BACKGROUND: Thiol-rich peptides and proteins possess a large number of biological activities and may serve as markers for numerous health problems including cancer. Metallothionein (MT), a small molecular mass protein rich in cysteine, may be considered as one of the promising tumour markers. The aim of this paper was to employ chronopotentiometric stripping analysis (CPSA) for highly sensitive detection of MT. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used adsorptive transfer stripping technique coupled with CPSA for detection of cysteine, glutathione oxidized and reduced, phytochelatin, bovine serum albumin, and metallothionein. Under the optimal conditions, we were able to estimate detection limits down to tens of fg per ml. Further, this method was applied to detect metallothioneins in blood serum obtained from patients with breast cancer and in neuroblastoma cells resistant and sensitive to cisplatin in order to show the possible role of metallothioneins in carcinogenesis. It was found that MT level in blood serum was almost twice higher as compared to the level determined in healthy individuals. CONCLUSIONS/SIGNIFICANCE: This paper brings unique results on the application of ultra-sensitive electroanalytical method for metallothionein detection. The detection limit and other analytical parameters are the best among the parameters of other techniques. In spite of the fact that the paper is mainly focused on metallothionein, it is worth mentioning that successful detection of other biologically important molecules is possible by this method. Coupling of this method with simple isolation methods such as antibody-modified paramagnetic particles may be implemented to lab-on-chip instrument.

• MeSH
• buněčné linie MeSH
• elektrochemie metody   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• králíci MeSH
• lidé MeSH
• metalothionein krev   moč   MeSH
• potenciometrie MeSH
• reprodukovatelnost výsledků MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• metalothionein 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