EP1960480A2 - Kovalent befestigte nilblau-derivate für optische sensoren - Google Patents
Kovalent befestigte nilblau-derivate für optische sensorenInfo
- Publication number
- EP1960480A2 EP1960480A2 EP06846279A EP06846279A EP1960480A2 EP 1960480 A2 EP1960480 A2 EP 1960480A2 EP 06846279 A EP06846279 A EP 06846279A EP 06846279 A EP06846279 A EP 06846279A EP 1960480 A2 EP1960480 A2 EP 1960480A2
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- EP
- European Patent Office
- Prior art keywords
- polymer
- monomer
- composition
- optode
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B69/00—Dyes not provided for by a single group of this subclass
- C09B69/10—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds
- C09B69/109—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds containing other specific dyes
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B19/00—Oxazine dyes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
Definitions
- the present invention is related to systems for detecting target ions in a sample and methods of use thereof in clinical laboratory instrumentation.
- Chromoionophores are normally lipophilic pH indicators used in ion selective optodes. Such dyes usually require sufficient lipophilicity (logP TLC > 10.6), a large molar extinction coefficient, a high chemical and photostability and a high selectivity to H + (1, &J Chromoionophores may be classified into neutral and charged chromoionophores, depending on the electrical charge of their unprotonated form.
- lipophilic H + selective chromoionophores of different basicities were synthesized (2) .
- the lipophilic H + selective chromoionophores may be combined with different ionophores to design optodes with different measuring ranges for specific analytical needs (2) .
- chromoionophores are highly lipophilic and suitable for general applications.
- optodes based on these chromoionophores exhibit short lifetime in lipophilic samples such as undiluted serum.
- the leaching of plasticizer and other sensor components is more dramatic when the size of the sensor is reduced to microparticles in the micro-meter range (3) or nanometer range (4, 5) .
- the sensing components including chromoionophores are immobilized onto the polymer matrix.
- chromoionophores have been covalently attached onto functionalized poly (vinyl chloride) (6, 7) and onto polyurethane matrices (8), but such polymers could not be used without plasticizer and lead to longer response time.
- immobilization of non-chromogenic ionophores onto polymers has been more widely studied. Ionophores selective for Na + , K + and Pb 2+ have been covalently grafted to a polysiloxane matrix and applied to the fabrication of CHEMFET sensors (9, 10) .
- Kimura introduced a sol-gel technique to immobilize ionophores and ion exchangers (11, 12, 13) .
- Another direction in ionophore grafting is to copolymerize the polymerizable ionophores with blank polymers by a one-step solution polymerization method.
- the present invention is directed to the synthesis of polymerizable compositions having a pH indicator, a spacer and a polymerizable group.
- the polymerizable group can be an acrylate or a methacrylate .
- the spacer can be an oxyalkyl group, an oxypropyl group, or an oxybenzoyl group.
- the composition can have the structure of NBl or NB2 shown in Fig. 1.
- the present invention is also directed to an indicating polymer comprising a copolymer of a composition having a pH indicator, a spacer and a polymerizable group; and a co-monomer.
- the co-monomer can comprise acrylate or methacrylate.
- the co-monomer comprises methyl methacrylate and decyl methacrylate.
- the co- monomer comprises poly (n-butyl acrylate).
- the present invention is also directed to an optode having an indicating polymer, the indicating polymer having: a copolymer of a composition having a pH indicator, a spacer and a polymerizable group; and a co- monomer.
- the optode can also have a self-plasticizing polymer.
- the optode can further have a grafted ionophore .
- the optode is a particulate optode.
- the present invention is also directed to a method of making a copolymer having selectivity for a target ion.
- the method includes: providing a monomer having a pH indicator, a spacer, and a polymerizable group; combining the monomer and a co-monomer; and polymerizing the monomer and co-monomer to form a copolymer.
- the monomer and the co-monomer can be polymerized by thermally initiated free radical solution polymerization.
