Download Report

ONE-STEP SOLID PHASE-BASED ON-CHIP SAMPLE PREPARATION AND
INTEGRATION WITH FLOW-THROUGH POLYMERASE CHAIN
REACTION
K.T.L. Trinh, H.H. Tran, Y. Zhang, J. Wu and N.Y. Lee*
Gachon University, KOREA
ABSTRACT
Sample preparation is one of the most labor-intensive and time-consuming processes yet indispensable for realizing a
truly integrated micro total analyses systems (μTAS). In this study, we introduce Chelex resin as a solid support for onestep sample preparation. A plastic microdevice was fabricated using poly(methylmethacrylate) (PMMA), and Chelex
resin was physically captured inside a microchannel. Using the fabricated microdevice, DNA was successfully extracted
and purified from Escherichia coli and from human hairs. Furthermore, the sample preparation system was functionally
integrated with flow-through polymerase chain reaction unit, and E. coli was successfully amplified in a seamless flow
using the integrated microdevice.
KEYWORDS: Sample Preparation, Chelex Resin, Flow-Through Polymerase Chain Reaction, Integrated Plastic
Microdevice
INTRODUCTION
Sample preparation is one of the most labor-intensive and time-consuming processes yet indispensable for realizing a
truly integrated system for micro total analyses on a miniaturized platform. Although many functional components have
already been miniaturized, such as amplification, separation, and detection, sample preparation is the least developed
among the functional components needed for realizing μTAS. Some researchers have reported on solid phase-based
sample preparation employing silica beads [1], sol-gel matrix [2], glass micropillars [3], and carboxyl-coated surfaces.
However, most of these processes involve repeated reactions with multiple solutions followed by centrifugation, making
sample preparation fundamentally cumbersome.
In this study, we introduce Chelex resin, a commercially available styrene divinylbenzene copolymer, as a solid support for one-step sample preparation in a continuous manner without requiring multiple operations. While there are many
types of commercially available styrene divinylbenzene copolymers, Chelex 100 is unique because it is a cation exchange resin manufactured to contain paired iminodiacetate ions, which have high affinity for polyvalent metal ions, including Ca2+ and Mg2+, abundant in cell lysis buffers. Besides its ion capturing capacity, the benzene rings comprising
the styrene and divinylbenzene functional groups can attract proteins via hydrophobic interaction, thereby removing contaminating protein from the DNA prepared from the cell lysate. Despite these advantages, there has been no report of
Chelex resin applied for on-chip sample preparation, probably due to difficulty in preconcentration of the target nucleic
acid within relatively large amount of sample. A plastic microdevice was fabricated using PMMA, and a weir structure
was formed and Chelex resin (75–150 μm) was physically captured inside a microchannel. Using the fabricated PMMA
microdevice, DNA was successfully extracted and purified from Escherichia coli and human hairs. Furthermore, the
sample preparation unit was functionally integrated with flow-through PCR unit, since both of the units were operated
by pressure. The integration of these two units would pave the way for a pressure-driven, simple one-step sample preparation and amplification on a monolithic plastic device with greatly decreased manufacture cost and enhanced device
disposability.
THEORY
Chelex is a styrene divinylbenzene copolymer containing paired iminodiacetate ions that have a high affinity for
polyvalent metal ions. Chelex resin aids in the extraction and purification of DNA in the following two ways. First,
Chelex captures proteins via hydrophobic interactions with the styrene, as well as divinylbenzene copolymers, for under
a heated condition, proteins unfold and can interact electrostatically with benzene functionalities comprising the resin,
eventually acting as a sieve for DNA concentration. Second, Chelex resin protects the DNA from the attack of divalent
metal ions such as Ca2+ and Mg2+ abundant in cell lysis buffers and prevent the degradation of DNA extracted in the
heating step by quenching the metal ions, which could damage the DNA.
978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001
341
17th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
27-31 October 2013, Freiburg, Germany
Figure 1. (a) Image showing the microchannel design. (b) Chelex resin packed inside a PMMA microdevice. (c) Mechanism showing the purification of DNA employing Chelex resin.
EXPERIMENTAL
Two PMMA substrates were bonded by ethanol treatment followed by UV irradiation as shown in Figure 2a–c [4].
The size of the overall microdevice was 40 × 40 mm (Figure 2d), and the lengths of microchannels A and B were 20
mm and 8 mm, respectively (Figure 2e). The lengths of the narrow microchannels between microchannels A and B,
functioning as a weir structure, were 3 mm.
Figure 2. (a-c) Bonding of PMMA assembly mediated by ethanol treatment followed by UV irradiation. (d) A photo
showing PMMA microdevice. (e) 3D image showing microchannel construction.
RESULTS AND DISCUSSION
As shown in Figure 3, the surface temperature of the PMMA substrate was measured to be approximately 95.5 ±
0.5°C, and temperature distribution was homogenous on the entire surface of PMMA. The thermal conductivity of
PMMA is relatively low (0.19 W K-1 m-1), which is comparable to that of poly(dimethylsiloxane) (PDMS) (0.16–0.2 W
K-1 m-1). For this reason, we can assume the surface temperature represents the inner temperature of the microchannel.
