhao a, Ying Ma a,*, Jianshan Ye a,*, and Jiye Jin b,*

College of Chemistry and Chemical Engineering, Key Laboratory of Fuel Cell

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Technology of Guangdong Province, South China University of Technology, Guangzhou 510641, P. R. China

Department of Chemistry, Faculty of Science, Shinshu University, 3-1-1 Asahi,

Corresponding author at: a

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Matsumoto, Nagano 390-8621, Japan

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b

College of Chemistry and Chemical Engineering, Key Laboratory of Fuel Cell

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Technology of Guangdong Province, South China University of Technology, Guangzhou 510641, P. R. China

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E-mail: [email protected] (J. Ye); [email protected] (Y. Ma) Department of Chemistry, Faculty of Science, Shinshu University, 3-1-1 Asahi,

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Matsumoto, Nagano 390-8621, Japan E-mail: [email protected] (J. Jin)

Highlights 

The ECL behaviors of two organic quantum dots (QDs) (CsPbBr3 QDs and CdSe/ZnS QDs) were studied. 1

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A new type of CBP-ECL sensing platform was set up using the QDs-modified electrode as the reporting element in the organic solvent and the sensing electrode in aqueous solution.

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A sensitive ECL sensor for the determination of H2O2 was developed based on the as-prepared CBP-ECL platform.

ABSTRACT Many quantum dots (QDs) are considered as excellent electrochemiluminescence (ECL)

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fluorophores with high quantum yields (QYs) and good stability. However, their ECL efficiency dramatically decreases in the aqueous solution compared to that in organic

phase, which significantly limits their applications in bioanalysis. In this study, we developed a new sensing strategy via the combination of a closed bipolar electrode and

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electrochemiluminescence (CBP-ECL) system, which allows the separation of sensing

in aqueous media and generation of ECL in organic media. Two kinds of QDs,

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CdSe/ZnS and CsPbBr3 were applied as ECL fluorophores in this system, and stable, high QYs ECL signals were achieved. The resulting sensing platform demonstrated

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high sensitivity for the detection of H2O2 with a linear range of 0.02-12 mM and the limit of detection (LOD) of 9.2×10-8 M when CdSe/ZnS QDs were used as ECL

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reagents. We envision that this system can be widely employed in any kind of bioanalysis which can produce the electrochemical currents in the detecting cell. More importantly, we expect this system could break through the limitation of the

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fluorophores, especially those with solvent dependent ECL signals such as aggregationinduced emission (AIE) molecules, or most of the hydrophobic dyes.

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Keywords: CBP-ECL; Quantum dots; H2O2; CdSe/ZnS QDs; CsPbBr3 QDs 1. Introduction Electrochemiluminescence (ECL) is a method of producing a light during

electrochemical reactions in solution or on electrode surface[1-3]. Since its first introduction into analytical fields in 1972, ECL has demonstrated significant merits in numerous sensors such as environmental monitoring[4, 5], food safety[6] and 2

bioanalysis[7-11] thanks to its high sensitivity and wide range of determination[12]. ECL reagents (luminophores) are the key factor to determine the performance of sensors, luminol, tris(2,2′-bipyridyl) ruthenium(II) (Ru(bpy)32+) and their analogs are the mostly used agents. Among them, Ru(bpy)32+ is most frequently used due to its available low oxidation potential, high emission yield, and low cost[13, 14]. Nevertheless, it is still important to develop innovative, stable and highly efficient luminophores for the sensitive, specific and rapid determination. Quantum dots (QDs) are a new type of luminescent fluorophores displaying

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remarkable optical properties, such as high quantum yields (QYs), excellent photochemical stability, size-tunable emission, and low photobleaching [15]. Especially the QDs prepared with organic ligands such as oleylamine (OAm) and tri-noctyl phosphine (TOP) presented excellent optical and chemical stability. In 2002, the

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ECL behavior of semiconductor QDs in the organic phase was reported by Bard[16]. Later, the corresponding aqueous QDs-based ECL sensors have been developed for

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various assays, including immunoassay[17], DNA detection[13], cytosensor[18] and so on[19, 20]. However, the aqueous semiconductor QDs mostly synthesized via ligand

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exchange of hydrophobic QDs or directly synthesized in aqueous phase suffered from the dramatically declined fluorescence QYs and stability[21]. The other example is

