BIOSENSORS eguquguqukayo heliX cyto ngokuPheleleyo oluzenzekelayo inkqubo yoHlahlelo lweLabhoratri

Iinkcukacha

• Igama lemveliso: heliX cyto
• Umenzi: Dynamic Biosensors GmbH
• Inguqulelo: 1.0

Ulwazi lweMveliso

• Inkqubo ye-cyto ye-heliX yenzelwe i-cell-cell Interaction Cytometry (scIC) ukulinganisa i-kinetics ebophelelayo yee-molecule ezibhalwe nge-fluorescently kwiithagethi kwiindawo zeseli ngexesha elixazululwe.

Imiyalelo yokusetyenziswa kwemveliso

Ukusebenzisa i-heliXcyto Chip

• I-chip ye-heliXcyto iqulethe i-channel eyodwa ye-microfluidic eneendawo ze-electrode zokubamba iiseli kunye nokulinganisa. Qinisekisa ukubekwa kakuhle kwetshiphu kwaye ulandele imiyalelo yokubambisa iiseli.

Chip yokuLondoloza

• Sebenzisa iChip yokuLondoloza ukucoca, ukuvavanya, kunye nemisebenzi yokulondoloza kuphela. Kuphephe ukuyisebenzisela imifuniselo yokuthintela ukosuleleka.

Ukuqhuba i-Buffer

• Sebenzisa i-Running Buffer 1 (RB 1) ngexesha lemilinganiselo. Jonga kwiphepha lokuhambelana kwe-scIC kwisikhokelo esiphambili ngolwazi oluthe kratya.

Uhlalutyo lwe-Sensorgram

• Beka iliso kwi-sensorgram ngexesha lokwenyani kwisoftware ye-heliOS ngexesha lemilinganiselo. Hlalutya impendulo ye-fluorescent ekuhambeni kwexesha ukukhupha amazinga e-kinetic.

Ukunyamezela

• Cinga ukusebenzisa i-passivation njengenyathelo lokuzikhethela ukukhusela i-adsorption ye-analyte ngokufaka i-reagent passivation kwi-chip.

Ukwenziwa kwesiqhelo

• I-normalization yinkqubo yokulinganisa idatha yokuthelekisa kunye nohlalutyo. Landela iinkqubo zesiqhelo ngokwencwadi yomsebenzisi.

INTSHAYELELO

• Esi sikhokelo sichazwa njenge-over emfutshaneview yenkqubo ye-heliXcyto kunye nobuchule bayo. Zonke izahluko zandisiwe kwisikhokelo esiphambili se-heliXcyto efumaneka kwi https://www.dynamic-biosensors.com/helios-download-latest/

heliXcyto Terminology single-cell Interaction Cytometry

• I-single-cell Interaction Cytometry (scIC) iteknoloji elinganisa i-kinetics ebophelelayo ye-molecule ebhalwe nge-fluorescently (i-analyte) kwithagethi (i-ligand) kwi-cell surface ngokurekhoda isignali ye-fluorescent ngexesha elisonjululwe.

Hlalutya

• I-Analyte yi-molecule ebhalwe nge-fluorescently kwisigaba esihambayo esinokubophelela kwithagethi kumphezulu weseli.

Ligand

• Ligand” ligama elisetyenziswa kwi-software ye-heliOS ukuchaza into ekujoliswe kuyo kumphezulu weseli apho umhlalutyi unokubophelela khona.” Oku kunokuba yi-molecule ye-receptor, i-lipid, iswekile okanye enye i-molecule ye-cell-surface.

Cell Trap

• Imigibe yeeseli iyakwazi ukuhamba-permeable, iikheji zepolymer ezihambelana ne-biocompatible kwi-chip ye-heliXcyto. Ziyilelwe ukubamba kwaye zingashukumi iiseli ezinobungakanani obahlukeneyo ukusuka malunga ne-6 ukuya kwi-25 µm kumjelo wokuhamba. Imigibe yeeseli ifumaneka kwi-3 yobukhulu obuhlukeneyo: encinci, ephakathi kunye nenkulu.

heliXcyto Chip

• Iitshiphusi ze-heliXcyto ziqulethe itshaneli enye ye-microfluidic enemingxuma kwicala ngalinye. Amabala amabini e-electrode egolide abekwe embindini wetshaneli.
• Indawo enye isebenza njengendawo yereferensi, kwaye indawo yesibini ithwala i-3D biocompatible polymer cages ukuze kubanjwe iiseli kwaye isebenza njengendawo yokulinganisa. Indawo yokulinganisa ingaba ne-1 okanye i-5 imigibe yohlobo olufanayo.
• Indawo yereferensi ivumela ireferensi yexesha langempela lophawu oluqokelelweyo lwefluorescent. I-heliXcyto chip iphinda isetyenziswe kwaye iyalahlwa.

Chip yokuLondoloza

• I-Chip yokuLondoloza yi-chip eyodwa ye-microfluidic chip esetyenziselwa yonke imisebenzi yokucoca, yokuvavanya kunye nokugcinwa.
• Le misebenzi ibandakanya i-Clean & Sleep routine, Vuka kunye ne-Prime routine, ukuhlamba kweNkqubo kunye novavanyo lwe-Fluidics. Le chip kufuneka ingasetyenziselwa imifuniselo yokwenyani kunye nezisombululo zeseli/zeprotheni ukunqanda ukungcoliseka ngakumbi kwetshiphu.

Ukuqhuba i-Buffer

• Ukuqhuba isithinteli sisithinteli esisetyenziswa kwi-heliXcyto ngexesha lomlinganiselo. Ukusetyenziswa kwe-Running Buffer 1 (RB 1) kuyacetyiswa ngokubanzi.
• Nceda ujonge kwiphepha lokuhambelana kwe-scIC elifunyenwe kwisikhokelo esiphambili ngolwazi oluthe kratya.

