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Microfluidic System for Detection of r-Thalassemia-1 Deletion Using Saliva Samples Kang-Yi Lien,† Chien-Ju Liu,‡ Pao-Lin Kuo,§ and Gwo-Bin Lee*,†,‡,| Institute of Nanotechnology and Microsystems Engineering, Department of Engineering Science, Department of Obstetrics and Gynecology, Medical College, National Cheng Kung University, Tainan 701, Taiwan, and Medical Electronics and Device Technology Center, Industrial Technology Research Institute, Hsinchu 310, Taiwan This current study presents a new miniature, integrated system capable of rapid detection of genetic deletion from saliva samples. Several critical modules including a genomic DNA (gDNA) extraction module, a polymerase chain reaction (PCR) module, and an external optical detection module are integrated into the system. Silicamodified magnetic beads are first incubated with saliva in an extraction chamber with a cell lysis solution. This is followed by the collection of released gDNA onto the surface of the microbeads, which is then further purified and concentrated utilizing a magnetic field generated by an on-chip array of microcoils. Then, genetic deletion of human genes can be specifically amplified by the on-chip PCR module and is immediately detected using the optical detection module. Experimental results show that highquality gDNA with an average concentration of 50.45 ng/ µL can be extracted from 100 µL of saliva. The detection of a mutated r-globin gene associated with r-thalassemia-1 of southeast Asian (SEA)-type deletion can be completed within less than 1 h. Moreover, the detection limit of the system is found to be 12.00 pg/µL with a high sensitivity up to 90%. Consequently, the proposed salivabased miniature system can provide a powerful platform for rapid DNA extraction and detection of genetic diseases. Recently, substantial improvements in preventive medicine and genetic analyses have been enabled thanks to advancements in molecular diagnosis. The deletion and mutation of various genes may cause serious illnesses such as cardiovascular disease or rare diseases that may interfere with a person’s physical and mental health. Therefore, the genotyping of nucleotide polymorphisms and the analysis of genetic sequences has become one of the most critical areas of focus for clinical diagnosis. Moreover, people with defective genes can be diagnosed earlier and even pretreated in advance. Therefore, there is a great need to develop a costeffective, accurate, and rapid protocol for these applications. Usually, detection of genetic diseases involves a series of timeconsuming procedures which require well-trained personnel, * To whom correspondence should be addressed. E-mail: gwobin@ mail.ncku.edu.tw. Phone: +886-6-2757575, ext 63347. Fax: +886-6-2761687. † Institute of Nanotechnology and Microsystems Engineering, National Cheng Kung University. ‡ Department of Engineering Science, National Cheng Kung University. § Medical College, National Cheng Kung University. | Industrial Technology Research Institute.
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delicate equipment, and organic reagents. Among them, extraction of genomic DNA (gDNA) from clinical biosamples such as whole blood, tissue, and ascites fluid is the key process for genetic analysis.1 Tedious purification steps for gDNA extraction from a clinical sample (usually human whole blood) are usually inevitable. In addition, an invasive and painful venepuncture is always needed to obtain the clinical whole blood samples. Alternatively, saliva has attracted considerable interest as a viable source of human gDNA in genetic epidemiology.2,3 A variety of biological substances including enzymes, hormones, immunoglobulin, and biomolecules have already been successfully quantified in saliva.4 The use of saliva samples is much more convenient compared with a whole blood sample if high-quality gDNA can be extracted and purified from the saliva for subsequent biological applications. Therefore, the methods of DNA extraction using magnetic beadbased techniques from clinical biosamples are promising.5,6 The surface-treated magnetic beads which may be modified with functional groups or specific probes can bind the target DNA onto the beads with a high affinity and limited human intervention.7,8 Nevertheless, the entire procedure of DNA extraction is still timeconsuming due to the incubation process. Moreover, a large-scale permanent magnet and pipettes are normally required. Also, additional block heaters and bulky polymerase chain reaction (PCR) machines are always needed to perform the subsequent nucleic acid amplification process for genetic analysis. As a result, there is a great need to develop a compact, automatic lab-on-achip (LOC) system that integrates