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perovskite QDs, their ECL signal in the organic phase can reach up to 5 times higher compared to the Ru(bpy)32+/tri-n-propylamine (TPrA) system[22-24]. They also encountered the stability problem in aqueous phase owing to their ionic nature, which

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leads them to dissociate or degrade in aqueous solution, resulting in the dramatical fluorescence quenching[25, 26]. Recently, aggregation-induced emission (AIE)

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molecules such as hexaphenylsilole (HPS) and tetraphenylethene (TPE) have also been used as sensitive ECL reagents with good performance, they suffered from the same problem like semiconductor QDs and perovskite QDs since their ECL signal dramatically decreases in aqueous phase compared to that in the organic phase[27, 28]. This dramatically restricts their applications in the ECL biosensors as most of the analytes were measured in aqueous solution. Therefore, it is still a big challenge to 3

maintain the ECL efficiency of the fluorophores and simultaneously provide a friendly environment for bioanalysis. The overcome of this problem could greatly broaden the applications of fluorophore species in ECL-based chemical and biological sensors. A bipolar electrode (BPE) is an electrical conductor that promotes electrochemical reactions at its extremities (poles) when it is immersed in one (open BPE) or two solutions (closed BPE)[29]. When a sufficiently high voltage is applied between two driving electrodes, an interfacial potential difference will be generated between BPE and the contacting solution, which leads to the cathodic and anodic overpotentials on

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opposite sides of the same object and triggers faradaic reactions[30]. The charge balance of BPE determines its detection performance because electrochemical signals

on one pole could be collected to indicate the events happened on the other pole[31].

In 2001, ECL was introduced as a reporting tool for BPE and this BP-ECL technique

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immediately attracted great attention in chemical and biosensors[32-36]. The CBP-ECL system allows the separation of the anode and cathode into two physically isolated

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compartments with current efficiency nearly 100% in theory[37, 38]. Recently, Scanlon group developed a thermodynamic framework to understand all possible applications

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of closed bipolar electrochemistry[39]. In particular, they reported the two-phase electron transfer from an organic redox couple to an aqueous redox couple by flowing

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along the BPE when closed bipolar electrochemical cells were loaded with immiscible aqueous–organic solutions. These features provide the possibility to generate the ECL signal in the organic phase and sense in the aqueous phase, which can ensure the strong

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ECL signal as well as a good environment for biosensors. In this study, we proposed a new method to apply the hydrophobic quantum dots

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to the ECL sensing through the combination of BPE and ECL techniques. We first studied the ECL behavior of two kinds of QDs modified electrodes (CsPbBr3 QDs and CdSe/ZnS QDs), illustrating that their ECL properties are poor in aqueous solution while excellent in organic solution in terms of fluorescence intensity and stability. Then we set up a new type of CBP-ECL sensing strategy with the QDs-modified electrode as the reporting electrode in the organic solvent, and the sensing electrode in aqueous 4

solution. Using this CBP-ECL sensing platform, we developed a H2O2 sensor with fast response, low limit of detection (LOD) and broad linear range. 2. Experiment section 2.1. Materials and chemicals Cesium carbonate (Cs 2CO3, 99.99%), lead bromide (PbBr 2, 99.9%), oleic acid (OA, 85%), oleylamine (OAm, 80–90%), octadecene (ODE, >90%), Tri-npropylamine (TPrA, >99%) were purchased from Aladdin (Shanghai, China). Tetra-n-butylammonium hexafluorophosphate (TBAPF 6, 99%) was bought from

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Innochem (Beijing, China). All other chemicals are reagent grade and used as received. CdSe/ZnS QDs (520 nm ± 10 nm) were purchased from Nanjing Mknano Tech. Co. Ltd. Phosphate buffer solution (PBS) (pH = 7.4, 0.1 M) was prepared

by using 0.1 M NaH2 PO4 and 0.1 M Na2HPO4. Ultrapure water obtained from

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EasyQ water purification system was used for all the aqueous experiments (18.25 MΩ•cm-1).

