Dielectrophoretic detection of electrical property changes of stored human red blood cells

The ability to transport and store a large human blood inventory for transfusions is an essential requirement for medical institutions. Thus, there is an important need for rapid and low-cost characterization tools for analyzing the properties of human red blood cells (RBCs) while in storage. In this study, we investigate the ability to use dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant, and stored in a blood bank refrigerator for a period of 20 days. Cells were removed from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction to “freeze” cells at their ionic equilibrium at that point in time and prevent ion leakage during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to measurements taken with a 2D quadrupole electrode array. The DEP analysis confirms that RBC electrical property changes occur during storage and are only discernable with the use of the cell crosslinking reaction above a glutaraldehyde fixation concentration of 1.0 w/v%. In particular, cytoplasm conductivity was observed to decrease by more than 75% while the RBC membrane conductance was observed to increase by more than 1000% over a period of 20 days. These results show that the presented combination of chemical crosslinking and DEP can be used as rapid characterization tool for monitoring electrical properties changes of human RBCs while subjected to refrigeration in blood bank storage.     

Dielectrophoresis microbial characterization and isolation of Staphylococcus aureus based on optimum crossover frequency

Characterization of antibiotic-resistant bacteria is a significant concern that persists for the rapid classification and analysis of the bacteria. A technology that utilizes the manipulation of antibiotic-resistant bacteria is key to solving the significant threat of these pathogenic bacteria by rapid characterization profile. Dielectrophoresis (DEP) can differentiate between antibiotic-resistant and susceptible bacteria based on their physical structure and polarization properties. In this work, the DEP response of two Gram-positive bacteria, namely, Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-susceptible S. aureus (MSSA), was investigated and simulated. The DEP characterization was experimentally observed on the bacteria influenced by oxacillin and vancomycin antibiotics. MSSA control without antibiotics has crossover frequencies ($f_{x0}$) from 6 to 8 MHz, whereas MRSA control is from 2 to 3 MHz. The $f_{x0}$ changed when bacteria were exposed to the antibiotic. As for MSSA, the $f_{x0}$ decreased to 3.35 MHz compared to $f_{x0}$ MSSA control without antibiotics, MRSA, $f_{x0}$ increased to 7 MHz when compared to MRSA control. The changes in the DEP response of MSSA and MRSA with and without antibiotics were theoretically proven using MyDEP and COMSOL simulation and experimentally based on the modification to the bacteria cell walls. Thus, the DEP response can be employed as a label-free detectable method to sense and differentiate between resistant and susceptible strains with different antibiotic profiles. The developed method can be implemented on a single platform to analyze and identify bacteria for rapid, scalable, and accurate characterization.     

Dielectric behaviour of binary dual-frequency nematics with low crossover frequencies

Binary dual-frequency nematic liquid crystals synthesised at the Military University of Technology were measured by dielectric means. All the investigated materials exhibit low crossover frequency. They were composed of only two molecules with longitudinal and transverse dipole moments. The main difference is the molecular weight of the molecules. Results of relaxation parameters of detected mode and crossover frequency are shown. The influence of structure on ion conductivity is discussed. The broad phase transition from an isotropic phase to a nematic one was observed.     

Dielectrophoretic characterization of macrophage phenotypes

Different macrophage phenotypes play important roles in diverse biological processes and diseases. In this study, we have characterized the dielectrophoretic responses of human monocytes and macrophage phenotypes: nonactivated (M0), pro-inflammatory (M1), and pro-healing (M2a). Dielectrophoretic responses of cells change as a function of frequency of the applied electric field. We measured the crossover frequency at which cells transition from negative to positive dielectrophoresis (DEP) or vice versa using interdigitated electrodes. For these characterization experiments, we also developed a new low-conductivity media formulation that retained 100% of the initial viability for 1 h. Human THP1 monocytes showed a distinguishable DEP response from mature macrophages. M1 macrophages also showed a distinct DEP response compared to M0 and M2a macrophages. No clear distinction could be drawn between M0 and M2a. The median values of the crossover frequencies of monocytes, M0, M1, and M2a were 38, 21, 11, and 23 kHz, respectively. Membrane capacitances of these cells were calculated consequently, and the values were 0.0111, 0.0128, 0.0244, and 0.0117 F/m² for monocytes, M0, M1, and M2a, respectively. These results show how bioelectric properties are influenced by changes in macrophage phenotype.     

