Electrochemiluminescence Imaging Research Articles

Stereolithography 3-D Printed Electrochemiluminescence Platform With Random Grade Graphite Electrodes: Detection of HO and Cholesterol Using a Smartphone

The growing demand for low-cost mini biosensing platforms combining important features, such as fast performance, rapid prototyping, portability, and easy integration in point-of-care settings, has proven their existence in the field of targeted biomolecule detection. This work encompasses a novel, simple, and ultrafast fabrication of a stereolithographic 3-D printed electrochemiluminescence (GP-SE-ECL) biosensor with a graphite pencil-based single electrode (SE) to detect H2O2 and cholesterol. Graphite pencil electrodes were extensively used as a replacement for screen-printing, laser-induced graphene-based electrodes due to their user-friendliness, ease of availability, excellent electrochemical properties, and cost-efficiency. Different graded graphite electrodes (F, H, HB, 3B, 6B, and 8B) were used to fabricate SE electrochemiluminescence (ECL) devices. Since the conductivity values and surface area for different graded pencils vary, the impact of conductivity and surface area of the electrodes on the effectiveness of the ECL device was confirmed with optimized parameters by sensing H2O2. The lab-made, portable 3-D printed dark room platform integrated with a smartphone was used to capture the ECL signals. The smartphone android application was developed which can not only capture the ECL images, but also calculates the intensities values for the region of interest. Finally, the performance of the fabricated GP-SE-ECL biosensor was corroborated for cholesterol sensing to obtain a linear range (LR) from 50 to <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$1000 ~\mu \text{M}$ </tex-math></inline-formula> and a detection limit of <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$15.71 ~\mu \text{M}$ </tex-math></inline-formula> . Based on the experimental data, it was confirmed that the fabricated device with random grade pencil possesses strong acceptability in the field of biomolecule detection.

Detection of COVID-19: A Smartphone-Based Machine-Learning-Assisted ECL Immunoassay Approach with the Ability of RT-PCR CT Value Prediction.

The unstoppable spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely threatened public health over the past 2 years. The current ubiquitously accepted method for its diagnosis provides sensitive detection of the virus; however, it is relatively time-consuming and costly, not to mention the need for highly skilled personnel. There is a clear need to develop novel computer-based diagnostic tools to provide rapid, cost-efficient, and time-saving detection in places where massive traditional testing is not practical. Here, we develop an electrochemiluminescence (ECL)-based detection system whose results are quantified as reverse transcriptase polymerase chain reaction (RT-PCR) cyclic threshold (CT) values. A concentration-dependent signal is generated upon the introduction of the virus to the electrode and is recorded with a smartphone camera. The ECL images are used to train machine learning algorithms, and a model using artificial neural networks (ANNs) for 45 samples was developed. The model demonstrated more than 90% accuracy in the diagnosis of 50 unknown real samples, detecting up to a CT value of 32 and a limit of detection (LOD) of 10-12 g mL-1 in the testing of artificial samples.

TiO2 nanoparticles modified graphitic carbon nitride with potential-resolved multicolor electrochemiluminescence and application for sensitive sensing of rutin.

Recently, nanocomposites with potential-resolved multicolor electrochemiluminescence (ECL) property have attracted new research interests. Herein, TiO2 nanoparticles modified graphitic carbon nitride (TiO2-NPs/g-C3N4) with inherent potential-resolved multicolor ECL emission was prepared via a simple synthesis method. The morphology and chemical composition of the synthesized TiO2-NPs/g-C3N4 were characterized. The obtained TiO2-NPs/g-C3N4 exhibited dual-peak multicolor ECL emission under cyclic voltammetry scanning by using K2S2O8 as co-reagent. The first ECL peak (ECL-1) is composed of turquoise blue ECL emission (471nm) located at -1.3V and olive green ECL emission (490nm) ranging from -1.4 to -2.0V. The second ECL peak (ECL-2) is composed of navy blue ECL emission (458nm) located at -3.0V. The ECL mechanism for the potential-resolved multicolor ECL emissionwas proposed. Furthermore, the first ECL imaging sensing method was fabricated for the sensitive quantitative detection of rutin based on the effective quenching effect of rutin on the ECL of TiO2-NPs/g-C3N4. The linear response range is 0.005-400µM with detection limit as low as 2nM. This work presents a simple way to prepare g-C3N4-based nanocomposites with potential-resolved multicolor ECL, which broadens the potential applications of g-C3N4-based nanocomposites for ECL imaging sensing and light-emitting devices.

