I HC QUC GIA H NI

TRNG I HC CNG NGH

1 Nguyn Ngc Hng

NGHIN CU CH TO HT NANO BC V KH
NNG ST KHUN CA N

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

2 Nguyn Ngc Hng

NGHIN CU CH TO HT NANO BC V KH
NNG ST KHUN CA N

KHO LUN TT NGHIP I HC H CHNH QUY

Ngnh: Vt l k thut

Cn b hng dn: TS. Nguyn c Quang

2.1 H NI - 2011
Li cm n

u tin, em xin by t lng bit n su sc ti TS. Nguyn c Quang (Khoa

VLKT HCN HQGHN) v TS. L Anh Tun (Vin tin tin Khoa hc v cng
ngh - H Bch Khoa H Ni) tn tnh hng dn, gip em trong sut thi gian
va qua.

Em xin gi li cm n ti cc anh ch trong Vin Vi sinh vt v Cng ngh sinh

hc HQG H Ni ch bo v to iu kin thun li nht em hon th nghim.

Em cng xin t lng bit n ti cc thy c v cc anh ch khoa Vt l k thut,

c bit l GS. L Trn Bnh, TS. Trn ng Khoa v TS. H Th Quyn v dy
bo v gip em nhng lc kh khn.

Cui cng, em xin gi li cm n n gia nh v bn b, nhng ngi lun ng

h v ng vin em trong sut thi gian va qua.

H Ni, Ngy 19/5/2011

Nguyn Ngc Hng

Li cam oan

Ti xin cam oan lun vn ny l cng trnh do ti t lm v nghin cu, cc trch dn ti

u ghi r trong phn ti liu tham kho. Nu c bt k vn g xy ra, ti xin chu
hon ton trch nhim

Ngi vit lun vn:

Nguyn Ngc Hng

 MC LC

Tm tt ni dung kha lun.......................................................................................- 1 -

Chng 1: TNG QUAN V HT NANO BC V NG DNG CA CHNG

TRONG SINH HC....................................................................................................- 2 -

1.1 Gii thiu v cng ngh nano...............................................................................- 2 -

1.1.1 Khi nim v ngun gc ca cng ngh nano................................................- 2 -

1.1.2 C s khoa hc ca cng ngh nano..............................................................- 2 -

1.1.3 ng dng ca cng ngh nano trong sinh hc v y hc.................................- 4 -

1.2 Ht nano bc.........................................................................................................- 5 -

1.2.1 Gii thiu v bc kim loi..............................................................................- 5 -

1.2.2 c tnh khng khun ca bc........................................................................- 6 -

1.2.3 C ch khng khun ca bc..........................................................................- 6 -

1.2.4 Cc phng php ch to ht nano kim loi...................................................- 8 -

1.2.5 Gii thiu v ht nano bc..............................................................................- 9 -

1.2.5.1 Cc phng php phn tch ht nano bc....................................................- 9 -

1.2.5.2 ng dng ca nano bc.............................................................................- 11 -

1.3 S sinh trng v pht trin ca vi khun..........................................................- 15 -

1.3.1 ng cong sinh trng..............................................................................- 15 -

1.3.1.1 Giai on Tim pht (Lag phase)..............................................................- 15 -

1.3.1.2 Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase)..........- 16 -

1.3.1.3 Giai on t vong (Death Phase)..............................................................- 17 -

1.3.2 Xc nh s sinh trng ca vi khun..........................................................- 17 -

1.3.2.1 Xc nh s lng t bo..........................................................................- 17 -

 1.3.2.2 Xc nh khi lng t bo.......................................................................- 18 -

Chng 2: NGHIN CU THC NGHIM.........................................................- 20 -

2.1 Phng php ch to ht nano bc.....................................................................- 20 -

2.1.1 Quy trnh cng ngh ch to dung dch nano bc s dng k thut kh ha hc
vi bc x UV kch thch......................................................................................- 20 -

2.1.1.1 Ha cht th nghim s dng:....................................................................- 20 -

2.1.1.2 Thit b s dng........................................................................................- 20 -

2.1.1.3 Quy trnh tng hp....................................................................................- 20 -

2.1.2 C ch hnh thnh ht nano bc...................................................................- 22 -

2.2 nh hng ca nano bc ln s pht trin ca vi khun....................................- 23 -

2.2.1 Phng php o OD.....................................................................................- 23 -

2.2.2 Cc trang thit b..........................................................................................- 24 -

2.2.3 Cc bc tin hnh th nghim.....................................................................- 24 -

2.2.3.1 Chun b mi trng.................................................................................- 24 -

2.2.3.2 Cy vi khun.............................................................................................- 25 -

2.2.3.3 o OD.......................................................................................................- 25 -

Chng 3: KT QU V THO LUN................................................................- 26 -

3.1 Phn tch dung dch nano bc.............................................................................- 26 -

3.2 Kt qu theo di s pht trin ca vi khun khi c mt ca dung dch nano bc- 28 -

3.3 So snh vi phng php o bn knh vng v khun......................................- 34 -

Chng 4: KT LUN.............................................................................................- 35 -
Tm tt ni dung kha lun

T xa xa, con ngi s dng bc lm cc dng c cha n, nc ung tr bnh.

