HOi NGH! KHOA HOC C O N G NGHE SINH HQC T O A N QUOC 2013
LAM GIAU SALMONELLA TYPHIMURIUM BANG HAT NANO TLfTlNH FesO^
G A N K H A N G THe IgY
N g u y l r \ T h j T r u n g \ NguySn HoSag Hal^, T r u - o ^ g Nam H a l ' " . ' VI0n Cbng ngh$ sinh hQc, Vi^n Hin lim Khoa hoc vi Cong ngh$ V/|f Nam 'Tmbng D^i hoc Khoa hgc ti/ nhien, Dai hgc Quoc gia Hi Ngi
T6IVITAT
Salmonella typonurium l i loai vi khuin giy nhilm d§c ihuc phim ph5 bien nhit ct nguiii. Cic phuong phip phit h i ^ vi khuin niy chi yhx dva tren phuong phap njiSi cay t^ bio t r u y ^ diong hoac xet n ^ e m DNA vi miln dich hgc. Bii bio niy trinh b i y phucmg phip lim giiu 5 Q/phtmurium bJmg hat nano t i tinh. Ifet nano til tlnh Fej04 dugc che tjo hing plucrag phSp d^ng Icet tiia voi kich thii6c khoing 10 nm. Be mat h^t nano dugc chiic nang h6a amino bing S-^aminopropyl-tdethoxy-silance dk gdn voi khing thS IgY khang S. typhimurium dixgc san xuSt lu trang gi. Khing the nay ^up cac h^t nano tfr tinh gSn djic hifu v6i cic kliang nguy€n d$c hifu khang thS tr€n be m^t ciia le bio vi khuin. K€t qui cho thiy, su' diing h^l nano tu tioh co g ^ khing the IgY, chung toi c6 th^ Ihu i?i tdi 87% vi khuin o6 mat trong dung djch.
7i>tAofl:Fe304,hatnanoti(tinh,khingtheIgY,5fl/mone//o/(pAiniKrnon, Idmgidu " "
MdoAU
Hi$n nay, nhu c l u v ^ b5 sinh pham phdt hien S. typhimurium gay ngp dgc thi/c p h i m trong ckc thuc p h i m rat lo'n.
Khfing chl co cac nhd quan ly V# sinh an toan thi,FC pham mk ca nh&ng n g u o i dan li£u dOng thi^c p h i m cOng rat c^n cflng cy nSy. 'Cac b? sinh p h i m du'o'c t^o ra thu-frng bj han che ve dO nhgy d l Okp irng bugc y%u c l u cCia T 6 chCrc Y t^
t h l gi&i Ik khSng cfl b i t kl mOt t l b^o Salmonella trong cSc m l u t h y c p h i m . Nhu* v§y, b i n g b i t kl phu'O'ng phap p h i t h i | n nao chOng ta d i u phai t i l n hanh l^m g i i u vi sinh v3t tnjo'c khi phat hi^n (Cudjoe, Krona 1997; Keller ef ai, 1993).
N i m 1989, Prusak-Sochaczewski v a Luong da sir dung enzyme danh dau bang avidin-biotin vk da phkt h i f n du'ip'c Salmonella trong ckc m l u t h y c pham vfl'i mire 25 cfu/g (Pmsak-Sochaczewski, Luong 1939),
Cdng ngh$ nano cung du'o'c iing- dung trong sinh hoc bk phat j i i ^ n cac ttpong t^c o' mi>c do phSn ti> (Jain 2003). K?
