Influence of AC electric field on the charge generation in albumin solution in a flow-based AFM-fishing system

The work was supported by the Russian Ministry of Education and Science, Agreement No. 14.613.21.0063, universal identifier RFMEFI61317X0063

Crystal structure of the ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus

The crystal structure of the ferritin from the archaeon, hyperthermophile and anaerobe Pyrococcus furiosus (PfFtn) is presented. While many ferritin structures from bacteria to mammals have been reported, until now only one was available from archaea, the ferritin from Archaeoglobus fulgidus (AfFtn). The PfFtn 24-mer exhibits the 432 point-group symmetry that is characteristic of most ferritins, which suggests that the 23 symmetry found in the previously reported AfFtn is not a common feature of archaeal ferritins. Consequently, the four large pores that were found in AfFtn are not present in PfFtn. The structure has been solved by molecular replacement and refined at 2.75-Å resolution to R = 0.195 and Rfree = 0.247. The ferroxidase center of the aerobically crystallized ferritin contains one iron at site A and shows sites B and C only upon iron or zinc soaking. Electron paramagnetic resonance studies suggest this iron depletion of the native ferroxidase center to be a result of a complexation of iron by the crystallization salt. The extreme thermostability of PfFtn is compared with that of eight structurally similar ferritins and is proposed to originate mostly from the observed high number of intrasubunit hydrogen bonds. A preservation of the monomer fold, rather than the 24-mer assembly, appears to be the most important factor that protects the ferritin from inactivation by heat

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ? 98 %). All samples were analyzed following injection (100 ?l) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 ?m, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients

Concentration Sensitivity of Nucleic Acid and Protein Molecule Detection Using Nanowire Biosensors

The concentration detection limit (DL) of biomacromolecules attainable using a nanowire detector has become a topical issue. A DL of 10−15 M is required to reveal oncological and infectious diseases at an early stage. This study discusses the DL experimentally attainable in the subfemtomolar concentration range, and possible mechanisms explaining such a low-concentration DL through the cooperative effect of biomacromolecular complexes formed on the surface of the nanowire (NW) chip near the nanowire

Interfacial Localization and Voltage-Tunable Arrays of Charged Nanoparticles

Experiments and computer simulations provide a new perspective that strong correlations of counterions with charged nanoparticles can influence the localization of nanoparticles at liquidliquid interfaces and support the formation of voltage-tunable nanoparticle arrays. We show that ion condensation onto charged nanoparticles facilitates their transport from the aqueous-side of an interface between two immiscible electrolyte solutions to the organic-side, but contiguous to the interface. Counterion condensation onto the highly charged nanoparticles overcomes the electrostatic barrier presented by the low permittivity organic material, thus providing a mechanism to transport charged nanoparticles into organic phases with implications for the distribution of nanoparticles throughout the environment and within living organisms. After transport, the nanoparticles assemble into a two-dimensional (2D) nearly close-packed array on the organic side of the interface. Voltage-tunable counterion-mediated interactions between the nanoparticles are used to control the lattice spacing of the 2D array. Tunable nanoparticle arrays self-assembled at liquid interfaces are applicable to the development of electro-variable optical devices and active elements that control the physical and chemical properties of liquid interfaces on the nanoscale

Effect of Spherical Elements of Biosensors and Bioreactors on the Physicochemical Properties of a Peroxidase Protein

