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Combined Immunocapture and Laser Desorption/ Ionization Mass Spectrometry on Porous Silicon Rachel D. Lowe,† Endre J. Szili,† Paul Kirkbride,‡ Helmut Thissen,§ Gary Siuzdak,| and Nicolas H. Voelcker*,† School of Chemical and Physical Sciences, Flinders University, Bedford Park, SA 5042, Australia, Australian Federal Police, Canberra, ACT, 2601, Australia, CSIRO Molecular and Health Technologies, Clayton, VIC, 3178, Australia, and The Scripps Center for Mass Spectrometry, The Scripps Research Institute, La Jolla, California 92037 There is considerable interest in the highly parallelized mass spectrometry analysis of complex sample mixtures without any time-consuming prepurification. Porous silicon-based laser desorption/ionization mass spectrometry (pSi LDI-MS) is enabling technology for such analysis. Previous studies have focused on pSi surface functionalization to enhance sensitivity of detection and engineer surfaces for sample capture and enrichment in LDI-MS analysis. In this report, we build on this work by showing that surface functionalization of thin pSi films can be extended to the covalent immobilization of antibodies, producing a porous immunoaffinity surface. We demonstrate highly selective mass spectrometric detection of illicit drugs (benzodiazepines) on pSi films displaying antibenzodiazepine antibodies covalently immobilized via isocyanate chemistry. The effects of antibody immobilization conditions, antibody concentration, and surface blocking on LDI-MS performance and selectivity were studied. X-ray photoelectron spectroscopy (XPS) was instrumental in characterizing surface chemistry and optimizing LDI-MS performance. Overall, our approach is suitable for rapid and sensitive confirmatory analysis in forensic toxicology requiring only minimal sample volume and may be applied to other areas requiring small molecular analysis such as metabolomics and pharmacology. Mass spectrometric analysis of complex mixtures such as body fluids or tissue homogenates is greatly facilitated by selective capture and enrichment approaches. The need for selectivity is particularly acute in the area of small molecule analysis for forensic purposes, food quality control, metabolomics, and pharmacology, where examples of complex and heterogeneous mixtures abound.1 Typically, an immunoaffinity isolation step is performed prior to and separate from downstream analysis to achieve selective analysis.2 In turn, direct mass spectrometric evaluation of analytes * Corresponding author. Tel: (+61 8) 8201 5338. Fax: (+61 8) 8201 2905. E-mail:
[email protected]. † Flinders University. ‡ Australian Federal Police. § CSIRO Molecular and Health Technologies. | The Scripps Research Institute. (1) Guzman, N. A.; Blanc, T.; Phillips, T. M. Electrophoresis 2008, 29, 3259– 3278. (2) Lin, P. C.; Tseng, M. C.; Su, A. K.; Chen, Y. J.; Lin, C. C. Anal. Chem. 2007, 79, 3401–3408. 10.1021/ac100455x 2010 American Chemical Society Published on Web 04/22/2010
selectively captured and enriched on a support surface is poised to impart more rapid and sensitive analyses. Efforts to achieve analyte enrichment and subsequent mass spectrometric characterization resulted in the development of surface enhanced laser desorption/ionization (SELDI) surfaces.3 Various affinity capture surfaces have been developed where the surface modification plays a critical role in the purification, modification, and amplification of the analyte prior to cocrystallization with a matrix compound before desorption/ionization.4,5 A broad range of surface chemistries and specific biomolecular capture agents including antibodies, enzymes, receptors, DNA fragments, and aptamers have been reported. However, the need for a matrix in SELDI means that scope for small molecule analysis remains limited. Porous silicon-based laser desorption/ionization mass spectrometry (pSi LDI-MS) is a technique well suited for small molecule analysis since it does not require matrix molecules for desorption/ionization to occur.6,7 pSi LDI-MS employs a porous semiconductor layer as an effective medium for trapping and desorbing analyte molecules. The porous substrate features a tremendous surface area (∼600 m2 cm-3) in combination with high ultraviolet absorptivity and substantial thermal conductivity, allowing the silicon structure to act as an energy receptacle for laser energy transfer to the analytes.8 Efficient analyte ionization is achieved with minimal fragmentation, limited surface degradation, and minimal chemical background.9,10 Since its development in 1999,11 the ease of surface fabrication, minimal sample preparation, and successful application to the detection of a wide selection of molecules12-17 have made pSi LDI(3) Xu, Y. D.; Bruening, M. L.; Watson, J. T. Mass Spectrom. Rev. 2003, 22, 429–440. (4) Tang, N.; Tornatore, P.; Weinberger, S. R. Mass Spectrom. Rev. 2004, 23, 34–44. (5) Merchant, M.; Weinberger, S. R. Electrophoresis 2000, 21, 1164–1167. (6) Guo, Z.; Ganawi, A. A. A.; Liu, Q.; He, L. Anal. Bioanal. Chem. 2006, 384, 584–592. (7) Peterson, D. S. Mass Spectrom. Rev. 2007, 26, 19–34. (8) Schmeltzer, J. M.; Buriak, J. M. In The Chemistry of Nanomaterials: Synthesis, Properties and Applications; Rao, C. N. R., Muller, A., Cheetham, A. K. , Eds.; Wiley VCH: Weinheim, 2004; Vol. 2. (9) Alimpiev, S J. Chem. Phys. 2008, 128, 014711–014719. (10) Kruse, R. A.; Li, X.; Bohn, P. W.; Sweedler, J. V. Anal. Chem. 2001, 73, 3639–3645. (11) Wei, J.; Buriak, J. M.; Siuzdak, G. Nature 1999, 399, 243–246. (12) Go, E. P.; Shen, Z.; Harris, K.; Siuzdak, G. Anal. Chem. 2003, 75, 5475– 5479.