- Fig. 1 contains schematic representations of commercially available Nile Blue derivates usable as H + -chromo and fluoroionophores and NBl and NB2 prepared according to embodiments of the present invention
- Fig. 2A is a graph of potentiometric pH responses of a NBl-MMA-DMA based PVC-DOS membrane prepared according to an embodiment of the present invention
- Fig. 2B is a graph of potentiometric pH responses of a NBl-MMA-DMA based PVC-NPOE membrane prepared according to an embodiment of the present invention
- Fig. 3A shows absorbance spectra of free NBl in its protonated and unprotonated form in MeOH;
- Fig. 3B shows fluorescence spectra of free NBl in its protonated and unprotonated form in MeOH;
- Fig. 4 is a fluorescence response curve of a Pb 2+ -selective optode film containing grafted NBl-MMA-DMA, Pb-ionophore (IV) , NaTFPB, PVC and DOS;
- Fig. 5A is a potentiometric pH response curve of a PVC-DOS membrane containing freely dissolved NB2;
- Fig. 5B is a potentiometric pH response curve of a PVC- NPOE membrane containing freely dissolved NB2
- Fig. 6A is a potentiometric response curve of a grafted NB-2-MMA-DMA based PVC-DOS membrane
- Fig. 6B is a potentiometric response curve of a grafted NB-2-MMA-DMA based PVC-NPOE membrane
- Fig. 7A shows the absorbance spectra of free NB2 in its protonated and unprotonated form in MeOH;
- Fig. 7B shows the fluorescence spectra of free NB2 in its protonated and unprotonated form in MeOH;
- Fig. 8 is a chart of fluorescence response curves and observed selectivity of optode films containing NB2-MMA-DMA, Na(X), and NaTFPB towards sodium (open circles), magnesium, potassium and calcium ions at pH 7.4;
- Fig. 9 is a chart of fluorescence response curves and selectivity of 10- ⁇ m diameter optode microspheres containing NB2-MMA-DMA, poly (n-butyl acrylate) , Na(X), and NaTFPB to sodium ions; and
- Fig. 10 is a chart of typical response times of a single sensing microsphere of Fig. 9 deposited on the bottom of a glass capillary cell and characterized in the flowing stream of changing sodium ion concentrations.
- the present invention is directed to synthesis of polymerizable Nile Blue derivatives with acrylic groups and the covalent attachment of the chromoionophores in MMA-DMA or nBA based polymers by simple one step solution polymerization.
- Chromoionophores typically have different absorbance and fluorescence spectra when protonated and unprotonated, acting as lipophilic pH indicators.
- the change of the protonation and deprotonation of the chromoionophore is related to the change of the activity of the target ions because of a competitive extraction of the two ions into the membrane phase.
- Bulk optodes contain long alkyl chains to provide sufficient lipophilicity for regular ion-selective sensors. However, to improve the lifetime of the sensors and. eliminate the leaching of the components into lipophilic samples during in-vivo measurements, it is important to covalently graft the chromoionopores .
- the present invention is directed to polymerizable chromoionophores having pH indicators in combination with spacers and polymerizable acrylic groups for preparing all—polymeric materials and eliminating this leaching problem.
- a pH indicator suitable for purposes of this invention is the highly basic oxazine-dye molecule Nile Blue.
- Nile Blue derivatives such as ETH 5294, ETH 2439, ETH 5350 and ETH 5418, which are shown in Fig. 1, are well known in the art.
- pH indicators that may be suitable for the present invention include neutral red (toluylene red) ; chromoionophore X (ETH4004), 4-dioctylamino-4' - (trifluoroacetyl) stilbene; chromoionophore IV is (ETH2412) 5-octadecanoiloxy-2- (4- nitophenylazo) phenol; chromoionophone VI (ETCH7075), 4', 5'- dibromofluorescein octadecyl ester; and chromoionophore VIII (TBTE), 3' ,3" ,5' ,5' ' -tetrabromophenylphthaleinethyel ester.
- Spacers suitable for purposes this invention include oxyalkyl, oxypropyl and oxybenzoyl groups. Generally, suitable spacers have carbon chains from about 2 to about 18 carbon atoms in length.
- polymerizable chromoionophore refers to a chromoionophore having a polymerizable group, which allows the chromoionophore to become covalently bonded to a copolymer.
- the polymerizable group is required to allow the chromoionophore to react with a reactive group of the copolymer, such as a carbon-carbon double bond, to form covalent linkages, whereby the chromoionophore becomes covalently grafted onto the copolymer.
- examples of such polymerizable groups include, but are not limited to, carbon- carbon double bonds, such as acrylic and methacrlyic groups, carbon-carbon triple bonds and carbonyl groups.
- covalently grafted chromoionophore refers to a chromoionophore that is attached to a polymer through covalent bonds .
