Multivariable Response of Semiconductor Nanocrystal-Dye Sensors

Nov 16, 2010 - Department of Chemistry, University of Illinois at Chicago, 845 West ... QDs from pH-Sensitive Single-Particle and Ensemble Fluorescenc...
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Multivariable Response of Semiconductor Nanocrystal-Dye Sensors: The Case of pH Joel D. Krooswyk, Christina M. Tyrakowski, and Preston T. Snee* Department of Chemistry, UniVersity of Illinois at Chicago, 845 West Taylor Street, Chicago, Illinois 60607-7061, United States ReceiVed: September 28, 2010; ReVised Manuscript ReceiVed: October 25, 2010

Emissive semiconductor nanocrystals can be efficient FRET donors to chemically responsive organic fluorophores; conjugating the two creates a ratiometric (or self-calibrating) sensor. Given that the organic component engenders chemical and/or biological sensitivity, it is natural to assume that the NC-dye response is reflective of the photochemical properties of the neat dye. In the case of a pH sensor like fluorescein, the dye emission can be analytically described by the Henderson-Hasselbalch equation. Our research demonstrates that the ratiometric sensing response of CdS/ZnS-fluorescein pH sensors, however, does not directly reflect the Henderson-Hasselbalch relationship and is difficult to predict. The reasons for this complexity are the alternations of the dye microenvironment within the nanoscale architecture and the nature of energy transfer from the NC donor to the acceptor dye. Introduction The use of water-soluble emissive semiconductor nanocrystals (NCs) for biological and chemical sensing applications has become a significant area of research in nanoscience.1-4 Once issues with water solubilizing hydrophobic colloidal core/shell CdSe/ZnS NCs were addressed in 1998,5,6 the large number of reports that followed amply demonstrated the robustness of cadmium chalcogenide NCs and other related materials7-9 for biological imaging applications.10 The most significant advantages for using NCs in bioimaging are NC photochemical stability and the fact that NCs do not require complex or costly excitation schemes. However, there are many challenges to address concerning the implementation of semiconductor nanotechnology for biological imaging applications. The difficulties with cellular delivery of NCs are well-known,11-13 although significant advances in this area are being made.14-16 Furthermore, it is not easy to engender an optical response toward specific analytes in the NCs’ local environment. This is due to the surface passivation of most nanomaterials, such as the ubiquitous CdSe/ZnS NCs.17,18 If properly synthesized, these materials have no direct interaction with their environment, which is actually the source of their optical durability. As fluorescent chemical and biological sensing with organic dyes is a well-developed technology,19-23 combining the photochemical stability of an inorganic NC material with the responding capability of an organic dye represents a novel solution toward creating NC-based chemical and biological sensors. Many groups have demonstrated that emissive NCs can be used as chemical and biological sensors by indirectly imparting an optical response to analytes through Fo¨rster Resonant Energy Transfer (FRET).3,24-28 In most schemes, the FRET efficiency from a NC donor to an acceptor is engineered to be a function of environmental variable(s) as summarized in several recent reviews.2-4 In our method, we conjugate pH-insensitive CdS/ ZnS NCs29,30 to pH-sensitive fluorescein dye as demonstrated in our earlier report.25,31 The emission of the NCs is designed to overlap the absorption of the fluorescein dye to ensure efficient energy transfer, although we note that the absorption * To whom correspondence should be addressed. E-mail: [email protected].

spectrum of fluorescein is pH dependent. Thus, the FRET efficiency from the NC donor to the acceptor dye is modulated by pH as the dye’s absorption moves into or out of resonance with the NC’s insensitive emission spectrum. As a result, we can quantitatively measure the pH as a function of the ratio of the NC-to-dye emission intensity.25 In contrast, organic sensing dyes alone generally display a single response (i.e., brighten or darken) in the presence of analytes, this may be difficult to quantify and calibrate in complex biological environments. As many organic sensing dyes have absorption spectra that are functions of their chemical environments, the NC-based FRET pH sensing motif we study may be generalized for many analytes. Our scheme may lead one to conclude that any particular NC-dye sensing system can be created as long as care is taken in choosing appropriate chemically responsive organic dyes that are FRET acceptors (under some condition) for bright, water-soluble semiconductor NC donors. With the materials in hand, the NC and dye must be conjugated using one of the many methods that have been developed for this purpose;31-33 the resulting NC-dye couple should exhibit an analyte-specific optical response dictated by the sensing organic component. We have studied this assumption by examining the emission of ratiometric CdS/ZnS-fluorescein pH sensors over incremental (0.1) changes in pH from 5.0 to 8.0 in a series of phosphate buffer solutions. We can demonstrate that the response of NC-dye FRET sensors is highly complex and cannot be easily predicted from the photochemical properties of the dye alone. Given that robust surface passivated NCs are generally insensitive to their environment, the response of a NC-dye sensor must be due to the organic component. In our example, the pH-dependent absorption and emission spectra of neat fluorescein are dictated by the dye’s pKa (∼6.4).34 We first study the pH-dependent fluorescence response of fluorescein iodoacetamide to compare to the same dye when conjugated to 40% octylamine modified RAFT poly(acrylic acid);31 this dyeconjugated polymer is also used to coat CdS/ZnS NCs to create ratiometrically reporting NC-dye pH sensors. We have also varied the size of the NC donor to ascertain the effect of donor emission on the pH sensing response.