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MS an extremely versatile tool.18-20 We have recently applied this tool to the simultaneous detection of multiple illicit drugs.21 In that study, we exploited the fact that the presence of certain functional groups and, in particular, fluorinated species, such as pentafluorophenyl groups on the pSi surface, enables the detection of small hydrophobic molecules from complex biological samples by means of selective capture of hydrophobic species from aqueous solution and removal of abundant hydrophilic species such as salts.22 While this development clearly sets the stage for selective analysis in pSi LDI-MS, further advances will depend particularly on surface modifications yielding specific molecular affinity. Inroads have certainly been made to incorporate affinity capture in pSi LDI-MS using immobilization of protein receptors23-25 or surface functionalization with fluoro silanes in combination with perfluoroalkyl affinity tagged analytes.26 However, these studies have not yet demonstrated sensitive and selective capture of target analytes from a mixture of structurally similar molecules in aqueous solution. A study by Meng et al.25 relied on cleavable linker chemistry to achieve desorption of the captured small molecule; while in the case of Zou et al.,23,24 analyte concentration exceeded the mg/mL level, and ethanol washes were required prior to mass spectrometric analysis. Finally, Hu et al.23,24 did show sensitive detection of captured small molecules but failed to challenge the protein-modified pSi spots with mixtures of small molecules. Another aspect of this affinity capture technology that has not yet been investigated in detail is the influence of the bioconjugation strategy on the pSi LDI-MS performance. Several recognition species including DNA,27 enzymes,28-30 antibodies,31 and other (13) Kinumi, T.; Shimomae, Y.; Arakawa, R.; Tatsu, Y.; Shigeri, Y.; Yumoto, N.; Niki, E. J. Mass Spectrom. 2006, 41, 103–112. (14) Thomas, J. J.; Shen, Z.; Crowell, J. E.; Finn, M. G.; Siuzdak, G. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4932–4937. (15) Arakawa, R.; Shimomae, Y.; Morikawa, H.; Ohara, K.; Okuno, S. J. Mass Spectrom. 2004, 39, 961–965. (16) Liu, Q.; Guo, Z.; He, L. Anal. Chem. 2007, 79, 3535–3541. (17) Vaidyanathan, S.; Jones, D.; Ellis, J.; Jenkins, T.; Chong, C.; Anderson, M.; Goodacre, R. Rapid Commun. Mass Spectrom. 2007, 21, 2157–2166. (18) Shen, Z.; Thomas, J. J.; Averbuj, C.; Broo, K. M.; Engelhard, M.; Crowell, J. E.; Finn, M. G.; Siuzdak, G. Anal. Chem. 2001, 73, 612–619. (19) Thomas, J. J.; Shen, Z.; Blackledge, R.; Siuzdak, G. Anal. Chim. Acta 2001, 442, 183–190. (20) Lewis, W. G.; Shen, Z.; Finn, M. G.; Siuzdak, G. Int. J. Mass Spectrom. 2003, 226, 107–116. (21) Lowe, R. D.; Guild, G. E.; Harpas, P.; Kirkbride, P.; Hoffmann, P.; Voelcker, N. H; Kobus, H. Rapid Commun. Mass Spectrom. 2009, 23, 3543–3548. (22) Trauger, S. A.; Go, E. P.; Shen, Z. X.; Apon, J. V.; Compton, B. J.; Bouvier, E. S. P.; Finn, M. G.; Siuzdak, G. Anal. Chem. 2004, 76, 4484–4489. (23) Zou, H. F.; Zhang, Q. C.; Guo, Z.; Guo, B. C.; Zhang, Q.; Chen, X. M. Angew. Chem., Int. Ed. 2002, 41, 646–648. (24) Hu, L. G.; Xu, S. Y.; Pan, C. S.; Zou, H. F.; Jiang, G. B. Rapid Commun. Mass Spectrom. 2007, 21, 1277–1281. (25) Meng, J.-C.; Siuzdak, G.; Finn, M. G. Chem. Commun. (Cambridge, U. K.) 2004, 2108–2109. (26) Go, E. P.; Uritboonthai, W.; Apon, J. V.; Trauger, S. A.; Nordstrom, A.; O’Maille, G.; Brittain, S. M.; Peters, E. C.; Siuzdak, G. J. Proteome Res. 2007, 6, 1492–1499. (27) Steinman, C.; Janshoff, A.; Lin, S.-Y. V.; Voelcker, N. H.; Ghadiri, M. R. Tetrahedron 2004, 60, 11259–11267. (28) Hart, B. R.; Letant, S. E.; Kane, S. E.; Hadi, M. Z.; Shields, S. J.; Reynolds, J. G. Chem. Commun. (Cambridge, U. K.) 2003, 322–323. (29) Thust, M.; Schoning, M. J.; Frohnhoff, S.; Arens-Fischer, R.; Kordos, P.; Luth, H. Meas. Sci. Technol. 1996, 7, 26–29. (30) Drott, J.; Lindstrom, K.; Rosengren, L.; Laurell, T. J. Micromech. Microeng. 1997, 7, 14–23. (31) Laurell, T.; Drott, J.; Rosengren, L.; Lindstrom, K. Sens. Actuators, B: Chem. 1996, 31, 161–166.
