pubs.acs.org/Langmuir © 2010 American Chemical Society
Improved Method for Counting DNA Molecules on Biofunctionalized Nanoparticles Filip Delport,† Ania Deres,‡ Jun-ichi Hotta,‡ Jeroen Pollet,† Bert Verbruggen,† Bert Sels,§ Johan Hofkens,‡ and Jeroen Lammertyn*,† †
Department of Biosystems, Division Mechatronics, Biostatistics and Sensors, ‡Department of Chemistry, Molecular and Nanomaterials, and §Department of Microbial and Molecular Systems, Centre for Surface Chemistry and Catalysis, KULeuven, Leuven, Belgium Received December 14, 2009
In order to accurately determine low numbers (1-100) of immobilized ssDNA molecules at a single, silica 250 nm nanoparticle surface, we hereby propose an integrated approach combining classic single molecule confocal microscopy (SMCM), that is, stepwise photobleaching of labeled ssDNA, with modified total internal reflection fluorescence microscopy (mTIRF). We postulate that SMCM alone is unable to exactly account for all labeled ssDNA because of inherent laser polarization effects; that is, perpendicularly oriented molecules to the sample surface are not (or are only slightly) susceptible to laser excitation and thus are invisible in a classic photobleaching experiment. The SMCM method accounts for at best two-thirds (68%) of the present ssDNA molecules. The principle of the mTIRF technique, which relies on the creation of highly inclined illumination combined with part of the laser remaining in normal K€ohler illumination, enables accurate counting of SMCM invisble molecules. The combined approach proposed here circumvents the polarization issue and allows a complete single molecule counting on individual nanoparticles, fully in line with bulk measurements, as will be demonstrated.
New developments in biofunctionalized nanomaterials today are the driving force for innovative future applications in the electronic, chemical, biotechnology, and medical industries. Particularly, functionalized nanoparticles (NPs) attract a lot of attention in the field of life sciences for their diagnostic and therapeutic properties, for example, as miniaturized biosensors or as drug delivery vehicles.1-4 NPs have for instance been conjugated with a variety of biomolecules such as proteins, enzymes, and antibodies.5-9 Recently, DNA functionalized NPs have been recognized as attractive nanotools in medical and food biomolecular diagnostics because of their inherent specific properties including DNA detection through hybridization, aptamer based ligand detection,10,11 and DNA mediated NP aggregation and disaggregation.12 While accurate quantification of low numbers of anchored DNA biomolecules on individual NPs is of utmost importance, it is a challenging task. Common direct detection techniques based *To whom correspondence should be addressed. E-mail: Jeroen.Lammertyn@ biw.kuleuven.be. (1) Quarta, A.; Ragusa, A.; Deka, S.; Tortiglione, C.; Tino, A.; Cingolani, R.; Pellegrino, T. Langmuir 2009, 25(21), 12614–12622. (2) Kim, J. S.; Rieter, W. J.; Taylor, K. M. L.; An, H.; Lin, W. L.; Lin, W. B. J. Am. Chem. Soc. 2007, 129(29), 8962–8963. (3) Fu, Y.; Zhang, J.; Lakowicz, J. R. Langmuir 2008, 24(7), 3429–3433. (4) Senarath-Yapa, M. D.; Phimphivong, S.; Coym, J. W.; Wirth, M. J.; Aspinwall, C. A.; Saavedra, S. S. Langmuir 2007, 23(25), 12624–12633. (5) Tan, J. S.; Butterfield, D. E.; Voycheck, C. L.; Caldwell, K. D.; Li, J. T. Biomaterials 1993, 14(11), 823–833. (6) Tansil, N. C.; Gao, Z. Q. Nano Today 2006, 1(1), 28–37. (7) Wang, J. Small 2005, 1(11), 1036–1043. (8) Wang, L.; Wang, K. M.; Santra, S.; Zhao, X. J.; Hilliard, L. R.; Smith, J. E.; Wu, J. R.; Tan, W. H. Anal. Chem. 2006, 78(3), 646–654. (9) You, C. C.; De, M.; Rotello, V. M. Curr. Opin. Chem. Biol. 2005, 9(6), 639– 646. (10) Ellington, A. D.; Szostak, J. W. Nature 2002, 355(6363), 850-852. (11) Jhaveri, S.; Rajendran, M.; Ellington, A. D. Nat. Biotechnol. 2000, 18(12), 1293–1297. (12) Hurst, S. J.; Hill, H. D.; Mirkin, C. A. J. Am. Chem. Soc. 2008, 130(36), 12192–12200.
