Functionalized Magnetic Nanoparticles for Small-Molecule Isolation

Academia Sinica, Taipei, Taiwan. Functionalized magnetic nanoparticles (MNPs) were syn- thesized to serve as laser desorption/ionization elements as w...
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Anal. Chem. 2007, 79, 3401-3408

Functionalized Magnetic Nanoparticles for Small-Molecule Isolation, Identification, and Quantification Po-Chiao Lin,† Mei-Chun Tseng,‡ An-Kai Su,‡ Yu-Ju Chen,*,‡ and Chun-Cheng Lin*,†

Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan and Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan, and Institute of Chemistry, Academia Sinica, Taipei, Taiwan

Functionalized magnetic nanoparticles (MNPs) were synthesized to serve as laser desorption/ionization elements as well as solid-phase extraction probes for simultaneous enrichment and detection of small molecules in matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Two laserabsorbing matrices were each conjugated onto MNP to give MNP@matrix which provided high ionization efficiency and background-free detection in MS leading to unambiguous identification of target small molecules in a complex mixture. MNP@matrix was demonstrated to serve as a general matrix-free additive in MALDI-TOF MS analysis of structurally distinct small molecules. Also, MNP@matrix provides a simple, rapid, and reliable quantitative assay for small molecules by mass without either the use of an internal standard or an isotopic labeling tag. Furthermore, the affinity extraction of small molecules from complex biofluid was achieved by probe protein-conjugated MNP@matrix without laborious purification. We demonstrated that a nanoprobe-based assay is a cost-effective, rapid, and accurate platform for robotic screening of small molecules. Many small molecules, such as synthetic/natural organic compounds and metabolites, are important agonists or antagonists of specific target proteins.1 As such, the small-molecule-biomacromolecule interaction constitutes one of the most important biological networks, encompassing protein-ligand, protein-drug, and enzyme-substrate complexes.2 Following the evolution of genomics, proteomics, and metabolomics, as well as advances in combinatorial chemistry and syntheses of natural products, the study of small-molecule-biomacromolecule interactions has progressed from simple objects to complex systems.2 Conventional characterization methods for collection, separation, identification, and/or quantification of small-molecule-protein interactions, such * To whom correspondence should be addressed. E-mail: [email protected] (C.-C.L.); [email protected] (Y.-J.C.). Fax: (+)886-3-5711082. † National Tsing Hua University and Taiwan International Graduate Program, Academia Sinica. ‡ Institute of Chemistry, Academia Sinica. (1) Gohlke, H.; Klebe, G. Angew. Chem., Int. Ed. 2002, 41, 2644-2676. (2) Tian, R.; Xu, S.; Lei, X.; Jin, W.; Ye, M.; Zou, H. Trends Anal. Chem. 2005, 24, 810-825. 10.1021/ac070195u CCC: $37.00 Published on Web 04/03/2007

