Patent Application: US-39610603-A

Abstract:
this invention relates to a quick method for preparation of photorcactive polymers and immobilization of biomolecules onto these polymers . particularly , this invention relates to a microwave - mediated rapid and efficient method for the preparation of photoreactive polymer having at least one photoactivable functional group capable of forming a covalent bond with the biomolecule in a photochemical reaction . any matrix either organic or inorganic having a nucleophilic group preferably , amino , hydroxyl or thiol group can be modified by the invented method . immobilization of biomolecules onto the photoreactive polymers was carried out by uv radiation . the method has applications for inmmobilization of bio - molecules irrespective of their functional groups in the fields of molecular biology , proteomics , genomics , diagnostics , chemical or biochemical industry and other related fields .

Description:
the present invention comprises a rapid and efficient method for modification of polymer surface for the introduction of latent reactive group onto it and immobilization of biomolecules onto said modified polymer surface by light energy . more specifically , polymer surface with latent photoreactive group is prepared rapidly by microwave - mediated reaction of 1 - fluoro - 2 - nitro4 - azidobenzene with a polymer having a reactive nucleophilic group . the modified polymer ( photoreactive polymer ) thus prepared has photoreactive azido group , which under uv light generates highly reactive nitrene capable of binding with the biomolecule . concentration of photolinker plays an important role in preparing photoreactive polymer . best result was obtained when the ratio of the photolinker ( fnab ) and polymer is 1 : 1 ( w / w ). further increase in fnab did not increase the photoreactive group in a polymer . the efficacy of the photoreactive surface was determined by immobilizing hrp onto it and subsequently assaying the immobilized enzyme . when untreated surface ( fnab : 0 mg ) was used , there was practically no immobilization of biomolecule onto it . ( fig1 fig2 and fig3 ). microwaves were found an excellent tool for making photoreactive surface . thus , photoreactive lcaa - cpg prepared in 70 microwave irradiation was found to give better results than the photoreactive surface obtained by prior ad i . e 37 ° c . for 20 hours ( table 1 ). microwave radiation for 60 seconds was also found to give photoreactive aminosilica with good results ( fig4 ). for aminopolystyrene also 60 seconds microwave irradiation was sufficient for the reaction of the polymer and fnab to produce photoreactive amino polystyrene . ( fig5 ). in contrast to amino polymer , hydroxyl group bearing polymer needed a basic catalyst for the reaction with fnab . optimization of amount of koh ( catalyst ) was carried out by performing the reaction of pva and fnab by stirring at room temperature for one hour in presence of toluene and different amount of koh in aqueous solution 6 . 5 μl of 30 % koh was found as optimum for the reaction of pva and fnab ( 50 mg each ) in presence of 5 ml toluene . ( fig6 ). the applicants also optimized the time required for making photoreactive pva using koh at room temperature . the optimum time was 60 minutes . ( fig7 ), however , comparable results were obtained by microwave irradiation in 10 minutes . in photoactivation procedure pva was activated by exposing fnab coated pva to uv light . the photoactivated pva was then used to immobilize horse radish peroxidase at 37 ° c . for 1 hour . in the invented procedure , photoreactive pva was prepared by the reaction of pva and fnab by microwave irradiation ( or by thermal incubation at 37 ° c .). horse radish peroxidase immobilization on this photoreactive pva was then carried out by exposing them to uv light . results in table 2 showed that photoreactive polymer ( surface activated thermally ) gave better immobilization of biomolecule than thermoreactive ( surface activated by light ) surface . hence , photochemical immobilization procedure ( thermochemical activation and photochemical immobilization ) as described in this invented procedure is better option than thermochemical immobilization ( photochemical activation and thermochemical immobilization ) procedure . the applicants have optimized the concentration of linker ( fnab ) and time of incubation with polymer ( pva ) for the preparation of photoreactive polymer . in the second step , enzyme ( hrp ) was immobilized onto the polymer by its photoreactive group