Silica-based solid phase extraction of DNA on a microchip

TSINGHUA SCIENCE AND TECHNOLOGY ISSN 1007-0214 03/21 pp379-383 Volume 9, Number 4, August 2004 Silica-Based Solid Phase Extraction of DNA on a Microchip* CHEN Xiaofang (陈晓芳)1,2, SHEN Keyue (沈科跃)1,2, LIU Peng (刘 鹏)1,2, GUO Min (郭 旻)2, CHENG Jing (程 京)1,2, ZHOU Yuxiang (周玉祥)1,2 ,** 1. Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China; 2. National Engineering Research Center for Beijing Biochip Technology, 18 Life Science Parkway, Changping District, Beijing 102206, China Abstract: Micro total analysis systems for chemical and biological analysis have attracted much attention. However, microchips for sample preparation and especially DNA purification are still underdeveloped. This work describes a solid phase extraction chip for purifying DNA from biological samples based on the ad- sorption of DNA on bare silica beads prepacked in a microchannel. The chip was fabricated with poly- dimethylsiloxane. The silica beads were packed in the channel on the chip with a tapered microchannel to form the packed bed. Fluorescence detection was used to evaluate the DNA adsorbing efficiency of the solid phase. The polymerase chain reaction was used to evaluate the quality of the purified DNA for further use. The extraction efficiency for the DNA extraction chip is approximately 50% with a 150-nL extraction volume. Successful amplification of DNA extracted from human whole blood indicates that this method is compatible with the polymerase chain reaction. Key words: DNA purification; sample preparation; solid phase extraction; microchip; silica the next level of disease detection and treatment. Introduction Traditional diagnostic procedures relying on DNA se- Miniaturization and integration of analytical methods quence information involve three steps: purification of and instruments for biomedical and clinical research is DNA from biological samples, amplification of DNA of great interest[1]. The reduction in size and the inte- by reactions such as the polymerase chain reaction gration of the analytical processes have three main ad- (PCR), and subsequent analysis using slab gel electro- vantages: automation of the whole process, reduction phoresis. This is a lengthy process. Great efforts have of the analysis time, and decreased cost. In many cases, been made to develop fast, cost-effective, and high- the integration often paves the way for high-throughput throughput methods for these steps. For the third step, applications of established technologies. capillary electrophoresis (CE) is an attractive alterna- With the successful completion of the Human Ge- tive over traditional slab gel electrophoresis for its nome Project, the analysis of mutations and polymor- speed and automation. In recent years, microchip- phisms in the human genome for individuals represents based CE has been developed to further miniaturize the DNA separation process, allowing the separation of Received: 2003-09-25 DNA fragments in a matter of minutes compared with ﹡ Supported by the National Key Basic Research and Develop- tens of minutes for CE and hours for slab gels[2,3]. Cap- ment (973) Program of China (No. G19990166) and the Na- tional High-Tech Research and Development (863) Program illary array electrophoresis has also been developed to of China (No. 2002AAZZ2011) improve throughput[4]. ﹡﹡ To whom correspondence should be addressed. Miniaturization and integration of the second step, E-mail: [email protected]; Tel: 86-10-80726786 PCR amplification, have also been reported. Recently,  380 Tsinghua Science and Technology, August 2004, 9(4): 379–383 several groups adapted the polymerase chain reaction 1 Experimental to chip format[5]. The rapid thermocycling of chip- based PCR resulted in successful PCR amplification in 1.1 Reagents less than 240 s[6]. Efforts on adapting both processes into a single microchip device have produced several Silica beads, 10-20 µm in diameter, were purchased successful methods which perform PCR and electro- from YMC (Kyoto, Japan). The loading buffer was 6 phoresis in a rapid manner with little user interven- mol/L guanidine hydrochloride (GuHCl) in TE buffer tion[7]. However, PCR requires relatively pure DNA (10 mmol/L Tris, 1 mmol/L EDTA), titrated to pH 6.0 template free from contaminants. Thus, microchip- by HCl. The washing buffer was 80% (V/V) ethanol. based DNA purification would be of great benefit for The eluting buffer was deionized water at pH 8.2 ti- the automation and integration of the whole DNA trated by NaOH. analysis process. DNA fragments (175 bp) amplified from HLA_A* Compared with the fast development of the latter 3401 were labeled with Cy5-dCTP, which was pur- two steps, the first step, the purification of DNA from chased from Amersham Pharmacia (Piscataway, NJ, biological samples is still