I HC QUC GIA H NI TRNG I HC CNG NGH

Trn Minh Hi

NGHIN CU V CH TO NANO BC NG DNG TRONG SINH HC

KHO LUN TT NGHIP I HC H CHNH QUY Ngnh: Vt l k thut

H NI - 2011

I HC QUC GIA H NI TRNG I HC CNG NGH

Trn Minh Hi

NGHIN CU V CH TO NANO BC NG DNG TRONG SINH HC

KHO LUN TT NGHIP I HC H CHNH QUY Ngnh: Vt l k thut

Cn b hng dn: TS. Trn ng Khoa

H NI - 2011

Li cm n
Trc tin, ti xin gi li cm n su sc ti TS. Trn ng Khoa tn tnh ch bo v hng dn ti trong qu trnh hc tp, nghin cu thc hin kha lun tt nghip ny. Ti xin chn thnh cm n TS. Nguyn Th Hoi H, Vin Vi sinh vt v Cng ngh Sinh hc, i hc Quc Gia H Ni, nhn xt, gp cho kha lun ca ti. Ti cng xin chn thnh cm n KS. Phm Th Bch o, Vin Vi sinh vt v Cng ngh Sinh hc, i hc Quc Gia H Ni. CN. Nguyn Th Ha, Khoa Vt l k thut v Cng ngh nano, i hc Cng ngh, i hc Quc Gia H Ni hng dn, to iu kin gip ti trong thi gian thc hin kha lun ny. Ti cng xin gi li cm n tp th cc Thy, C gio v cn b ca Khoa Vt l k thut v Cng ngh nano, i hc Cng ngh, i hc Quc Gia H Ni cung cp cc kin thc tin ti hon thnh kha lun ny. Cui cng, ti xin gi li cm n gia nh, bn b ng vin v gip ti rt nhiu trong sut thi gian hc tp, nghin cu v hon thnh kha lun.

Tm tt ni dung
Trong nhng nm gn y vic nghin cu ht nano rt c quan tm bi nhng tnh cht c bit v l th ca n. Trong s cc loi ht nano c nghin cu, ng dng th ht nano bc gy c s ch c bit bi tnh cht khng khun vt tri. Trn th gii nano bc c nghin cu, ch to v ng dng trong rt nhiu cc sn phm gn gi vi i sng nh: tm trn bng cu thng, ph ln cc loi si vi, s dng chng nhim khun nc sinh hot, cc dng cho tr em... Vit Nam, vic nghin cu, ch to vt liu nano ni chung, nano bc ni ring vn cn kh mi m v mi c tin hnh trong thi gian gn y. Vi mc ch nghin cu nhng tnh cht l th ca keo nano bc, ti thc hin ch to ht nano bc bng phng php ha kh bc PVP v PEG. Nghin cu, kho st tnh cht ca ht nano bc bng UV-vis v FE-SEM, tin hnh th nghim kh nng khng khun v tng tc ca nano bc ch to vi nm mc v vi to.

Li cam oan
Ti xin cam oan rng y l cng trnh nghin cu ca ti, vi s hng dn ca TS. Trn ng Khoa. Cc ni dung nghin cu v kt qu trong ti ny l trung thc v cha tng c ai cng b trong bt c cng trnh nghin cu no trc y. Nhng ni dung kha lun c tham kho v s dng cc ti liu, thng tin c ng ti trn cc tc phm, tp ch v cc trang web c lit k trong danh mc ti liu tham kho ca kha lun.

H Ni, ngy 10 thng 5 nm 2011

Sinh vin thc hin kha lun

Trn Minh Hi

Mc Lc
Trang H NI - 200<s cui ca nm bo v KLTN>..........................................................1 Mc Lc............................................................................................................... 6 .............................................................................................................................. 8 M U........................................................................................................................ 9 Chng 1. TNG QUAN TI LIU.........................................................................10
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1.1. Khi qut v cng ngh nano......................................................................10

1.1.1. Lch s hnh thnh ca cng ngh nano.....................................................10 1.1.2. C s khoa hc .........................................................................................10 1.1.3. Cc nghin cu v ht nano trong v ngoi nc.......................................11

1.2. Khi qut v keo nano bc..........................................................................12

1.2.1. S lc v tnh cht v c tnh ca bc....................................................12 1.2.2. Cc phng php ch to vt liu nano bc...............................................13 1.2.3. C ch n nh ht bc ca PVP v PEG...................................................16 1.2.4. ng dng ca keo nano bc.......................................................................18

1.3. Khi qut v vi khun..................................................................................20

1.3.1. Khi nim chung v vi khun.....................................................................20 1.3.2. Vi khun E.coli .........................................................................................20 1.3.3. Vi khun Staphylococcus...........................................................................21 1.3.4. Tnh dit khun ca keo nano bc..............................................................22

1.4. Khi qut v nm mc.................................................................................24 1.5. Khi qut v to...........................................................................................25

1.5.1. Khi nim chung v to lc........................................................................25 1.5.2. Vi to Chlorella..........................................................................................25

Chng 2. VT LIU, PHNG PHP NGHIN CU .....................................27 V THC NGHIM..................................................................................................27

2.1. Vt liu, trang thit b s dng...................................................................27

2.1.1. Cc dng c, thit b nghin cu................................................................27 2.1.2. Cc ho cht s dng.................................................................................28 2.1.3. Cc i tng nghin cu..........................................................................28

2.2. Phng php nghin cu v thc nghim.................................................28

2.2.1. Ch to keo nano bc.................................................................................28 2.2.2. Phn tch bng my quang ph hp th UV-vis.........................................30 2.2.3. Phn tch ht bng FE-SEM.......................................................................30 2.2.4. Kho st hiu qu khng khun ca nano bc............................................31 2.2.5. Th nghim s tng tc gia keo nano bc vi nm mc........................31 2.2.6. Nui cy vi to...........................................................................................32 2.2.7. S tng tc gia keo nano bc vi to vi to ...........................................34 Chng 3. KT QU V THO LUN.................................................................36

3.1. Ch to keo nano bc...................................................................................36 3.2. Phn tch ht bng tia UV-vis.....................................................................37 3.3. Phn tch ht bng FE-SEM.......................................................................38 3.4. Kh nng khng khun ca cc dung dch keo nano bc........................39 3.5. S tng tc gia keo nano bc vi nm mc ..........................................41 3.6. La chn mi trng nui cy vi to.........................................................41 3.7. S tng tc gia keo nano bc vi vi to.................................................44
3.7.1. ng cong chun ha gia mt quang vi mt to........................44 3.7.2. Kh nng tng tc ca keo nano bc vi vi to trong dung dch..............45 3.7.3. Kh nng tng tc ca keo nano bc vi vi to khi tm ln mng xp.....48 KT LUN.................................................................................................................. 49 HNG NGHIN CU TIP THEO......................................................................50

PH LC..................................................................................................................... 51 TI LIU THAM KHO...........................................................................................55

Danh mc ch vit tt
Da E.coli FE-SEM n v khi lng Danton Vi khun E.coli Knh hin vi trng in t qut Escherichia Coli Field emission scanning electron microscopy Polyvinyl pyrrolidon polyethylene Glycol Quang ph hp th trc quang UV - vis Ultraviolet-visible spectroscopy

PVP PEG UV - vis

M U
Khoa hc, cng ngh nano l mt lnh vc khoa hc v cng ngh mi, pht trin rt nhanh chng. Vt liu c ch to bng cng ngh ny th hin nhiu tnh cht mi l do hiu ng kch thc. Khoa hc v cng ngh nano trn c s kt hp a ngnh to nn cuc cch mng v khoa hc k thut. Hin nay, nhiu quc gia trn th gii xem cng ngh nano l mc tiu mi nhn u t pht trin. c tnh tng u t cho lnh vc cng ngh nano trn ton th gii xp x 3 t la v c hng trm sn phm ca cng ngh nano c thng mi, ng dng trong nhiu lnh vc nh in t, ha hc, y sinh, mi trng.[5,10]. Trong cng ngh nano th ht nano l mt vt liu quan trng. Mt trong nhng ht nano c s dng sm v rng ri nht l ht nano bc. kch thc nano bc th hin nhng tnh cht vt l, ha hc, sinh hc khc bit v v cng qu gi, c bit l tnh khng khun. Nghin cu ch ra rng khi kch thc nano, hot tnh st khun ca bc tng ln khong 50.000 ln so vi bc ion [15,9]. Nh kh nng khng khun tuyt vi m nano bc c ng dng trong rt nhiu sn phm nh: tm trn bng cu thng, ph ln cc loi si vi, s dng kh trng nc, cc dng cho tr em.... Trong nhng nm gn y, vic gia tng vi khun siu khng thuc khng sinh, cc loi nm gy bnh thiu thuc c tr th vic nghin cu ch to sn phm cha nano bc tiu dit chng l hng i mi v cp thit [17]. l l do m ti quyt nh chn ti Nghin cu v ch to nano bc ng dng trong sinh hc. Cc ni dung ca ti nh sau: + Ch to ht nano bc bng phng php ha kh, s dng NaBH 4 lm tc nhn kh, ht c bc bi PVP v PEG. + Nghin cu, kho st tnh cht ca keo bc nano ch to bng phn tch quang ph hp th UV-vis, v chp nh bng FE-SEM. + ng dng nghin cu kh nng dit khun v kh nng tng tc vi nm mc, vi to ca keo bc nano.

