Synthesis of a Red Fluorescent Dye-Conjugated Ag@SiO2 Nanocomposite for Cell Immunofluorescence Meicong Dong, Yu Tian, Dimitri Pappas* Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409 USA
In this work we describe a one-step approach for incorporating a red fluorophore (2SBPO) into core-shell nanoparticles for metalenhanced fluorescence immunolabels. The 2SBPO-MEF nanoparticles are particularly attractive as cell labels because their 670 nm emission has minimal overlap with cell autofluorescence and from overlap with many conventional probes. 2SBPO was incorporated through physical entrapment during the Sto¨ber process. Antibody-based cell labels were then synthesized using covalent linkage. The nanoparticle fluorescence was 7.5-fold higher than control nanoparticles lacking a metal core. We demonstrated labeling of CD4 þ HuT 78 T lymphocytes using anti-CD4– conjugated nanoparticle labels. Cells labeled with anti-CD4 nanoparticles showed a 35-fold fluorescence signal compared to antiCD4 coreless controls. This simple synthesis protocol can be applied to a variety of hydrophilic fluorophore types and has broad potential in bioanalytical and biosensing applications. Index Headings: Metal-enhanced fluorescence; Core-shell Ag@SiO2 nanoparticle; 2SBPO; Single molecule spectroscopy; Cell labeling.
INTRODUCTION Immunofluorescence approaches are commonly used to render an optically and electrochemically inactive species available for detection.1 Immunolabels are the primary reagent for many analyses such as flow cytometry, fluorescence microscopy, and confocal microscopy.2 Organic fluorophores are typically conjugated to antibodies or other ligands to produce immunolabels. The main problems of using organic fluorophores directly as fluorescent antibody labels include photobleaching, low signal intensities, and limited sensitivity.3 To overcome these issues, several types of nanomaterials, such as fluorescent dye-doped nanoparticles (NPs), quantum dots, and metallic NPs, have been explored as labels for bioanalysis. These probes present advantages such as uniform size, photostability, and facile functionalization. Among them, nanocomposites using metallic cores are an effective structure to increase the intensity of fluorophores and improved sensitivity.4 Certain metallic cores, such as silver, produce plasmon enhancement of the fluorescence signal, described as metal enhanced fluorescence (MEF) by Geddes.5 MEF leads to shorter lifetimes and increased molecular brightness, thus enhancing the fluorescence signal from individual dye molecules. MEF nanocomposites have been applied to many Received 5 June 2014; accepted 6 August 2014. * Author to whom correspondence should be sent. E-mail: d.pappas@ ttu.edu. DOI: 10.1366/14-07615
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systems, including single-cell imaging, protein detection, flow measurements, and other applications.6–16 In MEF, the fluorophore-metal distance is critical; at the same time many metal particles are not amenable to direct conjugation of dyes. Confining the fluorophore to a silica shell maintains a uniform distance from the metal core and provides a means of confining dye near the metal surface. Distances smaller than the optimum result in quenching, while distances longer than the optimum result in diminished plasmon enhancement.17,18 In our previous work, rhodamine B Ag@SiO2 nanoparticles, silver metal cores with silica sol-gel shells, were synthesized to produce green-fluorescent nanoparticle labels.19 Rhodamine B was incorporated into this nanocarrier through physical adsorption to form a bright fluorescent nanocomposite that is used in cell labeling and other applications. However, the green fluorescence of rhodamine B overlaps with cell autofluorescence, which may result in high background in cell labeling and imaging applications. To eliminate spectral overlap issues, we have developed a red-fluorescent nanoparticle label for cell immunofluorescence. We incorporated 9-di-3-sulfonyl-propylaminobenzo[a]phenoxazonium perchlorate (2SBPO) into the silica layer of Ag@SiO2 nanoparticles. 2SBPO is a red fluorescent, water-soluble dye suitable for biological labeling.20 The extinction coefficient for 2SBPO is higher than similar analogs (e = 36 000 M1 cm1) making it an attractive fluorophore for MEF. Using single-molecule/ single-particle fluorescence, we demonstrated an increase in fluorescence of 7.5 times compared to control particles lacking a silver core. We then successfully labeled cells with antibody-conjugated 2SBPO nanocomposites for bright cell labeling of CD4þ cells.
MATERIALS AND METHODS Materials. Silver nitrite and ammonium hydroxide were purchased from Acros Organics (Pittsburgh, PA). 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid 3sulfo-N-hydroxysuccinimide ester sodium salt (sulfoSMCC cross-linker), (3-mercaptopropyl) trimethoxysilane, sodium chloride, and ethanol were purchased from Sigma-Aldrich (Milwaukee, WI). Anti-CD4 was purchased from eBioscience. Trisodium citrate and tetraethyl orthosilicate (TEOS) were purchased from Alfa Aesar (Ward Hill, MA). All solutions and samples were freshly prepared before each experiment. Synthesis of 2SBPO. Benzo[a]phenoxazinium salts (2SBPO) were synthesized by heating arylazo-substituted aminophenols with 1-aminonaphthalene in DMF in the presence of perchloric acid. Two sulfonate acid groups were then added to increase water solubility.20 The
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FIG. 1. Synthesis scheme of 2SBPO Ag@SiO2 nanoparticles and antibody-conjugated nanoparticle labels. In scheme (a) silver ion was reduced to Ag colloids, and a sol-gel layer was grown over the silver core. 2SBPO dye molecules were encapsulated during the sol-gel step. To produce coreless controls, the 2SBPO Ag@SiO2 nanoparticles were exposed to NaCl to etch the silver core. In schemes (b)–(c), 2SBPO Ag@SiO2 or coreless nanoparticles were covalently conjugated to anti-CD4 antibodies. Thiol groups were added to the sol-gel surface using (3-mercaptopropyl) trimethoxysilane (MPS). Antibodies activated by sulfo-SMCC cross-linker were attached to the thiol-functionalized nanoparticles to produce cell labels.
