DK2864781T3 - En fremgangsmåde og et system til kvantitativ eller kvalitativ bestemmelse af en målkomponent - Google Patents

En fremgangsmåde og et system til kvantitativ eller kvalitativ bestemmelse af en målkomponent Download PDF

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DK2864781T3
DK2864781T3 DK13807718.5T DK13807718T DK2864781T3 DK 2864781 T3 DK2864781 T3 DK 2864781T3 DK 13807718 T DK13807718 T DK 13807718T DK 2864781 T3 DK2864781 T3 DK 2864781T3
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Denmark
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magnetic particles
fluorophores
flow channel
target component
liquid sample
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DK13807718.5T
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English (en)
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Martin Heller
Madsen Niels Kristian Bau
Bent Overby
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Scandinavian Micro Biodevices Aps
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Claims (20)

1. Fremgangsmåde til kvantitativ eller kvalitativ bestemmelse afen målkomponent, såsom et biomolekyle i en flydende prøve, hvor fremgangsmåden omfatter at tilvejebringe en flerhed af magnetiske partikler (17, 54, 64) omfattende et eller flere indfangningssites (55, 56) for målkomponenten (51, 61) på deres respektive overflader; at tilvejebringe en flerhed af fluorforer (18, 52, 62) konfigureret til at binde til nævnte indfangningssites (55, 66) af de magnetiske partikler (17, 54, 64); at anbringe nævnte fluorforer (18, 52, 62) og nævnte magnetiske partikler (17, 54, 64) med afstand til hinanden i en strømningskanal (11, 21) afen mikrofluidindretning (31) og nedstrøms fra et indløb (13, 23) til strømningskanalen (11, 21); at indføre den flydende prøve i nævnte strømningskanal (11, 21) via nævnte indløb (13, 23) og blande den med nævnte fluorforer (18, 52, 62) og nævnte magnetiske partikler (17, 54, 64); og mindst midlertidigt at immobilisere nævnte magnetiske partikler (17, 54, 64) tilstødende til et transparent vindue af strømningskanalen (11, 21) under anvendelse afen magnet (33), at emittere exciterende elektromagnetisk stråle (38a) mod nævnte immobiliserede magnetiske partikler (17, 54, 64), at læse signaler (39a) emitteret fra fluorforer (18, 52, 62) indfanget af nævnte immobiliserede magnetiske partikler (17, 54, 64) og at udføre en kvantitativ eller kvalitativ bestemmelse af nævnte målkomponent (51, 61) baseret på de læste signaler (39a), hvor fortrinsvis den flydende prøve omfatter en biologisk fluid eller en fraktion af en biologisk fluid, såsom menneske- eller dyre- eller plantefluid for eksempel blod, spyt, urin, mælk, cytosol (intracellulær fluid), interstitial fluid (vævsfluid) og/eller en eller flere fraktioner og/eller blandinger deraf, og/eller opslæmmede biologiske faststoffer, såsom væv eller faste fødevarer f.eks. kød eller grøntsager.
2. Fremgangsmåden ifølge krav 1, hvor målkomponenten omfatter en mikroorganisme såsom mindst en af bakterie-, virus- eller svampepatogener, f.eks. E-coli E. coli, Citrobacter spp, Aeromonas spp., Pasteurella spp., non-serogroup DI Salmonella, Camphylobacter Staphylococcus spp. og kombinationer deraf og/eller målkomponenten omfatter en celle, såsom en blodcelle, en stamcelle eller en tumorcelle og/eller målkomponenten omfatter proteiner, nukleotider, kulhydrater eller lipider, i særdeleshed et enzym, et antigen eller et antistof.
3. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor indfangningssites (55, 66) er specifikke for nævnte målkomponent (51, 61) eller specifikke for en gruppe af komponenter omfattende en eller flere målkomponenter (51, 61).
4. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor de magnetiske partikler er belagte magnetiske partikler omfattende en belægning omfattende indfangningssitesene, hvor indfangningssitesene vælges til at være indfangningssites for målkomponenten, idet belægningen for eksempel omfatter indfangningssitesene i formen af antigen, antistof, avidin, biotin eller ged-anti-mus-IgG.
5. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor fluorforerne er kvantepunkter eller aromatiske prober og/eller konjugerede prober, fortrinsvis hvor fluorforerne er konfigureret til at binde til nævnte indfangningssites af de magnetiske partikler ved at være koblet til en komponent, der kan binde til indfangningssitesene af de magnetiske partikler, hvor komponenten er fortrinsvis identisk eller homolog med nævnte målkomponent.
6. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor fluorforerne er kvantepunkter, der emitterer en eller flere diskrete frekvenser af lys, når stimuleret af en lyskilde, hvor hvert kvantepunkt omfatter en kerne af et exciterbart materiale, såsom en halvleder nanopartikel eller en sjælden jorddoteret oxid-kolloid-nanopartikel, fortrinsvis hvor kvantepunkterne hver omfatter en kerne med en størrelse på op til omkring 25 nm, såsom fra 2-10 nm, kvantepunkterne fortrinsvis omfatter en organisk belægning, såsom en polymerbelægning, hvor belægningen er koblet til en komponent, der kan binde til indfangningssitesene af de magnetiske partikler.
7. Fremgangsmåden ifølge krav 6, hvor nævnte fluorforer (18, 52, 62) og nævnte magnetiske partikler (17, 54, 64) midlertidigt immobiliseres i nævnte strømningskanal (11, 21) af mikrofluidindretningen (31) således at de ikke kan binde til hinanden forud for indføringen af nævnte flydende prøve til nævnte strømningskanal (11, 21).
8. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor fremgangsmåden omfatter at tillade indfangningssitesene af de magnetiske partikler (17, 54, 64) at indfange eventuel målkomponent i den flydende prøve og/eller fluorforer (18, 52, 62), hvor fremgangsmåden fortrinsvis omfatter at pulsere nævnte flydende prøve i nævnte strømningskanal eventuelt under anvendelse afen aktuator, hvor efter de magnetiske partikler (17, 54, 64) mindst midlertidigt immobiliseres tilstødende til nævnte transparente vindue under anvendelse afen magnet.
9. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor nævnte mindst midlertidigt immobiliserede magnetiske partikler (17, 54, 64) udsættes for nævnte elektromagnetiske stråle (38a) således at mindst en del af eventuelle fluorforer (18, 52, 62) indfanget af nævnte indfangningssites (55, 65) af de magnetiske partikler (17, 54, 64) exciteres, hvor efter det emitterede signal (39a) fra hvilke som helst indfangede fluorforer (18, 52, 62) læses og en kvantitativ eller kvalitativ bestemmelse af nævnte målkomponent (51, 61) baseret på det læste signal (30a) udføres, hvor fortrinsvis nævnte mindst midlertidigt immobiliserede magnetiske partikler (17, 54, 64) frigives fra magnetiske kræfter påført af magneten (33) forud for udsættelse for nævnte elektromagnetiske stråle (38a).
10. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor nævnte fremgangsmåde omfatter at udføre to eller flere parallelle assayer på den flydende prøve til kvantitativ eller kvalitativ bestemmelse af målkomponenten(s), idet hvert assay omfatter: - at bringe en del af den flydende prøve i kontakt med nævnte fluorforer (18, 52, 62) og nævnte magnetiske partikler (17, 54, 64) i en mikrofluidindretning (31) omfattende et transparent vindue; og - mindst midlertidigt at immobilisere nævnte magnetiske partikler (17, 54, 64) tilstødende til nævnte transparente vindue under anvendelse af en magnet (33), at emittere exciterende elektromagnetisk stråle (38a) mod nævnte immobiliserede magnetiske partikler (17, 54, 64), og at læse signaler (38b) emitteret fra fluorforer (18, 52, 62) indfanget af nævnte immobiliserede magnetiske partikler (17, 54, 64), fortrinsvis hvor fluorforerne og/eller de magnetiske partikler anvendt i et af de to eller flere parallelle assayer er forskellige fra fluorforerne og/eller de magnetiske partikler anvendt i et andet af de to eller flere parallelle assayer, fortrinsvis hvor fluorforerne og/eller de magnetiske partikler anvendt i et af de to eller flere parallelle assayer er forskellige fra fluorforerne og/eller de magnetiske partikler anvendt i et andet af de to eller flere parallelle assayer i forhold til type, størrelse, belægning, form og/eller mængde.
11. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor den kvantitative eller kvalitative bestemmelse af målkomponent(er) (51, 61) i en flydende prøve udføres ved sammenligning af det/de læste signal(er) med signaler opnået fra flydende prøver af kendt sammensætning og/eller ved at multiplekse det/de læste signal(er) fra forskellige grupper af fluorforer f.eks. fra det samme assay eventuelt under anvendelse af reference fluorforer med en forskellig excitationsbølgelængde, fra fluorforer fra parallelle assayer og/eller fra fluorforer i referencetests af kendte eller ukendte flydende prøver.
12. Mikrofluidindretning til anvendelse i fremstilling afen flydende prøve til optisk analyse til kvantitativ eller kvalitativ bestemmelse afen målkomponent (51, 61) i prøven, hvor mikrofluidindretningen (31) omfatter mindst en strømningskanal (11, 21) med et transparent vindue og et indløb (13, 23) for den flydende prøve, kendetegnet ved at mikrofluidindretningen (31) yderligere omfatter i sin strømningskanal (11, 21) - en flerhed af magnetiske partikler (17, 54, 64) omfattende indfangningssites (55, 65) for målkomponenten (51, 61) på deres overflader; og - en flerhed af fluorforer (18, 52, 62) konfigureret til at binde indfangningssitesene (55, 65) af de magnetiske partikler (17, 54, 64), hvor nævnte fluorforer (18, 52, 62) og nævnte magnetiske partikler (17, 54, 64) er anbragt nedstrøms til nævnte indløb (13, 23) og med afstand til hinanden i nævnte strømningskanal (11, 21), således at ved indføring af den flydende prøve i strømningskanalen (11, 21) via nævnte indløb (13, 23), blandes nævnte flydende prøve med nævnte magnetiske partikler (17, 54, 64) og nævnte fluorforer (18, 52, 62).
13. System til kvantitativ eller kvalitativ bestemmelse afen målkomponent (51, 61) i en flydende prøve, hvor systemet omfatter - mikrofluidindretningen (31) ifølge krav 12; - en magnet (33) anbragt til mindst midlertidigt at immobilisere nævnte magnetiske partikler (17, 54, 64) tilstødende til nævnte transparente vindue; - en emitter (38) til at excitere nævnte fluorforer (18, 52, 62), og - en læser (39) til at læse signaler emitteret fra nævnte fluorforer (18, 52, 62).
14. Systemet ifølge krav 13, hvor mikrofluidindretningen (31) er af polymer og/eller glas, og mikrofluidindretningen (31) omfatter et substrat 12, 22) med en rille til strømningskanalen (11, 21) og et folie (11a), der dækker strømningskanalen (11, 21) og mikrofluidindretningen (31) omfatter et indløb (13, 23) til sugning i den flydende prøve, fortrinsvis hvor mikrofluidindretningen (31) omfatter et fleksibelt vægafsnit (15) af strømningskanalen (11, 21) eller et sinkafsnit (14, 24) i fluidforbindelse med strømningskanalen (11, 21), hvor systemet fortrinsvis omfatter en aktuator, fortrinsvis hvor aktuatoren er anbragt til at bevæge det fleksible vægafsnit, hvor aktuatoren eventuelt er en step motordrevet aktuator.
15. Systemet ifølge krav 14, hvor strømningskanalen (11, 21) er i fluidforbindelse med et sinkafsnit (14, 24) af mikrofluidindretningen, fortrinsvis hvor mikrofluidindretningen (31) omfatter et excitations- og udlæsningsområde henvist til som et læseområde (16, 26) i formen af det transparente vindue, hvor vinduet er transparent for mindst de exciterende og emitterende bølgelængder af fluorforerne (18, 52, 62).