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2.2. Instruments

Scanning electron microscopy (SEM) was performed with a SU 8220 field

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emission scanning electron microscope (Hitachi, Japan). Ultraviolet-visible (UV– vis) absorption spectra were recorded on a UV 2600 spectrophotometer (Shimadzu,

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Japan). Fluorescence (FL) spectra were measured with an LS 55 fluorescence spectrometer (PerkinElmer, America). Electrochemical and ECL experiments were conducted on an ECL-20

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electrochemiluminescence instrument (Guangzhou Ingsens sensor Technology Co. Ltd., China). A three-electrode system was used with a glassy carbon electrode

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(GCE, Φ=3 mm) as the working electrode, a platinum wire as the counter electrode and a Ag/Ag+ as the reference electrode. ECL signals were measured with a photomultiplier tube (PMT) and a biased voltage of 850 V. 2.3. Preparation of CsPbBr3 QDs CsPbBr3 QDs were prepared according to the reported method[40]. Simply, 0.2035 g Cs2CO3, 625 µL OA, and 10 mL ODE were added into a 3-neck flask. After purging 5

with N2 for 0.5 h, the mixture was heated to 150 ºC to form a Cs-oleate solution after the complete reaction of CsCO3 with OA. In another 3-neck flask, 20 mL ODE and 0.267 g PbBr3 were loaded and purged with N2 at 100 ºC for 1 h, followed by the injection of dried OAm (2.0 mL) and OA (2.0 mL). After stirring and heating to 120 ºC under N2 protection, the temperature of the resulting clear solution was adjusted to 150 ºC, followed by the addition of 1.6 mL as-prepared Cs-oleate. The reaction was quenched by an ice-water bath after 5 s reaction to acquire CsPbBr3 QDs. The QDs were precipitated by adding excessive ethyl acetate (EA), and the pellet was collected

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and redispersed in toluene after centrifugation at 8000 rpm for 10 min. The resulting CsPbBr3 QDs were stored under dark conditions for further usage. 2.4. Modification of QDs on GCE

GCE was polished with 0.3 and 0.05 μm alumina slurry respectively,

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subsequently washed with 0.5 M H 2SO4, ethanol and water, and dried with a

stream of N 2 gas. 10 μL 0.01 g/mL CsPbBr 3 QDs solution was dropped onto the

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GCE surface and dried at room temperature to form the CsPbBr 3/GCE. The QDsmodified electrode was immersed in EA for several seconds and dried at room

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temperature. A similar procedure was used to modify CdSe/ZnS QDs on GCE (named as CdSe/ZnS/GCE).

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2.5. Fabrication of the CBP-ECL system

The setup of the CBP-ECL system is shown in Scheme 1. It consists of two electrochemical cells. In the reporting cell, acetonitrile was used as a solvent to generate

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a stable ECL signal. In the detecting cell, the driving electrode is a platinum electrode (Pt, Φ=1 mm), where the target molecule was oxidized to induce a detectable current

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change. The pole 1 of the BPE is a platinum electrode (Φ=2 mm). In the ECL reporting cell, a 10 mL quartz cell was charged with acetonitrile containing 0.05 M TBAPF6 and 10 mM TPrA. The driving electrode is a platinum electrode (Φ=2 mm), and the pole 2 of the BPE is a GCE (Φ=3 mm), where the QDs was oxidized to produce ECL signal. The power supply was connected to the Pt electrode in cell 1 and the other Pt electrode in cell 2. The Pt electrode in cell 1 and the GCE in cell 2 were connected via copper 6

wire to establish the connection and form a BPE. During the electrochemical process, the PMT is facing the bottom of the reporting cell to record ECL signals. 3. Result and discussion 3.1. Synthesis and characterization of QDs CsPbBr3 QDs have a cubic crystal structure. The six-coordinated lead cation forms a PbBr6 octahedron with six bromide ions, and the octahedron is co-pointed to form a three-dimensional structure with the cesium ions embedded in the cavity of the frame (Fig. 1A). The as-prepared CsPbBr3 QDs exhibit a yellow color and green fluorescence

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under UV irradiation (inset of Fig. 1B), and their corresponding first electronic absorption and FL spectra are located at 511 and 512 nm respectively (Fig. 1B). The CdSe/ZnS QDs display their first electronic absorption and FL spectra at 516 and 520

nm respectively (Fig. S1). The bandgap values Eg of CsPbBr3 and CdSe/ZnS QDs were

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calculated based on their absorption and FL spectra according to the empirical formula

Eg =hc /

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as follows[41]:

(1)

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where h is Planck’s constant (4.1357 × 10–15 eV·s), c is the speed of light (2.9979 × 108 m/s), and λ is the wavelength (m) of the maximum absorption and FL spectra. Table S1 shows the corresponding comparison of the two QDs, revealing that their

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luminescent peaks slightly shift from their absorption edge position, which is consistent with the reported Stokes shift effect[42].

