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DBBTAB covesicles
Sensors and Actuators B 119 (2006) 695–700
Glucose-responsive vesicular sensor based on boronic acid–glucose recognition in the ARS/PBA/DBBTAB covesicles Qiusheng Wang, Guangquan Li, Wenyi Xiao, Haixia Qi, Guowen Li ∗ Key laboratory for supramolecular structures and materials of ministry of education, Jilin University, Changchun 130012, China Received 29 September 2005; received in revised form 16 January 2006; accepted 16 January 2006 Available online 21 February 2006
Abstract The amphiphile, 4-(4-dodecyloxybiphenyl-4-yloxy) butyl trimethylammonium bromide (DBBTAB), has a cationic group with quaternary ammonium salt, which can self-organize into vesicles in dilute aqueous solutions. The alizarin red S (ARS) with a negatively charged group is attracted electrostatically to the positively charged surface on the DBBTAB vesicles to form covesicles. By taking advantages of the features, a vesicular fluorescent sensor was prepared based on phenylboronic acid (PBA)–glucose recognition in the aqueous ARS/PBA/DBBTAB covesicles. The sensor was constructed with three constituents: PBA, DBBTAB amphiphile and ARS which served as the detector. The vesicular sensor enhanced the sensitivity to glucose by about seven- to eight-fold, compared with the same aqueous PBA/ARS solution, which was ascribed to the increased local concentrations of glucose on the surface of vesicles. The vesicular sensor may be available for detection of saccharides in biological systems. © 2006 Elsevier B.V. All rights reserved. Keywords: Vesicular sensor; Glucose; Phenylboronic acid; Alizarin red S; Amphiphile
1. Introduction The design and synthesis of effective fluorescent chemosensors for biologically relevant analytes are of paramount interest in supramolecular chemistry [1]. Saccharides are of great importance in biological systems [2]. It is therefore unsurprising that receptors with the capacity to detect chosen saccharides selectively and signal this presence by altering their optical signature have attracted considerable interest in recent years [3–6]. To date, a wide variety of methods have been reported for glucose analysis in research literature, including electrochemistry [7], near infrared spectroscopy [8], optical rotation [9], colorimetric [10] and fluorescence detection [11]. The most commonly used technology for blood glucose determination is an enzymebased method [12], which requires frequent collection of blood samples. Although frequent “finger pricking” with a small needle to obtain the blood sample is a relatively painless process, this method does suffer from a few practical problems. The first one is inconvenience, which affects compliance by patients.
∗
Corresponding author. E-mail address: [email protected] (G. Li).
0925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2006.01.030
Second, this is not a continuous monitoring method. Recently, there is a great deal of interest in the development of continuous glucose monitoring systems, which would be able to provide patients with instantaneous feedback and should help to improve the management of proper glucose concentration in diabetic patients [13,14]. To develop a continuous monitoring system, it would be ideal to use an implantable device that is in constant contact with the biological fluid to give a continuous reading of glucose concentration. It is unlikely that the currently used enzyme-based method could be developed as an implantable device due to the instability issues associated with protein-based products [15]. Chemical sensors do offer the advantage of higher stability and relatively easy manufacturing. Such a concept has already been put into test by companies such as Sensors for Medicine and Science [16]. To develop a chemical sensor-based continuous monitoring device, one needs to develop glucose sensors that show high sensitivity and selectivity. Along this line, we are interested in the development of fluorescent sensors for glucose in the vesicular system. Following the pioneering works of Yoon and Czarnik [17] and later Sandanayake and Shinkai [18], boronic acid derivatives have been used in the recognition and sensing of vicinal
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diols, carbohydrates, and catechols for the past two decades, with many successful examples emerging during this time period [19, and references within]. Boronic acids are known to bind with compounds containing diol moieties with a high affinity through reversible ester formation [20]. Such tight bonding allows boronic acids to be used as the recognition moieties in the construction of sensors for saccharides [21, and references within]. However, the boronic acid compounds are not fluorescent and are only weakly chromophoric. Under such a circumstance, spectroscopic determination relying on the intrinsic spectroscopic property changes upon binding becomes very difficult. Springsteen and Wang [22] has developed a two component competitive assay containing the fluorescent compound alizarin red S (ARS) in the phenylboronic acid (PBA)/ARS system, in which ARS was used as a general fluorescent reading agent for studying the binding events between boronic acids and compounds containing diol moieties with a high affinity. It is known that the amphiphile, 4-(4-dodecyloxybiphenyl4-yloxy) butyl trimethylammonium bromide (DBBTAB), has a cationic group with quaternary ammonium salt, which can selforganize into vesicles in dilute aqueous solution [23,24]. The vesicles composed of bilayer structure may be used as membrane models [25]. The ARS with a negatively charged group can be attracted electrostatically to the positively charged surface of the DBBTAB vesicles to form covesicles. Herein, we have developed a novel vesicular sensor composed of three tunable components: PBA receptor, ARS “read-out” units and DBBTAB vesicular carrier, in which the DBBTAB vesicles were used as a novel carrier of device to bind to the ARS component. This fluorescence sensor designed is mainly based upon self-organization of DBBTAB amphiphile and vesicular functionallization as mimetic membranes.
