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Walxaha silica ee daloola leh waxaa lagu diyaariyey hab sol-gel ah iyadoo wax laga beddelayo si loo helo walxaha macroporous. Walxahaan waxaa laga soo saaray iyadoo la beddelayo polymerization silsiladda kala-goynta ee la beddeli karo (RAFT) oo leh N-phenylmaleemide-methylvinylisocyanate (PMI) iyo styrene si loo diyaariyo isku-darka N-phenylmaleemide ee wejiga taagan ee polystyrene (PMP). Tiirarka birta aan miridhku lahayn ee cidhiidhiga ah (100 × 1.8 mm id) waxaa lagu duubay baakad slurry ah. Kala-soocidda tiirka PMP ee isku-darka peptide oo ka kooban shan peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) waxqabadka chromatographic) iyo dheefshiidka trypsin ee albumin-ka serum-ka aadanaha (HAS). Xaaladaha ugu wanaagsan ee nadiifinta, tirada saxanka aragtiyeed ee isku-darka peptide-ka waa mid aad u sarreysa ilaa 280,000 oo saxan/m². Marka la barbardhigo waxqabadka kala-soocidda tiirka la horumariyey. Iyada oo la adeegsanayo tiirka ganacsiga ee Ascentis Express RP-Amide, waxaa la arkay in waxqabadka kala-soocidda ee tiirka PMP uu ka sarreeyay tiirka ganacsiga marka loo eego hufnaanta kala-soocidda iyo xallinta.
Sannadihii ugu dambeeyay, warshadaha dawooyinka bayoolojiga ah waxay noqdeen suuq caalami ah oo sii kordhaya iyadoo ay si weyn u kordheen saamiga suuqa. Iyadoo ay jirto kororka qarxa ee warshadaha dawooyinka bayoolojiga ah1,2,3, falanqaynta peptides iyo borotiinnada ayaa aad loo doonayaa. Marka laga soo tago peptide-ka bartilmaameedka ah, dhowr wasakh ayaa la soo saaraa inta lagu jiro isku-darka peptide, sidaas darteed waxay u baahan tahay nadiifinta chromatographic si loo helo peptides-ka daahirnimada la rabo. Falanqaynta iyo qeexidda borotiinnada ku jira dareerayaasha jirka, unugyada iyo unugyada waa hawl aad u adag sababtoo ah tirada badan ee noocyada laga yaabo in la ogaado ee hal muunad. In kasta oo spectrometry-ga tiro-badan uu yahay qalab wax ku ool ah oo loogu talagalay peptide iyo taxanaha borotiinka, haddii muunadaha noocaas ah lagu duro spectrometer-ka tiro-badan hal mar, kala-soociddu ma noqon doonto mid ku habboon. Dhibaatadan waxaa lagu yareyn karaa iyadoo la hirgelinayo kala-soocidda chromatography-ga dareeraha ah (LC) ka hor falanqaynta MS, taas oo yarayn doonta tirada falanqeeyayaasha galaya spectrometer-ka tiro-badan waqti cayiman4,5,6. Intaa waxaa dheer, inta lagu jiro kala-soocidda marxaladda dareeraha ah, falanqeeyayaasha waxaa diiradda lagu saari karaa gobollada cidhiidhiga ah, sidaas darteedna diiradda la saarayo falanqeeyayaashan iyo hagaajinta ogaanshaha MS. xasaasiyad. Koromagrafiga dareeraha ah (LC) ayaa si weyn u horumaray tobankii sano ee la soo dhaafay wuxuuna noqday farsamo caan ah oo ku saabsan falanqaynta borotiinka7,8,9,10.
