Electrochemiluminescence Imaging Research Articles

Electrochemiluminescence in Graphitic Carbon Nitride Decorated with Silver Nanoparticles for Dopamine Determination Using Machine Learning.

Electrochemiluminescence (ECL) luminophores with wavelength-tunable multicolor emissions are essential for multicolor ECL imaging detection and multiplexed analysis. In this work, silver nanoparticle (Ag NP)-decorated graphitic carbon nitride (g-CN@Ag) nanocomposites were synthesized. The morphology, chemical composition, structure, and ECL property of g-CN@Ag were investigated. The prepared g-CN, g-CN@Ag1, g-CN@Ag5, and g-CN@Ag10 can produce blue, blue-green, chartreuse, and yellow colored ECL emissions, respectively, by using K2S2O8 as the coreagent. The ECL emission wavelength of g-CN@Ag can be regulated from 460 to 565 nm by modulating the content of the immobilized Ag NPs. Then, a multicolor ECL detection array was fabricated by using g-CN, g-CN@Ag1, g-CN@Ag5, and g-CN@Ag10 as four ECL luminophores. Dopamine was detected based on its inhibition effect on the multicolor ECL emissions. The linear range is from 0.1 nM to 1 mM with the lowest detection limit of 44 pM. Then, machine learning-assisted multiparameter concentration prediction of dopamine was further carried out by combining the deep neural network (DNN) algorithm. This work provides a new avenue to regulate the ECL emission wavelength of g-CN by using the metal nanoparticle modification strategy and presents an effective machine learning-assisted multicolor ECL detection strategy for accurate multiparameter quantitative detection.

Electrochemiluminescence Microscopy.

Electrochemiluminescence (ECL) is rapidly evolving from an analytical method into an optical microscopy. The orthogonality of the electrochemical trigger and the optical readout distinguishes it from classic microscopy and electrochemical techniques, owing to its near-zero background, remarkable sensitivity, and absence of photobleaching and phototoxicity. In this review, we summarize the recent advances in ECL imaging technology, emphasising original configurations which enable the imaging of biological entities and the improvement of the analytical properties by increasing the complexity and multiplexing of bioassays. Additionally, mapping the (electro)chemical reactivity in space provides valuable information on nanomaterials and facilitates deciphering ECL mechanisms for improving their performances in diagnostics and (electro)catalysis. Finally, we highlight the recent achievements in imaging at the ultimate limits of single molecules, single photons or single chemical reactions, and the current challenges to translate the ECL imaging advances to other fields such as material science, catalysis and biology.

Electrochemiluminescent evaluation of GLUT4 expression in rat adipocytes induced by Ganoderma lucidum polysaccharides

Glucose transporter 4 (GLUT4) directly facilitates cellular uptake of glucose and plays a crucial role in mammalian adipose tissue glucose metabolism. In this work, we constructed a cytosensor for sensitive electrochemiluminescence (ECL) detection of GLUT4 in rat adipocytes (RA cells). A carbon nanotube sponge (CNTSP) was selected to fabricate a permeable electrode to overcome the steric hindrance of cells on the electrode. The expression of GLUT4 after treatment with Ganoderma lucidum polysaccharide (GLP) was assessed by analyzing the luminescence emitted from cell-surface ECL probes. Our preliminary results suggest that GLP promote the expression of GLUT4, thereby enhancing the uptake of the fluorescent glucose 2-NBDG. Treatment with GLP affected GLUT4 expression in RA cells in a dose-dependent manner. Additionally, the ECL cytosensor contributes to the development of ECL imaging of receptors on the cell surface for clinical drug evaluation.