Uluvo

• I-sensorgram ye-scIC yiplani yempendulo ye-fluorescence kumanani ngomzuzwana (cps) ekuhambeni kwexesha, ebonisa ukuqhubela phambili kokunxibelelana kwiiseli okanye kwiiseli. Le igophe ingaba viewed ngexesha langempela kwi-heliOS ngexesha lokulinganisa.
• Xa kuhlalutywa i-sensorgrams, eyona exponential ifaneleka kakhulu kwizalathi zefluorescent ezijongiweyo ziyamiselwa kwaye amazinga ekinetic (kon, koff) akhutshiwe.

Ukunyamezela

• Ukugqithiswa linyathelo lokuzikhethela ekubalekeni, apho i-reagent passivation ifakwe kwi-chip kwaye ifakwe kwi-block block. Oku kunokunceda ukuthintela i-adsorption ye-analytes kwizinto ze-chip.

Ukwenziwa kwesiqhelo

• Inyathelo lesiqhelo lisetyenziselwe ukulinganisa umahluko omncinci kwimiqondiso ngenxa yokuqokelela umqondiso ukusuka kwindawo nganye yokulinganisa ngesixhobo esahlukileyo. Idayi ye-fluorescent yoxinaniso oluchaziweyo (ngokusekwe kugxininiso oluphezulu lohlalutyi kunye neqondo lokuleyibhela kwe-analyte) itofwe ekuqaleni kovavanyo. Eli nyathelo lesiqhelo libandakanyiwe ngokuzenzekelayo kwindlela yokulinganisa, kwaye i-normalization peak elinganisiweyo isetyenziselwa ukulungisa ngokuzenzekelayo ukungafani phakathi kwee-detectors ngexesha lokuhlalutya idatha, ngaphambi kokubhekisela kwexesha langempela.

scIC iMigaqo yoMlinganiselo

Usetyenziso lwe-scIC

• I-single-cell Interaction Cytometry (scIC) ilinganisa amazinga e-kinetic kunye nokuhambelana kwe-fluorescently ebhalwe uhlalutyi ebophelelayo kunye nokungabopheleli ekujoliswe kuyo ngokuthe ngqo kwiiseli eziphilayo.
• Ukufunyanwa kwe-analytes kwi-scIC kubukhulu obuzimeleyo kwaye ngoko buxhomekeke kuyo nayiphi na i-molecule ukusuka kwi-sub-nm ukuya kwi-> 100 nm kwaye ihambelana nazo zonke iiklasi ze-molecule ukusuka kwiimolekyuli ezincinci, i-peptides, i-aptamers, i-nanobodies, i-affibodies, i-antibodies, iifomati ze-bispecific antibody, iiprotheni, iiprotheyini zeprotheyini, i-naxonosome virus

Itekhnoloji ye-scIC inokujongana nale mimandla yesicelo ilandelayo:

• umlinganiselo womqondiso ampiilitudes kwizikrini ezibophelelayo
• uhlalutyo lwekinetic lwamazinga e-kon kunye ne-koff
• ukuvandlakanywa kunye novavanyo lokukhuthala ngokusebenzisa iimodeli ze-koff kunye ne-biphasic fit
• iimvavanyo ezithile
• quantification isihlobo iiproteni inwebu kumphezulu weseli
• i-epitope binning kunye nezifundo zokhuphiswano
• inhibition assess
• Uvavanyo lokungena ngaphakathi kweeprotheni ezijoliswe kuyo

heliXcyto Hardware Ubunakho

Inkqubo yeFluidic

• Inkqubo ye-fluidic ye-heliXcyto yondliwa ukuya kuthi ga kwiibhotile ze-buffer ezi-3, ezenza iinguqu ekusebenzeni kunye nokugcinwa kwe-buffer. Iimpompo kwi-heliXcyto zinokusetwa ukuba zihambe phakathi kwe-20 kunye ne-500 µL/min, ukulungiselela ii-s ezahlukeneyo.ample kunye neemfuno zovavanyo. Umjelo wokuhamba we-chip udibanisa kwinkqubo ye-fluidic ngokusebenzisa izivulo ezimbini. Ukuvulwa kwecala lasekhohlo kuqhagamshelwe kwi-sampi-le, i-buffer, kunye nemigca yokuhlamba, ngelixa ukuvula icala lasekunene kudityaniswa nomgca wohlaziyo.
• Le nkqubo ye-fluidic yeendlela ezimbini yenza ukuba iseli lizinzile ngelixa liqhuba amanyathelo ahlukeneyo omlinganiselo kwaye ekugqibeleni i-chip ihlaziywe kwi-assay chip reusability.
• Umzobo 1. Ukuhlanganiswa kweChip kwinkqubo yeFluidic

Inkqubo yokubona

• Isignali ye-fluorescence ivuyiswa yi-diodes ebomvu kunye ne-green light-emitting diodes (i-LED) kwaye ifunyenwe ngamakhawunta amane e-photon enye, eqokelela izibonakaliso ezibomvu kunye neziluhlaza kwindawo nganye kubunzulu obugcweleyo beshaneli.
• Izibonakaliso ezimbini ezizimeleyo zinokujongwa ngaxeshanye kwindawo efanayo yokulinganisa, ukuvumela ukusebenzisana okubini ukuba kulinganiswe kanye kwisethingi yovavanyo lwemibala emibini.
• Umzobo 2. Ukuseta i-Optics
• Ingqokelela yemiqondiso kwindawo nganye yomlinganiselo iphathwa sisixhobo esahlukileyo, esinokubangela umahluko omncinane kwizibalo ezikrwada. Ukuphendula oku, inyathelo lesiqhelo lifakwe ngokuzenzekelayo kwiimvavanyo zokuvelisa idatha.
• Ukongeza kwi-counter-photon counters, isixhobo sixhotyiswe ngekhamera yeCCD kunye ne-microscope yokukhanya ebonakalisiweyo. Ezi zixhobo zivumela ukucinga kweeseli zexesha langempela, okwenza uvavanyo lwengqibelelo yeeseli kunye nokuhlalutya umgangatho kulo lonke umlinganiselo.
• Olu cwangciso ludibeneyo luqinisekisa ukuba zombini intsebenziswano kunye neemeko zebhayoloji zilandelwa, zibonelela ngovavanyo olubanzi lovavanyo.