resulting solid was purified on a reverse phase cartridge (Sep-Pak C18, Waters) and diluted to 10 mM for future use (see Supplemental Material for synthesis scheme and procedures). Preparation of 2SBPO-Labeled Ag@SiO2 Nanoparticles. To prepare the silver nanoparticles (Fig. 1a), 9 mg of silver nitrite was dissolved in 49 mL DI water and heated to boiling while stirring.1 We dissolved 10 mg of trisodium citrate in 1 mL DI water that was then added, and the mixture was kept boiling and stirring for 30 min until the solution became green–brown in color. After cooling to room temperature, the solution was centrifuged at 500 rpm for 1 h to remove large particles and was then dispersed in 200 mL ethanol. Afterward, 4 mL of ammonium hydroxide (28–30%) was added to adjust the pH to 9. At this point, a sol-gel coating was created to cover the Ag nanoparticles with SiO2. 2SBPO was added at the same time to encapsulate the fluorophores inside the sol-gel layer. We added 40 lL of 10 mM 2SBPO to the silver colloid solution, and then 22.5 lL of TEOS in 10 mL
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ethanol was added to the silver colloid solution at a rate of 2.5 mL/h under stirring. The solution was then kept still overnight to form 2SBPO core-shell Ag@SiO2 nanoparticles. Finally, the nanoparticle solution was centrifuged at 3500 rpm for 30 min three times and resuspended in 200 mL ethanol for future use. Transmission electron microscopy measurements showed that particles were 120 6 20 nm with a shell that was 15–20 nm thick.19 Synthesis of Coreless Nanoparticles. To prepare nanoparticle controls (Fig. 1a), 48 mg of sodium chloride was added to 500 lL of the red-fluorescent 2SBPO Ag@SiO2 nanoparticle solution. The final concentration of sodium chloride was 1.5 mM. The reaction mixture was kept stirring overnight to dissolve the silver within the nanoparticles while maintaining the fluorophore conjugation. When the reaction was completed, the resulting nanobubble sample was used for single molecule analysis directly.
FIG. 2. Fluorescence and white light images of 2SBPO Ag@SiO2 nanoparticles (a)–(b) and 2SBPO SiO2 coreless nanoparticles (c)–(d). The mean fluorescence intensity of 2SBPO nanoparticle clusters was 5800 6 500 counts, and the mean fluorescence intensity of 2SBPO control clusters was 1000 6 30 counts. Both fluorescent images are contrast enhanced for clarity by the same amount.
Covalent Immobilization of the Antibody onto 2SBPO Ag@SiO 2 Nanoparticles. We centrifuged 20 mL of 2SBPO Ag@SiO2 nanoparticle solution as discussed above. The resulting pellet was then resuspended in 8 mL ethanol, and 10 lL of (3-mercaptopropyl) trimethoxysilane was added to the nanocomposites, and the mixture was kept stirring for 8 h at room temperature. Then the dispersion was purified three times by centrifugation with the same parameters as given above and dispersed in 1 mL DI water. Sulfosuccinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (SulfoSMCC) was then used to cross-link antibodies to the nanoparticles. We activated 2 lL of 0.5 mg/mL human anti-CD4 with 500 lL 4 mM Sulfo-SMCC cross-linker at room temperature with stirring and reacted for 5 h. We added 1 mL of nanoparticle dispersion to the activated cross-linker and antibodies and reacted with stirring for 24 h at 4 8C. The resultant anti-CD4-conjugated nanoparticles were centrifuged twice and resuspended in 1 mL DI water. Anti-CD4–attached 2SBPO Ag@SiO2 nanoparticle controls were synthesized using the same protocol to immobilize antibodies onto the coreless nanoparticle surface (Fig. 1b–c). Single-Molecule Fluorescence Measurements. The confocal single-molecule fluorescence microscope used in this work has been described previously.21 Briefly, a red diode laser (635 nm, Edmund Optics) was used for excitation, and the beam was directed into the back port of a custom-modified inverted Olympus microscope (IX 51) via a mirror periscope. Laser radiation was then reflected onto the back aperture of an oil-immersion 1003 objective (1.3 NA, Olympus) by a long-pass dichroic
mirror (Semrock). The fluorescence emission was collected by the same objective, transmitted through the dichroic mirror, and spatially filtered using a 100 lm pinhole (Newport). The fluorescence was then spectrally filtered using a 676 nm interference filter (Omega Optical) matching the emission of 2SBPO and detected by a single-photon avalanche photodiode. Photon counts were recorded using a high-speed counter (National Instruments) and processed in LabView software. A handheld power meter (Laser Check, Edmund Optics) was used to measure laser power just before the sample. For fluorescence correlation spectroscopy (FCS) measurements the same microscopy system was used, but photon counts were measured in photon arrival mode and processed in LabView software.22–24 FCS curves were fitted to a 3D diffusion model of autocorrelation, G(s): !1=2 wxy 2 s 1 s 1 GðsÞ ¼ 1þ 1þ ð1Þ N sD w z sD where N is the average number of molecules/particles present in the probe volume, sD is the correlation time due to diffusion, and wxy and wz are the axial and lateral radii of the observation volume, respectively. For analysis, a 30 lL droplet of nanoparticle suspension was placed on a 150 lm coverslip. The coverslip was coupled to the microscope objective with immersion oil. This procedure was repeated for every measurement. Nanoparticle or control samples were then
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FIG. 3. Single-particle fluorescence of 2SBPO Ag@SiO2 nanoparticles (blue) and coreless nanoparticles (red) at 400 lW of excitation power. The nanoparticles were allowed to freely diffuse through the laser beam probe volume, generating fluorescence bursts. The lack of enhancement in the coreless 2SBPO nanoparticles indicates that the increased fluorescence is due to metal enhanced effects rather than dye aggregation.