multifunctional modules to perform rapid gDNA extraction from saliva and the nucleic acid amplification of genes. In addition, the enabling platform will be even more powerful if the gene can be optically analyzed, which avoids the need to perform slab-gel electrophoresis separation and detection. LOC systems capable of sample preparation, transportation, separation, reaction, and detection have attracted considerable (1) Aljanabi, S. M.; Martinez, I. Nucleic Acids Res. 1997, 25, 4692–4693. (2) Ng, D. P. K.; Koh, D.; Choo, S. G. L.; Ng, V.; Fu, Q. Clin. Chim. Acta 2004, 343, 191–194. (3) Walt, D. R. Science 2005, 308, 217–219. (4) Streckfus, C. F.; Bigler, L. R. Oral Dis. 2002, 8, 69–76. (5) Fuentes, M.; Mateo, C.; Rodriguez, A.; Casqueiro, M.; Tercero, J. C.; Riese, H. H.; Ferna´ndez-Lafuente, R.; Guisa´n, J. M. Biosens. Bioelectron. 2006, 21, 1574–1580. (6) Liu, Y.-J.; Yao, D.-J.; Chang, H.-Y.; Liu, C.-M.; Chen, C. Biosens. Bioelectron. 2008, 24, 558–565. (7) Archer, M. J.; Lin, B.; Wang, Z.; Stenger, D. A. Anal. Biochem. 2006, 355, 285–297. (8) Yeung, S. W.; Hsing, I.-M. Biosens. Bioelectron. 2006, 21, 989–997. 10.1021/ac900453d CCC: $40.75 2009 American Chemical Society Published on Web 05/06/2009
interest and have been extensively explored in recent years. Almost the entire process from extraction through detection can be performed with little human intervention in these LOC systems.9 These micro-total-analysis systems (µ-TAS) may have a great impact on the development of miniaturized instruments for biomedical analysis. Miniature LOC systems have several advantages over their large-scale counterparts, including portability, lower unit cost, disposability, parallel processing, lower reagent and sample consumption, and so on.10 Methods for DNA amplification and genetic identification such as PCR and DNA sequencing have also been integrated onto a single microfluidic chip.11,12 These microfabricated nucleic acid-based systems have shown great potential for target DNA/RNA amplification for the reason that a high heating and cooling rate can be achieved, which completes the whole diagnostic procedure in a shorter period of time. In addition, the target amplified DNA can be rapidly analyzed if a separation and an optical detection module such as a laserinduced fluorescence (LIF) capillary electrophoresis (CE) device can be used subsequently.13,14 However, there remain some offchip sample pretreatment processes (e.g., cell lysis and DNA extraction) to be carried out manually before the analytical process. Furthermore, monotonous washing steps and highvoltage operating conditions are inevitable during the CE analyses. Hence, a microfluidic system capable of leukocytes purification from the whole blood sample, gDNA extraction from the leukocytes, and the identification of genetic mutation by using slab-gel electrophoresis was therefore presented by the current research group.15 Nevertheless, the two-step process of gDNA extraction from human whole blood is still a lengthy process. Also, the sensitivity of the detection limit and diagnostic time of genetic analysis can be highly improved if the optical detection module can be integrated into the LOC system. The use of the whole blood sample still requires a tingling venepuncture process. Consequently, the current study presents an integrated, salivabased, microfluidic system utilizing surface-modified magnetic beads for rapid gDNA extraction and R-thalassemia-1 detection. Given the noninvasive and painless nature of saliva collection, high-quality gDNA can be extracted from saliva automatically, followed by amplifying the target region of genetic deletion using an on-chip PCR module. Subsequently, genetic deletion of the extracted gDNA can then be immediately analyzed by an optical detection module. With this approach, fast diagnosis of R-thalassemia-1 can be realized within 1 h with a high sensitivity and selectivity. MATERIALS AND METHODS Operational Procedure. A new microsystem capable of magnetic bead-based gDNA extraction and fast detection of R-thalassemia-1 has been developed in the current study. By using the adsorption/desorption between the silica-modified surface of (9) Chin, C. D.; Linder, V.; Sia, S. K. Lab Chip 2007, 7, 41–57. (10) Vilkner, T.; Janasek, D.; Manz, A. Anal. Chem. 2004, 76, 3373–3385. (11) Liu, P.; Seo, T. S.; Beyor, N.; Shin, K. J.; Scherer, J. R.; Mathies, R. A. Anal. Chem. 2007, 79, 1881–1889. (12) Zhang, C.; Xing, D. Nucleic Acids Res. 2007, 35, 4223–4237. (13) Hunt, H. C.; Wilkinson, J. S. Microfluid. Nanofluid. 2007, 4, 53–79. (14) Kaigala, G. V.; Hoang, V. N.; Stickel, A.; Lauzon, J.; Manage, D.; Pilarski, L. M.; Backhouse, C. J. Analyst 2008, 133, 331–338. (15) Lien, K.-Y.; Liu, C.-J.; Lin, Y.-C.; Kuo, P.-K.; Lee, G.-B. Microfluid. Nanofluid. 2009, 6, 539–555.
