ABSTRACT
Acute myocardial infarction (AMI) is a significant health problem owing to its high mortality rate. Heart-type fatty acid-binding protein (h-FABP) is an important biomarker in the diagnosis of AMI. In this work, an electrochemical h-FABP immunosensor was developed based on Cd0.5Zn0.5S/d-Ti3C2Tx MXene (MXene: Transition metal carbide or nitride) composite as signal amplificator and core-shell high-crystalline graphitic carbon nitride@carbon dots (hc-g-C3N4@CDs) as electrochemical sensor platform. Firstly, a facile calcination technique was applied to the preparation of hc-g-C3N4@CDs and immobilization of primary antibody was performed on hc-g-C3N4@CDs surface. Then, the conjugation of the second antibody to Cd0.5Zn0.5S/d-Ti3C2Tx MXene was carried out by strong π-π and electrostatic interactions. The prepared electrochemical h-FABP immunosensor was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), x-ray diffraction (XRD) method, Fourier-transform infrared spectroscopy (FTIR), x-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The prepared electrochemical h-FABP immunosensor indicated a good sensitivity with detection limit (LOD) of 3.30 fg mL-1 in the potential range +0.1 to +0.5 V. Lastly, low-cost, satisfactory stable, and environmentally friendly immunosensor was presented for the diagnosis of acute myocardial infarction.
Subject(s)
Fatty Acid Binding Protein 3/blood , Graphite/chemistry , Immunoassay/methods , Nitrogen Compounds/chemistry , Quantum Dots/chemistry , Acute Disease , Antibodies, Immobilized/immunology , Biomarkers/blood , Cadmium/chemistry , Electrochemical Techniques/methods , Fatty Acid Binding Protein 3/immunology , Humans , Limit of Detection , Myocardial Infarction/blood , Myocardial Infarction/diagnosis , Reproducibility of Results , Sulfur/chemistry , Titanium/chemistry , Zinc/chemistryABSTRACT
Cross-reactivity between allergens and human proteins could have a clinical impact in allergic diseases. Blo t 13 is an allergen from the mite Blomia tropicalis, which belongs to the fatty acid binding protein (FABP) family and has structural homology with human FABPs. This work aimed to map B cell epitopes on Blo t 13 and to identify epitopes involved in cross-reactivity with human heart FABP (FABP3) and adipocyte FABP (FABP4). Sera from 25 patients with house dust mite (HDM) allergy that were sensitized to Blo t 13 were used for testing the reactivity of immunoglobulin E (IgE) and IgG to FABP. The epitope mapping of Blo t 13 was performed using overlapping peptides, and cross-reactivity between Blo t 13 and human FABP was analyzed using human sera and anti-Blo t 13 monoclonal antibodies. IgE antibodies to all FABPs were detected in 14/25 serum samples, and IgG was detected in 25/25 serum samples. The cross-reactivity of Blo t 13 was 42% with FABP3 and 48% with FABP4. Two IgE-binding regions were identified in Blo t 13; one between residues 54 and 72 (the main cross-reacting region) and another between residues 111 to 129. Our results suggest that exposure to the Blo t 13 allergen could induce an auto-reactive response to endogenous FABP in allergic patients sensitized to Blo t 13.
Subject(s)
Allergens/metabolism , Epitopes, B-Lymphocyte/immunology , Fatty Acid Binding Protein 3/immunology , Fatty Acid-Binding Proteins/immunology , Fatty Acid-Binding Proteins/metabolism , Adipocytes/metabolism , Allergens/genetics , Allergens/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Cross Reactions , Epitope Mapping , Fatty Acid Binding Protein 3/chemistry , Fatty Acid Binding Protein 3/genetics , Fatty Acid-Binding Proteins/chemistry , Fatty Acid-Binding Proteins/genetics , Female , Humans , Hypersensitivity/blood , Hypersensitivity/pathology , Immunoglobulin E/immunology , Immunoglobulin G/immunology , Male , Mites/metabolism , Myocardium/metabolism , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Fatty-acid binding proteins (FABP) and myeloperoxidases (MPO) are associated with many chronic conditions in humans and considered to be important biomarkers for diagnosis of cardiac diseases. Here we assemble a new electrical biosensor platform based on graphene-coated interdigitated electrode arrays (IDE-arrays) towards ultrafast, label-free screening of heart type-FABP and MPO. Arrays of nanoscale (nanoIDE) and microscale (microIDE) electrode-arrays were fabricated on wafer-scale by combining nanoimprint and photolithography processes. Chemical vapor deposition grown multilayer graphene was transferred onto nano/microIDE-arrays and used as a high surface-to-volume ratio electrical transducer. Novel biofunctional layers of specially engineered anti-h-FABP and anti-MPO single-chain fragment variables (scFv) were immobilized onto graphene-coated IDE-array sensor platform for electrical detection of h-FABP and MPO in physiological saline. scFv fragments show increased sensitivity in comparison to the state-of-the-art competitive ELISA for their higher affinity towards target analytes. Deploying FABP and MPO specific scFvs as receptor molecules onto our high-sensitivity graphene-coated IDE-arrays with identical sensor characteristics and assays covering clinically relevant concentrations in physiological saline, we demonstrate realization of a simple and versatile biosensor platform capable of high performance cardiac-bioassays for point-of-care applications.