ScIC ukuhamba komsebenzi

Inkqubo yoMlinganiselo ye-scIC inokwahlulwa ibe ngamanyathelo ama-3 abalulekileyo

1. Uyilo loMfuniselo kwi-heliOS: Umlinganiselo ngamnye uqala ngokuyila umfuniselo kwisoftware ye-heliOS. Ukuhamba komsebenzi wokuvavanya kusetwa ngokukhetha iindlela ezichazwe kwangaphambili kunye nokulungelelanisa iiparamitha zokulinga, ezifana nomgca weseli osetyenzisiweyo, ugxininiso lwe-analyte, kunye neemeko ze-buffer. I-heliOS ibala ngokuzenzekelayo ubungakanani obuchanekileyo beeseli, abahlalutyi, i-buffers, kunye nezinye izisombululo eziyimfuneko kumlinganiselo, ukulungelelanisa inkqubo yokulungiselela.
2. Ukulungiswa kwezithinteli, abahlalutyi, kunye neeseli: Emva kokuba uyilo lokulinga lugqitywe, izinto ezifunekayo zilungiswa ngokweenkcukacha ezinikezelwe yi-heliOS. Ii-buffers, ii-analytes, kunye neeseli zilayishwa kwi-autos yesixhoboampler.
3. Umlinganiselo kunye noHlahlelo lweDatha: Isistim yenza uthotho olucwangcisiweyo lwemilinganiselo yentsebenziswano yexesha langempela ezenzekelayo efuna kungabikho kungenelela kumsebenzisi. Idatha inokuhlalutywa ngelixa umlinganiselo usasebenza ukufumana ulwazi olukhawulezileyo kwiziphumo.

Uvavanyo lwe-scIC kwi-heliOS

Ukongeza kwiimvavanyo zokuvelisa idatha, isofthiwe ikwabonelela ngeendlela zokuvavanya i-chip, ukucoca kunye nokugcinwa kwesixhobo.

Itheyibhile 1. Iimvavanyo zokuvelisa iDatha eziqinisekisiweyo

scIC Kinetics	Ilinganisa i-kinetics epheleleyo kwiiseli ezinombutho wohlalutyi ohlengahlengiswayo phakathi kwe-1 kunye ne-5 yoxinaniso, ilandelwa kunye nokuhlukana okukodwa emva koxinzelelo oluphezulu. Ingaba itshaneli eluhlaza okanye ebomvu. Iqulethe ukhetho lovavanyo lwe-Analyte kuphela.
I-scIC Kinetics – uMbala oMabini	Ilinganisa i-kinetics epheleleyo kwiiseli ezinombutho wohlalutyi ohlengahlengiswayo phakathi kwe-1 kunye ne-5 yoxinaniso, ilandelwa kunye nokuhlukana okukodwa emva koxinzelelo oluphezulu. Zombini itshaneli eluhlaza kunye nebomvu yenziwe ngaxeshanye. Iqulethe ukhetho lovavanyo lwe-Analyte kuphela.
Ukwahlulwa kwe-scIC-Umbala oMabini	Ilinganisa ukuchithwa kwe-analyte kwiiseli eziye zafakwa ngaphambili kunye ne-fluorescent ebhalwe i-analyte ene-green kunye / okanye i-channel ebomvu.

Amalungiselelo kunye noPhuhliso loVavanyo kuMlinganiselo we-scIC

Kukho amanqaku ambalwa okuqwalasela phambi kokuseta umlinganiselo we-scIC

• Hlalutya i-concentration, iDegree yokuLeyibhile, iipropati, kunye nomgangatho.
• Isisombululo sesiqhelo kunye namandla okuvuselela.
• Umgangatho weseli kunye nokuphathwa.

I-Analyte Concentration kunye neDegree yokuLeyibhile

• Ngokweendlela eziqhelekileyo zokulinganisa i-kinetic, sincoma ukuba ukhethe uluhlu lwe-analyte molar concentration ye-0.1x ukuya kwi-10x elindelekileyo ye-equilibrium dissociation constant (Kd) ye-multiple concentration kinetics. Kwi-kinetics eyodwa-concentration or dissociation measurements, i-analyte concentration kufuneka ihambe phakathi kwe-1x kunye ne-10x elindelekileyo ye-Kd. Umthamo wohlalutyi ofunekayo obalelwe ixesha le-10 lemizuzu yonxulumano kumyinge wokuhamba kwe-25 µL/min itotali malunga ne-300 µL.
• I-Degree of Labeling (DOL) yomhlalutyi kufuneka ngokufanelekileyo ibe yi-1, nangona ixabiso le-DOL ye-2 okanye yesi-3 isamkelekile. Nangona kunjalo, qaphela ukuba inani eliphezulu leelebhile kwi-analyte linokuthi libe nefuthe kwiimpawu zalo ezibophelelayo, kwaye linokunyusa amathuba okudityaniswa okanye ukubophelela okungacacanga. Ukufezekisa i-DOL efanelekileyo, landela iprotocol enikwe iiKits zokuLayibhile eziqukethe idayi ebomvu okanye eluhlaza ukusuka kumgca we-heliXcyto reagents.
  Ukwenziwa kwesiqhelo
• Ukwenziwa kwesiqhelo linyathelo lokuqala kuzo zonke iindlela zokuvelisa idatha. Ihlawulela utshintsho oluncinci lwesignali olubangelwa ukusebenzisa ii-detectors ezahlukeneyo kwindawo nganye yokulinganisa. Kule nyathelo, idayi ye-fluorescent kwi-concentration echazwe ngumsebenzisi ijojowe ekuqaleni kovavanyo.
• Uxinaniso lweSisombululo sokuQinisekisa sibalwa ngokuphinda-phinda olona xinzelelo luphezulu lwe-fluorescent oluleyibhile yohlalutyi olusetyenziswa kumlinganiselo ngohlalutyi lweDegree yokuLebula (DOL). Olu gxininiso lukhokela amandla okuvuselela i-LED asetyenziswa ngexesha lokulinganisa. Injongo yeyokuba iNgxaki yeSisombululo esiQinisekileyo sifikelele kumqondiso ofanayo we-fluorescence amplitude njengeyona nto iphezulu yoxinaniso lohlalutyi.
• Jonga kwiTshati yoxinaniso yesisombululo kwiSikhokelo esiphambili sokuseta amaxabiso okuqala. Qaphela ukuba itshathi ikhokela useto lokuqala, kodwa amandla okuvuselela i-LED anokulungiswa ngakumbi ngexesha lophuhliso lovavanyo.

