High-Throughput Trace Analysis Using SERS-Coated Microtiter Plates with a Raman Plate Reader - - Spectroscopy
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High-Throughput Trace Analysis Using SERS-Coated Microtiter Plates with a Raman Plate Reader

Raman spectroscopy has been widely used in a variety of analytical measurements because of its chemical specificity, its ability to produce spectral signatures that are unique, and its ability to obtain measurements conveniently with little sample preparation. Raman spectroscopy, though widely applied, is not inherently sensitive, with routine detection limits in the parts-per-thousand range. Although sensitivity can be improved by 10 to 50 times using a shorter laser excitation wavelength due to the inverse fourth power dependence of Raman scattering on wavelength, it also can cause fluorescence, which can overwhelm the weak Raman effect. A more dramatic method of increasing Raman scattering is surface-enhanced Raman spectroscopy (SERS), which can enhance scattering by six orders of magnitude or more (1), improving detection limits to the parts-per-billion (nanogram-per-milliliter) range. Since its discovery and recognition in the 1970s (2,3), the vast majority of research has been aimed at elucidating the mechanisms that promote this enhancement (4), which now includes detection of single molecules on "hot-spots" and enhancements as high as 14 orders of magnitude (5,6). Only in the last decade have efforts begun to exploit the ability of SERS to detect trace chemicals, such as chemical and biochemical agents and drugs (7–9). Furthermore, very little research has been reported on the routine use of SERS in high sample throughput applications that might benefit drug discovery or routine clinical analysis.

Farquharson and colleagues reported the use of SERS-active microtiter plates for drug discovery (10), while Sägmuller and colleagues described the preparation of silver halide dispersions in microplates for the analysis of illicit drugs (11). Bell and colleagues reported the deposition of silver sols onto glass slides and polymer-coated microtiter plates (12), with subsequent extension of the method to high-throughput detection of nicotine (13). However, all of these reported measurements employed unique SERS-active substrates prepared by the researchers, as well as custom-built Raman spectrometer systems or Raman microscope systems. Many laboratories that have the need to measure trace components in high throughput, however, might not have the expertise to synthesize the SERS substrates with high reproducibility, nor the budget to acquire the high performance Raman instrumentation described in the previous research. Here, we describe the use of commercially available SERS-active microtiter plates with a novel, low-cost Raman microtiter plate reader for high-throughput trace analysis measurements and show preliminary results for samples representative of explosives, nerve agents, pharmaceuticals, and biological compounds.

Experimental

Benzenethiol, benzoic acid, 2,4-dinitrotoluene, and methylphosphonic acid were obtained from Sigma Aldrich (Allentown, Pennsylvania), and were used as is or diluted in high performance liquid chromatography (HPLC)-grade water or methanol for normal Raman and SERS measurements.

A stock aqueous solution of ampicillin (molecular biology grade, Na salt, Shelton Scientific, Shelton, Connecticut) was prepared by weighing (Explorer PRO analytical balance, Ohaus, Pine Brook, New Jersey) dry ampicillin powder and dissolving into purified water (sequential purification of tap water with Diamond-RO and NANOpure Diamond systems, Barnstead, Dubuque, Iowa) to a final concentration of 100 mg/mL. The stock solution was serially diluted with purified water using precision pipettes and disposable pipette tips to achieve the 100 ppm solution.

A DNA stock solution was prepared by weighing a portion of herring sperm DNA (free acid, Sigma Co., St. Louis, Missouri, fragment size 50–200 base pairs as determined by gel electophoresis in a 1% agarose gel), allowing the dry DNA to hydrate with excess purified water in a centrifuge tube for 1 h at room temperature, after which the volume was adjusted with the addition of NANO-pure water to achieve a final concentration of 50 mg/mL, and the solution was manually shaken until a clear homogenous solution was obtained. A 100-ppm DNA solution was prepared by serial dilution as described earlier.


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