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Waxay soo bandhigaysaa sawir-qaadis saddex sawir-qaadis ah isku mar. Isticmaal badhamada Hore iyo kuwa Xiga si aad ugu dhex marto saddex sawir-qaadis markiiba, ama isticmaal badhamada sawir-qaadis ee dhammaadka si aad ugu dhex marto saddex sawir-qaadis markiiba.
Walxaha silica ee daloola leh waxaa lagu diyaariyey habka sol-gel iyadoo wax laga beddelayo si loo helo walxo daloola ballaaran. Walxahaan waxaa laga soo saaray N-phenylmaleemide-methylvinyl isocyanate (PMI) iyo styrene iyada oo loo marayo polymerization-ka wareejinta silsiladda dib u celinta (RAFT) si loo soo saaro polyamides-ka isku dhafan ee N-phenylmaleemide. Marxaladda Styrene (PMP). Tiirarka birta aan miridhku lahayn ee cidhiidhiga ah (100 × 1.8 mm dhexroor gudaha ah) waxaa lagu buuxiyey baakad slurry ah. Waxqabadka koromatografiga ee tiirka PMP ayaa la qiimeeyay si loo kala saaro isku darka peptides-ka synthetic-ga oo ka kooban shan peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) iyo tryptic hydrolyzate ee albumin-ka serum-ka aadanaha (HAS). Xaaladaha ugu wanaagsan ee lagu nadiifiyo, tirada aragtiyeed ee taarikada oo leh isku darka peptides waxay gaartay 280,000 oo saxan/sq.m. Marka la barbardhigo waxqabadka kala soocida tiirka la sameeyay iyo tiirka ganacsiga ee Ascentis Express RP-Amide, waxaa la arkay in hufnaanta kala soocida tiirka PMP ay ka sarreyso tiirka ganacsiga marka loo eego hufnaanta kala soocida iyo xallinta.
Warshadaha dawooyinka noolaha ayaa noqday suuq caalami ah oo sii kordhaya iyadoo saami weyn ku leh suuqa sannadihii ugu dambeeyay. Iyadoo ay jirto kororka baaxadda leh ee warshadaha dawooyinka noolaha1,2,3 waxaa jira baahi weyn oo loo qabo falanqaynta peptide iyo borotiinka. Marka laga soo tago peptide-ka bartilmaameedka ah, wasakhyo kala duwan ayaa la sameeyaa inta lagu jiro isku-darka peptide, sidaa darteed nadiifinta chromatographic ayaa loo baahan yahay si loo helo daahirnimada la rabo ee peptide-ka. Falanqaynta iyo qeexidda borotiinnada ku jira dareerayaasha jirka, unugyada, iyo unugyada waa hawl aad u adag sababtoo ah tirada badan ee noocyada la ogaan karo ee ku jira hal muunad. In kasta oo spectrometry-ga cufku uu yahay qalab wax ku ool ah oo loogu talagalay isku-xigxiga peptides iyo borotiinnada, haddii muunadaha noocaas ah si toos ah loogu soo bandhigo spectrometer-ka cufku, kala-soociddu ma noqon doonto mid aan ku qanacsanayn. Dhibaatadan waxaa lagu xallin karaa iyadoo la samaynayo chromatography dareere ah (LC) ka hor falanqaynta MS, taas oo yarayn doonta tirada falanqeeyayaasha galaya spectrometer-ka cufku waqti cayiman4,5,6. Intaa waxaa dheer, falanqeeyayaashu waxay ku ururin karaan gobol cidhiidhi ah inta lagu jiro kala-goynta marxaladda dareeraha ah, sidaas darteedna waxay xoogga saarayaan falanqeeyayaashan waxayna kordhinayaan xasaasiyadda ogaanshaha MS. Koromagrafiga dareeraha ah (LC) ayaa si weyn u horumaray tobankii sano ee la soo dhaafay wuxuuna noqday hab si ballaaran loo isticmaalo falanqaynta borotiinka7,8,9,10.
