I-Hielscher Ultrasound Technology

I-Sonication Lysis: Ukuphazamiseka Kwamaseli & Ukukhipha

Ukuhlukaniswa kwamagciwane noma i-lysis kuyinxenye evamile yokulungiselela isampula nsuku zonke kuma laboratories e-biotech. Umgomo we-lysis ukuphazamisa izingxenye zodonga lwamaseli noma iseli eliphelele ukukhulula ama-molecule ephilayo. Okubizwa okuthiwa i-lysate kungahlanganisa isib. I-plasmid, i-receptor assays, amaprotheni, i-DNA, i-RNA njll. Ukulandela izinyathelo ze-lysis yi-fractionation, isotioning organelle noma / iphrotheni kanye nokuhlanzwa. Izinto ezikhishwe (= lysate) kufanele zihlukaniswe futhi zihloselwe ukuphenywa okuqhubekayo noma izinhlelo zokusebenza, isb. Ucwaningo lweproteomic. I-homogenizers ye-Ultrasonic iyithuluzi elivamile le-cell lysis ephumelelayo. Njengoba ukuqina kwe-ultrasonic kungasungulwa ngokulungiswa kwemigomo yemigomo, ukuqina kwe-sonication okunamandla kakhulu kusukela kanzima kunzima kakhulu kungenziwa kubekwe ngabanye kuzo zonke izidakamizwa neziphakathi ukuhlangabezana nezidingo ezithile zokusebenza.

Ukwakhiwa kweselula

Amaseli avikelwe i-plasma engenakulinganiswa engxenyeni ye-plasma ene-phospho-lipid bilayer (futhi i-protein-lipid bilayer; eyakhiwe yi-hydrophobic lipids nama-molecule hydrophilic phosphorus ane-protein e-protein) futhi yenza umgoqo phakathi kwe-cell interiors (i-cytoplasm) imvelo engaphezulu. Amaseli ezitshalo nama cell prokaryotic azungezwe udonga lweseli. Ngenxa yamathemitha amaningi obukhulu besellu se-cellulose, amangqamuzana okutshala anzima kakhulu ukudlula i-cell cell. Ingaphakathi leseli, njenge-organelles, i-nucleus, i-mitochondrion, iqiniswa yi-cytoskeleton.
Ngokulenga amangqamuzana, kuhloswe ukukhipha nokuhlukanisa i-organelles, amaprotheni, i-DNA, i-MRNA noma amanye ama-biomolecules.

I-Sonication ijwayele ukusetshenziselwa ukulungiswa kwesampula kwendaba yeseli.

Umdwebo 1: Isizinda se-ultrasonic esivela kumaseli: isigaba esithintekayo esincane (TS) se-apical stem of mint (i-Mentha piperita) sibonisa indlela yezenzo phakathi kwesikhumba se-ultrasonic kusuka kumaseli (ukukhulisa 2000x) [isisetshenziswa: Vilkhu et al. 2011]

Izindlela

Kunezindlela eziningana zokulenga amangqamuzana, angahlukaniswa ngezindlela zomshini nezomakhemikhali, okubandakanya ukusetshenziswa kwe-detergents noma i-solvents, ukusetshenziswa komfutho ophezulu, noma ukusetshenziswa komshini we-bead noma umshini we-French. Ukungahleleki okunzima kakhulu kulezi zindlela ukulawulwa okunzima nokulungiswa kwemigomo yezinqubo futhi ngaleyo ndlela kuthinte.
Izinkinga eziyinhloko zezindlela ezivamile zelayisense:

Izindlela zokuhlukana ezahlukene

Ithebulethi: Izindlela ezivamile zel cell lysis zinokulimaza okukhulu

Ngokuphambene nalokho, i-sonication iyithuluzi eliphumelelayo kakhulu futhi elithembekile le-cell disintegration evumela ukulawula okuphelele phezu kwemingcele ye-sonication. Lokhu kuqinisekisa ukukhethwa okuphezulu ezintweni zokukhululwa nokuhlanzeka komkhiqizo. [Balasundaram et al. 2009] Kufanelekile kuzo zonke izinhlobo zamaseli futhi zisetshenziswe kalula ezincane nezinkulu. I-ultrasonicators kulula ukuyihlanza. I-homogenizer yama-ultrasonic ihlale ihlanzekile-in-place (CIP) futhi inomsebenzi oyinzalo (SIP). I-sonotrode iqukethe inqwaba ye-titanium phondo engasulwa noma igubungele emanzini noma nge-solvent (kuye ngokuthi isigaba sokusebenza). Ukugcinwa kwe-ultrasonicators kubangelwa ukuqina kwawo okungazelelwe.

lysis

I-Lysis iyinkqubo ebucayi. Ngesikhathi se-lysis ukuvikelwa kwe-membrane yeseli kubhujiswe, kodwa ukungasebenzi, ukuguqulwa nokuchithwa kwamaprotheni akhishwe yi-imvelo engavumelani nayo (ukuphambuka kusuka ku-pH-value) kumele kuvinjelwe. Ngakho-ke, ngokuvamile i-lysis yenziwa kusixazululo se-buffer. Izinkinga eziningi zivela ekulawuleni okungalawuliwe kwamangqamuzana okubangelwa ukukhululwa okungalindelekile kukho konke okufakwe ngaphakathi kwe-intracellular noma / nokukhonjiswa komkhiqizo ohlosiwe.

