WO2009023287A1 - Dérivés polymérisés du bleu de nil pour détecteurs à optodes spécifiques d'ions à base de microsphères fluorescentes, sans plastifiant - Google Patents

Dérivés polymérisés du bleu de nil pour détecteurs à optodes spécifiques d'ions à base de microsphères fluorescentes, sans plastifiant Download PDF

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WO2009023287A1
WO2009023287A1 PCT/US2008/052694 US2008052694W WO2009023287A1 WO 2009023287 A1 WO2009023287 A1 WO 2009023287A1 US 2008052694 W US2008052694 W US 2008052694W WO 2009023287 A1 WO2009023287 A1 WO 2009023287A1
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nile blue
formula
fluoroionophore
copolymer
matrix
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PCT/US2008/052694
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English (en)
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Eric Bakker
Wittaya Ngeontae
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Beckman Coulter, Inc.
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Publication of WO2009023287A1 publication Critical patent/WO2009023287A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/0008—Organic ingredients according to more than one of the "one dot" groups of C08K5/01 - C08K5/59
    • C08K5/0041—Optical brightening agents, organic pigments
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6434—Optrodes

Definitions

  • Ion-selective electrodes 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.
  • 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.
  • thermodynamics of ion extraction 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].
  • 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].
  • the sensing components are conventionally blended into a plasticized polymer matrix as a solid support.
  • 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].
  • leaching is an even more severe problem. It is therefore advisable to covalently attach the sensing ingredients to the polymer matrix.
  • a chromoionophore was grafted onto PVC-COOH for the fabrication of a Ca 2+ optode sensor [13] and onto plasticized polyurethane matrices [14].
  • AU-i Ca 2+ -ionophore
  • 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.
  • 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.
  • 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) 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.
  • 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.
  • Nile Blue derivatives are attractive H-selective chromo- and fluoroionophores that are used in optical [27] and potentiometric sensors [28].
  • 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.
  • 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 50 0 C, and often below about 20 0 C, 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.
  • 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).
  • a sensing component needs to be carefully designed so that the ion binding properties of the components are maintained after being grafted.
  • 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.
  • 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.
  • NB-Urea monomer methacrylate 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.
  • 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.
  • a first monomer of Formula (I) such as H-butyl acrylate monomer
  • 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.
  • 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 50 0 C, and preferably below about 20 0 C. 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 50 0 C, and preferably below about 20 0 C.
  • Tg glass transition temperature
  • 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.
  • these Nile Blue analogs include compounds of Formula III:
  • R 1 and R 2 independently represent a C ⁇ 4 alkyl group, and wherein R 1 and R 2 can be taken together with N to which they are attached to form a heterocyclic group selected from pyrrolidine, piperidine, morpholine, thiomorpholine, and N 4 - methylpiperazine.
  • copolymers useful for the invention include a first monomer of Formula I:
  • X is H or Me
  • 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.
  • copolymers of the invention also include a second monomer of Formula II:
  • X is H or Me
  • n is 1-7
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • leaving groups e.g., halide such as Cl, Br or I
  • alkylsulfonates such as mesylate or triflate
  • arylsulfonates such as phenylsulfonate or tosylate
  • 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.
  • 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.
  • 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.
  • a dehydrating agent such as a carbodiimide (DCC; DIPC; EDC)
  • formation of an activated ester such as with HOBt (N-hydroxybenzotriazole)
  • HOBt N-hydroxybenzotriazole
  • formation of an acid halide or mixed anhydride e.g., the linkage may be produced by using reductive alkylation conditions.
  • 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-(CH 2 ) 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.
  • 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.
  • fluoroionophore e.g., Nile Blue
  • 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.
  • the sensor includes a microbead that comprises or that consists essentially of the functionalized fluoroionophore- containing polymer composition described above.
  • 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.
  • 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.
  • L ⁇ , Indj and R ⁇ ⁇ 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 ⁇ is experimentally accessible as the ratio of the fluorescence intensity of the protonated (R P ) and deprotonated form (R D ) of the chromoionophore [32]:
  • the competition equilibrium of P + and H + is primarily described by K exch -
  • the Ke x c 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.
  • 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.
  • 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.
  • 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.
  • grafted polymers from Route 2 were dissolved in THF for UV- Vis absorbance measurements. Both grafted polymers exhibited well defined protonation and deprotonation spectra.
  • 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.
  • 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.
  • 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.
  • NB-urea — PnBA (R2) the emission spectra are shown in Fig.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • selection of the amide linking group provided a sensor with an operating range that includes normal Na + levels found in undiluted blood.
  • 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 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.
  • 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.
  • 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 CaH 2 and freshly distilled under nitrogen atmosphere prior to use. The H-butyl acrylate monomer, 99%, was obtained from Polysciences, Inc.
  • 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
  • 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 M ⁇ cm "1 ).
  • 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).
  • ISE membranes were prepared by dissolving Nile Blue grafted PnBA (10%w/w),
  • 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 ⁇ L 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- ⁇ m 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.
  • the particles were concentrated by removing some of the curing water. A 100 ⁇ L 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 interfering ions response curves were acquired by observing microsphere responses in 1 M KNO 3 , 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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Abstract

