Libuše Trnková

Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges

We review the progress achieved during the recent five years in immunochemical biosensors (immunosensors) combined with nanoparticles for enhanced sensitivity. The initial part introduces antibodies as classic recognition elements. The optical sensing part describes fluorescent, luminescent, and surface plasmon resonance systems. Amperometry, voltammetry, and impedance spectroscopy represent electrochemical transducer methods; electrochemiluminescence with photoelectric conversion constitutes a widely utilized combined method. The transducing options function together with suitable nanoparticles: metallic and metal oxides, including magnetic ones, carbon-based nanotubes, graphene variants, luminescent carbon dots, nanocrystals as quantum dots, and photon up-converting particles. These sources merged together provide extreme variability of existing nanoimmunosensing options. Finally, applications in clinical analysis (markers, tumor cells, and pharmaceuticals) and in the detection of pathogenic microorganisms, toxic agents, and pesticides in the environmental field and food products are summarized.

Electrochemical study of heavy metals and metallothionein in yeast Yarrowia lipolytica

The bioaccumulation of heavy metals (cadmium, nickel, cobalt and zinc) and the effect of these metals on the production of metallothionein and metallothionein-like proteins (MT) in Yarrowia lipolytica was studied by electrochemical methods. The concentrations of heavy metals were determined by differential pulse voltammetry (DPV). A combination of the constant current chronopotentiometric stripping analysis (CPSA) and adsorptive transfer stripping technique (AdTS) was used to determine the content of MT in cells. Both the bioaccumulation of heavy metals and the production of MT in different cell compartments of Y. lipolytica exposed to heavy metals were monitored. The LD(50) of each metal was determined from the number of viable cells in yeast cultures: LD(50)Cd (37.5 microM), LD(50)Ni (570 microM), LD(50)Co (700 microM), and LD(50)Zn (1800 microM). The highest concentrations of heavy metals were found in the cell wall and membrane debris while the lowest concentrations were detected in the cytoplasm. Cadmium and nickel showed the most significant effect on the production of MT. This study provides new insights into the ecophysiology of microorganisms and demonstrates the potential use of these electrochemical methods in biotechnology.

Fully automated spectrometric protocols for determination of antioxidant activity: advantages and disadvantages.

The aim of this study was to describe behaviour, kinetics, time courses and limitations of the six different fully automated spectrometric methods - DPPH, TEAC, FRAP, DMPD, Free Radicals and Blue CrO5. Absorption curves were measured and absorbance maxima were found. All methods were calibrated using the standard compounds Trolox® and/or gallic acid. Calibration curves were determined (relative standard deviation was within the range from 1.5 to 2.5 %). The obtained characteristics were compared and discussed. Moreover, the data obtained were applied to optimize and to automate all mentioned protocols. Automatic analyzer allowed us to analyse simultaneously larger set of samples, to decrease the measurement time, to eliminate the errors and to provide data of higher quality in comparison to manual analysis. The total time of analysis for one sample was decreased to 10 min for all six methods. In contrary, the total time of manual spectrometric determination was approximately 120 min. The obtained data provided good correlations between studied methods (R = 0.97 – 0.99).

Application of Elimination Voltammetry to Adsorptive Stripping of DNA

Adsorptive stripping voltammetry has been extended by elimination voltammetry with linear scan rate (EVLS). EVLS offers the elimination of selected currents from the linear scan voltammetry results. It can be achieved by an elimination function formed by a linear combination of total currents measured at different scan rates, because the basic idea of elimination of chosen currents (for example the charging current, I-c, the reversible current for substances transported only by linear diffusion, I-r, and the purely kinetic current, I-k) consists of the different dependence of voltammetric current on the scan rare. In this article, the problem of substance adsorption influence on the elimination voltammetry results has been studied. Theoretical curves of an irreversible current with adsorbed substance being electroactive were calculated for three elimination functions. A: the function eliminating I-k and I-c and conserving I-r B: the function eliminating I-c and I-r. conserving I-k; and C: the function eliminating I-r and I-k and conserving I-c, where I-c, I-r, and I-k are the charging current, the reversible diffusion current, and the kinetic current, respectively. A larger difference between diffusion and adsorption was exhibited by the elimination function A. It provides a substantial increase in resolution and sensitivity as compared to the linear scan voltammetry current peak and clearly marks the adsorption. In case of the adsorption of electroactive substances this elimination function A can be useful for analytical applications, particularly for the adsorptive stripping voltammetry with electrochemical irreversible systems. As a practical application, the adsorptive stripping voltammetry of thermally denatured DNA on a hanging mercury electrode has been developed. While the LSV signal of single-stranded DNA at low concentrations gives a slight hint of the cathodic peak (due to the reduction of adenine and cytosine residues), the elimination A provides quite a clear signal in the form of the peak-counterpeak. It is possible to determinate DNA at concentrations below micrograms per milliliter by using this elimination function. The results received by the adsorptive stripping voltammetry in the mode of linear scan (LSV), square-wave (SWV) and elimination with linear scan (EVLS) are discussed.