© 2007 American Chemical Society

as affinity chromatography,3 capillary electrophoresis,4 surface plasma resonance,5 nuclear magnetic resonance,6 and X-ray crystallography, are often labor intensive and time-consuming. Thus, development of a high-throughput assay that emphasizes accuracy, sensitivity, and reproducibility would facilitate the verification of candidate molecular targets as well as the discovery of target-oriented drugs.7 Recent reports on the use of nanoscale materials in biological systems have provided new access to understanding and manipulating biomolecular interactions.8 Medical diagnoses and recognition of molecular targets are two promising applications of biomolecule-conjugated nanoparticles (NPs).9 In particular, magnetic nanoparticles (MNPs) have been applied extensively in various biological applications, including magnetic resonance imaging,10 drug delivery,11 biomolecular sensors,12 magnetothermal therapy,13 and bioseparation.14 Recently, we have taken advantage of the large surface area to volume ratio and the magnetic property of MNPs to demonstrate that antibodyconjugated MNPs can serve as nanoprobes for the separation of target biomarkers from human plasma with high specificity15 and sensitivity.16 We developed nanoprobe-based affinity mass spectrometry (NBAMS)17 in combination with direct protein identification by matrix-assisted laser desorption/ionization time-of-flight (3) Mano, N.; Sato, K.; Goto, J. Anal. Chem. 2006, 78, 4668-4675. (4) Martinez-Gomez, M. A.; Sagrado, S.; Villanueva-Camanas, R. M.; MedinaHernandez, M. J. Electrophoresis 2006, 27, 3410-3419. (5) Lo ¨ssner, D.; Kessler, H.; Thumshirn, G.; Dahmen, C.; Wiltschi, B.; Tanaka, M.; Knoll, W.; Sinner, E.-K.; Reuning, U. Anal. Chem. 2006, 78, 45244533. (6) Feeney, J. Angew. Chem., Int. Ed. 2000, 39, 290-312. (7) Annis, D. A.; Nazef, N.; Chuang, C.-C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503. (8) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042-6108. (9) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547-1562. (10) Pankhurst, Q. A.; Connolly, J.; Jones, S. K.; Dobson, J. J. Phys. D: Appl. Phys. 2003, 36, R167-R181. (11) Gupta, A. K.; Curtis, A. S. G. J. Mater. Sci.: Mater. Med. 2004, 15, 493. (12) Graham, D. L.; Ferreira, H. A.; Freitas, P. P. Trends Biotechnol. 2004, 22, 455-462. (13) Hiergeist, R.; Andra, W.; Buske, N.; Hergt, R.; Hilger, I.; Richter, U.; Kaiser, W. J. Magn. Magn. Mater. 1999, 201, 420-422. (14) Gu, H.; Xu, K.; Xu, C.; Xu, B. Chem. Commun. (Cambridge, U.K.) 2006, 941-949. (15) Lin, P.-C.; Chou, P.-H.; Chen, S.-H.; Liao, H.-K.; Wang, K.-Y.; Chen, Y.-J.; Lin, C.-C. Small 2006, 2, 485-489. (16) Chou, P.-H.; Chen, S.-H.; Liao, H.-K.; Lin, P.-C.; Her, G.-R.; Lai, A. C.-Y.; Chen, J.-H.; Lin, C.-C.; Chen, Y.-J. Anal. Chem. 2005, 77, 5990-5997.

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Figure 1. Workflow of functionalized MNP-assisted MALDI-TOF MS. Option 1: MNP@matrix serves as a matrix-free additive. Option 2: bifunctional MNP@matrix-protein serves as an affinity matrix-free additive.

mass spectrometry (MALDI-TOF MS). NBAMS provides a novel approach for the study of targeted proteomics without the need for time-consuming purification and enrichment processes. Mass spectrometers use the characteristic mass-to-charge ratio (m/z) of ionized atoms or molecules to distinguish them from each other. For small organic molecules, the molecular mass can be measured to within an accuracy of 5 ppm or less, which is often sufficient to confirm the molecular formula of a compound.18 Furthermore, molecules have distinctive fragmentation patterns in a mass spectrum that provides structural information for the analyzed molecules.19 Thus, mass spectrometry has been considered as one of the gold standard methods for small-molecule identification due to its low detection limit, rich structural information, and, most importantly, high accuracy. Among various types of mass spectrometers, MALDI-TOF MS has been widely used for protein identification due to its sensitivity, high throughput, and applicability to complex mixtures with unknown structures.20 However, this approach is not suitable for detection of small molecules due to the interference from the matrix in the low molecular weight region of the mass spectrum.21 The matrix material is essential for the ionization of the analyte. However, the matrix will absorb laser energy to produce molecular ions (17) Huang, L.-S.; Chien, Y.-Y.; Chen, S.-H.; Lin, P.-C.; Wang, K.-Y.; Chou, P.-H.; Lin, C.-C.; Chen, Y.-J. In Nanomaterials for Cancer Diagnosis; Kumar Challa, S. S. R., Ed.; Wiley-VCH: New York, 2006; Vol. 7, pp 338-376. (18) (a) Bristow, A. W. T.; Webb, K. S. J. Am. Soc. Mass Spectrum. 2003, 14, 1086-1098. (b) Gross, M. L. J. Am. Soc. Mass. Spectrum. 1994, 5, 57-62. (c) Beynon, J. H. Adv. Mass Spectrom. 1959, 328-354. (19) (a) Tandem Mass Spectrometry; McLafferty, F. W., Ed.; Wiley-VCH: New York, 1983. (b) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry: Techniques and Application of Tandem Mass Spectrometry; Wiley-VCH: New York, 1988. (20) (a) Pasch, H.; Schrepp, W. MADLI-TOF Mass Spectrometry of Synthetic Polymers; Springer: New York, 2002. (b) Baar, B. L. M. V. FEMS Microbiol. Rev. 2000, 24, 193-219. (c) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349-451. (21) (a) Shu, Y.-R.; Su, A.-K.; Liu, J.-T.; Lin, C.-H. Anal. Chem. 2006, 78, 46974701. (b) Kinumi, T.; Saisu, T.; Takayama, M.; Niwa, H. J. Mass Spectrom. 2000, 35, 417-422. (c) McCombie, G.; Knochenmuss, R. Anal. Chem. 2004, 76, 4990-4997.

