Cell Imaging with Fluorescent Bi-Metallic Nanoparticles

Last decades various imaging techniques have been applied in biological and biomedical research, such as magnetic resonance imaging, different types of tomography, fluorescence/bioluminescence, ultrasound, as well as multimodality approaches. Fluorescence imaging, especially in combination with nanoscale materials, is a very prospective tool for experiments in vivo and clinical applications due to its high temporal and spatial resolutions. Fluorescent nanoparticles (NPs), having ability to interact with biomolecules both on the surface of and inside the cells, may revolutionize the cell imaging approaches for diagnostics and therapy. In our investigation we report about new method of cell imaging with fluorescent bi-metallic NPs synthesized by chemical reduction of the relevant ions. As the model of living organism, the cells of yeast Hansenula polymorpha were used. All NPs in minimal concentration (up to 0.05 mM) was proved to be non-toxic for yeast cells. The NPs and NPs-modified cells were characterized with the methods of UV-VIS spectroscopy, scanning electron microscopy, atom force microscopy, transmission electron microscopy and fluorescence microscopy. The bimetallic NPs, possessing the stable fluorescence in solution and inside the cells, allow to observe the phenomenon of NPs transferring from parental to daughter cells through at least three generations followed by releasing from the modified cells. The fluorescent NPs synthesized being small, non-toxic and fluorescent was shown to be perspective tool for cell imaging

Special Issue “World of Biosensing”

The broad definition of the term biosensing relates to practically all processes of molecular recognition [...

ARGINASE-BASED AMPEROMETRIC BIOSENSOR FOR MANGANESE IONS ANALYSIS

The development of simple cost-effective sensitive enzymatic methods for analysis of toxic metallic ions is an actual problem. Promising tools for elaboration of such methods are Mn2+-dependent enzymes. A novel manganese(II)-sensitive amperometric bi-enzyme biosensor based on of recombinant human arginase I (arginase) isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer with urease and apo-enzyme of arginase was placed onto a polyaniline-Nafion composite platinum electrode. The developed sensor revealed a high sensitivity to Mn2+-ions – 9200±20 A/(M∙m2)with the apparent Michaelis-Menten constant derived from Mn2+-ions calibration curve of 11.5±1.0 µM. A linear concentration range was observed from 1 µM to 6,5 µM MnCl2, a limit of detection being of 0.15 µM and a response time – 2.5 min. The proposed biosensor may be useful to monitor manganese compounds in laboratories of medicine, food industry and environmental control service

Towards a Self-Powered Amperometric Glucose Biosensor Based on a Single-Enzyme Biofuel Cell

This paper describes the study of an amperometric glucose biosensor based on an enzymatic biofuel cell consisting of a bioanode and a biocathode modified with the same enzyme—glucose oxidase (GOx). A graphite rod electrode (GRE) was electrochemically modified with a layer of Prussian blue (PB) nanoparticles embedded in a poly(pyrrole-2-carboxylic acid) (PPCA) shell, and an additional layer of PPCA and was used as the cathode. A GRE modified with a nanocomposite composed of poly(1,10-phenanthroline-5,6-dione) (PPD) and gold nanoparticles (AuNPs) entrapped in a PPCA shell was used as an anode. Both electrodes were modified with GOx by covalently bonding the enzyme to the carboxyl groups of PPCA. The developed biosensor exhibited a wide linear range of 0.15–124.00 mM with an R2 of 0.9998 and a sensitivity of 0.16 μA/mM. The limit of detection (LOD) and quantification (LOQ) were found to be 0.07 and 0.23 mM, respectively. The biosensor demonstrated exceptional selectivity to glucose and operational stability throughout 35 days, as well as good reproducibility, repeatability, and anti-interference ability towards common interfering substances. The studies on human serum demonstrate the ability of the newly designed biosensor to determine glucose in complex real samples at clinically relevant concentrations

ARGINASE-BASED AMPEROMETRIC BIOSENSOR FOR MANGANESE IONS ANALYSIS

The development of simple cost-effective sensitive enzymatic methods for analysis of toxic metallic ions is an actual problem. Promising tools for elaboration of such methods are Mn2+-dependent enzymes. A novel manganese(II)-sensitive amperometric bi-enzyme biosensor based on of recombinant human arginase I (arginase) isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer with urease and apo-enzyme of arginase was placed onto a polyaniline-Nafion composite platinum electrode. The developed sensor revealed a high sensitivity to Mn2+-ions – 9200±20 A/(M∙m2)with the apparent Michaelis-Menten constant derived from Mn2+-ions calibration curve of 11.5±1.0 µM. A linear concentration range was observed from 1 µM to 6,5 µM MnCl2, a limit of detection being of 0.15 µM and a response time – 2.5 min. The proposed biosensor may be useful to monitor manganese compounds in laboratories of medicine, food industry and environmental control service