A discrete dielectrophoresis device for the separation of malaria-infected cells

Malaria is a serious disease caused by Plasmodium parasites that infect red blood cells (RBCs). This paper presents the continuous separation of malaria-infected RBCs (iRBCs) from normal blood cells. The proposed method employed the discrete dielectrophoresis (DEP) in a microfluidic device with interdigitated electrodes. Our aim is to treat a sample having high concentration of cells to realize high throughput and to prevent the clogging of the microchannel with the use of the discrete DEP. The discrete DEP force for deflecting cells in the device was controlled by adjusting the magnitude, frequency, and duty cycle of the applied voltage. The effectiveness of the proposed method was demonstrated by separating the malaria-infected cells in samples having a cell concentration of 10⁶ cells/µl. From experimental results, we determined the enrichment that is needed to enhance the detection in the case of low parasitemia. The enrichment of the infected cells at the device output was 3000 times as high as that of the input containing 1 infected cell to 10⁶ normal cells. Therefore, the proposed method is highly effective and can significantly facilitate the detection of the infected cells for the identification of Malaria patients.     

SEM observation of the live morphology of human red blood cells under high vacuum conditions using a novel RTIL

In this study, we succeed in visualizing a ‘living’ morphology of red blood cells (RBCs) by a rapid and simple scanning electron microscope (SEM) pretreatment using a hydrophilic room temperature ionic liquid (RTIL) with asymmetrical structure. The conventional pretreatment induces size shrinkage of the cells, and the diameter observed in an SEM (less than 6 µm) was smaller than that in an optical microscope (8 µm). Though RBCs are well‐known to be easily deformed with the environmental conditions, the cells did not show the deformation when they were pretreated with our RTILs. Since the chemical structure of our RTIL resembles a ‘choline’, which is a typical bioactive material, and has biocompatibilities, they may make it avoid from the deformation of RBCs. In fact, only immersing RBCs, our novel RTIL can provide a clear SEM image without size shrinkage and deformation. Interestingly, the obtained SEM image shows 8 µm in the diameter, almost same size as that in optical microscope. Therefore, this method is expected for novel SEM pretreatment for various biological samples observation as ‘living’ matter. Copyright © 2014 John Wiley & Sons, Ltd.     

Novel microfluidic device for the continuous separation of cancer cells using dielectrophoresis

We describe the design, microfabrication, and testing of a microfluidic device for the separation of cancer cells based on dielectrophoresis. Cancer cells, specifically green fluorescent protein‐labeled MDA‐MB‐231, are successfully separated from a heterogeneous mixture of the same and normal blood cells. MDA‐MB‐231 cancer cells are separated with an accuracy that enables precise detection and counting of circulating tumor cells present among normal blood cells. The separation is performed using a set of planar interdigitated transducer electrodes that are deposited on the surface of a glass wafer and slightly protrude into the separation microchannel at one side. The device includes two parts, namely, a glass wafer and polydimethylsiloxane element. The device is fabricated using standard microfabrication techniques. All experiments are conducted with low conductivity sucrose‐dextrose isotonic medium. The variation in response between MDA‐MB‐231 cancer cells and normal cells to a certain band of alternating‐current frequencies is used for continuous separation of cells. The fabrication of the microfluidic device, preparation of cells and medium, and flow conditions are detailed. The proposed microdevice can be used to detect and separate malignant cells from heterogeneous mixture of cells for the purpose of early screening for cancer.     