Electrochemiluminescence nanoemitters for immunoassay of protein biomarkers

The family of electrochemiluminescent luminophores has witnessed quick development since the electrochemiluminescence (ECL) phenomenon of silicon nanoparticles was first reported in 2002. Moreover, these developed ECL nanoemitters have extensively been applied in sensitive detection of protein biomarker by combining with immunological recognition. This review firstly summarized the origin and development of various ECL nanoemitters including inorganic and organic nanomaterials, with an emphasis on metal-organic frameworks (MOFs)-based ECL nanoemitters. Several effective strategies to amplify the ECL response of nanoemitters and improve the sensitivity of immunosensing were discussed. The application of ECL nanoemitters in immunoassay of protein biomarkers for diagnosis of cancers and other diseases, especially lung cancer and heart diseases, was comprehensively presented. The recent development of ECL imaging with the nanoemitters as ECL tags for detection of multiplex protein biomarkers on single cell membrane also attracted attention. Finally, the future opportunities and challenges in the ECL biosensing field were highlighted.

Electrochemiluminescence microscopy: From single objects to living cells

Electrochemiluminescence (ECL) is a light-emitting phenomenon triggered by electrochemistry. The resulting optical signal enables spatiotemporal resolution of the luminescent signal that can be imaged with high accuracy. The field of ECL is evolving rapidly from an analytical method widely applied in biosensing to a novel microscopy technique. The efficient generation of ECL depends on the electrode materials, the luminophore/coreactant systems, and experimental configurations, focusing research on improving these parameters. Nowadays, ECL enables the analysis of transient events and micro/nanometric objects, surface boundaries, interfaces, and physical phenomena, as well as catalytic and biological processes. Since ECL microscopy offers many novel applications, this article will describe first the context and then the most recent advances in ECL imaging of single objects and living cells or subcellular structures.

O-Fluorobenzoic Acid-Mediated Construction of Porous Graphitic Carbon Nitride with Nitrogen Defects for Multicolor Electrochemiluminescence Imaging Sensing.

Graphitic carbon nitride (g-CN) is an attractive electrochemiluminescence (ECL) luminophore. However, g-CN with wavelength-tunable ECL emission is still limited, which limits its application in multicolor ECL sensing and imaging analysis. In this study, porous g-CN (PCN) with nitrogen defects was synthesized through the condensation of melamine by using o-fluorobenzoic acid (o-FBA) as an effective regulation reagent. A series of PCNs, including PCN-5%, PCN-10%, and PCN-30%, were obtained by changing the mass ratio of o-FBA and melamine. The porous structure and tunable chemical composition change of the PCNs were carefully characterized. The nitrogen defects and porous structure of the synthesized PCNs can enlarge the specific surface area, facilitate electron transfer, and generate various surface states with gradually changed energy bands, leading to wavelength-tunable multicolor ECL emissions. Accordingly, g-CN, PCN-5%, PCN-10%, and PCN-30% can generate navy blue, turquoise blue, turquoise green, and olive green ECL emissions, respectively, with the peak ECL wavelength varied from 465 to 550 nm. Then, a multicolor ECL sensing array was proposed for the discrimination of polyphenols based on the prepared g-CN and PCNs by using a smartphone as a portable detector for the first time. Five polyphenol substances including vitamin P, resveratrol, phloretin, phlorizin, and caffeic acid were discriminated by using principal component analysis and hierarchical cluster analysis. The present work provides a simple strategy to adjust the ECL wavelength of g-CN and presents a simple way to fabricate multicolor ECL sensing array, which has great application potential for multiplexed analysis and multicolor ECL imaging sensing.

