Single Human Red Blood Cells Research Articles

Thermo-Mechanical Fluid-Structure Interaction Numerical Modelling and Experimental Validation of MEMS Electrothermal Actuators for Aqueous Biomedical Applications.

Recent developments in MEMS technologies have made such devices attractive for use in applications that involve precision engineering and scalability. In the biomedical industry, MEMS devices have gained popularity in recent years for use as single-cell manipulation and characterisation tools. A niche application is the mechanical characterisation of single human red blood cells, which may exhibit certain pathological conditions that impart biomarkers of quantifiable magnitude that are potentially detectable via MEMS devices. Such applications come with stringent thermal and structural specifications wherein the potential device candidates must be able to function with no exceptions. This work presents a state-of-the-art numerical modelling methodology that is capable of accurately predicting MEMS device performance in various media, including aqueous ones. The method is strongly coupled in nature, whereby thermal as well as structural degrees of freedom are transferred to and from finite element and finite volume solvers at every iteration. This method therefore provides MEMS design engineers with a reliable tool that can be used in design and development stages and helps to avoid total reliability on experimental testing. The proposed numerical model is validated via a series of physical experiments. Four MEMS electrothermal actuators with cascaded V-shaped drivers are presented. With the use of the newly proposed numerical model as well as the experimental testing, the MEMS devices' suitability for biomedical applications is confirmed.

Hemoglobin conformation detection by Raman spectroscopy on single human red blood cells captured in a microfluidic chip

The Raman spectrum of a single erythrocyte captured by a microfluidic chip was recorded to determine the conformation of hemoglobin under conditions similar to the hemodynamics of a blood vessel. Amplitude changes in the Raman spectrum at 1355, 1375, 1552, 1620, 1585 and 1637 cm−1 reflect changes in pO2 due to O2 binding to hemoglobin heme.

Multimodal detection and analysis of a new type of advanced Heinz body-like aggregate (AHBA) and cytoskeleton deformation in human RBCs.

A new type of aggregate, formed in human red blood cells (RBCs) in response to glutaraldehyde treatment, was discovered and analyzed with the classical and advanced biomolecular imaging techniques. Advanced Heinz body-like aggregates (AHBA) formed in a single human RBC are characterized by a higher level of hemoglobin (Hb) degradation compared to typical Heinz bodies, which consist of hemichromes. The complete destruction of the porphyrin structure of Hb and the aggregation of the degraded proteins in the presence of Fe3+ ions are observed. The presence of such aggregated, highly degraded proteins inside RBCs, without cell membrane destruction, has been never reported before. For the first time the spatial differentiation of two kinds of protein mixtures inside a single RBC, with different phenylalanine (Phe) conformations, is visualized. The non-resonant Raman spectra of altered RBCs with AHBA are characterized by the presence of a strong band located at 1037 cm-1, which confirms that glutaraldehyde interacts strongly with Phe. The shape-shifting of RBCs from a biconcave disk to a spherical structure and sinking of AHBA to the bottom of the cell are observed. Results reveal that the presence of AHBA should be considered when fixing RBCs and indicate the analytical potential of Raman spectroscopy, atomic force microscopy and scanning near-field optical microscopy in AHBA detection and analysis.

A highly efficient introduction system for single cell- ICP-MS and its application to detection of copper in single human red blood cells

A method of simultaneous cell counting and determination of metals in single cells using time-resolved inductively coupled plasma-mass spectrometry (ICP-MS) was reported. A facile, low cost and highly efficient single-cell introduction system of time-resolved ICP-MS consists of a flow cell, a visual contrast calibration device, a customized nebulizer and a fabricated spray chamber. The flow cell includes a cell sample tube, a sheath liquid tube and a flow chamber. The visual contrast calibration device was composed of a microscope with a 16 × microscope objective (160 × total magnification). The flow chamber was used to combine a flow of red blood cell suspension (0.800 μL/min) and a flow of PBS (4.40 μL/min) into the nebulizer. The intact cells were directly introduced with the single-cell introduction system into the plasma via nebulizing, and then ion plumes corresponding to single cells were individually detected with mass spectrometer. The frequency of the spikes directly reflects the number of cells, and the intensity of spikes is proportional to the concentration of copper within one cell. The single-cell introduction system can be transported into the ICP-MS via a customized transport system with 100% efficiency. A high cell introduction efficiency into the plasma supports for a reduction of cell consumption. The Cu signal frequency was about 120 cell events per minute. This single-cell introduction system simplifies the introduction of individual and intact cells. The copper content in single red blood cell was 0.20–0.40 fg.

