Colorimetric detection
Dotaz
Zobrazit nápovědu
OBJECTIVES: Glutathione belongs to the family of small-molecular weight antioxidants like ascorbic acid, cysteine, α-tocopherol, uric acid, etc. These molecules play important role in the neutralization of free radicals and reactive oxygen species (ROS). Oxidative stress may lead to ageing and the development of large scale of pathological states of organism. This low molecular weight antioxidant´s level can alter under pathological conditions from reduced (GSH, thiols) to oxidized (oxidized glutathione -GSSG, disulfides) form. A GSSG-to-GSH ratio is indicative marker of oxidative stress. There is a large scale of methods how to determine this biomarker. The trend of the analysis is to minimalize the instrument equipment, sample application volume and analysis cost. DESIGN: Reduced glutathione (GSH) solutions were prepared in water in the concentration 0-16 mmol/L. Other small-molecular weight antioxidants like 0.25 mmol/L ascorbic acid, 0.15 mmol/L TROLOX and 0.02 mmol/L N-acetyl-cysteine (NAcCys) were studied as possible interferents. The samples were mixed with 5,5´-dithiobis-(2-nitrobenzoic) acid (DTNB) resulting in yellow colored drops forming. Coloration was assayed using camera integrated in a smartphone and color channels analysis. The total volume of 10 µl of sample was applied for one analysis. The smartphone-based data were compared with the reference Ellman assay. RESULTS: The calibration of glutathione was evaluated. The blue channel intensity data were decreasing according to the increasing glutathione concentration. Red and green channel intensities were stagnating during the whole concentration scale of glutathione. Limits of detection were 0.4 mmol/l for glutathione. Addition of 0.25 mmol/L of ascorbic acid, 0.15 mmol/L of TROLOX and 0.02mmol/L of N-acetylcysteine to GSH in final concentration 0-16 mmol/L had minimal influence on the assay. The results from smartphone-based analysis correlate with the standard Ellman method. The detection limit for GSH was 0.03 mmol/L. CONCLUSION: The smartphone-based assay seems to be promising because of simplicity, reliability, robustness and low cost. In spite of the fact that there is a large scale for approaches for the glutathione determination, the main advantage of our colorimetric method is portability and easibility to perform the assay in the field and publically availability of smartphones for home applications.
- MeSH
- antioxidancia analýza MeSH
- biologické markery MeSH
- biotest metody MeSH
- chytrý telefon využití MeSH
- glutathion analýza MeSH
- kolorimetrie MeSH
- kyselina dithionitrobenzoová MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The enzyme immobilization is a very important process providing an attachment of the enzyme to a certain type of the carrier. This allows to maintain the enzyme activity during the storage and it also simplifies the detection procedure. When designing a new detector based on the enzymatic reaction, the proper immobilization method must be chosen depending on the advantages and disadvantages of the available methods of immobilization. Examples of immobilization method for colorimetric detection include, e.g., adsorption to the insoluble carrier, covalent bonding, attachment to ion exchangers, incorporation into gels and foams, immobilization in the form of cross-linked aggregates or nanostructures or with the utilization of antibodies.
- Klíčová slova
- kovalentní vazba,
- MeSH
- adsorpce MeSH
- cholinesterasové inhibitory MeSH
- cholinesterasy MeSH
- enzymy * MeSH
- gely analýza chemie MeSH
- imobilizace * metody přístrojové vybavení MeSH
- Publikační typ
- práce podpořená grantem MeSH
A dipstick for fast assay of nerve agents and organophosphate pesticides was developed. Indicator pH papers were used as detectors. The principle of the assay is based on enzymatic hydrolysis of acetylcholine into acetic acid and choline by acetylcholinesterase. Acidification of the reaction medium due to accumulation of acetic acid was visible. The colour changed from dark red to yellow as the pH indicator recognized pH shift. Presence of an organophosphate pesticide or a nerve agent results in irreversible inhibition of acetylcholinesterase intercepted on the dipstick. The inhibition stops the enzymatic reaction. The inhibition appears as no change of the medium pH. Three compounds were assayed: paraoxon-ethyl as representative organophosphate pesticides and nerve agents sarin and VX. The achieved limit of detection was 5 x 10(-8)M for paraoxon-ethyl and 5 x 10(-9)M for sarin and VX. Dipsticks were found stable for at least one month. Suitability of these dipsticks for routine assay is discussed.