Koromagrafiga dareeraha ee wareega (RP-LC) si weyn ayaa loogu isticmaalaa nadiifinta iyo kala-soocidda isku-darka peptide iyadoo la adeegsanayo silica octadecyl-modified (ODS) oo ah wejiga taagan11,12,13. Si kastaba ha ahaatee, marxaladaha taagan ee RP ma bixiyaan kala-soocid ku filan oo peptides iyo borotiinno ah sababtoo ah qaab-dhismeedkooda adag iyo dabeecadda amphiphilic 14,15. Sidaa darteed, marxaladaha taagan ee si gaar ah loogu talagalay ayaa looga baahan yahay in lagu falanqeeyo peptides iyo borotiinnada leh qaybaha polar iyo kuwa aan polar ahayn si ay ula falgalaan oo ay u hayaan falanqeeyayaashan16. Koramagrafiga qaabka isku-dhafan, kaas oo bixiya isdhexgalka qaababka badan, wuxuu noqon karaa beddel RP-LC si loo kala saaro peptides, borotiinnada, iyo isku-darka kale ee adag. Dhowr marxaladood oo isku-dhafan oo taagan ayaa la diyaariyey, tiirarkana waxaa lagu buuxiyay marxaladahan ayaa loo isticmaalay kala-soocidda peptide iyo borotiinka17,18,19,20,21. Marxaladaha isku-dhafan ee qaabka isku-dhafan (WAX/RPLC, HILIC/RPLC, is-dhexgalka polar/RPLC) waxay ku habboon yihiin peptide iyo borotiin kala-soocid ay sabab u tahay joogitaanka labada kooxood ee kala-goysyada iyo kuwa aan kala-goysyada ahayn22,23,24,25,26,27,28. Sidoo kale, marxaladaha is-dhexgalka ee kala-goysyada oo leh kooxo isku-xidhan oo isku-xidhan ayaa muujinaya awood kala-soocid wanaagsan iyo xulasho gaar ah oo loogu talagalay falanqeeyayaasha kala-goysyada iyo kuwa aan kala-goysyada ahayn, maadaama kala-soociddu ay ku xiran tahay isdhexgalka u dhexeeya falanqaynta iyo wejiga taagan. Isdhexgalka qaababka badan 29, 30, 31, 32. Dhawaan, Zhang et al. 30 waxay diyaarisay marxalad polyamine ah oo go'an oo dodecyl ah waxayna si guul leh u kala saartay hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, iyo dhowr falanqeeye kale. Dhex-dhexaadiyaha polar-ka wuxuu leeyahay labadaba kooxo polar iyo kuwa aan polar-ka ahayn, sidaa darteed waxaa loo isticmaali karaa in lagu kala saaro peptides iyo borotiinno leh labadaba qaybo hydrophobic iyo hydrophilic ah. Tiirarka polar-ka ku jira (tusaale ahaan, tiirarka C18 ee amide-ku ku jira) ayaa ganacsi ahaan laga heli karaa magaca ganacsiga ee tiirarka Ascentis Express RP-Amide, laakiin tiirarkan waxaa loo isticmaalaa falanqaynta amine 33 oo keliya.
Daraasadda hadda socota, marxalad taagan oo polar-embedded stained (N-phenylmaleimide-embedded polystyrene) ayaa la diyaariyey oo lagu qiimeeyay kala soocidda peptides iyo dheefshiidka trypsin ee HSA. Marxaladda taagan waxaa la diyaariyey iyadoo la adeegsanayo istaraatiijiyadda soo socota. Qaybaha silica ee daloola leh ayaa la diyaariyey iyadoo la raacayo habkii lagu bixiyay daabacaaddeennii hore iyadoo wax laga beddelay hab-raaca diyaarinta. Saamiga urea, polyethylene glycol (PEG), TMOS, aashitada biyaha ayaa la hagaajiyay si loo diyaariyo walxaha silica ee leh cabbir weyn oo dalool ah. Marka labaad, ligand cusub, phenylmaleimide-methyl vinyl isocyanate, ayaa la sameeyay waxaana loo isticmaalay in laga soo saaro walxaha silica si loo diyaariyo marxalad taagan oo polar ah. Marxaladda taagan ee ka dhalatay waxaa lagu duubay tiir bir ah oo aan miridh lahayn (100 × 1.8 mm id) iyadoo la adeegsanayo nidaamka baakadka ee la hagaajiyay. Baakaynta tiirka waxaa lagu caawiyaa gariir farsamo si loo hubiyo in sariir isku mid ah lagu sameeyay tiirka. Qiimee kala soocidda tiirka xiran ee isku darka peptide oo ka kooban shan peptides; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) iyo Trypsin dheefshiidka albumin-ka serum-ka aadanaha (HAS). Isku darka peptide-ka iyo dheefshiidka trypsin ee HSA ayaa la arkay inay kala soocaan xallin iyo hufnaan wanaagsan. Waxqabadka kala-soocidda tiirka PMP waxaa la barbar dhigay kan tiirka Ascentis Express RP-Amide. Labada peptide-ka iyo borotiinnada waxaa la arkay inay si fiican u xallisan yihiin oo hufan yihiin tiirka PMP, kaasoo ka hufan tiirka Ascentis Express RP-Amide.