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proteins32,33 have been covalently bound to pSi during the pursuit of various applications such as biosensing and drug delivery. A variety of chemical routes for covalent biomolecule attachment to silicon and pSi surfaces is readily available.28,34,35 For the work presented here, we used antibodies since they are readily available for the target compounds. Antibodies are commonly attached to the substrate surface using bioconjugate approaches involving amine and thiol moieties present on the protein surface. The success of molecularly selective LDI-MS using antibodies (immunocapture) is arguably contingent on resolving several issues. The first relates to the preservation of antibody activity once attached to the surface. Furthermore, it is prima facie unclear whether LDI is still effective when the analyte is bound to an antibody and, therefore, located at a nanometric distance away from the surface. Finally, antibody coverage needs to be optimized to give both high analyte binding capacity and analyte ionization efficiency. These issues can be addressed through the optimization of antibody concentration during immobilization, pore dimensions,36 and linker chemistry.37 An area that would significantly benefit from a selective capture and analysis platform is that of illicit drug detection in forensic toxicology. Currently, extensive laboratory procedures involving multiple extraction procedures and long analysis times are required to detect a mixture of drugs in complex sample matrixes. In view of the demand for an adequate forensic mass spectrometry tool, we set out to develop a method for the selective detection of benzodiazepines by immunocapture pSi LDI-MS using a benzodiazepine antibody. Figure 1 shows a schematic of our approach. In essence, an aqueous sample containing a mixture of analytes is deposited on the antibody-modified pSi substrate and is incubated for a short period of time before being removed. Analyte molecules that bind to the immobilized antibody are captured and remain on the surface. Subsequent interrogation of the surface with a UV laser releases the captured analyte molecules from the surface and detects their mass by time-of-flight. Here, we demonstrate that this approach requires minimal sample volume for rapid and sensitive confirmatory analysis. We also investigate the influence of bioconjugation reaction and blocking conditions on the LDI-MS performance. The approach presented here significantly expands the repertoire of pSi LDI-MS for applications in forensic analysis but also has immediate applications in metabolomics, pharmacology, and many other areas requiring small molecule analysis. EXPERIMENTAL SECTION Chemicals. Methanol (99.9%) and hydrofluoric acid (48%) were purchased from Merck (Melbourne, Australia). Ethanol (100% undenatured) was purchased from Chem Supply (Gillman, (32) Dancil, K. P. S.; Greiner, D. P.; Sailor, M. J. J. Am. Chem. Soc. 1999, 121, 7925–7930. (33) Lin, V. S. Y.; Motesharei, K.; Dancil, K. P. S.; Sailor, M. J.; Ghadiri, M. R. Science 1997, 278, 840–843. (34) Letant, S. E.; Hart, B. R.; Kane, S. E.; Hadi, M. Z.; Shields, S. J.; Reynolds, J. G. Adv. Mater. 2004, 16, 689-693. (35) Xia, B.; Xiao, S. J.; Guo, D. J.; Wang, J.; Chao, M.; Liu, H. B.; Pei, J.; Chen, Y. Q.; Tang, Y. C.; Liu, J. N. J. Mater. Chem. 2006, 16, 570–578. (36) Janshoff, A.; Dancil, K.-P. S.; Steinem, C.; Greiner, D. P.; Lin, V. S.-Y.; Gurtner, C.; Motesharei, K.; Sailor, M. J.; Ghadiri, M. R. J. Am. Chem. Soc. 1998, 120, 12108–12116. (37) Davis, D. H.; Giannoulis, C. S.; Johnson, R. W.; Desai, T. A. Biomaterials 2002, 23, 4019–4027.
Figure 1. Immunocapture pSi LDI-MS analysis. An antibody-modified pSi surface is exposed to analyte solution via the solid-liquid extraction method, following solvent and unbound molecule removal. The captured analyte molecules are then desorbed and ionized using a pulsed laser into the time-of-flight (TOF) detector.
