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Determination of pKa Values of Hydrophobic Colorimetric pH Sensitive Probes in Nanospheres Xiaojiang Xie, Jingying Zhai, Zdenka Jarolimova, and Eric Bakker Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04671 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on February 27, 2016
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Analytical Chemistry
Determination of pKa Values of Hydrophobic Colorimetric pH Sensitive Probes in Nanospheres Xiaojiang Xie*, Jingying Zhai, Zdenka Jarolímová and Eric Bakker* Department of Inorganic and Analytical Chemistry, University of Geneva, 30 Quai Ernest Ansermet, Geneva, Switzerland. Abstract. A simple and novel method is proposed here for the first time to determine pKa values of chromogenic hydrophobic pH sensitive probes directly in nanospheres. pKa values can be obtained by measuring the pH response of the nanospheres (containing the probes and ion exchanger) followed with + measuring the pH and Na responses of the nanospheres (containing solvatochromic dyes and ion exchanger). The pKa values of four chromoionophores were successfully determined. This method is in principle also applicable to characterize colorimetric probes in other water immiscible nanomaterials.
Introduction Optical pH sensitive probes have received considerable attentions from researchers in various fields including chemical sensors, biochemistry and environmental 1-5 science. The basicity (pKa values) and hydrophilicity of these compounds are fundamentally important limiting factors for their applications. Hydrophilic pH sensitive probes with pKa values in the physiological pH range have 6-8 been widely used in bioimaging. The pKa values of hydrophilic pH sensitive probes can be easily determined with titrations in homogeneous solutions according to the 9 Henderson-Hasselbalch equation. On the other hand, various hydrophobic pH sensitive probes (sometimes also + coined H chromoionophores) have been used to design chemical sensors for ions based on thermodynamic equilibria involving ion exchange (for sensing cations) or 10-16 ion coextraction (for sensing anions). Although the pKa values can be measured in organic solvent, they provide little relevance because the microenvironments (such as 17,18 polarity) in the nanosensors are usually very different. Recently, a method based on solvatochromic dyes has been introduced to determine stability constants of ion17 carriers inside the nanospheres. The basicity of + tridodecylamine, a colorless H ionophore, has been successfully measured. However, the method is difficult to apply to colored pH sensitive probes unless there are large spectra separations between the solvatochromic dyes and the chromoionophores. Therefore, methods to determine the pKa values directly inside the nanoscale
matrices are still highly desirable. Herein, we report for the first time on a new methodology based on solvatochromic + dyes to determine the pKa values of H chromoionophores directly in nanospheres.
Experimental Section Reagents. Pluronic F-127 (F127), bis(2-ethylhexyl) sebacate (DOS), methanol, boric acid, sodium phosphate monobasic, + sodium tetrakis-[3,5-bis(trifluoromethyl)phenyl]borate (Na R ), chromoionophore I (CH I), chromoionophore III (CH III) and citric acid were purchased from Sigma-Aldrich. Ox R, Ox B and the solvatochromic dyes were synthesized in 19,20 house as previously described. The chemical structure of the solvatochromic dye SD2 used in Figure S1 is shown in Supporting Information. Nanosensor Preparation Typically, nanospheres were prepared by dissolving 8.0 mg of DOS, 5.0 mg of F127, and appropriate amounts of chromoionophore, ion exchanger and solvatochromic dyes if necessary (see table S1 in Supporting Information) in 3.0 mL of methanol to form a homogeneous solution. 0.5 mL of this solution was pipetted and injected into 4.5 mL of deionized water on a vortex with a spinning speed of 1000 rpm. The resulting clear mixture was blown with compressed air on the surface for at least 30 min to remove methanol, giving a clear particle suspension. Instrumentation and Measurements The absorbance was measured with a UV-Vis spectrometer (SPECORD 250 plus, Analytic Jena, AG, Germany) using disposable poly(methyl methacrylate) cuvettes with path length of 1 cm as sample container. Fluorescence responses of the nanosensors were measured with a fluorescence spectrometer (Fluorolog3, Horiba Jobin Yvon). For the calibration of the nanosensors, stock solutions were added stepwise to reach the desired concentration, causing negligible volume variation. Universal pH buffer was prepared by dissolving appropriate amounts (5 mM) of citric acid, boric acid and
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sodium phosphate monobasic. The nanosphere suspensions were mixed with the buffer solutions to reach a 2.5 mM buffer concentration.
Results and Discussion +
The H chromoionophores (Ind in Scheme 1) and ion exchanger sodium tetrakis-[3,5+ bis(trifluoromethyl)phenyl]borate (Na R ) were dissolved in the plasticizer bis(2-ethylhexyl) sebacate (DOS) and assembled inside the hydrophobic core of polymeric spheres composed of a triblock copolymer Pluronic F-127 21 by a precipitation method described by Xie et al. These nanospheres exhibit a typical radius from 20 to 50 nm. This matrix has been adopted to fabricate various ion+ + 2+ selective nanosensors including Na , K , Ca and 21-23 protamine. Here, the pH responses of the nanospheres arise from the ion transfer equilibrium as expressed in Scheme 1a (and Eqn. 1, where ns and aq designate the nanosphere and aqueous phase, respectively). The acid dissociation constant Ka is defined in Eqn. 2. Experimentally, the pH response of the nanospheres can be evaluated in absorption mode at a + fixed Na background. From this experiment, the overall
Figure 1. pH and Na+ responses of the nanospheres incorporating solvatochromic dye (SD+) and ion exchanger (R-) in fluorescence mode.