Reduction and oxidation of peptide nucleic acid and DNA at mercury and carbon electrodes

Abstract Peptide nucleic acid (PNA) is a DNA mimic that binds strongly and specifically to complementary DNA or RNA oligomers, but in contrast to DNA its backbone does not carry any electric charge. We used voltammetry in cyclic and square-wave modes to study reduction and oxidation signals of single stranded (ss)PNA and DNA decamers and pentadecamers with the same base sequences at mercury and carbon electrodes. The signals produced by the ssDNA and ssPNA oligomers at the hanging mercury drop electrode (HMDE), i.e. the cathodic peak CA (due to reduction of cytosine and adenine) and the anodic peak G (due to oxidation of the guanine reduction product) corresponded roughly to those observed earlier with ssDNAs. ssPNA peak potentials were more negative compared to DNA. Differences in the signals of ssPNA and ssDNA were explained primarily by different adsorption properties of these compounds. At an accumulation time of 5 min the detection limit of ssPNA was below 5 ng ml −1 . Constant current derivative chronopotentiometric stripping analysis (CPSA) at a pyrolytic graphite electrode produced two well-separated oxidation peaks of guanine and adenine residues in ssDNA and ssPNA in contrast to the poorly developed signals obtained by linear sweep (LS) and square wave (SW) voltammetries. The voltammetric signals were improved greatly as a result of application of a suitable baseline correction method. Using the polynomic method for LSV and moving average baseline correction for SWV, the ssDNA detection limits were comparable to those of CPSA at carbon electrodes as well to those obtained with peak G measurements at the HMDE.

Elimination voltammetry. Experimental verification and extension of theoretical results

Abstract The theoretical results of elimination voltammetry with linear scan have been verified experimentally for the case of reversible and irreversible processes and for the potential range expansion. All experiments were performed with a hanging mercury drop electrode. An experimental extension of elimination for cyclic voltammetry reverse scans of reversible and irreversible electrode processes and for forward and reverse scans of quasireversible electrode processes has been made. The equations for elimination of reversible, charging and kinetic currents, I r , I c and I k respectively, were tested for six cases: (A) simultaneous elimination of I k and I c with I r conservation; (B) simultaneous elimination of I c and I r with I k conservation; (C) simultaneous elimination of I r and I k with I c conservation; (D) elimination of I k with I r conservation; (E) simultaneous elimination of I c and a deeper suppression of the irreversible current foot with I r conservation; (F) elimination of I c with I r conservation. Scan rate ratios using the base number 2 were used. It was proved that the case C could be used with advantage for sensitive detection of the reversibility loss of the electrode process and for the distinction and quantitative measurement of the irreversible electrode process in the presence of a reversible one. The cases A, D and E were proved to be successful for the potential range expansion.

Utilizing of Square Wave Voltammetry to Detect Flavonoids in the Presence of Human Urine

About biological affecting of flavonoids on animal organisms is known less, thus we selected flavonoids, flavanones and flavones, and their glycosides, which were examined as potential inducers of cytochrome(s) P450 when administrated by gavages into experimental male rats. The study was focused on induction of CYP1A1, the major cytochrome P450 involved in carcinogen activation. The data obtained demonstrate the necessity 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 (including colon 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 %).

Zeptomole electrochemical detection of metallothioneins.

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.

Electrochemical Microsensors for the Detection of Cadmium(II) and Lead(II) Ions in Plants

Routine determination of trace metals in complex media is still a difficult task for many analytical instruments. The aim of this work was to compare three electro-chemical instruments [a standard potentiostat (Autolab), a commercially available miniaturized potentiostat (PalmSens) and a homemade micropotentiostat] for easy-to-use and sensitive determination of cadmium(II) and lead(II) ions. The lowest detection limits (hundreds of pM) for both metals was achieved by using of the standard potentiostat, followed by the miniaturized potentiostat (tens of nM) and the homemade instrument (hundreds of nM). Nevertheless, all potentiostats were sensitive enough to evaluate contamination of the environment, because the environmental limits for both metals are higher than detection limits of the instruments. Further, we tested all used potentiostats and working electrodes on analysis of environmental samples (rainwater, flour and plant extract) with artificially added cadmium(II) and lead(II). Based on the similar results obtained for all potentiostats we choose a homemade instrument with a carbon tip working electrode for our subsequent environmental experiments, in which we analyzed maize and sunflower seedlings and rainwater obtained from various sites in the Czech Republic.

Utilizing of Adsorptive Transfer Stripping Technique Brdicka Reaction for Determination of Metallothioneins Level in Melanoma Cells, Blood Serum and Tissues.

In the paper we utilized the adsorptive transfer stripping differential pulse voltammetry Brdicka reaction for the determination of metallothioneins (MT) in melanoma cells, animal melanoma tissues (MeLiM miniature pig) and blood serum of patients with malignant melanoma. Primarily we attempted to investigate the influence of dilution of real sample on MT electrochemical response. Dilution of samples of 1 000 times was chosen the most suitable for determination of MT level in biological samples. Then we quantified the MT level in the melanoma cells, the animal melanoma tissues and the blood serum samples. The MT content in the cells varied within the range from 4.2 to 11.2 μM. At animal melanoma tissues (melanomas localized on abdomen, back limb and dorsum) the highest content of MT was determined in the tumour sampled on the back of the animal and was nearly 500 μg of MTs per gram of a tissue. We also quantified content of MT in metastases, which was found in liver, spleen and lymph nodes. Moreover the average MT level in the blood serum samples from patients with melanoma was 3.0 ± 0.8 μM. MT levels determined at melanoma samples were significantly (p < 0.05) higher compared to control ones at cells, tissues and blood serum.