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ARTICLES Conjugation of (E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine to Hydrophilic Microspheres: Development of a Mobile Microscale UV Light Actinometer Shiyue Fang,‡,§,# Yousheng Guan,‡,4 Ernest R. Blatchley III,† Chengyue Shen,†,⊥ and Donald E. Bergstrom*,‡,§ School of Civil Engineering, Purdue University, West Lafayette, Indiana 47907-2051, Birck Nanotechnology Center, Department of Medicinal Chemistry and Molecular Pharmacology and the Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907, and The Walther Cancer Institute, Indianapolis, Indiana 46208. Received September 3, 2007; Revised Manuscript Received December 18, 2007
(E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine was biotinylated through a diisopropylsilylacetal linkage and attached to the surface of hydrophilic streptavidin-coated microspheres through the high-affinity noncovalent interaction between biotin and streptavidin. The functionalized microspheres form a stable suspension in water. Upon UV irradiation, the nonfluorescent (E)-5-[2-(methoxycarbonyl)ethenyl]cytidine on the microspheres undergoes photocyclization to produce highly fluorescent 3-β-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine. The fluorescence intensity of the microspheres can be correlated to the particle-specific UV doses applied at different suspension concentrations. The microspheres allow one to measure the UV dose (fluence) distribution in highthroughput water disinfection systems.
INTRODUCTION Several years ago, we initiated a multidisciplinary project aimed at developing a means for experimentally characterizing continuous-flow UV light disinfection photoreactors. For this project, a mobile UV light actinometer based on a microsphere that has the following three features is required: (i) Under UV irradiation at 254 nm (and other germicidally active wavelengths), the microsphere produces fluorescence when excited with a lower energy light, and the fluorescent intensity on the microsphere is correlated to the dose of germicidal UV light applied to the microspheres over a broad range. (ii) The functionalized microsphere has a hydrophilic surface and forms a relatively stable suspension in water. (iii) The physical properties of the microspheres such as size, shape, and density resemble those of relevant waterborne microbial pathogens. The use of microspheres for measurement of UV dose (fluence) distribution has been reported, but the microspheres were coated with a fluorescent dye whose absorption maximum fell at a much higher wavelength than required for germicidal action, and the measured effect was photobleaching (1). Moreover, the magnitude of the change in fluorescence signal was relatively small for the doses of UV radiation that are relevant in disinfection applications. In order to achieve maximum sensitivity and range, * Correspondence to Donald E. Bergstrom, Purdue University, Room 2042, Birck Nanotechnology Center, 1205 West State Street, West Lafayette, IN 47907-2057, Telephone: 765-494-6275, E-mail: bergstrom@ purdue.edu. † School of Civil Engineering, Purdue University. ‡ Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University. § The Walther Cancer Institute. 4 Current address: Anthill Technologies, Woburn, MA. ⊥ Current address: HydroQual, Inc., Johnstown, NY. # Current address: Department of Chemistry, Michigan Tech University, Houghton, MI.
a system in which the UV irradiation transforms a nonfluorescent molecule with an absorption maximum in the wavelength range of germicidal lamps to a light-stable fluorescent molecule would be preferable. There are many examples of fluorescent photochromic compounds (2–5), but relatively few examples of compounds that when irradiated with UV light of wavelength less than 300 nm lead to a single product that is both UV-stable and fluorescent. In 1982, we reported the preparation and characterization of (E)-5-[2-(methoxycarbonyl)ethenyl]cytidine (1) (6). This cytidine nucleoside analogue is not fluorescent; however, under UV irradiation at 254 nm, its trans-double bond is presumed to undergo trans–cis isomerization to form the intermediate 2, which cannot be isolated or even observed. In intermediate 2, the amino group would now be positioned in close proximity to the methyl ester group, which facilitates rapid cyclization through nucleophilic attack to give the stable and highly fluorescent product 3-β-D-ribofuranosyl-2,7-dioxopyrido[2,3d]pyrimidine (3, Scheme 1). We have previously reported the use of compound 1 as a chemical actinometer for germicidal UV irradiation (7). We reasoned that attaching 1 to 5 µm diameter hydrophilic microspheres would provide mobile actinometers with the desired properties for application in the measurement of UV fluence distribution in flowthrough UV disinfection systems. This manuscript describes the chemistry to accomplish the construction of the derivatized microsphere, while the dosimetry application is described elsewhere (8).
