Fluorescence anisotropic probe for sensing cardiac troponin-I antigen through target-specific antibody-conjugated gold nanoclusters.

Fluorescence anisotropy (FA) is a versatile and efficient platform for developing biosensors that rely on the rate of rotations of fluorescence molecular entities in biochemical systems. However, by virtue of its intricate complexity, FA is a neglected and less explored area for developing biosensors. Herein, we experimented with the possibility of developing a fluorescence anisotropic probe to detect cardiac troponin I (cTnI), the gold standard biomarker for acute myocardial infarction, via target-specific monoclonal antibody-conjugated gold nanoclusters. The successful detection of cTnI antigen in clinically relevant concentration with a low detection limit of 0.91 ng mL-1 was achieved. The specific molecular interaction between the cTnI antigen and its monoclonal antibody tagged at the surface of gold nanoclusters has restricted the free rotation of gold nanoclusters and increased the FA value. This incremental increase in FA can be correlated to the concentration of cTnI antigen in the sample, thereby achieving the quantitative linear detection of cTnI.

Controlled labelling of tracer antibodies for time-resolved fluorescence-based immunoassays.

Tracer antibodies, which are labelled with fluorescent or other type of reporter molecules, are widely employed in diagnostic immunoassays. Time-resolved fluorescence immunoassay (TRFIA), recognized as one of the most sensitive immunoassay techniques, utilizes tracers labelled with lanthanide ion (Ln) chelates. The conventional approach for conjugating isothiocyanate (ITC) Ln-chelates to antibodies involves random chemical targeting of the primary amino group of Lys residues, requiring typically overnight exposure to an elevated pH of 9-9.3 and leading to heterogeneity. Moreover, efforts to enhance the sensitivity of the assays by introducing a higher number of Ln-chelates per tracer antibody are associated with an elevated risk of targeting critical amino acid residues in the binding site, compromising the binding properties of the antibody. Herein, we report a method to precisely label recombinant antibodies with a defined number of Ln-chelates in a well-controlled manner by employing the SpyTag/SpyCatcher protein ligation technology. We demonstrate the functionality of the method with a full-length recombinant antibody (IgG) as well as an antibody fragment by producing site-specifically labelled antibodies for TRFIA for cardiac troponin I (cTnI) detection with a significant improvement in assay sensitivity compared to that with conventionally labelled tracer antibodies. Overall, our data clearly illustrates the benefits of the site-specific labelling strategy for generating high-performing tracer antibodies for TRF immunoassays.

Copper(II)-Tannic Acid@Cu with In Situ Grown Gold Nanoparticles as a Bifunctional Matrix for Facile Construction of Label-Free and Ultrasensitive Electrochemical cTnI Immunosensor.

Sensitive detection of cardiac troponin I (cTnI) is of great significance in the diagnosis of a fatal acute myocardial infarction. A redox-active nanocomposite of copper(II)-tannic acid@Cu (CuTA@Cu) was herein prepared on the surface of a glassy carbon electrode by electrochemical deposition of metallic copper combined with a metal stripping strategy. Then, HAuCl4 was in situ reduced to gold nanoparticles (AuNPs) by strong reductive catechol groups in the TA ligand. The AuNPs/CuTA@Cu composite was further utilized as a bifunctional matrix for the immobilization of the cTnI antibody (anti-cTnI), producing an electrochemical immunosensor. Electrochemical tests show that the immunoreaction between anti-cTnI and target cTnI can cause a significant reduction of the electrochemical signal of CuTA@Cu. It can be attributed to the insulating characteristic of the immunocomplex and its barrier effect to the electrolyte ion diffusion. From the signal changes of CuTA@Cu, cTnI can be analyzed in a wide range from 10 fg mL-1 to 10 ng mL-1, with an ultralow detection limit of 0.65 fg mL-1. The spiked recovery assays show that the immunosensor is reliable for cTnI determination in human serum samples, demonstrating its promising application in the early clinical diagnosis of myocardial infarction.