Umgangatho weSeli kunye noLungiselelo

• Ukusebenza kweeseli kufuneka kube ngaphezulu kwe-95%, kwaye iiseli kufuneka zibekwimeko entle ngokubanzi. Kwiiseli ezibambelelayo, sincoma ukusebenza kunye neeseli ezidibanisa i-50-70%.
• Kwiiseli zokunqunyanyiswa, sincoma ukuvuna kwinqanaba eliphakathi lokukhula. I-50 µl yokumiswa kweeseli kwi-1E + 06 iiseli/mL iyafuneka ngokubaleka.

Ukuvuna iiseli ezimisiweyo

1. Centrifuge malunga. Iiseli ze-2E + 06 kwi-300 g imizuzu eyi-7 ukususa imidiya yenkcubeko yeseli. Lahla amandla amakhulu.
2. Hlamba iiseli kube kanye ngokumisa kwakhona i-pellet yeseli malunga ne-approx. 1 mL ye-DPBS. I-Centrifuge ukunqunyanyiswa kwi-400 g imizuzu emi-5 ukukhupha iiseli eziphilayo. Lahla ngononophelo i-supernatant ukususa amaqhekeza kunye nemidiya.
3. Ukumisa ngokukhawuleza kwi-1 mL ye-DPBS, tshintshela ukumiswa phezulu kwesihlunu seseli kwaye ugxininise ukumiswa ngokuhamba komxhuzulane.
4. Ukulinganisa i-cell concentration ye-strained suspension and adjust to 1E + 06 cells / mL.
   • ICEBISO: Ezinye iiseli ezimisiweyo zikholisa ukwenza amaqela. Buyisela kwakhona amaqela ngobunono kwaye ubandakanye inyathelo lokutsala nzima phambi kwenyathelo lokuqala le-centrifugation.
   • Sebenzisa iingcebiso ezibanzi zepipette xa uphatha ukumiswa kweeseli ukuphepha amandla aphezulu okucheba ngexesha lokudluliselwa okanye ukumiswa.

Ukuvuna iiseli ezibambelelayo

1. Hlamba iiseli nge-DPBS engenazintsholongwane.
2. Hlukanisa iiseli kwiflaski yokulima usebenzisa ireyinti oyikhethayo yokuqhawula (umzekelo, i-TrypLE).
3. Phinda umise iiseli ezidityanisiweyo kumthamo okhethwayo wemidiya kunye nesihluzo nge-30 nm cell strainer.
4. Ngokuzithandela, yongeza i-EDTA kwimeko yeeseli ezibambelela ngokuqinileyo (i-5 mM yoxinzelelo lokugqibela).
5. I-Centrifuge iiseli kwi-300 g imizuzu eyi-7 ukususa imidiya yenkcubeko yeseli. Lahla amandla amakhulu.
6. Phinda umise iiseli kwi-1 mL ye-DPBS.
7. Ukulinganisa i-concentration yeseli kwaye ulungelelanise kwi-1E + 06 iiseli / mL.
   • ICEBISO: Imidiya yeSeli exutywe i-1: 4 kunye ne-DPBS ingasetyenziselwa ukongeza izondlo kwiiseliampngaphantsi kwimeko yeeseli ezinovakalelo okanye uhambo olude lovavanyo.

Ukulungiswa kweeSeli

1. I-Centrifuge ukuya kwiiseli ze-10E + 06 kwi-300 g yemizuzu ye-7 ukususa imidiya yenkcubeko yeseli. Lahla amandla amakhulu.
2. Phinda umise i-pellet yeseli kwi-1 mL PBS, i-centrifuge kwaye ulahle i-supernatant.
3. Phinda umise i-cell pellet kwi-500 µL PBS/2% PFA (62.5 µL ye-16% yesisombululo se-PFA + 437.5 µL PBS).
4. Faka iiseli kwiqondo lobushushu begumbi imizuzu eyi-10 (kunye nokungcangcazela okuthambileyo).
5. Yongeza i-500 µL ye-PBS kunye ne-centrifuge yokunqunyanyiswa kwi-400 g imizuzu emi-5 ukususa i-PFA.
6. Lahla amandla amakhulu.
7. Phinda umise iiseli kwi-1 mL PBS, hluza nge-30 µm i-cell strainer, i-centrifuge (400 g, 5 min) kwaye ulahle i-supernatant.
8. Phinda umise iiseli malunga ne-1 mL ye-PBS (ukhetho: lungelelanisa kwi-concentration yokugqibela ye-5E + 06 iiseli / mL).
9. Gcina iiseli ezimiselweyo kwi-2-8 ° C, sebenzisa kwinyanga.
   • Ukuba iiseli ezininzi kufuneka zilungiswe, nceda unyuse onke amanani ngokufanelekileyo.
   • PHAWULA: Isantya se-Centrifugation ngexesha lokuvunwa kweeseli sinokuhlengahlengiswa kuhlobo lweeseli, ukuba iiseli zibuthathaka kwaye i-g-force esezantsi isetyenziswa rhoqo.
   • Ugxininiso lwe-PFA lunokwahluka phakathi kwe-0.5 kunye ne-4%.

scIC ukuseta uvavanyo

Olu vavanyo lulandelayo lwe-scIC kufuneka lufakwe kuphuhliso lovavanyo kwaye lunokuphinda luphindwe ngaphakathi kokuhamba komsebenzi wokuvavanya kamva.

Ukuhamba komsebenzi wophuhliso lovavanyo lwahlulwe ngamanyathelo ama-3 ahlukeneyo alandelelanayo:

1. Uvavanyo lweCyto chip
2. Hlalutya uvavanyo kuphela
3. Uvavanyo lweKinetics

Uvavanyo lweCyto Chip

• Umgangatho wetshiphu entsha kufuneka uvavanywe phambi kokusebenzisa itshiphu yecyto kumlinganiselo. Qala ngokuqhuba uvavanyo lweCyto Chip ngokwahlukileyo. Jonga i-Helix cyto chip isahluko kwisikhokelo esiphambili malunga nendlela yokuseta kunye nokuvavanya uvavanyo lwe-chip.