Improved HPLC method for the determination of ribavirin concentration in red blood cells and its application in patients with COVID-19

Ribavirin is a synthetic, broad-spectrum antiviral drug. Ribavirin is recommended as an antiviral drug in the Interim Guidance for Diagnosis and Treatment (the seventh edition) of COVID-19. The ribavirin levels in red blood cells may be closely related to both its efficacy and adverse drug reactions. In this study, a simple and fast HPLC–UV method was established to determine the concentrations of total ribavirin in the red blood cells of 13 patients with COVID-19. Phosphorylated ribavirin was dephosphorylated by phosphatase incubation to obtain the total amount of ribavirin in red blood cells. The chromatographic column was an Atlantis C₁₈. The recoveries were 85.45–89.05% at three levels. A good linear response was from 1 to 200 μg/ml, with a correlation coefficient of r² = 0.9991. The concentration of total ribavirin in the red blood cells of the patients ranged from 30.83 to 133.34 μg/ml. The same samples without phosphatase incubation ranged from 4.07 to 20.84 μg/ml. About 85% of ribavirin was phosphorylated in red blood cells. In addition, we observed changes in these patients' hematological parameters and found that the erythrocyte, hemoglobin and hematocrit declined to the lowest levels on the fifth day after discontinuation of ribavirin (p < 0.05).     

活体小鼠中单个红细胞的拉曼光谱分析

应用拉曼光谱与光镊技术对活体小鼠毛细动脉和静脉血管中的全血以及毛细血管中的红细胞和体外的全血及单个红细胞进行研究;光谱分析显示,可以获得活体血液的拉曼光谱;在动脉毛细管和静脉毛细管中可以观察到清晰的携氧与去氧血液的不同光谱;体内血管中血红细胞的血红蛋白浓度比体外的更高,在动脉毛细管中的血红细胞的携氧态特征峰明显,且具有...     

以伯胺为模板剂合成含铁介孔分子筛FeHMS

以非离子型表面活性剂伯胺为模板剂合成了含铁介孔分子筛FeHMS,并对其结构和形态进行了表征。发现FeHMS的颗粒比SiHMS小,粒子表面更为光滑。除了2.5nm的介孔外,FeHMS还原10nm左右的孔。FeHMS介孔分子筛在脱除模板剂之前,铁主要处于四面体配位状态的骨架位,经焙烧脱除模板剂之后,部分铁由骨架位迁移到孔壁表面。用酸化的乙醇溶液抽提脱除模板剂,能更有效地让铁保留在四面体配位状态的骨架位上。高温脱除模板剂后,孔壁表面的铁物种处于一种高分散的状态。此外还研究了HMS类介孔分子筛材料的水热稳定性,发现铁的引入可改善介孔分子筛材料的水热稳定性。     

Determining the interaction and characterization of asphaltene in alkylbenzene solvents using nuclear-electron double resonance

This study uses ¹H dynamic nuclear polarization (DNP) methods to determine asphaltene aggregates and the interaction between asphaltene extracted from MC800 asphalt and alkylbenzene solvents, as well as elemental analysis for the characterization of asphaltene. The asphaltene sample was characterized using the elemental analysis of carbon (C), hydrogen (H), nitrogen (N), and sulfur (S). The results show that asphaltenes have the highest carbon content. The sulfur and hydrogen contents are nearly the same and nitrogen content is the smallest. The DNP data provided good results for characterizing asphaltene behavior in alkylbenzene solvents.     

Determination of different forms of aminothiols in red blood cells without washing erythrocytes

Detection and quantification of different aminothiols forms (reduced and total) in biological fluids are important for the investigation of oxidative stress‐related diseases and cell homeostasis study. The aim of this study was to optimize a HPLC method in order to determine both reduced and total thiol forms in red blood cells (RBC) at low temperature without washing erythrocytes. Analytical recoveries for total and reduced thiols were 91.6–98.5 and 94.9–98.2% respectively. The relative standard deviations intra‐assay for total and reduced thiols were 1.14–3.64 and 0.83–2.3% respectively and the relative standard deviations inter‐assay for total and reduced thiols were 1.12–3.54 and 0.84–2.03%, respectively. This method allows specific analysis of the aminothiol state inside the RBC, as a model of intracellular metabolism functioning. Copyright © 2013 John Wiley & Sons, Ltd.     

