Journal of Hazardous Materials 299 (2015) 412–416
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
A cyclodextrin-based polymer for sensing diclofenac in water Pu Xiao a,d,∗,1 , Nicolas Weibel a,1 , Yves Dudal b , Philippe F.-X. Corvini a,c , Patrick Shahgaldian a,∗ a
School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, CH-4132 Muttenz, Switzerland INOFEA GmbH, Hochbergerstrasse 60c, CH-4057 Basel, Switzerland State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, PR China d Centre for Advanced Macromolecular Design, School of Chemistry, University of New South Wales, Sydney, New South Wales 2052, Australia b c
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Tailor-made
cyclodextrin-based polymer can be utilized to assay diclofenac in water. • Fluorescence polarization displacement assay showed selectivity for diclofenac. • The developed approach can be used to quantify diclofenac in wastewaters
a r t i c l e
i n f o
Article history: Received 2 March 2015 Received in revised form 25 May 2015 Accepted 19 June 2015 Available online 24 June 2015 Keywords: Cyclodextrin-based polymer Diclofenac Fluorescent polarization Wastewater
a b s t r a c t An assay for the determination of diclofenac concentration, in the micromolar range in water, was developed. It is based on the use of a recently developed cyclodextrin-based polymer that possesses an inherent affinity for the target pharmaceutical. This competitive assay is exploiting the possibility to displace a fluorescent dye, adsorbed in the cyclodextrin-based polymer, by the target drug. This displacement is followed by measuring the increase in fluorescence polarization of the dye released in solution. The assay was successfully tested on a real wastewater sample with a limit of detection of 1 M. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding authors. Tel.: +41 61467436. E-mail addresses: [email protected] (P. Xiao), [email protected] (P. Shahgaldian). 1 These authors equally contributed to the work. http://dx.doi.org/10.1016/j.jhazmat.2015.06.047 0304-3894/© 2015 Elsevier B.V. All rights reserved.
The occurrence and persistence of pharmaceutical compounds in the aquatic environment has given rise to increasing attentions as some of them have been revealed to be harmful not only to environment but also to human health [1–4]. Diclofenac (DF)
P. Xiao et al. / Journal of Hazardous Materials 299 (2015) 412–416
is a widely used non-steroidal anti-inflammatory, analgesic and antipyretic drug for human that is mainly administered via the oral route. It is rapidly absorbed by the organism and has a halflife as short as 1–2 h [5]. Partially excreted unmetabolized, DF is known to be mainly recalcitrant to biological wastewater treatment and thus reaches the water cycle where DF is one of the most frequently detected pharmaceuticals [6–11]. State-of-the-art methods to detect DF are based on hyphenated chromatography techniques that are rather expensive and time-consuming for large scale screening purposes [12–14]. New methods based on non-separation approaches are needed: they necessitate the development of sorbent materials with a good affinity and selectivity for this pharmaceutical, which can be used to develop a rapid and inexpensive detection kit. The development of materials able of molecular recognition is an important challenge in modern chemical sciences. Among the different strategies developed to produce artificial recognition materials, molecular imprinting [15] has been demonstrated to be a suitable method to produce recognition materials for targets ranging from small organic molecules [15] and ions [16] to large biomolecules such as proteins [17] and viruses [18]. It has been successfully used to design materials with binding properties for DF [19–21] mainly applied in separation-based techniques (e.g., solid-phase extraction). Nevertheless, molecularly imprinted polymers (MIPs) suffer from the major drawback, especially from an industrial perspective, of the need of template removal after the polymerization reaction, which typically requires a thorough washing sequence with various solvents [22]. In addition to the high costs related to this tedious washing procedure, incomplete extraction of the template is likely to cause quantification inaccuracies in analytical applications [23]. A relevant alternative to circumvent these limitations is to design materials with monomeric building blocks possessing an inherent affinity for the target molecule. Owing