Efficient Electrochemiluminescence Signal Research Articles

High-efficiency electrochemiluminescence of host-guest ligand-assembled gold nanoclusters preoxidated for ultrasensitive biosensing of protein

Herein, host–guest ligand-assembled gold nanoclusters (AuNCs) with highly efficient electrochemiluminescence (ECL) as an emitter and a programable tripedal deoxyribonucleic acid (DNA) walker with a well-regulated DNA track as the signal amplifier were used to develop an ultrasensitive biosensing platform for determining soluble urokinase-type plasminogen activator receptor (suPAR) related to chronic kidney disease. Notably, the host–guest ligand-assembled AuNCs with 6-aza-2-thiothymine (ATT) as the ligand and guanidine butyric acid (Gba) as the model (Gba/ATT-AuNCs) exhibited a strong and stable ECL signal under preoxidation, 25.6 times higher than that of traditional ATT-protected AuNCs (ATT-AuNCs). This is because of the dual-enhancement ECL mechanism of the suppression of nonradiative transitions by host–guest recognition and the improvement of the electrochemical redox rate by preoxidation. Furthermore, the trace target protein suPAR could be efficiently converted into a tripedal DNA walker to walk along a well-regulated square DNA orbit with high movement efficiency and low background signal, thereby considerably improving detection sensitivity. Thus, the established ECL sensing platform achieved the ultrasensitive detection of suPAR with a linear range from 10 fg/mL to 10 ng/mL and a limit of detection of 3.15 fg/mL, whose detection sensitivity was five orders of magnitude higher than that of the gold-standard enzyme-linked immunosorbent assay with the lowest detection concentration of 1 ng/mL. This strategy provides a novel enhanced ECL mode of AuNCs for constructing a sensitive biosensing platform and is expected to be extended to the trace detection of other protein markers in human body fluids for disease diagnosis.

Visualized monitoring of radioactive iodide ions in environment using electrochemiluminescence device through free radical annihilation mechanism

The monitoring of radioactive iodide ions in environment is an apparent challenge in the field of environmental radiochemistry. Herein, we report an efficient electrochemiluminescence (ECL) array detection device to achieve visualized rapid monitoring and accurate warning of trace radioactive iodide in the environment. In detail, we prepared conjugated polymer poly[(9,9-dioctylfuor-enyl-2,7-diyl)-alt-co-(1,4-benzo-{2,1,3}-thiadiazole)] (PFBT) into polymer dots and modified it on ITO electrode to obtain an array ECL detection device, of which the ECL signal is quenched by iodide ions. This detection device gives a low limit of detection (17 ng/L) and an excellent selectivity to iodide ion, as well as realizing visualized rapid monitoring of trace iodide in natural water samples by using ECL imaging technology. A new iodide detection mechanism, free radical annihilation (FRA), is presented, which involves an ECL co-reactant reagent poisoning process by iodine free radical obtained on the surface of electrode and is further demonstrated by electrochemical experiments, DFT calculations and ESP analysis. This work provides an efficient strategy for the visualized, rapid and accurate monitoring and warning of trace radioactive iodide in water environment, showing significant meaningful prospects in the field of public security and health.

Selenium and nitrogen co-doped carbon dots with highly efficient electrochemiluminescence for ultrasensitive detection of microRNA

In this work, selenium and nitrogen co-doped carbon dots (SeN-CDs) possessing highly efficient electrochemiluminescence (ECL) and excellent biocompatibility were synthesized as a new emitter with S2O82− as a coreactant for constructing a biosensor to detect microRNA-221 (miRNA-221) sensitively. Notably, the SeN-CDs exhibited superior ECL performance compared with the N-doped CDs, in which selenium with excellent redox activity served as a coreaction accelerator for facilitating the electroreduction of S2O82− to significantly improve ECL efficiency. Furthermore, target-induced T7 exonuclease (T7 Exo)-assisted double cycle amplification strategy could convert traces of target miRNA-221 into large amounts of output DNA to capture three-dimensional (3D) nanostructures (DTN-Au NPs-DOX-Fc) loaded with large amounts of ECL signal quencher. The constructed biosensor could realize ultrasensitive detection of miRNA-221 and has a low detection limit reaching 2.3 aM, with a successful application to detect miRNA-221 in lysate of Hela and MHCC97-L cancer cell. This work explored a novel method to strengthen the ECL performance of CDs to construct an ECL biosensing platform with sensitive detecting of biomarkers and disease diagnosis.

