WO2009023287A1 - Polymerized nile blue derivatives for plasticizer-free fluorescent ion optode microsphere sensors - Google Patents

Abstract

Lipophilic H+-selective fluorophores such as Nile Blue derivatives are widely used in ISE-based pH sensors and bulk optodes, and are commonly dissolved in a plasticized matrix such as PVC. Unfortunately, leaching of the active sensing ingredients and plasticizer from the matrix dictates the life time of the sensors and hampers their applications in vivo, especially with miniaturized particle based sensors. We find that classical copolymerization of Nile Blue derivatives containing an acrylic side group gives rise to multiple reaction products with different spectral and H+-binding properties, making this approach unsuitable for the development of reliable sensor materials. This limitation was overcome by grafting Nile Blue to a self -plasticized poly(«-butyl acrylate) matrix via an urea or amide linkage between the Nile Blue base structure and the polymer. Optode leaching experiments into methanol confirmed the successful covalent attachment of the two chromoionophores to the polymer matrix. Both polymerized Nile Blue derivatives have satisfactory pH response and appropriate optical properties that are suitable for use in ion-selective electrodes and optodes. Plasticizer-free Na+-selective microsphere sensors using the polymerized chromoionophores were fabricated under mild conditions with an in-house sonic microparticle generator for the measurement of sodium activities at physiological pH. The measuring range for sodium was found as 10-1 - 10-4 M and 1 - 10-3 M, for Nile Blue derivatives linked via urea and amide functionalities, respectively, at physiological pH. The observed ion-exchange constant of the plasticizer- free microsphere were log Kexch = -5.6 and log KeXch = -6.5 for the same two systems, respectively. Compared with earlier Na+-selective bulk optodes, the fabricated optical sensing microbeads reported here have agreeable selectivity patterns, reasonably fast response times, and more appropriate measuring ranges for determination of Na activity at physiological pH in undiluted blood samples.

Description

POLYMERIZED NILE BLUE DERIVATIVES FOR PLASTICIZER-FREE FLUORESCENT ION OPTODE MICROSPHERE SENSORS

RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Patent Application Number 60/956,285 filed on August 16, 2007, which is incorporated herein by reference in its entirety.

GOVERNMENT RIGHTS

Part of the work during the development of this invention was made with government support from the National Science Foundation under grant number BIO8-004-001. The U.S. Government has certain rights in this invention.

BACKGROUND

Ion-selective electrodes (ISEs) and their optical counterparts, ion-selective bulk optodes, have the unique capability of sensing free ion activities instead of the total concentration. The former have been routinely used in clinical laboratories for blood analysis for decades. More recently, the detection limits of ISEs have been significantly improved down to subnanomolar levels [1], making ISEs attractive for trace analysis as well [2]. Bulk optodes belong to a newer class of sensors and are usually based on the competitive or cooperative extraction of the analyte ion with protons between the polymeric and aqueous phase. This true two-phase sensing mechanism has advantages in reaching a lower detection limit dictated by the thermodynamics of ion extraction, and may lead to the mass production of monodisperse ion sensing microbeads that can be flexibly coupled with analytical flow cytometry or optical-fiber based microsensor arrays [3-5]. The unique ion-sensing capabilities of ISEs and bulk optodes make them very useful for clinical diagnosis [6], drug analysis [7], and environmental monitoring [8].

In ISEs and bulk optodes, the sensing components are conventionally blended into a plasticized polymer matrix as a solid support. However, such components and the plasticizer may leach out, especially in contact with relatively lipophilic samples such as undiluted whole blood [9]. This limits the sensor life time, and the leaching of the plasticizer induces inflammatory effects [10]. For sensors with smaller sizes such as the recently developed fluorescent ion-sensing microbeads, leaching is an even more severe problem. It is therefore advisable to covalently attach the sensing ingredients to the polymer matrix. By linking all components to the backbone of a polymer with a low Tg, leachable components may be completely eliminated from the sensor composition. Several recent papers reported on such grafting of sensing ingredients, for example, a Na ionophore (calix[4]arene tetraethyl ester) onto a IDA-MMA copolymer [11] and a Pb²⁺ ionophore (4-tert-buty\ calix[4]arene-tetrakis(N,N'-dimethylthioacetamide)) to poly(tetrahydrofuran)diol and 2,2,4-trimethylhexamethylene diisocyanate [12] for ISE applications. In an early report, a chromoionophore was grafted onto PVC-COOH for the fabrication of a Ca²⁺ optode sensor [13] and onto plasticized polyurethane matrices [14]. In our group, the covalent attachment of a Ca²⁺-ionophore (AU-i) [15] and dodecacarborane anion as cation exchanger [16] onto a poly(methyl methacrylate — co — decyl methacrylate), poly (MM A-DM A), polymer matrix for ISE and optode sensors have been successfully developed. In most cases, this was accomplished by the copolymerization of the monomer with a suitably modified free ionophore or ion exchanger. However, this method may not be suitable if undesired reactions occur, leading to significant side products that are difficult to purify. Experimentally, it was found that classical copolymerization of Nile Blue derivatives containing an acrylate group gives rise to multiple reaction products with different spectral and H⁺-binding properties, making this approach unsuitable for the development of reliable sensor materials. Presumably the polymerization reaction conditions cause some degree of attack on the Nile Blue structure, producing a non-homogenous fluoroionophoric product.