Chng 1. TNG QUAN TI LIU

1.1. Khi qut v cng ngh nano
1.1.1. Lch s hnh thnh ca cng ngh nano
Thut ng cng ngh nano (nanotechnology) xut hin t nhng nm 70 ca th k XX, ch vic thit k, phn tch, ch to v ng dng cc cu trc, thit b v h thng bng vic iu khin hnh dng, kch thc trn quy m nanmt. Chng c chnh xc rt cao 0,1 - 100nm, tc l chnh xc n tng lp nguyn t, phn t. Tin t nano xut hin trong ti liu khoa hc ln u tin vo nm 1908, khi Lohman s dng n ch cc sinh vt rt nh vi ng knh 200nm. Nm 1974, Tanigushi ln u tin s dng thut ng cng ngh nano hm s lin kt cc vt liu cho k thut chnh xc trong tng lai. Hin ti trong khoa hc, tin t nano biu th con s 10-9 tc kch thc 1 phn t mt. Cho ti nay, vn cha c c mt nh ngha thng nht v cng ngh nano. Theo c quan Hng Khng V tr Hoa K (NASA), cng ngh nano l cng ngh ch to ra cc cu trc, vt liu, thit b v h thng chc nng vi kch thc o bng (khong t 1 n 100nm) v khai thc ng dng cc c tnh c o ca nhng sn phm ny. Cng ngh nano cng c th hiu l ngnh cng ngh da trn cc hiu bit v cc quy lut, hin tng, tnh cht ca cu trc vt l c kch thc c trng thang nano [4]. C th ni, trong thi im hin ti, tim nng pht trin ca mt cng ngh hay k thut mi r nht qua ngun ngn sch nghin cu hng nm v doanh thu em li t cc sn phm thng mi ca n. c ton th gii nghin cu v u t pht trin, ngn sch u t cho cng ngh nano ca cc t chc thuc chnh ph tng khong 7 ln t 430 triu nm 1997 ln 3 t USD nm 2003 [12,23].

1.1.2. C s khoa hc
Cng ngh nano da trn ba c s khoa hc chnh: + Chuyn tip t tnh cht c in n tnh cht lng t: khc vi vt liu khi, khi kch thc nano th cc tnh cht lng t c th hin rt r rng. V vy khi nghin cu vt liu nano chng ta cn tnh ti cc thng ging ngu nhin. Cng kch thc nh th cc tnh cht lng t cng th hin mt cch r rng hn. V d

mt chm lng t c th c coi nh mt i nguyn t, n c cc mc nng lng ging nh mt nguyn t [25]. + Hiu ng b mt: Cng mt khi lng nhng khi kch thc nano chng c din tch b mt ln hn rt nhiu so vi khi chng dng khi. iu ny, c ngha rt quan trng trong cc ng dng ca vt liu nano c lin quan ti kh nng tip xc b mt ca vt liu, nh trong cc ng dng vt liu nano lm cht dit khun. y l mt tnh cht quan trng lm nn s khc bit ca vt liu c kch thc nanomet so vi vt liu dng khi [8]. + Kch thc ti hn: Kch thc ti hn l kch thc m vt gi nguyn cc tnh cht v vt l, ha hc khi dng khi. Nu kch thc vt liu m nh hn kch thc ny th tnh cht ca n hon ton b thay i. Nu ta gim kch thc ca vt liu n kch c nh hn bc sng ca vng nh sng thy c (400 700 nm), theo Mie hin tng "cng hng plasmon b mt" xy ra v nh sng quan st c s thay i ph thuc vo bc sng nh sng xy ra hin tng cng hng. Hay nh tnh dn in ca vt liu khi ti kch thc ti hn th khng tun theo nh lut Ohm na. M lc ny in tr ca chng s tun theo cc quy tc lng t. Mi vt liu u c nhng kch thc ti hn khc nhau v bn thn trong mt vt liu cng c nhiu kch thc ti hn ng vi cc tnh cht khc nhau ca chng. Bi vy khi nghin cu vt liu nano chng ta cn xc nh r tnh cht s nghin cu l g. Chnh nh nhng tnh cht l th ca vt liu kch thc ti hn nn cng ngh nano c ngha quan trng v thu ht c s ch c bit ca cc nh nghin cu [24].

1.1.3. Cc nghin cu v ht nano trong v ngoi nc

Tnh hnh nghin cu trong nc: Ti Vit Nam trong nhng nm gn y cng ngh nano bt u c u t v thu ht s ch ca cc nh khoa hc. Tuy nhin cho n nay s lng cng trnh nghin cu v kim loi nano c cng b trn tp tr khoa hc trong nc cn rt hn ch. ti nghin cu v vng v platin nano xc tc chuyn ha CO thnh CO 2 c tc gi Nguyn Thit Dng Vin khoa hc Vt liu ng dng Vin khoa hc v cng ngh Vit Nam thc hin (2009 2010). V bc, nhm tc gi Nguyn c Ngha, Hong Mai H cng b trn Tp ch ha hc (2001) ch to c ht nano bc bng phng php kh cc ion bc s dng tc nhn oleate trong polyme n nh, thu c cc ht bc c kch thc t 4 7nm.

Cc nh khoa hc Vit Nam cng bt u trin khai ng dng cng ngh nano trong ch to thuc hng ch v kt hoch nghin cu ng dng ca cc ht nano trong y - sinh hc chn on v cha bnh. Bi bo ch to v ng dng ht nano t tnh trong y sinh hc ca nhm tc gi Nguyn Hu c, Nguyn Hoi H, Trn Mu Danh B mn Vt liu v Linh kin t tnh nano, khoa Vt l k thut v Cng ngh nano, trng i hc Cng ngh, i hc Quc Gia H Ni v Trung tm Khoa hc Vt liu, trng i hc Khoa hc t nhin, i hc Quc gia H Ni bo co ti hi ngh Vt l ton quc ln th VI (2005). Tuy nhin, cng ngh nano vn l mt iu g mi l Vit Nam. Ni chung, cng ngh nano ti Vit Nam hin ch mi ang t nhng vin gch mng u tin. Tnh hnh nghin cu ngoi nc: Phng php ch to ht kim loi nano ni chung v ch to nano bc ni ring c rt nhiu nh khoa hc trn th gii nghin cu. Phng php thng c s dng ch yu l: in ha, kh ha hc, kh nhit, sinh hc, kh do bc x ion ha Nguyn tc chung ca cc phng php ny l kh ion kim loi trong dung dch thnh nguyn t kim loi, sau cc nguyn t lin kt vi nhau thnh tp hp ri pht trin kch thc thnh cc ht nano v s dng polyme n nh ht. Hng nghin cu ng dng chnh ca nano bc tp trung vo kh nng khng li cc loi vi khun, virut, cc ng dng trong cc thit b y t v trong cc thit b dit khun, lc nc.v.v Theo nhn nh ca nhiu chuyn gia, cng ngh nano s to nn mt cuc cch mng t ph trong nhiu ngnh khoa hc v i sng, to tin cho mt th gii nh hn v thng minh hn [7].

1.2. Khi qut v keo nano bc

1.2.1. S lc v tnh cht v c tnh ca bc

T thi Alexander i (nm 356-323 trc cng nguyn), con ngi bit s dng cc dng c bng bc ng thc n v ung gp phn lm gim nguy c gy c. Qua thi gian nhng c tnh qu gi ca bc c con ngi khai thc v s dng to ra nhiu sn phm hu ch.

 Tnh cht vt l: + Bc l kim loi chuyn tip, mu trng, sng, d dng dt mng, c tnh dn in v dn nhit cao nht v in tr thp nht trong cc kim loi. + Nhit nng chy l 961.930C [18]. Tnh cht ha hc: + Bc c k hiu l Ag, s nguyn t 47 thuc phn nhm IB trong bng tun hon cc nguyn t ha hc, bc c khi lng phn t l 107.868 (n v C). + Cu hnh electron [Kr]4d105s1 , c s oxi ha l +1 v +2, ph bin nht l trng thi oxi ha +1. + Trong t nhin, bc tn ti hai dng ng v bn l Ag-107(52%) v Ag109(48%). Bc khng tan trong nc, mi trng kim nhng c kh nng tan trong mt s axit mnh nh axit nitric, sufuric c nng .v.v. Ngy nay nhng thuc tnh qu ca kim loi ny c th hin ti a khi chng c ch to bng cng ngh nano. V trn th trng cng xut hin nhiu sn phm cha nano bc nh bng gc y t, nc ty trng b mt, hay hin din ngay trong gia nh bn nh t lnh, my gt .v.v.

1.2.2. Cc phng php ch to vt liu nano bc

C 2 phng php iu ch ht nano kim loi bc: phng php t di ln v phng php t trn xung. Phng php t di ln bottom-up l phng php to ht nano t cc nguyn t hoc ion kt hp li vi nhau. Phng php t trn xung top-down l phng php to cc ht nano t vt liu khi ban u. i vi ht nano bc, ngi ta thng iu ch bng phng php t di ln. Nguyn tc l kh ion Ag+ thnh Ag. Cc ion ny sau lin kt vi nhau to thnh ht nano v cc ht nano ny s c bc bi cc cht n nh nh PVP, PVE, chitosan.v.v. Cc phng php t trn xung t c s dng v nano bc ch to bng phng php ny thng c kch thc ht ln v khng ng u. Hin nay cc vt liu kim loi nano nh vng (Au), St (Fe), ng (Cu), bc (Ag) di dng bt hay dung dch keo c ch to ch yu bng cc phng php sau:

 Phng php bay hi vt l

Bay hi vt l l phng php t trn xung, l mt cng c gp phn cho s pht trin ca cng ngh nano. Bay hi vt l bao gm k thut ngng t kh tr, ng ngng t v ngng t dng hi phun trn bia bn. + K thut ngng t kh tr: cho ha hi si dy bc tinh khit nhit cao trong iu kin chn khng, sau dng hi bc nguyn t qu bo ha c ngng t v pht trin thnh ht bc khi tip xc vi kh heli v c lm lnh bi nit lng. + K thut ng ngng t: tng t nh ngng t kh tr nhng qu trnh hnh thnh v pht trin ht xy ra trn lp bng dung mi thch hp. K thut ngng t kh tr v ng ngng t c thc hin nhit cao (>2.0000C), sn phm to ra c tinh khit cao, kch thc ht nano bc trung bnh 75nm(phng php ngng t kh tr), 12nm (phng php ng ngng t) [9].

