Troponin Immunoassays Research Articles

Exploring strategies to rapidly identify false positives in high-sensitivity cardiac troponin I assay: A prospective study

BackgroundCardiac troponin is the pivotal biomarker of myocardial injury, playing a central role in the diagnosis of acute coronary syndrome and various clinical situations. Nevertheless, challenges arise when patients exhibit elevated cardiac troponin levels in the absence of evident cardiac origin, as evidenced by extensive cardiac exploration, which suggests the presence of an interfering factor. Despite the high performance of high-sensitive cardiac troponin immunoassays, these tests remain susceptible to interferences that may lead to false-positives. MethodsIn the period between July 2021 and July 2024, 8129 patients were hospitalized in the cardiology departments of Bordeaux University Hospital with positive results for high-sensitivity cardiac troponin I. Among them, 15 patients were suspected of having false-positive results, based on clinical and biological observations. ResultsIn this subpopulation, we evaluated prospectively various techniques, including serial dilutions, antibody-binding tubes, and alternative immunoassays (for cardiac troponin I and T) with the objective of identifying any potential analytical interference in their high-sensitive cardiac troponin I measurements. Our investigations revealed that 12 out of 15 suspected cases exhibited an interference on the high-sensitive cardiac troponin I assay. ConclusionIn conclusion, we propose an original algorithm designed to identify high-sensitive cardiac troponin I false-positives. This algorithm can help clinicians to make prompt and informed decisions about patient care, and to avoid erroneous clinical interventions that may result from such interferences.

Photoelectrochemical immunoassay of cardiac troponin I based on ZnTCPP/CdIn2S4 type-II heterojunction and co-catalyzed precipitation biolabeling.

CdIn2S4 and zinc tetrakis(4-carboxyphenyl)porphyrin (ZnTCPP) were synthesized by hydrothermal method, and an organic dye-sensitized inorganic semiconductor ZnTCPP/CdIn2S4 type II heterojunction was constructed on a fluorine-doped tin oxide (FTO) substrate electrode. Asandwich immunostructure forsignal-attenuation photoelectrochemical (PEC) detection of cardiac troponin I (cTnI) was constructed using the ZnTCPP/CdIn2S4/FTO photoanode and a horseradish peroxidase (HRP)-ZnFe2O4-Ab2-bovine serum albumin (BSA) immunolabeling complex. The bioenzyme HRP and the HRP-like nanozyme ZnFe2O4 can co-catalyze the oxidation of 4-chloro-1-naphthol (4-CN) by H2O2 to produce an insoluble precipitate on the photoanode, thus notably reducing the anodic photocurrent for quantitative determination of cTnI. Under the optimal conditions, the photocurrent at 0V vs. SCE in 0.1M phosphate buffer solution (pH 7.40) containing 0.1M ascorbic acid was linear with the logarithm of cTnI concentration from 500fg mL-1 to 50.0 ng mL-1, and the limit of detection (LOD, S/N = 3) is 0.15 pg mL-1. Spiked recoveries were 95.1% ~ 104% for assay of cTnI in human serum samples.

Block-Polymer-Restricted Sub-nanometer Pt Nanoclusters Nanozyme-Enhanced Immunoassay for Monitoring of Cardiac Troponin I.

Noble-metal nanozymes have demonstrated great potential in various fields. However, aggregation of single-particle nanoparticles severely affects their exposed catalytically active sites to the extent of exhibiting weak enzyme-like activity. Here, we present an organic block surfactant (polyvinylpyrrolidone, PVP) to construct monodisperse water-stable Pt nanoclusters (Pt NCs) for an enhanced immunoassay of cardiac troponin I (cTnI). The PVP-modified Pt NC nanozyme exhibited up to 16.3 U mg-1 peroxidase-mimicking activity, which was mainly attributed to the ligand modification on the surface and the electron-absorbing effect of the ligand on the Pt NCs. The PVP-modified Pt NCs have a lower OH-transition potential, as determined by density functional theory. Under optimized experimental conditions, the enhanced nanozyme immunoassay strategy exhibited an ultrawide dynamic response range of 0.005-50 ng mL-1 for cTnI targets with a detection limit of 1.3 pg mL-1, far superior to some reported test protocols. This work provides a designable pathway for the design of artificial enzymes with high enzyme-like activity to further expand the practical range of enzyme alternatives.