Sol-gels [17], polyurethanes [18], polysiloxanes [19, 20], silicone rubber [21, 22], polythiophenes [23], polyacrylates [24], epoxy acrylate [25], and poly(MMA-DMA) [26], have been used as plasticizer-free matrices. However, because the response of bulk optode is given by the equilibration of the sensor with the bulk sample, the response time is a crucial parameter in choosing the polymer matrix, which depends on the diffusion coefficient and the thickness of the polymer. MMA-DMA copolymer has been used in ISEs, but as a matrix for the microsphere optodes, it requires a response time on the order of many hours, which is generally unacceptable. Poly(n-butyl acrylate) (PnBA) is a polyacrylate type of polymer with lower Tg than the previously used poly(MMA-DMA) copolymer, which may provide larger diffusion coefficient and a shortened response times. Thus PnBA and similar matrices that are "self plasticized" and have a suitable Tg below that of previously used MMA-DMA copolymers may be suitable for developing plasticizer-free microspheres as sensors. The present invention overcomes the limitations of polymerizing the matrix in the presence of Nile Blue, by preparing a suitably functionalized polymer matrix and grafting Nile Blue to it by reactions that do not affect the Nile Blue fluorescence properties. For example, using a poly(«-butyl acrylate) matrix linked via a urea or amide linkage between the Nile Blue base structure and the polymer has been shown to overcome these issues. These materials enable preparation of plasticizer-free fluorescent ion optode materials suitable for making microsphere sensors that can be used in contact with cells, such as for Na⁺ measurements in blood samples.

SUMMARY OF THE INVENTION

Lipophilized Nile Blue derivatives are attractive H-selective chromo- and fluoroionophores that are used in optical [27] and potentiometric sensors [28]. Here, new types of Nile Blue derivatives that are covalently linked, for example via a urea, carbamate or amide linkage, to a self -plasticized polymer or copolymer matrix having a suitably low Tg, such as poly(«-butyl acrylate), were synthesized and characterized. They were applied to thin film optodes as well as plasticizer-free microspheres with sodium detection as an example of their utility. These represent a class of novel fluoroionophores covalently linked to a copolymer by covalently linking the fluoroionophore to a functionalized copolymer matrix. The polymer or copolymer matrix is selected to be self- plasticizing, i.e., it is one that does not require an added plasticizer; and it is selected to have a Tg below about 50⁰C, and often below about 20⁰C, to provide a suitably short response time. These sensor materials are suitable for use in undiluted blood samples at physiological pH, for determining Na⁺ activity levels. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows synthesis routes to covalently attach the H⁺-fluoroionophore Nile Blue onto a poly-H-butylacrylate backbone. Route 1: Direct copolymerization of NB-Urea monomer with H-butyl acrylate monomer. Route 2: Covalent grafting of NB-Urea and NB-Amide onto PnBA containing linker functionalities.

Fig. 2 shows absorbance (in THF) and fluorescence spectra (in PVC-DOS films) of NB- Urea monomer (top) and NB-Urea- PnB A (via route 1, bottom) in their protonated (dashed line) and deprotonated forms (solid line).

Fig. 3 shows fluorescence sodium response curves and corresponding selectivity of optode films containing NB-Urea monomer, Na(X), and NaTFPB dissolved in PVC-DOS film at pH 7.4. The lines are according to theory (eq 2).

Fig. 4 shows absorbance (in THF) and fluorescence spectra (in PVC-DOS films) of NBUrea — PnBA (via route 2, top) and NB-Amide — PnBA (via route 2, bottom) in their protonated (dashed line) and deprotonated forms (solid line).

Fig. 5 shows fluorescence — time profile for continuous solvent extraction of the two grafted chromoionophores obtained via route 2 from PnBA films into methanol, and comparison with the commercial fluoroionophore ETH 5294 in the same matrix.

Fig. 6 shows fluorescence sodium response curves and corresponding selectivity for optode films containing the two fluoroionophores from route 2, NB-Urea — PnBA (A) or NBAmide— PnBA (B) and Na(X), NaTFPB in PVC-DOS films at pH 7.4. The lines are according to theory (eq T).

Fig. 7 shows fluorescence sodium response curves and corresponding selectivity for 10- im diameter optode microspheres containing the two fluoroionophores from route 2, NB- Urea— 3 PnBA (A) or NB-Amide— PnBA (B) and PnBA, Na(X), and NaTFPB at pH 7.4. The lines are according to theory (eq 2). Fig. 8 shows potentiometric pH response of PVC-DOS membranes containing the two types of grafted Nile Blue derivatives, (A) NB-Urea— PnBA (R2) and (B) NB-Amide- PnBA (R2).

DETAILED DESCRIPTION

The covalent attachment of a sensing component needs to be carefully designed so that the ion binding properties of the components are maintained after being grafted. Different from earlier work of grafting ionophores or ion exchangers, a H⁺-chromoionophore is a fluorescent dye, and maintaining its spectral properties is important. The UV- Vis absorbance and fluorescence spectra of the organic molecule may change drastically when the chemical environment is altered, e.g. the appearance of an unwanted absorbance peak may severely mask the pH response. Therefore, the present invention provides a method for attaching the fluoroionophore to a suitable polymer matrix by clean and selective reactions that are performed after the polymerization to form the polymer.

In a first, more traditional route, the Nile Blue structure was modified to contain a polymerizable moiety as methacrylate monomer (NB-Urea monomer) and this derivative was copolymerized with H-butyl acrylate monomer. This route has been widely used for grafting ISE and optode ingredient into the polymer backbone [11, 12]. It covalently links Nile Blue to a polymer without requiring a plasticizer, but this method led to the formation of a material having an unexpected additional absorbance band for the deprotonated state. Accordingly, this method was unsuitable for the present purposes because it produced a material that was not suitable for use in an optode sensor.

To overcome this problem, the invention provides a self-plasticized polymer matrix with a fluoroionophore such as Nile Blue covalently attached (grafted) to the matrix after the polymerization reaction used to form the polymer or copolymer matrix, to avoid reactions of the fluoroionophore core that could occur during polymerization. As an example, a first monomer of Formula (I), such as H-butyl acrylate monomer, was copolymerized with a second acrylate monomer of Formula (II) containing a functional group (R-NCO and R- COOH are examples) suitable for coupling the resulting polymer to Nile Blue. Use of the functional group to attach the fluoroionophore after polymerization avoids any undesirable reactions that could occur during the radical polymerization reaction if the fluoroionophore were present during the polymerization. By using covalent linkages such as the NB -urea and NB -amide linking groups described herein, the invention also provides new fluorescent dyes with high pKa values for purposes of measuring high analyte levels at near neutral pH, such as sodium ions in physiological samples.