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on thermogravimetric analysis, atom emission spectroscopy, infrared spectroscopy, and liquid chromatography13-16 are sometimes labor intensive and require relatively large amounts of sample, and hence, they are less suitable for the correct analysis of ultralow quantities of DNA, when attached on nanomaterials. Instead, measuring directly on the single NP is more accurate but also more complex. Recently, single molecule detection strategies, such as single molecule counting and single molecule bleaching, have been proposed to offer a solution for the problem of counting single proteins17 and quantum dots18,19 when coupled to silica, polystyrene, gold, silver, lipid, NPs, and so on.20,21 Although clear molecule-by-molecule counting has been demonstrated, a deviation from bulk measurements was always encountered.17 In order to accurately determine low numbers of immobilized ssDNA molecules at a single NP surface, we hereby propose an integrated approach combining classic single molecule confocal microscopy (SMCM) techniques, that is, stepwise photobleaching of labeled ssDNA, and modified total internal reflection fluorescence (mTIRF) illumination (vide infra and Supporting Information, Figure S4). We postulate that SMCM alone is unable to exactly account for all labeled tags because of laser polarization (13) Chen, S. H.; Kimura, K. Langmuir 1999, 15(4), 1075–1082. (14) Chen, Y.; Lu, Z. H. Anal. Chim. Acta 2007, 587(2), 180–186. (15) Li, Z. Z.; Wen, L. X.; Shao, L.; Chen, J. F. J. Controlled Release 2004, 98(2), 245–254. (16) Mao, Y.; Daniel, L. N.; Whittaker, N.; Saffiotti, U. Environ. Health Perspect. 1994, 102, 165–171. (17) Casanova, D.; Giaume, D.; Moreau, M.; Martin, J. L.; Gacoin, T.; Boilot, J. P.; Alexandrou, A. J. Am. Chem. Soc. 2007, 129(42), 12592–12593. (18) Stavis, S. M.; Edel, J. B.; Samiee, K. T.; Craighead, H. G. Lab Chip 2005, 5(3), 337–343. (19) Zhang, C. Y.; Johnson, L. W. J. Am. Chem. Soc. 2008, 130(12), 3750–3751. (20) Sonnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P. Nat. Biotechnol. 2005, 23(6), 741–745. (21) Kunding, A. H.; Mortensen, M. W.; Christensen, S. M.; Stamou, D. Biophys. J. 2008, 95(3), 1176–1188.
Published on Web 01/05/2010
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effects; that is, perpendicularly oriented molecules to the sample surface are not (or are only slightly) susceptible to laser excitation and thus are invisible in a classic photobleaching experiment. The mTIRF approach circumvents the polarization issue and allows a complete single molecule counting on individual NPs, fully in line with bulk measurements, as will be demonstrated. Immobilization of the ssDNA on the NPs was based on synthesis procedures reported by Hermanson:22 15-mer ssDNA tagged with both an amine group and a fluorescent ATTO 647N dye was coupled to 249 ((27) nm silica carboxyl functionalized nanoparticles using carbodiimide chemistry (Supporting Information, Scheme S1). A linear relationship was observed between the average number of ssDNA immobilized on the NP and the initial concentration of ssDNA in the reaction mixture in the range from 0.5 to 500 nM, as determined with bulk fluorescence measurement (Supporting Information, Figure S2). Early attempts to determine coupling efficiency revealed a discrepancy between direct measurements on the NP and indirect measurements on the supernatant. This discrepancy originates from handling mistakes and losses which influence the ssDNA content of the supernatant and thus the calculated bound fraction. As a reference, classic single molecule photobleaching was applied according to Casanova et al.17 to quantify the immobilized ssDNA molecules on a single NP with the aim of comparing these results to bulk fluorescence measurements. The SMCM setup is described in detail in the Supporting Information (Figure S3). Previous experiments have indicated that the maximum fluorophore labeled ssDNA on a single NP, that can be differentiated by discrete energy levels, is limited to six molecules. To match the immobilization conditions of the bulk measurement, samples for the SMCM experiment were prepared by immobilizing different mixtures of fluorescent and nonfluorescent ssDNA with the following