Koromagrafiga dareeraha ee dib-u-rogidda (RP-LC) waxaa si weyn loogu isticmaalaa in lagu nadiifiyo oo lagu kala saaro isku-darka peptides iyadoo la adeegsanayo silica octadecyl-modified (ODS) oo ah wejiga taagan11,12,13. Si kastaba ha ahaatee, sababtoo ah qaab-dhismeedkooda adag iyo dabeecadda amphoteric, 14,15 marxaladood oo RP ah oo taagan ma bixin karaan kala-soocid qancisa oo peptides iyo borotiinno ah. Sidaa darteed, falanqaynta peptides iyo borotiinnada leh jajabyada polar iyo kuwa aan polar ahayn waxay u baahan tahay marxalado si gaar ah loogu talagalay oo joogto ah si ay ula falgalaan oo ay u hayaan falanqeeyayaashan16. Koramagrafiga isku dhafan, oo bixiya is-dhexgal badan, wuxuu noqon karaa beddel RP-LC ah si loo kala saaro peptides, borotiinnada, iyo isku-darka kale ee adag. Dhowr marxaladood oo isku dhafan oo taagan ayaa la diyaariyey tiirarna waxaa laga buuxiyay marxaladahan taagan si loo kala saaro peptides iyo borotiinnada17,18,19,20,21. Sababtoo ah joogitaanka kooxaha polar iyo kuwa aan polar ahayn, marxaladaha isku dhafan ee qaabka joogtada ah (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) ayaa ku habboon kala-soocidda peptides iyo borotiinnada22,23,24,25,26,27,28., marxaladaha taagan ee polar intercalated oo leh kooxo polar ah oo isku xiran ayaa muujinaya awoodo kala-soocid wanaagsan iyo xulasho gaar ah oo loogu talagalay falanqeeyayaasha polar iyo kuwa aan polar ahayn sababtoo ah kala-soociddu waxay ku xiran tahay isdhexgalka u dhexeeya falanqeeyaha iyo wejiga taagan Isdhexgalka Multimodal 29,30,31,32. Dhawaan, Zhang et al. 30 waxay heleen marxalado taagan oo behenyl ah oo la joojiyay oo polyamines ah iyo si guul leh loo kala saaray hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, iyo qaar ka mid ah falanqeeyayaasha kale. Maaddada taagan ee polar ku dhex jirta waxay leedahay labadaba kooxaha polar iyo kuwa aan polar ahayn, sidaa darteed waxaa loo isticmaali karaa in lagu kala saaro peptides iyo borotiinnada qaybo hydrophobic iyo hydrophilic ah. Tiirarka gudaha ee polar-ka (tusaale ahaan, tiirarka C18 oo leh amide inline) ayaa laga heli karaa magaca ganacsiga ee tiirarka RP-Amide ee Ascentis Express, laakiin tiirarkan waxaa loo isticmaalay oo keliya falanqaynta amine 33.
Daraasaddan hadda socota, marxalad taagan oo lagu dhejiyo polar embedding stain (N-phenylmaleimide, embedding polystyrene) ayaa la diyaariyey oo lagu qiimeeyay kala soocidda peptide iyo kala-goynta tryptic HSA. Istaraatiijiyadda soo socota ayaa loo isticmaalay in lagu diyaariyo marxaladda taagan. Qaybaha silica ee daloola leh ayaa la diyaariyey iyadoo la raacayo habraacyada lagu sharraxay daabacaadihii hore, iyadoo isbeddello lagu sameeyay qorshayaasha diyaarinta 31, 34, 35, 36, 37, 38, 39. Saamiyada urea, polyethylene glycol (PEG), TMOS iyo aqueous-acetic acid ayaa la hagaajiyay si loo helo qaybo silica ah oo leh cabbirro waaweyn oo dalool ah. Marka labaad, ligand cusub oo phenylmaleimide-methylvinyl isocyanate ah ayaa la sameeyay waxaana loo isticmaalay walxaha silica ee la soo saaray si loo diyaariyo marxalado taagan oo polar ah. Marxaladda taagan ee la helay waxaa lagu duubay tiir bir ah oo aan miridh lahayn (dhexroor gudaha ah 100 × 1.8 mm) iyadoo loo eegayo nidaamka baakadaynta ee la hagaajiyay. Xidhmada tiirka waxaa ka caawiya gariir farsamo si loo hubiyo lakab isku mid ah oo ku jira tiirka. Tiirka la buuxiyay waxaa lagu qiimeeyay kala soocidda isku darka peptides oo ka kooban shan peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). iyo tryptic hydrolysates oo ah albumin serum aadanaha (HSA). Waxaa la arkay in isku darka peptide iyo dheefshiidka tryptic ee HSA ay kala soocaan xallin iyo hufnaan wanaagsan. Hufnaanta kala soocidda ee tiirka PMP waxaa la barbar dhigay tiirka Ascentis Express RP-Amide. Waxaa la arkay in peptides iyo borotiinadu ay leeyihiin xallin wanaagsan iyo hufnaan kala soocid sare oo ku taal tiirka PMP, hufnaanta kala soocidda ee tiirka PMP-na ay ka sarreyso tiirka Ascentis Express RP-Amide.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), styrene, 4-hydroxy- TEMPO, benzoyl peroxide (BPO), acetonitrile (ACN) oo loogu talagalay HPLC, methanol, 2-propanol iyo acetone. Shirkadda Sigma-Aldrich (St. Louis, Missouri, USA).