Trong chin tranh th gii th nht, ngi ta thm ch cn s dng cc sn phm t bc
iu tr nhim trng trc khi thuc khng sinh ra i. Ngy nay, cng vi s ra i v
pht trin ca cng ngh nano, con ngi ch to c bc kch thc nano. iu
ny lm tng ng k s ng dng ca bc. kch thc nano, bc th hin kh nng
khng khun mnh m khng gy nh hng ti con ngi v mi trng. Chnh v vy,
gii khoa hc ang u t nghin cu bc phc v cho cc ng dng trong y hc, nht
l khi hin tng vi khun khng khng sinh ngy cng ph bin nh ngy nay.

Chnh v vy, trong kha lun ny, chng ti tp trung vo cc nhim v sau:

1. Nghin cu quy trnh cng ngh v tham gia th nghim ch to ht nano bc

2. Nghin cu nh hng ca ht nano bc ln s pht trin (kh nng c ch s pht

trin) ca vi khun gy bnh ng rut E. coli

3. nh gi hiu qu c ch ca nano bc vi E. coli

1
 Chng 1: TNG QUAN V HT NANO BC V NG DNG CA CHNG
TRONG SINH HC

1.1 Gii thiu v cng ngh nano

1.1.1 Khi nim v ngun gc ca cng ngh nano

Cng ngh nano l ngnh cng ngh lin quan n 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 (nm, 1 nm = 10-9 m) [3]. kch thc nano, vt liu s c nhng
tnh nng c bit m vt liu truyn thng khng c c l do s thu nh kch thc
v vic tng din tch mt ngoi.

tng c bn v cng ngh nano c a ra bi nh vt l hc ngi M

Richard Feynman vo nm 1959, ng cho rng khoa hc i vo chiu su ca cu trc
vt cht n tng phn t, nguyn t vo su hn na. Nhng thut ng cng ngh
nano mi bt u c s dng vo nm 1974 do Nario Taniguchi mt nh nghin cu
ti trng i hc Tokyo s dng cp kh nng ch to cu trc vi hnh ca mch vi
in t[4].

1.1.2 C s khoa hc ca cng ngh nano

Cng ngh nano da trn nhng c s khoa hc ch yu sau:

-
Chuyn tip t tnh cht c in n tnh cht lng t: i vi vt liu v
m gm rt nhiu nguyn t, cc hiu ng lng t c trung bnh ha vi rt
nhiu nguyn t (1 m3 c khong 1012 nguyn t) v c th b qua cc thng
ging ngu nhin. Nhng cc cu trc nano c t nguyn t hn th cc tnh
cht lng t th hin r rng hn.
-
Hiu ng b mt: Khi vt liu c kch thc nm, cc s nguyn t nm trn b
mt s chim t l ng k so vi tng s nguyn t. Chnh v vy cc hiu ng
c lin quan n b mt, gi tt l hiu ng b mt s tr nn quan trng lm
cho tnh cht ca vt liu c kch thc nanomet khc bit so vi vt liu
dng khi.

2
-
Kch thc ti hn: Cc tnh cht vt l, ha hc ca cc vt liu u c mt
gii hn v kch thc. Nu vt liu m nh hn kch thc ny th tnh cht
ca n hon ton b thay i. Ngi ta gi l kch thc ti hn. Vt liu
nano c tnh cht c bit l do kch thc ca n c th so snh c vi kch
thc ti hn ca cc tnh cht ca vt liu[3].

Bng 1: di ti hn ca mt s tnh cht ca vt liu[3].

Lnh vc Tnh cht di ti hn

(nm)

Tnh cht Bc sng in t 10-100

in
Qung ng t do trung bnh khng n 1-100
hi

Hiu ng ng ngm 1-10

Tnh cht t dy vch men 10-100

Qung ng tn x spin 1-100

Tnh cht H lng t 1-100

quang
di suy gim 10-100

su b mt kim loi 10-100

Tnh siu di lin kt cp Cooper 0,1-100

dn
thm thu Meisner 1-100

Tnh cht c Tng tc bt nh x 1-1000

Bin ht 1-10

Bn knh khi ng t v 1-100

Sai hng mm 0,1-10

nhn b mt 1-10

3
 Xc tc Hnh hc topo b mt 1-10

Siu phn t di Kuhn 1-100

Cu trc nh cp 1-10

Cu trc tam cp 10-1000

Min dch Nhn bit phn t 1-10

1.1.3 ng dng ca cng ngh nano trong sinh hc v y hc

Do c nhiu tnh nng c o v kch thc tng ng vi cc phn t sinh

hc nn hin nay, cng ngh nano ang c u t nghin cu c bit l trong lnh vc
y sinh. Cc ng dng tiu biu ca cng ngh nano trong lnh vc ny l:

-
Chn on: S dng cc ht nano (ht nano vng, nano t, chm lng t)
nh du cc phn t sinh hc, vi sinh vt, pht hin cc chui gen nh vo
c ch bt cp b xung ca DNA hoc c ch bt cp khng nguyn khng
th.
-
Vn chuyn thuc: Cung cp thuc cho tng t bo c th bng cch s dng
cc ht nano nhm tit kim thuc v trnh cc tc dng ph.
-
M k thut: Cng ngh nano c th gip c th ti sn xut hoc sa cha
cc m b h hng bng cch s dng gin da trn vt liu nano v cc yu
t tng trng[7].

4
 Hnh 1: Ht nano vng s dng trong truyn dn thuc.

1.2 Ht nano bc

1.2.1 Gii thiu v bc kim loi

Cu hnh electron ca bc: 1s22s22p63s23p63d104s24p64d105s1

Bn knh nguyn t Ag: 0,288 nm

Bn knh ion bc: 0,23 nm

Bng 2: S nguyn t bc trong mt n v th tch[14].

