A bipolar electrochemiluminescence sensing platform based on pencil core and paper reservoirs

Abstract

In this work, a novel closed bipolar electrochemiluminescence (ECL) sensing platform based on a commercial pencil core and paper reservoirs was described for the first time. A pencil core functioned as a bipolar electrode and the Ru(bpy)₃²⁺/(NH₄)₂C₂O₄ was used as the electrochemical reaction system to generate the ECL signal in existing model targets.

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Electrochemiluminescence (ECL) is an energy-relaxation process, where electrochemical species are stimulated by electricity and then undergo electron-transfer reactions at an electrode surface to realize light emitting and luminescence imaging from an excited photon-emitting state. Notably, the ECL as a highly localized and time-triggered detection method has shown great importance for chemical and biological analysis due to its versatility, sensitivity, perfect temporal and spatial control.^1–3 Bipolar electrochemistry is a rather new technique with attractive features for application in the field of analytical electrochemistry.^4–6 It allowed analyte separation and detection without direct external connection that realizing the development of analytical chemistry.⁷ Using bipolar electrode (BPE), it has become a powerful tool for analysis, biosensing and screening of electrocatalysts.^8–12

A bipolar electrode is an electronic conductor with external electric supply,¹³ which can be performed in open cell that bipolar electrode is immersed into one solution or the close cell that composed of two half cell bridged by the bipolar electrode in two separated solutions.¹⁴ In comparison with traditional three electrode electrochemistry that obsesses with the electrochemical reactions at the working electrode,^15–17 the bipolar electrochemistry concentrates on two coupled redox reactions that are generated at two opposite poles.¹⁸ The ECL biosensor based on BPE with wireless contact, simplifies the experiment operation and offers the merits on economic reagent consumption, sensitivity and portability.^19,20 Given its excellent characteristics, the BPE-ECL sensing platform has been increasingly concerned since Manz's group proposed bipolar ECL in 2001.²¹ At the very beginning, the detection of analytes were confined to the coreactant or quencher related to the ECL system using this method. Then Crooks and co-workers demonstrated that any electroactive analytes can be detected based on the quantitative relationship between the reactions occurring at both poles of bipolar.²² Recently, the indium tin oxide (ITO) coated glass as bipolar or substrate was widely proposed in various electrochemistry due to the effective and superior transparency^23–26 For instance, Zhang's group constructed a large parallel bipolar array in 2013, which achieved screening image on microelectrodes regarding transient and heterogeneous electrochemical progresses.²⁷ Although each approach exhibited good performance on bipolar, there are several challenges including the fabrication of thousands bipolar array and the construction of addressable microelectrodes.²⁸ Meanwhile, the wet chemical acid etching process of ITO is tedious, complicated, time-consuming and high-cost.²⁹ Furthermore, the reduction of SnO₂ occurred at the BPE surface when the applied driving voltage was enough high, resulting the damage of ITO and then causing the instability of the output signals. This problem was always ignored in previous research.³⁰

To achieve stable and low-cost BPE-ECL detecting platform, here, we introduce a common and ubiquitous material pencil core to replace the conventional ITO. Compared to ITO and other conducting substrate, pencil core is more facile, simple and disposable which can be used as a substitute for the prospective bipolar electrode (Fig. S1†). On basis of this, it was not only a novel style of closed bipolar sensing platform but also eliminated the self-reduction of ITO as bipolar. This portable and applicable BPE-ECL sensing platform has been illuminated as Scheme 1.

Scheme 1 Structure and operational mechanism of BPE-ECL sensing platform based on pencil core-paper reservoirs.

The anodic and cathodic poles of pencil core are in two separated paper cells and the pencil core regards as bridge to connect different cells. The conventional electrolysis cells have been replaced by the paper reservoirs that connect the bipolar to form a complete circuit.

As shown in Scheme 1, the structure and operating mechanism of the BPE-ECL sensing platform based on pencil core-paper cells, which consist of four parts: pencil core, hydrophilic cell, hydrophobic wax and glass slide. The hydrophilic round cells were obtained from patterned chromatographic paper (6 mm radius for sensing and reporting cell, respectively) and pencil core acted as electronic conductors, connecting two above-mentioned hydrophilic cells. The existing hydrophobic wax was used to anchor the pencil core, and also as boundaries for reservoirs. Then this sensing platform with pencil core and paper-cells were placed on a glass slide, and the analytic solution and ECL reagent solution was dropped into the paper reservoirs, respectively. The Ru(bpy)₃²⁺ was chosen as ECL luminophore together with its co-reactant (NH₄)₂C₂O₄. Under optimal conditions, the proposed dual BPE-ECL imaging/amperometric sensing platform was successfully implemented for detecting model analytes such as K₃[Fe(CN)₆] and H₂O₂. Apparently, this disposal sensor based on pencil core and paper reservoirs can expand its practical applications in medical diagnosis and environmental protection. The sensing principle was illustrated by using the following equation:

ΔE_elec = (E_tot/2x) × (l₁ + l₂)

here, l₁ and l₂ represent the length of cathode and anode, respectively, that the part of pencil core electrode soaking in the solution cells. While x means the distance of driving electrode towards the intersection of bipolar electrode and paper cells. When a certain voltage is applied between two driving electrodes (E_tot), a linear gradient of potential drop will be formed on the bipolar electrode with electrolyte solution. On account of linear potential gradient, potential difference across the bipolar electrode (ΔE_elec) and the over-potential on anode and cathode will exist. When ΔE_elec is high enough, the oxidation–reduction reactions are triggered at both poles. Given no electron accumulation, two couple redox reactions occurred at sensing and reporting cell simultaneously. Basically, the ECL reaction mechanism of Ru(bpy)₃²⁺/(NH₄)₂C₂O₄ on BPE was listed as follows,

Ru(bpy)₃²⁺ − e → Ru(bpy)₃³⁺

Ru(bpy)₃³⁺ + C₂O₄²⁻ → Ru(bpy)₃²⁺ + C₂O₄²⁻˙

Ru(bpy)₃³⁺ + CO₂⁻˙ → [Ru(bpy)₃²⁺]˙ + CO₂

or

Ru(bpy)₃³⁺ + CO₂⁻˙ → Ru(bpy)₃⁺ + CO₂

Ru(bpy)₃⁺ + Ru(bpy)₃³⁺ → [Ru(bpy)₃²⁺]* + Ru(bpy)₃²⁺

C₂O₄²⁻˙ → C₂O₄⁻˙ + CO₂

[Ru(bpy)₃²⁺]* → Ru(bpy)₃²⁺ + hv

Here, specifically, the effects of the length and diameter of pencil core were investigated on the above BPE-ECL sensing platform. The cathodic cell was injected with 150 μL of 10 mM K₃[Fe(CN)₆] in 0.1 M PBS (pH 7.0) and the anode cell was filled with 150 μL of 3.0 mM Ru(bpy)₃²⁺ and 30 mM (NH₄)₂C₂O₄ in 0.1 M PBS (pH 7.0). ECL signals were recorded by photomultiplier tube (PMT). Fig. 1A showed the effect of the diameters of pencil cores on the ECL response.

Fig. 1 (A) The BPE-ECL performances with different diameters of pencil cores from 0.3 mm to 0.9 mm (a–d) in the sensing cell (0.1 M PBS with 10 mM K₃[Fe(CN)₆]) and in the reporting cell (0.1 M PBS with 3 mM Ru[(bpy)₃]²⁺ and 30 mM (NH₄)₂C₂O₄). PMT was biased at 500 V. (B) The BPE-ECL performances with different lengths of pencil cores immersed in the solution from 1–5 mm (a–e), the ECL measurement was carried out as same as above.

To study the effect of bipolar electrode diameters, four pencil cores with different diameters, namely 0.3, 0.5, 0.7 and 0.9 mm were used. The ECL intensity increases with the increment of the diameter from 0.3 mm to 0.9 mm (curve a to d) due to the increment of bipolar electrode surface area. Considering that the pencil cores with the diameter of 0.5 mm and 0.7 mm are common commodity, the pencil core with the diameter of 0.7 mm was chosen for the following experiment. Fig. 1A also showed the typical curves of ECL intensity vs. potential of E_tot applied on the driving electrodes from 1.5 V to 3.9 V. The onset potential of ECL located at 2.5 V and the ECL intensity increased with the increasing of E_tot from 2.5 V to 3.7 V and with the further increasing E_tot, the ECL intensity began to decrease. Note that the E_tot dropped at bipolar electrode, namely ΔE_elec is vital to the ECL response. The length of pencil core immersed in the solutions (l₁ and l₂) greatly influences the final ECL behaviors in the reporting cell. As shown in Fig. 1B, when l₁ and l₂ increased from 1 mm to 5 mm, the onset potential of ECL decreased with the increment of l₁ and l₂ (in our experiment, l₁ keeps equal to l₂). These results are consistent with the sensing principle above. The ΔE_elec drives the faradaic process occurring on the wireless electrochemical reaction. Under certain potential gradient, longer pencil core bipolar electrode undertook higher partial potential, leading to the lower ECL onset potential and stronger ECL intensity.

