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Cov txheej txheem tshiab immunological thiab mass spectrometric rau kev tshuaj xyuas nyuaj ntawm cov oligosaccharides nyob rau hauv cov pob kws stover pretreated nrog AFEX. Lignocellulosic biomass yog ib qho kev xaiv ruaj khov rau cov roj fossil thiab siv dav los tsim cov biotechnologies rau kev tsim cov khoom xws li zaub mov, pub, roj thiab tshuaj lom neeg. Qhov tseem ceeb rau cov thev naus laus zis no yog kev tsim cov txheej txheem sib tw nqi rau kev hloov cov carbohydrates nyuaj uas muaj nyob rau hauv cov phab ntsa cell ntawm cov nroj tsuag mus rau hauv cov suab thaj yooj yim xws li glucose, xylose thiab arabinose. Vim tias lignocellulosic biomass yog tawv ncauj heev, nws yuav tsum tau raug rau kev kho mob thermochemical (piv txwv li, ammonia fiber exfoliation (AFEX), dilute acids (DA), ionic kua (IL)) thiab kev kho mob biological (piv txwv li, enzymatic hydrolysis thiab microbial fermentation) ua ke kom tau txais cov khoom xav tau. Txawm li cas los xij, thaum cov enzymes fungal lag luam siv rau hauv cov txheej txheem hydrolysis, tsuas yog 75-85% ntawm cov suab thaj soluble tsim yog monosaccharides, thiab cov seem 15-25% yog soluble, intractable oligosaccharides, uas tsis yog ib txwm muaj rau cov kab mob me me. Yav dhau los, peb tau ua tiav cais thiab ntxuav cov oligosaccharides uas yaj tau yooj yim siv kev sib xyaw ua ke ntawm carbon thiab diatomaceous earth separation thiab size exclusion chromatography, thiab tseem tshawb nrhiav lawv cov khoom enzyme inhibitory. Peb tau pom tias oligosaccharides uas muaj cov polymerization (DP) methylated uronic acid substitutions ntau dua rau kev ua tiav nrog cov enzyme blends lag luam dua li DP qis thiab neutral oligosaccharides. Ntawm no peb tshaj tawm kev siv ntau txoj hauv kev ntxiv, suav nrog glycan profiling siv monoclonal antibodies (mAbs) tshwj xeeb rau cov nroj tsuag biomass glycans los piav qhia txog glycan bonds hauv cov nroj tsuag cell phab ntsa thiab enzymatic hydrolysates, matrix-assisted laser desorption ionization, time-of-flight mass-spectrometry. . MALDI-TOF-MS) siv cov qauv-informative diagnostic peaks tau los ntawm spectroscopy tom qab secondary decay ntawm negative ions, gas chromatography thiab mass spectrometry (GC-MS) los piav qhia txog oligosaccharide bonds nrog thiab tsis muaj derivatization. Vim yog qhov me me ntawm oligosaccharides (DP 4–20), cov molecules no nyuaj rau siv rau mAb khi thiab tus cwj pwm. Txhawm rau kov yeej qhov teeb meem no, peb tau siv txoj kev tshiab biotin conjugation-based oligosaccharide immobilization uas tau sau npe rau feem ntau ntawm cov DP qis soluble oligosaccharides ntawm qhov microplate nto, uas tom qab ntawd tau siv rau hauv lub kaw lus mAb siab throughput rau kev tshuaj xyuas ligation tshwj xeeb. Txoj kev tshiab no yuav pab txhawb kev txhim kho ntawm cov kev ntsuas glycome siab throughput ntau dua yav tom ntej uas tuaj yeem siv los cais tawm thiab tus cwj pwm oligosaccharides uas muaj nyob hauv biomarkers rau lub hom phiaj kuaj mob.
Cov khoom siv biomass Lignocellulosic, uas muaj cov khoom ua liaj ua teb, hav zoov, nyom thiab ntoo, yog cov khoom siv rau kev tsim cov khoom siv bio-based, suav nrog cov khoom noj, pub tsiaj, roj thiab cov tshuaj ua ntej los tsim cov khoom muaj nqis siab dua1. Cov Carbohydrates (xws li cellulose thiab hemicellulose) uas muaj nyob rau hauv cov phab ntsa cell ntawm cov nroj tsuag raug depolymerized rau hauv monosaccharides los ntawm kev ua cov tshuaj lom neeg thiab biotransformation (xws li enzymatic hydrolysis thiab microbial fermentation). Cov kev kho ua ntej feem ntau suav nrog ammonia fiber expansion (AFEX), dilute acid (DA), ionic kua (IL), thiab steam explosion (SE), uas siv kev sib xyaw ua ke ntawm cov tshuaj lom neeg thiab cua sov los txo cov khoom siv lignocellulose los ntawm kev qhib cov phab ntsa cell ntawm cov nroj tsuag3,4. kev tawv ncauj ntawm cov khoom, 5. Enzymatic hydrolysis yog ua tiav ntawm cov khoom khov siab siv cov enzymes uas muaj cov carbohydrate ua lag luam (CAZymes) thiab microbial fermentation siv cov poov xab transgenic lossis cov kab mob los tsim cov roj bio-based thiab cov tshuaj lom neeg 6.
CAZymes hauv cov enzymes lag luam yog tsim los ntawm cov enzymes sib xyaw ua ke uas sib txuas cov carbohydrate-qab zib sib txuas ua ke los tsim cov monosaccharides2,7. Raws li peb tau tshaj tawm ua ntej, lub network nyuaj ntawm cov polymers aromatic ntawm lignin nrog carbohydrates ua rau lawv nyuaj heev, uas ua rau kev hloov pauv qab zib tsis tiav, sau 15-25% ntawm cov oligosaccharides sib deev uas tsis tsim tawm thaum lub sijhawm enzymatic hydrolysis ntawm cov biomass pretreated. Qhov no yog qhov teeb meem tshwm sim nrog ntau txoj kev biomass pretreatment. Qee qhov laj thawj rau qhov bottleneck no suav nrog enzyme inhibition thaum lub sijhawm hydrolysis, lossis tsis muaj lossis qis cov enzymes tseem ceeb uas yuav tsum tau tawg cov qab zib sib txuas hauv cov nroj tsuag biomass. Kev nkag siab txog cov khoom sib xyaw thiab cov yam ntxwv ntawm cov qab zib, xws li cov qab zib sib txuas hauv oligosaccharides, yuav pab peb txhim kho kev hloov pauv qab zib thaum lub sijhawm hydrolysis, ua rau cov txheej txheem biotechnological pheej yig sib tw nrog cov khoom lag luam roj av.
