Blood separation on microfluidic paper-based analytical devices

A microfluidic paper-based analytical device (μPAD) for the separation of blood plasma from whole blood is described. The device can separate plasma from whole blood and quantify plasma proteins in a single step. The μPAD was fabricated using the wax dipping method, and the final device was composed of a blood separation membrane combined with patterned Whatman No.1 paper. Blood separation membranes, LF1, MF1, VF1 and VF2 were tested for blood separation on the μPAD. The LF1 membrane was found to be the most suitable for blood separations when fabricating the μPAD by wax dipping. For blood separation, the blood cells (both red and white) were trapped on blood separation membrane allowing pure plasma to flow to the detection zone by capillary force. The LF1-μPAD was shown to be functional with human whole blood of 24-55% hematocrit without dilution, and effectively separated blood cells from plasma within 2 min when blood volumes of between 15-22 μL were added to the device. Microscopy was used to confirm that the device isolated plasma with high purity with no blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was 2.62% (n = 10) and 5.84% (n = 30) for within-day and between day precision, respectively. Our proposed blood separation on μPAD has the potential for reducing turnaround time, sample volume, sample preparation and detection processes for clinical diagnosis and point-of care testing.     

Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.     

Direct measurement of lithium in whole blood using microchip capillary electrophoresis with integrated conductivity detection

The direct measurement of lithium in whole blood is described. Using microchip capillary electrophoresis (CE) with defined sample loading and applying the principles of column coupling, alkali metals were determined in a drop of whole blood. Blood collected from a finger stick was mixed with anticoagulant and transferred onto the chip without extraction or removal of components. The electrokinetic transport of red blood cells inside the channels was studied to find sample loading conditions suitable for the analysis of lithium without injecting cells into the separation channel. Both bare glass chips and chips coated with polyacrylamide were used showing the behavior of the cells under different electroosmotic flow conditions. In serum a detection limit for lithium of 0.4 mmol/L was reached. Proteins quickly contaminated untreated chip surfaces but devices with coating gave reproducible electropherograms. In addition, potassium and sodium were also detected in the same separation run. To our knowledge, this is the first device to directly measure ions in whole blood with the use of capillary zone electrophoresis on a microchip.     

Graphene Oxide Nanosheets Modified with Single‐Domain Antibodies for Rapid and Efficient Capture of Cells

Peripheral blood can provide valuable information on an individual’s immune status. Cell‐based assays typically target leukocytes and their products. Characterization of leukocytes from whole blood requires their separation from the far more numerous red blood cells. 1 Current methods to classify leukocytes, such as recovery on antibody‐coated beads or fluorescence‐activated cell sorting require long sample preparation times and relatively large sample volumes. 2 A simple method that enables the characterization of cells from a small peripheral whole blood sample could overcome limitations of current analytical techniques. We describe the development of a simple graphene oxide surface coated with single‐domain antibody fragments. This format allows quick and efficient capture of distinct WBC subpopulations from small samples (～30 μL) of whole blood in a geometry that does not require any specialized equipment such as cell sorters or microfluidic devices.     

A microfluidic device for continuous, real time blood plasma separation

A microfluidic device for continuous, real time blood plasma separation is introduced. The principle of the blood plasma separation from blood cells is supported by the Zweifach-Fung effect and was experimentally demonstrated using simple microchannels. The blood plasma separation device is composed of a blood inlet, a bifurcating region which leads to a purified plasma outlet, and a concentrated blood cell outlet. It was designed to separate blood plasma from an initial blood sample of up to 45% inlet hematocrit (volume percentage of cells). The microfluidic network was designed using an analogous electrical circuit, as well as analytical and numerical studies. The functionality of this device was demonstrated using defibrinated sheep blood. During 30 minutes of continuous blood infusion through the device, all the erythrocytes (red blood cells) traveled through the device toward the concentrated blood outlet while only the plasma was separated at the bifurcating regions and flowed towards the plasma outlet. The device has been operated continuously without any clogging or hemolysis of cells. The experimentally determined plasma selectivity with respect to blood hematocrit level was almost 100% regardless of the inlet hematocrit. The total plasma separation volume percent varied from 15% to 25% with increasing inlet hematocrit. Due to the device's simple structure and control mechanism, this microdevice is expected to be used for highly efficient continuous, real time cell-free blood plasma separation from blood samples for use in lab on a chip applications.     

