Advances in bipolar electrochemiluminescence for the detection of biorelevant molecular targets

Bipolar electrochemistry (BPE) contrasts very much with conventional electrochemistry because it is based on the control of the solution potential instead of the working electrode potential. In a typical setup, a piece of conducting materials is immersed in an electrolyte and submitted to an electric field. Such conditions split the interfacial nature of the materials into cathodic and anodic domains where electrochemical reactions can readily take place. BPE has many potential applications, and the present contribution aims to focus on recent analytical applications that involve electrogenerated chemiluminescence (ECL) detection. ECL is a special case of luminescence where the excited state of the luminophore is populated after a sequence of reaction that is triggered by an initial electron transfer step occurring at the electrode surface. The coupling between BPE and ECL is a powerful approach because it provides a unique opportunity to combine the intrinsic advantages of both techniques. BPE enables the spatial separation of sensing and reporting poles, whereas ECL provides a simple and sensitive visual readout. This opinion article will describe the experimental possibilities and the most recent applications of BPE/ECL coupling for the detection of biorelevant molecular targets.     

Design of an electrochemiluminescence detection system through the regulation of charge density in a microchannel

Rare electrochemiluminescence (ECL) sensors have been developed based on the direct regulation of ionic current because it is difficult to establish a relationship between ionic current and ECL reporting. Ionic current can be adjusted by the effective radius and charge density of a functionalized microchannel and is frequently adopted to develop electrical sensors. Here, we show a novel ECL sensing platform that combines the microchannel-based electrical sensing technology with an ECL reporting system for the first time. The target regulated the effective radius and charge density of a microchannel which in turn adjusted the ionic transport in it and finally caused the change of ECL reporting of a tris(1,10-phenanthroline)ruthenium(ii)/tripropylamine system. The developed system has already been applied to detect aflatoxin B1 for demonstration. This configuration separated the target sensing and reporting reactions to achieve direct regulation of ECL reporting by ionic current and expanded the application of the ECL detection technology to microanalysis.

The microchannel-based target sensing and ECL reporting reactions were combined for the first time. This configuration separated the sensing and reporting reactions to achieve ionic current direct regulating ECL reporting.     

Flexibly regulated electrochemiluminescence of all-inorganic perovskite CsPbBr3 quantum dots through electron bridge to across interfaces between polar and non-polar solvents

All-inorganic perovskite quantum dots (QDs) have attracted great interests due to its outstanding properties. But their poor stability in polar solvents seriously hampered wide applications in analytical chemistry. In this work, strong, stable and flexibly regulated the electrochemiluminescence (ECL) emission form CsPbBr₃ QDs was successfully obtained and applied in the analysis of polar solvents through the unique structure of closed bipolar electrode (BPE). To demonstrate the feasibility, it was successfully used in the detection of tetracycline (Tc) aqueous solution. CsPbBr₃ QDs was immersed into organic solution in anode microcell of closed BPE while Tc aqueous solution was added into cathode microcell. The two microcells were physically separated and would not interfere with each other. But the bio-recognition event between aptamer and Tc in cathode microcell would induce the ECL signal change in anode microcell through the electrons conducted by BPE as the bridge. The ECL emission can be flexibly regulated by environmental factors of both polar and non-polar solvents and the interface status of the BPE. Compared with traditional methods to overcome the intrinsic instability in polar medium, the reported method does not need any further surface modifications, has no limitations on the targets and can provide wide development space for further deep research, which may open a new direction for the ECL sensing of CsPbBr₃ QDs.     

Digital versatile disc bipolar electrode: A fast and low-cost approach for visual sensing of analytes and electrocatalysts screening

This work represents a new, extremely low cost and easy method for fabrication of bipolar electrode (BPE) for rapid and simultaneous screening of potential candidates for electrocatalytic reactions and sensing applications. Our method takes advantage of the silver reflective layer deposited on already available recordable digital versatile disc (DVD-R) polycarbonate substrate which acts as BPE. Oxidation of the reflective layer of the DVD-R in anodic pole of the BPE results in a permanent and visually measurable dissolute length. Therefore, one could correlate the electrocatalytic activity of the catalyst at the cathodic pole of the BPE, as well as the concentration of analyte in the solution, to the dissolution length of the BPE. To illustrate the promising applications of this new substrate as BPE, p-benzoquinone (BQ) and hydrogen peroxide were tested as model targets for the sensing application. Moreover, in order to show the feasibility of using DVD BPEs for screening applications, the electrocatalytic activity of Pt, Pd, Au, and pristine DVD substrate toward hydrogen evolution reaction (HER) were compared using an array of BPEs prepared on DVD substrate.     

