Electrogenerated Chemiluminescence Labels Research Articles

A conceptual framework for the development of iridium(iii) complex-based electrogenerated chemiluminescence labels.

Translation of the highly promising electrogenerated chemiluminescence (ECL) properties of Ir(iii) complexes (with tri-n-propylamine (TPrA) as a co-reactant) into a new generation of ECL labels for ligand binding assays necessitates the introduction of functionality suitable for bioconjugation. Modification of the ligands, however, can affect not only the photophysical and electrochemical properties of the complex, but also the reaction pathways available to generate light. Through a combined theoretical and experimental study, we reveal the limitations of conventional approaches to the design of electrochemiluminophores and introduce a new class of ECL label, [Ir(C^N)2(pt-TOxT-Sq)]+ (where C^N is a range of possible cyclometalating ligands, and pt-TOxT-Sq is a pyridyltriazole ligand with trioxatridecane chain and squarate amide ethyl ester), which outperformed commercial Ir(iii) complex labels in two commonly used assay formats. Predicted limits on the redox potentials and emission wavelengths of Ir(iii) complexes capable of generating ECL via the dominant pathway applicable in microbead supported ECL assays were experimentally verified by measuring the ECL intensities of the parent luminophores at different applied potentials, and comparing the ECL responses for the corresponding labels under assay conditions. This study provides a framework to tailor ECL labels for specific assay conditions and a fundamental understanding of the ECL pathways that will underpin exploration of new luminophores and co-reactants.

Highly selective electrogenerated chemiluminescence biosensor for simultaneous detection of matrix metalloproteinase-2 and matrix metalloproteinase-7 in cell secretions

Highly selective bioassays are extremely important in biological and chemical sensing. Herein, a selective biosensor incorporating sensitive electrogenerated chemiluminescence (ECL) is reported for simultaneous detection of two matrix metalloproteinases (MMPs) that are related to pathological development and progression of cancer. An Ir complex ((dfppy)2Ir(dcbpy)PF6, Ir1) and a Ru complex ((Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2, Ru1) were employed as potential resolved ECL labels. It was found that the Ru1 and Ir1 in the presence of tripropylamine could produce two well-separated strong ECL emissions at +0.9V and +1.4V vs Ag/AgCl, respectively, with a large difference of ECL peak potential (∼0.5V) at the gold electrode, which provided an access for potential-resolved detection of dual targets. A multiplexed biosensor was simply fabricated by co-immobilizing the Ir1 and Ru1 labeled peptides on a gold electrode via self-assembling technique. It was facile to determine MMP-2 and MMP-7 in a single run with detection limit as low as 5ng/mL and 10pg/mL, respectively. The advantages of the multiplexed ECL biosensor were further exemplified by analyzing two MMPs released from living HeLa, K562, HCT116 and macrophage cells. The multiplexity of the biosensor will open a new door for facile and rapid detection of multiple biomarkers.

An ultrasensitive electrogenerated chemiluminescence-based immunoassay for specific detection of Zika virus

Zika virus (ZIKV) is a globally emerging mosquito-transmitted flavivirus that can cause severe fetal abnormalities, including microcephaly. As such, highly sensitive, specific, and cost-effective diagnostic methods are urgently needed. Here, we report a novel electrogenerated chemiluminescence (ECL)-based immunoassay for ultrasensitive and specific detection of ZIKV in human biological fluids. We loaded polystyrene beads (PSB) with a large number of ECL labels and conjugated them with anti-ZIKV monoclonal antibodies to generate anti-ZIKV-PSBs. These anti-ZIKV-PSBs efficiently captured ZIKV in solution forming ZIKV-anti-ZIKV-PSB complexes, which were subjected to measurement of ECL intensity after further magnetic beads separation. Our results show that the anti-ZIKV-PSBs can capture as little as 1 PFU of ZIKV in 100 μl of saline, human plasma, or human urine. This platform has the potential for development as a cost-effective, rapid and ultrasensitive assay for the detection of ZIKV and possibly other viruses in clinical diagnosis, epidemiologic and vector surveillance, and laboratory research.