by light energy . thus , immobilization of enzyme onto the alkylamino silica gel was carried out by uv light in a uv stratalinker in different time . the optimum time for photoimmobilization of enzyme on photoreactive alkylamino silica gel was found as 10 minutes . however , increase in uv irradiation time beyond 10 minutes did not increase immobilization ( fig8 ). immobilization of hrp onto photoreactive lcaa - cpg was found optimum in 20 minutes ( fig9 ). however , photoreactive pva requires 60 minutes of uv irradiation for optimum immobilization of hrp . further increase in uv irradiation time decreases the absorbance , which may be due to the deactivation of some immobilized enzymes . ( fig1 ). enzyme concentration is also an important factor for immnobilization . 2 μg hrp ) 50 mg photoreactive pva was found optimum for the immobilization on pva and further increase did not appreciable increase its immobilization ( fig1 ). however , optimum amount of hrp was 4 μg / 50 mg support for immobilization on photoreactive lcaa - cpg ( fig1 ) and alkylamino silica gel ( fig1 ). total active enzyme immobilized on 20 mg lcaa - cpg and 20 mg alkylamino silica gel was found 160 u and 120 u respectively . thus , in the present invention , this method of preparation of photoreactive supports is simple , rapid and overcomes drawbacks of the reported methods . also , the immobilization of biomolecules onto the photoreactive surfaces are simple and fast . the advantages of the process of the invention are given in the table below : table ii present invention and its advantages s . no present invention advantage 1 the present invention provides a rapid , the invented method can activate the microwave - mediated method for amino - polymers in around 50 preparation of photoreactive polymers seconds and alcohol bearing polymers capable of forming a covalent bond with in around 10 minutes by microwave the biomolecules irrespective of the irradiation . hence , overcomes the functional group of the biomolecule . lengthy process of making photoreactive polymer . 2 the present invention also provides a in the present invention , the optimum rapid technique , which varies from 10 - 70 time for immobilization of minutes for immobilization of biomolecule is 10 - 70 minutes , which biomolecules on to the photoreactive is much less than the prior art ( 12 - 16 polymer . hours ) using the same photolinker . 2 ) the activated surfaces have a potential application in chromatographic separations , genomics and proteomics where immobilized molecules are required . 3 ) 1 - fluoro 2 - nitro - 4 - azido benzene used as a photolinker can be made easily . horse radish peroxidase , long chain alkyl amine - controlled pore glass ( normal diameter 500 a °, mesh size : 80 - 120 ), o - phenylenediamine were purchased from sigma , usa . silica gel lr ( 100 - 200 mesh ) was purchased from s . d fine chemicals . h 2 o 2 , methanol , di - methyformamide , toluene and 3 - aminopropyltriethoxysilane , sodium chloride , di - sodium hydrogen orthophosphate , sodium dihydrogen ortho phosphate , citric acid were of analytical grade purchaed from merck , india . fnab was prepared from 4 - fluoro - 3 - nitroaniline by diazotization reaction as reported earlier [ 15 ]. microwave mediated reactions were carried out in bpl sanyo microwave oven operating at a frequency of 2450 hz with a power output of 700 watts . photo - immobilization was carried out at a wavelength of 365 nm in an u . v stratalinker ; model 2400 , ( stratagene , usa ) fitted with five 15 - watt tubes . all the solutions were freshly prepared in triple - distilled water before use . phosphate buffered saline ( pbs ) was prepared by mixing 0 . 85 % nacl to 0 . 01 m phosphate buffer ( ph 7 . 2 ). wash buffer was prepared by adding ( 0 . 1 % tween 20 in pbs ). a freshly prepared substrate dye buffer contain 12 ml of citrate buffer ( 0 . 025 m citric acid and 0 . 5 m na 2 hpo 4 . 2h 2 o , ph 5 ), 5 μl of h 2 o 2 ( 30 % w / v ) and 4 mg of o - phenylenediamine . optimization of amount of 1 - fluoro 2 - nitro - 4 - azidobenzene for the preparation of photoreactive silica gel ( fig1 ) 5 g of dry ( moisture free ) silica gel mixed with 10 ml of 3 - aminopropyltriethoxysilane and 80 ml toluene was stirred at 28 ° c . for 3 hours . it was filtered and washed with methanol , water , methanol : water ( 1 : 1 ) and methanol respectively . the support was then dried and assayed for amino group by ninhydrin test . positive test for ninhydrin confirm the linking of amino group to silica gel . conical flask containing 50 mg alkylamino silica gel , 6 . 