火焰原子吸收光谱法测定人血红细胞中锌、铁、镁

人血红细胞样品经低渗Triton X-100溶液超声振荡溶血处理并稀释100倍后采用火焰原子吸收光谱法测定其中锌、铁和镁的含量。锌、铁和镁的检出限(3S/N)分别为2.44×10~(-3),2.85×10~(-2),1.56×10~(-3)mg·L~(-1),相对标准偏差(n=7)分别为0.69%～1.03%,2.85%～3.84%,0.92%～1.22%。应用该法分析同一混合人血红细胞样品,测定结果与酸消化法测得的结果一致,3种元素的加标回收率为95.0%～110.0%之间。     

Comparison of density functionals for the study of the high spin low spin gap in Fe(III) spin crossover complexes

A detailed investigation of the accuracy of different quantum mechanical methods for the study of iron(III) spin crossover complexes is presented. The energy spin state gap between the high and low spin states; ΔE (HS‐LS) of nine iron(III) quinolylsalicylaldiminate complexes were calculated with nine different DFT functionals, then compared. DFT functionals: B3LYP, B3LYP‐D3, B3LYP*, BH&HLYP, BP86, OLYP, OPBE, M06L, and TPSSh were tested with six basis sets: 3‐21G*, dgdzvp, 6‐31G**, cc‐pVDZ, Def2TZVP, and cc‐pVTZ. The cations from the X‐ray crystal structures of [Fe(qsal‐OMe)₂]Cl·MeCN·H₂O, [Fe(qsal‐OMe)₂]Cl·2MeOH·0.5H₂O, [Fe(qsal‐OMe)₂]BF₄·MeOH, [Fe(qsal‐OMe)₂]NCS·CH₂Cl₂, [Fe(qsal‐F)₂]NCS, [Fe(qsal‐Cl)₂]NCS·MeOH, [Fe(qsal‐Br)₂]NCS·MeOH, [Fe(qsal‐I)₂]OTf·MeOH, and [Fe(qsal)₂]NCS?CH₂Cl₂ were used as starting structures. The results show that B3LYP, B3LYP‐D3, OLYP, and OPBE with a 6‐31G**, Def2TZVP, and cc‐pVTZ basis set give reasonable results of ΔE (HS‐LS) compared with the experimental data. The enthalpy of [Fe(qsal‐I)₂]⁺ calculated with an OLYP functional and cc‐pVTZ basis set (1.48 kcal/mol) most closely matches the experimental data (1.34 kcal/mol). B3LYP* yields an enthalpy of 5.92 kcal/mol suggesting it may be unsuitable for these Fe(III) complexes, mirroring recent results by Kepp (Inorg . Chem ., 2016, 55 , 2717–2727).     

Evaluation of anticoagulant activity of two algal polysaccharides

Marine algae are important sources of phycocolloids like agar, carrageenans and alginates used in industrial applications. Algal polysaccharides have emerged as an important class of bioactive products showing interesting properties. The aim of our study was to evaluate the potential uses as anticoagulant drugs of algal sulphate polysaccharides extracted from Ulva fasciata (Chlorophyta) and Agardhiella subulata (Rhodophyta) collected in Ganzirri Lake (Cape Peloro Lagoon, north-eastern Sicily, Italy). Toxicity of algal extracts through trypan blue test and anticoagulant action measured by activated partial thromboplastin time (APTT), prothrombin time (PT) test has been evaluated. Algal extracts showed to prolong the PT and APTT during the coagulation cascade and to avoid the blood coagulation of samples. Furthermore, the algal extracts lack toxic effects towards cellular metabolism and their productions are relatively at low cost. This permits to consider the algae as the biological source of the future.     

