Bio-Sensing of Cadmium(II) Ions Using Staphylococcus aureus†

Cadmium, as a hazardous pollutant commonly present in the living environment, represents an important risk to human health due to its undesirable effects (oxidative stress, changes in activities of many enzymes, interactions with biomolecules including DNA and RNA) and consequent potential risk, making its detection very important. New and unique technological and biotechnological approaches for solving this problems are intensely sought. In this study, we used the commonly occurring potential pathogenic microorganism Staphylococcus aureus for the determination of markers which could be used for sensing of cadmium(II) ions. We were focused on monitoring the effects of different cadmium(II) ion concentrations (0, 1.25, 2.5, 5, 10, 15, 25 and 50 μg mL−1) on the growth and energetic metabolism of Staphylococcus aureus. Highly significant changes have been detected in the metabolism of thiol compounds—specifically the protein metallothionein (0.79–26.82 mmol/mg of protein), the enzyme glutathione S-transferase (190–5,827 μmol/min/mg of protein), and sulfhydryl groups (9.6–274.3 μmol cysteine/mg of protein). The ratio of reduced and oxidized glutathione indicated marked oxidative stress. In addition, dramatic changes in urease activity, which is connected with resistance of bacteria, were determined. Further, the effects of cadmium(II) ions on the metabolic pathways of arginine, β-glucosidase, phosphatase, N-acetyl β-d-glucosamine, sucrose, trehalose, mannitol, maltose, lactose, fructose and total proteins were demonstrated. A metabolomic profile of Staphylococcus aureus under cadmium(II) ion treatment conditions was completed seeking data about the possibility of cadmium(II) ion accumulation in cells. The results demonstrate potential in the application of microorganisms as modern biosensor systems based on biological components

Bio-Sensing of Cadmium(II) Ions Using Staphylococcus aureus†

Cadmium, as a hazardous pollutant commonly present in the living environment, represents an important risk to human health due to its undesirable effects (oxidative stress, changes in activities of many enzymes, interactions with biomolecules including DNA and RNA) and consequent potential risk, making its detection very important. New and unique technological and biotechnological approaches for solving this problems are intensely sought. In this study, we used the commonly occurring potential pathogenic microorganism Staphylococcus aureus for the determination of markers which could be used for sensing of cadmium(II) ions. We were focused on monitoring the effects of different cadmium(II) ion concentrations (0, 1.25, 2.5, 5, 10, 15, 25 and 50 μg mL−1) on the growth and energetic metabolism of Staphylococcus aureus. Highly significant changes have been detected in the metabolism of thiol compounds—specifically the protein metallothionein (0.79–26.82 mmol/mg of protein), the enzyme glutathione S-transferase (190–5,827 μmol/min/mg of protein), and sulfhydryl groups (9.6–274.3 μmol cysteine/mg of protein). The ratio of reduced and oxidized glutathione indicated marked oxidative stress. In addition, dramatic changes in urease activity, which is connected with resistance of bacteria, were determined. Further, the effects of cadmium(II) ions on the metabolic pathways of arginine, β-glucosidase, phosphatase, N-acetyl β-d-glucosamine, sucrose, trehalose, mannitol, maltose, lactose, fructose and total proteins were demonstrated. A metabolomic profile of Staphylococcus aureus under cadmium(II) ion treatment conditions was completed seeking data about the possibility of cadmium(II) ion accumulation in cells. The results demonstrate potential in the application of microorganisms as modern biosensor systems based on biological components

Specifická magnetická izolace E6 HPV16 upravených magnetizovatelných částic spolu s PCR a elektrochemickou detekcí

The majority of carcinomas that were developed due to the infection with human papillomavirus (HPV) are caused by high-risk HPV types, HPV16 and HPV18. These HPV types contain the E6 and E7 oncogenes, so the fast detection of these oncogenes is an important point to avoid the development of cancer. Many different HPV tests are available to detect the presence of HPV in biological samples. The aim of this study was to design a fast and low cost method for HPV identification employing magnetic isolation, polymerase chain reaction (PCR) and electrochemical detection. These assays were developed to detect the interactions between E6-HPV16 oncogene and magnetizable particles (MPs) using commercial Dynabeads M-280 Streptavidin particles and laboratory-synthesized “homemade” particles called MANs (MAN-37, MAN-127 and MAN-164). The yields of PCR amplification of E6-HPV16 oncogene bound on the particles and after the elution from the particles were compared. A highest yield of E6-HPV16 DNA isolation was obtained with both MPs particles commercial M-280 Streptavidin and MAN-37 due to reducing of the interferents compared with the standard PCR method. A biosensor employing the isolation of E6-HPV16 oncogene with MPs particles followed by its electrochemical detection can be a very effective technique for HPV identification, providing simple, sensitive and cost-effective analysis.Většina karcinomů, které byly vyvolané v důsledku infekce lidským papilomavirem (HPV), jsou způsobeny vysoce rizikovými typy HPV, HPV16 a HPV18. Tyto typy HPV obsahují E6 a E7 onkogeny, takže rychlá detekce těchto onkogenů je důležitý bod k zabránění rozvoje rakoviny. Mnoho různých HPV testů je k dispozici pro detekci přítomnosti HPV v biologických vzorcích. Cílem této studie bylo navrhnout rychlou a levnou metodu pro identifikaci HPV využívající magnetickou izolaci, polymerázovou řetězovou reakci (PCR) a elektrochemickou detekci. Tyto testy byly vyvinuty s cílem odhalit interakce mezi E6-HPV16 onkogenem a magnetizovatelnými částicemi (MPS) s použitím komerčních částic Dynabeads M-280 Streptavidin a laboratorně syntetizovaných "homemade" částic zvaných MAN (MAN-37, MAN-127 a MAN-164). Byly porovnány výnosy z PCR amplifikace onkogenu E6-HPV16 vázaného na částicích a po eluci z částic. Nejvyšším výnos z izolace E6-HPV16 DNA byl získán s oběma komerčními MPs částicemi M-280 Streptavidin a MAN-37 v důsledku snížení interferencí ve srovnání se standardní metodou PCR. Biosenzor používající izolaci E6-HPV16 onkogenu s MPs částicemi následovaný jeho elektrochemickou detekci může být velmi účinná technika pro identifikaci HPV, která umožňuje jednoduchou, citlivou a nákladově efektivní analýzu

