scholarly journals Label-Free Peptide-Based Biosensor for Express Detection of Protein Markers of Acute Cardiovascular Conditions in Biological Fluids

Proceedings ◽  
2020 ◽  
Vol 60 (1) ◽  
pp. 8
Author(s):  
Nikita Sitkov ◽  
Tatiana Zimina ◽  
Vladimir Karasev ◽  
Olesya Naretskaya ◽  
Margarita Kiseleva
Keyword(s):  
Uv Light ◽  
Detection System ◽  
Biological Fluids ◽  
Clinical Sample ◽  
Microfluidic Channel ◽  
Label Free ◽  
Protein Markers ◽  
Protein Fluorescence ◽  
Peptide Aptamers ◽  
Biosensor System

Acute cardiovascular conditions require prompt assistance, which depends on a timely and accurate diagnosis. This could be achieved by using biosensor systems based on peptide aptamers capable of selectively binding protein markers of diseases. In this work, a label-free biosensor system based on fluorometric registration of the formation of a “peptide aptamer—target protein” complex is considered. It comprises a microfluidic subsystem integrated with arrays of sites with immobilized peptide aptamers, coupled with an optical detection system. The clinical sample of the whole blood is loaded into the inlet basin, where the cells are separated and plasma flows into the microfluidic channel for analysis. Peptide aptamers were created using the molecular complement search technique based on the search for systems of conjugated ion-hydrogen bonds in the three-dimensional structures of target proteins. The technology for manufacturing a microfluidic chip is a combination of thick-film and photolithography technologies based on the SU-8 photoresist, for which the relief and surface morphology have been studied. The composition of the biochip layers is selected in such a way that ultraviolet light with a wavelength of 280 nm passes through an inlet window, excites fluorescence inside the channel, which passes through the glass window, which absorbs UV-light. This wavelength accounts for the maximum absorption of aromatic amino acids—tyrosine and tryptophan. In this case, one of the last layers is a luminophore layer for re-emission of protein fluorescence as a visible light. The reading platform includes a 280 nm LED, a video sensor, 3D-printed PLA tooling, and software for processing and analyzing the received signal.

scholarly journals Toward Development of a Label-Free Detection Technique for Microfluidic Fluorometric Peptide-Based Biosensor Systems

Micromachines ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 691
Author(s):  
Nikita Sitkov ◽  
Tatiana Zimina ◽  
Alexander Kolobov ◽  
Vladimir Karasev ◽  
Alexander Romanov ◽  
...  
Keyword(s):  
Troponin I ◽  
Troponin T ◽  
Detection System ◽  
Label Free ◽  
Protein Markers ◽  
Peptide Aptamers ◽  
Registration System ◽  
Biosensor System ◽  
Label Free Detection ◽  
Selection Of

The problems of chronic or noncommunicable diseases (NCD) that now kill around 40 million people each year require multiparametric combinatorial diagnostics for the selection of effective treatment tactics. This could be implemented using the biosensor principle based on peptide aptamers for spatial recognition of corresponding protein markers of diseases in biological fluids. In this paper, a low-cost label-free principle of biomarker detection using a biosensor system based on fluorometric registration of the target proteins bound to peptide aptamers was investigated. The main detection principle considered includes the re-emission of the natural fluorescence of selectively bound protein markers into a longer-wavelength radiation easily detectable by common charge-coupled devices (CCD) using a specific luminophore. Implementation of this type of detection system demands the reduction of all types of stray light and background fluorescence of construction materials and aptamers. The latter was achieved by careful selection of materials and design of peptide aptamers with substituted aromatic amino acid residues and considering troponin T, troponin I, and bovine serum albumin as an example. The peptide aptamers for troponin T were designed in silico using the «Protein 3D» (SPB ETU, St. Petersburg, Russia) software. The luminophore was selected from the line of ZnS-based solid-state compounds. The test microfluidic system was arranged as a flow through a massive of four working chambers for immobilization of peptide aptamers, coupled with the optical detection system, based on thick film technology. The planar optical setup of the biosensor registration system was arranged as an excitation-emission cascade including 280 nm ultraviolet (UV) light-emitting diode (LED), polypropylene (PP) UV transparent film, proteins layer, glass filter, luminophore layer, and CCD sensor. A laboratory sample has been created.