A Sensitive Electrochemiluminescence Immunosensor for Celiac Disease Diagnosis Based on Nanoelectrode Ensembles.

We report here the design of a novel immunosensor and its application for celiac disease diagnosis, based on an electrogenerated chemiluminescence (ECL) readout, using membrane-templated gold nanoelectrode ensembles (NEEs) as a detection platform. An original sensing strategy is presented by segregating spatially the initial electrochemical reaction and the location of the immobilized biomolecules where ECL is finally emitted. The recognition scaffold is the following: tissue transglutaminase (tTG) is immobilized as a capturing agent on the polycarbonate (PC) surface of the track-etched templating membrane. It captures the target tissue transglutaminase antibody (anti-tTG), and finally allows the immobilization of a streptavidin-modified ruthenium-based ECL label via reaction with a suitable biotinylated secondary antibody. The application of an oxidizing potential in a tri-n-propylamine (TPrA) solution generates an intense and sharp ECL signal, suitable for analytical purposes. Voltammetric and ECL analyses evidenced that the ruthenium complex is not oxidized directly at the surface of the nanoelectrodes; instead ECL is generated following the TPrA oxidation, which produces the TPrA•+ and TPrA• radicals. With NEEs operating under total overlap diffusion conditions, high local fluxes of these reactive radicals are produced by the nanoelectrodes in the immediate vicinity of the ECL labels, so that they efficiently generate the ECL signal. The radicals can diffuse over short distances and react with the Ru(bpy)32+ label. In addition, the ECL emission is obtained by applying a potential of 0.88 V versus Ag/AgCl, which is about 0.3 V lower than when ECL is initiated by the electrochemical oxidation of Ru(bpy)3(2+). The immunosensor provides ECL signals which scale with anti-tTG concentration with a linearity range between 1.5 ng·mL–1 and 10 μg·mL–1 and a detection limit of 0.5 ng·mL–1. The sensor is finally applied to the analysis of anti-tTG in human serum samples, showing to be suitable to discriminate between healthy and celiac patients.

Electrogenerated chemiluminescence biosensors for the detection of pathogenic bacteria using antimicrobial peptides as capture/signal probes

Two electrogenerated chemiluminescence (ECL) biosensors for selective and sensitive detection of Escherichia coli O157:H7 were first designed by employing the antimicrobial peptide Magainin I as a biological recognition element and the ruthenium complex (Ru1) as an ECL label. In a direct format, the biosensor (I) was fabricated by self-assembling the Ru1-labeled peptide taken as a capture and signal probe onto the gold electrode surface. This system showed that the decreased ECL intensity was logarithmically proportional to the concentration of E. coli O157:H7 in the range of 1.0×103–5.0×105CFU/mL, with a detection limit of 2.3×102CFU/mL. In a sandwich format, the biosensor (II) was fabricated by self-assembling the modified Magainin I taken as capture probe onto the gold electrode surface. After E. coli O157:H7 was captured by the capture probe on the biosensor (II) and then bound with the Ru1-labeled peptide as the signal probe, the increased ECL intensity was logarithmically proportional to the concentration of E. coli from 5.0×102CFU/mL to 5.0×105CFU/mL, with a detection limit of 1.2×102CFU/mL. Importantly, the designed ECL biosensors showed a satisfactory selectivity in discriminating the gram-negative E. coli O157:H7 from gram-positive bacteria, and pathogenic from nonpathogenic E. coli. The strategy of using a Ru1-labeled peptide as a capture/signal probe is a promising approach to designing ECL biosensors for highly sensitive and rapid detection of E. coli O157:H7; this approach could be extended to the design of ECL biosensors for other desired bacteria as well.

Color‐Tunable Electrogenerated Chemiluminescence of Ruthenium N‐Heterocyclic Carbene Complexes

AbstractRu(II) complexes 1–3 bearing various N‐heterocyclic carbene (NHC) ligands were synthesized, and their photophysical, electrochemical, and electrogenerated chemiluminescence (ECL) properties were discussed to evaluate a potential of their use as multicolor ECL labels. Interestingly, they exhibited ECL emission ranging from greenish‐yellow to red both in nonaqueous and mixed aqueous solutions, which might show the potential of the Ru(II) complexes as multicolor ECL labels.

