INTEGRATED NASBA ARRAY FOR DRUG SCREENING AND EXPRESSION PROFILING
Ivan K. Dimov1, Luke P. Lee1,2 1 Biomedical Diagnostics Institute, Dublin City University,
Glasnevin, Dublin 9, IRELAND 2Biomolecular Nanotechnology Center, Berkeley Sensor and Actuator Center Department of Bioengineering, University of California, Berkeley, CA, USA
ABSTRACT
We present the design and development of a novel integrated microfluidic NASBA array that includes microfluidic dynamic cell culture, cell stimulation, cell labeling, lysis, and real-time Nucleic-Acid-Sequence-Based-Amplification (NASBA) based RNA analysis on a single chip. By multiplexing this integrated functionality the device can be used for high content screening, gene expression pro-filing, and biomedical diagnostic applications. KEYWORDS: NASBA, microfluific, array, cell culture, gene profiling.
INTRODUCTION
Many groups have reported microfluidic cell culture arrays and high content screening with on-chip drug gradient generation [1-2] while others have demon-strated the effectiveness of gene profiling on a chip [3-4]. In this work we present a device that can merge these two cellular and molecular functionalities into a single chip enabling from sample-in to experiment and answer out capabilities with an in-tegrated NASBA amplification and detection system. NASBA is an isothermal reac-tion performed at 41°C, which obviates the need for a thermal cycler facilitating the design and operation of devices. In some cases the one step NASBA protocol can achieve levels of detection of extracted RNA a 100 times lower compared to the three step RT-PCR protocol. Furthermore NASBA has the unique ability to specifi-cally amplify RNA in a background of DNA of comparable sequence.
Figure 1. Integrated NASBA array device.(A) 3D render with the loading tips. (B) Microfluidic NASBA element. (C) 2D schematic of the full system. (D) photograph of a row of 8 chambers with perfusion pipette tips. (E) Design of each microfluidic NASBA, micrograph scale-bars 200µm.
Twelfth International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 12 - 16, 2008, San Diego, California, USA
EXPERIMENTAL A complete integrated and multiplexed device schematic is shown in figure 1, 64
identical modules are multiplexed with the same distribution as a 96 micro titter well plate, which enables sample loading to be done with a standard 8 channel pipette. Each module consists of a cell capture and culture reservoir, large and small particle filters, lysis and NASBA mixtures reservoir.
The reagent and sample loading is done with standard pipettes, while media per-fusion for cell culture is done with hydrostatic flow (Fig. 2). First cells are loaded and captured in the cell culture chamber (Fig. 3), where they can be cultured and stimulated with drugs mixed into the perfusing media and keeping the device in a 37ºC environment. Cell labeling can also be done at any time by pipetting the flo-rescent label into the perfusion medium and allowing it to hydrostatically flow over the cell culture. RNA profiling can be done by pumping in the lysis reagent and later the real-time NASBA reagents into the cell culture chamber. This is done by using a PDMS-XB composite that expands its volume by up to 200% when heated with the electrodes up to 80ºC. The reagents are mixed into the culture chamber by diffusion within 4 min. (Fig. 2). Once that is done the device is incubated at 41ºC in a thermally controlled inverted fluorescent microscope where the isothermal real-time NASBA reaction is fluorescently monitored. A real-time test NASBA reaction was successfully done on the device (Fig. 3). The positive and negative control NASBA reactions were done by flowing into the device, pre-mixed reagents that contained all the elements (enzymes, RNA target, molecular beacon probes, forward and reverse primers) required for the real-time NASBA reaction.
Figure 2. (A) Hydrostatic flow characterization through each culture chamber for different outlet tube inner diameters (0.02”-0.06”). (B) In chamber mixing charac-terization acquired micrographs and 2D-histograms of the fluid mixing. DISCUSSION
By utilising gravity driven passive pumping 64 individually addressable culture media reservoir and pumping units can be densly integrated. By varing the height of
(A) (B)
641
Twelfth International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 12 - 16, 2008, San Diego, California, USA
the outlet tubes the flow velocity in the culture chambers can be adjusted. The opened pipette tips alow for an easy and gentle insertion of cell for loading the device, quick insertion of the stimulation drugs and labelling dyes.
Figure 3. (A) 24 hrs cell culture demonstrating that several cell types can retain vi-ability after being cultured in the Poly-L-Lysine coated micro-device. (B) On-chip microfluidic real-time molecular beacon based NASBA. Insert: Reaction showing clear difference between a positive and a negative control after 90 min. CONCLUSIONS
In summary, we have designed and fabricated an integrated NASBA array. We have characterized each critical part of the device (Figure 2-3). At the current stage of this work in progress the device is being tested and optimized in the individual microfluidic cell-culture and NASBA elements before implementing the fully multi-plexed array.
ACKNOWLEDGEMENTS
The authors would like to thank Claus R. Poulsen, Sharon O'Toole, John O'Leary and Marek Radomski for providing the cancer cells and Justin O’Grady, Majella Maher and Terry J. Smith for providing the NASBA probes and primers. This work was supported by the Science Foundation Ireland under Grant No. 05/CE3/B754. REFERENCES [1] P. J. Lee, P. J. Hung, V. M. Rao, and L. P. Lee, Nanoliter scale microbioreactor array
for quantitative cell biology, Biotechnology and Bioengineering, 94, pp 5-14 (2005) [2] Ye, N., et al., Cell-based high content screening using an integrated microfluidic de-
vice, Lab on a Chip, 7(12): p, 1696-1704, (2007) [3] Bontoux, N., et al., Integrating whole transcriptome assays on a lab-on-a-chip for sin-
gle cell gene profiling, Lab on a Chip, 8(3): p, 443-450, (2008) [4] Warren, L., et al., Transcription factor profiling in individual hematopoietic progeni-
tors by digital RT-PCR, Proceedings of the National Academy of Sciences, 103(47): p, 17807-17812, (2006)
(A) (B)
642
Twelfth International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 12 - 16, 2008, San Diego, California, USA