PEG (Polyethylene Glycol), Urea, Acetic Acid, Trimethoxy Orthosilicate (TMOS), Trimethyl Chlorosisane (TMCS), Trypsin, Human Serum Albumin (HSA), Ammonium Chloride, Urea, Hexane Methyldisilazane (HMDS), Methacryloyl Chloride (MC), Styrene, 4-Hydroxy-TEMPO, Benzoyl Peroxide (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, iyo Acetone Waxaa laga soo iibsaday Sigma-Aldrich (St. Louis, MO, 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, ka dibna 24 mL oo TMOS ah ayaa lagu daray xaaladaha barafka qabow. 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. Biyaha ayaa la shubay, haraadigana waxaa lagu qalajiyey 70 ° C muddo 12 saacadood ah. Cufnaanta jilicsan ee la qalajiyey si siman ayaa loogu shiiday foorno waxaana lagu shiiday 550 ° C muddo 12 saacadood ah. Saddex dufcadood ayaa la diyaariyey oo lagu sifeeyay si loo baaro soo-saarista cabbirka walxaha, cabbirka daloolka iyo bedka dusha sare.
Wax ka beddelka dusha sare ee walxaha silica oo leh ligand phenylmaleemide-methylvinylisocyanate (PCMP) oo ay ku xigto polymerization radial oo leh styrene, waxaa la diyaariyey isku-darka koox-kooxeed oo polar ah. Marxaladda joogtada ah ee isku-darka iyo silsiladaha polystyrene. Habka diyaarinta waxaa lagu sharraxay hoos.
N-phenylmaleimide (200 mg) iyo methyl vinyl isocyanate (100 mg) ayaa lagu milmay toluene qalalan, 0.1 mL oo ah 2,2′-azoisobutyronitrile (AIBN) ayaa lagu daray dhalada falcelinta si loo diyaariyo phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP). Isku darka waxaa lagu kululeeyay 60°C muddo 3 saacadood ah, 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), la walaaqay oo lagu shubay dhalo hoose oo wareegsan oo 500 mL ah muddo 10 daqiiqo ah. PMCP (10 mg) ayaa lagu milmay toluene waxaana lagu daray hoos u dhac ku yimaada dhalada falcelinta iyada oo loo marayo dalool hoos u dhacaya. Isku darka waxaa dib loogu celiyey 100 ° C muddo 8 saacadood ah, la sifeeyay oo lagu dhaqay acetone waxaana lagu qalajiyey 60 ° C muddo 3 saacadood ah. Kadib, Walxaha silica ee PMCP-ku xiran (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, 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 60°C habeenkii. Nidaamka falcelinta guud waxaa lagu muujiyey Jaantuska 1.
Shaybaarrada waxaa laga saaray 393 K muddo 1 saac ah si loo helo cadaadis haraaga ah oo ka yar 10-3 Torr. Xaddiga N2 ee lagu nuugay cadaadis qaraabo ah oo ah P/P0 = 0.99 ayaa loo isticmaalay in lagu go'aamiyo mugga daloolka guud. Qaab-dhismeedka walxaha silica ee qaawan iyo kuwa ligand-ka ku xiran ayaa lagu baaray mikroskoob elektaroonik ah oo sawiraya (Hitachi High Technologies, Tokyo, Japan). Shaybaarrada qalalan (walxaha silica qaawan iyo walxaha silica ee ligand-ku xiran) ayaa la saaray tiir aluminium ah iyadoo la isticmaalayo cajalad kaarboon ah oo dhejis ah. Dahabka waxaa lagu dahaadhay muunadaha iyadoo la isticmaalayo dahaadh xoqan oo Q150T ah, lakabka 5 nm Au ahna waxaa lagu shubay muunadaha. Tani waxay wanaajisaa hufnaanta habka iyadoo la isticmaalayo danab hoose waxayna bixisaa hadhuudh fiican, xoqid qabow. Falanqeeye elektaroonik ah oo Thermo Electron ah (Waltham, MA, USA) Flash EA1112 ayaa loo isticmaalay falanqaynta elektaroonik. Falanqeeye cabbirka walxaha Malvern (Worcestershire, UK) Mastersizer 2000 ayaa loo isticmaalay in lagu helo qaybinta cabbirka walxaha. Silica qaawan Walxaha iyo walxaha silica ee ligand-ku xidhan (5 mg midkiiba) ayaa lagu kala firdhiyey 5 mL oo isopropanol ah, oo lagu shubay sonicated muddo 10 daqiiqo ah, lagu shubay 5 daqiiqo, ka dibna la dhigay kursiga indhaha ee Mastersizer. Falanqaynta thermogravimetric waxaa lagu sameeyay heer ah 5 °C daqiiqaddii heerkul u dhexeeya 30 ilaa 800 °C.