Kch thc ca ht S nguyn t cha

nano Ag (nm) trong

1 31

5 3900

5
 20 250000

Bc nano l vt liu c din tch b mt ring rt ln, c nhng c tnh c o

[14]
sau :
-
Tnh kh khun, chng nm, kh mi, c kh nng pht x tia hng ngoi i
xa, chng tnh.
-
Khng c hi cho sc khe con ngi vi liu lng tng i cao, khng c
ph gia ha cht.
-
C kh nng phn tn n nh trong cc loi dung mi khc nhau (trong cc
dung mi phn cc nh nc v trong cc dung mi khng phn cc nh
benzene, toluene).
-
bn ha hc cao, khng b bin i di tc dng ca nh sng v cc tc
nhn oxy ha kh thng thng.
-
Chi ph cho qu trnh sn xut thp.
-
n nh nhit cao.

1.2.2 c tnh khng khun ca bc

Bc v cc hp cht ca bc th hin tnh c i vi vi khun, virus, to v nm .

Tuy nhin, khc vi cc kim loi nng khc (ch, thy ngn) bc khng th hin tnh
c vi con ngi.

T xa xa, ngi ta s dng c tnh ny ca bc phng bnh. Ngi c i

s dng cc bnh bng bc lu tr nc, ru dm. Trong th k 20, ngi ta thng
t mt ng bc trong chai sa ko di ti ca sa. Bc v cc hp cht ca bc
c s dng rng ri t u th k XIX n gia th k XX iu tr cc vt bng v
kh trng[10].

Sau khi thuc khng sinh c pht minh v a vo ng dng vi hiu qu cao
ngi ta khng cn quan tm n tc dng khng khun ca bc na. Tuy nhin, t
nhng nm gn y, do hin tng cc chng vi sinh ngy cng tr nn khng thuc,
ngi ta li quan tm tr li i vi vic ng dng kh nng dit khun v cc ng dng
khc ca bc, c bit l di dng ht c kch thc nano.

6
 1.2.3 C ch khng khun ca bc

Hnh 2: Tc ng ca ion bc ln vi khun.

Cc c tnh khng khun ca bc bt ngun t tnh cht ha hc ca cc ion Ag +.

Ion ny c kh nng lin kt mnh vi peptidoglican, thnh phn cu to nn thnh t
bo ca vi khun v c ch kh nng vn chuyn oxy vo bn trong t bo dn n lm t
lit vi khun. Nu cc ion bc c ly ra khi t bo ngay sau , kh nng hot ng
ca vi khun li c th c phc hi. Do ng vt khng c thnh t bo,v vy chng ta
khng b tn thng khi tip xc vi cc ion ny.

C mt c ch tc ng ca cc ion bc ln vi khun ng ch c m t nh
sau: Sau khi Ag+ tc ng ln lp mng bo v ca t bo vi khun gy bnh n s i vo
bn trong t bo v phn ng vi nhm sunfuahydrin SH ca phn t enzym chuyn
ha oxy v v hiu ha men ny dn n c ch qu trnh h hp ca t bo vi khun [14].

Hnh 3: Ion bc v hiu ha enzym chuyn ha oxy ca vi khun

7
 Ngoi ra cc ion bc cn c kh nng lin kt vi cc base ca DNA v trung ha
in tch ca gc phosphate do ngn chn qu trnh sao chp DNA[15].

Hnh 4: Ion bc lin kt vi cc base ca DNA

1.2.4 Cc phng php ch to ht nano kim loi

- Phng php n mn laze: Phng php ny s dng chm tia laze vi b

sng ngn bn ln vt liu khi t trong dung dch c cha cht hot ha b mt. Cc ht
nano c to thnh vi kch thc khong 10 nm v c bao ph bi cht hot ha b
mt.

- Phng php kh ha hc: Phng php ny s dng cc tc nhn ha hc

kh ion kim loi thnh kim loi. cc ht phn tn tt trong dung mi m khng b kt
t thnh m, ngi ta s dng phng php tnh in lm cho b mt cc ht nano c
cng in tch v y nhau hoc dng phng php bao bc bng cht hot ha b mt.
Cc ht nano to thnh bng phng php ny c kch thc t 10 nm n 100 nm.

- Phng php kh vt l: Phng kh vt l dng cc tc nhn vt l nh in

t, sng in t nng lng cao nh tia gamm, tia t ngoi, tia laser kh ion kim loi
thnh kim loi. Di tc dng ca cc tc nhn vt l, c nhiu qu trnh bin i ca
dung mi v cc ph gia trong dung mi sinh ra cc gc ha hc c tc dng kh ion
thnh kim loi.

- Phng php kh ha l: y l phng php trung gian gia ha hc v vt

l. Nguyn l l dng phng php in phn kt hp vi siu m to ht nano.
Phng php in phn thng thng ch c th to c mng mng kim loi. Trc khi
8
xy ra s hnh thnh mng, cc nguyn t kim loi sau khi c in ha s to cc ht
nano bm ln in cc m. Lc ny ngi ta tc dng mt xung siu m ng b vi
xung in phn th ht nano kim loi s ri khi in cc v i vo dung dch.

- Phng php kh sinh hc: Dng vi khun l tc nhn kh ion kim loi. Ngi
ta cy vi khun MKY3 vo trong dung dch c cha ion bc thu c ht nano bc.
Phng php ny n gin, thn thin vi mi trng v c th to ht vi s lng
ln[6].