to their ability to entrap poorly water-soluble drugs in their hydrophobic cavity upon formation of inclusion complexes, cyclodextrins turn out to be promising candidates for the formulation of polymers with superior recognition properties [24], a large number of highly cross-linked cyclodextrin-based polymers (CDPs) have been designed and well-designed candidates have been shown to have a good potential for environmental applications [25]. This strategy rules out the use of the template necessary for the imprinting process. In this context, we recently developed template-free high-throughput combinatorial approaches to produce a series of highly cross-linked cyclodextrin-based polymers (CDPs) either based on polyurethanes [26] or photopolymers [27]. The composition of the CDPs turned out to have a major impact on their selective molecular recognition properties and those methods allowed for the design of a number of different CDPs with tunable affinities for a series of pharmaceuticals. In particular, the photopolymerization of an acryloyl-bearing -cyclodextrin, in the absence of any additional monomer or cross-linker, led to a polymeric material, namely CDP5, with enhanced affinity and selectivity for DF against other nine active pharmaceutical ingredients (APIs) in water [27]. In the present manuscript, we report on the utilization of CDP5 to develop an assay to measure DF concentration in water. This assay is based on the competitive displacement of a fluorescent dye (i.e., fluorescein: FS) by the target analyte (namely DF) monitored by fluorescence polarization (FP). FP is widely used to study interactions of small molecules with larger biomolecules [28]. It is based on the anisotropy increase of a rather small fluorophore upon interaction with a bulkier molecule or material, as depicted in Fig. 1. Unlike classical fluorescence assays, assays based on FP are homogeneous as no separation of free and bound material is required. Besides its mainstream use for the study of interactions of small molecules with larger biomolecules, FP has also been applied to study the interactions of MIP with their target analytes
413
Fig. 1. Schematic representation of the developed FP displacement assay. After the initial adsorption of FS onto the polymer, it is competitively displaced by DF to give rise to a decrease of the FP signal due to an increase of the isotropy of the system.
[29–31]. To our knowledge, the present manuscript reports for the first time on the use of FP for the development of an assay based on a CDP material.
2. Experimental 2.1. Materials Diclofenac sodium salt (DF) and fluorescein sodium salt (FS) were purchased from Sigma–Aldrich and used without further purification. The photopolymer CDP5 was prepared according to the previously reported procedure [27]. Briefly, CDP5 was synthesized by the photopolymerization of acryloyl -cyclodextrin in DMF using 1-hydroxycyclohexyl phenyl ketone as photoinitiator under the irradiation of UV light (365 nm). Nanopure water (resistivity ≥ 18 M cm) was produced with a Millipore Synergy purification system. Municipal wastewater samples were collected from a pilot-scale membrane bioreactor at the ARA Birs wastewater treatment plant located in Birsfelden (Switzerland).
2.2. Interaction studies by fluorescence intensity (FI) and fluorescence polarization (FP) Appropriate amounts of the CDP5 suspension and FS solution were mixed to get 1 mL of the CDP5–FS suspension in water. After incubation at 28 ◦ C for 10 min and centrifugation at 16,000 g for 10 min, 300 L of the supernatant was transferred to a black 96well microplate (Greiner Bio-One) and the FI was measured. A 300 L of the CDP5–FS or CDP5–FS–API suspension in water was prepared in a black 96-well microplate. After incubation at 28 ◦ C for 10 min, the FP was directly recorded. FI and FP values were measured with an Infinite F200 PRO (Tecan) or a Synergy H1 (BioTek Instruments) microplate reader. The measurements were carried out using 485/20 nm excitation and 535/25 nm emission filters. Each sample was prepared in triplicate to ensure reproducibility of the results. The concentrations of CDP5, FS and API are given in the figure captions.
2.3. Sorption kinetics of FS to CDP5 In a black 96-well microplate, 30 L of the FS stock solution (1 M) was added to 270 L of the CDP5 suspension to get 300 L of the CDP5–FS suspension in water with [CDP5] = 50 g mL−1 and [FS] = 100 nM. FP was monitored as a function of time after addition of FS.