Tiirar bir ah oo aan miridh lahayn oo muraayad lagu dahaadhay oo cabbirkoodu yahay (100 × 1.8 mm id) ayaa lagu duubay iyadoo la adeegsanayo habka baakadaha slurry, iyadoo la adeegsanayo isla habka loo isticmaalay Ref. 31. Tiir bir ah oo aan miridh lahayn (oo muraayad ku dahaaran, 100 × 1.8 mm id) oo leh qalab ka soo baxa oo ka kooban 1 µm frit ayaa lagu xiray baakad slurry ah (Alltech Deerfield, IL, USA). Diyaari slurry marxalad taagan adigoo ku xiraya 150 mg oo marxalad taagan ah 1.2 mL oo methanol ah oo u dir tiirka kaydinta. Methanol waxaa loo isticmaalay dareeraha slurry-ka iyo sidoo kale dareeraha riixitaanka. Buuxi tiirka si isdaba joog ah adigoo ku dhejinaya cadaadisyo ah 100 MP muddo 10 daqiiqo ah, 80 MP muddo 15 daqiiqo ah, iyo 60 MP muddo 30 daqiiqo ah. Inta lagu jiray baakadaynta, gariir farsamo ayaa lagu dabaqay laba ruxe oo tiir GC ah (Alltech, Deerfield, IL, USA) si loo hubiyo xidhmo isku mid ah oo tiirka ah. Xidh baakadka slurry-ka oo si tartiib ah u sii daa cadaadiska si looga hortago waxyeello kasta oo ku jirta tiirka. Ka jar tiirka qaybta baakadka slurry-ka oo ku xidh qalab kale oo ku habboon gelitaanka iyo nidaamka LC si aad u hubiso waxqabadkiisa.
Bamka LC (10AD Shimadzu, Japan), irbad (Valco (USA) C14 W.05) oo leh wareeg duritaan 50nL ah, degasser-ka xuubka (Shimadzu DGU-14A), daaqadda xididka UV-VIS ayaa la dhisay Qalabka ogaanshaha µLC ee Gaarka ah (UV-2075) iyo tiirar yar yar oo galaas ku dahaaran. Isticmaal tuubooyin isku xira oo aad u cidhiidhi ah oo gaaban si loo yareeyo saameynta ballaarinta xarigga dheeraadka ah. Ka dib markii la baakadeeyay, xididdada dhiigga (50 μm id 365 iyo xididdada dhiigga ee yareeya (50 μm) ayaa lagu rakibay meesha laga saaro 1/16″ ee midowga yareeya. Ururinta xogta iyo habaynta koromografiga waxaa lagu sameeyay iyadoo la adeegsanayo software Multichro 2000. La socodka 254 nm Analytes waxaa lagu tijaabiyay nuugista UV. Xogta koromografiga waxaa falanqeeyay OriginPro8 (Northampton, MA).
Albumin laga soo saaray seeramka aadanaha, budada lyophilized, ≥ 96% (electrophoresis gel agarose) 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 lagu damiyo 1 mL oo ah 0.1% TFA. Shaandhee xalka oo ku kaydi heerkul ka hooseeya 4°C.