Phng php n mn laze

y l phng php t trn xung. Vt liu ban u l mt tm bc c t trong mt dung dch c cha cht hot ha b mt. Mt chm laser dng xung c buc sng 532 nm, rng xung l 10 ns, tn s 10Hz, nng lng mi xung l 90mJ, ng knh vng kim loi b tc dng t 1nm - 3nm. Di tc dng ca chm laser xung, cc ht nano c kch thc khong 10 nm c hnh thnh v c bao ph bi cht hot ha b mt CnH2n+1SO4Na (vi n = 8, 10, 12, 14) nng t 0,001 n 0,1 M [16].

Phng php kh ha hc
Kh ha hc l mt phng php c s dng ph bin ch to nano bc theo phng thc t di ln. + C ch ca qu trnh kh ha hc: Phng php kh ha hc l dng cc tc nhn ha hc kh ion bc thnh bc kim loi. Thng thng, phn ng c thc hin trong dung dch lng nn cn gi l phn ng ha t. Ag+ + e- Ag0

Thng thng, ngun cung cp ion Ag + l cc mui ca bc nh AgNO 3. Cc tc nhn kh thng dng l: natri bohydrua, focmandehyt, xitrat, etylen glyxerol, NaBH4, ethanol,. Gn y c mt s cng trnh nghin cu ch to keo nano bc v bt nano bc t bc nitrat nhng sn phm trung gian l oxit bc (Ag 2O) ri t Ag2O tip tc kh v Ag0 nhm thu c keo bc c nng cao. cc ht nano bc phn tn tt trong dung mi m khng b kt t thnh m, ngi ta bao ph ht nano bc bng mt lp polyme, iu ny gip cho cc ht c bo v tt hn trnh hin tng kt ta, hn na phng php ny c th lm cho b mt ht nano c tnh cht cn thit. + Cc tc nhn kh: Tc nhn kh Sodium citrate C6H5O7Na3 Trong qu trnh kh, b mt ca ht nano bc hp th cc ion Ag+ to ra lp ion dng trn b mt. Tip cc ion m citrate c nghim v bm xung quanh cc ht nano bng lc ht tnh in ngn khng cho chng kt hp li vi nhau. Nh vy m b mt ca ht nano bc c mt lp keo citrate gip chng l lng v phn tn u trong dung dch. Citrate trong qu trnh va ng vai tr lm tc nhn kh ion Ag + to thnh ht nano bc, va ng vai tr lm cht n nh cho ht nano bc. Tc nhn kh NaBH4 khc vi phng php s dng Sodium citrate, phng php ny sau khi kt thc phn ng kh, ngi ta s dng cc polyme nh PVP, PVA, PEG, Chitosan, lm tc nhn n nh. Cc polyme ny bao bc ht nano bc, ngn chng kt t vi nhau, v vy m ht nano c bo v tt v trnh kt ta.

Phng php ha siu m

Phng php ha siu m l cc phn ng ha hc c h tr bi sng siu m cng c dng to ht nano. Ha siu m l mt chuyn ngnh ca ha hc, trong cc phn ng ha hc xy ra di tc dng ca sng siu m nh mt dng xc tc. Sng siu m l sng dc, l qu trnh truyn s co li v gin n ca cht lng. Tn s thng s dng trong cc my siu m l 20 kHz cao hn ngng nhn bit ca tai ngi (t vi Hz n 16 kHz). Khi sng siu m i qua mt cht lng, s gin n do siu m gy ra p sut m trong cht lng ko cc phn t cht lng ra xa nhau. Nu cng siu m mnh th s gin n ny s to ra nhng l hng trong cht lng. S pht trin ca cc l hng ph thuc vo cng siu m. Khi cng siu m cao, cc l hng nh c th pht trin rt nhanh. S gin n ca cc l hng nhanh trong na u chu k ca mt chu k sng siu m, nn n na sau chu

k th n khng c thi gian co li na. Di cc iu kin ny, kch thc ca mt l hng s dao ng theo cc chu k gin n v co li. Trong khi dao ng nh th lng kh hoc hi khuych tn vo hoc ra khi l hng ph thuc vo din tch b mt. Din tch b mt s ln hn trong qu trnh gin n v nh hn trong qu trnh co li. Do , s pht trin ca l hng trong qu trnh gin n s ln hn trong qu trnh co li. Sau nhiu chu k siu m, l hng s pht trin. L hng c th pht trin n mt kch thc ti hn m ti kch thc l hng c th hp th hiu qu nng lng ca sng siu m. Kch thc ny gi l kch thc cng hng, n ph thuc vo tn s ca sng m. V d, vi tn s 20 kHz, kch thc ny khong 170 mm. Lc ny, l hng c th pht trin rt nhanh trong mt chu k duy nht ca sng siu m. Mt khi l hng pht trin qu mc, ngay c trong trng hp cng siu m thp hay cao, n s khng th hp th nng lng siu m mt cch c hiu qu c na. V khi khng c nng lng tip ng, l hng khng th tn ti lu c. Cht lng xung quanh s vo v l hng b suy sp. S suy sp ca l hng to ra mt mi trng c bit cho cc phn ng ho hc - cc im nng (hot spot). Ha siu m c ng dng ch to rt nhiu loi vt liu nano nh vt liu nano xp, nano dng lng, ht nano, ng nano.

1.2.3. C ch n nh ht bc ca PVP v PEG C ch n nh ht bc ca PVP

Hnh 1. 1. Cng thc cu to PVP

PVP c tng hp t phn ng trng hp cc vinyl pyrolidon, l cc polyme a nc v ha tan trong nc, khng c, c s dng ph bin trong lnh vc y t [20].

Kt qu nghin cu ch ra rng, cc ht bc hp th mnh ln b mt ca PVP, chui polyvinyl pyrolidon to ra hiu ng khng gian, ngn cn s kt hp gia cc ht [20]. C ch n nh ht bc ca PVP gm cc giai on: + u tin, PVP chuyn mt cp electron t nguyn t oxi v nit trn mch sang cc orbital s v p cc ion bc to nn kin kt phi tr vi ion bc. + PVP thc y s hnh thnh nhn ca kim loi bc do phc ion Ag + PVP d b kh hn so vi ion Ag + t do trong dung dch v ion Ag + nhn in t t PVP. + Chui PVP ngn cn s kt t ca cc ht bc do hiu ng khng gian.

Hnh 1.2. C ch n nh ht nano bc ca PVP [20]

C ch n nh ht bc ca PEG
PEG c cng thc phn t l (C4H10O3)m, l mt polymer tr, ha tan trong nc, khng gy c, c to ra bng cch kt ni nhiu tiu n v ethylene oxide. PEG ha tan trong nc, methanol, benzen, diclorometan v khng ha tan trong ete diethyl v hexane. N cng vi cc phn t k nc dng sn xut b mt khng ion. Cc phn t PEG hin c khc nhau v hnh th ( dng thng hoc phn nhnh) v trng lng phn t. PEG c trng lng khc nhau s khc nhau v c tnh sinh l. Th d nh PEG dng tinh khit, khng kt hp, nh l dng u (nh PEG 200 Da), trong khi PEG ln hn (nh PEG 8000 Da) c dng rn nh sp [24].

Hnh 1.3. Cng thc cu to PEG

PEG c nhiu ng dng hu ch trong y sinh do PEG c nhiu c tnh ct li lin quan n nhng li ch i vi cc protein s dng trong iu tr v cn rt nhiu ng dng khc trong cc lnh vc khc nhau. PEG c s dng nh mt cht kh cho vic chun b cc ht kim loi nhit cao (>170 0C) v khng hot ng gim lng Ag+ 800C. ng ngc nhin hn, Ag +c th c gim xung u n to ht nano bc ti cng mt iu kin trong PEG 2000 Da. Nghin cu su hn chng minh rng t l gim ca Ag+ to thnh ht nano bc c tng cng ng k vi s gia tng chiu di chui polymer ca PEG. Kch c ht nano ph thuc vo nhit phn ng v nng ca tin cht v s gia tng nhit nh hng n s thay i ca phm vi kch thc ht t 10-80nm.

1.2.4. ng dng ca keo nano bc

Trong dn dng Vic sn xut cc mt hng tiu dng bng kim loi bc nguyn cht hoc ph chng bng bc l rt t. Tuy nhin, cc nghin cu pht hin ra rng, pha cc nguyn liu khc vi ht nano bc l mt phng php thch hp khai thc tnh cht ca bc. Cc ht nano siu nh lm cho cc ht c din tch b mt ln cn i vi khi lng ca chng. Trng hp bc dng ht nano, cho php chng tng tc d dng vi cc ht khc v tng hiu qu khng khun. Hiu qu ny ln ti mc 1 gam ht nano bc c th to tnh cht khng khun ti hng trm mt vung cht nn.