25 mg fnab and 10 ml dmf was exposed to microwaves for 60 sec . after which the support from the conical flask was washed by methanol , dried and kept in petridish . fnab concentration was optimizd by doing the same experiments with different amounts of fnab ( 12 . 5 , 25 , 50 , 75 and 100 mg respectively ). hrp ( 1 μg / 80 μl of pbs ) was then added to 50 mg of photoreactive silica gel and irradiated in an uv stratalinker at 365 nm for 20 minutes . the support was washed with washing buffer followed by the addition of 300 μl of substrate - dye buffer , the coloured solution was transferred to the respective polystyrene mictotiter wells and absorbance was recorded at 490 nm by an elisa reader . a control experiment was carried out with untreated alkylamino silica gel ( fnab : 0 mg ) in the similar way optimization of 1 - fluoro 2 - nitro 4 - azidobenzene concentration for the preparation of photoreactive long chain alkylamine controlled pore glass ( fig2 ) 50 mg lcaa - cpg , 6 . 25 mg fnab and 10 ml dmf were taken in a conical flask exposed to microwaves for 60 sec . fnab concentration was optimized by doing the same experiment with different amounts of fnab ( 12 . 5 , 25 and 50 mg respectively ). enzyme immobilization and its assay were carried out similarly as described in example 1 . a control experiment was carried out with untreated lcaa - cpg ( fnab : 0 mg ). optimization of 1 - fluoro 2 - nitro - 4 - azidobenzene concentration for preparation of photoreactive pva ( fig3 ) a conical flask containing 50 mg pva , 50 mg fnab , 6 . 5 μl of 30 % koh and 5 ml toluene were kept for stirring in dark at room temperature ( 28 ° c .) for 1 hour . three more conical flasks were taken and reaction mixtures were prepared similarly but with the fnab concentration of 12 . 5 , 25 and 75 mg respectively . after 1 hour , supports from conical flasks were washed by methanol , dried and kept in petridishes . hrp ( 16 μg / 80 μl of pbs ) was added to each photoreactive pva ( 50 mg ) and irradiated in uv stratalinker at 365 nm for 30 minutes . enzyme was assayed similarly as described in example 1 . a control experiment was carried out with untreated pva beads in the similar way . optimization of microwave exposure time for the activation of alkylamino silica gel ( fig4 ) four conical flasks , each containing 50 mg alkylamino silica gel , 50 mg fnab and 10 ml dmf were exposed to microwave for 30 , 50 , 60 and 70 seconds respectively . after the stipulated time , supports from each conical flask were washed separately by methanol , dried and kept on four petridishes . hrp was then immobilized similarly as described in example 1 . control experiment was carried out with untreated alkylamino silica gel beads in similar way , optimization of exposure time in microwave for the activation of aminopolystrene ( fig5 ) 20 mg aminopolystyrene , 20 mg fnab and 10 ml ethanol were taken in 50 ml conical flask and exposed to microwaves for 40 seconds . another two experiments were carried out in the similar manner but with the microwave exposure time of 50 and 60 seconds respectively . after the stipulated microwave exposure time the support from the conical flasks were washed by ethanol , dried and kept on petridishes . hrp ( 35 μg / 80 μl of pbs ) was added to each photoreactive aminopolystyrene ( 50 mg ), and irradiated in uv stratalinker at 365 nm for 20 minutes , enzyme was assayed similarly as described in example 1 . a control experiment was carried out with untreated aminopolystyrene in the similar manner . optimization of koh concentration for the thermoactivation of pva ( fig6 ) a conical flask containing 50 mg pva , 50 mg fnab , 1 . 62 μl of 30 % koh and 5 ml toluene were kept in dark for 1 hour with continuous stirring at room temperature . another four experiments were carried out similarly but with different quantity of 30 % koh solution ( 3 . 25 , 6 . 5 , 13 and 20 μl respectively ). after an hour the supports from the conical flasks were washed by methanol separately , dried and kept on petridishes . hrp ( 16 μg / 80 μl fit of pbs ) was added to each photoreactive pva ( 50 mg ) and irradiated in uv stratalinker at 365 nm for 30 minutes . the supports were washed with washing buffer followed by the addition of 300 μl of substrate - dye buffer . the coloured solution was transferred to the respective polystyrene mictotiter wells and absorbance was recorded at 490 nm by an elisa reader . a control experiment was carried out with untreated pva beads in the similar way . optimization of time for thermal activation of pva ( fig7 ) four conical flasks , each containing 50 mg pva , 50 mg fnab , 6 . 