Kala soocidda isku-darka peptide iyo dheefshiidka trypsin ee HSA ayaa si gaar ah loogu qiimeeyay tiirarka PMP. Hubi kala soocidda isku-darka peptide iyo dheefshiidka trypsin ee HSA iyadoo loo marayo tiirka PMP oo isbarbardhig natiijooyinka tiirka Ascentis Express RP-Amide. Lambarka saxanka aragtida waxaa loo xisaabiyaa sidan soo socota:
Sawirada SEM ee walxaha silica qaawan iyo walxaha silica qaawan ee ligand-ku xidhan ayaa lagu muujiyay Jaantuska 2. Sawirada SEM ee walxaha silica qaawan (A, B) waxay muujinayaan, marka la barbardhigo daraasadihii hore, walxahani waa kuwo wareegsan oo walxaha ay dheer yihiin ama ay leeyihiin isku dheelitir aan caadi ahayn. Dusha sare ee walxaha silica qaawan ee ligand-ku xidhan (C, D) way ka siman tahay tan walxaha silica qaawan, taasoo laga yaabo inay sabab u tahay dahaarka silsiladaha polystyrene ee dusha sare ee walxaha silica.
Sawirrada mikroskoobka elektarooniga ah ee walxaha silica ee qaawan (A, B) iyo walxaha silica ee ligand-ku xidhan (C, D).
Qaybinta cabbirka walxaha ee walxaha silica ee qaawan iyo walxaha silica ee ligand-ku xidhan ayaa lagu muujiyay Jaantuska 3(A). Qaloocyada qaybinta cabbirka walxaha ku salaysan mugga ayaa muujiyay in cabbirka walxaha silica uu kordhay ka dib wax ka beddelka kiimikada (Jaantuska 3A). Xogta qaybinta cabbirka walxaha ee walxaha silica ee daraasadda hadda jirta iyo daraasaddii hore ayaa lagu barbar dhigay Shaxda 1(A). Cabbirka walxaha ku salaysan mugga, d(0.5), ee PMP waa 3.36 μm, marka la barbar dhigo daraasaddeenna hore oo leh qiimaha ad(0.5) ee 3.05 μm (walxaha silica ee polystyrene-ku xidhan)34. Dufcaddani waxay lahayd qaybin cabbir walxaha ah oo cidhiidhi ah marka loo eego daraasaddeenna hore sababtoo ah saamiga kala duwan ee PEG, urea, TMOS, iyo acetic acid ee isku darka falcelinta. Cabbirka walxaha ee wejiga PMP wuxuu wax yar ka weyn yahay kan wejiga walxaha silica ee polystyrene-ku xidhan ee aan hore u barannay. Tani waxay ka dhigan tahay in shaqada dusha sare ee walxaha silica ee leh styrene ay kaliya dhigtay lakab polystyrene ah (0.97 µm) dusha sare ee silica, halka Marxaladda PMP dhumucda lakabka waxay ahayd 1.38 µm.
Qaybinta cabbirka walxaha (A) iyo qaybinta cabbirka daloolka (B) ee walxaha silica qaawan iyo walxaha silica ee ligand-ku xiran.
Cabbirka daloolka, mugga daloolka iyo bedka dusha sare ee walxaha silica ee daraasadda hadda socota waxaa lagu bixiyay Shaxda 1(B). Astaamaha PSD ee walxaha silica qaawan iyo walxaha silica xiran ee ligand-ka ku xiran waxaa lagu muujiyay Jaantuska 3(B). Natiijooyinku waxay la mid yihiin daraasaddeennii hore. Cabbirka daloolka ee walxaha silica qaawan iyo kuwa ligand-ka ku xiran waa 310 iyo 241, siday u kala horreeyaan, taas oo tilmaamaysa in cabbirka daloolka uu hoos u dhacayo 69 ka dib wax ka beddelka kiimikada, sida lagu muujiyey Shaxda 1(B), isbeddelka qaloocana waxaa lagu muujiyay Jaantuska 3(B). Sidoo kale, mugga daloolka ee walxaha silica wuxuu hoos uga dhacay 0.67 ilaa 0.58 cm3/g ka dib wax ka beddelka kiimikada. Bedka gaarka ah ee walxaha silica ee hadda la bartay waa 116 m2/g, taas oo la mid ah daraasaddeennii hore (124 m2/g). Sida lagu muujiyey Shaxda 1(B), bedka dusha sare (m2/g) ee walxaha silica sidoo kale wuxuu hoos uga dhacay 116 m2/g ilaa 105 m2/g ka dib markii wax laga beddelay kiimikada.
