Patent Application: US-201415028600-A

Abstract:
the present invention relates to a method for the production of a sol - gel based matrix . the method comprises the steps of : a ) providing a first alkoxysilane of the general formula : r 1 — si3 and a second alkoxysilane of the general formula : b ) preparing a first sol - gel component by polymerisation of the first alkoxysilane in the presence of an acid catalyst , c ) preparing a second sol - gel component by polymerisation of the second alkoxysilane in the presence of an lewis acid catalyst , d ) mixing the first sol - gel component and the second sol - gel component for the preparation of a sol - gel based matrix . the above method results 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 . methods for preparing these composites or sensors are provided as well .

Description:
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 . 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 3 ), phosphoric acid ( h 3 po 4 ), sulphuric acid ( h 2 so 4 ), hydrofluoric acid ( hf ), hydrobromic acid ( hbr ) and perchloric acid ( hclo 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 ”. 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 . 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 . 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 ehrlenmeyer 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 fig9 . compounds were used as received . sol - gel monomers were purchased from sigma - aldrich . sol - gel catalysts were purchased from sigma - aldrich and used as recieved . solvents used were analytical or hplc grade . an electronically controlled oven was used to cure the ormosil thin - films . 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 . 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 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 . a primary amine ( 20 eq , 40 mmol ) was added to a solution of dmb 3 c . bf 4 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 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 . 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 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 2 cl 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 over night . 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 . 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 . 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 . 10 , 11 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 . 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 . 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 3 . o ( ch 2 ch 3 ) 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 . 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 . 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 . 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 . a procedure analogous to that for the eteos gel component described above used to make xteos gel components , with x = pr and ph . 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 . 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 . 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 . 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 . 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 . general procedure for covalent entrapment of dyes into the gptms - eteos matrix 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 . 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 ( m n = 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 2 cl 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 . 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 . 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 . 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 . the tested sensors are prepared as illustrated in fig1 , 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 . 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 . 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 . fig2 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 ). fig2 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 fig2 . 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 . to evaluate the photostability of the sensor we performed a 16 - hour scan , see fig3 . 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 . fig3 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 . the performance of the sensors is shown as titration curves in fig4 . the spectra behind the titration curves are shown in fig5 . it is clear that a ph - dependent sensor action is achieved for these two sensor systems . for the examples given in fig4 and 5 the pka values are ˜ 5 , the data for all prepared sensors are compiled in table 1 , sensors with a pk a from 1 . 1 to 6 . 7 was made . fig4 a shows the ratiometric ph response of tmaacr - 4 immobilized in gptms - eteos matrix via lipophilic entrapment . fig4 b shows the ph response of tmaacr - 6 immobilized in the gptms - eteos matrix via covalent entrapment . the pk a values of tmaacr - 4 and tmaacr - 6 are determined to 4 . 9 ( lipophilic entrapment ) and 4 . 8 ( covalent entrapment ). fig5 shows spectra of the sensors in action . fig5 a 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 ). fig5 b 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 . 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 . fig6 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 fig7 . 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 ˜ 20s . 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 . fig6 shows the response time of ph - active dye daota - 2 in a phteos - gptms ( fig6 a ), an eteos - gptms ( fig6 b ) and prteos - gptms ( fig6 c ) matrices . high intensity : low ph (& lt ; 2 ). low intensity : high ph (& gt ; 10 ). fig7 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 (& lt ; 2 ). low intensity : high ph (& gt ; 10 ). table 3 . the response times ( t 90 and t 100 ) given in seconds ( s ) of the eteos - 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