Electrochemiluminescence Light Research Articles

(Invited) Multiplexed CRISPR-Based ECL Assay for Alzheimer’s Biomarkers

Currently, more than 6 million Americans live with Alzheimer’s disease (AD), and it is projected to be 13 million by 2050. AD has no permanent cure yet, and early detection is important to slow down the progress of the disease. Diagnosis is complicated by AD’s similarity to other geriatric diseases. Beta-amyloid peptide (Aβ42), tau proteins and neurofilament light (NFL) measured in the cerebrospinal fluid (CSF) is a reasonably effective method for diagnostics, but very painful for the patients. MicroRNA’s (miRNAs) expressed in the brain are promising biomarkers and our goal of this study is to develop a sensitive and selective point-of-care (POC) test to detect AD specific miRNA (miRNA 30e, 34c and 200c) based on the CRISPR-Cas system. The Cas 13a protein has the regular sequence specific RNase activity followed by a collateral RNase activity. We used this feature of Cas 13a to generate an electrochemiluminescence (ECL) signal by binding specific miRNA biomarkers, and then collaterally cleaving poly-ribose guanine (poly-rG). This cleaved strands of poly-rG and rG itself are coreactants for ECL, and react with ECL polymer Ruthenium Polyvinyl Pyridine (RuPVP) to generate ECL. RuPVP is deposited into pyrolytic graphite (PG) micro wells. Using a 3D-printed microfluidic device equipped with the Pt reference and Ag/AgCl counter electrodes, patient serum mixed with poly-rG is introduced to the RuPVP coated electrode at 1.3 V to provide ECL light measured by a CCD camera. The assay was multiplexed to detect 3 AD specific miRNAs, and obtain a separate calibration for each miRNA biomarker with linear ranges from 70 pg/ mL to 7 µg/mL, from 7.4 pg/ mL to 74 µg/ mL and from 7.4 fg/ mL to 74 µg/ mL for 30e, 34c and 200c respectively. Preliminary LODs were 42 pg/ mL, 0.074 fg/ mL and 0.15 fg/ mL for miRNA 30e, 34c and 200c respectively. Results confirm the sensitivity of the assay. Further, spike recovery, cross reactivity studies and patient sample assays will be reported.

Complex electrochemiluminescence patterns shaped by hydrodynamics at a rotating bipolar electrode.

Electrochemiluminescence (ECL) is a powerful analytical approach that enables the optical readout of electrochemical processes. Over the last few years, ECL has gained considerable attention due to its large number of applications, including chemical sensing, bioanalysis and microscopy. In these fields, the promotion of ECL at bipolar electrodes has offered unprecedented opportunities thanks to wireless electrochemical addressing. Herein, we take advantage of the synergy between ECL and bipolar electrochemistry (BE) for imaging light-emitting layers shaped by hydrodynamics, polarization effects and the nature of the electrochemical reactions taking place wirelessly on a rotating bipolar electrode. The proof-of-principle is established with the model ECL system [Ru(bpy)3]2+/tri-n-propylamine. Interestingly, the ECL-emitting region moves and expands progressively from the anodic bipolar pole to the cathodic one where ECL reactants should neither be generated nor ECL be observed. Therefore, it shows a completely unusual behavior in the ECL field since the region where ECL reagents are oxidized does not coincide with the zone where ECL light is emitted. In addition, the ECL patterns change progressively to an "ECL croissant" and then to a complete ring shape due to the hydrodynamic convection. Such an approach allows the visualization of complex light-emitting patterns, whose shape is directly controlled by the rotation speed, chemical reactivity and BE-induced polarization. Indeed, the bipolar electrochemical addressing of the electrode breaks the circular symmetry of the reported rotating system. This unexplored and a priori simple configuration yields unique ECL behavior and raises new curious questions from the theoretical and experimental points of view in analytical chemistry. Finally, this novel wireless approach will be useful for the development of original ECL systems for analytical chemistry, studies of electrochemical reactivity, coupling microfluidics with ECL and imaging.

Through-Space Electrochemiluminescence Reveals Bubble Forces at Remote Phase Boundaries.