The compositions of the invention comprise a polymer or copolymer matrix to which Nile Blue is covalently linked, and do not contain a plasticizer. Suitable polymer matrices include, for example, self-plasticized known sol-gels [17], polyurethanes [18], polysiloxanes [19, 20], silicone rubber [21, 22], polythiophenes [23], polyacrylates [24], epoxy acrylate [25], and poly(MMA-DMA) [26], that have suitable Tg below about 50⁰C, and preferably below about 20⁰C. Methods for making these polymer matrices and for determining Tg for the matrix are known in the art.

A preferred copolymer matrix comprises a first monomer and a second monomer, where the mixture provides a Tg (glass transition temperature) below about 50⁰C, and preferably below about 20⁰C. The Tg depends on the structure of each of the two monomers as well as the proportion of each monomer present in the copolymer. However, measurement of the Tg for such a copolymer is routine in the art, so selection of a specific mixture for use in the compositions of the invention is readily accomplished by one of ordinary skill. Nile Blue as used herein refers to the specific structure:

Nile Blue analogs are also suitable for use in the present invention, and include compounds having minor structural modifications of the Nile Blue core that do not substantially change its fluorescence properties. For purposes of the invention, these Nile Blue analogs include compounds of Formula III:

wherein R₁ and R₂ independently represent a C^₄ alkyl group, and wherein R₁ and R₂ can be taken together with N to which they are attached to form a heterocyclic group selected from pyrrolidine, piperidine, morpholine, thiomorpholine, and N⁴- methylpiperazine.

The copolymers useful for the invention include a first monomer of Formula I:

wherein X is H or Me, and R is a 3-10 carbon alkyl group, such as propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, n-hexyl, cyclopropylmethyl, and the like.

The copolymers of the invention also include a second monomer of Formula II:

wherein X is H or Me, n is 1-7, and Z is a functional group that can be used to alkylate or acylate Nile Blue on the exocyclic nitrogen shown as =NH in Formula III, or to form a covalent linkage to that exocyclic nitrogen.

X in Formula I can be H or Me; in some embodiments, X is H. Where X is Me, preferably R is a C4-C8 alkyl group to ensure that the copolymer has a suitable Tg. R can be a straight chain, branched chain, or cyclic alkyl group, or a combination of straight chain, branched chain and/or cyclic portions.

X in Formula II can be H or Me. Typically, the second monomer is used in smaller amounts than the first monomer, so its structure has less effect on the Tg of the copolymer than does the first monomer. As a result, X in Formula II can be either H or Me, i.e., the second monomer can be either an acrylate or a methacrylate, even when the alkyl group of its ester is small. Moreover, since the second monomer will be used to covalently attach Nile Blue, the length of its alkyl group is less important to the properties of the copolymer. Accordingly, n can be 1-7 for either acrylate or methacrylate embodiments of the second monomer. Frequently, n is 1, 2 or 3 for synthetic convenience.

Note that the composition requires a copolymer having at least two different monomers; the second monomer is one with a functional group attached to permit covalent linkage of the fluoroionophore Nile Blue. However, the performance of the material does not require limiting the composition to exactly two types of monomers, and it is also suitable for the first monomer to represent a mixture of materials, for example it could include two or more different ester groups, or it could include a mixture of acrylate and methacrylate monomers without departing from the inventive concept. Either the first monomer or the second monomer could include a mixture of materials within the scope of Formula I and II respectively.

Z is a functional group that permits easy attachment of Nile Blue to the polymer matrix after polymerization has been accomplished. Attachment to Nile Blue or an analog of Nile Blue is typically at the exocyclic nitrogen (=NH), so suitable functional groups are those that can be readily linked to this nitrogen with high efficiency. Such groups are well-known in the art. Z can be in a protected form if desired to ensure that it does not interfere with or participate in reactions during the polymerization step, but protection is frequently not needed — for example, when Z is -NCO or -COOH, the examples demonstrate that it can be carried through the polymerization reaction without protection. Other functional groups that Z can represent include hydroxyl, amino, epoxide, aldehyde, leaving groups (e.g., halide such as Cl, Br or I; alkylsulfonates such as mesylate or triflate; and arylsulfonates such as phenylsulfonate or tosylate) and thiol, each of which can be optionally protected. Methods for protection and deprotection of the functional groups that may require protection (hydroxyl, thiol, amino) are well known in the art, and suitable examples are disclosed in PGM Wuts and TH Greene, et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Wiley Interscience (2006), which is incorporated herein by reference.

Where Z is an epoxide or leaving group or -NCO, it may be linked to the Nile Blue analog by a direct reaction with the functionalized polymer, after polymerization has been done. Reaction is accomplished by contacting the polymerized material with Nile Blue or a Nile Blue analog under suitable conditions, typically in a solvent such as dichloromethane, chloroform, ethyl acetate, DME, DMF, NMP, DMSO, THF, or an alcohol such as t-butanol. Non-alcohol solvents are preferred if Z is -NCO, as is understood in the art. Where Z is COOH, reaction with Nile Blue is accomplished under conventional amide-bond forming conditions that are well known in the art. Typical examples include use of a dehydrating agent such as a carbodiimide (DCC; DIPC; EDC); formation of an activated ester such as with HOBt (N-hydroxybenzotriazole); or formation of an acid halide or mixed anhydride. Where Z is an aldehyde, the linkage may be produced by using reductive alkylation conditions.