10.1021/jp1093096  2010 American Chemical Society Published on Web 11/16/2010

Semiconductor Nanocrystal-Dye Sensors Experimental Methods All chemicals were obtained from commercial sources and were used as received unless noted otherwise (Supporting Information for details). CdS/ZnS NC Synthesis and Water Solubilization. Fluorescein functional CdS/ZnS NCs were synthesized by a modification of our previously reported procedure.31 Core CdS and core shell CdS/ZnS nanocrystals were synthesized according to published protocols.29,30 CdS/ZnS samples were processed by addition of excess isopropanol to precipitate the NCs. The slurry was centrifuged and the supernatant discarded. The CdS/ ZnS NCs were washed with excess methanol, centrifuged, and dried under reduced pressure. These NCs were then dissolved in ∼4 mL chloroform with a portion (5× relative weight) of amphiphilic 40% octylamine modified RAFT poly(acrylic acid) polymer.31 After drying, the sample was dissolved in basic buffer and purified by dialysis with Millipore 100K MW cutoff concentrating filters. The low molecular weight poly(acrylic acid) polymer used in this process was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization;35 approximately 40% of the carboxylic acid groups of the polymers are conjugated with octylamine to create an amphiphilic NC-solubilizing polymer. CdS/ZnS-Fluorescein Dye Conjugation. The pH-sensitive, thiol-reactive fluorescein iodoacetamide dye was synthesized according to recently published procedures.36-38 The iodoacetamide functionality can be conjugated to RAFT polymer encapsulated NCs as the RAFT method imparts a thiol at the head of each polymer. The presence of thiol groups on the polymers was confirmed with Ellman’s Reagent, 5-5′-dithiobis(2-nitrobenzoic acid). Previously, we functionalized CdS/ZnS with fluorescein maleimide;31 in our present report, fluorescein iodoacetamide was chosen due to its better reactivity with RAFT octylamine modified PAA polymers. We found that overnight mixing of fluorescein iodoacetamide with water-soluble CdS/ ZnS NCs, coated with 40% octylamine modified RAFT poly(acrylic acid), had a low conjugation yield. To improve the reaction efficiency, a milligram scale quantity of NaBH4 was added to freshly prepared aqueous CdS/ZnS NCs and stirred overnight; NaBH4 and byproduct were removed through dialysis with 100K MW cutoff concentrating filters. Fluorescein iodoacetamide was added such that the emission of the dye dominated that of the NC; generally this results in a near-even fluorescence from both chromophores after processing. The sample was stirred overnight and processed with 100K MW cutoff concentrating filters to remove excess dye. We found that the conjugation efficiency was improved significantly in this procedure. Using dithiothreitol in place of NaBH4 causes severe quenching of the CdS/ZnS NCs. A control sample of dye functionalized RAFT polymer was synthesized. The 40% octylamine modified RAFT poly(acrylic acid) polymer was dissolved in water along with a small (submilligram) portion of fluorescein iodoacetamide in DMF and stirred overnight. The polymer was precipitated out with acid (pH < 5) and redispersed in basic water in several cycles to remove unreacted dye. pH-Dependent Emission/Absorption Studies. The emission and absorption of each sample was studied in thirty different phosphate buffered solutions with a pH range of 5 to 8 in 0.1 increments. Each set of data was taken on the same day. For each pH, 100 µL of a stock solution of dye-labeled samples in deionized water was added to a quartz cuvette filled with ∼2.1 g of phosphate buffer. UV-vis absorbance spectra were taken using a Varian Cary 300 Bio UV-vis Spectrophotometer.

