In Situ SERS Sensing by a Laser-Induced Aggregation of Silver Nanoparticles Templated on a Thermoresponsive Polymer.

A stimuli-responsive (pH- and thermoresponsive) micelle-forming diblock copolymer, poly(1,2-butadiene)290-block-poly(N,N-dimethylaminoethyl methacrylate)240 (PB-b-PDMAEMA), was used as a polymer template for the in situ synthesis of silver nanoparticles (AgNPs) through Ag+ complexation with PDMAEMA blocks, followed by the reduction of the bound Ag+ with sodium borohydride. A successful synthesis of the AgNPs on a PB-b-PDMAEMA micellar template was confirmed by means of UV-Vis spectroscopy and transmission electron microscopy, wherein the shape and size of the AgNPs were determined. A phase transition of the polymer matrix in the AgNPs/PB-b-PDMAEMA metallopolymer hybrids, which results from a collapse and aggregation of PDMAEMA blocks, was manifested by changes in the transmittance of their aqueous solutions as a function of temperature. A SERS reporting probe, 4-mercaptophenylboronic acid (4-MPBA), was used to demonstrate a laser-induced enhancement of the SERS signal observed under constant laser irradiation. The local heating of the AgNPs/PB-b-PDMAEMA sample in the laser spot is thought to be responsible for the triggered SERS effect, which is caused by the approaching of AgNPs and the generation of "hot spots" under a thermo-induced collapse and the aggregation of the PDMAEMA blocks of the polymer matrix. The triggered SERS effect depends on the time of a laser exposure and on the concentration of 4-MPBA. Possible mechanisms of the laser-induced heating for the AgNPs/PB-b-PDMAEMA metallopolymer hybrids are discussed.

Human Angiotensin I-Converting Enzyme Produced by Different Cells: Classification of the SERS Spectra with Linear Discriminant Analysis.

Angiotensin I-converting enzyme (ACE) is a peptidase widely presented in human tissues and biological fluids. ACE is a glycoprotein containing 17 potential N-glycosylation sites which can be glycosylated in different ways due to post-translational modification of the protein in different cells. For the first time, surface-enhanced Raman scattering (SERS) spectra of human ACE from lungs, mainly produced by endothelial cells, ACE from heart, produced by endothelial heart cells and miofibroblasts, and ACE from seminal fluid, produced by epithelial cells, have been compared with full assignment. The ability to separate ACEs' SERS spectra was demonstrated using the linear discriminant analysis (LDA) method with high accuracy. The intervals in the spectra with maximum contributions of the spectral features were determined and their contribution to the spectrum of each separate ACE was evaluated. Near 25 spectral features forming three intervals were enough for successful separation of the spectra of different ACEs. However, more spectral information could be obtained from analysis of 50 spectral features. Band assignment showed that several features did not correlate with band assignments to amino acids or peptides, which indicated the carbohydrate contribution to the final spectra. Analysis of SERS spectra could be beneficial for the detection of tissue-specific ACEs.

Medical Evacuation and Liquidation of the Medical-Sanitary Aftereffects in Crisis.

The following article was cleared for publication following peer review and upon the Editor-in-Chief's decision. The manuscript is an addition to the global health literature. The manuscript reads uneven in the current English version, but the topic and concepts presented are of global interest and add to the disaster planning, response, and recovery knowledge base.Crisis situations (CS) are, unfortunately, happening in present days in each world side. And in this case, medical evacuation (ME) becomes necessary to save human lives. The presented study is regarding the description and analysis of the phases, peculiarities, and perspectives of ME organization and development. The study characterized the inter-sectoral method of the planning, and realization of crisis outcomes liquidation is provided. Four main ways of the realization of approaches that could guarantee the development of the ME system were found. Also has been identified the number of main problems which the ME system faces. Among them are lack of personnel/equipment in the medical crews; high time of transportation; the noise pollution and vibration in time of evacuation by air; the infection and exposure risk of the evacuation aircrews in the places of mass destruction weapons usage; the organizational and legislative problems of the foreign citizens' evacuation; and ME of the persons from the oil and gas production places.

Simultaneous express immunoassay of multiple cardiac biomarkers with an automatic platform in human plasma.