Vývoj magnetického elektrochemického barkodového pole pro analýzu bodových mutací v genu neuraminidazy H5N1

Since its first official detection in the Guangdong province of China in 1996, the highly pathogenic avian influenza virus of H5N1 subtype (HPAI H5N1) has reportedly been the cause of outbreaks in birds in more than 60 countries, 24 of which were European. The main issue is still to develop effective antiviral drugs. In this case, single point mutation in the neuraminidase gene, which causes resistance to antiviral drug and is, therefore, subjected to many studies including ours, was observed. In this study, we developed magnetic electrochemical bar code array for detection of single point mutations (mismatches in up to four nucleotides) in H5N1 neuraminidase gene. Paramagnetic particles Dynabeads with covalently bound oligo (dT)25 were used as a tool for isolation of complementary H5N1 chains (H5N1 Zhejin, China and Aichi). For detection of H5N1 chains, oligonucleotide chains of lengths of 12 (+5 adenine) or 28 (+5 adenine) bp labeled with quantum dots (CdS, ZnS and/or PbS) were used. Individual probes hybridized to target molecules specifically with efficiency higher than 60%. The obtained signals identified mutations present in the sequence. Suggested experimental procedure allows obtaining further information from the redox signals of nucleic acids. Moreover, the used biosensor exhibits sequence specificity and low limits of detection of subnanogram quantities of target nucleic acidsOd svého prvního oficiálního odhalení v provincii Guangdong v Číně v roce 1996, byl vysoce patogenní virus influenzy ptáků podtypu H5N1 (HPAI H5N1) údajně příčinou vyvolání choroby u ptáků ve více než 60 zemích světa, z nichž 24 bylo evropských. Hlavním problémem je i nadále rozvíjení efektivních antivirotik. V tomto případě byla pozorována jediná bodová mutace v genu neuraminidázy, která způsobuje odolnost vůči antivirovému léku, a je proto podroben mnoha studií, včetně té naší. V této studii jsme vyvinuli elektrochemické barkodové pole pro detekci jednotlivých bodových mutací (nesoulad až do čtyř nukleotidů) v genu neuraminidázy H5N1. Paramagnetické částice Dynabeads s kovalentně vázaným oligo dT (25) byly použity jako nástroje pro izolaci komplementárních řetězců H5N1 (H5N1 Zhejin, Čína a Aichi). Pro detekci řetězců H5N1 byly použity řetězce oligonukleotidů o délce 12 (5 adenin), nebo 28 (5 adenin) bp označené kvantovými tečkami (CdS, ZnS a / nebo PbS). Jednotlivé sondy se hybridizují k cílovým molekulám specificky s účinností vyšší než 60 %. Získané signály identifikovaly přítomnost mutací v sekvencích. Doporučený experimentální postup umožňuje získat další informace redoxních signálů nukleových kyselin. Použitý biosensor vykazuje sekvenční specificitu a nízké meze detekce v subnanogramových množství cílové nukleové kyseliny

ELECTROCHEMICAL SCIENCE Femtogram Electroanalytical Detection of Prostatic Specific Antigen by Brdicka Reaction

Prostatic-specific antigen is considered as the best marker for prostate cancer. Due to the importance of PSA for diagnostic purposes it is not surprising that there are tested and optimized various methods for its determination. In spite of such intensive research in the field of electrochemical detection of some by-products connected with concentration of PSA, electrochemical behaviour of PSA has not been studied yet. The aim of this study was to investigate electrochemical catalytic signals of PSA using differential pulse voltammetry Brdicka reaction. The catalytic signals were studied using adsorptive transfer stripping technique as well as directly in the electrochemical cell. Nevertheless, we primarily tested detection of PSA by standard immunoanalysis and by gel and capillary chip electrophoresis to investigate behaviour of this protein in electric field. Both electrophoretic methods showed that the most intensive band of PSA was determined at 37 kDa under reducing conditions and at 26 kDa under non-reducing. Band at 37 kDa corresponds to a reduced, and at 26 kDa to non-reduced PSA. Studying of basic electrochemical behaviour of PSA was primarily carried out using standard electrochemical cell and HMDE as a working electrode. Co(NH 3