exposed to laser light ranging from 150 lW to 1 5 mW. Evaporation of the sample droplet was not observed during the 2 min sample analysis time. Data were then exported as text files and analyzed with Origin software (version 8.0). Fluorescence bursts of 1 ms duration were counted above the background using a signal-to-noise ratio of 3.24,25 The average baseline intensity was calculated from 70 consecutive signal bins in the absence of single particles for each sample. Cell Imaging and Analysis. For cell staining with the anti-CD4 2SBPO nanoparticles, HuT 78 lymphocytes (American Type Culture Collection) were stained with the nanoparticles for 40 min in phosphate buffered saline (pH 7.4) and washed before analysis. Cells were analyzed using an inverted, epifluorescence microscope (IX71, Olympus) and a cooled CCD camera. Excitation was performed using a metal halide lamp (Prior Scientific) with appropriate excitation and emission filters for 2SBPO. Images were analyzed using ImageJ (v. 1.43, National Institutes of Health); image background was subtracted prior to cell intensity analysis.
RESULTS AND DISCUSSION Metal-Enhanced Fluorescence of 2SBPO Ag@SiO2 Nanoparticles. MEF nanoparticles and coreless control nanoparticles (Fig. 1) were compared to determine fluorescence enhancement and account for any dye aggregation effects. Antibody attachment via direct conjugation was then used to produce cell nanoparticle immunolabels and coreless controls. The method for preparing the new 2SBPO MEF nanoparticles is a modified Sto¨ber process. Compared to the other microemulsion-based methods commonly used, the Sto¨ber method is a one-pot, room-temperature synthesis. There are established techniques of encap-
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FIG. 4. Fluorescence correlation spectroscopy measurements of 2SBPO Ag@SiO2 nanoparticles. The mean diffusion time (sD) of the dye-doped nanoparticles was 0.4 6 0.2 ms, while the diffusion time of the free dye was 0.16 6 0.02 ms. The nanoparticle diffusion time is the convolution of the particle diffusion and diffusion of 2SBPO within the sol-gel layer.
sulating organic dyes into silica matrix through physical entrapment approach.25 The hydrophilic environment of silica sol-gel makes incorporating hydrophilic dyes such as 2SBPO more favorable.26–28 In addition to its water solubility, 2SBPO has a 0.3 quantum yield and a peak emission near 670 nm and is suitable for sensitive fluorescence assays.29,30 Fluorescence images of nanoparticle suspensions are shown in Fig. 2. The nanoparticles synthesized by these methods have 120 nm cores with 12–20 nm shells.19 The mean fluorescence intensity was 5800 6 500 counts (mean 6 standard deviation). The nanoparticles aggregated in these fluorescence images, but the mean fluorescence is independent of aggregate size. They verify that the fluorescence signal is enhanced by the metal core and not from concentrating effects in the solgel. We also measured the control nanoparticles with etched cores. The intensity of the coreless 2SBPO nanoparticles was 1000 6 30 counts, indicating the metal core enhancement occurred in the 2SBPO Ag@SiO2 nanoparticles. The emission spectra of the 2SBPO Ag@SiO2 nanoparticles and coreless 2SBPO nanoparticles were identical except for their intensities (see Supplemental Material). Single-Particle Spectroscopy of 2SBPO Ag@SiO2 Nanoparticles. Single nanoparticles diffusing through the confocal detection volume were measured (Fig. 3). The coreless control nanoparticles exhibited minimal fluorescence signal, while intense bursts of fluorescence were observed from individual 2SBPO Ag@SiO2 nanoparticles. In a typical 2 min scan, an average of two coreless nanoparticles were detected, as the fluorescence intensity was low (mean intensity ,20 counts). However, the metal core nanoparticles showed higher fluorescence, with nanoparticle signals as high as 254 counts. Each fluorescence burst corresponded to a single nanoparticle containing multiple fluorophores
FIG. 5. Fluorescence intensity distribution histograms of baselinesubtracted fluorescence bursts from 2SBPO Ag@SiO2 nanocomposites (blue) or SiO2 nanobubble (red) at 400 lW of excitation power show an increase in nanocomposite fluorescence when metal cores are present.