One-pot synthesis of AuPt@FexOy nanoparticles with excellent peroxidase-like activity for development of ultrasensitive colorimetric lateral flow immunoassay of cardiac troponin I

Detection of cardiac troponin I (cTnI) plays a critical role in diagnosing acute myocardial infarction (AMI). In this report, a new kind of spherical AuPt@FexOy core@shell nanoparticles (termed as AuPt@FexOy NPs) were one-pot synthesized by a redox interaction-engaged strategy (RIES) without the addition of any surfactants or reducing agents. The as-synthesized AuPt@FexOy NPs not only retain the plasmonic activity of gold nanoparticles (AuNPs), but also possess excellent catalytic activities of platinum nanoparticles (PtNPs) and FexOy nanoclusters. The features of AuPt@FexOy NPs enable greatly enhance the colorimetric detection sensitivity of lateral flow immunoassay (LFIA) through integrating AuPt@FexOy NPs labeling procedure and catalyzing oxidation of chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) signal amplification strategy. The as-developed colorimetric LFIA (termed as AuPt@FexOy-LFIA) exhibits the limit of detection (LOD) as 26.0 pg mL−1 cTnI under the TMB signal amplification mode. In particular, the detection results of cTnI in 40 clinical seral samples by AuPt@FexOy-LFIA are correlated well with those of cTnI in the same samples by commercial enzyme-linked immunosorbent assay (ELISA) detection kit (R2 = 0.97, slope = 1), demonstrating the highly reliable analytical performance and good application prospect of AuPt@FexOy-LFIA.

Automated and rapid chemiluminescence immunoassay for cardiac troponin I based on digital microfluidics

Rapid diagnosis of acute myocardial infarction (AMI) is critical in the clinical setting. An automated and rapid chemiluminescence immunoassay system based on digital microfluidics (DMF) is suggested for cardiac troponin I (cTnI). This method involves a double antibody sandwich structure. The cTnI antigen in the sample to be analyzed was captured by magnetic beads encapsulated with the cTnI antibody, which was subsequently recognized by the biotin-labeled secondary antibody. Horseradish peroxidase (HRP)-labeled streptavidin may be conjugated to biotin and used to mark the cTnI antigen because of the unique way that biotin binds to avidin. The simultaneous recognition of the cTnI antigen by primary and secondary antibodies in sandwich form, as well as the high specificity of the reaction between streptavidin and biotin, considerably improves the sensitivity and specificity of this technique for cTnI detection. This approach has decent stability. For cTnI samples, the full process took 40 min, with a limit of detection (LOD) of 0.34 ng mL−1. Additionally, there was a significant correlation range from 0.39 to 25 ng mL−1, with a correlation value of 0.994. The CV (Coefficient of variation) value was 2.26%. This system is anticipated to be used for the clinical measurement of cTnI in human serum.

Simultaneous SERS-based immunoassay of dual cardiac markers on pump-free hybrid microfluidic chip

We developed a new pump-free hybrid microfluidic chip for the simultaneous SERS-based immunoassay of creatine kinase MB isoenzyme (CK-MB) and cardiac troponin (cTnI) cardiac markers. As a main composition, the patterned SERS paper substrate was firstly fabricated by UV light curing method using water-based polyurethane acrylate resin to have the hydrophobic barriers of microchannel, and then in-situ synthesized AuNPs on hydrophilic paper microchannel. It took advantage of 3D fibre structure to provide the capillarity force and compact AuNPs deposition scaffold for sample flow and highly sensitive SERS detection. The paper substrate was attached to PDMS microchannel and enabled the aqueous samples to flow in PDMS microchannel, which driven by capillary force of paper materials. The proposed method successfully detected the concentrations of two cardiac markers ranging from 0.01 to 50 ng mL−1. These results reveal the simultaneous detection capability of SERS-based immunoassay on the pump-free hybrid microfluidic chip without heavy external pump. The integration of paper and PDMS microfluidic chip is portable, low-cost, and tremendously useful for the simultaneous detection of cardiac markers.

