Sol-gel based matrix

Abstract

A method for the production of a sol-gel based matrix resulting in a sol-gel based matrix with high stability and high porosity. The sol-gel based material may be used for the production of a composite or sensor suitable for monitoring analytes.

Claims

1. A sol-gel matrix comprising: a first polymer component prepared from polymerization of a first alkoxysilane of the general formula R.sup.1—Si(OR.sup.2).sub.3 and an additional alkoxysilane of the formula R.sup.5—Si(OR.sup.2).sub.3, a second sol-gel component comprising a Lewis acid and a second polymer component prepared from Lewis acid catalyzed polymerization of a second alkoxysilane of the general formula ##STR00005## wherein R.sup.1 is selected from the group consisting of a straight and branched C.sub.1-C.sub.6 alkyl, a C.sub.2-C.sub.6 alkenyl, a C.sub.3-C.sub.6 cycloalkyl, a C.sub.1-C.sub.6 aminoalkyl, a C.sub.1-C.sub.6 hydroxyalkyl, a C.sub.1-C.sub.6 cyanoalkyl, a phenyl, and a group of the formula —Y—(X—Y).sub.nH, with Y being independently selected from the group consisting of straight and branched C.sub.1-C.sub.6 alkylene, X being a hetero atom or group selected from the group consisting of O, S, and NH, and n being an integer of 1-5, or R.sup.1 represents a C.sub.1-C.sub.6 alkyl substituted with a group Z, with Z being independently selected form the group consisting of hydrogen, cyano, halogen, hydroxy, nitro, amide C.sub.1-C.sub.24-alkyl, C.sub.1-C.sub.24-haloalkyl, C.sub.2-C.sub.24-alkenyl, C.sub.2-C.sub.24-alkynyl, aryl, C.sub.1-C.sub.24-alkoxy, C.sub.1-C.sub.24-alkylsulfonyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester comprising a C.sub.1-C.sub.6 alkyl alcohol moiety, (carboxyl ester)amino comprising a C.sub.1-C.sub.6 alkyl alcohol moiety, (carboxyl ester)oxy comprising a C.sub.1-C.sub.6 alkyl alcohol moiety, sulfonyl, sulfonyloxy, thiol, thiocarbonyl, C.sub.1-C.sub.24-alkylthio, 5 or 6 membered heteroaryl, and a C.sub.3-C.sub.7 cycloalkyl; R.sup.2 independently represents a straight or branched C.sub.1-C.sub.6 alkyl; R.sup.3 represents a linker selected from a group of the formula —R.sup.4—(X—R.sup.4).sub.0-12—, wherein R.sup.4 is independently selected from straight or branched C.sub.2-C.sub.6 alkylene, C.sub.2-C.sub.24-haloalkylene, and X is a hetero atom or group selected from the group consisting of 0, S and NH; and R.sup.5 represents a group of the general formula
—R.sup.3—NH—C(═O)—X—R.sup.3-Q wherein R.sup.3 and X are as defined above and independently selected, and Q represents an indicator and/or a reference dye derived from triangulenium compounds, acridinium compounds, ruthenium doped sol-gel particles, ruthenium-based compounds with α-diimine ligands, porphorin with Pt or Pd as the central metal atom, Ru(bpy).sub.2(dpp)Cl.sub.2, Ru(bpy).sub.3Cl.sub.2 a lanthanide containing complex, or polymeric metal containing structure.

2. The sol-gel matrix according to claim 1, wherein the second polymer component is prepared from polymerization of the second alkoxysilane and the additional alkoxysilane.

3. The sol-gel matrix according to claim 1, wherein Q is an indicator dye derived from 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), fluorescein, or rhodamine B.

4. The sol-gel matrix according to claim 1, wherein the Lewis acid is a triagonal planar species.

5. The sol-gel matrix according to claim 4, wherein the Lewis acid is selected from the group consisting of TiCl.sub.3, AlCl.sub.3, and BF.sub.3, or solvates or etherates thereof.

6. The sol-gel matrix according to claim 1, wherein the first alkoxysilane is selected from the group consisting of ethyltriethoxysilane (ETEOS), methyltriethoxysilane (MTEOS), propyltriethoxysilane (PrTEOS), n-octyltriethoxysilane (n-octyl TEOS), methyltrimethoxysilane (MTMOS), aminopropyltrimethoxysilane (APTMOS), phenyltriethoxysilane (PhTEOS), and phenyl trimethoxysilane (PhTMOS).

