Integrated Plastic Microfluidic Devices with ESI-MS for Drug Screening

Mar 30, 2001 - Remote Radio-Frequency Controlled Nanoliter Chemistry and Chemical Delivery on Substrates. Hongke Ye , Christina L. Randall , Timothy G...
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Anal. Chem. 2001, 73, 2048-2053

Integrated Plastic Microfluidic Devices with ESI-MS for Drug Screening and Residue Analysis Yun Jiang,† Pen-Cheng Wang,† Laurie E. Locascio,‡ and Cheng S. Lee*,†

Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, and Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899

For this work, two different plastic microfluidic devices are designed and fabricated for applications in highthroughput residue analysis of food contaminants and drug screening of small-molecule libraries. Microfluidic networks on copolyester and poly(dimethylsiloxane) substrates are fabricated by silicon template imprinting and capillary molding techniques. The first device is developed to perform affinity capture, concentration, and direct identification of targeted compounds using electrospray ionization mass spectrometry. Poly(vinylidene fluoride) membranes sandwiched between the imprinted copolyester microchannels in an integrated platform provide continuous affinity dialysis and concentration of a reaction mixture containing aflatoxin B1 antibody and aflatoxins. The second microfluidic device is composed of microchannels on the poly(dimethylsiloxane) substrates. The device is designed to perform miniaturized ultrafiltration of affinity complexes of phenobarbital antibody and barbiturates, including the sequential loading, washing, and dissociation steps. These microfabricated devices not only significantly reduce dead volume and sample consumption but also increase the detection sensitivity by at least 1-2 orders of magnitude over those reported previously. Improvements in detection sensitivity are attributed to analyte preconcentration during the affinity purification step, limited analyte dilution in the microdialysis junction, minimal sample loss, and the amenability of ESI-MS to nanoscale sample flow rates. The area of microfluidics has developed into one of the most dynamic fields in analytical chemistry during the past decade.1-9 Miniaturized bioanalytical devices provide several advantages over * Corresponding author: (phone) (301) 405-1020; (fax) (301) 314-9121; (e-mail) [email protected]. † University of Maryland. ‡ National Institute of Standards and Technology. (1) Harrison, D. J.; Manz, A.; Fan, Z.; Ludi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926. (2) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895. (3) Chiem, N.; Harrison, D. J. Anal. Chem. 1997, 69, 373. (4) Effenhauser, S. F.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 2637. (5) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1995, 67, 2284. (6) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Warmakc, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107. (7) Waters, L. C.; Jacobsen, S. C.; Krotchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158. (8) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676.

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benchtop instruments, including smaller dead volume, smaller sample consumption, shorter analysis time, lower cost, greater portability, and potentially greater separation resolution for complex mixtures in an integrated format. For example, a glass microfluidic system for DNA amplification and detection was developed by Ramsey et al.7 The glass chip was thermally cycled to lyse cells and amplify the DNA, and the products were then analyzed on-chip using a sieving medium for size separation. The miniaturized system used very low sample volumes, decreased reaction times, and allowed for the integration of several steps in a complex assay. For this work, we have developed plastic microfluidic devices for high-throughput screening of small-molecule libraries. Previously, we reported on the development of an automated and continuous affinity capture and concentration system based on the use of dialysis hollow fibers.10 The system combined the strengths of hollow fiber dialysis, including the ease and speed of purification and concentration, with the specificity of affinity interactions in solution. The complexes between the lead compounds and the affinity binding proteins were separated from other chemical components inside a dialysis hollow fiber as a result of their differences in size. The affinity complexes were further concentrated inside a second dialysis fiber. The concentrated drug candidates were then liberated from the binding proteins in a microdialysis junction and directly identified using electrospray ionization mass spectrometry (ESI-MS). This work involves the miniaturization of affinity hollow fiber dialysis and concentration in a microfluidic format. Important advantages of miniaturizing the affinity-based system include significantly reduced dead volume and minimal sample consumption in a system designed to perform many sequential analytical steps. Microfluidic devices are fabricated in plastic substrates with polymeric membranes for performing affinity capture and analyte concentration. One advantage of using plastics for the development of a microfluidic system is that the pliability of plastics lends itself readily to inexpensive fabrication techniques such as casting,11-17 embossing, injection molding,18 imprinting,19 and laser ablation.20 (9) Woolley, A. T.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1997, 69, 2181. (10) Jiang, Y.; Lee, C. S. J. Mass Spectrom. submitted for publication. (11) Jackman, R. J.; Duffy, D. C.; Ostuni, E.; Willmore, N. D.; Whitesides, G. M. Anal. Chem. 1998, 70, 2280. (12) Duffy, D. C.; McDonald, J. C.; Schueller, O. J.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974. 10.1021/ac001474j CCC: $20.00

