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Uduce duto twa silika twateguwe nuburyo bwa sol-gel hamwe noguhindura kugirango tubone uduce duto twa macroporome.Ibice byavanywemo no guhinduranya byongeye guhinduranya urunigi rwimurwa (RAFT) polymerisation hamwe na N-phenylmaleimide-methylvinylisocyanate (PMI) na styrene kugirango utegure N-phenylmaleimide internalation ya polystire idahwitse. . , Isahani 000 / m². Ugereranije imikorere yo gutandukanya inkingi yateye imbere hamwe nubucuruzi bwa Ascentis Express RP-Amide, byagaragaye ko imikorere yo gutandukanya inkingi ya PMP yarutaga inkingi yubucuruzi muburyo bwo gutandukana no gukemura.
Mu myaka yashize, inganda zikomoka ku binyabuzima zahindutse isoko ry’isi yose ryiyongera ku buryo bugaragara ku mugabane w’isoko. Hamwe n’ubwiyongere bukabije bw’inganda zikomoka ku binyabuzima 1,2,3, isesengura rya peptide na poroteyine ryifuzwa cyane. Usibye peptide igamije, imyanda myinshi ikorwa mu gihe cyo guhuza peptide, bityo bikaba bisaba ko habaho isuku ya chromatografi kugira ngo haboneke peptide y’ubuziranenge bw’umubiri hamwe n’ingirangingo zikomeye ziterwa na poroteyine. icyitegererezo. Nubwo misa ya sprometrike ari igikoresho cyiza cyo gukurikiranya peptide na poroteyine, niba izo ngero zatewe muri sprometrike ya misa imwe, gutandukana ntibizaba byiza. Iki kibazo kirashobora kugabanywa no gushyira mu bikorwa itandukaniro rya chromatografiya (LC) mbere yo gusesengura kwa MS, bizagabanya umubare wa analyite winjira muri analytike mugihe cyo gutandukanya ibintu, hamwe no gusesengura ibintu, mugihe cyo gutandukanya, kugabanuka kwinshi, gutandukana. Amazi ya chromatografiya (LC) yateye imbere cyane mumyaka icumi ishize kandi yabaye tekinike izwi cyane mu isesengura rya proteomic 7,8,9,10.
Amazi ya chromatografiya yahinduwe (RP-LC) akoreshwa cyane mugusukura no gutandukanya imvange ya peptide ukoresheje silika yahinduwe na octadecyl (ODS) nkicyiciro gihagaze11,12,13.Nyamara, ibyiciro bya sitasiyo ya RP ntibitanga itandukaniro rishimishije rya peptide na proteyine bitewe na poroteyine zidahwitse hamwe na poroteyine zidahwitse hamwe na poroteyine zidahwitse hamwe na poroteyine zidahwitse hamwe na poroteyine zidahwitse hamwe na poroteyine zidasanzwe hamwe na peptide. hamwe no kugumana analyite16.Ivanga-moderi ya chromatografiya, itanga imikoranire ya multimodal, irashobora kuba inzira ya RP-LC yo gutandukanya peptide, proteyine, nizindi mvange zigoye. Hateguwe ibyiciro bitandukanye bivanze-byahagaritswe byateguwe, kandi inkingi zuzuyemo ibyo byiciro zakoreshejwe mugutandukanya peptide na proteine ​​RPLC / WALIQUE / birakwiriye gutandukana na peptide na proteyine bitewe nuko habaho amatsinda yombi ya polar na non-polar22,23,24,25,26,27,28 .Bisa nkibyo, polar ihuza intera ihagaze hamwe nitsinda rihuriweho hamwe ryerekana imbaraga nziza zo gutandukana hamwe no guhitamo bidasanzwe kuri polarite na polarite, kuko gutandukana biterwa nimikoranire hagati ya analyte nicyiciro gihagaze.Imikoranire myinshi 29, 30, 31, 32.Mu minsi ishize, Zhang n'abandi.30 yateguye icyiciro cya dodekile cyarangiye polyamine ihagaze kandi itandukanya neza hydrocarbone, antidepressants, flavonoide, nucleoside, estrogène, hamwe nizindi analyite nyinshi. Polar intercalator ifite amatsinda ya polar na non-polar, bityo irashobora gukoreshwa mugutandukanya peptide na proteyine zifite ubucuruzi bwa hydrophobi na hydrophilique. RP-Amide inkingi, ariko izi nkingi zikoreshwa mugusesengura amine 33 gusa.