the beads and gDNA of the buccal cells, the gDNA which is released from the epithelial cells in the saliva sample can be specifically bound onto the beads immersed in a high-salt buffer, followed by eluting the isolated gDNA from the beads utilizing a low-salt buffer for the purification of gDNA.16,17 Figure 1A shows a schematic illustration of the experimental procedure for the saliva-based microfluidic platform. The saliva sample is first loaded into a “DNA extraction chamber”. Then silica-coated magnetic beads along with a cell lysis solution are loaded in a “sample loading chamber” (see Figure 1B). These beads are then transported into the DNA extraction chamber by activating a micropump (Figure 1a-1). The gentle mixing can be generated within the DNA extraction chamber while the magnetic beads are pumped into the chamber. Cells in the saliva are then lysed, and the gDNA is released (Figure 1a-2). The target gDNA is then bonded onto the surface of the magnetic beads (Figure 1a-3), followed by collecting them onto the surface of the DNA extraction chamber utilizing the built-in circular array of microcoils. The other substances in the biological fluids such as cell debris are washed away into a “waste collection chamber” as a washing buffer is continuously pumped through the extraction chamber (Figure 1a-4). Meanwhile, another resuspension buffer is then pumped into the DNA extraction chamber to elute the bonded gDNA from the magnetic beads. The released gDNA is then resuspended and is transported into “PCR reaction chambers” (see Figure 1B) where the magnetic beads are still attracted onto the surface of the DNA extraction chamber by the magnetic field (Figure 1a-5). With this approach, the gDNA is totally isolated and purified from the clinical biological fluids and can be used for the subsequent detection of genetic diseases. Alternatively, gDNA can be also stored for further biomedical applications by extracting gDNA from the extraction chamber using pipettes. By incorporating a micro-PCR module, specific genes can be further amplified (Figure 1a-6). In this study, hybridization of a mutated R-globin gene associated with R-thalassemia-1 of southeast Asian (SEA)-type deletion with a fluorescence-labeled DNA probe is performed. Finally, excited fluorescent signals from the amplified genes are collected by using an external optical detection module consisting of a photomultiplier tube (PMT), a mercury lamp, a set of optical components including three fluorescence filters, one collimation lens, and one objective lens (Figure 1a-7). A photograph of the microfluidic system (Figure S-1) and the detailed operation conditions of the on-chip gDNA extraction and molecular diagnosis (Table S-1) can be found in the Supporting Information. Design and Fabrication of the Integrated System. In order to realize the operating process described above, a microfluidic system comprising three modules including a gDNA extraction module, a PCR module, and an external optical detection module has been designed and fabricated. The layout of the saliva-based microfluidic chip is schematically shown in Figure 1B. Detailed information of the working principles of each module and the scanning electron microscope (SEM) images of each component (16) Zeillinger, R.; Schneeberger, C.; Speiser, P.; Kury, F. BioTechniques 1993, 14, 202–203. (17) Taylor, J. I.; Hurst, C.; Davies, M.; Sachsinger, N.; Bruce, I. J. J. Chromatogr., A 2000, 890, 159–166.
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Figure 1. (A) Experimental procedure for using the integrated saliva-based microfluidic system. (B) Schematic diagram of the microfluidic system integrated with a DNA extraction module and the PCR module. (C) The experimental setup of the optical detection module.
can be found in the Supporting Information and our previous works.18-21 The dimensions of the microfluidic chip are measured to be 42 mm × 27 mm. Note that all the chambers including a sample loading chamber, a DNA extraction chamber, a waste collection chamber, PCR/optical detection chambers, and PCR reagent chambers are designed to be open chambers. Note that all the electric current is supplied and regulated by using an external digital controller, which is driven by a 9 V power provided by a transformer when the hand-held controller is plugged into a socket. Note that the average power consumption of the system (18) Lien, K.-Y.; Lee, W.-C.; Lei, H.-Y.; Lee, G.-B. Biosens. Bioelectron. 2007, 22, 1739–1748. (19) Lien, K.-Y.; Lin, J.-L.; Liu, C.-Y.; Lei, H.-Y.; Lee, G.-B. Lab Chip 2007, 7, 868–875. (20) Yang, Y.-N.; Hsiung, S.-K.; Lee, G.-B. Microfluid. Nanofluid. [Online early access]. DOI: 10.1007/s10404-008-0356-7. Published Online: Oct 28, 2008. http://www.springerlink.com/content/f34433r2704um112/?p) 77dcc2316cfc4e5d8bdf16b9e259b37dπ)0. (21) Hsieh, T.-M.; Luo, C.-H.; Wang, J.-H.; Lin, J.-L.; Lien, K.-Y.; Lee, G.-B. Microfluid. Nanofluid. [Online early access]. DOI: 10.1007/s10404-008-0353x. Published Online: Oct 7, 2008. http://www.springerlink.com/content/ 237834480k802450/?p)026321c9d26441c48ddb224a630c84cbπ)0.
