cTnT Detection Research Articles

Electrochemical detection strategies for cardiovascular disease biomarkers: Applications in troponin I and troponin T assays

Electrochemical biosensors have emerged as promising tools for the sensitive and rapid detection of cardiac troponin I (cTnI) and troponin T (cTnT), essential biomarkers for the diagnosis and management of cardiovascular diseases (CVD). This comprehensive review explores the recent advancements in electrochemical biosensing strategies for cTnI and cTnT detection, focusing on antibody-based immunosensors, aptamer-based biosensors, and the integration of nanomaterials and microfluidic platforms. The biological aspects, diagnostic and prognostic value, and evolution of troponin assays are discussed in detail. The review highlights the remarkable performance of electrochemical biosensors, achieving femtomolar to attomolar detection limits and clinically relevant linear ranges. Signal amplification strategies and the potential for non-invasive monitoring using alternative biofluids are also explored. Although challenges persist in clinical validation and commercialization, the opportunities for multiplexed analysis, integration with digital health technologies, and personalized CVD management are promising.

Smartphone-based colorimetric detection of cardiac troponin T via label-free aptasensing

We report an aptasensing platform for the detection of cardiac troponin T (cTnT) in the immediate and early phases of acute myocardial infarction (AMI). High-flow filter paper was used to fabricate a microfluidic paper-based analytical device (μ-PAD), which was further modified with gold-decorated polystyrene microparticles functionalized with a highly specific cTnT aptamer. Herein, cTnT detection is presented in two linear ranges (0.01-0.8 μg/ml and 6.25-50 μg/ml) with an LoD of 3.9X10−4 μg/ml, which is in agreement with reference values determined by the American Heart Association. The proposed platform showed remarkable selectivity against AMI-associated cardiac biomarkers such as TNF-alpha, interleukin-6, cardiac troponin I, and reactive protein-C. This aptasensor is a label-free assay that relies only on smartphone-based image analysis and takes less processing time in comparison with traditional methods like ELISA. Furthermore, it exhibits outstanding stability over 23 days when devices are stored at 4 °C. The reported platform is a stable and cost-effective method for the on-site and user-friendly detection of cTnT in normal saline buffer and diluted human serum.

Cardiac Troponins: Contemporary Biological Data and New Methods of Determination.

Laboratory diagnosis plays one of the key roles in the diagnosis of many diseases, including cardiovascular diseases (CVD). The methods underlying the in vitro study of many CVD biomarkers, including cardiac troponins (cTnI and cTnT), are imperfect and are continually being improved to enhance their analytical performance, with sensitivity and specificity being the most important. Recently developed improved cTnI and cTnT detection methods, referred to as highly sensitive methods (hs-cTnI, hs-cTnT), have changed many of our ideas about the biology of cardiac troponins and opened up a number of additional diagnostic capabilities for practical healthcare. This article systematizes some relevant data on the biology of cardiac troponins as well as on methods for determining cTnI and cTnT with an analysis of the diagnostic value of their analytical characteristics (limit of blank, limit of detection, 99th percentile, coefficient of variation, and others). Data on extracardiac expression of cTnI and cTnT, mechanisms of formation and potential clinical significance of gender, age, and circadian characteristics of hs-cTnI and hs-cTnT content in serum are discussed. Considerable attention is paid to the discussion of new diagnostic capabilities of hs-cTnI, hs-cTnT, including consideration of promising possibilities for their study in biological fluids that can be obtained by non-invasive methods. Also, some possibilities of using hs-cTnI and hs-cTnT as prognostic laboratory biomarkers in healthy people (for example, to assess the risk of developing CVD) and in patients suffering from a number of pathological conditions that cause damage to cardiomyocytes are examined, and the potential mechanisms underlying the increase in hs-cTnI and hs-cTnT are discussed.

Characterization and Clinical Serum Test of a Molecular Imprinted Polymer (MIP)-Based Cardiac Troponin T Sensing Electrode for Patient Monitoring Applications

This article presents a cardiac troponin T (cTnT) sensing electrode fabricated using a molecular imprinted polymer (MIP) for clinical studies for the first time. The MIP-based sensing electrode is an electrochemically polymerized o-phenylenediamine film, which can detect cTnT concentrations ranging from 0.017 to 10 ng/mL in 15 min and can be reused more than six times without any performance degradation. There are no size and thickness effects on the sensing electrode, showing its high potential to be a cost-effective cTnT sensor for clinical applications. Clinical blood serum testing results show that the electrode can provide the same cTnT detection levels as the levels measured using the existing electrochemiluminescence method in a tertiary university-affiliated hospital.