Local electrochemiluminescence imaging using scanning electrochemical cell microscopy

In this communication, local electrochemiluminescence (ECL) imaging is achieved using scanning electrochemical cell microscopy (SECCM) to create a localized region for the occurrence of ECL reaction. The system is demonstrated using a droplet at the micropipette that is contact with the prepared Ru-Silica@Au particles at indium tin oxide (ITO) slide. Tri-n-propylamine (TPrA) in the droplet reacts with ruthenium complex in a 10 μm-diameter-region to produce ECL emission, which is recorded by photo multiplier tube (PMT) underneath the ITO slide. By contacting each local region consecutively, the distribution of steady-state current and the related ECL emission at the ITO surface is imaged. The local ECL intensity show an approximately linear dependence with the current, but obvious deviation from the linear curve is observed at some regions. This result suggests that the ECL emission is not only determined by the transferred electron number in the electrochemical reaction, and also affected by local environments at ITO surface. As compared with the traditionally used ECL imaging in the bulk solution, this strategy avoids the cross-talking of ECL emission from nearby regions, and could offer a new way to unveil more molecular mechanism in the ECL reaction.

Electrochemiluminescence-Based Single-Particle Tracking of the Biomolecules Moving along Intercellular Membrane Nanotubes between Live Cells.

Electrochemiluminescence (ECL) imaging, a rapidly evolving technology, has attracted significant attention in the field of cellular imaging. However, its primary limitation lies in its inability to analyze the motion behaviors of individual particles in live cellular environments. In this study, we leveraged the exceptional ECL properties of quantum dots (QDs) and the excellent electrochemical properties of carbon dots (CDs) to develop a high-brightness ECL nanoprobe (CDs-QDs) for real-time ECL imaging between living cells. This nanoprobe has excellent signal-to-noise ratio imaging capabilities for the single-particle tracking (SPT) of biomolecules. Our finding elucidated the enhanced ECL mechanism of CDs-QDs in the presence of reactive oxygen species through photoluminescence, electrochemistry, and ECL techniques. We further tracked the movement of single particles on membrane nanotubes between live cells and confirmed that the ECL-based SPT technique using CD-QD nanoparticles is an effective approach for monitoring the transport behaviors of biomolecules on membrane nanotubes between live cells. This opens a promising avenue for the advancement of ECL-based single-particle detection and the dynamic quantitative imaging of biomolecules.

Osteogenic differentiation of mesenchymal stem cell spheroids: A microfluidic device and electrochemiluminescence imaging study

Human mesenchymal stem cells (MSCs) have attracted attention as a major cellular source for regenerative medicine and preclinical studies. Three-dimensional (3D) cell culture systems can be used to construct physiologically relevant MSC spheroids with enhanced tissue regeneration capacity. However, what aspect of the MSC spheroids retains the regeneration capacity is not yet fully understood. Here, we induced osteoblast differentiation of MSC spheroids and investigated their respiratory activity using electrochemiluminescence (ECL) imaging developed in our previous study. In parallel, the ability of MSC spheroids to induce angiogenesis was investigated in a microfluidic device with a co-culture of endothelial cells. We found that the osteogenic differentiation of MSCs induced changes in their metabolic activity (respiratory activity of the spheroids decreased during differentiation) and lead to the loss of their angiogenesis-inducing ability. In other words, it is suggested that maintaining the stem cell nature with in the MSC spheroids is an important factor for tissue regeneration. In this study, we demonstrated the effective use of ECL imaging and microfluidic systems for the evaluation of MSC spheroids, providing new insights into stem cell tissue engineering.

The carbon dots with near-infrared electrochemiluminescence emission and low excitation potential for ultrasensitive analysis of microRNA-222