2. Experimental 2.1. Materials The DBBTAB amphiphile was synthesized by this group previously [26]. Alizarin red S (Xinzhong Chemical Factory, A.R. grade), phenylboronic acid (Tianjin Taida Chemical Factory, A.R. grade), d (+)-glucose (Beijing Chemical Factory, A.R. grade) were used without further purification. Water was doubly distilled after passing through an ion-exchange resin column. Phosphate buffer was purchased from Shanghai Weiye Instrument Plant. The structure of the DBBTAB amphiphile and ARS is shown in Fig. 1.
2.2. Preparation of the DBBTAB-ARS-PBA covesicles [27] The DBBTAB and ARS were combined with spectroscopic grade CHCl3 in a 50 ml beaker. Solvent was removed under N2 flow, followed by evacuation at high vacuum for 2 h. The resulting amphiphilic film in the beaker was then covered with a pH 6.8 phosphate buffer solution, which was an ideal situation because we were most interested in searching for a sensor that was functional at physiological pH values. The concentrations of DBBTAB amphiphile and ARS were fixed at 4 × 10−3 M and 5 × 10−5 M, respectively. A clear solution was obtained by sonication (Bransonic 12 Ultrasonicator, water-bath type) for 4 h at about 50 ◦ C. The covesicles were stable for weeks in the aqueous solution in a pH range of 4–10. 2.3. Instrumentation Steady-state fluorescence spectra were recorded on a RF5301PC spectrophotometer. UV spectra were measured with a Shimadzu UV-3100 spectrophotometer. Since N2 -bubbled and air-saturated solutions gave the same results, the data presented here are all for air-saturated solutions. An aqueous solution was dripped on a silica plate which was then dried in a desiccator at room temperature. Scanning electron microscopy (SEM) was collected on a JEOL JSM-6700F electron microscope. The phase transition from crystal to liquid crystal (Tc ) of the covesicles (ca. 5 wt.%) was determined by means of differential scanning calorimetry (Netzsch DSC 204). 3. Results and discussion Stable aqueous vesicular morphologies were determined by SEM. The typical SEM image from a mixture solution containing 4 × 10−3 M of DBBTAB amphiphile, 5 × 10−5 M of ARS and 5.2 × 10−4 M of PBA is shown in Fig. 2. The diameter of the vesicles was estimated about 150–500 nm from Fig. 2. The phase transition from crystal to liquid crystal (Tc ) determined by means of differential scanning calorimetry was located at 45 ◦ C. These results confirmed that the system of ARS/PBA/DBBTAB formed covesicles [24]. A typical fluorescence spectrum is shown in Fig. 3 when a PBA buffer solution was dropwise added into an aqueous DBBTAB-ARS vesicular solution. It is obvious that the fluorescent intensities of ARS bound on the surfaces of DBBTAB vesicles centred at 560 nm increase dramatically with increasing the concentration of PBA from 0 M to 5.2 × 10−4 M, suggesting that the boronic acid formed a boronate ester with ARS [22].
Fig. 1. The structure of 4-(4-dodecyloxybiphenyl-4-yloxy) butyl trimethylammonium bromide (DBBTAB) and alizarin red S (ARS).
Q. Wang et al. / Sensors and Actuators B 119 (2006) 695–700
Fig. 2. SEM image of vesicles derived from the ARS/PBA/DBBTAB system: [DBBTAB] = 4 × 10−3 M, [PBA] = 5.2 × 10−4 M, [ARS] = 5 × 10−5 M.
In order to develop the applicability of using ARS for the determination of the binding events between a boronic acid and a carbohydrate in the vesicular system, we used glucose as a model compound. When a glucose buffer solution was dropwise added into the aqueous vesicular solution, the fluorescence intensities of ARS centred at 560 nm gradually decreased with increasing the concentration of glucose from 3.2 × 10−3 M to 43.3 × 10−3 M, which was the most physiologically relevant concentration range in terms of blood glucose detection, as shown in Fig. 4. The decrease of fluorescent intensity of ARS bound on the surface of DBBTAB vesicles implies that ARS molecules were replaced by glucose molecules from PBA-ARS ester (1) and then PBA-glucose ester (2) was gradually formed, as illustrated in Scheme 1. The free ARS replaced gradually from PBA-ARS ester (1) results in the decreasing fluorescence intensity of the vesicular system. It is known that an excited state proton transfer from the phenol hydroxyl group of ARS (1 in Scheme 1) to the ketone
Fig. 3. Fluorescence spectra in aqueous ARS/PBA/DBBTAB vesicles with adding PBA (0–5.2 × 10−4 M) at room temperature in an aqueous phosphate buffer at pH 6.8; [DBBTAB] = 4 × 10−3 M; [ARS] = 5 × 10−5 M; aqueous 0.1 M phosphate buffer; Exc. λ = 468 nm.