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As shown in Fig. S2A, the SEM image of the CsPbBr3 QDs demonstrates the uniform dispersion of QDs on the surface of GCE with a dense coverage and large grain

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size. After the treatment of the modified electrode in an EA solution, the film retained densely-packed morphology, while its grain size significantly decreased (Fig. S2B). This treatment with EA is essential as it can effectively eliminate the agglomeration problem of the degradation encountered by the perovskite QDs solution and remove the weakly absorbed CsPbBr3 QDs from the electrode surface, resulting in a pure and stable CsPbBr3 QDs film with the smaller grain size[24]. 3.2. ECL of QDs-modified electrodes 7

Next, we studied the electrochemical behavior of the QDs-modified electrode in acetonitrile. Fig. 2A shows the CV profiles acquired on the three-electrode system, TPrA displays a strong oxidation current on a bare GCE with an onset potential of +0.65 V and the peak potential of +0.98 V (curve a). The CsPbBr3/GCE shows a relative weak oxidation current without TPrA (curve b). However, in the presence of TPrA, the CsPbBr3/GCE presents a distinct oxidation signal with an onset potential of +0.80 V and peak potential of +1.18 V (curve c). The oxidation peak of TPrA at +0.98 V is hardly observed on CsPbBr3/GCE owing to the hindrance effect of CsPbBr3 QDs film

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to inhibit the oxidation of TPrA on GCE. As shown in Fig. 2B, no ECL signals were captured on the bare electrode or CsPbBr3/GCE in the absence of TPrA (curve a, curve

b), while an intense ECL signal was recorded on CsPbBr3/GCE in the presence of TPrA (curve c), revealing that TPrA is crucial as a co-reactant for the generation of ECL,

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which is similar to that of Ru(bpy)32+/TPrA. Notably, both the onset potential and the peak potential of of ECL are slightly lagging behind that of CV curve (onset potential

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+0.88 vs +0.80 V, and peak potential +1.26 vs +1.18 V). This retardation is ascribed to the accumulation of positive charges within the CsPbBr3 QDs film at a positive

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potential, and the high current density is unfavorable for radiative charge transfer and electric-field-induced light emission[43, 44]. Based on these observations, the ECL

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mechanism is proposed as follows:

(2)

TPrA-e  [TPrA ]  TPrA  H+

(3)

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CsPbBr3 -e  [CsPbBr3 ]+

CsPbBr3 +TPrA  [CsPbBr3 ]-

(4)

[CsPbBr3 ]+ +[CsPbBr3 ]-  [CsPbBr3 ]*

(5)

[CsPbBr3 ]*  [CsPbBr3 ]  hv

(6)

And the following route is also possible: [CsPbBr3 ] +TPrA  [CsPbBr3 ]*  Product

(7)

During the positive scanning process, CsPbBr3 is oxidized to form [CsPbBr3]+. 8

Simultaneously, the oxidation of TPrA generates TPrA•+ and subsequently undergoes a deprotonation process to produce TPrA•, which combines TPrA• with CsPbBr3 to form the negatively charged [CsPbBr3]-. The reaction of [CsPbBr3]- with [CsPbBr3]+ yields the excited state [CsPbBr3]*. Eventually, a strong ECL signal is generated when [CsPbBr3]* relaxes to its ground state via a radiative pathway. The stability and reproductivity of ECL are dependent on the oxidation potential applied. A very stable ECL signal was acquired when the potential window was set between 0 and 1.0 V, while its signal intensity suffered from quick drop under the even

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higher oxidation potentials (Fig. S3), especially its intensity drops to 11.12% after 50 times of scan when the oxidation potential is as high as 1.3 V. This oxidation potentialdependent ECL behavior is caused by the over oxidation of CsPbBr3 QDs, which may damage the sample owing to its irreversible process[24].