1.2.5 Gii thiu v ht nano bc

Ht nano bc l cc ht bc c kch thc t 1 nm n 100 nm. Do c din tch b

mt ln nn ht nano bc c kh nng khng khun tt hn so vi cc vt liu khi do
kh nng gii phng nhiu ion Ag+ hn.

Cc ht nano bc c hin tng cng hng Plasmon b mt. Hin tng ny to

nn mu sc t vng nht n en cho cc dung dch c cha ht nano bc vi cc mu
sc ph thuc vo nng v kch thc ht nano.

1.2.5.1 Cc phng php phn tch ht nano bc

a) S dng knh hin vi in t truyn qua

Knh hin vi in t truyn qua (TEM) hot ng trn nguyn tc ging thu knh
quang hc, ch khc l s dng sng in t thay cho bc sng nh sng nn c bc
sng rt ngn) v s dng cc thu knh in t - magnetic lens thay cho thu knh quang
hc[5].

9
 Hnh 5: M hnh nguyn l ca TEM so vi knh hin vi quang hc

nh ca knh hin vi in t truyn qua cho php ta quan st c hnh dng v

xc nh c kch thc ca cc ht nano.

10
 Hnh 6: nh TEM ca cc ht nano bc kch thc 10 nm[11].

b) Phn tch ph UV-VIS

UV-VIS (Ultravioletvisible spectroscopy) l phng php phn tch s dng ph

hp th hoc phn x trong phm vi vng cc tm cho ti vng nh sng nhn thy c.

Do cc thuc tnh quang hc ca dung dch cha ht nano ph thuc vo hnh

dng, kch thc v nng ca ht, nn ta c th s dng UV-VIS xc nh cc thuc
tnh trn.

11
 Hnh 7: nh UV-VIS ca cc ht nano bc[10].

Do ht nao bc c kch thc nh hn 20 nm ch c mt b mt plasmon duy nht

nn trong ph UV-VIS ca chng ch xut hin 1 nh duy nht. Ngi ta x dng tnh
cht ny xc nh hnh dng ca ht nano bc[12].

1.2.5.2 ng dng ca nano bc

Do th hin tnh khng khun tt nn nano bc thng c s dng lm cht

kh trng, khng khun, kh mi C th k mt vi sn phm cha ht nano bc nh:

-
Cc dng c cha thc phm: Nhng dng bng nha c pha thm ht
nano bc c tc dng kh trng. Qua kim tra cho thy chng c kh nng dit
99.9% vi khun.

12
 Hnh 8: Bnh sa lm bng nha c pha thm nano bc

-
may mc: ht nano bc c tm vo cc loi si dit khun v kh
mi.

Hnh 9: Tt lm bng si nilon c pha nano bc

13
 -
Cc thit b in t: iu ha, t lnh, my git

Hnh 10: iu ha s dng b lc nano bc

-
Y t:
+ Khu trang nano bc: c thit k vi 3-4 lp gm 2 lp vi, mt lp vt liu
tm nano bc v than hot tnh gia, loi khu trang ny c kh nng dit khun, dit
virus, lc khng kh rt tt. Lp vi tm nano bc c chc nng dit vi khun, virus, nm
b gi li trn khu trang ng thi c tc dng kh mi.

Hnh11: Khu trang nano bc do vin mi trng sn xut

14
 + Sn xut thuc cha bnh

Hnh 12: Cc dc phm s dng nano bc

+ Mng h hp: l mt tm mng mng c th cho kh v hi nc qua nhng

khng th cho cht lng i qua, c v s nhng l kh nh tn ti trong tm film. Cc ht
nano bc gn y c kt hp vi film polyolefin vi c tnh khng khun rt tt.

Hnh 13: nh SEM ca cc ht nano bc kt hp vi film polyolefin

-
Cc sn phm khc:

Hnh 14: cc sn phm c cha nano bc

15
 1.3 S sinh trng v pht trin ca vi khun

1.3.1 ng cong sinh trng

S sinh trng qun th vi sinh vt c nghin cu bng cch phn tch ng

cong sinh trng trong mt mi trng nui cy vi sinh vt theo phng php nui cy
theo m (batch culture) hoc trong mt h thng kn. C ngha l vi sinh vt c nui
cy trong mt thit b kn, trong qu trnh nui cy khng thay i mi trng v thi
gian nui cy cng ko di th nng cht dinh dng cng gim st, cc cht ph thi
ca trao i cht cng tng ln. Nu ly thi gian nui cy l trc honh v ly s logarit
ca s lng t bo sng lm trc tung s c th v c ng cong sinh trng ca cc
vi sinh vt sinh sn bng cch phn i. ng cong ny c 4 giai on (phases) khc
nhau[8].

Hnh 15: ng cong sinh trng trong h thng kn

1.3.1.1 Giai on Tim pht (Lag phase)

Khi cy vi sinh vt vo mt mi trng mi s lng thng khng tng ln ngay,

l giai on Tim pht hay pha Lag. Trong giai on ny t bo cha phn ct nhng
th tch v khi lng tng ln r rt do c s tng cc thnh phn mi ca t bo.

Giai on tim pht di hay ngn lin quan n bn thn tng loi vi sinh vt v
tnh cht ca mi trng. Nu tnh cht ha hc ca mi trng mi sai khc nhiu vi
16
mi trng c th giai on tim pht s ko di. Ngc li, nu cy t giai on logarit
vo mt mi trng c thnh phn tng t th giai on tim pht s rt ngn li. Nu
cy vi sinh vt t giai on tim pht hay t giai on t vong th giai on tim pht s
ko di[8].