Role of Antitroponin Antibodies and Macrotroponin in the Clinical Interpretation of Cardiac Troponin.

Cardiac troponin is extensively used as a biomarker in modern medicine due to its diagnostic capability for myocardial injury, as well as its predictive and prognostic value for cardiac diseases. However, heterophile antibodies, antitroponin antibodies, and macrotroponin complexes can be observed both in seemingly healthy individuals and patients with cardiac diseases, potentially leading to false positive or disproportionate elevation of cTn (cardiac troponin) assay results and introducing discrepancies in clinical interpretations with impact on medical management. In this review article, we describe the possible mechanisms of cTn release and the sources of variations in the assessment of circulating cTn levels. We also explore the pathophysiological mechanisms underlying antitroponin antibody development and discuss the influence exerted by macrotroponin complexes on the results of immunoassays. Additionally, we explore approaches to detect these complexes by presenting various clinical scenarios encountered in routine clinical practice. Finally, unsolved questions about the development, prevalence, and clinical significance of cardiac autoantibodies are discussed.

A novel integrated lateral flow immunoassay platform for the detection of cardiac troponin I using hierarchical dendritic copper-nickel nanostructures.

Cardiac troponin I (cTnI) is a critical biomarker for the diagnosis of acute myocardial infarction (AMI). Herein, we report a novel integrated lateral flow immunoassay (LFIA) platform for highly sensitive point-of-care testing (POCT) of cTnI using hierarchical dendritic copper-nickel (HD-nanoCu-Ni) nanostructures. The electrodeposited HD-nanoCu-Ni film (∼22 µm thick) on an ITO-coated glass substrate exhibits superior capillary action and structural integrity. These properties enable efficient sample transport and antibody immobilization, making it a compelling alternative to conventional multi-component paper-based LFIA test strips, which are often plagued by structural fragility and susceptibility to moisture damage. The biofunctionalized HD-nanoCu-Ni substrates were laser-etched with lateral flow channels, including a sample loading/conjugate release zone, a test zone, and a control zone. Numerical simulations were used to further optimize the design of these channels to achieve optimal fluid flow and target capture. The HD-nanoCu-Ni LFIA device utilizes a fluorescence quenching based sandwich immunoassay format using antibody-labeled gold nanoparticles (AuNPs) as quenchers. Two different fluorescent materials, fluorescein isothiocyanate (FITC) and CdSe@ZnS quantum dots (QDs), were used as background fluorophores in the device. Upon the formation of a sandwich immunocomplex with cTnI on the HD-nanoCu-Ni device, introduced AuNPs led to the fluorescence quenching of the background fluorophores. The total assay time was approximately 15 min, demonstrating the rapid and efficient nature of the HD-nanoCu-Ni LFIA platform. For FITC, both inner filter effect (IFE) and fluorescence resonance energy transfer (FRET) contributed to the AuNP-mediated quenching. In the case of CdSe@ZnS QDs, IFE dominated the AuNP-induced quenching. Calibration curves were established based on the relationship between the fluorescence quenching intensity and cTnI concentration in human serum samples, ranging from 0.5 to 128 ng/mL. The limits of detection (LODs) were determined to be 0.27 ng/mL and 0.40 ng/mL for FITC and CdSe@ZnS QDs, respectively. A method comparison study using Passing-Bablok regression analysis on varying cTnI concentrations in human serum samples confirmed the equivalence of the HD-nanoCu-Ni LFIA platform to a commercial fluorescence cTnI LFIA assay kit, with no significant systematic or proportional bias observed.

Establishment of a microspheres-based homogeneous fluorescence immunoassay for the rapid detection of cardiac troponin I.

