Electrochemiluminescence Bioassays with a Water‐Soluble Luminol Derivative Can Outperform Fluorescence Assays

The most efficient and commonly used electrochemiluminescence (ECL) emitters are luminol, [Ru(bpy)₃]²⁺, and derivatives thereof. Luminol stands out due to its low excitation potential, but applications are limited by its insolubility under physiological conditions. The water‐soluble m‐carboxy luminol was synthesized in 15 % yield and exhibited high solubility under physiological conditions and afforded a four‐fold ECL signal increase (vs. luminol). Entrapment in DNA‐tagged liposomes enabled a DNA assay with a detection limit of 3.2 pmol L^?1, which is 150 times lower than the corresponding fluorescence approach. This remarkable sensitivity gain and the low excitation potential establish m‐carboxy luminol as a superior ECL probe with direct relevance to chemiluminescence and enzymatic bioanalytical approaches.     

Determination of Hg2+ in Tap Water Based on the Electrochemiluminescence of Ru(phen)32+ and Thymine at Bare and Graphene Oxide‐Modified Glassy Carbon Electrodes

The electrochemiluminescence (ECL) of the Ru(phen)₃²⁺/thymine (T) system at bare and graphene oxide (GO)‐modified glassy carbon (GC) electrodes was utilized to determine Hg²⁺ in tap water. The ECL intensity of Ru(phen)₃²⁺ was considerably enhanced by the addition of thymine because of the occurrence of ECL reaction between them. Subsequently, the ECL intensity of Ru(phen)₃²⁺/T system rapidly decreased with the addition of Hg²⁺ because of the formation of a T‐Hg²⁺‐T complex. A linear response (R²=0.9914) was obtained over a Hg²⁺ concentration range of 1.0×10^?9 mol/L to 1.0×10^?5 mol/L with a detection limit of 3.4×10^?10 mol/L at a bare GC electrode in 0.1 mol/L phosphate buffer (pH=8.0). The detection limit can be further reduced to 4.2×10^?12 mol/L after modification of the GC electrode by GO. To verify its applicability, the proposed method was utilized to determine Hg²⁺ in tap water and simulated wastewater. The method exhibited good reproducibility and stability and thus reveals the possibility of developing a novel ECL detection method for Hg²⁺.     

Electrochemiluminescence Quenching by Halide Ions at Bipolar Electrodes

Quenching of Ru(bpy)₃²⁺ electrochemiluminescence (ECL) by Cl^?, Br^?, and I^? ions was studied as a function of halide concentration in a bipolar electrochemical cell. All of the halides investigated showed similar qualitative behavior: above a critical concentration, ECL intensity was found to decrease linearly as the halide ion concentration was increased, due to dynamic quenching of Ru(bpy)₃²⁺ ECL. Stern‐Volmer slopes (K_SV) of 0.111±0.003, 4.2±0.3, and 6.2±0.3 mM^?1 were measured for Cl^?, Br^? and I^?, respectively. The magnitude of K_SV correlates with halide ion oxidation potential, consistent with an electron transfer quenching mechanism. Using the bipolar platform described herein, aqueous, halide‐containing solutions could be quantified rapidly using the sequential standard addition method. The lower detection limit is determined by a complex mechanism involving the competitive electrooxidation of halide ions and the ECL co‐reactants, as well as the passivation of the surface of the bipolar electrode, and was found to be 0.20±0.01, 0.08±0.01 and 10±1 mM, respectively, for I^?, Br^?, and Cl^?. The performance of the bipolar ECL quenching assay is comparable to previously published fluorescence quenching methods for the determination of halide ions, while being much simpler and less expensive to implement.     

A Solid‐State Electrochemiluminescence Immunosensor Based on MWCNTs‐Nafion and Ru(bpy)32+/Nano‐Pt Nanocomposites for Detection of α‐Fetoprotein

A approach was successfully employed for constructing a solid‐state electrochemiluminescence (ECL) immunosensor by layer‐by‐layer self‐assembly of multiwall carbon nanotubes (MWCNTs)‐Nafion composite film, Ru(bpy)₃²⁺/nano‐Pt aggregates (Ru‐PtNPs) and Pt nanoparticles (PtNPs). The influence of Pt nanoparticles on the ECL intensity was quantitatively evaluated by calculating the electroactive surface area of different electrodes with or without PtNPs to immobilize Ru(bpy)₃²⁺. The principle of ECL detection for target α‐fetoprotein antigen (AFP) was based on the increment of resistance after immunoreaction, which led to a decrease in ECL intensity. The linear response range was 0.01–10 ng mL^?1 with the detection limit of 3.3 pg mL^?1. The immunosensor exhibited advantages of simple preparation and operation, high sensitivity and good selectivity.     