Hnh 1.4. Bnh sa nano silver

Hin nay, trn th gii sn xut nhiu sn phm tiu dng c cha nano bc nh hp ng thc n, bnh sa tr em lm bng nha c pha thm nano bc, hay cc thit b gia nh nh my git, t lnh.v.v. u s dng nano bc dit khun. ng dng trong y t Trong lnh vc y t nano bc c s dng tm vo cc loi bng, gc bng b vt thng nhm trnh nhim trng. Hay nh vic ch to khu trang c tm nano bc dng dit vi khun, bo v con ngi.

Hnh 1.5. Khu trang nano bc

ng dng trong cng nghip Sn khng khun: Bt nano bc c trn vi sn v ph ln cc phm in thoi di ng, tng nh v cc b mt cn c bo v. Vi kh nng khng khun

tuyt vi nano bc s gi cho b mt khng b nhim khun cng nh tiu dit nm mc lm tng tnh thm m v tui th cng trnh.

Hnh 1.6. Sn nano

Thit b in t: Cc nh khoa hc cng ang nghin cu ng dng nano bc sn xut linh kin in t phc v nhu cu ngy cng cao ca ngi tiu dng. Cc thit b in t ng dng cng ngh nano s c kch thc nh gn hn nhng li c tc x l v tui th cao hn cc thit b s dng cc cng ngh truyn thng

1.3. Khi qut v vi khun

1.3.1. Khi nim chung v vi khun
Vi khun l nhng sinh vt n bo, c cu trc t bo n gin khng c nhn (Prokaryote sinh vt nhn s). Vi khun hin din khp mi ni trong t, nc, khng kh, k c nhng ni c iu kin sng khc nghit nh trn ming ni la hay trn bng tuyt.v.v. C rt nhiu chng vi khun, v mi chng vi khun u c s khc nhau v c tnh v hnh thi. Vi khun c nhiu hnh dng: vi khun c nhiu hnh dng khc nhau v c gi vi tn gi theo hnh dng ca chng nh trc khun ( bacillus), hnh cu, xon khun (spirillum), hnh que, cu khun (coccus) hnh dng vi khun l mt c im quan trng nhn dng cc chi c t tn theo hnh dng. Vi khun c ch hoc c hi cho mi trng, thc vt v ng vt bao gm c con ngi. Mt s tc nhn gy bnh nh bnh un vn (tetanus), st thng hn (typhoid fover), giang mai (syphilis), t (cholera), lao (tuberculosis)

1.3.2. Vi khun E.coli

Phn loi khoa hc:

Ngnh: Proteobacteria Lp: Gamma Proteobacteria B : Enterobacteriales H : Enterobacteriaceae Chi : Escherichia Loi: E. coli

Hnh 1.7. Vi khun E.coli [25]

c im: E.coli hay cn gi l vi khun i trng, l mt trong nhng loi vi khun chnh k sinh trong ng rut ca ngi v ng vt mu nng. Chng c pht hin u tin vo nm 1885 do Escherich pht hin, thuc h vi khun Enterobacteriaceae. Chng l cc trc khun Gram m. Kch thc trung bnh (2-3m) x 0.5 m. Trong nhng iu kin khng thch hp vi khun c th di nh si ch.

1.3.3. Vi khun Staphylococcus

Phn loi khoa hc: Gii: Bacteria Ngnh: Firmicutes Lp: Bacilli B: Bacillales

H: Staphylococcaceae Chi: Staphylococcus

Hnh 1.8. Vi khun Staphylococcus [11]

c im: Staphylococcus hay t cu khun l nhng vi khun hnh cu, c ng knh t 0.8 1.0 m, v thng tp trung thnh tng chm nh nhng chm nho, bt mu Gram dng [3]. T cu khun c ba loi c ngha vi y hc l t cu vng (S.aureus), t cu da (S. epidermidis) v S. saprophyticus. Hai trong ba loi ny c th gy ra cc bnh nhim trng c hi: S.epidermidis gy nhim khun da v vim ni tm mc, S.saprophyticus gy nhim khun tit niu. S c tr trong c th v mi trng bnh vin gia cc loi t cu khun tit niu dn ti s truyn cho nhau kh nng khng thuc, lm cho s khng khng sinh tng ln, nht l trong bnh vin, cng lm cho vic iu tr chng bng khng sinh kh khn hn.

1.3.4. Tnh dit khun ca keo nano bc C ch dit khun ca keo nano bc
Hin nay, do vi khun ngy cng khng thuc khng sinh nn cc nh khoa hc ang tp trung i tm cc tc nhn mi dit chng v bc l mt trong nhng cht c tp trung nghin cu mnh nht. S d nano bc c nghin cu ng dng vo vic khng khun v bc l khng sinh t nhin v khng gy tc dng ph. Nano bc khng gy phn ng ph,

khng gy c cho ngi v vt nui khi nhim lng nano bc bng nng dit khun (khong nng <100ppm) [17]. Tuy nhin cho ti nay, c ch khng vi sinh vt ca nano bc vn cha c hiu bit r rng. Bng cc k thut chp nh knh hin vi in t c phng i cao (FE-SEM, FE-SEM), kt qu nghin cu cho thy, ht nano bc bm dnh vi cc thnh phn in tch m trn b mt t bo vi khun, virut lm thay i tnh thm v s h hp ca mng t bo. ng thi cc ht bc c kch thc nh chui vo trong t bo, kt hp vi cc enzym hay DNA c cha nhm sun phua hc pht pht gy bt hot enzym hay DNA dn n gy cht t bo [14]. Trc s gia tng ca dng vi khun khng thuc khng sinh in hnh l Staphylococcus aureus hay cc loi vi nm gy bnh thc vt thiu thuc c tr th vic la chn cc ch phm cha nano bc ang rt c quan tm. Thng thng nng bc s dng cho vic khng khun v st trng rt thp, v d nh khong 5ppm cho vic dit vi khun Esherichia Coli hiu qu n 999% v khun Staphylococcus aureus l hn 99%. V vy, nano bc rt hu ch cho vic s dng lm cht dit khun, dit nm bnh phc v cho nhu cu cuc sng con ngi. Cc yu t nh hng ti kh nng dit khun ca keo nano bc Kch thc, hnh dng ht, nng v s phn b l cc yu t nh hng trc tip n tnh khng khun ca keo nano bc. + Kch thc ht nano bc l yu t quan trng quyt nh kh nng dit khun ca chng. Ht nano bc c kch thc cng nh th kh nng dit khun ca chng cng mnh, v khi kch thc cng nh th t s gia din tch b mt v th tch cng ln v ht cng c th d dng tng tc vi vi khun hn. Tuy nhin cc ht c kch thc nh li c khuynh hng lin kt vi nhau trong qu trnh lu tr to thnh cc ht ln hn gy nh hng ti kh nng dit khun v bo qun keo nano bc. Do trong qu trnh ch to chng ta phi tm ra cc phng php va to ra ht nano bc c kch thc nh va bn vng. + Cc ht nano c th c rt nhiu hnh dng khc nhau nh hnh que, hnh cu, hnh tam gic, V s th hin ca cc ht nano bc vi cng nng , s phn b nhng vi cc hnh dng khc nhau l khng ging nhau. Cc ht nano bc c hnh tam gic ct tnh khng khun cao hn cc ht hnh cu v cc ht nano que c tnh khng khun thp nht.

+ Keo nano bc c nng cng cao v s phn b u th kh nng dit khun cng tt. Tuy nhin khi nng qu cao, do nng lng b mt ht nano ln, nn cc ht nano bc s va chm vo nhau v ph v cu trc nano. V vy chng ta cng cn tm nng thch hp cc ht phn b ng u, v trnh kt ta.

1.4. Khi qut v nm mc

Nm mc l vi sinh vt chn hch, th tn, t bo khng c dip lc t, sng d dng (hoi sinh, k sinh, cng sinh). Mt s t nm th n bo c hnh trng, a s c hnh si, si c ngn vch (a bo) hay khng c ngn vch (n bo). Si nm thng l mt ng hnh tr di c kch thc ln nh khc nhau ty loi. ng knh ca si nm thng t 3-5m, chiu di ca si nm c th ti vi chc centimet, Cc si nm pht trin di theo kiu tng trng ngn. Trn mi trng c v trn mt s c cht trong t nhin, bo t nm, t bo nm hoc mt on si nm c th pht trin thnh mt h si nm c hnh dng nht nh gi l khun lc nm. Hu ht cc loi nm mc khng cn nh sng trong qu trnh sinh trng. Nhit ti u nm mc pht trin l 25-28 0C v pH tt nht t 5-6.5. Nm mc ni chung c hai hnh thc sinh sn l sinh sn v tnh, nm hnh thnh bo t, hoc sinh sn hu tnh hnh thnh giao t c v giao t ci. Nm mc c nh hng xu n i sng con ngi mt cch trc tip nh lm h hng, gim phm cht thc phm. Nm mc cn gy h hi vt dng, qun o hay gy bnh cho ngi, ng vt v cy trng. Tuy nhin, cc quy trnh ch bin thc phn c lin quan n ln men u cn ti s c mt ca vi sinh vt trong c nm mc. Nm mc cng gip tng hp khng sinh, mt s enzym v cc hot cht khc dng trong cng nghip thc phm, y dc v s dng rng ri trn khp th gii.