EXPERIMENTAL PROCEDURES General. All reactions were performed under a blanket of dry dinitrogen or argon. Reagents and solvents available from commercial sources were used as received unless otherwise noted. Pyridine and triethylamine were distilled over CaH2. Acetone was dried over anhydrous Na2SO4 and filtered. Thin layer chromatography (TLC) was performed using silica gel on
10.1021/bc700336x CCC: $40.75 2008 American Chemical Society Published on Web 02/12/2008
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Scheme 1. Photochemical Reaction of Nucleoside 1
aluminum 60F-254, 200 µm thickness TLC plates. Flash column chromatography was performed using 40 µm silica gel. NMR spectra were obtained using 250 and 500 MHz NMR spectrometers. Chemical shifts (δ) are reported relative to residual CD3OH (δ ) 4.87 ppm for 1H and 49.15 ppm for 13C). Infrared spectra were recorded on a FTIR spectrophotometer. Melting points were determined using a Mel-Temp melting point apparatus and are uncorrected. Streptavidin-coated microspheres (1.0% solid content in 100 mM borate, pH 8.5 + 0.1% BSA + 0.05% Tween 20 + 10 mM EDTA buffer with 0.1% NaN3 antimicrobial agent; mean diameter 5.5 µm; number of microspheres per gram 2.038 × 1010; number of microspheres per milliliter 2.040 × 108; surface area 1.240 × 1012 µm2) were manufactured by Polysciences, Inc. (Warrington, PA). TTL buffer: 100 mM Tris-HCl, pH 8.0, and 0.1% Tween 20, 1.0 M LiCl. PBS buffer: 0.1 M, pH 7.0. 2′,3′-Propylidinonyl-(E)-5-[2-(methoxycarbonyl)ethenyl] cytidine (4). A one-neck round-bottom flask was charged with 1 (1.2 g, 3.6 mmol), p-toluenesulfonic acid monohydrate (2.1 g, 11.0 mmol), and acetone (20 mL). To this suspension was added DMF (about 5 mL) until insoluble materials were dissolved, forming a light yellow solution. After stirring at room temperature overnight, the reaction mixture was partitioned between saturated Na2CO3 solution (20 mL) and CH2Cl2 (20 mL × 3). The organic phase was washed with water (20 mL × 2), dried over MgSO4, and solvents were removed under reduced pressure. Flash column chromatography (9:1 EtOAc/MeOH) gave 4 as a white solid (1.16 g, 88%): mp 162–3 °C; Rf ) 0.45 (9:1 EtOAc/MeOH); IR (KBr, cm-1) ν 3350, 2984, 1646, 1625, 1207; 1H NMR (CD3OD, 500 MHz) δ 1.38 (s, 3H), 1.58 (s, 3H), 3.76–3.89 (m, 2H), 3.79 (s, 3H), 4.32 (s, 1H), 4.76–4.95 (m, 2H), 5.95 (s, 1H), 6.37 (d, 1H, J ) 15.8 Hz), 7.62 (d, 1H, J ) 15.8 Hz), 8.51 (s, 1H); 13C NMR (CD3OD, 500 MHz) δ 25.7, 27.7, 52.3, 63.1, 82.2, 87.1, 89.5, 95.7, 104.2, 115.0, 118.4, 137.0, 144.3, 157.2, 165.6, 168.8; HRMS (ESI, M + H+) Calcd for C16H22N3O7368.1458, found 368.1476. Biotinyl Alcohol 7. Biotin (2.00 g, 8.19 mmol), triethylamine (1.72 mL, 12.29 mmol), and DMF (10 mL) were combined in a one-neck round-bottom flask and cooled to 0 °C. To this white suspension, iso-butylchloroformate (1.70 mL, 13.10 mmol) was added via syringe, and the reaction mixture was stirred at 0 °C for 1 h. The white suspension was then cooled to -60 °C. A solution of 6-amino-1-hexanol (6, 1.55 g, 13.10 mmol) and triethylamine (1.37 mL, 9.83 mmol) in DMF (5 mL) was added slowly via syringe, and the suspension was