414
P. Xiao et al. / Journal of Hazardous Materials 299 (2015) 412–416
Fig. 3. Binding of FS to various amounts of CDP5 in water ([FS] = 100 nM). (a) % Bound calculated after centrifugation. (b) Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
Fig. 2. Sorption kinetics of FS to CDP5 in water ([CDP5] = 50 g mL−1 , [FS] = 100 nM). Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
2.4. Sorption kinetics of DF to CDP5-FS In a black 96-well microplate, 30 L of the DF stock solution (100 M) was added to 270 L of the CDP5–FS suspension to get 300 L of the CDP5–FS–DF suspension in water with [CDP5] = 50 g mL−1 , [FS] = 100 nM and [DF] = 10 M. The FP was monitored as a function of time after the addition of DF. 2.5. Sorption kinetics of DF to CDP5-FS in wastewater A 400 L of the CDP5 suspension (625 g mL−1 ) in a 3:5 wastewater/water mixture, 50 L of the FS stock solution (1000 nM) in water and 50 L of the DF stock solution (10−6 − 2 × 10−3 M) in water were mixed to get 0.5 mL of the CDP5–FS–DF suspension in wastewater with [CDP5] = 500 g mL−1 , [FS] = 100 nM and [DF] = 10−7 − 2 × 10−4 M. After incubation at 28 ◦ C for 10 min, 300 L of each suspension was transferred to a black 96-well microplate and the FP was measured (ten replicates each). 3. Results and discussion To establish the optimal conditions to utilize CDP5 for the assay of DF in water by fluorescence polarization, various factors including equilibration time, concentrations of CDP5 and FS were investigated. Based on a series of preliminary measurements, the results are presented and discussed organizedly as below. The sorption kinetics of FS onto CDP5 was first investigated by adding minute amounts of FS to a suspension of CDP5 in water (50 g mL−1 ) and monitoring the FP as a function of time (cf. Fig. 2). From Fig. 2, it could be seen that FP values rapidly increased as the fluorophore bound to the polymer, until equilibrium was reached after 10 min. It is noteworthy that such a short equilibration time is an asset for the performance of routine high-throughput assay. Next, the appropriate concentration of CDP5 to be used in the assay was determined. To that end, increasing amounts of CDP5 (0–300 g mL−1 ) were added to a fixed quantity of FS (100 nM) in water. The resulting aqueous suspensions were incubated at 28 ◦ C for 10 min to reach equilibrium and the fraction of bound FS was calculated by measuring the fluorescence of non-bound FS after removal of the polymer; the polarization was directly recorded after incubation; cf. Fig. 3.
Fig. 4. Binding of various amounts of FS to CDP5 in water ([CDP5] = 50 g mL−1 ). (a) % Bound calculated after centrifugation. (b) Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
As expected, the fraction of bound probe increased with increasing amounts of polymer. A CDP5 concentration of 300 g mL−1 allowed for binding approximately 95% of the FS initially present in solution. The polarization followed the same trend as a larger amount of bound FS results in a higher FP value. In order to choose the appropriate polymer concentration, one has to keep in mind that high polymer loadings are likely to lead to turbid suspensions and undesired excitation and emission light scattering phenomena. More specifically, with high polymer concentrations, the opaque suspensions can diminish the excitation light to reach FS and the emission light to arrive at the detector during the fluorescence polarization measurements [29,32]. Thus, the concentration of CDP5 was set to 50 g mL−1 for the following experiments in nanopure water. Then, the binding properties of CDP5 in presence of various amounts of FS (50–300 nM) were examined; cf. Fig. 4. Both the portion of bound probe and the polarization decreased as the initial concentration of the probe increased. The required conditions to perform the FP sorbent assay should correspond to a situation where approximately 50% of the fluorescent probe is bound to the polymer. Indeed, these conditions ensured a good balance between the amplitude of polarization and the sensitivity of the assay. Consequently, the optimal concentrations to use were 50 g mL−1 of CDP5 and 100 nM of FS (55% of FS bound to CDP5). Moreover, a Langmuir sorption model fitted well the experimental data (cf. Supplementary material). The capacity (Q, about 0.4 mg g−1 ) and affinity (k, about 4 × 105 L g−1 ) for fluorescein were derived from the intercept and slope of the linear regression line (R2 > 0.99). The kinetics of the competitive displacement of the fluorescent probe (FS) by the analyte (DF) was then investigated. DF was added to the aqueous CDP5–FS suspension and the polarization was monitored as a function of time (cf. Fig. 5). FP values sharply dropped as the probe was gradually displaced by the analyte and released in solution. Equilibrium was reached within 10 min. Note that this fast polarization decay correlated with the previously reported high binding affinity of CDP5 for DF in water [27].
