Chinese Chemical Letters Vol. 17, No. 8, pp 1101-1104, 2006 http://www.imm.ac.cn/journal/ccl.

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High Purity DNA Extraction with a SPE Microfluidic Chip Using KI as the Binding Salt

Xing CHEN, Da Fu CUI, Chang Chun LIU

State Key Laboratory of Transducer Tech., Institute of Electronics, Chinese Academy of Sciences, Beijing 100080 Abstract: Based on solid phase extraction method, a novel silicon-PDMS-glass microchip for high purity DNA extraction has been developed by using KI as the binding salt. The microfluidic chip fabricated by MEMS technology was composed of a silicon substrate with a coiled channel and a compounded PDMS-glass cover. With this microfluidic chip, the wall of the coiled channel was used as solid phase matrix for binding DNA and DNA was extracted by the fluxion of the binding buffer, washing buffer and elution buffer. KI as a substitute for guanidine, was used successfully as binding salt for purification DNA, obtaining higher purity of genomic DNA and about 13.9 ng DNA from 1 L rat whole blood in 35 minutes. Keywords: MEMS, microfluidic, KI, solid phase extraction (SPE), DNA purification.

With the development of the micro electro mechanical system (MEMS) technology, micro total analytical systems (-TAS) which has the potential for integrating sample pretreatment, target amplification, and detection, has been in progress. The micro analytical systems can minimize sample loss and contamination problems as well as reduce analytic times. SPE method can be applied in microdevices for extracting DNA, which is a crucial step for the development of -TAS. Tian et al. 1 firstly established a micro SPE DNA purification system in a capillary packed with silica resin to purify DNA. But the procedure of packing solid phase is complex and difficult to accurately handle on a microchip platform. In addition, a high packing density in the SPE microdevice might create problems with backpressure as well as clogging with crude samples. After that, Cady2 fabricated micropillars array in a silicon wafer as solid phase matrix for purification DNA. Guanidine was used as binding salt for extracting DNA with those previous SPE microchips, but it was proved it to be an inhibitor of polymerase chain reaction (PCR)2, moreover guanidine is toxic and it might contaminate environment. To look for a new binding salt is necessary. In this letter, we reported a high purify DNA extraction method with a SPE microfluidic chip using KI as the new binding salt.

E-mail: dfcui@mail.ie.ac.cn

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Experimental SPE Microfluidic Chip Fabrication The microchip consisted of a poly(dimethylsiloxane)(PDMS)-glass cover and a silicon substrate as shown in Figure 1a, which was 2 cm1.5 cm. The silicon substrate included an etched coiled channel 25 cm long, 200 m wide and 120 m deep, and the cover included two holes which were drilled according to the silicon substrate. The overview of the process for fabrication of the silicon substrate was as follows. Firstly, Al was evaporated on a silicon wafer as a mask for etching. The wafer was spin coated with a positive photoresist and patterned. The exposed photoresist was developed and the exposed Al was removed by phosphoric acid. Then the wafer was etched by DRIE technology to produce a coiled channel and two ports, after Al was removed, the wafer was thermally oxidized and then bonded to a PDMS-glass cover to form a close channel (Figure 1b). The fabrication process of the cover was as follows. A flat glass substrate was exposed to a vapor of trimethylchlorosilane for 5 min in order to facilitate the glass substrate to release. A mixture of PDMS oligomer and cross-linking agent, which had been degassed under vacuum, was poured onto the glass substrate. Then the glass with two holes was put on the PDMS prepolymer gently and cured in an oven at 80C for 1 h. After the silanized glass substrate was peeled off and the excessive PDMS in the holes was removed, the PDMS-glass cover was achieved. DNA Extraction Procedure Before experiments, two pipette tips were fixed at the two holes as connections, shown in Figure 1b. One was connected to a pump, and the other was connected to a centrifugation tube for collection the solutions. The DNA extraction procedure consisted of load, wash, elution steps. Chips were washed with HNO3 and TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8) for 10 min prior to each extraction. Then, 20 L load buffer (5 mol/L KI in TE, pH 6.4; or 4 mol/L GuSCN in TE, pH 6.4) containing 1% Triton X-100 and 2 L rat whole blood was introduced and passed through the microchannel by the pump and DNA was binding onto walls of the channel. Proteins and possible PCR inhibitors that were adsorbed onto the wall which were removed by passing 15 L wash buffer (70 % ethanol) twice through the microchannel. Finally, 5 L TE buffer was introduced and held in the chip for 10min at 55C and then 20 L TE buffer was continuously flowed through the microchannel. Genomic DNA from rat whole blood by a microchip could be extracted in 35 min.
Figure 1 Layout of the microchip (a) and photogragh of the microchip (b) a b