7. The sol-gel matrix according to claim 1, wherein second alkoxysilane is 3-glycidoxypropyltrimethoxysilane (GPTMS).

8. A composite comprising a layer of sol-gel matrix according to claim 1 and a platform comprising a microstructure area, wherein the sol-gel matrix is attached to the microstructure area.

9. The composite according to claim 8, wherein the microstructure area comprises a plurality of pillars having a height in the range of 0.1 μm to 500 μm and a distance between each pillar in the range of 0.1 μm to 500 μm.

10. The composite according to claim 8, wherein the distance between each pillar is in the range of 5 to 40 μm, the height of the pillars is in the range of 11 to 40 μm and the width of each pillar is in the range of 5 to 40 μm.

11. The composite according to claim 8, wherein the layer of the sol-gel matrix has a thickness smaller than the height of the microstructure.

12. The composite according to claim 8, comprising an array of sol-gel matrices attached to different areas on the microstructure area.

13. The composite according to claim 8, wherein sol-gel matrix comprises an indicator or reference dye.

14. The composite according to claim 13, wherein the sol-gel matrix includes at least two different indicator or reference dyes.

15. The composite according to claim 8, wherein the platform is an inner surface of a container or conduit for transporting a fluid.

16. The composite according to claim 15, wherein the container comprises an opening and cylindrical or tapered sides, and is closed opposite to the opening.

17. The composite according to claim 8, wherein the platform is an inner surface of a disposable container for transporting a fluid.

18. The composite according to claim 8, wherein the microstructure area has been prepared by a process selected from the group consisting of injection molding, hot embossing, laser microstructuring, micromachining, chemical etching, and photoresist layer structuring.

19. A method of monitoring of a bioculture, the method comprising the steps of: providing a composite according to claim 13, applying the bioculture to the platform, exposing the sol-gel matrix to light, and detecting light emitted from the sol-gel matrix.

Description

DRAWINGS

(1) FIG. 1 shows the general preparation steps for deposition of sensor material on a substrate,

(2) FIG. 2 shows the leakage over time of sensor spots.

(3) FIG. 3 shows the development over time for the ratiometric signal in four different buffer solutions,

(4) FIG. 4 shows the ratiometric responses; FIG. 4a shows TMAAcr-4 immobilised in the GPTMS-ETEOS matrix via lipophilic entrapment, and FIG. 4b shows TMAAcr-6 immobilised in the GPTMS-ETEOS matrix via covalent entrapment.

(5) FIG. 5 shows the emission spectra of the sensors in action; FIG. 5a shows the emission spectra of a GPTMS-ETEOS matrix with TMAAcr-4 and DMQA-1 lipophilic entrapped in the matrix, and FIG. 5b shows the emission spectra of the GPTMS-ETEOS matrix with TMAAcr-6 covalently and DMQA-1 lipophilic entrapped in the GPTMS-ETEOS matrix.

(6) FIG. 6 shows the response times of the pH active dye DAOTA-2; FIG. 6a shows the response time in PhTEOS-GPTMS matrix, FIG. 6b shows the response time for ETEOS-GPTMS matrix, and FIG. 6c shows the response time for the PrTEOS-GPTMS matrix.

(7) FIG. 7 discloses the response time of the pH active dye DAOTA-2 in an ETEOS-GPTMS matrix, compared to a matrix PVA and PEG-DA.

(8) FIG. 8 discloses the intensity ratio of lipophil bounded DQMA and a ruthenium kompleks (Tris(4,7-diphenyl-1, 10-phenanthroline)ruthenium (II) bis(hexafluorophosphate) kompleks CAS Nummer 123148-15-2) measured by ratiometic titration (I(Ru)/I(DMQA)) in aqueous solutions having different oxygen concentrations. The measurements are determined with an optical DO electrode from Mettler-Toledo.

(9) FIG. 9. Example of laboratory consumables comprising a sol-gel matrix according to the invention.

(10) FIG. 10 shows a preferred arrangement and geometry of the microstructure in the platform area.

DETAILED DESCRIPTION

(11) The sol-gel based matrix is usually deposited on a substrate as a part of a sensor. The substrate is generally selected to optimise the ability of the sol-gel to form an immobilized attachment to the substrate. Suitable substrates include glass, plastics, ceramics, and polymers. Suitable polymer substrates include polycarbonates, acrylics such as poly(methyl methacrylate), acrylonitrile-butadiene-styrene copolymer, polyvinylchloride, polyethylene, polypropylene, polystyrene, polyurethanes, silicones, and vinylidene fluoride-hexafluoropropylene copolymer.