Hlalutya Kuphela Uvavanyo

• Ukuphuhliswa kovavanyo lwe-scIC luqala ngoVavanyo lwe-Analyte Kuphela, oluvavanya ukuziphatha kwabahlalutyi ababhalwe nge-fluorescently kwindawo entsha ye-chip ngaphandle kweeseli.
• Olu vavanyo lunokuphinda luphindwe ngamaxesha ukuvavanya ukuzinza kwabahlalutyi ababhaliweyo. I-scIC Hlalutya Kuphela Uvavanyo lunokucwangciswa kwiimvavanyo ze-Kinetics ngokwenza i-Analyte kuphela ibhokisi yokukhangela phantsi kwezicwangciso zeSeli.
• Ukuvavanya umgangatho wokuhlaziya, ugxininiso oluphezulu olusetyenzisiweyo lwanele kwaye lunokulungiswa ngokukhubaza yonke into kodwa enye yemibutho kwi-assay.

I-Kinetics Assay Ukusetwa kokuhamba komsebenzi

• Uvavanyo lwekinetics luqhele ukusetyenziswa ngaphakathi kokuhamba komsebenzi okuzenzekelayo okuqulethe zombini iimvavanyo zokuvelisa idatha kunye nezinto zokulondoloza. Emva kokuba i-analyte iphumelele ulawulo lomgangatho we-Analyte uvavanyo kuphela, lunokusetyenziswa kumlinganiselo kwiiseli.
• Ukulinganisa amaxabiso e-kinetic ngokuthembekileyo, impendulo yomqondiso ngexesha lobudlelwane kufuneka ixhomekeke ekugxininiseni, inyuke ngogxininiso oluphezulu lwe-analyte.
• Olona gxininiso luphezulu lufanele lufikelele kwithafa, nto leyo ebonisa ukuba ukubophelela-zinzile kuphunyeziwe. I-dissociation kufuneka iqhutywe ixesha elide ngokwaneleyo ukukhulula inxalenye ebalulekileyo ye-analyte eboshiweyo (ngaphezulu kwe-5%) ukwenzela ukuba ukulungeleka okuthembekileyo kwejika.
• Sebenzisa iiparameters ezingagqibekanga kwiimeko zovavanyo njengendawo yokuqala kwaye uhlengahlengise ngakumbi ngokuxhomekeke kwiziphumo zakho.

Example 1. Ukuqhutywa kovavanyo olucetyiswayo

I-assay workflow ivumela abasebenzisi ukuba balandele iindlela zokulinganisa kunye nokugcinwa komgca ngokusekelwe kwiimfuno zabo zovavanyo oluthile.

Ukuqhutywa kovavanyo kunokubandakanya la manyathelo alandelayo ngendlela ebonisiweyo:

1. Prime (Itshiphu yesondlo)
2. I-scIC Kinetics (Iiseli A, i-Analyte A, i-Cyto chip)
3. Ukuhlamba inkqubo (Itshiphu yoLondolozo)
4. I-scIC Kinetics (Iiseli B, i-Analyte A, i-Cyto chip)
5. Ukuhlamba inkqubo (Itshiphu yoLondolozo)
6. Hlalutya eqwalaselweyo kuphela i-scIC Kinetics (Uhlahlelo A, itshiphu yeCyto)
   • I-Prime kunye ne-System yokuhlamba amanyathelo enziwa kwiChip yokuLondoloza. Ukuhlanjwa kweNkqubo kufakwe xa utshintshela kumgca weseli eyahlukileyo kwaye ngaphambi kokuba uqhube uvavanyo lwe-Analyte kuphela ekupheleni komsebenzi.
   • Ukufumana iinkcukacha ezithe kratya, jonga kwicandelo leCyto yokuhlamba inkqubo kwisikhokelo esiphambili.
   • Xa ufola iimvavanyo zekinetics kwiiseli, qwalasela ukusebenza kweseli, exhomekeke kumgca weseli.
   • Kwimigca yeeseli ebuthathaka, kuyacetyiswa ukuba kuqhutywe kuphela iimvavanyo ezi-1-2 ngokuqhutywa komsebenzi wovavanyo. Ukufumana imigca yeeseli ezomelele ngakumbi, iimvavanyo ezongezelelweyo zinokufakwa emgceni.
   • PHAWULA: Kwiimvavanyo ezongezelelweyo, lungiselela isisombululo seseli kwii-vials ezahlukeneyo ngokuthiya iiseli ngokwahlukileyo.
   • Qinisekisa ukuba zonke ii-reagents zintsha kwaye zihluzwa ngesihluzo se-0.22 µm. Ekugqibeleni, faka ii-reagents ezilungiselelwe kwisixhobo njengoko uyalelwe yi-heliOS.
   • Ukubaleka kunokuqalwa ngokucofa utolo oluluhlaza okwesibhakabhaka “Run” icon kwaye ulandele amanyathelo kwi-Assay Start Wizard.
   • Nje ukuba ubaleke uqalisile, isixhobo sisebenza ngokuzimeleyo de kube kugqitywe ukuqhutywa komsebenzi wovavanyo.

Uhlalutyo lwemifuniselo ye-scIC

scIC Uhlalutyo lokuhamba komsebenzi

• Idatha ye-scIC inokwahluka kwidatha ye-biosensor eqhelekileyo ngenxa yobunzima bendalo beziganeko ezenzeka kwiiseli ezifakwe kwi-heliXcyto chip surface, enokubangela ukungahambi kakuhle kwe-sensorgram.
• Ngoko ke, ukulawulwa komgangatho kugxininiswe kwimifanekiso ebanjwe kwinqanaba ngalinye lokulinganisa kunye neesensograms ezenziwe kwiphepha lokufika lohlalutyo oluzenzekelayo.
• Ukongeza, i-heliOS ibonelela ngezixhobo zokujongana nezitenxo ze-sensorgram ngexesha lohlalutyo lwedatha ye-scIC.