Kev txiav txim siab txog cov qauv ntawm cov carbohydrates yog qhov nyuaj thiab xav tau kev sib xyaw ua ke ntawm cov txheej txheem xws li kua chromatography (LC) 11, 12, nuclear magnetic resonance spectroscopy (NMR) 13, capillary electrophoresis (CE) 14, 15, 16 thiab mass spectrometry (MS) 17. , kaum yim. MS txoj kev xws li lub sijhawm-ntawm-dav hlau mass spectrometry nrog laser desorption thiab ionization siv matrix (MALDI-TOF-MS) yog ib txoj kev siv tau ntau yam rau kev txheeb xyuas cov qauv carbohydrate. Tsis ntev los no, Collision-Induced Dissociation (CID) tandem MS ntawm sodium ion adducts tau siv dav tshaj plaws los txheeb xyuas cov ntiv tes sib xws rau oligosaccharide txuas txoj haujlwm, anomeric configurations, sequences, thiab branching txoj haujlwm 20, 21.
Kev tshuaj xyuas Glycan yog ib qho cuab yeej zoo heev rau kev txheeb xyuas tob txog cov carbohydrate bonds22. Txoj kev no siv cov monoclonal antibodies (mAbs) uas tau coj mus rau cov nroj tsuag phab ntsa glycan ua cov probes kom nkag siab txog cov kev sib txuas ntawm cov carbohydrate nyuaj. Ntau tshaj 250 mAbs muaj thoob ntiaj teb, tsim tawm tsam ntau yam linear thiab branched oligosaccharides siv ntau yam saccharides24. Ntau cov mAbs tau siv dav los piav qhia txog cov qauv, cov khoom sib xyaw, thiab kev hloov kho ntawm cov nroj tsuag phab ntsa, vim tias muaj qhov sib txawv tseem ceeb nyob ntawm hom nroj tsuag, lub cev, hnub nyoog, theem kev loj hlob, thiab ib puag ncig kev loj hlob25,26. Tsis ntev los no, txoj kev no tau siv los nkag siab txog cov pej xeem vesicle hauv cov nroj tsuag thiab tsiaj txhu thiab lawv lub luag haujlwm hauv kev thauj mus los glycan raws li txiav txim siab los ntawm cov cim subcellular, theem kev loj hlob, lossis cov tshuaj ib puag ncig, thiab txiav txim siab txog kev ua haujlwm enzymatic. Qee cov qauv sib txawv ntawm glycans thiab xylans uas tau txheeb xyuas suav nrog pectin (P), xylan (X), mannan (M), xyloglucans (XylG), mixed bond glucans (MLG), arabinoxylan (ArbX), galactomannan (GalG), glucuronic acid-arabinoxylan (GArbX) thiab arabino-galactan (ArbG)29.
Txawm li cas los xij, txawm hais tias muaj tag nrho cov kev tshawb fawb no, tsuas yog ob peb txoj kev tshawb fawb tau tsom mus rau qhov xwm txheej ntawm oligosaccharide sib sau ua ke thaum lub sijhawm siab solids load (HSL) hydrolysis, suav nrog oligosaccharide tso tawm, oligomeric saw ntev hloov pauv thaum lub sijhawm hydrolysis, ntau yam qis DP polymers, thiab lawv cov nkhaus. kev faib tawm 30,31,32. Lub caij no, txawm hais tias kev tshuaj xyuas glycan tau ua pov thawj tias yog ib qho cuab yeej muaj txiaj ntsig rau kev tshuaj xyuas tag nrho ntawm cov qauv glycan, nws nyuaj rau kev soj ntsuam cov dej-soluble qis DP oligosaccharides siv cov txheej txheem antibody. Cov DP me dua nrog qhov hnyav molecular tsawg dua 5-10 kDa tsis khi rau ELISA phaj 33, 34 thiab raug ntxuav tawm ua ntej ntxiv antibody.
Ntawm no, thawj zaug, peb ua qauv qhia txog kev sim ELISA ntawm cov phaj avidin-coated siv cov tshuaj monoclonal antibodies, ua ke ib kauj ruam biotinylation rau cov soluble refractory oligosaccharides nrog kev tsom xam glycome. Peb txoj hauv kev rau kev tsom xam glycome tau lees paub los ntawm MALDI-TOF-MS thiab GC-MS raws li kev tsom xam ntawm kev sib txuas oligosaccharide siv trimethylsilyl (TMS) derivatization ntawm cov khoom qab zib hydrolyzed. Txoj hauv kev tshiab no tuaj yeem tsim los ua txoj hauv kev siab-throughput yav tom ntej thiab nrhiav kev siv dav dua hauv kev tshawb fawb biomedical35.
Kev hloov kho tom qab kev txhais lus ntawm cov enzymes thiab cov tshuaj tiv thaiv kab mob, xws li glycosylation,36 cuam tshuam rau lawv cov haujlwm biological. Piv txwv li, kev hloov pauv hauv glycosylation ntawm cov protein serum ua lub luag haujlwm tseem ceeb hauv kev mob caj dab, thiab kev hloov pauv hauv glycosylation yog siv ua cov cim kuaj mob37. Ntau yam glycans tau tshaj tawm hauv cov ntaub ntawv kom yooj yim tshwm sim hauv ntau yam kab mob, suav nrog cov kab mob inflammatory ntawm txoj hnyuv plab thiab siab, kab mob viral, ovarian, mis, thiab prostate cancer38,39,40. Kev nkag siab txog cov qauv ntawm glycans siv cov txheej txheem antibody-based glycan ELISA yuav muab kev ntseeg siab ntxiv rau kev kuaj mob yam tsis siv cov txheej txheem MS nyuaj.
