| DNA Hybridization Analysis in Microfluidic Devices
Hybridization analysis of nucleic acids using arrays of immobilized oligonucleotides or cDNAs is being developed in a number of laboratories for applications including gene mapping, detection of genetic diseases, and monitoring mRNA expression levels. Detection of hybridized sequences generally involves the covalent labeling of target nucleic acids with fluorescent labels prior to hybridization. In the present study, DNA probes were immobilized in the channels of a planar glass microfluidic device and exposed to unlabeled complementary or noncomplementary target sequences. Hybridization was determined using a dsDNA-specific intercalating fluorescent dye and fluorescence microscopy with CCD imaging. Target DNA samples and dye could be transported to the hybridization sites by either hydrostatic or electrokinetic pumping. Concentrations of target oligonucleotides as low as 50 nM were detected within 15-20 minutes by this method. These techniques demonstrate the ability to perform rapid hybridization assays on small DNA samples without the need to covalently label the target nucleic acids.
Oligonucleotide probes derivatized with 5'-hexylamine tails were covalently attached to microchannel surfaces coated with 3-aminopropyltriethoxysilane with glutaraldehyde as crosslinker. Channels were approximately 10 µm deep x 50 µm wide. In the experiment of Figure 1, the surfaces of a simple cross-channel chip were uniformly derivatized with a 16-mer probe. Complementary and noncomplementary DNA (50 µM, 16-mers) in PBS were simultaneously added via suction-induced flow to separate channels for 5 min, followed by washes with PBS and TE buffers for 1 min each. PicoGreen dye (Molecular Probes) in TE solution was then added for 5 min. Nonbound dye was washed out with TE buffer for approximately 1 min before fluorescence imaging of the chip (Figure 1b) with an epifluorescence microscope equipped with Hg lamp excitation, FITC filters and CCD camera. The image clearly demonstrates DNA hybrid formation in the flow path of the complementary DNA. Quantitative analysis of the image (Figure 2c) showed approximately 3-fold greater fluorescence in the channel expected to contain duplex DNA. Channels exposed to noncomplementary DNA gave background fluorescence levels essentially identical to those exposed to buffer only.
In a similar experiment using laser-induced fluorescence detection (argon ion laser, 488 nm) and longer hybridization time (35 min) an 8- to 9-fold difference in fluorescence intensity was observed between channels treated with complementary and noncomplementary DNA (Figure 2). The channel cross was imaged with PicoGreen solution present in all channels. Flushing the channels with TE buffer after treatment with PicoGreen/TE solution gave only modest improvements (~10%) in the fluorescence ratio of hybridized and nonhybridized channels.
In a variation of the above experiments, different probes were immobilized in separate channels of a cross-channel chip. A 50 nM solution of DNA complementary to one of the probes was flowed through both probe channels for a total of 15 minutes. The channels were washed with PBS and treated with PicoGreen/TE solution for two minutes. After a final 1-minute wash with TE buffer, the channels were examined for laser-induced fluorescence as described above. Quantitation by CCD imaging (Figure 3) showed approximately 3.5-fold greater fluorescence in the channel containing complementary probe than in other channels, as expected for sequence-specific hybridization to the complementary target DNA.
The microchip design shown in Figure 4 allows the electrokinetic addition of target DNA to a hybridization chamber containing immobilized probe, followed by washing and staining steps, as shown. Results using electrokinetic transport for both hybridization and staining steps have resulted in fluorescence signals equivalent to those obtained by hydrostatic transport (Figure 5). However, further experiments are needed to improve the reproducibility of electrokinetic transport in these devices.
Although no attempt was made to optimize either the rate or stringency of hybridization, these experiments indicate the feasibility of using microfluidic chips and fluorescent staining for microscale hybridization analysis of DNA. Chips designed for this purpose can incorporate channel manifolds with single or multiple probes. Low temperature microfabrication (1) would allow immobilization of probes in open channels prior to coverplate bonding. The hybridization and staining steps could potentially be combined with other on-chip functions, such as cell lysis and DNA amplification (2), to produce a fully integrated ëLaboratory-on-a-Chipí device for forensic or biomedical applications.
1. Wang, H. Y., Foote, R. S., Jacobson, S. C., Schneibel, J. H., and Ramsey, J. M. (1997) Sensors and Actuators B, 45, 199-207.