Anal. Chem. 2003, 75, 6124-6132
A Core-Shell-Type Fluorescent Nanosphere Possessing Reactive Poly(ethylene glycol) Tethered Chains on the Surface for Zeptomole Detection of Protein in Time-Resolved Fluorometric Immunoassay Takeshi Matsuya,† Shigeru Tashiro,† Nobuhiro Hoshino,*,† Naoya Shibata,‡ Yukio Nagasaki,§ and Kazunori Kataoka⊥
Research and Development Division, Mitsubishi Kagaku Iatron, Inc., 1460-6 Mitodai, Mito, Takomachi, Katori-gun, Chiba 289-2247, Japan, Research and Development Division, NanoCarrier Co., Ltd., Tokatsu Techno Plaza, 5-4-6 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan, Department of Materials Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan, and Department of Materials Science, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
To increase the sensitivity and to depress the nonspecific binding in biochemical assays, a new core-shell-type fluorescent nanosphere (106.7 nm) covalently conjugated with antibody was prepared. The core-shell-type nanosphere was constructed by dispersion radical polymerization of styrene in the presence of heterotelechelic poly(ethylene glycol) (PEG) macromonomer, which has a polymerizable vinylbenzyl group at one end and a primary amino group at the other chain end and used as well as a surfactant. The resulting nanosphere had PEG tethered chains on the surface, which possesses a primary amino group at the distal end of the PEG chain (NH2 nanosphere). The fluorescent NH2 nanosphere was constructed by incorporating fluorescent europium chelates with β-diketonate ligands in the core of the NH2 nanosphere by means of a physical entrapment method. The primary amino groups on the fluorescent NH2 nanosphere were then converted to maleimide groups using a hetero cross-linker. The resulting nanosphere had maleimide groups on the surface (maleimide nanosphere), onto which proteins having SH group in the molecule could be covalently conjugated quantitatively without any denaturation of the proteins under the milder reaction condition. The applicability of the fluorescent nanosphere was tested in a model sandwich immunoassay for r-fetoprotein (AFP) determination. Anti-human AFP Fab′ fragment was covalently conjugated onto the maleimide nanosphere (Fab′ nanosphere), and it was used for the solid-phase time-resolved fluorometric immunoassay of AFP. The detection limit (mean + 2 SD) was 0.040 pg/mL or 57.1 zmol (57.1 × 10-21 mol, Mw,AFP ) 70 000) for AFP. The * To whom correspondence should be addressed. Fax: +81-479-76-3663. E-mail:
[email protected]. † Mitsubishi Kagaku Iatron, Inc.. ‡ NanoCarrier Co., Ltd.. § Tokyo University of Science. ⊥ The University of Tokyo.
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imprecision (concentration CV) over the whole assay range was 1.1% (100 pg/mL) - 17.1% (0.1 pg/mL), even though with this conjugation of antibody to the nanosphere, the nonspecific binding was practically negligible (0.0008%) and even when ∼1.9 × 109 particles of the Fab′ nanosphere were applied to the microtitration well. In the present trend of applying nonradioactive labeling strategies, fluorescent molecules and enzymes are widely employed as labels. The measured optical signal is typically based on an accumulated sum of fluorescent labels present in a probe region. A new chelated Eu3+ ion-incorporated core-shell-type fluorescent nanosphere having poly(ethylene glycool) (PEG) chains on the surface is one of the fluorescent probes suitable for highly sensitive immunoassays. The sensitivity of time-resolved fluorometric immunoassay has been shown to be improved by attaching many fluorescent groups to the labeling reagents using macromolecules, such as polyvinylamine,1 thyroglobulin,2 and a conjugate of streptavidin and albumin molecules,3 since the multiple labeling of fluorescent lanthanide chelates is free of quenching.2 The sensitivity of the immunoassay was also improved by enzymatically amplified methods using horseradish peroxidase4 or alkaline phosphatase.5,6 The multiple labeling methods that are capable of measuring fluorescence directly from the solid phase are expected to be an applicable method for protein microarray and high-throughput screening assays. (1) Scorilas, A.; Bjartell, A.; Lilja, H.; Moller, C.; Diamandis, E. P. Clin. Chem. 2000, 46, 1450-1455. (2) Morton, R. C.; Diamandis, E. P. Anal. Chem. 1990, 62, 1841-1845. (3) Yuan, J.; Matsumoto, K.; Kimura, H. Anal. Chem. 1998, 70, 596-601. (4) Ioannou, P. C.; Christopoulos, T. K. Anal. Chem. 1998, 70, 698-702. (5) Bathrellos, L. M.; Lianidou, E. S.; Ioannou, P. C. Clin. Chem. 1998, 44, 1351-1353. (6) Christopoulos, T. K.; Diamandis, E. P. Anal. Chem. 1992, 64, 342-346. 10.1021/ac034346e CCC: $25.00
© 2003 American Chemical Society Published on Web 10/16/2003
Figure 1. Schematic illustration of the preparation of anti-human AFP Fab′ nanosphere with the chelated Eu3+ ions. M, maleimide group; SMCC, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate.