not well developed. Tradi- USA). HLA_A*3401 plasmid DNA and genomic tional DNA purification methods based on phenol ex- DNA were gifts from Mr. LAN Gengxin (Capital Bio- traction are incompatible with chip-based DNA purifi- chip Corporation, China). cation due to their complex steps and large solution 1.2 Solid phase extraction chip volumes. Historically, the time for DNA purification has been greatly reduced by the shift to solid phase ex- The solid phase extraction chips fabricated with PDMS traction on silica or ion exchange resins. This approach using established micro-machining techniques had a is more amenable to miniaturization, as demonstrated bottom plate and a cover plate. The bottom plate had a by Christel et al.[8] who reported the first DNA extrac- channel containing two segments with cross-sections tion chip. In their research, pillar-like structures which of 30 µm × 100 µm and 300 µm × 100 µm. These two greatly increased the adsorption area were fabricated in segments were connected by a tapered microchannel a silicon microchip using reactive ion etching. Al- which was used to pack the silica beads. The cover though the chip showed some potential, the use of this plate was prepared by punching an inlet and outlet chip was limited by the complex fabrication process and above the channel ends in the bottom plate. The two the cost. Landers’ group established a capillary-based plates were cleaned with water in an ultrasonic bath for DNA purification system containing a 500-nL chamber 15 min, rinsed with ethanol, dried with purified nitro- filled with silica particles[9]. This method was further gen, and then sealed together by adhesion. The sealing adapted into chip format with the silica beads packed was reversible so that it could be easily separated and into a microchannel etched in a glass substrate and im- cleaned when clogged. mobilized with sol-gel to provide a stable and repro- ducible solid phase[10, 11]. Although this approach had 1.3 Device preparation the potential of isolating PCR amplifiable DNA from The solid phase extraction apparatus consists of three several biological samples, the fabrication process was parts: a Kd Scientific Syringe Pump (New Hope, PA, also complex. USA), a 5-mL plastic syringe, and the solid phase ex- The aim of this study was to develop a convenient traction chip. The syringe was connected to the chip and highly efficient method to facilitate chip-based outlet through a plastic tubing. When packing, a drop DNA purification. In the current study, the DNA puri- of a suspension of the silica beads was loaded into the fication chip is fabricated with polydimethylsiloxane inlet and driven into the channel by applying a vacuum (PDMS) which is easy to handle and inexpensive. The at the outlet. The packed solid phase length was 5 mm. silica beads are packed in the microchannel to adsorb The column was then flushed with deionized water for DNA. 2 h and kept at room temperature overnight to stabilize the solid phase.  CHEN Xiaofang (陈晓芳) et al：Silica-Based Solid Phase Extraction of DNA on a Microchip 381 1.4 Solid phase extraction procedure 2 Results and Discussion Before each extraction, bubbles in the channel were 2.1 DNA adsorption on the silica surface carefully removed by a vacuum pump. Then the col- umn was flushed with deionized water and 6 mol/L Although the phenomenon of DNA adsorption on sil- GuHCl before loading the sample. All the DNA sam- ica surfaces in high ionic strength solutions is well ples were prepared in 6 mol/L GuHCl at a concentra- known and has been utilized to purify DNA from bio- tion of 1 ng/µL. For the blood sample, 2.5 µL of hu- logical samples for quite some time[13], the driving man whole blood was added to 100 µL of 6 mol/L forces for the adsorption are not completely known. GuHCl containing 2.5% Triton-X 100. The adsorption is the result of a number of interac- The extraction process included loading, washing, tions[9, 14] such as shielded intermolecular electrostatic and eluting steps. In the loading step, 10 µL of loading forces, dehydration of the DNA and the silica surface, buffer containing DNA was passed through the column. and intermolecular hydrogen bonds formed in the In the washing step, 10 µL of washing buffer was used DNA-silica contact layer. The type of salt, the concen- to wash out the proteinaceous materials and other pos- tration, and the solution pH were all found to signifi- sible PCR inhibitors that were adsorbed onto the solid cantly affect the adsorption. Lowering the pH from 8 phase during the loading step. Finally, the DNA was to 6 increased the saturation level (the maximum DNA eluted with 10 µL of deionized water at pH 8.2. The binding to a certain quantity of silica beads) by at least loading, washing, and eluting solutions were carefully a factor of 2, but further reduction of the pH produced loaded into the inlet one after another without a break no further changes in DNA adsorption capacity. Of in- to avoid inducing bubbles. All three steps were carried terest here is the solid phase extraction device, so pre- out with a vacuum applied at the outlet so that the flow viously published adsorption conditions were used rate in the solid phase extraction chip was controlled at without further optimization. The experiments demon- 50-100 µL/h. strate the capacity of the silica beads to adsorb DNA under these conditions. 