High Purity DNA Extraction with a SPE Microfluidic Chip

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DNA Detection DNA in the load, wash, elution buffers collected in different centrifugation tubes were quantified by using SYBR Green I dye in a fluorometer. Standard curves for DNA were generated using lambda DNA. The extracted genomic DNA as template was qualified by PCR for amplifying 203-bp-Gapd gene and gel electrophoresis of PCR products. Results and Discussion DNA purification is based on the solid phase extract method, which relies on adsorption of DNA onto a solid surface, followed by washing the impurities such as protein, and finally the purity DNA eluted in aqueous solution. The mechanism of DNA adsorption on a solid surface elucidated by Melzak3 was that the adsorption of highly charged duplex DNA to negatively charged silica was controlled by three competing effects: weak electrostatic repulsion forces, dehydration and hydrogen bond formation. It is well known that the binding of DNA to silica, celite or glass powder in the presence of low pH chaotropic agents, such as NaClO4, GuHCl and GuSCN, which can efficiently weaken the electrostatic repulsion force, dehydrate both DNA and the surface of matrix, and lead to form hydrogen bond3, 4. Finally DNA was eluted with solutions of low ionic strength and high pH. We have previously demonstrated a SPE microchip for DNA purification using guanidium hydrochloride5, however, the purity of DNA is low for the presence of the residual guanidium. Now we used KI to replace guanidium for extracting high purity DNA. To value the effect of DNA purification on KI in microchip, 4 mol/L of GuSCN and 5 mol/L of KI were used to bind DNA from whole blood with our SPE microfluidic chip. The concentration of both binding salts was established for maximal binding. The extracted DNA was detected by fluorescence array to evaluate the quantity, while the extracted DNA was used as the template in PCR and then the products of PCR were run through gel electrophoresis to test the purity. As seen in Figure 2, 2 L whole blood was mixed in 20L KI or GuSCN binding buffers, respectively, then loaded into the chip (fraction 1, 2), washed with ethanol (fractions 3-4), and eluted with TE buffer (fractions 5-10). About 13.9 ng genomic DNA was extracted from 1 L rat whole blood by KI. A 203-bp fragment of Gapd gene was amplified, illustrating that the eluted genomic DNA from blood was PCR-amplifiable, the purity of the extracted DNA was higher by
Figure 2 DNA elution profile for genomic DNA from rat whole blood
Recovered DNA (ng)
15 12 9 6 3 0 0 3 6 9 12 GuSCN KI

25 L Collected Fractions

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Figure 3 PCR amplification for DNA extracted using a microchip

1 and 2 showed the result of PCR for DNA extracted by using GuSCN and KI, respectively. Purified DNA by using a commercial kit was used for the positive control reaction.

KI than that by GuSCN (Figure 3). The reason probably was that guanidine binding salt, which is a stronger denaturant of protein, which would be an inhibitor of Taq polymerase, whereas KI binding salt is not. Moreover the residual GuSCN in the eluted solution would alter the pH by affecting the dissociation of the PCR solution. In additional the amplification efficiency of PCR for DNA extracted by KI was comparable to that by a commercial kit (Figure 3). Based on the centrifugal technology, SPE method for extracting DNA could remove dramatically various buffers and most contaminations. But in microchip, DNA was extracted by the fluxion of solutions. All the buffers and sample, which included some PCR inhibitors, such as guanidine, could not be removed drastically during the flowing process, and a small quantity of them would remain in the eluted solution. Although GuSCN binding salt could extract DNA a little more, the purity of the extracted DNA might be reduced by the presence of the residual GuSCN. Moreover using KI instead of guanidine to promote DNA binding to silica offers another advantage that KI is a nontoxic, safe and cheap salt. Acknowledgments
The authors greatly acknowledge the financial support from the National Natural Science Foundation of China (No. 20299030, 60427001 and 60501020).

References
1. 2. 3. 4. 5. H. Tian, A. F. R. Hhmer, J. P. Landers, Anal. Biochem., 2000, 283, 175. N. C. Cady, S. Stelick, C. A. Batt, Biosensors and Bioelectronics, 2003, 19, 59. K. A. Melzak, C. S. Sherwood, R. B. Turner, et al., J. Colloid Interf. Sc., 1996, 181, 635. R. Boom, C. J. Sol, M. M. Salimans, et al., J. Clin. Microbiol., 1990, 28, 495. X. Chen, D. F. Cui, L. Wanget, et al., Acta Microbiologica Sinica, 2004, 44(2), 251.

Received 4 January, 2006