Natiijooyinka falanqaynta walxaha ee marxaladda taagan waxaa lagu muujiyay Shaxda 2. Rarka kaarboonka ee marxaladda taagan ee hadda jirta waa 6.35%, taas oo ka hooseysa rarka kaarboonka ee daraasaddeenna hore (walxaha silica ee polystyrene ku xiran, 7.93%35 iyo 10.21%, siday u kala horreeyaan) 42. Rarka kaarboonka ee marxaladda taagan ee hadda jirta waa mid hooseeya, Sababtoo ah diyaarinta SP-ga hadda jira, marka lagu daro styrene, qaar ka mid ah ligands-ka polar sida phenylmaleemide-methylvinylisocyanate (PCMP) iyo 4-hydroxy-TEMPO ayaa la isticmaalay. Boqolkiiba miisaanka nitrogen ee marxaladda taagan ee hadda jirta waa 2.21%, marka la barbar dhigo 0.1735 iyo 0.85% miisaanka nitrogen ee daraasadihii hore, siday u kala horreeyaan. Tani waxay ka dhigan tahay in boqolkiiba boqol ee nitrogen ay ka sarreyso marxaladda taagan ee hadda jirta sababtoo ah phenylmaleemide. Sidoo kale, rarka kaarboonka ee alaabada (4) iyo (5) waxay ahaayeen 2.7% iyo 2.9%, siday u kala horreeyaan, halka rarka kaarboonka Badeecada ugu dambeysa (6) waxay ahayd 6.35%, sida ku cad Shaxda 2. Miisaanka oo yaraada waxaa lagu hubiyey marxaladda PMP ee taagan, qalooca TGA-na waxaa lagu muujiyey Jaantuska 4. Qalooca TGA wuxuu muujinayaa miisaan lumis ah oo ah 8.6%, kaas oo si fiican ula jaanqaadaya rarista kaarboonka (6.35%) sababtoo ah ligands-ku waxay ka kooban yihiin C oo keliya laakiin sidoo kale N, O, iyo H.
Ligand-ka phenylmaleemide-methylvinylisocyanate waxaa loo doortay wax ka beddelka dusha sare ee walxaha silica sababtoo ah waxay leedahay kooxo polar phenylmaleemide iyo kooxo vinylisocyanate ah. Kooxaha isocyanate ee Vinyl waxay si dheeraad ah uga fal celin karaan styrene iyagoo nool polymerization xagjir ah. Sababta labaad waa in la geliyo koox leh isdhexgal dhexdhexaad ah oo 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 waxayna beddeli kartaa kala-goynta wejiga taagan. Falanqaynta walxaha ayaa la sameeyay si loo hubiyo rarista kaarboon ee marxaladahan taagan. Waxaa la arkay in kordhinta xaddiga styrene iyo waqtiga falcelinta ay kordhisay rarista kaarboon ee marxaladda taagan iyo caksigeeda. SP-yada lagu diyaariyey isku-darka kala duwan ee styrene waxay leeyihiin rarista kaarboon oo kala duwan. Mar labaad, ku rid marxaladahan taagan tiirar bir ah oo aan daxal lahayn oo hubi Waxqabadka koromografiga (xulashada, xallinta, qiimaha N, iwm.). Iyada oo lagu saleynayo tijaabooyinkan, qaab-dhismeed la habeeyay ayaa la doortay si loo diyaariyo wejiga taagan ee PMP si loo hubiyo kala-goynta la xakameeyey iyo haynta falanqeeye wanaagsan.