Inkqubo yohlalutyo lwedatha ye-scIC:

1. Ukuhlolwa koMfanekiso:
   • Jonga yonke imifanekiso ethathiweyo kulo lonke umlinganiselo (Ithebhu yeMifanekiso kwifestile yovavanyo).
   • Zingaphi iiseli ezisele zizinzile kwimigibe kulo lonke umlinganiselo?
   • Injani imeko yeeseli? Jonga i-granularity kunye nokuthembeka kweseli.
   • Ngaba imigibe icocekile emva kwenyathelo lokuvuselela?
2. Ujongo lwedatha ekrwada:
   • Phonononga idatha ekrwada yereferensi yendawo 1 kunye nendawo yokulinganisa 2
   • Isiginali yencopho yesiqhelo kufuneka isondele okanye ibephezulu kunophawu lwedatha ekrwada iphezulu.
   • Ngaba umqondiso ubuyela kumgangatho wesiseko kuzo zombini iindawo, okanye kukho ubungqina bokubopha okungacacanga?
3. Ujongo lweDatha oluQhelekileyo:
   • Ingaba inaliti itsiba indawo yoku-1 kunye nebala lesi-2 eligqume ngokufanelekileyo?
4. UJongo lweDatha eQinisekisiweyo:
   • Ngaba inyathelo lokuhlaziya libuyisela umqondiso kwisiseko? Ukuba akunjalo, khangela ukuba kukho iiseli okanye i-debris kwimigibe emva kokuvuselelwa ngokutshaviewukujonga imifanekiso.
   • Ngaba kukho i-spikes, i-outliers, okanye ezinye izitenxo kwiigophe ezikhankanyiweyo? Ukuba kunjalo, review imifanekiso ukuchonga izizathu ezinokubakho kwaye uqwalasele uhlengahlengiso lwezandla.
5. Ujongo lweDatha yokuFina:
   • Ukwenza uvavanyo lomgangatho wokubonwayo
   • Ngaba ukulingana kuchaza ngokuchanekileyo ukuziphatha kweegophe, okanye ngaba umgca ofanelekileyo uwela i-sensorgram?
   • Ngaba ukulingana kuchaza ngokufanelekileyo ukugoba kwedatha?
   • Ingaba i amplitude iyahambelana nedatha ebhekiswayo?

scIC Uhlalutyo lwedatha oluzenzekelayo

• Uhlalutyo oluzenzekelayo lwe-scIC luqhuba idatha eluhlaza kwiiplani zokulawula umgangatho kunye nedatha edibeneyo ngokuchofoza nje okumbalwa.
  1. Vula umfuniselo we-scIC ngokucofa kabini kumfuniselo okhethiweyo kuLuhlu loMfuniselo
  2. Cofa iqhosha elikhulu elibhulowu Hlalutya ezantsi komfuniselo thebhu.
  3. Ukusuka kuvavanyo lomsebenzi, khetha isivavanyi ongathanda ukusihlalutya.
  4. Khetha iKinetics scIC kwiintlobo zohlalutyo ezikhoyo kwaye ucofe Okulandelayo.
     • PHAWULA: Ukuba uvavanyo lusaqhuba, kuphela iimvavanyo ezigqityiweyo ziya kuboniswa kuluhlu. Nje ukuba uvavanyo lukhethiwe, iwindow elandelayo iya kubonisa zonke iintlobo zohlalutyo olukhoyo oluthile kwindlela / uvavanyo olusetyenzisiweyo.
  5. Ukuba kuyimfuneko, qwalasela uhlalutyo kwifestile elandelayo. Imodeli ehlala ikho “yiKinetics – inqanaba lesiphelo sasimahla” kunye nebhokisi yokukhangela “Nyanzelela inqanaba lesiphelo ukuya kuZero” iyasebenza. Ukutenxa kule modeli kuphela ukuba ibhayoloji okanye idatha ifuna inkcazo entsonkothileyo.
  6. Nje ukuba uqwalaselo lugqitywe, cofa u- Hlalutya ukuze ubone zonke ii-snapshots ezifunyenweyo, iisensograms ezikrwada kunye nezicutshungulwayo, kunye namaxabiso e-kinetics.
• Ngenxa yokuntsonkotha okungokwemvelo kwesampI-lens isetyenziselwa uvavanyo lwe-scIC, i-sensorgrams ezibangelwayo zinokuba nezitenxo.
• Ukujongana nale nto, ukulawulwa komgangatho wemifanekiso kunye neesensograms kugxininiswe kuhlalutyo oluzenzekelayo.
• Ukuba izilungiso ezenziwe ngesandla okanye uhlalutyo olunzulu ngakumbi luyafuneka, izixhobo zokulungiswa kwe-artifact zibonelelwa ngaphakathi kohlalutyo lwesandla lweScratchpad.

i-heliXcyto Ukugcinwa kunye nokuNcitshiswa

• Ukugcinwa kwe-heliXcyto kubalulekile ukuqinisekisa ukusebenza kakuhle kwesixhobo kunye nokuphila ixesha elide. Ukugcinwa rhoqo kubandakanya iinkqubo zokucoca ezikhusela ukungcola kunye nokugcina ukunyaniseka kwenkqubo, okwenza iziphumo ezichanekileyo kunye nokusebenza kakuhle. Ukunyamekela okuhambelanayo kubalulekile ekuthembekeni kwesixhobo kunye nomgangatho weziphumo zovavanyo.
  Ukugcinwa kwe-heliXcyto kubandakanya iinkqubo ezimbini eziphambili: I-Cyto System Wash, enokuthi idibaniswe ngokuthe ngqo kwi-assay workflow, kunye ne-Coca & Sleep, eyenziwa njengendlela eyahlukileyo yokugcina isixhobo ngaphambi kokusetyenziswa emva kokulinganisa kwangaphambili kwexesha elide okanye xa ingasetyenziswanga ngaphezu kweentsuku ze-2.
• Zombini i-System yokuhlamba kunye nokuCoca kunye nokulala kufuna ubukho be-heliX® Maintenance Chip kwi-chip tray.