I-ultrasonic cell lysis ingasetshenziselwa ukulungiselela isampula ebhodini nokukhiqizwa kwemigomo emikhulu. Chofoza ukuze ukhulise!

Umdwebo 1: 200 Watts i-homogenizer ene-ultrasonic enamandla UP200Ht ngokulawulwa kwedijithali nokuqoshwa kwedatha okuzenzekelayo kweseli lysis elithembekile futhi elikhiqizayo.

I-Ultrasonic Lysis

Ngokuvamile, i-lysis yamasampuli ebhodini izothatha phakathi kwemizuzwana engu-15 nemizuzu engu-2. Njengoba ukuqina kwe-sonication kulula kakhulu ukulungiswa yi-amplitude ukubeka isikhathi sokunakekelwa kwengane kanye nokukhetha imishini efanele, kungenzeka ukuphazamisa izimbungulu zezinsiza ngokuzithoba noma ngokuzumayo, kuye ngokuthi isakhi selinye futhi ngenhloso yelysis ( isib. Ukukhipha i-DNA kudinga ukunakekelwa okuncane, ukukhishwa kweprotheyini egcwele yamabhaktheriya kudinga ukwelashwa okukhulu kwe-ultrasound). Ukushisa ngesikhathi sokucubungula kungabhekwa yi-sensor edidiyelwe yokushisa futhi kungenziwa ukulawulwa kalula ngokupholisa (ukugeza kwe-ice noma ukugeleza kwamangqamuzana ngamajezi okupholisa) noma ngokunikezwa kwimodi kwimodi evuthayo. Ngesikhathi kutholakala nge-pulse-mode sonication, imijikelezo emfushane ye-sonication ye-1-15 imizuzwana ubude buvumela ukushisa kokushisa nokupholisa phakathi nezinkathi eziphakathi kwesikhathi eside.
Zonke izinqubo eziqhutshwa nge-ultrasound zenziwe ngokugcwele futhi zihlukana ngokulinganayo.

I-homogenizer ye-ultrasonic VialTweeter ye-sampuli yesikhathi esifanayo yokulungiselela amathobhu ayi-10 wokuhlola. (Chofoza ukuze ukhulise!)

Idivayisi ye-ultrasonic I-VialTweeter ivumela ukulungiselela isampuli esisodwa ngesikhathi esisodwa.

Ultrasonic Homogenizers

Izinhlobo ezahlukene Amadivayisi we-Ultrasonic vumela ukufanisa umgomo wokulungiselela isampula kanye nokuqinisekisa induduzo yomsebenzisi nokusebenza kwenduduzo. Uhlobo lwe-probe-ultrasonicators yiwona amadivayisi avame kakhulu kulabhu. Zilungele kakhulu ukulungiswa kwamasampula amancane naphakathi nosayizi nge-0.1mL kuya ku-1000mL. Amandla amandla ahlukile kanye ne-sonotrodes avumela ukuvumelanisa i-ultrasonicator kumvolumu wesampula kanye nomkhumbi wemiphumela ephumelela kakhulu futhi ephumelelayo yokuveza indodana. Idivayisi ye-ultrasonic probe iyona engcono kakhulu uma amasampuli angashadile kufanele alungiselelwe.

Uma amasampuli amaningi kufanele alungiselelwe, isb. 8 izimbiza zesisombululo seseli, amadivaysi afana ne-VialTweeter noma i-cuphorn ye-ultrasonic yiyona homogenizer efanelekayo ye-lysis. Izimbiza eziningana zihanjiswa ngesikhathi esifanayo, ngamandla afanayo. Lokhu kusindisa isikhathi nje kuphela, kodwa kuqinisekisa nokuphathwa okufanayo kwazo zonke amasampula, okwenza imiphumela phakathi kwamasampuli athembeke futhi afaneke. Ngaphezu kwalokho, ukungcoliswa kwegazi ngokuzifaka nge-sonotrode ye-ultrasonic (eyaziwa nangokuthi i-probe ye-ultrasonic, uphondo, tip, noma umunwe) igwenywe. Njengoba kusetshenziswe izimbiza, ukulahleka isikhathi nokuhlanza kwesampula ngenxa yokunciphisa izitsha akushiyiwe.
Ukuze uthole imiqulu ephakeme, isib. Ukukhiqizwa kwezokuhweba kwamaskitholi esele, izinhlelo eziqhubekayo ze-ultrasonic ezine-streak cell reactor zifanele kakhulu. Ukuqhubeka nokuqhuma kwezinto ezicutshungulwa kuqinisekisa ngisho ne-sonication. Yonke imingcele yenqubo yokuhlukanisa i-ultrasonic ingahle ihlelwe futhi ihlelwe izidingo zesicelo kanye nemininingwane ethile yeselula.