scholarly journals Secretome Proteomic Approaches for Biomarker Discovery: An Update on Colorectal Cancer

Medicina ◽  
2020 ◽  
Vol 56 (9) ◽  
pp. 443
Author(s):  
Armando Cevenini ◽  
Stefania Orrù ◽  
Esther Imperlini
Keyword(s):  
Colorectal Cancer ◽  
Biomarker Discovery ◽  
Dynamic Range ◽  
Biological Fluids ◽  
Label Free ◽  
Protein Markers ◽  
Comprehensive Overview ◽  
Bioinformatic Tools ◽  
Complete Translation

Searching for new cancer-related biomarkers is a key priority for the early detection of solid tumors, such as colorectal cancer (CRC), in clinically relevant biological fluids. The cell line and/or tumor tissue secretome represents a valuable resource for discovering novel protein markers secreted by cancer cells. The advantage of a secretome analysis is the reduction of the large dynamic range characterizing human plasma/serum, and the simultaneous enrichment of low abundance cancer-secreted proteins, thereby overcoming the technical limitations underlying the direct search in blood samples. In this review, we provided a comprehensive overview of recent studies on the CRC secretome for biomarker discovery, focusing both on methodological and technical aspects of secretome proteomic approaches and on biomarker-independent validation in CRC patient samples (blood and tissues). Secretome proteomics are mainly based on LC-MS/MS analyses for which secretome samples are either in-gel or in-solution trypsin-digested. Adequate numbers of biological and technical replicates are required to ensure high reproducibility and robustness of the secretome studies. Moreover, another major challenge is the accuracy of proteomic quantitative analysis performed by label-free or labeling methods. The analysis of differentially expressed proteins in the CRC secretome by using bioinformatic tools allowed the identification of potential biomarkers for early CRC detection. In this scenario, this review may help to follow-up the recent secretome studies in order to select promising circulating biomarkers to be validated in larger screenings, thereby contributing toward a complete translation in clinical practice.

scholarly journals Differential Sorting of Microparticles Using Spiral Microchannels with Elliptic Configurations

Micromachines ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 412
Author(s):  
Kaan Erdem ◽  
Vahid Ebrahimpour Ahmadi ◽  
Ali Kosar ◽  
Lütfullah Kuddusi
Keyword(s):  
Cell Sorting ◽  
Microfluidic Channel ◽  
Major Axis ◽  
Label Free ◽  
Flow Rates ◽  
Inertial Focusing ◽  
Size Dependent ◽  
Curved Channels ◽  
Aspect Ratios ◽  
Dependent Cell

Label-free, size-dependent cell-sorting applications based on inertial focusing phenomena have attracted much interest during the last decade. The separation capability heavily depends on the precision of microparticle focusing. In this study, five-loop spiral microchannels with a height of 90 µm and a width of 500 µm are introduced. Unlike their original spiral counterparts, these channels have elliptic configurations of varying initial aspect ratios, namely major axis to minor axis ratios of 3:2, 11:9, 9:11, and 2:3. Accordingly, the curvature of these configurations increases in a curvilinear manner through the channel. The effects of the alternating curvature and channel Reynolds number on the focusing of fluorescent microparticles with sizes of 10 and 20 µm in the prepared suspensions were investigated. At volumetric flow rates between 0.5 and 3.5 mL/min (allowing separation), each channel was tested to collect samples at the designated outlets. Then, these samples were analyzed by counting the particles. These curved channels were capable of separating 20 and 10 µm particles with total yields up to approximately 95% and 90%, respectively. The results exhibited that the level of enrichment and the focusing behavior of the proposed configurations are promising compared to the existing microfluidic channel configurations.