The new core-shell-type nanosphere has the capacity to incorporate a large number of chelated Eu3+ ions into the core of the nanosphere. The chelated Eu3+ ions incorporated into the nanosphere have the high quantum yields of the known lanthanide chelators,7,8 since the chelated Eu3+ ions in the nanosphere are surrounded by polystyrene, and therefore, the polymer shell of the nanosphere deprives water efficiently, which could be a fluorescence-quencher, from the vicinity of the chelate by producing a hydrophobic environment. On the other hand, the degree of the nonspecific adsorption of fluorescent probe reagents to the cuvette wall is a drawback in the highly sensitive immunoassays. The fluorescent probes used in fluorescent immunoassays should possess groups for high water solubility and low nonspecific adsorption.9 The newly prepared fluorescent nanosphere has a hydrophilic surface that is covered with PEG tethered chains. Thereby, the fluorescent nanosphere showed little adsorption onto the solid phase, such as microtitration well. The fluorescent nanosphere was applied as a fluorescent probe for highly sensitive immunoassays. In the labeling method of the fluorophore to biomolecules, covalent binding of label molecules
using a primary amino group usually leads to a decrease in specific binding activity of biomolecules such as antibodies and streptavidin.10 As a milder modification method, the utilization of a SH group at the hinge region of the Fab′ fragment in the preparation of the conjugates is recommended to retain the original activities of the Fab′ fragment and gives lower nonspecific adsorption and higher specific binding. These methods gave a higher sensitivity than immunoglobulin G in immunoassays.11-13 In the present paper, the preparation method of the chelated Eu3+ ion-incorporated core-shell-type nanosphere having primary amino groups on the surface (fluorescent NH2 nanosphere) is described. To conjugate antibody onto the fluorescent NH2 nanosphere by a milder modification method, the primary amino groups on the fluorescent NH2 nanosphere were further converted to maleimide groups, which can be selectively bound to a SH group using a hetero cross-linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. The maleimide group-introduced nanosphere (maleimide nanosphere) synthesized was then covalently coupled with a Fab′ fragment of antibodies (Figure 1). Using the Fab′ fragment-conjugated fluorescent nanosphere (Fab′ nanosphere) as a fluorescent probe and a conventional time-
(7) Huang, H. G.; Hiraki, K.; Nishikawa, Y. Nippon Kagaku Kaishi 1981, 1, 66-73 (in Japanese). (8) Ullman, E. F.; Kirakossian, H.; Singh, S.; Wu, Z. P.; Irvin, B. R.; Pease, J. S.; Switchenko, A. C.; Irvine, J. D.; Dafforn, A.; Skold, C. N.; Wagner, D. B. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5426-5430. (9) Trau, D.; Yang, W.; Seydack, M.; Caruso, F.; Yu, N. T.; Renneberg, R. Anal. Chem. 2002, 74, 5480-5486.