Peb txoj kev tshawb fawb yav dhau los qhia tau hais tias cov oligosaccharides tawv ncauj tseem tsis tau hydrolyzed tom qab pretreatment thiab enzymatic hydrolysis (Daim Duab 1). Hauv peb cov haujlwm luam tawm yav dhau los, peb tau tsim ib txoj kev rho tawm cov pa roj carbon activated solid-phase los cais cov oligosaccharides los ntawm AFEX-pretreated pob kws stover hydrolyzate (ACSH) 8. Tom qab rho tawm thiab sib cais thawj zaug, cov oligosaccharides tau ntxiv fractionated los ntawm qhov loj me exclusion chromatography (SEC) thiab sau raws li qhov hnyav molecular. Cov monomers qab zib thiab oligomers tso tawm los ntawm ntau yam kev kho mob ua ntej tau soj ntsuam los ntawm kev tshuaj xyuas cov suab thaj. Thaum piv cov ntsiab lus ntawm cov suab thaj oligomers tau los ntawm ntau yam kev kho mob ua ntej, qhov muaj cov oligosaccharides tawv ncauj yog ib qho teeb meem tshwm sim hauv kev hloov pauv ntawm biomass mus rau monosaccharides thiab tuaj yeem ua rau txo qis cov suab thaj tsawg kawg 10-15% thiab txawm tias txog li 18%. Tebchaws Meskas. Txoj kev no yog siv rau kev tsim cov oligosaccharide fractions loj ntxiv. Cov ACH uas tshwm sim thiab nws cov feem tom qab nrog cov qhov hnyav molecular sib txawv tau siv los ua cov khoom siv sim rau tus cwj pwm ntawm oligosaccharides hauv cov haujlwm no.
Tom qab kev kho ua ntej thiab enzymatic hydrolysis, cov oligosaccharides uas nyob ruaj khov tseem tsis tau hydrolysed. Ntawm no (A) yog ib txoj kev sib cais oligosaccharide uas oligosaccharides raug rho tawm ntawm AFEX-pretreated pob kws stover hydrolysate (ACSH) siv lub txaj ntim ntawm cov pa roj carbon thiab diatomaceous av; (B) Txoj kev rau kev sib cais ntawm oligosaccharides. Cov oligosaccharides tau sib cais ntxiv los ntawm qhov loj me exclusion chromatography (SEC); (C) Saccharide monomers thiab oligomers tso tawm los ntawm ntau yam kev kho ua ntej (diluted acid: DA, ionic kua: IL thiab AFEX). Cov xwm txheej enzymatic hydrolysis: cov khoom khov siab ntawm 25% (w/w) (kwv yees li 8% glucan loading), 96 teev hydrolysis, 20 mg/g commercial enzyme loading (Ctec2:Htec2:MP-2:1:1 piv) thiab (D) Cov suab thaj monomers thiab oligomers ntawm glucose, xylose thiab arabinose tso tawm los ntawm AFEX pre-treated corn stover (ACS).
Kev tshuaj xyuas Glycan tau ua pov thawj tias yog ib qho cuab yeej pab tau rau kev tshuaj xyuas cov qauv ntawm glycans hauv cov extracts cais tawm ntawm cov khoom seem biomass khov. Txawm li cas los xij, cov saccharides uas yaj tau hauv dej tsis tshua muaj neeg sawv cev siv cov txheej txheem ib txwm no41 vim tias cov oligosaccharides uas hnyav molecular tsawg nyuaj rau immobilize ntawm ELISA phaj thiab raug ntxuav tawm ua ntej ntxiv cov tshuaj antibody. Yog li ntawd, rau kev khi thiab kev piav qhia ntawm cov tshuaj antibody, ib txoj kev biotinylation ib kauj ruam tau siv los txheej cov oligosaccharides uas yaj tau, tsis ua raws li cov phaj avidin-coated ELISA. Txoj kev no tau sim siv peb cov ACSH uas tau tsim ua ntej thiab ib feem raws li nws qhov hnyav molecular (lossis qib ntawm polymerization, DP). Ib kauj ruam biotinylation tau siv los ua kom oligosaccharide khi affinity los ntawm kev ntxiv biotin-LC-hydrazide rau qhov kawg txo qis ntawm carbohydrate (Daim duab 2). Hauv kev daws teeb meem, pawg hemiacetal ntawm qhov kawg txo qis reacts nrog pawg hydrazide ntawm biotin-LC-hydrazide los tsim cov hydrazone bond. Nyob rau hauv lub xub ntiag ntawm tus neeg sawv cev txo NaCNBH3, qhov hydrazone bond raug txo kom ruaj khov rau cov khoom kawg biotinylated. Nrog kev hloov kho ntawm qhov kawg txo qab zib, kev khi ntawm cov DP oligosaccharides qis rau ELISA phaj tau ua tau, thiab hauv peb txoj kev tshawb fawb qhov no tau ua tiav rau ntawm avidin-coated phaj siv glycan-targeted mAbs.
Kev tshuaj ntsuam cov monoclonal antibodies raws li ELISA rau biotinylated oligosaccharides. Ntawm no (A) ua ke biotinylation ntawm oligosaccharides thiab tom qab ntawd ELISA tshuaj ntsuam nrog glycan-targeted mAbs ntawm NeutrAvidin coated phaj thiab (B) qhia txog ib kauj ruam txheej txheem rau biotinylation ntawm cov khoom tshuaj tiv thaiv.
Cov phaj Avidin-coated nrog cov tshuaj oligosaccharide-conjugated tau ntxiv rau cov tshuaj thawj zaug thiab theem ob thiab ntxuav hauv lub teeb- thiab lub sijhawm-sensitive. Tom qab antibody binding tiav lawm, ntxiv TMB substrate los incubate lub phaj. Qhov tshuaj tiv thaiv thaum kawg tau nres nrog sulfuric acid. Cov phaj incubated tau soj ntsuam siv ELISA nyeem ntawv los txiav txim siab lub zog khi ntawm txhua lub antibody kom pom cov antibody-specific cross-linking. Rau cov ntsiab lus thiab cov kev ntsuas ntawm qhov kev sim, saib ntu sib xws "Cov Khoom Siv thiab Cov Txheej Txheem".
Peb ua qauv qhia txog qhov siv tau ntawm txoj kev tshiab no rau cov ntawv thov tshwj xeeb los ntawm kev piav qhia txog cov oligosaccharides uas yaj tau hauv ACSH nrog rau hauv cov feem pua ​​​​​​oligosaccharide nyoos thiab huv uas cais tawm ntawm lignocellulosic hydrolysates. Raws li pom hauv Daim Duab 3, cov xylans epitope-substituted feem ntau pom hauv ACSH siv cov txheej txheem bioacylated glycome assay feem ntau yog uronic (U) lossis methyluronic (MeU) thiab pectic arabinogalactans. Feem ntau ntawm lawv kuj tau pom hauv peb txoj kev tshawb fawb yav dhau los ntawm kev tshuaj xyuas cov glycans ntawm cov khoom tsis-hydrolyzed (UHS) 43.
