Bright and Monodispersed Phosphorescent Particles and Their

Xuedong Song,* Lei Huang, and Bin Wu. / Global Science & Technologies, Kimberly Clark Worldwide, 1400 Holcomb Bridge Road, Roswell, Georgia 30076...
0 downloads 0 Views 638KB Size
Download PDF
Anal. Chem. 2008, 80, 5501–5507

Bright and Monodispersed Phosphorescent Particles and Their Applications for Biological Assays Xuedong Song,* Lei Huang, and Bin Wu /

Global Science & Technologies, Kimberly Clark Worldwide, 1400 Holcomb Bridge Road, Roswell, Georgia 30076

Halogen-containing polymers and copolymers have been discovered to provide excellent encapsulation matrixes for making bright and monodispersed phosphorescent nanoparticles. The phosphorescent nanoparticles exhibit strong phosphorescence with long lifetime and large Stoke shift under ambient conditions. The cross-linked phosphorescent particles using halogen-containing copolymers have been found to be very stable and easily resuspendable in aqueous media. The surface functional groups have been demonstrated to allow covalent tagging of biological recognition molecules such as antibodies to make particleantibody conjugates. The conjugates can be used to provide very sensitive detection of analytes through timeresolved phosphorescence measurements. Time-resolved fluorescence detection technique has been proven to be capable of providing highly sensitive assays for chemical and biological samples because of its lower background and higher signal/noise ratio1,2 than conventional fluorescence technique. However, only a limited number of fluorescence probes have been discovered to possess fluorescence lifetime long enough to significantly differentiate from typical background fluorescence. Long fluorescence lifetime is also critical for constructing simple and low-cost instruments for time-resolved fluorescence measurement. Europium chelate-based probes are the best known ones and have been successfully commercialized for biological assays because of their high fluorescence quantum yield, large Stoke shift, and long lifetime.3–6 However, existing Eu chelates can be effectively excited only from ∼270 to ∼370nm. Powerful UV light sources at this wavelength range are not cheaply available. In addition, most of biological media have strong absorption at this region and significantly interfere with the measurement in many situations. Furthermore, most of the Eu chelates are often not very stable both chemically and photochemically. For example, many Eu chelates dissociate at a very low concentration and are subject to significant photobleaching * To whom correspondence should be addressed. E-mail: xuedong.song@ kcc.com. Tel: 770-587-8591. (1) Merio, L.; Pattersson, K.; Lovgren, T. Clin. Chem. 1996, 42, 1513–1517. (2) Harma, H.; Soukka, T.; Lovgren, T. Clin. Chem. 2001, 47, 561–568. (3) Qin, Q.; Peltola, O.; Pettersson, K. Clin. Chem. 2003, 49, 1105–1113. (4) Wang, G,.; Yuan, J.; Matsumoto, K.; Hu, Z. Anal. Biochem. 2001, 299, 169–172. (5) Hai, X.; Tan, M.; Wang, G.; Ye, Z.; Yuan, J.; Matsumoto, K. Anal. Sci. 2004, 2, 245. (6) Lopez-Crapez, E.; Bazin, H.; Andre, E.; Noletti, J.; Grenier, J.; Mathis, G. Nucleic Acids Res. 2001, 29, 70. 10.1021/ac800483n CCC: $40.75  2008 American Chemical Society Published on Web 05/30/2008

