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One-dimensional microfluidic beads array for multiple mRNAs expression detection
Biosensors and Bioelectronics 22 (2007) 2759–2762
Short communication
One-dimensional microfluidic beads array for multiple mRNAs expression detection Jianhui Wen, Xiaohai Yang, Kemin Wang ∗ , Weihong Tan, Leiji Zhou, Xinbing Zuo, He Zhang, Yunqing Chen State Key Laboratory of Chemo/Biosensing and Chemometrics, Biomedical Engineering Center, College of Chemistry and Chemical Engineering, Hunan University, Engineering Research Center for Bio-Nanotechnology of Hunan Province, Changsha 410082, PR China Received 17 September 2006; received in revised form 13 November 2006; accepted 21 November 2006 Available online 22 January 2007
Abstract A one-dimensional microfluidic beads array for in vitro rapid measurement of multiple mRNAs expression is presented in this paper. Gene specific capture DNA-functional beads were deposited along a microchannel to form an addressable beads array. We demonstrated that the one-dimensional beads array could perform simultaneous multiple nucleic acid targets detection and a DNA detection limit of 0.02 nM was obtained. Using this array, transcripts expression of three tumor-associated genes, including p53, H-ras, and NME1, both in CNE2 nasopharyngeal carcinoma cell lines and in normal human nasopharyngeal epithelial cells were evaluated. The responses of these three genes expression in CNE2 cells to 5-flouorouracil (5-Fu) stimuli were also assessed. Validation of these results was performed using reverse transcriptase-PCR. The presented methodology combines high throughput of microarrays and low sample consumption, convenient liquid handling of microfluidics. It enables rapid and facile determination of multi-gene expression and holds great potential in early cancer diagnostics and molecular biology. © 2006 Elsevier B.V. All rights reserved. Keywords: Microfluidic; Beads array; Transcriptional expression; Cancer cell
1. Introduction Many diseases have close association with genes expression variations (Chien et al., 2001; Fung et al., 2000). Techniques that can perform rapid and facile determination of multiple mRNAs expression concurrently are of great importance not only in disease diagnostics but also in fundamental molecular biological studies. Some traditional methods for mRNA analysis, including differential display PCR (Liang and Pardee, 1992), suppressive subtractive hybridization (Zhu et al., 2003), sequence analyses of gene expression (Polyak and Riggins, 2001), etc., still suffer from various undesirable features such as laborious, time consuming, poor sensitivity, etc. High-density DNA array chip, because of its high throughput capability, has become a focus of genomics research (Schena et al., 1995; Wong et al., 2004). While complex surface processing and high cost are barriers for its popularity. Microfluidic systems have numerous advan-
∗
Corresponding author. Tel.: +86 731 8821566; fax: +86 731 8821566. E-mail address: [email protected] (K. Wang).
0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.11.029
tages (Effenhauser et al., 1994; Sato et al., 2002; Medintz et al., 2001), but not adept in multianalyte assay. Therefore, to develop an efficient, inexpensive analytical system capable of multiple gene analysis should be of great interest. Recently, some attempts have been made along this direction for such an integrated platform that combines the merits of microfluidics for efficient sample handling and the advantages of microarrays for simultaneously multianalyte assessment. A “criss-cross” procedure for microarray patterning was utilized to develop a microfluidic based peptide arrays for multi-enzyme assays (Su et al., 2005). A whole-cell bioassay was performed using bacterial sensor strains immobilized in three-dimensional microfluidic network (Tani et al., 2004). An integrated microfluidic beads array was introduced for DNA hybridization and single-nucleotide mismatches detection (Ali et al., 2003), and the same beads array system was utilized for simultaneous detection of the cardiac risk factors (Christodoulides et al., 2002). Yet all of these systems adopted a two-dimensional microarray format for multiple assays. As our previous effort to develop a more effective platform of microfluidics and microarrays, a one-dimensional flow system
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has been shown for intracellular proteins profiling by a two-site “sandwich” assay (Zhou et al., 2006). The central component of this total analysis system is a linear beads array along the microchannel. It is versatile and can be adapted to different customized applications. In this paper, we describe a novel application of this technology for the simultaneous detection of multiple gene transcripts. Gene specific capture probes were immobilized on the beads surface by biotin–streptavidin linkage. After beads deposition and chip sealing, a one-dimensional nucleic acids-functional beads array with integrated microfluidics was formed for multiple transcripts expression detection. In order to realize label-free mRNA targets detection, a “sandwich” hybridization assay (Zhao et al., 2003) was used. We have examined the transcripts expression of p53, H-ras and NME1 in cultured cancer and normal cells, and expression changes of three transcripts mediated by an anti-tumor drug have also been determined. This report represents the initial application of the one-dimensional microfluidic beads array for transcriptional gene profiling. It was found that this DNA-functional beads array could realize minimal sample consumption, convenient liquid handling and rapid DNA hybridization kinetics. Array design endows the platform with multiplexing and encoding capability. Furthermore, the system is simple and cost favorable, makes it popular for various practical applications.