(10) Hemmila, I. A. Applications of fluorescence in immunoassays; J. Wiley & Sons: New York, 1991. (11) Hoshino, N.; Hama, M.; Suzuki, R.; Kataoka, Y.; Soe, G. J. Biochem. 1985, 97, 113-118. (12) Sugiyama, K.; Hoshino, N. J. Anal. Bio-Sci. 2000, 23, 69-74 (in Japanese). (13) Matsuya, T.; Hoshino, N.; Matsumoto, K. Bulletin of the Ogata Institute for Medical and Chemical Research 1999, 13-22 (in Japanese).
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resolved fluorometer, a highly sensitive heterogeneous sandwich immunoassay for R-fetoprotein (AFP), which was selected as a subject of the model assay, was developed. EXPERIMENTAL SECTION Materials. Commercial tetrahydrofuran (THF, Kanto Chemical Co., Inc., Tokyo, Japan), vinylbenzyl alcohol (VBA, Seimi Chemical Co., Ltd., Kanagawa, Japan), ethylene oxide (EO, Sumitomo-3M Ltd., Tokyo, Japan), triethylamine (Kanto), methane sulfonyl chloride (Kanto), styrene monomer (Kanto), R,R′-azobisisobutyronitrile (AIBN, Kanto) were purified by conventional methods. Potassium naphthalene was used as a THF solution, the concentration of which was determined by titration. Europium chloride (Nacalai Tesque, Kyoto, Japan), 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (TTA, Dojindo Laboratories, Kumamoto, Japan), and tri-n-octylphosphine oxide (TOPO, Dojindo) were used as purchased. Reagent N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC,) was obtained from Zieben Chemicals Co., Ltd. (Tokyo, Japan). Anti-human AFP monoclonal antibody was from Oriental Yeast Co., Ltd. (Tokyo, Japan). The 96-well microtitration plate used was from FluoroNunc Modules & Plates (Nalge Nunc International, NY). All other reagents were of analytical reagent grade. Instruments. (a) An Arcus 1234 time-resolved fluorometer (Wallac Oy., Turku, Finland) was used for measuring the fluorescence of Eu3+ chelate in the microtitration wells. The measurement conditions were delay time, 0.20 ms; window time, 0.40 ms; and flash rate, 1.00 ms. The time-resolved fluorescence excitation and emission spectra of the chelated Eu3+ ionincorporated nanosphere (30 µg/mL) and the Eu3+-TTA-TOPO complex in 0.1% Triton X-100 solution were measured by a Spectramax Gemini XS (Molecular Devices Corporation, CA). (b) The HPLC system consisted of a Hitachi HPLC system (Tokyo, Japan); a Hitachi L-7100 HPLC pump; a Hitachi L-7420 UV/VIS detector; a Hitachi L-7490 RI detector; a Reodyne (Cotati, CA) model 7725I syringe-loading sample injector valve equipped with 2 mL sample loop. (c) The average diameter and the polydispersity factor of the NH2 nanosphere were measured with a DLS-7000 dynamic light-scattering spectrophotometer equipped with an argon laser (Otsuka Electronics Co., Ltd., Osaka, Japan). Heterotelechelic PEG Macromonomer (Vinylbenzyl-PEGNH2). Heterotelechelic PEG macromonomer possessing a polymerizable vinylbenzyl group at one end and a primary amino group at the other chain end was prepared as follows.14 Reagent VBA in THF (2 mmol, 1 mL) and 0.374 M potassium naphthalene in THF (2 mmol, 5.4 mL) were added to 100 mL of dry THF to form vinylbenzylalkolate. After the mixture was stirred for several minutes, EO (319 mmol) was added via a cooled syringe to the potassium vinylbenzylalkolate (2 mmol) in THF solution. The mixture was stirred for 2 days at room temperature. The purity of the vinylbenzyl PEG macromonomer formed was confirmed by gel permeation chromatography (GPC) (column, TSKgel SuperHZ2500 + SuperHZ3000 + SuperHZ4000 (4.6 mm i.d. × 3, Tosoh, Tokyo, Japan)); mobile phase, THF containing 2% of triethylamine; flow rate, 1.0 mL/min; detection, RI. The molecular weight of the vinylbenzyl PEG macromonomer was calculated by the ratio of the PEG segment on the basis of the number average (14) Hayashi, H.; Iijima, M.; Nagasaki, Y.; Kataoka, K. To be published.
