Introduction

Electrochemiluminescence (ECL) combines electrochemical reactions and luminescence, converting electrical energy to light.[1] ECL differs from chemiluminescence; in ECL, the reactive species that produce the chemiluminescent reaction are electrochemically generated from stable precursors at the surface of an electrode.[2]

Luminophores are substances that emit light. In ECL, luminophores attain a high-energy state induced by electron transfer over the surface of an electrode via an oxidation-reduction reaction. The excited luminophores emit light as photons while returning to the ground state.[1] Luminophores can be used as labels for biomolecules; the biomolecules can be detected and quantified by measuring the amount of light emitted.[3]

ECL is an important diagnostic technique known for its versatility and numerous advantages. The applications of ECL include detecting, separating, and quantifying various intracellular and extracellular biomolecules, including proteins, enzymes, hormones, metabolites, and nucleic acids.[4] ECL is also used to visualize cells, study the functions of various intramembrane and transmembrane proteins, detect nucleic acids of interest, and assay drugs.[3]

ECL systems are broadly classified as ion annihilation or coreactant types.

Ion Annihilation ECL System

In ion annihilation ECL systems, a pulsed potential is applied over the electrode, generating radical cations and anions of the luminophore.[3][5] Electron transfer from anion to cation results in an excited cation; subsequent decay of this excited cation to the ground state liberates light. Ion annihilation systems typically utilize organic compounds dissolved in organic solvents, donor-acceptor conjugated molecules.[6] These organic compounds are usually poor candidates for biomolecular assay labels. Ion annihilation also generates highly reactive intermediates unsuitable for routine assays.[7]

Coreactant ECL System

Most of the ECL systems in current use are co-reactant systems.[6] These systems employ a high-efficiency coreactant added to the luminophore with one-directional potential scanning. Oxidation or reduction of both species at the electrode generates radicals. Intermediates from the co-reactant decompose, forming a robust species that reacts with the luminophore, producing excited states and emitting light. Coreactant systems are used for biomolecular assays due to the solubility of the co-reactant in the surrounding medium, low reduction-oxidation potential, and stability.[7] Ruthenium metal ions and luminol derivatives are the most commonly used lumiphores in coreactant ECL systems.[8] The coreactant commonly used with ruthenium metal ions is tripropylamine (TPA). Other widely utilized coreactants include 2-(Dibutylamino)ethanol, peroxydisulfate, and hydrogen peroxide.[9]

Most reported ECL applications for immunoassay or genetic analysis utilize tris(2,2′-bipyridyl)ruthenium as a label and TPA as a coreactant.[10] These systems are highly efficient;  the ruthenium compound is stable, highly soluble in polar and non-polar solvents, and is strongly luminescent. TPA undergoes oxidation with potential application, forming a TPA radical cation and a TPA radical. Both radicals generate excited bipyridyl-ruthenium, which emits orange-spectrum light at 600 to 640 nm as it relaxes to the ground state. The luminophore is regenerated after emission.[11] 

Luminol is an organic luminophore commonly used for cell imaging.[3] Luminol undergoes oxidation to generate a diazaquinone form. In the presence of hydrogen peroxide, this intermediate further oxidizes to 3-aminophthalate, emitting blue light. Hydrogen peroxide, generated in biological processes, is often detected alongside luminol. Reactive oxygen species can enhance luminol ECL emission. Luminol is irreversibly oxidized and requires alkaline conditions, limiting cellular analysis applications. However, it operates at a lower anodic potential than bipyridyl-ruthenium, providing advantages for imaging living cells.[12] 

Basic Instrumentation for Electrochemiluminescence

The basic instrumentation setup in an ECL includes an electrochemical cell, a detector, a signal amplification system, and a reagent and sample delivery system.[7] The electrochemical cell houses the working electrode, usually made of carbon or gold and serves as a site of the ECL reaction. A reference electrode is also present to maintain a stable potential for measurement. A photomultiplier tube (PMT) or a photodiode is commonly used to detect the light emitted during the ECL reaction. These detectors are highly sensitive to low light levels and convert the photons into electrical signals. The electrical signals generated by the light detection system are weak and must be amplified and processed for accurate measurement. Amplification circuits, such as transimpedance amplifiers, can boost signal strength, and signal processing units can filter and digitize the signal for analysis. The ECL assay requires the delivery of reagents and samples to the electrochemical cell. This can be achieved using a syringe pump or a microfluidic system that precisely delivers the necessary volumes of reagents and samples at specific time points.