under UV light.20 Recently, some new types of europium chelates using caged ligands have been synthesized to increase their association constants with better fluorescence quantum yield.7 Nevertheless, they still have photobleaching problem and still require expensive UV excitation light. Eu chelates have also been encapsulated inside polymeric particles to improve chemical stability and provide signal amplification. Eu chelate-based polystyrene particles have been commercialized (e.g., by Seradyn Inc., Indianapolis, IN). Yet, they still have severe photobleaching problems and require expensive UV excitation light source. Phosphorescence is well-known21,22 and has been proposed for analyte detection.7 It has been widely explored for its potential applications in chemical and biological detection and analysis.8–12 However, unlike a time-resolved fluorescence technique, timeresolved phosphorescence has not been very successfully commercialized in the field of biological detection because of oxygen quenching and limited availability of phosphorescent probes with good spectral properties. In almost all the cases, an oxygen-free environment is needed for generating strong phosphorescence. One way to prevent oxygen quenching is to encapsulate the phosphorescent molecules in a low oxygen or oxygen-free matrix. For example, phosphorescent molecules have been encapsulated inside polyacrylonitrile (PAN) to provide strong phosphorescence under ambient conditions due to its low oxygen permeability.13,14 Their phosphorescence intensity and lifetime have been reported to be similar to the corresponding phosphorescent molecules in an oxygen-free environment.13,14 However, the reported PAN(7) (a) Raymond, K.; Petoud, S.; Cohen, S.; Xu. J. U.S. Patent 7,018,850B2, 2006. (b) Raymond, K.; Petoud, S.; Cohen, S.; Xu. J. U.S. Patent 6,864,103 B2, 2005. (8) (a) Papkovsky, D. B.; O’Riordan, T.; Soini, A. Biochem. Soc. Trans. 2000, 28, 74–77. (b) Hendrix, J. L. U.S. Patent 5,464,741, 1995. (c) Sagner, G.; De Hass, R.; Gijlswijk. R.; Tanke, H. U.S. Patent 6,004,530 (1999). (d) Sun, B.; Yi, G.; Zhao, S.; Chen, D.; Zhou, Y.; Cheng, J. Anal. Lett. 2001, 34, 1627–1637. (9) Scholl, P. F.; Bargeron, C. B.; Phillips, T. E.; Wong, T.; Abubaker, S.; Groopman, J. D.; Strickland, P. T.; Benson, R. C. in-Vitro Diagn. Instrum. Proc. SPIE 2000, 3913, 204–213. (10) Christopoulos, T. K.; Diamandis, E. P. Anal. Chem. 1992, 64, 342–346. (11) Phimphivong, S.; Saavedra, S. S. Bioconjugate Chem. 1998, 9, 350–357. (12) Matveeva, E. G.; Gribkova, E. V.; Sanborn, J. R.; Gee, S. J.; Hammock, B. D.; Savitsky, A. P. Anal. Lett. 2001, 34, 2311–2320. (13) (a) O’Riordan, T. C.; Soini, A. E.; Papkovsky, D. B. Anal. Biochem. 2001, 290, 366–375. (b) O’Riordan, T. C.; Soini, A. E.; Soini, J. T.; Papkovsky, D. B. Anal. Chem. 2002, 74, 5845. (c) Ponomarev, G. V.; Vladimirovich, D.; Meltola, J. J.; Soini, A. E. U.S. Patent 6,582,930 B1, 2003. (14) Kuerner, J. M.; Klimant, I.; Krause, C.; Preu, H.; Kunz, W.; Wolfbeis, O. S. Bioconjugate Chem. 2001, 12, 883–889.

Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

5501

based phosphorescent particles are very small and difficult to manipulate because of severe aggregation. They are not crosslinked and have potential stability issues. Less successful matrixes include polystyrene and Sephadex15 that results in particles with relatively weak phosphorescence of short lifetime. Nevertheless, they still have been found to be useful for immunoassays.15 The ideal fluorescent or phosphorescent particles for timeresolved biological/chemical assays are desired to be bright with long lifetime, to be monodispersed with suitable sizes, to be excitable by low-cost light sources, to have surface functional groups to allow ligand immobilization, to be stable (chemically, thermally, mechanically, and photochemically), and to be inexpensive to manufacture. None of the existing phosphorescent particles can meet all those criteria. In this article, we report a new class of phosphorescent nanoparticles with excellent physical and spectral properties for time-resolved biological detection techniques. The particles exhibit strong phosphorescence with long lifetime and large Stoke shift under ambient conditions. They can be effectively excited by commercially available low-cost light sources such as light-emitting diodes (LEDs). We have discovered an excellent class of encapsulation matrixes for making bright phosphorescent nanoparticles that can prevent oxygen quenching and photobleaching. The size of the particles can be easily tailored. The phosphorescent particles can also be cross-linked and surface-functionalizable with longterm thermo- and mechanical stability. The particles have been successfully used to covalently tag a C-reactive protein (CRP) monoclonal antibody and provide a very sensitive time-resolved phosphorescent assay for CRP on a lateral flow assay device. The particles should find a number of applications, ranging from biological and chemical assays, clinical diagnostics, and detection of warfare agents to food and environmental monitoring. EXPERIMENTS Preparation of Phosphorescent Nanoparticles. Typically, 30 µg of platinum(II) tetra-meso-fluorophenylporphine and palladium(II) tetra-meso-fluorophenylporphine (Pt-TMPFP and PdTMPFP, respectively, from Frontier Scientific Inc.) and 3 mg of poly(vinyl fluoride) (PVF; MW 150K, from Aldrich), poly(vinyl chloride) (PVC, MW-110K from Aldrich), or polyacrylonitrile (PAN; MW 120K, Polysciences, Inc.) were completely dissolved in 0.6 mL of DMF through gentle heating at 50 °C. After cooling to room temperature, 3 mL of water was added to the solution, with vigorous stirring, through either a dropping funnel or a syringe pump. The solution became turbid and particles were formed. After stirring for ∼10 min, the particles were heated at 50 °C for 30 min. The heated particles were then washed with water four times through centrifugation (15K rmp, 20 min) and suspended in water for further analysis. Preparation of Surface-Functionalized Phosphorescent Nanoparticles. To a vial containing 625 µL of poly(vinyl chlorideco-ethyl acetate-co-maleic acid) (VERR, 5 mg/mL, under the trade name UCAR VERR-40 from Dow Chemical) and Pt-TMPFP (4 mg/ mL) in DMF was added with 3 mL of water under vigorous stirring. The solution was then stirred for another 5 min. The particles were heated at 50 °C for 30 min, then washed with water four times, and suspended in water for further analysis. (15) Klimant, I. U.S. Patent 6770220, 2004.