Studying Single Red Blood Cells Under a Tunable External Force by Combining Passive Microrheology with Raman Spectroscopy

The dynamic micromechanical and structural properties of single human red blood cells are studied using a combination of dual trap optical tweezers and confocal Raman spectroscopy. Such a combination permits us to show a direct relationship between the rheological properties and chemical structure conformation. The frequency dependence of the complex stiffness of the cells was measured using both one and two probe response functions under identical experimental conditions. Both the microrheology and Raman measurements were performed at different stretching forces applied to the cell. A detailed analysis of the auto- and cross-correlated probe motions allows exploring the local and overall viscoelastic properties of the cells over a controlled range of the deformations. The observed growth of the cell viscoelasticity with stretching was associated with structural changes in the cell membrane monitored via the Raman spectroscopy.

Absorption spectroscopy of single red blood cells in the presence of mechanical deformations induced by optical traps

The electronic properties of single human red blood cells under mechanical deformations were investigated using a combination of dual beam optical tweezers and UV-vis absorption spectroscopy. The mechanical deformations were induced by two near-infrared optical traps with different trapping powers and trap configurations. The deformations were applied in two ways: locally, due to the mechanical forces around the traps, and by stretching the cell by moving the traps in opposite directions. In the presence of local deformations, the single cell undergoes a transition from an oxygenated state to a partially deoxygenated state. This process was found to be reversible and strongly power-dependent. Stretching the cell caused an opposite effect, indicating that the electronic response of the whole cell is dominated by the local interaction with the trapping beams. Results are discussed considering light-induced local heating, the Stark effect, and biochemical alterations due to mechanical forces, and are compared with reports of previous Raman spectroscopy studies. The information gained by the analysis of a single red blood cell's electronic response facilitates the understanding of fundamental physiological processes and sheds further light on the cell's mechanochemistry. This information may offer new opportunities for the diagnosis and treatment of blood diseases.

Coupling chemiluminescence with capillary electrophoresis to analyze single human red blood cells

In this paper, we describe a new method for determination of hemoglobin of single red blood cells by coupling chemiluminescence with capillary electrophoresis (CL–CE). The chemiluminescent detection is based on the catalytic effects of hemoglobin on the luminol–hydrogen peroxide reaction. The conditions of chemiluminescent reaction and capillary electrophoresis were investigated. Hemoglobin in human blood samples was detected with the present method, the linear range from 1.7 μg mL −1 to 6.8 μg mL −1 was tested, and the correlation coefficient of 0.997 and low detection limit of 0.17 μg mL −1 (approximately 2.2 pg, S/N = 3) were obtained. Cell injection procedure was improved, and the method was successfully used to determine hemoglobin of single red blood cells and the statistical result of the average content of hemoglobin in 26 human red blood cells was 23.6 pg. Compared to other current methods, CE with CL system is simple, sensitive and will become an attractive alternative method for single cell analysis.

Study of the effect of alcohol on single human red blood cells using near-infrared laser tweezers Raman spectroscopy

The effect of alcohol solution on single human red blood Cells (RBCs) was investigated using near-infrared laser tweezers Raman spectroscopy (LTRS). In our system, a low-power diode laser at 785 nm was applied for the trapping of a living cell and the excitation of its Raman spectrum. Such a design could simultaneously reduce the photo-damage to the cell and suppress the interference from the fluorescence on the Raman signal. The denaturation process of single RBCs in 20% alcohol solution was investigated by detecting the time evolution of the Raman spectra at the single-cell level. The vitality of RBCs was characterized by the Raman band at 752 cm−1, which corresponds to the porphyrin breathing mode. We found that the intensity of this band decreased by 34.1% over a period of 25 min after the administration of alcohol. In a further study of the dependence of denaturation on alcohol concentration, we discovered that the decrease in the intensity of the 752 cm−1 band became more rapid and more prominent as the alcohol concentration increased. The present LTRS technique may have several potential applications in cell biology and medicine, including probing dynamic cellular processes at the single cell level and diagnosing cell disorders in real time. Copyright © 2005 John Wiley & Sons, Ltd.

Cellular absorption of electric field energy: influence of molecular properties of the cytoplasm

Molecular dispersions may significantly alter the frequency dependence of structural polarizations. Consequently, the molecular properties cannot be neglected when the energy absorption is calculated with a subcellular resolution. Our example presents calculations that explain the absorption in single human red blood cells. The molecular properties of the cytoplasm have been derived from literature data on the impedance of Hb suspensions. The resulting cell properties were then compared to own data obtained by single cell dielectric spectroscopy.

Ultra-micro analysis of liquids and suspensions based on laser-induced plasma emissions

Spectrochemical analysis of liquids and suspensions using laser-induced plasma emissions was investigated. Nd:YAG pulsed-laser (532-nm) ablation of aqueous samples produced plasmas that were hot (few eV) and extensively ionized, with electron density in the 10 18 cm −3 range. Analyte line signals were initially masked by intense plasma continuum emissions, and would only emerge briefly above the background when the plume temperature dropped below 1 eV during the course of its very rapid cooling. In contrast, 193-nm laser ablation at similar fluence generated plasmas of much lower (<1 eV) temperature but comparable electron density. The plasma continuum emissions were relatively weak and the signal-to-background ratio was a thousand times better. This `cold' plasma was ideal for sampling trace amounts of biologically important elements such as sodium and potassium. By ablating hydrodynamically focused jets in a sheath-flow, and with acoustic normalization for improved precision, the single-shot detection limits of sodium and potassium were 8 and 50 fg, respectively. Using the sheath-flow arrangement, the amounts of sodium and potassium inside single human red blood cells were simultaneously determined for the first time. The intracellular contents for a given blood donor were found to vary significantly, with only very weak correlation between the amounts of sodium and potassium in individual cells.