- Polymerizable chromoionophores in accordance with the invention can be synthesized, for example, by methods described in the examples below.
- the method of making the polymerizable chromoionophore will typically include: (1) attaching a spacer to a polymerizable group; and (2) attaching a pH indicating dye, such as a Nile Blue derivative, to the spacer, as described in Example 1.
- polymer and “copolymer” are used interchangeably and refer to a chemical compound or mixture of compounds formed by polymerization and comprising repeating monomer units, wherein the polymer can comprise one type of monomer unit or can contain two or more different monomer units.
- Preferred polymers will have adequate solubility in organic solvents so that they can be mixed with the other components and can be converted into coatings by conventional coating methods. They should furthermore be permeable to ions.
- the dielectric constant of the polymers is preferably from 2 to 25, particularly preferably from 5 to 15, at 100 Hz and room temperature.
- the optical transparency is preferably in the range of from about 400 to about 1200 nm, particularly preferably from about 400 to about 900 nm.
- Suitable polymers are known to the person skilled in the art. They can be homopolymers, copolymers, block polymers, graft polymers and polymer alloys .
- the components of a polymer alloy may be a combination of two or more polymer components, said components having high and low glass transition temperatures.
- the glass transition temperature can be adjusted, for example, by means of the polarity and the chain length and content of structural units.
- the glass transition temperature is preferably from -130 to 0 0 C.
- Polymers with very low T g values are normally much softer and more difficult to handle mechanically.
- the T g is typically determined experimentally with a differential scanning calorimeter, a standard instrument for this purpose.
- the polymers can be selected, for example, from the group consisting of polyolefins, polyesters, polyamides, polyethers, polyimides, polyesteramides, polyamideimides, polyurethanes, polyetherurethanes, polyesterurethanes, polyureas, polyurethaneureas and polysiloxanes, it being possible for the polymers to contain ionizable, basic groups (for example amino groups) or ionizable, acidic groups (for example carboxyl or sulfonyl groups) , which may be used as replacement for a counterion of lipophilic salts and can provide improved ion transport .
- ionizable, basic groups for example amino groups
- acidic groups for example carboxyl or sulfonyl groups
- Some examples of monomers for the preparation of polyolefins are C 2 -Ci 2 olefins, acrylic acid, methacrylic acid, maleic acid, maleic anhydride, Ci-C 30 esters of acrylic and methacrylic acid, Ci-C 30 amides of acrylic and methacrylic acid, acrylamide and methacrylamide, vinyl esters of Ci-C 2 O carboxylic acids, acrylonitrile, butadiene, isoprene, chlorobutadiene, styrene, ⁇ -ethylstyrene, vinyl chloride, vinyl fluoride, vinylidene chloride and vinyl ethers of Ci -C30 alcohols.
- Polyesters, polyesteramides and polyamides are preferably synthesized from C 2 -Ci 2 dicarboxylic acids and C 2 -C 18 diols or - diamines .
- Polyimides are preferably synthesized from C 2 -Ci 8 tetracarboxylic acids and C 2 -Ci 8 diamines .
- Polyethers are preferably synthesized from aliphatic C 2 -Ci 2 diols (1,2- or ⁇ , ⁇ -lining) or linear adducts of these diols and C 8 -C 30 diglycidyl ethers.
- Polyurethanes and polyureas are preferably synthesized from C 2 -Ci 8 diols or -diamines and C 2 -C 20 diisocyanates and/or triisocyanates .
- Polysiloxanes are preferably synthesized from di (C 1 -C 4 ) alkylsilyldichlorosilanes
- the polymers comprise a copolymer of methacrylate monomers with different pendant alkyl groups Ri and R 2 , wherein R 1 may be any of C x _ 3 alkyl group, and R 2 may be any of C 4 - I2 alkyl group.
- R 1 may be any of C x _ 3 alkyl group
- R 2 may be any of C 4 - I2 alkyl group.
- alkyl refers to a saturated linear or branched-chain monovalent hydrocarbon radical of one to twelve carbon atoms, wherein the alkyl radical may be optionally substituted independently with one or more substituents described below.
- alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like
- R 1 is a Ci_ 2 alkyl group
- R 2 is a C 8 -i 2 alkyl group.
- methyl methacrylate and decyl methacrylate monomers are used for forming a methyl methacrylate-decyl methacrylate (MMA-DMA) copolymer matrix of the present invention .