Kev nrhiav pom cov recalcitrant oligosaccharide epitopes siv cov monoclonal antibody uas taw rau lub cell phab ntsa glycan. Qhov "neutral" fraction yog ACN fraction thiab qhov "acidic" fraction yog FA fraction. Cov xim liab ci dua ntawm daim ntawv qhia kub qhia txog cov ntsiab lus epitope siab dua, thiab cov xim xiav ci dua qhia txog keeb kwm yav dhau dawb paug. Cov nqi xim ntawm qhov nplai yog raws li cov nqi OD raw rau cov qauv N = 2. Cov epitopes tseem ceeb uas cov tshuaj tiv thaiv pom tau pom ntawm sab xis.
Cov qauv tsis yog cellulose no tsis tuaj yeem raug txiav los ntawm cov cellulases thiab hemicellulases feem ntau hauv cov tshuaj sib xyaw ua ke enzyme, uas suav nrog cov enzymes lag luam feem ntau siv. Yog li ntawd, cov enzymes pabcuam tshiab yog qhov xav tau rau lawv cov hydrolysis. Yog tsis muaj cov enzymes ntxiv uas tsis yog cellulose tsim nyog, cov non-cellulose bonds no tiv thaiv kev hloov pauv tag nrho rau monosaccharides, txawm tias lawv cov polymers qab zib niam txiv tau hydrolyzed ntau rau hauv cov khoom luv luv thiab yaj siv cov tshuaj sib xyaw ua ke enzyme lag luam.
Kev kawm ntxiv txog kev faib tawm ntawm lub teeb liab thiab nws lub zog khi tau qhia tias kev khi epitopes qis dua hauv cov feem pua ​​​​​​qab zib siab DP (A, B, C, DP txog 20+) dua li hauv cov feem pua ​​​​​​DP qis (D, E, F, DP) hauv dimers) (Daim duab 1). Cov khoom siv acid muaj ntau dua hauv cov epitopes tsis yog cellulose dua li cov khoom tsis muaj zog. Cov xwm txheej no yog sib xws nrog tus qauv pom hauv peb txoj kev tshawb fawb yav dhau los, qhov twg DP siab thiab cov kua qaub moieties tiv taus enzymatic hydrolysis ntau dua. Yog li ntawd, qhov muaj cov epitopes tsis yog cellulose glycan thiab U thiab MeU hloov pauv tuaj yeem pab txhawb rau kev ruaj khov ntawm oligosaccharides. Nws yuav tsum tau sau tseg tias kev khi thiab kev tshawb pom zoo tuaj yeem ua teeb meem rau DP qis oligosaccharides, tshwj xeeb tshaj yog tias epitope yog dimeric lossis trimeric oligosaccharide. Qhov no tuaj yeem sim siv cov oligosaccharides lag luam ntawm qhov ntev sib txawv, txhua tus muaj tsuas yog ib qho epitope uas khi rau ib qho mAb tshwj xeeb.
Yog li, kev siv cov tshuaj tiv thaiv kab mob tshwj xeeb qhia txog qee hom kev sib txuas lus tsis sib haum xeeb. Nyob ntawm hom tshuaj tiv thaiv kab mob siv, tus qauv ligation tsim nyog, thiab lub zog ntawm lub teeb liab uas nws tsim tawm (feem ntau thiab tsawg tshaj plaws), cov enzymes tshiab tuaj yeem txheeb xyuas thiab ntxiv semi-quantitatively rau cov enzyme sib xyaw rau kev ua tiav glycoconversion. Ua piv txwv li, kev tshuaj xyuas ntawm ACSH oligosaccharides, peb tuaj yeem tsim cov ntaub ntawv ntawm glycan bonds rau txhua yam khoom siv biomass. Nws yuav tsum tau sau tseg ntawm no tias qhov sib txawv ntawm cov tshuaj tiv thaiv kab mob yuav tsum tau coj mus rau hauv tus account, thiab yog tias lawv qhov sib txawv tsis paub, qhov no yuav tsim qee qhov teeb meem thaum piv cov teeb liab ntawm cov tshuaj tiv thaiv kab mob sib txawv. Tsis tas li ntawd, kev sib piv ntawm glycan bonds yuav ua haujlwm zoo tshaj plaws ntawm cov qauv rau tib lub antibody. Cov kev sib txuas lus tawv ncauj no tuaj yeem txuas rau CAZyme database, uas peb tuaj yeem txheeb xyuas cov enzymes, xaiv cov enzymes sib tw thiab sim rau cov enzymes tawg, lossis tsim cov kab ke microbial los qhia cov enzymes no rau kev siv hauv biorefineries44.
Txhawm rau soj ntsuam seb cov txheej txheem tiv thaiv kab mob ua tiav lwm txoj hauv kev rau kev piav qhia txog cov oligosaccharides uas muaj qhov hnyav molecular tsawg uas muaj nyob hauv lignocellulosic hydrolysates li cas, peb tau ua MALDI (Daim Duab 4, S1-S8) thiab kev tshuaj xyuas ntawm TMS-derived saccharides raws li GC-MS ntawm tib lub vaj huam sib luag (Daim Duab 5) oligosaccharide ib feem. MALDI yog siv los sib piv seb qhov kev faib tawm ntawm cov molecules oligosaccharide puas phim cov qauv npaj tseg. Ntawm daim duab 4 qhia MC ntawm cov khoom nruab nrab ACN-A thiab ACN-B. ACN-A kev tshuaj xyuas tau lees paub ntau yam pentose suab thaj xws li DP 4–8 (Daim Duab 4) txog DP 22 (Daim Duab S1), uas nws qhov hnyav sib xws rau MeU-xylan oligosaccharides. ACN-B kev tshuaj xyuas tau lees paub cov koob pentose thiab glucoxylan nrog DP 8-15. Hauv cov ntaub ntawv ntxiv xws li Daim Duab S3, FA-C acidic moiety mass distribution maps qhia txog ntau yam (Me)U substituted pentose sugars nrog DP ntawm 8-15 uas sib xws nrog cov substituted xylans pom nyob rau hauv ELISA-based mAb screening. Cov epitopes yog sib xws.