A Label-free Cardiac Troponin T Electrochemiluminescence Immunosensor Enhanced by Graphene Nanoplatelets.

In this study, a direct and label-free immunosensor was designed and constructed by modifying the screen-printed electrode with graphene nanoplatelets (GNPs) for the detection of the cardiac troponin T (cTnT). Firstly, GNPs were drop-casted onto carbon working electrode. Monoclonal cTnT antibodies were then immobilized on the GNPs via physical adsorption; finally, BSA was introduced to block non-specific binding sites. The detection of cTnT was performed using an electrochemiluminescence (ECL) technique with tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) used as a luminophore and TPrA (tripropylamine) as a co-reactant. The ECL intensity was demonstrated to be directly proportional to the cTnT concentration where a linear range from 100 pg mL-1 to 5 fg mL-1 of the cTnT detection was established. An extremely low limit of detection was achieved to be 0.05 fg mL-1 with an outstanding specificity. Additionally, this immunosensor showed excellent percentage recovery for real samples analyses in artificially spiked human serum.

New Signal Probe Integrated with ABEI as ECL Luminophore and Ag Nanoparticles Decorated CoS Nanoflowers as Bis-Co-Reaction Accelerator to Develop a Ultrasensitive cTnT Immunosensor

In this work, Ag nanoparticles (Ag NPs) decorated CoS nanoflowers (CoS NFs) were firstly used as bis-coreaction accelerator for signal amplification to establish an highly sensitive electrochemiluminescence (ECL) immunosensor for cardiac troponin T (cTnT) detection. Due to the synergistic catalysis between CoS NFs and Ag NPs, the decomposition rate of the co-reaction reagent H2O2 was largely improved, generating more ROS for signal amplification. Using the N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as ECL luminophore, a new signal tag was acheived according to the assembly of ABEI functionalized Ag NPs (ABEI-Ag) on the CoS NFs via Ag-S bond, which integrated with the ECL luminophore and bis-co-reaction accelerator. In addition, to fabricate sensing interface, primary antibodies (Ab1) was immobilized on the glassy carbon electrode which decorated with Au nanoparticles (Au NPs), almost providing a zero background signal. As a result, this developed immunosensor for cTnT possessed a linear range from 0.1 fg mL−1 to 100 pg mL−1 and the limit of detection down to 0.03 fg mL−1 for ultrasensitive detection of cTnT, which was expected to be applicable to cTnT detection in clinic acute myocardial infarction.

Evaluation of a newly developed chemiluminescence immunoassay for detecting cardiac troponin T

BackgroundTo evaluate the performance of a chemiluminescence detection kit for cardiac troponin T (cTnT).MethodsAccording to the “Guiding principles on performance analysis of diagnostic reagents in vitro” and the Clinical and Laboratory Standards Institute (CLSI) Guidelines, we assessed the detection limit, linear range, reportable range, accuracy, precision, cross reactivity, interference factors, and matrix effect of the kit, and compared these parameters with that of the commercial electrochemiluminescence detection kit for cTnT (Roche).ResultsThe minimum detection limit of the kit was 0.01 ng/mL. The linear range was 0.01 ng/mL‐25 ng/mL. The reportable range was from 0.01 ng/mL to 100 ng/mL. Precision within the batch was 2.9%‐6.4%, and precision between batches was 6.0%; the accuracy was good and the recovery rate reached 96.2%. The cross‐reaction test demonstrated that there was no reactivity between cTnT and a variety of troponins. Test results deviated by less than ±10% in the presence of hemoglobin ≤1000 μg/mL, bilirubin ≤250 μg/mL, triglycerides ≤11.25 mg/mL, and rheumatoid factor ≤206 U/mL, suggesting that kit results were not significantly interfered with these factors. Results from the matrix‐effect assessment revealed absence of a matrix effect in the tested serum samples. Correlation study revealed that the performance of the kit was very consistent with that of the Roche electrochemiluminescence detection kit (Kappa = 0.900, P < .001) with a high correlation (r = .903, P < .001) and a total matching rate of 95.00%.ConclusionThe performance of the evaluated chemiluminescence immunoquantitation kit for cTnT detection was acceptable in all tested parameters, fulfilling requirements for clinical applications.