ratios: 1/20, 1/37.5, 1/75, and 1/150 with each time a total added ssDNA concentration of 25 nM. No significant effect of diluting fluorescent ssDNA with nonfluorescent ssDNA on the bioconjugation was observed for the bulk measurements, with respect to the dilution factor (Supporting Information, Figure S6). In a typical bleaching experiment, 20 μL of the ssDNA-NP sample was then dispensed on ozone treated cover glass and spincoated. The sample was scanned in the confocal setup to locate the NP in a 10 10 μm area, after which the laser (633 nm) was focused on a single NP (Figure 1A). For each sample with a different ratio labeled/unlabeled ssDNA, around 100 NPs were measured individually by stochastically photobleaching the fluorophores. The number of different energy levels in the stepwise decrease of the fluorescence intensity as a function of time are a measure for the number of fluorescent ssDNA molecules bound to the NP surface (Figure 1B). The fluorescence intensity steps are not equidistant and suggest a xyz component dependency of differently oriented dyes and hence a polarization effect. Based on these counting data, a histogram was created relating the number of NPs with their respective number of bound fluorescent ssDNA molecules (Figure 2A). Since the NPs without fluorescently labeled ssDNA are not visible on the confocal scan and, hence, not accounted for in the histogram, the number of empty NPs was determined by comparing the density of NPs on the fluorescence image to a regular optical transmission image of the same cover glass. No interfering impurities in the transmission or fluorescent (22) Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Elsevier: London, 2008. (23) Agresti, A. An Introduction to Categorical Data Analysis, 2nd ed.; WileyInterscience: New York, 2007.
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Figure 1. (A) 10 10 μm confocal scan of fluo-labeled ssDNA functionalized NPs. (B) Stepwise photobleaching of 3 fluorescent molecules on 1 NP until a baseline signal is reached.
Figure 2. (A) Histogram of the SMCM measurements showing the distribution of the number of NPs bioconjugated with 0-4 anchored fluorescent ssDNA molecules and corresponding fitted Poisson distribution (illustrated here for the dilution 1/37.5). (B) Average conjugated ssDNA per NP for the different fluorescence dilution ratios.
images were observed. A Poisson distribution23 was fitted to the histogram data, after correction for the nonfluorescent NPs, to estimate the average number (c) of immobilized fluorescent ssDNA molecules per NP using MATLAB software (The Mathworks). Using the ssDNA immobilization protocols mentioned above, the bulk fluorescence measurements reveal a total average number of 80 immobilized ssDNA molecules per NP (Supporting Information, Figure S2). However, the average number of fluorescent ssDNA molecules counted in the 1/20 ratio of fluorescent/ nonfluorescent ssDNA mixture with the SMCM setup was equal to 2.54, giving, accounting for a dilution factor of 20, a total of 51 DNA molecules on 1 NP. Similar experiments were repeated for the other dilution factors (Supporting Information, Table S5). The data are summarized in Figure 2B. As was suggested in the introduction, SMCM measurements with focused illumination are indeed unable to accurately account for the presence of all attached ssDNA. Instead, at best, two-thirds (68%) of the immobilized ssDNA could be counted as compared to the bulk fluorescence measurement. We believe the difference can be accounted for by polarization effects of the excitation laser light. Indeed, with an incoming light source perpendicularly oriented to the surface (along the z-axis), only ssDNA molecules, more specifically the orientation of the disklike fluorescent dye, oriented with a certain angle to the z-axis are appropriately susceptible to excitation (Figure 3A, gray cone) and are thus visible for counting. Because of the high N.A. (1.3) of DOI: 10.1021/la904702j
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Figure 4. (A) Stepwise photobleaching with mTIRF illumination of 5 fluorescent groups on 1 NP; fluoresence intensity expressed in kRFU per 0.28 μm2. (B) Histogram of K€ ohler photobleached NPs. (C) Histogram of mTIRF photobleached NPs.
