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

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.

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

Proceedings ◽

10.3390/iecb2020-07066 ◽

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.

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Microfluidic Impedance Biosensor Chips Using Sensing Layers Based on DNA-Based Self-Assembled Monolayers for Label-Free Detection of Proteins

Biosensors ◽

10.3390/bios11030080 ◽

2021 ◽

Vol 11 (3) ◽

pp. 80

Author(s):

Khaled Alsabbagh ◽

Tim Hornung ◽

Achim Voigt ◽

Sahba Sadir ◽

Taleieh Rajabi ◽

...

Keyword(s):

Troponin I ◽

Electrochemical Impedance ◽

Specific Binding ◽

Self Assembled Monolayers ◽

Significant Shift ◽

Label Free ◽

Label Free Detection ◽

Assembled Monolayers ◽

Self Assembled ◽

Biosensor Chip

A microfluidic chip for electrochemical impedance spectroscopy (EIS) is presented as bio-sensor for label-free detection of proteins by using the example of cardiac troponin I. Troponin I is one of the most specific diagnostic serum biomarkers for myocardial infarction. The microfluidic impedance biosensor chip presented here consists of a microscope glass slide serving as base plate, sputtered electrodes, and a polydimethylsiloxane (PDMS) microchannel. Electrode functionalization protocols were developed considering a possible charge transfer through the sensing layer, in addition to analyte-specific binding by corresponding antibodies and reduction of nonspecific protein adsorption to prevent false-positive signals. Reagents tested for self-assembled monolayers (SAMs) on gold electrodes included thiolated hydrocarbons and thiolated oligonucleotides, where SAMs based on the latter showed a better performance. The corresponding antibody was covalently coupled on the SAM using carbodiimide chemistry. Sampling and measurement took only a few minutes. Application of a human serum albumin (HSA) sample, 1000 ng/mL, led to negligible impedance changes, while application of a troponin I sample, 1 ng/mL, led to a significant shift in the Nyquist plot. The results are promising regarding specific detection of clinically relevant concentrations of biomarkers, such as cardiac markers, with the newly developed microfluidic impedance biosensor chip.

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Characterisation of Antibodies Against the Active Conformation of Gαi1 Using the SRU-BIND® Label-Free Detection System

Label-Free Technologies for Drug Discovery ◽

10.1002/9780470979129.ch17 ◽

2011 ◽

pp. 255-268

Author(s):

Melanie Leveridge ◽

Chun-Wa Chung ◽

Trevor Wattam

Keyword(s):

Detection System ◽

Active Conformation ◽

Label Free ◽

Label Free Detection

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Long-Range Surface Plasmon-Polariton Waveguide Biosensors for Human Cardiac Troponin I Detection

Sensors ◽

10.3390/s19030631 ◽

2019 ◽

Vol 19 (3) ◽

pp. 631 ◽

Cited By ~ 3

Author(s):

Oleksiy Krupin ◽

Pierre Berini

Keyword(s):

Long Range ◽

Surface Plasmon ◽

Cardiac Troponin ◽

Surface Plasmon Polariton ◽

Troponin I ◽

Biological Marker ◽

Cardiac Troponin I ◽

Label Free ◽

Label Free Detection ◽

Plasmon Polariton

Straight long-range surface plasmon-polariton (LRSPP) waveguides as biosensors for label-free detection are discussed. The sensors consist of 5-μm-wide 35-nm-thick gold stripes embedded in a low-index optical-grade fluoropolymer (CYTOPTM) with fluidic channels etched to the Au surface of the stripes. This work demonstrates the application of the LRSPP biosensors for the detection of human cardiac troponin I (cTnI) protein. cTnI is a biological marker for acute myocardial infarction (AMI), often referred to as a heart attack, which can be diagnosed by elevated levels of cTnI in patient blood. Direct and sandwich assays were developed and demonstrated over the concentration range from 1 to 1000 ng/mL, yielding detection limits of 430 pg/mL for the direct assay and 28 pg/mL for the sandwich assay (1 standard deviation), the latter being physiologically relevant to the early detection or onset of AMI. In addition, a novel approach for data analysis is proposed, where the analyte response is normalized to the response of the antibody layer.

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