Electrochemiluminescence of SDBS‐Ru(bpy)32+‐CPM System and Its Application

When the concentration of dodecyl benzene sulfonic acid sodium salt (SDBS) is 0.7 mmol·L^?1, the electrochemical and electrochemiluminescence (ECL) intensity of Ru(bpy)₃²⁺‐chlorpheniramine maleate (CPM) system at the Au electrode were studied. The results showed that compared with the absence of SDBS, enhancement of the ECL intensity was 14‐fold at Au electrode. Base on this, an ECL method was established for efficient and simple determination of CPM at Au electrode. Under the optimum experimental condition, the enhanced ECL intensities had good linear relationship with the concentration of CPM in the range of 1.0×10^?4–1.0×10^?7 mol·L^?1, and a linear regression equation was obtained as follows: I (counts)=48.805×10⁶c+394.03 (r=0.9975), the detection limit for CPM was 1.4×10^?8 mol·L^?1. The RSD for 5 times determinations of 1.0×10^?5 mol·L^?1 CPM was 3.2%. The results of recovery test were between 96.3%–102.5%, and the RSD of recovery test (n=5) was 2.7%. In addition, eleven kinds of tertiary amines‐Ru(bpy)₃²⁺ systems were investigated in the absence and presence of SDBS. The results showed that the enhancement of SDBS on ECL intensity of tertiary amines‐Ru(bpy)₃²⁺ systems was universal.     

Comparison of homoleptic and heteroleptic 2,2′-bipyridine and 1,10-phenanthroline ruthenium complexes as chemiluminescence and electrochemiluminescence reagents in aqueous solution

We have conducted a comprehensive comparative study of Ru(bipy)₃²⁺, Ru(bipy)₂(phen)²⁺, Ru(bipy)(phen)₂²⁺, and Ru(phen)₃²⁺ as chemiluminescence and electrochemiluminescence (ECL) reagents, to address several previous conflicting observations and gain a greater insight into their potential for chemical analysis. Clear trends were observed in many of their spectroscopic and electrochemical properties, but the relative chemiluminescence or ECL intensity with a range of analytes/co-reactants is complicated by the contribution of numerous (sometimes opposing) factors. Significantly, the reversibility of cyclic voltammetric responses for the complexes decreased as the number of phenanthroline ligands was increased, due to the lower stability of their ruthenium(III) form in the aqueous solvent. This trend was also evident over a longer timescale when the ruthenium(III) form was spectrophotometrically monitored after chemical oxidation of the ruthenium(II) complexes. In general, the greater stability of Ru(bipy)₃³⁺ resulted in lower blank signals, although this effect was less pronounced with ECL, where the reagent is oxidised in the presence of the co-reactants. Nevertheless, this shows the need to compare signal-to-blank ratios or detection limits, rather than the more common comparisons of overall signal intensity for different ruthenium complexes. Furthermore, our results support previous observations that, compared to Ru(bipy)₃²⁺, Ru(phen)₃²⁺ provides greater ECL and chemiluminescence intensities with oxalate, which in some circumstances translates to superior detection limits, but they do not support the subsequent generalised notion that Ru(phen)₃²⁺ is a more sensitive reagent than Ru(bipy)₃²⁺ for all analytes.     

Highly Sensitive Electrochemiluminescence Detection of Mercury(II) Ions Based on DNA‐Linked Luminol‐Au NPs Superstructure

A novel highly sensitive electrochemiluminescence (ECL) detection protocol for mercury(II) ions was developed. Based on the strong and stable thymine? thymine mismatches complexes coordination chemistry, mercury(II) ions can specifically bind to a designed DNA strand, leading to the release of the complimentary DNA strand. The released DNA strand was then captured by magnetic beads modified with specific DNA, and then through the formation of DNA‐linked luminol‐Au nanoparticles (NPs) superstructure, a specific ECL system for mercury(II) ions was developed. Using 3‐aminopropyl‐triethoxysilane as an effective enhancer, the ECL system can detect Hg²⁺ ion within a linear range from 2.0×10^?10 mol L^?1 to 2.0×10^?8 M, with a detection limit as low as 1.05×10^?10 M (3σ). Moreover, this ECL system is highly specific for Hg²⁺, without interference from other commonly coexisted metal ions, and it can be used for the analysis of real samples.     