J. Phys. Chem. C, Vol. 114, No. 49, 2010 21349 SCHEME 1: Relatively Dark Acidic and Brighter Basic Forms of Fluorescein Iodoacetamide Conjugated to a CdS/ZnS NCa

a Whereas the neat dye has a pKa of 6.4, our data demonstrate that binding the dye to a CdS/ZnS NC significantly changes the overall optical response to pH.

Fluorescence emission spectra were taken using a customized Fluorolog (HORIBA Jobin Yvon) modular spectrofluorometer. The absorbance of all solutions was kept near or below 0.1 OD at the excited wavelength to avoid inner-filtering effects. All data and fitting of spectra were performed with the Matlab suite of packages. Gaussian functions were used to fit the NC portion of the NC-dye spectra; the NC component was then subtracted out leaving the dye component. The dye component was then integrated and divided by the area of the NC Gaussian fits to calculate the ratio of the integrated dye emission divided by the integrated NC emission. Polymer Swelling. A FRET assay was used to ascertain whether the amphiphilic 40% octylamine modified RAFT poly(acrylic acid) polymer used to coat our NCs swells as a function of pH. First, a set of CdS/ZnS NCs was water solubilized according to the procedure discussed above. Next, rhodamine B piperazine and BODIPY TR cadaverine dyes were simultaneously attached to the water-soluble NC sample using our recently developed carbodiimide coupling protocol.32 The 580 centered emission of rhodamine B is well overlapped with the broad, 588 absorption of BODIPY TR such that the rhodamine B should be a good FRET donor to the BODIPY TR. After processing to remove excess dye, the emission of this sample of CdS/ZnS-dyes was measured in pH 6 and pH 8 buffer. The excitation wavelength of 560 nm was chosen such that the NC would not absorb energy; furthermore, the rhodamine B dye donor has a stronger absorption at this wavelength compared to the acceptor BODIPY although some direct excitation of the acceptor must be occurring due to the significant width of the acceptor dye absorption. Results and Discussion To develop a molecular-level description of NC-dye sensing, we first examined the neat dye emission as a function of pH as a control. While it is well-known that fluorescein is bright in base and dark in acid (Scheme 1), we noticed that the emission profile is also a function of pH. This fact allowed us to calculate the absolute emission spectra of the pure acid and base forms of fluorescein by first noting that the wavelength-dependent emission can be decomposed as:39

Itotal(ω,pH) ) [A]Ia(ω) + [B]Ib(ω) where Itotal (ω,pH) is the pH-dependent emission spectrum, Ia(ω) and Ib(ω) are the emission spectra of the pure acid and base forms of fluorescein, and [A] and [B] are the concentrations of

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Figure 1. A. Emission of fluorescein iodoacetamide (green) at pH 6.0. The pure acid emission Ia(ω) (red) and pure base Ib(ω) (blue), when weighted appropriately, can be summed (black dashed) to reproduce the data. B. The apparent pH calculated from decomposing the emission spectra into pure acid and base forms versus the pH of the buffer for neat and polymer-bound fluorescein dyes.

Figure 2. A. pH-dependent emission from sample 1 and B. sample 2. Inserts demonstrate the integrated dye/NC emission ratio as a function of buffer pH.

the pure acid and base forms of fluorescein, respectively. For the neat dye in buffer, [A] and [B] are calculated by the Henderson-Hasselbalch equation:40,41

pH ) pKa + log([B]/[A]) We decomposed the spectra of the neat dye at pH 5 and 8 into the pure acid and base components given the known concentrations of those species from the Henderson-Hasselbalch equation. Essentially, the two intensities from the pH 5 and 8 spectra at a given wavelength were used to solve a system of two equations with two unknowns; the pure acid and base spectra are calculated in this manner on a point-by-point basis. These spectra are shown in part A of Figure 1, where we also demonstrate that a weighted sum of the pure acid and base spectra can be used to fit the emission of fluorescein at any other pH. Further, the ratio of the weights can be used to calculate log([B]/[A]) in the Henderson-Hasselbalch equation to determine the apparent pH. As shown in part B of Figure 1, the buffer pH and apparent pH (calculated from spectral decomposition) for the neat dye are strongly correlated as demonstrated by the nearly unit slope (0.96 ( 0.02) of the best fit line. This analysis technique was used to probe the response of the sensing dye in other microenvironments as discussed below. To synthesize a CdS/ZnS-fluorescein pH sensor, the NCs must first be water solubilized either through cap exchange6 or polymer encapsulation,42,43 and subsequently be conjugated to the dye. We have used the encapsulation method to coat CdS/ ZnS NCs with 40% octylamine modified RAFT poly(acrylic acid),31,43 disperse them in water, and conjugate them to fluorescein iodoacetamide via the RAFT polymer’s thiol functionality.31 BeforeexaminingthecomplexresponseofNC-fluorescein