Isku darka urea (8 g), polyethylene glycol (8 g) iyo 8 ml oo ah 0.01 N. acetic acid ayaa la walaaqay 10 daqiiqo, waxaana lagu daray 24 ml oo TMOS ah iyadoo barafku qaboobo. Isku darka falcelinta waxaa lagu kululeeyay 40°C muddo 6 saacadood ah ka dibna heerkul ah 120°C muddo 8 saacadood ah oo lagu shubay bir aan miridh lahayn oo autoclave ah. Biyaha waa la nadiifiyay, haraagana waxaa lagu qalajiyay 70°C muddo 12 saacadood ah. Baloogyada jilicsan ee la qalajiyey si habsami leh ayaa loo shiiday oo lagu kariyey foorno heerkul ah 550°C muddo 12 saacadood ah. Saddex dufcadood ayaa la diyaariyey oo lagu sifeeyay si loo tijaabiyo soo-saarista cabbirrada walxaha, cabbirka daloolada iyo bedka dusha sare.
Kooxda polar iyo marxaladda taagan ee silsiladaha polystyrene. Habka diyaarinta ayaa hoos lagu sharaxay.
N-phenylmaleimide (200 mg) iyo methyl vinyl isocyanate (100 mg) ayaa lagu milmay toluene aan biyo lahayn, ka dibna 0.1 ml oo ah 2,2′-azoisobutyronitrile (AIBN) ayaa lagu daray dhalada falcelinta si loo helo copolymer phenylmaleimide iyo methyl vinyl isocyanate (PMCP).) Isku darka waxaa lagu kululeeyay 60°C muddo 3 saacadood ah, waxaana la sifeeyay oo lagu qalajiyay foorno heerkulkeedu yahay 40°C muddo 3 saacadood ah.
Walxaha silica ee qalalan (2 g) ayaa lagu kala firdhiyey toluene qalalan (100 ml), waxaana lagu walaaqay oo lagu walaaqay 10 daqiiqo weel wareegsan oo 500 ml ah oo hoose ah. PMCP (10 mg) waxaa lagu milmay toluene waxaana lagu daray hoos u dhac ku yimaada weelka falcelinta iyada oo loo marayo funnel lagu daray. Isku darka waxaa dib loogu celiyey 100°C muddo 8 saacadood ah, waa la sifeeyay, waxaa lagu dhaqay acetone waxaana lagu qalajiyey 60°C muddo 3 saacadood ah. Kadib, walxaha silica ee la xiriira PMCP (100 g) ayaa lagu milmay toluene (200 ml), waxaana lagu daray 4-hydroxy-TEMPO (2 ml) iyadoo la joogo 100 μl oo dibutyltin dilaurate ah oo ah kiciye. Isku darka waxaa lagu walaaqay 50°C muddo 8 saacadood ah, waxaana la sifeeyay oo lagu qalajiyey 50°C muddo 3 saacadood ah.
Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) iyo walxaha silica ee ku xiran TEMPO-PMCP (1.5 g) ayaa lagu kala firdhiyey toluene waxaana lagu nadiifiyey nitrogen. Polymerization-ka styrene waxaa lagu sameeyay 100°C muddo 12 saacadood ah. Badeecada ka dhalatay waxaa lagu dhaqay methanol waxaana lagu qalajiyey habeenkii heerkul ah 60°C. Qaabka guud ee falgalka waxaa lagu muujiyey Jaantuska koowaad.
