Fluorescent Nanoparticles for Multiplexed Bacteria Monitoring

Bioconjugate Chem. 2007, 18, 297−301

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Fluorescent Nanoparticles for Multiplexed Bacteria Monitoring Lin Wang, Wenjun Zhao, Meghan B. O’Donoghue, and Weihong Tan* Center for Research at the Bio/Nano Interface, Department of Chemistry, Shands Cancer Center and UF Genetics Institute, University of Florida, Gainesville, Florida 32611-7200. Received August 16, 2006; Revised Manuscript Received December 14, 2006

Rapid, sensitive, and selective detection of pathogenic bacteria is extremely important for proper containment, diagnosis, and treatment of diseases like foodborne illness, sepsis, and bioterrorism. Most current bacterial detection methods are time-consuming and laborious and can detect only one bacterial pathogen at a time. We have developed a method for sensitive, multiplexed monitoring of bacterial pathogens within 30 min using multicolored FRET (fluorescence resonance energy transfer) silica NPs (nanoparticles). By varying the ratio of three tandem dyes coencapsulated into the NPs, we have synthesized NPs that emit unique colors upon excitation with a single wavelength. When these NPs were conjugated to monoclonal antibodies specific for the pathogenic bacteria species Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and then incubated with small concentrations of the bacteria, simultaneous and sensitive detection of the multiple bacterial targets was achieved.

INTRODUCTION Under the appropriate conditions, bacterial pathogens can survive and spread easily in the environment. As a result, trace concentrations (∼10-100 cells) of bacterial pathogens in food, donated blood, or the environment can pose a serious threat to human health. An estimated 76 million illnesses, over 300000 hospitalizations, and over 5000 deaths are attributable to foodborne illness in the United States every year (1). In all of these pathogenic situations, there are several likely bacterial suspects of infection. For example, E. coli O157:H7, Salmonella spp., and Staphyllococcus spp. are the most common causes of foodborne illness and are responsible for 20% of deaths related to sepsis from blood transfusions (2, 3). Consequently, there has been a critical demand for techniques that can recognize numerous bacterial pathogens simultaneously in a single assay (multiplexing), thereby giving an early warning of bacteria contamination. However, the currently accepted bacteria detection methods require PCR amplification and/or cell culturing (4-6) which are slow, time-consuming, laborious, and have a limited ability to multiplex (7-13). Many attempts have been made to improve the sensitivity of bacterial detection of numerous strains simultaneously without the need for bacterial culturing or PCR (14-18) but have met with limited success. To address this issue, we have developed triple-dye-doped FRET silica nanoparticles (NPs) (19, 20) to serve as promising substrates for multiplexed bacteria monitoring. Previously, our group has used single-dye-doped NPs for in situ pathogen quantification down to a single bacterium (21). These dye-doped NPs, each encapsulating ∼10 000 dye molecules in a ∼60 nm silica sphere, provide an extremely strong fluorescent signal for bioanalysis and are easily conjugated to biorecognition molecules, such as antibodies, for fluorescence-based immunoassays. Now, by varying the ratio of three fluorescence energy transfer tandem dyes coencapsulated into the silica NPs, excitation with a single wavelength leads to emission of numerous different colors (20), thereby permitting simultaneous and sensitive detection of multiple targets. The work described here assesses the use of FRET NPs for multiplexed bacterial * Fax and Tel 352-846-2410; E-mail [email protected].

detection. Escherichia coli (E. coli), Salmonella typhimurium (ST), and Staphylococcus aureus (SA) were used as model pathogens in our system because they are major human pathogens responsible for foodborne illness and sepsis (22). Thus, if routine testing of food and blood products could rapidly detect these three pathogens, both the safety and the shelf life of these products could be increased (2, 3, 23). Our results show that FRET NPs can be used as highly sensitive and selective platforms for sensing multiple microorganisms simultaneously.