- Methacrylate monomers of the present invention are commercially available from, for example, Polysciences, Inc. (Warrington, Pa.). Alternatively, the methacrylate monomers can be prepared by standard methods known in the art or via thermally initiated free radical solution.
- the chromoionophore monomer comprises a polymerizable group, and the chromoionophore is covalently grafted onto a polymer chain by copolymerizing the chromoionophore monomer with methacrylate co-monomers such as MMA and DMA monomers.
- the copolymer may comprise a random distribution of immobilized chromoionophores within the MMA-DMA polymer chain .
- Graft copolymers of the present invention comprising a covalently grafted chromoionophore may be made in accordance with methods known in the art or the methods described herein.
- the graft copolymer is prepared by thermally initiated free radical solution polymerization of a mixture of methacrylate monomers and a polymerizable chromoionophore as described herein in detail in Example 3.
- a sol-gel technique may be used to prepare the graft copolymer.
- Another approach involves directly grafting the chromoionophore onto an existing polymer with active sites .
- Yet another approach involves blending two different polymers together, with one of them containing the grafted chromoionophore.
- a solution containing methacrylated monomers and the polymerizable chromoionophore can be irradiated with an electron beam to cause polymerization and covalent attachment of the polymerizable chromoionophore onto the methacrylate copolymer.
- a sufficient amount of polymerizable chromoionophore is combined with the copolymer to obtain the desired improvement in desired properties of the copolymer, such as ion selectivity, faster response and recovery times and extended lifetime. Such properties may be quantitatively measured by well-known test methods .
- the optimal amount of polymerizable chromoionophore required to produce a significant enhancement of such properties varies depending upon the chemical compositions, structures, and molecular weights of the components employed as well as the extent of grafting achieved.
- the graft copolymer typically contains the chromoionophore in, for example, an amount from about 0.01% to about 10% by weight, and preferably from about 0.1 to about 5% by weight, based on the amount of polymer. In general, however, it will be advantageous to use at least one part by weight of the polymerizable chromoionophore for every 1000 parts by weight of the copolymer.
- the conditions necessary to achieve at least partial grafting of the components of the polymer composition will vary depending upon the reactivities of the individual components.
- the grafting conditions may comprise a thermal or photoinitiated co-polymerization in a solvent such as EtOAc.
- the graft copolymer will expediently have a mean molecular weight of at least 5,000, preferably at least 10,000 and particularly preferably at least 20,000 daltons, for example from 20,000 to 200,000 daltons, preferably from 50,000 to 200,000 daltons.
- the graft copolymers of this invention may be blended, admixed, or combined with other polymers to obtain blends having improved properties or performance characteristics .
- the polymer composition when blended with poly (vinyl chloride) or polyurethane and a plasticizer, such as bis (2-ethylhexyl) sebacate (DOS) or o-nitrophenyloctylether (NPOE) has the beneficial effect of increased mechanical robustness.
- the relative proportion of PVC polymer : graft polymer composition may be varied as desired, preferably from about 95:5 to about 80:20 on a weight basis.
- the graft polymers and blended polymer compositions of the present invention can be adapted for use in a variety of anion- selective sensors capable of detecting the interaction of a target anion with the ionophore .
- the graft polymers and blended polymer compositions may be used to fabricate ion-selective membranes, films or particles, which include, but are not limited to, carrier-based ion-selective electrodes (ISEs) , thin film ion-specific optodes, particle- based optodes, and bulk optodes.
- ISEs carrier-based ion-selective electrodes
- thin film ion-specific optodes thin film ion-specific optodes
- particle- based optodes and bulk optodes.
- a graft polymer of this invention may be used to fabricate polymer membranes of an ion-selective electrode
- Polymers of this invention may also be used to fabricate thin films to be used in a thin film ion-specific optode or to fabricate microsphere particles to be used in particle-based optodes in accordance with methods known in the art .
- the electrodes and optodes may be prepared by solvent casting and spin coating technigues, as discussed in Examples 5 and 6.
- the chromoionophores according to the invention have suitable absorption and emission wavelength ranges that allow the use of known and inexpensive light sources, for example halogen or xenon lamps or light-emitting diodes. Examples of detectors which can be employed are photodiodes. In addition to fluorescence spectroscopy, other optical measurement methods may also be used, for example, absorption spectroscopy.
