Peptide-based biosensor for biochemical express-testing

Today, biochemical methods are one of the most precise ways to confirm a diagnosis. When it comes to biochemical diagnostics of cardiovascular diseases, medical professionals evaluate the level of cardiac markers in blood – special proteins that are hidden in cells of a cardiac muscle. There are several well-known cardiac markers that are used in clinical diagnostics - cardiac troponins, myoglobin, creatine kinase and many others.

Precise and quick disease detection requires multiparametric express-testing systems for preclinical diagnostics.  This is a task for biochips, i.e. integrated transducer-type biosensor devices that are capable of selective quantitative or semi-quantitative analysis, using biorecognition elements. Antibodies, nucleic acids and peptides can be used as spatially complementary bio-recognition elements (ligands or aptamers), selectively binding protein biomarkers. The result of the bio-recognition process is converted into a measurable response due to various types of transducers. The most common ones are optical, electrochemical, impedimetric, etc. In modern practice, quantitative detection of the target protein bound by biorecognizing ligands is typically achieved through attaching labels to target proteins. These are very costly and labile substances that require specific storage conditions and the attachment stage prolongs the analysis.

The researchers from Engineering Center for Microtechnology and Diagnostics, Department of Automation and Control Processes (ETU “LETI”) and Institute of Highly Pure Biopreparations proposed to carry out direct fluorescence detection of peptide markers, selectively bound by peptide aptamers, without the use of 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.’ – explained by Associate Professor Tatiana Zimina, research fellow at the Engineering Center for Microtechnology and Diagnostics, ETU “LETI”.

New-generation biochips are made for multiparametric express-testing based on molecular recognition and direct fluorimetric registration of the peptide aptamer-protein marker system. The biochips that consist of a microfluidic transport element and casing with inlet and outlet containing covalently bound peptide – aptamer systems. The peptide aptamers were designed using data from Protein Data Bank and Protein 3D software, developed at the Center of Microtechnology and Diagnostics of Saint Petersburg Electrotechnical University “LETI”. The biosensor was designed as a sandwich structure using thick film and photolithographic technology. The researchers excited fluorescence of protein markers using UV-LED of 275 nm wavelength. The device can be used for other purposes, not just cardiovascular disease diagnostics.

‘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. 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. 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.

Therefore, a microfluidic system proved to be effective for the protein troponin Т in proposed detection method. In the current configuration, background fluorescence reduced down to 30% and was caused by the fluorescence of the luminophore layer when first exposed to UV-lighting. The researchers are planning to increase effectiveness of the system by background fluorescence reduction, signal accumulation, software and digital processing, and also further spectral selection of the system.