EXPERIMENTAL PROCEDURES Reagents and Apparatus. Biotinylated rabbit anti-E. coli IgG, biotinylated rabbit anti-ST IgG, and goat anti-rabbit IgG were ordered from Biodesign International (Saco, ME). Biotinylated rabbit anti-SA IgG was bought from Cortex Biochem (San Leandro, CA). NanoOrange, DAPI (4′-6-diamidino-2phenylindole), and amine reactive dyes FAM-SE (5-carboxyfluorescein succinimidyl ester), R6G-SE (5-carboxyrhodamine 6G, succinimidyl ester), ROX-SE (6-carboxy-X-rhodamine, succinimidyl ester), and TMR-SE (carboxytetramethylrhodamine, succinimidyl ester) were ordered from Invitrogen Corporation (Carlsbad, CA). NHS-PEG5000-biotin was purchased from Nektar Therapeutics (San Carlos, CA). TEOS (tetraethyl orthosilicate) and APTS [(3-aminopropyl)triethoxysilane)] were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Sulfo-NHS (N-hydroxysulfosuccinimide sodium salt) and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) were ordered from Pierce Chemicals (Rockford, IL). Tween20 and MES (2-(N-morpholino)ethanesulfonic acid) were obtained from Sigma Chemical Co. (St. Louis, MO). Carboxylated silica microspheres (5.06 µm in diameter) were ordered from Bangs Laboratories (St. Louis, MO). All other chemicals were of analytical reagent grade. Distilled deionized water (Easy Pure LF) was used for the preparation of all aqueous solutions. A Hitachi S-4000 scanning electron microscope was used for imaging bacteria-NP conjugates. Optical and fluorescence images were obtained on a fluorescence microscope (Zeiss, U.S.A.) and a laser scanning confocal microscope (Olympus, Japan). NP Preparation. TMR-doped and triple-dye-doped silica NPs (diameter Φ ) 70 nm) were prepared through a modified Sto¨ber

10.1021/bc060255n CCC: $37.00 © 2007 American Chemical Society Published on Web 03/07/2007

