Oct 11, 2007 - method is based on on-chip freeze-drying of reagents for lab-on-a-chip applications. The stability of three enzymes, viz. Polymerase ...
A SIMPLE AND EFFICIENT METHOD FOR ON-CHIP STORAGE OF REAGENTS: TOWARDS LAB-ON-A-CHIP SYSTEMS FOR POINT-OF-CARE DNA DIAGNOSTICS Monica Brivio,1 Yiping Li,2 Annika Ahlford, 3 Bastian Gaardsvig Kjeldsen,1 Jakob Leffland Reimers,1 Minqiang Bu,1 Ann-Christine Syvänen, 3 Dang Duong Bang2 and Anders Wolff1 1
Dept. of Micro and Nanotechnology, Technical University of Denmark, DENMARK, 2 National Veterinary Institute, Technical University of Denmark, DENMARK and 3 Molecular Medicine, Dept. Medical Sciences, University Hospital, Uppsala, SWEDEN ABSTRACT A method for on-chip storage of reagents for point-of-care diagnostics is presented. The method is based on on-chip freeze-drying of reagents for lab-on-a-chip applications. The stability of three enzymes, viz. Polymerase, Exonuclease I and Shrimp Alkaline Phosphatase throughout freeze drying and storage were investigated. As a proof of concept, onchip DNA amplification was carried out using on-chip freeze-dried reagents. The results reported here are a key step towards the development of a ready to use tool for point-ofcare genetic diagnostics. Keywords: lab-on-a-chip, diagnostics, genotyping, freeze-drying. 1. INTRODUCTION Current state-of-the-art genotyping techniques used in centralized laboratories require bulky equipment and laborious procedures. By integrating miniaturized components and automating functionalities, lab-on-a-chip technologies offer a valuable tool to perform fast, cost-effective and efficient point-of-care DNA analysis [1]. In the EC-funded SMART-BioMEMS project a microfluidic platform for genotyping of single nucleotide polymorphisms (SNPs) is being developed. Genotyping is based on PCR-amplification followed by minisequencing [2]. Three independent enzymatic steps, i.e. PCR, PCR clean-up Figure 1. Schematic representation of a and minisequencing are carried out on generic polymer chip with reaction chambers polymer chips, where the freeze-dried reafor storage of freeze-dried reagents. gents are stored, see Figure 1. 2. EXPERIMENTAL A premixed solution of all PCR reagents was introduced into the reaction chambers of three different polymer chips. After filling, the chips were stored overnight at –80 ºC and then transferred to a freeze-drier. Photographs of three 200 m deep polymer chambers with freeze-dried PCR reagents are displayed in Figure 2.
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Figure 2. Freeze-drying for on-chip storage of reagents has been investigated in two European projects: OPTOLABCARD and SMART-BioMEMS. Pictures of PCR reagents freeze-dried in (a) a 7 L chamber milled in Topas, (b) a 10 L SU-8 chamber microfabricated on a Pyrex substrate and (c) a 6 L SU-8 chamber. The freeze-dried PCR reagents stored in the polymer chambers were redissolved upon addition of DNA solution. The chips were then transferred to a flat bed where PCR was carried out. Examples of results of on-chip freeze-drying and subsequent PCR are displayed in Figure 3.
Figure 3. Gel chromatogram of on-chip PCR carried out in the closed SU-8 PCR chips of (a and b) Figure 2b and (c) Figure 2c, respectively. 3. RESULTS AND DISCUSSION The stability of freeze-dried reagents during storage is a key issue. The activity of PCR reagents after six months of storage at room temperature (RT), at 4 ºC and at –20 ºC was measured by real-time PCR. No Polymerase activity was left after storage at room temperature. However, about half or complete polymerase activity was recovered after storage at 4 ºC and –20 ºC, respectively (Figure 4). Unlike the thermo-stable polymerrase, used for the PCR, the enzymes used for the PCR clean-up, Exonuclease I (Exo) and Shrimp Alkaline Phosphatase (SAP), are thermo-labile. Accelerated stability tests were carried out for these enzymes, using TaqMan probe and fluorescein diphosphate as substrates for Exo and SAP, respectively. Samples prepared with “low” and “high” lyoprotectant concentration (Table 1) were freezedried and incubated at 37 ºC and 60 ºC. The residual enzyme activity was Figure 4. Stability tests of freeze-dried PCR evaluated by end-point fluorescence reagents after six months storage at room measurements. With “low” temperature (RT), 4 °C and –20 ºC. (a) Gel lyoprotectant concentration the activity chromatogram and (b) and melting curve of both Exo (Figure 5) and SAP (Figure measurements of real-time PCR at various 6) was rapidly lost, regardless of the concentration of DNA template. storage temperature. With “high”
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lyoprotectants concentration no residual activity of SAP was measured whereas that of Exo decreased of about 20% after 5 days at 60 ºC. Remarkably, the activity of both Exo and SAP stored at 37 ºC for 5 days remained virtually unchanged, demonstrating the feasibility of the method for labile enzymes. Table 1. Lyoprotectants used to stabilize EXO and SAP during freeze-drying. Lyoprotactants concentration lyoprotactants (%w/vol) high low Threalose 5% 1% Mannitol 2% 0.5% PEG 1.5% 0.5% Dextran 1.5% 0.5%
Figure 5. Accelerated stability tests of freezeFigure 6. Accelerated stability tests of dried Exo. Samples with “low” (dashed lines) freeze-dried SAP. Samples with “low” and “high” (solid lines) lyoprotectant con(dashed lines) and “high” (solid lines) lyocentration (Table 1) were stored at both 37 ºC protectant concentration (Table 1) were () and 60 ºC (). stored at both 37 ºC () and 60 ºC (). 4. CONCLUSIONS Our results clearly demonstrate the feasibility of storing freeze-dried reagents on-chip for point-of-care diagnostics. After testing each step of the genetic analysis, we are currently working on the integration and automation of all steps on a single platform. ACKNOWLEDGEMENTS The authors would like to thank Ikerlan for providing the structures of Figure 2c and both SMART-BioMEMS and OPTOLABCARD European projects for financial support. REFERENCES [1] C. H. Mastrangelo et al., Microfabricated Devices for Genetic Diagnostics, Proceedings if IEEE, 86, 1769 (1998). [2] A. C. Syvanen, et al., Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA, Nucleic Acid Research, 31, 1 (2003).
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