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molecular weight (Mn) of PEG determined from the GPC result. Subsequently, 9.3 mmol of triethylamine was added to the reaction mixture, and the mixture was poured into 6.5 mmol of methane sulfonyl chloride in THF (10 mL) for the ω-end modification reaction. After the polymer was obtained, it was precipitated in ether, followed by drying in vacuo (intermediate, vinylbenzyl-PEGSO2CH3). The polymer was dissolved in distilled water (0.1 g/mL), and the solution was added dropwise with stirring to aqueous ammonia (500 mL; 25 wt %). The mixture was stirred for 2 days at room temperature to convert the ω-end group to a primary amine. The heterotelechelic PEG macromonomer, vinylbenzylPEG-NH2 (Mw ) 7257), thus obtained was purified by evaporating ammonia, and lyophilized after dialysis against water (2 L for 2 + 2 + 2 + 14 h; Mw,CO ) 3500). The 1H NMR spectrum was obtained using DMSO-d6 solution with a JEOL EX-400 spectrometer at 400 MHz (JEOL, Tokyo, Japan). Nanosphere. The anti-human AFP Fab′-conjugated fluorescent nanosphere (Fab′ nanosphere) was prepared as follows. The schematic illustration of the preparation process for the Fab′ nanosphere is shown in Figure 1. There are four steps to prepare the actually reactive Fab′ nanosphere: (I) the NH2 nanosphere is prepared by copolymerization of styrene monomer and heterotelechelic vinylbenzyl-PEG-NH2 macromonomers, (II) the fluorescent NH2 nanosphere is prepared by incorporating fluorescent europium chelates into the NH2 nanosphere, (III) the maleimide nanosphere is prepared by reacting a hetero crosslinking agent with the fluorescent NH2 nanosphere, and (IV) the Fab′ nanosphere is prepared by reacting Fab′ fragment with the maleimide nanosphere, which is applicable to the immunoassay as a tag antibody-conjugated fluorescent probe. (a) Preparation of NH2 Nanosphere. The core-shell-type NH2 nanosphere was prepared by dispersion radical polymerization of styrene monomer with heterotelechelic PEG macromonomer in water.15 Briefly, 1.04 mmol (7.5 g) of vinylbenzyl-PEG-NH2 and 8.7 mmol (1 mL) of styrene monomer containing 0.1 mmol (16 mg) of AIBN were dispersed in distilled water (260 mL). The mixture was heated at 60 °C for 18 h with stirring at 400 rpm and further for 6 h at 80 °C. After the polymerization, a solid mass of polystyrene was removed by filtering through filter paper (filter paper 2, Advantec, Tokyo, Japan), and the filtrate was obtained as the NH2 nanosphere suspension. (b) Preparation of Eu3+ Chelate Solution. The Eu3+-β-diketone-TOPO stock solution was prepared as follows: To 1 mL of EuCl3‚6H2O water solution (22 mg/mL) was added 1 mL of TTA solution (36.9 mg/mL, dissolved in acetone) and 2 mL of TOPO solution (22.3 mg/mL, dissolved in acetone). (c) Preparation of Chelated Eu3+ Ion-Incorporated NH2 Nanosphere (Fluorescent NH2 Nanosphere). The incorporation of Eu3+ chelate into the NH2 nanosphere was performed practically according to the previous publication.16,17 To 0.5 mL of the NH2 nanosphere suspension (21.37 mg/mL) was added 2 mL of the Eu3+ chelate solution in a 3-mL glass vial. The mixture was incubated at room temperature for 30 min in the dark, and then (15) Ogawa, R.; Nagasaki, Y.; Shibata, N.; Otsuka, H.; Kataoka, K. Polym. J. 2002, 12, 868-875. (16) Munebayashi, T.; Sawai, M. Japanese Patent publication no. 1996-304399. (17) Kataoka, K.; Nagasaki, Y.; Shibata, N.; Hoshino, N. Japanese Patent publication no. 2001-161788.