Where Z is an amine, hydroxyl, or thiol, the copolymer comprising Z can be treated with an acylating agent such as carbonyl diimidazole to provide a reactive species of the form [polymer]-O-(CH₂)_n-A-C(O)-LG, where A is O, N, or S from group Z; and LG represents a leaving group such as imidazolyl or halo. This reactive species can be used to acylate the nitrogen of Nile Blue, to provide a copolymer with Nile Blue or a Nile Blue analog covalently attached to it.

In another aspect, the invention provides a method for making compositions of the invention by polymerization of a first acrylate monomer and a second acrylate monomer as described above, followed by covalent attachment of the fluoroionophore (e.g., Nile Blue) to the polymer matrix using efficient, conventional acylation reactions.

In another aspect, the invention provides an optode sensor that comprises the composition described above, and methods of using this sensor for the determination of ion levels in samples such as blood samples. In certain embodiments, the sensor includes a microbead that comprises or that consists essentially of the functionalized fluoroionophore- containing polymer composition described above.

Route 1: Direct copolymerization of Nile Blue derivatives

In the first route, the synthesis of NB -Urea monomer with a methacrylate polymerizable group was performed by the reaction of 2-(methacryloyloxy)ethyl isocyanate with Nile Blue base to yield the urea linkage between Nile Blue and polymerizable group (Route 1 in Figure 1). The free NB-Urea monomer showed clearly distinguished spectra in the protonated and deprotonated form, both in absorbance and fluorescence mode. The characteristic absorption and fluorescence wavelengths of all synthetic compounds, along with the pKa values calculated from optode experiments (see below), are show in Table 1. The absorbance spectra (Fig. 2 top) in THF solution exhibited protonation and deprotonation peak maxima at 660 nm and 525 nm with molar absorptivities of 5,961 ± 2 and 2,610 + 4 L mol^"1 cm^"1, respectively. The fluorescence emission spectra (Fig. 2 top) were observed by microscopy in a PVC-DOS matrix and showed peak maxima in their protonated and deprotonated forms at 678 nm and 635 nm, respectively. The well-defined spectra in both protonation and deprotonation forms are comparable with commercial chromoionophores and may be used in ion optode systems.

Table 1 : Spectral characteristics and estimated acidity constants of the fluoroionophores synthesized in this work

a In THF. b InPVC-DOS films. c In PVC-DOS films, calculated from K_exch values in sodium-selective optode experiments (see text).

d By spectrophotometric titration in THF. e ε = 5961 + 2L mOl ¹Cm^"1 f ε =2610 + 4L mOl ¹Cm^"1

The ion-exchange equilibrium mechanism of a bulk optode is normally given by Eq (1) [6]:

ZlUdH⁺ + nL +l^z+(aq) + zK = zlnd+ IL_n ¹⁺ + zlT(aq)+ zR

(1)

where Ind denotes a neutral chromoionophore, L a neutral ionophore and R^" the cation exchanger, z is the charge of analyte ion I^z+, and n is the complex stoichiometry of ionophore to analyte for IL_n ^z+. Species in the aqueous sample phase are indicated with (aq), while all others are in the organic sensing phase. Equation 1 illustrates that the change between the protonated and deprotonated form of the chromoionophore depends on the activity of the ion I^z+ in the aqueous phase. It is measured by the ratio of peak intensities from the unprotonated form over that of the protonated form. The relationship between the activity of T⁺, a_Ϊ5 and the mole fraction of unprotonated form of the fluoroionophore, α, is given as [30, 31]: ai = (l/zK^H)(αa_H/(l-α))²(Rτϊl-α)/»^τ) (2)

(L_T - (n/Z)(R_T - (l-α)Ind_τ)ⁿ

where L_τ , Indj and R_τ ^~ are the total concentration of ionophore, chromoionophore and lipophilic ion-exchanger, respectively, and K_exch is the ion exchange constant that describes equilibrium 1. The parameter α is experimentally accessible as the ratio of the fluorescence intensity of the protonated (R_P) and deprotonated form (R_D) of the chromoionophore [32]:

α = [Ind] / Ind_τ = 1 + [(RP - R) / (R-Rd)]^"1 (3)

Consequently, the competition equilibrium of P⁺ and H⁺ is primarily described by K_exch- In order to measure Na⁺ in blood (135 - 150 mM) at physiological pH, for instance, the Ke_xc_h of the system is required to be relatively low, which may, for example, be accomplished by using a chromoionophore of high basicity.

A Na⁺ selective optode film was prepared with NB-Urea monomer as chromoionophore in PVC-DOS and gave satisfactory response characteristics in Na⁺ solutions at pH 7.4. As shown in Fig. 3, the experimental data correspond to theory with log K_exch = -3.75 and the sodium measuring range was 10^"3 - 10^" M. Note, however, that the effective measuring range at this pH does not cover the physiological concentration of sodium ions, suggesting a lower than desired chromoionophore pKa value.

Subsequently, the NB-Urea monomer was copolymerized with H -butyl acrylate monomer by radical polymerization with the initiator AIBN in a one-step reaction. Fig. 2, bottom, presents the absorbance spectra of the protonated and deprotonated form of the Urea- PnBA in THF. They are quite similar to the monomer, except that an additional peak now appears at 435 nm. The original maximum at 524 nm corresponding to the deprotonated form is not disturbed. This result suggests that the final polymer contains different species of Nile Blue derivative. It is plausible that a radical polymerization reaction designed to attack the double bonds of acrylic monomers may also react with a highly conjugated Nile Blue structure to form a variety of products, but no further studies were performed here to determine the exact structure of the resulting reaction products. A titration of the polymer was performed to observe the absorption spectrum change and to evaluate the existence of species with different pKa values. Indeed, the results indicate that the two deprotonation peaks exhibit different behaviors to changes in pH. In addition, the fluorescence spectra in PVC-DOS film, Fig. 2, bottom, show that the protonation spectra cannot easily reach the fully protonated form upon exposure to acidic solutions. Although this polymer was explored for the fabrication of Na⁺-selective optode films, the results were not satisfactory, with a response slope much smaller than theoretically expected (data not shown). This problem was likely to be caused by a change of the properties of Nile Blue base structure during radical polymerization. Route 2 was subsequently chosen to circumvent this limitation.