Several groups have reported on the curious chemistry and reaction acceleration in confined volumes. These complex multiphase systems most closely resemble natural processes, and new measurement tools are necessary to probe chemistry in such environments. Generally, electrochemiluminescence (ECL) reports on processes immediately near (within a few micrometers) the electrode surface. Here, we introduce through-space ECL, reporting on dynamics of processes far away (100s of μm) from the electrode surface. We achieved this by collecting reflected ECL light. During the heterogeneous oxidation of C2O42- in an aqueous phase adjacent to a 1,2-dichlorethane droplet, CO2 accumulates in the 1,2-dichloroethane droplet. Upon buildup, we demonstrate that a CO2 bubble forms in the nonaqueous phase and is surprisingly trapped at the water|1,2-dichloroethane interface and continues to grow. The co-oxidation of tris(bipyridine)ruthenium(II) in the aqueous phase lights up the electrode surface and reflects off the edges of the bubble, revealing the bubble growth over time even when the bubble is fractions of a millimeter from the surface. We extend our results to quantifying bubble forces at the water-oil interface at remote distances from the electrode surface.

Extending the Operational Lifetime of Electrochemiluminescence Devices by Installing a Floating Bipolar Electrode.

Electrochemiluminescence (ECL) holds significant promise for the development of cost-effective light-emitting devices because of its simple structure. However, conventional ECL devices (ECLDs) have a major limitation of short operational lifetimes, rendering them impractical for real-world applications. Typically, the luminescence of these devices lasts no longer than a few minutes during operation. In the current study, a novel architecture is provided for ECLDs that addresses this luminescence lifespan issue. The device architecture features an ECL active layer between two coplanar driving electrodes and a third floating bipolar electrode. The inclusion of the floating bipolar electrode enables modulating the electrical-field distribution within the active layer when a bias is applied between the driving electrodes. This, in turn, enables the use of opaque yet electrochemically stable noble metals as the driving electrodes while allowing ECL light to escape through the transparent floating bipolar electrode. A significant extension on operational lifetime is achieved, defined as the time required for the initial luminance (>100cdm-2) to decrease by 50%, surpassing 1h. This starkly contrasts the short lifetime (<1min) attained by ECLDs in a conventional sandwich-type architecture with two transparent electrodes. These results provide simple strategies for developing durable ECL-based light-emitting devices.

Polydopamine nanoparticles@MoS2 nanosheet aerogel-based ECL sensing system for MiRNA-126 detection

In this paper, we developed a novel electrochemiluminescence (ECL) sensing system based on polydopamine nanoparticles (PDA NPs) and MoS2 nanosheet aerogel. PDA NPs with phenylboronic acid (PBA) were synthesized as a novel kind of organic nano-luminophores. The PDA-PBA NPs had high ECL efficiency and regulable emitting wavelength (552 ~ 610 nm). MoS2 nanosheet aerogel played the important role in the improvement of ECL sensing performance. On the one hand, MoS2 nanosheet aerogel can load a large number of PDA-PBA NPs. The accommodation of PDA-PBA NPs in the MoS2 nanosheet aerogel kept the dispersed state, which prevented NPs aggregation and ECL quenching effect. On the other hand, the excellent conductivity of MoS2 nanosheet aerogel significantly facilitated the electron transfer. Electron injection with MoS2 nanosheet aerogel can effectively reduce the damage of PDA-PBA NPs by excessive hot electrons. As a result, the ECL signal of PDA-PBA NPs@MoS2 nanosheet aerogel have been amplified over 60 times than PDA NPs. Furthermore, the bright ECL light of PDA-PBA NPs@MoS2 nanosheet aerogel can be clearly observed with the smartphone. Finally, a target-catalyzed hairpin assembly (CHA) sensing mode was designed to visually detect MiRNA-126 (detection limit as 0.56 aM). The MoS2 nanosheet aerogel-based sensing system provided a new pathway in the development of ECL analysis research.