(12) The first sol-gel component is prepared by polymerisation of the first alkoxysilane defined above in the presence of an acid catalyst. The acid catalyst may be any suitable acid, such as an inorganic or organic acid. Suitable inorganic acids include hydrochloric acid (HCl), nitric acid (HNO.sub.3), phosphoric acid (H.sub.3PO.sub.4), sulphuric acid (H.sub.2SO.sub.4), hydrofluoric acid (HF), hydrobromic acid (HBr) and perchloric acid (HClO.sub.4). A preferred inorganic acid is hydrochloric acid. Suitable organic acids include lactic acid, acetic acid, formic acid, citric acid, oxalic acid, and malic acid. Furthermore, the acid catalyst may be any combination of the above compounds.

(13) To be suitable, the acid chosen must be able to hydrolyse the first alkoxysilane under the acidic conditions. The hydrolysis initiates the polymeric condensation reaction upon formation of a polymer silicon oxide network.

(14) The procedure for the preparation of the first sol-gel component generally include that the first alkoxysilane is dissolved in an organic solvent, usually an alcohol like ethanol prior to the addition of the acid catalyst. The amounts in mole of acid are generally at the same level or lower as the molar amount of the first alkoxysilane. The mixture of first alkoxysilane, organic solvent and acid catalyst is left until the reaction is completed. The reaction time may be several days.

(15) The second sol-gel component is prepared by either dissolving the second alkoxysilane in a solvent before the addition of the Lewis acid catalyst or by mixing the alkoxysilane and the Lewis acid and then adding the solvent. The molar amount of Lewis acid catalyst is generally lower than the molar amount of the second alkoxysilane. In a preferred embodiment the molar amount of Lewis acid to second alkoxysilane is 1:2, such as 1:3, preferably 1:4. The solvent is usually an alcohol like ethanol but may be chosen among various solvents assumed by the skilled person to be inert under the conditions.

(16) The Lewis acid is believed to attack the epoxy ring of the second alkoxysilane whereby a secondary carbocation is formed. This intermediate carbocation can then react with another molecule in the polymerisation process. The amount and the type of second alkoxysilane should be chosen so as to be able to participate in the intended chemical reaction within a reasonable time. The formation of the second sol-gel component normally proceeds much faster than the formation of the first sol-gel component. A typical reaction time for the second sol-gel component is between 10 min and 3 hours. After the reaction the second sol-gel component is typically allowed to rest for a few hours.

(17) After the two sol-gel components have been prepared separately, they are mixed. Typically, the molar amount of the first sol-gel component to the second sol-gel component is in the range of 5:1 to 1:5, such as 3:1 to 1:3, typically 2:1 to 1:2, and suitably approximately 1:1.

(18) If the sol-gel matrix is used for sensing, an indicator and/or reference dye may be incorporated in to the matrix by a number of methods to obtain either a non-covalent or a covalent attachment. If a non-covalent attachment is used it is preferred to anchor the dye in some way to the matrix to avoid excessive leakage. A preferred anchoring method is the so-called lipophilic entrapment, according to which the dye core is provided with one or more lipophilic linkers. The lipophilic linkers will engage with the lipophilic environment of the network formed by the sol-gel components and thereby retard the leakage. In a preferred method the dye core provided with one or more lipophilic linkers is added either to one or both of the sol-gel components or to the mixture of the first and the second sol-gel component. To ensure a sufficient maturation of the mixture it may be kept for 1 hour to 7 days before it is deposited on the substrate and cured.

(19) A covalent attachment of the dye is possible by linking the dye to one of the monomers before polymerisation. In a preferred method, an additional alkoxysilane is prepared as a derivative of the first alkoxysilane by attaching the dye thereto. The additional alkoxysilane may be incorporated into either the first sol-gel component or the second sol-gel component. In a preferred aspect the first or second alkoxysilane is allowed to polymerise a short time, such as at least 15 minutes, before the further alkoxysilane is added to avoid end positioning.

(20) The mixture of the first sol-gel component and the second sol-gel component may be allowed to mature before the deposition on a suitable substrate. The substrate is generally transparent at the wavelength used for monitoring the emitted light. The amount of the mixture used for deposition varies in dependence of the purpose and geometry of the sensor. In a certain aspect the amount is 100 μl or less, such as 50 μl or less, suitably 20 μl or less. The deposition may be referred to herein as a “spot”.

