Bang, D.D.

El-Ali, Jamil

Perch-Nielsen, Ivan Ryberg

Poulsen, Claus Riber

Wolff, Anders

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 16, 2017Removal of PCR inhibitors using dielectrophoresis for sample preparation in amicrofabricated systemWolff, Anders; Perch-Nielsen, Ivan R.; Poulsen, Claus R.; El-Ali, Jamil; Bang, D.D.Published in:Proceedings of the Seventh International Conference on Miniaturized Chemical and Biochemical AnalysisSystems (MicroTAS)Publication date:2003Document VersionPeer reviewed versionLink back to DTU OrbitCitation (APA):Wolff, A., Perch-Nielsen, I. R., Poulsen, C. R., El-Ali, J., & Bang, D. D. (2003). Removal of PCR inhibitors usingdielectrophoresis for sample preparation in a microfabricated system. In Proceedings of the SeventhInternational Conference on Miniaturized Chemical and Biochemical Analysis Systems (MicroTAS) (pp. 1137-1140)REMOVAL OF PCR INHIBITORS USING DIELECTROPHORESIS FOR SAMPLE PREPARATION IN A MICROFABRICATED SYSTEM.  A. Wolff1*, I. R. Perch-Nielsen1, C. R. Poulsen1, J. El-Ali1, D. D. Bang2 1 Mikroelektronik Centret (MIC), Technical University of Denmark (DTU),  Building 345 east,DK-2800 Kongens Lyngby, Denmark   2 Department of Poultry, Fish, and Fur Animals, Danish Veterinary Institute, 2, Hangøvej, DK-8200 Aarhus N, Denmark  Abstract  In this abstract we present the results of a novel method for sample preparation to remove inhibitory substances from samples that would prevent DNA amplification. The method is based on dielectrophoresis and is suitable for integration in PTAS.  Keywords: Sample preparation, Dielectrophoresis, PCR inhibitors, DEP, Cells   1. Introduction  Polymerase chain reaction (PCR) has been used to amplify specific DNA sequences and to detect microorganisms in clinical diagnostic and food industry. However, when conducting PCR with biological materials (blood, milk, feces etc.), residual materials in the sample may significantly or totally inhibit the PCR process [1, 2]. Conventional methods for sample preparation prior to PCR can involve intensive manual operations that may be very difficult or impossible to integrate in PTAS. We present a novel method for sample preparation based on dielectrophoresis (DEP). DEP is the movement of dielectric particles e.g. cells by the forces imposed upon them by an inhomogeneous electric field and DEP has been used for manipulating cells in microsystems [3].  2. Experimental  The microsystem was a DEP-chip fabricated on a silicon substrate. A 2 μm LPCVD (Low Pressure Chemical Vapour Deposition) nitride layer was deposited on the silicon substrate to insulate the electrodes from the substrate. On top of that a 180 μm LPCVD polysilicon layer was deposited. An 80 nm layer of titanium was deposited using E-beam evaporation and structured using a lift of process. To remove the natural oxide a 30 second HF (hydrofluoric acid) dip was applied just before titanium deposition. The silicide was formed using rapid thermal annealing for 1 minute at above 700qC in an argon atmosphere. The electrode design was an interdigitated-finger structure [4] with a characteristic dimension of 10 μm and a length of 2 mm (figure 1). The walls of the channel were made of SU-8 [5] and the channel was height  width = 70  400 μm. The total channel length in the microstructure was 11 mm.         Figure 1. Left: diagram of microfluidic structure with integrated silicide electrodes. Right: SEM picture of the electrodes   PCR was performed using the primers of a yeast housekeeping gene (ribosomal protein S3). PCR program: 10 minutes at 94qC, 35 cycles of 94qC for 30 seconds, 59qC for 30 seconds and 72qC for 1 minute. The final step was 72qC for 10 minutes.  3. Results and Discussions  In this study, we investigated the inhibitory effect of hemoglobin and heparin on PCR. Both hemoglobin and heparin are natural part of blood and heparin is furthermore often added to blood samples to prevent coagulation. Hemoglobin or heparin was added to PCR mixture. The target DNA for amplification was in the form of whole yeast cells (Saccharomyces cerevisiae) and the PCR reactions were performed in tubes using a conventional thermocycler (table 1). These initial experiments show that the PCR reactions were completely inhibited by a hemoglobin concentration of 0.1 mg/ml and by a heparin concentration of 1.3u10-3 mg/ml.  