© 2001 American Chemical Society Published on Web 03/30/2001

Figure 1. Side view schematic of miniaturized affinity dialysis and concentration system (device 1). I-III indicate top, middle, and bottom imprinted copolyester pieces, respectively. Piece II is imprinted on both sides. Two PVDF membranes separate the copolyester channels.

In this work, microchannels on copolyester and poly(dimethylsiloxane) (PDMS) substrates are fabricated by silicon template imprinting19 and capillary molding21 techniques, respectively. Online coupling of affinity capture and concentration with direct identification of captured compounds using ESI-MS are demonstrated in the integrated microfluidic system. Assays are performed for high-throughput screening of two model systems, including aflatoxin B1 antibody/aflatoxins and phenobarbital antibody/barbiturates. EXPERIMENTAL SECTION Materials and Reagents. Aflatoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin G2a) and barbiturates (barbital, allobarbital, butalbital, amobarbital, pentobarbital, phenobarbital, hexobarbital, secobarbital, thiopental) were purchased from Sigma (St. Louis, MO). Monoclonal antibodies against aflatoxin B1 and phenobarbital were obtained from Sigma and Biodesign International (Kennebunk, ME), respectively. Ammonium acetate, acetic acid, ammonium hydroxide, methanol, 2-propanol, and sodium mono- and diphosphate were acquired from Fisher Scientific (Fair Lawn, NJ). All solutions were prepared using water purified by a Nanopure II system (Barnstead, Dubuque, IA) and further filtered with a 0.22-µm membrane (Millipore, Bedford, MA). Plastic Microfluidic System for Affinity Dialysis and Concentration of Aflatoxin B1 Antibody/Aflatoxins. Copolyester microfluidic devices (see Figure 1) were constructed by a silicon template imprinting technique at room temperature as (13) Anderson, J. R.; Chiu, D. T.; Jackman, R. J.; Cherniavskaya, O.; McDonald, J. C.; Wu, H.; Whitesides, S. H.; Whitesides, G. M. Anal. Chem. 2000, 72, 3158. (14) Deng, T.; Wu, H.; Brittain, S. T.; Whitesides, G. M. Anal. Chem. 2000, 72, 3176. (15) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. A. Science 1997, 276, 779. (16) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. A. J. Am. Chem. Soc. 1998, 120, 500. (17) Effenhauser, C. S.; Bruin, G. J. M.; Paulus, A.; Ehrat, M. Anal. Chem. 1997, 69, 3451. (18) McCormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, D. J.; Hooper, H. H. Anal. Chem. 1997, 69, 2626. (19) Xu, J.; Locascio, L. E.; Gaitan, M., Lee, C. S. Anal. Chem. 2000, 72, 1930. (20) Roberts, M. A.; Rossier, J. S.; Bercier, P.; Girault, H. Anal. Chem. 1997, 69, 2035. (21) Xu, J.; Gao, J.; Locascio, L. E.; Lee, C. S. Anal. Chem., in press.