5 μl of 30 % koh and 5 ml toluene were kept in dark and stirred at room temperature for 5 , 10 , 60 , 180 and 300 minutes respectively . hrp was immobilized on the photoreactive pva and assayed as described in example 6 . control experiment was carried out with untreated pva beads similarly . optimization of uv irradiation time for immobilization of enzyme onto the microwave activated photoreactive alkylamino silica gel ( fig8 ) 300 mg alkylamino silica gel was activated using 300 mg fnab in 10 ml dmf by 70 seconds microwave irradiation . six petri plates , each containing 50 mg photoreactive alkylamino silica gel and hrp ( 1 μg / 80 μl of pbs ) were irradiated in uv stratalinker at 365 nm for 2 , 5 , 10 , 20 , 40 , 60 and 80 minutes respectively , the supports were washed with washing buffer followed by te addition of 300 μl of substrate - dye buffer . the coloured solution was transferred to the respective polystyrene microtiter wells and absorbance was recorded at 490 nm by an elisa reader , a control experiment was carried out with untreated alkylamino silica gel in the similar way . optimization of time of irradiation for the immobilization of enzyme onto the microwave activated photoreactive lcaa - cpg ( fig9 ) 300 mg lcaa - cpg was activated using 300 mg fnab in 10 ml dmf by 70 seconds microwave irradiation . six petri plates , each containing 50 mg photoreactive lcaa - cpg and hrp ( 1 μg / 80 μl of pbs ) were irradiated in uv stratalinker at 365 nm for 2 , 10 , 20 , 40 and 60 minutes respectively . immobilized enzyme was assayed as described in example 8 . a control experiment was carried out with untreated lcaa - cpg in the similar way . optimizg time of uv irradiation for enzyme immobilization onto the microwave activated photoreactive pva ( fig1 ) 300 mg pva was activated using 300 mg fnab dissolved in 10 ml dmf by 10 minutes microwave irradiation . six petri plates , each containing 50 mg photoreactive pva mixed with hrp ( 16 μg / 80 μl of pbs ) was irradiated in uv stratalinker at 365 nm for 5 , 20 , 30 , 60 , 120 and 180 minutes respectively . the supports were washed with washing buffer followed by the addition of 300 μl of substrate - dye buffer . the coloured solution was transferred to the respective polystyrene microtiter wells and absorbance was recorded at 490 nm by an elisa reader . control experiment was carried out with untreated pva similarly . optimization of enzyme concentration for its immobilization on photoreactive pva ( fig1 ) the stock solution was prepared by taking 1 mg hrp in 100 ml pbs . seven petri plates , each containing 50 mg photoreactive pva and varied hrp concentration ie 2 . 5 , 5 , 10 , 20 , 40 , 80 and 160 μl corresponding to 0 . 25 , 0 . 5 , 1 . 0 , 2 . 0 , 4 . 0 , 8 . 0 and 16 . 0 μg of hrp respectively were irradiated in uv stratalinker at 365 nm for 30 minutes . the support were washed with washing buffer followed by the addition of 300 μl of substrate - dye buffer . enzyme was assayed similarly as described in example 1 . a control experiment was carried out with untreated pva similarly . optimization of enzyme concentration for immobilization onto the microwave activated lcaa - cpg ( fig1 ) five petri plates each containing 50 mg photoreactive lcaa - cpg and 10 , 25 , 50 , 100 or 200 μl hrp solution corresponding to 0 . 2 , 0 . 5 , 1 . 0 , 2 . 0 or 4 . 0 μg respectively of hrp were irradiated in uv stratatinker at 365 nm for 20 minutes . enzyme immobilized on photoreactive lcaa - cpg was then assayed as described in example 8 . a control experiment was carried out with untreated lcaa - cpg . optimization of enzyme concentration for immobilization onto the microwave activated photoreactive alkylamino silica gel ( fig1 ) five petri plates each containing 50 mg microwave activated photoreactive alkylamino silica gel and 0 . 5 , 1 . 0 , 2 . 0 , 4 . 0 or 6 . 