P. Xiao et al. / Journal of Hazardous Materials 299 (2015) 412–416
415
Fig. 5. Competitive displacement kinetics of FS by DF in water ([CDP5] = 50 g mL−1 , [FS] = 100 nM, [DF] = 10 M). Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm. Fig. 7. Normalized standard curves for DF with the CDP5–FS suspension with [CDP5] = 500 g mL−1 in wastewater (empty circles) and [CDP5] = 50 g mL−1 in water (full squares) and wastewater (empty triangles). [FS] = 100 nM. Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
Fig. 6. Standard curves for DF (full squares), CHPH (empty circles), and PRO (empty triangles) with the optimal CDP5–FS suspension in water ([CDP5] = 50 g mL−1 , [FS] = 100 nM). Standard curves for DF in water (cross). Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
The optimal CDP5–FS suspension system was then incubated for 10 min with varying concentrations of DF (0.1–200 M), the polarization was measured and the corresponding standard curve was plotted (cf. Fig. 6). For low concentrations of target analyte (
Contents lists available at ScienceDirect
Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
A cyclodextrin-based polymer for sensing diclofenac in water Pu Xiao a,d,∗,1 , Nicolas Weibel a,1 , Yves Dudal b , Philippe F.-X. Corvini a,c , Patrick Shahgaldian a,∗ a
School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, CH-4132 Muttenz, Switzerland INOFEA GmbH, Hochbergerstrasse 60c, CH-4057 Basel, Switzerland State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, PR China d Centre for Advanced Macromolecular Design, School of Chemistry, University of New South Wales, Sydney, New South Wales 2052, Australia b c
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Tailor-made
cyclodextrin-based polymer can be utilized to assay diclofenac in water. • Fluorescence polarization displacement assay showed selectivity for diclofenac. • The developed approach can be used to quantify diclofenac in wastewaters
a r t i c l e
i n f o
Article history: Received 2 March 2015 Received in revised form 25 May 2015 Accepted 19 June 2015 Available online 24 June 2015 Keywords: Cyclodextrin-based polymer Diclofenac Fluorescent polarization Wastewater
a b s t r a c t An assay for the determination of diclofenac concentration, in the micromolar range in water, was developed. It is based on the use of a recently developed cyclodextrin-based polymer that possesses an inherent affinity for the target pharmaceutical. This competitive assay is exploiting the possibility to displace a fluorescent dye, adsorbed in the cyclodextrin-based polymer, by the target drug. This displacement is followed by measuring the increase in fluorescence polarization of the dye released in solution. The assay was successfully tested on a real wastewater sample with a limit of detection of 1 M. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding authors. Tel.: +41 61467436. E-mail addresses: [email protected] (P. Xiao), [email protected] (P. Shahgaldian). 1 These authors equally contributed to the work. http://dx.doi.org/10.1016/j.jhazmat.2015.06.047 0304-3894/© 2015 Elsevier B.V. All rights reserved.