undergoing metal-enhanced fluorescence. While typical data are shown in Fig. 3, there was a consistent enhancement effect at all laser powers (data not shown). The 2SBPO AgSiO2 nanoparticles exhibited slower diffusion than free 2SBPO, which is expected given the size difference between the nanoparticle and free dye. Fluorescence Correlation Spectroscopy measurements of free 2SBPO and 2SBPO Ag@SiO2 nanoparticles
showed longer diffusion times than the free dye (Fig. 4). The diffusion time for free 2SBPO dye was 0.16 6 0.02 ms, while the diffusion time for 2SBPO/nanoparticles was 0.4 6 0.2 ms (n = 3 scans each). The behavior of the nanoparticles in the laser beam probe volume is a convolution of nanoparticle motion as well as diffusion of 2SBPO through the sol-gel layer. It is likely that the diffusion time of the particle itself is slower, but movement of 2SBPO within the sol-gel can decrease the diffusion time. Measurements of diffusion times in coreless nanoparticles were statistically similar to the core-shell MEF nanoparticles. The intensity distribution of typical single nanoparticles excited with 400 lW of 635 nm laser light is shown in Fig. 5. Strong fluorescence signals were observed in the case of 2SBPO Ag@SiO2 nanoparticles, while the coreless control nanoparticles exhibited lower fluorescence intensities. The highest signal enhancement obtained was 7.5-fold for individual nanoparticles. While only 400 lW excitation power results are shown, the MEF effect was observed at all excitation powers used in this work (150–1500 lW). Cell Labeling Using Ag@SiO2 Nanoparticles. Redfluorescent nanoparticles are particularly attractive as cell labels due to reduced background signals. Since autofluorescence from cells at 660–680 nm is negligible, the interference from cell autofluorescence was minimized. In our previous work, rhodamine B nanoparticles were synthesized and conjugated with mouse anti-CD71, using a sandwich conjugation approach via non-covalent linkage.31 However, using the same conjugation approach for 2SBPO nanoparticles led to
FIG. 6. White light and fluorescence images of HuT 78 cells stained with 2SBPO nanoparticles conjugated to anti-CD4 (a)–(b), or 2SBPO nanobubble controls conjugated to anti-CD4 (c)–(d). The mean fluorescence intensity of nanoparticle labeled cells was 1400 6 400 counts, and the mean fluorescence intensity of nanobubble control labeled cells was 50 6 10 counts.
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fluorophore quenching. To overcome this issue, a covalent-linking method was used to attach the antibodies to thiol-functionalized nanoparticles.32 Covalent methods, while more complex than the sandwich-conjugation approach, preserved 2SBPO fluorescence in the nanoparticle. Figure 6 shows typical microscopy images of HuT 78 cells stained with 2SBPO Ag@SiO2 nanoparticles conjugated to antiCD4 antibodies. HuT 78 cells are CD4þ, and all experiments were performed on the same day to minimize differences in antigen expression. Control experiments were conducted using coreless 2SBPO nanoparticles conjugated to anti-CD4. Antibody-attached 2SBPO nanoparticles with metal cores showed brighter fluorescence than the nanobubble controls. The control particles showed a mean cell fluorescence intensity of 50 6 10 counts, while the core-shell particles had a mean cell fluorescence of 1400 6 400 counts. Blank cells (no particle labeling) had intensities of 40 6 4 counts, resulting in a signal difference between core-shell nanoparticles and coreless control immunolabels of 35-fold. The signal differences between the labeled cells and individual particles measured by single-molecule fluorescence could be due to several factors, including differences in 2SBPO incorporation during the sol-gel process or signal enhancement due to the large number of antibodies expressed on the cell surface.
CONCLUSION In this work, we synthesized a new Ag@SiO 2 nanoparticle by incorporating the red fluorescent dye 2SBPO in a simple, one-step method. The increase in fluorescence intensity due to the MEF was verified using core-etched particles as controls. Antibodies were immobilized to the nanoparticles through covalent linkage to produce bright cell labels. HuT 78 cells labeled with an anti-CD4 2SBPO nanoparticle demonstrated more than 30-fold enhancement of fluorescence intensity over the control nanobubbles. The facile synthesis, enhanced fluorescence properties, and excellent performance in cell labeling makes the 2SBPO Ag@SiO2 nanoparticle a promising tool for many bioanalytical and biosensing applications.
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ACKNOWLEDGMENT This work was supported by the National Institute of Heath (RR025782 and GM103550). SUPPLEMENTAL MATERIAL All supplemental material mentioned in the text, including one scheme and the synthesis procedures for the 2SBPO dye, can be found in the online version of the journal at http://www.s-a-s.org. 1. H. Yang, H. Qu, P. Lin, S. Li, M. Ding, J. Xu. ‘‘Nanometer Fluorescent Hybrid Silica Particle as Ultrasensitive and Photostable Biological Labels’’. Analyst. 2003. 128(5): 462-466. 2. D. Pappas. ‘‘Protocols, Probes, and Standards’’. In: D. Pappas, editor. Practical Cell Analysis. West Sussex, UK: John Wiley and Sons, 2010. Chapter 9. Pp. 247-282. 3. G. Canton, R. Ricco`, F. Marinello, S. Carmignato, F. Enrichi. ‘‘Modified Sto¨ber Synthesis of Highly Luminescent Dye-Doped Silica Nanoparticles’’. J. Nanopart. Res. 2011. 13(9): 4349-4356. 4. M.H. Chowdhury, K. Ray, S.K. Gray, J. Pond, J.R. Lakowicz. ‘‘Aluminum Nanoparticles as Substrates for Metal-Enhanced
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27. Sˇ.K. Ureka, A. Lobnikb. ‘‘Fluorescent-Based Chemical Sensor for Organophosphate Detection’’. Proc. SPIE 8073. 2011. doi:10.1117/12. 887051. 28. S. Shibata, T. Taniguchi, T. Yano, M. Yamane. ‘‘Formation of WaterSoluble Dye-Doped Silica Particles’’. J Sol–Gel Sci. Technol. 1997. 10(3): 263-268. 29. A. Burghart, H. Kim, M.B. Welch, L.H. Thoresen, J. Reibenspies, K. Burgess. ‘‘3,5-diaryl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) Dyes: Synthesis, Spectroscopic, Electrochemical, and Structural Properties’’. J. Org. Chem. 1999. 64(21): 7813-7819.