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and fragment peaks (MW < 500 Da),22 which cause ion suppression and ambiguous identification.23 Diverse matrix-free methods such as porous silicon (desorption/ionization on silicon, DIOS),24 sol-gel-derived matrix film,25 gold NPs,26 and silicon-coated nanostructures27 have been described as alternatives to the conventional MALDI matrix. However, most of these methods were not designed to specifically enrich the target analyte from a complex medium. This gap in methodology has inspired us to develop specific and cost-effective bifunctional NPs for future implementation in high-throughput screening of multiple small molecules from biological media. In this report, we have developed a straightforward method for simultaneous enrichment and detection of small molecules using MALDI-TOF MS and bifunctionalized MNPs that serve as laser desorption/ionization elements as well as solid-phase extraction probes. As shown in Figure 1, the dual covalent immobilization of the matrix without (MNP@matrix, option 1) or with probe protein (MNP@matrix-protein, option 2) on the same MNP is a unique feature of this approach. As shown in option 1, the MNP@matrix can be used as a matrix-free additive for MALDITOF MS analysis of small molecules. The entrapped matrix is covalently conjugated to the polymeric NP surface; thus, the MNP@matrix can potentially reduce the interference caused by matrix ion peaks normally found in the low molecular weight range for MALDI-TOF MS. As shown in option 2, the MNP@matrix-protein can alternatively be used to purify and (22) (a) Dally, J. E.; Gorniak, J.; Bowie, R.; Bentzley, C. M. Anal. Chem. 2003, 75, 5046-5053. (b) Choen, L. H.; Gusev, A. I. Anal. Bioanal. Chem. 2002, 373, 571-586. (c) Guo, Z.; Zhang, Q.; Zou, H.; Guo, B.; Ni, J. Anal. Chem. 2002, 74, 1637-1641. (23) Knochenmuss, R.; Zenobi, R. Chem. Rev. 2003, 103, 441-452. (24) (a) Wei, J.; Buriak, J. M.; Siuzdak, G. Nature 1999, 399, 243-246. (b) Lee, J.-C.; Wu, C.-Y.; Apon, J. V.; Siuzdak, G.; Wong, C.-H. Angew. Chem., Int. Ed. 2006, 45, 2753-2757. (25) Lin, Y.-S.; Chen, Y.-C. Anal. Chem. 2002, 74, 5793-5798. (26) Huang, Y.-F.; Chang, H.-T. Anal. Chem. 2006, 78, 1485-1493. (27) Go, E. P.; Apon, J. V.; Luo, G.; Saghatelian, A.; Daniels, R. H.; Sahi, V.; Dubrow, R.; Cravatt, B. F.; Vertes, A.; Siuzdak, G. Anal. Chem. 2005, 77, 1641-1646.