Hnh 1.9. Chng nm mc Aspergillus niger [28]

Chng nm mc c s dng nghin cu l Aspergillus cn gi l mc tng. Si nm c vch ngn, cung mang bo t bi phng ln ngn. Cc chui bo t bi t u phng mc ta khp mi hng. Bo t bi c th mu vng (Aspergillus flavus), mu en (Aspergillus niger). Nm Aspergillus oryzae l loi mc chnh trong qu trnh ch to tng. Hai loi khng c lm tng l Aspergillus oryzae v Aspergillus sojae c hnh thi v mu sc rt ging vi 2 loi rt nguy him l Aspergillus flavus v Aspergillus parasiticus sn sinh ra c t Aflatoxin gy bnh ung th.

1.5. Khi qut v to

1.5.1. Khi nim chung v to lc
To l nhng thc vt bc thp, c th cha c s phn ho thnh r, thn, l (nhng du hiu ca thc vt bc cao) nn c th chng c gi chung l tn. C th chng c cha sc t quang hp, chng c kh nng quang t dng, s dng nng lng nh sng mt tri, tng hp nn cc cht hu c t cc hp cht v c n gin. To lc c khong 6.000 loi [27]. To lc phn bit vi cc ngnh to khc c im u tin l mu lc thun khit ca c th ging nh mu lc ca cy ci xung quang chng ta . To lc gm cc t bo sng n c, cc t bo sng thnh tp on, a dng. Cu trc t bo vi to lc c tc tin ho khc nhau. Chng phn b rng khp t nc ngt ngho dinh dng n nc l, nc bin. Mt s b ch sng trn vt m hay sng ngay di mt t. Hnh dng cu trc chung ca t bo vi to lc rt a dng, c th c dng n c hay tp on: c dng si, dng ht, dng ng, dng mng,...Phn ln to lc c dng hnh cu, hnh bu dc, hnh vung, hnh ch nht, hay hnh li lim. To lc c vai tr quan trng trong t nhin v i sng nhn loi [6]. To lc l ngun thc phm giu cht dinh dng cho ngi v ng vt, chng c kh nng x l nhim mi trng, dng lm cht ch th sinh hc.v.v.

1.5.2. Vi to Chlorella
+ Phn loi khoa hc: Ngnh: Chlorophyta

Lp: Trebouxiophyceae B: Chlorellales H: Chlorellaceae Chi: Chlorella [21]

Hnh 1.10. Vi to Chlorella elipsoidea [22]

+ c im: Vi to Chlorella l mt chi to a dng, c Beijerinck m t ln u tin vo nm 1890, vi to Chlorella hin c tng cng 38 loi. Chng gm cc to t bo n bo c hnh cu hay hnh ovan, khng di ng, ng knh khng vt qu 15 m. Mng t bo vi to Chlorella mng cha cellulose, t bo cht l dch trong sut, cha th mu hnh chn cha chlorophyll a v b, th mu ln, chim phn ln th tch t bo vi mt ht to bt (pyrenoid). T bo ch c mt nhn, thng khng nhn thy r [1]. Vi to Chlorella c phng thc sinh sn v tnh nn tnh trng di truyn ca chng l n nh. Vi to Chlorella rt ph bin, sinh trng nhanh, h s s dng nng lng nh sng cao v khng i hi kht khe v iu kin sng [2]. Nhng c tnh ny khin cho vi to Chlorella c s dng rng ri trong nhiu nghin cu ng dng. V mt sinh l, qu trnh quang hp ca to vi to Chlorella cng din ra tng t nh vi cc thc vt bc cao. Vi to Chlorella c th quang hp tt trong iu kin bnh thng, h s s dng nng lng mt tri cn cao hn c thc vt bc cao. Vit Nam, vi to Chlorella c nhp ni nghin cu v ng dng t u nhng nm 1960 [2]. Chng c s dng ch yu vo mc ch x l nc thi.

Chng 2. VT LIU, PHNG PHP NGHIN CU V THC NGHIM

2.1. Vt liu, trang thit b s dng
2.1.1. Cc dng c, thit b nghin cu
S dng cc thit b sn c ti cc phng th nghim sau: + Phng th nghim micro-nano, khoa Vt l k thut & Cng ngh nano, trng i hc Cng ngh, i hc Quc gia H Ni: My khuy t c gia nhit RCT basic, my o pH bn sension3, my nh canon power shot G10, knh hin vi Zeiss, t nui cy Class II, bung u nhit c lc Shaking Incubator, l vi sng Microwave, cn phn tch GR-200, t m BD 115.

+ Phng th nghim quang t, khoa Vt l k thut & Cng ngh nano, trng i hc Cng ngh, i hc Quc gia H Ni: My o quang ph hp th UV/VIS/NIR V-570. + Phng Sinh hc to, Bo tng ging chun Vi sinh vt v cc phng khc thuc Vin Vi sinh vt v Cng ngh Sinh hc, i hc Quc Gia H Ni: Bung m Neubauer, knh hin vi quan hc (Zeiss), My o pH Osi. + My FE-SEM 4800, ti vin Khoa hc v Cng ngh Vit Nam.

2.1.2. Cc ho cht s dng

+ Cc ha cht s dng: AgNO3, NaBH4, PVP (40.000Da), PEG (4.000Da), HCl, NaCl, nc kh ion. + Cc mi trng nui cy: Nui cy to BBM, BG II, C (xem ph lc C), nui cy khun LB c, LB lng (xem ph lc A), nui cy nm mc Czapek (xem ph lc B).

2.1.3. Cc i tng nghin cu

Vi to dng trong nghin cu ny l loi Chlorella elipsoidea. c cung cp bi phng Sinh hc To, Vin Vi sinh vt v Cng ngh Sinh hc, i hc Quc Gia H Ni. Hai chng vi khun E.coli VTCCB 883, Staphylococus aureus VTCCB 484, v chng nm mc ASP.Nigger-F15. c cung cp bi Bo tng ging chun Vi sinh vt hc, Vin Vi sinh vt v Cng ngh Sinh hc, i hc Quc Gia H Ni.

2.2. Phng php nghin cu v thc nghim

2.2.1. Ch to keo nano bc
Chun b cc ho cht cn thit vi cc nng cho trc nh sau:

Bng 2.1. Cc ha cht s dng trong ch to keo nano bc

Ha cht NaBH4 AgNO3 PVP (40.000Da) PEG (4.000Da) NaOH

Nng 0,002M 0,001M 0,3% 0,3% 1M

Th tch 100ml 4ml 0,5ml 0,5ml 10ml

Cc bc tin hnh: + Cho 50ml dung dch NaBH4 0,002M vo l cho ln my khuy t vi tc 1000 vng/pht s dng nhit phng. + S dng pipet nh t t 3,33ml dung dch AgNO3 0,001M vo dung dch ang khuy t (1 git/giy). Dung dch dn chuyn sang mu vng ti. + Tip tc nh t t 0,83ml dung dch cht bc (PVP 0,3% hoc PEG 0,3%) vo dung dch ang khuy t (3 git/giy). Ta thy dung dch gi nguyn mu sc v c xut hin nhng bt nh trn b mt. + Sau dng my o pH bn chnh pH dung dch ti 7,0. + Kt thc khuy t, cho dung dch khy t xung v cho vo ng falcon 50ml bo qun ni kh mt, trnh nh sng. C ch phn ng: + Trong qu trnh phn ng trn NaBH4 ng vai tr l tc nhn kh, kh ion Ag+ thnh Ag0. Phng trnh phn ng nh sau:

2AgNO3 + 2NaBH4

2Ag0 + H2

+ B2H6 + 2NaNO3

+ Sau khi nh AgNO3 vo xy ra hin tng kh cc ion Ag + thnh Ag0, v vy dung dch chuyn sang mu vng nht, chng t c s xut hin ca nano bc. Sau khi ht nano bc c to thnh, chng ta s dng PVP hoc PEG bc cc ht Ag0 li, khng cho chng lin kt li vi nhau, iu ny gip tng kh nng bo v nano bc, trnh xut hin kt ta. Cc yu t nh hng ti cht lng ht nano bc:

Trong phng php ha kh, t l cht kh v nng ion bc, pH ca dung dch, nng polyme nh hng n hiu sut v kch thc ht bc. Phng php ha kh ha hc thu c ht c kch thc 10-20nm [9]. Nng AgNO3 nh hng ng k n kch thc ht. Cc nghin cu cho thy, nu tng nng AgNO3 th tc hnh thnh ht nano bc cng nhanh hn do c mt lng ln ht nano c hnh thnh trong mt thi gian ngn. Tuy nhin, khi nng AgNO3 tng, cc ht nano bc c hnh thnh sau s va chm vi nhau thng xuyn hn (do mt ln) v kt qu l chng s b ngng t li. Nng cht bao bc b mt polyme cng nh hng trc tip ti kch thc v tnh cht keo nano bc. Nu nng polyme qu ln, cc ht nano bc s phn tn khng u, b kt m v kt ta. Nu nng polyme thp, chng s khng bao ph c ht lng nano bc v vy cc ht nano bc s kt t li. pH nh hng trc tip ti cu trc v s bn vng cng nh mu sc ca ht. Khi pH cao hoc thp chng s nh hng trc tip ti qu trnh phn ng v nh hng ti lp polyme bao ph lm thay i tnh cht cht ca keo nano bc v kch thc ht ca chng.