couples, we first studied the pH-dependent emission of the dye bound to the polymer alone. The emissive response of the polymer-bound dye dissolved into excess phosphate buffer solution was measured as a function of pH. Each emission spectrum was decomposed into a weighted sum of the pure acid and pure base spectra; these weights were then used to calculate the apparent pH as discussed above. Unliketheneatdye,strongdeviationsfromHenderson-Hasselbalch behavior are observed in the emission from the polymer-bound dye as shown in part B of Figure 1. The slope of the best fit line (0.38 ( 0.01) is far from unity unlike the neat dye; these results demonstrate that the dye microenvironment has been significantly altered by attachment to the polymer. Thus, the dye’s sensitivity to the environment is no longer as simple as that expected from the neat dye alone. Obviously, this perturbation of the dye’s sensing properties must also alter the response of NC-dye coupled chromophores as well. These data show that the response of a sensing organic chromophore incorporated into a nanoscale architecture is not predictable based on the neat dye’s chemical and photophysical properties alone. The fact that the NC-to-dye FRET efficiency is tunable by altering the NC emission adds a second layer of complexity. We have synthesized two CdS/ZnS samples, one emitting at 460 nm (sample 1) and 480 nm (sample 2), that were water solubilized and conjugated to fluorescein iodoacetamide to explore the effect of the NC donor spectrum on the pH sensing response. Shown in parts A and B of Figure 2 are the emission spectra of these two CdS/ZnS NC-fluorescein coupled chromophores as a function of pH. A clean isosbestic point is observed in both cases, the inserts also demonstrate ratiometrically calibratable responses of the sensors to pH (i.e., integrated dye emission divided by the integrated NC emission). There is no evidence of sigmoidal shapes to the ratiometric

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Figure 3. A. Integrated neat dye emission and dye/NC emission ratio (sample 1) versus pH. The fit to the neat dye data is derived from the Henderson-Hasselbalch equation. B. A fit to the NC/dye emission ratio through Fo¨rster theory where the NC-to-dye distance is a function of pH as shown in the insert (Supporting Information for details).

Figure 4. A. Emission of the neat dye, polymer-bound dye, and the NC-dye conjugate (sample 1) at pH 6.4, the nominal pKa of fluorescein dye. B. A comparison of the normalized pure acid and pure base spectra and the dye spectrum from a 460 nm emitting NC-dye conjugate (pH 6.4, sample 1).

responses as one would expect if the sensing response is dictated solely by the photophysical properties of the neat dye. The ratiometric response of the 460 nm emitting CdS/ZnS NC-fluorescein dye couple (sample 1, part A of Figure 2) is very different compared to sample 2 as shown in part B of Figure 2. The shorter wavelength emission from sample 1’s CdS/ ZnS NC donor has greater overlap with the acidic form of fluorescein resulting in significant dye emission at lower pHs (Figure S1 of the Supporting Information). Conversely, this is also why sample 2 displays almost no dye emission below pH ∼5.5. Furthermore, the ratiometric response of sample 1 displays some curvature, whereas the response of sample 2 appears almost completely linear over the pH range under study. Consequently, the donor-emission-dependent FRET efficiency appears to have a significant impact on the response of the coupled chromophores. The pH-dependent neat dye integrated emission and dye/NC integrated emission ratio of sample 1 are plotted together in part A of Figure 3 to illustrate their significant differences. The neat dye integrated emission follows that predicted from the Henderson-Hasselbalch equation, whereas the ratiometric response of the CdS/ZnS-fluorescein sensor only has a generally increasing (and somewhat nonlinear) trend. We have found that simulating the dye/NC integrated emission ratiometric response using the pH-dependent NC-to-dye FRET efficiency from Fo¨rster theory does not predict the data shown in part A of Figure 3 regardless of whether we use the buffer pH or the apparent polymer-bound dye pH (part B of Figure 1). The data may be fit, however, if we take into account the fact that poly(acrylic acid) containing copolymers are known to (de)swell as a function of pH and ion content.44 We have also verified that the polymer coating we use to water solubilize CdS/ZnS NCs swells with increasing pH (Supporting Information). Thus, the NC-to-dye distance must be used as a parameter in fitting

