Polyacrylate Microspheres for Tunable Fluorimetric Zinc Ions Sensor

Dec 2, 2013 - ... Polypyrrole Nanospheres - Synthesis and Properties of “Wireless” ... Analytical advantages of copolymeric microspheres for fluor...
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Polyacrylate Microspheres for Tunable Fluorimetric Zinc Ions Sensor Emilia Woźnica, Krzysztof Maksymiuk, and Agata Michalska* Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland S Supporting Information *

ABSTRACT: A novel concept of optical fluorimetric sensing using polymeric microspheres is explored on example of zinc ions sensors. The novel approach proposed uses the advantage of concomitant presence in a microsphere of two compounds: a receptor, fluorescently silent complexing ligand and an optical transducer, fluorescent compound. Binding of the analyte by the ligand affects its absorption spectrum, leading to decrease of the free ligand absorption and increase of complex absorption band. The decrease of free ligand absorption exposes emission of the transducer, yielding increase in fluorescence intensity on analyte concentration increase. This approach was verified experimentally using Zn2+ as a model analyte, the fluorimetric sensor obtained uses 1-(2-pyridylazo)-2naphthol (PAN) as analyte sensitive receptor and pyrene as optical transducer. In the absence of zinc ions in the sample emission of pyrene embedded in the spheres was significantly quenched, whereas increase of Zn2+ ions concentration in the sample resulted in dependence of fluorescence intensity on logarithm of zinc ions concentration in extraordinary wide range, from 10−7 to 0.1 M. The response mechanism was explained by surface accumulation of zinc ion−PAN complex on the microsphere/ sample solution interface. It was also shown that introduction of cation-exchanging sites to the microspheres significantly alters the responses pattern leading to high sensitivity over relatively limited concentration range (3−4 orders of magnitude). In the latter case the observed responses can be tuned to occur in chosen concentration range, simply by adjusting sample pH.

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istic and an optical signal transducer.12 A nonfluorescent ligand of high affinity to Zn2+ has been linked with quantum dots leading to fluorometric zinc ions sensors.13,14 An azamacrocyle ligand conjugated with quantum dots13 has resulted in decrease of their fluorescence. Upon coordination of Zn2+ ions within the azacrown ligand the increase of fluorescence intensity was observed, allowing assessment of zinc ions concentration in samples with simulated physiological medium spiked with zinc, within the range 5−500 μM.13 Similarly, conjugation of quantum dots with zincon ligand14 resulted in quenching of nanoparticles fluorescence. Upon coordination of Zn2+ the fluorescence response of nanoparticles was observed, with linear dependence of fluorescence intensity on zinc ions concentration within the range 10−1000 μM.14 Zinc ions sensors employing quantum dots conjugates with water-soluble complexones as azamacrocyles or zincon, are rather nontypical examples of a large group of sensors employing for recognition highly selective (nonfluorescent) ligands developed for lipophilic environment of ion-selective electrodes membranes. These type sensors originate from membranes applied in spectropotentiometric detection mode, reported nearly 25 years ago.15 Apart from an ionophore highly selective for target ions, these type membranes contain also a chromoionophore, H+ selective ligand, of different absorption

inc ions are important analyte from the point of view of biochemistry and medicine. The need for reliable methods applicable for biological samples that will allow quantification of Zn2+ ions, especially in wide concentration range, stimulates the research. Taking into account envisaged applications, especially interesting are zinc ions sensors/probes operating in fluorimetric mode.1−3 Many of the research works reported require relatively challenging synthetic approach to prepare new probes or synthetically modify existing ones as, for example, shown in recently published papers, for example, refs 1−10. Some of proposed fluorescent probes were developed for direct application in solution analysis [e.g., refs 5, 7, and 10], others were intended for immobilization in polymeric films [e.g., refs 4 and 8]. Most often thus proposed systems cover relatively narrow zinc ions concentration range from micromolar to millimolar. This approach, although elegant, apart from demanding synthesis, is often difficult to follow/apply in other analytical systems (e.g., in other media) than originally proposed. Thus, an alternative way to prepare sensors is to try to employ together two fluorescent compounds, one of known, high selectivity toward zinc ions. For example, two fluorescent dyes, one sensitive to zinc ions and the other one used as a reference, were embedded within the polyacrylamide nanostructures11 resulting in a ratiometric sensor operating within micromolar concentration range. Other fluorimetric zinc ions sensors, incorporate a recognition element: a complexing ligand of known character© 2013 American Chemical Society

Received: July 10, 2013 Accepted: December 2, 2013 Published: December 2, 2013 411