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synthesis method (20, 24, 25). First, amine-reactive dye molecules (TMR-SE, FAM-SE, R6G-SE, ROX-SE) were individually dissolved in 1.5 mL of anhydrous DMF (dimethylformamide), combined with an excess of APTS (molar ratio of dye to APTS ) 1:2), and stirred under a dry nitrogen atmosphere for 24 h in the dark. Second, the TMR-APTS conjugate or the three tandem dye-APTS conjugates (FAM-APTS, R6GAPTS, ROX-APTS) mixed at desired ratios (1:0.5:1, 0.5:1:4, 0.5:0.5:3) were added to a clean glass reaction vessel containing 16.75 mL of ethanol and 1.275 mL of ammonium hydroxide, respectively. The mixture was stirred for 24 h. 0.71 mL of TEOS (0.00375 mol) was added afterward and stirred for another 24 h. After the reaction, the samples were centrifuged at 14 000 rpm for 30 min to collect the silica NPs. The NPs were further washed with ethanol and deionized water by centrifugation and decanted several times to remove unreacted chemicals. NP Surface Modification. Amine Group Modification. After washing the NPs, approximately 10 mg of NPs were dispersed in 1 mL of deionized water with sonication and mixed with 20 µL of glacial acetic acid. 20 µL of (3-trimethoxysilylpropyl)diethylenetriamine was added with sonication, and the suspension was stirred for 3-4 h. The particles were washed and dispersed in 10 mM MES buffer (pH ) 5.5). Carboxyl Group Modification. Carboxyl group modification was accomplished by mixing 10 mg of NPs in 1 mL of phosphate buffer (10 mM, pH 7.4) and 20 µL of carboxyethylsilanetriol sodium salt, 25% in water (Gelest, Inc., Kent, ME). The mixture was stirred for 3-4 h, washed with phosphate buffer, and dispersed in the same buffer. NP-Antibody Bioconjugation. Strategy A: Carbodiimide chemistry was used to cross-link free carboxylic acid groups on NPs with amine-containing antibodies. Briefly, 100 µL of a 0.22% (w/v) suspension of COOH-modified NPs was washed by centrifuging once with deionized water. The pellet was then resuspended in 1 mL of 0.1 M MES (pH 5.5). Aqueous solutions of 10 mM sulfo-NHS and 4 mM EDC dissolved in MES buffer were freshly prepared, and 0.5 mL of each solution was immediately added to the NP solution. The NPs were incubated at room temperature with gentle agitation. After 15 min, the NPs were centrifuged and washed with 10 mM phosphate buffer (pH 7.4), then resuspended in 1.5 mL phosphate buffer, followed by addition of 50 µL of antibodies (rabbit anti-ST-IgG) at a concentration of 1 mg/mL. The mixture was incubated at room temperature for 2 h with gentle end-to-end shaking. NPs were washed in 10 mM phosphate buffer and then resuspended in the quenching solution (40 mM Tris-HCl with 0.05% (w/v) BSA) for 60 min to block free carboxylates. Protein-coated NPs were purified by alternately centrifuging and resuspending in phosphate buffer (10 mM, pH 7.4) with 1% BSA and stored at 4 °C until use. Covalent coupling of secondary antibodies (goat anti-rabbit-IgG) to carboxylated silica microspheres followed the same procedures. Strategy B. Streptavidin conjugation to NPs used the above procedure except that the antibody was changed to streptavidin. After the centrifugation and washing steps, biotin-labeled antibody was added to the solution with a final concentration ratio of biotin-Ab:NP ) 1000:1. The solution was allowed to gently mix for 1 h, washed, and centrifuged to remove free biotin-antibody. Strategy C. Amine-modified NPs were reacted with the activated ester of biotin (NHS-PEG5000-biotin) in the phosphate buffer for 2 h to couple biotin molecules to the NPs. The complexes were centrifuged and washed several times with phosphate buffer to remove the unbound biotin. Streptavidin and NP-biotin were then mixed at a 1000:1 ratio to ensure sufficient saturation of streptavidin on the NP surface, and the mixture was slowly shaken for 2 h. The complexes were

Figure 1. Schematic representation of three antibody-labeling strategies on NPs.

centrifuged and washed several times with phosphate buffer to remove unbound streptavidin. Biotin-labeled antibody was further mixed into the above solution with a final concentration ratio of biotin-Ab to NP of 1000:1. The solution was allowed to gently mix for 1 h, washed, and centrifuged to remove free biotin-antibody. NP-Antibody Conjugation with Bacteria. Antibodylabeled NPs and bacteria were mixed at an experimentally optimized ratio (10 000:1), diluted in the incubation buffer (10 mM phosphate buffer, pH 7.4, 0.05% Tween-20), and gently shaken at room temperature for 30 min. The resultant product was washed three times by centrifugation (3000 rpm, 5 min) to remove all unbound antibody-conjugated NPs.