1.5 Detection methods The adsorption capacity of the silica beads was The quantity of the eluted Cy5-dCTP labeled HLA_A* tested using 0.25 mg beads added to 100 µL of 6 mol/L 3401 DNA fragments was analyzed using a ScanArray GuHCl containing varying amounts of DNA. The 4000 (Packard Biochip, Billerica, MA, USA). amount of DNA adsorbed on the silica was determined The whole extraction procedure was analyzed using by solution depletion as measured by the absorbance at laser-induced fluorescence detection with a detection 260 nm using a Beckman Coulter DU 800 Spectropho- system similar to that reported previously[12]. Briefly, a tometer (Beckman, Fullerton, CA, USA). Figure 1 632-nm He-Ne laser was used as the excitation source. shows the ascending adsorption isotherm for the silica The fluorescence of the Cy5-dCTP labeled DNA frag- beads. The result demonstrates that these beads, which ments was collected by PMT with a 670-nm band-pass were used to form the column, can be used to perform filter at a sampling frequency of 1 Hz. solid phase extraction. To evaluate the quality of the purified DNA, the eluted solution (5 µL) containing extracted plasmid DNA and genomic DNA were amplified by PCR using PTC-225 Thermal Cycler (MJ Research, South San Francisco, CA, USA) and a standard PCR protocol: 96℃ for 3 min, up to 35 cycles with 96℃ for 20 s, 60℃ for 20 s, 72℃ for 1 min followed by extension at 72℃ for 10 min. Fig. 1 Amount of DNA adsorbed on the surface of silica particles for different DNA concentrations. Silica beads (0.25 mg) were added to a polyethylene centrifuge tube containing 100 µL DNA solutions.  382 Tsinghua Science and Technology, August 2004, 9(4): 379–383 2.2 Channel packing relationship between the DNA concentration and the fluorescence intensity. Although the absolute fluores- The tapered microchannel in the chip used to retain the cence intensity of a given DNA concentration varies solid phase was similar to that described by Ceriotti[15]. slightly on different glass slides, their ratios are consis- The channel had no physical barrier, but the particles tent and could represent the ratio of the DNA concen- were blocked by the “keystone effect”. Upon packing, trations. The data in Fig. 3 have a correlation coeffi- the density of the particles increases and they aggre- cient of 0.99, indicating that the ratio of the DNA con- gate at the taper. These particles act as “keystones”, centrations could be determined by the ratio of their blocking the other beads and allowing the beads to fluorescence intensities. form a stable structure under pressure. One significant advantage of this structure is the simple fabrication process. However, the particle concentration in the suspension is critical. The beads would fill the 30-µm wide channel at lower concentrations and aggregate somewhere in the 300-µm wide channel at higher con- centrations. In these experiments, a 5% suspension (W/V) of 10-20 µm sized beads could be packed repro- ducibly. An image of a channel packed with silica Fig. 3 Fluorescence intensity calibration for various beads is shown in Fig. 2. DNA concentrations on glass slides Therefore, the concentration of the eluted DNA was measured by comparing the fluorescence intensity of the eluted solution with a standard solution containing the DNA fragments at a concentration of 1 ng/µL. The efficiency of 9 independent extractions using a single microchip is shown in Fig. 4. The average efficiency of 52% could be further improved by increasing the ad- Fig. 2 Image of the channel on the solid phase extraction sorption volume. chip packed with silica beads 2.3 Chip extraction efficiency The DNA extraction efficiency of the chips was inves- tigated using a fluorescence-based assay to quantify the HLA_A*3401 DNA fragments retained on the sil- ica beads. The DNA fragments were labeled with Cy5- dCTP in a PCR process with 100 bp containing 2 or 3 Cy5-dCTP molecules. After extraction, a 0.25-µL Fig. 4 Efficiency of solid phase extraction for HLA_ droplet of the eluted solution was spotted onto a glass A*3401 DNA fragments on a microchip filled with silica slide. After the droplet dried, the fluorescent intensity particles. Extraction conditions: loading solution con- of the spot was measured by a Scanarray 4000. The tained 1 ng/µL DNA in 6 mol/L GuHCl, pH 6.0. Loading, average of ten spots was used to determine the DNA washing, and eluting buffers (10 µL each) were allowed to pass through the device in succession at flow rates of 50- concentration of the eluted solution. 