Electrochemical fabrication of Ru[bpy]32+@Cu-MOF(-SH)/Au photo/electrochemical coupling interface and its DNA-based biosensing performance

An electrochemical fabrication method of Ru[bpy]32+@Cu-MOF(-SH)/Au (MOF = metal–organic framework) was developed with a photo/electrochemical information characterization and a DNA-based biosensing validation for the proposed interface. Applying an anodic synthesis method, a modification layer of Ru[bpy]32+ encapsulated with MOF was formed on the Au electrode surface to fabricate an interface with photo/electrochemical dual-signal response. Combining the scanning electrochemical microscopy (SECM) imaging technology with electrochemical and electrochemiluminescence (ECL) detection, the information about the interfacial photo/electrochemical bi-functional properties was explored from three aspects of the parameter variable, intensity variable, and morphology variable. The results show that the effect of parameter variables on the interfacial response follows an “interference effect” mechanism. Under the optimized conditions, the background and the saturation ECL intensities are 610.0 and 24.0 with a quenching efficiency of 96.1% and a detection limit of 30 fM. The saturation current is 48.8 µA with a detection limit of 330.0 nM. Both ECL and electrochemical imaging methods can corroborate each other, validating the logical correlation between the interface fabrication and the response performance in the regional interfacial distribution and laying a methodological foundation for evaluating the interface fabrication quality based on imaging morphology qualitatively. The proposed interface can construct photo/electrochemical dual-signal DNA-based biosensors.

Low-Triggering-Potential Electrochemiluminescence from a Luminol Analogue Functionalized Semiconducting Polymer Dots for Imaging Detection of Blood Glucose.

In recent years, semiconducting polymer dots (Pdots) as environmentally friendly and high-brightness electrochemiluminescence (ECL) nanoemitters have attracted intense attention in ECL biosensing and imaging. However, most of the available Pdots have a high ECL excitation potential in the aqueous phase (>1.0 V vs Ag/AgCl), which causes poor selectivity in actual sample detection. Therefore, it is particularly important to construct a simple and universal strategy to lower the trigger potential of Pdots. This work has realized the ECL emission of Pdots at low-trigger-potential based on the electrochemiluminescence resonance energy transfer (ERET) strategy. By covalently coupling the Pdots with a luminol analogue, N-(4-aminobutyl)-N-ethylisoluminol (ABEI), the ABEI-Pdots showed an anodic ECL emission with a low onset potential of +0.34 V and a peak potential at +0.45 V (vs Ag/AgCl), which was the lowest trigger potential reported so far. We further explored this low-triggering-potential ECL for imaging detection of glucose in buffer and serum. By imaging the ABEI-Pdots-modified screen-printed electrodes (SPCE) at +0.45 V for 16 s, the ECL imaging method could quantify the glucose concentration in buffer from 10 to 200 μM with detection limits of 3.3 μM, while exhibiting excellent selectivity. When applied to real serum, the results of our method were highly consistent with a commercial blood glucose meter, with the relative errors ranging from 3.2 to 13%. This work provided a universal strategy for constructing low potential Pdots and demonstrated its application potential in complex biological sample analysis.

Electrochemiluminescence imaging of cellular adhesion in vascular endothelial cells during tube formation on hydrogel scaffolds

This study describes a method for electrochemiluminescence (ECL) imaging of cellular adhesion in vascular endothelial cells during tube formation on hydrogel scaffolds. Human umbilical vein endothelial cells were cultured on a Matrigel-coated indium tin oxide (ITO) electrode to facilitate tubule growth. Once these tubes were formed, the culture medium was changed to an ECL solution containing [Ru(bpy)3]2+ and tripropylamine and then subjected to 1.2 V vs Ag/AgCl (sat. KCl) to produce ECL signals at the ITO electrode. This allowed for ECL imaging of cellular adhesion because these ECL signals were blocked in areas typified by strong cell–cell and cell–substrate adhesion. The imaging showed that there were two types of cellular adhesion during tube formation. To the best of our knowledge, this is the first report on ECL imaging for adhesion between cells and between cells and substrates during tube formation on hydrogel scaffolds. We believe that this ECL imaging technique will be useful for the evaluation of organs-on-a-chip and for drug screening targeting cell–cell and cell–substrate interactions in the future.