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Fig. 4. Fluorescence spectra in aqueous ARS/PBA/DBBTAB vesicles with adding glucose (0–43.3 × 10−3 M) at room temperature in an aqueous phosphate buffer at pH 6.8; [DBBTAB] = 4 × 10−3 M; [ARS] = 5 × 10−5 M; [PBA] = 5.2 × 10−4 M; aqueous 0.1 M phosphate buffer; Exc. λ = 468 nm.
oxygen results in the fluorescence quenching of free ARS [28]. Therefore, it was reasonable to expect that the boronate ester (2 in Scheme 1) formation would increase the fluorescence of the vesicular system through the removal of the fluorescence quenching mechanism [22]. There are two competing equilibria when glucose is added into the three component vesicular system of ARS/PBA/DBBTAB. The first equilibrium, between the phenylboronic acid and the fluorescent reporter ARS (K1 ), can directly be detected through fluorescence changes, as shown in Fig. 3. The addition of glucose sets up a second equilibrium between the boronic acid and glucose, to give boronate ester 3. This perturbs the ARS/boronic acid equilibrium, resulting in a change in the fluorescent intensity of the aqueous vesicular system [29]. The fluorescent intensity changes in the DBBTAB
Scheme 1. Schematic routine for competitive binding of a phenylboronic acid with ARS and glucose. ABS 1: weak fluorescence, boronate ester 2: strong fluorescence.
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Fig. 5. Fluorescence spectra in an aqueous ARS/PBA solution with adding glucose (0–43.3 × 10−3 M) at room temperature in aqueous phosphate buffer at pH 6.8; [ARS] = 5 × 10−5 M; [PBA] = 5.2 × 10−4 M; aqueous 0.1 M phosphate buffer; Exc. λ = 468 nm.
vesicular system can be used as a sensor for the determination of concentrations of target carbohydrates such as glucose. The sensor might be prepared as an implantable device which can be in constant contact with the biological fluid to give a continuous reading of glucose concentration. In the vesicular sensor, alizarin red S has been used as a fluorescent reading agent since the active protons of hydroxyanthraquinones in ARS are responsible for a large fluorescence quenching [14]. This result is consistent with phenomenon in aqueous solution [29]. Also, the fluorescence intensities were determined in an aqueous ARS/PBA solution for comparison, as shown Fig. 5. When a glucose buffer solution was dropwise added into the aqueous ARS/PBA solution, the fluorescence intensities of ARS gradually decreased with increasing the concentration of glucose from 3.2 × 10−3 M to 43.3 × 10−3 M. It needs to be noted that the fluorescence intensity is weak and their changes were less than that in the ARS/PBA/DBBTAB vesicular solution. It should also be noted that the vesicular sensor showed the most sensitive fluorescence intensity changes to glucose in the region of 3.2 × 10−3 M to 43.3 × 10−3 M (Fig. 4). In contrast, the dependence of the relative fluorescent intensities in the aqueous ARS/PBA/DBBTAB vesicular system and the aqueous ARS/PBA solution on the concentration of glucose, from Figs. 4 and 5, respectively, are shown in Fig. 6. Compared with the sensors in the aqueous PBA/ARS solution, the vesicular sensor showed higher sensitivity to glucose and was enhanced by about seven- to eight-fold from Fig. 6. The most significant result of the present work is the demonstration of the capacity to improve the sensitivity to glucose in the DBBTAB vesicular system. Due to the DBBTAB molecule with an ammonium hydrophilic head group, the surface of the vesicles is thought, reasonably, to be positively charged. Hence, ARS anions were attracted and then anchored on the surface of vesicles by their electrostatical action, which resulted in the increased local concentrations of ARS on the surface of covesicles. It is well known that the fluorescent intensity is dependent
Fig. 6. Relative fluorescent intensities changed with increasing the glucose concentration from 3.2 × 10−3 M to 43.3 × 10−3 M in an aqueous ARS/PBA/DBBTAB vesicular solution and a PBA/ARS solution.
on the concentration of the fluorephores in the system [24]. Therefore, it is reasonable that the sensitivity was enhanced in the DBBTAB vesicular system. The selectivity of the ARS/PBA/DBBTAB vesicular system is similar to that in an aqueous boronic acid solution [22]. The sensor showed decreased fluorescent intensity after binding with carbohydrates for glucose over lactose and ethylene glycol in the biological concentration, respectively, as shown in Fig. 7. By comparison, the selectivity for glucose in the vesicular system is higher than that for lactose, which is ascribed to the different isomerization of the saccharides [30]. As we known, the phenyl-
Fig. 7. Saccharide concentration (3.2 × 10−3 to 43.3 × 10−3 M) vs. relative fluorescence intensity profile for the PBA/ARS/DBBTAB vesicular sensor at pH 6.8 in an aqueous phosphate buffer solution; Exc. λ = 468 nm.
