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Ned Tijdschr Klin Chem Labgeneesk 2004; 29: 295-296 Direct measurement of lithium in whole blood using a capillary electrophoresis
microchip
E.X. VROUWE1, P. KÖLLING2, R. LUTTGE1 and A. van den BERG1 Introduction
Receiving clinically relevant blood parameters even Blood was obtained performing the finger stick for off-chip cleaned up sample is a nontrivial analy- method on a volunteer using Haemolance (HaeMedic tical problem. Few devices have been developed that AB, Sweden) disposable lancets. Approximately 30 fully exploit combining multiple functions in so- µl of blood was collected with a pipette and trans- called micro total analysis systems (1, 2). Instead of ferred to a plastic tube. For experiments requiring advanced component integration on chip, here we whole blood 10 µl of 0.0806 mol/l sodium citrate demonstrate that the measurement of alkali metals (Sigma) was added to stop coagulation whereas when can be performed in a drop of whole blood using an serum was required the sample was allowed to clot established type of capillary electrophoresis mi- for 10 minutes. Serum was collected after centrifug- crochip (3-7) by applying the principles of moving ing the sample for 10 minutes at 11,500 g. From each boundary electrophoresis. The emphasis will be on sample an aliquot of 18 µl was spiked with 2 µl of 20 the determination of lithium, which is used in the mmol/l lithium just before the start of an experiment treatment of manic-depressive mood disorders. The in order to obtain a concentration of 2 mmol/l.
low therapeutic index of lithium in blood is criticalthroughout the treatment and is therefore an ideal candidate for the demonstration of a microchip for Performing capillary electrophoresis on the microchip the sample was diluted on-chip in the BGE during thesample loading step. Due to electrophoretic mobility Materials and methods
difference between the cells and the analytes of interesta cell-free sample plug was formed in the double-T.
Because of the EOF suppression, electromigration The inset of Figure 1 shows the CE chip made of dominates and therefore the blood cells, which carry glass. Electrodes were integrated in the device for a negative charge, migrate in the opposite direction of conductivity detection. To reduce both the electro- the cations. We observed that the formed sample plug osmotic flow (EOF) and the adsorption of proteins to matches the conductivity of the BGE, which can be the chip surface the channels were coated with poly- explained by moving boundary electrophoresis dur- acrylamide according to the procedure of Hjertén for ing sample loading. In figure 1, the control run using fused silica capillaries (8). During the experiments (a) whole blood without lithium is compared with (b) the chip was placed in a custom-made holder, which the same blood sample spiked with lithium. Separa- was placed on an inverted microscope to follow the tion of the blood sample plug resulted in three peaks filling of the channels with the buffer as well as for originating from potassium, sodium and lithium. For tracking the cells. For the CE experiments a back- determining the lithium concentration, the sodium ground electrolyte (BGE) consisting of 50 mmol/l 2- peak was used as internal standard assuming a fixed (N-morpholino)-ethanesulfonic acid (Sigma, Ger- concentration of 140 mmol/l. By doing so an error of many) and 50 mmol/l histidine (Fluka, Switzerland) less than 4% will be introduced in the calculated with a pH of 6.1 was used. When whole blood was lithium concentration due to variations within the used as sample 200 mmol/l glucose (Sigma) was normal limits for sodium. For quantitation of whole added to the BGE to adjust the osmotic strength. For blood samples prepared by manual spiking with the separations a computer controlled high voltage lithium there is an uncertainty in the exact concentra- power supply with four independently controllable tion of lithium in the plasma since it is not known positive voltage outputs was used together with a how fast the cells take up the lithium. The actual con- custom-made conductivity detector. An optimized centration of the spiked sample can therefore be in voltage scheme was applied to form a sample plug in the range of 3.4 mmol/l (no lithium uptake) to 2.0 the double-T (9). The separation was performed using mmol/l (equal distribution between plasma and cells).