(21) The addition of the mixture to the substrate may be allowed deliberately to solidify or the added amount of mixture may be spread on the substrate to form a film with an essentially uniform thickness. After the deposition of the mixture on the substrate it is cured. The curing may be performed in a number of ways, including heating at elevated temperatures so as to form a solid film attached to the substrate. The temperature of the curing is suitably 70° C. or above, such as 90° C. or above, and suitably 100° C. or above. Usually, the curing temperature does not exceed 150° C. to avoid degradation of the materials, i.e. to maintain the porous three dimensional polymer networks, which allow for fast diffusion of the analyte, such as a proton. The relatively unhindered diffusion of the analyte in the porous network is believed to be the reason for the observed fast response time.

(22) The research reported herein suggests that covalent attachment of the dye to the polymer network is preferred when a long-time stability is of importance. Even when the dyes are provided with lipophilic linkers to retard the leakage from the film, the leakage is still too high for a product stabile over a longer time period to be obtained. For short-time use, such as in non-reusable sensors, non-covalently attached dyes may be acceptable.

(23) An aspect of the invention relates to the manufacturing of a container or laboratory equipment with a composite, i.e. a container or laboratory equipment respectively comprising one or more platforms. Particularly suited laboratory equipment is Erlenmeyer flasks, beaker glasses, tissue culture flasks, tissue culture dishes, tissue culture plates and storage systems. Examples of laborative equipments comprising the composite of the invention are shown on FIG. 9.

EXAMPLES

(24) Methods and Materials

(25) Compounds were used as received. Sol-gel monomers were purchased from Sigma-Aldrich. Sol-gel catalysts were purchased from Sigma-Aldrich and used as received. Solvents used were analytical or HPLC grade. An electronically controlled oven was used to cure the ORMOSIL thin-films.

(26) Synthesis

(27) The synthesis of compounds 1.BF.sub.4 and 2.PF6 are reported elsewhere..sup.18

(28) General Preparation of Tetramethoxyamino-Acridinium (TMAAcr)

(29) 2 (162 mg, 0.23 mmol) was dissolved in 15 ml acetonitrile and n-octylamine (26 ml, 0.14 mmol) was added to the solution. The reaction mixture was heated to slight reflux temperature and stirred in 5 h. The reaction mixture was allowed to cool down when the color of the mixture had changed from blue to red-brown and MALDI-TOF analysis indicated that a mass corresponding to that of the starting material was not present any more. The reaction mixture was washed with heptane (3×50 ml). The crude product was isolated by evaporation and recrystallized from ethanol, and the product was washed with ether and heptane several times. The product was isolated as a red-purple powder, which was metallic-green when filtered.

(30) General Approach to Activate TMAAcr for Covalent Attachment

(31) TMAAcr (100 mg, 0.11 mmol) was dissolved in 20 ml acetonitrile and then triethoxy(3-isocyanatopropyl)silane (1.1 ml, 0.45 mmol) was added dropwise using a syringe at room temperature. The mixture was stirred for 1 h, when MALDI-TOF analysis indicated that 9 was not present. The reaction mixture was washed with heptane (3×50 ml) and then the acetonitrile phase was mixed with a 0.2 M KPF.sub.6 solution. The slurry was stirred for 20 min and then gently filtered. The precipitate was washed with water several times. The product was dissolved in dichloromethane through the filter and the non-dissolved solid in the filter was discarded. The product is collected by removal of the solvent yielding metallic-green flakes.

(32) General Preparation of Dimethoxyquinacridinium (DMQA)

(33) A primary amine (20 eq, 40 mmol) was added to a solution of DMB.sub.3C.BF.sub.4.sup.19 in NMP (1.0 g, 2 mmol in 8 mL). The solution was warmed to 140° C. for 10-20 minutes (the degree of reaction is followed by MALDI-TOF mass spectroscopy). After cooling to RT the reaction mixture was poured on to 0.2 M KPF.sub.4(aq) (200 mL). The precipitate was collected, washed and dried. The crude can be recrystallized from methanol, reprecipitated from dichloromethane with ethylacetate or reprecipitated from acetonitrile with ether depending on the how lipophile the side chains are.