2.3. Chip fabrication and assembly The microfluidic beads array was fabricated according to our previous work (Zhou et al., 2006). Briefly, the PDMS replica was peeled from the master after curing, and the inner channel wall was coated successively with 5% polymethobromide and 3% dextran sulphate solution (Katayama et al., 1998). The functional beads were transferred into micro-chambers by the vacuum tweezers (Nikon Narishige, Nikon Corp., Japan) under Leica DMIRB microscope (Leica Corp., Germany). After manipulation, PDMS replica was sealed by conformal contact with a slide with pre-designed reservoirs for sample introduction and liquid outlet and the linear beads array chip was constructed. 2.4. Total RNA extraction Total RNAs of CNE2, 5-Fu mediated CNE2 and normal human nasopharyngeal epithelial cells (HNE) were extracted using Trizol reagent with a standard total RNA isolation protocol. Finally, three total RNA samples were diluted in 20 l DEPC treated distilled water. After dilution, the total RNA concentration of these samples was measured using an UV/vis spectrophotometer (Beckman Coulter) and all samples were diluted to have the same RNA concentration. 2.5. Reverse transcriptase-PCR
2. Materials and methods 2.1. Materials Silica beads, polymethobromide (PB), biotinylated BSA and streptavidin were purchased from Sigma–Aldirch, PDMS prepolymer and curing agent was obtained from Dow Corning Corporation. Dextran sulphate (DS) was from Tianjin H&Y Bio Co. Ltd. All DNA oligonucleotides were synthesized by Takara Dalian Corporation and purified using HPLC method. AMV reverse transcriptase, RNase inhibitor, DNA Taq polymerase and dNTPs mixtures were obtained from Takara Dalian Corporation. Trizol reagent kit for total RNA extraction was from invitrogen. Except noted, all other reagents used were commercially available and of analytical grade. 2.2. Bead modification The DNA capture sequences were immobilized on the surface of silica beads through biotin–streptavidin conjugation. Briefly, about 10 mg silica beads were activated by suspending them in 1.5 ml of 2.0 M Na2 CO3 for 30 min followed by washing three times using 10 mM PBS buffer. Subsequently, the silica beads were resuspended in 250 l of 0.3 mg/ml biotinylated BSA and were incubated for 48 h in a low temperature shaker (4.0 ◦ C, >300 rpm). After washed three times with PBS, the beads were incubated with 200 l of 2.0 mg/ml streptavidin for 4 h. The streptavidin-functionalized beads were then divided into three parts and incubated separately with three biotinylated capture sequences (3.5 M) for 6 h. After washing three times with PBS, the beads were stored at −20 ◦ C prior to use.