Determination of Reduced Nicotinamide Adenine Dinucleotide Based on Immobilization of Tris(2,2′‐bipyridyl) Ruthenium(II) in Multiwall Carbon Nanotubes/Nafion Composite Membrane

Abstract

An electrochemiluminescence (ECL) method for reduced nicotinamide adenine dinucleotide (NADH) was proposed by immobilizing tris(2,2′‐bipyridyl) ruthenium(II) (Ru(bpy)₃ ²⁺) in multiwall carbon nanotubes (MWCNTs)/Nafion composite membrane that was formed on glassy carbon electrode surface. The electrochemical and ECL behaviors of the immobilized Ru(bpy)₃ ²⁺ were investigated. The cyclic votammogram of the modified electrode in pH 7.0 phosphate buffer solution showed a couple of redox peaks at +1190 and +1060 mV at 100 mV/s. The composite film had a more open structure and a large surface area allowing faster diffusion of Ru(bpy)₃ ²⁺. The presence of MWCNTs resulted in the improved ECL sensitivity and longer‐term stability of the modified electrode. The modified electrode showed a linear response to NADH in the concentration range of 1.0×10^?6 to 1.6×10^?5 M with a detection limit of 8.2×10^?7 M.     

Highly Sensitive and Selective Detection of Pb(II) by NH2−SiO2/Ru(bpy)32+−UiO66 based Solid‐state ECL Sensor

In this study, a novel electrochemiluminescence (ECL) sensor for highly sensitive and selective detection of Pb(II) was developed based on Ru(bpy)₃²⁺ encapsulated UiO66 metal‐organic‐framework (Ru(bpy)₃²⁺?UiO66 MOF) and ?NH₂ group functionalized silica (NH₂?SiO₂). The NH₂?SiO₂ with large surface area provided an excellent platform for the ECL sensor. As numerous exposed carboxyl groups were present on UiO66 backbone, the Ru(bpy)₃²⁺?UiO66 could be steadily immobilized to NH₂?SiO₂ by forming amide bonds. Meanwhile, the introduced UiO66 MOF which used for the encapsulation of Ru(bpy)₃²⁺, significantly enhanced the ECL efficiency of the proposed sensor, as it possessed a large specific surface area and porosity for the loading of Ru(bpy)₃²⁺. Moreover, a high quenching effect on ECL intensity was obtained in the presence of Pb(II) in the electrolyte. Under the optimal conditions, the quenched ECL intensity showed a good linear relationship within Pb(II) concentration in the range from 1.0×10^?6 to 1.0×10² μM, with a detection limit of 1.0×10^?7 μM (S/N=3). The proposed sensor for Pb(II) detection was simple in operation, rapid in testing, stable in signal, and showed a good anti‐interference ability to some other metal ions. Besides, its application for detecting Pb(II) in a real sample was also investigated here. This work provides a potential platform for metal ions detection in environmental monitoring field.     

Enhanced Anodic Electrochemiluminescence of Dissolved Oxygen with 2‐(Dibutylamino) ethanol at TiO2 Nanoparticles Modified Platinum Electrode for Dopamine Detection

The anodic electrochemiluminescence (ECL) of dissolved oxygen with 2‐(dibutylamino) ethanol (DBAE) on platinum electrode has been reported previously by our group. Interestingly, the ECL intensity can be greatly amplified at TiO₂ nanoparticles modified platinum electrode (TiO₂/Pt), which is due to the catalytic effect of TiO₂ nanoparticles to electrochemical oxidation of DBAE. It is the first case to obtain the enhanced ECL from luminophor by electrochemical catalysis of co‐reactant. The enhanced anodic ECL intensity can be quenched by dopamine sensitively. And the ECL intensity versus the logarithm of concentration of dopamine was linear over the 4.0×10^?12–1.8×10^?8 M (R²=0.9957), with the limit of detection of 2.7×10^?12 M (S/N=3).     