stirred overnight while gradually warming to room temperature. Anhydrous ether (100 mL) was added; the resulting white precipitate washed with ether (10 mL × 2) and then dissolved in methanol (50 mL) and stirred with Rexyn 201 (OH) resin (10 g) for 1 h. After filtration to remove the resin, the light yellow solution was concentrated under reduced pressure. Addition of ether (30 mL) produced the product as a white solid (2.40 g, 85%): mp 159–60 °C; IR (thin film, cm-1) ν 3301, 2931, 2854, 1684, 1633; 1H NMR (CD3OD, 500 MHz) δ 1.36–1.75 (m, 14H), 2.20 (t, 2H, J ) 7.4 Hz), 2.71 (d, 1H, J ) 12.7 Hz), 2.93 (dd, 1H, J ) 12.8, 5.0 Hz), 3.15–3.31 (m, 3H), 3.54 (t, 2H, J ) 6.6 Hz),
4.31 (dd, 1H, J ) 7.8, 4.4 Hz), 4.50 (dd, 1H, J ) 7.8, 5.0 Hz); C NMR (CD3OD, 500 MHz) δ 26.6, 26.9, 27.8, 29.5, 29.8, 30.4, 33.6, 36.8, 40.3, 41.0, 57.0, 61.6, 62.9, 63.4, 166.1, 175.9; EIMS m/z 343 (M+), 299, 283, 226, 179, 124; HRMS (EI) Calcd for C16H29N3O3S 343.1930, found 343.1914. Biotinylated Nucleoside Derivative 8. To a suspension of 7 (1.20 g, 3.48 mmol) in DMF (7.0 mL) and pyridine (2.0 mL) was added diisopropyldichlorosilane (630 µL, 3.48 mmol) via syringe at -60 °C. The colorless solution was stirred for about 5 h while warming to room temperature, and then cooled to -60 °C again. A solution of 4 (427 mg, 1.16 mmol) in DMF (3.0 mL) was added via syringe, and stirred overnight while warming to rt. Addition of ether (50 mL) gave a white precipitate, which was partitioned between sat NaHCO3 (25 mL) and n-butanol (20 mL × 5). The organic phase was dried over MgSO4, solvents removed, and the product purified by flash column chromatography (9:1 CH2Cl2/MeOH). The product was obtained as a light yellow foam (433 mg, 45%): Rf ) 0.25 (9:1 CH2Cl2/MeOH); IR (thin film, cm-1) ν 2928, 2860, 1699, 1648; 1 H NMR (CD3OD, 500 MHz) δ 0.88–1.77 (m, 34H), 2.19 (t, 2H, J ) 6.6 Hz), 2.71 (d, 1H, J ) 12.7 Hz), 2.92 (dd, 1H, J ) 12.7, 5.0 Hz), 3.10–3.21 (m, 4H), 3.31 (d, 2H, J ) 1.4 Hz), 3.54 (t, 1H, J ) 6.6 Hz), 3.75 (t, 2H, J ) 6.3 Hz), 3.77 (s, 3H, -OMe), 3.97–4.06 (m, 2H), 4.30 (dd, 1H, J ) 7.8, 4.4 Hz), 4.36–4.37 (m, 1H), 4.48–4.51 (m, 1H), 4.95 (dd, 1H, J ) 6.2, 1.8 Hz), 5.81 (d, 1H, J ) 1.7 Hz), 6.30 (d, 1H, J ) 16.3 Hz), 7.60 (d, 1H, J ) 15.8 Hz), 8.19 (s, 1H); 13C NMR (CD3OD, 500 MHz) δ 13.3, 13.4, 17.9, 17.9, 18.0, 25.7, 26.8, 27.1, 27.6, 28.0, 29.7, 29.9, 30.6, 33.9, 37.0, 40.5, 41.2, 52.4, 57.2, 61.8, 63.5, 64.4, 64.8, 82.6, 87.2, 90.0, 96.8, 104.1, 114.8, 118.6, 137.3, 144.1, 157.1, 165.6, 166.2, 168.6, 176.1; HRMS (ESI, M + H+) Calcd for C38H63N6O10SSi 823.4096, found 823.4087. Biotinyl Nucleoside Derivative 10. Following the procedure for preparation of 8, biotin derivative 9 (255 mg, 0.52 mmol), DMF (6 mL), imidazole (87 mg, 1.25 mmol), diisopropyldichlorosilane (113 µL, 0.63 mmol) and 4 (230 mg, 0.63 mmol) gave 10 as a white foam after flash column chromatography (9:1 CH2Cl2/MeOH, 255 mg, 51%): Rf ) 0.3 (9:1 CH2Cl2/ MeOH); IR (thin film, cm-1) ν 3187, 2941, 2862, 1648, 1068; 1 H NMR (CD3OD, 500 MHz) δ 0.94–1.04 (m, 14H), 1.29–1.31 (m, 6H), 1.36 (s, 3H), 1.43–1.45 (m, 2H), 1.56 (s, 3H), 1.64–1.68 (m, 2H), 1.69–1.80 (m, 2H), 1.82 (t, 2H, J ) 