Mu bushakashatsi burimo gukorwa, hateguwe icyiciro gihagaze (N-phenylmaleimide-yashizwemo polystirene) cyateguwe kandi gisuzumwa kugirango habeho gutandukanya peptide na trypsin igogora rya HSA.Icyiciro gihagaze cyateguwe hifashishijwe ingamba zikurikira. Ingano ya ore. Icya kabiri, ligand nshya, phenylmaleimide-methyl vinyl isocyanate, yashizwemo kandi ikoreshwa mu kuvoma uduce duto twa silika kugirango hategurwe inkingi ya sitasiyo ihagaze. Icyiciro cyahagaritswe cyapakiwe mu nkingi y’icyuma (100 × 1.8 mm id) ikoresheje uburyo bwo gupakira ibintu mu buryo bwuzuye. igizwe na peptide eshanu;. gukemurwa neza no gukora neza kumurongo wa PMP, wari mwiza cyane kuruta Ascentis Express RP-Amide.
PEG. eroxide (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, na Acetone Yaguzwe muri Sigma-Aldrich (St. Louis, MO, USA).
Uruvange rwa urea (8 g), polyethylene glycol (8 g), na 8 mL ya acide acetike ya 0.01 N yashizwemo iminota 10, hanyuma mL 24 ya TMOS yongerwaho muri yo mu gihe cy'ubukonje bukabije. kandi ubarwa kuri 550 ° C mumasaha 12. Hateguwe ibyiciro bitatu kandi birangwa no gusuzuma imyororokere yubunini buke, ubunini bwa pore nubuso bwubuso.
Mugihe cyo guhindura ibice bya silika hamwe na ligand phenylmaleimide-methylvinylisocyanate (PCMP) yabanje gukomatanya hakurikiraho polymerisiyumu ya radiyo hamwe na styrene, hateguwe uruganda rufite polar.Icyiciro gihagaze kumurongo hamwe na polystirene iminyururu.Imyiteguro yasobanuwe hepfo.
N. Amasaha 3.
Ibice bya silika byumye (2 g) byakwirakwijwe muri toluene yumye (100 mL), bikabyutsa kandi bigashyirwa muri flask ya 500 mL yo hepfo kuminota 10 ) yashongeshejwe muri toluene (200 ml) hanyuma 4-hydroxy-TEMPO (2 mL) yongerwamo imbere ya 100 µL ya dibutyltin dilaurate nka catalizator. Uruvange rwavanze kuri 50 ° C mu masaha 8, rwungururwa kandi rwumishwa kuri 50 ° C mu masaha 3.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), hamwe na silika ya TEMPO-PMCP yometse kuri silika (1.5 g) yakwirakwijwe muri toluene hanyuma isukurwa na azote. Polimerisation ya styrene yakorewe kuri 100 ° C mu masaha 12. Ibicuruzwa byavuyemo byogejwe na methanol kandi byumye kuri 60 ° C ijoro ryose.
Izi ngero zamanuwe kuri 393 K mu gihe cyisaha 1 kugirango haboneke umuvuduko usigaye uri munsi ya 10-3 Torr.Umubare wa N2 wagabanijwe kumuvuduko ugereranije wa P / P0 = 0.99 wakoreshejwe kugirango hamenyekane ingano ya pore yose. Morfologiya y’ibice bya silika yambaye ubusa kandi ihujwe na ligandike ya elegitoronike ya elegitoronike yakozwe na silike ya elegitoronike ya silike (Tokyo, Yapani). karubone uduce duto twa silika duhujwe na ligand (5 mg buri umwe) twatatanye muri mL 5 ya isopropanol, ikorwa iminota 10, ikazunguruka iminota 5, igashyirwa ku ntebe ya optique ya Mastersizer. Isesengura rya termogravimetric ryakozwe ku gipimo cya 5 ° C ku munota hejuru y’ubushyuhe buri hagati ya 30 na 800 ° C.