equilibrium constant K can be readily obtained. However, to determine Ka independently, one still has to know the +
+
exchange constant k H , Na which is the equilibrium constant + + for the ion exchange between uncomplexed H and Na at the nanosphere-water interface. For that purpose, solvatochromic dyes previously reported for ionophore20 As shown in based nanosensors were employed. Scheme 1b, in the next step, the nanospheres were + initially loaded with solvatochromic dye (SD ) and R . The + + aqueous concentrations of the Na or H surrounding the nanospheres were then gradually increased while the fluorescence of the suspension was monitored. In such a + + + process, Na or H gradually displaces the SD in the nanospheres. As shown in Figure 1, this results in a + decrease in fluorescence intensity because the SD inside the nanospheres is much brighter than in aqueous phase. The fluorescence response ranges were dictated by the +
+
+
+
ion exchange constants k SD , Na and k SD , H , which were obtained by fitting the experimental data. Here, the value +
0.9
+
of k H , Na was calculated as 10 according to Eqn. 3 where + + + + + + aNa , aH and aSD are the activity of Na , H and SD in the aqueous phase, respectively, and the bracketed terms are the concentrations in the nanospheres.
+
Ka =
k Na
+
,H +
=
[ H ns+ ][ Ind ns ] k H , Na = [ HInd ns+ ] K
[ H ns+ ] aNa + [ SDns+ ]aNa + = aH + [ Nans+ ] aSD + [ Nans+ ]
+
(2) +
+
[ SDns+ ]aH + k SD , Na = + + aSD + [ H ns+ ] k SD , H
(3)
In principle, the methodology is also applicable to other water immiscible matrices including decyl methacrylate, 24-26 poly(vinyl chloride) (PVC) and polycaprolactone. Scheme 1. (a): Schematic illustration for the acid dissociation and pH response for nanospheres incorporating chromoionophores (Ind) and ion exchanger (not shown for simplicity). (b): Schematic illustration for the ion exchange processes for the nanospheres incorporating solvatochromic dye (SD+) and ion exchanger with changing background in Na+ concentration or pH.
Figure 2 shows the UV-visible absorption spectra and the calibration curves of the nanospheres in the presence of 0.1 M NaCl, where 1–α is the mole fraction of protonated chromoionophore. Fitting the experimental data to 27 classical ion exchange theory then allows one to obtain the overall equilibrium constant K.
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Analytical Chemistry
(a)
(b)
(f)
(e)
(c)
(d)
(g)
(h)
Figure 2. (a, b, c, d): UV-visible absorption spectra measured at 0.1 M NaCl background and various pH for pH sensitive nanospheres incorporating ion exchanger and various chromoionophores as indicated. Arrows indicate increasing pH values. (e, f, g, h): pH calibration using 1α (degree of protonation) calculated from the corresponding maximum absorbance. The blue sigmoidal curves were simulated from classical ion exchange theory with a single pKa value while the red curves were obtained by further considering multiple pKa values for the chromoionophores.
It was noted that the theoretical response curves modelled from classical ion exchange theory (blue curves in Figure 2b) apparently deviated from the experimental calibration data. This does not mean that the method introduced here is wrong. Classical ion exchange optode theory was developed based on relatively thick (micrometer scale) polymeric films in which the chromoionophores were 27 assumed to exhibit a single pKa value. However, at the nanoscale, the chromoionophores may exhibit a range of pKa values depending on their distribution in the nanospheres (close to the surface or in the interior). It has been well recognized that pKa shifts do exist for various dyes, proteins and drug molecules distributed in micellar 28-30 Here, improved fits (red curves in Figure structures. 2b, see Supporting Information for parameters) were obtained by accounting for multiple pKa values for the chromoionophores in the nanospheres. These observations indicated that the chromoionophores could + indeed have different affinities for H depending on the actual locations in the nanospheres. Table 1. pKa values of the chromoionophores determined in the nanospheres A
B
Chromoionophore pKa pKa CH I 8.0 ± 0.1 7.9 ± 0.2 CH III 9.2 ± 0.1 9.3 ± 0.2 Ox R 6.3 ± 0.1 6.4 ± 0.2 Ox B 6.3 ± 0.1 6.5 ± 0.2 A Calculated according to the red curves in Figure 2 B Calculated according to the blue curves in Figure 2 With this in mind, the pKa values of the chromoionophores were readily calculated from Eqn. 2, see values presented
in Table 1. Notably, the pKa values are much smaller than 31 those determined in plasticized PVC membranes. In a previous study with other receptors, the stability constants for ion-carriers were also found to be much smaller compared with the values determined in PVC membranes and the result was attributed to the more polar nature of 17 the nanospheres compared with PVC membranes. The observation here of reduced pKa values further confirms this. Among the chromoionophores investigated here, Ox R exhibited a minimum spectra overlap with the solvatochromic dye (SD2 in Supporting Information). Therefore, the pKa value of Ox R was also measured with the previously described method and found as 6.1 ± 0.2 (See Figure S1 in Supporting Information). Even so, the analysis of the emission spectrum showed some difficulty in a certain pH range (See Supporting Information for discussion). The proposed method, however, does not suffer from this drawback.
Conclusions In conclusion, a methodology was proposed for the first time to determine the pKa values of hydrophobic colorimetric pH sensitive probes (such as chromoionophores) directly in nanospheres. The method is in principle applicable in various water immiscible nanomaterials. A comparison between the experimental pH response and the theoretical response curves of the nanospheres also indicates that the chromoionophores may have a range of basicities in the nanospheres.
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Associated Content Supporting Information Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
Author Information Corresponding Author *
[email protected] *
[email protected] Notes The authors declare no competing financial interest.
Acknowledgements The authors thank the Swiss National Science Foundation (SNF) and the University of Geneva for financial support of this study. J.Z. gratefully acknowledges the Chinese Scholarship Council.
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