Bipolar Electrochemiluminescence Imaging: A Way to Investigate the Passivation of Silicon Surfaces

This work depicts the original combination of electrochemiluminescence (ECL) and bipolar electrochemistry (BPE) to map in real-time the oxidation of silicon in microchannels. We fabricated model silicon-PDMS microfluidic chips, optionally containing a restriction, and monitored the evolution of the surface reactivity using ECL. BPE was used to remotely promote ECL at the silicon surface inside microfluidic channels. The effects of the fluidic design, the applied potential and the resistance of the channel (controlled by the fluidic configuration) on the silicon polarization and oxide formation were investigated. A potential difference down to 6 V was sufficient to induce ECL, which is two orders of magnitude less than in classical BPE configurations. Increasing the resistance of the channel led to an increase in the current passing through the silicon and boosted the intensity of ECL signals. Finally, the possibility of achieving electrochemical reactions at predetermined locations on the microfluidic chip was investigated using a patterning of the silicon oxide surface by etched micrometric squares. This ECL imaging approach opens exciting perspectives for the precise understanding and implementation of electrochemical functionalization on passivating materials. In addition, it may help the development and the design of fully integrated microfluidic biochips paving the way for development of original bioanalytical applications.     

A Resettable Keypad Lock with Visible Readout Based on Closed Bipolar Electrochemistry and Electrochromic Poly(3‐methylthiophene) Films

A closed bipolar electrode (BPE) system was developed with electrochromic poly(3‐methylthiophene) (PMT) films electropolymerized on the ITO/rGO electrode as one pole of BPE in the reporting reservoir and the bare ITO electrode as another pole of BPE in the analyte reservoir, in which rGO represents reduced graphene oxide. Under a suitable driving voltage (V_tot), the electrochemical reduction/oxidation of electroactive probes, such as H₂O₂/glutathione (Glu), in the analyte reservoir could induce the reversible color change of PMT films in the reporting reservoir between blue and red. Based on this, a keypad lock with H₂O₂, Glu, and V_tot=?3.0 V as the three inputs and the color change of PMT films as the visible output was established. This system was easily operated and did not need to synthesize the complex compounds or DNA molecules. The security system was easy to reset and could be used repeatedly.     

Electrochemical sensing in microfluidic systems using electrogenerated chemiluminescence as a photonic reporter of redox reactions

This paper describes a microfluidics-based sensing system that relies on electrochemical detection and electrogenerated chemiluminescent (ECL) reporting. The important result is that the ECL reporting reaction is chemically decoupled from the electrochemical sensing reaction. That is, the electrochemical sensing reaction does not participate directly in the ECL process, but because electrochemical cells require charge balance, the sensing and ECL reactions are electrically coupled. This provides a convenient and sensitive means for direct photonic readout of electrochemical reactions that do not directly participate in an ECL reaction and thus broadens the spectrum of redox compounds that can be detected by ECL. The approach can be implemented in either a two-electrode or bipolar (single-electrode) configuration. By manipulating the placement and dimensions of the conductors, the photonic response can be enhanced. The system is used to electrochemically detect benzyl viologen present in solution and report its presence via Ru(bpy)(3)(2+) (bpy = 2,2'-bipyridine) luminescence.     

Electrochemiluminescent detection of the hybridization of oligonucleotides using an electrode modified with nanocomposite of carbon nanotubes and gold nanoparticles

We report on an electrochemiluminescent (ECL) sensing technique for the detection of the hybridization between oligonucleotides. A glassy carbon electrode was first functionalized with a composite prepared from gold nanoparticles and carbon nanotubes, and a sensor was then constructed by immobilizing the probing oligonucleotide. The ECL of luminol acts as the sensing signal. It is quenched, to a different degree, by the hybridized double strands of the oligonucleotide depending on the match status. The slope of the ECL response as a function of the status of hybridization drops with increasingly matched hybridization. The response is attributed to the interaction between luminol and the strands of oligomers, and also related to the reduction of reactive oxygen species.