- the ion-detecting sensors of the present invention may be used for detecting ions of all types of body fluid samples.
- the samples include, but are not limited to, whole blood, spinal fluid, blood serum, urine, saliva, semen, tears, etc.
- the fluid sample can be assayed neat or after dilution or treatment with a buffer.
- Ethyl acetate, dichloromethane and 1,4-dioxane were reagent grade and obtained from Fisher.
- Inhibitors were removed from the monomers by the reported method (19) . AIBN was recrystallized from warm methanol prior to use.
- Aqueous solutions were prepared by dissolving the appropriate chloride salts in Nanopure purified water (18 MQ cm) .
- Step two 9.6 g (0.05 mol) of 4-acryloyloxybenzoic acid was reacted at 45-50 0 C with 30 mL of thionyl chloride containing a few drops of N, N-dimethylfomamide for 10 h, and then the excess thionyl chloride was removed under reduced pressure to give the corresponding acid chloride (yield 85%) .
- Step three To a solution of 317 mg (1 mmol) of basic Nile Blue in 13 mL of CH 2 Cl 2 , 242 mg (0.8 mmol) of 4-acryloyloxybenzoyl chloride dissolved in 2 mL of CH 2 C12 was added.
- EXAMPLE 3 Polymer Synthesis and Characterizations. All polymers were synthesized via thermally initiated free radical solution polymerization. The amount of methyl methacrylate and n-decyl methacrylate used was the same as reported previously (16, 20) .
- 1 wt % of Nile Blue derivatives (20 mg) , 0.78 g MMA and 0.20 g DMA were dissolved in anhydrous EtOAc. The solution was purged with N 2 for 20 minutes before adding 5.1 mg AIBN. The homogeneous solution was continuously stirred and the temperature was ramped to 90 0 C, which was maintained for 16 h. After the reaction was complete, the solvent was evaporated and the polymer was redissolved in 10 mL of dioxane .
- the solvent was evaporated and the resultant polymer was washed with methanol to ensure the complete removal of the free ionophores .
- the polymer was dried under ambient laboratory conditions (yield 80%) .
- the grafted polymers were characterized by H 1 NMR as reported (21, 22) .
- the concentrations of grafted dyes were estimated from the H 1 NMR spectrum to be 40 mmol/kg.
- the EtOAc solution containing 1 g n-BA was heated at 90 0 C for 30 min.
- the reaction temperature was decreased to 60 0 C and 25 mg NB2 was added to the solution for another 16 h. This modified procedure was used to prevent the decomposition of the dye.
- the polymer (yield 60%) was purified and characterized as described above.
- the concentration of grafted NB2 was estimated from the 1 HNMR spectrum to be 40 mmol/kg.
- ISE Membrane Preparation and Measurements. ISE membranes were prepared by dissolving chromoionophore (10 mmol/kg) , NaTFPB (2 mmol/kg), PVC and plasticizer (DOS or NPOE) to give a total cocktail mass of 140 mg in 1.5 mL of THF.
- the cocktail of the membrane with grafted chromoionophore contained 2 mmol/kg NaTFPB, 10 wt% MMA-DMA polymer with grafted indicators, PVC and plasticizer (DOS or NPOE) . Cocktails were poured into glass rings (2.2 cm i.d.) affixed onto glass microscope slides. The solvent was evaporated overnight to give a transparent membrane.
- buffer solutions are 10 mM citric acid and 10 mM boric acid with the indicated concentrations of NaCl (either 10 mM or 0.1 mM) adjusted to pH 1.5 by 1 M standard HCl, or 10 mM phosphate buffer solution with NaCl as background.
- the solution was titrated with a 0.1 M standard NaOH solution and a pH electrode was used to monitor the sample pH. All experimental results given are the average of at least three electrodes, with calculated standard deviations.
- the sandwich membrane method (23, 24) was used to measure the pKa of the grafted chromoionophores .
- One single parent membrane contained 10 mmol/kg chromoionophore and 5 mmol/kg NaTFPB in PVC-DOS, PVC-NPOE or MMA-DMA polymer while the other segment contained only NaTFPB in the same matrix.
- For the grafted ionophore one single membrane contained 1.5 mmol/kg NaTFPB and 10 wt% MMA-DM ⁇ -NB polymer in PVC-DOS or blank MMA- DMA while the other one contained the same amount of NaTFPB in PVC-DOS or blank MMA-DMA polymer.