enrich the target small molecule from the complex mixture through protein-small-molecule interactions and subsequent magnetic separation. The captured MNP@matrix-protein-target molecule complex was directly analyzed by MALDI-TOF MS without additional elution/desalting or addition of matrix molecules. We demonstrate that the developed method facilitates simultaneous detection and quantification of small-molecule drugs. Furthermore, we implemented the functionalized MNP to create a target-specific biomolecular probe for the rapid enrichment and detection of low-abundance small molecules in human serum. EXPERIMENTAL SECTION Materials. Tetraethyl orthosilicate (TEOS, Aldrich), 3-aminopropyltrimethoxysilane (APS, Aldrich), 1-propanol (Acros), 25% ammonia solution (Acros), 2,5-dihydroxybenzoic acid (DHB, Sigma), R-cyano-4-hydroxycinnamic acid (CHCA, Sigma), and suberic acid bis-N-hydroxysuccinimide ester (DSS, Sigma) were used as received. Transmission electron microscopy (TEM) images were obtained by a JEM-2100F electron microscope (JEOL Co. 200 KV). Scanning electron microscopy (SEM) images were obtained by a Hitachi S-4200 scanning microscope. Fabrication of MNP@matrix. The Fe3O4 MNPS (30 mg) were prepared as previously reported15,16 and suspended in 1-propanol (3 mL) by sonication for 30 min at room temperature. Then, NH4OH (25% w/w, 0.408 mL), ddH2O (0.3 mL), and TEOS (0.1 mL) were added to the above solution, and the mixture was stirred at 50 °C for 1 h. After filtration, the black precipitate (MNP@SiO2) was washed three times with 1-propanol and then added to a mixture of APS (0.1 mL) and DHB (70 mg) in ddH2O (3 mL) or saturated CHCA aqueous solution (3 mL) followed by the addition of 0.01 N HCl (1.5 mL). The mixture was shaken at room temperature for 12 h and then centrifuged (8000 rpm, 15 min) to separate the precipitate. The resulting black precipitate was washed with ddH2O to give the MNP@matrix. Fabrication of MNP@DHB-Probe Protein. To activate the MNP surface, MNP@DHB (1 mg) and DSS (10 mg) were incubated in dimethyl sulfoxide (DMSO, 250 µL) for 1 h to give the N-hydroxysuccinimide (OSu)-activated MNP. The activated MNP@DHB-OSu was washed three times with DMSO (200 µL), and then the MNP was incubated with concanavalin A (Con A) (50 µL at 10 mg/mL) at 4 °C for 12 h. The resulting MNP@DHBCon A was isolated by simple magnetic separation and then purified by washing with PBS buffer (0.1 M, pH 7.4) to give MNP@DHB-Con A as a black powder. Quantification of Flufenamic Acid using MALDI-TOF MS and MNP@CHCA. Flufenamic acid (1.0 mg) was dissolved in 1.0 mL of methanol to yield a 1000 ng/µL stock solution. A 0.1 mL aliquot of the stock solution was diluted with 0.9 mL of methanol to give a 100 ng/µL standard solution that was subsequently diluted with methanol (to give 1, 2, 5, 10, 25, and 50 ng/µL) to establish a working curve for quantification. A 1.0 µL sample of MNP@CHCA matrix solution (2000 ppm) was mixed with a 1.0 µL standard solution at each concentration, and 1.0 µL of the resulting mixture was loaded onto the MALDI plate for the quantification analysis as described in the MALDI-TOF MS analysis. In the working curve, each spot was given by three samples and got the relative standard deviation as 0.2095, 0.1175, 0.1903, 0.1319, 0.0533, and 0.1796 for 1, 2, 5, 10, 25, and 50 ng/ mL.

Enrichment of Small-Molecule Analyte by MNP@DHBProbe Protein. The model study was performed on the mannose-Con A interaction. To increase the normally low concentration of mannose present in the serum, the MNP@DHB-Con A (0.2 mg) was incubated with human plasma (20 µL) containing mannose (20 µg) at room temperature for 30 min. The MNP@DHB-Con A-mannose complex was magnetically separated and then washed three times with 25 mM NH4HCO3 buffer (100 µL). The MNP@DHB-Con A-mannose complex was analyzed directly by MALDI-TOF MS. MALDI-TOF MS Analysis. All mass spectra were acquired using a MALDI-TOF mass spectrometer (Voyager DE-PRO, Applied Biosystems, Foster City, CA) equipped with a 337 nm nitrogen laser. The spectra were recorded in the linear mode using an accelerating voltage of 25 kV, a 90% grid voltage, 0.1% guide wire voltage, and 100 ns delay time. For MALDI MS analysis in small-molecule quantification, the MNP@CHCA was directly mixed with 1 to ∼2 µL of target, spotted onto the sample plate, air-dried, and analyzed. For the extracted mannose analysis, the extracted sample (MNP@DHB-Con A-mannose) was directly deposited onto the sample plate and then analyzed without further isolation or purification. RESULTS AND DISCUSSION Fabrication and Characterization of Functionalized Nanoparticles. As shown in Scheme 1, two types of functionalized NPs were fabricated for use as matrix in either direct identification of small molecules or specific enrichment and simultaneous detection of small molecules in biofluid. It has been well-established that various matrices are needed for different types of analytes because the ionization efficiency resulting from proton transfer is analyte dependent. In this report, two commonly used matrices, DHB and CHCA, were conjugated to the MNP@SiO2 to give MNP@matrix as detailed in the Experimental Section. To characterize functionalized MNPs, FT-IR spectroscopy was used to ensure proper fabrication of the surface modification. As shown in Figure 2, FT-IR spectroscopy provided clear evidence for the step-by-step surface modification including silanation and DHB/amine functionalization. There was no significant absorption signal in the FT-IR spectrum (Figure 2A) for the starting Fe3O4 NP. After TEOS polymerization, the unique Si-O absorption band was observed in the range from 1000 to 1100 cm-1 (Figure 2B), indicating successful silanation on the MNP surface. DHB conjugation on the MNP surface was confirmed by the newly present absorption peak at 1730 cm-1, which corresponds to the CdO absorption band from the carboxylic acid group (Figure 2C). The MNPs were analyzed by electron microscopy to obtain information on size, shape, and distribution on a solid support. As shown in Figure 3A, the TEM image of MNP@DHB clearly shows the global shape of core MNP with ∼10 nm diameter. In addition to the TEM image, SEM was applied to profile the MNP distribution on a solid support. The MNP@DHB used in this study was directly deposited onto the MALDI plate; thus, we investigated whether the MNPs distributed homogeneously on the solid support without aggregation, as this aspect is important for reducing spot-to-spot variation. The SEM image in Figure 3B suggests that MNPs were evenly distributed on a solid surface (also see Figure S2 in the Supporting Information). The homogeneous deposition suggests that the MNP@matrix could help Analytical Chemistry, Vol. 79, No. 9, May 1, 2007