Myocardial infarction occurs rapidly, and thus the rapid detection of cTnI levels is the key to its diagnosis. Most current assays take 10-30 min. In this study, we developed a method for accurately measuring cardiac troponin I (cTnI) levels in human sera with amplified luminescence neighborhood homogeneous assay (AlphaLISA). The method involves coupling two cTnI antibodies targeting different epitopes to the surface of carboxylated donor and acceptor beads. The final signal values were detected by the double-antibody sandwich method, and the best reaction conditions were obtained by optimizing the experimental conditions. The sensitivity, specificity, accuracy, and precision of the method were evaluated. Results showed that the method requires only 3 min to produce the results, the detection sensitivity is 27.06 ng L-1, and the measurement range is 34.56-62 500 ng L-1. cTnI-AlphaLISA has an intra-assay precision of 2.18-4.57% (<10%) and an inter-assay precision of 5.60-6.95% (<10%). The relative recovery rates are within reasonable limits. In addition, the serum assay results of the method were compared with chemiluminescence immunoassay, and the results are in agreement with one another (ρ = 0.8803; P < 0.0001). The method is expected to be developed as a routine method, but further studies and evaluations are needed.

Increase of Cardiac Autoantibodies Against Beta-2-adrenergic Receptor During Acute Cellular Heart Transplant Rejection.

BACKGROUND: Acute cellular rejection (ACR) in heart transplant (HTx) recipients may be accompanied by cardiac cell damage with subsequent exposure to cardiac autoantigens and the production of cardiac autoantibodies (aABs). This study aimed to evaluate a peptide array screening approach for cardiac aABs in HTx recipients during ACR (ACR-HTx). METHODS: In this retrospective single-center observational study, sera from 37 HTx recipients, as well as age and sex-matched healthy subjects were screened for a total of 130 cardiac aABs of partially overlapping peptide sequences directed against structural proteins using a peptide array approach. RESULTS: In ACR-HTx, troponin I (TnI) serum levels were found to be elevated. Here, we could identify aABs against beta-2-adrenergic receptor (ß-2AR: EAINCYANETCCDFFTNQAY) to be upregulated in ACR-HTx (intensities: 0.80 versus 1.31, P = 0.0413). Likewise, patients positive for ß-2AR aABs showed higher TnI serum levels during ACR compared with aAB negative patients (10.0 versus 30.0 ng/L, P = 0.0375). Surprisingly, aABs against a sequence of troponin I (TnI: QKIFDLRGKFKRPTLRRV) were found to be downregulated in ACR-HTx (intensities: 3.49 versus 1.13, P = 0.0025). A comparison in healthy subjects showed the same TnI sequence to be upregulated in non-ACR-HTx (intensities: 2.19 versus 3.49, P = 0.0205), whereas the majority of aABs were suppressed in non-ACR-HTx. CONCLUSIONS: Our study served as a feasibility analysis for a peptide array screening approach in HTx recipients during ACR and identified 2 different regulated aABs in ACR-HTx. Hence, further multicenter studies are needed to evaluate the prognostic implications of aAB testing and diagnostic or therapeutic consequences.

Ternary BiOI/Bi2S3/Au Nanosheet Arrays as a Photoelectrochemical Signal Converter for the Detection of Cardiac Troponin I.

Nanosheet arrays with stable signal output have become promising photoactive materials for photoelectrochemical (PEC) immunosensors. However, an essential concern is the facile recombination of carriers in one-component nanoarrays, which cannot be readily prevented, ultimately resulting in weak photocurrent signals. In this study, an immunosensor using gold nanoparticle-anchored BiOI/Bi2S3 nanosheet arrays (BiOI/Bi2S3/Au) as a signal converter was fabricated for sensitive detection of cardiac troponin I (cTnI). The ternary nanosheet arrays were prepared by a simple method in which Bi2S3 was well-coated on the BiOI surface by in situ growth, whereas the addition of Au further improved the photoelectric conversion efficiency and could link more antibodies. The three-dimensional (3D) ordered sheet-like network array structure and BiOI/Bi2S3/Au ternary nanosheet arrays showed stable and high photoelectric signal output and no significant difference in signals across different batches under visible light excitation. The fabricated immunosensor has a sensitive response to the target detection marker cTnI in a wide linear range of 500 fg/mL to 50 ng/mL, and the detection limit was 32 fg/mL, demonstrating good stability and selectivity. This work not only shows the great application potential of ternary heterojunction arrays in the field of PEC immunosensors but also provides a useful exploration for improving the stability of immunosensors.