RESULTS AND DISCUSSION Antibody Labeling Strategy Optimization. Before applying FRET NPs for multiplexed detection of pathogenic bacteria, single dye (TMR)-doped NPs were used in the initial assay to test bacteria binding efficiency and specificity. TMR-doped NPs were prepared using the Sto¨ber method (24, 25). The aminereactive dye TMR-SE was first chemically bound to an aminecontaining silane agent APTS, and then APTS and TEOS were allowed to hydrolyze and co-condense in a mixture of water, ammonia, and ethanol, resulting in TMR-doped silica NPs. The NPs were then modified with surface functional groups and covalently conjugated to antibodies specific to three bacteria individually. Considering the effects of strong steric hindrance between the rigid NP and the bacterial surface, different antibody labeling strategies were designed (Figure 1). In strategy A, we directly immobilized monoclonal antibody onto COOH-NP surface using carbodiimide chemistry (19, 26, 27) to cross-link free carboxylic acid groups on NPs with amine-containing antibodies; in strategy B, we first linked COOH-NP surface with streptavidin as a bridge molecule (28), then immobilized biotinantibody onto streptavidin; in strategy C, we immobilized NHSPEG-biotin molecules onto NH2-modified NPs (20); after incubation with streptavidin, biotin-labeled antibody was anchored onto NP surface. For strategy A, we assumed that, due to the random attachment of antibody onto the NPs, some of the antigen binding sites of the antibody may be blocked. In strategy B, the bridge molecule streptavidin was bound to the biotin attached to the antibody ensuring that the antibody recognition sites were oriented away from the NP surface, so that they do not lose their ability to bind to a target bacterium. The third strategy added hydrophilic PEG linkers between NP and antibody to further improve the NP solubility and dispersivity, and allow the conjugated biotin-antibody to extend out from the NP surface, reducing the binding steric hindrance and benefiting the binding efficiency. To verify the functionality of conjugating antibody onto NPs and mimic the bacteria-NP interaction, goat-anti-rabbit IgG-coated microspheres and rabbit

Communications

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Figure 3. Fluorescence images of all three bacteria species incubated with NPs labeled with antibody to ST. (A) All three bacteria species were stained with DAPI. (B) Target bacteria ST were prestained with NanoOrange. (C) Only target bacteria ST fluorescedsthe color of the NPs. (D) Combination of the three channels.

Figure 2. (A-D) ST bacteria conjugated with antibody-labeled TMR NPs and stained with DAPI and NanoOrange. (A) Blue channel, DAPI; (B) green channel, NanoOrange; (C) red channel, TMR; (D) combinatorial color of the three detection channels. (E) SEM image of an ST bacterium coated with PEG-ST-biotin-antibody labeled NPs.

anti-ST-IgG conjugated NPs prepared with the three strategies were mixed and allowed to react for 30 min. As expected, we found that NPs prepared with the third strategy exhibited the best colloidal stability and binding performance. NP-Antibody Binding with Bacteria. ST was used for the initial bacteria monitoring. Anti-ST-IgG conjugated TMR-NPs were allowed to bind to ST bacteria. The resultant product was washed three times by centrifugation to remove all unbound antibody-conjugated NPs. To clarify the bacterial configuration and differentiate bacteria from NP aggregates, the ST bacteria were stained with DAPI (4′-6-diamidino-2-phenylindole, which stains double-stranded DNA) and NanoOrange (protein stain). Fluorescence images of the NP-coated ST were taken by exciting them with a 100 W mercury lamp, while three orthogonal detection channels monitored fluorescence for DAPI, NanoOrange, and TMR, respectively. Both the fluorescence and SEM images (Figure 2) display the successful binding between NPs and ST bacteria. NP-Bacteria Binding Specificity. One critical issue when detecting large numbers of targets simultaneously is specificity of binding. Cross-reactivity in highly multiplexed assays will lead to potentially disastrous false positives. The next experiment was designed to specifically detect the target bacteria ST in the presence of other pathogenic bacteria. Before incubation with NP-antibody conjugates, only target bacteria ST were stained