30. A. Loudet, K. Burgess. ‘‘BODIPY Dyes and Their Derivatives: Syntheses and Spectroscopic Properties’’. Chem. Rev. 2007. 107(11): 4891-4932. 31. K. Wang, B. Cometti, D. Pappas. ‘‘Isolation and Counting of Multiple Cell Types Using an Affinity Separation Device’’. Anal. Chim. Acta. 2007. 601(1): 1-9. 32. D. Kozlova, S. Chernousova, T. Knuschke, J. Buer, A.M. Westendorf, M. Epple. ‘‘Cell Targeting by Antibody-Functionalized Calcium Phosphate Nanoparticles’’. J. Mater. Chem. 2012. 22(2): 396-404.
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In this work we describe a one-step approach for incorporating a red fluorophore (2SBPO) into core-shell nanoparticles for metalenhanced fluorescence immunolabels. The 2SBPO-MEF nanoparticles are particularly attractive as cell labels because their 670 nm emission has minimal overlap with cell autofluorescence and from overlap with many conventional probes. 2SBPO was incorporated through physical entrapment during the Sto¨ber process. Antibody-based cell labels were then synthesized using covalent linkage. The nanoparticle fluorescence was 7.5-fold higher than control nanoparticles lacking a metal core. We demonstrated labeling of CD4 þ HuT 78 T lymphocytes using anti-CD4– conjugated nanoparticle labels. Cells labeled with anti-CD4 nanoparticles showed a 35-fold fluorescence signal compared to antiCD4 coreless controls. This simple synthesis protocol can be applied to a variety of hydrophilic fluorophore types and has broad potential in bioanalytical and biosensing applications. Index Headings: Metal-enhanced fluorescence; Core-shell Ag@SiO2 nanoparticle; 2SBPO; Single molecule spectroscopy; Cell labeling.
INTRODUCTION Immunofluorescence approaches are commonly used to render an optically and electrochemically inactive species available for detection.1 Immunolabels are the primary reagent for many analyses such as flow cytometry, fluorescence microscopy, and confocal microscopy.2 Organic fluorophores are typically conjugated to antibodies or other ligands to produce immunolabels. The main problems of using organic fluorophores directly as fluorescent antibody labels include photobleaching, low signal intensities, and limited sensitivity.3 To overcome these issues, several types of nanomaterials, such as fluorescent dye-doped nanoparticles (NPs), quantum dots, and metallic NPs, have been explored as labels for bioanalysis. These probes present advantages such as uniform size, photostability, and facile functionalization. Among them, nanocomposites using metallic cores are an effective structure to increase the intensity of fluorophores and improved sensitivity.4 Certain metallic cores, such as silver, produce plasmon enhancement of the fluorescence signal, described as metal enhanced fluorescence (MEF) by Geddes.5 MEF leads to shorter lifetimes and increased molecular brightness, thus enhancing the fluorescence signal from individual dye molecules. MEF nanocomposites have been applied to many Received 5 June 2014; accepted 6 August 2014. * Author to whom correspondence should be sent. E-mail: d.pappas@ ttu.edu. DOI: 10.1366/14-07615
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systems, including single-cell imaging, protein detection, flow measurements, and other applications.6–16 In MEF, the fluorophore-metal distance is critical; at the same time many metal particles are not amenable to direct conjugation of dyes. Confining the fluorophore to a silica shell maintains a uniform distance from the metal core and provides a means of confining dye near the metal surface. Distances smaller than the optimum result in quenching, while distances longer than the optimum result in diminished plasmon enhancement.17,18 In our previous work, rhodamine B Ag@SiO2 nanoparticles, silver metal cores with silica sol-gel shells, were synthesized to produce green-fluorescent nanoparticle labels.19 Rhodamine B was incorporated into this nanocarrier through physical adsorption to form a bright fluorescent nanocomposite that is used in cell labeling and other applications. However, the green fluorescence of rhodamine B overlaps with cell autofluorescence, which may result in high background in cell labeling and imaging applications. To eliminate spectral overlap issues, we have developed a red-fluorescent nanoparticle label for cell immunofluorescence. We incorporated 9-di-3-sulfonyl-propylaminobenzo[a]phenoxazonium perchlorate (2SBPO) into the silica layer of Ag@SiO2 nanoparticles. 2SBPO is a red fluorescent, water-soluble dye suitable for biological labeling.20 The extinction coefficient for 2SBPO is higher than similar analogs (e = 36 000 M1 cm1) making it an attractive fluorophore for MEF. Using single-molecule/ single-particle fluorescence, we demonstrated an increase in fluorescence of 7.5 times compared to control particles lacking a silver core. We then successfully labeled cells with antibody-conjugated 2SBPO nanocomposites for bright cell labeling of CD4þ cells.