the acetone was evaporated by a N2 gas stream with stirring in the dark. The suspension was filtered through a nitrocellulose membrane (pore size, 0.2 µm, Advantec) in order to remove the excessive Eu3+ chelate precipitate. The filtrate was dialyzed against 0.1 M phosphate buffer of pH 8.0 in the dark. The fluorescent NH2 nanosphere was stored at 4 °C in the dark prior to use. (d) Preparation of Maleimide Nanosphere. The primary amino groups on the fluorescent NH2 nanosphere were converted to maleimide groups using a hetero cross-linking reagent. To 1.0 mL of the fluorescent NH2 nanosphere suspension (concentration of nanosphere, 3.7 mg/mL in 0.1 M phosphate buffer of pH 6.9) was added 94.6 µg of SMCC (4.73 µL of 20 mg/mL dimethylformamide), and the mixture was incubated for 1 h at room temperature in the dark. The suspension was dialyzed against 0.1 M phosphate buffer of pH 7.0 at 4 °C in the dark. (e) Preparation of Anti-Human AFP Fab′-Conjugated Fluorescent Nanosphere (Fab′ Nanosphere). Anti-human AFP Fab′ fragments were covalently conjugated onto the maleimide nanosphere. To 209 µL of the anti-human AFP Fab′ solution (2.09 mg/0.21 mL) dissolved in 0.05 M phosphate buffer of pH 7.0 containing 1 mM EDTA was added the maleimide nanosphere suspension (2.5 mg/ 1.35 mL). After the mixture was incubated for 72 h at 4 °C in the dark, 50 µL of 2-mercaptoethylamine solution (10 mg/mL in distilled water) was added to the mixture in order to block the remaining maleimide groups on the nanosphere. After incubation for 1 h at room temperature in the dark, the reaction mixture was filtered through a nitrocellulose membrane (pore size, 0.2 µm), and the suspension (1 mL) was injected onto a GPC column (TSK-gel G3000SW, 21.5 mm i.d. × 300 mm, Tosoh). The eluent was monitored by the absorbance at 280 nm. Each fraction of 1.0 mL was collected and was diluted 50-fold with 0.1 M Tris-HCl buffer of pH 9.1 containing 0.05% Tween-20. One hundred microliters of the respective diluted solution was placed into the microtitration well, and the time-resolved flourescence intensity of the Eu3+ chelate was determined by the Arcus 1234 fluorometer. Preparation of Fab′ Fragment. The Fab′ fragment of antihuman AFP antibody was prepared approximately as described previously.11-13 To 3 mL of 0.05 M acetate buffer of pH 4.5 containing 5 mg of anti-human AFP rabbit IgG antibody (purified fraction, Mitsubishi Kagaku Iatron Inc., Tokyo, Japan), 0.125 mg of pepsin (2.5 w/w% of IgG, Boehringer Mannheim, Germany) was added. The solution was incubated for 20 h at 37 °C, and the digestion was stopped by adding 0.135 mL of 1 M Tris (4.5 v/v % of reaction solution). The F(ab′)2 fraction of antibody was collected by a Sephacryl S-200 HR column with 0.05 M acetate buffer, pH 5.0, as an eluent. To 2 mg of F(ab′)2 dissolved in 2 mL of 0.05 M acetate buffer of pH 5.0, 2.84 mg of 2-mercaptoethylamine was added, and the solution was incubated for 90 min at 37 °C. The reaction mixture was loaded on a Sephadex G-25 SP column with 0.05 M phosphate buffer of pH 7.0 containing 1 mM EDTA and eluted with the same buffer solution, and then the fractions of anti-human AFP Fab′ were collected. The SH group of Fab′ fragment was stable for 3 days at 4 °C in the above eluted solution. Preparation of AFP Sample Solution.13 Standard AFP was obtained from Dako Japan Co., Ltd. (Kyoto, Japan). The stock solution of the standard AFP (1 µg/mL) was prepared in 0.02 M phosphate buffer of pH 7.4 containing 0.15 M NaCl, 5% bovine serum albumin (BSA), and 0.05% NaN3 and stored at 4 °C prior