Figure 3. (A) 3D artist impression and fluorescent microscopy image of NPs with K€ ohler illumination (gray cone represents unexcitable orientation, blue labels are fluorescing, red labels in the gray cone are not fluorescing). (B) 3D artist impression and fluorescent microscopy image of the same area as in (A) now excited with mTIRF after complete photobleaching with K€ ohler illumination. Size of the microscopy image is 24.6 24.6 μm2.
the objective, some z-axis polarization (up to 12.8%) might occur in the confocal setup.24-26 Nevertheless, the fluorescence intensity caused by excited z-axis oriented fluorophores remains small compared to the noise on the signal of xy-oriented fluorophores. If this hypothesis is valid, the missing fluorescence should be visible using the wide field fluorescence imaging technique with mTIRF. The principle of this technique is based on the creation of a highly inclined illumination with a substantial z-component combined with part of the laser beam remaining in K€ohler illumination. This total excitation contains all directions of polarization components. As a result, every fluorophore is excited irrespective of their orientation toward the laser.27 This mTIRF setup (Supporting Information, Figure S4) allows a depth of field excitation of several micrometers.28,29 Figure 3 illustrates the envisioned polarization effect. First, a laser with a high power beam (50 mW) was employed to illuminate the sample in K€ohler illumination modus (Figure 3A) to image the molecules with a substantial x-axis or y-axis component orientation. Imaging was continued until complete photobleaching of the dye molecules (24) Dedecker, P.; Muls, B.; Hofkens, J.; Enderlein, J.; Hotta, J. I. Opt. Express 2007, 15(6), 3372–3383. (25) Richards, B.; Wolf, E. Proc. R. Soc. London, Ser. A 1959, 253(1274), 358– 379. (26) Wolf, E. Proc. R. Soc. London, Ser. A 1959, 253(1274), 349–357. (27) Schneckenburger, H. Curr. Opin. Biotechnol. 2005, 16(1), 13–18. (28) Tokunaga, M.; Imamoto, N.; Sakata-Sogawa, K. Nat. Methods 2008, 5(2), 159–161. (29) Rocha, S.; Hutchison, J. A.; Peneva, K.; Herrmann, A.; Muellen, K.; Skjot, M.; Jorgensen, C. I.; Svendsen, A.; de Schryver, F. C.; Hofkens, J.; Uji, I. ChemPhysChem 2009, 10(1), 151–161.
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was achieved, leaving no detectable signal from the screened region above the background level. Then the angle of laser incidence was increased above the critical angle to establish a partial objective based mTIRF with the creation of the highly inclined illumination as a result. As can clearly be seen from Figure 3B, under the mTIRF conditions, some fluorescence indeed reappears, confirming our hypothesis. Thus, by counting the SMCM nonbleached fluorescent NPs in the mTIRF mode, we account for between 13% and 30% of the fluorescent ssDNA NPs with respect to the bulk measurements, respectively, for the 1/20 and 1/10 dilution factors. To further quantify the exact number of SMCM invisible fluorophores, a “counting-by-photobleaching experiment” was performed with confocal and mTIRF microscopy on the same sample with a dilution ratio of 1/25 (Figure 4A). The fluorescence intensity of multiple NPs on a 24.6 24.6 μm2 image was recorded simultaneously with a highly sensitive cooled CCD camera and integrated over a square region of 0.28 μm2, comprising all emitted light of one NP. By fitting a Poisson distribution on the histogram of the counted ssDNA molecules per NP in the combined confocal and mTIRF (Figure 4B, C) photobleaching measurements, we determined the mean of the distribution (c) to be 2.33 and 3.37, respectively. This amounts to a 31% difference in counted ssDNA. Since an average of 68% of fluorescent ssDNA was counted using the SMCM experiment compared to the bulk measurement, the combined experiment fairly adds up the missing ssDNA. A simple calculation reveals that 99% of the ssDNA is traced when combining both single molecule techniques. In conclusion, we have demonstrated a combined, general approach for accurate quantification of low numbers of bioconjugated ssDNA molecules on single nanoparticles. The single molecule microscopy approach was compared to bulk fluorescence measurements which are commonly applied to determine the density of bioconjugated molecules on the nanoparticles. The presented improved counting method is based on a simple stepby-step photobleaching of single molecules both in confocal and wide field mTIRF mode. The use of the latter technique accounts for the missing number of fluorescent molecules that are not excited and bleached in the confocal technique as a result of polarization effects. Adding up the measured ssDNA numbers resulting from both bleaching techniques fairly accounts for the total number of attached target biomolecules such as ssDNA. This counting method allows quantification, through Poisson modeling, of the number of bioconjugated molecules per NP in Langmuir 2010, 26(3), 1594–1597