Electochemiluminescence of CdTe/CdS Quantum Dots with Triproprylamine as Coreactant in Aqueous Solution at a Lower Potential and Its Application for Highly Sensitive and Selective Detection of Cu2+

Anodic electrochemiluminescence (ECL) of 3‐mercaptopropionic acid (MPA)‐ capped CdTe/CdS core‐shell quantum dots (QDs) with tripropylamine (TPrA) as the co‐reactant were studied in aqueous (Tris buffer) solution for the first time. The results suggest that the oxidation of TPrA at a glassy carbon electrode (GCE) surface participated in the ECL of QDs, and the onset potential and the intensity of ECL of CdTe/CdS QDs were affected seriously by TPrA, as the co‐reactant, in Tris buffer solution. The onset potential of ECL in this new system was about +0.5 V (vs. Ag/AgCl) and the ECL intensity greatly enhanced when TPrA was present. Various influencing factors, such as the electrolyte, pH, QDs concentration, potential range and scan rates on the ECL were studied. Based on the selective quenching by Cu²⁺ to the light emission from CdTe/CdS QDs/TPrA system, a highly sensitive and selective method for the determination of Cu²⁺ was developed. At the optimal conditions, the relative ECL intensity, I₀/I, was proportional to the concentration of Cu²⁺ from 14 nM to 0.21 μM with the detection limit of 6.1 nM based on the signal‐to‐noise ratio of 3. The possible ECL mechanism of QDs and the quenching mechanism of ECL were proposed.     

Study on Electrochemiluminesce of Ru(bpy3)2+ Immobilized in a Titania Sol‐Gel Membrane

The immobilization of tris(2,2′‐bipyridyl)ruthenium(II), Ru(bpy)₃²⁺, at a glassy carbon electrode was achieved by entrapping the Ru(bpy)₃²⁺ in a vapor deposited titania sol‐gel membrane. The electrogenerated chemiluminescence (ECL) of the immobilized Ru(bpy)₃²⁺ was studied. The Ru(bpy)₃²⁺ modified electrode showed a fast ECL response to both oxalate and proline. The ECL intensity was linearly related to concentrations of oxalate and proline over the ranges from 20 to 700 μmol L^?1 and 20 to 600 μmol L^?1, respectively. The detection limits for oxalate and proline at 3σ were 5.0 μmol L^?1 and 4.0 μmol L^?1, respectively. This electrode possessed good precision and stability for oxalate and proline determinations. The electrogenerated chemiluminescence mechanism of proline system was discussed. This work provided a new way for the immobilization of Ru(bpy)₃²⁺ and the application of titania sol‐gel membrane in electrogenerated chemiluminescence.     

Enhanced electrochemiluminescence from Os(phen)2(dppene) (phen=1,10-phenanthroline and dppene=bis(diphenylphosphino)ethene) in the presence of Triton X-100 (polyethylene glycol tert-octylphenyl ether)

The effects of the non-ionic surfactant Triton X-100 (polyethylene glycol tert-octylphenyl ether) on the properties of Os(phen)₂(dppene)²⁺ (phen=1,10-phenanthroline and dppene=bis(diphenylphosphino)ethene) electrochemiluminescence (ECL) have been investigated. The anodic oxidation of Os(phen)₂(dppene)²⁺ produces ECL in the presence of tri-n-propylamine (TPrA) in aqueous surfactant solution. Increases in ECL efficiency (≥3-fold) and TPrA oxidation current (≥2.0-fold) have been observed in surfactant media with experiments supporting adsorption of surfactant on the electrode surface.     

Electrochemiluminescence of Supramolecular Nanorods and Their Application in the “On–Off–On” Detection of Copper Ions

In this work, an “on–off–on” switch system has been successfully applied through the construction of an electrochemiluminscent biosensor for copper ion (Cu²⁺) detection based on a new electrochemiluminescence (ECL) emitter of supramolecular nanorods, which was achieved through supramolecular interactions between 3,4,9,10‐perylenetetracarboxylic acid (PTCA) and aniline. The initial “signal‐on” state with strong and stable ECL emission was obtained by use of the supramolecular nanorods with a new signal amplification strategy involving a co‐reaction accelerator. In addition, ECL quencher probes (Fc‐NH₂/Cu‐Sub/nano‐Au) were fabricated by immobilizing aminoferrocene (Fc‐NH₂) on Cu‐substrate strand modified Au nanoparticles. The quencher probes were hybridized with the immobilized Cu‐enzyme strand to form Cu²⁺‐specific DNAzyme. Similarly, the “signal‐off” state was obtained by the high quenching effect of Fc‐NH₂ on the ECL of the excited‐state PTCA (¹PTCA*). As expected, the second “switch‐on” state could achieved by incubating with the target Cu²⁺, owing to the Cu²⁺‐specific DNAzyme, which was irreversibly cleaved, resulting in the release of the quencher probes from the sensor interface. Herein, on the basis of the ECL intensity changes (ΔI_ECL) before and after incubating with the target Cu²⁺, the prepared Cu²⁺‐specific DNAzyme‐based biosensor was used for the determination of Cu²⁺ concentrations with high sensitivity, excellent selectivity, and good regeneration.     

