Agata Kowalczyk

Nanogravimetric and voltammetric DNA-hybridization biosensors for studies of DNA damage by common toxicants and pollutants

Electrochemical and nanogravimetric DNA-hybridization biosensors have been developed for sensing single mismatches in the probe-target ssDNA sequences. The voltammetric transduction was achieved by coupling ferrocene moiety to streptavidin linked to biotinylated tDNA. The mass-related frequency transduction was implemented by immobilizing the sensory pDNA on a gold-coated quartz crystal piezoresonators oscillating in the 10MHz band. The high sensitivity of these sensors enabled us to study DNA damage caused by representative toxicants and environmental pollutants, including Cr(VI) species, common pesticides and herbicides. We have found that the sensor responds rapidly to any damage caused by Cr(VI) species, with more severe DNA damage observed for Cr(2)O(7)(2-) and for CrO(4)(2-) in the presence of H(2)O(2) as compared to CrO(4)(2-) alone. All herbicides and pesticides examined caused DNA damage or structural alterations leading to the double-helix unwinding. Among these compounds, paraoxon-ethyl and atrazine caused the fastest and most severe damage to DNA. The physico-chemical mechanism of damaging interactions between toxicants and DNA has been proposed. The methodology of testing voltammetric and nanogravimetric DNA-hybridization biosensors developed in this work can be employed as a simple protocol to obtain rapid comparative data concerning DNA damage caused by herbicide, pesticides and other toxic pollutants. The DNA-hybridization biosensor can, therefore, be utilized as a rapid screening device for classifying environmental pollutants and to evaluate DNA damage induced by these compounds.

Progress in Targeting Tumor Cells by Using Drug-Magnetic Nanoparticles Conjugate

To limit cytotoxicity of anticancer drugs against healthy cells, an appropriate carrier should be synthesized to deliver the drug to the tumor tissue only. A good solution is to anchor a magnetic nanoparticle to the molecule of the drug and to use a properly directed external magnetic field. The synthesis of the conjugate of doxorubicin with magnetic nanoparticles (iron oxide) modified by us resulted in a substantial depression of the aggregation process of the nanoparticles and therefore allowed the correct examination of cytotoxicity of the modified drug. It has been shown, by performing the electrochemical microbalance measurements, that the use of magnetic field guaranteed the efficient delivery of the drug to the desired place. The change in the synthesis procedure led to an increase in the number of DOX molecules attached to one magnetic nanoparticle. The release of the drug took place at pH 5.8 (and below it), which pH characterizes the cancer cells. It has also been found that while the iron oxide magnetic nanoparticles were not cytotoxic toward human urinary bladder carcinoma cells UM-UC-3, the tumor cell sensitivity of the DOX-Np complex was slightly higher in comparison to the identical concentration of doxorubicin alone.

A novel type of electrochemical sensor based on ferromagnetic carbon-encapsulated iron nanoparticles for direct determination of hemoglobin in blood samples

An effective, fast, facile and direct electrochemical method of determination of hemoglobin (Hb) in blood sample without any sample preparation is described. The method is accomplished by using the ferromagnetic electrode modifier (carbon-encapsulated iron nanoparticles) and an external magnetic field. The successful voltammetric determination of hemoglobin is achieved in PBS buffer as well as in the whole blood sample. The obtained results show the excellent electroactivity of Hb. The measurements are of high sensitivity and good reproducibility. The detection limit is estimated to be 0.7 pM. The electrochemical determination data were compared with the gravimetric data obtained with a quartz crystal microbalance. The agreement between these results is very good. The changes of the electrode surface morphology before and after Hb detection are monitored by electron microscopy. The functionality of the electrochemical sensor is tested with human and rat blood samples. The concentration of hemoglobin in the blood samples determined by using voltammetric/gravimetric detection is in perfect agreement with the data obtained from typical clinical analysis.