Nanomaterials as Redox Mediators in Laccase-Based Amperometric Biosensors for Catechol Assay

Laccase is a copper-containing enzyme that does not require hydrogen peroxide as a co-substrate or additional cofactors for an enzymatic reaction. Nanomaterials of various chemical structures are usually applied to the construction of enzyme-based biosensors. Metals, metal oxides, semiconductors, and composite NPs perform various functions in electrochemical transformation schemes as a platform for the enzyme immobilization, a mediator of an electron transfer, and a signal amplifier. We describe here the development of amperometric biosensors (ABSs) based on laccase and redox-active micro/nanoparticles (hereafter—NPs), which were immobilized on a graphite electrode (GE). For this purpose, we isolated a highly purified enzyme from the fungus Trametes zonatus, and then synthesized bi- and trimetallic NPs of noble and transition metals, as well as hexacyanoferrates (HCF) of noble metals; these were layered onto the surfaces of GEs. The electroactivity of many of the NPs immobilized on the GEs was characterized by cyclic voltammetry (CV) experiments. The most effective mediators of electron transfer were selected as the platform for the development of laccase-based ABSs. As a result, a number of catechol-sensitive ABSs were constructed and characterized. The laccase/CuCo/GE was demonstrated to possess the highest sensitivity to catechol (4523 A·M−1·m−2) among the tested ABSs. The proposed ABSs may be promising for the analysis of phenolic derivatives in real samples of drinking water, wastewater, and food products

Amperometric Biosensors for L-Arginine Determination Based on L-Arginine Oxidase and Peroxidase-Like Nanozymes

There are limited data on amperometric biosensors (ABSs) for L-arginine (Arg) determination based on oxidases that produce hydrogen peroxide (H2O2) as a byproduct of enzymatic reaction, and artificial peroxidases (POs) for decomposition of H2O2. The most frequently proposed Arg-sensitive oxidase-based ABSs contain at least two enzymes in the bioselective layer; this complicates the procedure and increases the cost of analysis. Therefore, the construction of a one-enzyme ABS for Arg analysis is a practical problem. In the current work, fabrication, and characterization of three ABS types for the direct measurement of Arg were proposed. L-arginine oxidase (ArgO) isolated from the mushroom Amanita phalloides was co-immobilized with PO-like nanozymes (NZs) on the surface of graphite electrodes. As PO mimetics, chemically synthesized NZs of CeCu (nCeCU) and NiPtPd (nNiPtPd), as well as green-synthesized hexacyanoferrate of copper (gCuHCF), were used. The novel ABSs exhibited high sensitivity and selectivity to Arg, broad linear ranges and good storage stabilities. Two ABSs were tested on real samples of products containing Arg, including the pharmaceutical preparation “Tivortine”, juices, and wine. A high correlation (R = 0.995) was demonstrated between the results of testing “Tivortine” and juice using nCeCU/GE and nNiPtPd/GE. It is worth mentioning that only a slight difference (less than 1%) was observed for “Tivortin” between the experimentally determined content of Arg and its value declared by the producer. The proposed ArgO-NZ-based ABSs may be promising for Arg analysis in different branches of science, medicine, and industry

Peroxidase-Like Metal-Based Nanozymes: Synthesis, Catalytic Properties, and Analytical Application

Nanozymes (NZs) are nanostructured artificial enzymes that mimic catalytic properties of natural enzymes. The NZs have essential advantages over natural enzymes, namely low preparation costs, stability, high surface area, self-assembling capability, size and composition-dependent activities, broad possibility for modification, and biocompatibility. NZs have wide potential practical applications as catalysts in biosensorics, fuel-cell technology, environmental biotechnology, and medicine. Most known NZs are mimetics of oxidoreductases or hydrolases. The present work aimed to obtain effective artificial peroxidase (PO)-like NZs (nanoPOs), to characterize them, and to estimate the prospects of their analytical application. NanoPOs were synthesized using a number of nanoparticles (NPs) of transition and noble metals and were screened for their catalytic activity in solution and on electrodes. The most effective nanoPOs were chosen as NZs and characterized by their catalytic activity. Kinetic parameters, size, and structure of the best nanoPOs (Cu/CeS) were determined. Cu/CeS-based sensor for H2O2 determination showed high sensitivity (1890 A·M−1·m−2) and broad linear range (1.5–20,000 µM). The possibility to apply Cu/CeS-NZ as a selective layer in an amperometric sensor for hydrogen-peroxide analysis of commercial disinfectant samples was demonstrated