MATERIALS AND METHODS Materials. Silver nitrite and ammonium hydroxide were purchased from Acros Organics (Pittsburgh, PA). 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid 3sulfo-N-hydroxysuccinimide ester sodium salt (sulfoSMCC cross-linker), (3-mercaptopropyl) trimethoxysilane, sodium chloride, and ethanol were purchased from Sigma-Aldrich (Milwaukee, WI). Anti-CD4 was purchased from eBioscience. Trisodium citrate and tetraethyl orthosilicate (TEOS) were purchased from Alfa Aesar (Ward Hill, MA). All solutions and samples were freshly prepared before each experiment. Synthesis of 2SBPO. Benzo[a]phenoxazinium salts (2SBPO) were synthesized by heating arylazo-substituted aminophenols with 1-aminonaphthalene in DMF in the presence of perchloric acid. Two sulfonate acid groups were then added to increase water solubility.20 The
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FIG. 1. Synthesis scheme of 2SBPO Ag@SiO2 nanoparticles and antibody-conjugated nanoparticle labels. In scheme (a) silver ion was reduced to Ag colloids, and a sol-gel layer was grown over the silver core. 2SBPO dye molecules were encapsulated during the sol-gel step. To produce coreless controls, the 2SBPO Ag@SiO2 nanoparticles were exposed to NaCl to etch the silver core. In schemes (b)–(c), 2SBPO Ag@SiO2 or coreless nanoparticles were covalently conjugated to anti-CD4 antibodies. Thiol groups were added to the sol-gel surface using (3-mercaptopropyl) trimethoxysilane (MPS). Antibodies activated by sulfo-SMCC cross-linker were attached to the thiol-functionalized nanoparticles to produce cell labels.
resulting solid was purified on a reverse phase cartridge (Sep-Pak C18, Waters) and diluted to 10 mM for future use (see Supplemental Material for synthesis scheme and procedures). Preparation of 2SBPO-Labeled Ag@SiO2 Nanoparticles. To prepare the silver nanoparticles (Fig. 1a), 9 mg of silver nitrite was dissolved in 49 mL DI water and heated to boiling while stirring.1 We dissolved 10 mg of trisodium citrate in 1 mL DI water that was then added, and the mixture was kept boiling and stirring for 30 min until the solution became green–brown in color. After cooling to room temperature, the solution was centrifuged at 500 rpm for 1 h to remove large particles and was then dispersed in 200 mL ethanol. Afterward, 4 mL of ammonium hydroxide (28–30%) was added to adjust the pH to 9. At this point, a sol-gel coating was created to cover the Ag nanoparticles with SiO2. 2SBPO was added at the same time to encapsulate the fluorophores inside the sol-gel layer. We added 40 lL of 10 mM 2SBPO to the silver colloid solution, and then 22.5 lL of TEOS in 10 mL
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ethanol was added to the silver colloid solution at a rate of 2.5 mL/h under stirring. The solution was then kept still overnight to form 2SBPO core-shell Ag@SiO2 nanoparticles. Finally, the nanoparticle solution was centrifuged at 3500 rpm for 30 min three times and resuspended in 200 mL ethanol for future use. Transmission electron microscopy measurements showed that particles were 120 6 20 nm with a shell that was 15–20 nm thick.19 Synthesis of Coreless Nanoparticles. To prepare nanoparticle controls (Fig. 1a), 48 mg of sodium chloride was added to 500 lL of the red-fluorescent 2SBPO Ag@SiO2 nanoparticle solution. The final concentration of sodium chloride was 1.5 mM. The reaction mixture was kept stirring overnight to dissolve the silver within the nanoparticles while maintaining the fluorophore conjugation. When the reaction was completed, the resulting nanobubble sample was used for single molecule analysis directly.
FIG. 2. Fluorescence and white light images of 2SBPO Ag@SiO2 nanoparticles (a)–(b) and 2SBPO SiO2 coreless nanoparticles (c)–(d). The mean fluorescence intensity of 2SBPO nanoparticle clusters was 5800 6 500 counts, and the mean fluorescence intensity of 2SBPO control clusters was 1000 6 30 counts. Both fluorescent images are contrast enhanced for clarity by the same amount.