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is only 1.2 W, which includes the heating power for the microPCR module, the power for magnetic field generation by the microcoils array, and the power consumption of the controller. It has also been demonstrated that the microcontroller and the microfluidic system can be operated by a commercially available 9 V battery. gDNA Extraction Module. The gDNA extraction module consists of new normally closed pneumatic micropumps with a poly(dimethylsiloxane) (PDMS, Dow Corning Corp., U.S.A.)-based floating block structure, multiple microfluidic channels, a circular microcoil array, a sample loading chamber, a waste collection chamber, and PCR reaction chambers. In addition, the microcoil arrays made of copper (Cu) resistors have also been integrated. The required magnetic field can be generated to attract the magnetic beads onto the surface of the extraction chamber for purification so that the other suspended substances can be washed away when the washing buffer flows through the chamber. Self-Compensated PCR Module. The thermal uniformity of the temperature distribution within a PCR reaction chamber is crucial
in performing accurate thermal cycling, particularly in maintaining the uniform temperature distribution inside a chamber with a larger reaction area.21,22 Therefore, a self-compensated PCR module integrated with two array-type microheaters with symmetrical layouts and three temperature sensors is adopted to improve the temperature uniformity in the reaction chambers. Note that multiple PCR reaction chambers are both used to perform nucleic acids amplification for genetic identification. Both of the fluorescent signals from the amplified genes can be detected by the optical detection module. Optical Detection Module. The external optical detection module is designed for rapid detection of the fluorescent signals from the amplified nucleic acids of the genes. The experimental setup of the optical detection module is schematically shown in Figure 1C. The light source from the mercury lamp is first directed through a band-pass (BP) filter (470/20BP, Nikon Corp., Japan) and is used to excite the reporter dye in the amplified PCR products at an excitation wavelength of 488 nm. The emitted fluorescent signals from the reporter dye are then directed through a long-pass filter (505LP, Nikon Corp., Japan), followed by filtering out other fluorescent signals excited from the PCR product utilizing another BP filter (522/16BP, Nikon Corp., Japan). Only signals with wavelengths ranging from 506 to 538 nm can pass through and be detected by the PMT. With this approach, high-sensitivity detection of the genes can be achieved and the corresponding genetic disease is analyzed in a shorter period of time by utilizing the optical detection module. Sample Preparation and Nucleic Acid Amplification. gDNA Extraction Materials and Process. The major contribution of the proposed system is that the high-quality gDNA can be directly extracted from saliva in an automatic format, followed by amplifying target DNA fragment associated with genetic mutation in a short period of time. The entire process of gDNA extraction can be completed in approximately 10 min in an automatic manner. Briefly, a large volume of saliva samples would be (∼1 mL) spit by the individuals and mixed in the Eppendorf tube at first, followed by loading 100 µL saliva samples, a 200 µL solution of silica-coated, DNA-specific magnetic beads (diameter ) 4.5 µm, concentration ) 6 × 107 beads/mL, DynabeadsDNA-DIRECTUniversal, Invitrogen Corporation, U.S.A.) containing cell lysis buffer (Invitrogen Corp., U.S.A.) with 2 µL of proteinase-K (Viogene, U.S.A.) into the reaction chambers, respectively. The magnetic beads solution are then transported into the DNA extraction chamber at a pumping rate of 200 µL/min. Note that a flow disturbance is generated inside the chamber to gently mix the biosamples and the magnetic beads. Next, 100 µL of a washing buffer (10× washing buffer (100 mM Tris-HCl, pH ) 7.5), Invitrogen Corp., U.S.A.) is loaded into the sample loading chamber, and the washing process is completed by continuously pumping the buffer through the extraction chamber while the magnetic complex is still restrained by the generated magnetic field. The other substances are washed away and blocked within the waste collection chamber by the normally closed valve. Next, the resuspended buffer (10 mM Tris-HCl, pH ) 8.0, Invitrogen Corp., U.S.A.) with a volume of 125 µL is loaded into the sample loading chamber and (22) Wang, Z.; Sekulovic, A.; Kutter, J. P.; Bang, D. D.; Wolff, A. Electrophoresis 2006, 27, 5051–5058.