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spectra of protonated and deprotonated form.15 Deprotonation of H+ ionophore upon binding of analyte cation within the sensor phases (for constant number of ion-exchanging sites within the polymeric moiety together with keeping the pH of the sample constant to ensure stable degree of protonation of chromoionophore in the absence of analyte) yields change of absorption spectra and allows quantification of the analyte.15 This concept was further developed yielding fluorimetric sensors, taking the advantage of the increase of fluorescence emission accompanying deprotonation of chromoionophore.12,16 Using this approach, different fluorimetric sensors have been prepared including microspheres based ones, for example, potassium17 or sodium18 ones. Many of those have used polacrylate moiety as the lipophilic environment of choice [e.g., ref 17], applying micro- or nanospheres. The latter seems to be especially useful for development of small fluorescent probes,12,17 as their preparation is much easier than preparation of poly(vinyl chloride) beads.18 In this work, we introduce a novel concept of preparation of microspheres based fluorimetric sensors, targeting zinc ions as analyte. Instead of using H+ ions sensitive chromoionophore/ fluorophore and ionophore in the presence of ionic sites as an optical transducer and recognition element, respectively, we have applied a fluorescent dye as a transducer and a commercial complexometric ligand as a recognition element. As the absorption spectrum of the ligand−analyte complexes is different from that of free complexon, the appropriate choice of fluorophore transducer showing emission within the range coinciding with absorption spectra of free ligand, opens the possibility of fluorimetric quantification of the analyte. The important benefit of the proposed approach is possibility of performing determination in a wide concentration range or tuning the sensitivity of determination through change of the sample pH exploiting equilibrium complexation dependence on pH. However, to explore this new possibility the optical transducer used is required to have spectra practically insensitive for sample pH changes, unlike the transducer applied in optrodes/microspheres.15,17 On the other hand, similarly as in the case of optrodes or microspheres benefiting from optrode approach, the selectivity of determination is dependent on the selectivity of applied ligand. The advantage of herein proposed approach is also elimination of tedious and complicated synthetic approaches, which require analyte tailored specific fluorophores, needed to prepare fluorimetric sensors. Analytical chemistry offers a broad range of ligands originally developed for colorimetric analysis, that have distinctly different spectra in free and bound form, and that are able to react with analytes in the lipophilic moiety of a polyacrylate microsphere. Last but not the least, the spectropotentiometric approach is not readily available for zinc ions determination, as potentiometric determination of Zn2+ using conventional solvent polymeric membrane based ionselective electrodes is difficult. Thus, there is no system available that can be applied directly to prepare fluorescent polymeric beads sensitive to this biologically important analyte. Therefore, the aim of this work is to verify analytical applicability of the above proposed novel approach allowing fluorimetric quantification, using as a model analyte zinc ions. Among ligands available for colorimetric determination of zinc ions, that can be used as receptor within the lipophilic polymeric microspheres, 1-(2-pyridylazo)-2-naphthol (PAN) was chosen. The high selectivity of this ligand for Zn2+ over possible interferents like Ca2+ or Na+ ions important from

biological point of view is a clear advantage. Moreover, this ligand was recently successfully applied to prepare potentiometric sensors for zinc ions using reduced graphene oxide as the membrane material 19. As a model transducer, fluorophore pyrene was chosen.