Single Cell Imaging of Electrochemiluminescence‐Driven Photodynamic Therapy

AbstractWe report a photodynamic therapy driven by electrochemiluminescence (ECL). The luminescence generated by Ru(bpy)32+ and co‐reactant tripropylamine (TPA) pair acts as both optical readout for ECL imaging, and light source for the excitation of photosensitizer to produce reactive oxygen species (ROS) in photodynamic therapy (PDT) system. The ECL‐driven PDT (ECL‐PDT) relies on the effective energy transfer from ECL emission to photosensitizer chlorin e6 (Ce6), which sensitizes the surrounding O2 into ROS. The dynamic process of gradual morphological changes, the variation of cell‐matrix adhesions, as well as the increase of cell membrane permeability in the process of ECL‐PDT were monitored under ECL microscopy (ECLM) with good spatiotemporal resolution. Combining real‐time imaging with ECL‐PDT, this new strategy provides not only new insights into dynamic cellular processes, but also promising potential of ECL in clinical applications.

Single Cell Imaging of Electrochemiluminescence‐Driven Photodynamic Therapy

We report a photodynamic therapy driven by electrochemiluminescence (ECL). The luminescence generated by Ru(bpy)32+ and co-reactant tripropylamine (TPA) pair acts as both optical readout for ECL imaging, and light source for the excitation of photosensitizer to produce reactive oxygen species (ROS) in photodynamic therapy (PDT) system. The ECL-driven PDT (ECL-PDT) relies on the effective energy transfer from ECL emission to photosensitizer chlorin e6 (Ce6), which sensitizes the surrounding O2 into ROS. The dynamic process of gradual morphological changes, the variation of cell-matrix adhesions, as well as the increase of cell membrane permeability in the process of ECL-PDT were monitored under ECL microscopy (ECLM) with good spatiotemporal resolution. Combining real-time imaging with ECL-PDT, this new strategy provides not only new insights into dynamic cellular processes, but also promising potential of ECL in clinical applications.

Indium tin oxide bipolar electrodes modified with Pt nanoparticles encapsulated inside dendrimers as sensitive electrochemiluminescence platforms

• Both poles of a closed bipolar electrode (BPE) were modified with catalytic Pt nanoparticles. • The dual-modified BPE showed amplified electrochemiluminescence (ECL) of Ru(bpy) 3 2+ . • The amplified ECL was ascribed to simultaneous catalysis on the both BPE poles. • The dual-modified BPE was utilized for bipolar ECL imaging analysis of H 2 O 2 . Here, we report highly enhanced electrochemiluminescence (ECL) from a closed bipolar electrode (BPE) system consisting of an indium tin oxide (ITO) microband BPE whose both poles were modified with catalytic Pt nanoparticles encapsulated inside dendrimers. The closed BPE-based ECL sensing system generated sensitive and stable ECL emission under a mild driving voltage owing to simultaneous catalysis of both anodic ECL and cathodic sensing reactions on the reporting pole and the detecting pole of the dual-modified ITO microband BPE, respectively. We demonstrated much amplified ECL of Ru(bpy) 3 2+ /tripropylamine with the closed BPE-based ECL systems having the dual-modified ITO BPEs; specifically, ∼4 times higher sensitivity than that of the ECL systems having unmodified ITO BPEs for bipolar ECL imaging analysis of H 2 O 2 as a model marker of metabolism. In addition, the closed BPE-based ECL systems exhibited stable and steady ECL emission even upon 50 consecutive applications of a driving voltage of 2.3 V for robust and extended bipolar ECL imaging analysis of H 2 O 2 .