Signal amplification aptamer biosensor for thrombin based on a glassy carbon electrode modified with graphene, quantum dots and gold nanoparticles

A novel electrogenerated chemiluminescence (ECL) assay for sensitive determination of thrombin is designed employing CdSe/ZnS quantum dots served as an ECL label. This ECL sensor is fabricated on graphene modified glassy carbon electrode which is then covered with a low surface coverage of gold nanoparticles (AuNPs). An aptamer is used to selectively recognize the target. The thiol-terminated aptamer is first immobilized on AuNPs/graphene modified electrode, and then thrombin is imported to form the aptamer–thrombin complexes. After blocking the nonspecifically bound oligonucleotides with MCH solution, another CdSe/ZnS quantum dots modified aptamer is hybridized with the free thiol-terminated aptamer to form a DNA complexe. A decreased ECL signal is observed upon recognition of the target thrombin. The integrated ECL intensity versus the concentration of thrombin is linear in the range from 0.01 to 50nM. The detection limit is 10fM. The present aptasensor also exhibits excellent selectivity, stability and reusability. This sensing system can provide a promising label-free model for aptamer-based compounds sensitive detection.

Improving the signal-to-noise ratio of an ECL-based sensor using ad hoc carbon nanotube electrodes

In this paper, we demonstrate that mechanically modified cylinder-shaped carbon nanotube (CNT) working electrodes (WEs), combined with an averaging processing algorithm, can increase electrogenerated chemiluminescence (ECL) limit of detection by more than one order of magnitude, compared to gold electrodes. With CNT WEs, we obtained a stable light emission that lasts for hundreds of voltammetric cycles. This stability was further exploited to increase the detection limit with a simple algorithm, based on mean calculation. Ad hoc fabricated sensors are characterized with a full-custom potentiostat testbed and software platform, using tris(2,2-bipyridyl)ruthenium (II) as ECL labels. Our measurement results show that the signal-to-noise ratio (SNR) improves by a factor of larger than 20 compared to standard gold WEs to reach a detection limit up to 40 pg μl−1.

Homogeneous electrogenerated chemiluminescence peptide-based method for determination of troponin I

A novel homogeneous electrogenerated chemiluminescence (ECL) peptide-based method for the determination of troponin I (TnI) was developed by using peptide (FYSHSFHENWPSK) as a molecular recognition element and ruthenium bis(2,2′-bipyridine) (2,2′-bipyridine-4,4′-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy)2(dcbpy)NHS) as an ECL label. Ru(bpy)2(dcbpy)NHS was covalently labeled onto the peptide via acylation reaction and was utilized as an ECL probe. In the presence of TnI, a decrease in ECL signal was observed upon the binding event between peptide and target TnI. The ECL intensity versus the concentration of TnI was linear in the range from 7.8 × 10−10 g mL−1 to 7.8 × 10−8 g mL−1 with a detection limit of 1.2 × 10−10 g mL−1. The relative standard deviation for 2.6 × 10−9 g mL−1 was 3.8%. The proposed method has been applied to assay TnI in human control serum with satisfactory results. This work demonstrates that the combination of small peptide with a highly sensitive ECL technique for homogeneous ECL peptide-based bioassay is a great promising approach for simple, sensitive and selective determination of proteins.

Ultrasensitive Eletrogenerated Chemiluminescence Immunoassay by Magnetic Nanobead Amplification

AbstractAn ultrasensitive electrogenerated chemiluminescence (ECL) immunoassay was proposed by using magnetic nanobeads (MNBs) as the carrier of ECL labels for ECL emission amplification. Carcinoembryonic antigen (CEA) and MNBs were initially immobilized on a platform in 1 : 1 molar ratio via sandwich immunoreaction. Subsequently, the MNBs were released from the platform and labeled with Ru(bpy)32+ species. After the MNBs with Ru(bpy)32+ were immobilized on an Au electrode, ECL of the Ru(bpy)32+ was measured for CEA determination. A linear relation between the ECL intensity and CEA concentration was obtained in a range of 1×10−14 to 3×10−13 mol/L (2.0 to 60 pg/mL) with a limit of detection of 8.0×10−15 mol/L (1.6 pg/mL).