Incremental Interface Surface Potential Measured with a Nano-Gap Coplanar Device Structure and Its Applications

Because of detection characteristics in traditional capacitive-based biomolecular sensing technologies, it is important to have a uniform and defect-free interface layers. However, efforts to achieve the interface layer are tedious and labor intensive. To conquer this problem and enhance the sensing characteristics of capacitive-based biomolecular sensing device, in our report, we use co-planar electrode with nano-gap structure and placed the sensing element on the gap surface, which is different from traditional capacitive putting antigen/antibody on the electrode surface. This arrangement leads to gap-surface charge changes as molecular binding occurring. And the altered gap-surface charges modulate the electric double layer (EDL) of electrode sidewalls and result in the change of nano-gap capacitance. Utilizing surface modification with amine-terminated and aldehyde-terminated groups, we demonstrates the proposed electrode sidewall EDL contraction phenomena induced by gap-surface ion redistribution. Based on our experimental results of surface modifications, the capacitance variance of 1 µm and 100 nm gap width are 8% and 17%, respectively. In addition, we show that both gap width and detection environment play important roles in sensitivity and detection limit of the developed nano-gap device. With a demonstration of cTnT detection, a demonstrated detection dynamic range of the proposed method is from 10 pg/ml to 1 µg/ml, which is around 100 fold higher than that of traditional capacitive biosensors. The limit of detection is 10 pg/ml, which is the cut-off level in clinical examinations.

Development, identification and application of 33 monoclonal antibodies against cardiac troponin T

The aim of this study is to prepare and characterize cardiac troponin T (cTnT) monoclonal antibodies (mAb), and further develop a chemiluminescence quantitative detection assay for cTnT. BALB/c mice were immunized with recombinant cTnT antigen, and specific mAbs were prepared using conventional hybridoma technique and screened by indirect ELISA method. To identify the epitopes, several cTnT peptide fragments were synthesized or expressed by genetic engineering. A double antibody sandwich ELISA method was used to screen the mAb pairs for cTnT detection, and the automatic chemiluminescence detection assay for cTnT was developed. In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay; further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples. We successfully screened 33 strains of hybridoms against cTnT, and the mAbs' epitopes were identified. Mab E16H8 and C8G11 with a detection limit of 10 pg/mL cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay. The correlation coefficient of our reagent with Roche's was 0.959 9, with a coincidence rate of 95%. The assay presented a sensitivity of 97.5%, and a specificity of 99.15% in detection of clinical samples. The cTnT concentration was less than 0.080 6 ng/mL in 99% of general population, which agrees with the definition of WHO on patients with acute myocardial infarction (AMI). In summary, we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT, and an automatic tubular chemiluminescence quantitative detection assay was further developed, which presents a high coincidence rate with Roche's.

SPR detection of cardiac troponin T for acute myocardial infarction

A surface plasmon resonance (SPR) sensor developed for the rapid, sensitive and specific detection of cardiac troponin T (cTnT) in serum samples is reported in this work. An extensive optimisation of assay parameters was conducted to achieve optimal detection strategy. Both direct and sandwich immunoassay formats were investigated and optimised. The response obtained was enhanced further by the use of gold nanoparticles (AuNPs) conjugated to the anti-cTnT detection antibody. A regeneration method was developed to enable the reuse of the SPR sensor for multiple sample application. The SPR immunosensor showed good reproducibility for cTnT detection in the concentration range of 25–1000ngmL−1 and 5–400ngmL−1 for the direct and sandwich assays in buffer, respectively. The linear regression analysis was performed and R2 value was found as 0.99 for both assays. In order to optimise the sensor for serum analysis, nonspecific binding of serum proteins was reduced through the use of additives in the dilution buffer. To achieve greater sensitivity, the performance of the cTnT immunosensor sandwich assay in human serum was evaluated using non-modified and AuNP modified detector antibodies. A detection limit (LOD) for the immunosensor in 50% serum was assessed as 5ngmL−1 cTnT for the standard sandwich assay and 0.5ngmL−1 cTnT when using AuNP conjugated detector antibodies with a linear dynamic range of 0.5–40ngmL−1. The dissociation constant was found as 3.28 × 10−9M using Langmuir binding model which indicates high affinity between cTnT and its antibody. The proposed SPR immunosensor has a promising potential to be developed for point-of-care testing for the early diagnosis of acute myocardial infarction (AMI). This method can also be used for the rapid detection of biomarkers in central nervous system diseases.