Total RNA samples were firstly purified with RNase-free DNase I and RNase inhibitor to remove DNA contaminant. The reverse transcription reaction was performed in 20 l volumes with 4 l 5× reverse transcriptase buffer, 1 g RNA template, 1.0 mM each nucleotide mixture, 30 U RNase inhibitor, 5 U AMV RTase, 50 pmol oligo(dT)18 primer at 42 ◦ C for 1 h, followed by inactivation of RTase at 94 ◦ C for 2 min. PCR was performed in 50 l volumes containing reaction mixture of 5 l 10× PCR buffer, 200 M each nucleotide, 0.4 M each primer, 2.5 U of Taq DNA polymerase per reaction and 0.5 l of sample per RT reaction. Amplification reactions were carried out in a thermocycler (GeneAmp. PCR system 2700, Applied Biosystems) with preliminary denaturation for 5 min at 94 ◦ C, followed by 30 cycles of denaturation at 94 ◦ C for 50 s, annealing at 55 ◦ C for 50 s, primer extension at 72 ◦ C for 60 s, and a final extension at 72 ◦ C for 5 min. PCR products were electrophoresed and visualized by UV transillumination. 2.6. On-chip detection of DNA and specific RNA The purified DNA and the total RNA samples were hybridized and detected on-chip, respectively. Gravity driven model was used throughout for the samples and reagents introduction. For the purified DNA samples, various concentrations of samples were pre-hybridized with 20 nM report probe (3× SSC, 0.1% SDS) for 20 min, then 2 l sample and report probe containing buffer was added to the sample reservoir. After 20 min hybridization, 2 l elution buffer (1.5× SSC, 40% formamide, 0.1% SDS) was introduced and the elution lasted for 15 min. For RNA samples, Total RNA samples were denatured
J. Wen et al. / Biosensors and Bioelectronics 22 (2007) 2759–2762
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at 90 ◦ C for 10 min and flash cooled in ice water. Then, 40 g of each total RNA fraction and report probes mixture (100 nM) were added to SSC stock solution to make 40 g/100 l RNA containing hybridization buffer (6× SSC, 50% deionized formamide, 0.1% SDS). After 30 min hybridization, elution buffer (1.5× SSC, 10% deionized formamide, 0.1% SDS) was introduced and the elution lasted for 15 min under the same conditions for the purified DNA samples. Fluorescence images of the beads were captured using a Leica DMIRB fluorescence microscope equipped with a cooled CCD camera (Leica DC 300F) and the data were analyzed using Leica QWin analysis software. 3. Results and discussion 3.1. Design of the DNA-functional microfluidic beads array and hybridization assay Fig. 1a was a scheme illustration of the one-dimensional microbeads array. Setting hundreds of chambers along the microchannel featured this novel one-dimensional microbeads array chip. Briefly, PDMS was used to fabricate the microchannel with a series of chambers (80 m width × 60 m deep) and these chambers were linked in series with narrow flow channels (25 m width × 60 m deep). Biotinylated DNA capture probes were immobilized on the surface of beads through biotin–streptavidin linkage. Then different DNA-functional beads (50 m diameter) were put into chambers using a vacuum tweezers under the microscope and each chamber holds one bead. Here a “sandwich” hybridization assay is adopted to detect specific DNA or RNA sequences. As shown in Fig. 1b, when samples flow through the microchannel, targets (DNA or RNA) are specifically recognized and captured by the corresponding beads, report probes labeled with fluorescence dye and com-
Fig. 2. Fluorescence intensity enhancement of DNA target bound report probe as a function of concentration of target DNA (three-fold redundant beads array).
plementary to part of target sequences are added at last to realize fluorescent detection of targets. A distinct advantage of this beads array based “sandwich” assay is that there is no need for the targets labeling or modification (see supplemental information for “sandwich” probes of p53, H-ras and NME1). 3.2. On-chip detection of purified DNA samples Sensitivity characteristic in nucleic acid assays is an important parameter for a new approach. To determine the assay sensitivity, different concentrations of p53 target DNA were firstly hybridized with 20 nM p53 report DNA. The p53 capture probe functionalized beads array was exposed to p53 target/report DNA complex solution for 20 min. Fluorescence images were taken after 15 min buffer rinse. Control experiment without DNA sample was also performed. On the basis of signal-to-noise of 3, the achieved detection limit is 0.02 nM, and a linear dynamic range from 0 to 1.0 nM was obtained, as shown in Fig. 2. The detection limit corresponds to an absolute of ∼10−17 mol of target molecules in 2 l sample. The signal does not increase significantly for target concentration higher than 1.0 nM, suggesting that 1.0 nM is the saturation concentration for the microbead surface. 3.3. Applications to multiple transcripts expression analysis
Fig. 1. Schematic illustrations of microfluidic beads array chip assembly and bioconjugated microbeads for DNA/RNA detection. (a) Beads were confined within tandem chambers along the microchannel, followed by sealing with a top plate to form linear microfluidic beads array. (b) Principle of beads modification with biotinylated DNA capture probe and sandwich hybridization for target gene detection.