The occurrence and persistence of pharmaceutical compounds in the aquatic environment has given rise to increasing attentions as some of them have been revealed to be harmful not only to environment but also to human health [1–4]. Diclofenac (DF)
P. Xiao et al. / Journal of Hazardous Materials 299 (2015) 412–416
is a widely used non-steroidal anti-inflammatory, analgesic and antipyretic drug for human that is mainly administered via the oral route. It is rapidly absorbed by the organism and has a halflife as short as 1–2 h [5]. Partially excreted unmetabolized, DF is known to be mainly recalcitrant to biological wastewater treatment and thus reaches the water cycle where DF is one of the most frequently detected pharmaceuticals [6–11]. State-of-the-art methods to detect DF are based on hyphenated chromatography techniques that are rather expensive and time-consuming for large scale screening purposes [12–14]. New methods based on non-separation approaches are needed: they necessitate the development of sorbent materials with a good affinity and selectivity for this pharmaceutical, which can be used to develop a rapid and inexpensive detection kit. The development of materials able of molecular recognition is an important challenge in modern chemical sciences. Among the different strategies developed to produce artificial recognition materials, molecular imprinting [15] has been demonstrated to be a suitable method to produce recognition materials for targets ranging from small organic molecules [15] and ions [16] to large biomolecules such as proteins [17] and viruses [18]. It has been successfully used to design materials with binding properties for DF [19–21] mainly applied in separation-based techniques (e.g., solid-phase extraction). Nevertheless, molecularly imprinted polymers (MIPs) suffer from the major drawback, especially from an industrial perspective, of the need of template removal after the polymerization reaction, which typically requires a thorough washing sequence with various solvents [22]. In addition to the high costs related to this tedious washing procedure, incomplete extraction of the template is likely to cause quantification inaccuracies in analytical applications [23]. A relevant alternative to circumvent these limitations is to design materials with monomeric building blocks possessing an inherent affinity for the target molecule. Owing to their ability to entrap poorly water-soluble drugs in their hydrophobic cavity upon formation of inclusion complexes, cyclodextrins turn out to be promising candidates for the formulation of polymers with superior recognition properties [24], a large number of highly cross-linked cyclodextrin-based polymers (CDPs) have been designed and well-designed candidates have been shown to have a good potential for environmental applications [25]. This strategy rules out the use of the template necessary for the imprinting process. In this context, we recently developed template-free high-throughput combinatorial approaches to produce a series of highly cross-linked cyclodextrin-based polymers (CDPs) either based on polyurethanes [26] or photopolymers [27]. The composition of the CDPs turned out to have a major impact on their selective molecular recognition properties and those methods allowed for the design of a number of different CDPs with tunable affinities for a series of pharmaceuticals. In particular, the photopolymerization of an acryloyl-bearing -cyclodextrin, in the absence of any additional monomer or cross-linker, led to a polymeric material, namely CDP5, with enhanced affinity and selectivity for DF against other nine active pharmaceutical ingredients (APIs) in water [27]. In the present manuscript, we report on the utilization of CDP5 to develop an assay to measure DF concentration in water. This assay is based on the competitive displacement of a fluorescent dye (i.e., fluorescein: FS) by the target analyte (namely DF) monitored by fluorescence polarization (FP). FP is widely used to study interactions of small molecules with larger biomolecules [28]. It is based on the anisotropy increase of a rather small fluorophore upon interaction with a bulkier molecule or material, as depicted in Fig. 1. Unlike classical fluorescence assays, assays based on FP are homogeneous as no separation of free and bound material is required. Besides its mainstream use for the study of interactions of small molecules with larger biomolecules, FP has also been applied to study the interactions of MIP with their target analytes
413
Fig. 1. Schematic representation of the developed FP displacement assay. After the initial adsorption of FS onto the polymer, it is competitively displaced by DF to give rise to a decrease of the FP signal due to an increase of the isotropy of the system.
[29–31]. To our knowledge, the present manuscript reports for the first time on the use of FP for the development of an assay based on a CDP material.