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EXPERIMENTAL SECTION Reagents. 1,6-Hexanedioldiacrylate (HDDA), 2,2-dimethoxy-2-phenylacetophenone (DMPP), methacrylic acid (MA), n-butyl acrylate (nBA), hexylacrylate, poly(vinyl alcohol) (PVA), tris(hydroxymethyl)aminomethane (Tris), tetrahydrofuran (THF) pyrene (puriss p.a., for fluorescence) and 1-(2-pyridylazo)-2-napthol (PAN) were from SigmaAldrich (Germany). All salts used, bovine serum albumins (BSA) as well as hydrochloric acid, acetic acid and NaOH were of analytical grade and were obtained from POCh (Gliwice, Poland). Doubly distilled and freshly deionized water (resistance 18.2 MΩcm, Milli-Qplus, Millipore, Austria) was used throughout this work. The following pH buffers were used: 0.1 M acetate (0.1 M sodium acetate + acetic acid) buffer, pH = 5.2 or 0.1 M Tris (adjusted with HCl) buffer, pH = 7.5. Apparatus. The fluorimetric experiments were performed using a spectrofluorimeter Cary Eclipse (Varian). After exposure at an excitation wavelength of 337 nm emission intensity was recorded within the range from 350 to 500 nm. Unless otherwise stated the slits used were 5 nm both for excitation and emission, while the detector voltage was maintained at 690 V. The UV/vis experiments were performed using LAMBDA 25 UV/vis Spectrophotometer (PerkinElmer). Emulsions for photopolymerization of microspheres were prepared using Ultrasonic Processor (homogenizer) Hielscher, model UP 200S. The obtained microspheres were separated from solutions using centrifuge MPW-251 Centrifuge (MPW Med. Instruments). To obtain SEM images of prepared microspheres FE-SEM Merlin (Zeiss) apparatus was used, the obtained nanoparticles were characterized using Zetasizer Nano ZS Malvern (scattering angle 173 degrees). UV/vis Spectra. Spectra of PAN in solution (0.0027 mg/ mL) were recorded in 2 ethanol: 1 (aqueous) Tris (pH = 7.5) (volume) mixture. The spectra were recorded 5 min after introduction of Zn2+ ions to the sample. UV/vis absorption spectra were recorded following calibration of the instrument according to manufacturer recommendation, for polyacrylate films (on polyacetate foil) loaded with PAN, placed in solution (in cuvette). Polyacrylate Films Synthesis and Incorporation of PAN in These Layers. If polyacrylate films were used, they were prepared by 5 min photopolymerization of 10 μL of membrane cocktail (mixture of 300 μL, that is, 268 mg/2.1 mmol, of monomer with 3.6 mg, that is, 0.014 mmol, DMPP and 0.3 μL, that is, 0.3 mg/0.0013 mmol HDDA) applied on top of a laser printer type polyacetate foil. The photopolymerization was performed under argon atmosphere. The ready layers were dipped in 2 mL of water/THF mixture (volume ratio 1:1) containing dissolved 0.5 mg of PAN for 30 min. After this procedure, the layers were well rinsed with water and tested. 412

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Synthesis and Loading of Polyacrylate Microspheres with Pyrene and/or PAN. Synthesis of polyacrylate spheres has been carried out according to Hall method.17 A mixture of n-butyl acrylate (nBA) (480 μL, i.e. 429 mg/3.35 mmol), HDDA (220 μL, i.e. 222 mg/0.98 mmol), and DMPP (25 mg, i.e. 0.098 mmol) was prepared and then it was dispersed in 5 mL of aqueous 1% (w/v) PVA solution, using ultrasound bath (5 min, cycle 0.5, power 70%). The polymerization step was conducted under argon atmosphere for 5 min using vigorous stirring of the emulsion (UV lamp of peak output 360 nm). Following polymerization, the spheres were separated using centrifugation at 5800 rpm for 15 min and were resuspended in 5 mL portion of PVA. The (dry) mass of obtained microspheres was close to 490 mg, pointing out to ∼70% yield of polymerization. Absorption of dyes (either pyrene or PAN) was performed by adding to 1 mL of the nBA microspheres suspension 200 μL of THF solution containing 0.12 mg (4.9 × 10−7 mol) of PAN and one of the following amounts of pyrene: 0.1 mg (4.9 × 10−7 mol) (resulting in 1:1 molar ratio), 0.05 mg (2.4 × 10−7 mol) or 0.025 mg (1.2 × 10−7 mol), typically 1 to 1 molar ratio of PAN to pyrene was used. The mixture was left for 20 min in the ultrasound bath for absorption to occur. Then the microspheres were centrifuged and the resulting microspheres were suspended in 1 mL of 1% (w/v) PVA solution. Thus obtained microspheres were kept in fridge until use. The concentration of PAN and pyrene in microspheres (1:1 molar ratio) was estimated taking into account incorporation effectiveness determined, amount of microspheres used and their volume (assuming that volume of spheres is equal to the volume of monomers used taking into account effectiveness of polymerization procedure) to be equal to 4.8 × 10−3 and 5 × 10−3 M, respectively. If not stated otherwise, polyacrylate microspheres (prepared from n-butyl acrylate and cross-linker) were used. For some experiments microspheres prepared form copolymer of n-butyl acrylate and methacrylic acid (nBA−MA) were used. To prepare copolymer nBA−MA microspheres, the mixture of nbutyl acrylate (480 μL, i.e. 429 mg/3.35 mmol), methacrylic acid (48 μL, i.e. 49 mg/0.57 mmol), HDDA (220 μL, i.e. 222 mg/0.98 mmol), and DMPP (25 mg, i.e. 0.098 mmol) was prepared and then it was dispersed in 5 mL of aqueous 1% (w/ v) PVA solution. The procedure of copolymer microspheres preparation, dyes introduction, was the same as for the poly(nbutyl acrylate−HDDA) copolymer. For fluorescence emission measurements 20 μL of microspheres were mixed with 3 mL of buffer (the concentration of microspheres in the range of 5 × 10−4 mg/mL), then small volume of Zn2+ (ZnSO4 solution) standards of different concentrations (≤30 μL) were introduced to the sample and the mixture was shortly stirred to ensure mixing of components. The 0.1 M samples were obtained by zinc sulfate solution in Tris buffer, to these solution microspheres were introduced. The fluorescence emission was read after 5 min time.