In this work, nitrogen and sulfur codoped near-infrared emission carbon dots (N,S-NIR-CDs) were used as neoteric electrochemiluminescence (ECL) emitters with high efficiency to construct a biosensor for ultrasensitive analysis of microRNA-222 (miRNA-222), which has been regarded as prospective biomarkers for early cancer diagnosis. Impressively, compared to conventional CDs with short ECL wavelength and large excitation voltage, N,S-NIR-CDs possessed wide sp2 domains and narrow band gap, leading to the near-infrared ECL emission at 680 nm and low excitation potential from −1.0 to 0 V, which not only decreased the photochemical damage for biomolecules but also reduced the side reaction. Moreover, an improved T7 Exonuclease induced cascade amplification (EICA) strategy was designed to transform trace miRNA-222 into plentiful output DNA in dual-cycle for greatly improving the target conversion efficiency. Therefore, by combining the N,S-NIR-CDs as neoteric ECL emitters and EICA strategy as signal amplifier, the constructed ECL biosensor could realize the ultrasensitive analysis of miRNA-222 with a identification limit of 5.65 aM and successfully employed in miRNA-222 analysis from liver cancer cell (HCCLM3 and MHCC-97L) lysate. This strategy developed a kind of neoteric CDs with NIR ECL emission and low excitation potential to establish a highly efficient biosensor for biomarker analysis, which prepared a road for early diseases diagnosis and lied a foundation for biological ECL imaging.

Coupled Poly(ethylenimine) Coreactant to Enhance Electrochemiluminescence of Polymer Dots for Array Imaging of Protein Biomarkers.

Traditional electrochemiluminescent (ECL) bioanalysis suffers from the demand for excessive external coreactants and the damage of reaction intermediates. In this work, a poly(ethylenimine) (PEI)-coupled ECL emitter was proposed by covalently coupling tertiary amine-rich PEI to polymer dots (Pdots). The coupled PEI might act as a highly efficient coreactant to enhance the ECL emission of Pdots through intramolecular electron transfer, reducing the electron transfer distance between emitter and coreactant intermediates and avoiding the disadvantages of traditional ECL systems. Through modification of the PEI-Pdots with tDNA, a sequence partially complementary to cDNA that was complementary to the aptamer of target protein biomarker (aDNA), tDNA-PEI-Pdots were obtained. The biosensors were produced using Au/indium tin oxide (ITO) with an aDNA/cDNA hybrid, and an ECL imaging biosensor array was constructed for ultrasensitive detection of protein biomarkers. Using vascular endothelial growth factor 165 (VEGF165) as a protein model, the proposed ECL imaging method containing two simple incubations with target samples and then tDNA-PEI-Pdots showed a detectable range of 1 pg mL-1 to 100 ng mL-1 and a detection limit of 0.71 pg mL-1, as well as excellent performance such as low toxicity, high sensitivity, excellent selectivity, good accuracy, and acceptable fabrication reproducibility. The PEI-coupled Pdots provide a new avenue for the design of ECL emitters and the application of ECL imaging in disease biomarker detection.

Near-IR Photoinduced Electrochemiluminescence Imaging with Structured Silicon Photoanodes.

Infrared (IR) imaging devices that convert IR irradiation (invisible to the human eye) to a visible signal are based on solid-state components. Here, we introduce an alternative concept based on light-addressable electrochemistry (i.e., electrochemistry spatially confined under the action of a light stimulus) that involves the use of a liquid electrolyte. In this method, the projection of a near-IR image (λexc = 850 or 840 nm) onto a photoactive Si-based photoanode, immersed into a liquid phase, triggers locally the photoinduced electrochemiluminescence (PECL) of the efficient [Ru(bpy)3]2+-TPrA system. This leads to the local conversion of near-IR light to visible (λPECL = 632 nm) light. We demonstrate that compared to planar Si photoanodes, the use of a micropillar Si array leads to a large enhancement of local light generation and considerably improves the resolution of the PECL image by preventing photogenerated minority carriers from diffusing laterally. These results are important for the design of original light conversion devices and can lead to important applications in photothermal imaging and analytical chemistry.

Bipolar Clark-Type Oxygen Electrode Arrays for Imaging and Multiplexed Measurements of the Respiratory Activity of Cells.