0 μg of hrp were irradiated in an uv stratalinker at 365 nm for 20 minutes . immobilized enzyme was assayed as described in example 8 . a control experiment was carried out with untreated alkylamino silica gel in the similar way . optimization of microwave exposure time for attachment of photolinker to lcaa - cpg ( table 1 ) four conical flasks , each containing 50 mg lcaa - cpg , 50 mg fnab and 10 ml dmf were exposed to microwave for 30 , 50 , 60 and 70 seconds respectively . after the stipulated time , the support from each conical flask were washed separately by methanol dried and kept on four petridishes . hrp was then immobilized similarly as described in example 1 . control experinment was carried out with untreated lcaa - cpg beads similarly . optimization of activation time for lcaa - cpg at 37 ° c . ( table 1 ) four conical flasks , each containing 50 mg lcaa - cpg , 50 mg fnab and 10 ml dmf were stirred for 5 , 10 , 15 and 20 hours respectively at 37 ° c . after the stipulated time , the support from each conical flask were washed separately by methanol , dried and kept on a petridish . hrp was then immobilized and assayed similarly as described in example 1 . a control experiment was carred out with untreated lcaa - cpg beads in the similar way . photochemical activation of pva — 250 mg pva and 250 mg fnab in 2500 μl methanol were mixed in a petriplate . after evaporating methanol , reaction mixture was irradiated for 60 minutes in uv stratalinker at 365 nm . after which support was washed with methanol and dried . four petri plates , each containing 50 mg thermoreactive pva were mixed with 2 , 4 , 8 and 16 μg hrp respectively and incubated at 37 ° c . for 60 minutes . enzyme was assayed as in example 1 . a control experiment was carried out with untreated pva in the similar way . thermochemical activation of pva — thermal activation of pva and hrp immobilization onto this photoreactive pva were carried out by using same amount of pva , fnab and hrp as above and according to procedure as described in example 3 . determination of activity of immobilized hrp onto the photoreactive lcaa - cpg and alkylamino silica gel hrp was immobilized onto the photoreactive lcaa - cpg by irradiating reaction mixture ( 1 μg hrp / 50 μl pbs and 50 mg photoreactive lcaa - cpg ) in uv stratalinker for 20 minutes . activity of immobilized hrp was determined by measuring the rate of color development at 490 nm , using dianisidine as hydrogen donor and hydrogen peroxide as substrate . standard curve was drawn using different amount of hrp ( 0 . 5 , 1 , 2 , 4 , 8 , 16 , 32 and 64 ng ). the absorbance was recorded at 1 - minute intervals for 10 minutes and rate of change of absorbance per minute was determined . activity of immobilized hrp onto the photoreactive alkylamino silica gel was determined as above . pva ( 50 mg ) was mixed with fnab ( 50 mg ), toluene ( 5 ml ) and 6 . 5 μl of 30 % koh and kept for stirring at room temperature for 1 hour . similar reaction mixture was exposed to microwave radiation for 10 minutes . after the reaction the support was washed with methanol and dried . 50 mg photoreactive alkylamino silica gel prepared at room temperature was mixed with 1 μl of hrp in a petriplate and irradiated in uv stratalinker at 365 nm for 30 minutes . similar experiment was conducted by the photoreactive pva prepared by microwaves . immobilized hrp assayed as described above . a control experiment was carried out with untreated pva in the similar way . photoreactive lcaa - cpg was prepared in different microwave exposure time ( 30 , 50 , 60 and 70 seconds ) and different incubation time ( 5 , 10 , 15 and 20 hours ) at 37 ° c . photoreactive surface was checked by photoirradiating 1 μg of hrp with 50 mg support and assaying the immobilized enzyme colorimetrically . table 2 . comparison of the efficacy of immobilization onto thermochemically and photochemically activated pva ( example 16 ) photoreactive and thermoreactive pvas were prepared by thermochemical and photochemical reactions respectively . their efficacy of immobilization was compared by immobilizng different amounts of hrp ( 2 , 4 , 8 and 16 μg ) onto them and subsequently assaying the immobilized enzyme . 1 . a simple , rapid and efficient method is invented for the preparation of a photoreactive polymer and rapid immobilization of biomolecule by uv light on to this surface . 2 . any polymer having reactive nucleophilic group can be activated by the invented method . 3 . activation of polymer can be carried out in 50 seconds for amino bearing polymers and in 10 minutes for hydroxyl group alcohol bearing polymers by the invented method . 4 . in the invented method , activation is carried out by microwave radiation . 5 . the invented photoreactive surface can potentially be used for immobilization of biomolecules used in diagnostics , affinity chromatography , proteomics , gemomics , drug screening and related fields . 1 . krysteva , al ., “ covalent binding of enzymes to synthetic membranes containing acrylamide units , using formaldehyde ”, biotechnol . appl . biochem . 13 , 106 - 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