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Uduce duto twa silika twateguwe nuburyo bwa sol-gel hamwe na bimwe byahinduwe kugirango tubone uduce duto twa macroporome.Ibice byavanywemo no guhinduranya byongeye kugabanywa kwihererekanyabubasha (RAFT) polymerisation hamwe na N-phenylmaleimide-methylvinylisocyanate (PMI) na styrene kugirango utegure N-phenylmaleimide intercalation ya polystyrene. . ya peptide ivanze ni hejuru ya plaque 280.000 / 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 yagenewe, imyanda myinshi ikorwa mu gihe cyo guhuza peptide, bityo bikaba bisaba ko habaho isuku ya chromatografi kugira ngo ibone peptide y’isuku yifuzwa. amoko yamenyekana murugero rumwe.Nubwo misa ya sprometrike nigikoresho cyingirakamaro mugukurikirana peptide na proteyine, niba izo ngero zatewe mumashanyarazi ya pasiporo imwe, gutandukana ntibizaba byiza. Iki kibazo gishobora kugabanywa mugushira mu bikorwa gutandukana kwa chromatografiya (LC) mbere yo gusesengura kwa MS, bizagabanya umubare wa analyite winjira mugice cya analyse. kwibanda kuri analyite no kunoza sensibilité ya MS.Cromatografiya y'amazi (LC) yateye imbere cyane mumyaka icumi ishize kandi yabaye tekinike izwi cyane mubisesengura 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 kubera imiterere yabyo hamwe na poroteyine zidakorwa hamwe na proteine ​​zidahwitse hamwe na poroteyine zidasanzwe hamwe na proteine ​​zakozwe na peptide hamwe na poroteyine zidahwitse hamwe na peptide. moieties yo gukorana 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-bihagaze byateguwe, kandi inkingi zuzuyemo ibyo byiciro byakoreshejwe mu gutandukanya peptide na proteine17,18,19,20,20,22 HILIC / RPLC, polar intercalation / RPLC) ikwiranye no gutandukanya peptide na proteyine bitewe nuko habaho amatsinda yombi ya polar na non-polar22,23,24,25,26,27,28 .Bisa nkibyo, polar ihuza ibice byimyanya ihagaze hamwe na polarike ihuriweho hamwe byerekana imbaraga zo gutandukana hamwe no guhitamo icyiciro hagati ya analyite na polarite. Imikoranire myinshi 29, 30, 31, 32.Mu minsi ishize, Zhang n'abandi. 30 yateguye icyiciro cya dodecyl 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. izina Ascentis Express RP-Amide inkingi, ariko izi nkingi zikoreshwa mugusesengura amine 33 gusa.
Mu bushakashatsi burimo gukorwa, hateguwe icyiciro gihagaze (N-phenylmaleimide-yashyizwemo polystirene) cyateguwe kandi gisuzumwa kugira ngo habeho gutandukanya peptide na trypsin igogora rya HSA.Icyiciro gihagaze cyateguwe hifashishijwe ingamba zikurikira. ibice bya silika bifite ubunini bunini bwa kabiri. Icya kabiri, ligand nshya, fenylmaleimide-methyl vinyl isocyanate, yashizwemo kandi ikoreshwa mu kuvoma uduce duto twa silika kugirango hategurwe icyuma gishyizwemo icyiciro. Icyiciro cyahagaritswe cyapakiwe mu nkingi y’icyuma (100 × 1.8 mm id) ikoresheje uburyo bwo gupakira ibikoresho bya tekinike. inkingi. Suzuma inkingi zipakiye gutandukanya peptide ivanze igizwe na peptide eshanu; . peptide na proteyine byagaragaye ko byakemuwe neza kandi neza ku nkingi ya PMP, byakoraga neza kurusha Ascentis Express RP-Amide.
PEG. 4-Hydroxy-TEMPO, Benzoyl Peroxide (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. Ubutaka bworoshye mu ziko kandi ubarwa kuri 550 ° C mu masaha 12. Ibice bitatu byateguwe kandi birangwa no gusuzuma imyororokere mu bunini, ubunini bwa pore n'ubuso.
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. kuri 40 ° C mu masaha 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. PMCP (mg 10) yashongeshejwe muri toluene hanyuma ikongerwaho ibitonyanga kuri flask ya reaction ikoresheje umuyonga wamanutse. ibice bya silika (100 g) byashongeshejwe muri toluene (200 ml) hanyuma hongerwaho 4-hydroxy-TEMPO (2 mL) imbere ya 100 µL ya dibutyltin dilaurate nka catalizator.