2.2.2. Phn tch bng my quang ph hp th UV-vis

Phng php ny da vo vic do cng dng nh sng cn li sau khi i qua dung dch cht phn tch. Phng php o UV-vis dng nh tnh, nh lng cc cht c mu v dung dch keo da vo chiu cao gi tr bc sng hp th cc i (max), mt quang (E) v b rng hp th. Vi mu keo nano bc ch to, n nh trong iu kin bnh thng 24 gi, pha long i 2 ln bng nc, a vo cuvet thch anh c chiu dy 1cm, ghi ph UVvis trong di bc sng t 300-700nm. Da trn kt qu cng hp th E v bc sng hp th cc i ( max) ca dung dch keo nano bc, suy on s thay i kch thc ht bc.

2.2.3. Phn tch ht bng FE-SEM

FE-SEM: L loi knh hin vi in t c th to ra nh ca b mt vt liu vi phn gii cao, bng cch s dng mt chm in t hp qut ln b mt vt liu. Vic to nh ca vt liu c thc hin qua vic ghi nhn v phn tch cc bc x pht ra t tng tc ca chm in t vi b mt.

Cu to: Gm mt sng phng in t theo chiu t di ln, ba thu knh tnh in v h thng cc cun qut in t t gia thu knh th hai v th 3, v ghi nhn chm in t th cp bng mt ng nhn quang in. Nguyn l hot ng: in t c pht ra t sng phng in t, sau c tng tc, v hi t thnh chm in t hp nh h thng thu knh t, sau qut trn b mt vt liu nh cc cun qut tnh in. phn gii ca FE-SEM c xc nh t kch thc ca chm in t hi t, kch thc ca chm in t ny c hn ch bi quang sai, do FE-SEM khng c c phn gii tt nht. phn gii cn ph thuc vo tng tc gia b mt vt liu v in t.

2.2.4. Kho st hiu qu khng khun ca nano bc

Th nghim hiu qu khng khun ca 2 mu nano bc (bc PEG, PVP) vi hai loi vi khun E.coli v Staphylococcus c tin hnh trn a LB c. S dng 6 a LB c cho th nghim ny, 3 a nui E.coli v 3 a nui Staphylococcus th nghim kh nng khng khun nano bc bng phng php c l, nh vo mi l 50l nano bc, v nui cy sau 12 gi bung nui cy, nhm tm ra mu keo nano bc c tnh khng khun mnh hn, tip tc s dng mu cho cc th nghim tip theo. Cc bc tin hnh th nghim: Cy tri vi khun + + + S dng pipet cho 200 l dch khun ln b mt a LB agar c. S dng que cy tri ( kh trng) tri u vi khun ra ton b mt a. Kt thc cy tri.

Phng php c l + Sau khi cy tri vi khun, s dng dng c c 1 l nh ti tm a + Dng pipet nh 50l nano bc vo l i 15 pht cho nano bc khuch tn ra ton b mt a. + Cho vo t nui qua m nhit 370C.

2.2.5. Th nghim s tng tc gia keo nano bc vi nm mc

Nm mc c s dng trong th nghim ny l chng Aspergillus niger do Vin vi sinh vt v Cng ngh Sinh hc, i hc Quc Gia H Ni cung cp.

Chng ti tin hnh th nghim kh nng tng tc ca nm mc Aspergillus niger vi mu nano bc bc PEG. Nm mc c nui trn a thch bng mi trng Czapek. Tin hnh th nghim vi 2 a, mt a i chng v mt a th nghim vi nano bc. Cc bc tin hnh nh sau:

+ +

Vi mu i chng: dng que cy thu cc bo t nm t a gi ging sau chm thnh 11 im trn a nui cy. Vi mu th nghim vi nano bc: chun b ng eppendorf 1ml c 0.8ml nc ct v 0.2ml keo nano bc, dng que cy thu cc bo t nm t a ging sau cho vo ng eppendorf lc u. i 30 pht cc bo t nm mc lng xung. Sau dng que cy tri thu cc bo t nm mc v chm thnh 11 im trn a nui cy. C hai a c nui trong t gi nhit nhit 260C.

2.2.6. Nui cy vi to
Ngun ging, mi trng dinh dng v ch nui cy to Trong nghin cu, s dng cc mi trng BG 11, C, BBM cho nui cy vi to Chlorella elipsoidea . To c nui trong iu kin nhit phng di nh sng n neon vi cng sng l 10.000-20.000 Lux theo chu k quang l 10 gi chiu sng v 14 gi ti. m mt t bo bng bung m Neubauer [29] - Cu to bung m Neubauer Bung m Neubauer l mt tm thy tinh dy khong 3mm, c chia ra lm 3 phn. Cc phn bn ngn cch vi phn gia bi hai rnh dc. Phn gia c chia i do mt rnh ngang v thp hn hai phn bn 0.1mm, to ra hai ngn m ging nhau. Mi ngn m c chia thnh 9 vung ln mi c din tch 1mm 2. Mi ngn m hnh vung, c chia thnh 16 ln. Ring ngn m hnh vung chnh gia c chia thnh 16 trung bnh, mi trung bnh li c chia thnh 16 nh. Tng s nh ngn m chnh gia l 16x16=256 nh. Mi nh ny c din tch l 1/400mm2 , v chiu cao l 1/10mm2.

Nh vy th tch ca nh ny l: 1/400 x 1/10 = 25.10-5 mm2 Th tch ca mt ngn m l: 25.10-5 x 256 = 64.10-3 mm-3 (=64.10-6ml) Mt t bo trong 1ml c tnh theo cng thc: D = (A/64).106 Trong : + D: mt t bo (s t bo/ml). + A : l s t bo trong bung m.

a
Hnh 2.1. : Bung mNeubauer a- Bung m nhn thng v nhn nghing b- Bung m i knh hin vi

- Thao tc m Bung m v lamelle c lau sch bng cn v c thm kh trc khi cho dch to vo. Lamelle c dt trong bung m sao cho khi nhn nghing thy c s giao thoa nh sng v tr tip xc gia bung m v lamen. a bung m ln

knh hin vi, th knh 10 dng vt knh 10 iu chnh m tm m. Sau dng pipet ht mt t dch to c lc u v chm vo cnh ca lamelle. Dch to s trn vo bung m. Bung m cha to c a ln knh hin vi v quan st vt knh 40, th knh 10. i vi nhng mu m qu c hoc khng th m chnh xc th pha long trc khi m v nhn h s pha long khi tnh kt qu Lp chu k sinh trng ca to Trong nui cy to, s gia tng v kch thc ca cc t bo l khng ng k. Do khi s dng t sinh trng chng ta s hiu l s gia tng sinh khi, ch yu l do s gia tng v s lng t bo trong dung dch. Trong nui cy tnh, tc l ngun cung cp dinh dng l gii hn, khng b sung thm cng khng ly bt, sinh trng ca to tri qua bn pha c bn: + Pha thch ng. + Pha sinh trng. + Pha n nh. + Pha suy vong.

lp chu k sinh trng ca to, chng ta s tin hnh m mt t bo to bng bung m Neubauer. Bt u m t ngy tin hnh nui cy (ngy s 0), tin hnh m theo chu k 2 ngy 1 ln cho ti khi mt t bo to t gi tr cc i. Trn c s d liu thu c qua cc ngy m, lp ng cong sinh trng bng cch t trn trc tung ch s mt t bo, t trn trc honh ch s thi gian (ngy) nui to.

2.2.7. S tng tc gia keo nano bc vi to vi to

Lp ng chun ha gia hp th quang vi mt vi to Mt vi to Chlorella elipsoidea trong mi trng nui cy t l tuyn tnh vi mt quan hp th nn khi bit c h s t l chng ta c th bit c mt vi to thng qua mt quang hp th, v ngc li. xy dng c ng chun ha chng ta s tin hnh o gi tr mt quang hp th bc sng 650nm ng vi cc ngy m mt . T xc nh c cc im trn ng chun ha.

 Tin hnh thc nghim

- Kh nng tng tc ca keo nano bc vi vi to trong dung dch Da vo ng cong sinh trng ca vi to Chlorella elipsoidea tin hnh th nghim kh nng tng tc ca vi to vi keo nano bc, ti ba pha sinh trng ca vi to l pha thch ng, pha sinh trng, v pha suy vong nh bng 2.1. Vi to c nui trong 4 bnh tam gic vi th tch vi to Chlorella elipsoidea trong mi bnh 50ml, di iu kin nhit phng, nh sng n neon vi cng sng l 10.000-20.000 Lux, theo chu k quang l 10 gi chiu sng v 14 gi ti. mi mt chng ta tin hnh th nghim kh nng tng tc vi to ca keo nano bc vi 4 hm lng nano bc khc nhau l 0ml, 0.1ml, 1ml, 5ml nh bng sau:

Bng 2.2. Bng th nghim tng tc ca cc th tch keo nano bc vi vi to ti cc pha sinh trng

Th nghim

Bnh T1-1 T1-2

Th tch nano bc 0 ml 0.1ml 1ml 5ml 0 ml 0.1ml 1ml 5ml 0 ml 0.1ml 1ml 5ml

TN1

T1-3 T1-4 T2-1 T2-2

TN2

T2-3 T2-4 T3-1 T3-2

TN3

T3-3 T3-4

- Kh nng tng tc ca keo nano bc vi vi to khi tm ln mng xp

Thm t tm mng xp bng 30ml keo nano bc ri em sy kh bng t sy. Sau dng 30ml dung dch to vi mt 10.5 x 10 6 tb/ml ln mng sao cho to c gi li trn mng. Tin hnh nui to di iu kin nhit phng, nh sng n neon vi cng sng l 10000-20000 Lux theo chu k quang l 10 gi chiu sng v 14 gi ti.