MALDI-MS spectrum ntawm cov soluble non-compliant oligosaccharides uas muaj nyob hauv ACS. Ntawm no, (A) ACN-A qhov hnyav qis fractions uas muaj methylated uronic acid (DP 4-8) hloov glucuroxylan oligosaccharides thiab (B) ACN-B xylan thiab methylated uronic acid oligosaccharides hloov nrog glucuroxylan (DP 8-15).
Kev tshuaj xyuas cov khoom sib xyaw ntawm cov glycan residue ntawm refractory oligosaccharides. Ntawm no (A) TMS saccharide muaj ntau yam oligosaccharide fractions tau txais los ntawm kev siv GC-MS tsom xam. (B) Cov qauv ntawm ntau yam TMS-tau los ntawm cov suab thaj uas muaj nyob rau hauv oligosaccharides. ACN - acetonitrile fraction uas muaj cov oligosaccharides nruab nrab thiab FA - ferulic acid fraction uas muaj cov kua qaub oligosaccharides.
Lwm qhov xaus lus nthuav tau kos los ntawm LC-MS kev tshuaj xyuas ntawm cov feem oligosaccharide, raws li pom hauv Daim Duab S9 (cov txheej txheem tuaj yeem pom hauv cov khoom siv hluav taws xob ntxiv). Cov khoom tawg ntawm hexose thiab -OAc pawg tau pom ntau zaus thaum lub sijhawm ligation ntawm ACN-B feem. Qhov kev tshawb pom no tsis yog tsuas yog lees paub qhov kev tawg uas pom hauv glycome thiab MALDI-TOF kev tshuaj xyuas, tab sis kuj muab cov ntaub ntawv tshiab txog cov khoom siv carbohydrate hauv cov biomass lignocellulosic ua ntej kho.
Peb kuj tau tshuaj xyuas cov suab thaj ntawm cov feem oligosaccharide siv TMS suab thaj derivatization. Siv GC-MS, peb tau txiav txim siab cov khoom sib xyaw ntawm neural (tsis yog derivative) thiab acidic suab thaj (GluA thiab GalA) hauv feem oligosaccharide (Daim Duab 5). Glucuronic acid pom muaj nyob rau hauv cov khoom acidic C thiab D, thaum galacturonic acid pom muaj nyob rau hauv cov khoom acidic A thiab B, ob qho tib si yog cov khoom DP siab ntawm cov suab thaj acidic. Cov txiaj ntsig no tsis yog tsuas yog lees paub peb cov ntaub ntawv ELISA thiab MALDI, tab sis kuj sib xws nrog peb cov kev tshawb fawb yav dhau los ntawm oligosaccharide accumulation. Yog li ntawd, peb ntseeg tias cov txheej txheem niaj hnub immunological siv biotinylation ntawm oligosaccharides thiab tom qab ELISA kev tshuaj ntsuam txaus los ntes cov soluble recalcitrant oligosaccharides hauv ntau yam qauv biological.
Vim tias cov txheej txheem tshuaj ntsuam mAb raws li ELISA tau raug lees paub los ntawm ntau txoj kev sib txawv, peb xav tshawb nrhiav ntxiv txog lub peev xwm ntawm txoj kev ntsuas tshiab no. Ob lub oligosaccharides lag luam, xylohexasaccharide oligosaccharide (XHE) thiab 23-α-L-arabinofuranosyl-xylotriose (A2XX), tau yuav thiab sim siv txoj hauv kev mAb tshiab uas tsom mus rau phab ntsa cell glycan. Daim duab 6 qhia txog kev sib raug zoo ntawm cov cim biotinylated binding thiab cov cav concentration ntawm oligosaccharide concentration, qhia txog qhov ua tau Langmuir adsorption qauv. Ntawm cov mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, thiab CCRC-M151 sib raug zoo nrog XHE, thiab CCRC-M108, CCRC-M109, thiab LM11 sib raug zoo nrog A2XX dhau ntawm ntau yam ntawm 1 nm txog 100 nano. Vim muaj tsawg cov tshuaj tua kab mob thaum lub sijhawm sim, kev sim tsawg tau ua nrog txhua qhov kev sib xyaw ntawm oligosaccharide. Nws yuav tsum tau sau tseg ntawm no tias qee cov tshuaj tua kab mob teb sib txawv heev rau tib oligosaccharide ua ib qho substrate, tej zaum vim lawv khi rau cov epitopes sib txawv me ntsis thiab tuaj yeem muaj cov kev sib txuas sib txawv heev. Cov txheej txheem thiab cov txiaj ntsig ntawm kev txheeb xyuas epitope kom raug yuav nyuaj dua thaum txoj hauv kev mAb tshiab raug siv rau cov qauv tiag tiag.
Ob lub oligosaccharides lag luam tau siv los txiav txim siab qhov ntau ntawm kev kuaj pom ntawm ntau yam glycan-targeting mAbs. Ntawm no, linear correlations nrog log concentration ntawm oligosaccharide concentration qhia Langmuir adsorption qauv rau (A) XHE nrog mAb thiab (B) A2XX nrog mAb. Cov epitopes sib xws qhia txog cov qauv ntawm cov oligosaccharides lag luam siv ua substrates hauv qhov kev sim.
Kev siv cov glycan-targeted monoclonal antibodies (glycocomic analysis lossis ELISA-based mAb screening) yog ib qho cuab yeej muaj zog rau kev piav qhia tob txog feem ntau ntawm cov glycans tseem ceeb ntawm phab ntsa cell uas ua rau cov nroj tsuag biomass. Txawm li cas los xij, kev tshuaj xyuas glycan classical tsuas yog piav qhia txog cov glycans loj dua ntawm phab ntsa cell, vim tias feem ntau oligosaccharides tsis tau immobilized zoo ntawm ELISA phaj. Hauv kev tshawb fawb no, AFEX-pretreated pob kws stover tau enzymatically hydrolyzed ntawm cov khoom khov siab. Kev tshuaj xyuas qab zib tau siv los txiav txim siab qhov sib xyaw ntawm cov recalcitrant cell phab ntsa carbohydrates hauv hydrolyzate. Txawm li cas los xij, kev tshuaj xyuas mAb ntawm cov oligosaccharides me me hauv hydrolysates raug kwv yees qis, thiab cov cuab yeej ntxiv yog qhov xav tau los ua kom zoo immobilize oligosaccharides ntawm ELISA phaj.