to use. The stock solution was diluted with the same buffer to prepare the sample solution. The dilution buffer was used as the zero calibrator. Solid-Phase Sandwich Immunoassay of AFP.13 (a) Preparation of Anti-Human AFP Antibody-Immobilized Microtitration Well. The anti-human AFP monoclonal antibody solution (50 µL) dissolved in 0.1 M carbonate buffer of pH 9.6 was placed into the microtitration well (300 ng of antibody per well). The wells were incubated for 24 h at 4 °C, and then the capture antibodies were immobilized onto the well. After incubation, the antibody solution was removed by aspiration, and 100 µL of 0.1 M NaHCO3 solution containing 1% BSA, 2% sucrose and 0.05% NaN3 was placed into the wells. The wells were sealed with the above solution and stored at 4 °C prior to use. The NaHCO3 solution containing BSA and sucrose was used as the blocking solution in order to cover the unbound area of the antibodies in the microtitration well. (b) Immunoassay Procedure. The principle of a sandwich immunoassay in this study is shown in Figure 2. Following the removal of the blocking solution, 100 µL/well of the prepared standard AFP solution was added to the well (Figure 2A) and incubated at 37 °C for 1 h. The assay plate was rinsed twice with each 300 µL of 0.02 M phosphate buffer of pH 7.0 containing 0.15 M NaCl and 0.1% Tween-20. After that, 100 µL of the Fab′ nanosphere suspension in 0.02 M phosphate buffer of pH 7.0 containing 0.15 M NaCl, 0.2% BSA, and 0.05% NaN3 was added to the well (Figure 2B) and incubated at 37 °C for 1 h. Then the wells were rinsed four times with each 300 µL of 0.1 M Tris-HCl buffer of pH 9.1 containing 0.05% Tween-20 (Figure 2C), and the time-resolved fluorescence intensity in the solid phase was measured with the Arcus 1234 fluorometer. Determination of NH2 Group on the Nanosphere. The determination of the NH2 group on the fluorescent NH2 nanosphere and the maleimide nanosphere were performed by trinitrobenzene sulfonic acid (TNBS) method.18 Reagent monoethanolamine was used as the standard of NH2 group measurement. Prior to the analysis, 1.5 mL of 0.1 M Na2SO3 was mixed with 98.5 mL of 0.1 M NaH2PO4 (reagent A). To 50 µL of the nanosphere suspension was added 50 µL of 0.1 M Na2B4O7/0.1 M NaOH, and 10 µL of TNBS solutions (0.22 M in distilled water). After incubation for 5 min at room temperature, 200 µL of reagent A was added to the mixture. Absorbance at 420 nm of the reaction solution was measured with an Ultraspec 3100pro spectrophotometer (Amersham Pharmacia Biotech). Nonspecific Binding of Fab′ Nanosphere to the Microtitration Well. The dilution series of the Fab′ nanosphere prepared in 0.02 M phosphate buffer of pH 7.0 containing 0.15 M NaCl, 0.2% BSA, and 0.05% NaN3 were placed into the anti-human AFP antibody-immobilized microtitration well. The wells were incubated for 1 h at 37 °C in the dark and were rinsed four times with each 300 µL of 0.1 M Tris-HCl buffer of pH 9.1 containing 0.05% Tween-20. The time-resolved fluorescence intensity of the Fab′ nanosphere adsorbed nonspecifically to the well was measured with the Arcus 1234 fluorometer. RESULTS AND DISCUSSION Synthesis of Heterotelechelic PEG Macromonomer (Vinylbenzyl-PEG-NH2). There are two routes to synthesize PEG (18) Okuyama, T.; Satake, K. J. Biochem. 1960, 47, 454.