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boronic acid preferentially binds with the hydroxyls of glucose in the furanose form, compared with the hydroxyls of lactose in pyranose forms [31]. 4. Conclusions A vesicular fluorescent sensor was prepared based on phenylboronic acid–glucose recognition in aqueous ARS/PBA/DBBTAB vesicles. The sensor was constructed with three components: DBBTAB vesicles, PBA and ARS which served as the detector. The vesicular sensor enhanced the sensitivity to glucose by about seven- to eight-fold, compared with the corresponding aqueous solution, which was ascribed to the increased local concentrations of ARS on the surface of the DBBTAB covesicles. The vesicular sensor may be applicable to the detection of saccharides in biological systems. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 20274015, 50473005). References [1] P.A. Gale, Pyrrolic and polypyrrolic anion binding agents, Coord. Chem. Rev. 240 (2003) 17–55. [2] S. Hurtley, R. Service, P. Szuromi, Cinderella’s coach is ready, Science 291 (2001) 2337. [3] T.D. James, S. Shinkai, Artificial receptors as chemosensors for carbohydrates, Top. Curr. Chem. 218 (2002) 159–200. [4] S. Striegler, Selective carbohydrate recognition by synthetic receptors in aqueous solution, Curr. Org. Chem. 7 (2003) 81–102. [5] W. Ni, H. Fang, G. Springsteen, B. Wang, The design of boronic acid spectroscopic reporter compounds by taking advantage of the pKa -lowering effect of diol binding: nitrophenol-based color reporters for diols, J. Org. Chem. 69 (2004) 1999–2007. [6] M. Granda-Valdes, R. Badia, G. Pina-Luis, M.E. Diaz-Garcia, Photoinduced eletron transfer systems and their analytical application in chemical sensing, Quim. Anal. 19 (2000) 38–53. [7] D.J. Claremont, I.E. Sambrook, C. Penton, J.C. Pickup, Subcutaneous implantation of a ferrocene-mediated glucose sensor in pigs, Diabetologa 29 (1986) 817–821. [8] M.R. Robinson, R.P. Eaton, D.M. Haaland, G.W. Koepp, E.V. Thomas, B.R. Stallard, P.L. Robinson, Noninvasive glucose monitoring in diabetic patients: a preliminary evaluation, Clin. Chem. 38 (1992) 1618– 1622. [9] B. Rabinovitch, W.F. March, R.L. Adams, Noninvasive glucose monitoring of the aqueous humor of the eye: Part I. Measurement of very small optical rotations, Diabetes Care 5 (1982) 254–258. [10] G.M. Schier, R.G. Moses, I.E.T. Gan, S.C. Blair, An evaluation and comparison of reflolux II and glucometer II, two new portable reflectance meters for capillary blood glucose determination, Diabetes Res. Clin. Pract. 4 (1988) 177–181. [11] S. D’Auria, N. Dicesare, Z. Gryczynski, I. Gryczynski, M. Rossi, J.R. Lakowicz, A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor, Biochem. Biophys. Res. Commun. 274 (2000) 727– 731. [12] E.R. Kenneth, K.J. Ernest, Issues and implications in the selection of blood glucose monitoring technologies, Diabetes Technol. Ther. 1 (1999) 3–11. [13] T. Koschinsky, L. Heinemann, Sensors for glucose monitoring: technical and clinical aspects, Diabetes Met. Res. Rev. 17 (2001) 113–123, 17.
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[14] P. Atanasov, S. Yang, C. Salehi, A.L. Ghindilis, E. Wilkins, D. Schade, Implantation of a refillable glucose monitoring-telemetry device, Biosens. Bioelectron. 12 (1997) 669–680. [15] M. Gerritsen, J.A. Jansen, J.A. Lutterman, Performance of subcutaneously implanted glucose sensors for continuous monitoring, Neth. J. Med. 54 (1999) 167–179. [16] G.Y. Daniloff, Continuous glucose monitoring: long-term implantable sensor approach, Diabetes Technol. Ther. 1 (1999) 261–266. [17] J. Yoon, A.W. Czarnik, Fluorescent chemosensors of carbohydrates. A means of chemically communicating the binding of polyols in water based on chelation-enhanced quenching, J. Am. Chem. Soc. 114 (1992) 5874–5875. [18] K.R.A.S. Sandanayake, S. Shinkai, Novel molecular sensor for saccharides based on the interaction of boronic acid and amine and saccharide sensing in neutral water, J. Chem. Soc. Chem. Commun. (1994) 1083–1084. [19] A. Robertson, S. Shinkai, Coord, Cooperative binding in selective sensors, catalysts and actuators, Chem. Rev. 205 (2000) 157–199 (and references therein). [20] J.P. Lorand, J.O. Edwards, Polyol complexes and structure of the benzeneboronate ion, J. Org. Chem. 24 (1959) 769–774. [21] T.D. James, K.R.A.S. Sandanayake, S. Shinkai, Saccharide sensing with molecular receptors based on boronic acid, Angew. Chem. Int. Ed. Engl. 35 (1996) 1910–1922. [22] G. Springsteen, B. Wang, A detailed examination of boronic acid–diol complexation, Tetrahedron 58 (2002) 5291–5300. [23] Y. Okahata, T. Kunitake, Self-assembling behavior of single-chain amphiphiles with the biphenyl moiety in dilute aqueous solution, Ber. Bunsenges. Phys. Chem. 84 (1980) 550–556. [24] G. Li, Y. Tian, Y. Liang, Photoexcitation energy transfer of binary and ternary systems mediated by a bilayer membrane containing biphenyl moiety, J. Chem. Soc. Faraday Trans. 89 (1993) 1365–1371. [25] J.H. Fendler, Membrane Mimetic Chemistry, John Wiley & Sons, 1982, p. 113. [26] G. Li, Z. Wang, Y. Tian, Y. Liang, Synthesis of a DBBTAB amphiphile and self-organization of the amphiphiles in dilute aqueous solution, Chem. J. Chin. Univ. 10 (1989) 1024–1025. [27] R.A. Moss, G. Li, J.-M. Li, Enhanced dynamic stability of macrocyclic and bolaamphiphilic macrocyclic lipids in liposomes, J. Am. Chem. Soc. 116 (1994) 805–806. [28] D. Palit, H. Pal, T. Mukherjiee, J. Mittal, Photodynamics of the S1 state of some hydroxy- and amino-substituted naphthoquinones and anthraquinones, J. Chem. Soc. Faraday Trans. 86 (1990) 3861– 3869. [29] G. Springsteen, B. Wang, Alizarin red S as a general optical reporter for studying the binding of boronic acids with carbohydrates, Chem. Commun. (2001) 1608–1609. [30] C.R. Cooper, T.D. James, Selective fluorescence signalling of saccharides in their furanose form, Chem. Lett. (1998) 883– 884. [31] J.C. Norrid, H. Eggert, Evidence for mono- and bisdentate boronate complexes of glucose in the furanose form. Application of 1JC-C coupling constants as a structural probe, J. Am. Chem. Soc. 117 (1995) 1479–1484.