Electrochemiluminescence Advantages and Limitations

ECL systems have several advantages. The luminophores used in ECL are small, stable substances that can label a wide range of molecules and haptens without cross-reaction. There is minimal background interference in ECL because the luminophore has the inherent capacity to emit light, and no additional light source is required. ECL is highly sensitive due to multiple excitation cycles and offers a wide range of detection with lower detection limits of 200 fmol/L. ECL also offers improved reagent stability.[4][7]

ECL is susceptible to light leaks and background luminescence from reagents. The high sensitivity offered by ECL requires pure reagents and solvents. Additionally, high-intensity light emission may lead to pulse pile-up, resulting in underestimating light emission.[13][14]

Specimen Requirements and Procedure

ECL assays in the clinical setting require careful sample handling to ensure accurate and reliable results. Sample handling includes the following steps:

Sample Collection 

The first step in an ECL assay is collecting a clinical sample from the patient, such as blood, urine, or serum. Proper collection techniques and appropriate sample containers are essential to maintain sample integrity and prevent contamination. A 200- to 400-microlitre sample is required for any ECL assay.[15] Adequate samples should be available for single-sample and entire-run repeat analyses in case of QC failure. 

Sample Preparation 

Samples containing precipitates should be centrifuged before testing. Do not use heat-inactivated samples. Samples should be stored frozen upon receipt unless they are to be analyzed immediately and should be assayed without delay after thawing. Multiple freeze-thaw cycles should be avoided; samples may lose their integrity.[16] For the nucleic acid assay, specimens are processed to extract the DNA or RNA of interest using appropriate isolation methods. The purity and concentration of the nucleic acids are determined using spectrophotometry or fluorometry.[17]

Sample Dilution 

In some cases, the concentration of the target analyte in the clinical sample may be too high or too low for accurate measurement. In such instances, appropriate sample dilutions may be necessary to bring the analyte concentration within the assay's linear range.[15] Dilution protocols should be followed precisely to ensure accurate results.

Reagent Mixing

ECL assays often involve adding specific reagents to the sample to initiate the ECL reaction. Proper mixing of the sample and reagents is essential to ensure uniform distribution and complete reaction. Depending on the assay requirements, mixing can be achieved by vortexing, pipetting, or using automated mixing devices.

Incubation

Some ECL assays may require a specific incubation period to allow the ECL reaction to proceed wholly. During this time, the sample and the added reagents are kept at a controlled temperature to promote the reaction. The duration and temperature of the incubation should be precise to achieve optimal assay performance.

Calibration Standards and Controls

Calibration standards and controls are typically included to validate the accuracy and precision of the ECL assay. Calibration standards with known target analyte concentrations are used to generate a calibration curve, which relates the ECL signal to the analyte concentration.[16] Control samples with known values monitor the assay performance and detect potential issues.

Diagnostic Tests

Electrochemiluminescence (ECL) methods are used in the following specialized clinical assays: [4][17] 

Hormones

• Thyroid-stimulating hormone, T3, and T4
• Progesterone
• Testosterone
• Follicle-stimulating hormone
• Luteinizing hormone
• Prolactin

Tumor Markers

• Carcinoembryonic antigen
• Carbohydrate antigen-125, -19.9, and -15.3
• Prostrate specific antigen
• Beta-human chorionic gonadotropin

Therapeutic Drug Monitoring 

• Carbamazepine
• Digoxin
• Phenytoin
• Vancomycin 

Vitamins

• Vitamin B12
• Folic acid 
• Vitamin D 

Proteins

• Ferritin
• D-dimer
• Brain natriuretic polypeptide
• Atrial natriuretic polypeptide
• Insulin
• C-peptide 

Inflammatory Markers

• C-reactive Protein
• Interleukins
• Tumor necrosis factor
• Growth factors 
• Complements