16. Systemet ifølge et hvilket som helst af kravene 13-15, hvor systemet omfatter en temperaturregulator til at regulere temperaturen af den flydende prøve i strømningskanalen, hvor temperaturregulatoren eventuelt omfatter et peltier-element, et tyndfilm-opvarmningselement og/eller andre resistive opvarmningselementer.
17. Systemet ifølge et hvilket som helst af kravene 13-16, hvor de magnetiske partikler (17, 54, 64) er belagte magnetiske partikler omfattende en belægning omfattende indfangningssitesene, fortrinsvis hvor fluorforerne er kvantepunkter eller aromatiske prober og/eller konjugerede prober, fluorforerne er fortrinsvis kvantepunkter, mere fortrinsvis fluorforerne er konfigureret til at binde til nævnte indfangningssites af de magnetiske partikler ved at være koblet til en komponent, der kan binde til indfangningssitesene af de magnetiske partikler, hvor komponenten er fortrinsvis identisk eller homolog med nævnte målkomponent.
18. Systemet ifølge et hvilket som helst af kravene 13-17, hvor nævnte emitter omfatter mindst en optisk fiber (91) med en outputende (93) til at emittere nævnte elektromagnetiske stråling og nævnte læser omfatter mindst en optisk fiber (96) med en inputende (95) til at modtage nævnte signaler emitteret fra fluorforer (18, 52, 62) indfanget af magnetiske partikler (17, 54, 64), hvor fortrinsvis nævnte optiske fiber (91) af emitteren og nævnte optiske fiber (96) af læseren er anbragt tilstødende til hinanden i mindst respektive længdeafsnit (92) tilstødende til henholdsvis outputenden (93) og inputenden (95), fortrinsvis nævnte emitter omfatter en flerhed af optiske fibre (91), hver med en outputende (93) til at emittere nævnte elektromagnetiske stråling og nævnte læser omfatter en flerhed af optiske fibre (96), hver med en inputende (95) til at modtage nævnte signaler emitteret fra fluorforer (18, 52, 62) indfanget af magnetiske partikler, fortrinsvis nævnte optiske fibre (91) af emitteren og nævnte optiske fibre (96) af læseren er anbragt tilstødende til hinanden i mindst respektive længdeafsnit (92) tilstødende til henholdsvis outputenden og inputtet, mere fortrinsvis outputenderne og inputenderne er anbragt i et forudbestemt mønster.
19. Systemet ifølge et hvilket som helst af kravene 13-18, hvor nævnte system omfatter en signalprocessor omfattende en computer til at udføre den kvantitative eller kvalitative bestemmelse af målkomponent(er) i en flydende prøve baseret på det/de læste signal(er), hvor signalprocessoren er konfigureret til at multiplekse signaler fra forskellige grupper af fluorforer, fra fluorforer fra parallelle assayer og/eller fra fluorforer i referencetests af kendte eller ukendte flydende prøver, fortrinsvis signalprocessoren er konfigureret til at multiplekse signaler fra forskellige grupper af fluorforer påført i det samme assay.
20. Mikrofluidindretning ifølge krav 12, hvor indretningen omfatter et substrat (12, 22) med en rille til strømningskanalen (11, 21) og et folie (11a), der dækker strømningskanalen (11, 21), idet strømningskanalen (11, 21) omfatter det transparente vindue og indløbet (13, 23) til sugning i den flydende prøve, hvor mikrofluidindretningen (31) omfatter et fleksibelt vægafsnit (15) af strømningskanalen (11, 21) eller af et sinkafsnit (14, 24) i fluidforbindelse med strømningskanalen (11, 21), hvor det fleksible vægafsnit (15) kan bevæges således at luft bliver presset ud afstrømningskanalen (11, 21) hvor efter den fleksible væg (15) vender tilbage til sin oprindelige position, hvor fortrinsvis strømningskanalen (11, 21) er i fluidforbindelse med et sinkafsnit (14, 24) af mikrofluidindretningen (31).
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AU2013279833A1 (en) 2015-01-22
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