High-density, microsphere-based fiber optic DNA microarrays

Abstract

A high-density fiber optic DNA microarray has been developed consisting of oligonucleotide-functionalized, 3.1-μm-diameter microspheres randomly distributed on the etched face of an imaging fiber bundle. The fiber bundles are comprised of 6000–50 000 fused optical fibers and each fiber terminates with an etched well. The microwell array is capable of housing complementary-sized microspheres, each containing thousands of copies of a unique oligonucleotide probe sequence. The array fabrication process results in random microsphere placement. Determining the position of microspheres in the random array requires an optical encoding scheme. This array platform provides many advantages over other array formats. The microsphere-stock suspension concentration added to the etched fiber can be controlled to provide inherent sensor redundancy. Examining identical microspheres has a beneficial effect on the signal-to-noise ratio. As other sequences of interest are discovered, new microsphere sensing elements can be added to existing microsphere pools and new arrays can be fabricated incorporating the new sequences without altering the existing detection capabilities. These microarrays contain the smallest feature sizes (3 μm) of any DNA array, allowing interrogation of extremely small sample volumes. Reducing the feature size results in higher local target molecule concentrations, creating rapid and highly sensitive assays. The microsphere array platform is also flexible in its applications; research has included DNA–protein interaction profiles, microbial strain differentiation, and non-labeled target interrogation with molecular beacons. Fiber optic microsphere-based DNA microarrays have a simple fabrication protocol enabling their expansion into other applications, such as single cell-based assays.

Introduction

Optical fibers consist of an inner core surrounded by a clad material of lower refractive index. The differences in refractive index cause total internal reflection of light. A fiber optic bundle consists of thousands of individual fibers fused together such that each fiber retains its ability to transmit light independently of its neighbors (Fig. 1). By selectively etching the fiber core, an array of microwells is formed (Pantano and Walt, 1996). These microwells may be filled with oligonucleotide-functionalized microspheres. The array dimensions can be tailored to suit any size of oligonucleotide-functionalized microsphere. The well diameters are equal to those of the fiber cores, and the depths are dependent on the etchant concentration, the exposure time, and the fiber composition. Because each microsphere is optically wired to a fiber, the specific interactions on each microsphere surface can be independently monitored. Fiber optic microsphere-based DNA detection is a viable alternative to other high throughput microarray methods. Microarrays employ thousands of single-stranded oligonucleotide sequences immobilized to discrete sensing regions on a solid substrate.(Fodor et al., 1991, Schena et al., 1995, Schena et al., 1996) The tethered sequences, or probes, hybridize with their complementary sequences, or target molecules, which are detected in the hybridization solution. Thousands of discrete sensing regions are patterned on a solid substrate so that each different sensing region simultaneously detects a unique target sequence that may be related to a disease state or expression profile. The fiber optic array platform uses probe sequences coupled to the microspheres as the oligonucleotide sensing regions (Epstein et al., 2002, Ferguson et al., 2000, Michael et al., 1998, Walt, 2000). The desired oligonucleotide sequences are attached to individual microspheres and added to the etched wells on the fiber optic bundle face.

The detection scheme combines the intrinsic recognition abilities of nucleic acids with fluorescence-based detection methods. Fluorescence-based assays are more desirable than traditional radiolabeled methods due to their increased safety and experimental versatility. Fluorescence can be incorporated into microarray assays by fluorescent intercalating dyes, fluorescently-labeled targets, or label-less methods employing fluorescence resonance energy transfer (FRET). Fluorescence-based assays enable the measurement of multiple wavelengths independently and simultaneously. The use of multiple fluorophores enables parallel interrogation schemes.

Parallel analysis can be further realized with microarrays that incorporate numerous sensing elements. The microspheres are fabricated in a batch process, where 10⁹ identical microsensors can be made simultaneously in 1 ml, and used to make a microsphere stock for multiple arrays. The placement of different microsphere types on the etched fiber face is random, so the exact position of each microsphere must be determined after assembly of the array. An optical decoding scheme was developed whereby each microsphere-type was impregnated with a fluorescent dye or combination of dyes, creating a dye ‘bar code’ that can be used to locate and identify the microsensors in an array (Ferguson et al., 2000, Michael et al., 1998, Walt, 2000).

The fiber-optic microsphere-based platform provides a number of advantages over other array-based methods (Epstein et al., 2002, Ferguson et al., 2000, Walt, 2000). This platform provides a high-density array with the smallest individual feature sizes available. A higher packing density corresponds to more sensing elements per array, enabling simultaneous measurements and higher throughput. The reduced array size also enables smaller volumes to be interrogated, and since many array interactions are diffusion dependent, more rapid responses are possible with reduced volumes, further increasing throughput. The miniature feature sizes also provide a detection advantage. The same number of target molecules confined to a smaller volume yields a higher local concentration. For example, a few hundred target molecules limited to one 3 micron well of 30 fl volume corresponds to nanomolar concentrations. Nanomolar concentrations of fluorophores are readily detectable (Epstein et al., 2002, Ferguson et al., 2000). In addition, the platform allows for multiple assays with one array while most other array-based systems are single use. We have demonstrated the use of these microsphere-based arrays over 100 times with less than 8% S.D. (Ferguson et al., 2000). Reusable arrays have an impact on overall assay cost and preparation time. Furthermore, they eliminate issues regarding array-to-array reproducibility. This platform also allows flexibility in design as research needs evolve. A microsphere sensor pool used to fabricate an existing array can be combined with additional microsphere probes to fabricate a new array with additional capabilities. Because of the random assembly process, these arrays are fabricated containing multiple copies of each microsphere type. Such redundancy reduces the occurrence of false positives and false negatives. Identical sensors provide consensus-based analysis, where individual responses from redundant counterparts in the array must agree. The fiber optic array platform is also flexible in its ability to incorporate different nucleic acid detection schemes, such as FRET-based molecular beacon assays (Steemers et al., 2000) and aptamers (Lee and Walt, 2000).