Antibodies 

• Anticardiolipin antibodies 
• Anti-thyroid peroxidase antibody
• Anti-thyroglobulin antibody

Infectious Diseases 

• Herpes simplex virus antibody
• Rubella virus antibody
• Toxoplasma circulating antibody
• Hepatitis E antigen and antibody
• Coxsackievirus antibody
• SARS-Co-V antigen and antibody

Testing Procedures

Electrochemiluminescence Immunoassay

The electrochemiluminescence immunoassay (ECLIA) relies on the interaction between an antibody and its corresponding antigen. The ECLIA utilizes specialized reagents, including a capture antibody against the antigen, usually a biomolecule, and a labeled antibody to detect the interaction. The capture antibody is immobilized on a solid support, such as a microplate or magnetic bead. In contrast, the labeled antibody is conjugated with a luminescent marker and an electrochemically active molecule.[18] Many studies have proven ECLIA superior to conventional radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA); ECLIA is highly sensitive and uses neither a radioisotope nor enzymes with limited stability.[19][20]

ECLIA uses 3 basic interaction principles to detect analytes: direct, competitive, and sandwich. In a direct ECLIA system, a single antibody is immobilized on the electrode. The electrode can capture the target analyte (Ag) within the specimen and act as an electrochemiluminescent probe. The Ag competes with the coreactant for binding sites on the antibody; an increase in bound Ag decreases the ECL signal. The quantifiable decrease in the ECL signal directly correlates to the concentration of the Ag.[21]  In a competitive interaction, an antigen analog is labeled with a luminophore, and the antibody is immobilized on the surface of the electrode.[21] The Ag in the analyte competes with the analog to bind to the antibody; the concentration of labels in the system decreases. The concentration of the Ag is inversely proportional to the measured ECL signal. The competitive ECLIA is used for smaller Ag such as thyroid hormones, cortisol, and testosterone.[22] Lastly, a sandwich interaction uses 2 different antibodies. The primary antibody is usually immobilized on the electrode surface as a capture probe for the target antigen. The secondary antibody also targets the antigen and is labeled with a luminophore. The target antigen binds to form an immunocomplex with the capture probe and the ECL probe, and the measured ECL signal intensity is proportional to the concentration of the target antigen.[23] The sandwich ECLIA is used for larger analytes such as thyroid-stimulating hormone, follicle-stimulating hormone, and luteinizing hormone.[22]

Bioconjugation

Bioconjugation involves linking the luminophore with biological samples such as antigens or antibodies. There are various linking groups suitable for ruthenium complexes to conjugate with biological samples, such as phosphoramidite for conjugation with oligonucleotides, N-hydroxysuccinimide ester for linking with amines on proteins, hydrazide for carbohydrates, maleimide for thiols, and amines as linking groups for reactions with carboxylic acids on proteins.[24][25] Surface modification with biotin-streptavidin is performed to capture antibodies or antigens on the surface support. The binding between streptavidin and biotin provides a strong and specific interaction immobilizing the antibody on the surface. The subsequent formation of a sandwich complex and the generation of the ECL signal upon electrochemical stimulation enable the sensitive detection of the target analyte in the ECL immunoassay.[26]

Nucleic Acid Detection

Specific DNA probes or primers are designed to target the desired DNA or RNA sequences. Labeled with luminophores, these probes can hybridize with the complementary target DNA, bringing the ECL-active molecule close to the electrode surface and generating luminescence.[27] These assays are used in genetic analysis, mutation detection, pathogen identification, and other molecular biology and diagnostics fields.

Cellular Imaging

Living cells are typically labeled with ECL-active probes or antibodies specific to cellular markers. The ECL-based detection allows visualization and analysis of cellular processes, protein-protein interactions, and intracellular signaling pathways.[28]

Interfering Factors

Endogenous Factors

Most commercially available ECLIA-based immunoassays are generally unaffected by endogenous interfering substances.[29] The most common endogenous interfering substances are hemoglobin, bilirubin, and lipids. However, serum bilirubin up to 64 mg/dL, lipemia up to 1900 mg/dL, and serum hemoglobin up to 1 g/dL do not interfere with ECLIA-based immunoassays.