Route 2: Coupling Nile Blue derivatives to a functionalized polymer

This route is illustrated in Figure 1 as Route 2 and was designed to avoid undesired side reactions of Nile Blue in the radical polymerization process. In the first step, PnBA was modified to exhibit isocyanate and carboxylic moieties for reaction with Nile Blue via urea or amide linkages. This step still employed AIBN via a radical polymerization reaction. The NMR spectra of both polymers were identical: characteristic peaks for the monomer containing the functional group of interest were not observed. The unreacted monomer characteristic with a high coupling constant of the acrylate group at about 5.5- 6.5 ppm was also absent, suggesting that the grafted polymer no longer contained appreciable residual monomers.

The grafted polymers from Route 2 were dissolved in THF for UV- Vis absorbance measurements. Both grafted polymers exhibited well defined protonation and deprotonation spectra. For the NB -Urea spectrum, its protonated form is shown in Fig 4 (top) with peaks at 618 and 666 nm, while the deprotonated form gives one broad peak at 534 nm. The absorption spectrum of NB-Amide is similar to that of NB-Urea and is shown in Fig. 4 (bottom) with 608 nm and 656 nm for the protonated and 542 rim for the deprotonated form. The NB-Urea grafted PnBA absorption spectra obtained in the second route were compared with the Urea-PnBA from the first route, and the spectra were indeed different. In the deprotonated form, NB-Urea from the second route showed only one well defined peak and when titrated with HCl solution the spectral change exhibited the expected isosbestic point. The fluorescence spectra of the two adducts obtained via route 2 were compared as blends in PVC — DOS films. For NB-urea — PnBA (R2), the emission spectra are shown in Fig. 4 (top) with peaks at 679 nm (protonated) and 630 nm (deprotonated), quite similar to the properties of NB-Amide — PnBA (R2) (680 and 650 nm, respectively; see Fig. 4 bottom). No shoulder was present in the spectrum of the protonated form as for the first synthesis route. This again suggests that route 2 yields cleaner reaction products with fewer undesired side reactions that otherwise results in a mixture of fluorescing pH indicator dyes with a range of effective pKa values.

The amount of the grafted moieties in the polymer was determined by spectrophotometric titration with HCl in THF. NB-Urea — PnBA and NB-Amide — PnBA obtained via route 2 contained 45+1 and 26.6+0.5 mmol kg^"1, respectively. In other embodiments, the amount of fluoroionophore grafted to the copolymer can be between about 10 and 100 mmol/kg.

A leaching experiment was performed for the polymer-grafted dyes and non-grafted dyes by using methanol as a lipophilic solvent that should aid in the rapid extraction of unbound fluoroionophore. The fluorescence was monitored by continuously flushing methanol over a polymeric membrane deposited on a quartz slide in a flow cell. As shown in Fig. 5, the fluorescence intensity did not change significantly over 60 min for both polymer grafted dyes synthesized via route 2. In the control experiment with a PnBA film containing freely dissolved fluoroionophore ETH 5294, fluorescence dropped instantaneously, and the fluorescence signal completely disappeared within 5 min. This confirms that grafting effectively retains the dye in the polymer even under harsh solvent conditions.

The grafted polymers were applied to thin film optodes. We prepared Na⁺-selective bulk optodes with Na⁺ ionophore X , NaTFPB and grafted Nile Blue derivatives. Figure 6 shows the response curves of this experiment and the agreement with the theoretical curve according to eq 2. This confirmed that both grafted polymers in the second synthesis route behave in analogy to commercial, freely dissolved chromoionophores. The observed ion-exchange constant was log K_exch = -4.45 for NB-Urea — PnBA (R2) which is lower than for the NB-Urea monomer (log K_exch = -3.75) at the same composition. This result points to a difference in basicity between grafted and non grafted chromoionophore. The pKa values shown in Table 1 were calculated from the difference between the log K_eXCh values determined experimentally and the corresponding value obtained for the same optode system [3], but containing the same concentration of chromoionophore I (ETH 5294) with an apparent pKa value of about 11.4 [33]. The NB- Amide — PnBA (R2) showed log K_exch = -5.25, lower than that of the NB-Urea system, suggesting a higher basicity than NB-Urea grafted PnBA. This result may be explained by the effect of the difference in the linkage groups between the two Nile Blue base structures. The urea linkage exhibits a stronger electron withdrawing character than the amide linkage group, resulting in a lower electron density on the basic nitrogen.

Furthermore, the basicity of NB-Amide — PnBA (R2) is comparable to that of ETH 5294 (see Table 1). With a lower log K_exch of the Amide derivative, the measuring range appears to be suitable for the determination of Na⁺ in undiluted blood samples.

PnBA was used together with the synthesized NB-Urea or NB-amide (route 2) for preparing plasticizer-free ion-sensing microsphere optodes. As shown in Fig. 7 A and B the data for both NB-Urea — PnBA and NB-Amide — PnBA are in good agreement with the theoretically predicted curve (eq T). The observed ion-exchange constant were log Ke_xc_h = -5.55 and log Ke_XCh = -6.50 for NB-Urea and NB-Amide grafted PnBA, respectively. When compared with the thin film system both log K_eXCh were lower and the measuring range are shifted to a higher Na+ concentration range under the same conditions and with the same sensing components. Clearly, the apparent pKa values of the chromoionophores are also dependent on the solvent matrix. The selectivities of Na⁺ over K⁺, Ca²⁺ and Mg²⁺ were characterized as in the thin film experiment. The selectivity values of the plasticizer-free micro spheres agree with the thin film experiments (see table 2) and with previous studies using the same sodium ionophore [3]. The plasticizer-free microspheres also exhibited reasonably rapid response times, with 30 min equilibration times adequate for their functioning.