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Scheme 1. Fabrication of MNP@matrix Probes

to reduce the MALDI signal fluctuation, which is the most critical factor for accurate and robust quantification. The MNP@matrix was stable at 4 °C for at least 6 months without any decomposition (data not shown).

Figure 2. Step-by-step characterization of the fabricated MNPs by FT-IR spectroscopy: FT-IR spectra of (A) Fe3O4 MNP, (B) MNP@SiO2, and (C) MNP@DHB.

Figure 3. Electron microscopy images of MNP@DHB by (A) TEM and (B) SEM. 3404

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Optimization of Ionization Efficiency for the MNP@matrix. We initially evaluated the performance of MNP@matrix as a background-free ionization element. A small-molecule drug, flufenamic acid, was chosen as a model. We compared the MALDITOF mass spectra of using conventional free DHB, MNP@SiO2, and MNP@DHB. As shown in Figure 4A, the mass spectrum of the commercial matrix DHB with flufenamic acid exhibited many DHB-derived peaks (marked with an “/”) that masked the identification of the drug. It has been reported that DIOS has emerged as a successful alternative for conventional MALDI.24 Besides organic materials, SiO2 is an effective MALDI matrix for small-molecule detection.28 Thus, we examined the performance of MNP@SiO2 as a MALDI matrix to explore the possibility of SiO2 polymer on an NP surface as matrix. As shown in Figure 4B, the flufenamic acid peak was the most intense peak, but there was also an abundance of MNP@SiO2-derived peaks (marked with “B”). These peaks may derive from fragile Si skeleton networks and may also potentially interfere with the spectra interpretations.28 On the other hand, the use of MNP@DHB, formed by covalently conjugating DHB to the polymeric NP surface, dramatically reduced the matrix-derived interference in the low molecular weight range (Figure 4C). Most importantly, flufenamic acid was detected as a single prominent peak with excellent signal-to-noise ratio, demonstrating superior background-free detection when compared with that of the conventional MALDI MS matrix and higher ion intensity when compared with that of MNP@SiO2. Based on these results, we speculate that the observed superior detection performance may be the collective result of ionization with the matrix and SiO2 on the MNP@matrix surface. A previous report indicated that the smaller pore size of silicon yields better MS performance in DIOS when compared with that of a smoothed surface silicon,29 implying large surface provides better ionization (28) Go´recka-Drzazga, A.; Bargiel, S.; Walczak, R.; Dziuban, J. A.; Kraj, A.; Dylag, T.; Silberring, J. Sens. Actuators, B 2004, 103, 206-212. (29) Finkel, N. H.; Prevo, B. G.; Velev, O. D.; He, L. Anal. Chem. 2005, 77, 1088-1095.

Figure 5. Intensity of prometryn (10 ng) desorbed from MNP@DHB with various trapped DHB concentrations (50, 100, 500, 1000, 5000, and 10 000 ppm).

Figure 4. Comparison of (A) DHB, (B) MNP@SiO2, and (C) MNP@DHB as the matrix for the detection of flufenamic acid by MALDI-TOF MS. The flufenamic acid (“4”), DHB-derived (“/”), and MNP@SiO2-derived (“B”) peaks are shown.