Biographies Qiusheng Wang is a PhD candidate student in chemistry. His interest is in synthesis of functional amphiphile and vesicular sensor and sensor applications. Guangquan Li is an MSc candidate student in chemistry. His interest is in synthesis of functional compounds and study on the photochemical and phtophysical properties of their aggregates. Wenyi Xiao is an MSc candidate student in chemistry. Her interest is in synthesis of functional amphiphiles and molecular recognition in vesicles.
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Haixia Qi received her MSc degree in chemistry at Jilin University in 2005. Her interest is in photochemical and photophysical properties of vesicles. Guowen Li is a professor of chemistry at Jilin University. He received his PhD in chemistry in 1990 from Jilin University and did his postdoctoral research at Rutgers University from 1991 to 1993 and University of Wyoming from
1993 to 1995, respectively. His research interests are in chemistry and soft materials. His research programs focus on the synthesis of organic and polymeric compounds such as amphiphiles, and surfactants and preparation of functional aggregates. Recent works include the molecular recognition in vesicles, micelles and polymeric aggregates; fluorescence sensor and morphologies of aggregates.
Glucose-responsive vesicular sensor based on boronic acid–glucose recognition in the ARS/PBA/DBBTAB covesicles Qiusheng Wang, Guangquan Li, Wenyi Xiao, Haixia Qi, Guowen Li ∗ Key laboratory for supramolecular structures and materials of ministry of education, Jilin University, Changchun 130012, China Received 29 September 2005; received in revised form 16 January 2006; accepted 16 January 2006 Available online 21 February 2006
Abstract The amphiphile, 4-(4-dodecyloxybiphenyl-4-yloxy) butyl trimethylammonium bromide (DBBTAB), has a cationic group with quaternary ammonium salt, which can self-organize into vesicles in dilute aqueous solutions. The alizarin red S (ARS) with a negatively charged group is attracted electrostatically to the positively charged surface on the DBBTAB vesicles to form covesicles. By taking advantages of the features, a vesicular fluorescent sensor was prepared based on phenylboronic acid (PBA)–glucose recognition in the aqueous ARS/PBA/DBBTAB covesicles. The sensor was constructed with three constituents: PBA, DBBTAB amphiphile and ARS which served as the detector. The vesicular sensor enhanced the sensitivity to glucose by about seven- to eight-fold, compared with the same aqueous PBA/ARS solution, which was ascribed to the increased local concentrations of glucose on the surface of vesicles. The vesicular sensor may be available for detection of saccharides in biological systems. © 2006 Elsevier B.V. All rights reserved. Keywords: Vesicular sensor; Glucose; Phenylboronic acid; Alizarin red S; Amphiphile
1. Introduction The design and synthesis of effective fluorescent chemosensors for biologically relevant analytes are of paramount interest in supramolecular chemistry [1]. Saccharides are of great importance in biological systems [2]. It is therefore unsurprising that receptors with the capacity to detect chosen saccharides selectively and signal this presence by altering their optical signature have attracted considerable interest in recent years [3–6]. To date, a wide variety of methods have been reported for glucose analysis in research literature, including electrochemistry [7], near infrared spectroscopy [8], optical rotation [9], colorimetric [10] and fluorescence detection [11]. The most commonly used technology for blood glucose determination is an enzymebased method [12], which requires frequent collection of blood samples. Although frequent “finger pricking” with a small needle to obtain the blood sample is a relatively painless process, this method does suffer from a few practical problems. The first one is inconvenience, which affects compliance by patients.
∗
Corresponding author. E-mail address: [email protected] (G. Li).