1.3.1.2 Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase)

Trong giai on ny vi sinh vt sinh trng v phn ct vi tc ti a nu gp

mi trng v iu kin nui cy thch hp. Nhp sinh trng ca chng l khng thay
i trong sut giai on ny, cc t bo phn i mt cch u n. Do cc t bo sinh ra
ch khc nhau rt t cho nn ng cong sinh trng l mt ng trn nhn ch khng
gp khc Hnh dng ca ng cong hu nh phn nh tc hp thu cht dinh dng
nh s chuyn vn protein ca vi sinh vt[8].

Hnh 16: Nng cht dinh dng sinh trng

(a ) nh hng ca s hn ch cht dinh dng i vi sn lng chung ca vi sinh vt.

Lc nng cao th sn lng chung s t ti n nh.

(b)- nh hng ca s hn ch cht dinh dng ti tc sinh trng

1.3.1.3 Giai on t vong (Death Phase)

17
 Vic tiu hao cht dinh dng v vic tch ly cc cht thi c hi s lm tn tht
n mi trng sng ca vi sinh vt, lm cho s lng t bo sng gim xung. l c
im ca giai on t vong.

Ging nh giai on logarit, s t vong ca qun th vi sinh vt cng c tnh

logarit (t l t bo cht trong mi gi l khng i). Tng s t bo sng v t bo cht
khng thay i v cc t bo cht cha b phn hy. Mun xc nh s lng t bo sng
phi pha long ra ri cy ln thch a v a vo iu kin thch hp xc nh s
khun lc xut hin.

Mc du phn ln vi sinh vt t vong theo phng thc logarit nhng sau khi s
lng t bo t nhin gim xung th tc cht ca t bo chm li. l do mt s c
th sng li nh c tnh khng c bit mnh. V iu ny v nhng nguyn nhn khc
lm cho ng cong ca giai on t vong c th kh phc tp[8].

1.3.2 Xc nh s sinh trng ca vi khun

1.3.2.1 Xc nh s lng t bo

Phng php n gin nht xc nh s lng t bo l m trc tip di knh

hin vi. Dng cc phng m m va nhanh chng, d dng, li r tin nht, li c
th quan st thy kch c v hnh dng t bo.

Vi t bo vi khun cn nhum mu hoc l dng knh hin vi tng phn pha hay
knh hin vi hunh quang (phase-constrast or fluoresence microscope) d quan st
hn. Phng m c cu trc c mt su nht nh li c chia ra thnh cc nh.
Khi m s lng ta a dch pha long vo phng m, y l knh (lamelle/ cover
glass) ln trn, sau tin hnh m s lng di knh hin vi.

Khuyt im ca phng php ny l khng xc nh c vi cc mu c s

lng vi khun qu nh, chnh xc cng khng cao v khng phn bit c gia t
bo sng v t bo cht[8].

18
 Hnh 17: Phng m Petroff-Hauser:

(a)- Mt nhn nghing ca phng m

(b)- Gia phin knh c phng m vi cc nh

(c) phng i khong x 400-500 tin hnh m s lng vi

khun trong cc nh.

1.3.2.2 Xc nh khi lng t bo

S sinh trng ca vi sinh vt khng ch biu hin s lng t bo m cn c

s tng trng ca tng khi lng t bo. Phng php trc tip nht l xc nh trng
lng kh ca t bo. Trc ht cn ly tm thu nhn sinh khi t bo. Sau ra t
bo ri lm kh trong l sy ri cn trng lng kh. Phng php ny thch hp xc
nh s sinh trng ca nm. Phng php ny tn thi gian v khng tht mn cm. i
vi vi khun v trng lng tng c th l rt nh, thm ch phi ly tm ti vi trm ml
mi s lng xc nh trng lng sinh khi kh.

Phng php nhanh hn, mn cm hn l dng phng php o mt quang

(OD). Mc tn x nh sng t l thun vi nng t bo. Lc nng vi khun t
n 107 t bo/ml th dch nui cy s vn c, nng cng tng th c cng tng
theo v lm cn tr nh sng i qua dch nui. C th o tn x nh sng bng quang
ph k (spectrophotometer). mt mc hp th nh sng thp, gia nng t bo v
gi tr hp th nh sng c quan h tuyn tnh. Ch cn nng vi sinh vt t ti nng
c th o c l u c th dng phng php o OD trn quang ph k xc nh
s sinh trng ca vi sinh vt. Nu hm lng mt s vt cht trong mi t bo l ging

19
nhau th tng lng cht trong t bo c tng quan trc tip vi tng sinh khi vi
sinh vt[8].

Hnh18: Phng php o OD

20
 Chng 2: NGHIN CU THC NGHIM
2.1 Phng php ch to ht nano bc

Trong nghin cu ny, dung dch nano bc c cung cp bi Vin tin tin Khoa
hc v cng ngh (AIST) ca i Hc Bch Khoa H Ni. Dung dch nano bc c
ch to bng k thut kh ha hc s dng bc x UV kch thch vi cht hot ng b
mt l axit oleic.

2.1.1 Quy trnh cng ngh ch to dung dch nano bc s dng k thut kh
ha hc vi bc x UV kch thch.

2.1.1.1 Ha cht th nghim s dng:

- AgNO3 (bc nitrat): sch 99%, Trung Quc.

- NaOH (natri hydroxyt): Ha cht th nghim, Trung Quc.

- NH4OH (amoni hydroxyt): Ha cht th nghim, Trung Quc.