Electron Transfer Processes Enabling Genotoxicity Sensor Arrays

Design of biosensor arrays for drug development and diagnostic applications has become a major activity in bioelectrochemistry. Achieving efficient electron transfer processes to produce reliable, reproducible, and sensitive output signals is a critical goal for such devices. This paper describes key electron transfer processes in our development of sensors for the chemistry of genotoxicity, defined here as resulting from the damage and oxidation of DNA by chemicals and their metabolites. This research is driven mainly by the need to screen for the potential genotoxicity of new drug and environmental chemical candidates, about 30% of which have toxicity issues that do not manifest until human clinical trials. A major issue is metabolic chemistry driven mainly by cytochrome (cyt) P450s and other enzymes, and we needed to produce metabolites in our arrays. Metabolic chemistry involves electron-transfer catalysis of organic reactions, usually involving oxygen transfer in the case of cyt P450s. We have found ways to efficiently transfer electrons into cyt P450s in thin films via their natural reductase electron-transfer partners to catalyze metabolic reactions electrochemically via mechanisms that are the same as in our livers and other organs. Optimizing the electrode-driven electron transfer chain from electrode to reductase to cyt P450 has allowed us to replace expensive NADPH as reducing agent and simplify our genotoxicity arrays. We also needed a way to detect the resulting metabolite-related DNA damage in sensors combining thin LbL films of metabolic enzymes and DNA in which the metabolites are generated and react with DNA. We found that guanines in DNA chains can react in an electrocatalytic process involving electron transfer to a Ru or Os metallopolymer (RuPVP) to output visible light via electrochemiluminescence (ECL). A complex electron transfer sequence drives an electrocatalytic process that oxidizes RuII to RuIII to oxidize guanine moieties in the DNA and produces visible light. The OsII/OsIII redox couple can be used in a similar ways to design sensor arrays selective for DNA oxidation. Electrocatalytic Cyt P450 oxidations, ECL generation and microfluidics are combined in our most sophisticated genotoxicity sensor arrays. When DNA is adducted by metabolites, its double-stranded structure is disrupted and RuIII sites in the film gain better access to guanine moieties to produce more ECL light. The primary oxidation product in DNA oxidation is 8-oxo-guanine, which in a second step is selectively oxidized by OsIII complexes to give ECL at a lower applied potential that for RuIII/RuII. Examples of metabolite-related DNA damage and oxidation, including the genotoxic potential of e-cigarettes, will be discussed.

Electrochemistry and electrochemiluminescence of copper metal cluster

The electrochemistry and electrochemiluminescence (ECL) of copper metal cluster were studied in this work. The redox behavior observed was originated from the couples of Cu8+/Cu82+, Cu8/Cu82+, Cu8/Cu8+ at the cluster metal core. ECL light emissions were observed under both annihilation and tripropylamine co-reactant conditions and only one excited state was involved in this ECL process and this excited state was also at the metal core. Theoretical investigation found that this excitation corresponds to an electron transfer from the metal core to the ligands. And the energy released from excited sate was approximate to the LUMO→HOMO gap energy of the cluster.

Organic Electrochemistry Goes to Work: Genotoxicity Sensor Arrays

This paper focuses on fundamental concepts derived from the technical evolution of organic electrochemistry that have helped us to develop sensors for the chemistry of genotoxicity, which is essentially the damage of DNA by chemicals and their metabolites. This research is driven by the need to screen for the possibility of genotoxicity of new environmental chemicals and drug candidates, about 30% of which have toxicity issues that do not manifest until clinical trials on humans. A major issue is metabolic chemistry driven mainly by cytochrome (cyt) P450s and other metabolic enzymes. Metabolic chemistry involves catalysis of organic reactions, oxidations in the case of cyt P450s. We found ways to inject electrons into cyt P450s in thin films via their native reductase electron-transfer partners to catalyze metabolic reactions electrochemically via mechanisms that are the same as in our livers. This is organic electrochemistry. For the sensors, we needed a way to detect the resulting metabolite-related DNA damage in sensors combining thin LbL films of metabolic enzymes and DNA in which the metabolites are generated and react with DNA. We found that guanines in DNA chains can react in an electrocatalytic process involving a Ru metallopolymer (RuPVP) and provide a strong visible light output via electrochemiluminescence (ECL). This electrocatalytic process oxidizes RuII in the polymer to RuIII to oxidize guanine moieties in the DNA, and a complex redox reaction sequence produces the light. The catalytic oxidation current can also be measured. Organic electro-oxidations are responsible for these outputs. Similar approaches can be used to design sensor arrays that monitor DNA oxidation. Electrocatalytic Cyt P450 oxidations and ECL generation are combined in our most sophisticated genotoxicity sensor arrays. When DNA is damaged, its double-stranded structure is disrupted and RuIII sites in the film gain better access to guanine moieties to produce more ECL light, or more catalytic current. Examples, including the genotoxic potential of e-cigarettes, will be discussed.