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contrast to the bulk measurements that only give an average estimate for a complete batch of NPs. As the precise detection of small numbers of bioconjugated molecules is a prerequisite for many particle based theranostic applications, including cancer detection and treatment, single virus detection, and nucleic acid based biosensors, we believe the presented improved counting methodology is of value for many biorelated fields. Our combined counting approach has been demonstrated here for labeled ssDNA, but it certainly contributes to the characterization of a broader range of bioconjugated nanomaterials (e.g., gold, silver, silica, polysterene) and nanostructures (e.g., nanorods, nanoshells, and branched nanoparticles) potentially having a large impact on the development of new technologies with applications in medical and food diagnostics, drug delivery systems, and biopolymer mediated catalysis.
Materials and Methods Sample Preparation. Before functionalization of the silica NPs (Micromod, Germany) with ssDNA (Eurogentec, Belgium), the carboxyl modified NPs were washed in a 25 mM MES buffer (pH = 5, Sigma Aldrich), centrifuged, and decanted. A common method in biomolecule immobilization was used to covalently link ssDNA molecules to the cleaned NPs. Clean NPs were contacted with 12.5 mg/mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, Pierce Biotechnology) and 12.5 mg/mL N-hydroxysuccinimide (NHS, Sigma Aldrich), yielding active N-hydroxy succinimide pendant groups on the surface of the NPs, which spontaneously react with the amine of ssDNA to form covalent links in the next step. Variable ssDNA loadings were prepared simply by varying the concentration (in the nM range) of 50 -amine functionalized, 30 -Atto647N dye labeled ssDNA (15 nucleotides). The ssDNA modified NPs were collected and washed thoroughly in several solutions (once in a SSC-SDS buffer (pH = 7.0) supplemented with 0.5% SDS and
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twice in the MES buffer) to remove unbound ssDNA. As the ssDNA does not form a dense layer around the NP, nonspecific binding sites at the NP surface were blocked by incubating the ssDNA NPs in a new mixture of EDC/NHS together with 25 mM ethanolamine, resulting in a hydrophilic uncharged surface of pendant alcohol groups. A final washing step (once in SSC-SDS buffer and twice in PBS buffer) was applied before storage of the final ssDNA NPs in PBS buffer until measurement. Measurement. The supernatant was collected after each washing step, diluted to 1 mL, and stored for further analysis. Bulk fluorescence measurements were carried out to quantify the amount of unbound ssDNA in the supernatant after the different washing steps and of the bound DNA on the NPs. Samples were transferred to a microtiterplate and measured on a spectrophotometer (Spectramax M2e, molecular Probes) at an excitation wavelength of 600 nm, cutoff wavelength of 630 nm, and emission wavelength of 664 nm. Calibration curves linking the Atto-647N labeled ssDNA concentration to the fluorescence intensity in solutions were carefully set up taking into account possible buffer effects. A detailed description of the SMCM and mTIRF setup can be found in the Supporting Information.
Acknowledgment. This work was financially supported by the Institute for the Promotion and Innovation by Science and Technology (IWT)-Flanders (63437-63384), the Fund for Scientific Research (FWO)-Flanders (Grants G.0337.08 and G.0298.06), the Industrial Research Fund (IOF) KULeuven, and the Research Fund KULeuven (Centre of Excellence CECAT). Long term structural funding “Methusalem” by the Flemmish government is gratefully acknowledged. Supporting Information Available: List of abbreviations, experimental details, bulk fluorescence data, microscopy setups, and widefield fluorescence data summarizing table and bulk dilution data. This material is available free of charge via the Internet at http://pubs.acs.org.
DOI: 10.1021/la904702j
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