Covalent Immobilization of the Antibody onto 2SBPO Ag@SiO 2 Nanoparticles. We centrifuged 20 mL of 2SBPO Ag@SiO2 nanoparticle solution as discussed above. The resulting pellet was then resuspended in 8 mL ethanol, and 10 lL of (3-mercaptopropyl) trimethoxysilane was added to the nanocomposites, and the mixture was kept stirring for 8 h at room temperature. Then the dispersion was purified three times by centrifugation with the same parameters as given above and dispersed in 1 mL DI water. Sulfosuccinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (SulfoSMCC) was then used to cross-link antibodies to the nanoparticles. We activated 2 lL of 0.5 mg/mL human anti-CD4 with 500 lL 4 mM Sulfo-SMCC cross-linker at room temperature with stirring and reacted for 5 h. We added 1 mL of nanoparticle dispersion to the activated cross-linker and antibodies and reacted with stirring for 24 h at 4 8C. The resultant anti-CD4-conjugated nanoparticles were centrifuged twice and resuspended in 1 mL DI water. Anti-CD4–attached 2SBPO Ag@SiO2 nanoparticle controls were synthesized using the same protocol to immobilize antibodies onto the coreless nanoparticle surface (Fig. 1b–c). Single-Molecule Fluorescence Measurements. The confocal single-molecule fluorescence microscope used in this work has been described previously.21 Briefly, a red diode laser (635 nm, Edmund Optics) was used for excitation, and the beam was directed into the back port of a custom-modified inverted Olympus microscope (IX 51) via a mirror periscope. Laser radiation was then reflected onto the back aperture of an oil-immersion 1003 objective (1.3 NA, Olympus) by a long-pass dichroic
mirror (Semrock). The fluorescence emission was collected by the same objective, transmitted through the dichroic mirror, and spatially filtered using a 100 lm pinhole (Newport). The fluorescence was then spectrally filtered using a 676 nm interference filter (Omega Optical) matching the emission of 2SBPO and detected by a single-photon avalanche photodiode. Photon counts were recorded using a high-speed counter (National Instruments) and processed in LabView software. A handheld power meter (Laser Check, Edmund Optics) was used to measure laser power just before the sample. For fluorescence correlation spectroscopy (FCS) measurements the same microscopy system was used, but photon counts were measured in photon arrival mode and processed in LabView software.22–24 FCS curves were fitted to a 3D diffusion model of autocorrelation, G(s): !1=2 wxy 2 s 1 s 1 GðsÞ ¼ 1þ 1þ ð1Þ N sD w z sD where N is the average number of molecules/particles present in the probe volume, sD is the correlation time due to diffusion, and wxy and wz are the axial and lateral radii of the observation volume, respectively. For analysis, a 30 lL droplet of nanoparticle suspension was placed on a 150 lm coverslip. The coverslip was coupled to the microscope objective with immersion oil. This procedure was repeated for every measurement. Nanoparticle or control samples were then
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FIG. 3. Single-particle fluorescence of 2SBPO Ag@SiO2 nanoparticles (blue) and coreless nanoparticles (red) at 400 lW of excitation power. The nanoparticles were allowed to freely diffuse through the laser beam probe volume, generating fluorescence bursts. The lack of enhancement in the coreless 2SBPO nanoparticles indicates that the increased fluorescence is due to metal enhanced effects rather than dye aggregation.
exposed to laser light ranging from 150 lW to 1 5 mW. Evaporation of the sample droplet was not observed during the 2 min sample analysis time. Data were then exported as text files and analyzed with Origin software (version 8.0). Fluorescence bursts of 1 ms duration were counted above the background using a signal-to-noise ratio of 3.24,25 The average baseline intensity was calculated from 70 consecutive signal bins in the absence of single particles for each sample. Cell Imaging and Analysis. For cell staining with the anti-CD4 2SBPO nanoparticles, HuT 78 lymphocytes (American Type Culture Collection) were stained with the nanoparticles for 40 min in phosphate buffered saline (pH 7.4) and washed before analysis. Cells were analyzed using an inverted, epifluorescence microscope (IX71, Olympus) and a cooled CCD camera. Excitation was performed using a metal halide lamp (Prior Scientific) with appropriate excitation and emission filters for 2SBPO. Images were analyzed using ImageJ (v. 1.43, National Institutes of Health); image background was subtracted prior to cell intensity analysis.
RESULTS AND DISCUSSION Metal-Enhanced Fluorescence of 2SBPO Ag@SiO2 Nanoparticles. MEF nanoparticles and coreless control nanoparticles (Fig. 1) were compared to determine fluorescence enhancement and account for any dye aggregation effects. Antibody attachment via direct conjugation was then used to produce cell nanoparticle immunolabels and coreless controls. The method for preparing the new 2SBPO MEF nanoparticles is a modified Sto¨ber process. Compared to the other microemulsion-based methods commonly used, the Sto¨ber method is a one-pot, room-temperature synthesis. There are established techniques of encap-
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FIG. 4. Fluorescence correlation spectroscopy measurements of 2SBPO Ag@SiO2 nanoparticles. The mean diffusion time (sD) of the dye-doped nanoparticles was 0.4 6 0.2 ms, while the diffusion time of the free dye was 0.16 6 0.02 ms. The nanoparticle diffusion time is the convolution of the particle diffusion and diffusion of 2SBPO within the sol-gel layer.
sulating organic dyes into silica matrix through physical entrapment approach.25 The hydrophilic environment of silica sol-gel makes incorporating hydrophilic dyes such as 2SBPO more favorable.26–28 In addition to its water solubility, 2SBPO has a 0.3 quantum yield and a peak emission near 670 nm and is suitable for sensitive fluorescence assays.29,30 Fluorescence images of nanoparticle suspensions are shown in Fig. 2. The nanoparticles synthesized by these methods have 120 nm cores with 12–20 nm shells.19 The mean fluorescence intensity was 5800 6 500 counts (mean 6 standard deviation). The nanoparticles aggregated in these fluorescence images, but the mean fluorescence is independent of aggregate size. They verify that the fluorescence signal is enhanced by the metal core and not from concentrating effects in the solgel. We also measured the control nanoparticles with etched cores. The intensity of the coreless 2SBPO nanoparticles was 1000 6 30 counts, indicating the metal core enhancement occurred in the 2SBPO Ag@SiO2 nanoparticles. The emission spectra of the 2SBPO Ag@SiO2 nanoparticles and coreless 2SBPO nanoparticles were identical except for their intensities (see Supplemental Material). Single-Particle Spectroscopy of 2SBPO Ag@SiO2 Nanoparticles. Single nanoparticles diffusing through the confocal detection volume were measured (Fig. 3). The coreless control nanoparticles exhibited minimal fluorescence signal, while intense bursts of fluorescence were observed from individual 2SBPO Ag@SiO2 nanoparticles. In a typical 2 min scan, an average of two coreless nanoparticles were detected, as the fluorescence intensity was low (mean intensity ,20 counts). However, the metal core nanoparticles showed higher fluorescence, with nanoparticle signals as high as 254 counts. Each fluorescence burst corresponded to a single nanoparticle containing multiple fluorophores
FIG. 5. Fluorescence intensity distribution histograms of baselinesubtracted fluorescence bursts from 2SBPO Ag@SiO2 nanocomposites (blue) or SiO2 nanobubble (red) at 400 lW of excitation power show an increase in nanocomposite fluorescence when metal cores are present.