Shaybaarrada waxaa laga saaray 393 K muddo 1 saac ah ilaa laga helay cadaadis haraaga ah oo ka yar 10-3 Torr. Tirada N2 ee lagu nuugay cadaadiska qaraabada ah P/P0 = 0.99 ayaa loo isticmaalay in lagu go'aamiyo mugga daloolka guud. Qaab-dhismeedka walxaha silica ee saafiga ah iyo kuwa ligand-ka ku xiran ayaa la baaray iyadoo la adeegsanayo mikroskoob elektaroonik ah oo sawiraya (Hitachi High Technologies, Tokyo, Japan). Shaybaarrada qalalan (walxaha silica saafiga ah iyo walxaha silica ee ligand-ka ku xiran) ayaa lagu dhejiyay ulaha aluminium iyadoo la isticmaalayo cajalad kaarboon. Dahabka ayaa lagu shubay muunadda iyadoo la isticmaalayo qalab buufin Q150T ah, waxaana la dhigay lakab Au ah oo 5 nm qaro weyn ah oo muunadda ah. Tani waxay hagaajinaysaa hufnaanta habka danab hooseeya waxayna bixisaa buufin qabow oo fiican. Falanqaynta walxaha waxaa lagu sameeyay iyadoo la adeegsanayo falanqeeye isku-darka walxaha ee Thermo Electron (Waltham, MA, USA) Flash EA1112. Falanqeeye cabbirka walxaha ee Malvern (Worcestershire, UK) Mastersizer 2000 ayaa loo isticmaalay in lagu helo qaybinta cabbirka walxaha. Walxaha silica ee aan dahaarka lahayn iyo walxaha silica ee ligand-ku xiran (5 mg midkiiba) ayaa lagu kala firdhiyey 5 ml oo isopropanol ah, waxaana lagu shubay sonicated muddo 10 daqiiqo ah, waxaana la walaaqay muddo 5 daqiiqo ah, ka dibna waxaa la saaray kursiga indhaha ee Mastersizer. Falanqaynta thermogravimetric waxaa lagu sameeyaa heer ah 5 °C daqiiqaddii heerkulka u dhexeeya 30 ilaa 800 °C.
Tiirar bir ah oo aan lahayn bir aan lahayn oo cidhiidhi ah oo leh cabbirro (ID 100 × 1.8 mm) ayaa lagu duubay habka buuxinta slurry iyadoo la raacayo isla nidaamka sida ku xusan tixraaca 31. Tiirka birta aan lahayn (muraayadda dahaarka leh, ID 100 × 1 .8 mm) iyo meel laga soo galo oo ka kooban 1 µm frit ayaa lagu xiray mashiinka baakadaha slurry (Alltech Deerfield, IL, USA). Diyaari joojinta marxaladda taagan adigoo ku dhejinaya 150 mg oo wejiga taagan 1.2 ml oo methanol ah oo ku shubaya tiirka kaydka. Methanol waxaa loo isticmaalay sidii dareeraha slurry iyo dareeraha xakamaynta. Ku xidh tiirka adigoo ku dhejinaya taxane cadaadis ah oo ah 100 MP muddo 10 daqiiqo ah, 80 MP muddo 15 daqiiqo ah, iyo 60 MP muddo 30 daqiiqo ah. Habka baakaduhu wuxuu isticmaalay laba gariiriye tiirka koromatografi gaas ah (Alltech, Deerfield, IL, USA) si loogu sameeyo gariir farsamo si loo hubiyo xidhitaanka tiirka midaysan. Xidh baakadaha slurry-ka oo si tartiib ah u sii daa cadaadiska si looga hortago waxyeelada xarigga. Tiirka ayaa laga jaray afka slurry-ka, qalab kale ayaa lagu xiray albaabka waxaana lagu xiray nidaamka LC si loo tijaabiyo shaqadiisa.
MLC gaar ah ayaa la sameeyay iyadoo la adeegsanayo bamka LC (10AD Shimadzu, Japan), muunad qaade leh wareeg duritaan 50 nL ah (Valco (USA) C14 W.05), degaser xuubka (Shimadzu DGU-14A), iyo daaqad xidid UV-VIS ah. Qalabka ogaanshaha (UV-2075) iyo tiir yar oo dhaldhalaal leh. Isticmaal tuubooyin isku xira oo aad u cidhiidhi ah oo gaaban si aad u yareyso saameynta ballaarinta tiirka dheeraadka ah. Ka dib markaad buuxiso tiirka, ku rakib xidid xidid (50 µm id 365) meesha laga soo galo isgoyska yaraynta 1/16″ oo ku rakib xidid xidid (50 µm) oo ah isgoyska yaraynta. Ururinta xogta iyo habaynta koromatogram waxaa lagu sameeyaa iyadoo la adeegsanayo barnaamijka Multichro 2000. 254 nm, nuugista UV ee falanqeeyayaasha mowduucyada waxaa lagu kormeeray 0. Xogta koromatografiga waxaa lagu falanqeeyay iyadoo la adeegsanayo OriginPro8 (Northampton, MA).
Albamin serum ah oo laga sameeyay Human, budada lyophilized, ≥ 96% (agarose gel electrophoresis) 3 mg oo lagu qasay trypsin (1.5 mg), 4.0 M urea (1 ml) iyo 0.2 M ammonium bicarbonate (1 ml). Xalka waxaa la walaaqay 10 daqiiqo waxaana lagu hayaa biyo qubeys ah 37°C muddo 6 saacadood ah, ka dibna waxaa lagu damiyay 1 ml oo ah 0.1% TFA. Shaandhee xalka oo ku kaydi heerkul ka hooseeya 4°C.