(34) General Approach to Activate DMQA for Covalent Attachment

(35) DMAQ (70 mg, 0.143 mmol) was dissolved in 8 ml anhydrous acetonitrile and then 3-(triethoxysilane)propyl isocyanate (cold, 100 ul, d=0.999 g/ml, 0,404 mmol) was added. The flask was fitted with a stopper and stirred at room temperature for 4 h. After 4 h MALDI-TOF analysis indicated that the reaction mixture only contained starting material. Then excess of isocyanate (1 ml) was added together with approx. 1 ml of triethylamine. The mixture was heated to 65° C. and stirred for 1.5 h. Then MALDI-TOF analysis indicated that the reaction mixture contained a compound with a mass of 649 m/z, which is the mass of the desired product and no mass corresponding to that of the starting material was present. The reaction mixture was washed (still warm) with heptane (2×50 ml) and then dried over MgSO.sub.4 for 10 min. The solvent was removed by evaporation at 50° C. in vacuum and the crude product was dissolved in a minimum of CH.sub.2Cl.sub.2 and then diethyl ether (200 ml) was added and a green precipitate was allowed to form. The dark product was collected and dried in vacuum over KOH overnight.

(36) Spectroscopy

(37) Emission spectroscopy was performed in front-face set-up for sensor spot samples and in a conventional L-shape set-up for measurements in solution. A Perkin-Elmer LS50B and a Horiba Fluorolog 3 were used interchangeably. Intensity based sensor measurements were only performed on the LS50B platform. Fluorescence lifetime based sensor measurements were only performed on the Fluorolog 3. Absorption spectroscopy was performed on a Perkin Elmer Lambda 1050, with integrating sphere (for sensor spots) and with a 3-detector module for solution samples.

(38) Sol-Gel Preparation

(39) The procedure includes preparation of two separate gel components of the organic modified silanes: Ethyltriethoxysilane (ETEOS) or a similar alkyl or aryl trialkoxy silane (XTEOS) and 3-(glycidoxy)propyltrimethoxysilane (GPTMS). All the different preparations and combinations are compiled in table 1, and the detailed procedures are as follows.

(40) TABLE-US-00001 TABLE 1 The different compositions of sol-gels tested in this work; variations can be seen in the alkyltrialkoxy silane part, the Lewis acid, and the dye additives. The pKa of the resulting sensor is included. Entry Monomer 1 Monomer 2 Catalyst Dye 1 Dye 2 pKa 1 GPTMS ETEOS BF.sub.3 TMAAcr-1 — 3.8 2 GPTMS ETEOS BF.sub.3 TMARh — 1.1 3 GPTMS ETEOS.sup.1 BF.sub.3 TMAAcr-3 — 2.6 4 GPTMS ETEOS.sup.2 BF.sub.3 TMAAcr-3 — 3.1 5 GPTMS ETEOS BF.sub.3 TMAAcr-4 DMQA-1 4.9 6 GPTMS ETEOS.sup.1 BF.sub.3 TMAAcr-6 DMQA-1 4.8 7 GPTMS ETEOS BF.sub.3 DAOTA-1 DMQA-2 6.5 8 GPTMS ETEOS BF.sub.3 DAOTA-2 DMQA-2 6.5 9 GPTMS PrTEOS BF.sub.3 DAOTA-2 DMQA-2 6.5 10 GPTMS PhTEOS BF.sub.3 DAOTA-2 DMQA-2 6.7 11 GPTMS ETEOS TiCl.sub.4 DAOTA-2 DMQA-2 — 12 GPTMS ETEOS AlCl.sub.3 DAOTA-2 DMQA-2 — .sup.1Dye 6 pre-mixed with ETEOS component, .sup.2Dye 6 pre-mixed with GPTMS component.

(41) ETEOS

(42) The ETEOS gel component is prepared from polymerization of the silicon network under acidic conditions. ETEOS is hydrolysed under acidic conditions, which initiates a polymeric condensation reaction upon formation of a polymer silicon oxide network. The presented procedure is an equivalent to the procedure reported by Wencel et al..sup.10, 11

(43) Procedure for preparation of ETEOS gel component: 5 ml ETEOS (0.02 mol) is dissolved in 8 ml absolute ethanol (0.14 mol) upon stirring. Hereafter, 1.6 ml of 0.1 M HCl solution (0.16 mmol) is added dropwise. This mixture is then left on a stirring table for a minimum of 7 days to allow the polymerization process to proceed.

(44) GPTMS Gel Component.

(45) The GPTMS gel component is prepared from polymerization of the organic linker using a Lewis acid as initiator. In this procedure we use boron trifluoride diethyletherate as the Lewis acid. The Lewis acid attacks the epoxy ring that allows for ring opening of the epoxy ring upon formation of a secondary carbocation. This intermediate carbocation can then react with another GPTMS molecule, initiating a polymerization reaction. Due to the acidic environment a polymerization of the silicon network equivalent to that described for the ETEOS component will proceed alongside.