- the parent membranes were conditioned in citric acid-boric acid buffer solution overnight .
- Optode Film Preparation and Measurements Optode thin films were prepared by spin-coating.
- the cocktails contained the free or grafted chromoionophores, PVC and plasticizer dissolved in THF.
- a 200- ⁇ L aliquot of the cocktail was transferred with a syringe onto a quartz disk placed in a spin- coating device (1) .
- the resulting 2-3 ⁇ m-thick films were equilibrated in different solutions.
- the absorbance spectra were recorded with an HP 8452 A diode array spectrophotometer and the fluorescence spectra were recorded by fluorescence microscopy and spectroscopy as previously reported (3, 25) .
- Molar absorption coefficients (e) of NBl and NB2 were measured in methanol.
- a total of 300 mg of membrane components containing 5 wt% NBl-MMA-DMA, 8.1 mmol/kg NaTFPB, 18.6 mmol/kg Pb (IV) ionophore and PVC/DOS (1:2) were dissolved in 1.75 mL of THF.
- a total of 300 mg of membrane components containing 5 wt% NB2- MMA-DMA, 10 mmol/kg NaTFPB, 20 mmol/kg Na (X) ionophore and PVC/DOS (1:2) were dissolved in 1.75 mL of THF.
- the thin films were prepared and characterized by the same spin-coating device and fluorescence microscope described above. All the data points are the average of five measurements, with calculated standard deviations.
- Plasticizer-free microspheres were prepared with a high-throughput particle casting apparatus previously described (25) .
- the cocktail was prepared by weighing out -2.09 rag (53 mmol/kg) Na ionophore (X), 0.75 mg (21 mmol/kg) NaTFPB, 11 mg grafted NB2 in PnBA (10 mmol/kg) and 25.4 mg blank PnBA dissolved in 1.5 mL cyclohexanone .
- the mixture was shaken in a vortex mixer for 30 min and than added dropwise to 25 mL dichloromethane under gentle stirring.
- the solution was filtered through a 0.45 ⁇ m Gelman filter and was transferred to a gastight Hamilton syringe.
- the syringe containing the polymer core solution was mounted on a syringe pump (Stoelting, Wood Dale, IL) and set to flow at a rate of 0.29 mL/min.
- Deionized water used as the sheath liquid stream flowing at a rate of 43 mL/min was controlled via a pressure regulator.
- the frequency generator was operated at a setting of 11.9-12.3 kHz.
- a borosilicate glass microcapillary of 1.0 mm i.d. and 0.15 mm wall thickness was used for response time measurements.
- a 50 L aliquot of collected particles was pipetted inside the capillary cell, and the particles were then left to settle for a couple of hours.
- the capillary was attached at each end with polyethylene tubing, and connected to a peristaltic pump operated at a rate of 0.1 mL/min. Measurements with a fluorescence microscope (3, 26) were taken every 30-60 s or longer thereafter. Discussion of the Examples
- the first polymerizable chromoionophore NBl is a derivative of the highly basic oxazine-dye molecule Nile Blue attached to a acryloyloxybenzoyl group.
- the pH responses of NBl based ion selective electrode ("ISE") membranes were investigated first. ISE membranes containing free NBl were prepared. However, the chromoionophore was found to leach from the membrane during the conditioning step in contact with buffer due to insufficient lipophilicity . Consequently, an NBl grafted polymer was prepared by solution polymerization of NBl, MMA and DMA in ethyl acetate and with AIBN as an initiator.
- the purified polymers were washed, with methanol until the solution remained colorless and indicated the removal of unreacted free NBl in the polymer.
- the blending of an ionophore grafted MMA-DMA polymer with other polymers, such as plasticized PVC exhibits no significant influence on the sensor response but may improve the mechanical stability of the ISE membranes (16) .
- the same blending method was employed for initial evaluation in ISE membranes.
- 10 wt% grafted NBl-MMA-DMA was mixed with 90 wt% PVC and DOS (1:2 by mass) to prepare the ISE membranes. No leaching of the dyes was observed during the conditioning and measurements.
- Figs . 2A and 2B show the recorded pH responses of the resulting membranes.
- a Nernstian response was only found at pH lower than 3.
- a Nernstian response was found at a pH range of about 1 to 5.
- the wider response range for NPOE membranes is in agreement with a higher pKa value of the dye in the NPOE based membranes .