pumped into the DNA extraction chamber for gDNA elution for another 3 min. The gDNA is then released from the magnetic beads and resuspended into the buffer solution, followed by transporting them into the subsequent PCR reaction chambers. PCR Materials and Process. Extracted gDNA with a volume of 3 µL is transported in parallel into the subsequent multiple PCR reaction/optical detection chambers (A and B), respectively, followed by pumping 22 µL of PCR reaction mixture along with 10 µL of mineral oil which are preloaded in the PCR reagent chambers into the PCR reaction chambers. Note that the 10 µL mineral oil is used to avoid the evaporation of PCR reaction mixture during the amplification process. A fluorescent-based PCR technique is employed for the detection of genetic diseases (R-thalassemia-1) by using a TaqMan DNA probe, which is a duallabeled fluorochrome DNA segment that will emit specific fluorescent signals from the 5′-end reporter dye (i.e., carboxyfluorescein, FAM) by the Taq DNA polymerase during the PCR process.23 The detail information of the on-chip operation protocol of PCR process and primer sequence can be found in the Supporting Information. Note that two specific primer sets are used to verify the R-thalassemia deletion in the multiple PCR reaction chambers. The primer set of S1/S2 with a 287 bp fragment is designed for the wild-type R-globin gene alleles in the PCR reaction chamber A, whereas a primer set of S1/S3 with a 194 bp fragment is used for the detection of R-thalassemia-1 SEA-type deletions in the PCR reaction chamber B. Finally, the fluorescent signals from the target amplified gene indicating genetic deletion is immediately detected by the optical detection module. RESULTS AND DISCUSSION Characterization of the Integrated Platform. Microfluidic Devices. A normally closed pneumatic micropump has been demonstrated with a high pumping rate. Figure 2a shows the relationship between the flow pumping rate and the driving frequency (fd) of the electromagnetic valves (EMVs, SMC Inc., S070M-5BG-32, Japan) at a supplied air pressure of 20.0 psi. Four sets of micropumps are investigated including a micropump without a valve in the single microchannel (as a baseline control), a micropump with a normally closed valve in the single microchannel (normally closed pump no. 1), a normally closed micropump in the multichannels located between the DNA extraction chamber and PCR reaction chambers (normally closed pump no. 2), and a normally closed micropump in the multichannels located between the PCR reaction chambers and PCR reagent chambers (normally closed pump no. 3). It can be clearly seen from the results that the pumping rate increases with an increase in driving frequency. A flow rate as high as 866 µL/min (normally closed pump no. 1) is achieved (fd ) 81.7 Hz), while the maximum pumping rate of the pneumatic micropump without a valve is only 288 µL/min (fd ) 34.8 Hz). The pumping rate of the pneumatic micropump starts to decrease after it reaches an optimum value. In addition, these results also show that a higher flow pumping rate can (23) Kutyavin, I. V.; Afonina, I. A.; Mills, A.; Gorn, V. V.; Lukhtanov, E. A.; Belousov, E. S.; Singer, M. J.; Walburger, D. K.; Lokhov, S. G.; Gall, A. A.; Dempcy, R.; Reed, M. W.; Meyer, R. B.; Hedgpeth, J. Nucleic Acids Res. 2000, 28, 655–661.
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Figure 2. (a) Relationship between the flow pumping rate and the driving frequency of the EMV at a supplied air pressure of 20.0 psi. (b) The relationship between the localized magnetic fields generated by the circular microcoils array and the applied dc currents. The temperature generated by the circular microcoils array is also presented. (c) An IR image of the temperature distribution that is generated by the on-chip self-compensated PCR module at a set temperature of 95.0 °C. (Note that the smooth curves presented in the figure are splines.)
be achieved by integrating multiple microfluidic channels. The maximum value of the flow pumping rate generated by the normally closed micropumps (normally closed pump no. 2) is measured to be 1381 µL/min (fd ) 72.0 Hz). Circular Microcoils Array. The magnetic field generated by the built-in circular microcoil array is investigated by measuring the magnetic field and the temperature utilizing a Tesla meter (7010 Gauss/Tesla meter, F. W. Bell, U.S.A.) and a temperature detector (model-3003, DER-EE Inc., Taiwan) (Figure 2b). As expected, experimental data show a quadratic relationship between the magnetic field and the applied direct current (dc) currents. However, the temperature also increases with the increasing 4506
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applied dc currents. Hence, in order to avoid thermal damage to the biosamples during the gDNA extraction process, a magnetic field of 65 G generated at an applied electric current of 150 mA is used, thus resulting in a maximum temperature of only 39.2 °C, which may prevent damaging the biosamples during the DNA extraction process. PCR Module. The temperature uniformity of the generated temperature field by the PCR module is verified at a setup temperature of 95.0 °C by an infrared (IR) imaging system (infrared-thermography TVS-200N, Nippon Avionics Co. Ltd., Japan) as shown in Figure 2c. According to the IR image, microheaters with self-compensated heating grids provide additional heating power against the thermal loss at the edges of the microheaters such that a uniform temperature distribution around the microheaters can be achieved. The variation of the temperature uniformity at a set point can be controlled