Australia). Water was purified in a Labconco Milli-Q water system (Kansas City, USA). Pentafluorophenylpropyl dimethylchlorosilane (PFPDCS) was purchased from Gelest Inc. (Morrisville, USA). 3-Isocyanatopropyl triethoxysilane (ICPTES) was obtained from Fluka (Castle Hill, Australia). Monoethanolamine (MEA), Tween 20, sodium chloride, bovine serum albumin (BSA), aminotelechelic poly(ethylene glycol) with a MW of 2000 Da (PEG-2000), and sodium hydroxide were purchased from Sigma-Aldrich (Castle Hill, Australia). Chem Supply (Gillman, Australia) provided sodium chloride (NaCl) and disodium hydrogen phosphate (Na2HPO4 · 2H2O). Potassium dihydrogen orthophosphate (KH2PO4) was purchased from Southern Cross Scientific (Melrose Park, Australia). Solutions of diazepam, alprazolam, and cocaine were supplied by Forensic Science South Australia. Buffers and Solutions. Phosphate buffered saline (1× PBS), 10 mM, was prepared by dissolving 8 g of NaCl, 0.2 g of KCl, 1.12 g of Na2HPO4 · 2H2O, and 0.24 g of KH2PO4 in 1 L of water and adjusting the pH to 7.4 with 0.1 M HCl. Tween 20 washing solution was prepared at 0.05% v/v in water. The solutions employed as blocking agents included 1 or 10 mM MEA in water, 1 mM BSA in water, and 1 mM PEG-2000 in water. Small molecule mixture 1 (SMM-1) contained equal concentrations of 100 µM for all three molecules. Small molecule mixture 2 (SMM-2) contained 100 µM diazepam concentration and 10 µM cocaine and alprazolam concentrations. Antibodies. Affinity purified monoclonal benzodiazepine antibodies were purchased from Arista Biologicals (Allentown, USA). Monoclonal antibenzodiazepine was purified by Protein A affinity chromatography by 98% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified antibody was supplied in a PBS stock solution (pH 7.2) with 0.05% sodium azide. Fabrication of pSi. Porous films were prepared in a setup similar to that previously described.11,18 A conventional singletank Teflon electrochemical etching cell was used. Squares of (∼3.5 × 3.5 cm) pieces of monocrystalline (0.008-0.02 Ω · cm) antimony doped n-type Si (100) from Silicon Quest International (Santa Clara, USA) were cut and placed into the etching cell on top of a piece of gold foil of 3N5 purity obtained from Electronic Space Products International (Ashland, USA). The cathode utilized was a 0.5 mm diameter platinum wire (99.9%) purchased from Aldrich (Milwaukee, USA) that was placed in the cell cavity, and the cell assembly was then filled with 10 mL of etching electrolyte (25% hydrofluoric acid in ethanol). The printed mask was made in Adobe Photoshop (San Jose, USA) and consisted of 10 × 10
transparent circles (diameter )1 mm) in a black square of 4 cm2. The mask was printed on a standard laser printable transparency film and placed between the light source and the pair of focusing lenses.18 A Keithley (2425 100 W) SourceMeter (Cleveland, USA) was utilized and controlled by a custom-built LabView, version 6.1, National Instruments (Austin, USA) module. Illumination was supplied by a Schott KL 2500 LCD (Mainz, Germany) fiber optic light source hosting a 24 V, 250 W General Electric Quartzline Lamp (Cleveland, USA). Light was directed through a single branch light guide, and a focusing lens was also supplied by Schott. Aspheric lenses, f ) 80 mm, were supplied by OptoSigma (Santa Ana, USA) and placed between the mask. Anodization was carried out for 2 min under illumination through a photomask at a constant applied current of 4 mA. The hydride-terminated chip was then washed with methanol and dried with a stream of N2. The chip was then initially oxidized with ozone using an Ozon-Generator 500 (Fischer, Germany) at a flow rate of 3.25 g h-1 of ozone for 30 s. Following this, chips were then re-etched for 30 s in a 5% v/v HF/H2O solution to expand pore size and were once again ozone-oxidized to obtain a hydroxyl-terminated surface. Silanization and Antibody Attachment. Following ozone oxidation, one batch of pSi surfaces were prepared by silanization with neat PFPDCS silane (80 µL) for 15 min at 90 °C and washed with copious amounts of methanol to generate the fluorinated pSi array surface. The functionalized substrates were then placed in clean containers and stored in air until required. A second batch of pSi samples was silanized with ICPTES in toluene (50 mM) for 30 min at room temperature. Samples were then washed extensively with toluene and dried with a stream of N2. The isocyanate-functional surfaces were immediately incubated for 2 h in benzodiazepine antibody solution in water (10 µg mL-1), followed by a thorough water wash and dried with a stream of N2 before being stored at 4 °C for up to 1 week. Antibody concentrations of 1 and 100 µg mL-1 were used as well. In addition, immobilization of antibody was also performed in PBS (pH 7.4). Variations to the washing procedure for the removal of excess protein included the use of PBS and PBS with 0.05% Tween 20. Quenching of Residual Isocyanate Groups. After antibody immobilization on isocyanate-functional surfaces, quenching of unreacted isocyanate groups was performed using MEA, BSA, or PEG-2000 (1 mM) for 1 h. The quenching solution was then washed off the sample with water and dried with a stream of N2. Analytical Chemistry, Vol. 82, No. 10, May 15, 2010
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Figure 2. (a) Mass spectrum of SMM-1 containing 50 pmol of diazepam [MH]+ ) 285 (b), cocaine [MH]+ ) 304 (f), and alprazolam [MH]+ ) 309 (2) deposited on a PFPDCS-modified pSi surface. (b) Plot of the corresponding S/N values from three replicates.