Based on the results obtained using the purified samples, we have further applied the beads array to multiple transcripts expression detection in various cells extracts. p53, H-ras and NME1 are tumor-associated genes and their expression products play key roles in cell growth, differentiation and malignant transformation. We selected CNE2 tumor cell and normal HNE cell as a model for the transcripts expression analysis. At the same time, the responses of three genes expression in CNE2 cells to 5-flouorouracil (5-Fu) stimuli were also assessed. Fig. 3a shows the fluorescence images of the beads array for three cellular RNA samples in HNE, 5-Fu mediated CNE2 and CNE2 cells. Fig. 3b is the corresponding fluorescence intensity quantification for the beads array. According to the results, NME1 and H-ras
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two-dimensional chip-based microbeads array (Ali et al., 2003), reagent consumption has been reduced by three orders magnitude. Finally, compared with common planar DNA array technologies, the microbeads array methodology provides a number of unique advantages: high area-to-volume ratio of porous beads offers high density of immobilized capture probes, thus enabling a obvious sensitivity enhancement with comparison to the planar array, furthermore, DNA hybridization kinetics of beads array (<20 min) is several times faster than planar DNA array (typically >1 h). We concluded that this linear beads array is expected to find its practical applications in gene expression profiling and disease diagnosis. Acknowledgments This work was partially supported by the National Key Basic Research Program of China (2002CB513110), Key Project of Natural Science Foundation of China (90606003), Major International (Regional) Joint Research Program of Natural Science Foundation of China (20620120107), Natural Science Foundation of China (20475015), and the China National Key Projects (05FK5029). Appendix A. Supplementary data Fig. 3. (a) Fluorescence images of beads array detecting three cellular transcripts in three cells. Control images were obtained using the same experimental conditions without adding RNA samples (three-fold redundant beads array). Bar, 50 m. (b) Fluorescence intensity quantification of the beads array images.
have relative low expression in normal cells but high expression in carcinoma cells. After 5-Fu inducement to the CNE2 cells, p53 expression shows obvious upregulation, while expressions of H-ras and NME1 decrease obviously. At last, these results were validated by reverse transcriptase-PCR (see supplemental information for PCR primers and RT-PCR results). 4. Conclusions In conclusion, this work serves to extend the application of one-dimensional microfluidic beads array for multiple transcripts assay. With the use of “sandwich” hybridization assay, a detection limit of 0.02 nM with DNA samples was obtained, transcriptional expression of three tumor-associated genes in normal and tumor cells and expression changes of these genes in CNE2 cells before and after drug stimuli were also assessed. Some advantages of the microfluidic beads array are documented as follows: first, the beads array offers the capability of multiplexed oligonucleotide assays. This DNA multiplexing capacity extends the prior multiplexing application for protein profiling (Zhou et al., 2006). Second, the microfluidic design minimizes the sample and reagent consumption. The total volume of a 2 cm long one-dimensional beads array is less than 100 nl, thus only 2–3 l of sample is sufficient in a test. Compared with 1.5 ml sample consumption per test in