efficiency.Thus,theexcellentdetectionperformancebyMNP@matrix may also be attributed to the increase in accessible surface area for analyte absorption, resulting in enhanced MS signals. We believe that a larger NP surface area is the key factor to enhance the thermal conductivity for direct ionization from SiO2. Additionally, the larger surface area promotes better reaction of primary matrix ions with the analyte, which intensifies the analyte ion signal. The amount of matrix examined using MALDI has a significant effect on the sensitivity and background of the resulting spectrum.23 Thus, the effect of entrapped matrix on the ionization efficiency was studied by applying prometryn (10 ng) with MNP@DHB (1 µL). A series of diluted MNP@DHB was tested in optimization experiments as shown in Figure 5. Because the DHB molecules were covalently bonded to the polymeric SiO2 on the particle surface, all the mass spectra using MNP@DHB showed no background matrix ion peaks. The signal intensity of prometryn increased when high concentrations of MNP@DHB were applied to the sample plate. Using MNP@DHB concentra-

tions ranging from 50 to 10 000 ppm of DHB, as shown in Figure 5, we found that at least 1000 ppm was required to provide stable laser desorption/ionization efficiency, and the signal gradually plateaued after 1000 ppm. In comparison with a commercial DHB matrix, no prometryn signal could be observed when the same amount of free DHB was examined (data not shown). In general, up to 10 000 ppm of free DHB was required to obtain comparable signal intensity. Therefore, the use of covalent matrix-encapsulated MNPs not only eliminates the background signals for unambiguous identification of small molecules but also provides high ionization for enhanced detection sensitivity. Detection of Target Small-Molecule Drugs by MNP@matrix and MALDI-TOF MS. We analyzed six drugs with distinct chemical structures, including salicylamide, mefenamic acid, ketoprofen, flufenamic acid, sulindac, and prednisolone (100 ppm), to demonstrate the general applicability for use of MNP@matrix in MALDI-TOF MS (see Table S1 in the Supporting Information for structures). These drugs are commonly used to reduce pain and fever and are often illegally added to traditional Chinese herbs.30 As shown in Figure 6, the MNP@CHCA generated very clean spectra (spectra G-L in Figure 6) that show the presence of only the drug-related peak(s) for each analyte, including the cationic parent, sodium adduct, and the dehydrated form. The presence of the sodium adduct form of the target molecule may have been caused by the use of NaOH(aq) during the MNP@matrix fabrication process, resulting in capping of the sodium cation on the MNP. Notably, MNP@DHB also produced clean mass spectra with signal-to-noise ratios comparable to those of MNP@CHCA (data not shown). By contrast, the use of the conventional free CHCA matrix with each of these drugs yielded numerous strong CHCA-derived ion peaks (marked with an “/”) that obscured the low molecular weight peaks (spectra A-F in Figure 6), thereby seriously interfering with the assignment of the target analyte. Interference due to the use of conventional matrices in MALDTOF MS often results in substantial signal suppression of target molecules, especially very low abundance analytes in complex biofluids. Thus, we used a mixture of the six aforementioned drugs as a representative complex fluid to examine the matrix-free feature of MNP@CHCA. As expected, the use of the conventional (30) Cheng, H. L.; Tseng, M.-C.; Tsai, P.-L.; Her, G. R. Rapid Commun. Mass Spectrom. 2001, 15, 1473-1480.

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Figure 6. MALDI-TOF MS for each drug (100 ppm). Spectra A-F correspond to salicylamide (1), mefenamic acid (2), ketoprofen (3), flufenamic acid (4), sulindac (5), and prednisolone (6), respectively, using CHCA as the matrix. Spectra G-L correspond to the same analytes; however, MNP@CHCA was used as the matrix. The peaks marked with an “/” in spectra A-F correspond to matrix-derived ions of CHCA.

Figure 7. Comparison of the performance of free CHCA, DHB, and MNP@matrix as the matrix in MALDI-TOF MS analysis of six drugs and six herbicides. Numbers 1-6 represent molecules as shown in Figure 7. The six herbicides shown are simazine (a), simetryn (b), prometon (c), atrazine (d), propazine (e), and terbutryne (f). Mass spectra of the six-drug mixture obtained using (A) free CHCA and (B) MNP@CHCA are shown. Mass spectra of the six-herbicide mixture obtained using (C) free DHB and (D) MNP@DHB are shown. The peaks marked an “/” in the spectra A and C correspond to matrixderived ions of CHCA and DHB, respectively. The peak at m/z 379.33 could not be determined as either a matrix ion or target molecule.