In situ growth of metal-organic layer on ultrathin Ti3C2Tx MXene nanosheet boosting fast electron/ion transport for electrochemiluminescence enhancement

Ultrathin metal-organic layers (MOLs) have attracted substantial attention in fabricating highly efficient electrochemiluminescence (ECL) materials due to their porous structure, small diffusion blockage, and short electron/ion-diffusion pathway, yet MOLs suffer from the inherent poor electrical conductivity that astricted the electrochemical activation, resulting in the unsatisfactory utilization ratio of ECL emitters. Herein, to address this limitation, we in situ hybridized Zr-based ultrathin MOL (Zr-TCBPE-MOL, H4TCBPE = 1,1,2,2-tetra(4-carboxylbiphenyl)ethylene) with the highly conductive Ti3C2Tx MXene nanosheet to obtain a unique 2D-2D hybrid nanocomposite (Zr-TCBPE-MOL/MXene). Benefiting from the above-mentioned attractive virtues of ultrathin MOLs and the superior conductivity of Ti3C2Tx MXene nanosheet, the resulting Zr-TCBPE-MOL/MXene nanocomposite permitted fast electron/ion transport across the whole framework of Zr-TCBPE-MOL/MXene, which efficiently boosted the electrochemical activation of TCBPE luminophores and thus improved the utilization ratio of luminophores to realize a remarkable ECL emission. Gratifyingly, we found that the ECL signal of Zr-TCBPE-MOL/MXene nanocomposite was greatly enhanced by around 4.1 times in contrast to that of pure Zr-TCBPE-MOL. On basis of the prominent ECL performance of Zr-TCBPE-MOL/MXene nanocomposite, a novel “off-on” ECL biosensor was proposed to sensitively analyze microRNA-141, which possessed a wide response range (100 aM–1 nM) and a low detection limit of 16.2 aM. Overall, this work puts forward a rational strategy to construct high-performance ECL materials and sheds new light on developing sensitive ECL sensing platforms.

Highly Efficient PTCA/Co3O4/CuO/S2O82- Ternary Electrochemiluminescence System Combined with a Portable Chip for Bioanalysis.

Herein, we reported an efficient electrochemiluminescence (ECL) biosensor chip for sensitive detection of neuron-specific enolase (NSE). First, 3,4,9,10-perylenetetracarboxylic acid with good luminescence characteristics was used as a luminophore to obtain a stable ECL signal. Subsequently, hollow porous Co3O4/CuO concave polyhedron nanocages (CPNCs) were designed as co-reaction promoters to amplify the luminescence signals for highly sensitive trace detection of NSE. In brief, the rapid cyclic conversion of Co3+/Co2+ and Cu2+/Cu+ redox pairs could continuously catalyze the reduction of persulfate (S2O82-), thus providing a large number of essential active intermediates (SO4•-) for ECL emission. Meanwhile, the unique structure of Co3O4/CuO CPNCs possessed a large specific surface area, which greatly improved its catalytic efficiency. Third, NKFRGKYKC was developed as an affinity ligand for specific antibody fixation, which improved incubation efficiency and protected bioactivity of antibodies. Finally, we independently designed a microchip and applied it for ECL detection to improve the practical application ability of the sensor. The developed biosensor exhibited good sensitivity with a wide linear range (10 fg/mL to 100 ng/mL) and a low detection limit (3.42 fg/mL), which played an active role in the clinical application of sensing analysis.

Signal-enhanced electrochemiluminescence strategy using iron-based metal-organic frameworks modified with carboxylated Ru(II) complexes for neuron-specific enolase detection

The preparation of highly efficient electrochemiluminescence (ECL) illuminants is an effective method to improve the sensitivity and repeatability of ECL immunoassay. In this study, we prepared an ECL immunoassay for efficient and sensitive detection of neuron-specific enolase (NSE) by linking carboxylated Ru(bpy)32+ to an iron-based metal-organic framework (NH2-MIL-88 (Fe)) via an amide bond as an ECL signal probe. NH2-MIL-88 (Fe) possesses a large number of amino groups that can catalyze the co-reactant S2O82−, which generates abundant reaction intermediates SO4•- around Ru(dcbpy)32+, reduces the loss of material transport and energy transfer between SO4•- and Ru(dcbpy)32+, and significantly enhances the ECL signal. We used polyaniline-intercalating vanadium oxide (PVO) nanosheets as the substrates to capture NSE owing to the large specific surface area and extraordinary conductivity of the nanosheets. Similarly, PVO nanosheets also possess abundant amino groups, which can act as co-reaction promoters to catalyze the reaction of S2O82− to SO4•-, enhancing the ECL signal of the immunoassay. Therefore, we constructed a dual-enhanced ECL immunoassay with Ru(dcbpy)32+/NH2-MIL-88 (Fe) and PVO as the signal probe and substrate, respectively, which exhibited excellent sensitivity and selectivity for detecting NSE. This study offers an effective strategy for ultrasensitive detection of trace proteins using ECL immunoassays.