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2006.11.029. References Ali, M.F., Kirby, R., Goodey, A.P., Rodriguez, M.D., Ellington, A.D., Neikirk, D.P., McDevitt, J.T., 2003. Anal. Chem. 75, 4732–4739. Chien, G., Yuen, P.W., Kwong, D., 2001. Cancer Genet. Cytogenet. 126, 63– 67. Christodoulides, N., Tran, M., Floriano, P.N., Rodriguez, M., Goodey, A., Ali, M., Neikirk, D., McDevitt, J.T., 2002. Anal. Chem. 74, 3030–3036. Effenhauser, C.S., Paulus, A., Manz, A., Widmer, H.M., 1994. Anal. Chem. 66, 2949–2953. Fung, L.F., Lo, A.K., Yuen, P.W., 2000. Life Sci. 67, 923–936. Katayama, H., Ishihama, Y., Asakawa, N., 1998. Anal. Chem. 70, 5272–5277. Liang, P., Pardee, A.B., 1992. Science 257, 967–971. Medintz, I.L., Paegel, B.M., Mathies, R.A., 2001. J. Chromatogr. A 924, 265–270. Polyak, K., Riggins, G.J., 2001. J. Clin. Oncol. 19, 2948–2958. Sato, K., Yamanaka, M., Takahashi, H., Tokeshi, M., Kimura, H., Kitamori, T., 2002. Electrophoresis 23, 734–739. Schena, M., Shalon, D., Davis, R.W., Brown, P.O., 1995. Science 270, 467–470. Su, J., Bringer, M.R., Ismagilov, R.F., Mrksich, M., 2005. J. Am. Chem. Soc. 127, 7280–7281. Tani, H., Maehana, K., Kamidate, T., 2004. Anal. Chem. 76, 6693–6697. Wong, K.K., Tsang, Y.T.M., Shen, J.H., Cheng, R.S., Chang, Y.M., Man, T.K., Lau, C.C., 2004. Nucl. Acids Res. 32, e69. Zhao, X.J., Tapec-Dytioco, R., Tan, W.H., 2003. J. Am. Chem. Soc. 125, 11474–11475. Zhou, L.J., Wang, K.M., Tan, W.H., Chen, Y.Q., Zuo, X.B., Wen, J.H., Liu, B., Tang, H.X., He, L.F., Yang, X.H., 2006. Anal. Chem. 78, 6246–6251. Zhu, N.S., Yang, J.L., Wang, Y.M., Guan, X.F., 2003. Exp. Mol. Med. 35, 243–248.
Short communication
One-dimensional microfluidic beads array for multiple mRNAs expression detection Jianhui Wen, Xiaohai Yang, Kemin Wang ∗ , Weihong Tan, Leiji Zhou, Xinbing Zuo, He Zhang, Yunqing Chen State Key Laboratory of Chemo/Biosensing and Chemometrics, Biomedical Engineering Center, College of Chemistry and Chemical Engineering, Hunan University, Engineering Research Center for Bio-Nanotechnology of Hunan Province, Changsha 410082, PR China Received 17 September 2006; received in revised form 13 November 2006; accepted 21 November 2006 Available online 22 January 2007
Abstract A one-dimensional microfluidic beads array for in vitro rapid measurement of multiple mRNAs expression is presented in this paper. Gene specific capture DNA-functional beads were deposited along a microchannel to form an addressable beads array. We demonstrated that the one-dimensional beads array could perform simultaneous multiple nucleic acid targets detection and a DNA detection limit of 0.02 nM was obtained. Using this array, transcripts expression of three tumor-associated genes, including p53, H-ras, and NME1, both in CNE2 nasopharyngeal carcinoma cell lines and in normal human nasopharyngeal epithelial cells were evaluated. The responses of these three genes expression in CNE2 cells to 5-flouorouracil (5-Fu) stimuli were also assessed. Validation of these results was performed using reverse transcriptase-PCR. The presented methodology combines high throughput of microarrays and low sample consumption, convenient liquid handling of microfluidics. It enables rapid and facile determination of multi-gene expression and holds great potential in early cancer diagnostics and molecular biology. © 2006 Elsevier B.V. All rights reserved. Keywords: Microfluidic; Beads array; Transcriptional expression; Cancer cell
1. Introduction Many diseases have close association with genes expression variations (Chien et al., 2001; Fung et al., 2000). Techniques that can perform rapid and facile determination of multiple mRNAs expression concurrently are of great importance not only in disease diagnostics but also in fundamental molecular biological studies. Some traditional methods for mRNA analysis, including differential display PCR (Liang and Pardee, 1992), suppressive subtractive hybridization (Zhu et al., 2003), sequence analyses of gene expression (Polyak and Riggins, 2001), etc., still suffer from various undesirable features such as laborious, time consuming, poor sensitivity, etc. High-density DNA array chip, because of its high throughput capability, has become a focus of genomics research (Schena et al., 1995; Wong et al., 2004). While complex surface processing and high cost are barriers for its popularity. Microfluidic systems have numerous advan-
∗
Corresponding author. Tel.: +86 731 8821566; fax: +86 731 8821566. E-mail address: [email protected] (K. Wang).