0925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2006.01.030
Second, this is not a continuous monitoring method. Recently, there is a great deal of interest in the development of continuous glucose monitoring systems, which would be able to provide patients with instantaneous feedback and should help to improve the management of proper glucose concentration in diabetic patients [13,14]. To develop a continuous monitoring system, it would be ideal to use an implantable device that is in constant contact with the biological fluid to give a continuous reading of glucose concentration. It is unlikely that the currently used enzyme-based method could be developed as an implantable device due to the instability issues associated with protein-based products [15]. Chemical sensors do offer the advantage of higher stability and relatively easy manufacturing. Such a concept has already been put into test by companies such as Sensors for Medicine and Science [16]. To develop a chemical sensor-based continuous monitoring device, one needs to develop glucose sensors that show high sensitivity and selectivity. Along this line, we are interested in the development of fluorescent sensors for glucose in the vesicular system. Following the pioneering works of Yoon and Czarnik [17] and later Sandanayake and Shinkai [18], boronic acid derivatives have been used in the recognition and sensing of vicinal
696
Q. Wang et al. / Sensors and Actuators B 119 (2006) 695–700
diols, carbohydrates, and catechols for the past two decades, with many successful examples emerging during this time period [19, and references within]. Boronic acids are known to bind with compounds containing diol moieties with a high affinity through reversible ester formation [20]. Such tight bonding allows boronic acids to be used as the recognition moieties in the construction of sensors for saccharides [21, and references within]. However, the boronic acid compounds are not fluorescent and are only weakly chromophoric. Under such a circumstance, spectroscopic determination relying on the intrinsic spectroscopic property changes upon binding becomes very difficult. Springsteen and Wang [22] has developed a two component competitive assay containing the fluorescent compound alizarin red S (ARS) in the phenylboronic acid (PBA)/ARS system, in which ARS was used as a general fluorescent reading agent for studying the binding events between boronic acids and compounds containing diol moieties with a high affinity. It is known that the amphiphile, 4-(4-dodecyloxybiphenyl4-yloxy) butyl trimethylammonium bromide (DBBTAB), has a cationic group with quaternary ammonium salt, which can selforganize into vesicles in dilute aqueous solution [23,24]. The vesicles composed of bilayer structure may be used as membrane models [25]. The ARS with a negatively charged group can be attracted electrostatically to the positively charged surface of the DBBTAB vesicles to form covesicles. Herein, we have developed a novel vesicular sensor composed of three tunable components: PBA receptor, ARS “read-out” units and DBBTAB vesicular carrier, in which the DBBTAB vesicles were used as a novel carrier of device to bind to the ARS component. This fluorescence sensor designed is mainly based upon self-organization of DBBTAB amphiphile and vesicular functionallization as mimetic membranes.
2. Experimental 2.1. Materials The DBBTAB amphiphile was synthesized by this group previously [26]. Alizarin red S (Xinzhong Chemical Factory, A.R. grade), phenylboronic acid (Tianjin Taida Chemical Factory, A.R. grade), d (+)-glucose (Beijing Chemical Factory, A.R. grade) were used without further purification. Water was doubly distilled after passing through an ion-exchange resin column. Phosphate buffer was purchased from Shanghai Weiye Instrument Plant. The structure of the DBBTAB amphiphile and ARS is shown in Fig. 1.
2.2. Preparation of the DBBTAB-ARS-PBA covesicles [27] The DBBTAB and ARS were combined with spectroscopic grade CHCl3 in a 50 ml beaker. Solvent was removed under N2 flow, followed by evacuation at high vacuum for 2 h. The resulting amphiphilic film in the beaker was then covered with a pH 6.8 phosphate buffer solution, which was an ideal situation because we were most interested in searching for a sensor that was functional at physiological pH values. The concentrations of DBBTAB amphiphile and ARS were fixed at 4 × 10−3 M and 5 × 10−5 M, respectively. A clear solution was obtained by sonication (Bransonic 12 Ultrasonicator, water-bath type) for 4 h at about 50 ◦ C. The covesicles were stable for weeks in the aqueous solution in a pH range of 4–10. 2.3. Instrumentation Steady-state fluorescence spectra were recorded on a RF5301PC spectrophotometer. UV spectra were measured with a Shimadzu UV-3100 spectrophotometer. Since N2 -bubbled and air-saturated solutions gave the same results, the data presented here are all for air-saturated solutions. An aqueous solution was dripped on a silica plate which was then dried in a desiccator at room temperature. Scanning electron microscopy (SEM) was collected on a JEOL JSM-6700F electron microscope. The phase transition from crystal to liquid crystal (Tc ) of the covesicles (ca. 5 wt.%) was determined by means of differential scanning calorimetry (Netzsch DSC 204). 3. Results and discussion Stable aqueous vesicular morphologies were determined by SEM. The typical SEM image from a mixture solution containing 4 × 10−3 M of DBBTAB amphiphile, 5 × 10−5 M of ARS and 5.2 × 10−4 M of PBA is shown in Fig. 2. The diameter of the vesicles was estimated about 150–500 nm from Fig. 2. The phase transition from crystal to liquid crystal (Tc ) determined by means of differential scanning calorimetry was located at 45 ◦ C. These results confirmed that the system of ARS/PBA/DBBTAB formed covesicles [24]. A typical fluorescence spectrum is shown in Fig. 3 when a PBA buffer solution was dropwise added into an aqueous DBBTAB-ARS vesicular solution. It is obvious that the fluorescent intensities of ARS bound on the surfaces of DBBTAB vesicles centred at 560 nm increase dramatically with increasing the concentration of PBA from 0 M to 5.2 × 10−4 M, suggesting that the boronic acid formed a boronate ester with ARS [22].
Fig. 1. The structure of 4-(4-dodecyloxybiphenyl-4-yloxy) butyl trimethylammonium bromide (DBBTAB) and alizarin red S (ARS).
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Fig. 2. SEM image of vesicles derived from the ARS/PBA/DBBTAB system: [DBBTAB] = 4 × 10−3 M, [PBA] = 5.2 × 10−4 M, [ARS] = 5 × 10−5 M.