X-ray Photoelectron Spectroscopy Analysis. X-ray photoelectron spectroscopy (XPS) analysis of pSi surfaces was performed on an AXIS HSi spectrometer (Kratos Analytical Ltd., UK) equipped with a monochromatic Al KR X-ray source. The elemental composition of samples was obtained from survey spectra, collected at a pass energy of 320 eV. High-resolution spectra of the C1s peak were collected at a pass energy of 40 eV, and binding energies were referenced to aliphatic carbon at 285.0 eV. Vision 1.5 software (Kratos Analytical Ltd.) was used for peak fitting and quantification of XPS spectra. Sample Deposition on pSi Spots. The pSi surfaces were directly attached via Scotch tape to a MALDI target plate that was machined to the depth of the silicon wafer (550 µm). Aliquots of analyte mixtures SMM-1 (0.5 µL corresponding to 50 pmol per analyte) or SMM-2 (0.5 µL corresponding to 50 pmol of diazepam and 5 pmol each of cocaine and alprazolam) were deposited onto the individual spots of pSi for 30 s, and the remaining liquid was then removed with a pipet, leaving behind captured molecules. Surfaces were not subjected to further washing steps. The sample was subsequently analyzed by LDI-MS, and each spot was employed for a one-time use. Mass Spectrometry and Acquisition Parameters. Mass spectra were acquired using an UltraFlex III MALDI TOF/TOF (Bruker Daltronics, Germany) equipped with a neodymium-doped yttrium aluminum garnet (Nd:YAG) pulse laser, operated at 50 Hz frequency and laser attenuator offset of ∼68% in positive reflectron mode. Acquisition parameters included 25 kV acceleration voltages and pulsed ion extraction at 100 ns. Each recorded mass spectrum was generated by averaging data from 500 individual laser shots. Data acquisition used FlexControl 3.0 (Bruker Daltonics) software, and data analysis was performed using Flex Analysis 3.0 (Bruker Daltonics) software. Signal-tonoise (S/N) ratios were expressed as the signal intensity relative to the noise. Noise was calculated by taking the mean of the top 5% of intensity values over a 50 Da region.
surface with high LDI-MS performance was obtained yielding excellent limits of simultaneous detection for a range of illicit drugs (50-0.5 pmol) with minimal background noise, as previously described.21 Here, small molecule mixture 1 (SMM-1) containing equal amounts of diazepam, cocaine, and alprazolam (50 pmol) was deposited onto the PFPDCS-modified pSi surface. LDI-MS analysis (Figure 2) shows good detection for all three compounds and the absence of background signal. However, the cocaine signal dominates the mass spectrum (Figure 2a). The benzodiazepines, diazepam, and alprazolam, appear to be less efficiently ionized relative to the cocaine signal. In addition to signal intensity, a representative way of scoring the performance of an LDI-MS surface is to determine S/N values (Figure 2b). Here, diazepam detection in the mixture is close to the threshold of detection, S/N ) 5, which may be due to analyte suppression. Such analyte suppression, while useful in some instances, is generally undesirable. The SMM-1 was, therefore, an appropriate model mixture to tackle the challenge of overcoming the analyte suppression issue and achieving selective detection of the suppressed analytes using immunocapture and direct mass spectrometry analysis. Benzodiazepine antibodies were immobilized on oxidized pSi surfaces by employing an isocyanato silane linker (ICPTES) as shown in Scheme 1. This chemistry involves the formation of stable urea bonds between the primary amines on the surface of the antibody and the highly reactive isocyanates of the ICPTES. This particular bioconjugation reaction was chosen since it is fast and does not require any further activation steps. In addition, ICPTES is a commercially available, inexpensive compound. Due to the relative large size of antibodies (approximate MW ) 150 kDa, 4-14 nm length),38 pore diameters of 50 nm or larger were required to allow antibodies ready access to the pores.36,39,40 pSi surfaces fabricated for this work had an average pore size of 100 nm with an average pore depth of 125 nm. An earlier study reported that pore radii of 100 nm were filled with antibody within 3 minutes,36 indicating that pore size and the time of 2 h chosen
RESULTS AND DISCUSSION When pSi surfaces were modified with the organosilane pentafluorophenylpropyl dimethylchlorosilane22 (PFPDCS), a
(38) Taylor, A. E.; Parker, J. C. J. Physiol. (London) 2003, 553, 333. (39) Ouyang, H.; Christophersen, M.; Viard, R.; Miller, B. L.; Fauchet, P. M. Adv. Funct. Mater. 2005, 15, 1851–1859. (40) Bonanno, L. M.; DeLouise, L. A. Langmuir 2007, 23, 5817–5823.
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Scheme 1. Covalent Attachment of Antibodies to pSi Surface via an Isocyanato Silanea
a
Note that the antibody is not drawn to scale.