(46) Procedure for preparation of GPTMS gel component: 6 ml of GPTMS (0.027 mol) is mixed with 11 ml of absolute ethanol (0.19 mol) upon stirring. Then 0.75 ml of cold borontrifluoride diethyletherat (BF.sub.3.O(CH.sub.2CH.sub.3).sub.2, 5.8 mmol) is added dropwise. The mixture is left with stirring for 30 min in a sealed container until the temperature of the mixture has dropped to room temperature. After 30 min 2 ml of MilliQ water (0.11 mol) is added to the solution. The resulting mixture was left with stirring for 4 h.

(47) When the two gel components have been prepared they are mixed in 1:1 molar ratio and left for a minimum of 3 days to allow the networks to mix. This is referred to as the GPTMS-ETEOS mixture.

(48) GPTMS-ETEOS Mixture

(49) When the GPTMS and ETEOS components have been prepared they are mixed to obtain a 1:1 molar ratio (1.1 ml GPTMS+1 ml ETEOS) and the dyes are added in order to obtain a concentration of approx. 0.1 mM. The resulting mixture is then allowed to further mix for a minimum of 3 days.

(50) The GPTMS-ETEOS mixture with the dye entrapped can now be deposited onto a glass or plastic surface. When deposited it has to be cured at 110 degrees for 3-4 h. The result is a porous and transparent matrix.

(51) XTEOS Variations

(52) A procedure analogous to that for the ETEOS Gel component described above used to make XTEOS gel components, with X=Pr and Ph.

(53) Preparation of XTEOS Gel Components

(54) X=Phenyl (Ph): Phenyltriethoxyilane (PhTEOS, 10 ml, M=240.14 g/mol, d=0.996 g/ml, 0.041 mol) and absolute ethanol (15 ml, d=0.789 g/ml, 0.26 mol) was mixed and the freshly prepared 0.1 M HCl solution (2.8 ml, 0.28 mmol) was added. The solution was stirred for 15 min in the sealed vial, and then left at a vibration table for 20 days in the dark at room temperature.

(55) X=Propyl (Pr): Propyltriethoxyilane (PrTEOS, 10 ml, M=206.13 g/mol, d=0.892 g/ml, 0.043 mmol) and absolute ethanol (16 ml, d=0.789 g/ml, 0.27 mol) was mixed and then freshly prepared 0.1 M HCl solution (3.2 ml, 0.32 mmol) was added. The solution was stirred for 15 min in the sealed vial, and then left at a vibration table for 20 days in the dark at room temperature.

(56) Lipophilic Entrapment

(57) In the lipophilic entrapment method the dyes in entrapped in the GPTMS-ETEOS network requires that the dye has one or several lipophilic linker(s) attached to the dye to prevent leakage from the resulting matrix material.

(58) General Procedure for Lipophilic Entrapment of Dyes

(59) The ETEOS and GPTMS gel components are prepared and mixed as described above with the addition of the dye such that a final concentration of 0.1 mM is obtained. The resulting GPTMS-ETEOS-dye mixture is then left at a stirring table for at least 3 days before deposition and curing at 110° C. for 3-4 hours.

(60) Covalent Method:

(61) This procedure requires that the dye has been activated by linking to a trialkoxysilane group that can mix into the silicon network of either the ETEOS or GPTMS gels.

(62) General Procedure for Covalent Entrapment of Dyes into the GPTMS-ETEOS Matrix

(63) The ETEOS and GPTMS gel components are prepared and mixed as described above, with the exception that the silane functionalized dye is mixed into the either the ETEOS or the GPTMS gel component after 1 h after mixing of the materials described to mix the ETEOS or the GPTMS gel components. The GPTMS and ETEOS components are left for polymerization reaction time described in the general procedure. The two components are then mixed in the described 1:1 molar ratio and left at a stirring table for no less than 3 days. The dye should be added in an amount so that a final concentration of 0.1 mM of dye is obtained in the final GPTMS-ETEOS mixture. The resulting GPTMS-ETEOS-dye mixture is then deposited and cured at 110° C. for 3-4 hours.

(64) Fabrication of Sensor-Spots

(65) The sensor spots were drop coated on a glass or polycarbonate substrate and then cured. The substrate material appears to be inconsequential as long as thin films can be prepared. For comparison sensor spots were prepared from direct incorporation of the dyes in PVA (from 10% w/w solutions in water) which were subsequently drop coated on glass. PEG-DA hydrogel with dye entrapped was prepared by mixing PEG-DA (Mn=700) and ethanol in a 1:1 v/v ratio and then the dye was added to obtain 1 mM. Then a catalytic amount of a solution of 2,2′-azobis(2-methylpropionitrile) in CH.sub.2Cl.sub.2 (25 mg/ml) was added. The mixture was spread out on a petri dish, the dish was equipped with a glass lid, and the mixture was baked in the oven at 110° C. for 1 h. A thin piece of the resulting hydrogel was immobilized on a clean glass slide using double-sided tape and the regular tape.

