Biosensor
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Cholinesterase inhibitors are widely used as pesticides in agriculture, but also form a group of organophosphates known as nerve chemical warfare agents. This calls for close attention regarding their detection, including the use of various biosensors. One such biosensor made in the Czech Republic is the Detehit, which is based on a cholinesterase reaction that is assessed using a colour indicator-the Ellman's reagent-which is anchored on cellulose filter paper together with the substrate. With the use of this biosensor, detection is simple, quick, and sensitive. However, its disadvantage is that a less pronounced yellow discoloration occurs, especially under difficult light conditions. As a possible solution, a new indicator/substrate carrier has been designed. It is made of glass nanofibres, so the physical characteristics of the carrier positively influence reaction conditions, and as a result improve the colour response of the biosensor. The authors present and discuss some of the results of the study of this carrier under various experimental conditions. These findings have been used for the development of a modified Detehit biosensor.
- MeSH
- biosenzitivní techniky metody MeSH
- cholinesterasové inhibitory chemie MeSH
- nanovlákna chemie MeSH
- sklo chemie MeSH
- Publikační typ
- časopisecké články MeSH
Colorimetric biosensors of cholinesterase inhibitors are ideal for fast, reliable, and very simple detection of agents in air, in water, and on surfaces. This paper describes an innovation of the Czech Detehit biosensor, which is based on a biochemical enzymatic reaction visualized by using Ellman's reagent as a chromogenic indicator. The modification basically consists of a much more distinct color response of the biosensor, attained through optimization of the reaction system by using Guinea Green B as the indicator. The performance of the modified biosensor was verified on the chemical warfare agents (sarin, soman, cyclosarin, and VX) in water. The detection limits ascertained visually (with the naked eye) were about 0.001 µg/mL in water (exposure time 60 s, inhibition efficiency 25%).
Biosensors containing cholinesterase are analytical devices suitable for the assay of neurotoxic compounds. In the research on biosensors, a new platform has appeared some years ago. It is the digital photography and scoring of coloration (photogrammetry). In this paper, a colorimetric biosensor is constructed using 3D-printed multiwell pads treated with indoxylacetate as a chromogenic substrate and gold nanoparticles with the immobilized enzyme butyrylcholinesterase. A smartphone camera served for photogrammetry. The biosensor was tested for the assay of carbofuran and paraoxon ethyl as two types of covalently binding inhibitors: irreversible and pseudoirreversible. The biosensor exerted good sensitivity to the inhibitors and was able to detect carbofuran with a limit of detection for carbofuran 7.7 nmol/l and 17.6 nmol/l for paraoxon ethyl. A sample sized 25 μl was suitable for the assay lasting approximately 70 minutes. Up to 121 samples can be measured contemporary using one multiwell pad. The received data fully correlated with the standard spectrophotometry. The colorimetric biosensor exerts promising specifications and appears to be competitive to the other analytical procedures working on the principle of cholinesterase inhibition. Low-cost, simple, and portable design represent an advantage of the assay of the biosensor. Despite the overall simplicity, the biosensor can fully replace the standard spectroscopic methods.
- Publikační typ
- časopisecké články MeSH
Simple analytical devices suitable for the analysis of various biochemical and immunechemical markers are highly desirable and can provide laboratory diagnoses outside standard hospitals. This study focuses on constructing an easily reproducible do-it-yourself ELISA plate reader biosensor device, assembled from generally available and inexpensive parts. The colorimetric biosensor was based on standard 96-well microplates, 3D-printed parts, and a smartphone camera as a detector was utilized here as a tool to replace the ELISA method, and its function was illustrated in the assay of TNFα as a model immunochemical marker. The assay provided a limit of detection of 19 pg/mL when the B channel of the RGB color model was used for calibration. The assay was well correlated with the ELISA method, and no significant matrix effect was observed for standard biological samples or interference of proteins expected in a sample. The results of this study will inform the development of simple analytical devices easily reproducible by 3D printing and found on generally available electronics.
- MeSH
- 3D tisk * MeSH
- biosenzitivní techniky * MeSH
- ELISA * MeSH
- kolorimetrie MeSH
- lidé MeSH
- TNF-alfa * analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In early stages of Alzheimer's disease (AD), amyloid beta (Aβ) accumulates in the mitochondrial matrix and interacts with mitochondrial proteins, such as cyclophilin D (cypD) and 17β-hydroxysteroid dehydrogenase 10 (17β-HSD10). Multiple processes associated with AD such as increased production or oligomerization of Aβ affect these interactions and disbalance the equilibrium between the biomolecules, which contributes to mitochondrial dysfunction. Here, we investigate the effect of the ionic environment on the interactions of Aβ (Aβ1-40, Aβ1-42) with cypD and 17β-HSD10 using a surface plasmon resonance (SPR) biosensor. We show that changes in concentrations of K+ and Mg2+ significantly affect the interactions and may increase the binding efficiency between the biomolecules by up to 35% and 65% for the interactions with Aβ1-40 and Aβ1-42, respectively, in comparison with the physiological state. We also demonstrate that while the binding of Aβ1-40 to cypD and 17β-HSD10 takes place preferentially around the physiological concentrations of ions, decreased concentrations of K+ and increased concentrations of Mg2+ promote the interaction of both mitochondrial proteins with Aβ1-42. These results suggest that the ionic environment represents an important factor that should be considered in the investigation of biomolecular interactions taking place in the mitochondrial matrix under physiological as well as AD-associated conditions.
- MeSH
- 17-hydroxysteroidní dehydrogenasy chemie genetika MeSH
- Alzheimerova nemoc diagnóza genetika patologie MeSH
- amyloidní beta-protein chemie MeSH
- biosenzitivní techniky metody MeSH
- ionty chemie MeSH
- lidé MeSH
- mitochondriální proteiny chemie MeSH
- mitochondrie chemie MeSH
- peptidové fragmenty chemie genetika MeSH
- peptidylprolylisomerasa F chemie genetika MeSH
- povrchová plasmonová rezonance metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Src kinase plays an important role in a multitude of fundamental cellular processes and is often found deregulated in tumors. Active Src adopts an open conformation, whereas inactive Src is characterized by a very compact structure stabilized by inhibitory intramolecular interactions. Taking advantage of this spatial regulation, we constructed a fluorescence resonance energy transfer (FRET)-based Src biosensor and analyzed conformational changes of Src following Src activation and the spatiotemporal dynamics of Src activity in cells. We found that activatory mutations either in regulatory or kinase domains induce opening of the Src structure. Surprisingly, we discovered that Src inhibitors differ in their effect on the Src structure, some counterintuitively inducing an open conformation. Finally, we analyzed the dynamics of Src activity in focal adhesions by FRET imaging and found that Src is rapidly activated during focal adhesion assembly, and its activity remains steady and high throughout the life cycle of focal adhesion and decreases during focal adhesion disassembly.
- MeSH
- biosenzitivní techniky metody MeSH
- fokální adheze metabolismus MeSH
- FRAP MeSH
- HEK293 buňky MeSH
- lidé MeSH
- mutageneze MeSH
- rezonanční přenos fluorescenční energie MeSH
- skupina kinas odvozených od src-genu antagonisté a inhibitory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Biosensors using cholinesterases as the biorecognition component have been used to assay organophosphates and carbamates for a long time. In this review, some strategies convenient for biosensor construction are presented. Solutions for cholinesterase immobilization and output signal monitoring are presented as the basic presumptions for successful biosensor construction.
- MeSH
- biosenzitivní techniky metody MeSH
- cholinesterasy metabolismus MeSH
- enzymy imobilizované metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- přehledy MeSH