CHCA matrix resulted in a complex and overlapping mass spectrum (Figure 7A). The peaks of target drugs were partially buried in the strong matrix-derived background peaks, resulting in uncertain identification of the target drugs. For example, the peak m/z 379.33 in Figure 7A could be attributed to either the CHCA-related species or the sodiated form of sulindac, leading 3406 Analytical Chemistry, Vol. 79, No. 9, May 1, 2007

to ambiguity in target molecule identification. Furthermore, prednisolone (drug 6) is the most difficult ionizable molecule among the six-drug mixture and was thus barely observed because of strong signal suppression by other drugs. By contrast, a clean spectrum with prominent sodium adduct peaks was obtained using MNP@CHCA as the matrix (Figure 7B), indicating excellent ionization efficiency of MNP@CHCA for multiple analytes in a matrix-free background. We also extended the application of MNP@DHB to the detection of six common triazine herbicides, simazine, simetryn, prometon, atrazine, propazine, and terbutryne, to evaluate the general use of MNP@matrix to other small molecules (see Supporting Information Table S2 for structures). Triazine derivatives are the most commonly used herbicides for agriculture and land management in Europe and the United States. The U.S. Environmental Protection Agency has cited concerns that longterm exposure to these chemicals in food and drinking water may pose a cancer risk to the U.S. population.31 These compounds are usually applied as a pre- and postemergent weed control to improve the yield and quality of agricultural products. Due to their widespread use and relative high resistance to degradation, the ability to qualitatively and quantitatively monitor triazines that remain in the environment is of great importance for environmental and water control. Figure 7, parts C and D, shows the comparison of mass spectra between the conventional free matrix and the NP-derived matrix. The use of the conventional DHB results in a convoluted mass spectrum comprising matrix, matrix adducts, and the herbicide mixture (Figure 7C). Alternatively, all six herbicides were clearly detected without any background ions using MNP@DHB as the matrix (Figure 7D). Our examination of the two structurally distinct groups (drugs and triazine-derived herbicides) demonstrates the advantages of the MNP@matrix for the detection and identification of complex small-molecule mixtures by MALDI-TOF MS in biomedical and environmental applications. Quantification of Target Molecules by MNP@matrix and MALDI-TOF MS. Quantitative analysis by MALDI-TOF MS has been considered a challenge because of sample/matrix heterogeneity and the ion suppression effect,23 both of which result in poor reproducibility of analyte signals and a limited dynamic detection range. An alternative solution for quantitative MALDI analysis is to spike the sample with an isotopic internal standard to compensate for the inherently poor shot-to-shot reproducibility. As depicted in Figure 3B, MNP@DHB showed uniform deposition on the solid surface, which may potentially improve the homogeneity of sample deposition on the MALDI sample plate. Therefore, we further investigated the use of matrix-free MNP@matrix for quantitative measurement in MALDI-TOF MS. A quantitative standard curve of flufenamic acid was established with MNP@CHCA. Without an internal standard, a linear correlation response was observed in the 1-50 ng range with an excellent correlation coefficient, r2 ) 0.9989 (Figure 8). On the other hand, flufenamic acid with the conventional CHCA matrix did not show a linear response because of poor spectra reproducibility (data not shown). These results demonstrate that MNP@matrix provides a simple, rapid, and reliable quantitative (31) Whalen, M. M.; Loganathan, B. G.; Yamashita, N.; Saito, T. Chem.-Biol. Interact. 2003, 145, 311-319.

Figure 8. Working curve for the quantification of flufenamic acid by MALDI-TOF MS with MNP@CHCA.

Figure 9. MALDI-TOF mass spectra of various concentrations of mannose extracted by MNP@DHB-Con A.

assay for small molecules without the use of an internal standard or isotopic labeling tag. Biofunctionalized MNP for Enrichment and Detection of Target Molecules in Human Serum. Additionally, we explored the function of MNP@matrix-protein as a biomolecular affinity probe (option 2 in Figure 1) to investigate ligand-protein interactions. The molecular recognition between carbohydrates and lectins plays an important role in cell-cell communication, proliferation, and differentiation, yet the study of these interactions remains difficult due to their relatively weak binding. To examine the ability of MNP@matrix-protein to probe a weak biomolecular interaction, the well-defined recognition between mannose and Con A was chosen as a proof-of-concept experiment. As shown in Scheme 1, the probe protein, Con A, was covalently conjugated to MNP@matrix through amide bond formation with the diactivated ester linker, DSS, to generate MNP@DHB-Con A. Final mannose concentrations ranging from 0.5 to 1000 µg mL-1 were used as tested samples. The MNP@DHB-Con A was used for affinity extraction of the mannose to form a MNP@DHB-Con A-mannose complex. This complex was then directly analyzed by MALDI-TOF MS without further elution. As shown in Figure 9, strong sodium adduct mannose signals were observed in all spectra, and the intensities of mannose peaks decreased in a concentration-dependent manner. Affinity-based methods for smallmolecule target identification are more likely to succeed with stronger binding affinity and an abundance of target analytes. Although the binding between Con A and mannose is very weak (Kd ∼ 10-3 M), our results suggest that the native structure of