Table 2: Experimental ion-exchange constants and selectivity coefficients, normalized to pH 74a of sodium-selective optodes containing the indicated H-selective fluoroionophore

Calculated at α = 0.5, see [27]

This illustrates that the invention provides a method to modulate the K_eXCh of a fluoroionophore that is grafted to a polymer matrix, by selection of the linking group. The selection of the linking group affects electron density on the nitrogen of Nile Blue or its analog to which the polymer is linked. By selecting a linking group that is more electron withdrawing, e.g. using the urea instead of the amide linker, that nitrogen has a lower electron density; the result is a lower basicity and a higher K_exch- The Ke_XCh can thus be adjusted to provide a sensor having a measuring range that is suitable for a particular application as illustrated by the example above, where selection of the amide linking group provided a sensor with an operating range that includes normal Na⁺ levels found in undiluted blood. Those of skill in the art are aware of the relative electron- withdrawing strength of the linkers that are disclosed herein, and can accordingly modulate K_e -4.2 -16.4

_xch of the fluoroionophore in the optodes of the invention by appropriate selection of Z in Formula II. Ion-selective electrode membranes containing grafted polymers in PVC-DOS containing the anion exchanger NaTFPB were also prepared to assess the potentiometric response toward H⁺ and the selectivity of H⁺ over Na⁺, see Figure 8. The pH response range of such membranes was between pH 2.5 — 7 and pH 2 - 8, with a gradually decreasing slope with increasing pH. The apparent selectivity coefficient for H⁺ over Na⁺ was found as — 7.1 and — 7.6, for NB-Urea and NB-Amide grafted PnBA, respectively. This would point to a lower basicity of the resulting grafted polymer adduct compared to the evidence gathered by the optode experiments (c.f. Table 1). While this study was not sufficiently detailed to fully understand this discrepancy, it appears that covalent attachment may impact on the observed sensor response to a lesser extent with bulk optodes than with potentiometric sensors.

This invention provides a new strategy for covalent grafting of fluorophores into a self- plasticized polymer matrix, and a plasticizer-free bulk optode microsphere sensor for sodium using the polymer-fluorophore composite. Two types of Nile Blue derivatives were synthesized by covalently grafting the Nile Blue structure into self-plasticized poly(n-butyl acrylate) via urea (NB-Urea) or amide (NB-Amide) linkers. Initially, NB- urea monomer was synthesized, and the optical characterization and the consequent experiments with bulk optode films showed that the free NB-Urea derivative can be successfully used as a fluorescent pH indicator. However, copolymerization of the fluorophore with n-butyl acrylate monomer altered the Nile Blue structure, and affected the response of the bulk optodes. Via a second route, we modified H -butyl acrylate with a suitable functional group into the polymer backbone for polymerization, followed by the reaction with Nile Blue. This route turned out to be successful for the synthesis of two polymer grafted fluorophores. The grafted NB-urea and NB-amide polymers from this route 2 can be used as fluoroionophore in ion selective optodes in the same way as commercial chromoionophores, but with improved lifetimes. Indeed, plasticizer-free fluorescent ion-sensing microspheres were prepared using the two polymer-fluorophore composite for sodium, and showed good selectivity toward potassium, calcium and magnesium. The measuring ranges of sodium ions were found as 10^"1 - 10^"4 M and 1 - 10^~3 M, for NB-Urea and NB-Amide — PnBA, respectively, at physiological pH. The method reported here is not only suitable for fabricating bulk optodes, but also can be used generally in any sensors based on fluorescence transductions, and may be promising for applying this type of sensor in vivo.

The following examples illustrate specific embodiments in further detail. These examples are provided for illustrative purposes only and should not be construed as limiting the invention or the inventive concept to any particular physical configuration in any way.

EXAMPLES

Reagents

2-carboxyethyl acrylate, 2-(methacryloyloxy)ethyl isocyanate, triethylamine and bis(2- oxo-3-oxazolidinyl)-phosphinic chloride (BOP-Cl) were reagent grade from Aldrich (Milwaukee, WI). Nile Blue chloride salt was obtained from Acros Organic (Newburyport, MA). Ethyl acetate, dichloromethane, methanol and 1,4-dioxane were reagent grade and obtained from Fisher. Dichloromethane was dried over CaH₂ and freshly distilled under nitrogen atmosphere prior to use. The H-butyl acrylate monomer, 99%, was obtained from Polysciences, Inc. (Warrington, PA). Inhibitors were removed from the monomers by the method previously reported [29]. The polymerization initiator 2, 2'-azobisisobutyrohltrile, 98%, (AIBN) was obtained from Aldrich and recrystallized from warm methanol prior to use. A-tert- butylcalix[4]arene-tetraacetic acid tetraethyl ester (sodium ionophore X), sodium tetrakis [3, 5- bis(trifluoromethyl)phenyl]borate (NaTFPB), bis(2-ethylhexyl)sebacate (DOS), high molecular weight poly(vinyl chloride) (PVC), tetrahydrofuran (THF) and all salts were purchased in Selectophore or puriss quality from Fluka (Milwaukee, WI). Aqueous solutions were prepared by dissolving in Nanopure purified water (18.2 MΩ cm^"1).