complex [e.g., ref 20]. PAN dissolved in an organic solvent (or mixture of solvent with water) is yellow, on its absorption spectra recorded in water−ethanol mixtures there is a broad peak with absorption maximum at 470 nm and a low intensity shoulder from 525 to 560 nm. Figure 1A. Upon complexation

Figure 1. UV/vis absorption spectra (a) of PAN (0.0027 mg/mL) in solution (mixture of water and ethanol) in the presence of Tris buffer, pH = 7.5 (black line), and the same solution spiked with Zn2+ ions to reach (total) 10−7 M ZnSO4 (blue line) and 10−5 M ZnSO4 (red line) and (b) polyacrylate membranes loaded with PAN and recorded in Tris buffer, pH = 7.5, (black line) measurement was taken after 1 h contact time with 10−5 M ZnSO4 (red line)

with Zn2+ ions the absorption peak at 470 nm peak diminishes, even introduction of zinc ions in concentration 10−7 M to PAN containing solution causes significant decrease of free PAN absorption band. A new peak is formed at 550 nm, characteristic for the complex formed, Figure 1A. Further increase of Zn2+ concentration in the solution leads to even more pronounced changes in Vis absorption spectra, the peak at 470 nm attributed to free PAN disappears on the expense of formation of absorption peak of two maxima at 520 and 550 nm, seen as a change of color to red-pink, Figure 1A. PAN can be introduced to polyacrylate polymer (regardless if it is in the form of a film or a microsphere) in course of simple absorption procedure, earlier applied to introduce chromoionophore and ionophore to microspheres.17 The absorption procedure was performed from water−THF solution containing ligand (as such or alternatively together with pyrene). Absorption Vis spectra recorded for polyacrylate membranes following incorporation of the ligand are characterized with a broad absorption peak ranging from 350 to 500 nm, with

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RESULTS AND DISCUSSION To prepare zinc ions sensitive fluorescent microspheres we have chosen to adapt a novel approach and to use as a receptor commercially available zinc ions complexing ligand, 1-(2pyridylazo)-2-naphthol (PAN). Its typical application is complexation coupled with extraction (due to practical insolubility of PAN in water) followed by colorimetric determination taking into account absorption spectra of the 413

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maximum at 465 nm, Figure 1. Despite incorporation into polyacrylate moiety (free from any ion-exchanger), PAN ligand is able to form complexes with Zn2+ ions, as seen in Figure 1B. Reaction of PAN incorporated in polyacrylate film with zinc cations (Tris buffer, pH 7.5) results in change of the Vis spectrum of the layer. The PAN absorption peak at 465 nm decreases and a new band, attributed to complex formation, is seen with maxima at 560 nm, Figure 1B. As it can be seen in Figure 1B, the change in the free ligand absorption band is even more pronounced compared to formation of a new absorption peak characteristic for the complex, similarly as in the case of PAN solutions. It should be also stressed that under experimental conditions no fluorescence of PAN was observed. Among different fluorophores emitting light within the wavelength typical for absorption of PAN free ligand, pyrene was chosen because of its lipophilic character as well as high quantum yield and lifetime of emitted fluorescence. Pyrene fluorescence is also relatively insensitive to pH change and this compound can be easily introduced to polyacrylate microspheres in the absorption procedure. The absorption procedure was extremely effective as shown by comparison of the Vis absorption (PAN) (at the absorption maxima 470 nm) or fluorescence (pyrene) intensity at emission maxima at 393 nm, respectively (1:1 molar ratio of the compounds in solution was used for this experiment). The signals recorded for solution used to load the microspheres with dyes and the supernatants following separation of the microspheres post process, have shown that incorporation effectiveness in cases of PAN was equal to 96% and in the case of pyrene it was equal to 98%. The dyes absorption procedure applied has resulted in temporary and reversible increase of microspheres size, as expected for contact of polyacrylate cross-linked polymer with organic solvent-water mixture [e.g., ref 21]. According to dynamic light scattering (DLS) measurements mean diameter of as prepared microspheres was close to 950 nm, which is in good agreement with SEM results showing for majority of obtained microspheres diameter close to 1 μm (Supporting Information Figure S1). Contact of microspheres with water/ THF mixture resulted in significant increase in particles diameter, which in this mixture was close to 2200 nm (DLS measurement). This result suggests that the structure of microspheres becomes significantly loose and thus permeable for dyes. Interestingly, after separation of microspheres from dyes containing solution, the contraction to size close to initial value was observed, the mean diameter determined under DLS conditions was close to 1050 nm. The slight increase of size observed can result from, for example, deprotonation of some of the incorporated PAN molecules (measurement was performed in Tris buffer) leading to creation of positive charges and some repulsion within the microspheres. This conclusion seems to be supported by the results of DLS measurement performed for microspheres containing pyrene and PAN following their contact with 10−2 M Zn2+ ions for 30 minuts (DLS measurement was performed in the absence of Zn2+ in solution), that is, post formation of zinc complexes. In this experiment the determined mean spheres diameter was close to 1150 nm. As it can be seen in Figure 2, emission spectra of pyrene in microspheres (recorded in Tris buffer) are observed for wavelengths ranging from 360 to 430 nm. Thus, the emission range of pyrene overlaps with absorption peak of (free) PAN embedded within polyacrylate polymer. Upon incorporation of both PAN and pyrene to the microsphere, the emission of

