Fiber Optics

Fiber biosensor analyzes multiple DNA samples

Feb. 1, 1997

FIGURE 1. Proximal ends of probe fibers are epoxied into a stainless steel tube that is placed in the fiber chuck - a harness to hold together the bundle - of the imaging system. Protective rubber tubing around each fiber's distal end enables individual detection. The bundle used for the experiment was 3 ft long, though the cable can be any length. The probe can also be used for remote sensing.

A technology for simultaneous DNA analysis using an optical fiber bundle has been developed by Christopher Adams and associates at Mosaic Technologies (Natick, MA). The disposable fiberoptic device allows a researcher to both amplify and detect a target DNA sequence. Present detection techniques require a high sample DNA concentration and lengthy assay times, whereas an optical fiber biosensor facilitates sensitive, quantitative fluorescence detection and requires very small sample volumes for analysis. Mosaic's Bridge amplification chemistry is a solid-phase application of the conventional liquid-phase polymerase chain reaction (PCR) process, used in infectious disease and cancer diagnosis, forensics, basic research, and microbiology.

To form the multiplex DNA sensor, synthetic oligonucleotide hybridization probes were covalently immobilized on one end of an optical fiber 200 µm in diameter, though each fiber can range from 100 ?m to 1 mm in diameter. Several such fibers were bundled together (see Fig. 1 on p. 34).¹ "The implication is that one can take any number of target bundles—from 2 to 2000—and do multiple tests with a single disposable device," says Adams.

Immobilized probe concentrations limit the amount of target DNA that hybridizes and generates a signal. The feasibility of detecting fluorescein-labeled target DNA after hybridization to the immobilized probe on the fiber surface was tested using a 500-µm-diameter single-core fiber with an immobilized interleukin-4 (IL4) probe. The tips of the bundle were placed directly in the target solution, which can be as little as 3 µl. The detection system consisted of a modified epifluorescence microscope with the optics optimized to couple with an optical fiber.

The specificity of the sensor was evaluated by placing the sensor in both complementary and noncomplementary target solutions. The fluorescent signal increases when exposed to the complementary target. A complete absence of signal confirmed hybridization specificity.

FIGURE 2. Images of fiberoptic biosensor bundle in buffer solution taken by a charged-coupled device camera show varying degrees of fluorescence of individual fibers reacting to their target sample. White light indicates high intensities. Signals obtained in the buffer solution before hybridization were subtracted from the signals obtained after hybridization.

Probes specific for human cytokine mRNA sequences were immobilized on the tips of single-core fibers to demonstrate a DNA biosensor. Cytokines are powerful immune-system hormones whose expression is stimulated in response to inflammatory stimuli or infection. Each fiber of the seven-fiber bundle was functionalized with different cytokine oligonucleotide probes, creating a multiple-target sensor. Each fiber tip was placed in a solution containing one or more 5-fluorescein-labeled cytokine sequences for 5 min. Fluorescence signals were acquired using an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Commercially available software analyzed the signal in less than 30 s. Only those fibers with probes complementary to the added target emitted a signal (see Fig. 2 on p. 37).

"The system can analyze tissue, blood, or urine—any biological sample," says Adams. "It is fast, highly sensitive, disposable, inexpensive." The complete analysis of multiple DNA sequences can be performed in less than 5 min. The fiberoptic biosensor is scheduled to be on the market in 1997.

REFERENCE

1. Jane Ferguson et al., Nature Biotech. 14, 1681 (Dec. 1996).