0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.11.029
tages (Effenhauser et al., 1994; Sato et al., 2002; Medintz et al., 2001), but not adept in multianalyte assay. Therefore, to develop an efficient, inexpensive analytical system capable of multiple gene analysis should be of great interest. Recently, some attempts have been made along this direction for such an integrated platform that combines the merits of microfluidics for efficient sample handling and the advantages of microarrays for simultaneously multianalyte assessment. A “criss-cross” procedure for microarray patterning was utilized to develop a microfluidic based peptide arrays for multi-enzyme assays (Su et al., 2005). A whole-cell bioassay was performed using bacterial sensor strains immobilized in three-dimensional microfluidic network (Tani et al., 2004). An integrated microfluidic beads array was introduced for DNA hybridization and single-nucleotide mismatches detection (Ali et al., 2003), and the same beads array system was utilized for simultaneous detection of the cardiac risk factors (Christodoulides et al., 2002). Yet all of these systems adopted a two-dimensional microarray format for multiple assays. As our previous effort to develop a more effective platform of microfluidics and microarrays, a one-dimensional flow system
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has been shown for intracellular proteins profiling by a two-site “sandwich” assay (Zhou et al., 2006). The central component of this total analysis system is a linear beads array along the microchannel. It is versatile and can be adapted to different customized applications. In this paper, we describe a novel application of this technology for the simultaneous detection of multiple gene transcripts. Gene specific capture probes were immobilized on the beads surface by biotin–streptavidin linkage. After beads deposition and chip sealing, a one-dimensional nucleic acids-functional beads array with integrated microfluidics was formed for multiple transcripts expression detection. In order to realize label-free mRNA targets detection, a “sandwich” hybridization assay (Zhao et al., 2003) was used. We have examined the transcripts expression of p53, H-ras and NME1 in cultured cancer and normal cells, and expression changes of three transcripts mediated by an anti-tumor drug have also been determined. This report represents the initial application of the one-dimensional microfluidic beads array for transcriptional gene profiling. It was found that this DNA-functional beads array could realize minimal sample consumption, convenient liquid handling and rapid DNA hybridization kinetics. Array design endows the platform with multiplexing and encoding capability. Furthermore, the system is simple and cost favorable, makes it popular for various practical applications.
2.3. Chip fabrication and assembly The microfluidic beads array was fabricated according to our previous work (Zhou et al., 2006). Briefly, the PDMS replica was peeled from the master after curing, and the inner channel wall was coated successively with 5% polymethobromide and 3% dextran sulphate solution (Katayama et al., 1998). The functional beads were transferred into micro-chambers by the vacuum tweezers (Nikon Narishige, Nikon Corp., Japan) under Leica DMIRB microscope (Leica Corp., Germany). After manipulation, PDMS replica was sealed by conformal contact with a slide with pre-designed reservoirs for sample introduction and liquid outlet and the linear beads array chip was constructed. 2.4. Total RNA extraction Total RNAs of CNE2, 5-Fu mediated CNE2 and normal human nasopharyngeal epithelial cells (HNE) were extracted using Trizol reagent with a standard total RNA isolation protocol. Finally, three total RNA samples were diluted in 20 l DEPC treated distilled water. After dilution, the total RNA concentration of these samples was measured using an UV/vis spectrophotometer (Beckman Coulter) and all samples were diluted to have the same RNA concentration. 2.5. Reverse transcriptase-PCR
2. Materials and methods 2.1. Materials Silica beads, polymethobromide (PB), biotinylated BSA and streptavidin were purchased from Sigma–Aldirch, PDMS prepolymer and curing agent was obtained from Dow Corning Corporation. Dextran sulphate (DS) was from Tianjin H&Y Bio Co. Ltd. All DNA oligonucleotides were synthesized by Takara Dalian Corporation and purified using HPLC method. AMV reverse transcriptase, RNase inhibitor, DNA Taq polymerase and dNTPs mixtures were obtained from Takara Dalian Corporation. Trizol reagent kit for total RNA extraction was from invitrogen. Except noted, all other reagents used were commercially available and of analytical grade. 2.2. Bead modification The DNA capture sequences were immobilized on the surface of silica beads through biotin–streptavidin conjugation. Briefly, about 10 mg silica beads were activated by suspending them in 1.5 ml of 2.0 M Na2 CO3 for 30 min followed by washing three times using 10 mM PBS buffer. Subsequently, the silica beads were resuspended in 250 l of 0.3 mg/ml biotinylated BSA and were incubated for 48 h in a low temperature shaker (4.0 ◦ C, >300 rpm). After washed three times with PBS, the beads were incubated with 200 l of 2.0 mg/ml streptavidin for 4 h. The streptavidin-functionalized beads were then divided into three parts and incubated separately with three biotinylated capture sequences (3.5 M) for 6 h. After washing three times with PBS, the beads were stored at −20 ◦ C prior to use.