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Figure 2. A sandwich immunoassay process using anti-AFP Fab′ nanosphere. The analyte was first immobilized on the solid phase by the preadsorbed capture antibody (A) and then exposed to the Fab′ nanosphere (B). After incubation and washing out of the excess Fab′ nanosphere, the remaining time-resolved fluorescence intensity of the chelated Eu3+ ion (615 nm) was measured by irradiating of the pulsed excitation light at around 350 nm (C), and the intensity was recorded.
possessing a polymerizable vinylbenzyl group at one end and a primary amino group at the other chain end. One of the methods is to introduce a vinylbenzyl group at the initiation chain end and the other is to introduce an ω-chain end modification. We have chosen the former method, that is, the potassium 4-vinylbenzyl alkoxide initiation system, because when doing ω modification with vinylbenzyl bromide, it is somewhat difficult to maintain the R-primary amino group as intact. The purity of vinylbenzyl-PEG macromonomers synthesized was confirmed by GPC. The number molecular weight (Mn) of the PEG segment was calculated to be 7124 from the GPC results. The molecular weight distribution (weight-average molecular weight (Mw)/number-average molecular weight (Mn)) was determined to be 1.20. The Mn of vinylbenzyl-PEG-NH2 was calculated to be 7257, which was in good accordance with the initial monomer/initiator ratio ([EO]0/[VBA]0). The vinylbenzyl-PEGNH2 macromonomer was thus obtained, and the yield was 54.7%. However, a small amount of high molecular weight materials along with PEG with anticipated molecular weight was observed. When potassium naphthalene was used as a methylation agent for VBA, vinyl oligomerization took place to some extent. The resulting VBA oligomers can also initiate the polymerization of EO to form higher Mw byproducts. Fortunately, such byproducts should not have any polymerizable double bond, and it must not be incorporated into the nanosphere by the dispersion radical polymerization. Thus, the products could be used without further fractionation. Figure 3 shows the 1H NMR spectra of vinylbenzylPEG-NH2 (Figure 3B) compared with vinylbenzyl-PEG-SO2CH3 (polymer intermediate, Figure 3A). From the 1H NMR spectrum of methane sulfonate-ended PEG, it is clear that both vinylbenzyl proton signals (5-8 ppm) and methane sulfonate proton signals (3.2 and 4.3 ppm) are appearing. After the amination reaction, the signals based on methane sulfonate disappeared completely, and new signals based on an aminomethylene proton signal appeared at 2.8 ppm. 6128 Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
Preparation of NH2 Nanosphere. We have already reported on the synthesis of the core-shell-type nanosphere possessing aldehyde-PEG tethered chains on the surface.15 The aldehyde end group can be utilized as a conjugation with primary amino groups on proteins under the reduced condition. No charge variation was observed during the reductive amination, since the primary amino groups on proteins were converted to sec-amino groups via the reduction of the Shiff base formed. As a milder modification method, the utilization of a SH group at the hinge region of antibody in the conjugation reaction does not lead to a decrease of the specific binding activity of antibody.11,19 To conjugate Fab′ fragments of immunoglobulin on the nanosphere surface, the primary amino group at the PEG chain end is rather suitable, because the primary amino group can be easily converted to maleimide group with the hetero cross-linking agent, followed by conjugation with the Fab′ fragment via a Michael reaction with SH group. As is well-known that macromonomers can work as both surfactant and comonomer, the size and dispersion stability of the nanosphere possessing amino-PEG tethered chain can be controlled by the ratio of the PEG macromonomer to the styrene monomer. Table 1 lists the results of the preparation of styrene nanosphere in the presence of vinylbenzyl-PEG-NH2. Using vinylbenzyl-PEG-NH2 as comonomer, nanosized NH2 nanospheres were obtained easily as well as by the method of aldehyde-ended macromonomers. The size of the sphere was in the range from 100 to 200 nm in this experiments, keeping a fairly low size dispersion factor. One of the typical SEM views of the prepared NH2 nanosphere is shown in Figure 4. The average diameter and the polydispersity factor (µΓ-2) of the NH2 nanosphere are listed in Table 1. We have already reported that the occupied area of each PEG chain on the aldehyde nanosphere surface prepared by the (19) Ishikawa, E.; Hashida, S.; Kohno, T.; Hirota, K. Clin. Chim. Acta 1990, 194, 51-72.
Figure 3.
