Single Human Erythrocytes Research Articles

Interactions of ultrashort laser pulses with hemoglobin: Photophysical aspects and potential applications

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

Nonlinear indentation of single human erythrocytes under application of a localized mechanical force

Despite the accepted notion that erythrocytes are uniquely deformable cells, the apparent Young’s modulus values reported in the literature do not differ so much from those of other cells. We devised to measure the local deformability of living immobilized human erythrocytes at a low force, in contact-free mode, using an application of Scanning Ion Conductance Microscopy (SICM) previously developed in our laboratory. Reversible indentations were induced by forces of up to few hundreds pN. The indentation did not grow linearly with the force. The apparent Young’s modulus varied from 0.2 to 1.5 kPa applying forces from 20 to 500 pN on a cell surface area of about 0.2 μm2, exhibiting a progressive stiffening at increasing force. Control measurements showed that A549 cells exhibit a constant value of the apparent Young’s modulus (about 2 kPa) for forces up to about 800 pN. These findings show that SICM is a suitable tool to investigate cell mechanical properties, when forces in the range of tens of pN are required, in the absence of mechanical contact between probe and sample. The nonlinear deformation of the erythrocyte has to be taken into account in modeling the complex regulation mechanism of the microvascular beds.

Mass spectrometry based detection of glutathione with sensitivity for single-cell analysis.

Glutathione (GSH) is a very important molecule that participates in various physiologically important events. Depletion of cellular GSH has been suggested as a biomarker of early stage of cell injury. Assessment of GSH with high selectivity and sensitivity is in demand in many fields of life sciences. Cell lysates were deproteinated by 5-sulfosalicylic acid. Glutathione was derivatized with monobromobimane to form a GSH conjugate. S-Hexylglutathione was employed as internal standard. The resulting samples were analyzed using a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) system. The limit of detection was as low as 500 amol with high selectivity. The approach was sensitive enough to detect GSH content in a single human erythrocyte. The technique is the most sensitive approach among the reported MS-based GSH assays. A monobromobimane derivatization and mass spectrometry based method was developed for the assessment of GSH. The derivatized GSH was chemically stable against autooxidation. With the great selectivity and sensitivity, the approach is anticipated to serve as a routine method for assessment of GSH and possibly other important endogenous low molecular weight thiols.

Effect of 4-aminoantipyrine on oxidative stress induced by glutathione depletion in single human erythrocytes using a microfluidic device together with fluorescence imaging

The effects of 4-aminoantipyrine (AAP) on oxidative stress induced by glutathione (GSH) depletion in single human erythrocytes were investigated using microfluidic technique and fluorescence imaging. Most cell-based toxicity evaluations on GSH are performed with bulk experiments based on analysis of cell populations. This work established a single-cell toxicity evaluation method to statistically analyze the GSH amount in single erythrocytes incubated with AAP in different concentrations. The experimental conditions of cell flow rate and cell concentration were optimized. The GSH contents in erythrocytes decreased with increasing dose of AAP. At low concentration, AAP had a little effect on GSH; while at high concentration, AAP led to GSH depletion reaching a maximum of 14.53%. The depletion of GSH leads to a significant shift to a more oxidizing intracellular environment. This study provides basic data for presenting the effect of AAP on GSH in erythrocytes and is helpful for understanding its toxicity during the blood transportation process. In addition, it will also complement studies on the environmental risk assessment of AAP pollution.

Determination of reactive oxygen species in single human erythrocytes using microfluidic chip electrophoresis

Reactive oxygen species (ROS) are known to not only mediate the damage of cellular constituents but also to regulate cellular signaling. Analysis of ROS is essential if we wish to understand the mechanisms of cellular alterations. In this paper, a microfluidic chip-based approach to the determination of ROS in single erythrocyte was developed by using a simple crossed-channel glass chip with integrated operational functions, including cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser-induced fluorescence (LIF) detection. Non-fluorescent dihydrorhodamine 123 (DHR 123), which can be oxidized intracellularly by ROS to the fluorescent rhodamine 123 (Rh 123), was used as the fluorogenic reagent. The effect of pH on the migration time of Rh 123 and detection sensitivity was discussed. The present method minimized dilution of intracellular ROS during reaction with DHR 123 and determination. As a result, an extremely low detection limit of 0.8 amol has been achieved. The time required for complete analysis of one human erythrocyte was less than 3 min. A migration time precision of 4.1% RSD was obtained for six consecutively-injected cells. Upon stimulation with 4 mmol/l H2O2 for 10 min, the intracellular ROS concentration was found to increase on average by about a factor of 8.4.

Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip.

A microfluidic system was developed for the analysis of single biological cells, with functional integration of cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser induced fluorescence (LIF) detection in microfabricated channels of a single glass chip. Channels were 12 microm deep and 48 microm wide, with a simple crossed-channel design. The effective separation channel length was 35 mm. During sampling with a cell suspension (cell population 1.2 x 10(5) cells per mL in physiological salt solution), differential hydrostatic pressure (created by adjusting liquid levels in the four reservoirs) was used to control cell flow exclusively through the channel crossing. Single cell loading into the separation channel was achieved by electrophoretic means by applying a set of potentials at the four reservoirs, counteracting the hydrostatic flow. A special docking (adhering) procedure for the loaded cell was applied before lysis by repeatedly connecting and disconnecting a set of low potentials, allowing precise positioning of the cell within the separation channel. Cell lysis was then effected within 40 ms under an applied CE separation voltage of 1.4 kV (280 V cm(-1)) within the working electrolyte (pH 9.2 borate buffer) without additional lysates. The docked lysing approach reduced dispersion of released intracellular constituents, and significantly improved the reproducibility of CE separations. Glutathione (GSH) was used as a model intracellular component in single human erythrocyte cells. NDA derivatized GSH was detected using LIF. A throughput of 15 samples h(-1), a retention time precision of 2.4% RSD was obtained for 14 consecutively injected cells. The average cellular concentration of GSH in human erythrocytes was found to be 7.2 [times] 10(-4)+/- 3.3 x 10(-4) M (63 +/- 29 amol per cell). The average separation efficiency for GSH in lysed cells was 2.13 x 10(6)+/- 0.4 x 10(6) plates per m, and was about a factor of 5 higher than those obtained with GSH standards using pinched injection.

Electroporation Introduction of Diclofenac Sodium into Human Erythrocytes and Its Determination

A method of electroporation for introducing diclofenac sodium (a drug) into human erythrocytes was developed. The conditions (the pulse strength, pulse duration and pulse number) for the introduction of diclofenac sodium into erythrocytes by electroporation were investigated. The diclofenac sodium in single human erythrocytes introduced by the electroporation was analyzed qualitatively and quantitatively by capillary zone electrophoresis (CZE) with end-column ampermetric detection at a carbon microdisk array electrode.

Electroporation Introduction of Diclofenac Sodium into Human Erythrocytes and Its Determination

A method of electroporation for introducing diclofenac sodium (a drug) into human erythrocytes was developed. The conditions (the pulse strength, pulse duration and pulse number) for the introduction of diclofenac sodium into erythrocytes by electroporation were investigated. The diclofenac sodium in single human erythrocytes introduced by the electroporation was analyzed qualitatively and quantitatively by capillary zone electrophoresis (CZE) with end-column ampermetric detection at a carbon microdisk array electrode.

Monitoring diclofenac sodium in single human erythrocytes introduced by electroporation using capillary zone electrophoresis with electrochemical detection.

A method for determination of the drug diclofenac sodium introduced into individual human erythrocytes by electroporation using capillary zone electrophoresis with electrochemical detection at a carbon fiber array microelectrode was developed. In this method, the whole cell was injected into the separation capillary by electromigration. Cell lysis was accomplished by injecting a plug of the separation buffer (1.25 x 10(-2) mol/L Na2B4O7-3.13 x 10(-3) mol/L NaOH). The optimum conditions of separation and detection were 20 kV for the separation voltage and 1.0 V for the detection potential. The concentration of diclofenac sodium in the single cells was quantified by a calibration curve. The mean concentration of diclofenac sodium introduced into the cell was 4.21 micromol/L. The relative standard deviation of the concentration of diclofenac sodium introduced into ten cells is 10%.

Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection

Glutathione (GSH) in single human erythrocytes is determined by capillary zone electrophoresis with end-column amperometric detection at a gold/mercury amalgam microelectrode. A capillary of 10 microm inner diameter is suitable for determination of GSH in an individual erythrocyte with a good signal-to-noise ratio. The limit of detection is 1 x 10(-7) mol/L or 26 amol and the linear dynamic range is 2 x 10(-7) to 2 X 10(-5) mol/L for the capillary. In this method, the calibration line is obtained with a capillary adsorbed before a certain amount of hemoglobin can be used for the quantification of GSH in the external standardization. The whole cell injection and the lack of necessity of a derivatization reaction lead to more accurate and precise results, which are closer to the macroscopic values of glutathione in human red blood cell (i.e., hemolysate) than those determined by indirect laser-induced fluorescence detection.

Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection

Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection

Dual-enzyme assay of glutamate in single cells based on capillary electrophoresis.

A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Glutamate dehydrogenase and glutamic pyruvic transaminase were used to catalyze the glutamate reaction. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. Glutamate dehydrogenase catalyzed the formation of NADH, and glutamic pyruvic transaminase drives the glutamate dehydrogenase reaction by removing a reaction product and regenerating glutamate. The detection limit of glutamate is down to the 10(-8) M level, which is 1 order of magnitude lower than previously reported detection limits based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most amino acids. The glutamate content in single human erythrocytes and baby rat brain neurons were determined with this method and the results agreed well with literature values.

Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection.

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electroosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The approximately 90 fl injection volume for individual cells is 7 x 10(4) times lower than those previously reported. Adult (normal and elevated A1), sickle (heterozygous), and fetal erythrocytes were analyzed with the amounts of hemoglobins AO, A1c, S and F determined. The pH gradient for cIEF was linear (r = 0.9984), which allowed tentative identification of Hb Fac. Variants differing by as little as 0.025 pl units were resolved.

Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: dispersion of the cytoplasm

Usually dielectrophoretic and electrorotation measurements are carried out at low ionic strength to reduce electrolysis and heat production. Such problems are minimized in microelectrode chambers. In a planar ultramicroelectrode chamber fabricated by semiconductor technology, we were able to measure the dielectric properties of human red blood cells in the frequency range from 2 kHz to 200 MHz up to physiological ion concentrations. At low ionic strength, red cells exhibit a typical electrorotation spectrum with an antifield rotation peak at low frequencies and a cofield rotation peak at higher ones. With increasing medium conductivity, both electrorotational peaks shift toward higher frequencies. The cofield peak becomes antifield for conductivities higher than 0.5 S/m. Because the polarizability of the external medium at these ionic strengths becomes similar to that of the cytoplasm, properties can be measured more sensitively. The critical dielectrophoretic frequencies were also determined. From our measurements, in the wide conductivity range from 2 mS/m to 1.5 S/m we propose a single-shell erythrocyte model. This pictures the cell as an oblate spheroid with a long semiaxis of 3.3 microns and an axial ratio of 1:2. Its membrane exhibits a capacitance of 0.997 x 10(-2) F/m2 and a specific conductance of 480 S/m2. The cytoplasmic parameters, a conductivity of 0.4 S/m at a dielectric constant of 212, disperse around 15 MHz to become 0.535 S/m and 50, respectively. We attribute this cytoplasmic dispersion to hemoglobin and cytoplasmic ion properties. In electrorotation measurements at about 60 MHz, an unexpectedly low rotation speed was observed. Around 180 MHz, the speed increased dramatically. By analysis of the electric chamber circuit properties, we were able to show that these effects are not due to cell polarization but are instead caused by a dramatic increase in the chamber field strength around 180 MHz. Although the chamber exhibits a resonance around 180 MHz, the harmonic content of the square-topped driving signals generates distortions of electrorotational spectra at far lower frequencies. Possible technological applications of chamber resonances are mentioned.