External electromagnetic fields are known to be able to concentrate inside the construction elements of biosensors and bioreactors owing to reflection from their surface. This can lead to changes in the structure of biopolymers (such as proteins), incubated inside these elements, thus influencing their functional properties. Our present study concerned the revelation of the effect of spherical elements, commonly employed in biosensors and bioreactors, on the physicochemical properties of proteins with the example of the horseradish peroxidase (HRP) enzyme. In our experiments, a solution of HRP was incubated within a 30 cm-diameter titanium half-sphere, which was used as a model construction element. Atomic force microscopy (AFM) was employed for the single-molecule visualization of the HRP macromolecules, adsorbed from the test solution onto mica substrates in order to find out whether the incubation of the test HRP solution within the half-sphere influenced the HRP aggregation state. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was employed in order to reveal whether the incubation of HRP solution within the half-sphere led to any changes in its secondary structure. In parallel, spectrophotometry-based estimation of the HRP enzymatic activity was performed in order to find out if the HRP active site was affected by the electromagnetic field under the conditions of our experiments. We revealed an increased aggregation of HRP after the incubation of its solution within the half-sphere in comparison with the control sample incubated far outside the half-sphere. ATR-FTIR allowed us to reveal alterations in HRP’s secondary structure. Such changes in the protein structure did not affect its active site, as was confirmed by spectrophotometry. The effect of spherical elements on a protein solution should be taken into account in the development of the optimized design of biosensors and bioreactors, intended for performing processes involving proteins in biomedicine and biotechnology, including highly sensitive biosensors intended for the diagnosis of socially significant diseases in humans (including oncology, cardiovascular diseases, etc.) at early stages

AFM and FTIR Investigation of the Effect of Water Flow on Horseradish Peroxidase

Atomic force microscopy (AFM)-based fishing is a promising method for the detection of low-abundant proteins. This method is based on the capturing of the target proteins from the analyzed solution onto a solid substrate, with subsequent counting of the captured protein molecules on the substrate surface by AFM. Protein adsorption onto the substrate surface represents one of the key factors determining the capturing efficiency. Accordingly, studying the factors influencing the protein adsorbability onto the substrate surface represents an actual direction in biomedical research. Herein, the influence of water motion in a flow-based system on the protein adsorbability and on its enzymatic activity has been studied with an example of horseradish peroxidase (HRP) enzyme by AFM, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and conventional spectrophotometry. In the experiments, HRP solution was incubated in a setup modeling the flow section of a biosensor communication. The measuring cell with the protein solution was placed near a coiled silicone pipe, through which water was pumped. The adsorbability of the protein onto the surface of the mica substrate has been studied by AFM. It has been demonstrated that incubation of the HRP solution near the coiled silicone pipe with flowing water leads to an increase in its adsorbability onto mica. This is accompanied by a change in the enzyme’s secondary structure, as has been revealed by ATR-FTIR. At the same time, its enzymatic activity remains unchanged. The results reported herein can be useful in the development of models describing the influence of liquid flow on the properties of enzymes and other proteins. The latter is particularly important for the development of biosensors for biomedical applications—particularly for serological analysis, which is intended for the early diagnosis of various types of cancer and infectious diseases. Our results should also be taken into account in studies of the effects of protein aggregation on hemodynamics, which plays a key role in human body functioning

AFM and FTIR Investigation of the Effect of Water Flow on Horseradish Peroxidase

Atomic force microscopy (AFM)-based fishing is a promising method for the detection of low-abundant proteins. This method is based on the capturing of the target proteins from the analyzed solution onto a solid substrate, with subsequent counting of the captured protein molecules on the substrate surface by AFM. Protein adsorption onto the substrate surface represents one of the key factors determining the capturing efficiency. Accordingly, studying the factors influencing the protein adsorbability onto the substrate surface represents an actual direction in biomedical research. Herein, the influence of water motion in a flow-based system on the protein adsorbability and on its enzymatic activity has been studied with an example of horseradish peroxidase (HRP) enzyme by AFM, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and conventional spectrophotometry. In the experiments, HRP solution was incubated in a setup modeling the flow section of a biosensor communication. The measuring cell with the protein solution was placed near a coiled silicone pipe, through which water was pumped. The adsorbability of the protein onto the surface of the mica substrate has been studied by AFM. It has been demonstrated that incubation of the HRP solution near the coiled silicone pipe with flowing water leads to an increase in its adsorbability onto mica. This is accompanied by a change in the enzyme’s secondary structure, as has been revealed by ATR-FTIR. At the same time, its enzymatic activity remains unchanged. The results reported herein can be useful in the development of models describing the influence of liquid flow on the properties of enzymes and other proteins. The latter is particularly important for the development of biosensors for biomedical applications—particularly for serological analysis, which is intended for the early diagnosis of various types of cancer and infectious diseases. Our results should also be taken into account in studies of the effects of protein aggregation on hemodynamics, which plays a key role in human body functioning