100 µL/h. However, when quantifying DNA concentrations, this method cannot determine the exact number of The DNA eluting profile of the three steps is shown Cy5-dCTP contained in one DNA molecule and cannot in Fig. 5. A peak appeared soon after the eluting buffer accurately measure the DNA concentrations repre- was added, with most of the DNA eluted in 15 s in a sented by the fluorescent intensity. To improve the ac- volume of approximately 0.5 µL. The relatively short curacy, a series of DNA solutions with concentrations eluting time and the small volume indicate that sample from 0.1 ng/µL to 1.0 ng/µL were measured to test the enrichment is achieved in the microfluidic chip.  CHEN Xiaofang (陈晓芳) et al：Silica-Based Solid Phase Extraction of DNA on a Microchip 383 the device is human whole blood which is a complex mixture containing many PCR inhibitors. To extract PCR amplifiable DNA from whole blood, the device should extract enough DNA and remove most of the inhibitors. The results shown in Fig. 6 indicate that an adequate amount of genomic DNA is extracted by the microfluidic chip and the extracted DNA can be suc- cessfully amplified with PCR reaction. Fig. 5 Extraction profile of HLA_A*3401 DNA on solid phase extraction chip. Extraction conditions: loading so- 3 Conclusions lution contained 1 ng/µL of DNA fragments in 6 mol/L GuHCl, pH 6.0. Loading, washing, and eluting buffers (10 A chip-based device was developed to extract DNA µL each) were allowed to pass through the device in suc- using silica beads packed in a tapered microchannel. cession at flow rates of 50-100 µL/h. The extraction efficiency is reasonably high. DNA pu- 2.4 PCR amplification of the extracted DNA rification from a complex biological sample of human whole blood is also demonstrated. The successful am- One important criterion in evaluating the DNA purifi- plification of the DNA purified from human whole cation capacity of the solid phase extraction chip is its blood proves the compatibility of this chip-based DNA effect on PCR amplification. The eluted solutions con- purification method with the subsequent PCR amplifi- taining HLA_A*3401 plasmid DNA were subjected to cation. This chip-based device with its miniaturized PCR with the results shown in Fig. 6. volume, simplified process, and acceptable efficiency will be helpful for the development of a practical micro total analysis system. Acknowledgements The authors would like to thank Mr. LAN Gengxin for provid- ing the DNA samples and GAO Huafang and WANG Dong for Fig. 6 Agarose gel electrophoresis of the PCR products helpful discussions about the experiments. for the HLA_A*3401 fragments. Lane 1 and Lane 9: DL2000 marker (Takara Bio-Company, Shiga, Japan). References Lane 2: 5 ng of HLA_A*3401 plasmid DNA directly used for PCR amplification. Lane 3: HLA_A*3401 plasmid [1] Manz A, Graber N, Widmer H M. Miniaturized total DNA eluted from the solid phase. Lane 4 and Lane 6: 5 chemical analysis systems: A novel concept for chemical ng of genomic DNA directly used for PCR amplification. sensing. Sensors and Actuators, 1990, B1: 244-248. Lane 5: genomic DNA eluted from the solid phase. Lane 7: [2] Harrison D J, Fluri K, Seiler K, Fan Z, Effenhauser C S, DNA purified from human whole blood. Lane 8: negative control, no DNA was added to the PCR process. Manz A. Micromachining a miniaturized capillary electro- phoresis-based chemical analysis system on a chip. Science, The current study sought to extract DNA directly 1993, 261: 895-896. from a biological matrix. The solid phase extraction [3] Wooley A, Mathies R. Ultra-high-speed DNA sequencing device must be capable of extracting human genomic using capillary electrophoresis chips. Analytical Chemistry, DNA fragments with reasonable efficiency. The effi- 1995, 67: 3676-3680. [4] Wang Y, Ju J, Carpenter B, Atherton J, Sensabaugh G, ciency was investigated by extracting human genomic Mathies R. Rapid sizing of short tandem repeat alleles us- DNA from a prepared solution with the HLA_A*3401 ing capillary array electrophoresis and energy-transfer fragment amplified from the eluted DNA. fluorescent primers. Analytical Chemistry, 1995, 67: The PCR results demonstrate that the DNA eluted 1197-1203. from the solid phase is PCR amplifiable, but the proc- [5] Kopp M, Mello A J, Manz A. Chemical amplification: ess is meaningless unless it can extract DNA from a Continuous-flow PCR on a chip. Science, 1998, 280: real biological sample. Since the eventual device may 1046-1048. be used for clinical diagnosis, the ideal sample to test (Continued on page 405)