scholarly journals Cellular lensing and near infrared fluorescent nanosensor arrays to enable chemical efflux cytometry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Soo-Yeon Cho ◽  
Xun Gong ◽  
Volodymyr B. Koman ◽  
Matthias Kuehne ◽  
Sun Jin Moon ◽  
...  
Keyword(s):  
Near Infrared ◽  
Single Cells ◽  
Microfluidic Channel ◽  
Human Monocyte ◽  
Cellular Heterogeneity ◽  
Label Free ◽  
Nanotube Array ◽  
Chemical Cytometry ◽  
Rich Information ◽  
Non Destructive

AbstractNanosensors have proven to be powerful tools to monitor single cells, achieving spatiotemporal precision even at molecular level. However, there has not been way of extending this approach to statistically relevant numbers of living cells. Herein, we design and fabricate nanosensor array in microfluidics that addresses this limitation, creating a Nanosensor Chemical Cytometry (NCC). nIR fluorescent carbon nanotube array is integrated along microfluidic channel through which flowing cells is guided. We can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR profiles and extract rich information. This unique biophotonic waveguide allows for quantified cross-correlation of biomolecular information with various physical properties and creates label-free chemical cytometer for cellular heterogeneity measurement. As an example, the NCC can profile the immune heterogeneities of human monocyte populations at attomolar sensitivity in completely non-destructive and real-time manner with rate of ~600 cells/hr, highest range demonstrated to date for state-of-the-art chemical cytometry.

scholarly journals An Aptamer-Based Capacitive Sensing Platform for Specific Detection of Lung Carcinoma Cells in the Microfluidic Chip

Biosensors ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 98 ◽  
Author(s):  
Ngoc-Viet Nguyen ◽  
Chun-Hao Yang ◽  
Chung-Jung Liu ◽  
Chao-Hung Kuo ◽  
Deng-Chyang Wu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Lung Carcinoma ◽  
Early Stage ◽  
A549 Cells ◽  
Microfluidic Channel ◽  
Electrical Impedance Spectroscopy ◽  
Label Free ◽  
Early Stage Lung Cancer ◽  
Sensing Platform ◽  
Label Free Detection

Improvement of methods for reliable and early diagnosis of the cellular diseases is necessary. A biological selectivity probe, such as an aptamer, is one of the candidate recognition layers that can be used to detect important biomolecules. Lung cancer is currently a typical cause of cancer-related deaths. In this work, an electrical sensing platform is built based on amine-terminated aptamer modified-gold electrodes for the specific, label-free detection of a human lung carcinoma cell line (A549). The microdevice, that includes a coplanar electrodes configuration and a simple microfluidic channel on a glass substrate, is fabricated using standard photolithography and cast molding techniques. A procedure of self-assembly onto the gold surface is proposed. Optical microscope observations and electrical impedance spectroscopy measurements confirm that the fabricated microchip can specifically and effectively identify A549 cells. In the experiments, the capacitance element that is dominant in the change of the impedance is calculated at the appropriate frequency for evaluation of the sensitivity of the biosensor. Therefore, a simple, inexpensive, biocompatible, and selective biosensor that has the potential to detect early-stage lung cancer would be developed.

scholarly journals Utilization of Pharmaceutical Technology Methods for the Development of Innovative Porous Metasilicate Pellets with a Very High Specific Surface Area for Chemical Warfare Agents Detection

Pharmaceutics ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1860
Author(s):  
Jiří Zeman ◽  
Sylvie Pavloková ◽  
David Vetchý ◽  
Adam Staňo ◽  
Zdeněk Moravec ◽  
...  
Keyword(s):  
Specific Surface ◽  
Surface Area ◽  
Specific Surface Area ◽  
Uv Light ◽  
Detection System ◽  
Chemical Warfare Agents ◽  
High Specific Surface Area ◽  
Ammonium Bicarbonate ◽  
Toxic Substances ◽  
Pharmaceutical Technology