Comparison study of electrochemiluminescence of boron-dipyrromethene (BODIPY) dyes in aprotic and aqueous solutions

Boron-dipyrromethene (BODIPY) dye molecules are well known for their excellent absorption and emission optical properties. Here, we report the results of an electrochemical and electrochemiluminescence (ECL) study of an organic-soluble and a water-soluble BODIPY dye to explore their effectiveness as ECL luminophores. A combination of UV–visible, fluorescence, and spooling ECL spectroscopy was used to study the mechanisms of ECL light emission. It was discovered that excimers, the excited states of analyte dimers, contributed significantly to the production of ECL. These BODIPY molecules exhibited relatively weak ECL efficiencies following the annihilation mechanism. However, ECL was enhanced with co-reactants such as persulfate, and tri-n-propylamine. Spooling ECL spectroscopy was utilized to gain analytical insight into light emission mechanisms. It is shown that while these two BODIPY compounds can form photoinduced excited monomers, excimers were generated mainly in their co-reactant ECL pathways.

3D-printed supercapacitor-powered electrochemiluminescent protein immunoarray.

Herein we report a low cost, sensitive, supercapacitor-powered electrochemiluminescent (ECL) protein immunoarray fabricated by an inexpensive 3-dimensional (3D) printer. The immunosensor detects three cancer biomarker proteins in serum within 35 min. The 3D-printed device employs hand screen printed carbon sensors with gravity flow for sample/reagent delivery and washing. Prostate cancer biomarker proteins, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA) and platelet factor-4 (PF-4) in serum were captured on the antibody-coated carbon sensors followed by delivery of detection-antibody-coated Ru(bpy)3(2+) (RuBPY)-doped silica nanoparticles in a sandwich immunoassay. ECL light was initiated from RuBPY in the silica nanoparticles by electrochemical oxidation with tripropylamine (TPrA) co-reactant using supercapacitor power and ECL was captured with a CCD camera. The supercapacitor was rapidly photo-recharged between assays using an inexpensive solar cell. Detection limits were 300-500f gmL(-1) for the 3 proteins in undiluted calf serum. Assays of 6 prostate cancer patient serum samples gave good correlation with conventional single protein ELISAs. This technology could provide sensitive onsite cancer diagnostic tests in resource-limited settings with the need for only moderate-level training.

Automated multiplexed ECL Immunoarrays for cancer biomarker proteins.

Point-of-care diagnostics based on multiplexed protein measurements face challenges of simple, automated, low-cost, and high-throughput operation with high sensitivity. Herein, we describe an automated, microprocessor-controlled microfluidic immunoarray for simultaneous multiplexed detection of small protein panels in complex samples. A microfluidic sample/reagent delivery cassette was coupled to a 30-microwell detection array to achieve sensitive detection of four prostate cancer biomarker proteins in serum. The proteins are prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interlukin-6 (IL-6). The six channel system is driven by integrated micropumps controlled by an inexpensive programmable microprocessor. The reagent delivery cassette and detection array feature channels made by precision-cut 0.8 mm silicone gaskets. Single-wall carbon nanotube forests were grown in printed microwells on a pyrolytic graphite detection chip and decorated with capture antibodies. The detection chip is housed in a machined microfluidic chamber with a steel metal shim counter electrode and Ag/AgCl reference electrode for electrochemiluminescent (ECL) measurements. The preloaded sample/reagent cassette automatically delivers samples, wash buffers, and ECL RuBPY-silica-antibody detection nanoparticles sequentially. An onboard microcontroller controls micropumps and reagent flow to the detection chamber according to a preset program. Detection employs tripropylamine, a sacrificial reductant, while applying 0.95 V vs Ag/AgCl. Resulting ECL light was measured by a CCD camera. Ultralow detection limits of 10-100 fg mL(-1) were achieved in simultaneous detection of the four protein in 36 min assays. Results for the four proteins in prostate cancer patient serum gave excellent correlation with those from single-protein ELISA.

