Fe3O4@PDA immune probe-based signal amplification in surface plasmon resonance (SPR) biosensing of human cardiac troponin I.

This work reports immunomagnetic separation technology-assisted surface plasmon resonance (SPR) biosensing for human cardiac troponin-I (cTnI), a well-known diagnostic marker for myocardial damage. Au film modified by Au nanoparticles (AuNPs) and polydopamine (PDA) was employed as the platforms for immobilizing capture antibody (cAb) and SPR sensing. Magnetic immune probe was prepared by attaching detection antibody (dAb) on the surface of Fe3O4 nanoparticles (Fe3O4 NPs) coated by PDA for precise capture, magnetic separation and enrichment of target analyte (cTnI) from samples. This extraction process greatly improves the sensitivity and effectively reduces the nonspecific interference from complex matrixes. The analyte cTnI collected via Fe3O4@PDA-dAb immune probe can be specially recognized by cAb immobilized on the sensing platform. By introducing secondary antibody (Ab2) conjugated with multi-walled carbon nanotube-PDA-AgNPs (MWCNTs-PDA-AgNPs/Ab2) to the sensing system, the residual binding sites of cTnI were occupied, and the SPR response signals were further amplified. The obtained detection limit for cTnI is 3.75 ng mL-1, which is 320-folds lower than that achieved by PDA-based sensing strategy. The present method was applied to the examination of serum samples spiked with cTnI, and the good recoveries demonstrate its future applicability in clinical diagnosis.

Long-Range Surface Plasmon-Polariton Waveguide Biosensors for Human Cardiac Troponin I Detection.

Straight long-range surface plasmon-polariton (LRSPP) waveguides as biosensors for label-free detection are discussed. The sensors consist of 5-µm-wide 35-nm-thick gold stripes embedded in a low-index optical-grade fluoropolymer (CYTOPTM) with fluidic channels etched to the Au surface of the stripes. This work demonstrates the application of the LRSPP biosensors for the detection of human cardiac troponin I (cTnI) protein. cTnI is a biological marker for acute myocardial infarction (AMI), often referred to as a heart attack, which can be diagnosed by elevated levels of cTnI in patient blood. Direct and sandwich assays were developed and demonstrated over the concentration range from 1 to 1000 ng/mL, yielding detection limits of 430 pg/mL for the direct assay and 28 pg/mL for the sandwich assay (1 standard deviation), the latter being physiologically relevant to the early detection or onset of AMI. In addition, a novel approach for data analysis is proposed, where the analyte response is normalized to the response of the antibody layer.

An integrated microfluidic platform to perform uninterrupted SELEX cycles to screen affinity reagents specific to cardiovascular biomarkers.

As cardiovascular diseases (CVD) are responsible for millions of deaths annually, there is a need for rapid and sensitive diagnosis of CVD at earlier stages. Aptamers generated by systematic evolution of ligands by exponential enrichment (SELEX) processes have been shown to be superior to conventional antibody-based cardiac biomarker detection. However, SELEX is a complicated, lengthy procedure requiring multiple rounds of extraction/amplification and well-trained personnel. To circumvent such issue, we designed an automated, miniaturized SELEX platform for the screening of aptamers towards three protein biomarkers associated with CVDs: N-terminal pro-peptide of B-type natriuretic peptide, human cardiac troponin I, and fibrinogen. The developed microfluidic platform was equipped with microfluidic devices capable of sample transport and mixing along with an on-chip nucleic acid amplification module such that the entire screening process (5 rounds of selection in 8 h.) could be performed consecutively on a single chip while consuming only 35 µL of reagents in each cycle. This system may therefore serve as a promising, sensitive, cost-effective platform for the selection of aptamers specific for CVD biomarkers.

Studies towards hcTnI Immunodetection Using Electrochemical Approaches Based on Magnetic Microbeads.