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 na ligand yasuzumwe hamwe na microscopi ya elegitoronike (Hitachi High Technologies, Tokiyo, Ubuyapani). nkingi ukoresheje kaseti ya karubone. Zahabu yashyizwe ku cyitegererezo ikoresheje ikote rya Q150T, hanyuma igashyirwa kuri 5 nm Au igashyirwa kuri izo ngero.Ibyo bizamura imikorere neza ukoresheje voltage nkeya kandi bitanga ingano nziza, imbeho ikonje. Ikintu cya Thermo Electron (Waltham, MA, USA) Isesengura ryibanze rya Flash EA1112 ryakoreshwaga mu gusesengura ibice. Gukwirakwiza ingano ya selile.Ibice bya silika byambaye ubusa hamwe na silika ihujwe na ligande (5 mg buri umwe) byakwirakwijwe muri mL 5 ya isopropanol, ikabyara iminota 10, ikabikwa mu minota 5, igashyirwa ku ntebe ya optique ya Mastersizer. Isesengura rya termogravimetric ryakozwe ku gipimo cya 5 ° C ku munota hejuru y’ubushyuhe bwa 30 kugeza 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) .Tegura icyiciro cya sitasiyo uhagarika mg 150 zicyiciro cya stasiyo muri 1.2 mL ya methanol hanyuma ukohereza muburyo bwo kubika.Methanol yakoreshejwe nkibisubizo. igitutu cya MP 100 muminota 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 yihuta hanyuma urekure igitutu gahoro gahoro kugirango wirinde kwangirika kwa sisitemu kugirango uhuze na sisitemu kugirango uhuze na sisitemu.
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. 365 no kugabanya capillaries yubumwe (50 μ m) yashyizwe kumurongo wa 1/16 ″ ugabanya ubumwe bwikusanyamakuru hamwe no gutunganya chromatografique byakozwe hakoreshejwe software ya Multichro 2000 Analytes zapimwe kugirango UV yinjire.
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 igabanya ingano y’ibice bya silika yerekanaga ko ingano y’ibice bya silika yiyongereye nyuma yo guhindura imiti (Igishusho 3A) .Ibipimo byerekana ingano yo gukwirakwiza ibice bya silika bivuye mu bushakashatsi bwakozwe kandi ubushakashatsi bwabanjirije ugereranije na Imbonerahamwe ya 1 (A) .Ubunini bwa d. ubushakashatsi hamwe na ad (0.5) bifite agaciro ka 3.05 mm (uduce duto twa silisike duhujwe na polystirene) 34.Iki cyiciro cyari gifite ubunini buke bwagabanijwe ugereranije nubushakashatsi bwatubanjirije kubera ibipimo bitandukanye bya PEG, urea, TMOS, na acide acetike bivanze na reaction. yashyize polystirene (0,97 µm) hejuru ya silika, mugihe mugice cya PMP umubyimba wari 1.38 µm.
Ingano nini yo gukwirakwiza (A) no gukwirakwiza ingano ya pore (B) ya silika yambaye ubusa hamwe na silika ihuza ligande.
Ingano ya pore, ingano ya pore nubuso bwubuso bwa silika yubushakashatsi bugezweho butangwa mu mbonerahamwe ya 1 (B) .Imyirondoro 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. kandi ihinduka ryumurongo ryerekanwe ku gishushanyo cya 3 (B) .Nkuko bimeze, ingano ya pore yibice bya silika yagabanutse kuva kuri 0,67 igera kuri 0.58 cm3 / g nyuma yo guhindura imiti. Ubuso bwihariye bwubuso bwa silika yizwe ubu ni 116 m2 / g, ibyo bikaba bigereranywa nubushakashatsi twabanje (124 m2 / g) .Byerekanwe kumeza 1 (B), g / g / g). m2 / g nyuma yo guhindura imiti.
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 ya silika, 7.93% 35 na 10.21%), 42. phenylmaleimide-methylvinylisocyanate (PCMP) na 4-hydroxy-TEMPO yakoreshejwe.Uburemere bwa azote ijanisha ryicyiciro gihagaze ni 2,21%, ugereranije na 0.1735 na 0,85% kuburemere bwa azote mubushakashatsi bwabanje, ibi bivuze ko wt% ya azote iri hejuru yicyiciro cya karubone. .
