Comparison of Metal Nanoparticles (Au, Ag, Eu, Cd) Used for Immunoanalysis Using LA-ICP-MS Detection.

Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1-14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.

Prebiotic Route to Thymine from Formamide-A Combined Experimental-Theoretical Study.

Synthesis of RNA nucleobases from formamide is one of the recurring topics of prebiotic chemistry research. Earlier reports suggest that thymine, the substitute for uracil in DNA, may also be synthesized from formamide in the presence of catalysts enabling conversion of formamide to formaldehyde. In the current paper, we show that to a lesser extent conversion of uracil to thymine may occur even in the absence of catalysts. This is enabled by the presence of formic acid in the reaction mixture that forms as the hydrolysis product of formamide. Under the reaction conditions of our study, the disproportionation of formic acid may produce formaldehyde that hydroxymethylates uracil in the first step of the conversion process. The experiments are supplemented by quantum chemical modeling of the reaction pathway, supporting the plausibility of the mechanism suggested by Saladino and coworkers.

Molecularly imprinted polymers and capillary electrophoresis for sensing phytoestrogens in milk.

Dairy cow feed contains, among other ingredients, soybeans, legumes, and clover, plants that are rich in phytoestrogens. Several publications have reported a positive influence of phytoestrogens on human health; however, several unfavorable effects have also been reported. In this work, a simple, selective, and eco-friendly method of phytoestrogen isolation based on the technique of noncovalent molecular imprinting was developed. Genistein was used as a template, and dopamine was chosen as a functional monomer. A layer of molecularly imprinted polymers was created in a microtitration well plate. The binding capability and selective properties of obtained molecularly imprinted polymers were investigated. The imprinted polymers exhibited higher binding affinity toward chosen phytoestrogen than did the nonimprinted polymers. A selectivity factor of 6.94 was calculated, confirming satisfactory selectivity of the polymeric layer. The applicability of the proposed sensing method was tested by isolation of genistein from a real sample of bovine milk and combined with micellar electrokinetic capillary chromatography with UV-visible detection.

A Novel Ruthenium Based Coordination Compound Against Pathogenic Bacteria.

The current epidemic of antibiotic-resistant infections urges to develop alternatives to less-effective antibiotics. To assess anti-bacterial potential, a novel coordinate compound (RU-S4) was synthesized using ruthenium-Schiff base-benzimidazole ligand, where ruthenium chloride was used as the central atom. RU-S4 was characterized by scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDS), and Raman spectroscopy. Antibacterial effect of RU-S4 was studied against Staphylococcus aureus (NCTC 8511), vancomycin-resistant Staphylococcus aureus (VRSA) (CCM 1767), methicillin-resistant Staphylococcus aureus (MRSA) (ST239: SCCmecIIIA), and hospital isolate Staphylococcus epidermidis. The antibacterial activity of RU-S4 was checked by growth curve analysis and the outcome was supported by optical microscopy imaging and fluorescence LIVE/DEAD cell imaging. In vivo (balb/c mice) infection model prepared with VRSA (CCM 1767) and treated with RU-S4. In our experimental conditions, all infected mice were cured. The interaction of coordination compound with bacterial cells were further confirmed by cryo-scanning electron microscope (Cryo-SEM). RU-S4 was completely non-toxic against mammalian cells and in mice and subsequently treated with synthesized RU-S4.

Gold nanoparticles as labels for immunochemical analysis using laser ablation inductively coupled plasma mass spectrometry.

In this paper, we describe the labelling of antibodies by gold nanoparticles (AuNPs) with diameters of 10 and 60 nm with detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Additionally, the AuNPs labelling strategy is compared with commercially available labelling reagents based on MeCAT (metal coded affinity tagging). Proof of principle experiments based on dot blot experiments were performed. The two labelling methods investigated were compared by sensitivity and limit of detection (LOD). The absolute LODs achieved were in the range of tens of picograms for AuNP labelling compared to a few hundred picograms by the MeCAT labelling.

Escape of Tick-Borne Flavivirus from 2'-C-Methylated Nucleoside Antivirals Is Mediated by a Single Conservative Mutation in NS5 That Has a Dramatic Effect on Viral Fitness.

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-C-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

Upconversion nanoparticle bioconjugates characterized by capillary electrophoresis.