Table 1. PCR inhibition effects of hemoglobin and heparin, respectively. (+): PCR positive  (-): no PCR product, (n.t.): not tested. Pos/neg: positive and negative controls.    To test whether DEP can be used to remove the PCR inhibitors, we used a microfluidic structure with integrated silicide electrodes (figure 1). The yeast cells were mixed with hemoglobin (10 mg/ml) or heparin (1.3 mg/ml) and these inhibitor/yeast samples were prepared for PCR in 3 steps (figure 2 and 3): First the sample is sucked into the microstructure. During this step the yeast cells are captured on the electrodes by the electric field using positive dielectrophoresis (DEP) (figure 4). Secondly, the inhibitor is wash away while the cells remain captured on the electrodes. Finally, the electric field is turned off, and cells are released and collected. The PCR were performed directly on Hemogl. 10 mg/ml 1 mg/ml 100 μg/ml 10 μg/ml 1 μg/ml 100 ng/ml pos neg PCR result – – + + + + + – Heparin 1.3 mg/ml 130  μg/ml 13  μg/ml 1.3  μg/ml 130 ng/ml 13 ng/ml pos neg PCR result – – – + + + + – these yeast cells. The initial hemoglobin or heparin concentration was 3 orders of magnitudes higher than the PCR inhibition level (table 1). A washing for 5 and 10 minutes for hemoglobin and heparin could remove the inhibitory effect, respectively (table 2).   Figure 2. Schematic overview shows the first step in which the sample is sucked into the microstructure and cells captured on the electrodes   Figure 3. The 3 steps in the sample preparation: collection of cells, washing, and sample release    Figure 4. A series of 3 images showing the build up of cells as they collect on the electrodes under the influence of positive DEP.  4. Conclusions In conclusion, in the present study we showed that yeast could be selectively withheld while PCR inhibitors are removed by using dielectrophoresis in a microsystem. The required volumes (sample and wash) are small, and in the order of 30 μl. The dielectrophoretic filter can be used to collect any cell type, without altering the set up much. In contrast to conventional methods the sample preparation method presented here is suited for integration with microstructures for PCR reaction and DNA analysis. This work is an important contribution towards sample preparation, PCR reaction and DNA analysis in a micro total analysis system for molecular diagnostics.            Table 2. Sample preparation in the microstructure. Result of the PCR reaction after washing with a flow rate of 0.2 ml/h for various time. (+): PCR positive (-): no PCR product, (n.t.): not tested. Pos/neg: positive and negative controls.  Acknowledgements  We thank Mrs Mette Hansen and Annie Brandstrup (Danish Veterinary Institute, Aarhus) for their technical assistance. This work was supported by the Danish Technical Research Council (grant no. 9901659).  References 1. Abu Al-Soud, W., L.J. Jonsson, and P. Radstrom, Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. Journal of Clinical Microbiology, 2000. 38(1): p. 345-350. 2. Lantz, P.G., R. Knutsson, Y. Blixt, W. Abu Al-Soud, E. Borch, and P. Radstrom, Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components. International Journal of Food Microbiology, 1998. 45(2): p. 93-105. 3. Goater, A.D. and R. Pethig, Electrorotation and dielectrophoresis. Parasitology, 1998. 117: p. S177-S189. 4. Burt, J.P.H., R. Pethig, and M.S. Talary, Microelectrode devices for manipulating and analysing bioparticles. Transactions of the Institute of Measurement and Control, 1998. 20(2): p. 82-90. 5. Cui, L. and H. Morgan, Design and fabrication of travelling wave dielectrophoresis structures. Journal of Micromechanics and Microengineering, 2000. 10(1): p. 72-79. 6. Huang, Y., E.L. Mather, J.L. Bell, and M. Madou, MEMS-based sample preparation for molecular diagnostics. Analytical and Bioanalytical Chemistry, 2002. 372(1): p. 49-65.  min. hemogl. 1 hemogl. 2 heparin 1 heparin 2 0 – – – – 2.5 – – – – 5 + + – – 7.5 n.t. + n.t. – 10 + + + + 30 + n.t. + n.t. pos + + + + neg – – – – 

Removal of PCR inhibitors using dielectrophoresis for sample preparation in a microfabricated system

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Removal of PCR inhibitors using dielectrophoresis for sample preparation in a microfabricated system

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