with NanoOrange. Free NanoOrange unbound to ST was removed by centrifugation, mixed with the other two bacteria species (E. coli and SA), and incubated with rabbit anti-STIgG-NPs. After 30 min, the sample was washed three times by centrifugation to remove free antibody-conjugated NPs that did not bind to the bacteria, and the sample was stained together with DAPI before imaging. The fluorescence image demonstrates that only those bacteria stained with NanoOrange fluoresced red due to the bound NPs, verifying that the antibodyconjugated NPs specifically associated with ST cell surfaces (Figure 3). Simultaneous Monitoring of Multiple Bacteria. Simultaneous multiple bacteria monitoring was achieved using colorencoded FRET NPs. To prepare the FRET NPs, the three aminereactive energy-transfer tandem dyes (FAM-SE, R6G-SE, ROXSE) were first covalently linked to the silane coupling agent APTS. The three APTS-dye conjugates were then mixed at desired ratios (1:0.5:1, 0.5:1:4, 0.5:0.5:3), and each mixture was added to a clean glass reaction vessel containing pure ethanol and ammonium hydroxide. After stirring for 24 h, TEOS was added, and the mixture was stirred for 24 h. When the reaction was complete, the solution was centrifuged to collect the silica NPs. The NPs were further washed with ethanol and deionized water to remove the unreacted chemicals. During the NP preparation process, the three APTS-dye conjugates were first allowed to prehydrolyze for 24 h before adding TEOS to start the colloid formation. During the prehydrolysis step, the three tandem dyes were covalently attached to one another and shortened the distance between them to the benefit of the FRET efficiency (20). In the FRET NPs, FAM was used as a common donor for R6G and ROX, while R6G acted as both an acceptor for FAM and a donor for ROX. By exciting at the maximum excitation wavelength for FAM (488 nm), efficient energy transfer between the three dyes in the same NP occurred, and NPs exhibited three emission peaks corresponding to the three encapsulated dyes, while the combinatorial color varies due to the different dye doping ratio. Figure 4A-C displays the fluorescence images of the three types of NPs taken under a confocal microscope. Circles a, b, and c display the fluorescence images of NPs close to the three emission peaks, while circle d exhibits the resultant combina-

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Figure 4. (A-C) Fluorescence images of three types of FRET NPs taken under a confocal microscope. (a) FAM emission channel; (b) R6G emission channel; (c) ROX emission channel; (d) combinatorial color of the three channels. The three types of NPs exhibit blue, orange, and purple colors. (D) Confocal image of three bacteria species specifically covered with the corresponding antibody labeled NPs.

torial color of the three detection channels. When excited at 488 nm, the three types of NPs fluoresce blue, orange, and purple. These three types of FRET NPs were further conjugated with the PEG-streptavidin-biotin-IgG complex. Blue NPs were conjugated with antibody to E. coli; orange NPs were coated with antibody to SA; while the purple NPs were linked with antibody to ST. These three antibody-labeled NPs and three bacteria species were mixed together in the buffer (10 mM phosphate buffer, pH 7.4, 0.05% Tween-20) and incubated for 30 min. After the centrifugation and washing steps, the confocal image of the resultant bacteria-NP complexes shows that three bacteria species were specifically covered with their corresponding antibody-labeled NPs and exhibit the coding colors of the attached NPs (Figure 4D). Furthermore, using the recently established two NP assay (29), it is feasible to use both antibodyconjugated magnetic NPs and fluorescent NPs to achieve multiple bacteria extraction and detection. In summary, we have been the first to apply multicolor FRET NPs for multiplexed monitoring of bacteria species. This method is fast and accurate with high sensitivity and specificity. By changing the dye doping ratios of the energy-transfer dye series, a large FRET NP bar-coding library can be built permitting multiplexed detection of numerous bacterial pathogens. By eliminating the sample enrichment and amplification steps required with current cell culture and PCR-based methods, our method can achieve the diagnosis of disease at the earliest stage of its development. Routine bacteria testing of food or blood using this method could allow for increased safety and shelf life (especially important for blood platelets whose current shelf life is 3-5 days). FRET silica NPs hold great promise to develop multiplexed bacterial detection kits.

ACKNOWLEDGMENT This work was supported by NSF NIRT and NIH grants. The bacteria were kindly provided by Dr. Samuel Farrah of the Microbiology and Cell Science Department at University of Florida. L.W. receives support as an ACS Division of Analytical Chemistry Graduate Student Fellow sponsored by GlaxoSmithKline.

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