(66) Titrations

(67) To perform titrations rapidly a set-up employing an epi-fluorescence microscopy equipped with a halogen light source and an Ocean Optics spectrometer for detection. The sensor spot was attached to a homemade holder, which kept the spot in place in a large chamber filled with water, where pH was externally monitored with a pH meter. Alternatively the sensor spot was affixed on the wall of a cuvette and the titration was performed in a Perkin Elmer LS50B, controlling the pH between measurements.

(68) Stability Testing

(69) The photostability was followed by constant illumination of the sensor spot with wavelength selected light from a xenon lamp. The physical stability was tested by immersing the sensor spot in low or high pH aqueous solution, and monitoring the fluorescence from the solution.

(70) Response Analysis

(71) The signal from the sensor is monitored after inducing a significant (more than 4 pH units) jump in pH. The time it takes to obtain a full (100%) and partial (90%) response, compared to the equilibrium signal is recorded.

(72) FIG. 1 shows the general preparation of sensor spots.

(73) ##STR00002##

(74) Results

(75) The tested sensors are prepared as illustrated in FIG. 1, on glass and polycarbonate substrates. The five components are mixed in a fashion that allows for the formation of a porous covalently linked 3D polymer network, which allows for fast diffusion of protons.

(76) Scheme 2 shows the structure of the pH-responsive and the reference dyes used in this study. The pKa values of the resulting sol-gel based sensors are compiled in table 1. Cursory inspections of the structures, which are physically immobilized in the sol-gel show that a long alkyl chain is required to prevent leakage, while the molecules covalently linked to the matrix can have either a long or a short linker.

(77) ##STR00003## ##STR00004##

(78) Stability

(79) TABLE-US-00002 TABLE 2 Leaking of 5(6)-carboxyfluorescein (CF), DMQA-2, DAOTA-1, and 6-stearamido-fluorescein (AF18) from the ETEOS-GPTMS matrix given as fluorescence intensity measured from a PBS solution at pH 7 surrounding a glass slide coated with ETEOS- GPTMS-dye matrix using maximum sized slit widths at the emission and excitation sites of the spectrometer. Dye Intensity (a.u.) Leaking Period pH pK.sub.a CF >>800 2 h 7.0 6.5 DMQA-2 0 15 h 7.0 — DAOTA-1 80 15 h 7.0 6.5 AFC18 100 4 d 7.0 6.5

(80) FIG. 2 shows leakage over time of sensor spots: non-bound 5(6)-carboxyfluorescein (plus-signs), covalently bound DAOTA-1 (crosses), and covalently bound DMQA-2 (dots).

(81) FIG. 2 shows the performance in leakage studies, against the performance of molecules without anchoring groups, and the data are collected in table 2. Leaking of the dyes entrapped or bound to the matrix was investigated by measuring the emission intensity from a PBS solution at pH 7.0 surrounding a non-bound dye (5(6)-carboxy fluorescein, CF), covalently bound (DMQA-2 and DAOTA-1) using the largest possible slit widths on the excitation and emission sites of the spectrometer and an excitation wavelength of 450 nm, these data are shown in FIG. 2. While the physically bound dye and 6-stearamido-fluorescein (AF18) was also tested, we did not record the transient curve. All the leakage data is compiled in table 2. The results reveal that DAOTA-1 leaked to a small extend, which we, based on NMR data, can assign to a fraction of un-linked dye in the ETEOS-GPTMS matrix, this issue has previously been reported for fluorescein, which was only partially activated. The compound DMQA-2 could based on NMR data be shown to be 100% activated and did as a consequence not show any leakage. This shows that effective binding can indeed be obtained in the ETEOS-GPTMS matrix and leakage can be avoided completely by fully activating the dye for polymerization. The unbound CF showed extensive leakage and the data in table 2 is obtained using half the sizes of the slit widths as those used for DMQA-2 and DAOTA-1.

(82) To evaluate the photostability of the sensor we performed a 16-hour scan, see FIG. 3. No perceivable slope of the curves could be seen in this time interval, which proves that this system has a very high long-term stability under constant irradiation.