1H
NMR spectra of vinylbenzyl-PEG-SO2CH3 (A) and vinylbenzyl-PEG-NH2 (B) at 20 °C (solvent, DMSO-d6)
Table 1. Results of Dispersion Polymerization of Styrene in the Presence of Heterotelechelic Vinylbenzyl-PEG-NH2 Macromonomers
run
styrene, mL
PEG macromonomer, g
1 2 3
25 3.2 1
2 5.8 7.5
yield, %
diam, nm
PDFa (µ Γ-2)
amineb nm2
76.8 93.6 54.7
216.3 119.9 106.7
8.86 × 10-3 4.82 × 10-2 4.65 × 10-2
8.1 (0.06 7.4 (1.50 5.4 (0.05
a Polydispersity factor. b Occupation area of each amino group on the nanosphere surface.
Figure 4. Scanning electron micrograph of NH2 nanosphere (run 3 in Table 1). The SEM view was observed by a field emission scanning electron micrograph (FE-SEM, Hitachi S-4200B).
dispersion radical polymerization with PEG macromonomers is ∼35 nm2, regardless of the size of the nanosphere.15 The amino groups on the surface of the nanosphere were determined by the TNBS method and are listed in Table 1. Incorporation of Eu3+ Chelate into the NH2 Nanosphere. The number of chelated Eu3+ ions in a single fluorescent NH2 nanosphere was determined as described previously.20 The (20) Ha¨rma¨, H.; Soukka, T.; Lo ¨vgren, T. Clin. Chem. 2001, 47, 561-568.
method is efficient for protecting the quenching effect of water. The signals obtained by the Arcus 1234 fluorometer were compared with a Eu3+ chelate calibrator in the enhancement solution. The number of chelated Eu3+ ions in a single fluorescent NH2 nanosphere was determined by comparing the signal of a known number of particles in the 0.1 M carbonate buffer of pH 9.1 containing 0.05% Tween-20 or the enhancement solution to the Eu3+ chelate calibrator (in the enhancement solution). The fluorescence intensity was equivalent to 6300 chelated Eu3+ ions in a single fluorescent NH2 nanosphere. The excitation and emission maximums of the fluorescent NH2 nanosphere were 346 and 617 nm, respectively, which have spectra similar to that of the free Eu3+-TTA-TOPO complex. It was clear that polystyrene and PEG did not affect the fluorescence properties of the Eu3+TTA-TOPO complex. The detection limit of the fluorescent NH2 nanosphere was measured in 50 µL of 0.1 M Tris-HCl buffer of pH 9.1 containing 0.05% Tween-20 with the Arcus 1234 fluorometer. As shown in Figure 5, the number of fluorescent NH2 nanosphere at the detection limit was determined to be 2000 particles in one well. The detection limit of the fluorescent NH2 nanosphere was defined as the number of particles corresponding to a signal 3 SD above the mean of five replicates of the dilution buffer. Preparation of Maleimide Nanosphere and Fab′ Nanosphere. The amino groups on the nanosphere were converted to maleimide groups using a hetero cross-linking agent, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and the anti-human AFP Fab′ fragments were conjugated with the maleimide groups on the maleimide nanosphere. The amino groups on the nanosphere before and after the reaction of SMCC were determined to be 4.3 × 10-8 mol/mL and 3.5 ×10-8 mol/ mL, respectively. It was suggested that the 19% of amino groups on the nanosphere were reactive with SMCC. After the remaining maleimide groups that were not reacted with the Fab′ fragment were blocked with 2-mercaptoethylamine, the conjugate was introduced onto a GPC column. Figure 6 shows the gel permeation chromatograms of the Fab′ nanosphere. As shown in Figure 6A, the peak for the anti-human AFP Fab′ fragment, which was not Analytical Chemistry, Vol. 75, No. 22, November 15, 2003
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Figure 5. Dilution curve of chelated Eu3+ ion-incorporated 106.7nm nanosphere measured with a conventional time-resolved fluorometer. The arrow indicates the lower limit of detection.
conjugated onto the nanosphere, was observed at 23 min when the reaction mixture of the fluorescent NH2 nanosphere and the Fab′ fragment was loaded onto the column in the absorbance measurement (dotted line in Figure 6A). Since the absorbance of fluorescent NH2 nanosphere itself was little observed in the nanosphere concentration eluted from the column (