The role of antibody-based troponin detection in cardiovascular disease: A critical assessment.

Cardiovascular disease has remained the world's biggest killer for 30 years. To aid in the diagnosis and prognosis of patients suffering cardiovascular-related disease accurate detection methods are essential. For over 20 years, the cardiac-specific troponins, I (cTnI) and T (cTnT), have acted as sensitive and specific biomarkers to assist in the diagnosis of various types of heart diseases. Various cardiovascular complications were commonly detected in patients with COVID-19, where cTn elevation is detectable, which suggested potential prognostic value of cTn in COVID-19-infected patients. Detection of these biomarkers circulating in the bloodstream is generally facilitated by immunoassays employing cTnI- and/or cTnT-specific antibodies. While several anti-troponin assays are commercially available, there are still obstacles to overcome to achieve optimal troponin detection. Such obstacles include the proteolytic degradation of N and C terminals on cTnI, epitope occlusion of troponin binding-sites by the cTnI/cTnT complex, cross reactivity of antibodies with skeletal troponins or assay interference caused by human anti-species antibodies. Therefore, further research into multi-antibody based platforms, multi-epitope targeting and rigorous validation of immunoassays is required to ensure accurate measurements. Moreover, in combination with various technical advances (e.g. microfluidics), antibody-based troponin detection systems can be more sensitive and rapid for incorporation into portable biosensor systems to be used at point-of care.

Change in troponin concentrations in patients with macrotroponin: An in vitro mixing study.

INTRODUCTION: Macrotroponin is a complex formed between endogenous cardiac troponin autoantibodies (cTnAABs) and circulating cardiac troponin (cTn). The potential effect of macrotroponin on current high sensitivity cTn assays has not been fully explored but has recently been identified as a major cause of discrepancy in cTn results between assays. In this study we investigated the effects of mixing troponin (cTn) standards to specimens with and without macrotroponin. METHOD: Macrotroponin was identified in specimens by a recovery of cTnI < 40% following protein A immunoglobulin depletion. Troponin standards containing cTn-IC and cTn-TIC complexes were mixed with serum samples, with (n = 20) and without (n = 10) the presence of macrotroponin. Specimens were tested for cTn before and after mixing by three commercially available high sensitivity cTn assays. Gel filtration chromatography was carried out on five specimens with macrotroponin and each fraction was analzyed by multiple cTn assays. FINDINGS: Following mixing with cTn-TIC standard, all specimens with macrotroponin had a markedly reduced absolute increase in cTnI, indicating negative analytical interference due to macrotroponin. Following mixing with the cTn-IC standard, specimens with macrotroponin demonstrated highly variable changes in cTnI, suggesting significant heterogeneity in macrotroponin complex reactivity between individuals. When the ratio of change, calculated by dividing the absolute change between two cTn assays, was compared between specimens with and without macrotroponin, significant differences were observed (p < 0.001). These findings were supported by variable migration of peak cTn activity on gel filtration chromatography. CONCLUSION: Macrotroponin leads to assay dependent analytical interference affecting current high sensitivity troponin I assays. Furthermore, endogenously occurring cTnAABs are conformationally specific and the analytical effects vary between assays and individuals.

A liquid-crystal-based immunosensor for the detection of cardiac troponin I.

Cardiac troponin I (cTnI) is one of the most sensitive and specific markers of myocardial cell injury, which can detect even minor myocardial damages. It is recognized as the main biochemical marker of the rapid diagnosis of acute myocardial infarction (AMI) and acute coronary syndrome (ACS). In this study, a label-free biosensor that utilizes the birefringence property of a nematic liquid crystal (LC) for the detection of cTnI is demonstrated. A chemically sensitive film with specific molecular recognition ability was decorated on the surface of a substrate, and the LC molecules were arranged in a vertically oriented order under the influence of the sensitive film, and a dark background signal was obtained using a polarizing optical microscope. When the antigen-antibody specifically binds to form a stronger acting force, the orientation of the LC molecules changes, resulting in a bright optical appearance. This LC-based immunosensor not only has the advantages of a facile structure, low cost and excellent specificity but also high sensitivity (a low detection limit of 1 pg ml-1), and has a promising future in biomedical related fields.

