There has been a great deal of interest in the development of optical techniques for genomics analysis such as nonradioactive DNA probes for use in biomedical diagnostics, pathogen detection, gene identification, gene mapping, and DNA sequencing. Hybridization of a nucleic acid probe to DNA biotargets (e.g., gene sequences, bacteria, viral DNA) permits a very high degree of accuracy in identifying DNA sequences complementary to that probe. The possibility of using SERS for low-level detection of DNA bases and oligonucleotides was demonstrated in several studies in the 1980s (123-126). More recently, the possibility of using Raman and/or SERS labels for extremely sensitive detection of DNA has been demonstrated. For example, Graham et al. (113) have claimed adequate sensitivity for single-molecule DNA detection via sur face-enhanced resonance Raman scattering (SERRS). With the use of labeled DNA as gene probes, the SERS technique has been recently applied to the detection of DNA fragments of the human immunodeficiency virus (112) as well as B-cell lymphoma 2 gene (110).
A critical aspect of sequencing the entire human genome involves defining and identifying large insert clones of DNA corresponding to specific regions of the human genome. An approach that facilitates large-scale genomic sequencing involves developing maps of human chromosomes into maps based on large-insert bacterial clones, such as bacterial artificial chromosomes (BACs) (127-129). A time-saving method for detecting multiple BAC clone-labeled probes simultaneously would be especially appealing for the BAC approach to genome sequencing and mapping. The SERS technique can provide this label-multiplex capability. The SERS gene probes described in this section preclude the need for radioactive labels and have great potential to provide sensitivity, selectivity, and label multiplexing for DNA sequencing as well as clinical assays.
A detection system used for recording the SERS spectrum of individual spots is illustrated in Fig. 3. An individual spot could correspond to an individual microdot on a hybridization platform. This system can readily be assembled using commercially available or off-the-shelf components. A focused, low-power laser beam is used to excite an individual spot on the hybridization platform. In our studies, a helium-neon laser is used to provide 632.8-nm excitation with approx 5 mW of power. A bandpass filter is used to isolate the 632.8-nm line prior to sample excitation. A signal collection optical module is used to collect the SERS signal at 180° with respect to propagation of the incident laser beam. The collection module includes a Raman holographic filter, which rejects the Rayleigh scattered radiation before it enters the collection fiber. Finally, the collection fiber is coupled to a spectrograph (Instruments S4 Inc., Edison, NJ, HR-320) which is equipped with a Princeton, Trenton, NJ, RE-ICCD-576S detection system. The signal collection module can be coupled directly to the spectrograph, bypassing the optical fiber. However, the optical fiber greatly simplifies measurements and improves reproducibility by minimizing critical optical alignment steps.
Because the SERS gene probe hybridization platform consists of a two-dimensional (2D) array of DNA hybridization spots, a method for recording signals from all spots simultaneously would be highly advantageous, reducing analysis time as well as precluding the need for platform scanning. For analysis of SERS gene probe-based assays, this feat can be accomplished through multispectral imaging (MSI). The concept of MSI is illustrated in Fig. 4. With conventional imaging, the optical emission from every pixel of an image is without the capability for tunable wavelength selection (Fig. 4A). With con-
ventional spectroscopy, the signal at every wavelength within a spectral range can be recorded, but for only a single analyte spot. This condition is the basis for the single-point analysis system described above in the previous paragraph. The MSI concept (Fig. 4C) combines these two recording modalities, thereby allowing the acquisition of a Raman spectrum for every hybridization spot on the assay platform, provided that the entire platform can be included in the instrument's field of view. Critical to the success of this concept is an imaging spectrometer (Fig. 5). In our studies, a rapid-scanning solid-state device, an acoustooptic tunable filter (AOTF), is used for tunable wavelength selection
with image-preserving capability. This compact solid-state device has an effective wavelength range of 450 to 700 nm with a spectral resolution of 2 A and a diffraction efficiency of 70%. Wavelength tuning is achieved simply by supplying the AOTF with a tunable radiofrequency signal.
We have recently developed in our laboratory a SERS-active DNA hybridization platform that greatly enhances the utility of the SERS gene probe technology (106). In previous work, hybridization of labeled DNA probes was performed on conventional commercially available platforms (e.g., DNA-bind plates), followed by SERS activation of the platform (24,112). To make the SERS gene probe technology more practical, we have developed an inherently SERS-active hybridization platform (106). No SERS activation of the assay is required following the hybridization step. The new DNA assay platform is an array of oligonucleotide capture probes immobilized directly on a silver island-based substrate prepared on glass. The capture probes were anchored directly to the silver surface via reaction with an SAM of alkyl mercaptans. The coupling approach involved esterification under mild conditions of a carboxylic acid (immobilized mercaptoundecanoic acid) with a labile group; an N-hydroxysuccinimide (NHS) derivative; and further reaction with a 5'-amine-labeled DNA capture probe, producing a stable amide (130). Our recent study demonstrated the potential of this technology in cancer gene detection (BRCA1 and BAX genes) (106).
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