Nejvíce citovaný článek - PubMed ID 15456317
Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutations in DNA
Electrochemical methods can be used not only for the sensitive analysis of proteins but also for deeper research into their structure, transport functions (transfer of electrons and protons), and sensing their interactions with soft and solid surfaces. Last but not least, electrochemical tools are useful for investigating the effect of an electric field on protein structure, the direct application of electrochemical methods for controlling protein function, or the micromanipulation of supramolecular protein structures. There are many experimental arrangements (modalities), from the classic configuration that works with an electrochemical cell to miniaturized electrochemical sensors and microchip platforms. The support of computational chemistry methods which appropriately complement the interpretation framework of experimental results is also important. This text describes recent directions in electrochemical methods for the determination of proteins and briefly summarizes available methodologies for the selective labeling of proteins using redox-active probes. Attention is also paid to the theoretical aspects of electron transport and the effect of an external electric field on the structure of selected proteins. Instead of providing a comprehensive overview, we aim to highlight areas of interest that have not been summarized recently, but, at the same time, represent current trends in the field.
- Klíčová slova
- Electrode, Microdevice, Peptide, Protein, Sensor,
- MeSH
- elektrochemické techniky * metody MeSH
- elektrochemie MeSH
- oxidace-redukce MeSH
- proteiny * MeSH
- transport elektronů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- proteiny * MeSH
- MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- elektrochemické techniky přístrojové vybavení metody MeSH
- glykomika přístrojové vybavení metody MeSH
- glykoproteiny analýza metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- proteiny analýza metabolismus MeSH
- sacharidové sekvence MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- glykoproteiny MeSH
- proteiny MeSH
An Electrochemical Detection of Metallothioneins at the Zeptomole Level in Nanolitre VolumesWe report on improvement of the adsorptive transfer stripping technique (AdTS) coupled with the differential pulse voltammetry Brdicka reaction to determine a thiol-protein. The current technique has been unable to generate reproducible results when analyzing very low sample volumes (nanolitres). This obstacle can be overcome technically by modifying the current transfer technique including cooling step of the adsorbed analyte. We tested the technique on determination of a promising tumour disease marker protein called metallothionein (MT). The detection limit (3 S/N) of MT was evaluated as 500 zeptomoles per 500 nL (1 pM) and the quantification limit (10 S/N) as 1,500 zeptomoles per 500 nL (3 pM). Further, the improved AdTS technique was utilized to analyze blood serum samples from patients with breast cancer. Based on the results obtained it can be concluded that the improved technique can be used to detect a thiolprotein in very low sample volumes and can also prevent interferences during the washing and transferring step.
- Klíčová slova
- Brdicka reaction, Proteomics, adsorptive transfer stripping technique, differential pulse voltammetry, human blood serum, metallothionein, thiols, tumour disease, zeptomole,
- Publikační typ
- časopisecké články MeSH
Lactoferrin is a multifunctional protein with antimicrobial activity and others tohealth beneficial properties. The main aim of this work was to propose easy to usetechnique for lactoferrin isolation from cow colostrum samples. Primarily we utilizedsodium dodecyl sulphate - polyacrylamide gel electrophoresis for isolation of lactoferrinfrom the real samples. Moreover we tested automated microfluidic Experionelectrophoresis system to isolate lactoferrin from the collostrum sample. The welldeveloped signal of lactoferrin was determined with detection limit (3 S/N) of 20 ng/ml. Inspite of the fact that Experion is faster than SDS-PAGE both separation techniques cannotbe used in routine analysis. Therefore we have tested third separation technique, ionexchange chromatography, using monolithic column coupled with UV-VIS detector (LCUV-VIS). We optimized wave length (280 nm), ionic strength of the elution solution (1.5M NaCl) and flow rate of the retention and elution solutions (0.25 ml/min and 0.75 ml/min.respectively). Under the optimal conditions the detection limit was estimated as 0.1 μg/mlof lactoferrin measured. Using LC-UV-VIS we determined that lactoferrin concentrationvaried from 0.5 g/l to 1.1 g/l in cow colostrums collected in the certain time interval up to 72 hours after birth. Further we focused on miniaturization of detection device. We testedamperometric detection at carbon electrode. The results encouraged us to attempt tominiaturise whole detection system and to test it on analysis of real samples of humanfaeces, because lactoferrin level in faeces is closely associated with the inflammations ofintestine mucous membrane. For the purpose of miniaturization we employed thetechnology of printed electrodes. The detection limit of lactoferrin was estimated as 10μg/ml measured by the screen-printed electrodes fabricated by us. The fabricatedelectrodes were compared with commercially available ones. It follows from the obtainedresults that the responses measured by commercial electrodes are app. ten times highercompared with those measured by the electrodes fabricated by us. This phenomenonrelates with smaller working electrode surface area of the electrodes fabricated by us(about 50 %) compared to the commercial ones. The screen-printed electrodes fabricatedby us were utilized for determination of lactoferrin faeces. Regarding to fact that sample offaeces was obtained from young and healthy man the amount of lactoferrin in sample wasunder the limit of detection of this method.
About biological affecting of flavonoids on animal organisms is known less,thus we selected flavonoids, flavanones and flavones, and their glycosides, which wereexamined as potential inducers of cytochrome(s) P450 when administrated by gavages intoexperimental male rats. The study was focused on induction of CYP1A1, the majorcytochrome P450 involved in carcinogen activation. The data obtained demonstrate thenecessity of taking into account not only ability of flavonoids to bind to Ah receptor(induction factor) but also to concentrate on their distribution and metabolism (includingcolon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode. Primarily we aimed on investigation of their basic electrochemical behaviour. After that we have optimized frequency, step potential and supporting electrolyte. Based on the results obtained, we selected the most suitable conditions for determination of the flavonoids as follows: frequency 180 Hz, step potential 1.95 mV/s and phosphate buffer of pH 7 as supporting electrolyte. Detection limits (3 S/N) of the flavonoids were from units to tens of nM except diosmin, where the limit were higher than μM. In addition, we attempted to suggest a sensor for analysis of flavonoids in urine. It clearly follows from the results obtained that flavonoids can be analysed in the presence of animal urine, because urine did not influence much the signals of flavonoids (recoveries of the signals were about 90 %).
- Klíčová slova
- antioxidant, cancer, carbon paste electrode, cytochrome P450, flavonoids, square wave voltammetry, western blot analysis,
- Publikační typ
- časopisecké články MeSH
Presence of mutated and/or structurally modified (e.g., denatured, aggregated) protein p53 form is associated with several disorders such as Alzheimer's disease, Parkinson's disease, prion diseases, and many types of tumours. The aim of this work was to distinguish native, denatured and aggregated form of full-length p53 by flow injection analysis coupled with electrochemical detector (FIA-ED). Firstly FIA-ED method used for protein native form determination was optimized (detection limit 45.8 amol per 5 mul injection; 3 x S/N). In addition the technique was applied to identify p53 structural forms (denatured and aggregated). It was found out that denatured form provides about three times higher electrochemical response (protein structure unfolding, approach of more electroactive centers - aminoacid residues - towards electrode surface) in comparison with native form. On the other hand, aggregated form offers lower response (steric eclipse of electroactive protein parts) when compared with the signal of native form. The obtained data show that we are not only able to sensitively determine native, denatured, and aggregated structural forms of p53 protein but also to distinguish them.
- MeSH
- denaturace proteinů MeSH
- elektrochemie MeSH
- konformace proteinů MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie MeSH
- průtoková injekční analýza metody MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- nádorový supresorový protein p53 MeSH