Upconversion nanoparticles (UCNPs) are an emerging class of optical materials with high potential in bioimaging due to practically no background signal and high penetration depth. Their excellent optical properties and easy surface functionalization make them perfect for conjugation with targeting ligands. In this work, capillary electrophoretic (CE) method with laser-induced fluorescence detection was used to investigate the behavior of carboxyl-silica-coated UCNPs. Folic acid, targeting folate receptor overexpressed by wide variety of cancer cells, was used for illustrative purposes and assessed by CE under optimized conditions. Peptide-mediated bioconjugation of antibodies to UCNPs was also investigated. Despite the numerous advantages of CE, this is the first time that CE was employed for characterization of UCNPs and their bioconjugates. The separation conditions were optimized including the background electrolyte concentration and pH. The optimized electrolyte was 20 mM borate buffer with pH 8.

Capillary electrophoresis-driven synthesis of water-soluble CdTe quantum dots in nanoliter scale.

'Green nanotechnology' is a term used for the design of nanomaterials and processes that reduce or eliminate the use and/or generation of hazardous substances. In this paper, a capillary electrophoresis (CE)-driven synthesis of CdTe quantum dots (QDs) and their subsequent conjugation with a metal-binding protein metallothionein (isofom MT1) is reported. Even though the toxic materials (cadmium and potassium borohydride) were used for synthesis, the proposed method can be labeled as 'environmentally friendly' because the whole process (synthesis of QDs and MT1 conjugation) was carried out under mild conditions: ultra-low volume (nanoliter scale), relatively low temperature (50 °C), atmospheric pressure, and completed in a short time (under 90 s). Prepared QDs were also characterized by classical fluorescence spectroscopy and transmission electron microscopy. This study opens up new possibilities for the utilization of classical CE in the synthesis of nanoparticles and on-line labeling of biomolecules in the nanoliter scale in short period of time.

CE and nanomaterials - Part II: Nanomaterials in CE.

The scope of this two-part review is to summarize publications dealing with CE and nanomaterials together. This topic can be viewed from two broad perspectives, and this article is trying to highlight these two approaches: (i) CE of nanomaterials, and (ii) nanomaterials in CE. The second part aims at summarization of publications dealing with application of nanomaterials for enhancement of CE performance either in terms of increasing the separation resolution or for improvement of the detection. To increase the resolution, nanomaterials are employed as either surface modification of the capillary wall forming open tubular column or as additives to the separation electrolyte resulting in a pseudostationary phase. Moreover, nanomaterials have proven to be very beneficial for increasing also the sensitivity of detection employed in CE or even they enable the detection (e.g., fluorescent tags of nonfluorescent molecules).

Capillary electrophoresis and nanomaterials - Part I: Capillary electrophoresis of nanomaterials.

Nanomaterials are in analytical science used for a broad range of purposes, covering the area of sample pretreatment as well as separation, detection, and identification of target molecules. This part of the review covers capillary electrophoresis (CE) of nanomaterials and focuses on the application of CE as a method for characterization used during nanomaterial synthesis and modification as well as the monitoring of their properties and interactions with other molecules. The heterogeneity of the nanomaterial family is extremely large. Depending on different definitions of the term Nanomaterial/Nanoparticle, the group may cover metal and polymeric nanoparticles, carbon nanomaterials, liposomes and even dendrimers. Moreover, these nanomaterials are usually subjected to some kind of surface modification or functionalization, which broadens the diversity even more. Not only for purposes of verification of nanomaterial synthesis and batch-to-batch quality check, but also for determination the polydispersity and for functionality characterization on the nanoparticle surface, has CE offered very beneficial capabilities. Finally, the monitoring of interactions between nanomaterials and other (bio)molecules is easily performed by some kind of capillary electromigration technique.

DNA interaction with platinum-based cytostatics revealed by DNA sequencing.

The main mechanism of action of platinum-based cytostatic drugs - cisplatin, oxaliplatin and carboplatin - is the formation of DNA cross-links, which restricts the transcription due to the disability of DNA to enter the active site of the polymerase. The polymerase chain reaction (PCR) was employed as a simplified model of the amplification process in the cell nucleus. PCR with fluorescently labelled dideoxynucleotides commonly employed for DNA sequencing was used to monitor the effect of platinum-based cytostatics on DNA in terms of decrease in labeling efficiency dependent on a presence of the DNA-drug cross-link. It was found that significantly different amounts of the drugs - cisplatin (0.21 µg/mL), oxaliplatin (5.23 µg/mL), and carboplatin (71.11 µg/mL) - were required to cause the same quenching effect (50%) on the fluorescent labelling of 50 µg/mL of DNA. Moreover, it was found that even though the amounts of the drugs was applied to the reaction mixture differing by several orders of magnitude, the amount of incorporated platinum, quantified by inductively coupled plasma mass spectrometry, was in all cases at the level of tenths of µg per 5 µg of DNA.