8.3 Hz), 2.62 (t, 2H, J ) 7.4 Hz), 2.34–2.37 (m, 2H), 2.76 (d, 1H, J ) 12.7 Hz), 2.95 (dd, 1H, J ) 12.7, 7.9 Hz), 3.22–3.24 (m, 1H), 3.38 (t, 4H, J ) 5.4 Hz), 3.57 (t, 4H, J ) 5.7 Hz), 3.63 (s, 4H), 3.78 (s, 3H), 4.00–4.05 (m, 2H), 4.36–4.38 (m, 2H), 4.56 (dd, 1H, J ) 7.7, 5.1 Hz), 4.88 (dd, 1H, J ) 6.3, 3.3 Hz), 5.03 (dd, 1H, J ) 6.4, 1.4 Hz), 5.90 (s, 1H), 6.35 (d, 1H, J ) 15.7 Hz), 7.63 (d, 1H, J ) 15.5 Hz), 8.22 (s, 1H); 13C NMR (CD3OD, 500 MHz) δ 14.5, 14.7, 18.27, 18.30, 18.33, 18.4, 25.7, 26.9, 27.6, 29.6, 29.8, 30.3, 30.5, 32.5, 35.5, 36.8, 40.3, 40.4, 41.2, 41.6, 50.0, 52.4, 52.5, 57.0, 61.7, 63.4, 64.5, 70.7, 71.3, 74.9, 82.2, 86.7, 89.5, 95.8, 104.7, 115.0, 119.4, 136.7, 143.6, 165.2, 168.2, 176.2, 176.3; HRMS (ESI, M + H+) Calcd for C41H74N7O13SSi 968.4835, found 968.4830. 13
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Scheme 2. Strategies for Directly Linking Compounds 1 and 3 to Microspheres
Attachment of Biotinylated Nucleoside Analogues to Streptavidin-Coated Microspheres. The microsphere suspension (1.0 mg, 0.1 mL) was transferred to a centrifuge tube. The storage buffer was removed by first centrifuging and removing the supernatant, leaving the white microspheres, which were washed with 200 µL TTL buffer. The biotinylated nucleosides (8 or 10, ∼80µg in 25 µL TTL buffer) were added to the microspheres, and incubated at room temperature for 20 min with occasional gentle shaking. The buffer was removed after centrifuging, and the microspheres were washed with PBS buffer (1 mL × 3) and water (1 mL × 3) and stored as a suspension in water at 0 °C in the dark. Microspheres coated with 8 were found to be unstable, even after repeated additional washings with PBS buffer (1 mL × 2) and water (1 mL × 2), fluorescence in the buffer or water was observed under UV light, and fluorescence on the washed microspheres became less and less intense. Microspheres coated with 10 were stable under these washing conditions, after storing at 0 °C for several days in the dark, and no adverse effects were observed in fluorescence studies. Following UV irradiation, the excitation maximum of the fluorescent microsphere suspension in water was 336 nm and the emission maximum fell at 381 nm, which closely matches the excitation and emission maxima of compound 3 (6). The determination of the relationship between UV dose and fluorescent intensity of the microsphere suspension in water is reported elsewhere (8).
RESULTS AND DISCUSSION To tether nucleosides 1 and 3 to microspheres, we initially explored the strategies outlined in Scheme 2. Microspheres with nucleoside 1 directly attached would serve as the sensor, but we also required microspheres with compound 3 for calibration. It became clear after considerable effort that simple direct methods for tethering would not yield microspheres with the fluorescence response required for quantification of UV dose. In all cases, the conjugation reaction was either inefficient or there was considerable quenching of the photoreaction and/or fluorescence. Problems associated with quenching when fluorophores are closely spaced within a matrix or on a surface are well-recognized (9–11).
