Description And Application Of Several Methods For Detection Of Human Cardiac Troponin I

Cardiac troponin I (cTnI) is a subunit of cardiac troponin complex, which is comprised of troponin I, troponin T and troponin C. Following the myocardial damage, the troponin complex is broken up and the individual protein components are released into the bloodstream. Cardiac troponin I has high tissue specificity because of it's structural difference from the corresponding skeletal isoforms in its amino acid composition. Therefore, cTnI in the serum of patients has been considered as a "gold standard" for diagnosis of myocardial injury.Many methods have been developed for detecting cTnI, which include radioactive immunoassay (RIA), enzyme-linked immuno sorbent assay (ELISA), chemiluminescent immunoassay (CLIA), fluorescent immunoassay (FIA), and gold chromatography immunoassay (GCIA). Nowadays some detection kits based on these principles are commercial available. However, there are some disadvantages in these methods, such as high cost of the instruments and the detection kits, long detection period, and low sensitivity.In this study we developed several methods for the detection of human cTnI, which combine the excellent properties of nano-materials, the high sensitivity of electrochemical and chemiluminescent detection techniques.The major contributions are as follows:1. Colorimetric method based on nanogold probe and silver enhancement for the detection of human cardiac troponin IGold nanoparticles (nano-gold) possess many unique physical and chemical properties and, therefore, have been intensively studied in the protein and DNA detection. They can strongly interact with biomolecules and do not degrade the bioactivity of them seriously during the labeling. Furthermore, the signal of nanogold can be largely amplified by the reduction of silver promoted by the gold particles. In this work we presented a rapid, inexpensive, reliable, and flexible quantitative immunoassay for cTnI. The assay was based on the concepts of one-step dual monoclonal antibody "sandwich" principle, the low density protein array, the nanogold probe, and the silver enhancement on the gold particles. The capture antibody (IgG₁) coated supporting nitrocellulose membrane and the colloidal gold-labeled detection antibody (cAu-IgG₂) were prepared before the detection. The detection procedure involved two steps, i.e., immunoreaction and silver amplification. The assay needs only small amounts of serum samples of patients. The whole detection procedure of the assay could be fulfilled within 40 min (much faster than ELISA that takes usually at least 3 hours for a turnaround test). The detection results could be easily imaged with a simple flatbed scanner or even observed with the naked eye. The assay showed good specific response to cTnI with very little cross-reactivity to the skeletal isoforms of troponin I (sTnI), cardiac troponin T (cTnT), and myoglobin (Mb). A cut-off value of 0.3 ng/mL was obtained from a reference control group (200 normal serum samples). 588 patients' serum samples were assayed simultaneously by routine ELISA and this colloidal gold method to test the validity of the method. The data were analyzed using the statistical package SPSS version 11.0 (SPSS Inc.). There was no significant difference between these two assays (P=0.66>0.05). The agreement between this method (≥ or <0.3 ng/mL) and ELISA was 86%.2. Molecular sieves materials modified carbon paste electrodes for the determination of cardiac troponin I by anodic stripping voltammetryThe fixation of biomacromolecule (protein) on inorganic materials is very useful for practical application. Mesoporous materials possess many unusual properties such as large pore size, uniform pore structure, high surface areas, and high loading capacity, which make them attractive candidates for the immobilization of biomacromolecule (protein). Recently, mesoporous materials have been used as electrode surface modifiers. However, the potential still remains practically unexplored. In this section we present a novel electrochemical immunoassay of cTnI based on the concepts of the dual monoclonal antibody "sandwich" principle, the nanogold particle promoted precipitation of silver, the modified carbon paste electrode, and the anodic stripping voltammetry. Three different kinds of molecular sieves (mesoporous materials SBA-15 (9.19 nm in diameter) and MCM-41 (3.10 nm in diameter), microporous zeolite Y (0.74 nm in diameter)) have been used as the modifiers to fabricate the modified carbon paste electrodes (MCPEs). Four main steps were carried out to obtain the analytical signal, i.e., electrode preparation, immunoreaction, silver enhancement, and the anodic stripping voltammetric detection. Among the molecular sieves selected in this study, the performance of SBA-15 was the best probably due to it's rather larger pore size. A linear relationship between the anodic stripping peak current and the concentration of cTnI from 0.5 to 5.0 ng/mL and a limit of detection of 0.2 ng/mL were obtained using SBA-15 modified CPE (SBA-MCPE). The established method was tested by determining cTnI in acute myocardial infarction (AMI) samples using enzyme-linked immunoadsorbent assay (ELISA) for comparison analysis, and good results were obtained.3. Development of a chemiluminescent enzyme immunoassay for cardiac troponin I detectionChemiluminescent immunoassays (CLIA) have been intensively studied in antigen/antibody detection. The sensitivity of CLIA is much higher than that of the traditional ELISA, which is widely used in cTnI detection, due to the extremely sensitive chemiluminescent detection techniques. Here we present a sensitive immunoassay for cTnI based on this concept. The assay was executed as thetraditional ELISA except that two major modifications were made to obtain a rapid test and a high sensitivity. The first modification is that the two-step dual monoclonal antibody "sandwich" principle of traditional ELISA was substituted by a one-step immunoreaction mode which decreased the turnaround time greatly. The second modification is that a chemiluminescent substrate (AMPPD) and chemiluminescent detection techniques was used to replace the colorimetric substrate and colorimetric detection method, which elevated the sensitivity remarkably. The detection procedure of the assay could be fulfilled within 75 min. A linear range between the luminescent signal current and the concentration of cTnI from 0.04 to 36.2 ng/mL and a detection limit of 0.02 ng/mL were obtained. The established method was tested by determining cTnI in real samples using ELISA for comparison analysis, and good results were obtained.