Nanomaterials for sample pretreatment prior to capillary electrophoretic analysis.

Nanomaterials are, in analytical science, used for a broad range of purposes, covering the area of sample pretreatment as well as separation, detection and identification of target molecules. This review covers the application of nanomaterials for sample pretreatment in capillary electrophoresis. It targets the utilization of nanomaterials for sample purification, preconcentration and/or extraction coupled both off-line and on-line with capillary electrophoretic analysis. Especially due to their large surface area, nanoparticles and nanomaterials are exceptionally helpful in making up for the limited concentration detection limits provided by capillary electrophoresis. This method possesses excellent separation power; however, its sensitivity may be problematic in some cases. Therefore, this review is focused on utilization of nanomaterials as a powerful tool for sample preconcentration, which is so often required prior to capillary electrophoretic analysis.

Amalgam Electrode-Based Electrochemical Detector for On-Site Direct Determination of Cadmium(II) and Lead(II) from Soils.

Toxic metal contamination of the environment is a global issue. In this paper, we present a low-cost and rapid production of amalgam electrodes used for determination of Cd(II) and Pb(II) in environmental samples (soils and wastewaters) by on-site analysis using difference pulse voltammetry. Changes in the electrochemical signals were recorded with a miniaturized potentiostat (width: 80 mm, depth: 54 mm, height: 23 mm) and a portable computer. The limit of detection (LOD) was calculated for the geometric surface of the working electrode 15 mm² that can be varied as required for analysis. The LODs were 80 ng·mL-1 for Cd(II) and 50 ng·mL-1 for Pb(II), relative standard deviation, RSD ≤ 8% (n = 3). The area of interest (Dolni Rozinka, Czech Republic) was selected because there is a deposit of uranium ore and extreme anthropogenic activity. Environmental samples were taken directly on-site and immediately analysed. Duration of a single analysis was approximately two minutes. The average concentrations of Cd(II) and Pb(II) in this area were below the global average. The obtained values were verified (correlated) by standard electrochemical methods based on hanging drop electrodes and were in good agreement. The advantages of this method are its cost and time effectivity (approximately two minutes per one sample) with direct analysis of turbid samples (soil leach) in a 2 M HNO3 environment. This type of sample cannot be analyzed using the classical analytical methods without pretreatment.

Quantification of nanomaterial bioconjugation based on electrophoretic mobility shift.

Electrophoretic mobility shift assay (EMSA) is a well-established technique to monitor interactions between biomolecules particularly DNA and proteins. Even though numerous variations of this method have been presented, challenges in the form of detection sensitivity and/or variations in the stability of the formed complex still remain. With advances in the area of nanomaterials improvements in EMSA have been also suggested. Recently, Zhang and Wang (Electrophoresis 2015, 36, 1011-1015) presented electrophoretic mobility shift method for determination of number of DNA molecules conjugated to quantum dots (QDs), which was further utilized for calculation of enzymatic activity, sequence specific DNA detection, and neutral molecule quantification.

Doxorubicin interactions with bovine serum albumin revealed by microdialysis with on-line laser-induced fluorescence detection at subpicogram level.

Doxorubicin (DOX) is an effective antitumor drug employed for treatment of a wide range of cancers types such as neuroblastoma, osteosarcoma, breast and esophageal carcinomas. On the other hand, the cumulative dose is restricted (300-550 mg/m(2)) and its amount administered to a patient has to be closely controlled due to its cardiotoxicity. To understand the mechanisms of the DOX side effects as well as to reveal the ways how to reduce its adverse impact on cardiomyocytes, the interactions with particular components of the blood and tissues have to be studied in greater detail. In this work, microdialysis technique was optimized to extract DOX from samples and subsequently monitor its interaction with BSA. Finally, the microdialysis probe was connected on-line to the LIF detector to ensure the real-time detection. The best flow rate was 1 µL/min and after 120 min of microdialysis 28% of the DOX was dialyzed out from the sample. The results from investigation of the DOX-BSA interaction indicate that the interaction occurs in less than 30 min, causing marked decrease in the amount of DOX extracted by microdialysis.

Capillary electrophoresis of quantum dots: minireview.

It has been already three decades, since the fluorescent nanocrystals called quantum dots (QDs) appeared and attracted attention of a broad scientific community. Their excellent not only optical but also electronic properties predetermined QDs for utilization in a variety of areas. Besides lasers, solar cells, and/or computers, QDs have established themselves in the field of (bio)chemical labeling as well as medical imaging. However, due to the numerous application possibilities of QDs, there are high demands on their properties that need to be precisely controlled and characterized. CE with its versatile modes and possibilities of detection was found to be an effective tool not only for characterization of QDs size and/or surface properties but also for monitoring of their interactions with other molecules of interest. In this minireview, we are giving short insight in analysis of QDs by CE, and summarizing the advantages of this method for QDs characterization.