Peb tshaj tawm ntawm no ib txoj kev tshiab thiab ua tau zoo oligosaccharide immobilization rau mAb kev tshuaj ntsuam los ntawm kev sib xyaw oligosaccharide biotinylation ua raws li ELISA kev tshuaj ntsuam ntawm NeutrAvidin ™ coated phaj. Cov immobilized biotinylated oligosaccharides tau qhia txog kev sib raug zoo txaus rau cov tshuaj antibody kom ua tau sai thiab ua tau zoo nrhiav tau cov recalcitrant oligosaccharides. Kev tshuaj xyuas cov khoom sib xyaw ntawm cov oligosaccharides tawv ncauj no raws li kev ntsuas huab hwm coj tau lees paub cov txiaj ntsig ntawm txoj hauv kev tshiab no rau kev tiv thaiv kab mob. Yog li, cov kev tshawb fawb no qhia tau tias kev sib xyaw ua ke ntawm oligosaccharide biotinylation thiab ELISA kev tshuaj ntsuam nrog glycan-targeted monoclonal antibodies tuaj yeem siv los ntes cov crosslinks hauv oligosaccharides thiab tuaj yeem siv dav hauv lwm cov kev tshawb fawb biochemical uas piav qhia txog cov qauv ntawm oligosaccharides.
Txoj kev tshawb nrhiav glycan raws li biotin no yog thawj daim ntawv tshaj tawm uas muaj peev xwm tshawb nrhiav cov carbohydrate bonds ntawm cov oligosaccharides uas yaj tau hauv cov nroj tsuag biomass. Qhov no pab kom nkag siab tias vim li cas qee qhov ntawm biomass thiaj li tawv ncauj thaum nws los txog rau kev tsim cov roj biofuel. Txoj kev no sau qhov sib txawv tseem ceeb hauv cov txheej txheem tshuaj xyuas glycome thiab nthuav nws daim ntawv thov mus rau ntau yam substrates dhau ntawm cov nroj tsuag oligosaccharides. Yav tom ntej, peb yuav siv cov neeg hlau rau biotinylation thiab siv txoj kev uas peb tau tsim rau kev tshuaj xyuas cov qauv siab siv ELISA.
Cov quav ciab pob kws (CS) uas cog los ntawm cov noob Pioneer 33A14 hybrid tau sau xyoo 2010 los ntawm Kramer Farms hauv Ray, Colorado. Nrog kev tso cai ntawm tus tswv av, cov biomass no tuaj yeem siv rau kev tshawb fawb. Cov qauv tau khaws cia qhuav < 6% noo noo hauv lub hnab zip-lock hauv chav tsev kub. Cov qauv tau khaws cia qhuav < 6% noo noo hauv lub hnab zip-lock hauv chav tsev kub. Образцы хранились сухими при влажности < 6% в пакетах с застежкой-молнией при комнатной температуре. Cov qauv tau khaws cia kom qhuav ntawm <6% av noo hauv cov hnab zippered ntawm chav tsev kub.样品在室温下以干燥< 6% 的水分储存在自封袋中。Cov nyiaj tau los <6% Образцы хранят в пакетах с застежкой-молнией при комнатной температуре с влажностью < 6%. Cov qauv khaws cia rau hauv lub hnab zipper ntawm chav tsev kub nrog cov av noo < 6%.Txoj kev tshawb fawb no ua raws li cov lus qhia hauv zos thiab lub tebchaws. Kev tshuaj xyuas cov khoom sib xyaw tau ua tiav los ntawm kev siv cov txheej txheem NREL. Cov khoom sib xyaw tau pom tias muaj 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein thiab 13.4% tshauv.
Cellic® CTec2 (138 mg protein/ml, ntau VCNI 0001) yog ib qho sib xyaw ua ke ntawm cellulase, β-glucosidase thiab Cellic® HTec2 (157 mg protein/ml, ntau VHN00001) los ntawm Novozymes (Franklinton, NC, USA)). Multifect Pectinase® (72 mg protein/mL), ib qho sib xyaw ua ke ntawm cov enzymes uas rhuav tshem pectin, tau pub dawb los ntawm DuPont Industrial Biosciences (Palo Alto, CA, USA). Cov protein enzyme concentration tau txiav txim siab los ntawm kev kwv yees cov protein content (thiab rho tawm qhov kev pab ntawm cov nitrogen uas tsis yog protein) siv Kjeldahl nitrogen analysis (AOAC method 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). Diatomaceous earth 545 tau yuav los ntawm EMD Millipore (Billerica, MA). Cov pa roj carbon ua kom muaj zog (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, thiab lwm yam tshuaj lom neeg tau yuav los ntawm Sigma-Aldrich (St. Louis, MO).
Kev kho ua ntej ntawm AFEX tau ua tiav ntawm GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Kev kho ua ntej tau ua tiav ntawm 140° C. rau 15 feeb. Lub sijhawm nyob ntawm 1:1 piv ntawm anhydrous ammonia rau biomass ntawm 60% (w/w) thauj khoom hauv lub tshuab hluav taws xob stainless hlau (Parr Instruments Company). Nws siv sijhawm 30 feeb. Lub tshuab hluav taws xob tau coj mus rau 140°C thiab cov ammonia tau tso tawm sai sai, ua rau cov biomass rov qab mus rau qhov kub thiab txias hauv chav tsev sai sai. Cov khoom xyaw ntawm AFEX pre-treated corn stover (ACS) zoo ib yam li cov untreated corn stover (UT-CS).