2. Experimental 2.1. Materials Diclofenac sodium salt (DF) and fluorescein sodium salt (FS) were purchased from Sigma–Aldrich and used without further purification. The photopolymer CDP5 was prepared according to the previously reported procedure [27]. Briefly, CDP5 was synthesized by the photopolymerization of acryloyl -cyclodextrin in DMF using 1-hydroxycyclohexyl phenyl ketone as photoinitiator under the irradiation of UV light (365 nm). Nanopure water (resistivity ≥ 18 M cm) was produced with a Millipore Synergy purification system. Municipal wastewater samples were collected from a pilot-scale membrane bioreactor at the ARA Birs wastewater treatment plant located in Birsfelden (Switzerland).
2.2. Interaction studies by fluorescence intensity (FI) and fluorescence polarization (FP) Appropriate amounts of the CDP5 suspension and FS solution were mixed to get 1 mL of the CDP5–FS suspension in water. After incubation at 28 ◦ C for 10 min and centrifugation at 16,000 g for 10 min, 300 L of the supernatant was transferred to a black 96well microplate (Greiner Bio-One) and the FI was measured. A 300 L of the CDP5–FS or CDP5–FS–API suspension in water was prepared in a black 96-well microplate. After incubation at 28 ◦ C for 10 min, the FP was directly recorded. FI and FP values were measured with an Infinite F200 PRO (Tecan) or a Synergy H1 (BioTek Instruments) microplate reader. The measurements were carried out using 485/20 nm excitation and 535/25 nm emission filters. Each sample was prepared in triplicate to ensure reproducibility of the results. The concentrations of CDP5, FS and API are given in the figure captions.
2.3. Sorption kinetics of FS to CDP5 In a black 96-well microplate, 30 L of the FS stock solution (1 M) was added to 270 L of the CDP5 suspension to get 300 L of the CDP5–FS suspension in water with [CDP5] = 50 g mL−1 and [FS] = 100 nM. FP was monitored as a function of time after addition of FS.
414
P. Xiao et al. / Journal of Hazardous Materials 299 (2015) 412–416
Fig. 3. Binding of FS to various amounts of CDP5 in water ([FS] = 100 nM). (a) % Bound calculated after centrifugation. (b) Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
Fig. 2. Sorption kinetics of FS to CDP5 in water ([CDP5] = 50 g mL−1 , [FS] = 100 nM). Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
2.4. Sorption kinetics of DF to CDP5-FS In a black 96-well microplate, 30 L of the DF stock solution (100 M) was added to 270 L of the CDP5–FS suspension to get 300 L of the CDP5–FS–DF suspension in water with [CDP5] = 50 g mL−1 , [FS] = 100 nM and [DF] = 10 M. The FP was monitored as a function of time after the addition of DF. 2.5. Sorption kinetics of DF to CDP5-FS in wastewater A 400 L of the CDP5 suspension (625 g mL−1 ) in a 3:5 wastewater/water mixture, 50 L of the FS stock solution (1000 nM) in water and 50 L of the DF stock solution (10−6 − 2 × 10−3 M) in water were mixed to get 0.5 mL of the CDP5–FS–DF suspension in wastewater with [CDP5] = 500 g mL−1 , [FS] = 100 nM and [DF] = 10−7 − 2 × 10−4 M. After incubation at 28 ◦ C for 10 min, 300 L of each suspension was transferred to a black 96-well microplate and the FP was measured (ten replicates each). 3. Results and discussion To establish the optimal conditions to utilize CDP5 for the assay of DF in water by fluorescence polarization, various factors including equilibration time, concentrations of CDP5 and FS were investigated. Based on a series of preliminary measurements, the results are presented and discussed organizedly as below. The sorption kinetics of FS onto CDP5 was first investigated by adding minute amounts of FS to a suspension of CDP5 in water (50 g mL−1 ) and monitoring the FP as a function of time (cf. Fig. 2). From Fig. 2, it could be seen that FP values rapidly increased as the fluorophore bound to the polymer, until equilibrium was reached after 10 min. It is noteworthy that such a short equilibration time is an asset for the performance of routine high-throughput assay. Next, the appropriate concentration of CDP5 to be used in the assay was determined. To that end, increasing amounts of CDP5 (0–300 g mL−1 ) were added to a fixed quantity of FS (100 nM) in water. The resulting aqueous suspensions were incubated at 28 ◦ C for 10 min to reach equilibrium and the fraction of bound FS was calculated by measuring the fluorescence of non-bound FS after removal of the polymer; the polarization was directly recorded after incubation; cf. Fig. 3.
