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CONTRACT NUMBER Lab-on-a-Chip Analysis of Explosives 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION Naval Research Laboratory,Chemistry Division,4555 Overlook Avenue REPORT NUMBER SW,Washington,DC,20375 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF ABSTRACT OF PAGES RESPONSIBLE PERSON a. REPORT b. ABSTRACT c. THIS PAGE Same as 3 unclassified unclassified unclassified Report (SAR) Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 Lab-on-a-Chip Analysis of with improved separation effi ciencies, therefore, are Explosives very attractive for addressing some of the security needs currently facing our troops. Th ese needs include G.E. Collins,1 J.D. Ramsey,2 B.C. Giordano,3 and the sensitive and selective detection of explosives, M.P. Chatrathi4 whether in oceanic environments (e.g., mines), or 1Chemistry Division as improvised explosive devices (IED). Despite the 2GeoCenters, Inc. advantages aff orded by these devices, the reduced 333AASSEEEE PPoossttddoocc microfl uidic channel dimensions directly aff ect the 444AArriizzoonnaa SSttaattee UUnniivveerrssiittyy ((PPoossttddoocc)) sensitivity of most traditional detection technologies and require integrating additional sample preconcen- Introduction: Laboratory analysis of complex tration techniques onto the microchip platform. We “real-world” samples typically requires a series of discuss the successful application of LOC to the analy- time-consuming and labor-intensive steps that include sis of explosive mixtures. We examine the integration sample preparation, separation, and detection. Th e of on-chip solid phase extraction (SPE) techniques for emerging technology of microfl uidic analytical devices, dramatic enhancements in sensitivity. or “Lab-on-a-Chip” (LOC), allows these functions to be integrated onto a single compact platform. Such Microchip Design: Figure 1(a) shows the micro- devices, due to the design simplicity available through chip design for integrating solid phase extraction advanced microfabrication technologies, permit the on a packed bed of beads, the subsequent elution of integration of various functional elements such as analytes using an organic eluent, injection, separation, sample preparation and handling, sample loading, and detection. Th e channels (20-µm deep by 60-μm separation, and detection, onto a single microchip wide) are etched in a glass substrate and bonded with platform. Typical analytical microsystems rely on a cover glass plate to close the microfl uidic network. electrokinetic fl uid “pumping” of the sample through All fl uid fl ow is controlled electro-osmotically by the a network of channels patterned in a planar (glass or application of desired voltages to the individual reser- plastic) substrate, eliminating the need for external voirs. A typical SPE injection sequence consists of fi rst pumps or valves. Microchip capillary electrophoresis directing the sample across the micro-SPE column to (CE) has been shown to provide high-speed analysis the sample waste reservoir. Th is is followed by elution (b) (a) (c) (d) FIGURE 1 (a) SPE microchip design; (b) close-up of packed bed; (c) fl uorescence microscope image of Rhodamine B loading onto packed bed; and (d) electropherogram for micro-SPE of 100 pM Rhodamine B. chemical/biochemical research 2005 NRL Review 121 of a concentrated band of analytes via subsequent onto a micro-SPE bed, the electropherogram shown in application of voltage to the eluent reservoir. Th e Fig. 1(d) for the on-chip SPE extraction and elution eluted sample plug is introduced into the separation of a 100-pM sample dye indicates that the detection channel by application of another voltage sequence, limits can be lowered into the femtomolar range. leading to its separation into individual analyte bands Th e quantitative nature of the extraction process is using the appropriate run buff er. illustrated in Fig. 3(a). As expected, a linear increase in the fl uorescence intensity of the eluted Rhodamine Explosives Analysis on a Microchip: Because of B was observed with increasing extraction time. Th e their electrical neutrality, the electrophoretic separa- preconcentration factors range from 20 to 300 times, tion of nitroaromatic explosives requires the introduc- but it can be much larger, depending on the total tion of a pseudo-stationary phase in the form of a extraction time. When coupled to the separation step, surfactant added to the run buff er. Figure 2 shows a it is possible to concentrate and separate a series of CE microchip separation of fi ve diff erent nitroaro- neutral fl uorescent dyes (Fig. 3(b)) in an experiment matic explosives in the absence of any SPE. Th e elec- analogous to that needed for the detection of nitroaro- trochemical activity of nitro-functional groups permits matic explosives. amperometric detection of the separated bands as they elute from the end of the microchip. Despite the Summary: We have demonstrated success- similarity in structure between these diff erent aromatic ful direct coupling of micro-SPE enrichment with explosives, the high resolving power of the LOC advanced separation techniques for model dye permits near-baseline resolution in just 100 s. compounds. Eff orts are underway to couple enrich- ment technologies with microseparation devices for Solid Phase Extraction (SPE) on a Microchip: the separation of nitroaromatic explosive mixtures, Although TNT (2,4,6-trinitrotoluene) and DNB investigations which should ultimately lead to power- (1,3-dinitrobenzene) detection limits have been achieved down to 60 ppb,1 the demanding sensitivity ful detection schemes for explosives and other toxic analytes of concern to the DoD and analytical com- requirements of explosive sensors in oceanic environ- munity alike. ments prompted us to investigate the incorporation of SPE techniques2 directly on the microchip platform. [Sponsored by ONR and MIPT] Figure 1(b) is a close-up image of the micro-SPE References bed. To contain the packing material, 5-μm C18 1J. Wang, M. Pumera, M.P. Chatrathi, A. Escarpa, M. Musameh, coated silica beads, within a microfl uidic platform, a G. Collins, A. Mulchandani, Y. Lin, and K. Olsen, “Single- porous poly(methacrylate) polymer was synthetically Channel Microchip for Fast Screening and Detailed Identifi cation crosslinked inside the channel by ultraviolet (UV) of Nitroaromatic Explosives or Organophosphate Nerve Agents,” Anal. Chem. 74, 1187-1191 (2002). photoinitiation. Although the fl uorescence image 2Q. Lu, G.E. Collins, M. Smith, and J. Wang, “Sensitive Capillary shown in Fig. 1(c) visually demonstrates the successful Electrophoretic Microchip Determination of Trinitroaromatic extraction of Rhodamine B (a model dye compound Explosives in Nonaqueous Electrolyte Following Solid Phase with similar hydrophobicity to aromatic explosives) Extraction,” Anal. Chim. Acta469, 253-260 (2002). FIGURE 2 Microchip capillary electrophoretic separation and amperometric detection of fi ve nitroaromatic explosives (2 ppm each). DNB = 1,3-dinitroben- zene; TNT = 2,4,6-trinitrotoluene; 2,4-DNT = 2,4-dinitrotoluene; 2,6-DNT = 2,6-dinitrotoluene. 122 2005 NRL Review chemical/biochemical research (a) (b) FIGURE 3 (a) Microchip SPE of 250-nm Rhodamine B for sequentially increasing load times; (b) microchip separation of two neutral dyes following on-chip SPE. chemical/biochemical research 2005 NRL Review 123