Ikirahuri cyometseho ibyuma bidafite ibyuma bigufi-bore inkingi zingana (100 × 1.8 mm id) zapakishijwe hakoreshejwe uburyo bwo gupakira ibintu, hakoreshejwe uburyo bumwe bwakoreshejwe muri Ref.31.Inkingi idafite ibyuma (umurongo wikirahure, 100 × 1.8 mm id) hamwe nisoko isohokamo ifiriti ya 1 µm yahujwe nudupaki twa shitingi (Alltech Deerfield, IL, USA) .Gutegura icyuma gihagaze uhagarika mg 150 yicyiciro gihagaze muri 1,2 mL ya methanol hanyuma ukohereza muburyo bwo kubika.Methanol yakoreshejwe nkibisubizo bya MPF. kuminota 10, MP 80 muminota 15, na MP 60 muminota 30. Mugihe cyo gupakira, kunyeganyeza imashini byakoreshejwe hamwe na shitingi ebyiri za GC (Alltech, Deerfield, IL, USA) kugirango habeho gupakira inkingi imwe. Funga ipaki ya slurry hanyuma urekure igitutu gahoro gahoro kugirango wirinde kwangirika kwinkingi zose hanyuma uhuze ikindi kintu gikwiranye na LC.
Pompe ya LC (10AD Shimadzu, Ubuyapani), inshinge (Valco (USA) C14 W.05) hamwe na 50nL yo gutera inshinge, membrane degasser (Shimadzu DGU-14A), idirishya rya capillary ya UV-VIS ryubatswe ryihariye rya µLC ibikoresho byerekana ibikoresho (UV-2075) hamwe na microcolumns zifunga ibirahuri. illaries (50 μ m) zashyizwe kumurongo wa 1/16 ″ cyo kugabanya ubumwe. Gukusanya amakuru no gutunganya chromatografique byakozwe hakoreshejwe software ya Multichro 2000. Gukurikirana kuri 254 nm Analytes zapimwe kugirango UV yinjire. Amakuru ya Chromatografi yasesenguwe na OriginPro8 (Northampton, MA).
Albumin ivuye muri serumu yumuntu, ifu ya lyofilize, ≥ 96% (agarose gel electrophoreis) mg 3 ivanze na trypsin (1.5 mg), 4.0 M urea (1 mL), na 0.2 M ammonium bicarbonate (1 mL) .Umuti washyizwe muminota 10 hanyuma ubikwa mubwogero bwamazi kuri 37 ° C mumasaha 6F.
Gutandukanya imvange ya peptide hamwe na HSA trypsin byapimwe byasuzumwe ukundi ku nkingi za PMP.Reba itandukaniro rya peptide ivanze na trypsin igogora ya HSA ninkingi ya PMP hanyuma ugereranye ibisubizo ninkingi ya Ascentis Express RP-Amide. Umubare wibyapa bya teoretiki ubarwa kuburyo bukurikira:
SEM amashusho yibice bya silika yambaye ubusa hamwe na silika ya ligand-ihuza silika irerekanwa muri FIG.2 .SEM ishusho yibice bya silika yambaye ubusa (A, B) byerekana ko, bitandukanye nubushakashatsi bwatubanjirije, utwo duce ni serefegitire aho uduce twinshi turambuye cyangwa dufite uburinganire budasanzwe. Ubuso bwibice bya silika bihujwe na ligande (C, D) biroroshye cyane kuruta ibice bya silika yambaye ubusa, bishobora guterwa no gutwikisha iminyururu ya polyisirene.
Gusikana microscope ya electron yerekana uduce duto twa silika yambaye ubusa (A, B) hamwe na silika ihuza ligande (C, D).
Ingano yubunini bwikwirakwizwa rya silika yambaye ubusa hamwe na silika ya ligande ihujwe na silika yerekanwa ku gishushanyo cya 3 (A) .Imirongo ikwirakwiza ingano y’ibice bya silika yerekanaga ko ingano y’ibice bya silika yiyongereye nyuma yo guhindura imiti (Ishusho 3A) .Ibipimo byerekana ingano y’ibice bya silika bivuye mu bushakashatsi bwakozwe hamwe n’ubushakashatsi bwabanjirije ugereranije na Imbonerahamwe ya 0 (A) .Ubunini bwa d. . 7 µm) hejuru ya silika, mugihe mugice cya PMP uburebure bwa layer bwari 1.38 µm.
Ingano nini yo gukwirakwiza (A) no gukwirakwiza ingano ya pore (B) ya silika yambaye ubusa hamwe na silika ihujwe na ligande.