Figure 2. Fluorescence emission spectra of pyrene loaded microspheres (black line), microspheres loaded with both pyrene and PAN (red line) and microspheres loaded with pyrene and PAN recorded in Tris buffer following contact with 10−3 M Zn2+ ions in solution (blue line). (Exciation and emission slits 2.5 nm).

pyrene was quenched, Figure 2. This conclusion is supported by observation that despite pyrene fluorescence lifetime in microspheres was shorter compared to that observed for pyrene in solution, both for solution and for microspheres comparable values of fluorescence lifetimes were recorded in the presence and in the absence of PAN (results not shown). It should be stressed that the ratio of the intensities of individual peaks observed on pyrene emission spectra was not affected by PAN incorporation to the microspheres; thus it can be assumed that the chemical environment of pyrene was not changed.22 Thus the overall fluorescence emitted from microspheres is lowered for this system compared to pyrene only present in the polymer. As it can be seen in Figure 2, when Zn2+ ions are introduced to the sample, their complexation by PAN (and extraction to the microspheres) results in change of PAN absorption spectra, shift toward longer wavelengths, resulting ultimately in increase of the fluorescence emission from the microsphere. It should be stressed that absorption spectra recorded for the solution (supernatant resulting from centrifugation of microspheres that has been in contact with Zn2+ containing sample for about 30 min) showed only small absorption band at 510 and 540 nm characteristic for PAN zinc complex and only for high concentrations of Zn2+ ions, equal to 10−2 and 10−1 M (results not shown). Thus, it can be assumed that the complex formed is predominantly retained within the microspheres. Moreover, even if the complex is leaking into the sample, this process is not expected to affect the responses significantly as the emission increase is coupled with decrease of amount of free ligand and related to decrease of absorbance at the pyrene emission wavelength, that is, at 390 nm. The shape of the emission spectra of pyrene is unchanged, and the ratio of intensities of peaks is unaffected, just the overall intensity is higher. Moreover, for above-described supernatants, emission at pyrene characteristic wavelength was equal to about 1% of that observed for microspheres containing solution. Thus it can be assumed that pyrene practically does not leak to the solution and the fluorescence observed is related to processes occurring in the microspheres. It should be also stressed that when polymeric microspheres loaded with pyrene only were introduced to the samples of different concentration of Zn2+ ions (ranging from 10−8 to 10−1 M, Tris buffer pH 7.5), no change in fluorescence intensity of 414

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determination was equal only to about 40% of that obtained for 1:1 pyrene to PAN ratio. Clearly the amount of pyrene used affects the sensitivity of Zn2+-sensor, however, the dependence of sensitivity of fluorescence responses on pyrene loading is not linear. The receptor used, PAN, is known to react with zinc ions; however, it does not form complexes with common alkali or alkali earth metals cations including for example sodium or calcium cations. Indeed, in the presence of Na+ or Ca2+ (in Tris buffer) no change of the fluorescence intensity was observed within the concentration range of interfering ions from 10−7 to 10−1 M, and the overall fluorescence intensity was much lower compared to that of zinc ions in solution, Supporting Information Figure S3A. Moreover, similar linear responses within the wide concentration range were observed if the bovine serum albumin was present in the tested solution, Supporting Information Figure S2B. These results clearly originate from properties of the applied ligand and are highly promising for practical application of the proposed sensor, especially in biomedical samples known to contain significantly different amounts of Zn2+ depending on studied system (tissue or protein, uptake, release, or inhibition) 23 . Moreover, as shown earlier,20 the complexation of zinc ions by PAN occurs in a wide pH range. Discussion on Observed Response Range−Response Mechanism. The analyte, zinc ions, is present in the aqueous solution, while the free ligand, PAN (in its neutral form HL) is present within polyacrylate microspheres. Taking into account properties of divalent zinc ions − high hydrophilicity − it is justified to assume that spontaneous extraction of free zinc ions from the sample solution to the microsphere is insignificant. On the other hand, concentration of neutral PAN ligand form HL in the aqueous phase − the partition of HL into the solution is insignificant. Therefore, the complexation reaction most probably occurs at the microsphere/solution interface, with formation of neutral complex ZnL2 (formed between zinc ion and PAN), coupled with release of H+ ions:

pyrene was observed. Accordingly, when the above-described experiment was repeated using microspheres loaded with PAN only, no emission was observed within the whole Zn2+ concentration range. Thus the presence of both PAN (receptor) and pyrene (optical transducer) in the microsphere is necessary to obtain the fluorescence emission change upon introduction of Zn2+ ions to the sample. The observed increase of emission intensity was accompanying change of zinc ions concentration within a broad range (Figure 2) resulting in linear dependence of recorded signal on logarithm of zinc ions concentration within the range from 10−7 to 10−1 M, regardless the molar ratio PAN, pyrene in the microspheres, Figure 3. It

Figure 3. Change of relative fluorescence intensity (read at 393 nm) of pyrene and PAN loaded microspheres recorded in Tris buffer (pH = 7.5) solutions of different zinc ions concentrations, different molar ratios of pyrene and PAN were introduced to the spheres: (black) 1:1, (red) 1:2, (green) 1:4. Inset: Gradual change of fluorescence emission spectra as a result of Zn2+ concentration increase in the sample.

should be stressed, to our best knowledge, the achieved broad responses range is unique among proposed zinc ions fluorimetric sensors. It should be added that linear increase of fluorescence emission accompanying increase of zinc ions concentration recorded for spheres prepared (synthesis + dyes incorporation) in 4 different batches resulted in similar responses, the standard deviation of mean relative fluorescence intensity value recorded was, close or lower than 4% for all concentrations tested, despite the test was performed within the zinc ions concentration range from 10−7 to 10−1 M, Supporting Information Figure S2. The molar ratio PAN to pyrene affected the sensitivity of obtained dependences. For pyrene to PAN molar ratio (in the solution used to saturate the microspheres) equal to 1:1, a very good linear correlation between fluorescence intensity and logarithm of zinc ions concentration was observed (R2 = 0.998) within the range from 10−7 to 0.1 M. Decreasing the amount of pyrene (in the solution used to introduce dyes to microspheres) and keeping the amount of PAN constant, the mole ratio pyrene to PAN equal 1:2, results in about 60% of sensitivity observed for equal amounts of both dyes. However, the linear responses range was not affected, the correlation coefficient of dependence shown in Figure 3 is equal to 0.993 for zinc ions concentration range 10−7 to 10−1 M. Further decrease of amount of pyrene, mole ratio of dyes: pyrene to PAN equal to 1:4, resulted again in linear dependence within above given range (R2 = 0.986), however, the sensitivity of

Zn 2 +aq + 2(HL)pBA → ZnL 2 pBA + 2H+aq

(1)

It should be noted that PAN molecule is functioning (adapting ion-selective electrodes nomenclature) as charged ionophore, thus in principle formation of complex does not require presence of cation-exchanger in the microsphere. Taking into account stability constants of complexes ZnL+ and ZnL2 (log β1 = 11.2, log β2 = 21.7, respectively) and dissociation constant of HL (pKa = 12.2),24 the “conditional” stability constant (β′) will be pH dependent, for example, at pH = 7, the corresponding stability constants will be: log β1′ = 6.0 and log β2′ = 11.3. The ratio of conditional stability constants β2′/β1′ is in this case sufficiently high to obtain predominance of ZnL2 over ZnL+ for ligand concentration higher than 10−5 M. The formed neutral ZnL2 complex diffuses then inside the microsphere. The expected time required for the complex to access the whole microsphere can be estimated using a simple formula: t = d 2/2D

(2)

where d is the microsphere diameter (∼1 μm)25 and D is diffusion coefficient of the complex (assumed to be in order of 10−11 cm2 s−1 [e.g., refs 26−28]). For the above data, the 415