Fig. 4. Binding of various amounts of FS to CDP5 in water ([CDP5] = 50 g mL−1 ). (a) % Bound calculated after centrifugation. (b) Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
As expected, the fraction of bound probe increased with increasing amounts of polymer. A CDP5 concentration of 300 g mL−1 allowed for binding approximately 95% of the FS initially present in solution. The polarization followed the same trend as a larger amount of bound FS results in a higher FP value. In order to choose the appropriate polymer concentration, one has to keep in mind that high polymer loadings are likely to lead to turbid suspensions and undesired excitation and emission light scattering phenomena. More specifically, with high polymer concentrations, the opaque suspensions can diminish the excitation light to reach FS and the emission light to arrive at the detector during the fluorescence polarization measurements [29,32]. Thus, the concentration of CDP5 was set to 50 g mL−1 for the following experiments in nanopure water. Then, the binding properties of CDP5 in presence of various amounts of FS (50–300 nM) were examined; cf. Fig. 4. Both the portion of bound probe and the polarization decreased as the initial concentration of the probe increased. The required conditions to perform the FP sorbent assay should correspond to a situation where approximately 50% of the fluorescent probe is bound to the polymer. Indeed, these conditions ensured a good balance between the amplitude of polarization and the sensitivity of the assay. Consequently, the optimal concentrations to use were 50 g mL−1 of CDP5 and 100 nM of FS (55% of FS bound to CDP5). Moreover, a Langmuir sorption model fitted well the experimental data (cf. Supplementary material). The capacity (Q, about 0.4 mg g−1 ) and affinity (k, about 4 × 105 L g−1 ) for fluorescein were derived from the intercept and slope of the linear regression line (R2 > 0.99). The kinetics of the competitive displacement of the fluorescent probe (FS) by the analyte (DF) was then investigated. DF was added to the aqueous CDP5–FS suspension and the polarization was monitored as a function of time (cf. Fig. 5). FP values sharply dropped as the probe was gradually displaced by the analyte and released in solution. Equilibrium was reached within 10 min. Note that this fast polarization decay correlated with the previously reported high binding affinity of CDP5 for DF in water [27].
P. Xiao et al. / Journal of Hazardous Materials 299 (2015) 412–416
415
Fig. 5. Competitive displacement kinetics of FS by DF in water ([CDP5] = 50 g mL−1 , [FS] = 100 nM, [DF] = 10 M). Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm. Fig. 7. Normalized standard curves for DF with the CDP5–FS suspension with [CDP5] = 500 g mL−1 in wastewater (empty circles) and [CDP5] = 50 g mL−1 in water (full squares) and wastewater (empty triangles). [FS] = 100 nM. Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
Fig. 6. Standard curves for DF (full squares), CHPH (empty circles), and PRO (empty triangles) with the optimal CDP5–FS suspension in water ([CDP5] = 50 g mL−1 , [FS] = 100 nM). Standard curves for DF in water (cross). Fluorescence polarization measured at 535/25 nm after excitation at 485/20 nm.
The optimal CDP5–FS suspension system was then incubated for 10 min with varying concentrations of DF (0.1–200 M), the polarization was measured and the corresponding standard curve was plotted (cf. Fig. 6). For low concentrations of target analyte (