Interactions between CdTe quantum dots and DNA revealed by capillary electrophoresis with laser-induced fluorescence detection.

Quantum dots (QDs) are one of the most promising nanomaterials, due to their size-dependent characteristics as well as easily controllable size during the synthesis process. They are promising label material and their interaction with biomolecules is of great interest for science. In this study, CdTe QDs were synthesized under optimal conditions for 2 nm size. Characterization and verification of QDs synthesis procedure were done by fluorimetric method and with CE. Afterwards, QDs interaction with chicken genomic DNA and 500 bpDNA fragment was observed employing CE-LIF and gel electrophoresis. Performed interaction relies on possible matching between size of QDs and major groove of the DNA, which is approximately 2.1 nm.

Joint forces of mass spectrometric techniques (ICP-MS and MALDI-TOF-MS) and fluorescence spectrometry in the study of platinum-based cytostatic drugs interactions with metallothionein MT2 and MT3.

Herby, the interaction of metallothioneins with commonly used Pt-based anticancer drugs - cisplatin, carboplatin, and oxaliplatin - was investigated using the combined power of elemental (i.e. LA-ICP-MS, CE-ICP-MS) and molecular (i.e. MALDI-TOF-MS) analytical techniques providing not only required information about the interaction, but also the benefit of low sample consumption. The amount of Cd and Pt incorporated within the protein was determined for protein monomers and dimer/oligomers formed by non-oxidative dimerization. Moreover, fluorescence spectrometry using Zn2+-selective fluorescent indicator - FluoZin3 - was employed to monitor the ability of Pt drugs to release natively occurring Zn from the protein molecule. The investigation was carried out using two protein isoforms (i.e. MT2, MT3), and significant differences in behaviour of these two isoforms were observed. The main attention was paid to elucidating whether the protein dimerization/oligomerization may be the reason for the potential failure of the anticancer therapy based on these drugs. Based on the results, it was demonstrated that the interaction of MT2 (both monomers and dimers) interacted with Pt drugs significantly less compared to MT3 (both monomers and dimers). Also, a significant difference between monomeric and dimeric forms (both MT2 and MT3) was not observed. This may suggest that dimer formation is not the key factor leading to the inactivation of Pt drugs.

Electrophoretic study of peptide-mediated quantum dot-human immunoglobulin bioconjugation.

The bioconjugation of quantum dots (QDs) is a key process in their application for bioanalysis as well as imaging. The coupling of QDs with biologically active molecules such as peptides, nucleic acids, and/or antibodies enables their fluorescent labeling, and therefore, selective and sensitive tracking during the bioanalytical process, however, the efficiency of the labeling and preservation of the biological activity of the bioconjugate have to be considered. In this study, a new approach of the bioconjugation of CdTe-QDs and human immunoglobulin employing a small peptide is described. The heptapeptide (HWRGWVC) was synthesized and characterized by mass spectrometry, liquid chromatography, and capillary electrophoresis. Moreover, the peptide was used as a capping agent for QDs synthesis. The CdTe-QDs were synthesized by microwave synthesis (600 W, 20 min) using 3.2 mM CdCl2 and 0.8 mM Na2TeO3. The bioconjugation of QDs capped by this peptide with immunoglobulin was investigated by capillary electrophoresis and magnetic immunoextraction coupled with electrochemical detection by differential pulse voltammetry. Furthermore, the applicability of prepared bioconjugates for fluorescent immunodetection was verified using immobilized goat antihuman IgG antibody.

Integrated chip electrophoresis and magnetic particle isolation used for detection of hepatitis B virus oligonucleotides.

Rapid and sensitive detection is a key step in the effective and early response to the global hazard of various viral diseases. In this study, an integrated isolation of hepatitis B virus (HBV)-specific DNA fragment by magnetic nanoparticles (MNPs) and its immediate analysis by microchip CGE was performed. Microfluidic CE chip was used to accommodate the complete process of viral DNA isolation by MNPs including hybridization and thermal denaturation followed by CE separation. Beforehand, calibration curves of HBV fragments were constructed. For isolation by MNPs, specific streptavidin-biotin interaction was used to bind complementary HBV fragment to magnetic particles. After analysis of isolated HBV by regular MNPs method, innovative approach was performed. The commercial CE chip (Bio-rad) was successfully used to execute HBV fragment isolation. Detection using LIF with detection limit of 1 ng/mL was accomplished.