C-reactive protein, cystatin C, myoglobin, and D-dimer represent the inflammatory or thromboembolic status of the patient and play important roles in early diagnostics of acute myocardial infarction. Each protein can indicate some health problems, but their simultaneous detection can be crucial for differential diagnostics. The express analysis of these proteins in a small drop of plasma was developed using magnetic beads. The suggested method is based on immunomagnetic extraction of the target analyte from plasma samples and its simultaneous labelling by fluorescent dye. Reaction time was optimized for quantification of cardiac biomarkers in the spike solutions and human plasma samples. In this paper, we developed a one-protein detection technique for each cardiac biomarker and united it to a four-protein facility using an automatic platform. The proposed technique requires only 17 µL of the human plasma and takes 14 min for four-protein measuring. The suggested technique covers concentration difference by more than two orders of magnitude and demonstrates analytical applicability by measurements of human plasma samples of 16 volunteers.

Surface-enhanced Raman spectroscopy in tandem with a gradient electric field from 4-mercaptophenylboronic acid on silver nanoparticles.

The surface-enhanced Raman spectroscopy (SERS) signal of a reporter on silver nanoparticles can be effectively gained by gradient electric field application. The external electric field initiates the dielectrophoresis of nanoparticles and their electrically induced dipole-dipole interaction. Owing to dielectrophoresis, the nanoparticles are concentrated in the area of high electrical field strength. The induced dipole-dipole interaction leads to additional coagulation of nanoparticles and formation of hotspots. Both dielectrophoresis and induced dipole-dipole interaction increase the number of hotspots, which leads to a SERS signal growth. These two mechanisms of SERS signal amplification are explained by the dielectrophoresis and Derjaguin-Landau-Verwey-Overbeek theories. The benefits of the surface-enhanced Raman spectroscopy in tandem with the gradient electric field are experimentally confirmed using a SERS-active reporter, 4-mercaptophenylboronic acid which has a characteristic peak at Raman shift of 1586 cm-1, conjugated to silver nanoparticles of 32, 52, 58, and 74 nm in diameter. The SERS signal gain depends on the silver nanoparticle stability, size, and electric field strength. The limit of detection for 4-mPBA in the system under study can be calculated from the concentration plot and equals to 63 nM. The enhancement factor calculated for SERS in tandem with the gradient electric field can reach 106.Graphical abstract.

Multiscale flaked silver SERS-substrate for glycated human albumin biosensing.

Original multiscale flaked silver SERS-substrate (MFSS substrate) was applied for glycated albumin (GA) biosensing. The substrate is composed from silver flakes that have three orders of magnitude size dispersion: from 50 nm to 2 µm. The multiscale silver structure refracts the incident light and various surface plasmons are excited. Some of the internal plasmons are localized and give rise of the large local electric field. It was demonstrated that Raman scattering signal strongly depends: a) on "hot spots" formation at the edges and points of contact of silver plates, and b) on the angle of incidence. As a result the silver structure operates as an effective SERS substrate. To achieve the selectivity to glycated part, the surface of SERS-substrate was modified with 4-mercaptophenylboronic acid (4-mPBA). Various saccharides (Fru, Glc, Suc, Dex) were taken as model compounds for the glycated proteins determination. The saccharides contain cis-diol groups that form five- or six-membered ethers with boronic acid. Spectrum of SERS-substrate changes after sugar/glycated albumin treatment. Main differences in the SERS-spectra of sugar/glycated albumin treated SERS-substrate and control are referred to phenylboronic acid vibrations (999, 1021, 1072 and 1589 cm-1). Principal component analysis (PCA) and Partial Least Squares Regression (PLS-R) were used to discriminate spectra and to construct calibration curve, as well as to measure GA values in real samples of human plasma. Multiscale flaked silver SERS-substrate modified with 4-mPBA allows quantitative one-step biosensing of glycated albumin in 15 µl of human plasma.

Single short-term signal that enhances cooperative activity of old rat hepatocytes acts for several days.

Primary cultures of rat hepatocytes were studied in serum-free medium. Ultradian protein synthesis rhythm was used as a marker of overall cell synchronization and cooperation amongst the population. The level of synchronization was determined by amplitudes of the rhythm. Low synchronization of old rat hepatocytes can be enhanced by addition of either gangliosides or phenylephrine to the medium. Incubation of cultures with gangliosides lasted for 2.5 h, while action of phenylephrine was only for 2 min. The amplitude of protein synthesis rhythm was increased 1.5-2 times. In cultures transferred to a fresh normal medium, this increased amplitude was observed for at least 2-3 days. Thus, both gangliosides and phenyleprine are triggers, which, as shown earlier, initiated calcium-dependent processes in the cytoplasm. The results are discussed in the light of concept of the cell self-organization by a direct cell-cell communication.

