Robert Ziółkowski

Electrochemical oligonucleotide-based biosensor for the determination of lead ion

The possibility of utilization of gold electrodes modified with short guanine-rich ssDNA probes for determination of Pb(2+) was examined. Interaction between guanine residues and lead ion followed by formation of G-quadruplex structures was confirmed by electrochemical impedance spectroscopy investigations. An external cationic redox label, methylene blue, was employed in voltammetric measurements for analytical signal generation. It was shown that due to the G-quadruplex formation, the oligonucleotides in the recognition layer fold, which enhances the electron transfer between methylene blue and the electrode surface. The MB current signal rises proportionally to the lead ion concentration in the range from 0.05 to 1μmol/L. The developed biosensor demonstrated high selectivity towards Pb(2+) ion, with only minor response towards interfering metal cations. The calculated limit of detection was of 34.7nmol/L. The utilization of the biosensor for Pb(2+) determination in real samples of water was also tested.

Electrochemical detection of DNA hybridization using metallacarborane unit.

The evaluation of novel electrochemically active label for electrochemical detection of DNA hybridization is presented. Metallacarborane units modified with iron, cobalt or chromium were investigated. The value of redox potential and relatively strong current signal facilitate usage of Fe-carborane as marker covalently attached to the ssDNA. In electrochemical genosensor the sequence complementary to UL55 gene was labeled and used as a target for biosensor device. Interactions were investigated using electrochemical and piezoelectric methods. Obtained results confirm usefulness of the designed label in electrochemical detection of DNA hybridization.

Application of mass fabricated silicon-based gold transducers for amperometric biosensors

The backside contact, silicon-based transducers with vacuum-deposited gold layer (BSC) are evaluated as the base for electrochemical biosensors construction. Their comparison with commercially available transducers with screen printed gold and traditional gold disc electrode is reported. To determine the advantages and disadvantages of each of gold surfaces mentioned above, the 6-(ferrocenyl)-hexanethiol was used as the indicator. The results revealed the usefulness of BSC chips for the formation of stable self-assembled monolayers (SAMs). After the preliminary analysis, the SAM of thiol-ssDNA was formed on the BSC transducers as recognition layer. The electrochemical analysis in methylene blue solution was carried out after ssDNA immobilization and DNA-DNA hybridization. It is shown that prepared sensors are able to recognize complementary DNA sequence, based on the change in height and the potential shifts of reduction peaks of methylene blue. Obtained results are in full agreement with literature data. The compact size of silicone-based transducers allows to significantly reduce the required volume of tested solutions.

Electrochemical uranyl cation biosensor with DNA oligonucleotides as receptor layer.

The present study aims at the further development of the uranyl oligonucleotide-based voltammetric biosensor, which takes advantage of strong interaction between UO2(2+) and phosphate DNA backbone. Herein we report the optimization of working parameters of previously elaborated electrochemical DNA biosensor. It is shown that the sensor sensitivity is highly dependent on the oligonucleotide probe length and the incubation time of sensor in a sample solution. Consequently, the highest sensitivity was obtained for 10-nucleotide sequence and 60 min incubation time. The lower detection limit towards uranyl cation for developed biosensor was 30 nM. The influence of mixed monolayers and the possibility of developing a non-calibration device were also investigated. The selectivity of the proposed biosensor was significantly improved via elimination of adenine nucleobases from the DNA probe. Moreover, the regeneration procedure was elaborated and tested to prolong the use of the same biosensor for 4 subsequent determinations of UO2(2+).

Development of silicon-based electrochemical transducers

Novel silicon-based three-electrode voltammetric transducers were fabricated using the integrated circuit technology. The electrochemical properties of the developed planar structures were tested by cyclic voltammetry and chronoamperometry, revealing the high surface smoothness of the gold working electrodes. The unmodified structures were introduced into a miniaturized voltammetric electronic tongue – the chemometric analysis of data provided by several electrochemical techniques allowed the monitoring of the biotechnological process of beer fermentation. The three-electrode transducers were also applied for the electrochemical determination of dopamine in the presence of ascorbic acid. Efficient peak separation, enabling the quantification of dopamine, was achieved by modifying the surface of gold working electrodes with N-acetyl-L-cysteine self-assembled monolayers.

Isothermal DNA amplification combined with lateral flow dipsticks for detection of biothreat agents

The recently developed methods of nucleic acids isothermal amplification are promising tools for point-of-care diagnostics and in the field detection of pathogenic microorganisms. However, application of these methods outside a laboratory faces some challenges such as the rapid and sensitive detection of amplified products and the absence of cross-reactivity with genetically related microorganisms. In the presented study we compared three methods of isothermal DNA amplification loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA), for detection of highly dangerous pathogens, such as Bacillus anthracis, Francisella tularensis and Yersinia pestis, and combined them with lateral flow dipsticks for the rapid visualization of amplified products. We observed low specificity of the three methods for B. antharcis, medium for Y. pestis and high for F. tularensis detection. Sensitivity and the detection limit were high and comparable for all the methods. We concluded that the lateral flow dipsticks have been a very useful tool for product detection of the isothermal amplification methods and enable reading the results without the use of any equipment. However, our results showed that the use of isothermal amplification methods is strongly related to the risk of false positive results.