Reviewed by Alex SmithAug 25 2021
Biochemical methods are one of the most accurate ways to confirm a diagnosis nowadays. In the case of biochemical diagnostics of cardiovascular diseases, medical professionals assess the level of cardiac markers in blood — unique proteins hidden in the cells of a cardiac muscle.
Image Credit: Shutterstock.com/ Marcin Janiec
Many familiar cardiac markers are used in clinical diagnostics, such as creatine kinase, myoglobin, cardiac troponins, etc.
Fast and precise detection of disease necessitates multiparametric express-testing systems for preclinical diagnostics. This task can be performed by biochips, that is, integrated transducer-type biosensor devices capable of performing selective quantitative or semi-quantitative analysis with the help of biorecognition elements.
Peptides, antibodies and nucleic acids can be employed as spatially complementary bio-recognition elements (aptamers or ligands), selectively binding protein biomarkers. The outcome of the bio-recognition process is transformed into a measurable response by different types of transducers.
Optical, electrochemical and impedimetric transducers are the most common ones. In the modern world, labels are attached to target proteins for the quantitative detection of the target protein bound by biorecognizing ligands. These labels are highly expensive and labile substances that need specific storage conditions. Moreover, the attachment stage prolongs the analysis.
Researchers from the Engineering Center for Microtechnology and Diagnostics, Department of Automation and Control Processes (ETU “LETI”), and Institute of Highly Pure Biopreparations suggested direct fluorescence detection of peptide markers that are selectively bound by peptide aptamers, without using special fluorescent labels.
The technology is based on molecular and direct fluorescence detection through the peptide aptamer-protein interaction system, including a microfluidic transport element and casing with inlet and outlet that contain covalently bound peptide – aptamer systems.
Tatiana Zimina, Associate Professor and Research Fellow, Engineering Center for Microtechnology and Diagnostics, ETU “LETI”
New-generation biochips have been designed for multiparametric express-testing based on molecular recognition as well as direct fluorimetric registration of the peptide aptamer-protein marker system. These biochips include a microfluidic transport element and casing with an inlet and an outlet holding covalently bound peptide-aptamer systems.
The researchers designed the peptide aptamers using data from Protein Data Bank and Protein 3D software that was created at the Center of Microtechnology and Diagnostics of Saint Petersburg Electrotechnical University “LETI”.
The biosensor was developed as a sandwich structure with thick film and photolithographic technology. The researchers used 275 nm-wavelength UV-LED to excite the fluorescence of protein markers. Apart from cardiovascular disease diagnostics, the device can be used for other purposes.
The amino acid residue sequences of peptide aptamers were designed using data from Protein Data Bank and Protein 3D. Preparation of peptides was carried out by solid state synthesis using Applied Biosystems 430A instrument and in situ method with Nα-Boc-protected amino acid residues. The batches complimentary to troponin Т proved to be the most selective.
Tatiana Zimina, Associate Professor and Research Fellow, Engineering Center for Microtechnology and Diagnostics, ETU “LETI”
“Plus, to exclude background fluorescence and enable direct detection of immobilized proteins, we replaced aromatic (fluorescent) amino acid in peptides. They retained their 3D structure nevertheless,” Zimina continued.
In most proteins, fluorescence is excited at the ultraviolet range of the spectrum of λ = 280 nm, because they contain tryptophan (Trp), an amino acid that demonstrates the highest quantum yield of fluorescence. About 90% of it causes protein fluorescence in the range of λ = 320 ... 350 nm.
Tatiana Zimina, Associate Professor and Research Fellow, Engineering Center for Microtechnology and Diagnostics, ETU “LETI”
Thus, a microfluidic system was found to be effective for the protein troponin Т in the suggested detection method. In the existing configuration, there was a decrease in background fluorescence down to 30% due to the fluorescence of the luminophore layer upon its first exposure to UV lighting.
The team has been looking forward to increasing the effectiveness of the system through software and digital processing, signal accumulation, background fluorescence reduction and further spectral selection of the system.