Spatially-resolved multicolor bipolar electrochemiluminescence

Here we describe a new aspect of multicolor potential-resolved electrochemiluminescence (ECL) based on bipolar electrochemistry (BPE). BPE involves a potential gradient established along a polarized conducting object which thus acts as a bipolar electrode (BE). The resulting driving force can induce electron-transfer reactions, necessary for processes such as ECL occurring at different longitudinal locations along the same BE. In this work, we exploit the entire spatial domain where anodic polarization occurs to demonstrate, for the first time, how the potential gradient along a BE may be used to simultaneously resolve the emissions of ECL-active luminophores with differing oxidation potentials. The control of both size and position of the ECL-emitting domains was achieved by tuning the applied electric field. Multicolor light-emission was analyzed in detail to demonstrate spatial and spectral resolution of a solution containing different emitters.     

双极电极-电致化学发光技术在生物分析中的应用

在微管道两端施加一定的电压时,置于这个电场中的金属或半导体带与溶液的界面电势差达到一定的数值后,就会引起溶液电活性物质在其两端发生氧化还原反应,该金属或半导体带即称为双极电极（BPE）. BPE与电致化学发光（ECL）技术的完美结合有诸多优点,如浓度富集、灵敏度高、成本低、装置轻便且不需要外加光源等,极适合生物分析检测. 本文综述了双极电极-电致化学发光技术在生物分析中的应用,并展望相关发展趋势.     

Control of Interfacial Potentials and Redox Reactions on Bipolar Electrodes Using Ag/AgCl

The interfacial potential difference on the surface of bipolar electrodes was controlled by placing Ag/AgCl on part of the electrode. Oxygen reduction on the cathodic pole was coupled with an electrochemiluminescence (ECL) reaction on the anodic pole. In an open bipolar system, the ECL intensity depended on the location of Ag/AgCl and the concentration of Cl⁻ ions. A current flowed through Ag/AgCl and the ratio of currents generated at the anodic and cathodic poles was affected by the position of Ag/AgCl. Further, the effect of Ag/AgCl placement was also demonstrated in a closed bipolar system using hydrogen peroxide (H₂O₂) and glucose as analytes. Ag/AgCl was also effective in adjusting the sensitivity to these analytes to achieve the best performance. This method of interfacial potential control is expected to contribute toward the development of reliable sensing devices and applications such as redox cycling, which require precise potential control.     

Highly stable ECL active films formed by the electrografting of a diazotized ruthenium complex generated in situ from the amine

The electrodeposition of the electrochemiluminescent (ECL) ruthenium complex, bis(2,2'-bipyridyl)(4'-(4-aminophenyl)-2,2'-bipyridyl)ruthenium(II), [Ru(bpy)(2)(apb)](2+), via the in situ formation of a diazonium species from aqueous media is reported. Surface characterization undertaken using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) determined that the layer is bound to the substrate via azo bonding. The layer displays good ECL activity and is stable over a long period of time. The excellent potential of this system for ECL sensing applications is demonstrated using the well-known ECL coreactant 2-(dibutylamino)ethanol (DBAE) as a model analyte, which can be detected to a level of 10 nM with a linear range between 10(-8) and 10(-4) M.     

Electrochemiluminescence of Dendritic Magnetic Quantum Dots Nanostructure and Its Quenching by Gold Nanoparticles for Cancer Cells Assay

A novel dendritic CdS‐ZnS‐Quantum Dots (QDs) nanocomposite with intense electrochemiluminescence (ECL) and excellent magnetism was prepared, which was applied to the cancer cells assay based on ECL quenching of QDs by gold nanoparticles (NPs). DNA conjugation, gold NPs linking and sensing target cells can be directly performed on the magnetic nanocomposites, which is more rapid, convenient, and has better reproducibility than the conventional methods. So far, this is the first report on magnetic electrochemiluminescent QDs nanocomposites for cell detection based on ECL quenching, which opens a new approach for developing multifunctional QDs nanocomposite for ECL assays of cancer cells.     