to within ±0.2 °C. In addition, typical thermal cycles for the developed selfcompensated PCR module are explored and the heating and cooling rates of the module are measured to be approximately 22.3 °C/s and 13.5 °C/s, respectively. Hence, these rapid heating and cooling rates can ensure fast nucleic acid amplification. Performance of the gDNA Extraction. The extraction of gDNA from saliva is first performed and compared by three methods, including a traditional gDNA extraction method using an organic kit (Gentra Systems Inc., U.S.A.), a manual gDNA extraction with the magnetic beads, and an automatic extraction by the developed microfluidic system. The quality of the extracted gDNA is then verified by measuring the optical intensity (OD) of ultraviolet (UV) absorbance at wavelengths of 230 nm/260 nm/ 280 nm utilizing a spectrophotometer (NanoDrop spectrophotometer ND-1000, U.S.A.). Experimental data show that gDNA with an average concentration of 50.45 ± 2.31 ng/µL (OD260/280 ) 1.81 ± 0.09, OD260/230 ) 1.19 ± 0.12, n ) 25) can be extracted automatically from 100 µL of saliva samples utilizing the developed microfluidic system, which is comparable to the quality extracted from saliva using the traditional method (54.34 ng/µL, OD260/280 ) 1.83, OD260/230 ) 1.39) and the manual extraction process (50.15 ng/µL, OD260/280 ) 1.80, OD260/230 ) 1.14). The yield of extracted gDNA from clinical samples has been reported in the literature.24-27 Various methods of gDNA extraction from two different samples including the whole blood (24) Chan, V. T.-W.; Fleming, K. A.; McGee, J. O. D. Anal. Biochem. 1988, 168, 16–24. (25) van Schie, R. C. A. A.; Wilson, M. E. J. Immunol. Methods 1997, 208, 91– 101. (26) Ng, D. P.; Koh, D.; Choo, S.; Chia, K. S. Clin. Chim. Acta 2006, 367, 81– 85. (27) Rogers, N. L.; Cole, S.; Lan, H.-C.; Crossa, A.; Demerath, E. W. Am. J. Hum. Biol. 2007, 19, 319–326. (28) Breadmore, M. C.; Wolfe, K. A.; Arcibal, I. G.; Leung, W. K.; Dickson, D.; Giordano, B. C.; Power, M. E.; Ferrance, J. P.; Feldman, S. H.; Norris, P. M.; Landers, J. P. Anal. Chem. 2003, 75, 1880–1886. (29) Sun, C.-F.; Lee, C.-H.; Cheng, S.-W.; Lin, M.-H.; Wu, T.-L.; Tsao, K.-C.; Chiu, D. T. Y.; Liou, J.-D.; Chu, D.-C. Clin. Genet. 2001, 60, 305–309. (30) Huang, F.-C.; Liao, C.-S.; Lee, G.-B. Electrophoresis 2006, 27, 3297–3305. (31) Hu, H.; Li, C.; Xiong, Q.; Gao, H.; Li, Y.; Chang, Q.; Liang, Z. Prenatal Diagn. 2008, 28, 222–229. (32) Wang, L.; Hirayasu, K.; Ishizawa, M.; Kobayashi, Y. Nucleic Acids Res. 1994, 22, 1774–1775. (33) Witek, M. A.; Llopis, S. D.; Wheatley, A.; McCarley, R. L.; Soper, S. A. Nucleic Acids Res. 2006, 34, e74. (34) Wen, J.; Guillo, C.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2007, 79, 6135–6142.
Table 1. Comparison of gDNA Extraction from Whole Blood and Saliva with Different Methods sample saliva saliva saliva saliva whole blood whole blood whole cell lysates whole blood
method magnetic bead-based microfluidic platform QIAamp blood kit alcohol precipitation phenol-chloroform two-step magnetic bead-based microfluidic system 2-propanol fractionation photoactivated polycarbonate microfluidic chip two-stage, dual-phase microchip
used sample volume (µL)
yield of extracted DNA (ng/µL)
purity (OD260/280)
ref
100
50.45 ± 2.31
1.81 ± 0.09
Lien et al. (the presented study)
1000 2000 2000 200
25.2 ± 13.70 17.75 90.94 33.26 ± 2.50
1.53 ± 0.02 1.77 ± 0.08 1.74 ± 0.07 1.81 ± 0.12
van Schie and Wilson (ref 25) Ng et al. (ref 26) Rogers et al. (ref 27) Lien et al. (ref 15)
500 10
22.00 7.60 ± 1.60
1.92 1.89 ± 0.10
Wang et al. (ref 32) Witek et al. (ref 33)
10
24.00 ± 0.20
and saliva are compared and listed in Table 1. For example, an average DNA yield from 1 mL of saliva was reported to be 25.20 ± 13.70 ng/µL.25 Also, a mean concentration of gDNA extracted from saliva by alcohol precipitation was found to be 17.75 ng/µL (with a wide range from 4.25 to 42.60 ng/µL).26 Another saliva DNA collection method showed that the whole saliva method (with a sample of 2 mL) provided a significantly greater DNA yield (90.94 ng/µL) than the other methods (oral rinse ) 18.28 ng/ µL; swab ) 5.36 ng/µL; cytobrush ) 6.61 ng/µL).27 Still, the difference of the yield regarding the extracted gDNA might be caused by the numbers of buccal cells in the saliva samples spit from individuals. It can be concluded that reasonable extraction of gDNA from the saliva performed in the developed microfluidic system can be obtained. In addition, it can be seen from Table 1 that the average yield of gDNA extracted from saliva is superior to the yield of gDNA extracted from the whole blood sample. More importantly, the entire extraction procedure performed in the proposed magnetic bead-based microfluidic platform can be completed in approximately 10 min which is faster than our previous work by using the two-step gDNA extraction process from the whole blood sample.15 Note that after loading the samples such as saliva and reaction reagents into the proposed miniature system, the whole extraction process of gDNA can be completed in a shorter period of time automatically without any manual operation. gDNA Extraction from Saliva with Different Storage Conditions. The gDNA extracted from different volumes of saliva samples is also investigated. Besides, gDNA extracted from saliva samples that are stored at different conditions have also been explored. Figure 3a shows the relationship between the total extracted gDNA and the volumes of saliva at three different storage conditions. A fixed amount of magnetic particles with 1.2 × 107 beads suspended in a lysis buffer is mixed with saliva, followed by washing away the biological fluids and eluting the purified gDNA into the resuspension buffer. The maximum amount of total extracted gDNA is 11.02 µg, which starts to saturate when the volume of saliva samples is higher than 200 µL, indicating that the surface of the magnetic beads might be entirely covered with released gDNA from the saliva. Therefore, an excessive amount of magnetic beads (∼1.2 × 107 beads) is applied to extract the gDNA from saliva (100 µL) to ensure that all the released gDNA can be extracted. In addition, gDNA can also be automatically purified from a small sample volume of saliva in the developed microfluidic system. The minimum limit of sample handling in the gDNA extraction module is