Les fluorophores lipophiles spécifiques de H+ tels que les dérivés du Bleu de Nil sont largement utilisés dans les détecteurs de pH à base de ISE et dans les optodes de substrat, et sont couramment dissous dans une matrice plastifiée telle que le PVC. Malheureusement, la lixiviation des ingrédients de détection actifs et du plastifiant à partir de la matrice régit la durée de vie des détecteurs et empêche leurs applications in vivo, notamment dans le cas de détecteurs à base de particules miniaturisées. Nous observons que la copolymérisation classique des dérivés du Bleu de Nil contenant un groupe latéral acrylique donne de multiples produits réactionnels ayant des propriétés spectrales et de liaison à H+ différentes, invalidant ainsi cette approche pour le développement de matériaux de détecteurs fiables. Cette limitation a été surmontée en greffant le Bleu de Nil à une matrice polyacrylate de butyle auto-plastifiante via une liaison urée ou amide entre la structure de base du Bleu de Nil et le polymère. Des expériences de lixiviation des optodes dans du méthanol ont confirmé l'attachement covalent solide des deux chromoionophores à la matrice polymère. Les deux dérivés du Bleu de Nil polymérisés ont une réponse au pH satisfaisante et des propriétés optiques convenables qui permettent leur utilisation dans les électrodes et les optodes spécifiques d'ions. Des détecteurs à base de microsphères spécifiques de Na+, sans plastifiant, utilisant lesdits chromoionophores polymérisés ont été fabriqués dans des conditions douces à l'aide d'un générateur de microparticules à ultrasons conçu en interne pour mesurer les activités sodium au pH physiologique. La plage de mesure pour le sodium s'est avérée être de 10-1 - 10-4 M et 1 - 10-3 M, pour les dérivés du Bleu de Nil liés par des fonctionnalités urée et amide, respectivement, au pH physiologique. La constante d'échange d'ions observée pour la microsphère sans plastifiant était de log Kexch = -5,6 et log KeXch = -6,5 pour ces mêmes deux systèmes, respectivement. Par rapport aux optodes de substrat spécifiques de Na+ antérieures, les microbilles de détection optique fabriquées mentionnées ici ont des profils de sélectivité intéressants, des temps de réponse raisonnablement rapides, et des plages de mesures plus appropriées pour déterminer l'activité Na au pH physiologique dans des échantillons de sang non dilués.
PCT/US2008/052694 2007-08-16 2008-01-31 Dérivés polymérisés du bleu de nil pour détecteurs à optodes spécifiques d'ions à base de microsphères fluorescentes, sans plastifiant WO2009023287A1 (fr)

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US60/956,285 2007-08-16

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CN102516988A (zh) * 2011-11-28 2012-06-27 大连理工大学 一类可聚合荧光染料、其制备方法及应用
WO2014013507A1 (fr) 2012-07-16 2014-01-23 Council Of Scientific & Industrial Research Procédé de préparation d'une composition de polymère fluorescent ajustable

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US6294390B1 (en) * 1996-07-22 2001-09-25 Novartis Ag Covalently immobilized fluoroionophores for optical ion sensors
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102516988A (zh) * 2011-11-28 2012-06-27 大连理工大学 一类可聚合荧光染料、其制备方法及应用
CN102516988B (zh) * 2011-11-28 2014-04-09 大连理工大学 一类可聚合荧光染料、其制备方法及应用
WO2014013507A1 (fr) 2012-07-16 2014-01-23 Council Of Scientific & Industrial Research Procédé de préparation d'une composition de polymère fluorescent ajustable
US20150166709A1 (en) * 2012-07-16 2015-06-18 Council Of Scientific & Indsutrial Research Process for the preparation of tunable fluorescent polymer composition
US9290601B2 (en) 2012-07-16 2016-03-22 Council Of Scientific & Industrial Research Process for the preparation of tunable fluorescent polymer composition

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