Scheme 3. Preparation of Compound 4
As outlined in Scheme 2, the conjugation methods attempted included periodate oxidation followed by borohydride (12) or cyanborohydride (13) mediated reductive amination to aminecoated polystyrene and silica microspheres. This method has been shown to enable the conjugation of ribonucleotides, including cytidine derivatives, to solid supports (12, 14, 15). However, we were not able to obtain fluorescent microspheres either by attachment of compound 1 to give derivatized beads 1-p followed by UV irradiation (254 nm) or by direct attachment of fluorescent nucleoside 3. The final product in both cases should be 3-p. A variety of solvents (H2O, DMF, and DMSO), both sodium borohydride and sodium cyanoborohydride, and a number of different silica and polystyrene amine-substituted microbeads were investigated without success. Seela and coworkers have attached ribonucleosides to sepharose beads by first synthesizing 2′,3′-ethylidene derivatives from levulinic acid and then coupling the pendant carboxylic group to 6-aminohexylagarose using the water-soluble carbodiimide EDC (16–18). We attempted a number of variations on this approach (not shown), but were not able to obtain fluorescent microspheres. Alcohols can be directly linked to polystyrene trityl chloride resin (19). However, fluorescent nucleoside 3 did not yield fluorescent microspheres with a polystyrene trityl resin. It is possible that resin 3-t was formed but we were unable to detect fluorescence. Finally, we attempted to link nucleoside 3 by way of a succinic acid linker (Scheme 2) to give 3-s, but this approach also failed to give fluorescent microspheres. As a result of these failures, we chose to pursue a biotin-streptavidin conjugation strategy that would significantly reduce the density of nucleoside 1 while at the same time placing each molecule
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Scheme 4. Preparation of Biotinyl Cytidine Analogue 8
further from the surface of the microsphere. Streptavidin-coated microspheres with a wide range of physical properties are commercially available; and these microspheres are hydrophilic because streptavidin is relatively hydrophilic. The microspheres chosen for this study have a diameter of ∼5.5 µm and a specific gravity of 1.05, which would allow them to mimic the movement and trajectories of waterborne microbial pathogens. A study by Buranda et al. on 6.2 µm diameter streptavidin-coated microspheres yielded an estimate of 106 binding sites per microsphere (20). This translates to an average density of less than one binding site per 100 nm2. However, there was yet another issue to be resolved. It has been observed that biotin-fluorophore conjugates are also subject to quenching. When Gruber et al. examined biotin conjugates of fluorescein, tetramethylrhodamine, Cy3, and Cy5, they found that fluorescein and tetramethyrhodamine bound through a 14 atom spacer showed considerable quenching (21). With longer spacers (PEG800 and PEG1900), there was considerably less quenching. We chose to investigate a 20 atom spacer that was both more rigid and sterically hindered than simple PEG spacers, rationalizing that the steric bulk and rigidity would function more effectively to separate chromophores than simple incorporation of very long, but highly flexible spacers. It was necessary to link the spacer through the ribose group, since the methoxycarbonyl and 4-amino groups are required for the photochemical reaction. Moreover, the 2′- and 3′-OH groups are conveniently protected by acetal formation, which leaves a single site, the 5′-hydroxy available for attachment of the spacer to biotin. At the same time, a 2′,3′-acetal would be anticipated to considerably increase solubility in organic solvents thereby facilitating the chemistry necessary to functionalize the molecule at the 5′-OH. When nucleoside 1 was stirred in a mixture of acetone and DMF in the presence of p-toluenesulfonic acid, compound 4 was obtained in 83% yield as a white solid (Scheme 3) (22). In order to achieve selective coupling between a biotin containing moiety and compound 4 at the 5′-hydroxyl group in the presence of the unprotected cytosine amino group, we pursued a strategy based on the generation of silyl ether linkers. We first prepared biotinyl alcohol 7 by coupling biotin (5) to aminoalcohol 6 using iBuOC(O)Cl as the carboxylic acid activating agent (23). Then, 1 equiv of dichlorodiisopropylsilane was allowed to react with the hydroxyl group in the biotinyl alcohol 7. The intermediate was not isolated but allowed to then react directly with the cytidine analogue 4 through the formation of a diisopropylsilylacetal functionality as shown in Scheme 4 to give the biotinylated cytidine analogue 8. Next, attempts were made to attach 8 to streptavidin-coated microspheres. The microspheres were first washed with two portions of TTL buffer (100 mM Tris-HCL, pH 8.0, and 0.1%
Scheme 5. Conjugation of Biotin Derivative 9to Nucleoside 4