Implementation of a Chloride‐selective Electrode Into a Closed Bipolar Electrode System with Fluorimetric Readout

Applicability of a bipolar electrode system was tested for arrangements containing a typical ion‐selective electrode (ISE) connected with an electrode coated by a conducting polymer characterized by electroluminescence. In this case a selective response of the ISE membrane at one pole of the bipolar electrode is transduced to a fluorimetric signal obtained by reduction of the conducting polymer at the second pole. This signal transformation mode was studied on example of a simple closed bipolar electrode system composed of all‐solid‐state chloride‐selective electrode with polypyrrole transducer as the sensing pole and the reporting pole represented by electrode coated by poly(3‐octylthiophene) (POT) layer characterized by fluorescence in the neutral state. In this system selective and linear dependences of fluorimetric signal on logarithm of chloride ions concentration in turn‐on mode were recorded for optimized external voltage applied. Alternatively, a concept of cascade bipolar electrode system with incorporation of additional bipolar electrode being a polarization source for the sensing bipolar electrode with ISE and POT layer was also tested. A significant advantage of the cascade system is its possibility to work spontaneously without external polarization. For this case also linear calibration plots of fluorimetric signal vs. logarithm of analyte concentration were recorded.     

Advanced Pt hollow nanospheres/rubrene nanoleaves coupled with M-shaped DNA walker for ultrasensitive electrochemiluminescence bioassay

Herein, an intense electrochemiluminescence (ECL) was achieved based on Pt hollow nanospheres/rubrene nanoleaves (Pt HNSs/Rub NLs) without the addition of any coreactant, which was employed for ultrasensitive detection of carcinoembryonic antigen (CEA) coupled with an M-shaped DNA walker (M-DNA walker) as signal switch. Specifically, in comparison with platinum nanoparticles (Pt NPs), Pt HNSs revealed excellent catalytic performance and pore confinement-enhanced ECL, which could significantly amplify ECL intensity of Rub NLs/dissolved O₂ (DO) binary system. Then, the tracks and M-DNA walker were confined on the Pt HNSs simultaneously to promote the reaction efficiency, whose M-structure boosted the interaction sites between walking strands and tracks and reduced the rigidity of their recognition. Once the CEA approached the sensing interface, the M-DNA walker was activated based on highly specific aptamer recognition to recover ECL intensity with the assistance of exonuclease Ⅲ (Exo Ⅲ). As proof of concept, the “on-off-on” switch aptasensor was constructed for CEA detection with a low detection limit of 0.20 fg/mL. The principle of the constructed ECL aptasensor also enables a universal platform for sensitive detection of other tumor markers.     

Organic nanoparticles for electrogenerated chemiluminescence assay

Organic nanoparticles (ONPs) are one type of nanoparticles assembled by the organic compounds with one dimension smaller than 100 nm. ONPs are alternative nanomaterials in organic light-emitting diode and analytical applications due to their unique optical and electrochemical properties. In electrogenerated chemiluminescence (ECL) assays, ONPs are generally taken as signal reporters for chemical sensing and biosensing. In this opinion, we focus on recent developments of ONPs as ECL luminophores in analytical application. The types and ECL mechanisms of ONPs systems and the approaches of ONPs-based ECL methods are briefly introduced. New advances on the improvement of the ECL efficiency of ONPs are highlighted. The challenges and perspectives of ONPs-based ECL methods are discussed.     

Ultrasensitive Electrochemiluminescence Biosensor for mRNA Based on Polymerase Assisted Signal Amplification

A novel biosensor for ultra‐trace mRNA sensing was constructed based on isothermal circular strand‐replacement polymerization (CSRP) to amplify the electrochmeiluminescence (ECL) signal by combining quantum dots (CdTe) as luminophore. After the hairpin‐like capture DNA was opened by hybridization with target mRNA, the additive primer (DNA1) was able to get access to its complementary sequence which is partially belong to the stem part and triggered a polymerization of DNA strand, leading to the release of target mRNA and another polymerization cycle. The remaining sequence of the stem part continued to hybridize with QDs labeled DNA, accomplishing ECL signal amplification. Target mRNA could be specifically assayed with a linear relationship between the signal intensity and the logarithm of concentrations of target DNA in the range of 1.0×10⁻¹⁴∼5.0×10⁻¹⁰ M, with a low detection limit of 1.4×10⁻¹⁵ M. The signal could discriminate perfect matched target mRNA from 1‐base mismatch sequence. This proposed ECL biosensor exhibited an efficient performance in serum sample, opening new opportunities for genetic target analysis in diagnostic and clinic biomedical fields.     