Cov khoom khov siab ACSH 25% (w/w) (kwv yees li 8% dextran loading) tau npaj ua cov khoom pib rau kev tsim cov oligosaccharides loj. Enzymatic hydrolysis ntawm ACS tau ua tiav los ntawm kev siv cov khoom sib xyaw enzyme lag luam suav nrog Cellic® Ctec2 10 mg protein/g glucan (hauv biomass pretreated), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, thiab Multifect Pectinase (Genencor Inc, USA). ), 5 mg protein/g dextran. Enzymatic hydrolysis tau ua tiav hauv 5-liter bioreactor nrog lub ntim ua haujlwm ntawm 3 liv, pH 4.8, 50°C thiab 250 rpm. Tom qab hydrolysis rau 96 teev, cov hydrolyzate tau sau los ntawm centrifugation ntawm 6000 rpm rau 30 feeb thiab tom qab ntawd ntawm 14000 rpm rau 30 feeb kom tshem tawm cov khoom khov tsis tau hydrolysed. Tom qab ntawd, cov hydrolyzate raug lim kom huv los ntawm lub beaker lim 0.22 hli. Cov hydrolyzate uas tau lim lawm tau khaws cia rau hauv lub raj mis huv ntawm 4 ° C. thiab tom qab ntawd muab faib ua feem ntawm cov pa roj carbon.
Kev tshuaj xyuas cov khoom sib xyaw ntawm cov qauv biomass raws li cov txheej txheem tshuaj xyuas hauv chaw kuaj mob NREL: kev npaj cov qauv rau kev tshuaj xyuas cov khoom sib xyaw (NREL/TP-510-42620) thiab kev txiav txim siab ntawm cov carbohydrates thiab lignin hauv biomass (NREL/TP-510 - 42618)47.
Kev tshuaj xyuas oligosaccharide ntawm cov dej hydrolyzate tau ua tiav ntawm 2 ml nplai siv txoj kev autoclave-based acid hydrolysis. Sib tov cov qauv hydrolyzate nrog 69.7 µl ntawm 72% sulfuric acid hauv lub raj mis kab lis kev cai 10 ml thiab incubate rau 1 teev ntawm lub rooj zaum ntawm 121 ° C, txias rau ntawm dej khov thiab lim rau hauv lub raj mis ua kua chromatography siab (HPLC). Qhov concentration ntawm oligosaccharides tau txiav txim siab los ntawm kev rho tawm qhov concentration ntawm monosaccharides hauv cov qauv tsis-hydrolyzed los ntawm tag nrho cov concentration qab zib hauv cov qauv acid-hydrolyzed.
Cov piam thaj, xylose, thiab arabinose concentration hauv cov biomass hydrolysed acid tau soj ntsuam siv Shimadzu HPLC system nruab nrog lub autosampler, lub tshuab cua sov kem, lub twj tso kua mis isocratic, thiab lub tshuab ntsuas refractive index ntawm Bio-Rad Aminex HPX-87H kem. Lub kem tau khaws cia ntawm 50 ° C thiab eluted nrog 0.6 ml / min 5 mM H2SO4 hauv dej. ntws.
Cov hydrolyzate supernatant tau diluted thiab soj ntsuam rau monomer thiab oligosaccharide cov ntsiab lus. Monomeric qab zib tau txais tom qab enzymatic hydrolysis tau soj ntsuam los ntawm HPLC nruab nrog Bio-Rad (Hercules, CA) Aminex HPX-87P kem thiab ib tug tshauv tiv thaiv kem. Lub kem kub tau khaws cia ntawm 80 ° C, dej tau siv ua theem mobile nrog tus nqi ntws ntawm 0.6 ml / min. Oligosaccharides tau txiav txim siab los ntawm hydrolysis hauv dilute acid ntawm 121 ° C raws li cov txheej txheem piav qhia hauv refs. 41, 48, 49.
Kev tshuaj xyuas Saccharide tau ua tiav rau ntawm cov khoom seem biomass raw, AFEX uas tau kho ua ntej thiab txhua yam tsis tau hydrolyzed (suav nrog kev tsim cov tshuaj rho tawm ntawm phab ntsa cell thiab lawv cov kev tshuaj ntsuam mAb) siv cov txheej txheem piav qhia ua ntej 27, 43, 50, 51. Rau kev tshuaj xyuas glycome, cov khoom seem uas tsis yaj hauv cawv ntawm cov khoom siv phab ntsa cell cog tau npaj los ntawm cov khoom seem biomass thiab raug rho tawm ntau zaus nrog cov tshuaj reagents uas nce ntxiv xws li ammonium oxalate (50 mM), sodium carbonate (50 mM thiab 0.5% w/v), CON. (1M thiab 4M, ob qho tib si nrog 1% w/v sodium borohydride) thiab acid chlorite raws li tau piav qhia ua ntej 52,53. Cov tshuaj rho tawm tom qab ntawd tau raug ELISA tawm tsam ib pawg ntawm mAb50s uas tau taw qhia rau phab ntsa cell glycan, thiab cov tshuaj tiv thaiv mAb tau nthuav tawm ua daim ntawv qhia cua sov. mAbs uas tsom mus rau phab ntsa cell cog glycan tau yuav los ntawm cov khoom lag luam hauv chaw kuaj mob (CCRC, JIM thiab MAC series).
Ib kauj ruam biotinylation ntawm oligosaccharides. Kev sib txuas ntawm cov carbohydrates nrog biotin-LC-hydrazide tau ua tiav siv cov txheej txheem hauv qab no. Biotin-LC-hydrazide (4.6 mg / 12 μmol) tau yaj hauv dimethyl sulfoxide (DMSO, 70 μl) los ntawm kev sib xyaw ua ke thiab cua sov ntawm 65 ° C rau 1 feeb. Glacial acetic acid (30 µl) tau ntxiv thiab cov khoom sib xyaw tau nchuav rau ntawm sodium cyanoborohydride (6.4 mg / 100 µmol) thiab yaj tag nrho tom qab cua sov ntawm 65 ° C rau li 1 feeb. Tom qab ntawd, los ntawm 5 txog 8 μl ntawm cov tshuaj sib xyaw tau ntxiv rau cov oligosaccharide qhuav (1-100 nmol) kom tau txais 10-npaug lossis ntau dua molar ntau dhau ntawm daim ntawv lo rau ntawm qhov kawg txo qis. Cov tshuaj tiv thaiv tau ua tiav ntawm 65 ° C rau 2 teev, tom qab ntawd cov qauv tau ntxuav tam sim ntawd. Tsis muaj sodium cyanoborohydride siv rau hauv kev sim sau ntawv yam tsis muaj kev txo qis, thiab cov qauv tau ua rau muaj kev cuam tshuam ntawm 65 ° C rau 2.5 teev.