Tween 20, 1.0 M LiCl), and then suspended in a TTL buffer containing 8. The suspension was incubated at room temperature for 20 min with occasional gentle shaking in the dark. The microspheres were then collected by centrifuging, and washed sequentially with PBS buffer and deionized water, and then suspended in deionized water. After exposure of the microspheres to UV light, fluorescence was observed on the microspheres. However, fluorescence was also observed in the washing buffer and continued to appear in the wash solution on repeated washing. Concomitantly, the fluorescence on the microspheres became less and less intense with washing. From these observations, it appeared that the silyl acetal linkage in 8 was not stable in aqueous buffer and water. This was confirmed by shaking 8 in a mixture of water and n-butanol, whereupon after 2 h, 8 could no longer be detected in the n-butanol phase by TLC. Since the stability of silyl acetal linkage is dependent on the bulkiness of the alkyl groups attached to silicon (24, 25), we also investigated tBu(Ph)SiCl2 as a reagent for linking the nucleoside to biotin (24, 25). However, this strategy generated complex mixtures that may be attributed to the generation of a new chiral silicon center as well as less complete transformation because of the lower reactivity of tBu(Ph)SiCl2. Attempts to isolate the desired product in pure form failed. This led us to ultimately design compound 10. The silyl acetal linkage in this molecule is expected to be more stable than that in 8, because of the bulky tertiary carbon attached to one of the two oxygen atoms in the linkage. Moreover, in comparison to tBu(Ph)SiCl2, iPr2SiCl2 is more reactive, and does not generate a new chiral silicon center. As shown in Scheme 5, under reaction conditions similar to those used to prepare compound 8, 4 was linked to biotin derivative 9 (26) with iPr2SiCl2to give the target molecule 10 in 51% yield after flash chromatography purification. We found that only 1.2 equiv of compound 9 were required to achieve effective conversion of nucleoside 4 to compound
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Supporting Information Available: UV, IR, 1H-NMR, and C-NMR spectra for compounds 8 and 10 (Figures S1–S8). This material is available free of charge via the Internet at http:// pubs.acs.org.
13
LITERATURE CITED
Figure 1. Mean fluorescence intensity (FI) of microspheres as a function of applied UV254 dose. Reported FI values represent the mean of measurements collected for roughly 10 000 particles by flow cytometry.
10. The biotinylated nucleoside 10 was obtained as a clean white foam and its structure established by 1H, 13C NMR, UV, IR, and high-resolution mass spectroscopy. It is stable in air, water, and other commonly used organic solvents, and no decomposition was observed during chromatography on silica gel and storage in desiccator for several months. With the more stable 10 in hand, attachment to streptavidincoated microspheres was investigated. Using a procedure similar to that used for attaching 8 to the microspheres, 10 was found to be irreversibly attached. Even after washing extensively with PBS buffer and water, UV irradiation of the microspheres still promoted the cyclization reaction to yield fluorescent microspheres when excited at 320 nm. The relationship between the fluorescence intensity (FI) of a population of microspheres (∼10 000) was determined by flow cytometery (Figure 1). Flow cytometry was performed on an Epics Altra cell sorter (Beckman-Coulter) using a 351 nm line of the Argon 5 W Enterprise laser (Coherent, Inc.) with power set at 60 mW for the UV line. FI measurements were collected in linear mode with a bandpass filter at 380 nm. As illustrated in Figure 1, the FI of the population of microspheres increased monotonically with increasing UV dose. The cause of the lag in FI in the limit of low UV dose is unknown; however, this lag is overcome in application of microspheres for measurement of UV dose distribution delivery by preirradiation of microspheres at a dose of 50 mJ/cm2 or more (8). In conclusion, by using the unique diisopropylsilyacetal linkage and the strong noncovalent interaction between biotin and streptavidin, we successfully attached the highly functionalized cytidine analogue to microspheres. Because of their hydrophilic surface, the microspheres can form a stable suspension in water. The fluorescence intensity of the suspension of these microspheres responds monotonically to applied UV light dose, which demonstrates their suitability for use as a mobile UV light actinometer.
ACKNOWLEDGMENT This work was funded by the Water Environment Research Foundation (Grant 99-CTS-2-UR) the Showwalter Trust, and the National Institutes of Health (GM53155). Assistance from the Purdue University Cytometry Laboratories and from Eric Cox is greatly appreciated. Support from the National Cancer Institute Grant (P30 CA23168) awarded to Purdue University is also gratefully acknowledged
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