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electrostatic repulsion effect) a very wide linear range of the fluorimetric response on logarithm of zinc ions concentration can be recorded, as observed experimentally. Thus the effects that in some cases hinder application of polyacrylates in potentiometric sensors, for example, ref 29, are herein used for the benefit of optical sensing. As the unique broad response range of polyacrylate microspheres presented is related to surface phenomena of polyacrylate polymers, it seems justified to expect that even change of polymer, for example, using hexylacrylate instead of n-butyl acrylate, in the absence of ion-exchanging sites in the polymeric microsphere, should result in similar responses. Indeed, as shown in Supporting Information Figure S4, for other polyacrylate polymers linear responses within zinc ions concentration range from 10−8 to 10−1 M were observed. Thus the high robustness of the herein proposed system for variation of experimental conditions of sensors preparation was confirmed. On the other hand, the above considerations highlight that a simple possibility to tune (increase) sensitivity (however, limiting the linear responses range) is to introduce cationexchanging sites to the microsphere. Tuning the Analytical Performance of Zinc Ions Fluorimetric Sensors. From the above presented considerations, it follows that introduction of ion-exchange sites into the microspheres can affect observed responses. The usual choice for ion-exchanger when the microspheres are considered, are compounds applied routinely for ion-selective electrodes [e.g., refs 16 and 17], thus in the case of cationexchanging microspheres this would be for example potassium tetrakis(4-chlorophenyl)borate. However, for the purpose of this study, the alternative approach was preferred. As cationexchanger carboxylic groups of methacrylic acid copolymerized with n-butyl acrylate (and croslinker) were used. Application of carboxylic groups as cation-exchanger offers additional possibility of controlling the number of sites by control of solution pH, that is, offers the possibility of “switching” on the cation-exchange through pH change. The pKa value of methacrylic acid is close to 4.7, whereas the pKa of polymethacrylic acid is higher and it is close to 5.7 30. Thus it seems justified to assume that in Tris buffer of pH = 7.5 the carboxylic groups of copolymer microspheres will be dissociated, that is, active as cation-exchanger. Indeed as it can be seen in Figure 4 the fluorescence intensities recorded for microspheres from copolymer of methacrylic acid and n-butyl acrylate containing pyrene to PAN molar ratio 1:1 are significantly different from those obtained for polyacrylate (copolymer of n-butyl acrylate and cross-linker only) microspheres. For copolymer of n-butyl acrylate and methacrylic acid (and cross-linker) microspheres only slight increase of fluorescence intensity was observed for low concentration of Zn2+ ions. However, the change of zinc ions concentration from 10−6 to 10−4 M resulted in pronounced increase of fluorescence intensity, as expected in above presented considerations on the mechanism of responses of proposed fluorimetric sensor. This effect can be attributed to enhanced incorporation of Zn2+ ions to the microsphere and zinc−PAN complex formation occurring within the whole volume of the microsphere. Further increase of Zn2+ ions concentration in the sample did not result in pronounced changes in intensity of recorded fluorescence. It should be stressed that due to applied system, where observed fluorescence is attributed to the transducer, not to the receptor: fluorescence quenching was not observed in high concen-

expected time required is below 10 min. This means that under conditions of fluorimetric measurements the volume of the microspheres is accessible for the complex. Because fluorescence intensity of pyrene is constant (resulting from constant concentration of pyrene in the spheres), increasing concentration of the complex on expense of lowering concentration of free ligand, HL, results in increase of observed pyrene fluorescence. Therefore, one can expect that transmittance under these conditions, proportional to recorded fluorescence intensity, will be exponential function of PAN concentration in the spheres ([PAN]pBA) (Lambert−Beer equation) I = I0 exp( −ε[HL]pBA l) = I0 exp( −ε([PAN]pBA − 0.5[ZnL 2]pBA )l) = I0 exp( −ε([PAN]pBA l) exp(0.5ε[ZnL 2]pBA l) = const1 exp(0.5ε[ZnL 2]pBA l)

(3)