Rapid electrophoretic monitoring of the anaesthetic ketamine and its metabolite norketamine in rat blood using a contactless conductivity detector to study the pharmacokinetics

A method of capillary electrophoresis with contactless conductivity detection has been developed for non‐enantioselective monitoring the anaesthetic ketamine and its main metabolite norketamine. The separation is performed in a 15 μm capillary with an overall length of 31.5 cm and length to detector of 18 cm; inner surface of the capillary is covered with a commercial coating solution to reduce the electroosmotic flow. In an optimised background electrolyte with composition 2 M acetic acid + 1% v/v coating solution under application of a high voltage of 30 kV, the migration time is 97.1 s for ketamine and 95.8 s for norketamine, with an electrophoretic resolution of 1.2. The attained detection limit was 83 ng/mL (0.3 μmol/L) for ketamine and 75 ng/mL (0.3 μmol/L) for norketamine; the number of theoretic plates for separation of an equimolar model mixture with a concentration of 2 μg/mL was 683 500 plates/m for ketamine and 695 400 plates/m for norketamine. Laboratory preparation of rat blood plasma is based on mixing 10 μL of plasma with 30 μL of acidified acetonitrile, followed by centrifugation. A pharmacokinetic study demonstrated an exponential decrease in the plasma concentration of ketamine after intravenous application and much slower kinetics for intraperitoneal application.     

Irradiation techniques for the release of bound heavy metals in natural waters and blood

Irradiation techniques are compared with conventional acid digestion procedures for the release of bound heavy metals in natural waters and in blood, before their determination by anodic stripping voltammetry. Ultra-violet irradiation of acidified water with a 550-W mercury vapour lamp releases bound zinc, cadmium, lead and copper after 4 h. The same results can be achieved with a 30-Mrad dose of high-energy γ-irradiation. These techniques are also effective for the release of metals in whole blood and blood plasma, where sample volumes as small as 200 μl are adequate in analyses for zinc, copper and lead. By comparison with acid digestion and solvent extraction methods, irradiation treatments offer the advantages of minimum sample manipulation and negligible reagent blanks.     

Determination of Ca, Mg, Fe, Cu, and Zn in blood fractions and whole blood of humans by ICP-OES

Inductively coupled plasma – optical emission spectrometry (ICP-OES) was applied to the determination of the elements Ca, Mg, Fe, Cu, and Zn in blood plasma, erythrocytes, lymphocytes, and whole blood to obtain reliable data on their distribution in blood fractions. The samples were carefully collected to avoid contamination. Two different nebulizers (Babington and Meinhard) were tested and optimized for this analytical problem. Line selections for all elements of interest were performed (LODs were 0.8 μg/L for Ca, 1.7 μg/L for Cu, 3.0 μg/L for Fe, 1.1 μg/L for Mg, and 4.2 μg/L for Zn). Recoveries were determined as approx. 100%, and standard reference material was analyzed to obtain reliable data on element distribution. The optimized method was applied to the determination of Ca, Mg, Fe, Cu, and Zn in the course of a clinical study on blood and blood fractions of two groups of humans of differing health. The concentrations measured in blood fractions were verified by balancing with the values found in whole blood.     

An HPLC method with diode array detector for the simultaneous quantification of chloroquine and desethylchloroquine in plasma and whole blood samples from Plasmodium vivax patients in Vietnam,using quinine as an internal standard

A sensitive, simple method for quantification of chloroquine (CQ) and desethylchloroquine (MCQ) in whole blood and plasma from Plasmodium vivax patients has been developed using HPLC with diode array detection (DAD). Solid‐phase extraction on Isolute‐96‐CBA was employed to process 100 μL of plasma/whole blood samples. CQ, MCQ and quinine were separated using a mobile phase of phosphate buffer 25 mm , pH 2.60–acetonitrile (88:12, v/v) with 2 mm sodium perchlorate on a Zorbax SB‐CN 150 × 4.6 mm, 5 μm column at a flow rate of 1.2 mL/min, at ambient temperature in 10 min, with the DAD wavelength of 343 nm. The method was linear over the range of 10–5000 ng/mL for both CQ and MCQ in plasma and whole blood. The limit of detection was 4 ng/mL and limit of quantification was 10 ng/mL in both plasma and blood for CQ and MCQ. The intra‐, inter‐ and total assay precision were <10% for CQ and MCQ in plasma and whole blood. In plasma, the accuracies varied between 101 and 103%, whereas in whole blood, the accuracies ranged from 97.0 to 102% for CQ and MCQ. The method is an ideal technique with simple facilities and instruments, bringing about good separation in comparison with previous methods. © 2016 The Authors Biomedical Chromatography Published by John Wiley & Sons Ltd     