Total RNA samples were firstly purified with RNase-free DNase I and RNase inhibitor to remove DNA contaminant. The reverse transcription reaction was performed in 20 l volumes with 4 l 5× reverse transcriptase buffer, 1 g RNA template, 1.0 mM each nucleotide mixture, 30 U RNase inhibitor, 5 U AMV RTase, 50 pmol oligo(dT)18 primer at 42 ◦ C for 1 h, followed by inactivation of RTase at 94 ◦ C for 2 min. PCR was performed in 50 l volumes containing reaction mixture of 5 l 10× PCR buffer, 200 M each nucleotide, 0.4 M each primer, 2.5 U of Taq DNA polymerase per reaction and 0.5 l of sample per RT reaction. Amplification reactions were carried out in a thermocycler (GeneAmp. PCR system 2700, Applied Biosystems) with preliminary denaturation for 5 min at 94 ◦ C, followed by 30 cycles of denaturation at 94 ◦ C for 50 s, annealing at 55 ◦ C for 50 s, primer extension at 72 ◦ C for 60 s, and a final extension at 72 ◦ C for 5 min. PCR products were electrophoresed and visualized by UV transillumination. 2.6. On-chip detection of DNA and specific RNA The purified DNA and the total RNA samples were hybridized and detected on-chip, respectively. Gravity driven model was used throughout for the samples and reagents introduction. For the purified DNA samples, various concentrations of samples were pre-hybridized with 20 nM report probe (3× SSC, 0.1% SDS) for 20 min, then 2 l sample and report probe containing buffer was added to the sample reservoir. After 20 min hybridization, 2 l elution buffer (1.5× SSC, 40% formamide, 0.1% SDS) was introduced and the elution lasted for 15 min. For RNA samples, Total RNA samples were denatured
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at 90 ◦ C for 10 min and flash cooled in ice water. Then, 40 g of each total RNA fraction and report probes mixture (100 nM) were added to SSC stock solution to make 40 g/100 l RNA containing hybridization buffer (6× SSC, 50% deionized formamide, 0.1% SDS). After 30 min hybridization, elution buffer (1.5× SSC, 10% deionized formamide, 0.1% SDS) was introduced and the elution lasted for 15 min under the same conditions for the purified DNA samples. Fluorescence images of the beads were captured using a Leica DMIRB fluorescence microscope equipped with a cooled CCD camera (Leica DC 300F) and the data were analyzed using Leica QWin analysis software. 3. Results and discussion 3.1. Design of the DNA-functional microfluidic beads array and hybridization assay Fig. 1a was a scheme illustration of the one-dimensional microbeads array. Setting hundreds of chambers along the microchannel featured this novel one-dimensional microbeads array chip. Briefly, PDMS was used to fabricate the microchannel with a series of chambers (80 m width × 60 m deep) and these chambers were linked in series with narrow flow channels (25 m width × 60 m deep). Biotinylated DNA capture probes were immobilized on the surface of beads through biotin–streptavidin linkage. Then different DNA-functional beads (50 m diameter) were put into chambers using a vacuum tweezers under the microscope and each chamber holds one bead. Here a “sandwich” hybridization assay is adopted to detect specific DNA or RNA sequences. As shown in Fig. 1b, when samples flow through the microchannel, targets (DNA or RNA) are specifically recognized and captured by the corresponding beads, report probes labeled with fluorescence dye and com-
Fig. 2. Fluorescence intensity enhancement of DNA target bound report probe as a function of concentration of target DNA (three-fold redundant beads array).