Different electrochemical strategies based on the use of magnetic beads are described in this work for the detection of human cardiac troponin I (hcTnI). hcTnI is also known as the gold standard for acute myocardial infarction (AMI) diagnosis according to the different guidelines from the European Society of Cardiology (ESC) and the American College of Cardiology (ACC). Amperometric and voltamperometric sandwich magnetoimmunoassays were developed by biofunctionalization of paramagnetic beads with specific antibodies. These bioconjugates were combined with biotinylated antibodies as detection antibodies, with the aim of testing different electrochemical transduction principles. Streptavidin labeled with horseradish peroxidase was used for the amperometric magnetoimmunoassay, reaching a detectability of 0.005 ± 0.002 µg mL-1 in 30 min. Cadmium quantum dots-streptavidin bioconjugates were used in the case of the voltamperometric immunosensor reaching a detectability of 0.023 ± 0.014 µg mL-1.

Ultra-sensitive, rapid gold nanoparticle-quantum dot plexcitonic self-assembled aptamer-based nanobiosensor for the detection of human cardiac troponin I.

Acute myocardial infarction (AMI) is one of the leading causes of death throughout the world. Usual methods for detecting AMI are expensive, time-consuming and using blood samples as biological samples. Therefore, creating an ultra-fast, sensitive and non-invasive diagnostic test is necessary. Herein, a novel ultra-sensitive, fluorescent, plasmon-exciton coupling hybrid of a gold nanoparticle-quantum dot (PQ)-based aptamer nanobiosensor is presented for the detection of human cardiac troponin I (cTnI), the golden biomarker of AMI, and a preclinical test is performed within saliva. The binding of the cTnI protein to aptamer leads to a fluorescence enhancement of the plexcitonic hybrid system. The response range of this nanobiosensor is 0.4-2500 fM and the limit of detection is 0.3 fM. It seems that this novel design of nanobiosensor in the form of the PQ plexcitonic hybrid system can presents new opportunities for nanobiosensor progress.

Scale-up production and characterization of anti-human cardiac troponin I monoclonal antibody in ascitic fluid of balb/c mice.

The Human Cardiac Troponin I (hcTnI) is a 210 amino acid protein, 23 kDa in molecular weight. This biomarker is commonly used to diagnose myocardial infarction, micro injury, and acute coronary syndrome (ACS) in patients referring to emergency departments. The American Heart Association (AHA) and European Society of Cardiology (ESC) proposed troponin I as the gold biomarker for early detection of heart attack, especially in myocardial infarction (MI). Therefore, developing monoclonal antibodies against this biomarker could help in for early detection of heart attack. Hybridoma technology is a well-known technique introduced to produce monoclonal antibodies in specialized cells. The aim of this study was to produce large scale of monoclonal antibody against human cardiac troponin I using Hybridoma technology in order to design a diagnostic kit. The monoclonal antibody was produced using conventional Hybridoma technology in ascitic fluid of mouse and characterized for its ability to detect Human Cardiac Troponin I in a rapid test system. The results indicate the successful detection of Troponin I using the obtained monoclonal antibody. According to the achieved results it seems that ascites production of monoclonal antibody is very versatile, inexpensive, and economically useful for monoclonal antibody production.

Selection of DNA aptamers against Human Cardiac Troponin I for colorimetric sensor based dot blot application.

Troponin T and I are ideal markers which are highly sensitive and specific for myocardial injury and have shown better efficacy than earlier markers. Since aptamers are ssDNA or RNA that bind to a wide variety of target molecules, the purpose of this research was to select an aptamer from a 79bp single-stranded DNA (ssDNA) random library that was used to bind the Human Cardiac Troponin I from a synthetic nucleic acids library by systematic evolution of ligands exponential enrichment (Selex) based on several selection and amplification steps. Human Cardiac Troponin I protein was coated onto the surface of streptavidin magnetic beads to extract specific aptamer from a large and diverse random ssDNA initial oligonucleotide library. As a result, several aptamers were selected and further examined for binding affinity and specificity. Finally TnIApt 23 showed beast affinity in nanomolar range (2.69nM) toward the target protein. A simple and rapid colorimetric detection assay for Human Cardiac Troponin I using the novel and specific aptamer-AuNPs conjugates based on dot blot assay was developed. The detection limit for this protein using aptamer-AuNPs-based assay was found to be 5ng/ml.