Ultrasensitive detection of TNT in soil, water, using enhanced electrogenerated chemiluminescence

The ultrasensitive detection of 2,4,6-trinitrotoluene (TNT) was accomplished on the basis of sandwich-type TNT immunoassay combined with electrogenerated chemiluminescence (ECL) technology. Biotinylated anti-TNT species were attached to the surface of 1-μm diameter streptavidin-coated magnetic beads (MB) and 10-μm diameter avidin-coated polystyrene microspheres/beads (PSB) pre-loaded with ECL labels (∼7 billion hydrophobic ruthenium(II) tris(2,2′-bipyridine) (Ru II) molecules per bead) to form anti-TNT ↔ MB and anti-TNT ↔ PSB(Ru II) conjugates, respectively. Sandwich-type PSB(Ru II) ↔ anti-TNT < TNT > anti-TNT ↔ MB aggregates were formed when PSB(Ru II) ↔ anti-TNT was mixed with anti-TNT ↔ MB conjugates in the presence of analyte TNT and 2.0% bovine serum albumin blocking agent. The newly formed aggregates were magnetically separated from the aqueous reaction media and dissolved in acetonitrile containing 0.10 M tri- n-propylamine ECL coreactant–0.055 M trifluoroacetic acid–0.10 M tetrabutylammonium tetrafluoroborate electrolyte. ECL as well as cyclic voltammetric measurements were carried out with a potential scan from 0 to 2.8 V vs Ag/Ag +, and the integrated ECL intensity was found to be linearly proportional to the analyte TNT concentration over the range of 0.10–1000 ppt (pg mL −1). The limit of detection (≤0.10 ± 0.01 ppb) is about 600× lower as compared with the most sensitive TNT detection method in the literature, and the absolute detection limit in mass (∼0.1 pg) is only ∼0.5% of that from mass spectroscopy. The approach coupled with the standard addition method was applied to measure the TNT contaminations in soil and creek water samples collected from a military training base.

Electrogenerated chemiluminescence detection for deoxyribonucleic acid hybridization based on gold nanoparticles carrying multiple probes

A novel sensitive electrogenerated chemiluminescence (ECL) method for the detection deoxyribonucleic acid (DNA) hybridization based on gold nanoparticles carrying multiple probes was developed. Ruthenium bis(2,2′-bipyridine)(2,2′-bipyridine-4,4′-dicarboxylic acid)- N-hydroxysuccinimide ester (Ru(bpy) 2(dcbpy)NHS) was used as a ECL label and gold nanoparticle as a carrier. Probe single strand DNA (ss-DNA) was self-assembled at the 3′-terminal with a thiol group to the surface of gold nanoparticle and covalently labeled at the 5′-terminal of a phosphate group with Ru(bpy) 2(dcbpy)NHS and the resulting conjugate, (Ru(bpy) 2(dcbpy)NHS)-ss-DNA-Au, was taken as a ECL probe. When target analyte ss-DNA was immobilized on a gold electrode by self-assembled monolayer technique and then hybridized with the ECL probe to form a double-stranded DNA (ds-DNA), a strong ECL response was electrochemically generated. The ECL intensity was linearly related to the concentration of the complementary sequence (target ss-DNA) in the range from 1.0 × 10 −11 to 1.0 × 10 −8 mol L −1, and the linear regression equation was S = 57301 + 4579.6 lg C (unit of C is mol L −1). A detection limit of 5.0 × 10 −12 mol L −1 for target ss-DNA was achieved. The ECL signal generated from many reporters of ECL probe prepared is greatly amplified, compared to the convention scheme which is based on one reporter per hybridization event.