A Rapid, Direct, Quantitative, and Label‐Free Detector of Cardiac Biomarker Troponin T Using Near‐Infrared Fluorescent Single‐Walled Carbon Nanotube Sensors

Patients with chest pain account for 10% of US emergency room visits according to data from the Center for Disease Control and Prevention (2013). For triage of these patients, cardiac biomarkers troponin I and T are endorsed as standard indicators for acute myocardial infarction (AMI, or heart attack). Thus, there is significant interest in developing a rapid, point-of-care (POC) device for troponin detection. In this work, a rapid, quantitative, and label-free assay, which is specific for cardiac troponin T (cTnT) detection, using fluorescent single-walled carbon nanotubes (SWCNTs), is demonstrated. Chitosan-wrapped carbon nanotubes are cross-linked to form a thin gel that is further functionalized with nitrilotriacetic acid (NTA) moieties. Upon chelation of Ni(2+) , the Ni(2+) -NTA group binds to a hexa-histidine-modified troponin antibody, which specifically recognizes the target protein, troponin T. As the troponin T binds to the antibody, the local environment of the sensor changes, allowing direct troponin detection through intensity changes in SWCNT bandgap fluorescence. This platform represents the first near-infrared SWCNT sensor array for cTnT detection. Detection can be completed within 5 min, demonstrating a linear response to cTnT concentration and an experimental detection limit of 100 ng mL(-1) (2.5 nm). This platform provides a promising new tool for POC AMI detection in the future. Moreover, the work presents two new methods of quantifying the number of amines and carboxylic groups, respectively, in a carbon hydrogel matrices.

The Influence of a Half-Marathon Race Upon Cardiac Troponin T Release in Adolescent Runners

Post-exercise cardiac troponin T (cTnT) release has been widely reported in adult athletes but limited data is available for adolescents. The aim of this study was to determine the incidence and magnitude of cTnT appearance in a large group of adolescent runners, and to determine any association between cTnT release and personal characteristics of adolescents. We recruited 63 adolescent runners (mean±SD: age 16.4±1.5 years; 10 females) who all completed a simulated half-marathon race (an all-out 21-km run) during routine training. Personal data collected included age, training history, 21-km run performance as well as pre-post exercise serum cTnT levels. Serum cTnT was assayed using a 3rd generation assay. At pre-exercise, cTnT concentrations were below the 0.01 µg/L cTnT detection limit of assay in 58/63 runners. The post-exercise cTnT level (range: <0.01-1.36 µg/L) was significantly (p<0.001) greater than that of the pre-exercise (range: <0.01-0.02 µg/L). After the exercise, 57 (90%) and 44 (70%) subjects had cTnT concentrations above the detection: 0.01, and clinical thresholds: 0.05 µg/L, respectively. Post-exercise cTnT was inversely correlated with training years (r=-0.25, p<0.05) and age (r=-0.31, p<0.05). Compared with runners who had trained for ≥ 3 years, runners with less training experience demonstrated increased post-race cTnT levels (p<0.01). cTnT increases are virtually universal among adolescent runners following a 21-km run during routine training and can reach levels typically diagnostic for acute myocardial infarction potentially initiating diagnostic dilemmas. Adolescents with less training experience had higher post-exercise cTnT.

Cardiac troponin T detection using polymers coated quartz crystal microbalance as a cost-effective immunosensor

Cardiac troponin T (cTnT) detection has been the focus of increased interest due to its role in myocardial infarction diagnosis. In this study, we report a relatively low coat technique to detect cTnT using a quartz crystal microbalance (QCM) sensor. A sensitive detection is achieved by introducing a QCM surface with a carboxylic polyvinyl chloride immobilization layer. The surface morphologies of this polymer film under varied deposition thickness have been investigated by field emission scanning electron microscopy and atomic force microscopy. A cTnT detection result from a modified QCM surface can be obtained within a short response time by a direct detection of the immunoreaction and a direct conversion of mass accumulation into a frequency shift, representing a measurable electrical signal. The relationship between the cTnT concentration and the response current from a QCM sensor shows detectability at the concentration of cTnT as low as 5 ng/ml.