- This trend has been reported earlier: the measuring range and the pKa values of chromoionophores change with different plasticizers (2, 24) .
- the cation selectivity of membranes based on grafted NBl was determined according to the fixed interference method (27) by measuring the pH response curves in a 0.1 M NaCl background.
- the relatively small ion selectivity and narrow pH response range are very different from other reported Nile Blue derivatives. This may be explained by a low pKa value of the chromoionophore or a significant binding affinity to the sodium ion interference.
- the pKa value of the new dye NBl is expected to be lower than commonly used chromoionophores such as ETH 5294.
- the carboxyl and benzoyl groups in NBl have a strong electron withdrawing effect and decrease the electron density on the nitrogen of the diethylamino group. The electron on this nitrogen is more delocalized than ETH 5294 and decreases the basicity of the dye.
- Fig. 3A shows the absorbance spectra of NBl in its unprotonated and protonated form in THF solution, exhibiting maxima at 510 and 560/640 nm respectively.
- the absorbance spectra of NBl are very similar to those for ETH 5294.
- ⁇ molar absorption coefficient
- the molar absorption coefficients ( ⁇ ) were found as 2.9 x 10 4 L mol ⁇ 1 cm “1 (660 nm, protonated form) and 2.2 x 10 4 L mol ⁇ 1 cm “1 (510 nm, deprotonated form) in methanol, measured according to the reference (2) . These values are typical for Nile Blue derivatives.
- the low pKa value of NBl makes it very attractive in optical sensors for measuring transition metal ions at low sample pH. For instance, the fluorescence detection of lead ions at pH 4 aqueous solution had been reported earlier with bulk optodes (26) .
- the optical sensors based on fluorescence measurements have a similar composition as the optodes based on absorbance mode, but require a fluorescence active pH indicator .
- the azo dye ETH 5315 exhibits a pKa of 5.5 in optode membranes and would be suitable for heavy metal detection in acidic solution. Unfortunately, it shows very poor fluorescence characteristics. Since Nile Blue derivatives are normally fluorescent, ETH 5418 with a pKa value 8.8 was chosen as an alternative for lead measurement . Unfortunately, the use of ETH 5418 for fluorescence-based measurements requires the development of an inner filter approach since its basic form is not light emitting. Thus, an additional lipophilic reference dye is required.
- Fig. 3B shows the fluorescence spectra of grafted NBl in MMA-DMA polymer in contact with 0.01 M HCl and 0.01 M NaOH solutions, respectively. The emission peaks at 640 nm and 680 nm correspond to the deprotonated and protonated forms of the dye .
- the Pb + optode contained Pb-ionophore (IV), ion-exchanger NaTFPB, grafted NBl in MMA-DMA (5 wt%) and PVC-DOS (1:2 by mass, 90 wt%) .
- Ind is a neutral chromoionophore
- L is an ionophore
- R the cation-exchanger, respectively.
- the organic sensing phase and the aqueous phase are indicated as (org) and (aq) .
- the optode film comes in contact with lead ions they are extracted into the film and in the process expel hydrogen ions to conserve electroneutrality within the film.
- the change in the degree of protonation of the fluoroionophore which is a result of proton release from the film, leads to a measurable change in its fluorescence properties.
- k exC h is the ion-exchange constant (to describe eq 2) .
- Figure 4 shows that the experimental data correspond well to the theoretical response curve calculated with eq 2 (solid line).
- the dynamic lead(II) measuring range at pH 4.7 was found between 10 ⁇ 7 M to 10 "3 M with a log K exch for Pb(II) of -4.3.
- NB2 based membranes gave Nernstian response ranges from pH 6 to 10 for DOS membranes and from pH 5 to 11 for NPOE membrane.
- the logarithmic selectivity coefficients for H + over Na + were found as -10.3 for DOS membranes and ⁇ -ll for NPOE membranes containing free NB2.
- the pKa values of free NB2 in DOS and NPOE membranes determined by the sandwich method were determined as 10.10 ⁇ 0.02 (DOS) and 12.49 ⁇ 0.02 (NPOE), which are just somewhat smaller than those of ETH 5294 (11.4 and
- NB2 was covalently grafted into a MMA-DMA polymer matrix by the same solution polymerization method used above.