A novel nanosponge-hydrogel system-based electrochemiluminescence biosensor for uric acid detection.

In this work, a highly efficient electrochemiluminescence (ECL) biosensor was developed based on the nanosponge-hydrogel system for uric acid (UA) detection. First, the nanosponge consisted of polylactic acid glycolic acid (PLGA) nanoparticles immobilized with MoS2 quantum dots (QDs) and urate oxidase (UAO). The marked loading capability of PLGA nanoparticles enables loading many biomolecules and QDs for the specific recognition of UA. Urate oxidase on the nanosponge can catalyze UA to generate H2 O2 in situ, which further triggers the ECL signal for the MoS2 QDs. Furthermore, the biocompatible acrylamide-based hydrogel not only effectively retained the functionalities of the chimeric nanosponge-hydrogel, but also provided structural integrity and engineering flexibility on the electrode for ECL sensing applications. In addition, there were many ester groups and amide bonds in the nanosponge-hydrogel structure. Therefore, many electron can be excited in the ECL process due to the large number of lone electron pairs on oxygen and nitrogen atoms. This resulted in a seven-fold ECL enhancement of the MoS2 QDs. Finally, the nanosponge-hydrogel structure-based ECL biosensor was successfully used in real clinical serum assays. This showed a good analytical performance for UA detection (100-500 μmol/L) with a limit of detection of 20 μmol/L.

The combination of highly efficient resonance energy transfer in one nanocomposite and ferrocene-quenching for ultrasensitive electrochemiluminescence bioanalysis

Sensitive and accurate detection of prostate-specific antigen (PSA) is of great significance since it is regarded as a biomarker for prostate diseases. Herein, a facile strategy for the design of highly efficient electrochemiluminescence (ECL) sensor was proposed for PSA assay. Carboxylated graphitic carbon nitride (g-C3N4) nanosheet (CCN) and tris (2, 2’-Bipyridyl) ruthenium (II) (Ru(bpy)32+) encapsulated in silica nanospheres (RuSi NPs) were employed as the donor and acceptor, respectively. CCN and RuSi NPs were covalently bound within one nanocomposite (CCN@RuSi) through the amide bond, which greatly shortened the electron-transfer path. Thus, the resonance energy transfer (RET) efficiency was remarkably increased, providing a high initial ECL intensity for the ECL assay. After the successive introducing of aptamer, PSA, and ferroceneboronic acid (FcBA) on the surface of CCN@RuSi modified electrode, the ECL signal remarkably decreased, which was caused by the steric hindrance of PSA and electron transfer quenching between Fc+ and excited-state Ru(bpy)32+*. Therefore, a highly efficient ECL platform was constructed, which achieved the ultrasensitive detection of PSA with a linear range and a limit of detection of 100 fg/mL - 50 ng/mL and 1.2 fg/mL, respectively. Furthermore, the dual-affinity of the aptamer and FcBA to PSA endowed the sensor with a high selectivity for the determination of PSA in human serum samples. The present work provides an important reference for the integration of RET and quenching strategy in the ECL study with rapid, ultrasensitive, and highly selective detection performances.

G-quadruplex-selective iridium(III) complex as a novel electrochemiluminescence probe for switch-on assay of double-stranded DNA.

In this work, we synthesized an iridium(III) complex and studied its selective ability to interact with a specific G-quadruplex DNA sequence (GTGGGTAGGGCGGGTTGG). Results showed that the iridium(III) complex exhibits high selectivity for the G-quadruplex DNA and could be used as an efficient electrochemiluminescence (ECL) probe in a switch-on assay format for the detection of double-stranded DNA (dsDNA). To construct the assay, a hairpin-structured capture probe (CP) which was modified by thiol at its 3' end and contained the G-quadruplex sequence at its 5' end was firstly immobilized on a gold electrode. Upon the specific recognition of the dsDNA sequence with the corresponding CP, the hairpin structure of the CP was opened to free G-quadruplex sequence, forming the G-quadruplex structure with the assistance of K+. Then, the iridium(III) complex was able to specifically interact with the G-quadruplex to produce an obvious ECL signal that was proportional to the dsDNA concentration. Notably, this iridium(III) complex/G-quadruplex-based strategy was universal and was not limited to the analysis of DNA using specific sequences, thus opening a new avenue for the application of the G-quadruplex-selective iridium(III) complex in the field of ECL.