Historically, the cross-reaction of endogenous substances, such as luteinizing hormone interfering with human chorionic gonadotropin, was problematic. However, with the advent of more specific antibodies, cross-reaction is minimal. Still, cross-reactivity can be a problem during the detection of drugs and their metabolites.[30] 

Heterophilic antibodies such as rheumatoid factor, anti-thyroid antibodies, and anti-animal antibodies may interfere via noncompetitive mechanisms resulting in falsely high or low values; the incidence varies from 0.5% to 6%.[31][32] Many cases of antibodies against streptavidin and ruthenium complexes have been reported.[33]

Exogenous Factor

ECLIA-based immunoassays should be avoided in patients taking therapeutic biotin of > 5 mg/day.[34] Temporary discontinuation of biotin is advised before performing the assay. When sodium citrate is used as an anticoagulant, the results must be corrected by more than 10%.[35] Samples or assay reagents contaminated with substances might interfere with the measurement of the label.

High-dose Hook Effect

The high-dose hook effect, or prozone phenomenon, is particular to the sandwich immunoassay. This effect is characterized by a false-negative or underestimated result at high target analyte concentrations. In the high-dose hook effect, an extremely high concentration of the target analyte can exceed the binding capacity of the capture and labeled antibodies.[24] As a result, the excess analyte saturates both the captured and labeled antibody binding sites, preventing the formation of the sandwich complex. This leads to decreased signal or signal intensity, resulting in a false-negative or underestimated result. Dilution of the sample is a commonly employed strategy to mitigate this effect.[36]

Results, Reporting, and Critical Findings

ECL provides highly sensitive and specific measurements, enabling the detection and quantification of analytes with exceptional precision. Signals are displayed as relative light units (RLU), representing the amount of light emitted.[4] The RLU is converted into concentration units by a calibration curve plotted using various analyte concentrations and signals obtained.

When reporting results, it is essential to provide accurate and comprehensive information, including the measured analyte concentration, units of measurement, and any necessary interpretation or reference ranges.[37] Critical findings, defined as values outside established thresholds or diagnostic cutoffs, require immediate attention and notification to the healthcare team. These findings may indicate significant health risks or the need for urgent medical interventions.[18]

Clinical Significance

Endocrinopathies

The high sensitivity, wide dynamic range, multiplexing capability, precision, and high throughput analysis of ECL make it a valuable tool for detecting and monitoring hormonal disorders. With fully automated analyzers and commercially available kits, ECLIA-based systems are superior to conventional RIA and ELISA. These assays are used when diagnosing and managing conditions such as hypothyroidism and hyperthyroidism to quantify circulating levels of thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3).[38] Insulin and C-peptide estimation assists in diagnosing and managing type 1 and type 2 diabetes mellitus. ECL is utilized to detect adrenal disorders, like adrenal insufficiency and the hypercortisolism of Cushing syndrome. Reproductive hormone assays, including luteinizing hormone (LH), follicle-stimulating hormone (FSH), estrogen, progesterone, and testosterone, aid in the evaluation and management of polycystic ovary syndrome (PCOS), infertility, and hormonal imbalances.[39][40]

Detection and Monitoring of Neoplasms

Prostate-specific antigen (PSA) is a widely used biomarker for prostate cancer. Tumor antigens such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) assist in diagnosing, prognosis, and monitoring disease progression in colorectal and pancreatic adenocarcinomas.[41]

Infectious Processes

ECL-based assays detect HIV antibodies or viral antigens, such as p24, hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibodies, or viral antigens.[42] ECL-based assays aid in detecting Mycobacterium tuberculosis infections by measuring antibodies or antigens specific to the bacillus, such as interferon-gamma release assays (IGRAs) or TB-antigen 85 complexes (Ag85).[43] Antigen-based ECL assays for the diagnosis of coronavirus infection are also available.[44]

Biosensors and Point-of-care Testing

Biosensors are miniaturized analytical systems incorporating a biological recognition element that selectively interacts with a target analyte. This recognition element can be an enzyme, antibody, nucleic acid, or other biomolecule that exhibits a specific binding affinity toward the target analyte. Recently, using Ru-loaded silica and gold nanoparticles, integrating the microfluidic system, and using screen print nanoelectrodes have enabled the integration of biosensors as point-of-care devices.[8] 