Contactless Photoelectrochemical Biosensor Based on the Ultraviolet-Assisted Gas Sensing Interface of Three-Dimensional SnS2 Nanosheets: From Mechanism Reveal to Practical Application.

This work reports a contactless photoelectrochemical biosensor based on an ultraviolet-assisted gas sensor (UV-AGS) with a homemade three-dimensional (3D)-SnS2 nanosheet-functionalized interdigitated electrode. After rigorous examination, it was found that the gas responsiveness accelerated and the sensitivity increased using the UV irradiation strategy. The effects of the interlayer structure and the Schottky heterojunction on the gas-sensitive response of O2 and NH3 under UV irradiation were further investigated theoretically by 3D electrostatic field simulations and first-principles density functional theory to reveal the mechanism. Finally, a UV-AGS device was developed to quantify the blood ammonia bioassay in a small-volume whole blood sample by alkalizing blood to release gas-phase ammonia with a linear range of 25-5000 μM with a limit of detection (LOD) of 29.5 μM. The device also enables a rapid immunoassay of human cardiac troponin I (cTnI) with a linear range of 0.4-25.6 ng/mL and an LOD of 0.37 ng/mL using a urease-labeled antibody as the immune recognition molecule. Both analyses showed satisfying specificity and stability, suggesting that the device can be applied to practical assays and is of great potential to increase the value of gas-sensitive sensors in chemical biosensing.

Complement C1q in plasma induces nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticle antibody conjugates

Upconverting nanoparticles are attractive reporters for immunoassays, because their high specific activity and lack of autofluorescence background enable their detection at extremely low concentrations. However, the sensitivity achieved with heterogeneous sandwich immunoassays using nanoparticle reporters is generally limited by the nonspecific binding of nanoparticle antibody conjugates to solid supports. In this study, we characterized plasma components associated with elevated nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticles in heterogeneous two-step sandwich immunoassays. Plasma was consecutively fractionated using various chromatographic methods by selecting after each step the fractions producing the highest nonspecific binding of upconverting nanoparticle conjugates in an immunoassay for cardiac troponin I. Finally, the proteins in the fractions associated with highest amount of nonspecific binding were separated by gel electrophoresis and identified with mass spectrometry. The results indicated that complement component C1q was present in the fractions associated with the highest signal from nonspecific binding. The interference was not limited to only poly(acrylic acid)-coated nanoparticles or certain antibody combination, but occurred more generally. The interference was removed by increasing the ionic strength of the assay buffer in the sample incubation step or by adding a negatively charged blocker to bind on positively charged C1q, suggesting that the interaction is mostly electrostatic. Hence, we assume that the interference is likely to affect various negatively charged nanoparticles. The identification of complement component C1q as the major interfering protein allows for more rational design of countermeasures in future immunoassay development utilizing nanoparticle reporters.Graphical abstract

Reagent Filming for Universal Point-of-Care Diagnostics.

Simplifying assays while maintaining the robustness of reagents is a challenge in diagnostics. This problem is exacerbated when translating quality diagnostic assays to developing countries that lack resources and infrastructure such as trained health workers, high-end equipment, and cold-chain systems. To solve this problem, in this study, a simple solution that films assay reagents to simplify the operation of diagnostic assays and preserve the stability of diagnostic reagents without using cold chains is presented. A polyvinyl-alcohol-based water-soluble film is used to encapsulate premeasured and premixed reagents. The reagent film, produced through a simple and scalable cast-drying process, provides a glassy inner matrix with abundant hydroxyl groups that can stabilize various reagents (ranging from chemicals to biological materials) by restricting molecular mobility and generating hydrogen bonds. The reagent film is applied to an enzymatic glucose assay, a high-sensitivity immunoassay for cardiac troponin, and a molecular assay for viral RNA detection, to test its practicability and universal applicability. The film-based assays result in excellent analytical/diagnostic performance and stable long-term reagent storage at elevated temperatures (at 25 or 37°C, for six months), demonstrating clinical readiness. This technology advances the development and distribution of affordable high-quality diagnostics to resource-limited regions.

Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin.

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL-1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals.

Label-free electrochemical immunosensor for ultrasensitive determination of cardiac troponin I based on porous fluffy-like AuPtPd trimetallic alloyed nanodendrites

Enhancement in the signals of electrochemical immunosensors via advanced nanomaterials is beneficial for developing immunoassay of cardiac troponin I (cTnI) with high sensitivity and selectivity. By virtue of K3[Fe(CN)6] as a signal probe, an advanced label-free electrochemical immunosensor was fabricated for ultrasensitive detection of cTnI with trimetallic alloyed AuPtPd porous fluffy-like nanodendrites (AuPtPd FNDs), followed by assembly of the antibody (Ab). By virtue of the structural advantages, electronic effects and synergetic catalysis of the trimetals, such nanostructures showed large specific surface area to increase the loading of the cTnI Ab and amplify the electrochemical signals of the probe. The sensor displayed a wide linear range (0.01 ~ 100.0 ng mL−1) and a low detection limit (LOD = 3 pg mL−1) under the optimal conditions, which showed great potential for detecting cTnI in serum samples. Furthermore, this biosensor provides an effective detection platform for analysis of other cardiac markers in practical samples.

High-sensitivity cardiac troponins: detection and central analytical characteristics

Recently, there have been some important changes in the laboratory diagnosis of patients with acute coronary syndrome, due to the introduction into routine practice of new high- and ultra-sensitive techniques for detection of myocardial damage biomarkers — cardiac troponins. Each method for cardiac troponins’ detection, among the existing wide variety of troponin immunoassays, has different analytical characteristics and allows the detection of different concentrations of troponins in the same patient. With an increasing number of companies developing high-sensitivity troponin immunoassays receiving regulatory approval, there is an urgent need for independent analytical and clinical evaluation of each method. This article discusses high- and ultrasensitive techniques for detection of cardiac troponins. The modern data on biochemical and metabolic characteristics of troponins, obtained using high- and ultra-sensitive techniques, are described: sex, age, circadian features and potential for detecting troponins in other biological fluids. Considerable attention is paid to the analytical characteristics of troponin immunoassays: limit of blank, limit of detection and limit of quantitation, coefficient of variation, as well as the 99th percentile and factors influencing it.

Prussian blue-doped PAMAM dendrimer nanospheres for electrochemical immunoassay of human plasma cardiac troponin I without enzymatic amplification

Rapid and accurate identification of cardiac troponin I (cTnl) in biological fluids is very essential for judging acute myocardial infarction (AMI).

Cardiac Troponins: Analytical Characteristics and Diagnostic Capabilities of Modern (High-sensitive) Determination Methods

Cardiovascular diseases have a leading role in terms of morbidity, mortality, and disability of the population, causing significant socio-economic damage to all countries of the world. This circumstance requires researchers to constantly seek for new biomarkers and improve methods for determining existing biomarkers, and search for new therapeutic targets to improve diagnostic and treatment strategies. Recently, there have been some important changes in laboratory diagnostics of patients with acute coronary syndrome, due to the introduction into the routine practice of new high and ultrasensitive methods for the determination of biomarkers of injury, specific to cardiac muscle tissue, namely cardiac troponins. A key advantage of highly sensitive immunochemical assays is the ability to detect cardiac troponins in the early stages of myocardial infarction. This allows making the optimal decision on the early choice and conduct of reperfusion therapy, which significantly improves the further prognosis of patients. Among the most significant generally recognised disadvantages of highly sensitive determination methods are low specificity and a huge variety of troponin immunoassays. The decrease in specificity is reflected in the fact that cardiac troponins are no longer considered the “gold standard” of diagnosis related to Acute Myocardial Infarction (AMI) (irreversible ischaemic damage to cardiomyocytes). As a result, any damage to the myocardium, even insignificant and reversible under physiological state (physical activity, stress) and several pathological conditions, can lead to an increase in serum levels of cardiac troponins and affect the accuracy of the diagnosis. Each method for the determination of cardiac troponins, among the existing wide variety of troponin immunoassays, possesses different analytical characteristics, and detects different concentrations of troponins in the same patient. This article provides a view of current data on the biology of cardiac troponins, and defines the analytical characteristics of new high-sensitive methods for the determination of cardiac troponins.