described previously.19 For imprinting microchannels with a silicon template, pieces of plastic substrates in the dimension of 10 cm by 5 cm were cut from the copolyester sheet (DSM Engineering Plastic Products, Sheffield, MA). Holes were drilled through the plastic pieces ∼5.5 cm apart. The copolyester substrate was placed over the silicon template, and the whole assembly was sandwiched between two polished aluminum plates. A hydraulic press (Carver Inc., Wabash, IN) was employed to apply an imprinting pressure of 1600 psi (1.1 × 107 N/m2) for 5 min at room temperature. The imprinted channels on the copolyester substrates were revealed after the pressure was released and the assembly was dismounted. The dimensions (30 µm in depth, 100 µm in width, and 5.5 cm in length) of imprinted microchannels were examined using a WYKO RST optical profilometer (WYKO Co., Tucson, AZ). Four 5-cm-long fused-silica capillaries with the dimensions of 50 µm i.d. × 350 µm o.d. (Polymicro Technologies, Phoenix, AZ) were inserted into the holes on the plastic substrates containing channels 1 and 4 (see Figure 1) and served as the inlets/outlets for the dialysis buffer and the dry air. Epoxy adhesive (Loctite, Cleveland, OH) was then applied around the outside of the capillary/hole boundary. After the epoxy cured, two poly(vinylidene fluoride) (PVDF) membranes (80 000 molecular weight cutoff, Spectrum Medical Industries Inc., Dallas, TX) and three plastic substrates containing four microchannels were clamped together between two polished aluminum plates. Besides fluid microchannels, additional position markers were imprinted on the plastic substrates for aligning the channels on the opposite sides of the membrane. The center section of aluminum plates was removed to expose capillary connections and view the microchannels. Through a capillary connection, one end of channel 1 was coupled to a Harvard Apparatus 22 syringe pump (South Natick, MA) for the introduction of a dialysis buffer at a flow rate of 2 µL/min. The dialysis buffer containing 10 mM ammonium acetate at pH 7.4 flowed countercurrently against the reaction sample. One end of channel 2 was connected to a sample inlet, a 5-cm-long fusedsilica capillary (50 µm i.d. × 192 µm o.d.), by a “zero dead volume” chromatographic fitting (Microtight union, Upchurch Scientific, Oak Harbor, WA) machined into the edge of the center plastic substrate. The reaction mixture containing aflatoxin B1 antibody and aflatoxins was prepared in 10 mM sodium phosphate buffer at pH 7.4 and was introduced through the sample inlet using a Harvard Apparatus 22 syringe pump. Channels 2 and 3 were connected via the hole drilled through the center copolyester substrate. One end of channel 3 was connected to a microdialysis junction by another “zero dead volume” chromatographic fitting machined into the edge of the center substrate. The capillary (10 cm × 50 µm i.d. × 192 µm o.d.) extending out of the fitting and a 2-cm-long ESI emitter capillary were butted together inside a 2-cm length of polysulfone dialysis tubing (200 µm i.d. × 250 µm o.d., nominal molecular weight cutoff of 10 000) for the formation of a microdialysis junction.22,23 The polysulfone dialysis tubing was purchased from A/G Technology (Needham, MA). The electrospray emitter was made from a 2-cm-long 50 µm i.d. × 192 µm o.d. fused-silica capillary. After the polyimide coating of the capillary was removed, (22) Severs, J. C.; Smith, R. D. Anal. Chem. 1997, 69, 2154. (23) Yang, L.; Lee, C. S.; Hofstadler, S. A.; Smith, R. D. Anal. Chem. 1998, 70, 4945.

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Figure 2. Procedures for fabricating PDMS microchannels used in miniaturized affinity ultrafiltration system (device 2).