Shan isku-darka peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) ayaa sidoo kale lagu qiimeeyay iyadoo la adeegsanayo tiir PMP ah iyadoo la adeegsanayo marxalad guurguurta; 60/40 (v/v) acetonitrile/biyo (0.1% TFA) oo ah heerka socodka 80 μL/daqiiqo. Xaaladaha ugu wanaagsan ee qulqulka, tirada saxanka aragtiyeed (N) tiirkiiba (100 × 1.8 mm id) waa 20,000 ± 100 (200,000 saxan/m²). Shaxda 3 waxay bixisaa qiimayaasha N ee saddexda tiir ee PMP iyo chromatogram-yada waxaa lagu muujiyay Jaantuska 5A. Falanqaynta degdega ah ee tiirka PMP ee heerka socodka sare (700 μL/daqiiqo), shan peptides ayaa la soo saaray hal daqiiqo gudahood, qiimayaasha N aad bay u fiicnaayeen, 13,500 ± 330 tiirkiiba (100 × 1.8 mm id), Waxay la mid tahay 135,000 saxan/m (Jaantuska 5B). Saddex tiir oo isku cabbir ah (100 × 1.8 mm id) waxaa lagu buuxiyay saddex marxaladood oo kala duwan oo PMP ah oo taagan si loo hubiyo Dib-u-soo-saarid. Isu-ururinta falanqaynta tiir kasta waxaa lagu duubay iyadoo la adeegsanayo xaaladaha ugu wanaagsan ee soo-saarid iyo tirada taarikada aragtiyeed N iyo waqtiga haynta si loo kala saaro isku-darka tijaabada ee tiir kasta. Xogta soo-saarista tiirarka PMP waxaa lagu muujiyay Shaxda 4. Soo-saarista tiirka PMP waxay si fiican ula xiriirtaa qiimayaasha %RSD ee aadka u hooseeya, sida ku cad Shaxda 3.
Kala soocidda isku darka peptide ee tiirka PMP (B) iyo tiirka Ascentis Express RP-Amide (A); marxaladda guurguura 60/40 ACN/H2O (TFA 0.1%), cabbirrada tiirka PMP (100 × 1.8 mm id); falanqayn Nidaamka soo saarista ee isku-dhafka: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) iyo 5 (leucine) acid enkephalin)).
Tiir PMP ah (100 × 1.8 mm id) ayaa lagu qiimeeyay kala soocidda dheefshiidka tryptic ee albumin-ka serum-ka aadanaha ee ku jira koromografiga dareeraha ee waxqabadka sare leh. Koromografiga Jaantuska 6 wuxuu muujinayaa in muunaddu si fiican u kala soocantahay oo xallintu aad u wanaagsan tahay. Qodobbada HSA waxaa lagu falanqeeyay iyadoo la isticmaalayo heerka socodka 100 µL/daqiiqo, wejiga wareega 70/30 acetonitrile/biyo iyo 0.1% TFA. Sida lagu muujiyay koromografiga (Jaantuska 6), dheefshiidka HSA waxaa loo qaybiyay 17 meelood oo u dhigma 17 peptides. Waxtarka kala soocida ee ugu sarreeya ee ku jira dheefshiidka HSA ayaa la xisaabiyay qiimayaashana waxaa lagu bixiyay Jadwalka 5.
Jaantuska tijaabada ah ee HSA (100 × 1.8 mm id) ayaa lagu kala saaray tiirka PMP; heerka socodka (100 µL/daqiiqo), wejiga wareega 60/40 acetonitrile/biyo oo leh 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, waxaana la isticmaalay 60/40 v/v ACN/biyo. Daxalka tiirka PMP (100 × 1.8 mm id) wuxuu la mid ahaa kii daraasaddeenna hore Tixraaca 34. Daxalka tiirka oo ay ku jiraan walxo daloolo dusha sare leh waa: 1.7 × 10-15 walxaha 1.3 μm, 3.1 × 10-15 walxaha 1.7 μm, 5.2 × 10-15 iyo 2.5 × 10-14 m2 walxaha 2.6 μm Wixii walxo 5 μm ah 43. Sidaa darteed, daxalka marxaladda PMP waxay la mid tahay walxaha 5 μm ee qolofta xudunta ah.