A cardiac troponin I photoelectrochemical immunosensor: nitrogen-doped carbon quantum dots-bismuth oxyiodide-flower-like SnO2.

A novel photoelectrochemical (PEC) immunosensor for the determination of cardiac troponin I (cTnI) was constructed. The flower-like stannic oxide (SnO2) with large specific surface area was prepared by hydrothermal synthesis. Nitrogen-doped carbon quantum dots (NCQDs) with excellent surface property were used as a sensitizer for SnO2. Bismuth oxyiodide (BiOI) is a narrow band gap (1.83 eV) nanomaterial, which was firstly modified on NCQDs-sensitized SnO2 through in situ growth method. After NCQDs with small size and BiOI nanoparticles are successively combined with SnO2, the SnO2/NCQDs/BiOI microflower was obtained, which possessed good photochemical properties. Using visible light as excitation source and ascorbic acid (AA) as electron donor, the ultrasensitive and quantitative determination of cTnI was realized by detecting the changes of photocurrent under different concentrations of cTnI. The PEC immunosensor showed a large-scaled response (0.001-100 ng mL-1) and a low detection limit (0.3 pg mL-1) under optimised experimental conditions. The sensor has potential clinical value in the prediction and diagnosis of cardiovascular diseases in elderly patients with diabetes. Graphical abstract.

Interleukin-10 delivered by mesenchymal stem cells attenuates experimental autoimmune myocarditis.

BACKGROUNDS: Autoimmune myocarditis is characterized by over-activated immune system attacking the cardiomyocytes, resulting in heart function decline. In the current study, we investigated the therapeutic advantages of delivering Interleukin-10 (IL-10) by mesenchymal stem cells (MSCs), both of which had immune suppression functions, in treating experimental autoimmune myocarditis. METHODS: The mouse model of autoimmune myocarditis was established by subcutaneous injection of troponin I in A/J mice. Mouse bone marrow derived mesenchymal stem cells (BM-MSCs) with or without IL-10 overexpression, or the recombinant IL-10 protein were delivered into the mice via tail-vein injection. The inflammation and fibrosis levels of the heart were evaluated with qPCR, ELISA and histological staining. Serum level of anti-troponin-I was assessed by ELISA. Heart function analysis was conducted with echocardiography. RESULTS: BM-MSCs overexpressing IL-10 had enhanced immune suppression functions. They also showed improved therapeutic effects from the perspective of heart function and cardiac fibrosis. The anti-troponin-I level was significantly reduced by MSCs overexpressing IL-10 when comparing with the MSCs or IL-10 protein injection. CONCLUSION: IL-10 delivered by MSCs showed therapeutic advantages in treating experimental autoimmune myocarditis.

Electrochemiluminescent Detection of Proteins Based on Fullerenols Modified Gold Nanoparticles and Triple Amplification Approaches.

In this work, fullerenols were found to be able to enhance the ECL signals of the luminol and H2O2 system and were employed for the first time as a reducing, catalyzing, and stabilizing agent in the one-step fast synthesis of fullerenols@AuNPs in only 5 min. First, the prepared fullerenols@AuNPs were applied to fabricate a label-free immunosensor for the detection of human cardiopathy biomarker (cardiac troponin I, cTnI). Second, using the fullerenols@AuNPs as biolabels to establish a sandwich-type immunosensor and catalyzing in situ copper-stained reaction to generate Cu particles capped on the fullerenols@AuNPs, and then a novel electrochemical stripping chemiluminescent (ESCL) method was developed for detection of cTnI and IgG with about 20 times more sensitive than the former one. At the process of ESCL detection, Cu2+was stripped from Cu@fullerenols@AuNPs with significant increase of the ECL signals. This can be attributed to the fact that the fullerenols@AuNPs nanoparticles and the Cu2+ have excellent conductivity and could facilitate the decomposition of H2O2 to generate various reactive oxygen species (ROSs), thereby accelerating the ECL process. Both immunosensors show high sensitivity and selectivity to cTnI and IgG detection with a wide linear range from fg/mL to ng/mL and the low limits of detection down to fg/mL for cTnI and IgG, respectively.