To expand our ECL-BPE sensing platform for bioassay, hydrogen peroxide (H₂O₂) was chosen to be detected as a model biochemical reagent since H₂O₂ is not only a product of oxidase but also a substrate of peroxidase. Considering that the electrochemical reduction of H₂O₂ at carbon electrode requires high over-potential with low reaction rate, platinum nanoparticles (Pt NPs) were electrodeposited onto the pencil core to electro-catalyze the reduction of H₂O₂. Signal amplification was achieved by modifying Pt NPs, while only a weak ECL was observed on the naked pencil core. The electrodeposition of Pt NPs on the cathodic pole of pencil core was achieved by bipolar electrochemistry. Fig. 2A was the ECL intensity–potential responses towards 5 mM fresh H₂O₂ without (curve a) and with (curve b) Pt NPs. The existing of Pt NPs represented excellent conductivity, large apparent surface area to facilitate the electro-reduction of H₂O₂. The morphology of Pt NPs onto pencil core was characterized by SEM (Fig. 2B) and EDAX (Fig. S1†). From Fig. 2B and S2,† the Pt NPs have been typical anchored and distributed well on the surface of pencil core. Accordingly, the initial ECL potential decreased about 1.0 V, indicating the easier generation of ECL signal at lower E_tot. The bipolar electrodeposited time of Pt NPs was optimized (Fig. S3†). Further prolonging electrodeposition time, the ECL intensity declined. This phenomenon maybe attribute to the destruction of platinum nanostructures with extending electro-deposited time. Moreover, due to the notable electrocatalysis of Pt NPs to H₂O₂ and the enlarged electrode surface area, the ECL signal was greatly amplified. For the Pt NPs modified pencil core-paper cells sensing platform, the driving voltage was also investigated for the measurement of H₂O₂. When the driving voltage was below 2.3 V, the ECL intensity was not obvious, while the ECL response enhanced dramatically with the driving voltage from 3.5 V to 4.0 V. Since high voltage could induce background reactions that interfere with ECL emission.³¹ Therefore, 3.8 V was chosen for driving voltage to detect H₂O₂. It should be noticed that the dissolved oxygen might be reduced at cathode that interfering the detection of H₂O₂, so the solution should be prepared with saturated nitrogen to eliminate the interference for further ensuring the sensitivity and stability of this sensing platform. The concentration of H₂O₂ was correlated with the ECL signals allowing the establishment of calibration curves. With the increasing of H₂O₂ concentration, the ECL intensity became stronger gradually (Fig. S4†). Furthermore, as shown in Fig. 3A, the ECL behavior of the Pt NPs modified pencil core-paper cells BPE-ECL platform was quite stable. With this ingenious device, the analysis of H₂O₂ was achieved by naked eyes (Fig. S5†). The ECL images were captured the anodic pole of pencil core electrode in the concentration range from 1 mM to 500 mM (Fig. 3B). In accordance with increasing the concentration of H₂O₂, the ECL images changed from faint to legible in the ECL solution containing 10 mM Ru(bpy)₃²⁺ and 100 mM (NH₄)₂C₂O₄. Fig. 3C exhibited the corresponding derived calibration curve relating to concentration of H₂O₂ in the range from 10⁻² M to 10⁻⁸ M (R² = 0.991) with the limit of detection of 3.2 × 10⁻⁹ M (S/N = 3). As shown in Fig. S6,† the durability, stability and an accepted reproducibility between pencil cores were obtained, and the relative standard deviation was 3.78% (N = 5) when the concentration of H₂O₂ was 1.0 × 10⁻⁴ M. The above result showed that the dual imaging/amperometric bipolar electrochemical chip possessed outstanding sensitivity for the targets of detection. Thus, the mass production, stability and disposability of this sensing platform may be forming a promising technology for chemical and biochemical analysis.

Fig. 2 (A) The ECL intensity–potential responses towards 5 mM H₂O₂ without (a) and with Pt NPs (b). (B) SEM image of Pt nanoparticles on the bipolar electrode.

Fig. 3 (A) Typical ECL-time signals of 0.7 mm bipolar electrode/Pt NPs in 0.1 M PBS (pH 7.0) that containing 1.0 × 10⁻⁴ M H₂O₂ with a scan rate of 100 mV s⁻¹. PMT was biased at 600 V. (B) ECL images of the ECL-BPE sensing platform for various concentrations of H₂O₂: 1 mM, 5 mM, 50 mM, 100 mM, 200 mM, 500 mM (from left to right). (C) ECL profiles as a function of H₂O₂ concentration in 0.1 M PBS (pH 7.0) and calibration curve with 0.7 mm pencil core as bipolar electrode. PMT was biased at 600 V.

Conclusions

In summary, a very simple, portable and stable BPE-ECL sensing platform has been successfully fabricated using commercial pencil core and paper cells. A model analyte, K₃[Fe(CN)₆] has been used to prove the feasibility of this sensing platform and optimize the sensing conditions. Meanwhile, the introduction of Pt NPs onto the BPE-ECL system leads to the successful detection of H₂O₂ at low potential, which expands its application in bioanalysis. Obviously, it might be feasible to be used as detectors by combing with flowing separation techniques such as electrophoresis, microchip electrophoresis, HPLC and etc. Or it might be expanded as multi-array BPE-ECL system for high-throughput multi-components analysis.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21575022, 21535003, 21375006), the National High Technology Research and Development Program (“863” Program) of China (2015AA020502), the Open Research Fund of Key Laboratory of Energy Thermal Conversion and Control of Ministry of Education, Southeast University, and the Fundamental Research Funds for the Central Universities.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra01460g

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