Chng 3. KT QU V THO LUN

3.1. Ch to keo nano bc

Hnh 3.1. Kt qu ch to keo nano bc a - Mu bc PEG b - Mu bc PVP

Kt qu ch to thc nghim mu keo nano bc s dng NaBH 4 lm tc nhn kh bc PVP v bc PEG bng phng php ha kh cho thy dung dch keo bc c

mu vng ti ng nht khng xut hin kt ta vi pH=7.0 (Hnh 3.1). Ht nano bc c bao bc tt bi PVP v PEG. S dng phng php o quang ph hp th UV-vis v phng php chp nh FE-SEM nghin cu tnh cht ca keo nano bc ch to.

3.2. Phn tch ht bng tia UV-vis

Hnh 3.2. Kt qu o UV-vis thc nghim keo nano bc a - Mu bc PEG b - Mu bc PVP

S dng my quang ph hp th (UV/VIS) xc nh tnh cht quang ca dung dch keo nano bc. Kt qu hnh 3.2. cho thy cng hp th ca keo nano bc c bc bi PEG sau khi pha long 2 ln c gi tr khong 0,72 ti bc sng 400nm. Vi mu keo nano bc bc PVP sau khi pha long 2 ln c gi tr thp hn khong 0,66 ti bc sng 413nm . Nh vy, kch thc ht nano bc mu bc PVP ln hn lm gim mt quang v tng bc sng hp th UV-vis so vi dung dch keo bc c bc bi PEG. Kt qu o quang ph hp th UV-vis ca keo nano bc c nh hp th cc i trong khong 400-413nm ph hp vi cc kt qu nghin cu

c cng b. T kt qu ny c th nhn nh ht nano bc c bao bc bi PEG c kch thc ht nh hn v phn tn tt hn so vi ht nano bc bc bi PVP.

3.3. Phn tch ht bng FE-SEM

b Hnh 3.3. Kt qu chp nh FESEM mu nano bc a - Mu bc PEG b - Mu bc PVP

Kt qu chp FESEM hnh 3.3 cho thy kch thc ht nano bc bc PVP khong 25-50nm (Hnh 3.3.-b). Ht nano bc phn tn tt trong dung dch. Vi hnh 3.3.-a mu nano bc bc PEG ta ta c th xc nh c kch thc ca ht nano bc khong 15-50nm. Ht tng i ng u v phn tn tt trong dung dch.

3.4. Kh nng khng khun ca cc dung dch keo nano bc

Hnh 3.4. Kt qu th nghm khng khun vi dung dch nano bc a - E.coli cho nano bc bc PEG b - E.coli cho nano bc bc PVP c - E.coli khng cho nano bc d - Staphylococcus cho nano bc bc PVG e - Staphylococcus cho nano bc bc PVP f - Staphylococcus khng cho nano bc

T kt qu th nghim khng khun hnh 3.4 ca keo nano bc i vi Staphylococcus v E.coli ta thy c hai mu nano bc bc PVP v PEG u cho kt qu khng khun vi c hai loi vi khun. Tuy nhin mu nano bc bc PEG th hin kh nng khng khun tt hn mu nano bc bc PVP. Sau mt m nui cy vi mu nano bc bc PEG to vng khng khun khong 2.2cm vi E.coli v khong 2.0cm vi Staphylococcus. Mu nano bc bc PVP to vng khng khun tng ng l 1.5cm vi E.coli v 1.3cm vi Staphylococcus. T th nghim ny, chng ti s dng

mu keo nano bc bc PEG cho cc th nghim tip theo v kho st kh nng tng tc ca nano bc vi nm mc v to.

3.5. S tng tc gia keo nano bc vi nm mc

Hnh 3.5. Kt qu th nghm khng khun vi dung dch nano bc sau 4 ngy nui cy a Mu khng c nano bc b Mu c nano bc

Qua hnh 3.5 ta thy vi mu th nghim khng c nano bc nm pht trin tt u khp b mt a. Vi mu c nano bc nm mc b c ch v pht trin rt chm. Kt qu ny chng t kh nng tng tc ca nano bc vi nm mc.

3.6. La chn mi trng nui cy vi to

Mt trong nhng yu t hng u nh hng ti s sinh trng v pht trin ca vi to l mi trng dinh dng. Mc d l sinh vt quang t dng, nhu cu cht dinh dng khng phi qu kht khe, c bit l dinh dng hu c, song do c tnh sinh l ca vi to d thay i theo s thay i ca mi trng. Chnh v vy trong nui cy vi to vic chn la mi trng ph hp nht cho sinh trng v ph trin ca to l rt cn thit. Trong nghin cu ny, tin hnh nui tnh vi to Chlorella elipsoidea trn ba mi trng: BG 11, C, BBM vi mt ban u l 1.35 x 10 6 tb/ml. Kt qu thu c bng 3.1. v hnh 3.6.

Bng 3.1. Bng kt qu m thc nghim

Thi gian
(ngy)

Mt t bo (x 106/ml) trn cc nui trn bn mi trng nui cy khc nhau BG II 1.35 1.3 2.05 3.35 4.95 6.3 8.2 10.5 15.45 21.15 27.15 35 43.8 53.05 58.35 55.3 52.4 C 1.35 1.35 1.75 2.85 3.4 4.2 5 7.4 10.8 14.5 19.05 26.35 34.65 40.2 37.45 34.8 33.55 BBM 1.35 1.25 1.95 3.1 3.75 5.25 6.35 9 13.15 17.45 21.65 29.9 38.8 44.65 48.35 43.85 41.55

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32

Hnh 3.6. th kt qu m vi to Chlorella elipsoidea

T kt qu bng 3.1 v hnh 3.6 Trong iu kin nui cy tnh, thi gian nui cy t ti nh sinh trng l tng i di (sau 26-28 ngy). Sinh trng ca vi to Chlorella elipsoidea trong cc mi trng dinh dng khc bit nhau mt cch ng k, vi to sinh trng tt nht trong mi trng BG II, mt ti nh sinh trng t 58.35 x 106 tb/ml, vi mi trng BBM mt ny t 48.35 x 10 6 tb/ml, cn trong mi trng C mt ny l thp nht 40.2 x 10 6 tb/ml. To Chlorella elipsoidea c 3 mi trng u sinh trng mnh khi bc vo ngy th 6, c s phn bit r rt gia cc mi trng mt vi to trong cc mi trng BG 11, BBM, C ln lt l 3.35 x 106 tb/ml, 3.1 x 106 tb/ml v 2.85 x 106 tb/ml. T kt qu ny chng ti s dng mi trng BG 11 nui vi to Chlorella elipsoidea cho cc th nghim tip theo.

a b c Hnh 3.7. To Vi to Chlorella elipsoidea nui ngy th 22 a Mi trng BG11 b Mi trng C c Mi trng BBM

3.7. S tng tc gia keo nano bc vi vi to

3.7.1. ng cong chun ha gia mt quang vi mt to
Bng 3.2. Bng tng quan gia mt vi to v gi tr OD650nm qua cc ngy

OD650nm
6.80 5.97 4.99 4.12 2.97 2.58 1.82 1.36 0.77 0.58 0.38 0.24
0.15 0.00

Mt
58.35 53.50 43.80 35 27.15 21.15 15.45 10.50 6.30 4.95 3.35 2.05
1.35 0.00

Hnh 3.8. ng chun ha mt to v mt quang hp th ti cc pha sinh trng ca vi to Chlorella elipsoidea

T s liu o gi tr mt quang hp th ti cc pha sinh trng ca vi to Chlorella elipsoidea.Ta xy dng c ng chun ha gia mt t bo vi OD650nm tng ng ca vi to trong cc pha ca vi to Chlorella elipsoidea.

3.7.2. Kh nng tng tc ca keo nano bc vi vi to trong dung dch

Bng 3.3. Bng thc nghim o OD650nm ca vi to
T1 (Pha thch ng) T1-1 0ml 0 2 4 6 8 0.17 0.16 0.25 0.40 0.57 T1-2 0.5m l 0.18 0.14 0.19 0.30 0.42 T1-3 1ml 0.17 0.12 0.09 0.12 0.14 T1-4 5ml 0.16 0.08 0.04 0.02 0.01 Ngy T2 (Pha sinh trng) T2-1 T2-2 0ml 0.5ml 0.95 1.21 1.77 2.43 3.12 0.92 1.16 1.68 2.31 2.98 T2-3 1ml 0.97 1.09 1.46 1.77 2.19 T2-4 5ml 0.91 0.84 0.77 0.58 0.51 T3-1 0ml 6.55 6.08 5.80 5.72 5.57 T3 (Pha suy vong) T3-2 0.5ml 6.52 5.77 5.31 5.08 4.49 T3-3 1ml 6.50 5.25 4.68 4.12 3.61 T3-4 5ml 6.56 4.49 3.02 1.87 1.09

Hnh 3.9. Kh nng tng tc cc nng Nano bc vi vi to ti pha thch ng

Qua bng 3.3 v hnh 3.9 ta thy vi th nghim T1-1 khng c nano bc trong mi trng nui, vi to pht trin theo ng ng cong sinh trng, sau 8 ngy nui cy mt vi to tng t 1,48 x 106tb/ml ln 4,96 x 106tb/ml. Vi th nghim T1-4 cho 5ml nano bc ta thy gi tr OD 650nm gim mnh qua cc ngy v ngy th 8 nui cy gi tr ny l 0,014 tng ng vi mt vi to khong 0,12 x 10 6tb/ml. Cn vi hai

th nghim T1-2 v T1-3 cho tng ng 0,5ml v 1ml nano bc ng gi tr mt quang hp th thp hn so vi th nghim T1-1, sau 8 ngy nui cy mt vi to th nghim T1-2 khong 4,42 x 106tb/ml, v th nghim T1-3 khong 2,00 x 106tb/ml.