undergoing metal-enhanced fluorescence. While typical data are shown in Fig. 3, there was a consistent enhancement effect at all laser powers (data not shown). The 2SBPO AgSiO2 nanoparticles exhibited slower diffusion than free 2SBPO, which is expected given the size difference between the nanoparticle and free dye. Fluorescence Correlation Spectroscopy measurements of free 2SBPO and 2SBPO Ag@SiO2 nanoparticles
showed longer diffusion times than the free dye (Fig. 4). The diffusion time for free 2SBPO dye was 0.16 6 0.02 ms, while the diffusion time for 2SBPO/nanoparticles was 0.4 6 0.2 ms (n = 3 scans each). The behavior of the nanoparticles in the laser beam probe volume is a convolution of nanoparticle motion as well as diffusion of 2SBPO through the sol-gel layer. It is likely that the diffusion time of the particle itself is slower, but movement of 2SBPO within the sol-gel can decrease the diffusion time. Measurements of diffusion times in coreless nanoparticles were statistically similar to the core-shell MEF nanoparticles. The intensity distribution of typical single nanoparticles excited with 400 lW of 635 nm laser light is shown in Fig. 5. Strong fluorescence signals were observed in the case of 2SBPO Ag@SiO2 nanoparticles, while the coreless control nanoparticles exhibited lower fluorescence intensities. The highest signal enhancement obtained was 7.5-fold for individual nanoparticles. While only 400 lW excitation power results are shown, the MEF effect was observed at all excitation powers used in this work (150–1500 lW). Cell Labeling Using Ag@SiO2 Nanoparticles. Redfluorescent nanoparticles are particularly attractive as cell labels due to reduced background signals. Since autofluorescence from cells at 660–680 nm is negligible, the interference from cell autofluorescence was minimized. In our previous work, rhodamine B nanoparticles were synthesized and conjugated with mouse anti-CD71, using a sandwich conjugation approach via non-covalent linkage.31 However, using the same conjugation approach for 2SBPO nanoparticles led to
FIG. 6. White light and fluorescence images of HuT 78 cells stained with 2SBPO nanoparticles conjugated to anti-CD4 (a)–(b), or 2SBPO nanobubble controls conjugated to anti-CD4 (c)–(d). The mean fluorescence intensity of nanoparticle labeled cells was 1400 6 400 counts, and the mean fluorescence intensity of nanobubble control labeled cells was 50 6 10 counts.
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fluorophore quenching. To overcome this issue, a covalent-linking method was used to attach the antibodies to thiol-functionalized nanoparticles.32 Covalent methods, while more complex than the sandwich-conjugation approach, preserved 2SBPO fluorescence in the nanoparticle. Figure 6 shows typical microscopy images of HuT 78 cells stained with 2SBPO Ag@SiO2 nanoparticles conjugated to antiCD4 antibodies. HuT 78 cells are CD4þ, and all experiments were performed on the same day to minimize differences in antigen expression. Control experiments were conducted using coreless 2SBPO nanoparticles conjugated to anti-CD4. Antibody-attached 2SBPO nanoparticles with metal cores showed brighter fluorescence than the nanobubble controls. The control particles showed a mean cell fluorescence intensity of 50 6 10 counts, while the core-shell particles had a mean cell fluorescence of 1400 6 400 counts. Blank cells (no particle labeling) had intensities of 40 6 4 counts, resulting in a signal difference between core-shell nanoparticles and coreless control immunolabels of 35-fold. The signal differences between the labeled cells and individual particles measured by single-molecule fluorescence could be due to several factors, including differences in 2SBPO incorporation during the sol-gel process or signal enhancement due to the large number of antibodies expressed on the cell surface.
CONCLUSION In this work, we synthesized a new Ag@SiO 2 nanoparticle by incorporating the red fluorescent dye 2SBPO in a simple, one-step method. The increase in fluorescence intensity due to the MEF was verified using core-etched particles as controls. Antibodies were immobilized to the nanoparticles through covalent linkage to produce bright cell labels. HuT 78 cells labeled with an anti-CD4 2SBPO nanoparticle demonstrated more than 30-fold enhancement of fluorescence intensity over the control nanobubbles. The facile synthesis, enhanced fluorescence properties, and excellent performance in cell labeling makes the 2SBPO Ag@SiO2 nanoparticle a promising tool for many bioanalytical and biosensing applications.
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