Wen et al. (ref 34)
found to be 5 µL, and approximately 0.32 µg of gDNA can be purified from 5 µL of saliva samples in the microfluidic platform. It is also found that fresh saliva yields the maximum amount of gDNA. The developed system can still extract a reasonable amount of gDNA from saliva stored at -20 °C or even at 4 °C for 3 days. Elution Effect of Extracted gDNA. The amount of elution buffer to elute gDNA from the surface of the magnetic beads is also explored. The gDNA eluted from the magnetic beads is used to
Figure 3. (a) gDNA is purified from different volumes of saliva samples stored at different conditions with a fixed amount of silicacoated magnetic beads. (b) Elution efficiency between the amount of resuspension buffer and the amount of eluted gDNA. (Note that the smooth curves presented in the figure are splines.) Analytical Chemistry, Vol. 81, No. 11, June 1, 2009
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perform the subsequent PCR and optical detection steps for the reason that the presence of magnetic beads might interfere with the optical detection process. Therefore, the elution efficiency between the amount of resuspension buffer and the eluted gDNA is investigated, as shown in Figure 3b. The gDNA is extracted from 100 µL saliva, and the resulting rinsed gDNA-bound magnetic complex is mixed with different volumes of resuspension buffer for three consecutive stages. The first stage is performed to collect the supernatant in the extraction chamber after gDNA is eluted from the purified DNA-bound magnetic complex when a magnetic field is generated such that beads can be attracted to the bottom of the chamber. Then, the second elution process is performed by mixing the same volume of resuspension buffer with the bead complex collected from the first stage to elute gDNA that still remains on the surface of the magnetic beads. The final stage is then carried out by eluting the residual gDNA on the magnetic beads that are collected from the second stage. Note that each elution process is performed for 3 min. All the eluted gDNA from the three stages are then collected and analyzed using the spectrophotometer. Experimental results show that almost 95% of the gDNA extracted from 100 µL of saliva can be eluted and is collected from the magnetic beads in the first stage when using a volume of resuspension buffer higher than 200 µL. In addition, the high DNA recovery ratio performed by the proposed magnetic bead-based microfluidic platform is reasonable when compared with the microchip-based DNA extraction method utilizing the silica-modified surface,28 which ∼80% of extracted DNA can be eluted and collected within 10 µL. Therefore, the experimental data indicate that most of the gDNA trapped on the silica-modified magnetic beads can be eluted from the magnetic beads and collected within the extraction chamber for subsequent biological analysis or further biomedical applications. Detection of r-Thalassemia-1 Deletion. Optical Analysis of R-Thalassemia-1 Deletion. Instead of using the conventional slabgel electrophoresis or CE, the proposed microfluidic platform presents an end point, fluorescent detection approach for the rapid detection of R-thalassemia-1 with the incorporation of an optical detection module and a fluorescence-based DNA probe. For the cases without the SEA-type deletion, only normal R-globin gene clusters will be amplified with the primer set S1/S2 and no signals will be produced from the primer set S1/S3. Saliva samples from a healthy person (negative case) and another person with R-thalassemia (positive case) are used. The amplified PCR products are both separated and detected by using a traditional slab-gel electrophoresis method (Figure 4a) and the developed optical detection module (Figure 4b) for comparison, respectively. Figure 4b shows the optical signals from the optical detection module, indicating that the developed microfluidic system can successfully detect these genes. The gDNA is purified and extracted from saliva utilizing the protocols described above, followed by pumping 3 µL of purified gDNA into the PCR reaction chambers to perform PCR and the optical detection. The experimental results show that the specific fluorescent signals with a wavelength of 518 nm (with the maximum emitted fluorescent intensity) is emitted and detected by the PMT when the filter of the objective is turned on for 30 s. Note that the no template control (NTC) is used for the negative control case and the optical signal from it (with an amplitude of 25 mV) can be regarded as a 4508
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Figure 4. Two types of patients including a healthy person and the other person a carrier with R-thalassemia-1 SEA-type deletion are used to test the performance of the PCR module, followed by immediate analysis utilizing both (a) the traditional slab-gel electrophoresis and (b) the optical detection module, respectively.