the outside of the capillary was etched in a 30% hydrofluoric acid solution for 20 min with nitrogen blowing. Epoxy adhesive was applied around the outside of the dialysis tubing/capillary boundaries. After the epoxy cured, the capillary was inserted into a 250-µL Eppendorf pipet tip containing an electrolyte solution. The pipet tip was attached to an x-y-z motion manipulator inside a microion spray source of Perkin-Elmer Sciex (Foster City, CA) API 150EX single quadrupole mass spectrometer. A platinum wire was inserted in the electrolyte reservoir and connected to a CZE 1000R high-voltage power supply (Spellman High Voltage Electronics, Plainview, NY). The use of an open reservoir rather than an enclosed/limited reservoir, and plastic rather than metal, avoids problems due to gas bubbles in the liquid circuit. Plastic Microfluidic System for Affinity Ultrafiltration of Phenobarbital Antibody/Barbiturates. As shown in Figure 2, a capillary molding technique21 was employed for the fabrication of PDMS microchannels. First, a hole was punched on one side of an aluminum foil dish (Fisher) using a syringe needle. A 30cm-long fused-silica capillary (50 µm i.d. × 100 µm o.d.) was then inserted through the hole and kept at the bottom of the dish. The hole was sealed with epoxy and then the PDMS prepolymer was poured into the dish. The capillary was used as a template for the fabrication of microchannel, as well as the fluid inlet/outlet. After the PDMS prepolymer was cured at 200 °C for 2 h inside a GC oven, the aluminum foil dish was carefully peeled off to obtain a PDMS substrate with the embedded capillary. A microchannel was formed by the removal of the last 0.5 cm of capillary tubing in the PDMS substrate. As shown in Figure 3, a symmetrically configured affinity ultrafiltration system consisted of two aluminum plates, two copolyester plates, two PDMS substrates containing the microchannels and the capillaries, and one PVDF membrane. To assemble the system, a PVDF membrane was first sandwiched between two PDMS substrates with microchannels facing the membrane. Microchannels were aligned using the capillaries extending out of PDMS substrates. Two copolyester plates were used to provide the additional support and were clamped between two aluminum plates. The capillary in the upper PDMS substrate 2050 Analytical Chemistry, Vol. 73, No. 9, May 1, 2001

Figure 3. Assembly of complete miniaturized affinity ultrafiltration system (device 2) containing preformed PDMS microchannels. Channels are fabricated in the PDMS substrates. The copolyester pieces are not imprinted but are used to provide structural support to the soft PDMS substrates.

was connected to a Harvard Apparatus 22 syringe pump for the introduction of a reaction mixture containing phenobarbital antibody and barbiturates prepared in 10 mM sodium phosphate buffer at pH 7.4. The capillary extending out of the lower PDMS substrate was coupled to a microdialysis junction prior to ESIMS. Electrospray Ionization Mass Spectrometry. The Eppendorf pipet tip housing the microdialysis junction contained a solution of 50% methanol, 49% water, and 1% acetic acid (v/v/v) at pH 2.6 for measuring aflatoxins eluted from the affinity dialysis and concentration system. The solution offered postseparation acidification and increased organic solvent content via the polysulfone dialysis tubing. The buffer exchange allowed the dissociation of affinity complexes (between aflatoxin B1 antibody and aflatoxins) and enhanced the protonation and the ionization efficiency of aflatoxins in the positive ESI mode. In contrast, a solution of 40% 2-propanol, 40% water, and 20% ammonium hydroxide (v/v/v) at pH 12 was employed for performing the negative ESI of barbiturates eluted from the affinity ultrafiltration system. Electrospray voltages of 4.5 and -3.0 kV were employed during positive and negative ESI modes, respectively. Safety Considerations. As a safety precaution, crystalline aflatoxins were handled in a glovebox for the preparation of a reaction mixture containing aflatoxin B1 antibody and aflatoxins. RESULTS AND DISCUSSION Smith and co-workers reported the construction of a miniaturized dual-microdialysis device using laser micromachining techniques.24 The device allowed rapid and efficient fractionation and cleanup of complex biological samples, including protein mixtures and cell lysates, prior to the ESI-MS analysis. In addition to its compactness, negligible dead volume, and robustness, the device significantly reduced sample consumption and improved sensitivity with ESI-MS. (24) Xiang, F.; Lin, Y.; Wen, J.; Matson, D. W.; Smith, R. D. Anal. Chem. 1999, 71, 1485.