Syntheses

NS- Urea monomer

Into a 100 mL two-necked round bottom flask equipped with a reflux condenser, (0.25 mL, 1.76 mmol) of 2-(methacryloyloxy)ethyl isocyanate and dried dichloromethane (30 mL) were mixed and stirred under nitrogen atmosphere at ice bath temperature. Nile Blue base (0.2 g, 0.63 mmol) was dissolved in dried dichloromethane (20 mL) in an addition funnel. The solution was then added dropwise for 30 min, followed by continuous stirring for 2 h. Dichloromethane was evaporated and the crude product was purified using flash chromatography (pure dichloromethane as eluent). The dark violet solid was obtained in 70 % yield. The structure was confirmed by ¹H-NMR and ESI-MS. Η-NMR spectrum (CDCl₃, 200 MHz.): δ (in ppm) 8.54 (d, J_H-_H = 7.4 Hz, IH), 8.36 (d, J_H-_H = 7.2 Hz, IH), 7.56 (m, 3H), 6.63 (m, 2H), 6. 36 (dd, IH), 6.17 (s, IH), 5.77 (s, IH), 5.59 (dd, IH), 4.37 (t, J_H-_H = 4.8 Hz, 2H), 3.74 (d, J_H-_H = 5 Hz, 2H), 3.47 (m, 4H), 1.97 (m, 3H), 1.26 (t, J_H-_H = 6.7 Hz, 6H). Mass spectrometry: ESI-MS m/z = 473.0 [C₂₇H₂₈N₄0₄+H⁺].

General procedure for polymerization

After mixing H -butyl acrylate monomer with co-polymerized monomer and dry ethyl acetate (except AIBN), the solutions were purged with N₂ for 20 min to remove dissolved oxygen prior to adding AIBN. The reactions were immersed in a liquid paraffin bath and continuously stirred at 90 ⁰C under N₂ atmosphere for 16 h. The ethyl acetate was then evaporated by rotary evaporator under reduced pressure. The polymers were redissolved in 20 mL of 1,4-dioxane. This solution was added dropwise to 500 mL deionized water under vigorous stirring. The white precipitate was collected and dissolved in 25 mL of dichloromethane. Water was removed by anhydrous Na₂SO₄, and the solution was filtered. Dichloromethane was again evaporated under reduced pressure.

Copolymerization ofNB-Urea monomer with n-butyl acrylate 2.2 g H-butyl acrylate and 29.8 mg NB-Urea monomer were added into a 100 mL round- bottom flask containing 15 mL of ethyl acetate. Then, following the general procedure (with 10.3 mg AIBN) but using only 10 mL of 1,4-dioxane to dissolve crude polymer, the product was obtained. The copolymers synthesized in this manner are referred to as NB- Urea — PnBA (Rl) in this work. Copolymerization ofn-butyl aery late with — CNO and — COOH groups 5 g of H-butyl acrylate and 100 mg of 2-(methacryloyloxy)ethyl isocyanate or 2- carboxyethyl acrylate were added into a 100 mL round-bottom flask containing 15 mL of ethyl acetate. Then, followed by the general procedure, the sticky transparent polymers were obtained and are referred to as PnBA-ΝCO and PnBA-COOH, respectively.

NB-Urea-PnBA (R2)

Into a 100 mL two-neck round bottom flask equipped with a reflux condenser, 2.5 g PnBA-ΝCO and dried dichloromethane (20 mL) were mixed and stirred under nitrogen atmosphere at room temperature. 25 mg of purified Nile Blue base was dissolved in dried dichloromethane (20 mL) in an addition funnel. The solution was then added dropwise for 30 min. The resulting mixture was constantly stirred for 3 h. Dichloromethane was evaporated to obtain a pink polymer. The polymer was dissolved in 10 mL of dichloromethane and 300 mL of methanol was then slowly added, precipitating NB- Urea — PnBA (R2). The polymer was washed with methanol to ensure the complete removal of non-grafted Nile Blue. The product was dried under ambient temperature and kept in the dark.

NB-Amide-PnBA (R2)

Into a 100 rnL two-neck round bottom flask equipped with a reflux condenser, PnBA-

COOH (2.5 g), BOP-Cl (0.2 g), triethylamine (0.5 mL) and dried dichloromethane (50 mL) were mixed and stirred under nitrogen atmosphere at 40 ⁰C. 50 mg of purified Nile Blue was dissolved in dried dichloromethane (20 mL) in an addition funnel. The solution was then added dropwise for 30 min. The solution was constantly stirred for 3 h at 70 ⁰C. After that, the reaction mixture was washed with 30 mL of saturated NaHCU₃, 50 mL of saturated NaCl (2 times), and finally washed with 100 mL of water. The polymer solution was dissolved in dichloromethane, dried with anhydrous Na₂SO₄ and filtered over cotton. The dichloromethane was evaporated to obtain a violet polymer. The polymer was dissolved in 50 mL of dichloromethane and 300 mL of methanol was then slowly added, precipitating NB -Amide — PnBA (R2). The polymer was washed with methanol to ensure the complete removal of non-grafted Nile Blue. The product was dried under ambient temperature and kept in the dark. Instrumentation

A Pariss Imaging Spectrometer (Light From, Belle Mead, NJ) combined with a Nikon Eclipse E400 microscope equipped with an epifluorescence attachment (Southern Micro Instruments, Marietta, GA) was used to observe the fluorescence change for optode films and particles as previously described [9]. The absorbance spectra for the protonated and deprotonated forms of the grafted polymers and monomers in the THF solution were carried on a HP8452 diode array spectrophotometer. The EMF of potentiometric titration experiment was recorded by a PCI MIO 16XE data acquisition board (National instruments, Austin, TX) utilizing a four- channel high Z interface (WPI, Sarasota, FL) with a Ag/ AgCl reference electrode with 1 M LiOAc liquid junction (type 6.0729.100, Metrohm AG, CH-9101 Herisau, Switzerland).

Potentiometric measurements

ISE membranes were prepared by dissolving Nile Blue grafted PnBA (10%w/w),

NaTFPB (2 mmollkg), PVC and DOS (1:2 by mass) to give a total cocktail mass of 140 mg in 1.5 mL of THF. Cocktails were poured into a glass ring (2.2 cm i.d.) affixed onto a glass slide. The solvent was evaporated overnight to give a transparent membrane. The membranes were then conditioned overnight in 10 mM citric acid solution + 10 mM boric acid solution with 10 mM NaCl solution as background (buffer solution) and this buffer solution was also used as inner filling solution. A Philips body electrode was used to perform all potentiometric experiments. In the calibration curve, 100 mL of universal buffer solution was used and the pH was first adjusted to 1.5 with 1 M HCl before titrating with 1 M standard NaOH solution. The solution pH was simultaneous monitored with a commercial pH electrode.