Analysis of single cells with capillary electrophoresis electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Recent results with on-line capillary electrophoresis (CE) electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry suggest that CE/ESI-FTICR can provide a powerful technique for micro-sample analyses owing to the inherent sensitivity of the technique and the enhanced information content derived from high-performance mass measurements. Using micro-sampling methods and ion accumulation techniques based on quadrupolar excitation, we demonstrate that adequate sensitivity exists to characterize the hemoglobin from a single human erythrocyte (approximately 450 amol). In these studies mass spectra with average mass resolution in excess of 45 000 (FWHM) were obtained for both the alpha- and beta chain of hemoglobin following in-column lysing of a single erythrocyte.

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.

Simultaneous Determination of Enzyme Activity and Enzyme Quantity in Single Human Erythrocytes

An integrated laser-based fluorescence enzyme assay and particle-counting immunoassay system with zeptomole sensitivity has been developed for simultaneous determinations of activity and concentration of enzymes in single erythrocytes. The product NADPH of the reaction between glucose-6-phosphate and NADP+ catalyzed by glucose-6-phosphate dehydrogenase (G6PDH) is monitored. Simultaneously, the agglutination of antibody-coated particles in the presence of G6PDH produces large particles which are counted by light scattering. The correlation between the two parameters indicates that on average only about 35% of the enzyme in individual cells is active. There is more than a 10-fold cell-to-cell variation in the ratios of the two parameters. This indicates that there are multiple mechanisms for the degradation of intracellular enzymes as a function of cell age.

Chemical Analysis of Single Human Erythrocytes

ADVERTISEMENT RETURN TO ISSUEPREVArticleChemical Analysis of Single Human ErythrocytesEdward S. YeungCite this: Acc. Chem. Res. 1994, 27, 12, 409–414Publication Date (Print):December 1, 1994Publication History Published online1 May 2002Published inissue 1 December 1994https://doi.org/10.1021/ar00048a004RIGHTS & PERMISSIONSArticle Views143Altmetric-Citations38LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (802 KB) Get e-Alerts Get e-Alerts

Laser-Based Particle-Counting Microimmunoassay for the Analysis of Single Human Erythrocytes

A particle-counting immunoassay system for ultrasensitive analysis of proteins in a capillary environment has been developed. The assay is based on the agglutination of antibody-coated particles in the presence of an antigen (usually a protein). The particles were electrophoretically migrated in a 20-microns-i.d. capillary past a detection window where a laser beam irradiates continuously. The light scattering events generated by the agglutinated particles were counted while those produced by unreacted particles were electronically rejected. Glucose-6-phosphate dehydrogenase (G6PDH) was chosen as a test compound for the off-column as well as for the on-column versions of this method. A limit of detection of 620 molecules of G6PDH (1 zmol) was found in the on-column assay. The standard deviation between runs was approximately 6%, which is comparable to that of standard immunoassay methods. The application to the determination of G6PDH levels in individual human erythrocytes is presented. A 14-fold cell-to-cell variation was found which can be explained by the age distribution in the red blood cells.

Variability of intracellular lactate dehydrogenase isoenzymes in single human erythrocytes.

Trace amounts of enzymes within single human erythrocytes can be quantified by a combination of on-column reaction and capillary electrophoresis. A detection limit of 1.3 x 10(-21) mol of LDH was achieved with laser-induced fluorescence by monitoring the product of the enzyme-catalyzed reaction between lactate and NAD+. Single erythrocyte analysis clearly isolates the major forms of LDH. The variation of total LDH activity in a population of cells from a single individual is large, but the relative activities of the isoenzymes LDH-1 and LDH-2 are fairly constant. This can be explained by the distribution of cell age in the population. A lower enzyme activity is indicative of senescence. The efficient separation of different LDH forms and the high detection sensitivity opens up the possibility of multiple-enzyme assays with a single mammalian cell.