Ingano ya pore, ingano ya pore nubuso bwubuso bwa silika yubushakashatsi bugezweho butangwa mu mbonerahamwe ya 1 (B) .Imiterere ya PSD yerekana uduce duto twa silika yambaye ubusa hamwe nuduce twa silika duhujwe na ligandike twerekanwe ku gishushanyo cya 3 (B) .Ibisubizo biragereranywa n’ubushakashatsi bwatubanjirije. umurongo werekanwe ku gishushanyo cya 3 (B) .Nkuko, ingano ya pore yibice bya silika yagabanutse kuva kuri 0,67 ikagera kuri 0.58 cm3 / g nyuma yo guhindura imiti. Ubuso bwihariye bwubutaka bwa silika yizwe ubu ni 116 m2 / g, ibyo bikaba bigereranywa nubushakashatsi bwatubanjirije (124 m2 / g) .Nkuko bigaragara mu mbonerahamwe ya 1 (B), ubuso bwa m2 / g2.
Ibisubizo by'isesengura ry'ibanze by'icyiciro gihagaze byerekanwe mu mbonerahamwe ya 2.Ibikoresho bya karubone byo mu cyiciro gihagaze ni 6.35%, ibyo bikaba biri munsi ya karuboni yapakiye ubushakashatsi twabanje (polystirene ihujwe na silika ibice, 7.93% 35 na 10.21%, 42. . 35%, nkuko bigaragara mu mbonerahamwe ya 2.Gutakaza ibiro byagenzuwe hamwe na PMP ihagaze, naho umurongo wa TGA ugaragara ku gishushanyo cya 4.Umurongo wa TGA werekana gutakaza ibiro 8,6%, ibyo bikaba bihuza neza no gupakira karubone (6.35%) kubera ko ligande zitarimo C gusa ariko na N, O, na H.
Ligand ya fenylmaleimide-methylvinylisocyanate yatoranijwe kugirango ihindurwe hejuru yubutaka bwa silika kuko ifite amatsinda ya polar fenylmaleimide hamwe nitsinda rya vinylisocyanate. Amatsinda ya vinyl isocyanate arashobora gukomeza kwitwara neza hamwe na styrene mubuzima bwa polymerisiyonike. iety ntabwo yishyurwa muburyo busanzwe kuri pH.Uburinganire bwicyiciro gihagaze burashobora kugenzurwa numubare mwiza wa styrene nigihe cyo kwitwara cya polymerisime yubusa.Intambwe yanyuma ya reaction (free-radical polymerisation) irakomeye kandi irashobora guhindura polarite yicyiciro gihagaze. Isesengura ryibanze ryakozwe kugirango harebwe ibipimo bya karuboni hamwe nibyiciro bya stasiyo. styrene ifite imitwaro itandukanye ya karubone. Ongera wongere, shyira ibyo byiciro bihagaze mumashanyarazi yibyuma hanyuma urebe imikorere ya chromatografiya (guhitamo, gukemura, N agaciro, nibindi.). Hashingiwe kuri ubwo bushakashatsi, hatoranijwe uburyo bwiza bwo gutegura icyiciro cya PMP kugirango harebwe polarite igenzurwa neza kandi igumane analyte nziza.
Imvange eshanu za peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) nazo zasuzumwe hifashishijwe inkingi ya PMP ikoresheje icyiciro kigendanwa;60/40. umunota umwe, N indangagaciro zari nziza cyane, 13.500 ± 330 kurinkingi (100 × 1.8 mm id), Bihuye na plaque 135,000 / m (Igicapo 5B) .Inkingi eshatu zingana (100 × 1.8 mm id) zari zipakiye hamwe nibice bitatu bitandukanye bya PMP bihagaze kugirango barebe imyororokere. Kwibanda kuri analyte kuri buri nkingi byanditse kuri progaramu ya progaramu ya progaramu ya progaramu ya progaramu ya progaramu ya progaramu ya progaramu ya buri gihe. mu mbonerahamwe ya 4.Ibyororoka byinkingi ya PMP bifitanye isano neza nagaciro gake cyane RSD, nkuko bigaragara mu mbonerahamwe ya 3.
Gutandukanya peptide ivanze kumurongo wa PMP (B) na Ascentis Express RP-Amide inkingi (A);icyiciro kigendanwa 60/40 ACN / H2O (TFA 0.1%), ibipimo bya PMP (100 × 1.8 mm id);isesengura Urutonde rwo gukuraho ibice: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) na 5 (leucine) aside enkephalin)).
