Patent Application: US-47929304-A

Abstract:
novel photoresist materials , which can be photolithographically processed in biocompatible conditions are presented in this invention . suitable lithographic scheme for the use of these and analogous resists for biomolecule layer patterning on solid substrates are also described . the processes described enable micropatterning of more than two different proteins on solid substrates without denaturation of the proteins . the preferred resist materials are based on acrylate copolymers that contain at least one acid cleavable ester group and at least one hydrophilic group such as an alcoholic or a carboxylic group .

Description:
in the first photolithographic scheme ( fig2 ) two proteins can be patterned on silicon surface . initially the silicon wafer surface is treated with 3 - aminopropyl - triethoxy - silane ( aptes ) in order to become hydrophobic for the subsequent physisorption of proteins . then , the photoresist solution is used for the creation of the photoresist film onto the treated silicon surface . the photoresist solution constists of two compounds in an appropriate solvent : one component is the copolymer invented and the other is a triphenyl or triaryl sulfonium salt acted as photosensitiser . this solution is casted onto the treated silicon wafer surface and then the wafer is spinned in order to form a homogenic thickness film . thermal treatment is followed for the vaporization of the solvent remained in the polymer matrix and the rearrangement of the polymer chains . although thermal treatment is not appropriate , usually it takes place for the formation of a high quality polymer film . in the second step defined areas of the polymer film are exposed to radiation via a two - dimension photolithographic mask : a mask with a transparent pattern on its surface , which is transferred to the resist film . this mask must be kept in contact to the film ( contact printing ) to prevent phenomena of light diffraction , which would generate unwanted exposed areas . also the radiation wavelength is selected in order to control effectively the photo - induced reactions ( the radiation wavelength must be in the photosensitiser absorption region ). thus , radiation band filters or cut - off filters are placed over the mask for the selection of deep uv or near uv radiation region , respectively . for the chemically amplified resist as the current one , a thermal treatment step usually takes place after the exposure . but this photoresist , due to the ( meth ) acrylate copolymer invented , does not need to be postexposure treated and this is one of the main differences with our previously introduced biocompatible photoresist ( 31 , 32 ). the third step is the removal of the previously exposed resist areas using a very dilute aqueous base , which can be tolerated by the proteins . more particularly we had reported formerly ( 31 , 32 ) that the aqueous base solutions of 0 . 27 n tetramethyl ammonium hydroxide ( tmah concentration ( standard aqueous base developers used in semiconductor technology ) denature almost completely the proteins , while the 100 times diluted of this aqueous base solution ( 2 . 7 × 10 3 n tmah concentration ) generates less than 10 % decline of their immunoreactivity ; this was considered by us as a limit of protein denaturation and so this base concentration was the maximum value used . actually the exposed areas of the current ( meth ) acrylate based photoresist can be dissolved in even more diluted aqueous base in comparison with our previous biocompatible photoresist ( 31 , 32 ). in the fourth step the patterned resist film is covered with the active protein solution for the adsorption of this protein on the substrate . actually the protein is physisorbed not only on the substrate areas , where the photoresist areas were previously exposed and removed , but also onto the remained unexposed film regions . the last one must not be considered as an inefficiency of the process , because the unexposed resist areas along with the protein monolayer adsorbed on them will be exposed and removed in the following steps . in this process the proteins are adsorbed on the hydrophobic substrate , but it would be the same if the proteins were covalently bound on the surface without any changes in the lithographic process . in addition it would cause no changes to the process sequence and conditions if other biomolecules were used instead of proteins , such as enzymes , nucleic acid chain parts , etc . the fifth step is consisted in the “ flood ” exposure of both the remained photoresist film and substrate areas where the proteins have been adsorbed . the “ flood ” exposure is carried out without using a mask , because the whole film must be exposed , and not specific regions . the radiation wavelength is the same with that in the previous exposure and is selected again using band filters or cut - off filters . it is necessary for this step that the radiation used must not affect the adsorbed proteins , because in the “ flood ” exposure they are irradiated together with the remained photoresist . thus , radiation in near uv or visible is