Dynamic Electrochemiluminescence Imaging of Single Giant Liposome Opening at Polarized Electrodes

In this work, the characterization of release events from liposomes has been addressed quantitatively by an electrochemiluminescence (ECL) imaging strategy. First, ECL reagents ([Ru(bpy)3]2+ and tripropylamine) were encapsulated in sealed giant asymmetrical liposomes (100 μm in diameter) made of DOPG/DOPC phospholipids. After sedimentation on an indium tin oxide electrode material, the opening of liposomes was triggered by polarization of the surface. Under these conditions, amperometry, epifluorescence imaging, and ECL imaging were combined and synchronized to monitor and image the rupture of giant liposomes during the release and subsequent ECL emission of their redox content. Amperometry allowed the quantification of the content released from single liposomes. The location and status of liposomes (closed or opened) were assessed by epifluorescence imaging. ECL provided the image of the efflux of matter after liposome opening. This original ECL imaging approach favorably compares with strictly photoluminescent or electrochemical techniques and appears to be adapted for the investigation of membrane rupture/permeation events.

Confined electrochemiluminescence imaging microarray for high-throughput biosensing of single cell-released dopamine

The quantitative detection of single cell secretions is always limited by their accurate collection and the heterogeneity of different cells. In this work, a confined electrochemiluminescence (ECL) imaging microarray (CEIM) chip was designed to capture single or a few cells in each cylindrical microwell for high-throughput quantitation of cell-secreted dopamine (DA). The ITO surface at the bottom of microwells was functionalized with the film of DA aptamer modified coreactant-embedded polymer dots (Pdots), which endowed the chip with the abilities to both in situ recognize the target DA secreted from the cells and emit the ECL signal for responding the secreted target without need of any additional coreactant. At the applied potential of +1.4 V, the Pdots in the film emitted strong ECL signal, which could be quenched by the electrochemical oxidation product of DA in individual microwell for sensitive detection of single cell-released DA. The practicability of the proposed CEIM chip along with the ECL imaging and biosensing strategy was demonstrated by evaluating the amounts of single cell-released DA in different microwells under hypoxia stimulation. This protocol revealed the heterogeneity of cell secretion, and could be extended for quantitation of other secretions from different kinds of single cells.

Front Cover: Label‐Free Electrochemiluminescence Imaging of Single‐Cell Adhesions by Using Bipolar Nanoelectrode Array (Chem. Eur. J. 3/2022)

Label-free electrochemiluminescence imaging of single cells has been achieved through a bipolar nanoelectrode array. The picture is divided into two parts by a strip of film, indicating that the bipolar nanoelectrode array separates cells and imaging. Oxygen is shown as the electrochemical probe and Ru(bpy)32+ as the luminescence molecule. More information can be found in the Research Article by X.-H. Xia et al. (DOI: 10.1002/chem.202103964).

Label-Free Electrochemiluminescence Imaging of Single-Cell Adhesions by Using Bipolar Nanoelectrode Array.

A label-free and fast approach for positive electrochemiluminescence (ECL) imaging of single cells by bipolar nanoelectrode array is proposed. The reduction of oxygen at a platinized gold nanoelectrode array in a closed bipolar electrochemical system is coupled with an oxidative ECL process at the anodic side. For elevating the ECL imaging contrast of single cells, a driving voltage of -2.0 V is applied to in situ generate oxygen confined beneath cells that is subsequently used for ECL imaging at 1.1 V. High oxygen concentration in the confined space resulting from steric hindrance generates prominent oxygen reduction current at the cathodic side and higher ECL intensity at the anodic side, allowing positive ECL imaging of the cells adhesion region with excellent contrast. Cell morphology and adhesion strength can be successfully imaged with high image acquisition rate. This approach opens a new avenue for label-free imaging of single cells.