ELISA txheej thiab ntxuav cov qauv ntawm biotinylated oligosaccharides. 25 μl ntawm biotinylated qauv (100 μl ntawm txhua tus qauv concentrated diluted hauv 5 ml ntawm 0.1 M Tris buffer solution (TBS)) tau ntxiv rau txhua lub qhov ntawm avidin-coated phaj. Cov qhov tswj tau coated nrog 50 μl ntawm biotin ntawm qhov concentration ntawm 10 μg / ml hauv 0.1 M TBS. Dej deionized tau siv ua txheej rau kev ntsuas dawb paug. Lub ntsiav tshuaj tau incubated rau 2 teev ntawm chav tsev kub hauv qhov tsaus ntuj. Ntxuav lub phaj 3 zaug nrog 0.1% skimmed mis nyuj hauv 0.1 M TBS siv qhov kev pab cuam naj npawb 11 rau Grenier tiaj tus 3A.
Ntxiv thiab ntxuav cov tshuaj tiv thaiv kab mob thawj zaug. Ntxiv 40 µl ntawm cov tshuaj tiv thaiv kab mob thawj zaug rau txhua lub qhov. Muab lub microplate tso rau 1 teev ntawm chav tsev kub hauv qhov tsaus ntuj. Tom qab ntawd ntxuav cov phaj 3 zaug nrog 0.1% mis nyuj hauv 0.1M TBS siv qhov kev pab cuam ntxuav #11 rau Grenier Flat 3A.
Ntxiv cov tshuaj tiv thaiv kab mob thib ob thiab ntxuav. Ntxiv 50 µl ntawm cov tshuaj tiv thaiv kab mob thib ob ntawm nas/nas (diluted 1: 5000 hauv 0.1% mis nyuj hauv 0.1 M TBS) rau txhua lub qhov. Incubate lub microplate rau 1 teev ntawm chav tsev kub hauv qhov tsaus ntuj. Cov microplates tom qab ntawd tau ntxuav 5 zaug nrog 0.1% mis nyuj hauv 0.1 M TBS siv Grenier Flat 5A phaj ntxuav program # 12.
Ntxiv ib lub substrate. Ntxiv 50 µl ntawm 3,3′,5,5′-tetramethylbenzidine (TMB) rau hauv lub substrate (los ntawm kev ntxiv 2 tee ntawm buffer, 3 tee ntawm TMB, 2 tee ntawm hydrogen peroxide rau 15 ml ntawm dej deionized). Npaj lub TMB substrate. thiab vortex ua ntej siv). Incubate lub microplate ntawm chav tsev kub rau 30 feeb. Hauv qhov tsaus ntuj.
Ua kom tiav cov kauj ruam thiab nyeem cov ntsiav tshuaj. Ntxiv 50 µl ntawm 1 N sulfuric acid rau txhua lub qhov thiab sau cov absorbance ntawm 450 txog 655 nm siv ELISA nyeem ntawv.
Npaj 1 mg/ml cov tshuaj ntawm cov analytes no hauv dej deionized: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine. (glcNAc), N-acetylgalactosamine (galNAc), inositol (tus qauv sab hauv). Ob tus qauv tau npaj los ntawm kev ntxiv cov tshuaj qab zib 1 mg/mL uas tau qhia hauv Rooj 1. Cov qauv raug khov thiab lyophilized ntawm -80° C. kom txog thaum tag nrho cov dej raug tshem tawm (feem ntau li ntawm 12-18 teev).
Ntxiv 100–500 µg ntawm cov qauv rau hauv cov raj uas muaj hau ntswj rau ntawm lub ntsuas ntsuas. Sau cov nqi ntxiv. Nws yog qhov zoo tshaj plaws los yaj cov qauv rau hauv ib qho concentration tshwj xeeb ntawm cov kuab tshuaj thiab ntxiv rau hauv lub raj ua kua aliquot. Siv 20 µl ntawm 1 mg/ml inositol ua tus qauv sab hauv rau txhua lub raj qauv. Qhov ntau ntawm tus qauv sab hauv ntxiv rau cov qauv yuav tsum zoo ib yam li qhov ntau ntawm tus qauv sab hauv ntxiv rau lub raj qauv.
Ntxiv 8 ml ntawm anhydrous methanol rau hauv lub raj mis uas muaj hau ntswj. Tom qab ntawd ntxiv 4 ml ntawm 3 N. methanolic HCl kua, kaw thiab co. Cov txheej txheem no tsis siv dej.
Ntxiv 500 µl ntawm 1 M HCl methanol kua rau cov qauv oligosaccharide thiab cov raj TMS txheem. Cov qauv tau incubated ib hmos (168 teev) ntawm 80 ° C. hauv lub thermal block. Qhuav cov khoom methanolysis ntawm chav tsev kub siv lub tshuab ziab manifold. Ntxiv 200 µl MeOH thiab qhuav dua. Cov txheej txheem no rov ua ob zaug. Ntxiv 200 µl ntawm methanol, 100 µl ntawm pyridine thiab 100 µl ntawm acetic anhydride rau cov qauv thiab sib tov zoo. Cov qauv tau incubated ntawm chav tsev kub rau 30 feeb. thiab qhuav. Ntxiv 200 µl ntawm methanol thiab qhuav dua.
Ntxiv 200 µl ntawm Tri-Sil thiab lub raj cua sov uas kaw rau 20 feeb. 80°C, tom qab ntawd txias rau qhov kub hauv chav. Siv lub tshuab ziab kom ziab cov qauv kom txog li 50 µl. Nws yog ib qho tseem ceeb uas yuav tsum nco ntsoov tias peb tsis tau cia cov qauv qhuav tag.
Ntxiv 2 ml ntawm hexane thiab sib tov kom zoo los ntawm vortexing. Sau cov lus qhia ntawm Pasteur pipettes (5-8 hli) nrog ib daim iav wool los ntawm kev ntxig cov iav wool rau saum lub pipette 5-3/4 nti txoj kab uas hla. Cov qauv tau centrifuged ntawm 3000 g rau 2 feeb. Txhua yam insoluble residues yog precipitated. Qhuav cov qauv mus rau 100-150 µl. Ib qho ntim ntawm kwv yees li 1 μl tau txhaj rau hauv GC-MS ntawm qhov kub pib ntawm 80 ° C thiab lub sijhawm pib ntawm 2.0 feeb (Rooj 2).