- Figure 6 shows the pH responses of the grafted NB2 based membranes blended with PVC-DOS or PVC-NPOE. The Nernstian pH response ranges were found between pH 3 and 8 for DOS membranes and pH 4 and 9 for NPOE membranes. The apparent pKa values of free NB2 in DOS and NPOE membranes determined by the sandwich method were 9.2 ⁇ 0.02 (DOS) and 11.24 ⁇ 0.02 (NPOE).
- NB2-MMA-DMA membrane For grafted NB2-MMA-DMA membrane the logarithmic selectivity coefficient for H + over Na + were found as -8.8 for DOS membrane and -9.3 for NPOE membrane.
- the somewhat diminished selectivity compared to membranes containing free NB2 may be partly explained by the influence of the methacrylate type copolymer, which possesses some ion binding properties originating from its ester functionalities.
- the absorbance properties of NB2 were characterized in THF solutions as shown in Fig. 7A. Similar to NBl and ETH 5294, NB2 also exhibits peaks at 550 nm and 610/670 nm for its deprotonated and protonated form, respectively.
- ⁇ for NB2 in methanol were determined as 3.5 x 10 (660 nm, protonated form) and 2.8 x 10 4 (550 nm, deprotonated form), which are quite close to the reported values for ETH 5294 (2) .
- Fig. 7B presents the fluorescence spectra of free NB2 based PVC-DOS films in contact with HCl and NaOH solutions .
- the peaks at 640 nm and 680 nm correspond to the emission maxima of the deprotonated and protonated forms of the dye, respectively.
- the fluorescence spectrum of NB2 in the polymer matrix is also nearly identical to that of ETH 5294 (3, 30) .
- grafted NB2 may be used as a HH—fluoroionophore for the detection of fluorescence ratios with optodes measured at neutral pH.
- Na-selective optode thin films were prepared. The films contained NB2-MMA-DMA, sodium ionophore Na- X and NaTFPB.
- the response curve of an optode film towards Na + and the corresponding selectivity behavior are presented in Figure 8.
- the solid lines describe the theoretical curves according to eq 2.
- the response curve observed with NB2-MMA-DMA films corresponds well to the theoretically predicted behavior, which confirms that the fluoroionophore remains fully functional in its covalently bound state.
- poly(n-butyl acrylate) with grafted NB2 was prepared by copolymerization of NB2 and n-butyl acrylate, in EtOAc.
- the resulting PnBA particles were mass fabricated using a high-throughput particle casting apparatus previously described (25).
- Fig. 9 presents the fluorescence response of the PnBA particles to sodium ions.
- the particles contained the ionophore Na-X, NaTFPB, NB2-PnBA and blank PnBA.
- the response of the particles is compared to that of analogous spin coated films discussed above. Each data point is the average of 10 measurements and solid lines are the theoretically predicted responses according to eq 2.
- Fig. 10 displays typical response times of a single sensing particle deposited on the bottom of a glass capillary cell and characterized in the flowing stream of changing sodium ion concentrations. As seen in Fig. 10, the equilibrium response for a lO ⁇ m particle is reached essentially within 10 min, which is dramatically decreased compared to the earlier reported 12 hours and acceptable for many clinical and biomedical applications.
- Nile Blue derivatives Two polymerizable Nile Blue derivatives (NBl and NB2) were synthesized. They exhibit similar absorbance and fluorescence spectra as the established Nile Blue derivative ETH 5294. The structural variations between the two dyes results in widely different pKa values and a large shift in the measuring range of the resulting sensing films. Upon covalent attachment of the dyes in the polymer matrix, the new materials may be successfully used as pH indicators and fluoroionophores in ion- selective optodes .
- NBl-MMA-DMA exhibits a low pKa value and two fluorescence emission peaks, and appears to be a better suited chromoionophore than ETH 5418 for the fabrication of transition metal selective optodes operating at low pH values and/or low analyte concentrations.
- NB2-MMA-DMA blended with PVC-DOS or NPOE is suitable for preparing ion-optodes used at near-neutral pH for the measurement of more abundant ions (especially alkali and alkaline earth metal ions) .
- NB2 can also be copolymerized with PnBA to prepare polymers with faster response times.
- the first reported hydrophobic bulk optodes containing a grafted NB2 and no plasticizer were prepared using NB2-MMA-DMA and exhibited a functional Na + response according to classical optode theory, with fast response times .
- the selectivity observed over common interferences such as Ca, K + , and Mg + was found to be high.
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