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Biochemical methods are one of the most accurate diagnostic methods today. In the biochemical diagnosis of cardiovascular diseases, medical experts evaluate the levels of cardiac markers in the blood-unique proteins hidden in the myocardial cells.
Image Credit: Shutterstock.com/Marcin Janiec
Many familiar cardiac markers are used for clinical diagnosis, such as creatine kinase, myoglobin, and cardiac troponin.
Rapid and accurate disease detection requires a multi-parameter rapid test system for preclinical diagnosis. This task can be accomplished by a biochip, that is, an integrated transducer-type biosensor device that can perform selective quantitative or semi-quantitative analysis with the help of biometrics.
Peptides, antibodies and nucleic acids can be used as spatially complementary biological recognition elements (aptamers or ligands) to selectively bind protein biomarkers. The results of the biometric process are converted into measurable responses by different types of sensors.
Optical, electrochemical and impedance sensors are the most common sensors. In the modern world, markers are attached to the target protein to quantitatively detect the target protein bound by the biometric ligand. These labels are very expensive and unstable substances and require specific storage conditions. In addition, the attachment phase lengthens the analysis time.
Researchers from the Microtechnology and Diagnostic Engineering Center, the Department of Automation and Control Processes (ETU “LETI”), and the Institute of High-Purity Biological Preparation recommend direct fluorescent detection of peptide markers selectively bound by peptide aptamers instead of using special fluorescent markers .
The technology is based on molecular and direct fluorescence detection through a peptide aptamer-protein interaction system, including a microfluidic transport element and a shell with inlet and outlet, which contains a covalently bound peptide-aptamer system.
Tatiana Zimina, Associate Professor and Researcher, ETU "LETI" Microtechnology and Diagnostic Engineering Center
The new generation of biochips has been designed for multi-parameter expression testing based on molecular recognition and direct fluorescence registration of peptide aptamer-protein labeling systems. These biochips include microfluidic transport elements and housings with inlets and outlets, and are equipped with a covalently bound peptide-aptamer system.
The researchers designed peptide aptamers using data from the protein database and protein 3D software, which was created at the "LETI" Microtechnology and Diagnostic Center of St. Petersburg Electrotechnical University.
The biosensor was developed as a sandwich structure with thick film and photolithography technology. The researchers used a 275 nm wavelength UV-LED to excite the fluorescence of the protein marker. In addition to the diagnosis of cardiovascular diseases, the device can also be used for other purposes.
The amino acid residue sequence of the peptide aptamer was designed using data from the protein database and protein 3D. The peptides were prepared using Applied Biosystems 430A instrument through solid-state synthesis and in situ method using Na-Boc protected amino acid residues. The batches complementary to troponin Т proved to be the most selective.
Tatiana Zimina, Associate Professor and Researcher, ETU "LETI" Microtechnology and Diagnostic Engineering Center
“In addition, in order to exclude background fluorescence and be able to directly detect the immobilized protein, we replaced the aromatic (fluorescent) amino acids in the peptides. Despite this, they retained the 3D structure,” Zimina continued.
In most proteins, fluorescence is excited in the ultraviolet range of the λ = 280 nm spectrum because they contain tryptophan (Trp), an amino acid with the highest fluorescence quantum yield. About 90% of these will cause protein fluorescence in the range of λ = 320 ... 350 nm.
Tatiana Zimina, Associate Professor and Researcher, ETU "LETI" Microtechnology and Diagnostic Engineering Center
Therefore, in the proposed detection method, the microfluidic system was found to be effective for the protein troponin Т. In the existing configuration, since the luminous body layer emits fluorescence when exposed to ultraviolet light for the first time, the background fluorescence is reduced to 30%.
The team has been looking forward to improving the effectiveness of the system through software and digital processing, signal accumulation, background fluorescence reduction, and further spectral selection of the system.
Source: https://etu.ru/en/university/
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