Based on calibration runs a concentration of 2.7mmol/l (2.1% RSD, n=3) was calculated from theelectropherograms. This was compared to experi-ments performed on serum in which the lithium con- MESA+ Research institute1, University of Twente, En-schede; Hospital Group, Medisch Spectrum Twente2, centration was exactly 2.0 mmol/l (Fig. 1c). In that case the concentration was determined at 1.8 mmol/l Ned Tijdschr Klin Chem Labgeneesk 2004, vol. 29, no. 5 with a detection limit of 0.4 mmol/l. The measure-ment error in the size of the peak area does not suffi-ciently explain the error of approximately 10% on therecovery for the serum sample. Depending on thechoice of buffer system the concentration profile ofions in the sample plug may be influenced by the ma-trix composition of sample and does not necessarilyrepresent the original concentration. To investigatethe sources of error in detail more experiments are Conclusion
The experiments demonstrated that lithium was sepa-
rated from a drop of whole blood with capillary elec-
Figure 1. Results of a separation of a) whole blood without
trophoresis on a microchip within two minutes. Cur- lithium, b) whole blood spiked with 2 mmol/l lithium and c) rently further investigations are conducted studying blood serum spiked with 2 mmol/l lithium. The inset shows a the process of concentration adjustment during the photograph of the CE chip with a capillary length of 2 cm(Micronit Microfluidics, The Netherlands) with a blow-up sample loading in order to improve on the accuracy of the end-column conductivity detection electrodes and the for quantitation. In addition, potassium was detected double-T injection region defining the size of the sample plug by this method and more study is currently dedicated dispensed into the separation channel.
to separate calcium and magnesium in order to utilizethe full potential of these microchips for ‘point ofcare’-testing. 5. Lichtenberg J, Rooij NF de, Verpoorte E. Electrophoresis References
6. Tanyanyiwa J, Abad-Villar EM, Fernandez-Abedul MT, Costa-Garcia A, Hoffmann W, Guber AE, Herrmann D, 1. Verpoorte E. Electrophoresis, 2002; 23: 677-712.
Gerlach A, Gottschlich N, Hauser PC. Analyst 2003; 128: 2. Berg A van den, Lammerink TSJ. Topics Curr Chem 1998; 7. Berthold A, Laugere F, Schellevis H, Boer CR de, Laros M, 3. Guij RM, Baltussen E, Steen G van der, Schasfoort RBM, Guijt RM, Sarro PM, Vellekoop MJ. Eelectrophoresis Schlautmann S, Billiet HAH, Frank J, Dedem GWK van, Berg A van den. Electrophoresis 2001; 22: 235-241.
8. Hjertén S. J Chromatogr 1985; 347: 191-198.
4. Pumera M, Wang J, Opekar F, Jelinek I, Feldman J, Lowe 9. Vrouwe EX, Luttge R, Berg A van den. Electrophoresis, H, Hardt S. Anal Chem 2002; 74: 1968-1971.
Ned Tijdschr Klin Chem Labgeneesk 2004; 29: 296-297 Apoptosis induced kinetic changes in autofluorescence of HL60 cells –
application for single cell analysis on chip
F. WOLBERS1,2, A. VALERO2, H. ANDERSSON2, R. LUTTGE2, A. van den BERG2 and I. VERMES1 Introduction
household of the cell. This paper presents a new Natural cellular autofluorescence (AF) can be a use- method using AF to study apoptosis. Apoptosis or ful tool to unravel intracellular pathophysiological programmed cell death plays an important role in processes and distinguish normal from diseased tis- maintaining a homeostatic equilibrium between cell sue. Many cellular metabolites exhibit autofluores- proliferation and cell death. Induction of apoptosis cence, e.g. NAD(P)H and flavins, which colocalizes results in shrinkage of the cell and fragmentation into strongly within the mitochondria and in some extent apoptotic bodies (7). AF intensity is first measured to the lysosomes (1-6). Both components are actively conventionally at the flow cytometer (FCM) and fi- involved in a number of metabolic processes within nally the results will be translated on to a micro- the cell and play an important role in the energy fluidic chip to perform single-cell analysis.
Autofluorescence measurements
Department of Clinical Chemistry1, Medisch Spectrum Human promyelocytic leukemic HL60 cells were in- Twente, Hospital Group and Department of Sensorsys- cubated with camptothecin (CPT), tumour necrosis tems for Biomedical and Environmental Applications2,MESA+ Institute, University of Twente, Enschede, The factor (TNF)-a in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gy, during varying Ned Tijdschr Klin Chem Labgeneesk 2004, vol. 29, no. 5

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