preferred with nearly no limitation in exposure time , while exposure duration in deep uv region must be kept relatively small . in addition the duration of the second exposure is usually increased in comparison with the first one , because a small part of the photoresist surface is deactivated in a way by the preceded covering of the film with the protein solution ; a surface dissolution of the film &# 39 ; s photosensitiser is a possible explanation to this phenomenon . as with the second step no thermal treatment is needed after the photoresist exposure , again because of the incomparable dissolutive convenience of the exposed areas of our photoresist . in the sixth step the dissolution of the previously exposed resist areas is taken place . the tmah concentration of the aqueous base solution that is used in this step may be slightly increased in comparison with the first dissolution made ( third step ), but it remains sufficiently below the base concentration level that is not tolerated by the proteins . at the end of this step a pattern of protein molecules is created on the substrate regions where the initially exposed resist areas ( in the second step ) were located . in the seventh step the surface is covered by an inert protein solution . therefore the inert protein molecules are adsorbed physically onto all free surface binding sites : not only on the binding sites of the substrate regions uncovered with lithography in steps ( 5 ) and ( 6 ), but also on the free binding sites that were not covered by the active protein molecules . the reason is to prevent non specific binding of the analyte : the analyte ( antibody ) must be bound only onto the active protein ( antigen ) patterned and not adsorbed on the surface , in order to confer the concentration of the analyte ( use of the device in immunoassay format ). at the end of this step and of the whole process two proteins are patterned on the aptes - treated silicon surface : an active protein that can be recognized by its antibody and an inert protein that does not interact with this antibody . this pattern can be visualized by the covering of the surface with the appropriate solution containing the antibody labeled with a fluorescence substance and consequently identification of the immunocomplex in an epifluorescence microscope ( example 1 ). the process that allows the patterning of three proteins is the same with that of patterning two proteins in relation to the general idea : the biocompatible photolithography of the ( meth ) acrylate based photoresist . the processing conditions of each step may have been altered and even the sequence of the steps , but this does not reduce the biocompatibility and the effectiveness of the method . about the differentiation of the process steps sequence , it concerns only the “ blocking ” steps that is the steps whose purpose is to introduce molecules of an inert protein in order to occupy any uncovered substrate sites . this step is taken place once at the end of the process , when two proteins have to be patterned . now for the process of patterning three proteins the “ blocking ” step is taken place twice , not necessarily in the same conditions : once immediately after the deposition of the first active protein and then again at the end of the process . similarly if four proteins have to be patterned the “ blocking ” step must take place three times : firstly immediately after the deposition of the first active protein , secondly after the deposition of the second active protein and then again at the end of the process . about the change of the process conditions , if the number of proteins that have to be patterned is increased this alteration is necessary for the effective removal of the resist film . the adsorption of the proteins onto the substrate is achieved , when the patterned photoresist is immersed in slightly base solutions of proteins for a considerable time . although these solutions are more diluted basic solutions than those used after every film exposure , they affect the resist film significantly , because they cover it for a long time ( may be more than an hour is necessary ). this causes deactivation of the polymer film surface layer and subsequent decrease in photoresist sensitivity , because of possible dissolution of the photoacid generator there , as it was previously mentioned . also the unwanted slightly exposed photoresist areas are more deactivated by the protein adsorption . that is why the limitation of the light diffraction and consequently the exposure of the unwanted regions is an essential matter to the current photolithographic approach . the radiation absorption by the protein monolayer physisorbed on the resist film must not contribute significantly to the polymer film surface deactivation , because the thickness of the protein monolayer is extremely small ( in the order of 10 a ) and consequently its absorption is very small in comparison to the resist film . to address the problem of surface deactivation of the polymer film that is its sensitivity decline , less mild - though biocompatible - process conditions must be used for the removal of the film . thus higher exposure duration is needed and / or slightly more concentrated base solutions for the development of the film ; usually it is preferred the first one and only if it is inevitable the second one . consequently for the three - protein patterning process the first exposure time might be smaller than the second one and the second one smaller than the third one . similarly the first aqueous developer solution might have smaller base ( tmah ) concentration than the second one , the second one smaller than the third one and the third one less to the one tolerated by the proteins ( only 10 % of immunoreactivity reduction is accepted ; see above ). the three - protein pattern on the aptes - treated silicon substrate can be visualized by two alternative ways : a ) introduction of an appropriate solution containing both antibodies of the two active proteins at the end of the photolithographic process or b ) introduction of the two antibodies separately : at first deposition of the first active protein - antibody after the corresponding “ blocking ” step ( step 5 ) and then deposition of the second active protein - antibody after the second “ blocking ” step ( step 11 ). in both ways the antibodies are labeled with a different fluorescent substance so that each immunocomplex formed can be identified in the epifluorescence microscope . usually it is preferred the first way of the above in order to avoid the effect of the antibody solution on polymer film , but this depends on the antibodies too , e . g . if they can bind their corresponding antigens in the same ph solution , etc ( example 2 ). when the biocompatible photolithographic process is applied for the patterning of e . g . two proteins onto the plastic capillary internal surface , extra limitations have been arisen , except the biocompatible one . the solvent of the copolymer - based photoresist must not influence the plastic material of the capillary tube , which is usually polystyrene , poly ( methyl pentene ), poly ( methyl methacrylate ), etc . also the capillary walls act as cut - off filters and allow passing only radiation in near uv or visible region ; consequently the photoresist film should be sensitive to this radiation region . the last requirement posed by the cylindrical geometry of the substrate forces us to use the appropriate photosensitisers , a problem that is not faced to the silicon substrate where deep uv exposure could be used , but in low doses in order not to denature the patterned proteins . moreover practical problems concerning the application itself of the photolithographic process are efficiently overcome . thus the photoresist solution is introduced in the capillary using a syringe , it is left in the horizontal position for 1 - 2 min and then it is extracted by turning the tube in the perpendicular position . the photoresist film formed can be thermally treated by introduction of the capillary into aluminum plate holes of similar size with the capillary . after that , specific areas of the capillary external surface are exposed and the other areas are covered with a non - transparent tape ; the use of radiation filters is not necessary , since the capillary walls act as cut - off filters . also for the homogeneous exposure of the photoresist film the capillary is turned by 60 ° around its axis at the end of each irradiation and consequently the tube is turned five times in order to achieve irradiation of the whole film . then the dissolution of the irradiated resist areas is taken place by introducing the dilute aqueous base solution with a syringe repeatedly ( at least twice from both capillary ends in order to ensure that fresh developer solution is continuously added ). in a following step the protein solution is added , incubated for the necessary period , and then rinsed . afterwards , the whole resist film is irradiated with the preceding homogeneous way ( turning the capillary by 60 ° after each exposure ). then the dilute aqueous base is introduced into the capillary by a syringe ( see above ). finally , the inert protein solution is introduced into the tube and incubated for a certain period of time . for the visualization of the two protein locations onto the capillary inner surface the specific antibody labeled with a fluorescent substance is introduced into the capillary and subsequent fluorescence scanning of the tube is taken place by an optical set - up constructed in our laboratory . thus the locations containing the immunocomplex formed are indicated by different signals ( example 3 ). the current photoresist is based on the copolymer : 2 - hydroxy - ethyl - methacrylate , isobornyl - methacrylate , t - butyl - methacrylate and acrylic acid . the chemical behavior of the resist is the same with the previously introduced photoresist based on the homopolymer t - butyl acrylate . the reaction mechanism is the chemical amplification mechanism , in which the acid generated by the photosensitizer during the exposure acts as a catalyst for the subsequent dissociation of the ester groups of the copolymer components in a way that for every ester pendant group that is broken with the aid of acid