Ligand ya fenylmaleimide-methylvinylisocyanate yatoranijwe kugirango ihindurwe hejuru yuduce twa silika kuko ifite amatsinda ya polar phenylmaleimide hamwe nitsinda rya vinylisocyanate. Amatsinda ya vinyl isocyanate arashobora kongera kwitwara hamwe na styrene mubuzima bwa polymerisiyonike. phenylmaleimide moiety ntamafaranga yishyurwa isanzwe kuri pH isanzwe.Uburinganire bwicyiciro gihagaze burashobora kugenzurwa numubare mwiza wa styrene hamwe nigihe cyo kwitabira cya polymerisime yubusa. Intambwe yanyuma ya reaction (polymerisation yubusa) irakomeye kandi irashobora guhindura polarite yicyiciro gihagaze. icyiciro gihagaze naho ubundi.SPs zateguwe hamwe nubushuhe butandukanye bwa styrene zifite imitwaro itandukanye ya karubone. Ongera wongere, shyira ibi byiciro bihagaze mumashanyarazi yibyuma hanyuma urebe imikorere ya chromatografiya (guhitamo, gukemura, N agaciro, nibindi.). Hashingiwe kuri ubwo bushakashatsi, hatoranijwe uburyo bunoze bwo gutegura icyiciro cya PMP kugirango habeho polarite igenzurwa neza.
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. peptide yavuzwe mu minota imwe, N indangagaciro zari nziza cyane, 13.500 ± 330 kuri buri nkingi (100 × 1.8 mm id), Ihuza amasahani 135.000 / m (Igicapo 5B) .Inkingi eshatu zingana (100 × 1.8 mm id) zari zuzuyemo ibice bitatu bitandukanye bya PMP bihagaze kugirango harebwe imyororokere. amakuru yimyororokere yinkingi ya PMP yerekanwa mumeza ya 4.Ibyororoka byinkingi ya PMP bifitanye isano neza nagaciro gake cyane RSD, nkuko bigaragara mumbonerahamwe 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 itandukanyirizo ryibiryo bya tryptic ya serumu yumuntu ya chromatografiya ikora neza. Chromatogramu yo ku gishushanyo cya 6 yerekana ko icyitegererezo cyatandukanijwe neza kandi imyanzuro ikaba nziza cyane. Ibyokurya bya HSA byasesenguwe hakoreshejwe umuvuduko wa 100 µL / min, icyiciro cya mobile 70/30 acetonitrile / amazi na 0.1% TFA.As yerekanwe kuri chromat kuri peptide 17.Ubushobozi bwo gutandukanya buri mpinga muri digest ya HSA bwabazwe 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 kigendanwa, 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 bwakorewe kuri PMP (100 × 1.8 mm). inkingi ipakiye hamwe nuduce duto duto cyane ni: 1.7 × 10-15 kuri 1,3 mμm, 3.1 × 10-15 kuri 1.7 μ mm, 5.2 × 10-15 na 2.5 × 10-14 m2 kuri 2.6 μ m ibice 5 μm 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, na ρ nubucucike bwumuti.Ubucucike bwa methanol (ρ = 0.7866) na chloroform (ρ = 1.484) .Ubwinshi bwinkingi za SILICA PARTICLES-C18 twari twize (100 × 1.8 mm id) muburyo bukurikira.Ibyo bivuze ko kuba urea ligands igabanya ubwikorezi bwicyiciro gihagaze. Kurundi ruhande, ubwinshi bwinkingi ya PMP (100 × 1.8 mm id) ni 0.60.Ubushobozi bwinkingi za PMP buri munsi ugereranije ninkingi zipakiye C18 zifatanije nuduce twinshi twa selile ya siline hamwe na parike ya C18. igipimo cyinshi cya polymer gikozwe hafi yacyo.Mu bushakashatsi busanzwe, inkingi ya porosity ibarwa nka:
Igicapo 7A, B cerekana inkingi ya PMP (100 × 1.8 mm id) hamwe 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. Imvange zatoranijwe za peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) zateguwe muri 20 µL / Igipimo ntarengwa cyo gutembera ku nkingi zombi ni 800 µL / min. 2.6 µm na 3.9 µm, uko bikurikirana. Indangagaciro za HETP zerekana ko uburyo bwo gutandukanya inkingi ya PMP (100 × 1.8 mm id) ari bwiza cyane kuruta ubucuruzi bwa Ascentis Express RP-Amide (100 × 1.8 mm id) Inkingi ya RP-Amide ishingiye ku kunoza imiterere yimiterere, ingano, hamwe nuburyo bukomeye bwo gupakira inkingi zikoreshwa mubikorwa byubu34.
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Icyiciro cya polystyrene 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 za PMP ivanze na peptide ninziza muburyo bwo gutandukana no gukemura. Gupakira inkingi zigoye. Usibye gukora neza cyane gutandukana, umuvuduko winkingi winyuma kumuvuduko mwinshi ni iyindi nyungu yiki cyiciro gihagaze.Inkingi ya PPP yerekana imyororokere myiza kandi irashobora gukoreshwa mugusesengura imvange ya peptide hamwe na trypsin igogorwa rya poroteyine zitandukanye. Turashaka gukoresha iyi nkingi mugutandukanya ibicuruzwa karemano, ibinyabuzima bikomoka kumiti hamwe na fonctionnement ya proteine. antibodies.
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