Electrochemiluminescent Reaction between Ru(bpy)32+ and Oxygen in Nafion Film

For years, dissolved oxygen (O2) has been regarded as a quenching species in Ru(bpy)32+ electrochemiluminescent (ECL) systems; however, in the present study, O2 was found to act as a coreactant for Ru(bpy)32+ ECL in Nafion film, resulting in a strong ECL light emission. ECL experiments were carried out at a Ru(bpy)32+/Nafion film-modified glassy carbon electrode (GCE) immersing in air-saturated phosphate buffer solution (pH 7.4). Scanning in the potential range of +1.5 to −1.0 V resulted in three luminescent processes, including two potential-dependent luminescence peaks (ECL-1 and ECL-2), and one potential-independent persistent luminescence emission (CL-P). Therein, ECL-2 occurring at potential less than −0.5 V was demonstrated to be a new chemiluminescent reactions between O2 and Ru(bpy)32+. In ECL-2, Nafion film plays important roles in stabilizing Ru(bpy)33+ and O2•− radical essentially for producing the new ECL. Unlike previously reported ECL processes, ECL-2 peak potential is dependent on the reduction potential of the coreactant (i.e., O2) rather than the redox potentials of the luminopore, Ru(bpy)32+, which would provide a useful way to probe O2, O2•− radical, and their stabilities in electrochemical reactions.

Coreactant Enhanced Anodic Electrochemiluminescence of CdTe Quantum Dots at Low Potential for Sensitive Biosensing Amplified by Enzymatic Cycle

This work used sulfite as a coreactant to enhance the anodic electrochemiluminescence (ECL) of mercaptopropionic acid modified CdTe quantum dots (QDs). This strategy proposed the first coreactant anodic ECL of QDs and led to a sensitive ECL emission of QDs in aqueous solution at relatively low potential. In the presence of dissolved oxygen, the stable ECL emission resulted from the excited QDs. Thus, an ECL detection method was proposed at +0.90 V (vs Ag/AgCl) based on the quenching of excited QDs by the analyte. Using tyrosine as a model compound, whose electrooxidized product could quench the excited QDs and thus the ECL emission, an analytical method for detection of tyrosine in a wide concentration range was developed. Furthermore, by combining an enzymatic cycle of trace tyrosinase to produce the oxidized product with an energy-transfer process, an extremely sensitive method for ECL detection of tyrosine with a subpicomolar limit of detection was developed. The sulfite-enhanced anodic ECL emission provided an alternative for traditional ECL light emitters and a new methodology for extremely sensitive ECL detection of mono- and dihydroxybenzenes at relatively low anodic potential. This strategy could be easily realized and opened new avenues for the applications of QDs in ECL biosensing.

The potential-resolved electrochemiluminescent behavior of calcein in aqueous alkaline solutions at a glassy carbon electrode

In aqueous alkaline solutions, the potential-resolved electrochemiluminescent (ECL) behavior of calcein at a glassy carbon electrode was investigated. A sharp ECL light emission peak appeared near 0.8 V (vs. Ag/AgCl) under cyclic voltammogram (CV) conditions. In order to obtain a better understanding of the light emission, we investigated the effects of supporting electrolytes, the pH value, the presence of O 2 or N 2, and the concentration of calcein. The oxidative form of calcein reacted with hydroxyl radicals and oxygen, producing singlet excited oxygen that was identified as the emitter using the ECL, UV–vis absorption and fluorescence spectra. A possible mechanism for the behavior of calcein in this system was also proposed.

Disposable Screen‐Printed Carbon Electrodes for Dual Electrochemiluminescence/Amperometric Detection: Sequential Injection Analysis of Oxalate

AbstractThis work describes the development and application of an electrochemical cell specifically designed for disposable screen printed carbon electrodes (SPCE) suitable for simultaneous electrochemiluminescence (ECL) and amperometric detection in sequential injection analysis. The flow system with facility for photomultiplier tube via a fiber optic facing the SPCE is user‐friendly and makes the detection process very easy to operate. Instead of the need to constant deliver the chemiluminescence (CL) reagents to the reaction zone, sequential injection analysis allows a considerable reduction in the consumption of the sample and expensive CL reagents (such as Ru(bpy)$\rm{ _3^{2 + } }$ salts). The utility of the analyzer was demonstrated for the detection of oxalate based on the ECL reaction with Ru(bpy)$\rm{ _3^{2 + } }$. Under optimized conditions, in the presence of 100 μM Ru(bpy)$\rm{ _3^{2 + } }$, the linear ranges of peak current and ECL light intensity for oxalate distinctly varied from 10 μM to 5 mM and 0.1 μM to 100 μM, respectively. In other words, the linear detection can be covered over a four‐order range with the combination of these two signals.