CDs assembled metal-organic framework: Exogenous coreactant-free biosensing platform with pore confinement-enhanced electrochemiluminescence

Despite the various synthesis approachs to obtain luminous carbon dots (CDs), it is still quite challenging to construct the efficient electrochemiluminescence (ECL) owing to their low ECL reactivity and easy agglomeration. Herein, an efficient and concise ECL system was skillfully constructed by taking advantage of the nitrogen and sulfur co-doped CDs (N,S-CDs) with surfaces rich in hydrazide groups as luminophors to emit intense ECL, and metal-organic framework (MOF) as the matrix to confine CDs in its nanospace. Surprisingly, the proposed CDs assembled MOF (CDs/ZIF-8) enhanced anodic ECL signal up to 250% of pure CDs under the exogenous coreactant-free condition. As a proof of concept, the highly sensitive detection of uric acid (UA) was realized by the constructed ECL platform with a low detection limit of 3.52 nmol/L ranging from 10 nmol/L to 50 µmol/L. This work expanded ideas for the application of pore confinement effect, and provided references for the detection of disease biomarkers of gout and hyperuricemia.

CRISPR-Cas12a-based efficient electrochemiluminescence biosensor for ATP detection

CRISPR-Cas12a system exhibits tremendous potential in accurate recognition and quantitation of nucleic acids and non-nucleic-acid targets thanks to the discovery of its cleavage capability toward single-stranded DNA (ssDNA). In this study, we developed an efficient electrochemiluminescence (ECL) sensing platform based on CRISPR-Cas12a for the analysis of adenosine triphosphate (ATP). In the presence of the target, the successful release of the DNA activator is specially recognized by Cas12a-crRNA duplex and activates the cleavage of ferrocene (Fc) labeled–ssDNA (Fc-ssDNA) modified on the cathode of bipolar electrode (BPE), resulting in a decrease of ECL intensity of [Ru(bpy)3]2+/TPrA in the anodic cell of BPE. By means of the unique combination of Cas12a with ECL technique based on BPE, it can convert the recognition of target ATP into a detectable ECL signal. The detection limit of ATP was determined to be 0.48 nM under the optimal conditions. This work will expand the application of CRISPR-Cas detection system and propose a potential method for the analysis of non-nucleic-acid targets.

Electrochemiluminescence resonance energy transfer system fabricated by quantum state complexes for cardiac troponin I detection

Electrochemiluminescence resonance energy transfer (ECL-RET) as an energy transfer model is used to explain signal quenching or enhancement phenomena. Establishing this model requires specific conditions including efficient electrochemiluminescence (ECL) emitters and energy donors with highly overlapping spectra and sufficient distance to complete the energy transfer. In this work, a novel ECL-based biosensor was proposed by utilizing graphene quantum dots (GQDs)-coupled gold nanoclusters (Au NCs) nanocomposites supported on a well-ordered Co3O4 nanoarrays (NAs) sensing substrate as signal labels. The as-synthesized Au NCs with controllable size by using glutathione as ligand showed stable ECL signal in triethylamine (TEA) aqueous solution. After coupling with GQDs, the obtained quantum state complexes (Au NCs-GQDs) are compact in space relation, and benefiting from resonance energy transfer and synergetic effect, thus producing higher ECL response. The prepared Co3O4 NAs exhibit consistent density orientation, which can not only work as an ideal substrate for capturing more immune molecules, but also act as a conductor to accelerate electron transport. This rational designed biosensor has been constructed for detecting cardiac troponin I (cTnI), a pivotal biomarker of acute myocardial infarction (AMI). Under optimal conditions, the biosensor showed a good ECL response of cTnI with a satisfactory linear range from 500 fg/mL to 20 ng/mL and an ultralow limit of detection of 354.2 fg/mL. With the excellent repeatability and stability, this method provides a clue for the expansion of the enhance-type ECL-RET model.

Aggregation-Induced Electrochemiluminescence of Conjugated Pdots Containing a Trace Ir(III) Complex: Insights into Structure-Property Relationships.