Figure 10. Mannose extraction from human plasma by MNP@DHBCon A: (A) the background signal of MNP@DHB-Con A; (B) the human serum profile; the black arrow indicates the spiked mannose; (C) the mass spectrum of mannose [mannose + Na]+ extracted by MNP@DHB-Con A.

immobilized Con A on MNP@DHB remained unchanged as it retained a weak interaction with mannose.32 These results demonstrate that biofunctional MNPs can be used for simultaneous isolation, concentration, and characterization of low-level analytes. Aberrant glycosylation or failure of the carbohydrate recognition system have been associated with many diseases,33 and further understanding of the biological roles of glycans and their receptors must be derived from naturally occurring diseases and disease phenotypes created in animal models. The effective and rapid study of carbohydrate-protein interactions is currently a challenging and critical step prior to further clinical validation to unravel carbohydrate-disease correlations. Thus, we pursued the potential applicability of the developed method to direct detection of low-level target small molecules in a physiological biofluid. Human plasma containing a target carbohydrate was chosen as a complex model system to evaluate the specificity of detection of the MNP@matrix-protein probe. The MNP@DHB-Con A was incubated with human plasma (20 µL) spiked with mannose (20 µg). No background signal was observed from the MNP@DHBCon A (Figure 10A). Due to the presence of many plasma components, the mannose-spiked human plasma profile (200-fold dilution, 1 µL) identified several small molecules within the sample in which mannose (designated by an arrow) was barely detectable prior to affinity extraction (Figure 10B). The complex spectrum obviously reveals the challenge of small-molecule identification in human plasma. Following affinity capture by MNP@DHB-Con A, the MALDI spectrum prominently displayed the sodium adduct mannose [mannose + Na]+ at m/z 203 (Figure 10C) without observation of any nonspecifically bound component. Thus, we can conclude that the use of MNP@DHB-Con A combined with rapid identification of analytes by MALDI-TOF MS provides a convenient, effective, and specific bioassay for small-molecule detection from an extremely complex medium. In comparison with conventional microarray methods using planar solid supports, these NP-based “suspension arrays” should (32) Lin, C.-C.; Yeh, Y.-C.; Yang, C.-Y.; Chen, G.-F.; Chen, Y.-C.; Wu, Y.-C.; Chen, C.-C. Chem. Commun. (Cambridge, U.K.) 2003, 2920-2921. (33) Taylor, M. E.; Drickamer, K. Introduction to Glycobiology; Oxford University Press: New York, 2003.

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improve assay homogeneity and decrease assay time due to radial diffusion and decreased planar hindrance. In this study, the assay time from incubation of MNP@DHB-Con A with mannose to magnetic separation and subsequent detection by MALDI-TOF MS could be completed in 30 min. Thus, this rapid and sensitive approach may be amenable to a biomolecular interaction assay with high throughput capability. Furthermore, we believe that high surface area to volume ratio and homogeneity of the nanoprobe in buffer will increase the amount of probe protein on the particle surface and thus facilitate concomitant enhancement of biocapture. CONCLUSION Through a combination of our previous work on epitope mapping34 and target protein identification15,16 using the NBAMS technique, we further demonstrated NBAMS as a new tool for biorecognition assays. Both the pharmaceutical industry and academia could potentially benefit from NBAMS to discover and to characterize small-molecule antagonists or agonists. The advantages of functionalized MNPs for MALDI MS are multifold: (34) Chen, Y.-J.; Chen, S.-H.; Chien, Y.-Y.; Chang, Y.-W.; Liao, H.-K.; Chang, C.Y.; Jan, M.-D.; Wang, K.-T.; Lin, C.-C. ChemBioChem 2005, 6, 1169-1173.

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(1) simultaneous enrichment, detection, and quantification of target small molecules, (2) generic ionization method for matrixfree studies utilizing MALDI mass spectrometers from different manufactures, and (3) cost-effective material that is 1/10 the price compared with that of the conventional matrix for each experiment. Moreover, miniaturization of analytical techniques to afford high-throughput screening will greatly facilitate the study of smallmolecule-mediated inhibition of target enzymes. ACKNOWLEDGMENT This work was financially supported by the National Tsing Hua University, Academia Sinica Research Project on Nanoscience and Technology, and the National Science Council, Taiwan. We also thank Miss C.-Y. Chen for helping prepare the manuscript. SUPPORTING INFORMATION AVAILABLE Structures of six triazine herbicides. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 31, 2007. Accepted February 27, 2007. AC070195U