Construction of DNA biosensor at glassy carbon surface modified with 4-aminoethylbenzenediazonium salt

A simple, label-free electrochemical impedance-spectroscopy method for sequence-specific detection of DNA using a 4-aminoethylbenzenediazonium (AEBD) salt as a binder for amino-modified probe DNA is reported. This novel method simplifies the anchoring of DNA at the GC surface and opens new ways for the detection of hybridization. The hybridization of target DNA, without and with mismatches, with the probe DNA anchored at the GC surface modified with AEBD, greatly increases the interfacial electron transfer resistance at the double-stranded DNA modified electrodes for the redox couple Fe(CN)(6)(3-/4-). The resistance was measured using electrochemical impedance spectroscopy. The sensor response increased linearly with logarithm of concentration of target DNA in the range 2×10(-12)÷2×10(-6) M. The obtained quantification limit was circa 6.5×10(-17) mole in a 7 μL droplet and corresponded to a concentration of 9.2×10(-12) M of target DNA in the sample. This limit is equivalent to the detection of circa 4×10(7) copies of DNA in a 7 μL droplet or circa 5.7×10(12) DNA copies in one litre of sample.

Influence of a Magnetic Nanoparticle As a Drug Carrier on the Activity of Anticancer Drugs: Interactions of Double Stranded DNA and Doxorubicin Modified with a Carrier

Magnetic drug targeting executed by nanoparticles as carriers is a promising cancer treatment which avoids the side effects of conventional chemotherapy. We have modified doxorubicin with magnetic nanoparticles. Doxorubicin (adriamycin) is a potential anticancer drug yet nonspecific in its antibiotic action. Therefore, carriers for tissue-specific, targeted drug delivery are of utmost importance. We report here on the interaction of doxorubicin covalently bound via tether molecules to colloidal magnetic nanoparticles (ferrofluid) with calf thymus double stranded DNA (dsDNA). By means of spectroscopic and electrochemical techniques, we have shown that appropriate length and flexibility of tether molecules allows the preservation of essentially intact intercalation capabilities of free doxorubicin in the solution. In order to evaluate these capabilities, we have studied the binding constant of doxorubicin attached to nanoferrites with dsDNA as well as the binding site size on the dsDNA molecule. The binding constant decreased slightly compared to that of free doxorubicin while the binding site size, describing the number of consecutive DNA lattice residues involved in the binding, increased. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) images were also obtained to support the conclusion on the interactions between doxorubicin-modified magnetic nanoparticles and dsDNA.

Oxidation of DNA followed by conformational change after OH radical attack.

Examination of the attack of OH radicals produced in the Fenton way on DNA molecules is important from biological, biochemical, and biosensor points of view. Calf thymus DNA was selected for the investigation, since this natural oligonucleotide is often used in examination of drug-DNA interactions. Particularly useful was the coherent application of five techniques: electrochemical quartz crystal microbalance (EQCM), square wave voltammetry (SWV), circular dichroism (CD), atomic force microscopy (AFM), and UV-vis spectroscopy. These techniques differ in sensitivity to radical concentration and layer thickness of DNA. EQCM appeared to be the most sensitive in monitoring the consequences of OH radical actions; radical activities corresponding to nanomolar concentrations of H(2)O(2) could be detected. SWV and AFM detection gave noticeable signal for higher than 1 μM H(2)O(2) concentrations. EQCM data led to a conclusion that at higher than 1 μM H(2)O(2) concentrations the DNA strands were locally disintegrated. The corresponding DNA loss was ca. 16%. It has been shown that in the presence of α-tocopherol, a strong antioxidant, the damage caused by OH radicals was practically prevented.

Hydrogel with chains functionalized with carboxyl groups as universal 3D platform in DNA biosensors.