Electrochemiluminescent detection of mucin 1 protein and MCF-7 cancer cells based on the resonance energy transfer

A novel sensing strategy for sensitive detection of mucin 1 protein (MUC1) and MCF-7 cells based on electrochemiluminescence (ECL) resonance energy transfer (ERET) from bis(2,2'-bipyridine)-(5-aminophenanthroline)ruthenium(II) (Ru1) to graphene oxide (GO) was proposed. The MUC1 aptamer was covalently combined with Ru1 (Ru1-aptamer) using aqueous carbodiimide coupling chemistry. Due to the strong noncovalent interaction between the Ru1-aptamer and GO, the ECL of Ru1 was efficiently quenched because of the ERET. In the presence of a target MUC1 protein, the binding between the Ru1-aptamer and MUC1 disturbed the interaction between the Ru1-aptamer and GO. These interactions led to the release of the Ru1-aptamer from GO, and resulted in the restoration of Ru1 ECL. This was shown to detect MUC1 protein sensitively in a linear range from 64.9 to 1036.8 nM with a detection limit of 40 nM. With further application in the detection of MCF-7 cells, the presented method could respond at concentrations as low as 30 cancer cells per mL. By substituting the aptamer and the corresponding target, this method could be conveniently extended for the sensitive detection of other biomolecules.     

A solid-state electrochemiluminescence sensing platform for detection of adenosine based on ferrocene-labeled structure-switching signaling aptamer

A solid-state electrochemiluminescence sensing platform based on ferrocene-labeled structure-switching signaling aptamer (Fc-aptamer) for highly sensitive detection of small molecules is developed successfully using adenosine as a model analyte. Such special sensing platform included two main parts, an electrochemiluminescence (ECL) substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Au nanoparticle and Ruthenium (II) tris-(bipyridine) (Ru(bpy)₃²⁺-AuNPs) onto Au electrode. An anti-adenosine aptamer labeled by ferrocene acted as the ECL intensity switch. A short complementary ssDNA for the aptamer was applied to hybridizing with the aptamer, yielding a double-stranded complex of the aptamer and the ssDNA on the electrode surface. The introduction of adenosine triggered structure switching of the aptamer. As a result, the ssDNA was forced to dissociate from the sensing platform. Such structural change of the aptamer resulted in an obvious ECL intensity decrease due to the increased quenching effect of Fc to the ECL substrate. The analytic results were sensitive and specific.     

Electrochemical and Electrochemiluminescence Determination of Cancer Cells Based on Aptamers and Magnetic Beads

The electrochemical and electrochemiluminescence (ECL) detection of cell lines of Burkitt’s lymphoma (Ramos) by using magnetic beads as the separation tool and high‐affinity DNA aptamers for signal recognition is reported. Au nanoparticles (NPs) bifunctionalized with aptamers and CdS NPs were used for electrochemical signal amplification. The anodic stripping voltammetry technology employed for the analysis of cadmium ions dissolved from CdS NPs on the aggregates provided a means to quantify the amount of the target cells. This electrochemical method could respond down to 67 cancer cells per mL with a linear calibration range from 1.0×10² to 1.0×10⁵ cells mL^?1, which shows very high sensitivity. In addition, the assay was able to differentiate between target and control cells based on the aptamer used in the assay, indicating the wide applicability of the assay for diseased cell detection. ECL detection was also performed by functionalizing the signal DNA, which was complementary to the aptamer of the Ramos cells, with tris(2,2‐bipyridyl) ruthenium. The ECL intensity of the signal DNA, replaced by the target cells from the ECL probes, directly reflected the quantity of the amount of the cells. With the use of the developed ECL probe, a limit of detection as low as 89 Ramos cells per mL could be achieved. The proposed methods based on electrochemical and ECL should have wide applications in the diagnosis of cancers due to their high sensitivity, simplicity, and low cost.