Small cooperative activity of old rat hepatocytes may depend on composition of the intercellular medium.

Ultradian oscillations of protein synthesis were used as a marker of hepatocyte synchronous cooperative activity producing a common rhythm in vitro; amplitude of the rhythm defines expression of the cell cooperation. Dense synchronous and sparse non-synchronous rat hepatocyte cultures on slides in a serum-free incubation medium 199 supplemented with 0.2 mg/ml albumin and 0.5 microg/ml insulin have been studied. The amplitude of the rhythm averaged approximately 2x in dense cultures of young (3 month old) rats than in old (2 year old) rats. But some cultures of young rats had the amplitude patterns similar to cultures of old rats, and vice versa. Addition to the medium of either 0.3 microM bovine brain gangliosides or 2 microM phenylephrine resulted in increase of the oscillation amplitude in dense cultures of old rats to the level inherent in young ones. Addition to the medium of 10% rat blood serum in non-synchronous sparse cultures from young rats resulted in detection of a protein synthetic rhythm. Although after serum from young rats, the rhythm expression was high, the rhythm after serum from old rats had been given was weak. Addition of gangliosides to old-rat serum resulted in synchronization of sparse cultures with amplitudes inherent of young-rat serum. The data tend to the conclusion that cell cooperation depends to a greater extent on the composition of the medium rather than on the age of the cell or animal.

Calcium ions as a factor of cell-cell cooperation in hepatocyte cultures.

Ultradian protein synthesis rhythm was used as a marker of cell cooperation in synchronous dense and non-synchronous sparse hepatocyte cultures. Phenylephrine (2 microM, 2 min), an alpha (1)-adrenoreceptor agonist, which exerts [Ca(2+)](cyt)elevation from intracellular stores, affected protein synthesis rhythm in sparse cultures, i.e. initiated cooperative activity of the cells. The same effect was produced by 2,5-di(tertiary-butyl)-1,4-benzohydroquinone (10 microM, 2 min), which increases [Ca(2+)](cyt)by a non-receptor pathway. Pretreatment of dense cultures with the intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'- tetraacetic acid (acetoxymethyl) ester (BAPTA-AM) at 10-20 microM for, 30-60 min resulted in loss of the rhythm of protein synthesis, i.e. loss of cooperative activity between the cells. The medium conditioned by control dense cultures initiated rhythm in sparse cultures, whereas the conditioned medium of cultures pretreated with BAPTA-AM did not. [Ca(2+)](cyt)increase is known to occur with monosialoganglioside GM1 treatment. By ELISA estimation, the GM1 content in 3 h conditioned medium was similar in control dense cultures to that in cultures pretreated with BAPTA-AM. Bearing in mind data on the Ca(2+)-dependence of vesicle formation and shedding, the conditioned medium was separated by 150000 g centrifugation to supernatant containing monomers and micelles, and a pellet containing vesicular form of gangliosides. Only the latter initiated cooperative activity of the cells of sparse cultures. These cultures were also synchronized by GM1-containing liposomes at lower concentrations than added free GM1, 0.0003 and 0.06 microM respectively. Thus, GM1 and calcium are both involved in cell-cell synchronization. Activation of gangliosides, including GM1 and elevation of [Ca(2+)](cyt,)is known to lead to changes of protein kinase activity and protein phosphorylation resulting in modulation of oscillations in protein metabolism.

Loss of hepatocyte co-operative activity after inhibition of ganglioside GM1 synthesis and shedding.

Pretreatment of hepatocyte cultures with 1 microM d-l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCL (PPPP) for 24 h decreased the ganglioside GM1 content of the cells by approximately 50% and that of the conditioned medium by 90%. No rhythm in the rate of protein synthesis was detected in dense cultures pretreated with PPPP, but was observed in control dense cultures. Conditioned medium from control dense cultures induced synchrony in sparse cultures, which were non-synchronous in their own medium. In contrast, conditioned medium from dense cultures pretreated with PPPP did not synchronize sparse cultures. Since protein synthesis rhythm is a marker of cell synchronization, i.e. their co-operative activity, then non-oscillatory behavior means loss of cell co-operation. The protein synthesis rhythm was restored 24 h after hepatocytes were transferred to PPPP-free medium. Restoration was more rapid when 0.9 microM gangliosides (standard mixture from bovine brain) were added to the medium just after the withdrawal of PPPP. These novel results concerning the loss of rhythm of protein synthesis in low GM1 ganglioside medium support the conclusion that ganglioside is implicated in the regulation of cell co-operative activity.