Hnh 3.10. Kh nng tng tc cc nng nano bc vi vi to ti pha sinh trng

Qua bng 3.3 v hnh 3.10 ta thy s tng tc ca nano bc th nghim T2 khi vi to pha sinh trng c s khc bit vi T1 khi to pha thch ng. Vi mu th nghim T2-4 cho 5ml nano bc ta thy gi tr OD650nm gim qua cc ngy (t 0,91 xung 0,51), tuy nhin t l gim khng nhanh nh T1-4 th hin qua dc ca th. V sau 8 ngy nui cy mt vi to th nghim T2-4 khong 4,44 x 106tb/ml.

Hnh 3.11. Kh nng tng tc cc nng nano bc vi vi to ti pha thch ng

Qua bng 3.3 v hnh 3.11 ta thy th nghim T3 khi vi to pha suy vong v vy th nghim T3-1 khng cho nano bc gi tr OD650nm gim sau 8 ngy (t 6,55 xung 5,57), tng ng mt vi to gim t 57,13 x 10 6tb/ml xung 48,52 x 106tb/ml. Vi cc th nghim T3-4 tc gim l nhanh nht mt vi to gim t 57,14 x 106tb/ml xung 9,5 x 106tb/ml. hai mu th nghim cn li ta thy tc gim mt vi to chm hn so vi th nghim T3-4, th nghim T3-2 gim t 56,78 x 106tb/ml xung 39,11 x 106tb/ml v T3-3 mt vi to gim t 56,61 x 10 6tb/ml xung 31,44 x 106tb/ml. Nh vy c ba th nghim T1, T2 v T3 ta thy c s tng tc gia nano bc vi vi to lm mt vi to gim thp hn so vi mu khng cho nano bc. S tng tc ny nhn thy r rt nht vi lng nano bc l 5ml. Vi hm lng ny c ba giai on sinh trng u c s suy gim ng k v mt vi to so vi thi im bt u cho nano bc. Tuy nhin s suy gim ny cc pha khc nhau l khng ging nhau. S gim mt vi to din ra nhanh nht vi th nghim T3 khi vi to ang pha suy vong v din ra chm hn vi th nghim T2 khi vi to pha sinh trng.

Hnh 3.12. nh to Chlorella elipsoidea mu th nghim T1-4 trc v sau khi cho nano bc 8 ngy (vt knh 40) a - Bnh khng cho nano bc b - Bnh cho 5ml keo nano bc

Hnh 3.12 cho ta thy sau khi cho nano bc vo trong mi trng nui to, cc ht nano bc tng tc vi to lm t bo vi to mt mu cht dip lc chlorophyll t c ch qu trnh quang hp ca to. Tuy nhin hnh dng ca t bo vi to khng b ph v nh vi khun.

3.7.3. Kh nng tng tc ca keo nano bc vi vi to khi tm ln mng xp

Hnh 3.13. Kh nng tng tc ca nano bc vi vi to khi tm ln mng xp a- Ngy th 0 b- Ngy th 6

Qua hnh 3.13 ta thy nano bc th hin kh nng tng tc vi vi to khi tm vo mng xp. Sau 6 ngy nui cy ta thy vi to vn sng trn ming xp khng c nano bc th hin qua mu xanh dip lc trong khi vi ming xp tm nano bc chng ta khng cn nhn thy mu xanh trn ming xp. Nh vy nano bc c c nh trn si ca mng xp tc ng tr tip ti s pht trin ca vi to.

KT LUN
ch to c keo nano bc bng phng php ha kh, s dng PVP v PEG lm cht bc. kho st c cc tnh cht ca keo nano bc bng phn tch quang ph hp th UV-vis v chp nh bng knh hin vi in t FESEM. Keo nano bc bc PVP v PEG cho thy kh nng khng khun nh vi khun E.coli v vi khun Staphylococcus. Kt qu th nghim kh nng tng tc ca keo nano bc vi nm mc Aspergillus niger cho thy nano bc c kh nng c ch sinh trng ca nm mc. Kt qu th nghim kh nng tng tc ca keo bc nano vi vi to Chlorella elipsoidea cho thy nano bc nh hng ti s pht trin ca vi to.

HNG NGHIN CU TIP THEO

Cn tip tc hon thin cc phng php ch to ht nano bc thu c cc ht nano bc nh hn, ng u hn. Tip tc nghin cu c ch nh hng ca nano bc mc phn t. Tip tc nghin cu ng dng kh nng khng nm mc v to ca nano bc trong iu kin thc t.

PH LC

A. Ph lc 1: Thnh phn cc mi trng LB nui cy vi khun STT 1 2 3 4 5 Ha cht 10g Peptone 5g Yeast extract 10g Sodium citrate 1L Nc ct B xung thm 15g Agar vi mi trng LB c Hm lng/l

B.Ph lc 1: Thnh phn cc mi trng nui cy nm mc STT 1 2 3 4 5 6 7 8 9 Ha cht Saccroza NaNO3 K2HPO4 MgSO4 KCl FeSO4.7H2O Thch Nc ct pH 6.0 Hm lng/l 30g 3g 1g 0.5g 0.5g 0.05g 15g 1l

C. Ph lc 1: Thnh phn cc mi trng nui cy vi to Mi trng C STT 1 2 3 4 5 6 7 8 9 10 11 Ha cht Ca(NO3)2.4H2O KNO3 -Na2glicerolphosphate.5H2O MgSO4.7H2O PIV metals Tris (hydroximethyl) aminoethanol Vitamin B12 Biotin Thiamine HCL Nc ct pH 7.5 Hm lng/100ml 15mg 10mg 5mg 4mg 0.3ml 50mg 0.01g 0.01g 1g 99.7ml

PIV metals

STT 1 2 3 4 5 6 7

Ha cht FeCl3.6H20 MnCl2.4H2O ZnSO4.7H2O CoCl2.6H2O Na2MoO4.2H2O Na2EDTA.2H2O Nc ct 100ml

Hm lng/l 19.6mg 3.6mg 2.2mg 0.4mg 0.25mg 100mg 100ml

 Mi trng BBM

STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 NaNO3 KH2PO4 K2HPO4 MgSO4.7H2O CaCl2.2 H2O NaCl KOH FeSO4.7H2O H3BO3 ZnSO4.7H2O MnCl2.7H2O MoO3 CuSO4.5H2O

Ha cht

Hm lng/100l 25mg 17.5mg 10mg 7.5ml 2.5mg 2.5mg 31mg 0.498mg 1.142mg 0.882mg 0.144mg 0.071mg 0.157mg 0.049mg 5mg 100ml

Co(NO3)2.6H2O Na2EDTA Distilled water

Mi trng BG11

STT 1 2 3 4 5 6 7 8 9 10 11
NaNO3 K2HPO4.3H2O MgSO4.7H2O CaCl2.2 H2O Citric acid

Ha cht

Hm lng/100ml
150mg 4mg 7.5mg 3.6mg 0.6mg 0.6mg 0.1mg 2mg 0.1ml 99.9ml

Ferric ammonium citrate Na2ETDA.Mg Na2CO3 Trace metal mix A5 + Co Distilled water

pH 7.4

Trace metal mix A5 + Co

STT 1 2 3 4 5 6 H3BO3 MnCl2 ZnSO4 Na2MoO4

Ha cht

Hm lng/l 2.86g 1.81g 0.222g 0.4mg 0.079g 0.049g

Na2MoO4.2H2O Na2EDTA

TI LIU THAM KHO

Ting Vit [1] [2] [3] [4] [5] [6] [7] [8] Dng c Tin, V Hnh, To nc ngt Vit Nam-Phn loi b to lc (chlorococcales), NXB nng nghip, 1998. ng nh Kim v ng Hong Phc Hin, Cng ngh sinh hc vi to, NXB Nng nghip, 1998. L Huy Chnh(ch bin), Vi sinh y hc, Nxb y hc, 2003. L Th Thu Hin, Nng Vn Hi, L Trn Bnh, Bi tng qun cng ngh sinh hc nano, Tp ch cng ngh sinh hc 2(2), 2004, Tr. 133-148. Nguyn c Ngha , ha hc nano-Cng ngh nn v vt liu ngun, NXB Khoa hc T nhin v Cng ngh, H Ni, 2007. Nguyn Ln Dng, Nguyn Th Hoi H, Vi to (Microalgae), 2006 Uldrich.J v Newberry.D, Cng ngh nano-u t & u t mo him, Sch dch, NXB Tr, 2006. V nh C, Nguyn Xun Chnh, Cng ngh nano iu khin n tng nguyn t, phn t, Nh xut bn khoa hc k thut, 2004.

Ting Anh [9] Badr.Y, mahmoud.M.A. Enhancement of the optical propertied of poly vinyl alcohol by doping with silver nanoparticles, J. Appl. Polym. Sci, 99, 2006, pp.3068-3614. Chmielewski A.G, Worldwide developments in the field of radiation processing of materials in the down of 21 st century, Nukleotika, 51(supplement 1): S3-S9, 2006 Liuchia-I, ph.D, Wen-Yih Jeng, Ph.D, and Andrew H.-j. Wang, ph.D, Mary E. Hensler, Ph.D, and Yongcheng Song, Ph.D, and Fenglin Yin, Ph.D, Novel Approach Strips Staphylococcus aureus of Virulence, National Institute of General Medical Sciences.

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