(83) FIG. 3 shows the development in the ratiometric signal in four different buffer solutions at pH 3 during 16 h of irradiation at 525 nm of a DAOTA/DMQA based sensor.

(84) Sensor Action

(85) The performance of the sensors is shown as titration curves in FIG. 4. The spectra behind the titration curves are shown in FIG. 5. It is clear that a pH-dependent sensor action is achieved for these two sensor systems. For the examples given in FIGS. 4 and 5 the pKa values are ˜5, the data for all prepared sensors are compiled in table 1, sensors with a pK.sub.a from 1.1 to 6.7 was made.

(86) FIG. 4a shows the ratiometric pH response of TMAAcr-4 immobilized in GPTMS-ETEOS matrix via lipophilic entrapment. FIG. 4b shows the pH response of TMAAcr-6 immobilized in the GPTMS-ETEOS matrix via covalent entrapment. The pK.sub.a values of TMAAcr-4 and TMAAcr-6 are determined to 4.9 (lipophilic entrapment) and 4.8 (covalent entrapment).

(87) FIG. 5 shows spectra of the sensors in action. FIG. 5a shows the emission spectra of a GPTMS-ETEOS matrix with TMAAcr-4 and DMQA-1 lipophilic entrapped in the GPTMS-ETEOS matrix at different pH values between 3 (black) and 7.5 (red). FIG. 5b shows emission spectra of a GPTMS-ETEOS matrix with TMAAcr-6 covalently and DMQA-1 lipophilic entrapped in the GPTMS-ETEOS matrix at different pH values between 2 (black) and 7.5 (red). Excitation at 475 nm±25 nm.

(88) In order to evaluate the response time the temporal evolution of the detected signal (intensity ratio) was monitored, when the sensor was monitoring a solution where the pH was changed drastically as well as moderately. FIG. 6 shows the result, each panel shows the response of different matrices. It is clear that the response of the Lewis acid catalyzed sol-gel is much faster than the others tested. To highlight the differences an overlay is shown in FIG. 7. All the data are compiled in table 3. The alkyltrialkoxy-GPTMS matrixes have by far the fastest response times, showing some hysteresis, with a response going from high pH to low pH of ˜10 s and going from low pH to high pH of ˜20 s. Propyltrialkoxy silane derived matrices are faster responding than the ethyltrialkoxy silane derived matrices when the signal level of 90% is considered, while the full response occur on a similar timescale for both matrices.

(89) FIG. 6 shows the response time of pH-active dye DAOTA-2 in a PhTEOS-GPTMS (FIG. 6a), an ETEOS-GPTMS (FIG. 6b) and PrTEOS-GPTMS (FIG. 6c) matrices. High intensity: Low pH (<2). Low intensity: High pH (>10).

(90) FIG. 7 shows the response time of pH active dye DAOTA-2 in an ETEOS-GPTMS (red), PVA (blue) and PEG-DA (green) matrix. High intensity: Low pH (<2). Low intensity: High pH (>10).

(91) TABLE-US-00003 TABLE 3 The response times (t.sub.90 and t.sub.100) given in seconds (s) of the ETEOS-GPTMS, PrTEOS-GPTMS, and PhTEOS-GPTMS matrices with of the pH-active dye DAOTA-2 incorporated, and the response times of PVA film and PEG-DA hydrogel with the pH-active dye TMAAcr-4 incorporated. H-L refers to the response time going from high (H) pH (>10) to a low (L) pH (<2) value, L-H has the opposite meaning. Numbers in parentheses refer to response times measured in the ETEOS-GPTMS matrix with the dyes DAOTA-1 and DMQA-2 covalently bound. t.sub.90 (H- t.sub.100 (H- t.sub.90 (L- t.sub.100 (L- Matrix Dye L)/s L)/s H)/s H)/s PhTEOS- DAOTA-2 59 171 108 271 GPTMS ETEOS- DAOTA-2 9 (10) 24 (30) 19 (23) 40 (47) GPTMS (DAOTA-1 DMQA-2) PrTEOS- DAOTA-2 6 34 7 31 GPTMS PVA TMAAcr-4 6 50 25 55 PEG-DA TMAAcr-4 40 98 192 262

CONCLUSION

(92) We have shown that our system has a shorter or comparable response time than what was previously reported and a high degree of photostability. Furthermore, with this sensor we have solved the leaking issue, using either covalent attachment or lipophilic entrapment of the active components. We have also shown that the activation of the active component is important in making leakage free film.

REFERENCES AND FOOTNOTES

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