Deciphering electrochemiluminescence generation from luminol and hydrogen peroxide by imaging light emitting layer

Electrochemiluminescence (ECL) of luminol is a luminescence process that proceeds in the presence of reactive oxygen species (e.g. hydrogen peroxide (H2O2)) at a suitable electrode potential, the reaction mechanism of which is complicated and remains ambiguous. In this work, we report a visualization approach for measuring the thickness of the ECL layer (TEL) of the luminol/H2O2 system to decipher the reaction process by combined use of the microtube electrode, ECL microscopy, and finite element simulations. With the increase of solution pH, the ECL image captured with the microtube electrode tends to vary from spot to ring, corresponding to the decrease of TEL from >9.1 μm to ca. 4.3 μm. We propose that different intermediates are involved in the course of ECL reaction. At a low pH (e.g. pH < 9), a relatively large TEL is most likely determined by the diffusion of oxidized and deprotonated luminol intermediate that is neutral and has a long lifetime. While at a high pH (e.g. pH in the range of 10 to 12), the ECL reaction is controlled by short-lived radical intermediates of both luminol and superoxide anion. The proposed mechanism is proved theoretically by finite element simulations and experimentally by the apparent effect of concentration ratio of luminol/H2O2.

Visualized uranium rapid monitoring system based on self-enhanced electrochemiluminescence-imaging of amidoxime functionalized polymer nanoparticles

The development of uranyl ion detection technology has exhibited its significance in public security and environmental fields for the radioactivity and chemical toxicity of uranyl ion. The WHO standard of uranyl ion makes it necessary to develop highly sensitive uranyl rapid warning system in drinking water monitoring. Herein, a visualized rapid warning system for trace uranyl ion is carried out based on electrochemiluminescence (ECL) imaging technology to give an ultra-low limit of detection (LOD) and high selectivity. Amidoxime, a bi-functional group with both uranyl ion capturing and co-reactive functions, is modified on a conjugated polymer backbone with strong ECL signal to be prepared into three-in-one polymer nanoparticles (PNPs) with self-enhanced ECL property. The captured uranyl ion can enhance the ECL signal of PNPs via resonance energy transfer process to give the LOD as 0.5 ng/L, which is much lower than the known luminescent uranyl sensors. Furthermore, ECL imaging technology is introduced into realizing visualized uranyl rapid warning, and can be successfully applied on natural water samples. This study provides a novel strategy for uranyl rapid warning, and shows its potential meaning in public security and environmental fields.

Thermally‐Drawn Multi‐Electrode Fibers for Bipolar Electrochemistry and Magnified Electrochemical Imaging

AbstractImaging systems using closed bipolar electrode (cBPE) arrays and electrochemiluminescence (ECL) have attracted great attention in recent years as a 2D imaging platform with high spatiotemporal resolution. However, the fabrication techniques for cBPE arrays involve complicated procedures. Therefore, a new fabrication scheme enabling the mass production of cBPE arrays with high precision, reproducibility, and yield, is desired. Here, the use of a versatile and scalable thermal drawing process as a novel fabrication method for fiber‐based cBPEs with feature sizes down to micro‐/nanoscales is proposed. First, a single‐electrode fiber consisting of a carbon‐based composite as the electrode material is produced by thermal drawing. The fundamental electrical properties of the single‐electrode fiber are characterized, and its applicability to the cBPE‐ECL system is demonstrated. A multielectrode fiber is fabricated by subjecting a bundle of 104 single‐electrode fibers to thermal drawing. Its usability as a cBPE array for ECL imaging is confirmed with a functional rate of 99%. Further the multielectrode fiber, utilizing the principle of thermal drawing, for magnified electrochemical imaging is tapered. This work establishes a novel mass‐production method for cBPE arrays, as well as a proof of concept for magnified electrochemical imaging using a thermally‐drawn electrode array fiber.

Single Biomolecule Imaging by Electrochemiluminescence.

Herein, a single biomolecule is imaged by electrochemiluminescence (ECL) using Ru(bpy)32+-doped silica/Au nanoparticles (RuDSNs/AuNPs) as the ECL nanoemitters. The ECL emission is confined to the local surface of RuDSNs leading to a significant enhancement in the intensity. To prove the concept, a single protein molecule at the electrode is initially visualized using the as-prepared RuDSN/AuNPs nanoemitters. Furthermore, the nanoemitter-labeled antibody is linked at the cellular membrane to image a single membrane protein at one cell, without the interference of current and optical background. The success in single-biomolecule ECL imaging solves the long-lasting task in the ultrasensitive ECL analysis, which should be able to provide more elegant information about the protein in cellular biology.