halkaasoo Wx uu yahay miisaanka tiirka ay ku jiraan kloroform, Wy waa miisaanka tiirka ay ku jiraan methanol, iyo ρ waa cufnaanta dareeraha. Cufnaanta methanol (ρ = 0.7866) iyo kloroform (ρ = 1.484). Wadarta daloolada tiirarka SILICA PARTICLES-C18 (100 × 1.8 mm id) 34 iyo C18-Urea tiirarka 31 ee aan hore u barannay waxay ahaayeen 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 daloolada tiirka PMP (100 × 1.8 mm id) waa 0.60. Gudbinta tiirarka PMP way ka hooseysaa tiirarka ay ku jiraan walxaha silica ee C18-ku xiran sababtoo ah marxaladaha taagan ee nooca C18 ligands-ka C18 waxay ku xiran yihiin walxaha silica sida silsilado toosan, halka nooca polystyrene-ka ah ee taagan marxaladaha, lakabka polymer-ka A ee qaro weyn ayaa lagu sameeyaa hareeraha. Tijaabo caadi ah, daloolka tiirka waxaa loo xisaabiyaa sidan:
Jaantuska 7A,B wuxuu muujinayaa tiirka PMP (100 × 1.8 mm id) iyo tiirka Ascentis Express RP-Amide (100 × 1.8 mm id) iyadoo la adeegsanayo xaaladaha isku midka ah ee qulqulka (tusaale ahaan, 60/40 ACN/H2O iyo 0.1% TFA). ) ee sawirka van Deemter. Isku darka peptide-ka ee la xushay (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) ayaa lagu diyaariyey 20 µL/ Heerka socodka ugu yar ee labada tiir waa 800 µL/daqiiqo. Qiimaha HETP ugu yar ee heerka socodka ugu fiican (80 µL/daqiiqo) ee tiirka PMP iyo tiirka Ascentis Express RP-Amide waxay ahaayeen 2.6 µm iyo 3.9 µm, siday u kala horreeyaan. Qiimaha HETP wuxuu tilmaamayaa in hufnaanta kala soocidda tiirka PMP (100 × 1.8 mm id) ay aad uga fiican tahay tiirka Ascentis Express RP-Amide ee ganacsiga laga heli karo (100 × 1.8 mm id). Jaantuska van Deemter ee Jaantuska 7(A) wuxuu muujinayaa in hoos u dhaca qiimaha N oo leh socodka sii kordhaya uusan muhiim ahayn marka la barbar dhigo daraasaddeenna hore. Hufnaanta kala soocidda sare ee tiirka PMP (100 × 1.8 mm id) marka la barbar dhigo tiirka RP-Amide ee Ascentis Express wuxuu ku salaysan yahay horumarinta qaabka walxaha, cabbirka, iyo hababka xidhitaanka tiirarka ee adag ee loo isticmaalo shaqada hadda jirta34.
(A) sawirka van Deemter (HETP iyo xawaaraha toosan ee wejiga moobaylka) oo laga helay iyadoo la isticmaalayo tiir PMP ah (100 × 1.8 mm id) oo ku jira 60/40 ACN/H2O oo leh 0.1% TFA.(B) sawirka van Deemter (HETP iyo xawaaraha toosan ee wejiga moobaylka) oo laga helay iyadoo la isticmaalayo tiir Ascentis Express RP-Amide ah (100 × 1.8 mm id) oo ku jira 60/40 ACN/H2O oo leh 0.1% TFA.
Marxalad taagan oo polystyrene ah oo polar-ku dhex jira ayaa la diyaariyey oo la qiimeeyay si loo kala saaro isku-darka peptide-ka ee synthetic-ga ah iyo dheefshiidka trypsin ee albumin-ka serum-ka aadanaha (HAS) ee chromatography-ga dareeraha ah ee waxqabadka sare leh. Waxqabadka chromatography-ga ee tiirarka PMP ee isku-darka peptide-ka waa mid aad u fiican hufnaanta kala-soocidda iyo xallinta. Waxqabadka kala-soocidda ee la hagaajiyay ee tiirarka PMP waxay sabab u tahay sababo kala duwan, sida cabbirka walxaha iyo cabbirka daloolka ee walxaha silica, isku-darka la xakameeyey ee wejiga taagan, iyo xidhitaanka tiirarka adag. Marka lagu daro hufnaanta kala-soocidda sare, cadaadiska dambe ee tiirka hooseeya ee heerarka socodka sare waa faa'iido kale oo wejigan taagan ah. Tiirarka PMP waxay muujiyaan dib-u-soo-saar wanaagsan waxaana loo isticmaali karaa falanqaynta isku-darka peptide-ka iyo dheefshiidka trypsin ee borotiinnada kala duwan. Waxaan rabnaa inaan u isticmaalno tiirkan kala-soocidda alaabada dabiiciga ah, isku-dhafka noolaha ee dhirta daawada iyo soosaarka fungal ee chromatography-ga dareeraha ah. Mustaqbalka, tiirarka PMP sidoo kale waxaa lagu qiimeyn doonaa kala-soocidda borotiinnada iyo unugyada difaaca jirka ee monoclonal.
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