plementary to part of target sequences are added at last to realize fluorescent detection of targets. A distinct advantage of this beads array based “sandwich” assay is that there is no need for the targets labeling or modification (see supplemental information for “sandwich” probes of p53, H-ras and NME1). 3.2. On-chip detection of purified DNA samples Sensitivity characteristic in nucleic acid assays is an important parameter for a new approach. To determine the assay sensitivity, different concentrations of p53 target DNA were firstly hybridized with 20 nM p53 report DNA. The p53 capture probe functionalized beads array was exposed to p53 target/report DNA complex solution for 20 min. Fluorescence images were taken after 15 min buffer rinse. Control experiment without DNA sample was also performed. On the basis of signal-to-noise of 3, the achieved detection limit is 0.02 nM, and a linear dynamic range from 0 to 1.0 nM was obtained, as shown in Fig. 2. The detection limit corresponds to an absolute of ∼10−17 mol of target molecules in 2 l sample. The signal does not increase significantly for target concentration higher than 1.0 nM, suggesting that 1.0 nM is the saturation concentration for the microbead surface. 3.3. Applications to multiple transcripts expression analysis
Fig. 1. Schematic illustrations of microfluidic beads array chip assembly and bioconjugated microbeads for DNA/RNA detection. (a) Beads were confined within tandem chambers along the microchannel, followed by sealing with a top plate to form linear microfluidic beads array. (b) Principle of beads modification with biotinylated DNA capture probe and sandwich hybridization for target gene detection.
Based on the results obtained using the purified samples, we have further applied the beads array to multiple transcripts expression detection in various cells extracts. p53, H-ras and NME1 are tumor-associated genes and their expression products play key roles in cell growth, differentiation and malignant transformation. We selected CNE2 tumor cell and normal HNE cell as a model for the transcripts expression analysis. At the same time, the responses of three genes expression in CNE2 cells to 5-flouorouracil (5-Fu) stimuli were also assessed. Fig. 3a shows the fluorescence images of the beads array for three cellular RNA samples in HNE, 5-Fu mediated CNE2 and CNE2 cells. Fig. 3b is the corresponding fluorescence intensity quantification for the beads array. According to the results, NME1 and H-ras
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two-dimensional chip-based microbeads array (Ali et al., 2003), reagent consumption has been reduced by three orders magnitude. Finally, compared with common planar DNA array technologies, the microbeads array methodology provides a number of unique advantages: high area-to-volume ratio of porous beads offers high density of immobilized capture probes, thus enabling a obvious sensitivity enhancement with comparison to the planar array, furthermore, DNA hybridization kinetics of beads array (<20 min) is several times faster than planar DNA array (typically >1 h). We concluded that this linear beads array is expected to find its practical applications in gene expression profiling and disease diagnosis. Acknowledgments This work was partially supported by the National Key Basic Research Program of China (2002CB513110), Key Project of Natural Science Foundation of China (90606003), Major International (Regional) Joint Research Program of Natural Science Foundation of China (20620120107), Natural Science Foundation of China (20475015), and the China National Key Projects (05FK5029). Appendix A. Supplementary data Fig. 3. (a) Fluorescence images of beads array detecting three cellular transcripts in three cells. Control images were obtained using the same experimental conditions without adding RNA samples (three-fold redundant beads array). Bar, 50 m. (b) Fluorescence intensity quantification of the beads array images.
have relative low expression in normal cells but high expression in carcinoma cells. After 5-Fu inducement to the CNE2 cells, p53 expression shows obvious upregulation, while expressions of H-ras and NME1 decrease obviously. At last, these results were validated by reverse transcriptase-PCR (see supplemental information for PCR primers and RT-PCR results). 4. Conclusions In conclusion, this work serves to extend the application of one-dimensional microfluidic beads array for multiple transcripts assay. With the use of “sandwich” hybridization assay, a detection limit of 0.02 nM with DNA samples was obtained, transcriptional expression of three tumor-associated genes in normal and tumor cells and expression changes of these genes in CNE2 cells before and after drug stimuli were also assessed. Some advantages of the microfluidic beads array are documented as follows: first, the beads array offers the capability of multiplexed oligonucleotide assays. This DNA multiplexing capacity extends the prior multiplexing application for protein profiling (Zhou et al., 2006). Second, the microfluidic design minimizes the sample and reagent consumption. The total volume of a 2 cm long one-dimensional beads array is less than 100 nl, thus only 2–3 l of sample is sufficient in a test. Compared with 1.5 ml sample consumption per test in
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