Improved efficiency and cost reduction in the emergency department by replacing contemporary sensitive with high-sensitivity cardiac troponin immunoassay.

The Authors planned this study to evaluate the impact of replacing a contemporary-sensitive with HS cTnI immunoassay on hospital and laboratory workload. The authors say that, 'Despite some evidence, the clinicians are still hesitant to replace the former so-called contemporary-sensitive methods with HS-cTn techniques, justifying this reluctance with concerns of overutilization, possible over diagnosis of cardiac injuries, overcrowding of emer-gency departments (EDs), and excess of cardiac invasive testing. Several factors have lead clinicians to use terms such as "troponin leak", "false-positive" troponin elevation, or "troponinemia". The results of this study show substantial organizational and economic benefits by replacing con-temporary-sensitive with HS cTnI immunoassays. This is very important question because there are some areas such as acute non-ST elevation coro-nary syndromes (NSTEMI) and in elderly patients, the specificity is very low for the simultaneous presence of factors that can alter the dosage of HS-cTnI values.

Kardiyak Troponinlerin Belirlenmesine Yönelik Yöntemlerin Tanısal Önemi ve Analitik Özellikleri: Geleneksel Derleme

The laboratory methods for the determination of cardiac troponins (cTnI, cTnT) used nowadays are extremely diverse, which has a significant impact on our understanding of the biology and diagnostic value of cTnI and cTnT as biomarkers. The main classification of methods for the determination of cTnI and cTnT is based on the sensitivity of the immunoassay. Low- and moderately sensitive detection methods are known to be relatively low sensitive, which leads to a relatively late confirmation of cardiomyocyte death. Due to new highly sensitive methods used for the determination of cTnI and cTnT, designated as highly or ultrasensitive immunoassays (hs-TnT and hsTnT), we received new, revised data about the biology of cardiac troponin molecules. In particular, it became clear that they can be considered products of normal myocardium metabolism since hs-TnT and hs-TnT are detected in almost all healthy patients. It also turned out that hs-TnT and hs-TnT are differ by gender (in men, troponin concentration in the blood is higher than in women), age (in elderly patients, the levels of troponins are higher than in young ones) and circadian cycles (morning concentrations of troponins are higher than in the evening). A huge variety of methods for determining cTnI and cTnT, differing in their diagnostic capabilities, creates the need for tests in order to perform an unbiased assessment of analytical characteristics of each method. This review focuses on the most pressing issues related to the discussion of the biological characteristics of cardiac troponins and the analytical characteristics of troponin immunoassays from a historical and contemporary point of view.

Clinical relevance of biological variation of cardiac troponins.