Liquid‐phase microextraction combined with liquid chromatography‐mass spectrometry. Extraction from small volumes of biological samples

Liquid‐phase microextraction (LPME) is a sample preparation technique based on disposable polypropylene hollow fibres, which results in efficient sample clean‐up and high preconcentration. The present paper describes the combination of LPME with LC‐MS utilising electrospray ionisation for high sensitivity. Nine antidepressant drugs were extracted from 50 or 500 μL of plasma or whole blood samples, through a thin layer of dodecyl acetate immobilised in the pores of the hollow fibre, and into 15 μL of 200 mM formic acid as acceptor solution inside the hollow fibre. Analyte recoveries in the range 12–68% and 9–52% were obtained from 50 μL of plasma and whole blood respectively. The acceptor solution (15 μL) was diluted with 60 μL of 5 mM ammonium formate pH = 2.7 prior to injection into the LC‐MS system. The system was qualitatively investigated for matrix effects utilising a post‐column infusion system. Whole blood from 5 different persons was cleaned‐up by LPME and injected onto the analytical column while a solution of the 9 model compounds was continuously infused post‐column. No signs of ion suppression were seen for any of the model compounds. Limits of quantification (S/N = 10) were in the low ng/mL range for 6 of the 9 model compounds utilising a whole blood sample volume of only 50 μL. The repeatability of the extractions was investigated utilising paroxetine as internal standard. Acceptable RSDs (%) were obtained (< 20%) for 5 of the antidepressants. By increasing the sample volume from 50 to 500 μL of whole blood RSDs below 20% (3–16%) were observed for all 8 antidepressants.     

Magnetically-actuated blood filter unit attachable to pre-made biochips

We present a novel blood filter unit that is designed to separate blood plasma from whole blood by simple magnetic actuation. A non-diluted blood sample is dropped into the filter unit and magnetic attraction is applied to squeeze out only blood plasma while blood particles are filtered by membranes stacked in the filter unit. The new filter device yields good filtering performance with nearly perfect filtering efficiency (～99.999%), high plasma recovery (～30%), low blood consumption (<50 μl), and fast operation (～1 min). Because it is simple to operate and is attachable to any kind of pre-made biochip, it has commercial potential in various lab-on-a-chip applications for blood tests.     

ETAAS method for the determination of Cd, Cr, Cu, Mn and Se in blood fractions and whole blood

An electrothermal atomic absorption method (ETAAS) for the direct determination of trace elements (Cd, Cr, Cu, Mn, Se) both in blood fractions (erythrocytes, plasma and lymphocytes) and whole blood was developed. Zeeman background correction and graphite tubes with L’vov platforms were used. Samples were ¶diluted with HNO₃/Triton X-100 and pipetted directly ¶into the graphite tube. Ashing, pretreatment and atomization steps were optimized carefully for the different fractions and elements applying different matrix modifiers ¶for each element. For the lymphocyte fraction a multi-fold injection technique was applied. Low detection limits ¶of the ETAAS method (Cd 0.13 μg/L, Cr 0.11 μg/L, ¶Cu 0.52 μg/L, Mn 0.13 μg/L, Se 0.7 μg/L of whole blood) combined with small quantities of sample necessary for analysis allow determination of trace elements in this matrix. Verification of possible differences in the trace element status of humans was performed with statistical significance (P < 0.05). In addition, a contribution to the determination of normal values of essential elements was achieved. The method was applied for determination of trace elements in human blood and blood fractions of two groups (n = 50) different in health status.     

Direct analysis of whole blood by internal surface reversed-phase chromatography: an examination of the binding and metabolism of technetium dioxime complexes.