- C6H12O6 (ng glucozo): Ha cht th nghim.

- C17H33COOH (axit oleic): Ha cht th nghim v ha cht cng nghip

Axit oleic c cng thc cu to: CH3-(CH2)7-CH=CH-(CH2)7-COOH l cht khng

mu, khng mi, khng v, nng chy 14 0C. Khi thy phn cht bo ngi ta thu c
c axit oleic cng vi cc loi axit no v khng no khc.

2.1.1.2 Thit b s dng

- Nc ct sch.

- Cc bnh ng ha cht, ng ong, pipet.

- Phim v giy lc.

- My khuy t v rung siu m phn tn v lm ng u.

- n pin bc x UV, cng sut 35W.

2.1.1.3 Quy trnh tng hp

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 Hnh 19: S quy trnh iu ch ht nano bc s dng k thut kh hoc hc vi bc
x UV kch thch[1]

- Ly 1.7 g bc nitrat AgNO3 (dng mui kt tinh mu trng) ha tan trong 100ml
nc ct.

- Thm vo dung dch 0.62 g natri hydroxyt, NaOH, to kt ta Ag 2O c mu

en.

- Ha tan kt ta bng mt lng va amoni hydroxyt, NH 4OH, to ra dung

dch phc bc Ag(NH3)2OH trong sut.

- Cho thm mt lng cht hot ng b mt axit oleic v khuy u trn my

khuy t trong 2 gi nhit phng to ra dung dch ng nht v c nht cao.

- Sau thm 2.0g ng glucozo v khuy u trn my khuy t thc hin

phn ng kh. Trong qu trnh kh s dng n bc x UV kch thch phn ng kh
v iu khin phn b kch thc ht nano bc.

22
- Phn ng kh c thc hin nhit phng trong khong 8 gi.

2.1.2 C ch hnh thnh ht nano bc

Qu trnh hnh thnh ht nano bc c gii thch nh sau:

-
u tin, bc x UV kch thch cc ion [Ag(NH 3)2]+ hot ng. Khi cc
phn t ng glucose nhng in t cho Ag + v to ra ht nhn nguyn t
bc Ag0

h
[Ag(NH3)2]+ + RCHOH Ag0 + 2NH3 + H+ + RCOH

nAg0 (Agn)0
-
Tip theo, cc ion Ag+ trong dung dch hp ph ln trn b mt nguyn t bc
v to ra mt lp in tch dng. Cc in tch dng Ag + s ht cc ion
RCOO- mang in tch m tri du v to ra lp bo v th nht. Do nhm
carboxyl ca ion oleate hng v pha b mt ca bc nn u k nc c
hng ra pha ngoi. Khi cc ion oleate trong dung dch tip tc gn kt
vi lp bo v u v hnh thnh lp bo v th hai vi u a nc hng ra
ngoi. Chnh cu trc ny khin cc ht nano bc phn tn u trong nc v
khng b kt m[1].

Hnh 20: S hnh thnh ca 2 lp ion oleate trn b mt ht nano bc

23
 2.2 nh hng ca nano bc ln s pht trin ca vi khun

2.2.1 Phng php o OD

OD (Optical Density) l phng php dng o nng t bo c trong dung

dch huyn ph. Phng php ny c u im ln l c th thc hin trong thi gian ngn
v khng gy ph hy t bo.

Nguyn l ca phng php ny da trn hin tng tn x nh sng: Khi truyn

qua dung dch huyn ph, nh sng s b tn x. S tn x ph thuc vo nng t bo
trong dung dch huyn ph. Nng t bo cng cao th s tn x cng ln.

Thng thng, xc nh nng t bo, ta cn phi xc nh mt quang

(OD). Mt quang c xc nh da vo t s gia cng nh sng trc khi qua
mu v sau khi qua mu:

Trong :
-
A l mt quang ti bc sng .
-
I0 l cng sng trc khi qua mu.
-
I l cng sng sau khi qua mu.

Ngi ta s dng bc sng = 600 nm xc nh nng t bo theo cng

thc sau:

[S t bo/ml] = A600x(H s chuyn i)x(H s pha long)

H s chuyn i c mc nh l 5x108.

Khng ging nh phng php o

2.2.2 Cc trang thit b

24
 - My quang ph k Beckman Coulter DU 730 (Vin vi sinh vt v cng ngh
sinh hc HQG H Ni)

Hnh 21: My quang ph k Beckman Coulter DU 730

- T nui cy vi sinh vt (Vin vi sinh vt v cng ngh sinh hc HQG H

Ni)

- Bnh tam gic, pipetman

2.2.3 Cc bc tin hnh th nghim

2.2.3.1 Chun b mi trng

- Ly 12,5g bt LB (Luria-Bertani) pha vi 0.5 ml nc ct, un nng cho bt tan

ht.

- Rt dung dch LB vo 10 bnh tam gic, mi bnh 30 ml. Cc bnh c chia

thnh 2 nhm M1 v M2.

- em kh trng cc bnh nhit 1800C.

2.2.3.2 Cy vi khun

25
- a vo mi bnh 1 ml dung dch E. coli (Vin vi sinh vt v cng ngh sinh
hc HQG H Ni)

- Cho ln lt vo mi bnh 0 ml, 1.5 ml, 3.0 ml 4.5 ml, 6.0 ml v 7.5 ml dung
dch cha ht nano bc 100 ppm (ppm = 1/1 000 000 = 10-4%) c cc dung
dch vi nng ht nano bc 0 g/ml, 5 g/ml, 10 g/ml, 15 g/ml, 25 g/ml.