Various miniature Clark-type oxygen electrodes (COEs), which are typically used to measure dissolved oxygen (DO) concentration in cellular respiration, have been developed since the 1980s. Arrays with individually addressable electrodes that constitute the sensor were used for various applications. However, the large number of leads and contact pads required for connecting the electrodes and the external instrument complicate the electrode layout and make the operation of integrated COE arrays challenging. Here, we fabricated closed bipolar electrochemical systems comprising 6 × 8 and 4 × 4 arrays of COEs for imaging and multiplexed detection. The cathodic compartment was sealed with a hydrophobic oxygen-permeable membrane to separate the internal electrolyte solution from the sample solutions. Using the bipolar Clark-type oxygen electrode (BCOE) arrays and electrochemiluminescence (ECL), we measured the DO concentration at each cathode. The results revealed that the ECL intensity changed linearly with the DO concentration. In addition, we used ECL imaging to investigate the respiratory activity of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) in suspensions with different cell densities. The ECL images showed that the ECL intensity changed noticeably with the bacterial density. The bacterial respiratory activity was then qualitatively analyzed based on the ECL images acquired successively over a time duration. Further, we measured the antibiotic efficacy of piperacillin, oxacillin, gentamicin, and cefmetazole against E. coli and P. aeruginosa using the BCOE. We found that the ECL intensity increased with the antibiotic concentration, thus indicating the suppression of the bacterial respiratory activity.

Electrochemiluminescent imaging of a NADH-based enzymatic reaction confined within giant liposomes.

Herein, transient releases either from NADH-loaded liposomes or enzymatic reactions confined in giant liposomes were imaged by electrochemiluminescence (ECL). NADH was first encapsulated with the [Ru(bpy)3]2+ luminophore inside giant liposomes (around 100µm in diameter) made of DOPC/DOPG phospholipids (i.e., 1,2-dioleolyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycerol-3-phospho-(1'-rac-glycerol) sodium salt) on their inner- and outer-leaflet, respectively. Then, membrane permeabilization triggered upon contact between the liposome and a polarized ITO electrode surface and ECL was locally generated. Combination of amperometry, photoluminescence, and ECL provided a comprehensive monitoring of a single liposome opening and content release. In a second part, the work is focused on the ECL characterization of NADH produced by glucose dehydrogenase (GDH)-catalyzed oxidation of glucose in the confined environment delimited by the liposome membrane. This was achieved by encapsulating both the ECL and catalytic reagents (i.e., the GDH, glucose, NAD+, and [Ru(bpy)3]2+) in the liposome. In accordance with the results obtained, NADH can be used as a biologically compatible ECL co-reactant to image membrane permeabilization events of giant liposomes. Under these conditions, the ECL signal duration was rather long (around 10s). Since many enzymatic reactions involve the NADH/NAD+ redox couple, this work opens up interesting prospects for the characterization of enzymatic reactions taking place notably in artificial cells and in confined environments.

Infrared photoinduced electrochemiluminescence microscopy of single cells

Photo-induced electrochemiluminescence imaging of single cells is reported using an infrared light stimulus.

Electrochemiluminescence of hot exciton nanomaterial with boosted efficiency for visual bioanalysis

The electrochemiluminescence (ECL) efficiency (ΦECL) of organic ECL emitters is greatly influenced by the excited state property and component. Here we design a hot exciton molecule (BCzP-BT) with hybridized local and charge-transfer (HLCT) excited state property to prepare the first hot exciton organic nanorods (BB NRs). The BB NRs exhibit both sensitive annihilation and intense coreactant ECL emissions with a band gap emission model. Due to the reverse intersystem crossing among high-lying excited states (hRISC), the HLCT excited state property leads to highly efficient utilization of triplet excitons and thus high photoluminescence quantum yields (ΦPL) of more than 80% at 554 nm, which is demonstrated by solvatochromic experiments and quantum chemical calculations. The high ΦPL endows BB NRs with superior ΦECL over other nanoemitters, even the thermally activated delayed fluorescence materials, and thus offers highly-efficient nanoemitters for the design of ECL imaging strategy. As a proof of concept, an ECL probe is constructed by assembling BHQ2-ssDNA on BB NRs to integrate CRISPR/Cas12a system for target recognition, which produces a rapid and high-throughput ECL imaging platform. The proposed imaging method for HPV16 DNA detection shows excellent performance with a detection limit of 0.6 fM. This work broadens the application of hot exciton materials in ECL field, and opens a new avenue for developing next-generation ECL devices.