Confocal Microscopy Imaging of Electrochemiluminescence at Double Band Microelectrode Assemblies: Numerical Solution of the Inverse Optical Problem

A realistic theoretical model describing the outcome of confocal microscopic imaging of electrochemiluminescence (ECL) light emission is derived for a two parallel band microelectrodes assembly operated under steady state. The model takes into account the experimental distortions ensuing from a) the specific finite shape of the sampling volume in confocal microscopy, b) the light arising directly from out-of-focus area but transmitted through the microscope diaphragm or c) transmitted after reflection from the polished platinum band electrodes. The model is based on a detailed optical, physico-mathematical and numerical analysis of the problem at hand, and on simulations of the concentration distribution of the species giving rise to the ECL generation. Its outcome allows the reconstruction of the real spatial distribution of ECL light emission based on the confocal microscopy measurements upon correcting for the effect of experimental distortions using numerical fitting procedure.

Development of an ordered microarray of electrochemiluminescent nanosensors

A microarray of electrochemiluminescent (ECL) nanosensors for remote detection is reported. Such nanosensor arrays were created on the distal face of coherent optical fibre bundles by adapting near-field optical probe and nanoelectrode methodologies. The fabrication process allows the production of high-density microarrays of nanosensors where each optical aperture is surrounded by a gold nanoring electrode. The initial architecture of the optical fibre bundle is retained and thus the microarray keeps its imaging properties. The electrochemical response of the array displays a steady-state current. This feature indicates that the nanoelectrodes forming the array can be considered as diffusively independent. In other words, each ring-shaped electrode of the array probes electrochemically a different micro-environment. We also show that this microdevice can be used as an ECL nanosensor microarray. Indeed, ECL light is initiated by the gold nanoring electrode in the presence of a co-reactant biospecies, NADH. A fraction of the isotropically electrochemically generated light is collected by the same aperture, transmitted by the corresponding fibre core and eventually imaged by a CCD camera. The gold coating therefore acts as an electrode material and also to confine the ECL light in each etched core. Such nanostructured microdevice integrates ECL-light generation, collection and imaging in a microarray format.

Remote NADH imaging through an ordered array of electrochemiluminescent nanoapertures

In this report, we present an ordered array comprising thousands of nanoapertures for the electrochemiluminescent (ECL) detection of NADH. It was fabricated on the distal face of a coherent optical fiber bundle. Such a high-density array of nanoapertures combines optical, imaging and electrochemical properties. Indeed, each nanoaperture is surrounded by a gold nanoring, which acts as an electrode material. The behavior of the array was characterized by cyclic voltammetry and it shows excellent electrochemical performances. NADH is the analyte, which is measured in presence of Ru(bpy) 3 2+. The ruthenium complex mediates the NADH oxidation and this coenzyme acts as a co-reactant in the ECL mechanism. ECL light is generated at the distal face of the array by each gold ring electrode. A fraction of this ECL light is collected by the corresponding nanoaperture, transmitted through the optical fiber bundle and finally imaged on the proximal face with a CCD camera. In this work, we show that NADH concentration is remotely detected by an oxidative-reductive ECL mechanism. We present also some preliminary results about the ECL process of NADH with Ru(bpy) 3 2+. The ECL behavior of NADH on gold surface is reported. The influence of the applied potential on the collected light intensity was investigated. The variation of the ECL intensity measured through the nanoaperture array with NADH concentration is linear. Remote ECL detection of NADH is spatially resolved over a large area with a micrometer resolution through the array. Therefore, such array integrates several complementary functions: ECL light generation, collection, transmission and remote imaging in an array format.