Table 1. XPS Elemental Compositions of Oxidized pSi (pSi-O), Silanized pSi with ICPTES (pSi-O-ICPTES), and Antibody Immobilized pSi (pSi-O-ICPTES-Ab) Surface Functionalization
O (at.%)
N (at.%)
C (at.%)
Si (at.%)
pSi-O pSi-O-ICPTES pSi-O-ICPTES-Ab
61.5 45.6 49.4
0 1.9 4.9
7.3 16.6 23.1
31.2 36.0 22.6
here for immobilization were sufficient for entry and covalent attachment of the antibody molecules into the porous network. XPS was used to characterize the relative chemical composition of the pSi surfaces at each step of the antibody immobilization process. Table 1 summarizes the XPS results from these surfaces. Ozone oxidized pSi contained no nitrogen and 7.3 atomic percent (at.%) of residual carbon. At the same time, 31.2 at.% silicon was found, along with 61.5 at.% oxygen from the oxide layer. Upon silanization with ICPTES, nitrogen was increased to 1.9 at.% due to the presence of the isocyanate group. Furthermore, the carbon content also increased from 7.3 at.% to 16.6 at.%, which is expected as a result of the addition of the short hydrocarbon chain in the linker. After the addition of the antibody (10 µg mL-1), the nitrogen signal further increased to 4.9 at.%. This is indicative of successful antibody immobilization. However, due to the fact that the information depth of XPS is approximately 10 nm, 22.6 at.% silicon was still detected after antibody immobilization. From the XPS results, we estimate submonolayer coverage of the antibody, which we attribute to the combination of the high pSi surface area and the relatively low concentration of protein used in this study. Higher antibody coverage using higher concentrations of antibody had a negative impact on detection sensitivity (vide infra). High-resolution C 1s XPS analysis was also used to further investigate the chemical nature of the silanized and the antibody-modified surfaces (Figure 3 and Table S-1 in the Supporting Information). All spectra were deconvoluted and fitted with four components that were assigned to different chemical environments,41,42 Component C1 represents hydrocarbons (C-H and C-C) at 285.0 eV, C2 represents ether and amine groups (C-O or C-N) at 286.5 eV, C3 represents carbonyl and amide groups (CdO or N-CdO) at 288.2 eV, (41) Cole, M. A.; Thissen, H.; Losic, D.; Voelcker, N. H. Surf. Sci. 2007, 601, 1716–1725. (42) Bullett, N. A.; Talib, R. A.; Short, R. D.; McArthur, S. L.; Shard, A. G. Surf. Interface Anal. 2006, 38, 1109–1116.
and C4 represents ester, carboxyl, or urea groups (O-CdO or N-C(dO)-N)) at 289.1 eV. The relative contributions from each component are tabulated in Table S-1 in the Supporting Information. The C 1s spectra for the isocyanate-modified surface and the antibody-modified surface both show a strong C1 component at ∼285.0 eV corresponding to hydrocarbons. The small contributions of C3 and C4 for the isocyanate functionalized surface (Figure 3a) indicate that some of the isocyanates may have crosslinked during silanization or storage (before analysis), forming urea bonds via carbamate intermediates.43 In comparison, the C 1s spectrum after antibody immobilization seen in Figure 3b shows typical contributions expected for immobilized proteins, with increased contributions in particular from the components C2 and C3.41 However, the XPS analysis cannot exclude the possibility that some isocyanate groups remain intact after the immobilization reaction. A representative LDI-MS spectrum from the antibody-modified surface incubated with SMM-1 is shown in Figure 4 along with the S/Ns ratios. The reduction of a cocaine signal at m/z 304 (shown by the star) is strikingly apparent. However, two strong peaks are present for the target benzodiazepines, diazepam, and alprazolam (m/z 285 and m/z 309, respectively). This result is in stark contrast to the data presented in Figure 2. It confirms that the antibodies, whose presence we detected by XPS, have retained their recognition properties. In addition, the spectrum shows that the antibody-modified pSi surface not only maintained but also increased desorption/ionization characteristics for target benzodiazepines as compared to the standard PFPDCS-modified pSi. The selectivity of the benzodiazepine antibody pSi substrate resulted in a greater than 3-fold increase in S/N of diazepam and slight increase in the S/N of alprazolam. While none of the three analytes carries functional groups that could possibly react with residual isocyanate groups on the pSi surface, there is nevertheless potential for nonspecific adsorption on the pSi, particularly in the light of the submonolayer surface coverage of the antibody. We, therefore, investigated if quenching of residual isocyanate groups with chemical entities reducing nonspecific surface adsorption (blocking) has an impact on LDIMS performance. Monoethanol amine (MEA) is used as amines are common blocking agents for quenching unreacted electrophiles such as isocyanates.44 Bovine serum albumin (BSA) was (43) Xia, B.; Li, J.; Xiao, S.-J.; Guo, D.-J.; Wang, J.; Pan, Y.; You, X.-Z. Chem. Lett. 2005, 34, 226–228. (44) Tassel, X.; Barbry, D.; Tighzert, L. Eur. Polym. J. 2000, 36, 1745–1751.
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Figure 3. High-resolution XPS C 1s spectra with curves fitted with four components of (a) an isocyanate-functionalized surface and (b) a surface with immobilized antibenzodiazepine antibody.