a new acid is generated . from the dissociation of the ester group methacrylic acid is generated , which is soluble by diluted aqueous base solutions . the extension of the ester groups dissociation defines the following dissolution of the exposed photoresist areas . the difference between this photoresist and our previous one ( 31 , 32 ) is that now the dissolution of the exposed photoresist areas can be achieved without thermal treatment of the polymer film . the photosensitizers used are mainly two : a ) triphenyl sulfonium hexafluoroantinonate for the irradiation at deep uv ( 254 nm ) or b ) a 50 % w / w solution in propylene carbonate of a 1 : 1 mixture of two triarylsulfonium hexafluoroantimonate salts ( one having diphenylthioether as an aryl substituent and the other is a thiodimer of triphenyl sulfonium hexafluoroantimonate salt ), for exposure at near uv ( λ & gt ; 300 nm ). the whole photolithographic strategy invented ( both the copolymer - based photoresist and the biocompatible photolithographic “ lift - off ” technique ) is a unique method for patterning biomolecules on solid surface . it is a general methodology independent of the biomolecules that have to be patterned : proteins , enzymes or oligonucleotides can be similarly patterned . furthermore it is independent of the substrate material and geometry : it can be applied on silicon or polymeric substrate , in planar or cylindrical surface . moreover , whatever way the biomolecules are bound onto the substrate — adsorbed or covalently bound — it is equally functional . no expensive or complicated instruments are needed ; just the photoresist and the usual irradiation sources used in deep uv or near uv exposure . and finally it can pattern in micro scale easily more than one biomolecules on the same substrate . in relation to our former invented photolithographic strategy ( 31 ) the main differences are summarized to the following : a ) the photoresist is based now on the copolymer synthesized by us consisted of 2 - hydroxyethyl - methacrylate , isobornyl - methacrylate , t - butyl - methacrylate and acrylic acid in a 30 / 40 / 20 / 10 weight ratio , while the previous one was based on the homopolymer t - butyl acrylate , b ) the photolithographic process fulfills better the biocompatibility requirements since using the copolymer synthesized lower exposure times are necessary , no thermal treatment is needed after the exposure and even more diluted aqueous bases can be used for the removal of the exposed photoresist areas . to show the effectiveness of this methodology some characteristic examples are presented . in examples 1 and 2 microstructures of two and three different proteins photolithographically patterned on aptes - treated silicon substrate are shown , correspondingly . in example 3 two “ bands ” of proteins photolithographically defined on the capillary inner surface are presented . initially the silicon wafer surface is treated with 3 - aminopropyl - triethoxysilane ( aptes ) solution in order to make it hydrophobic ( amino groups are formed ) and susceptible to physical binding by the proteins . thus a clean silicon wafer is immersed in a “ pyranha ” solution , for 1 hr , in ambient temperature ; this solution is a 1 : 1 mixture of 31 % v / v h 2 o 2 aqueous solution and 97 % v / v h 2 so 4 aqueous solution . then the wafer is washed very well with deionized water and it is immersed in a bath with continuously refreshing deionized water ( until its special resistance take the value of 12 mω ). the water is removed from the wafer surface under a nitrogen stream . afterwards , it is immersed in 2 % v / v aptes aqueous solution for 20 min and is quickly immersed and taken out from a bath with fresh deionized water , and dried under a nitrogen stream ; the reason that the wafer surface is immersed now in deionized water is to rinse it from the aminosilane salts that are not bound to the surface . subsequently the wafer is thermally treated at 120 ° c . for 20 min . finally it is immersed in deionized water in ultrasonic bath for 5 min , dried under a nitrogen stream and thermally treated at 95 ° c . for 5 min ( in order to remove completely the water from the surface ). the photoresist solution is prepared as following : a 10 % w / w solution in ethyl lactate , of our synthesized copolymer consisted of 2 - hydroxyethyl - methacrylate , isobornyl - methacrylate , t - butyl - methacrylate and acrylic acid in a 30 / 40 / 20 / 10 weight ratio , is formulated . the solution is stirred at least for 1 hr with parallel mild thermal treatment ( 50 ° c .). then triphenylsulfonium hexafluoroantimonate salt , provided by general electric , is added as photosensitizer , so that the final concentration of the salt to be 10 % w / w in solids . the final solution is stirred for ½ hr and after that filtered ( with filters of 0 . 