Separation of hemoglobin variants in single human erythrocytes by capillary electrophoresis with laser-induced native fluorescence detection

Single human red blood cells, in which the hemoglobin (Hb) molecules exist in their native, tetrameric states, were analyzed. Upon injection and lysis of a cell, the tetramers were dissociated on-column into their respective polypeptide chains, separated, and detected by laser-induced native fluorescence detection with 275-nm excitation. This technique was applied to the determination of hemoglobin variants as found in adult (normal and elevated Hb A 1) and fetal erythrocytes. Normal adult cells contained 9.6% and 4.8% glycated β- and α-chains, respectively.] Cells with elevated Hb A 1 gave 30% and 12%, respectively. The amounts of glycated Hb and total Hb in a given cell were found to be uncorrelated.

Analysis of iron content in individual human red blood cells by electron microprobe and scanning electron microscope

The iron content of single human red blood cells has been assessed using electron microprobe analysis and scanning electron microscope. Cells for microanalysis consist of a fixed-washed and freeze-dried preparation. This preparative procedure improves stability and spatial resolution of the analytic method. The reference standard employs compressed pellets of purified iron-containing human hemoglobin and human albumin, respectively. The cell preparation and reference standard remain stable for long periods of time. Differences in Fe content and its distribution among individual red cells from both normal subjects and sickle cell anaemia patients have been measured by the quantitative and semi-quantitative techniques (wavelength and energy dispersive X-ray spectrometry). There are several points which can be made from this study. One concerns the estimate of iron on a cell by cell basis, the average variation among cells being slightly over 13% ranging from 6% to 19.4% by individuals (samples) or from 11% to 14% by ethnic groups. These differences are thus closely comparable across ethnic grouping with a possibility of high level in Asians and lower level in sickle cell anaemia patients.

A study of the applicability of the electron microprobe to a quantitative analysis of K and Na in single human red blood cells

A study of the applicability of the electron microprobe to a quantitative analysis of K and Na in single human red blood cells

Viscoelastic Properties of the Human Red Blood Cell Membrane: I. Deformation, Volume Loss, and Rupture of Red Cells in Micropipettes

Single human red blood cells suspended in buffered Ringer's solution were rapidly drawn, at recorded pressures, into glass micropipettes of diameter 0.6-3.2 mum. Cells could enter micropipettes of diameter >/= 2.9 mum with minimal pressure. In micropipettes of 0.9-2.9 mum, the pressure required increased linearly with decreasing diameter. For diameters 2.5-2.9 mum, pressures ranged up to 7 cm Hg, and the cells returned to normal biconcave shape on release. For diameters 1.9-2.5 mum, the required pressures ranged from 7 to 17 cm Hg. The released cells were crenated. In micropipettes 0.9-1.9 mum, the pressures required ranged from 17 to 34 cm Hg. The cells hemolyzed on entry. As diameter decreased from 0.9 to 0.6 mum, cells were drawn into dumbbell shapes and parts of the cells were pinched off without complete hemolysis of the cell. Using an accepted value of 138 mum(2) for the mean cell area, the mean volume of the human red cell was calculated to be 94 mum(3). Under mechanical stress, about 12% of this volume is rapidly exchangeable with the external medium. The cell volume may further decrease by 20% which is not reversible.

Sedimentation of single human red blood cells. Differences between normal and glutaraldehyde fixed cells.

AbstractThe sedimentation behaviour of single human red blood cells fixed with glutaraldehyde at pH 7.4 and 6.4 was studied and compared to resiflts previously reported for normal fresh cells. The cells fixed at pH 7.4 were observed to have normal shapes while those fixed at pH 6.4 were more spherical and less disc‐like. Fixation of glutaraldehyde removed “membrane flicker,” resulting in increased stability as indicated by a decrease in the number of orientation changes per minute from 2.93 ± 0.16 (SEM) to 1.74 ± 0.10 (SEM). An orientation change was defined as a change of 45° in any direction. Fixation also increased the edge‐to‐flat time preference ratio from 2.5 to 4.7, and increased the sedimentation velocities in all three orientations, despite a measured 2.5% decrease in mean cell density.Fixation of cells at pH 6.4 showed that the decrease in stability and preference for the on‐edge orientation was associated with an increase in the sphericity of the cell.