Antigen retrieval assisted electrochemiluminescence imaging of surface antigens at single tissue section

Previously, our group has established an electrochemiluminescence (ECL) molecular imaging method to detect carcinoembryonic antigen (CEA) at single tissue sections of carcinoma patients. However, the tissue fixation blocks the binding sites of surface antigens affecting the antigen-antibody interaction and the resultant visualization sensitivity. Herein, we report an antigen retrieval assisted ECL imaging strategy that utilizes heat-induced epitope retrieval (HIER) to re-expose the binding sites of the antigens for more efficient interaction between surface antigen and ruthenium-labeled antibody. With more ruthenium complexes tagged with surface antigens, the ECL intensity at the tissue sections increases significantly which could more accurately reflect the amount and the distribution of surface CEA. Accordingly, the different distribution of CEA at the carcinoma and paracancerous regions of tissue sections from two patients with lung adenocarcinoma (LUAD) is visualized, which could not be easily distinguished without the HIER treatment. Eventually, the combination of antigen retrieval and ECL molecular imaging could improve the visualization sensitivity at single tissue section and minimize the false negative phenomenon, which will greatly advance the development of ECL-based tissue imaging for future application in pathological diagnosis.

Highly selective monitoring of trace radioactive I2 over organic iodides using NH2-DNA based electrochemiluminescence device

The selective determination of radioactive I2 over organic iodides is beneficial for the efficient treatment of spent fuel and nuclear emergency. We herein develop an efficient strategy for trace I2 selective detection over organic iodide for the first time by using electrochemiluminescence (ECL) device. In detail, an I2 selective sensor is prepared by Au nanoparticles and dsDNA with -NH2 group, and then modified on indium tin oxide (ITO) electrode to obtain I2 detection device. -NH2 acts as ECL co-reactant as well as I2 selective response group, which can specifically combine with I2 in organic iodide atmosphere to quench the signal of ECL emitter (Ru(bpy)32+). This device gives a low limit of detection (5 ng/L) and high selectivity to I2 under high humidity, acidity, temperature as well as CH3I atmosphere. ECL imaging technology is applied to realize visualized accurate monitoring to trace I2 in atmosphere. Electrochemical measurement and theory calculation are further employed to demonstrate the selective mechanism of I2 detection over organic iodides. This work provides an efficient strategy and device for trace I2 specific monitoring, which exhibits a promising prospect in the fields of environment and nuclear energy.

T Cell Antigen Recognition and Discrimination by Electrochemiluminescence Imaging.

Adoptive T lymphocyte (T cell) transfer and tumour-specific peptide vaccines are innovative cancer therapies. An accurate assessment of the specific reactivity of T cell receptors (TCRs) to tumour antigens is required because of the high heterogeneity of tumour cells and the immunosuppressive tumour microenvironment. In this study, we report a label-free electrochemiluminescence (ECL) imaging approach for recognising and discriminating between TCRs and tumour-specific antigens by imaging the immune synapses of T cells. Various T cell stimuli, including agonistic antibodies, auxiliary molecules, and tumour-specific antigens, were modified on the electrode's surface to allow for their interaction with T cells bearing different TCRs. The formation of immune synapses activated by specific stimuli produced a negative (shadow) ECL image, from which T cell antigen recognition and discrimination were evaluated by analysing the spreading area and the recognition intensity of T cells. This approach provides an easy way to assess TCR-antigen specificity and screen both of them for immunotherapies.