5502

Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

Preparation of Cross-Linked, Surface Functionalized Phosphorescent Nanoparticles. An appropriate amount of Pt (or Pd)-TMPFP, VERR, and poly(vinyl chloride-co-vinyl acetateco-epoxy monomer) (VMCA, under the trade name of UCAR VMCA from Dow Chemical) were completely dissolved in a watermiscible organic solvent such as THF, acetone, DMF, or dimethyl sulfoxide (DMSO) through gentle heating at 50 °C to prepare a solution with an appropriate weight ratio of VERR/VMCA. With vigorous stirring, an appropriate amount of water was added through either a dropping funnel or a syringe pump. The particles were washed three times by water through centrifugation and were resuspended in water. The washed particles were then heated overnight at 85 °C overnight or at 150 °C for 20 min to cross-link the phosphorescent nanoparticles. Measurements of Spectral Properties. All the spectral property measurements were carried out under ambient conditions, unless specified, using a Fluorolog III fluorometer (JoBin YVon, Horiba Group) with time-resolved phosphorescence measurement capability (Fluorolog SPEX 1934D Phosphorimeter). Typically, the speciment was either dissolved or suspended in an appropriate solvent in a glass-walled cell for spectral measurement. An appropriate amount of sodium sulfite was added to the solution to remove oxygen. For Pt-TMPFP-based samples, 390-400 nm was typically used for excitation, and phosphorescence at 650 nm was typically collected. For Pd-TMPFP-based samples, 400-410 nm was typically used for excitation, and phosphorescence at 670 nm was typically collected. For all the time-resolved measurements, the following parameters were used: typical delay time for data collection 40 µs; typical time-per-flash 50 ms; typical number of flash per measurement 20; slit width 5 nm for both excitation and emission. Usually, only one scan was recorded. Phosphorescence decay is typically obtained using the same parameters with the exception of initial delay time of 10 µs. Particle Analysis. Dynamic light scattering (Brookhaven instrument’s ZetaPlus) were used to analyze and characterize the particles. For typical light scattering measurement, suspension of the particles in 0.1 M KCl aqueous solutions was used. Two major parameters, average size and polydispersity, are recorded. For SEM analysis, the particles were washed first and suspended in water by bath sonication before sample preparation. Typically, on freshly cleaved mica was added a drop of particle suspension and air-dried at room temperature. The sample was then sputter-coated with a thin film of chromium and loaded into the measurement chamber of Hitachi S-4500 field emission scanning electron microscopy for imaging collection. Covalent Conjugation of Antibody to Phosphorescent Particles. Six milliliters of VERR/Pt-TMF particles (VERR/PtTMF ) 120, 1 mg/1.2 mL, 133 nm in diameter) prepared with water/THF was washed once with water and then with phosphatebuffered saline (PBS, from Polysciences, Inc.) through centrifugation. The washed particles were suspended in 0.5 mL of PBS by bath sonication (10 min) followed by 10 s of probe sonication. To the particle suspension was added with 3 mg of carbodiimide (from Polysciences, Inc.) in 50 µL of 0.1 M PBS, and the mixture was shaken for 15 min. The particles were then washed twice with 100 mM borate buffer through centrifugation, and the washed particles were suspended in 400 µL of 100 mM borate buffer. To the particle suspension was added with 35 µL of CRP monoclonal