Coca & Ukulala Umgaqo

• Indlela yokuCoca nokulala yenzelwe ukucoca kunye nokuvalwa kwesixhobo/ukugcinwa kwesixhobo. Indlela yokuhlamba iityhubhu ye-fluidic yesixhobo se-heliX® kuqala ngamanzi acocekileyo kwaye emva koko nge-ethanol engama-70%. Ekugqibeleni, zonke iityhubhu zikhutshwa ngomoya.
• Le nkqubo yokucoca izenzekelayo ngokupheleleyo kwaye ithatha malunga nemizuzu engama-40. Ngexesha lenkqubo, i-ethanol ikhutshwa kwibhotile yamanzi, ngoko iziqulatho zebhotile kufuneka zilahlwe emva koko.
• Emva kokuCoca kunye nokuLala, isixhobo singena kwindlela yokulala kwaye asinakusetyenziswa de i-Wake Up & Prime yenziwe. I-heliX® Maintenance Chip iyafuneka kuyo yomibini imitsi.
• OKUBALULEKILEYO: Ukuthintela ukungcoliswa ityhubhu, qiniseka ukuba ulahle iziqulatho zebhotile yamanzi (umxube wamanzi / we-ethanol) kwaye ukhuphe isitya senkunkuma emva kwenkqubo yokucoca.

Cima kunye nokugcinwa kwexesha elide

• Ngexesha elongeziweyo lokungasebenzisi, nceda ususe ibhotile enamanzi kunye ne-ethanol emva kwenkqubo yokuCoca & yokulala kwi-tray ye-buffer kwaye ushiye i-tubing ibonakale emoyeni.
• Kwakhona susa i-heliX® Maintenance Chip, kwaye uyigcine kwisingxobo sayo sokuqala. Oku kuthintela ukubuyela umva kunye nokungcoliseka. Cima isixhobo.

Ukuhlamba inkqubo yeCyto

• I-heliXcyto System Wash ihlambulula kakhulu inkqubo ye-microfluidic usebenzisa i-Cleaning Solution 3 (CS3) ekuhambeni kwemizuzu engama-45. I-CS3 icholwa ngemizuzwana emibinitages. Inaliti yokuqala kunye no-sampi-loop iyazaliswa kwaye incubated ukususa nakuphi na ungcoliseko.
• PHAWULA: Qalisa iCyto System Hlamba ubuncinci yonke imihla xa isixhobo sisetyenziswa. Sincoma ukongeza iCyto System Wash indlela kwi assay workflow nokuba emva kovavanyo ngalunye (xa utshintshela kumgca weseli entsha okanye ukuba sampumgangatho we-le ungowona mgangatho uphezulu) okanye ukuya ekupheleni kokuhamba komsebenzi wovavanyo. Tshintshelanisa ukugcinwa kwetshiphu emva kobuninzi be-50 yokuhlamba kweSistim ye-Cyto eboniswe njengesibalo soHlaziyo kwi-Chip tray. view yolawulo lweSixhobo.

Qhagamshelana

• Dynamic Biosensors GmbH
• Perchtinger Str. 8/10
• 81379 eMunich
• Jemani
• Bruker Scientific LLC
• 40 Manning Road, Manning Park
• Billerica, MA 01821

i-USA

• Ulwazi lweodolo order.biosensors@bruker.com
• Uxhaso lobuchwepheshe support.dbs@bruker.com
• www.dynamic-biosensors.com
• Izixhobo kunye neetshiphusi zenziwe ngobunjineli kwaye zenziwe eJamani.
• ©2025 Dynamic Biosensors GmbH
• Ukusetyenziswa koPhando kuphela. Ayinakusetyenziswa kwiinkqubo zokuxilonga zonyango.

FAQ

scIC FAQs

Ndingayisebenzisa kwakhona itshiphu ye-heliXcyto?

Ewe, itshiphu ye-heliXcyto iyasebenziseka kwakhona kwaye iyalahlwa. Landela izikhokelo ezifanelekileyo zokucoca kunye nokugcinwa.

Yintoni injongo yeChip yokuLondoloza?

I-Chip yokuLondoloza isetyenziselwa ukucoca, ukuvavanya, kunye nemisebenzi yokulondoloza ukuqinisekisa ukusebenza kakuhle kwenkqubo.

Kufuneka ndicoce kangaphi isixhobo?

Inkqubo ye-fluidic ye-heliXcyto igcinwe icocekile kunye neCyto System Wash kunye neendlela zokuCoca & zokulala. Inkqubo ye-microfluidic iyacetyiswa ukuba icocwe ngeCyto System Wash kwi-Chip yokuLondoloza emva kovavanyo ngalunye (ingakumbi xa utshintsha uhlobo lweseli) okanye yakutshanje ekupheleni kovavanyo lokuhamba komsebenzi. Inkqubo yokuCoca nokulala kufuneka isetyenziswe kanye phambi kokusetyenziswa emva komlinganiselo omde wangaphambili (umzekelo, kusasa emva kovavanyo lokulala) okanye xa i-heliXcyto ingasayi kusetyenziswa ngaphezulu kweentsuku ezi-2 zokuvala.

Kuze kube nini izisombululo: RB 1 / amanzi / CS 1 / CS 3 / isisombululo se-Normalization sigcinwe kwisixhobo?

Ngokubanzi, phambi komlinganiselo ngamnye, zonke izisombululo kufuneka zivavanyelwe umthamo oseleyo kunye ne-turbidity enokubakho okanye imvula. Kwimeko enjalo isisombululo kufuneka sitshintshwe ngokukhawuleza. Amanzi kunye ne-RB 1 kufuneka itshintshwe yonke imihla kwaye i-RB 1 kufuneka ihluzwe phambi komlinganiselo ngamnye. Isisombululo se-Diluted Normalization singasetyenziselwa iintsuku ezi-2 zilandelelana. Ukucoca Izisombululo zizinzile malunga neentsuku ezi-3.

Ndingayisebenzisa ixesha elingakanani itshiphu ye-heliXcyto?