Kala soocidda isku darka peptides iyo tryptic dheefshiidka HSA ee tiirka PMP ayaa si gaar ah loo qiimeeyay. Hubi hydrolysis-ka tryptic ee isku darka peptides iyo HSA oo ay kala sooceen tiirka PMP oo isbarbardhig natiijooyinka tiirka Ascentis Express RP-Amide. Tirada taarikada aragtiyeed waxaa lagu xisaabiyaa iyadoo la adeegsanayo isla'egta soo socota:
Sawirada SEM ee walxaha silica saafiga ah iyo walxaha silica ee isku xiran ligand ayaa lagu muujiyay Jaantuska 2. Sawirada SEM ee walxaha silica saafiga ah (A, B) waxay muujinayaan qaab wareegsan oo walxaha ay dheer yihiin ama ay leeyihiin isku dheelitirnaan aan caadi ahayn marka loo eego daraasadihii hore. Dusha sare ee walxaha silica ee ay ku xiran tahay ligand (C, D) way ka siman tahay kuwa walxaha silica saafiga ah, taasoo laga yaabo inay sabab u tahay silsiladaha polystyrene ee daboolaya dusha sare ee walxaha silica.
Sawir-qaadista mikrogaraafyada elektarooniga ee walxaha silica saafiga ah (A, B) iyo walxaha silica ee ligand-ka ku xiran (C, D).
Qaybinta cabbirka walxaha ee walxaha silica saafiga ah iyo walxaha silica ee ku xiran ligand waxaa lagu muujiyay Jaantuska 2. 3(A). Qalloocyada qaybinta cabbirka walxaha silica waxay muujiyeen in cabbirka walxaha silica uu kordhay ka dib wax ka beddelka kiimikada (Jaantuska 3A). Xogta qaybinta cabbirka walxaha silica ee daraasadda hadda jirta iyo daraasaddii hore waxaa lagu barbar dhigay Shaxda 1(A). Cabbirka walxaha mug leh d(0.5) ee PMP wuxuu ahaa 3.36 µm, marka la barbar dhigo qiimaha ad(0.5) ee 3.05 µm ee daraasaddeenna hore (walxaha silica ee ku xiran polystyrene)34. Sababtoo ah isbeddelka saamiga PEG, urea, TMOS iyo acetic acid ee isku darka falcelinta, qaybinta cabbirka walxaha ee dufcaddan ayaa ka cidhiidhi badnayd marka loo eego daraasaddeenna hore. Cabbirka walxaha ee wejiga PMP wuxuu wax yar ka weyn yahay kan wejiga walxaha silica ee ku xiran polystyrene ee aan hore u barannay. Taas macnaheedu waa in shaqada dusha sare ee walxaha silica ee leh styrene ay ku shubtay oo keliya lakab polystyrene ah (0.97 µm) dusha sare ee silica, halka marxaladda PMP dhumucda lakabku ay ahayd 1.38 µm.
Qaybinta cabbirka walxaha (A) iyo qaybinta cabbirka daloolka (B) ee walxaha silica saafiga ah iyo walxaha silica ee ligand ku xiran.
Cabbirka daloolka, mugga daloolka, iyo bedka dusha sare ee walxaha silica ee loo isticmaalay daraasaddan waxaa lagu muujiyay Shaxda 1 (B). Astaamaha PSD ee walxaha silica saafiga ah iyo walxaha silica ee ku xiran ligand waxaa lagu muujiyay Jaantusyada 3(B). Natiijooyinku waxay la mid ahaayeen daraasaddeenii hore34. Cabbirka daloolka ee walxaha silica ee saafiga ah iyo kuwa ku xiran ligand waxay ahaayeen 310 Å iyo 241 Å, siday u kala horreeyaan, taasoo tilmaamaysa in ka dib wax ka beddelka kiimikada, cabbirka daloolka uu hoos u dhacay 69 Å, sida lagu muujiyay Shaxda 1 (B), qalooca isbeddelkana waxaa lagu muujiyay Jaantuska. Bedka dusha sare ee walxaha silica ee daraasadda hadda socota waa 116 m2/g, taas oo la mid ah daraasaddeenna hore (124 m2/g). Sida lagu muujiyay Shaxda 1(B), bedka dusha sare (m2/g) ee walxaha silica ka dib wax ka beddelka kiimikada ayaa sidoo kale hoos uga dhacay 116 m2/g ilaa 105 m2/g.