ECL-based point-of-care-testing (ECL-POCT) biosensors aid in detecting biomarkers such as cardiac troponin I (cTnI) and C-reactive protein (CRP), enabling early diagnosis of acute myocardial infarction and monitoring of postoperative recovery.[36] Integrating ECL with mobile devices and highly integrated systems ensures portability, simplicity, and accessibility for effective disease control and surveillance in public health. ECL biosensors based on DNA hybridization principles are used for genetic analysis and genotyping. Aptamers, synthetic single-stranded DNA or RNA sequences with high affinity and specificity for target molecules, are recognition elements in ECL biosensors.[45]

Quality Control and Lab Safety

Quality control (QC) and lab safety are essential while employing the electrochemiluminescence (ECL) system for clinical assays. As a part of quality control procedures, regular calibration and verification using lyophilized control materials of multiple levels are run to check the precision and accuracy of the system.[46] For non-waived tests, laboratory regulations require, at the minimum, analysis of at least 2 levels of quality control materials once every 24 hours. If necessary, laboratories can assay QC samples more frequently to ensure accurate results.[47]

Quality control samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance.[48] To minimize QC when performing tests for which manufacturers' recommendations are less than those required by the regulatory agency, such as once per month, labs can develop an individualized quality control plan (IQCP) that involves performing a risk assessment of potential sources of error in all phases of testing and putting in place a QC plan to reduce the likelihood of errors.[49]

The design of a QC plan must consider the analytical performance capability of a measurement procedure and the risk of harm to a patient if an erroneous laboratory test result is used for a clinical care decision. An erroneous laboratory test result is a hazardous condition that may or may not cause harm to a patient, depending on what action or inaction a clinical care provider takes based on the erroneous result.[50]

The acceptable range and rules for interpreting QC results are based on the probability of detecting a significant analytical error condition with an acceptably low false alert rate.[48] The desired process control performance characteristics must be established for each measurement before selecting the appropriate QC rules.[50] Westgard multi-rules are usually used to evaluate the quality control runs. If a run is declared out of control, investigate the system to determine the cause of the problem. Do not perform any analysis until the issue has been resolved.[51]

Changing reagent lots can have an unexpected impact on QC results. Careful reagent lot crossover evaluation of QC target values is necessary. Because the matrix-related interaction between a QC material and a reagent can change with a different reagent lot, QC results may not be a reliable indicator of a measurement procedure's performance for patient samples after a reagent lot change.[52] It is necessary to use clinical patient samples to verify the consistency of results between old and new lots of reagents because of the unpredictability of a matrix-related bias being present for QC materials.[53]

The laboratory must participate in the external quality control or proficiency testing (PT) program; this is a regulatory requirement published by the Centers for Medicare and Medicaid Services (CMS) in the Clinical Laboratory Improvement Amendments (CLIA) regulations.[54] It is helpful to ensure the accuracy and reliability of the laboratory concerning other laboratories performing the same or comparable assays. Required participation and scored CMS and voluntary accreditation organizations monitor results. The PT plan should be included as an aspect of the quality assessment (QA) plan and the overall quality program of the laboratory.[55]

Simultaneously, maintaining lab safety by running the daily maintenance, preventive maintenance, proper disposal of waste, using personal protective equipment, following proper safety protocols, including the appropriate handling and storage of hazardous reagents, and adhering to safety guidelines is essential. By prioritizing quality control and lab safety, laboratories can deliver precise and dependable clinical assay results while safeguarding the well-being of staff and patients.[56]

Enhancing Healthcare Team Outcomes

Optimal team performance is required to apply an electrochemiluminescence (ECL) system in patient-centered care. Healthcare workers handling ECL systems should possess expertise in instrumentation, sample handling, report interpretation, and troubleshooting which can be attained through continuous training and skill development.

Factors such as sensitivity, specificity, and cost-effectiveness should be considered for each clinical setting and be a collaborative decision between healthcare practitioners, lab professionals, and technicians. Clear and concise communication regarding patient history, test requirements, and interpretation of ECL results promotes accurate diagnosis, appropriate treatment decisions, and coordinated care.