Monitoring casein kinase II at subcellular level via bio-bar-code-based electrochemiluminescence biosensing method

A highly sensitive electrochemiluminescence (ECL) biosensing method was developed for monitoring casein kinase II (CK2) at subcellular level via bio-bar-code assay. A bio-bar-code probe (h-DNA/AuNPs/p-DNA) prepared by conjugating phosphorylated DNA (p-DNA) and hairpin DNA (h-DNA) onto gold nanoparticles (AuNPs) was used as a carrier for ECL signal reagent (Ru(phen)₃²⁺) while a specific peptide was used as a recognition substance. A gold ultramicroelectrode with a diameter of 400 nm was fabricated and then modified with the specific peptide via self-assembly technique to obtain peptide modified gold ultramicroelectrode. The peptide on gold ultramicroelectrode was phosphorylated in the presence of CK2 and adenosine 5′-triphosphate, and then the phosphorylated peptide was integrated with the h-DNA/AuNPs/p-DNA through a process mediated by zirconium cations (Zr⁴⁺), and finally Ru(phen)₃²⁺ was intercalated into h-DNA. A “signal on” ECL method was developed for the detection of CK2 in the range of 0.005–0.2 U/mL with a detection limit of 0.001 U/mL. Additionally, combined efficient subcellular phosphorylation in vivo with bio-bar-code-based ECL biosensing method, the ECL method was further applied to monitor CK2 at subcellular level without tedious subcellular fractionation. It was found that the concentration of CK2 by inserting the peptide modified gold ultramicroelectrode into the nucleus was higher than that into cytoplasm of HeLa cells. A distinct heterogeneity among CK2 concentrations in single cells was observed for cellular heterogeneity assessment.     

Electrochemiluminescent Detection Method for Glyphosate in Soybean on Carbon Fiber-ionic Liquid Paste Electrode

A carbon fiber paste electrode using ionic liquid as the binder (CFILE) was fabricated. The electrochemical characteristics of the electrode was examined in ferro‐/ferricyanide solution and showed better conductivity and reversibility when compared with graphite paste‐ionic liquid electrode (GPILE) and a little better than that on the carbon nanotube paste‐ionic liquid electrode (CNTILE). Glyphosate (GLY), a pesticide, exhibited excellent catalysis to the oxidation of Ru(bpy)²⁺₃ on CFILE and brought an obvious enhancement to the electrochemiluminescence (ECL) intensity of Ru(bpy)²⁺₃. Based on the catalytic ability of GLY, a simple ECL method for GLY detection had been established. Under optimum conditions, the enhanced ECL intensities were found to had linearly respond to the GLY concentration between 3.0×10^?7 and 3.0×10^?5 mol/L, and the detection limit (S/N=3) was 2.0×10^?7 mol/L. The electrode also showed excellent sensitivity in detecting GLY‐spiked soybean samples. The linear range for GLY in soybean samples was 1.0×10^?6–4.0×10^?5 mol/L and the detection limit was 5.0×10^?7 mol/L, equal to 8.45 µg GLY in per gram of soybean. The detection limit in soybean sample was lower than the USA, EU regulation and so on. If the method is coupled with the separation technology, it can be applied to detect the GLY in the contaminated samples.     

Application of capillary electrophoresis coupling with electrochemiluminescence detection to estimate activity of leucine aminopeptidas

A new method to estimate the leucine aminopeptidase (LAP, EC 3.4.11.1) activity using capillary electrophoresis coupled with electrochemiluminescence (ECL) is described. The liberated proline produced by LAP catalyzing the hydrolysis reaction of leucin–proline was used as an ECL coreagent to enhance Ru(bpy)₃²⁺ ECL signals efficiently. The detection limit for proline was 2.88 × 10^?6 m (signal‐to‐noise ratio 3), which was equal to 9.60 × 10^?8 units of LAP being used to catalyze leucin–proline for 1 min. The Michaelis constant K_m (2.07 × 10^?2 mol/L) and the maximum reaction velocity V_max (1.06 × 10^?5 mol/L/min) of LAP for leucin–proline are reported. The reaction conditions including the concentration of metal ions, incubation temperature and pH were optimized. This method was successfully applied to detect LAP activity in plasma and the results were in good agreement with that obtained by the clinical method. Copyright © 2013 John Wiley & Sons, Ltd.     