The high-sensitivity immunoassays for cardiac troponin I (hs-cTnI) and cardiac troponin T (hs-cTnT) are recommended by all the most recent international guidelines as gold standard laboratory methods for the detection of myocardial injury and diagnosis of acute myocardial infarction (AMI). In this review article, the Authors aimed at discussing the relevant biochemical, physiological, and clinical issues related to biological variability of cTnI and cTnT. Cardiac troponins, measured with hs-cTn methods, show a better clinical profile than the other cardio-specific biomarkers (such as the natriuretic peptides, BNP and NT-proBNP). In particular, the hs-cTn methods are characterized by a low intra-individual index of variation (<0.6) and reduced analytical imprecision (about 5% CV) at the clinical cut-off value (i.e.,the 99th percentile URL value). Moreover, recent studies have reported that differences between two hs-cTn measured values (RCV)>30% can be considered statistically significant. These favourable biological characteristics and analytical performance of hs-cTn methods significantly improved the accuracy in the diagnostic process of acute coronary syndromes (ACS) in patients admitted to emergence department. In addition, several studies have demonstrated the clinical usefulness of cardiovascular risk evaluation with hs-cTn methods in some groups of patients with clinical conditions at high cardiovascular risk (such as systemic hypertension, severe obesity, diabetes mellitus, renal insufficiency, and chronic obstructive pulmonary disease). However, screening programs in the general population with hs-cTn methods for cardiovascular risk stratification require further investigation to define the optimal target populations, timing of measurement, and preventive interventions.

Small‐Volume Plasmonic Microwell Array with 3D Hierarchical Nanomaterials for Plasmon‐Enhanced Fluorescence Immunoassay

Developing a small‐volume bioassay using microliter‐scale biofluids is crucial for achieving a rapid, sensitive, and high‐throughput screening and medical diagnosis. Herein, a new plasmonic microwell array (PMA) containing 3D hierarchical nanomaterials for multiplexed bioassays is presented. The microwell array is formed by performing a sequential bottom‐up fabrication involving dispensing UV‐curable photopolymers and plasmonic Ag nanowires (NWs) on a flat substrate. The plasmonic Ag NWs are developed into a multilayer stack of Ag@Au core‐shell NWs having surfaces densely decorated with Au nanoparticles. This multi‐stacked hybrid plasmonic nanostructure provided 3D multiscale hotspots and large effective surfaces for molecular binding sites. Highly improved sensitivities are demonstrated for a plasmon‐enhanced fluorescence‐based immunoassay of cardiac troponin I (cTnI), a diagnostic biomarker for acute myocardial infarction, using a small volume of 5 μL. The limit of detection is determined to be 2.02 pg mL−1 for cTnI, representing a 30‐fold greater sensitivity than the clinical cut‐off value. Furthermore, 3D PMA chips exhibit a highly uniform fluorescence intensity with a coefficient of variation of 3.4%. The 3D PMA provides a sensitive and reliable detection in a high‐throughput manner for point‐of‐care bioanalysis.

Sensitive immunoassay of cardiac troponin I using an optimized microelectrode array in a novel integrated microfluidic electrochemical device.

A sensitive and portable microfluidic electrochemical array device (μFED) was developed for the immunoassay of trace amounts of human cardiac troponin I (cTnI), which is an attractive biomarker for acute myocardial infarction (AMI). The classical "sandwich" method was adopted for the immunoassay. The capture antibody was immobilized using a self-assembled monolayer (SAM) technique, and the process was reorganized to be compatible with the bonding process. The detection antibody was labeled with alkaline phosphatase (AP) for signal amplification. The performance of the μFED was improved by eliminating the shielding effect of the microelectrode array (MEA) integrated in the μFED. The effects of the interstice and the width of the MEA on the response peak current were analyzed and simulated. The concentration gradient, about 3% of the gradient at the surface, was considered as the criterion for estimation of the optimal interstice between electrodes, and its effectiveness was proved. A stable and miniaturized reference electrode was integrated in the μFED, and its potential deviation was less than 5mV in 15min. These efforts resulted in the enhanced immunoassay performance of the μFED. A low limit of detection of about 5pg/mL was obtained in serum samples, and the response current was proportional to the logarithm of concentration from 50pg/mL to 1μg/mL. The immunoassay process was accomplished in 15min. The μFED was thus qualified and is a promising candidate for point-of-care immunoassay of cTnI. Graphical abstract.

Biotin interference in cardiac troponin immunoassay – where the wild things are?

Biotin interference in cardiac troponin immunoassay – where the wild things are?