Pharmaceutical technology offers various dosage forms that can be applied interdisciplinary. One of them are spherical pellets which could be utilized as a carrier in emerging second-generation detection tubes. This detection system requires carriers with high specific surface area (SSA), which should allow better adsorption of toxic substances and detection reagents. In this study, a magnesium aluminometasilicate with high SSA was utilized along with various concentrations of volatile substances (menthol, camphor and ammonium bicarbonate) to increase further the carrier SSA after their sublimation. The samples were evaluated in terms of physicochemical parameters, their morphology was assessed by scanning electron microscopy, and the Brunauer–Emmett–Teller (BET) method was utilized to measure SSA. The samples were then impregnated with a detection reagent o-phenylenediamine-pyronine and tested with diphosgene. Only samples prepared using menthol or camphor were found to show red fluorescence under the UV light in addition to the eye-visible red-violet color. This allowed the detection of diphosgene/phosgene at a concentration of only 0.1 mg/m3 in the air for samples M20.0 and C20.0 with their SSA higher than 115 m2/g, thus exceeding the sensitivity of the first-generation DT-12 detection tube.

Hybrid-Integrated Biosensor for Express Determination of Protein Markers of Diseases based on Molecular Recognition and Direct Fluorimetric Detection

2021 ◽  
Vol 23 (6) ◽  
pp. 326-332
Author(s):  
N.O. Sitkov ◽  
◽  
T.M. Zimina ◽  
V.V. Luchinin ◽  
A.A. Kolobov ◽  
...  
Keyword(s):  
Molecular Recognition ◽  
Spatial Configuration ◽  
Limit Of Detection ◽  
Troponin T ◽  
Protein Structures ◽  
Microfluidic Channel ◽  
Protein Markers ◽  
Protein Marker ◽  
Aromatic Amino Acid Residue ◽  
Peptide Probes

Ways of creating new generation biosensors for multiparametric express diagnostics based on molecular recognition and direct fluorimetric registration of a peptide aptamer — protein marker complex were considered. The biosensor platform comprises a microfluidic channel for delivery sample solutions, coupled with flow-through zones containing covalently attached arrays of peptide probes — aptamers. An outer glass window of the biochip assembly contains a layer of luminophore ZnS:Cu, bound on it via an acrylic lacquer and intended for the re-emitting native fluorescence of bound proteins into the longer wavelength range, more efficient in registering signals with CMOS sensors. The aptamers were designed using "Protein 3D" program for analysis of spatial complementarity of protein structures. The peptide, complementary to Troponin T, was modified by replacement of aromatic amino acid residue while maintaining the spatial configuration. The complementarity of peptide and Troponin T was confirmed using a capillary electrophoresis-on-a-chip. Biosensors are manufactured using thick-film technology and photolithography. The fluorescence of marker proteins was excited using UV-LED with a radiation wavelength of 275 nm. The limit of detection achieved for Troponin T was 6 ng/ml.

Monitoring the effects of chemical stimuli on live cells with metasurface-enhanced infrared reflection spectroscopy

2021 ◽  
Author(s):  
Steven H. Huang ◽  
Jiaruo Li ◽  
Zhiyuan Fan ◽  
Robert Delgado ◽  
Gennady Shvets
Keyword(s):  
Intracellular Signaling ◽  
Strong Field ◽  
Detection System ◽  
Field Enhancement ◽  
Chemical Imaging ◽  
Live Cells ◽  
Label Free ◽  
Reflection Spectroscopy ◽  
Infrared Reflection ◽  
Infrared Reflection Spectroscopy

Infrared spectroscopy has found wide applications in the analysis of biological materials. A more recent development is the use of engineered nanostructures, or plasmonic metasurfaces, as substrates for metasurface-enhanced infrared reflection spectroscopy (MEIRS). Here, we demonstrate that strong field enhancement from plasmonic metasurfaces enables the use of MEIRS as a highly informative analytic technique for real-time monitoring of cells. By exposing live cells cultured on a plasmonic metasurface to chemical compounds, we show that MEIRS can be used as a label-free phenotypic assay for detecting multiple cellular responses to external stimuli: changes in cell morphology, adhesion, lipid composition of the cellular membrane, as well as intracellular signaling. Using a focal plane array detection system, we show that MEIRS also enables spectro-chemical imaging at the single-cell level. The described metasurface-based all-optical sensor opens the way to a scalable, high-throughput spectroscopic assay for live cells.

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