Figure 4. (a) Mass spectrum of SMM-1 containing 50 pmol of diazepam [MH]+ ) 285 (b), cocaine [MH]+ ) 304 (f), and alprazolam [MH]+ ) 309 (2) deposited on pSi with antibenzodiazepine immobilized via isocyanate chemistry. (b) Plot of the corresponding S/N values from three replicates.
also used because (a) it is commonly used as a blocker in immunoassays, (b) the protein’s amine groups can react with any remaining isocyanate functionalities, and (c) previous studies have shown that the immobilization of BSA is compatible with LDIMS on pSi.23 Finally, poly(ethylene glycol) (PEG) was grafted onto the surface. Here, an amino-telechelic, isocyanate-reactive PEG with an average molecular weight of 2000 Da was used. Surfaces with grafted PEG layers are known to show resistance toward nonspecific adsorption45 due to volume-exclusion effects.46,47 Mass spectra of all three tested blocking methods are shown in Figure S-3 in the Supporting Information. For this experiment, another small molecule mixture 2 (SMM-2) was used, which contained cocaine and alprazolam at 5 pmol and diazepam at 50 pmol. Variable analyte concentrations were used to study the response of the antibody-modified surfaces at concentrations close to the respective limit of detection for the three molecules. Both MEA and BSA blocked surfaces show retention of the benzodiazepine analyte selectivity, relative to cocaine. Intensities of the three analyte peaks roughly correspond to their concentration in SMM-2. However, introduction of MEA into the antibody-modified pSi surface did not show an increase in signal over the unblocked surface, indicating that any unreacted isocyanate linker molecules (45) Zhang, M. Q.; Desai, T.; Ferrari, M. Biomaterials 1998, 19, 953–960. (46) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Curr. Opin. Colloid Interface Sci. 2001, 6, 3–10. (47) Kim, Y. P.; Oh, Y. H.; Kim, H. S. Biosens. Bioelectron. 2008, 23, 980–986.
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did not negatively impact signal generation. Also, the noise level for BSA is visibly higher by a factor of ∼3. The reduction of performance when the BSA blocking agent was used indicates that the adsorbed BSA protein does interfere with analyte desorption/ionization, in contrast to a previous report.23 Surprisingly, grafting of PEG appears to have suppressed benzodiazepine selectivity completely. Obviously, the presence of the PEG in the pores negatively impacts the antibody activity. Interestingly, the surface maintains the relative difference in the benzodiazepine concentrations. However, despite cocaine being present in solution at a lower concentration than diazepam, the former analyte still dominates the mass spectra. Indeed, the surface displays a similar ionization profile observed on the PFPDCS-modified pSi surface. Given that quenching of isocyanates with MEA shows the best performance out of the three blocking agents tested, the effect of varying the concentration of the MEA blocker (1-10 mM) during quenching was investigated with SMM-1 (Figure 5). In addition, a control surface made by first quenching the ICPTES-modified surface with MEA before introducing the benzodiazepine antibody was also tested. The cocaine signal on the antibody-modified surfaces increases in relation to the benzodiazepine signal with increasing concentration of MEA during the blocking step. The ratio of alprazolam to diazepam signal also increases with increasing MEA concentration. The control surface shows an ionization profile similar to the PFPDCS-modified pSi. Taken together, these
Figure 5. Mass spectra of SMM-1 on a antibenzodiazepine-modified surface where residual isocyanate groups were quenched with different concentrations of MEA: (a) no MEA, (b) 1 mM MEA, and (c) 10 mM. In (d), the isocyanate groups were quenched with 1 mM MEA before antibody immobilization. Peaks identified as diazepam [MH]+ ) 285 (b), cocaine [MH]+ ) 304 (f) and alprazolam [MH]+ ) 309 (2).
results suggest that blocking of the residual isocyanates is not necessary and in fact worsens both LDI-MS performance and selectivity. Given the high reactivity of isocyanates, we are confident that most will have reacted either with water or with antibody during the 2 h incubation period and subsequent washing. However, the importance of the isocyanate for antibody immobilization is underscored by the control result presented in Figure 5d where the isocyanates were first quenched with MEA before antibody immobilization. The resulting surface shows overall reduced ionization and loss of selectivity, further confirming that the observed molecular selectivity is a result of the immunocapture properties of the antibody-modified surface. Several additional aspects of the isocyanate linker chemistry were evaluated in order to improve signal response. An important factor in the detection capabilities of affinity-capture surfaces is their surface capacity.48 Surface capacity is defined as the exposed surface area that will allow molecules to be bound.4 The remarkable increase in surface area as a result of electrochemical etching of silicon leads to a potential high surface capacity, assuming the surface area is covered by capture molecules. However, high surface area is also critically important for LDI-MS performance since the porous network acts as an energy receptacle and transfer medium for the laser energy.49 The exact mechanism for LDIMS has not been elucidated; however, water molecules residing within the porous scaffold have been suggested to act as “pseudomatrixes” and proton sources for ionization.10,50 Therefore, it is not immediately obvious whether high surface coverage of (48) DeLouise, L. A.; Miller, B. L. Anal. Chem. 2004, 76, 6915–6920. (49) Luo, G.; Chen, Y.; Siuzdak, G.; Vertes, A. J. Phys. Chem. B 2005, 109, 24450–24456. (50) Alimpiev, S.; Nikiforov, S.; Karavanslcii, V.; Minton, T.; Sunner, J. J. Chem. Phys. 2001, 115, 1891–1901.