noise. It is then found that the signal-to-noise (S/N) ratio is measured to be higher than 150. From these results, patients with R-thalassemia-1 SEA-type deletion can be rapidly diagnosed automatically with a high sensitivity using the developed microfluidic system. In addition, the entire diagnostic procedure including PCR and the optical detection can be completed in approximately 46 min. Sensitivity. The detection limit of the end point microfluidic PCR platform has also been explored. Experimental data about the optical signals (Figure S-5 and Table S-2) can be found in the Supporting Information. These results represents the fluorescent intensity of the amplified PCR products from different gDNA samples with concentrations ranging from 65.15 ng/µL (sample 1) to 0.11 pg/µL (sample 9), which are performed with 5 time serial dilution to verify the performance of the microfluidic system (n ) 10 for each concentration). From the experimental results, gDNA with a concentration of 12.00 pg/µL (sample 6, S/N ) 9) can be successfully amplified and fluorescently detected with a high sensitivity up to 90% by using the developed microfluidic system. However, it only has a sensitivity of 30% for a concentration of 2.56 pg/µL (sample 7, S/N ) 3). In addition, when compared with traditional methods using PCR machines or CE chips,29,30 the proposed microfluidic system provides an automatic platform for performing rapid detection of R-thalassemia-1 deletion. For example, a commercial lab-scale, real-time quantitative PCR analysis for R-thalassemia-1 of SEA-type deletion has been
demonstrated by using a dual-labeled probe.29 The detection limit of the study was perform by using the serial dilution of DNA sample to proceed the real-time PCR-based protocol. The detection limit of the system was determined to be around 0.28 ng of DNA, which is approximately 10-fold higher than the proposed microfluidic platform (∼36.00 pg of total gDNA can be detected in the developed microfluidic platform). Another study regarding prenatal diagnosis of β-thalassemia by chip-based CE was demonstrated with the detection limit of 1 ng of gDNA sample.31 Consequently, the proposed microfluidic platform capable of end point optical analysis of R-thalassemia deletion with the incorporation of fluorescence-labeled DNA probe may provide a potential tool for accurate and fast diagnosis. CONCLUSION A new saliva-based microfluidic system integrated with several functional modules utilizing magnetic beads for rapid gDNA extraction and R-thalassemia-1 detection has been successfully demonstrated in the present study. By using the silica-modified, DNA-specific magnetic beads, gDNA released from clinical saliva samples were specifically bonded onto the magnetic beads, followed by purification and enrichment of the gDNA with the incorporation of micropumps and a circular microcoil array. A uniform temperature distribution inside the PCR reaction chambers can provide a high yield of nucleic acid amplification to proceed the multiple PCR processes, followed by the detection of the amplified PCR products by utilizing an optical detection module. The microfluidic system has been demonstrated to extract gDNA with the average yield of 50.45 ng/µL with an OD260/280 ratio of 1.81 ± 0.09 from 100 µL of saliva. The isolation and purification process of the microfluidic platform was demonstrated to provide a high-quality gDNA samples that could be used for a variety of molecular diagnosis, such as PCR and the detection of genetic deletion. In addition, a high elution ratio (∼95%) of gDNA from the surface of magnetic beads can be achieved and the extracted gDNA can be collected for subsequent biological application. Furthermore, experimental results of the detection limit of the proposed system also showed that patients with R-thalassemia-1 SEA-type deletion with a minimum gDNA concentration of 12.00 pg/µL can be detected successfully with a high sensitivity up to 90%. When compared with the real-time PCR apparatus or other miniature devices, the proposed magnetic bead microfluidic platform can provide an efficient tool to automate all whole biomedical diagnosis processes from extraction through detection with little human intervention. Therefore, the microfluidic system
may provide a promising platform for rapid detection of R-thalassemia-1 deletion and various molecular diagnostic processes in the further biomedical applications. ACKNOWLEDGMENT The authors express thanks for the financial support from the National Science Council in Taiwan (NSC 96-2120-M-006-008). The authors also acknowledge Dr. Tsung-Min Hsieh for his assistance on the digital microcontrollers and for discussion on the design of the micro-PCR module. GLOSSARY Nomenclature
Au BP CE Cu dc DNA fd FAM gDNA EMV IR LIF LOC LP OD PCR PDMS PMT Pt RNA SEM S/N SEA UV µ-TAS
gold band-pass capillary electrophoresis copper direct current DNA driving frequency carboxyfluorescein genomic DNA electromagnetic valve infrared laser-induced fluorescence lab-on-a-chip long-pass optical intensity polymerase chain reaction poly(dimethylsiloxane) photomultiplier tube platinum ribonucleic acid scanning electron microscope signal-to-noise southeast Asian ultraviolet micro-total-analysis systems
SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 2, 2009. Accepted April 19, 2009. AC900453D
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