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The ECL behavior of CdSe/ZnS QDs is similar to that of CsPbBr3 QDs, while its signal is stronger with higher stability. As shown in Fig. 3B, CdSe/ZnS/GCE displays

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a strong ECL signal with an onset potential at +0.87 V and peak potential at +1.16 V (curve c) and only 3.51% signal decline after 90 times of scan, revealing its outstanding

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stability (Fig. 4). Besides, the solvent used for ECL generation is also crucial to the ECL performance, and the nonpolar solvent is favorable for the ECL, very weak or

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invisible ECL signals were observed when polar solvent such as PBS was used as a media (Fig. S4A). This is reasonable as the surface ligand of QDs is highly hydrophobic OAm and OA, their presence significantly blocks the electron transfer between the

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electrode and the electrolytes in hydrophilic media, preventing the generation of ECL signal. These results clearly indicate that the application of hydrophobic QDs as ECL

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fluorophores for the detection in the aqueous phase is limited. Table S2 shows the detailed CV and ECL behaviors of CsPbBr3/GCE and CdSe/ZnS/GCE in acetonitrile containing 0.05M TBAPF6 during the anodic process. 3.3. The ECL of QDs via a CBP-ECL system Since the CBP-ECL provides the possibility for the generation of ECL and sensing in two different cells, we set up a CBP-ECL system. When a uniform electric field 9

across the solution by applying a voltage between two driving electrodes, the bipolar electrode exhibits two distinct poles of opposite polarization with respect to the solution. This allows the separation of the sensing elements from the reporting species. Therefore, we can utilize organic solvents such as acetonitrile and ethyl acetate in the reporting cell, where the QYs of the QDs can be maintained. In the detecting cell, biocompatible PBS as the media is favorable for biomolecular detection. As shown in Fig. 5, a strong bipolar ECL signal was observed when CdSe/ZnS/GCE was scanned in the potential range of 0-4.2 V. Although this signal is

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slightly lower than that using the three-electrode system in the organic solvent, its intensity is much stronger than that of ECL acquired in PBS. The ECL behavior of

CsPbBr3 QDs presents similar results (Fig. S5). These findings demonstrate that the application of CBP-ECL system can significantly improve the ECL efficiency and

decomposition in aqueous solution.

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3.4. Detection of H2O2 via a CBP-ECL system

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avoid the signal decline caused by the hydrophobic properties of QDs or their possible

The sensing mechanism of H2O2 based on CBP-ECL system is shown in Scheme

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1. In the detecting cell, the oxidation of H2O2 on electrode 1 generates an electric current, and the concentration of H2O2 determines its intensity. In the reporting cell, QDs were

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oxidized to produce an ECL signal on the pole 1 of BPE, which intensity is dependent on the corresponding electrochemical signal generated in the detecting cell of the closed BPE system. Therefore, an ECL sensing strategy of H2O2 can be established.

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Fig. 6A presents that the ECL intensity gradually increases with the increased concentration of H2O2 when CdSe/ZnS/GCE was used as a reporting electrode. A good

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linear range of 0.02-12 mM with R2 of 0.993 was achieved (Fig. 6B), and the corresponding limit of detection (LOD) was calculated to be 9.2×10-8 M (S/N=3). A linear range of 0.1 to 10 mM, an R2 of 0.998 and the corresponding LOD of 1.56×10-7 M (S/N = 3) were acquired using CsPbBr3/GCE as the ECL reporting electrode (Fig. S6). The LOD and linear range is not good as some of the reported electrochemical sensors for H2O2 detection, however, we believe that its performance can be greatly 10

improved if the sensing electrode was modified some materials to generate strong oxidation current during the H2O2 oxidation, such as graphene/carbon nanotube, the metallic nanomaterials or even the biomolecules. The research in this direction is still under investigation. 4. Conclusions In summary, we studied the ECL behavior of two hydrophobic QDs and illustrated that the solvent and oxidation potential were the key factors to determine their ECL intensity and stability. Based on these findings, a CBP-ECL platform was set up using

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QDs as ECL fluorophores. Two separated cells containing two different solvents enabled the excellent ECL signals in an organic solvent and the electrochemical sensing

in the aqueous solvent. The determination of H2O2 with broad linear range and low

LOD confirmed the applicability of this sensing strategy. More importantly, this method

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can be widely applied for any kind of ECL fluorophores, especially those with solvent

dependent ECL behavior such as AIE dyes. Although we only proved the feasibility of

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the aqueous/organic phase in CBP-ECL, the possibility of an organic/organic phase can also be expected. Therefore, such a system can be widely applied for the detection in

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the organic phase or aqueous phase, and the generation of ECL in organic/aqueous phase as well.