Structural study of the thermal and photochemical spin states in the spin crossover complex [Fe(phen)2(NCSe)2

The first structural data for [Fe(phen)(2)(NCSe)(2)] (obtained using the extraction method of sample preparation) in its high-spin, low-spin and LIESST induced metastable high-spin states have been recorded using synchrotron radiation single crystal diffraction. The space group for all of the spin states was found to be Pbcn. On cooling from the high-spin state (HS-1) at 292 K through the spin crossover at about 235 K to the low-spin state at 100 K (LS-1) the iron coordination environment changed to a more regular octahedral geometry and the Fe-N bond lengths decreased by 0.216 and 0.196 A (Fe-N(phen)) and 0.147 A (Fe-N(CSe)). When the low-spin state was illuminated with visible light at about 26 K, the structure of this LIESST induced metastable high-spin state (HS-2) was very similar to that of HS-1 with regards to the Fe-phen bond lengths, but there were some differences in the bond lengths in the Fe-NCSe unit between HS-1 and HS-2. When HS-2 was warmed in the dark to 50 K, the resultant low-spin state (LS-2) had an essentially identical structure to LS-1. In all spin states, all of the shortest intermolecular contacts (in terms of van der Waals radii) involved the NCSe ligand, which may be important in describing the cooperativity in the solid state. The quality of the samples was confirmed by magnetic susceptibility and IR measurements.     

Dependence of the pure quadrupole resonance frequency on temperature for KBrO3

The pure quadrupole resonance frequency of KBrO₃ in the temperature range 4.17–300.16 K was determined experimentally and compared with the model developed by Stahl which contains three adjustable constants. Excellent agreement between the experimental data and the model was obtained when considering only the temperature range 4.17–52 K. As a result of the comparison, the following values are suggested for KBrO₃: the resonance frequency at 0 K is 179.50373 MHz, the Debye temperature, Θ_d, is 108 K, and the rotational energy, Iω_l², of each of the first two modes of oscillation of the BrO₃ group is 1.707 × 10^?18 kg m². Here I is the moment of inertia and ω_l is the lattice vibration frequency. Comparison of model and experimental data over the entire temperature range from 4.17 to 300.16 K shows that the suggested theory reflects very well the general trend of the experimental data but lacks in some detail. Copyright © 2003 John Wiley & Sons, Ltd.     

Comprehensive study of the macropore and mesopore size distributions in polymer monoliths using complementary physical characterization techniques and liquid chromatography

Poly(styrene‐co‐divinylbenzene) monolithic stationary phases with two different domain sizes were synthesized by a thermally initiated free‐radical copolymerization in capillary columns. The morphology was investigated at the meso‐ and macroscopic level using complementary physical characterization techniques aiming at better understanding the effect of column structure on separation performance. Varying the porogenic solvent ratio yielded materials with a mode pore size of 200 nm and 1.5 μm, respectively. Subsequently, nano‐liquid chromatography experiments were performed on 200 μm id × 200 mm columns using unretained markers, linking structure inhomogeneity to eddy dispersion. Although small‐domain‐size monoliths feature a relatively narrow macropore‐size distribution, their homogeneity is compromised by the presence of a small number of large macropores, which induces a significant eddy‐dispersion contribution to band broadening. The small‐domain size monolith also has a relatively steep mass‐transfer term, compared to a monolith containing larger globules and macropores. Structural inhomogeneity was also studied at the mesoscopic level using gas‐adsorption techniques combined with the non‐local‐density‐function‐theory. This model allows to accurately determine the mesopore properties in the dry state. The styrene‐based monolith with small domain size has a distinctive trimodal mesopore distribution with pores of 5, 15, and 25 nm, whereas the monolith with larger feature sizes only contains mesopores around 5 nm in size.