antibody (6.4 mg/mL, C-reactive protein monoclonal antibody from BioSpacific, Catalog No. A5811), and the mixture was shaken overnight. To the mixture was then added 200 µL of 0.1 M ethanoamine (Polysciences, Inc.) and 50 µL of bovine serum albumin (BSA, 1 mg/mL), and the mixture was shaken for 0.5 h. Then the particles were washed twice with 50 mM Hepes buffer (N-[2-hydoxyethyl]piperazine-N′-(2-ethanesulfonic acid) from Sigma). The washed particles were suspended in 1 mL of 50 mM Hepes containing 0.02 mg/mL BSA through 10-min bath sonication, followed by 10-s probe sonication. The suspended particle conjugate, designated as KCP-CRP Mab1, was stored at 4 °C. Lateral Flow Devices. A nitrocellulose membrane on a plastic supporting card (obtained from Millipore Co.) was striped with 1 mg/mL CRP monoclonal antibody (Catalog No. A5804, from BioSpacific Inc.) to form a detection line by a striping instrument from Kinametic Inc. The striped membrane was then dried at 37 °C for 1 h. A cellulose wicking pad (from Millipore Co.) was laminated with a ∼2-mm overlapping region to form a wicking zone. The portion of the supporting card for laminating a conjugate pad and a sample pad was cut off. The assembled card was then cut into 4-mm-wide half-dipsticks. See Pall Inc.’s Web site, www.pall.com, for general instructions to assemble lateral flow devices. Detection of C-Reactive Protein. To each of eight wells on a microtiter plate was added 20 µg of KCP-CRP Mab1 conjugate and a different amount of CRP (from Biodesign Inc.) in 50 µL of 100 mM Hepes buffer containing 0.5% Tween 20 from Sigma. The amount of CRP in each well ranges from 0, 10, 25, 50, 100, 250, 500, to 1000 pg, respectively. A half-dipstick was inserted into each well as prepared above. The dipsticks were allowed to develop for 20 min. The time-resolved phosphorescence on the detection line of each device was then measured. Front face mode was used for time-resolved phosphorescence measurement on lateral flow devices. For phosphorescence measurement of the detection line, a thin black paper cardboard with a central rectangular hole matching the size and shape of the detection line was used to block the rest portion of the device while exposing the detection line to the excitation beam. For background measurement, the hole of the card was moved to the background position close to the detection line and was aligned to be parallel to the detection line so that the excitation beam can be shined on the background position only. The lateral flow device was placed on a sample holder, which can be rotated to adjust the relative angles of the device. The device was aligned so that the detection line was positioned in the center of the excitation beam. For the measurement, the angle between the sample surface and excitation beam is 60 °C and the emission collection angle is 90 °C relative to the sample surface. The following parameters were used for the time-resolved phosphorescence measurement: phosphorescence at 650 nm when excited at 390 nm, delay time at 40 µs; time-per-flash at 50 ms, number of flash per measurement at 20, and slit width at 5 nm for both excitation and emission. RESULTS AND DISCUSSION Phosphorescent Molecules. Commonly encountered phosphorescent molecules are metal chelates, such as platinum, palladium, and ruthenium chelates, with various ligands such as

Figure 1. Structure of Pt-TMPFP.