We have developed a method using internal surface reversed-phase (ISRP) packing for rapid on-line separation of small hydrophobic compounds from cellular whole blood components. This is achieved by the use of 75-microns ISRP chromatographic material packed into a small high-performance liquid chromatographic (HPLC) column, in conjunction with column switching. We have applied this analytical method to study the in vitro metabolism of 99mTc-BATO (boronic acid adducts of technetium dioxime) cerebral and myocardial perfusion tracers in whole blood. The results from the ISRP procedure were compared with a conventional centrifugation method of analysis. This novel HPLC methods provides a rapid, convenient and reliable method for the analysis of radioactive and non-radioactive lipophilic components in whole blood.     

Using a co-culture microsystem for cell migration under fluid shear stress

We have successfully developed a microsystem to co-cultivate two types of cells with a minimum defined gap of 50 μm, and to quantitatively study the impact of fluid shear stress on the mutual influence of cell migration velocity and distance. We used the hydrostatic pressure to seed two different cells, endothelial cells (ECs) and smooth muscle cells (SMCs), on opposite sides of various gap sizes (500 μm, 200 μm, 100 μm, and 50 μm). After cultivating the cells for 12 h and peeling the co-culture microchip from the culture dish, we studied the impacts of gap size on the migration of either cell type in the absence or presence of fluid shear stress (7 dyne cm(-2) and 12 dyne cm(-2)) influence. We found that both gap size and shear stress have profound influence on cell migration. Smaller gap sizes (100 μm and 50 μm) significantly enhanced cell migration, suggesting a requirement of an effective concentration of released factor(s) by either cell type in the gap region. Flow-induced shear stress delayed the migration onset of either cell type in a dose-dependent manner regardless of the gap size. Moreover, shear stress-induced decrease of cell migration becomes evident when the gap size was 500 μm. We have developed a co-culture microsystem for two kinds of cells and overcome the conventional difficulties in observation and mixed culture, and it would have more application for bio-manipulation and tissue repair engineering.     

Preparation and properties of plasma-initiated graft copolymerized membranes for blood plasma separation

A hydrophilic composite membrane for blood plasma separation has been prepared by surface graft copolymerization initiated by low-temperature plasma (LTP). After short LTP pre-irradiation onto a microporous polypropylene (PP) membrane, N-N-dimethylacrylamide (DMAA) vapor was introduced for grafting. The PP membrane had a 0.45 μm effective pore size and a 130 μm thickness. The rate of DMAA grafting onto PP was very high, even in vapor-solid phase reaction under reduced pressure; DMAA 1 mm Hg (133Pa). The percentage of grafted poly-DMAA (PDMAA) reached 15% within 5 min post graft polymerization, and the membrane surface, including the interior surface of pores, became completely hydrophilic. There was no apparent change observed in the membrane morphology in the dry state after the PDMAA-grafted layer was formed. However, water flux significantly decreased, probably due to swelling of the PDMAA-grafted layer. With a grafting yield below 17%, the PDMAA-grafted PP (PP-g-PDMAA) membrane showed a good separation capability of plasma from whole blood. The PP-g-PDMAA membrane exhibited low complement activating potential, high sieving coefficient for plasma proteins and high blood compatibility. Decreases in adsorption of blood cells, plasma proteins, and other biomolecules may be the reason for the membrane performance.     

Determination of vitamin C and preservatives in beverages by conventional capillary electrophoresis and microchip electrophoresis with capacitively coupled contactless conductivity detection

The separation and detection of commonly used preservatives (benzoate, sorbate) and vitamin C by both conventional CE and microchip electrophoresis with capacitively coupled contactless conductivity detection is presented. The separation was optimized by adjusting the pH-value of the buffer and the use of hydroxypropyl-beta-CD (HP-beta-CD) and CTAB as additives. For conventional CE, optimal separation conditions were achieved in a histidine/tartrate buffer at pH 6.5, containing 0.025% HP-beta-CD and 0.1 mM CTAB. LOD ranged from 0.5 to 3 mg/L (S/N = 3) and the RSDs for migration time and peak area were less than 0.1 and 2%, respectively. A considerable reduction of analysis time can be accomplished by using microchip electrophoresis without significant loss in sensitivity under optimal separation conditions. A histidine/tartrate buffer at pH 6.5, incorporating 0.06% HP-beta-CD and 0.25 mM CTAB, gave detection limits ranging between 3 and 10 mg/L and satisfactory reproducibilities of < or =0.4% for the migration time and < or =3.5% for the peak area. The methods developed are useful for the quantitative determination of food additives in real samples such as soft drinks and vitamin C tablets.     