In order to develop the applicability of using ARS for the determination of the binding events between a boronic acid and a carbohydrate in the vesicular system, we used glucose as a model compound. When a glucose buffer solution was dropwise added into the aqueous vesicular solution, the fluorescence intensities of ARS centred at 560 nm gradually decreased with increasing the concentration of glucose from 3.2 × 10−3 M to 43.3 × 10−3 M, which was the most physiologically relevant concentration range in terms of blood glucose detection, as shown in Fig. 4. The decrease of fluorescent intensity of ARS bound on the surface of DBBTAB vesicles implies that ARS molecules were replaced by glucose molecules from PBA-ARS ester (1) and then PBA-glucose ester (2) was gradually formed, as illustrated in Scheme 1. The free ARS replaced gradually from PBA-ARS ester (1) results in the decreasing fluorescence intensity of the vesicular system. It is known that an excited state proton transfer from the phenol hydroxyl group of ARS (1 in Scheme 1) to the ketone
Fig. 3. Fluorescence spectra in aqueous ARS/PBA/DBBTAB vesicles with adding PBA (0–5.2 × 10−4 M) at room temperature in an aqueous phosphate buffer at pH 6.8; [DBBTAB] = 4 × 10−3 M; [ARS] = 5 × 10−5 M; aqueous 0.1 M phosphate buffer; Exc. λ = 468 nm.
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Fig. 4. Fluorescence spectra in aqueous ARS/PBA/DBBTAB vesicles with adding glucose (0–43.3 × 10−3 M) at room temperature in an aqueous phosphate buffer at pH 6.8; [DBBTAB] = 4 × 10−3 M; [ARS] = 5 × 10−5 M; [PBA] = 5.2 × 10−4 M; aqueous 0.1 M phosphate buffer; Exc. λ = 468 nm.
oxygen results in the fluorescence quenching of free ARS [28]. Therefore, it was reasonable to expect that the boronate ester (2 in Scheme 1) formation would increase the fluorescence of the vesicular system through the removal of the fluorescence quenching mechanism [22]. There are two competing equilibria when glucose is added into the three component vesicular system of ARS/PBA/DBBTAB. The first equilibrium, between the phenylboronic acid and the fluorescent reporter ARS (K1 ), can directly be detected through fluorescence changes, as shown in Fig. 3. The addition of glucose sets up a second equilibrium between the boronic acid and glucose, to give boronate ester 3. This perturbs the ARS/boronic acid equilibrium, resulting in a change in the fluorescent intensity of the aqueous vesicular system [29]. The fluorescent intensity changes in the DBBTAB
Scheme 1. Schematic routine for competitive binding of a phenylboronic acid with ARS and glucose. ABS 1: weak fluorescence, boronate ester 2: strong fluorescence.
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Fig. 5. Fluorescence spectra in an aqueous ARS/PBA solution with adding glucose (0–43.3 × 10−3 M) at room temperature in aqueous phosphate buffer at pH 6.8; [ARS] = 5 × 10−5 M; [PBA] = 5.2 × 10−4 M; aqueous 0.1 M phosphate buffer; Exc. λ = 468 nm.
vesicular system can be used as a sensor for the determination of concentrations of target carbohydrates such as glucose. The sensor might be prepared as an implantable device which can be in constant contact with the biological fluid to give a continuous reading of glucose concentration. In the vesicular sensor, alizarin red S has been used as a fluorescent reading agent since the active protons of hydroxyanthraquinones in ARS are responsible for a large fluorescence quenching [14]. This result is consistent with phenomenon in aqueous solution [29]. Also, the fluorescence intensities were determined in an aqueous ARS/PBA solution for comparison, as shown Fig. 5. When a glucose buffer solution was dropwise added into the aqueous ARS/PBA solution, the fluorescence intensities of ARS gradually decreased with increasing the concentration of glucose from 3.2 × 10−3 M to 43.3 × 10−3 M. It needs to be noted that the fluorescence intensity is weak and their changes were less than that in the ARS/PBA/DBBTAB vesicular solution. It should also be noted that the vesicular sensor showed the most sensitive fluorescence intensity changes to glucose in the region of 3.2 × 10−3 M to 43.3 × 10−3 M (Fig. 4). In contrast, the dependence of the relative fluorescent intensities in the aqueous ARS/PBA/DBBTAB vesicular system and the aqueous ARS/PBA solution on the concentration of glucose, from Figs. 4 and 5, respectively, are shown in Fig. 6. Compared with the sensors in the aqueous PBA/ARS solution, the vesicular sensor showed higher sensitivity to glucose and was enhanced by about seven- to eight-fold from Fig. 6. The most significant result of the present work is the demonstration of the capacity to improve the sensitivity to glucose in the DBBTAB vesicular system. Due to the DBBTAB molecule with an ammonium hydrophilic head group, the surface of the vesicles is thought, reasonably, to be positively charged. Hence, ARS anions were attracted and then anchored on the surface of vesicles by their electrostatical action, which resulted in the increased local concentrations of ARS on the surface of covesicles. It is well known that the fluorescent intensity is dependent
Fig. 6. Relative fluorescent intensities changed with increasing the glucose concentration from 3.2 × 10−3 M to 43.3 × 10−3 M in an aqueous ARS/PBA/DBBTAB vesicular solution and a PBA/ARS solution.
on the concentration of the fluorephores in the system [24]. Therefore, it is reasonable that the sensitivity was enhanced in the DBBTAB vesicular system. The selectivity of the ARS/PBA/DBBTAB vesicular system is similar to that in an aqueous boronic acid solution [22]. The sensor showed decreased fluorescent intensity after binding with carbohydrates for glucose over lactose and ethylene glycol in the biological concentration, respectively, as shown in Fig. 7. By comparison, the selectivity for glucose in the vesicular system is higher than that for lactose, which is ascribed to the different isomerization of the saccharides [30]. As we known, the phenyl-
Fig. 7. Saccharide concentration (3.2 × 10−3 to 43.3 × 10−3 M) vs. relative fluorescence intensity profile for the PBA/ARS/DBBTAB vesicular sensor at pH 6.8 in an aqueous phosphate buffer solution; Exc. λ = 468 nm.