Hnh 22: Cc bnh nui cy

2.2.3.3 o OD
-
cc bnh trong t lc nhit 300C trong vng 30 pht.
-
Ly 100 ml dung dch mi bnh em o OD bc sng = 600 nm.
-
Lp li cc bc trn 10 ln

Chng 3: KT QU V THO LUN

3.1 Phn tch dung dch nano bc

26
 Hnh 23: Dung dch cha ht nano bc cc nng khc nhau

Phn tch ph UV-VIS ca mu dung dch c nng 100ppm cho thy s xut
hin nh hp th cc i ti bc sng 422nm. iu ny cho thy s c mt ca
cc ht nano bc trong dung dch.

27
 Hnh24: Ph UV-VIS ca dung dch nano bc nng 100 ppm

Kt qu phn tch t nh TEM cho thy cc ht nano trong dung dch c hnh cu vi
kch thc trung bnh khong 9-10 nm. Cc ht nano bc phn tn u, khng b kt m
cho thy hiu qu ca vic s dng axit oleic lm cht hot ng b mt.

28
 Hnh 25: nh TEM ca mu dung dch nano bc

3.2 Kt qu theo di s pht trin ca vi khun khi c mt ca dung dch

nano bc

Theo di s pht trin ca vi khun E. coli bng phng php o OD, ta c bng
kt qu sau:
Bng 3: Kt qu o OD

29
 KT QU O OD

0(g/ml) 5(g/ml) 10(g/ml) 15(g/ml) 25(g/ml)

M1 M2 M1 M2 M1 M2 M1 M2 M1 M2

0.5 0.154 0.173 0.129 0.121 0.130 0.118 0.145 0.133 0.151 0.148

1.0 0.181 0.209 0.123 0.124 0.129 0.133 0.139 0.138 0.148 0.149

1.5 0.252 0.301 0.125 0.120 0.127 0.119 0.142 0.136 0.151 0.152

2.0 0.421 0.444 0.131 0.124 0.126 0.131 0.141 0.145 0.150 0.162

2.5 0.610 0.673 0.117 0.129 0.128 0.121 0.143 0.128 0.151 0.154

3.0 0.749 0.877 0.126 0.122 0.125 0.119 0.144 0.140 0.151 0.155

3.5 1.262 1.170 0.409 0.127 0.128 0.122 0.146 0.140 0.155 0.154

4.0 1.372 1.389 0.144 0.135 0.128 0.123 0.145 0.141 0.156 0.155

4.5 1.490 1.521 0.142 0.135 0.137 0.121 0.153 0.149 0.156 0.157

5.0 1.612 1.645 0.143 0.139 0.133 0.121 0.148 0.151 0.155 0.157

5.5 1.675 1.713 0.107 0.107 0.081 0.077 0.104 0.010 0.111 0.108

hnh 26, ta thy ng cong sinh trng ca mu c nng ht nano l

0(g/ml) (khng c mt ca cc ht nano bc) c dng tng t nh giai on logarit
trong l thuyt. giai on ny vi khun pht trin rt nhanh do gp mi trng nui
thun li, cc t bo phn chia u n. Sau 11 gi, mt vi khun trong mu xp x l:

1.7x5x108 = 8.5x108 (t bo/ml)

30
OD

t (gi)

Hnh 26: ng cong sinh trng ca E. coli khi khng c mt ca nano bc

a)
OD

t (gi)

Hnh 27: ng cong sinh trng ca E. coli khi c s xut hin ca nao bc

a) Nng 5(g/ml)

31
 b)
OD

t (gi)

b) Nng 10(g/ml)

c)
OD

t (gi)

c) Nng 15(g/ml)

32
 d)
OD

t (gi)

d) Nng 25(g/ml)

OD

t (gi)

Hnh 28: ng cong sinh trng ca E. coli trong tt c cc mu

T hnh 27, tt c cc mu c nano bc, pha lag ko di hn bnh thng, pha ch

s hu nh khng xut hin chng t vi khun khng th pht trin c mc d c
cung cp y cht dinh dng v c nhit thch hp. Sau 10 gi, qun th vi
khun trong tt c cc mu ny u t ti giai don t vong ngay c khi qun th vi

33
khun trong mu i chng cha t ti pha cn bng. iu ny chng t hiu qu khng
khun ca nano bc.

Nguyn nhn ca cc hin tng trn l do cc ht nano bc lin kt vi

peptidoglican thnh t bo ca vi khun gy c ch kh nng vn chuyn oxy vo bn
trong t bo dn n lm t lit vi khun. V sau khi tc ng ln mng t bo vi khun,
cc ht nano bc s thm nhp vo bn trong t bo, tng tc vi cc enzym tham gia
vo qu trnh h hp dn n c ch qu trnh h hp ca vi khun.

iu ng ch l nng t bo trong tt c cc mu c cha ht nano bc u

xp x nhau tt c cc mc thi gian (mc d trong mu c nng nano bc cao hn,
nng vi khun s thp hn). Sau 11 gi, nng vi khun trong tt c cc mu c
cha ht nano bc u xp x l:

0.1x5x108 = 0.5x108 (t bo/ml)

Chng t nng t bo trong cc mu c nano bc nh hn 17 ln nng t

bo trong mu khng c nano bc.

T kt qu trn ta thy rng vi mi nng nano bc t 5 g/ml n 25 g/ml

u th hin tnh khng khun mnh vi E. coli.