Inkambiso ye-ultrasonic lysing yama-bacterial cells:

  • Ukulungiswa kokumiswa kwamaseli: Amapulangwe eselula kufanele asamiswe ngokuphelele kwisisombululo se-buffer ngokuhlobisa i-homogenizing (khetha isisombululo sakho se-buffer ehambisana nokuhlaziywa okulandelayo, isib. Indlela ethile ye-chromatography). Engeza i-lysozymes kanye / noma ezinye izithasiselo, uma kudingeka (kumele nazo zihambisana nokuhlukaniswa / ukuhlanza izindlela). Hlanganisa / uhlaziye kahle isixazululo ngobumnene ngaphansi kwe-sonication emnene kuze kuphele ukupheliswa okuphelele.
  • I-ultrasonic lysis: Faka isampula ebhodini lokugeza. Ukuze uthole ukuphazanyiswa kwamangqamuzana, vumela ukumiswa kokushisa kwe-60-90 kwesibili (usebenzisa i-mode yakho ye-pulsometer ye-ultrasonicator).
  • Ukuhlukaniswa: I-Centrifuge i-lysate (isb. 10 iminithi ku-10,000 xg; ku-4degC). Hlukanisa ngokucophelela ukunqotshwa okuphezulu kusuka epelletini yeseli ngokucophelela. I-supernatant yi-cell lysate esiphelele. Ngemuva kokuhlanzwa kwe-supernatant, uthola uketshezi olucacile lwesiprotheni lesisunxele.

Izinhlelo ezivame kakhulu ze-ultrasonicators kwi-biology ne-biotechnology yizo:

  • Iseli lisusa ukulungiselela
  • Ukuphazanyiswa imvubelo, amabhaktheriya, izitshalo zokutshala, ezithambile & izicubu zeseli ezinzima, impahla ye-nucleic
  • Izakhi zeprotheyini
  • Ukulungiselela nokuhlukaniswa kwama-enzyme
  • Ukukhiqizwa kwama-antigens
  • Ukuhlukaniswa kwe-DNA kanye / noma ukuhlukaniswa okuhlosiwe
  • ukulungiselela i-liposome
Ama-homogenizers wamandla amakhulu aphezulu njenge-UIP1000hd asetshenziselwa ukuphazanyiswa kwamangqamuzana kanye nokukhipha ku-batch noma i-flow mode-though mode. (Chofoza ukuze ukhulise!)

Ama-ultrasonic benchtop izinhlelo ezifana ne- I-UIP1000hd (1kW) ingakwazi ukucubungula izinto eziningana zezinto eziphilayo.

Izicelo eziningi ze-ultrasound zivela emkhakheni we-biotechnology, i-bioengineering, i-microbiology, i-biology yemvelo, i-biochemistry, i-immunology, i-bacteriology, i-virology, i-proteomics, i-genetics, i-physiology, i-biology, i-hematology, ne-botany.

Ukuze kuhlolwe futhi kusetshenziswe izinhlelo zokusebenza zamakhasimende, i-Hielscher Ultrasonics inikeza ukuhlonywa ngokugcwele Ultrasonic Process Laboratory. Sicela uxhumane nathi ukuze uthole olunye ulwazi!

Izincwadi / Izinkomba

  • I-Balasundaram, B .; UHarrison, uS .; I-Bracewell, i-DG (2009): Intuthuko emikhakheni yokukhululwa komkhiqizo nomthelela kumklamo we-bioprocess. Amathrendi ku-Biotechnology 27/8, 2009. kk. 477-485.
  • I-Vilkhu, K .; UManase, R .; Mawson, R .; U-Ashokkumar, M. (2011): Ukubuyiswa kwe-ultrasonic nokuguqulwa kokudla izithako. Ku: Feng / Barbosa-Cánovas / Weiss (2011): Ubuchwepheshe be-Ultrasound for Food and Bioprocessing. I-New York: Springer, 2011. iphe. 345-368.

Xhumana Nathi / Cela Ukwaziswa Olwengeziwe

Khuluma nathi mayelana nezidingo zakho zokucubungula. Sizosikisela ukusetha nokulungiswa kwemingcele efanele yephrojekthi yakho.





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