Hot electron-induced electrogenerated chemiluminescence of SYBR ® Green I

Hot electrons can be injected from conductor/insulator/electrolyte (C/I/E) junctions into an aqueous electrolyte solution. Injected hot electrons induce electrogenerated chemiluminescence (ECL) of various luminophores in fully aqueous solutions. Such ECL gives the basis of electrochemiluminoimmunoassays and DNA-probe assays where different luminophores can be used as electrochemiluminescent labels. This work shows that SYBR ® Green I is suitable as an ECL label in detection methods based on C/I/E tunnel-emission electrodes such as oxide-coated aluminium, magnesium and silicon electrodes.

Electrogenerated chemiluminescence. 77. DNA hybridization detection at high amplification with [Ru(bpy)3]2+-containing microspheres.

An ultrasensitive DNA hybridization detection method based on electrogenerated chemiluminescence (ECL) using polystyrene microspheres/beads (PSB) as the carrier of the ECL labels, namely, tris(2,2'-bipyridyl)ruthenium(II) tetrakis(pentafluorophenyl)borate (Ru(bpy)3[B(C6F5)4]2), is reported. Probe single-stranded DNA (p-ssDNA) was attached to the surface of magnetic beads (MB) and hybridized with target-ssDNA (t-ssDNA) with immobilized PSB containing a large number of water insoluble Ru(bpy)3[B(C6F5)4]2 species (approximately 7.5 x 10(9) molecules/bead). With this approach a large amplification factor of Ru(bpy)3[B(C6F5)4]2 molecules for each t-ssDNA can be achieved, when each PSB is attached to a limited number of t-ssDNA. The p-ssDNA-MB <--> t-ssDNA-PSB/Ru(bpy)3(2+) conjugates formed were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine (TPrA) as an ECL coreactant. ECL was produced with a potential scan from 0 to 3.0 V versus Ag/Ag+, and the integrated ECL intensity was found to be linearly proportional to the t-ssDNA concentration in a range of 1.0 fM to 10 nM under optimized conditions. ECL signals associated with two base pair mismatched ssDNA and noncomplementary ssDNA can be distinguished well from the ECL signal related to the complementary DNA hybridization. A Poisson distribution is followed when a large number of MB reacts with PSB, and the minimum number of 1.0- and 2.8-microm diameter MB required to bind and magnetically separate a single 10-microm diameter PSB from the reaction solution was estimated to be three and one, respectively. The principle described in this paper could be also applied to many other ECL analyses, such as immunoassays.

Electrogenerated chemiluminescence. 72. Determination of immobilized DNA and C-reactive protein on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) labels.

Anodic electrogenerated chemiluminescence (ECL) with tri-n-propylamine (TPrA) as a coreactant was used to determine DNA and C-reactive protein (CRP) by immobilizations on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) labels. A 23-mer synthetic single-stranded (ss) DNA derived from the Bacillus anthracis with an amino-modified group at the 5' end position was covalently attached to the Au(111) substrate precoated with a self-assembled thiol monolayer of 3-mercaptopropanoic acid (3-MPA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and then hybridized with a target ssDNA tagged with Ru(bpy)(3)(2+) ECL labels. Similarly, biotinylated anti-CRP species were immobilized effectively onto the Au(111) substrate precovered with a layer of avidin linked covalently via the reaction between avidin and a mixed thiol monolayer of 3-MPA and 16-mercaptohexadecanoic acid on Au(111) in the presence of EDAC and N-hydroxysuccinimide. CRP and anti-CRP tagged with Ru(bpy)(3)(2+) labels were then conjugated to the surface layer. ECL responses were generated from the modified electrodes described above by immersing them in a TPrA-containing electrolyte solution. A series of electrode treatments, including blocking free -COOH groups with ethanol amine, pinhole blocking with bovine serum albumin, washing with EDTA/NaCl/Tris buffer, and spraying with inert gases, were used to reduce the nonspecific adsorption of the labeled species. The ECL peak intensity was linearly proportional to the analyte CRP concentration over the range 1-24 microg/mL. CRP concentrations of two unknown human plasma/serum specimens were measured by the standard addition method based on this technique.