The surface temperature was stabilized after heating the substrate for 5 min.
Figure 3. Infrared camera images showing time-dependent temperature variations at (a) 0 min, (b) 5 min, and (c) 70
min, when a PMMA substrate was heated.
Figure 4 shows the results of optical density (OD) measurement. The ratio of 260 nm to 280 nm was measured to
check the purity of DNA. Figure 4a–c shows the results of sample preparation when E. coli was simply heated in water at
95°C off-chip (Figure 4a), heated in water at 95°C mixed with Chelex resin off-chip (Figure 4b), and heated at 95°C in
the microchannel mixed with Chelex resin on chip (Figure 4c). The measured average ODs for Figure 4a–c were 1.42,
1.54, and 1.54, respectively. Although it is commonly accepted that the ratio of 260 nm to 280 nm should be between 1.6
and 2.0 to guarantee the purity of DNA, all the spectra had their peaks at 260 nm, which could reflect that the sample was
purified up to certain level prior to going through subsequent amplification process. However, among two methods utilized, the ratio was the highest when Chelex resin was involved in the preparation process, and the on-chip preparation
results were almost identical with the off-chip results when Chelex resin was employed.
342
Figure 4. Optical density spectra obtained when E. coli was (a) heated in water at 95°C off-chip, (b) heated in water
at 95°C with Chelex resin off-chip, and (c) heated at 95°C with Chelex resin on chip.
Figure 5a shows the photo of the functionally integrated PMMA microdevice for performing sample preparation and
amplification in one step, composed of Chelex-based sample preparation unit and flow-through PCR unit. The mixture
of E. coli culture solution and PCR reagent was introduced from the inlet and was driven to the outlet by pressure in a
continuous manner without being stopped in the middle. Figure 5b shows the enlarged image of the rectangle marked in
Figure 5a showing the Chelex resin packed inside the microchannel. Figure 5c shows the results for PCR for the
amplification of 230 bp target amplicon. Lane 1 shows the target amplicon obtained using commercially available
pGEM-3Zf(+) plasmid vector and a thermal cycler. Lane 2 shows the result of a negative control experiment. Lane 3
shows the target amplicon obtained using the integrated microdevice. Although the intensity of the target amplicon
shown in lane 3 was approximately 83% of that obtained off-chip, the band was clearly distinguishable. Using the
integrated microdevice, a complicated valve control was completely eliminated, enabling the whole process with one
seamless flow simply by applying pressure.
Figure 5. (a) A photo showing an integrated PMMA microdevice for performing sample purification and amplification in one step. (b) Enlarged image of the rectangle shown in (a) displaying Chelex resin packed inside the microchannel. (c) Result of agarose gel electrophoresis for E. coli extraction and purification performed off-chip (lane 1), negative control experiment (lane 2), and on chip (lane 3). Lane M shows 100 bp DNA size marker.
CONCLUSION
The integrated PMMA microdevice composed of Chelex-resin packed, solid phase-based sample preparation unit
combined with flow-through PCR unit successfully amplified E. coli in a seamless flow, paving the way for a pressuredriven, simple one-step sample preparation and amplification on a monolithic plastic device with greatly decreased manufacture cost and enhanced device disposability.
ACKNOWLEDGEMENTS
This work was supported by the GRRC program of Gyeonggi province (GRRC Gachon 2013-B04, Development of
sample preparation using wireless heating methods) and the Public welfare & Safety research program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF2012M3A2A1051681).
REFERENCES
[1] H. Tian, A.F.R. Hühmer and J.P. Landers, “Evaluation of silica resins for direct and efficient extraction of DNA
from complex biological matrices in a miniaturized format,” Anal. Biochem., vol. 283, p.175 (2000).
[2] Q. Wu, J.M. Bienvenue, B.J. Hassan, Y.C. Kwok, B.C. Giordano, P.M. Norris, J.P. Landers and J.P. Ferrance,
“ Microchip-based macroporous silica sol-gel monolith for efficient isolation of DNA from clinical samples”, Anal.
Chem., vol. 78, p. 5704 (2006).
[3] Q. Wu, J W. Jin, C. Zhou, S. Han, W. Yang, Q. Zhu, Q. Jin and Y. Mu, “Integrated glass microdevice for nucleic
acid purification, loop-mediated isothermal amplification, and online detection”, Anal. Chem., vol. 83, p. 3336
(2011).
[4] H.H. Tran, W. Wu and N.Y. Lee, “Ethanol and UV-assisted instantaneous bonding of PMMA assemblies and tuning in bonding reversibility”, Sens. Actuators B., vol. 181, p. 955 (2013).
CONTACT
*N.Y. Lee, tel: +82-31-7508556; nylee@gachon.ac.kr
343