Umhla wokuphelelwa kwe-chip ye-heliXcyto unokutshekishwa kwi-Chip tray tab kwicandelo leSixhobo kwi-heliOS. Akukho siqinisekiso sokuthembeka kwetshiphu esinokunikwa emva komhla wokuphelelwa. Nangona kunjalo, nje ukuba izingibe zihlala zizinzile, umphezulu ucocekile, kwaye imvelaphi ye-fluorescence ingaphantsi kwe-cut-off eboniswe kwi-Chip Test, i-chip ithathwa njengesetyenziselwa imilinganiselo. Ukucocwa rhoqo kwetshiphu yangaphandle (Ikhithi yokuCoca iChip CCK-1-1 iyacetyiswa ukuba yenziwe emva kokusetyenziswa kwetshiphu ukwandisa ixesha lokuphila kwetshiphu kakhulu.

Ndingayisebenzisa ixesha elingakanani itshiphu yeSondlo?

I-chip yokuLondoloza iyadingeka ukuba itshintshwe emva kokuhlamba kweSistim ye-50 (ibalwe njengohlaziyo kwi-Chip tray tab yolawulo lweSixhobo kwi-heliOS).

Kufuneka lwenziwe kangaphi uvavanyo lwe-heliXcyto Chip?

Uvavanyo lweChip luqokelela ulwazi malunga nemvelaphi ye-fluorescence, ucoceko, kunye nokuthembeka kwetshiphu phambi kokuqala umfuniselo omtsha. Kufuneka yenziwe kanye xa kusetyenziswa itshiphu entsha, kodwa kuyacetyiswa ukuba yenziwe phambi okanye ekuqaleni kovavanyo ngalunye ukujonga ubume betshiphu. Oku kwenza ukuba iimeko zokulinganisa zilungelelaniswe kunye nokungaqhelekanga kwi-sensorgrams ukuba zilandelwe kwi-chip okanye isixhobo.

Yintoni ikhemistri yokuleyibhela i-analyte?

Idayi ye-fluorescent ebomvu okanye eluhlaza, ngokulandelelanayo, idityaniswa ziikiti zethu zokuleyibhela nge-NHS-esters ukuya kwii-amine eziphambili kuhlalutyi lweprotheyini (umz. i-lysines okanye i-N-terminus). Iinkcukacha kunye necandelo lokusombulula ingxaki inokufumaneka kwi-heliXcyto yethu yokuLayibhisa idayi ebomvu (i-Labeling Kit idayi ebomvu 2 CY-LK-R2-1) kunye ne-heliXcyto ye-Labeling Kit idayi eluhlaza (i-Labeling Kit idayi eluhlaza CY-LK-G1-1 ) iincwadi ezivela kuthi webivenkile.

Apho ukufumana iziqinisekiso heliOS kunye nolwazi lwelayisensi?

Inkqubo yokufaka iziqinisekiso kunye nolwazi lwelayisenisi ichazwe kwicandelo loFakelo lwe-heliOS kwisikhokelo se-heliXcyto esivela kuthi. webindawo (isikhokelo se-heliXcyto). Ulwazi lwakho lwelayisensi kufuneka lugcinwe kwifolda ye-scIC kwi-HeliXcyto PC Desktop, ethe yaqulunqwa yiNgcali yethu yeSicelo ngexesha loqeqesho.

Luluphi uchulumanco/ukukhutshwa koluhlu lwe-heliXcyto ?

I-excitation ebomvu yi-605-625 nm, ukukhutshwa (ukufunyanwa) ngu-655-685 nm. I-excitation eluhlaza 490-510 nm, ukukhutshwa kohlaza ngu-525-575 nm.

Mangaphi amaxesha ekufuneka ndilinganise umfuniselo ofanayo wokuthembeka kweenkcukacha-manani?

Ukufezekisa imilinganiselo ye-kinetic ethembekileyo, ukuphindaphinda kwe-3-4 ukuphindaphinda kwe-3-concentration ye-Kinetics imilinganiselo iyanconywa ukuba yenziwe kwi-cell population. Kwiseti yedatha ehambelanayo, ukudibanisa kunye nokwahlukana kweereyithi zokuphindaphinda kufuneka kube ngaphakathi kwefestile ye-2-4 amaxesha.

Yintoni uvakalelo lwemilinganiselo ye-scIC?

Umda osezantsi wokufumanisa ngokubhekisele kwinkcazo ekujoliswe kuyo kuxhomekeke kwileyibhile yohlalutyi, kodwa ngokuqhelekileyo i-scIC inokuthi sele ilinganise i-kinetics kwii-molecule ze-1000-10000 ngeseli nganye. Ukwandisa uvakalelo, ubuncinane iiseli ezi-5 zicetyiswa ukuba zibanjwe, kwaye uhlalutye i-DOL ye-5-10 kunye namandla e-LED kufuneka alungelelaniswe ukuze afezekise amanani aphezulu e-fluorescence.

Yiyiphi imida yokubona i-heliXcyto ngokwemilinganiselo ye-kinetic?

Nceda ubhekisele kwiiNkcazo zezoBuchwephesha ze-heliXcyto kwimida yobugcisa obuphezulu, enokufumaneka kwi-heliXcyto guide (i-heliXcyto guide). Ukwahlukana rhoqo Kd: 10 pM ukuya 1 mM Umbutho ireyithi rhoqo kon: 1E3 ukuya 1E7 M-1s-1 Ireyithi yokwahlukana rhoqo koff: 1E-6 ukuya 1 s-1 Ngolwazi oluneenkcukacha nceda ujonge isikhokelo sethu se-heliXcyto kwisikhokelo sethu webindawo.

Amaxwebhu / Izibonelelo

BIOSENSORS eguquguqukayo heliX cyto ngokuPheleleyo oluzenzekelayo inkqubo yoHlahlelo lweLabhoratri [pdf] Incwadi yokusebenzisa I-heliX cyto i-Automated Laboratory Analysis System, i-heliX cyto, iNkqubo yokuHlalutya iLebhu yeLabhoratri, iNkqubo yokuHlalutya iLabhoratri, iNkqubo yokuHlalutya iLabhoratri, iNkqubo yokuHlalutya, iNkqubo

Iimbekiselo

• Incwadi yokusebenzisa