Successful establishment of MEKC with electrochemiluminescence detection based on one functionalized ionic liquid

Herein, one water‐soluble functionalized ionic liquid, 1‐butyl‐3‐methylimidazolium dodecyl sulfate ([BMIm⁺][C₁₂H₂₅SO^?₄]), was designed and its superiorities either used as supporting electrolytes or as additives for successful establishment of MEKC with electrochemiluminescence (ECL) detection (MEKC‐ECL) method were investigated. Compared with the common supporting electrolytes such as phosphate solution, 1‐butyl‐3‐methylimidazolium dodecyl sulfate solution used as running buffers led to greatly enhanced ECL intensities and column efficiencies for negative targets, a little increase for neutral‐charge ones while maintained nearly unchanged for positive ones due to the electrostatic forces between the large cation BMIm⁺ and the solutes and the hydrophobic interactions resulting from the large anion C₁₂H₂₅SO^?₄. Moreover, resolution efficiency between analytes was significantly improved. As the existence of ionic liquid moiety, BMIm⁺, accelerated the electron transfer at the electrode surface, the poisoning effect of long chain C₁₂H₂₅SO^?₄ on the electrode surface was eliminated and reproducible ECL intensities with relative standard derivation of 1.02% (n=6) were obtained. The proposed novel MEKC‐ECL system was again validated by the baseline separated two similar amino acids of proline and hydroxyproline with lower detection limits of 0.5 and 0.8 μM (S/N=3), respectively.     

A Novel Surface‐tethered Analysis Method for Mercury (II) ion Detection via Self‐assembly of Individual Electrochemiluminescence Signal Units

A novel analysis strategy based on the analyte‐induced surface‐tethered (AIST) of electrochemiluminescence (ECL) signal nanoparticles was first proposed for detection of Mercury (II) ion (Hg²⁺). In this work, luminol@Au‐cysteamine‐thymine (luminol@Au‐Cys‐T) multifunctional nanoarchitecture was designed as ECL signal units in the experimental process. Through a specific T‐Hg²⁺‐T coordination, luminol@Au‐Cys‐T composites were gravitationally self‐assembled to the electrode surface, which was coated with tris (2‐aminoethyl) amine functionalized graphene oxide@perylene‐3,4,9,10‐tetracarboxylic acid‐thymine (GO@PTCA‐TAEA‐T) complex film. This simple strategy of AIST of signal molecules could amplify the response signal and vastly enhance the sensitivity. Under the optimum condition, the linear relationship of Hg²⁺ concentration variation from 0.005 nM to 5 nM with a limit of detection (LOD) down to 0.002 nM (S/N=3), which also offered an alternative analytical approach with excellent performance of stability and selectivity. The regression equation was y=−414.52+2305.02 lgC_(Hg²⁺₎ (pM). This Hg²⁺ ECL sensor had a good application prospects for the analysis of real samples.     

Dual Intramolecular Electron Transfer for In Situ Coreactant‐Embedded Electrochemiluminescence Microimaging of Membrane Protein

The demand for transporting coreactant to emitter and short lifetime of the radicals in electrochemiluminescence (ECL) emission inhibit greatly its application in cytosensing and microscopic imaging. Herein we designed a dual intramolecular electron transfer strategy and tertiary amine conjugated polymer dots (TEA‐Pdots) to develop a coreactant‐embedded ECL mechanism and microimaging system. The TEA‐Pdots could produce ECL emission at +1.2 V without need of coreactant in test solution. The superstructure and intramolecular electron transfer led to unprecedented ECL strength, which was 132 and 45 times stronger than those from the mixture of Pdots with TEA at equivalent and 62.5 times higher amounts, respectively. The ECL efficiency was even higher than that of typical [Ru(bpy)₃]²⁺ system. Therefore, this strategy and coreactant‐embedded ECL system could be used for in situ ECL microimaging of membrane protein on single living cells without additional permeable treatment for transporting coreactant. The feasibility and validity were demonstrated by evaluating the specific protein expression on cell surface. This work opens new avenues for ECL applications in single cell analysis and dynamic study of biological events.