Figure 2. Absorption and time-resolved phosphorescence spectra of Pt-TMPFP and Pd-TMPFP in oxygen-free benzene solutions.

porphyrins, porphine, and their derivatives.16–18 Pt-TMPFP and Pd-TMPFP, respectively, Figure 1, have excellent phosphorescence spectral properties under oxygen-free conditions. While the two chelates have very weak phosphorescence under ambient conditions, they exhibit very strong phosphorescence at 650 nm for Pt-TMPFP (excited at 390 nm) and 670 nm for Pd-TMPFP (excited at 410 nm) in the absence of oxygen as shown in Figure 2. Their phosphorescence is very weak in solid state due to aggregation. They can be effectively excited by low-cost LEDs, and their phosphorescence can be efficiently detected by lostcost photodetectors such as photodiodes. Those two phosphorescent molecules are soluble in many organic solvents such as toluene, benzene, methanol, DMF, and DMSO, but not soluble in water. The phosphorescence lifetimes are estimated to be about 100 and 500 µs for Pt-TMPFP and Pd-TMPFP, respectively. Their long phosphorescence lifetimes are ideal for time-resolved phosphorescence measurements, which can eliminate unwanted interfering (16) Hennink, E. J.; Haas, R.; Verwoerd, N. P.; Tanke, H. J. Cytometry 1996, 24, 312–320. (17) Roza-Fernandez, M.; Valencia-Gonzalez, M. J.; Diaz-Garcia, M. E. Anal. Chem. 1997, 69, 2406–2410. (18) Martsev, S. P.; Preygerzon, V. A.; Mel’nikova, Y. I.; Kravchuk, Z. I.; Ponomarev, G. V.; Lunez, V. E.; Savitsky, A. P. J. Immun. Methods 1995, 186, 293–304. (19) Soini, A. E.; Yashunsky, D. V.; Meltola, N. J.; Ponomarev, G. V. J. Porphyrins Phthalocyanines 2001, 5, 735–741. (20) The result was obtained from authors’ laboratory. Typically, the fluorescence intensity of Eu particles from Molecular Probes, Inc., drops more than 30% upon exposure to 365-nm UV light for 20 min. (21) Park, E. J.; Reid, K.; Tang, W.; Kennedy, R. T.; Kopelman, R. J. Mater. Chem. 2005, 15, 2913. (22) Osamu, O.; Kaizu, Y.; Kobayashi, H. J. Chem. Phys. 1985, 82, 1779.

Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

5503

background for high-sensitivity assays. Their long phosphorescence lifetimes make it feasible to develop low-cost, time-resolved phosphorescence readers. Desired Encapsulation Matrixes. An ideal encapsulation matrix to make phosphorescent particles for biodetection should have several key properties. First, the matrix should have low oxygen permeability and solubility to shield the complex from phosphorescence quenching. This property is critical for strong phosphorescence, photostability, and long phosphorescence lifetime. Second, the matrix should be compatible with the encapsulated phosphorescent molecules so that a high loading can be achieved. The good compatibility is also vital to prevent aggregation of the phosphorescent molecules because many phosphorescent molecules have high tendency to form aggregates of weak phosphorescence. Finally, the matrix should be able to form monodispersed particles with surface functional groups to allow covalent attachment of recognition molecules. A few types of polymeric matrixes have been reported for encapsulation of phosphorescent molecules to form phosphorescent particles, including polystyrene and PAN.15 However, those polymer systems have many drawbacks for making desirable phosphorescent particles. We have discovered through this study that halogencontaining polymers are excellent matrixes for encapsulating PtTMPFP and Pd-TMPFP to make bright phosphorescent nanoparticles of long lifetime, providing an excellent probe for timeresolved luminescent chemical and biological assays. Phosphorescent Particles Using Simple Halogen-Containing Polymers (HCPs). In order to demonstrate the superiority of HCPs as encapsulating matrixes for phosphorescent molecules, PAN is selected as a benchmark matrix for comparison since PAN has been reported to be a good matrix for encapsulating phosphorescent molecules. PVF and PVC are selected as examples of simple HCPs for encapsulating Pt-TMPFP and PdTMPFP. Those particles were prepared under the same conditions. However, the particle sizes are different. The average particles sizes were estimated by light scattering techniques to be 330, 260, and 170 nm in diameter, respectively, for PAN, PVF. and PVC particles. Very weak phosphorescence was observed when Pt-TMPFP was dispersed in water. In contrast, much stronger phosphorescence was obtained when it was encapsulated in PVF, PVC, and PAN particles. With the same encapsulation loading (1%) of the dye, PVF, PVC, and PAN particles show 21, 10, and 5 times stronger phosphorescence than the phosphorescence in water, respectively. The phosphorescence decay profiles for PAN, PVC, and PVF particles containing Pt-TMPFP and Pd-TMPFP have been found to be very similar to their corresponding individual molecules in oxygen-free organic solvents (e.g., benzene), suggesting that those particles have similar low oxygen permeability. The higher relative phosphorescence intensity of PVF and PVC particles than a PAN particle may be attributed to the better solubility of PVF and PVC for Pt-TMPFP and Pd-TMPFP, which can reduce the dye aggregation. Phosphorescence intensity continues to increase when the loading of Pt-TMPFT in PVF and PFC particles increases up to >3%. However, the phosphorescence intensity of a PAN particle starts to level off at ∼1% loading for the same phosphorescent molecule under the same conditions. Similar results have been 5504

Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

Figure 3. Molecular structures of VERR and VMCA.

observed for particles containing Pd-TMPFP. The plateau of the phosphorescence intensity is ascribed to dye aggregation. The highest phosphorescence intensity for PVF particles and PVC particles is 6 and 3 times higher, respectively, than that achieved by PAN particles. The results suggest better dye solubility in PVF and PVC matrixes than PAN particles. Although the particles formed by simple HCPs with phosphorescent molecules exhibit strong phosphorescence and long phosphorescence lifetime, the particles are found to be highly polydispersed. For example, the particles of PVF with Pt-TMPFP (PVF/Pt-TMPFP ) 60, prepared with THF and water) were analyzed by a dynamic light scattering technique to have an average size of 260 nm in diameter with a high polydispersity of 0.23. Particle distribution analysis suggests the existence of at least two populations, probably due to the aggregation caused by hydrophobic surfaces. The particles formed by simple HCPs can not be readily suspended in water and also have no surface functional groups. Nevertheless, the results have laid a foundation to seek better encapsulation matrixes. Phosphorescent Particles Formed from Halogen-Containing Copolymers. From the positive results obtained from simple HCPs, more complex HCPs were tested to develop surface functionalized and monodispersed phosphorescent particles. In order for the particles to be surface-functionalized, halogencontaining copolymers with a portion of functional monomers such as carboxylic acid should be good candidates. For this reason, VERR (Figure 3) was selected for the study. VERR has 86% vinyl chloride, 13% vinyl acetate, and 1% maleic acid. The major content of vinyl chloride should provide low oxygen permeability and good solubility for phosphorescent dyes. The portion of vinyl acetate (after hydrolysis) and maleic acid can provide surface functional groups. To make the particles, VERR and Pt-TMPFP were typically dissolved in water-miscible organic solvents such as DMF, and water was then added to coprecipitate the polymer and phosphorescent molecules. Because water was used as a precipitating solvent, the hydrophilic groups such maleic acid should be preferred to sit at the water/particle interface to provide surface functional groups. The carboxylic acid groups on particle surface are indirectly supported by the fact that those particles are remarkably easy to be resuspended into water after centrifugation. In contrast, the particles formed from PVF and PVC are very difficult to be resuspended once aggregated. The particles exhibit strong phosphorescence and long lifetime, almost identical to the individual molecules under oxygen-free solutions. The phosphorescence intensity of the particles shows almost linear increase as a function of dye loading up to 3.33% for Pt-TMPFP in PVCEM when they are prepared in a number of solvents including THF, DMF, and acetone. In comparison, the phosphorescence intensity of PAN particle starts to level off with only 1% Pt-TMPFP loading. The phosphorescence intensity of VERR particles with 0.83% Pt-

Figure 4. SEM micrograph of VERR particles prepared in THF with 1% Pt-TMPFP. Table 1. Particle Analysis Results for VERR/Pt-TMPFP Particles VERR/PtTMPFP

DMF (nm)

after bathsonication (nm)

60 120 200

67.8

79.7

70.7

THF 140 163 163

after bath sonication (nm)

after probe sonication (nm)