Cholinesterases, specifically acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), play critical roles in neurotransmission and are key targets for inhibitors with therapeutic and toxicological significance. This review focuses on the development and application of fluorometric and colorimetric biosensors for the detection of cholinesterase inhibitors. These biosensors take advantage of the unique properties of AChE and BChE to provide sensitive and selective detection methods essential for environmental monitoring, food safety, and clinical diagnostics. Recent advances in assay techniques, including the use of gold nanoparticles, pseudoperoxidase nanomaterials, and innovative enzyme-substrate interactions, are highlighted. This review also discusses challenges and future directions for optimizing these biosensors for practical applications, emphasizing their potential to enhance public health and safety.
- MeSH
- acetylcholinesterasa chemie metabolismus MeSH
- biosenzitivní techniky * metody MeSH
- butyrylcholinesterasa chemie metabolismus MeSH
- cholinesterasové inhibitory * analýza MeSH
- fluorometrie * metody MeSH
- kolorimetrie * metody MeSH
- kovové nanočástice chemie MeSH
- lidé MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can serve as biochemical markers of various pathologies like liver disfunction and poisonings by nerve agents. Ellman's assay is the standard spectrophotometric method to measure cholinesterase activity in clinical laboratories. The authors present a new colorimetric test to assess AChE and BChE activity in biological samples using chromogenic reagents, treated 3D-printed measuring pads and a smartphone camera as a signal detector. Multiwell pads treated with reagent substrates 2,6-dichlorophenolindophenyl acetate, indoxylacetate, ethoxyresorufin and methoxyresorufin were prepared and tested for AChE and BChE. In the experiments, 3D-printed pads containing indoxylacetate as a chromogenic substrate were optimal for analytical purposes. The best results were achieved using the red (R) channel, where the limit of detection was 4.05 µkat/mL for BChE and 4.38 µkat/mL for AChE using a 40 µL sample and a 60 min assay. The major advantage of this method is its overall simplicity, as samples are applied directly without any specific treatment or added reagents. The assay was also validated to the standard Ellman's assay using human plasma samples. In conclusion, this smartphone camera-based colorimetric assay appears to have practical applicability and to be a suitable method for point-of-care testing because it does not require specific manipulation, additional education of staff or use of sophisticated analytical instruments.
- MeSH
- acetylcholinesterasa * MeSH
- butyrylcholinesterasa * MeSH
- cholinesterasové inhibitory MeSH
- chytrý telefon MeSH
- kolorimetrie MeSH
- lidé MeSH
- spektrofotometrie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Glucose is a typical marker of diabetes mellitus but it corresponds also with metabolic syndrome, immune disorders and some types of poisoning. Hence fast, low cost and simple method for its determination is required. Bubble wrap, cheap and available material, was used for immobilization of glucose oxidase (GOx) and peroxidase (POx), the enzymes necessary for colorimetric determination of glucose. Method is based on reaction of enzymes (immobilized in sol-gel membrane inside the bubbles) with substrates glucose and o-phenylenediamine dihydrochloride (o-PD) providing intensive coloration. The color change can be easily tracked by phone integrated camera. Color intensity expressed in red-green-blue (RGB) color model was used for displaying of photos and for gaining numeric data representing concentrations of glucose. The assay exerted good correlation of color intensity with the concentration of glucose, adequately low limit of detection (750 mmol/l) for glucose blood assay, no influence of interferents or matrix substances and by sufficient long term stability of sol-gel membrane. The sensor was found as low-cost simple way to analyze glucose blood levels with promising prospects in the field of portable devices.
Rostoucí význam kvasinek schopných tvořit biofilm sebou nese nutnost testování citlivosti jak pro jejich planktonické formy, tak pro formy biofilmové. Průkaz životaschopnosti, případně metabolické aktivity kvasinkových buněk po expozici vůči antimikrobiální látce má klíčovou úlohu při hodnocení výsledků. K tomuto účelu se s výhodou používají kolorimetrická média, která na přítomnost metabolicky aktivních buněk reagují změnou barvy. V této práci byla ověřena možnost využít kolorimetrického média s redoxním indikátorem resazurinem ke sledování vlivu hustoty kvasinkového inokula na rychlost redukce tohoto indikátoru. Bylo zjištěno, že rychlost barevné změny odpovídá hustotě inokula a tímto způsobem lze orientačně kvantifikovat množství životaschopných buněk. Test by bylo možno s výhodou použít nejen pro zjišťování citlivosti planktonických forem kvasinek k antimykotikům, ale i forem biofilmových.
The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.
- Klíčová slova
- Alamar blue,
- MeSH
- biofilmy MeSH
- Candida růst a vývoj MeSH
- financování organizované MeSH
- indikátory a reagencie MeSH
- infekce spojené se zdravotní péčí mikrobiologie MeSH
- kolorimetrie metody MeSH
- kvasinky růst a vývoj MeSH
- mikrobiální testy citlivosti metody MeSH
- oxaziny diagnostické užití MeSH
- xantheny diagnostické užití MeSH
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