where I is observed fluorescence intensity, I0 is fluorescence intensity of pyrene, ε is molar absorption coefficient of HL, and l is optical path length. This equation predicts exponential dependence of fluorescence intensity on ZnL2 concentration in the microspheres. Increase of ZnL2 concentration in the microspheres following rising concentration of zinc ions in the solution would be possible in the case of microspheres containing a cationexchanger. The influence of ion exchanger results from minimizing electrostatic repulsion in course of ZnL2 complex formation through transient ZnL+ form, as well as from facile transport of H+ from the microsphere. Thus, it seems reasonable to expect that facile incorporation of Zn2+ to the microsphere will result in significant increase of sensitivity, however, (due to exponential nature of the dependence) it would be observed in relatively narrow concentration range. Under experimental conditions, Figure 3, fluorescence intensity recorded is linearly dependent on logarithm of analyte concentration (not on exponent) and covers 7 orders of magnitude. The broad linear responses range of such sensor is achieved on the expense of sensitivity per decade concentration change. Such performance of the sensors can be explained by significant role of surface phenomena on the microsphere/ solution interface in the overall processes. This effect is wellknown for the polyacrylate receptor moiety, and has been observed predominantly for divalent ions.29 The reaction occurs at the microsphere/solution interface, where a saturation effect related to surface concentration of zinc ions and formed complex can be expected. Such saturation is highly probable due to positive charge of zinc ions, resulting in electrostatic repulsion between these ions on the microsphere surface (i.e., the incorporation of zinc ions to the microsphere is electrostatically controlled). Therefore, practically a plateau of ZnL2 concentration in the microspheres can be easily achieved, even for low concentration of zinc ions in the solution. In this case, a significant change of zinc ions concentration in the sample solution, reaching even a few orders of magnitude, would result in only relatively small increase of ZnL2 complex concentration in the microspheres giving rise to possible fluorescence intensity dependence on the wide range of concentration of analyte in the solution. In this way, because of the role of surface phenomena, in the absence of ion exchanger in the microspheres (which would diminish the 416

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Analytical Chemistry

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opt(r)odes concept, the optical transducer emission was not driven by the deprotonation of the dye, thus allowing finetuning of the sensor performance because of sample pH change. As shown on example of zinc ions sensors, this concept not only offers a tunable sensor that can either show a broad range of fluorescence intensity dependence on logarithm of concentration of analyte from 10−7 to 10−1 M or exhibit high sensitivity over more narrower range. In the latter case the high sensitivity range can be shifted along the concentration abcissa simply by change of the pH of the sample.

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ASSOCIATED CONTENT

S Supporting Information *

Additional material as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 4. Change of relative fluorescence intensity (read at 393 nm) of pyrene and PAN loaded microspheres (pyrene to PAN ratio 1:1) prepared from copolymer of n-butyl acrylate and methacrylic acid (and cross-linker) recorded in different pH buffers: (red) Tris, pH = 7.5, (black) acetic, pH = 5.2.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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trations of zinc ions in the sample. Thus the sensitivity of determination was significantly increased on the expense of the linear range of recorded dependence. Moreover, similarly as described above, also this type micropsheres were characterized with highly reproducible responses. The standard deviation of mean relative fluorescence intensity value recorded for microspheres prepared from crosslinked copolymer of n-butyl acrylate and methacrylic acid in 4 different batches was below 4.5%, Supporting Information Figure S5. Interestingly, the sensor proposed herein can be further tuned: the high sensitivity range can be shifted to cover significantly different concentrations of the analyte, Figure 4. For higher pH of a sample, that is, when acetic buffer of pH = 5.2 was used, the increase of zinc ions concentration in the sample up 10−4 M did not result in pronounced change of fluorescence intensity. However, within the concentration range from 10−4 to 10−2 M a pronounced increase in fluorescence intensity was observed. Further increase of zinc ions concentration from 10−2 to 10−1 M resulted only in small fluorescence intensity increase, similarly as observed in high concentration range in Tris buffer. It should be stressed that due to pH induced change in complexation equilibrium, the high sensitivity region of copolymeric microspheres was shifted for lower pH (lower value of “conditional binding constant”); the high sensitivity was observed for higher concentrations of Zn2+. The sensitivity of the sensor, expressed as change of the signal per concentration unit, practically was not affected. This effect is clearly promising for analytical application of herein proposed spheres. Thus it is clearly seen that depending on foreseen application, herein proposed sensor properties can be tailored either to show long linear response range or to expose the high sensitivity of the system.

ACKNOWLEDGMENTS Authors are grateful to DSc Maciej Mazur for comments and discussion on the manuscript. Authors are grateful to Dr M. Gniadek for SEM imaging and to MSc Z. Głeb̨ icka for help with DLS characterization of obtained microspheres. Financial support from National Science Centre (NCN, Poland), project 2011/03/B/ST4/00747, in the years 2012-2015, is gratefully acknowledged.

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CONCLUSIONS A novel type fluorimetric sensor is proposed: the sensing mechanism requires concomitant presence of a receptor (a ligand of different absorption spectra of free and analyte bound form) and a transducer (a fluorescent dye, of emission spectra overlapping with absorption of free ligand) in the polyacrylate microsphere. In contrary to classical spectropotentiometric 417

dx.doi.org/10.1021/ac4033142 | Anal. Chem. 2014, 86, 411−418

Analytical Chemistry

Article

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dx.doi.org/10.1021/ac4033142 | Anal. Chem. 2014, 86, 411−418