158

163

TMPFP is almost eight times stronger than the PAN particle with the same dye loading when they are prepared under the same conditions. Apparently, VERR is a better matrix than PAN for making bright phosphorescent nanoparticles of long lifetime. Similar results were obtained for Pd-TMPFP in VERR particles. The VERR phosphorescent particles were analyzed by a dynamic light scattering technique and scanning electron microscopy. Results from both techniques indicate that the VERR phosphorescent particles are monodispersed as shown in Figure 4. Typically, the particle sizes range from 40 to 300 nm in diameter, depending upon the preparation conditions, such as solvents and precipitation speed. The polydispersity of the particles by light scattering technique is typically in the range of 0.005-0.050. The particle sizes can be readily tailored by various parameters. One of the parameters used to control the particle sizes is solvent. By using different solvents, particles of different sizes can be obtained. For example, small particles are obtained when DMF or DMSO is used as a solvent, while larger particles were obtained with THF as shown by the data in Table 1. Another parameter useful for controlling size is the precipitation speed of the polymer/dye solution with water. Faster precipitation normally results in smaller particles. Size analysis of the particles indicates that particles from ∼40 to ∼300 nm in diameter can be easily obtained. The VERR phosphorescent particles are stable under normal aqueous environment and can tolerate multiple cycles of washings and resuspensions, as well as bath sonication and probe sonication as shown in Table 1. Heating of the particles at 50 °C for 0.5 h does not affect the particle sizes, particle size distribution, and phosphorescent properties. The phosphorescent particles are also found to be extremely stable photochemically. Irradiation by 390-

nm light for 1 h causes very little phosphorescence degradation, likely due to low oxygen exposure. Cross-Linked and Surface Functionalized Phosphorescent Particles. Although the phosphorescent particles made of VERR have strong phosphorescence of a long lifetime and have carboxylic acid surface functional groups as well as excellent photostability23 and good stability in aqueous suspensions, they can not tolerate solutions containing a high percentage of organic solvents and surfactants. As expected, the particles were found to fall apart with treatment by organic solvents such as THF and DMF and aqueous solutions containing a relatively high concentration of surfactants such as Tween-20. In order to make phosphorescent particles more stable, a halogen-containing crosslinking polymer, such as VMCA (Figure 3), was added. The epoxy groups of VMCA can react with the carboxylic acid groups of VERR to cross-link the polymer matrix. The phosphorescent particles made from VERR/VMCA have very similar spectral properties and physical properties to their non-cross-linked counterparts, with much better stability against organic solvents and surfactants. The VERR/VCMA phosphorescent particles had much higher phosphorescence intensity than PAN phosphorescent particles at the same level of dye loading ranging from 0.1 to 3.0% with almost identical phosphorescence decay profile. For instance, the phosphorescence intensity of the VERR/VCMA particle containing 2% Pt-TMPFP is five times stronger than that of the same amount of PAN particle with the same Pt-TMPFP loading. In order to confirm that the cross-linking reaction has indeed occurred after heating, FT-IR spectra were collected from the two polymers and cross-linked particles. Two peaks at 841 and 3052 cm-1 for the epoxy group of VMCA were found to disappear after cross-linking and two new peaks at 3501 (attributed to -OH) and 1772 cm-1 (attributed to lactone) appeared. FT-IR results strongly suggest that the epoxy group from VMCA had reacted with a COOH group from VERR during the cross-linking process. The cross-linked phosphorescent particles are very stable even in a solution with a high percentage of organic solvent. Dynamic light scattering results show that the size and polydispersity of the cross-linked particles increase only slightly after the particles are treated with an aqueous solution with a high percentage of organic solvents. In contrast, the non-cross-linked counterpart particles show a much larger increase in particles and polydispersity. For example, the cross-linked particles of VERR/VMCA ) 80/20 containing 2% Pt-TMPFP was found to change from 132 nm with 0.05 PD to 139 nm with 0.088 after the particles are suspended in a 1:1 THF/water solution. Its non-cross-linked particles change from 134 nm with 0.05 PD to 174 nm with PD 0.233. Apparently, the non-cross-linked particles remain largely monodispersed while the non-cross-linked counterparts have degraded into polydispersed particles after exposure to THF/water mixing solvents. The similar results were observed for other mixing solvents such as DMSO/water. The high stability of cross-linked phosphorescent particles has also been confirmed by SEM studies as shown in Figure 5. The (23) Under the same irradiation, the emission intensity of platinum-based particles (F-20887, Molecular Probes) and platinum-based particles (PDPt, Chromeon, Germany) actually increased 5 and 20%, respectively. A total 23% of the emission at 615 nm for F-30881 particles from Molecular Probes was lost when irradiated at 370 nm.

Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

5505

Table 2. Particle Sizes of Cross-Linked VMCA/VERR Particles with 2% Pt-TMPFP Using Different Organic Solvents for Precipitation VMCA/VERR (nm) THF DMF acetone

Figure 5. (a) SEM images of cross-linked particles after THF/water (1/1) treatment. (b) SEM images of non-cross-linked particles after THF/water (1/1) treatment.

cross-linked particles (Figure 5a) show very uniform and stabilized round particle morphology after treatment of THF/water solution, while the non-cross-linked particles (Figure 5b) aggregated and fused together, resulting in large and nonuniformed aggregates. The cross-linked phosphorescent particles are also very stable in a solution containing surfactants. For example, the cross-linked particles of VERR/VMCA ) 80/20 containing 2% Pt-TMPFP were minimally affected with size change from132 to 123 nm after the particles were bath sonicated for 10 min in a 2% Tween-20 aqueous solution. Its non-cross-linked counterparts were found to experience a large size decrease from 134 to 40 nm. Apparently, the non-cross-linked particles remain largely intact while the noncross-linked counterparts have degraded into much smaller particles. As observed for all the phosphorescent particles prepared in this study, the cross-linked phosphorescent particles also show remarkable photostability. Continuously irradiation with 390-nm light (Fluorolog III fluorometer, slit width 10 nm) results in less than 2% phosphorescence decrease at 650 nm for 30 min. In comparison, the fluorescence intensity of europium-based polystyrene particles decreases ∼50% after 30-min irradiation at 370 nm. 5506

Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

20/80

50/50

80/20

133 78 105

97 73 72

73 89 92

Similar to the VERR/Pt (or Pd)-TMPFPT system, the particle sizes of the VERR/VMCA/Pt (or Pd)-TMPFP system can also be easily tailored by the same parameters discussed above. For instance, the particles of different sizes can be obtained through selection of solvents as shown in Table 2 when the precipitation conditions are identical. Those particles are also easily resuspended into water after aggregated through vortexing or sonication. Detection of C-Reactive Protein. In order to demonstrate the application potential of the particles for time-resolved phosphorescent assays, an antibody for C-reaction protein, an inflammatory biomarker, was covalently tagged with the particles using well-established conjugation chemistry to provide an antibodyparticle conjugate (designated as Mab 1-KCP). Another monoclonal antibody (designated as Mab 2) for a different epitope of CRP was immobilized on a nitrocellulose membrane of a lateral flow device to form a detection zone. When CRP in a liquid sample contacts with Mab1-KCP, it binds to form a complex CRP-Mab1KCP. When the complex flows through the membrane to contact with the immobilized Mab2, the complex will be captured by the Mab2 in the detection zone. The remaining sample will continue to flow to a wicking zone. The amount of the particles captured on the detection zone is proportional to the amount of CRP in the sample. No particle should be captured when no CRP is present in the sample. The phosphorescence intensity is proportional to the amount of the captured particles, therefore, the amount of CRP. Figure 6 shows the dose response for a series of samples containing different amount of CRP. The time-resolved phosphorescence immunoassay using the phosphorescent particles can detect less than10 pg CRP. CONCLUSIONS Halogen-containing polymers and copolymers have been found to be excellent encapsulating matrixes for phosphorescent mol-

Figure 6. Relative phosphorescence intensities as a function of the amount of CRP in the sample. The results are the average of three duplicates.

ecules to provide phosphorescent nanoparticles. The particle phosphorescence is substantially inert to phosphorescence quencher such as oxygen at ambient conditions. The particles can have a high loading of the encapsulated phosphorescent molecules to provide strong phosphorescence of long lifetime and high photostability. Methods and processes to produce monodispersed and cross-linked phosphorescent particles with surface functional groups have also been developed. The sizes of the particles can be readily tailorable by various conditions. Those phosphorescent nanoparticles have been demonstrated to be very useful for immunoassays to provide high detection sensitivity. They should

find a wide variety of applications for detection of chemical and biological molecules and species. ACKNOWLEDGMENT The authors thank Dr. Stephan Quirk and Michael O’Shea for suggestions and proof-reading the manuscript. Help from Joel Brostin in collecting SEM images and discussions with Rosann Kaylor are greatly appreciated. Received for review March 7, 2008. Accepted April 23, 2008. AC800483N

Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

5507