2 μm pore size ). to pattern two proteins — an active and an inert protein — onto the treated silicon surface the first photolithographic process is followed ( fig2 ). thus the above photoresist solution is cast on the center of the treated silicon wafer covering about the ¾ of its surface . the lithographic resist is coated on the whole silicon surface by spinning the wafer at 3000 rpm for 30 sec . the coated wafer is baked in an oven at 70 ° c . for 5 min . selected areas from the resist film are exposed with an oriel hg — xe 500 w ( operated at 450 w ) radiation source . the selection of the resist areas that are going to be patterned is done with a quartz mask , which is placed over the polymer film and in contact with it ( through vacuum ) for preventing light diffraction . the selection of the wavelength radiation 254 nm ( deep uv ) is made with a broadband filter ( 50 nm bandwidth at half maximum ), which is placed over the mask . the exposure dose is 36 mj / cm 2 . subsequently the exposed areas are dissolved by immersion of the photoresist film in a 1 . 35 × 10 − 3 n aqueous solution of tetramethyl ammonium hydroxide ( tmah for 2 min , washed with deionized water and dried under a nitrogen stream . then the wafer surface is covered with a 20 mg / l rabbit igg solution in 0 . 04 m phosphate buffer , ph 6 . 5 , for 30 min . after that the surface is washed with deionized water and dried under a nitrogen stream . subsequently the whole surface is exposed (“ flood ” exposure ) using the same exposure tool and filter ( 254 nm broadband filter ); the exposure dose is now 109 mj / cm 2 . the remained film areas are dissolved by immersion of the photoresist in a 2 . 7 × 10 − 3 n tmah aqueous solution for 2 min , washed with deionized water and dried under a nitrogen stream . finally the whole surface is covered with a 10 g / l bovine serum albumin solution in 0 . 1 m nahco 3 buffer , ph 8 . 5 , for 1 hr and dried with nitrogen stream . at the end of the process two proteins are patterned onto the aptes - treated silicon surface : rabbit igg ( active protein ) and bovine serum albumin ( inert protein ). to visualize the created protein pattern the substrate is immersed in a 20 mg / l goat anti - rabbit igg - fluorescein isothiocyanate conjugate solution in 0 . 15 m tris - hcl buffer , ph 8 . 25 , containing 1 g / l bovine serum albumin , 0 . 5 g / l bovine igg , 1 m kcl and 0 . 2 g / l ethylmercury - thiosalicylic acid sodium salt , and incubated for 3 days , at 4 ° c . then the substrate is taken out , washed with a 0 . 01 m tris - hcl buffer , ph 8 . 25 , containing 0 . 05 % ( v / v ) tween 20 , and observed in epifluorescence microscope . microstructures of 3 . 75 μm lines / spaces of two proteins : rabbit - igg ( green lines ) and bovine serum albumin ( black lines ) are obtained ( fig5 ). in the beginning the silicon wafer surface is treated with 3 - aminopropyl - triethoxysilane ( aptes ) as it is described in example 1 . after that the photoresist solution is prepared . a 10 % w / w solution in ethyl lactate , of our synthesized copolymer consisted of 2 - hydroxyethyl - methacrylate , isobornyl - methacrylate , t - butyl - methacrylate and acrylic acid in a 30 / 40 / 20 / 10 weight ratio , is formulated . the solution is stirred at least for 1 hr with parallel mild thermal treatment (− 50 ° c .). then a 50 % w / w solution in propylene carbonate of a 1 : 1 mixture of two triarylsulfonium hexafluoroantimonate salts ( one having diphenylthioether as an aryl substituent and the other is a thiodimer of triphenyl sulfonium hexafluoroantimonate salt ), provided by union carbide with the name uvi 6974 , is added to the copolymer solution , so that the final concentration of the two salts totally to be 30 % w / w in solids . the ultimate solution is stirred for 30 min and then filtered ( with filters of 0 . 2 μm pore size ). the second photolithographic process ( fig3 ) is followed , because three different proteins ( two active and one inert ) are going to be patterned . thus the photoresist film is coated on the treated silicon wafer by spinning it at 3000 rpm for 30 sec . the film is baked in an oven at 95 ° c . for 5 min . the film areas that have to be exposed are selected by placing a quartz mask over the film and in contact ( through vacuum ) with it . also the required radiation region for λ & gt ; 300 nm ( near uv ) is done by using a pyrex cut - off filter over the mask . the exposure tool used is a karl suss aligner and the irradiation time is 5 min . then the exposed areas are developed by immersion of the wafer in a 1 . 35 × 10 − 3 n aqueous solution of tetramethyl ammonium hydroxide ( tmah ) for 2 min , washing with deionized water and drying under a nitrogen stream . afterwards , the surface is covered with a 50 mg / l mouse igg solution in 0 . 04 m phosphate buffer , ph 6 . 5 , for 30 min ; it is washed with deionized water and dried under a nitrogen stream . then it is covered with a 10 g / l bovine serum albumin solution in 0 . 04 m phosphate buffer , ph 6 . 5 , for 55 min , washed with deionized water and dried under a nitrogen stream . areas of the patterned film are selected for exposure by covering some of the unexposed areas and leaving others to irradiation , but always using the pyrex cut - off filter . the exposure lasts again 5 min . the exposed areas are removed by immersion of the wafer in a 2 . 