Sensitive detection of cardiac troponin-I protein using fabricated piezoresistive microcantilevers by a novel method of asymmetric biofunctionalization.

Microcantilever-based sensor platform has attracted a lot of attention over the time in detection of a variety of molecules due to their miniaturized dimensions. Sensitivity enhancement is an important aspect of such sensors, especially when used for point-of-care diagnostic purpose. However, the major concern while operating these sensors in deflection mode is their sensitivity which mainly relies on selective chemical modification protocols employed on these sensor surfaces. One of the ways of getting better sensitivity is through asymmetric (one side) biofunctionalization of the sensor surface. In the presented work here, we have demonstrated a novel approach of asymmetric biofunctionalization of proteins in overall sensitivity enhancement of piezoresistive silicon nitride-oxide microcantilever sensor platform inside a flow chamber. Herein, using our developed surface chemistry, asymmetrically biofunctionalized microcantilevers first exhibited a greater electrical response in terms of piezoresistance change than their symmetric counterpart in the detection of human immunoglobulins (HIgGs) protein. Finally, these microcantilevers were employed to exhibit the enhanced sensitivity towards the detection of a crucial cardiac marker protein, i.e. Troponin-I (cTnI) down to 250 ng ml-1 using asymmetric biofunctionalization process. This study shows that the developed asymmetric biofunctionalization methodology may be used as a general protocol to detect other important biomarkers of clinical applications with improved sensitivity.

Transposing Lateral Flow Immunoassays to Capillary-Driven Microfluidics Using Self-Coalescence Modules and Capillary-Assembled Receptor Carriers.

Point-of-care (POC) immunodiagnostic tests play a crucial role in enabling rapid and correct diagnosis of diseases in prehospital care, emergency, and remote settings. In this work, we present a silicon-based, capillary-driven microfluidic chip integrating two microfluidic modules for the implementation of highly miniaturized immunoassays. Specifically, we apply state-of-the-art microfluidic technology to demonstrate a one-step immunoassay for the detection of the cardiac marker troponin I in human serum using sample volumes of ∼1 µL and with a limit of detection (LOD) of ∼4 ng mL-1 within 25 min. The microfluidic modules discussed here broadly map functionalities found in standard lateral flow assays. We implement a self-coalescence module (SCM) for the controlled reconstitution and delivery of inkjet-spotted and dried detection antibodies (dAbs). This allows for homogeneous dissolution of 1.3 ng of fluorescently labeled dAbs in 416 nL of the sample used for the assay. We also show how to immobilize receptors inside closed microfluidic devices in <30 s using bead lane modules inside which microbeads functionalized with capture antibodies (cAbs) are self-assembled. The resulting bead lane module, with a volume of ∼3 × 10-5 mm3, is positioned across the flow path and holds ∼300 5 µm-diameter microbeads. Altogether, these capillary-driven elements allow for the manipulation of samples and reagents with an unprecedented precision and control, paving the way for the next generation of POC immunodiagnostics.

Reduced graphene oxide-gold nanoparticles-catalase-based dual signal amplification strategy in a spatial-resolved ratiometric electrochemiluminescence immunoassay.