Natiijooyinka falanqaynta walxaha ee marxaladda taagan waxaa lagu soo bandhigay Shaxda. 2. Kaarboonka ku jira marxaladda taagan ee hadda jirta waa 6.35%, taas oo ka hooseysa daraasaddeenna hore (walxaha silica ee la xiriira polystyrene, 7.93%35 iyo 10.21%, siday u kala horreeyaan) 42. Kaarboonka ku jira marxaladda taagan ee hadda jirta ee hoose, maadaama qaar ka mid ah ligands-ka polar sida phenylmaleemide methyl vinyl isocyanate (PCMP) iyo 4-hydroxy-TEMPO loo isticmaalay marka lagu daro styrene diyaarinta SP. Boqolleyda miisaanka ee nitrogen ee marxaladda taagan ee hadda jirta waa 2.21% marka la barbar dhigo 0.1735 iyo 0.85% daraasadihii hore42. Taas macnaheedu waa in marxaladda taagan ee hadda jirta ay leedahay boqolkiiba miisaan sare oo nitrogen ah oo ay ugu wacan tahay phenylmaleemide. Sidoo kale, alaabada (4) iyo (5) waxay leeyihiin kaarboon ah 2.7% iyo 2.9%, siday u kala horreeyaan, halka badeecada ugu dambeysa (6) ay leedahay kaarboon ah 6.35%, sida ku cad Shaxda 2. Falanqaynta Thermogravimetric (TGA) ayaa loo isticmaalay marxaladda taagan ee PMP si loo tijaabiyo miisaanka oo yaraada, qalooca TGA-na waxaa lagu muujiyay Jaantuska 4. Qalooca TGA wuxuu muujinayaa miisaan lumis ah 8.6%, kaas oo si fiican ula jaanqaadaya kaarboonka (6.35%), maadaama ligands-ku aysan ku jirin oo keliya C , laakiin sidoo kale N, O iyo H.
ligand phenylmaleemide-methylvinyl isocyanate waxaa loo doortay inuu wax ka beddelo dusha sare ee walxaha silica sababtoo ah kooxaha phenylmaleemide iyo vinylisocyanate ee polar. Kooxaha Vinyl isocyanate waxay si dheeraad ah ula falgeli karaan styrene iyagoo ku nool polymerization xagjir ah. Sababta labaad waa in la geliyo koox leh isdhexgal dhexdhexaad ah oo la leh analyte oo aan lahayn isdhexgal koronto oo xooggan oo u dhexeeya analyte iyo marxaladda taagan, maadaama qaybta phenylmaleemide aysan lahayn kharash dalwaddeed oo pH caadi ah. Kala-goynta wejiga taagan waxaa lagu xakameyn karaa xaddiga ugu habboon ee styrene iyo waqtiga falcelinta ee polymerization xagjirka xorta ah. Tallaabada ugu dambeysa ee falcelinta (polymerization xagjirka xorta ah) waa mid muhiim ah maadaama ay beddesho kala-goynta wejiga taagan. Falanqaynta curiyaha ayaa la sameeyay si loo hubiyo waxa ku jira kaarboonka marxaladahan taagan. Waxaa la arkay in kordhinta xaddiga styrene iyo waqtiga falcelinta ay kordhiso waxa ku jira kaarboonka marxaladda taagan iyo caksigeeda. SP-yada lagu diyaariyey isku-darka kala duwan ee styrene waxay leeyihiin culaysyo kaarboon oo kala duwan. Sidoo kale, marxaladahaan taagan waxaa la saaray tiirar bir ah oo aan miridh lahayn waxaana la hubiyay astaamahooda koromograafiga (xulashada, xallinta, qiimaha N, iwm.). Iyada oo lagu saleynayo tijaabooyinkan, waxaa la doortay hal-abuur la hagaajiyay oo loogu talagalay diyaarinta wejiga taagan ee PMP si loo bixiyo kala-goyn la xakameeyey iyo haynta wanaagsan ee falanqeeyaha.