Affinity Dialysis and Concentration of Aflatoxin B1 Antibody/Aflatoxins. In this study, copolyester microfluidic devices fabricated by the silicon template imprinting technique were employed for high-throughput residue analysis of aflatoxins. Aflatoxins are mycotoxins produced by the food-borne molds Aspergillus flavus and Aspergillus parasiticus that can infest a large variety of commodities. The four major aflatoxins produced by the molds are aflatoxins B1, B2, G1, and G2, which are carcinogenic and toxic compounds.25 Determination of aflatoxins in the sample was accomplished by solution-phase immunoassay with separation of affinity complexes (aflatoxins/aflatoxin B1 antibody) from other compounds by dialysis. The aflatoxin B1 antibody employed in this study is raised against aflatoxin B1 and cross-reacts with many aflatoxin compounds (except aflatoxin G2a). During operation, the reaction mixture containing aflatoxin B1 antibody and aflatoxins was introduced through the sample inlet in the second copolyester substrate and was exposed to a countercurrent flow of dialysis buffer (see Figure 1). Unbound compounds diffused through the PVDF membrane with a nominal molecular weight cutoff of 80 000 and eluted from the dialysis buffer outlet in the first substrate. Affinity complexes between aflatoxin B1 antibody and aflatoxins were retained in channel 2 and flowed through the hole into channel 3. The solution containing affinity complexes was exposed to a countercurrent flow of dry air for water evaporation and analyte concentration. The concentrated complexes then flowed directly to a microdialysis junction for complex dissociation and ESI-MS detection. The dialysis efficiency for the removal of unbound aflatoxins was dependent on the residence time of analytes in channel 2, which was a direct function of flow rate. In the absence of aflatoxin B1 antibody, a control experiment was carried out to ensure that all aflatoxins had sufficient time to diffuse across the membrane for removal by the dialysis buffer. It was determined that the sample flow rate had to be less than or equal to 100 nL/min in order for the complete removal of all aflatoxins (data not shown). The required 2.0-min residence time (∼0.2 µL solution volume inside the channel 2/100 nL/min) for complete dialysis was mostly due to the restricted diffusion of aflatoxin compounds across the membrane media. The reaction mixture, which consisted of 1 × 10-6 M antibody and 5 × 10-7 M of each aflatoxin, was infused at a flow rate of 100 nL/min. At a 4:1 molar binding ratio of antibody to each aflatoxin, all aflatoxins except aflatoxin G2a were retained during membrane dialysis by the formation of antibody/aflatoxin complexes and were detected using ESI-MS (see Figure 4a). The molecular weights and corresponding (M + H)+ values of aflatoxins in the positive ESI mode are summarized in Table 1. The miniaturized affinity dialysis and concentration system can be reused by simply introducing a 10-min phosphate buffer flush at a flow rate of 0.5 µL/min between the analyses. The robustness of the system is proven to be quite high as evidenced by continuous and repeated analysis of aflatoxins for at least two weeks. Day-to-day variation of aflatoxin analysis is about 5-10% and is mostly attributed to the differences in the electrospray condition. Furthermore, the miniaturized affinity dialysis and (25) Ellis, W. O.; Smith, J. P.; Simpson, B. K.; Oldham, J. H. Crit. Rev. Food Sci. Nutr. 1991, 30, 403.

Figure 4. Positive ESI mass spectra of aflatoxin B1 antibody/ aflatoxins reaction mixture eluted from a miniaturized affinity dialysis and concentration system: (a) 4:1 and (b) 1:4 molar binding ratios of antibody to each aflatoxin. Table 1. Molecular Weights and Corresponding (M + H)+ Values of Aflatoxins compound

MW

(M + H)+

aflatoxin B1 aflatoxin B2 aflatoxin G1 aflatoxin G2 aflatoxin G2a

312.3 314.3 328.3 330.3 346.3

313.3 315.3 329.3 331.3 347.3

concentration system can be regenerated by simply replacing the old or clogged membrane with a new PVDF strip. To demonstrate the screening power of the miniaturized affinity dialysis and concentration system, urinary metabolite lyophilizate (from human male urine) was purchased from Sigma and was added to the reaction mixture with a final concentration of 1% (w/v). No additional m/z ions were detected by scanning between m/z 100 and 700 in ESI-MS (data not shown), leading to the conclusion that low molecular weight urine components were completely removed during the membrane dialysis process. Through capillary connections, a stream of dry air from a compressed air cylinder was introduced into channel 4 under a pressure of 5 psi (3.5 × 104 N/m2). The solution containing retained affinity complexes in channel 3 was thus exposed to air, allowing for water evaporation and analyte concentration. The sample flow rate at the exit of channel 3 (before entering a microdialysis junction) was too difficult to measure accurately; therefore, no estimation was made of the final concentration factor. To investigate the competitive binding of aflatoxins toward the antibody, the molar binding ratio of antibody to each aflatoxin was reduced from 4:1 to 1:4 by decreasing the antibody concentration to 6.25 × 10-8 M. As shown in Figure 4b, only aflatoxin B1, which exhibits the strongest binding strength toward antibody among aflatoxins, was detected using ESI-MS. Due to the lower Analytical Chemistry, Vol. 73, No. 9, May 1, 2001