Inkingi ya PMP (100 × 1.8 mm id) yasuzumwe kugirango itandukane ryibiryo bya tryptic ya serumu yumuntu ya chromatografiya ikora neza. Chromatogramu yo ku gishushanyo cya 6 irerekana ko icyitegererezo cyatandukanijwe neza kandi imyanzuro ikaba nziza cyane. Hasesenguwe hifashishijwe igipimo cya 100 µL / min, icyiciro kigendanwa 70/30 acetonitike / amazi hamwe na 0.1% TFA.As. Gutandukanya imikorere ya buri mpinga muri digest ya HSA yabazwe kandi indangagaciro zitangwa mumeza 5.
Inzira ya tryptic ya HSA (100 × 1.8 mm id) yatandukanijwe kumurongo wa PMP;umuvuduko (100 µL / min), icyiciro kigendanwa 60/40 acetonitrile / amazi hamwe na 0.1% TFA.
aho L ni uburebure bwinkingi, η nubukonje bwicyiciro cya mobile, ΔP nigitutu cyinyuma cyinkingi, na u numuvuduko wumurongo wicyiciro cya mobile. Ubushobozi bwinkingi ya PMP bwari 2.5 × 10-14 m2, umuvuduko wamazi wari 25 μL / min, naho 60/40 v / v ACN / amazi yakoreshejwe.Ubushakashatsi bwimbaraga za PMP (100 × 1.8 mm id). icyegeranyo cyinshi ni: 1.7 × 10-15 kuri 1,3 μ m ibice, 3.1 × 10-15 kuri 1,7 mμm, 5.2 × 10-15 na 2.5 × 10-14 m2 kuri 2,6 μ mm zingana na 5 μ m ibice 43. Kubera iyo mpamvu, ubwikorezi bwicyiciro cya PMP burasa nubwa 5 μm core-shell.
aho Wx nuburemere bwinkingi ipakiye chloroform, Wy nuburemere bwinkingi yuzuye methanol, naho ρ nubucucike bwumuti. Ubucucike bwa methanol (ρ = 0.7866) na chloroform (ρ = 1.484) .Ubwinshi bwinkingi za SILICA PARTICLES-C18 twabigenzaga (100 × 1.8 mm id) 34 bivuze ko kuba urea ligands igabanya ubworoherane bwicyiciro gihagaze.Ku rundi ruhande, ubwinshi bwinkingi ya PMP (100 × 1.8 mm id) ni 0.60.Icyegeranyo cyinkingi za PMP kiri munsi yicy'inkingi zipakiye hamwe na silika ya C18 ihujwe na parike ya C18 igizwe na chaine ya ligne. .Mu bushakashatsi busanzwe, inkingi ya porosity ibarwa nka:
Igicapo 7A, B herekana inkingi ya PMP (100 × 1.8 mm id) na Ascentis Express RP-Amide inkingi (100 × 1.8 mm id) ukoresheje uburyo bumwe bwo gukuraho (urugero, 60/40 ACN / H2O na 0.1% TFA).) yimodoka ya Deemter.Uruvange rwa peptide rwatoranijwe (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) rwateguwe muri 20 µL / Igipimo ntarengwa cyo gutembera ku nkingi zombi ni 800 µL / min. 3.9 µm. le imiterere, ingano, hamwe nuburyo bugoye bwo gupakira bukoreshwa mubikorwa byubu34.
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Icyiciro cya polystirene cyashyizwemo polar cyateguwe kandi gisuzumwa kugirango hatandukanywe imvange ya peptide ya peptide hamwe na trypsin igogora ya serumu yumuntu yumuntu (HAS) mumikorere ya chromatografiya ikora neza. Imikorere ya chromatografique yinkingi ya PMP ivanze na peptide ninziza muburyo bwo gutandukana no gukemura.Imikorere itandukanye yo gutandukanya inkingi za parike hamwe nubunini bwa pine ya pisine biterwa nubunini butandukanye. Usibye uburyo bwiza bwo gutandukana, umuvuduko winkingi winyuma kumuvuduko mwinshi ni iyindi nyungu yiki cyiciro gihagaze. Inkingi zaPMP zigaragaza imyororokere myiza kandi irashobora gukoreshwa mugusesengura imvange ya peptide hamwe na trypsin igogorwa rya poroteyine zitandukanye. Turashaka gukoresha iyi nkingi mugutandukanya ibicuruzwa karemano, ibinyabuzima bioaktique biva mubihingwa bivura hamwe na fonctionnement ya antibodi ya PMP.
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