T Cell Antigen Recognition and Discrimination by Electrochemiluminescence Imaging

AbstractAdoptive T lymphocyte (T cell) transfer and tumour‐specific peptide vaccines are innovative cancer therapies. An accurate assessment of the specific reactivity of T cell receptors (TCRs) to tumour antigens is required because of the high heterogeneity of tumour cells and the immunosuppressive tumour microenvironment. In this study, we report a label‐free electrochemiluminescence (ECL) imaging approach for recognising and discriminating between TCRs and tumour‐specific antigens by imaging the immune synapses of T cells. Various T cell stimuli, including agonistic antibodies, auxiliary molecules, and tumour‐specific antigens, were modified on the electrode's surface to allow for their interaction with T cells bearing different TCRs. The formation of immune synapses activated by specific stimuli produced a negative (shadow) ECL image, from which T cell antigen recognition and discrimination were evaluated by analysing the spreading area and the recognition intensity of T cells. This approach provides an easy way to assess TCR‐antigen specificity and screen both of them for immunotherapies.

Nanoconfined Cathodic Electrochemiluminescence for Self-Sensitized Bioimaging of Membrane Protein.

Self-enhanced electrochemiluminescence (ECL) can be achieved via the confinement of coreactants and ECL emitters in a single nanostructure. This strategy has been used for the design of anodic ECL systems with amine compounds as coreactants. In this work, a novel confinement system was proposed by codoping positively charged ECL emitter tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+) and negatively charged coreactant peroxydisulfate (S2O82-) in silica nanoparticles. The codoping process could be performed by introducing S2O82- in cationic poly(diallyldimethylammonium chloride) (PDDA) to form PDDA@S2O82- and then encapsulating it and Ru(bpy)32+ in the Triton X-100 vesicle followed by the hydrolysis of tetraethyl ortosilicate, surface modification, and demulsification. The obtained RuSSNs exhibited good homogeneity, excellent monodispersity, acceptable biocompatibility, and 2.9-fold stronger ECL emission than Ru(bpy)32+-doped silica nanoparticles at an equal amount of nanoparticles in the presence of 0.1 M K2S2O8. Thus, an in situ self-sensitized cathodic ECL imaging method was designed for the monitoring of glycoprotein on living cell membranes. This work provides a new way for the modification, enhancement, and application of nano-ECL emitters in biological analysis.

Electrochemiluminescence Detection and Imaging of Biomolecules at the Single-Cell Level

Electrochemiluminescence (ECL) is an electrochemically induced light produced by the excitation of luminophores in redox reactions. For the past twenty years, ECL analysis has been continuously developed and applied for the sensitive detection of biomolecules at the single-cell level due to its low background interference and the resultant high sensitivity. In recent times, ECL-based microscopy has combined the elements of imaging and has thus emerged as a fast-developed imaging tool to visualize biomolecules in single cells. The surface-confined features of ECL imaging provide detailed information about cell membranes that is not easily obtained using classical fluorescence microscopy. In this review, we summarize the recent works on the detection and imaging of biomolecules at the single-cell level using ECL and discuss the development prospects and challenges in the biological application of this technology in the field of cell analysis.

Dynamic Mapping of Electrochemiluminescence Reactivity in Space: Application to Bead-Based Assays.

As an electrochemical technique offering an optical readout, electrochemiluminescence (ECL) evolved recently into a powerful microscopy technique with the visualization of a wide range of microscopic entities. However, the dynamic imaging of transient ECL events did not receive intensive attention due to the limited number of electrogenerated photons. Here, the reaction kinetics of the model ECL bioassay system was revealed by dynamic imaging of single [Ru(bpy)3]2+-functionalized beads in the presence of the efficient tripropylamine coreactant. The time profile behavior of ECL emission, the variations of the ECL layer thickness, and the position of maximum ECL intensity over time were investigated, which were not achieved by static imaging in previous studies. Moreover, the dynamics of the ECL emission were confronted with the simulation. The reported dynamic ECL imaging allows the investigation of the ECL kinetics and mechanisms operating in bioassays and cell microscopy.