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boronic acid preferentially binds with the hydroxyls of glucose in the furanose form, compared with the hydroxyls of lactose in pyranose forms [31]. 4. Conclusions A vesicular fluorescent sensor was prepared based on phenylboronic acid–glucose recognition in aqueous ARS/PBA/DBBTAB vesicles. The sensor was constructed with three components: DBBTAB vesicles, PBA and ARS which served as the detector. The vesicular sensor enhanced the sensitivity to glucose by about seven- to eight-fold, compared with the corresponding aqueous solution, which was ascribed to the increased local concentrations of ARS on the surface of the DBBTAB covesicles. The vesicular sensor may be applicable to the detection of saccharides in biological systems. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 20274015, 50473005). References [1] P.A. Gale, Pyrrolic and polypyrrolic anion binding agents, Coord. Chem. Rev. 240 (2003) 17–55. [2] S. Hurtley, R. Service, P. Szuromi, Cinderella’s coach is ready, Science 291 (2001) 2337. [3] T.D. James, S. Shinkai, Artificial receptors as chemosensors for carbohydrates, Top. Curr. Chem. 218 (2002) 159–200. [4] S. Striegler, Selective carbohydrate recognition by synthetic receptors in aqueous solution, Curr. Org. Chem. 7 (2003) 81–102. [5] W. Ni, H. Fang, G. Springsteen, B. Wang, The design of boronic acid spectroscopic reporter compounds by taking advantage of the pKa -lowering effect of diol binding: nitrophenol-based color reporters for diols, J. Org. Chem. 69 (2004) 1999–2007. [6] M. Granda-Valdes, R. Badia, G. Pina-Luis, M.E. Diaz-Garcia, Photoinduced eletron transfer systems and their analytical application in chemical sensing, Quim. Anal. 19 (2000) 38–53. [7] D.J. Claremont, I.E. Sambrook, C. Penton, J.C. Pickup, Subcutaneous implantation of a ferrocene-mediated glucose sensor in pigs, Diabetologa 29 (1986) 817–821. [8] M.R. Robinson, R.P. Eaton, D.M. Haaland, G.W. Koepp, E.V. Thomas, B.R. Stallard, P.L. Robinson, Noninvasive glucose monitoring in diabetic patients: a preliminary evaluation, Clin. Chem. 38 (1992) 1618– 1622. [9] B. Rabinovitch, W.F. March, R.L. Adams, Noninvasive glucose monitoring of the aqueous humor of the eye: Part I. Measurement of very small optical rotations, Diabetes Care 5 (1982) 254–258. [10] G.M. Schier, R.G. Moses, I.E.T. Gan, S.C. Blair, An evaluation and comparison of reflolux II and glucometer II, two new portable reflectance meters for capillary blood glucose determination, Diabetes Res. Clin. Pract. 4 (1988) 177–181. [11] S. D’Auria, N. Dicesare, Z. Gryczynski, I. Gryczynski, M. Rossi, J.R. Lakowicz, A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor, Biochem. Biophys. Res. Commun. 274 (2000) 727– 731. [12] E.R. Kenneth, K.J. Ernest, Issues and implications in the selection of blood glucose monitoring technologies, Diabetes Technol. Ther. 1 (1999) 3–11. [13] T. Koschinsky, L. Heinemann, Sensors for glucose monitoring: technical and clinical aspects, Diabetes Met. Res. Rev. 17 (2001) 113–123, 17.
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Biographies Qiusheng Wang is a PhD candidate student in chemistry. His interest is in synthesis of functional amphiphile and vesicular sensor and sensor applications. Guangquan Li is an MSc candidate student in chemistry. His interest is in synthesis of functional compounds and study on the photochemical and phtophysical properties of their aggregates. Wenyi Xiao is an MSc candidate student in chemistry. Her interest is in synthesis of functional amphiphiles and molecular recognition in vesicles.
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Haixia Qi received her MSc degree in chemistry at Jilin University in 2005. Her interest is in photochemical and photophysical properties of vesicles. Guowen Li is a professor of chemistry at Jilin University. He received his PhD in chemistry in 1990 from Jilin University and did his postdoctoral research at Rutgers University from 1991 to 1993 and University of Wyoming from
1993 to 1995, respectively. His research interests are in chemistry and soft materials. His research programs focus on the synthesis of organic and polymeric compounds such as amphiphiles, and surfactants and preparation of functional aggregates. Recent works include the molecular recognition in vesicles, micelles and polymeric aggregates; fluorescence sensor and morphologies of aggregates.