Tiirka PMP waxaa sidoo kale lagu qiimeeyay falanqaynta shan isku-darka peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) iyadoo la adeegsanayo awoodda marxaladda guurguurta. 60/40 (v/v) ACN/biyo (0.1% TFA) oo ah heerka socodka 80 µl/daqiiqo. Xaaladaha ugu wanaagsan ee soo saarista (200,000 saxan/m), tirada saxannada aragtiyeed (N) tiirkiiba (100 × 1.8 mm) waa 20,000 ± 100. Qiimaha N ee saddexda tiir ee PMP waxaa lagu muujiyay Shaxda 3, chromatogram-yadana waxaa lagu muujiyay Jaantuska 5A. Falanqayn degdeg ah oo ku saabsan heerka socodka sare (700 µl/daqiiqo) tiir PMP ah, shan peptides ayaa la soo saaray hal daqiiqo gudahood, qiimaha N ee aadka u fiican oo ah 13,500 ± 330 tiirkiiba (100 x 1.8 mm dhexroor), oo u dhiganta 135,000 saxan/ m (Jaantuska 5B). Saddex tiir oo isku cabbir ah (dhexroor gudaha ah oo ah 100 x 1.8 mm) ayaa lagu buuxiyay saddex dufcadood oo kala duwan oo ah marxalad taagan oo PMP ah si loo tijaabiyo soo-saarista. Falanqeeyayaasha waxaa lagu duubay tiir kasta iyadoo la kala saarayo isku-darka tijaabada ee isku midka ah tiir kasta iyadoo la adeegsanayo xaaladaha ugu wanaagsan ee soo-saarista, tirada taarikada aragtiyeed ee N, iyo waqtiga haynta. Xogta soo-saarista ee tiirarka PMP waxaa lagu muujiyay Shaxda 4. Soo-saarista tiirka PMP waxay si fiican ula xiriirtay qiimayaasha %RSD ee aadka u hooseeya sida ku cad Shaxda 3.
Kala soocidda isku-darka peptide-ka ee tiirka PMP (B) iyo tiirka Ascentis Express RP-Amide (A), marxaladda moobaylka 60/40 ACN/H2O (TFA 0.1%), cabbirrada tiirka PMP (100 x 1.8 mm id), falanqaynta Kala soocidda isku-dhafka: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) iyo 5 (leucic acid enkephalin).
Tiir PMP ah (dhexroor gudaha ah 100 x 1.8 mm) ayaa lagu qiimeeyay kala soocidda tryptic hydrolyzate ee albumin-ka serum-ka aadanaha iyadoo loo marayo HPLC. Jaantuska koromatograamka ee Jaantuska 6 wuxuu muujinayaa in muunadaha si fiican loo kala saaray iyadoo xal aad u wanaagsan la helayo. Xalalka HSA waxaa lagu falanqeeyay iyadoo la isticmaalayo heerka socodka 100 μl/daqiiqo, marxalad guurguura oo ah 70/30 acetonitrile/biyo iyo 0.1% TFA. Kala-goynta HSA waxaa loo qaybiyay 17 meelood, sida lagu muujiyay chromatograamka (Jaantuska 6), oo u dhiganta 17 peptides. Waxtarka kala-soocidda meelaha ugu sarreeya ee shaqsiga ah iyo hydrolyzate-ka HSA ayaa la xisaabiyay qiimayaashana waxaa lagu muujiyay Shaxda 5.
HSA tryptic hydrolysates waxaa lagu kala saaray tiir PMP ah (dhexroor gudaha ah 100 x 1.8 mm), heerka socodka (100 μl/daqiiqo), wejiga wareega 60/40 acetonitrile/biyo, iyo 0.1% TFA.
halkaasoo L uu yahay dhererka tiirka, η waa daxalka wejiga guurguura, ΔP waa cadaadiska dambe ee tiirka, iyo u waa xawaaraha toosan ee wejiga guurguura. Daxalka tiirka PMP wuxuu ahaa 2.5 × 10–14 m2, heerka socodka wuxuu ahaa 25 µl/daqiiqo, 60/40 v/v ayaa la isticmaalay. ACN/biyo. Daxalka tiirka PMP (ID 100 × 1.8 mm) wuxuu la mid ahaa kii daraasaddeennii hore ee Ref.34. Daxalka tiirka ka buuxa walxaha dusha sare ee daloola waa 1.7 × 10 .6 µm, 2.5 × 10-14 m2 walxaha 5 µm43. Sidaa darteed, daxalka marxaladda PMP waxay la mid tahay daxalka walxaha xudunta u ah qolofta oo cabbirkoodu yahay 5 μm.