34
 Hnh 29: Cc bnh nui sau 8 gi: Ta thy bnh khng c cha ht nano (ngoi cng bn
phi) b vn c chng t vi khun pht trin mnh. Cc bnh cn li c cha cc ht nano bc
bnh gn nh khng i mu.

Trong 10 gi u, mt vi khun hu nh khng i. Trong giai on ny vi

khun b c ch do tc dng ca cc ion bc c gii phng t ht nano bc. Tuy nhin
cu trc t bo ca vi khun vn cha b ph hy.

Sau 10 gi, nng vi khun gim mnh cho thy cc t bo bt u b ph

hy. Tuy nhin trong bnh nui cy cng c th c cc c th c kh nng khng li nano
bc tn ti.

3.3 So snh vi phng php o bn knh vng v khun

35
 Trong phng php o bn knh vng v khun, ta tm dung dch nano bc vo mt
a giy nng nht nh, ri t a c tm ln mt mi trng thch c cy vi
khun th nghim trc . Dung dch nano bc s nhanh chng khuch tn trn mi
trng thch c ch s sinh trng v pht trin ca vi khun, to vng v khun. Tnh
khng khun mnh hay yu tu thuc vo ng knh vng v khun ln hay nh [11]. u
im ca phng php ny l n gin, d thc hin. Tuy nhin, phng php ny ch
gip ta bit c mc khng khun cui cng ca dung dch nano bc (th hin qua
bn knh vng v khun)[11].

Phng php o OD tuy c phc tp hn nhng li gip ta bit c ng thi

pht trin ca qun th vi khun di tc dng ca dung dch nano bc. C th hn, ta c
th bit c nh hng ca ht nano bc ln tng pha sinh trng ca vi khun, t
xc nh xem giai on no qun th vi khun d b nh hng bi ht nano bc
nht[2,9].

Chng 4: KT LUN

Trong khun kh kha lun ny, chng ti :

36
1. Th nghim ch to ht nano bc dng dung dch theo cc nng t 5 g/ml
n 25 g/ml.
2. Nghin cu tc ng ca nano bc ln s pht trin ca vi khun bng k thut
OD (trong 11 gi).

3. Chng minh c rng cc mu nano bc dng dung dch u th hin kh

nng khng khun mnh vi vi khun E. coli.

nh hng, xut cho cc nghin cu tip theo:

1. Th nghim tnh khng khun ca nano bc vi cc chng vi khun gy bnh

khc.
2. Xc nh nng ti thiu ca ht nano bc dung dch c tc dng khng
khun

3. Nghin cu c ch tc ng ca ht nano bc ln vi khun.

TI LIU THAM KHO

Ting vit

37
[3] Cng ngh nano. Vi.wikipedia.org

http://vi.wikipedia.org/wiki/C%C3%B4ng_ngh%E1%BB%87_nano

[4] Cng ngh nano v nhng ng dng trong thc tin, Trang 1

www.prt.vn/upload/Nghiencuu/Congnghenanovaungdung.doc

[5] Gii thiu v Knh hin vi. Svtunhien.net, Trang 1

http://svtunhien.net/mybb/printthread.php?tid=693

[6] Nguyn Hong Hi, Trung tm Khoa hc Vt liu, Trng i hc Khoa hc

T nhin, i hc Quc gia H Ni. Cc ht nano kim loi. Tp ch
http://vatlyvietnam.org, 2007. Trang 9.

[8] Sinh trng v pht trin ca vi sinh vt. Maxreading.com

http://maxreading.com/sach-hay/vi-sinh-vat/sinh-truong-va-phat-trien-cua-vi-sinh
vat-37171.html

[13] Nguyn Minh Tr, Mt phng php nh gi kh nng khng khun ca

Chitozan, Trang 1

[14] Nguyn Ngc T. Nghin cu gel nc thng minh nhy pH lai nano bc.
Kha lun tt nghip i hc chnh quy 2009. Trang 8-9.

Ting Anh

[1] Anh-Tuan Le*, P.T. Huy, Phuong Dinh Tam, Tran Quang Huy, Phung Dac
Cam, A.A. Kudrinskiy, Yu A. Krutyakov, Green synthesis of finely-dispersed
38
highly bactericidal silver nanoparticles via modified Tollens technique, Current
Applied Physics, Vol. 10, pp. 910-916.

[2] Ales Pana cek, Milan Kola r, Renata Vecerova, Robert Prucek, Jana
Soukupova, Vladimr Krystof , Petr Hamal, Radek Zboril, Libor Kvtek,
Antifungal activity of silver nanoparticles against Candida spp, Biomaterials 30
(2009), pp. 63336340.

[7] List of nanotechnology applications. en.wikipedia.org

http://en.wikipedia.org/wiki/List_of_nanotechnology_applications

[9] Siddhartha Shrivastava, Tanmay Bera, Arnab Roy, Gajendra Singh, P

Ramachandrarao and Debabrata Dash, Characterization of enhanced antibacterial
effects of novel silver nanoparticles, Nanotechnology 18 (2007) 225103, pp 9.

[10] Silver. En.wikipedia.org

http://en.wikipedia.org/wiki/Silver

[11] Silver Nanoparticles: A Case Study in Cutting Edge Research. Cnx.org

http://cnx.org/content/m19597/latest/

[12] Preparation of Silver Nanoparticles and Their Characterization. Azonano.com

http://www.azonano.com/article.aspx?ArticleID=2318

[15] http://www.ncbi.nlm.nih.gov/pubmed/3807907

39