An approach to the design of iridium(III)-contained polytetraphenylethene Pdots that could exhibit highly efficient electrochemiluminescence (ECL) was proposed. The relationships of ECL performance between the iridium complex-embedded and end-capped aggregation-induced emission (AIE) active Pdots in aqueous media were investigated for the first time. The iridium complexes with cyclometalated ligand 6-phenylphenanthridine (pphent) were incorporated into the copolymers by either embedding (P0, P2-P5) or end-capping (P1) into the backbone via an ancillary 2,2'-bipyridine (bpy) ligand. Subsequently, the corresponding Pdots of P0-P5 encapsulated with poly(styrene-co-maleicanhydride) could be obtained by the nanoprecipitation method. Compared to Pdots0, Pdots2-Pdots5 with (pphent)2Ir(bpy) (M1) complex embedding, as the iridium complex content increases, ECL signals were decreased in the order of Pdots0 > Pdots2 > Pdots3 > Pdots4 > Pdots5; whereas among these Pdots of P0-P5, Pdots1 with M1 complex end-capping exhibited the highest ECL efficiency (relative to a Ru(bpy)32+ system of 18.9%) and 4.7-fold enhancement of the ECL signal compared to the parent Pdots of P0, which was mainly attributed to the good film conductivity of the completely conjugated architectures, thus prompting the intramolecular electron transfer. This work opened new avenues for designing highly efficient ECL emitters.

High-Efficiency CNNS@NH2-MIL(Fe) Electrochemiluminescence Emitters Coupled with Ti3C2 Nanosheets as a Matrix for a Highly Sensitive Cardiac Troponin I Assay.

Synthesis of a highly efficient electrochemiluminescence (ECL) luminescent material is one of the effective means to improve the sensitivity of the sensor. In this study, an efficient ECL luminescent nanomaterial, carbon nitride nanosheet (CNNS) decorated amino-functional metal-organic frameworks (CNNS@NH2-MIL(Fe)) were synthesized for sensitive ECL detection of cardiac troponin I (cTn-I). The synthesized CNNS@NH2-MIL(Fe) realized the effective mass loading of CNNS, and more importantly, the NH2-MIL(Fe) could expedite the reduction of coreactant S2O82- to produce abundant ECL reaction intermediate SO4•- near CNNS, thus, shortening the distance between SO4•- and the excited state of CNNS with less energy loss to extremely enhance the ECL signal of CNNS. Furthermore, the ECL signal of the immunosensor could be further enhanced when the Ti3C2 nanosheet was used as the matrix to capture primary anti-cTn-I due to the reason that Ti3C2 not only exhibited a large surface area and excellent metallic conductivity, but also could act as a coreaction accelerator to speed up the reduction of S2O82- with plenty of SO4•- generated. Therefore, this proposed ECL immunosensor using CNNS@NH2-MIL(Fe) as a signal probe and Ti3C2 as a sensing matrix exhibited a significantly enhanced ECL signal and had a high sensitivity and excellent selectivity for cTn-I. Consequently, this multiple signal amplification strategy provided an effective method for trace protein ultrasensitive detection in ECL bioanalysis.

A novel high efficient electrochemiluminescence sensor based on reductive Cu(I) particles catalyzed Zn-doped MoS2 QDs for HPV 16 DNA determination

In this work, we explored a high efficient electrochemiluminescence (ECL) sensor based on the synergistic enhancement strategy of Zn-doped MoS2 quantum dots (QDs) and reductive Cu(I) particles. On the one hand, Zn-doped MoS2 QDs with sulfur vacancies were designed to improve the ECL activity of QDs. The regulated sulfur vacancies with zinc doping resulted in adsorption and coordination with transition metals of H2O2 as coreactant. On the other hand, reductive Cu(I) particles were prepared to further catalyze the coreactant in the ECL system. The tight combination of glutathione (GSH) and copper in reductive Cu(I) particles can perfectly stabilize Cu(I) with outstanding catalytic activity in neutral pH condition. Under reduction of the cathode, the reductive Cu(I) particles acted as the catalytic role continuously. As a result, more ·OH were generated from H2O2. The signal of Zn-doped MoS2 QDs had 4.5-fold enhancement with the assistance of reductive Cu(I) particles. Furthermore, the DNA walker cycle was designed in presence of T7 exonuclease for HPV 16 DNA detection. The biosensor realized sensitive determination of HPV 16 DNA from 0.1 nmol L−1 to 200 nmol L−1 with the LOD of 0.03 nmol L−1. Interestingly, the entire sensing system can be reproduced on a simple family-friendly device powered by batteries. The ECL signal captured by a smartphone can be processed into high-resolution imaging by self-developed software, which provides great possibility of point-of-care HPV 16 DNA determination in the future.