the data, which is fortunately simplified by the fact that NC-donor/dye-acceptor FRET efficiency is known to have a normal 1/r6 efficiency dependence despite the fact that the NC is not a point dipole.45 Shown in part B of Figure 3 is the fit to the data where the NC-to-dye distance is altered with pH as shown in the insert; the accuracy of the fit demonstrates that an environmentally sensitive NC-to-dye distance represents a third layer of complexity in the response of NC-dye sensors. The same observation was made in the fit to the optical response of sample 2; these data and further details on the fitting procedures can be found in the Supporting Information. The most interesting aspect of these data is that our fitting requires the NC-to-dye distance to decrease with increasing pH, despite the fact that the polymer swells in base. While difficult to reconcile, we propose the dye becomes embedded within interior pockets of the polymer that form from the polymer’s swelling. Such a mechanism may be possible due to the repulsive negative charges of both the polymer and dye, and the fact that the dye lies solely at the head of the RAFT synthesized polymer; we plan to make further investigations on this phenomenon in the future. As a final illustration of the complex behavior of NC-dye coupled chromophores, we compare the normalized dye emission from the neat dye, the polymer-bound dye, and the NC-dye (sample 1, where the 460 nm emitting NC fluorescent component has been removed by subtracting out the blank NC spectrum) at the nominal fluorescein pKa of 6.4. These data are shown in part A of Figure 4. The spectra are not identical despite the common pH of all three solutions; the dye bound to the polymer displays characteristics that appear too basic, whereas the opposite is true for the dye bound to the NC. In fact, when compared to the pure acid and pure base fluorescein spectra, the dye emission component of sample 1 at pH 6.4 appears to be entirely from the pure acid form as illustrated in part B of

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Figure 4. This may be accounted for by the overlap of the ∼460 nm emitting CdS/ZnS NC with the pure acid form of fluorescein (Figure S1 of the Supporting Information), which has a blueshifted absorption compared to the more emissive, pure base form of the same dye.46 Conclusions To summarize, we have studied the pH response of CdS/ ZnS-fluorescein coupled FRET sensors in comparison to the neat dye. The results demonstrate that the intrinsic dye properties are not a good metric for accurately predicting the response of coupled NC-chromophore sensors. First, conjugating the organic dye into a nanoscale architecture significantly alters the response of the dye to its environment. We contend that this is a result of electrostatic properties of the highly anionic NC water solubilizing polymer that effectively screens the sensing dye from its environment. Second, the complex nature of the overlap of the NC emission with the acceptor dye absorption that defines the FRET efficiency has a significant effect. Not only is the ratiometric response a function of the NC-dye spectral overlap, the dye spectrum itself is altered by the same factor. Third, the micelle that encapsulates the NC may (de)swell due to environmental factors in such a way as to alter the NC-to-dye distance and thus FRET efficiency; although the same might not be true using other polymer NC coating techniques.47,48 Overall, these results show that one cannot assume that the photochemical properties of a pH sensing dye as described by the Henderson-Hasselbalch formula will translate into the same response in a NC-dye ratiometric sensor. Whereas we have used a pH reporting construct as an example, similar results may be demonstrated in other NC-dye sensor systems. Acknowledgment. We thank the University of Illinois at Chicago for financial support of this work. Supporting Information Available: Data analysis procedures, overlap of the emission of samples 1 and 2 with the absorption of fluorescein, absorption of samples presented in part A of Figure 4, and additional spectral data on sample 2. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Zhang, F.; Ali, Z.; Amin, F.; Riedinger, A.; Parak, W. J. Anal. Bioanal. Chem 2010, 397, 935–942. (2) Somers, R. C.; Bawendi, M. G.; Nocera, D. G. Chem. Soc. ReV. 2007, 36, 579–591. (3) Willard, D. M.; Mutschler, T.; Yu, M.; Jung, J.; Van Orden, A. Anal. Bioanal. Chem 2006, 384, 564–571. (4) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435–446. (5) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013–2016. (6) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016–2018. (7) Bharali, D. J.; Lucey, D. W.; Jayakumar, H.; Pudavar, H. E.; Prasad, P. N. J. Am. Chem. Soc. 2005, 127, 11364–11371. (8) Kim, S. W.; Zimmer, J. P.; Ohnishi, S.; Tracy, J. B.; Frangioni, J. V.; Bawendi, M. G. J. Am. Chem. Soc. 2005, 127, 10526–10532. (9) Pradhan, N.; Battaglia, D. M.; Liu, Y. C.; Peng, X. G. Nano Lett. 2007, 7, 312–317. (10) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538–544. (11) Delehanty, J. B.; Mattoussi, H.; Medintz, I. L. Anal. Bioanal. Chem. 2009, 393, 1091–1105.

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