7 × 10 − 3 n aqueous tmah solution for 1 min ; the substrate is washed with deionized water and dried under a nitrogen stream . after that the surface is covered with a 20 mg / l biotinylated - bovine serum albumin solution in 0 . 04 m phosphate buffer , ph 6 . 5 , for 45 min ; it is washed with deionized water and dried under a nitrogen stream . accordingly the whole surface is exposed (“ flood ” exposure ) for 10 min with the pyrex filter . it is immersed in a 2 . 7 × 10 13 n aqueous tmah solution for 5 min , rinsed with deionized water and dried under a nitrogen stream . thus , all the remained photoresist areas are dissolved . finally the silicon surface is covered with a 10 g / l bovine serum albumin solution in 0 . 1 m nahco 3 buffer , ph 8 . 5 , for 2 hr , washed with deionized water and dried with nitrogen stream . at the end of the photolithographic process three proteins are patterned on the aptes - treated silicon surface : mouse igg ( first active protein ), biotinylated - bovine serum albumin ( second active protein ) and bovine serum albumin ( inert protein ). the protein pattern is visualized by incubation of the wafer in a solution of 20 mg / l goat anti - mouse igg - fluorescein isothiocyanate conjugate and 5 mg / l streptavidin - r - phycoerytirin conjugate in 0 . 15 m tris - hcl buffer , ph 8 . 25 , containing 5 g / l bovine serum albumin , 0 . 5 g / l bovine igg , for 2 days , at 4 ° c . after that the substrate is taken out , washed with a 0 . 01 m tris - hcl buffer , ph 8 . 25 , containing 0 . 05 % ( v / v ) tween 20 and observed using epifluorescence microscope . microstructures of 22 . 5 μm lines / spaces of three different proteins : mouse - igg ( green lines ), biotinylated bovine serum albumin ( red lines ) and bovine serum albumin ( black lines ) are obtained ( fig6 ). for the definition of protein “ bands ” onto the internal surface of a plastic capillary tube the third biocompatible photolithographic process ( fig4 ) is used . the characteristics of the capillary , which is used in immunoanalysis are the following : 6 cm length , 1 mm internal diameter and 1 mm walls thickness ; the material is poly ( methyl pentene ). the photoresist solution used is the same with the example 2 : a 10 % ( w / w ) solution of our synthesized copolymer in ethyl lactate with 30 % ( w / w in solids ) of the 1 : 1 mixture of the two triarylsulfonium hexafluoantimonate salts named uvi 6974 . thus , the photoresist solution is introduced by a syringe into the capillary , is left horizontally for 2 min and then is extracted by turning the capillary perpendicularly . the photoresist film formed has a medium thickness of ˜ 3 μm ; a gentle nitrogen stream is used to remove the solution that blocks both edges of the capillary , while its external surface is cleaned with 2 - propanol . then a karl suss aligner exposure tool is used to expose two locations of the external capillary surface . for the selection of the exposed regions , two “ rings ” of 0 . 5 cm length each one ( 1 cm distant each other and at 2 cm distance of each one from the capillary edge ) are not covered and the other regions of the external capillary surface are covered with a non - transparent tape . thus , the capillary is exposed for 1 min , then turned 60 ° around its axis , exposed again for 1 min , turned 60 °, and so on until the whole film is irradiated ( the capillary is turned 6 times by 60 ° each time and exposed 1 min for each time , too ). afterwards , the opaque tape is removed and the tube is baked at 60 ° c . for 5 min into aluminum plate holes of similar size with the capillary . then a 2 . 7 × 10 − 3 n aqueous solution of tetramethyl ammonium hydroxide ( tmah ) is introduced in a continuous way into the capillary : 4 times by 2 ml solution each time , twice from each edge and with 5 min total time of the development . the washing of the capillary with deionized water is followed with the same continuous way ( 4 × 2 ml for 5 min ) and the tube is dried under a nitrogen stream . then , a 20 mg / l rabbit igg solution in 0 . 04 m phosphate buffer , ph 6 . 5 , is introduced into the capillary and incubated for 30 min . the capillary is washed with deionized water as described above ( 4 × 2 ml for 5 min ) and dried with nitrogen stream . subsequently the whole capillary is exposed with the previous manner ( 6 × 1 min exposure dose with 60 ° turn of the capillary each time ). after that it is baked at 60 ° c . for 5 min in the aluminum plate with the holes . next a 2 . 7 × 10 − 3 n aqueous tmah solution is introduced into the capillary as described above ( 4 × 2 ml for 5 min ), it is washed with deionized water with the same manner and dried under a nitrogen stream . then a 10 g / l bovine serum albumin in 0 . 1 m nahco 3 buffer , ph 8 . 5 , is incubated in the tube for 1 hr ; subsequently the capillary is dried with nitrogen . at the end of the lithographic process two distinct “ zones ” of rabbit igg ( active protein ) are formed and the other areas of capillary inert surface are covered with bovine serum albumin ( inert protein ). for the visualization of the above “ bands ” the capillary is filled with a 5 mg / l goat anti - 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