Figure 6. S/N ratios for SMM-2 on pSi surfaces modified with solutions containing different antibody concentrations: 1, 10, and 100 µg mL-1.
antibody affording high surface capacity,51 which potentially suppresses LDI-MS efficiency, or reduced surface capacity but enhanced LDI-MS efficiency is preferable. To investigate the influence of antibody concentration in the immobilization solution on the selectivity and the S/N ratio, three different antibody concentrations (1, 10, and 100 µg mL-1) were studied. Performance was evaluated using the analyte S/N ratios obtained from SMM-2 analysis. Figure 6 shows the S/N ratio values for three different antibody concentrations in the immobilization solution. At a concentration of 100 µg mL-1, the resulting surface showed a 50% decrease in S/N ratio relative to the surface (51) DeLouise, L. A.; Kou, P. M.; Miller, B. L. Anal. Chem. 2005, 77, 3222– 3230.
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where a 10 µg mL-1 antibody concentration was used. Analyte selectivity was maintained for both 10 µg mL-1 and 100 µg mL-1 concentrations since the benzodiazepine S/N ratio values relative to cocaine are similar. The S/N ratio values for all analytes on the 1 µg mL-1 antibody-modified surface were below 3. To determine the surface chemistry of surfaces exposed to different antibody concentrations used in the immobilization solutions, XPS measurements were carried out (Table S-2 in the Supporting Information). As expected, an increase in antibody concentration is accompanied by an increase in antibody coverage on the pSi surface, judging from the nitrogen and carbon content and the concomitant decrease in oxygen atomic percentage. However, the silicon percentage remains relatively stable, which is again due to the fact that the information depth of XPS is on the order of 10 nm. While the 100 µg mL-1 concentration appears to produce a surface with a greater antibody density than the 10 µg mL-1 surface (Table S-2 in the Supporting Information), the increase of the antibody appears to be offset by reduced analyte ionization. This may well be the result of excessive antibody on the surface suppressing the transfer of energy from the laser to the analyte, limiting LDI efficiency. We cannot exclude steric hindrance effects where antigen-binding capacities decrease above a certain antibody concentration as a result of surface crowding. This effect is well-known in immunoaffinity chromagraphy,52 biosensors,53 and SELDI,54 However, such steric hindrance effects are less likely given the moderate coverage levels observed here. At the same time as a low coverage (low concentration of antibody during immobilization), S/N ratios and selectivity deteriorated, presumably due to the limited immunocapture capability of this surface. Hence, our results suggest that a surface capacity and LDI efficiency are indeed conflicting requirements and that a balance between the two is needed for optimal performance. This was achieved here at an antibody concentration of 10 µg mL-1. To investigate the effect of immobilization conditions on surface performance, antibodies were also immobilized from 1× PBS instead of Milli-Q water. The primary objective here was to investigate if more physiological immobilization conditions would increase antibody activity leading to higher LDI-MS performance of the surface. In all previous preparations, immobilization and washing steps involved Milli-Q water only. Here, after immobilization of the antibody in 1× PBS, alternative washing solutions were (52) Matson, R. S.; Little, M. C. J. Chromatogr. 1988, 458, 67–77. (53) Cooper, M. A.; Fiorini, M. T.; Abell, C.; Williams, D. H. Bioorg. Med. Chem. 2000, 8, 2609–2616. (54) Neubert, H.; Jacoby, E. S.; Bansal, S. S.; Iles, R. K.; Cowan, D. A.; Kicman, A. T. Anal. Chem. 2002, 74, 3677–3683.
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used, namely 1× PBS and 1× PBS with 0.05% Tween 20. Both of these procedures are commonly employed to remove loosely bound proteins in immunoassays. Following completion of alternative washes, all surfaces were rinsed in water before analyte deposition in an attempt to remove salt. Omitting the water wash, detection of analytes using pSi LDI-MS was not possible, presumably due to the negative impact of salt on ion generation efficiency. Immobilization from 1× PBS solution shows a reduction in benzodiazepine analyte signal, by 50% for diazepam and alprazolam (Figure S-4 in the Supporting Information). Interestingly, inclusion of the mild detergent, 0.05% Tween 20 in the PBS washing solution, resulted in reduction of signal for all three analytes, to be just above noise level. While it is unlikely that the Tween 20 wash reduces antibody activity, it is possible that some detergent molecules remain physisorbed to the surface, hindering ion generation in LDI-MS. CONCLUSION We have demonstrated the selective detection of benzodiazepines in the presence of cocaine. The results are consistent with successful immunocapture of analytes from a mixed analyte aqueous solution. Benzodiazepine antibody-modified surfaces not only achieved targeted selectivity but also demonstrated greater sensitivity than previously published affinity-based LDI-MS platforms. XPS-based surface analysis of the modified pSi confirmed the success of the antibody immobilization and assisted in the optimization of the surface chemistry for LDI performance. Although the antibody-modified pSi surface is less sensitive than PFPDCS-modified pSi, the approach described here holds considerable potential for the fast-response and selective mass spectrometry detection of illicit drugs, as it minimizes ion suppression for targeted analytes. The platform can be easily expanded to an array format with a panel of antibodies. At the same time, these results should spawn research into the use of immunocapture pSi LDI-MS in other areas such as metabolomics and pharmacology. ACKNOWLEDGMENT Funding from the Australian Research Council and the South Australian Forensic Science Centre is kindly acknowledged. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review February 19, 2010. Accepted April 5, 2010. AC100455X