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credit author statement

Wenyuan Zhao: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Validation; Writing - original draft

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Ying Ma: Project administration; Conceptualization; Methodology; Writing - review & editing.

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Jianshan Ye: Funding acquisition; Methodology; Writing - review & editing. Jiye Jin: Funding acquisition; Methodology; Writing - review & editing.

Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments This work was supported by the Science and Technology Program of Guangdong

Province

(Nos.

2019A1515011983,

2019B020219002,

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2018A050506006), and the National Natural Science Foundation of China (Nos.

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21875070, 21974052).

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Author Biographies Wenyuan Zhao received his BS degree in Chemical Engineering and Technology from Xi’an University of Architecture & Technology (XAUAT), Shanxi, China. He is now pursuing his Master degree in Chemical Engineering in the College of Chemistry and Chemical

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Engineering at South China University of Technology (SCUT) under the supervision of Prof. Jianshan Ye. His research is mainly focused on electrochemiluminescence sensors.

Ying Ma is currently an associate professor in the College of Chemistry and Chemical

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Engineering at SCUT, Guangzhou, China. He received his Ph.D. from State Key Lab of

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Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy

electrochemical biosensors.

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of Sciences, Changchun, China. His research interest is in nanomaterials, optical and

Jianshan Ye is currently a professor in the College of Chemistry and Chemical Engineering

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at SCUT, Guangzhou, China. He received his Ph.D. from Hong Kong University of Science

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& Technology, Hong Kong, China. He has already published more than 50 high-quality research papers and has mentored 17 Ph.D. students. His current interest is in the fields

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of electrochemistry.

Jiye Jin is currently a tenured professor, in the Department of Chemistry at Shinshu University, Nagano, Japan. He received his Ph.D. from Nagoya University, Nagoya, Japan in 1993. He is an expert in new methods and instruments for electrochemical analysis.

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Figure captions Scheme 1. CBP-ECL: the separated configuration between reporting and sensing sites allow the use of different solvents in two cells.

Fig. 1. (A)The schematic cubic crystal structure of CsPbBr 3 QDs. (B)Fluorescence and UV-vis absorbance spectra of CsPbBr 3 QDs dispersed in hexane. The inset

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shows photographs of CsPbBr 3 QDs under visible and UV light.

Fig. 2. (A) CV and (B) ECL of (a) bare GCE and (b, c) CsPbBr 3 QD|GCE in 0.05M TBAPF6-acetonitrile electrolyte, containing (a, c) 10.0 and (b) 0.0 mM TPrA at

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0.05V/s with a potential window between 0.0 V and 1.4V.

Fig. 3. (A) CV and (B) ECL of (a) bare GCE and (b, c) CdSe/ZnS QD|GCE in 0.05

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M TBAPF6-acetonitrile electrolyte, containing (a, c) 10.0 and (b) 0.0 mM TPrA at

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0.05 V/s with a potential window between 0.0 V and 1.2 V.

Fig. 4. (A)The interception of the ECL-time curve of CdSe/ZnS QD film with a

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potential window between 0.0 V and 1.2V at 0.2 V/s (B) Relation ECL attenuation curve of Cyclic test.

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Fig. 5. ECL testing of CBP-ECL system: (A) ECL and (B) CV of bare GCE and CdSe/ZnS QDs|GCE and 0.05 M TBAPF 6-acetonitrile electrolyte containing 10.0

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mM TPrA in reporting cell and 0.1 M PBS in detecting cell at 0.1 V/s with a potential window between 0.0 V and 4.2 V.

Fig. 6. (A)CBP-ECL profiles as a function of H 2O2 concentration in 0.1 M PBS (pH=7.4) and (B)calibration curve. PMT was biased at 850 V. Error bars were obtained from three experiments. 18

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