A novel spatial-resolved electrochemiluminescent (ECL) ratiometry for cardiac troponin I (cTnI) analysis was developed using resonance energy transfer (RET) and a coreactant consumption strategy for signal amplification. Specifically, the spatial-resolved dual-disk glassy carbon electrodes were modified with CdS nanowires (CdS NWs) and luminol-gold nanoparticles (L-Au NPs) as potential-resolved ECL emitters, respectively. After stepwise immobilization of anti-cTnI and bovine serum albumin on the dual-disk electrodes, the CdS NWs-based electrode, with varied concentrations of cTnI, was used to provide a working signal, whereas the L-Au NPs-based electrode, with a fixed amount of cTnI, was employed to provide the reference signal. To efficiently amplify the working signal on the CdS NWs-based electrode, an anti-cTnI-reduced graphene oxide-gold nanoparticles-catalase probe (anti-cTnI-rGO-Au NPs-CAT) was loaded onto the electrode to form a sandwich immunocomplex. The RET from CdS NWs to Au NPs and the coreactant (i.e. H2O2) consumption by the CAT generate a significant ECL decrease on the CdS NWs-based electrode in the presence of cTnI. This novel and sensitive ratiometric detection mode for cTnI was achieved using the ratio values of the working signal of the CdS NWs-based electrode and the reference signal of the L-Au NPs-based electrode. The integration of RET and coreactant consumption strategy in the designed spatial-resolved ratiometric platform endows the immunosensor with a wide linear range of 5.0 × 10-13 - 1.0 × 10-7 g mL-1 and a low detection limit of 0.10 pg mL-1 for cTnI. Furthermore, the method exhibits high accuracy and sensitivity for cTnI determination in human serum samples.

A wavelength-resolved electrochemiluminescence resonance energy transfer ratiometric immunosensor for detection of cardiac troponin I.

In this study, a wavelength-resolved electrochemiluminescence resonance energy transfer (ECL-RET) ratiometric immunosensor from Au nanoparticle functionalized graphite-like carbon nitride nanosheets (Au-g-C3N4) to Au nanoclusters (Au NCs) has been constructed for the first time. At a working voltage of 0 to -1.2 V, Au-g-C3N4 showed a strong cathodic ECL emission with a peak at 460 nm, which overlapped well with the absorption spectra of Au NCs thus stimulating the fluorescence emission of Au NCs at 610 nm. Moreover, within this voltage range, the Au NCs showed no ECL signal; therefore, they would not interfere with the detection of the system. We used cardiac troponin I (cTnI) as an analytical model to construct a sandwich immunosensor based on the ECL-RET ratiometric strategy. By measuring the responses of the ECL460 nm/FL610 nm ratio at different cTnI concentrations, the sensitive detection of cTnI with a wide range of 50 fg mL-1 to 50 ng mL-1 and a low detection limit of 9.73 fg mL-1 can be achieved. This work enriches the wavelength-resolved ECL-RET system and provides an innovative reference for the development of more efficient and sensitive ECL-RET ratiometry.

Electrochemiluminescence Immunosensor Based on Au Nanocluster and Hybridization Chain Reaction Signal Amplification for Ultrasensitive Detection of Cardiac Troponin I.

Measurement of cardiac troponin I in the blood is crucial for the early diagnosis of acute myocardial infarction. Herein, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor has been developed for determination of cardiac troponin I (cTnI) by using Au nanoclusters and hybridization chain reaction (HCR) signal amplification. In this ECL immunosensor, Au nanoclusters were dual-labeled at each end of hairpin DNA (H1 and H2) and acted as the luminophore. DNA initiator strands (T1) and secondary antibody (Ab2) were conjugated on Au nanoparticles (AuNPs) to obtain a smart probe (Ab2-AuNP-T1). In the presence of target cTnI, the sandwiched immunocomplex composed of cTnI, Ab1, and Ab2-AuNP-T1 was formed. Then the initiator strands T1 of Ab2-AuNP-T1 opened the hairpin DNA structures and triggered a cascade of hybridization events. Consequently, a large number of Au NCs were indirectly modified on the surface of the electrode, which could react with the coreactant (K2S2O8) and emit a strong ECL signal. Under the optimal conditions, the immunosensor exhibited a wide detection range for cTnI from 5 fg/mL to 50 ng/mL and a low detection limit of 1.01 fg/mL (S/N = 3). Because of the excellent specificity, stability, and reproducibility of the proposed ECL-HCR sensor, it has a great application prospect for cTnI detection in clinical diagnosis.