Toward Development of a Label-Free Detection Technique for Microfluidic Fluorometric Peptide-Based Biosensor Systems
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein-Markers
- Cardiac troponin I (cTpI), T9924, Sigma-Aldrich (St. Louis, MO, USA).
- Cardiac troponin T (cTpT), T0175, Sigma-Aldrich (St. Louis, MO, USA).
2.2. Construction Materials
2.3. Inorganic Luminophores
2.4. Apparatus
2.5. Peptide Aptamers
2.6. Microfluidic Chip Fabrication Process
3. Results and Discussion
3.1. Designing Peptide Aptamers for Label-Free Detection
3.2. Optical Scheme of Protein Detection in Biosensor Channels
3.3. Preparation of Biosensor Model and Its Testing
4. Conclusions
- The fluorescence of a number of polymer materials was studied under excitation in the range of 260–280 nm, in order to select materials with minimal background fluorescence under radiation. The results showed that the selected materials meet the necessary requirements.
- A material for the preparation of the inlet window of the biosensor, transparent for UV radiation at 275 nm, was selected. The most fitting proved to be a PP film, 100 µm thick.
- A peptide aptamer constructed using the «Protein 3D» software complementary to troponin T was designed. On its basis, its non-fluorescent digital twin was designed, which retained its three-dimensional complementarity to target protein, which has been shown with capillary electrophoresis-on-a-chip experiments.
- For the outlet window transmitting fluorescence of troponin T for the channel depth of 50 µm in the range of 300–350 nm and absorbing incidental UV radiation at 275 nm, the most fitting material was glass. The cover glass plates were used for the preparation of this element.
- Comparison of solid-state inorganic luminophores in order to select the optimal material for use in a planar optical element with an excitation range of 300–350 nm (Troponin T emission range) and an emission range of 450–550 nm (optimal for a receiving device) showed that a luminophore with a ZnS:Cu composition was the most consistent with the tasks.
- A laboratory sample of the biosensor was designed and manufactured. Technology for applying a luminophore to glass was developed.
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Name of Peptide | Structure | Number of Amino Acid Residues |
---|---|---|
LETI-2 | HLNEDQLREKAKELWQTIYNLEAEKFDLQEKFKQQKE | 38 |
LETI-7 | TLNEDQLREKAKELAQTIANLEAEKIDLQEKAKQQKYE |
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Sitkov, N.; Zimina, T.; Kolobov, A.; Karasev, V.; Romanov, A.; Luchinin, V.; Kaplun, D. Toward Development of a Label-Free Detection Technique for Microfluidic Fluorometric Peptide-Based Biosensor Systems. Micromachines 2021, 12, 691. https://doi.org/10.3390/mi12060691
Sitkov N, Zimina T, Kolobov A, Karasev V, Romanov A, Luchinin V, Kaplun D. Toward Development of a Label-Free Detection Technique for Microfluidic Fluorometric Peptide-Based Biosensor Systems. Micromachines. 2021; 12(6):691. https://doi.org/10.3390/mi12060691
Chicago/Turabian StyleSitkov, Nikita, Tatiana Zimina, Alexander Kolobov, Vladimir Karasev, Alexander Romanov, Viktor Luchinin, and Dmitry Kaplun. 2021. "Toward Development of a Label-Free Detection Technique for Microfluidic Fluorometric Peptide-Based Biosensor Systems" Micromachines 12, no. 6: 691. https://doi.org/10.3390/mi12060691