Optode thin film preparation A cocktail mixture was made with 1 mL THF, which consisted of 5 mmol/kg NB grafted chromoionophore (as calculated from a spectrophotometric titration) or NB -Urea monomer, 10 mmol/kg NaTFPB as ion exchanger, 20 mmol/kg of sodium ionophore (Na(X)) and PVC-DOS (1:2 by mass) with total mass 45 mg. A 50 μL aliquot of cocktail was pipetted onto a 22-mm (No. 1) square cover glass slide. The freshly prepared films were dried in ambient air for at least 30 min prior to use. Particle preparation

Particles were prepared using a particle casting apparatus that has previously been described [9]. A cocktail (total mass of 45 mg) containing 5 mmol/kg of NB-grafted PnBA, 10 mmol/kg NaTFPB, 20 mmol/kg of sodium ionophore (Na (X)) and PnBA was dissolved in 1.25 mL of cyclohexanone. The cocktail was poured into a 50 mL ethyl acetate and with 0.5 mL of xylene. The solution was stirred to mix well. The solution was filtered through a 0.45-μm Gelman filter by a gas tight Hamilton syringe. The filtrate was poured into another gas tight syringe which was mounted on a syringe pump (Stoelting, Wood Dale, IL) and the flow rate was set to 0.3 mL/min. The deionized water was used as the sheath liquid stream at a flow rate 70 mL/min. The frequency generator was adjusted to 12.3-12.7 kHz. The microspheres were collected in a glass vial and left to cure for 4 d before use.

Particle characterization

After curing the particles for 4 d, the particles were concentrated by removing some of the curing water. A 100 μL of particle suspension was pipetted onto a microscope cover glass. After the particles settled down on the slide, 30 mL of water was added to remove any retained organic solvent in the particles. These slides were immersed in the calibration solution for 1 h before measurement. The Na⁺ calibration solutions for both film and microparticle were prepared from 1 M of NaNU₃ in 1 mM of TRIS-HCl buffer and the concentrations are 0.01 mM - 1 M. All calibration solutions were adjusted to pH 7.4 with 1 M HCl. The selectivity was determined using the horizontal distance between the response curves of the primary and interfering ions at α = 0.5 [30, 31]. The interfering ions response curves were acquired by observing microsphere responses in 1 M KNO₃, 1 M Ca(NU3)₂ and 1 M Mg(NO3)₂ at the same pH. Mean and standard deviations of fluorescence intensities of films and microspheres were determined from more than five discrete sampling points (or particles).

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Claims

CLAIMS:

1. A composition comprising a fluoroionophore grafted to a self-plasticized polymer matrix.

2. The composition of claim 1, wherein the fluoroionophore is lipophilic and H⁺-selective.

3. The composition of claim 1, wherein the fluoroionophore is Nile Blue or an analog thereof.

4. The composition of claim 1, wherein the matrix comprises poly(n-butyl acrylate).

5. The composition of claim 1, wherein the fluoroionophore is grafted to the matrix via a functional group, wherein the matrix comprises an acrylate copolymer, and the fluoroionophore is covalently linked to the matrix through a functional group on the acrylate copolymer.

6. The composition of claim 5, wherein the fluoroionophore is Nile Blue or an analog thereof.

7. The composition of claim 6, wherein the acrylate copolymer comprises a first monomer of Formula I:

wherein X in Formula I is H or Me, and R is a 3-10 carbon alkyl group,

and a second monomer of Formula II:

wherein X in Formula II is H or Me, n is 1-7, and Z is a functional group that can be used to covalently link to the exocyclic nitrogen (=NH) of Nile Blue or a Nile Blue analog;

and wherein the copolymer comprising Z is reacted with Nile Blue or a Nile Blue analog to covalently link Nile Blue or a Nile Blue analog to the copolymer matrix.

8. The composition of claim 7, wherein X in Formula I is H and R is n-butyl.

9. The composition of claim 7, wherein Z in Formula II is a functional group selected to modulate the K_exch of a fluoroionophore.

10. The composition of claim 8, wherein X in Formula II is Me and n is 1.

11. The composition of claim 10, wherein the fluoroionophore is Nile Blue or a Nile Blue analog, and is linked to the matrix by a reaction with Z in the copolymer comprising monomer of Formula I and monomer of Formula II to form an amide, urea or carbamate linker between the copolymer and the Nile Blue or Nile Blue analog.

12. A fluorescent ion optode sensor comprising the composition of claim 1.

13. The fluorescent ion optode sensor of claim 12, which is a microbead.

14. A method of measuring Na+ concentration of a liquid sample, comprising contacting the sample with the fluorescent ion optode sensor of claim 12.

15. A method to make a fluorescent ion optode sensor comprising:

a. Forming a copolymer by reaction of a first monomer of Formula I:

wherein X in Formula I is H or Me, and R is a 3-10 carbon alkyl group,

with a second monomer of Formula II:

wherein X in Formula II is H or Me, n is 1-7, and Z is a functional group that can be used to covalently link Nile Blue or a Nile Blue analog to the exocyclic nitrogen (=NH); and

b. covalently linking a fluoroionophore to the copolymer by reaction of the fluoroionophore with the copolymer comprising functional group Z.

16. The method of claim 15, wherein the fluoroionophore is Nile Blue or a Nile Blue analog.

17. The method of claim 16, wherein the copolymer has a Tg of less than about 50⁰C.

18. The method of claim 17, wherein the copolymer has a Tg of less than about 2O⁰C.

19. The method of claim 16, wherein X in Formula I is H, R is n-butyl, and X in Formula II is Me.