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Table 2. Molecular Weights and Corresponding (M H)- Values of Barbiturates compound

MW

(M - H)-

barbital allobarbital butalbital amobarbital pentobarbital phenobarbital hexobarbital secobarbital (()-thiopental

184.2 208.2 224.3 226.3 226.3 232.2 236.3 238.3 242.3

183.2 207.2 223.3 225.3 225.3 231.2 235.3 237.3 241.3

complex concentration, the ion intensity observed in Figure 4b for retained aflatoxin B1 was therefore smaller than that in Figure 4a. The sample flow rate (100 nL/min) was at least 1 order of magnitude lower than that achieved previously in an affinity hollow fiber dialysis and concentration system.10 By assuming a complex concentration of 6.25 × 10-8 M at a sample flow rate of 100 nL/ min, the retained aflatoxin B1 measured during the 5-s mass scan (see Figure 4b) was estimated to be ∼0.4 pg based on two binding sites per antibody. Therefore, our detection sensitivity is at least 1-2 orders of magnitude better than that reported in the literature.26 Significant enhancement in detection sensitivity is mainly due to the use of the concentration unit in conjunction with the microdialysis junction in the integrated system. Affinity Ultrafiltration of Phenobarbital Antibody/Barbiturates. Barbiturates are a class of compounds with a sixmembered heterocycle containing nitrogen. High doses of barbiturates are used to lower intercranial pressure caused by head trauma or metabolic coma, reduce ischemic brain damage, and treat refractory status epilepticus.27,28 The phenobarbital antibody employed in this study is raised against phenobarbital and crossreacts with many barbiturate compounds. The molecular weights and corresponding (M - H)- values of barbiturates in the negative ESI mode are summarized in Table 2. As an alternative to continuous dialysis and concentration, the affinity complexes can be separated from unbound compounds using membrane ultrafiltration.29-33 The complexes can then be dissociated with appropriate buffers for analysis by MS or liquid chromatography-MS. On-line coupling of membrane ultrafiltration with MS has also been demonstrated for the screening of drug metabolites.34,35 In this study, PDMS microfluidic devices were fabricated by the capillary molding technique described (26) Kussak, A.; Nilsson, C.-A.; Andersson, B.; Langridge, J. Rapid Commun. Mass Spectrom. 1995, 9, 1234. (27) Michenfelder, J. D. Anesthesiology 1986, 64, 140. (28) Lobato, R. D.; Sarabia, R.; Cordobes, F. J. Neurosurg. 1988, 68, 417. (29) van Breemen, R. B.; Huang, C. R.; Nikolic, D.; Woodbury, C. P.; Zhao, Y. Z.; Venton, D. L. Anal. Chem. 1997, 69, 2159. (30) Wieboldt, R.; Zweigenbaum, J.; Henion, J. D. Anal. Chem. 1997, 69, 1683. (31) Nikolic, D.; van Breemen, R. B. Comb. Chem. High Throughput Screening 1998, 1, 47. (32) Gu, C. G.; Nikolic, D.; Lai, J.; Xu, X. Y.; van Breemen, R. B. Comb. Chem. High Throughput Screening 1999, 2, 353. (33) Nikolic, D.; Habibi-Goudarzi, S.; Corley, D. G.; Gafner, S.; Pezzuto, J. M.; van Breemen, R. B. Anal. Chem. 2000, 72, 3853. (34) van Breemen, R. B.; Nikolic, D.; Bolton, J. L. Drug Metab. Dispos. 1998, 26, 85. (35) Nikolic, D.; Fan, P. W.; Bolton, J. L.; van Breemen, R. B. Comb. Chem. High Throughput Screening 1999, 2, 165.