Ultrasensitive detection of miRNA based on efficient immobilization of probe and electrochemiluminescent quenching of Ru(bpy)32+ by methylene blue

A high performance miRNA biosensor based on effective click chemistry assembly of a Ru(bpy)32+ labeled DNA probe and efficient electrochemiluminescence (ECL) quenching of the Ru(bpy)32+/BDEA (BDEA = N-butyldiethanolamine) system by surface-confined electroactive methylene blue (MB) dye is reported. When the target miRNA was present, the ECL signal instantly changed from “light off” to “light on” status. Using the specific miRNA let-7d as the target analyte, this biosensor provided sensitive detection over approximately six orders of magnitude (10 fM-10 nM), with a limit of detection of 10 fM (S/N = 3). Detailed study of the ECL quenching behavior of the Ru(bpy)32+/BDEA system by MB in solution suggested that the ECL quenching involves a combination of photoluminescence dynamic quenching and quenching processes directly associated with the redox reactions, as well as resonance energy transfer. A large binding constant of 4.7 × 1011 M−1 between let-7d and the DNA hairpin was estimated using an ECL-based extended Langmuir isotherm model, suggesting remarkably strong binding of the target to the probe. Furthermore, our biosensor exhibited excellent specificity and reproducibility. Using the developed system, the concentration of the target miRNA extracted from the A549 cell line could be obtained, demonstrating the potential application of the developed biosensor to practical biological sample analysis.

Amplified electrochemiluminescence detection of CEA based on magnetic Fe3O4@Au nanoparticles-assembled Ru@SiO2 nanocomposites combined with multiple cycling amplification strategy

In this work, we designed a new strategy for ultrasensitive detection of CEA based on efficient electrochemiluminescence (ECL) quenching of Ru(bpy)32+-doped SiO2 nanocomposite by ferrocene using target recycling amplification technique. A large number of Ru@SiO2 ECL signal probe were firstly assembled on the novel magnetic core−shell Fe3O4@Au nanoparticles (NPs), then the ferrocene-labeled ECL quenching probe (Fc-probe) was linked to the magnetic NPs. Finally, numerous DNA1 sequences were produced by target CEA-triggered multiple recycling amplification and displaced the Fc-probe on the magnetic NPs, leading to significantly enhanced ECL signal for CEA detection. Because of the designed cascade signal amplification strategy, the newly developed method achieved a wide linear range of 10 fg/mL to 10 ng/mL with a low detection limit of  3.5 fg/mL. Furthermore, taking advantages of the magnetic Fe3O4@Au NPs for carring abundant signal probes, sensing target and ECL detection, the developed ECL strategy is convenient, rapid and displayed high sensitivity for CEA detection, which has great potential for analyzing the clinical samples in practical disease diagnosis applications.

Synthesis, labeling and bioanalytical applications of a tris(2,2'-bipyridyl)ruthenium(II)-based electrochemiluminescence probe.

Assays using probes labeled with electrochemiluminescent moieties are extremely powerful analytical tools that are used in fields such as medical diagnostics, environmental analysis and food safety monitoring, in which sensitive, reliable and reproducible detection of biomolecules is a requirement. The most efficient electrochemiluminescence (ECL) reaction to date is based on tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) with tripropylamine (TPrA) as the co-reactant. Here we present a detailed protocol for preparing Ru(bpy)3(2+) probes and their bioanalytical applications. This protocol includes (i) the synthesis of a biologically active Ru(bpy)3(2+)-N-hydroxysuccinimide (NHS) ester, (ii) its covalent labeling with both antibodies and DNA probes and (iii) the detection and quantification of ECL in a microfluidic system with a paramagnetic microbead solid support. In our magnetic bead-based ECL system, two probes are required: a capture probe (labeled with biotin to be captured by a streptavidin-coated magnetic bead) and a detector probe (labeled with Ru(bpy)3(2+)). The complex consisting of the analyte, the capture probe, the detector probe and the magnetic bead is brought into contact with the electrode by using a magnetic field. The Ru(bpy)3(2+) reacts with TPrA in solution to generate the ECL signal. The full protocol, including the synthesis and labeling of the bioactive Ru(bpy)3(2+), requires 5-6 d to complete. ECL immunoassays or nucleic acid tests only require 1.5-2 h, including the sample preparation time.