Application of hydrogel based on N-isopropylacrylamide with carboxyl groups grafted to the chains enabled the immobilization of DNA at an extent exceeding that for flat surfaces by at least one order of magnitude. The probe DNA strands in the 3D platform were fully available for the hybridization process. The examination of the gels containing different amounts of grafted carboxyl groups (1-10%) was done using quartz crystal microbalance, electrochemical impedance spectroscopy, chronoamperometry and ionic coupled plasma with laser ablation. The optimal carboxyl group content was determined to be 5%. A very good agreement of the data obtained with independent techniques on content of DNA in the gel was obtained. In comparison to the other methods of immobilization of DNA the new platform enabled complete removal of DNA after the measurements and analysis and, therefore, could be used many times. After a 10-fold exchange of the DNA-sensing layer the efficiency of hybridization and analytical signal did not change by more than 5%. The sensor response increased linearly with logarithm of concentration of target DNA in the range 1×10(-13)-1×10(-6) M. The obtained detection limit was circa 8×10(-13) M of target DNA in the sample which is a substantial improvement over the planar sensing layers.

Tight and uniform layer of covalently bound aminoethylophenyl groups perpendicular to gold surface for attachment of biomolecules.

Strongly adhered layers of the compound with the primary amino group directed toward the solution were obtained at the gold surface by chronoamperometric electroreduction of 4-aminoethylobenzenodiazonium salt (AEBD) in acetonitrile solution at appropriately selected potential. The used techniques (EQCM, AFM, EIS, PM, IRRAS) showed that the nature and thickness of formed aminoethylophenyl layer strongly depend on the potential applied to the electrode. Electroreduction of AEBD salt at a potential more negative than -0.6 V (vs Ag/AgCl) leads to about monolayer on the gold surface. Additionally, such a layer was very tight and uniform. The electrochemical measurements indicate that the efficient and precise attachment of biomolecules to the aminoethylophenyl layer is only possible when this layer is formed at appropriate potential. This was shown for ss- and dsDNA.

Improved cytotoxicity and preserved level of cell death induced in colon cancer cells by doxorubicin after its conjugation with iron-oxide magnetic nanoparticles

A promising strategy for overcoming the problem of limited efficacy in antitumor drug delivery and in drug release is the use of a nanoparticle-conjugated drug. Doxorubicin (Dox) anticancer chemotherapeutics has been widely studied in this respect, because of severe cardiotoxic side effects. Here, we investigated the cytotoxic effects, the uptake process, the changes in cell cycle progression and the cell death processes in the presence of iron-oxide magnetic nanoparticles (Nps) and doxorubicin conjugates (Dox-Nps) in human colon HT29 cells. The amount of Dox participated in biological action of Dox-Nps was determined by cyclic voltammetry and thermogravimetric measurements. The cytotoxicity of Dox-Nps was shown to be two/three times higher than free Dox, whereas Nps alone did not inhibit cell proliferation. Dox-Nps penetrated cancer cells with higher efficacy than free Dox, what could be a consequence of Dox-Nps aggregation with proteins in culture medium and/or with cell surface. The treatment of HT29 cells with Dox-Nps and Dox at IC50 concentration resulted in G2/M arrest followed by late apoptosis and necrosis. Summing up, the application of iron-oxide magnetic nanoparticles improved Dox-Nps cell penetration compared to free Dox and achieved the cellular response to Dox-Nps conjugates similar to that of Dox alone.

Selective and sensitive electrochemical device for direct VB2 determination in real products.

The developed by us electrochemical device for vitamin B2 (VB2; riboflavin) determination, without preconcentration step, in real products exhibits high sensitivity, selectivity, stability and low detection limit compared to those described in the literature. The determination procedure was based on the monitoring of the reduction current signal of VB2 bound with dsDNA anchored to the electrode surface through intermediary - carboxyphenyl layer. The application of such intermediary layer formed during electroreduction of appropriate diazonium salt at CV peak potential guarantees high efficiency of hybridization process and thus fully available places for VB2 interaction. Moreover, such intermediary layer provides good electrical contact, what is very important in the case of electrochemical sensors. The analytical range of work of the proposed VB2 sensor was between 0.08-1µM (30-377μgL-1) of riboflavin concentration. The obtained detection (LOD) and quantification limits (LOQ) were 24±2 and 55±5μgL-1, respectively. The proposed VB2 detection method was used for determination of riboflavin content in commercially available dietary supplements and yolk of hen egg samples. The accuracy of the obtained data was proved using comparison with an independent method (HPLC FLD).