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Figure 5. Total ion current monitoring of reaction mixture (containing phenobarbital antibody and barbiturates) eluted from miniaturized affinity ultrafiltration system using the negative ESI mode. The inset representing the mass spectrum of phenobarbital taken from the average scans under the peak.

previously and were employed for demonstrating affinity ultrafiltration of a reaction mixture containing phenobarbital antibody and barbiturates. The reaction mixture, containing all nine barbiturates (4 × 10-7 M for each barbiturate compound) and antibody (5 × 10-8 M), was prepared in 10 mM sodium phosphate buffer at pH 7.4. A total volume of 4 µL of the reaction sample was infused into the upper PDMS microchannel at a flow rate of 0.4 µL/min. Unbound barbiturates were forced to permeate across a PVDF membrane (see Figure 3) with a nominal molecular weight cutoff of 80 000 while affinity complexes were retained in the upper microchannel. Immediately after sample infusion, a 10-min phosphate buffer flush at a flow rate of 1.0 µL/min was followed by the introduction of 0.12 µL of a dissociation buffer containing 40% 2-propanol, 40% water, and 20% ammonium hydroxide (v/v/v) at pH 12. The amount of dissociation buffer introduced during the dissociation step was just enough to fill the connecting capillary and the entire upper microchannel. It was retained in the affinity ultrafiltration device for 5 min. Barbiturates liberated from the antibody were eluted into the lower microchannel and then to the microdialysis junction for ESIMS detection by continuously pumping the dissociation buffer at a flow rate of 0.4 µL/min. As shown in Figure 5, the total ion current obtained from MS scanning between m/z 150 and 250 indicated a 12-s solution plug containing only phenobarbital. At a 1:4 molar binding ratio of antibody to each barbiturate, only phenobarbital, which exhibits the greatest binding strength toward phenobarbital antibody, was detected using ESI-MS. By comparing with the initially injected sample volume of 4 µL, this 80-nL elution plug (0.4 µL/min × 12 s) demonstrated a concentration factor of 50 times for captured phenobarbital during the affinity ultrafiltration and elution procedures. A concentration factor of 45 times was measured by comparing the ion intensities of phenobarbital obtained from affinity ultrafiltration versus direct infusion of phenobarbital into ESI-MS at the same flow rate. The miniaturized affinity ultrafiltration system can be reused by simply introducing a 10-min phosphate buffer flush at a flow rate of 0.4 µL/min between the analyses. Even though it takes a total of 30 min to complete each analysis, it only consumes 1.6 pmol of each barbiturate compound (4 µL × 4 × 10-7 M for each barbiturate compound). Most importantly, the miniaturized system offers a concentration factor of 50 times by reducing the sample

size from 4 µL to an 80-nL elution plug for ESI-MS analysis. In comparison, the sizable ultrafiltration chamber (1 mL) used in pulsed ultrafiltration mass spectrometry29-33 not only requires large sample size but also dilutes the ligands released from the ligand/receptor complexes during the dissociation process. Thus, a small reversed-phase cartridge is employed by Nikolic et al.33 as a trapping column to concentrate and desalt the dissociated ligands eluting from the chamber. CONCLUSION A microfabricated affinity dialysis and concentration system was developed and demonstrated for rapid and sensitive analysis of a reaction mixture containing aflatoxin B1 antibody and aflatoxins. Competitive selection of a tighter binder of aflatoxin B1 over other aflatoxins was achieved by manipulating the molar binding ratios of antibody to each aflatoxin. By performing the assay in an integrated and self-contained microfluidic system, we achieved a significant reduction in dead volume, sample consumption, and sample loss. Ultrasensitive detection of aflatoxin B1 was also attributed to the use of the concentration unit in conjunction with the microdialysis junction. Affinity ultrafiltration of phenobarbital antibody/barbiturates provided affinity capture, concentration (a concentration factor of

50 times), and direct identification of phenobarbital using ESIMS. The combination of “nanoscale” sample manipulation in microfabricated devices with ESI-MS detection is particularly attractive due to the speed and sensitivity that can be achieved with MS, as well as the amenability of ESI-MS to the low sample flow rates. Support for this work by the Joint Institute for Food Safety and Applied Nutrition at the University of Maryland and the Analytical Chemistry Division of the National Institute of Standards and Technology are gratefully acknowledged. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

Received for review December 12, 2000. Accepted February 17, 2001. AC001474J

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