蛋白质的微芯片电泳分离及其与脱氧核糖核酸迁移规律的比较

在自制的聚二甲基硅氧烷(PDMS)微芯片上,使用十二烷基磺酸钠(SDS)无胶筛分电泳分离体系(10 g/L的羟乙基纤维素(HEC), 1 g/L的SDS, 40 mmol/L磷酸盐缓冲溶液,pH 7.0),采用在线自校正激光诱导荧光检测方法,在6.4 min内高效分离了异硫氰酸荧光素(FITC)衍生的6种蛋白质标准样品,连续6次电泳所得迁移时间的相对标准偏差(RSD)均小于10%。用自主建立的脱氧核糖核酸(DNA)定量分离模型对蛋白质迁移数据进行拟合,发现SDS-蛋白质复合物迁移规律与DNA相似,但迁移淌度与相对分子质量及电场强度之间的线性关系明显变差,可见原DNA分离模型要扩展到蛋白质范围必须对一些参数进行校正。     

A simple and demountable capillary microflow nebulizer with a tapered tip for inductively coupled plasma mass spectrometry

A simple and demountable capillary microflow nebulizer (d-CMN) was developed for inductively coupled plasma mass spectrometry (ICP-MS). It consisted of a nebulizer body, a fused-silica capillary with a tapered tip and a polytetrafluoroethylene (PTFE) adapter. The gas orifice i.d., the solution capillary tip i.d. and its wall thickness were 200, 30, and 5 μm, respectively. The sensitivities, detection limits, precisions and the long-term stability with the d-CMN were evaluated. The experimental results indicated that its performances at low uptake rates were similar or better than those obtained with the conventional concentric nebulizer at 820 μL/min and the micromist nebulizer at 200 μL/min. The demountable construction of the d-CMN permitted that the blocked or broken solution capillary could be conveniently renewed. The low self-aspiration rate (4.77 μL/min) and the analytical characteristics comparable to commercial microflow nebulizers made the d-CMN a good choice for coupling capillary electrophoresis and microbore high-performance liquid chromatograph to ICP-MS. The proposed d-CMN was successfully applied in the iodine speciation by coupling microchip capillary electrophoresis (MCE) to ICP-MS. The absolute detection limits for iodide and iodate of 0.20 and 0.29 fg were achieved with satisfactory resolution.     

High-performance liquid chromatographic determination of cocaine and benzoylecgonine by direct injection of human blood plasma sample into an alkyl-diol-silica (ADS) precolumn

A column-switching high-performance liquid chromatographic method with UV detection for the determination of cocaine (COC) and benzoylecgonine (BZE) in human blood plasma samples is described. The method uses an alkyl-diol-silica ADS-C18 extraction precolumn. A 50- micro L plasma sample was introduced to the ADS precolumn in order to separate the analytes from proteins and endogenous compounds. The fraction containing COC and BZE was back-flushed and transferred to an Alltech mixed-mode C(18)/cation-exchange analytical column for final separation. The validation of the method revealed quantitative recoveries from 95.0 to 99.0% for COC at three different concentrations (0.5, 1.0 and 2.0 micro g mL(-1)), and from 96.0 to 99.0% for BZE at the same concentration levels with coefficients of variation <4.00% (n=5). The detection limit (signal to noise ratio (S/N)>3) was 0.03 micro g mL(-1) for all the compounds with an injection volume of 50 micro L. However, it was possible to enhance the sensitivity further by injecting larger plasma volumes, up to 200 micro L, at the same optimal conditions. The overlap of sample preparation, analysis and reconditioning of the extraction column, increase the overall sample throughput to 5 samples h(-1). The developed method has been applied to human blood plasma samples from subjects suspected of cocaine abuse.     

On-Line Coupling of Separation Techniques to NMR

The hyphenation of chromatographic separation techniques with NMR spectroscopy is one of the most powerful and time-saving methods for the separation and structural elucidation of unknown compounds and molecular compositions of mixtures. Most of the routinely used NMR flow-cells have detection volumes between 40–180 μL for conventional separations with analytical columns, and the newest designs employ detection volumes in the order of 200 nL for capillary separations. The low flow rates used in capillary chromatography permit the use of deuterated solvents. Unequivocal structural assignment of unknown chromatographic peaks is possible by two-dimensional stopped-flow capillary HPLC-NMR experiments.