halkaasoo Wx uu yahay cufka tiirka oo ay ka buuxaan kloroform, Wy waa cufka tiirka oo ay ka buuxaan methanol, ρna waa cufnaanta dareeraha. Cufnaanta methanol (ρ = 0.7866) iyo kloroform (ρ = 1.484). Wadarta daloolka tiirka walxaha silica-C18 (100 × 1.8 mm ID)34 iyo tiirkayaga hore loo bartay ee C18-urea31 wuxuu ahaa 0.63 iyo 0.55, siday u kala horreeyaan. Tani waxay ka dhigan tahay in joogitaanka ligands-ka urea uu yareeyo marin-u-helka wejiga taagan. Dhanka kale, wadarta daloolka tiirka PMP (dhexroorka gudaha 100 × 1.8 mm) waa 0.60. Tiirarka PMP way ka yar yihiin marin-u-helka tiirarka ay ka buuxaan walxaha silica ee C18 sababtoo ah nooca C18 marxaladaha taagan, ligands-ka C18 waxay ku xiran yihiin walxaha silica ee silsiladaha toosan, halka noocyada polystyrene marxaladaha taagan, polymer qaro weyn ayaa laga sameeyaa hareeraha walxaha. Lakabka A. Tijaabo caadi ah, daloolka tiirka waxaa loo xisaabiyaa sidan soo socota:
Jaantuska 7A, B waxay muujinayaan jaantusyada Van Deemter ee tiirka PMP (id 100 x 1.8 mm) iyo tiirka Ascentis Express RP-Amide (id 100 x 1.8 mm) xaaladaha isku midka ah ee qulqulka, 60/40 ACN/H2O iyo 0 .1% TFA 20 µl/daqiiqo ilaa 800 µl/daqiiqo labada tiir. Qiimaha HETP ugu yar ee heerka socodka ugu fiican (80 µl/daqiiqo) waxay ahaayeen 2.6 µm iyo 3.9 µm tiirka PMP iyo tiirka Ascentis Express RP-Amide, siday u kala horreeyaan. Qiimaha HETP waxay muujinayaan in hufnaanta kala soocidda tiirka PMP (100 x 1.8 mm id) ay aad uga sarreyso tiirka Ascentis Express RP-Amide ee ganacsiga laga heli karo (100 x 1.8 mm id). Garaafka van Deemter ee Jaantuska 7(A) wuxuu muujinayaa in hoos u dhaca qiimaha N uusan si weyn uga sarreyn qulqulka sii kordhaya marka la barbar dhigo daraasaddeennii hore. Waxtarka kala-soocidda sare ee tiirka PMP (id 100 × 1.8 mm) marka la barbar dhigo tiirka Ascentis Express RP-Amide waxay ku salaysan tahay qaabka iyo cabbirka walxaha la hagaajiyay iyo habka xirxirida tiirka casriga ah ee loo isticmaalo shaqada hadda jirta34.
(A) Sawirka Van Deemter (HETP iyo xawaaraha toosan ee wejiga moobaylka) oo laga helay tiirka PMP (id 100 x 1.8 mm) ee 60/40 ACN/H2O oo leh 0.1% TFA. (B) Sawirka Van Deemter (HETP iyo xawaaraha toosan ee wejiga moobaylka) oo laga helay tiirka Ascentis Express RP-Amide (id 100 x 1.8 mm) ee 60/40 ACN/H2O oo leh 0.1% TFA.
Marxalad taagan oo ka mid ah polystyrene isku dhafan ayaa la diyaariyey oo la qiimeeyay si loo kala saaro isku darka peptides-ka synthetic-ga ah iyo tryptic hydrolyzate-ka tryptic ee albumin-ka serum-ka aadanaha (HSA) ee chromatography-ga dareeraha ah ee waxqabadka sare leh. Waxqabadka chromatography-ga ee tiirarka PMP ee isku darka peptide-ga waa mid aad u fiican marka la eego hufnaanta kala-soocidda iyo xallinta. Hufnaanta kala-soocidda ee la hagaajiyay ee tiirarka PMP waxay sabab u tahay dhowr sababood sida cabbirka walxaha silica iyo cabbirka daloolka, isku-darka la xakameeyey ee marxaladaha taagan, iyo agabka baakadaha tiirarka ee adag. Marka lagu daro hufnaanta kala-soocidda sare, faa'iido kale oo ka mid ah wejigan taagan waa cadaadiska dambe ee tiirka hoose ee heerarka socodka sare. Tiirarka PMP aad bay u soo saari karaan waxaana loo isticmaali karaa in lagu falanqeeyo isku-darka peptides-ka iyo dheefshiidka tryptic ee borotiinnada kala duwan. Waxaan damacsannahay inaan u isticmaalno tiirkan kala-soocidda isku-dhafka noolaha firfircoon ee alaabada dabiiciga ah, soo-saarista dhirta daawada iyo boqoshaada ee chromatography-ga dareeraha ah. Mustaqbalka, tiirarka PMP sidoo kale waxaa lagu qiimeyn doonaa kala-soocidda borotiinada iyo unugyada difaaca jirka ee monoclonal.
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