t h u | t n i y cfl kha nSng phan tlch n h i l u lac nhSn cfl mat trong mau t?i mot thfri d i l m . Ckc khdng t h l danh d l u bang ckc h?t nano dfl d u ^ c dOng 6k phkt t r i l n ckc thir n g h i f m phkt h i f n lac nhSn gSy b f n h , ckc phSn ti> h o l e cdc c l u tnic d$c h i f u (Emerlch, Thanos 2003; Jianrong ef al., 2004; Zhao et al., 2004). Trong cflng trinh ndy, chting tfli Irinh biy cac k i t qud nghign c i i u v l d i l u che hgt nano 10 tlnh F e j O * (ggi tat Id hat nano tir tinh) c6 kich thud'c 10 - 30 nm d l i i n g d u n g ' trong nghiSn ciiu sinh hpc. Hgt nano t i r tfnh sau khi ducrc che tgo se du'pc chi>c nang hfla be m | t b o i c h i t ARTS (3- aminopropyl-triethoxy-siiancB). Sau khi dup'c c h i i c nang hfla be m g l thl tren b l mgt hgt nano ICr tlnh cfl nhflm chiic - NH2. Nhflm -NH2 ndy s€ l u a n g Idc-dug-c vfl'i cSc phan tCr sinh hpc n h u khdng nguyen, khdng t h l hoac DNA. Cac hgt nano ICr Unh da d u g c chiic n i n g hoa bia mgt dup'c s u dung de gan v o i khdng t h l IgY khdng vi khuan S. typhimurium n h i m tap trung vi k h u i n tnsng cac m l u nghign c t i u . B i n g phucmg p h i p nay, vi k h u i n 5. typhimurium 6ugc lam giau m i khflng can sCr dyng phuong phdp nufli c l y vi sinh trong mfli trufl'ng g i i u dinh duo'ng va mfli trufrng chgn Ipc.
NGUYEN L i e u V A P H l / O N G P H A P NGHIEN C1>U Nguyen ll$u
K h i n g t h l IgY nhin b i l t dac h l f u k h i n g nguySn roi cua vi fthuin S. typhimuriurn flo Phflng Ky t h u | t di t K ^ I r i , V i f n Cflng"
nghf sinh hpc tgo ra dup'c diing d l g i n vfl'i cac hat nano s i t 10.
Vi k h u i n S. typhimurium do W f n thii y cung d p dup'c dung de nghien cO'u s y l i n g k i t vfli c i c hgt nano 10" Hnh Fe304 g i n khdng t h l d^c h i f u IgY.
PhiFong p h a p
Chi tao h^t nano tO- " 7 - - , - -
Hgi nano tir tinh cfl kich l h u 6 c 10 nm - 1 5 nm dug'c c h l tao b i n g p h u a n g p h i p dong k i t tua ion Fe^" va Fe^* bang O H ' - Igi nhipt d f phong tnang mfli trufl'ng khi Nz d l trdnh fl xi hoa (Thach ef al, 2008V Lay 4,'if'^ •FeCl3.6H20 v i 1,52 g F B C I 2 . 4 H Z O (tlie Id tl p h l n mol Fe^/Fe^* = 2) hfla trong 80 ml n u a c d t hai l l n (nong dO cua Fe^* Id 0.1 M) bang mdy k h u i y tu". N h d dung djch nay v i o m f t^dung dich khdc cfl c h i i a 6 ml NH4OH 35% vfl'i toe dp nho 1 m f t giot/giay tgi n h i f t dd phflng dufl'i d i l u k i f n k h u i y d^u bang m a y k h u l y tir. K i t liia Fe304 m i u den d u g c hinh thanh ngay khi hai dung d|ch flip xOc vbi nhau. D f ibn kich thyfl'C hgt nano cfl t h l d u g c ffleu k h i l n b i n g t i c dp k h u i y , n h i f t flf phan l i n g . pH ciia
• dung dich v i nong flf c h i t Uiam gia phdn i i n g . Tdch Ipc hgt nano l i i tfnh bdng t i i trudng hoac m i y li t i m , Ipc n i a sdn p h i m 5 l l n b i n g nufl'C d l logi bfl cdc hfla c h i t efln d u ta thu d u g c cdc hgt nano tCr t l n h .
C/)i>c nang hba be m^t h$t nano OF
C i n 400 m g hgt nano tii'tfnh cho vao 100 ml nubc c l t hai l l n roi dCing may siSu dm phdn ldn hgt vdo dung dich de tho d u g c m f l t h l h u y l n phi3 o n dinh. Nhfl 1 ml dung djch APTS vao frong dung djch huyen phii nfli frSn va k h u i y ti> trong thW gian 8 gib d l qud trinh chiic n i n g hda be m | t x i y ra hoan t o i n . San p h i m fliu d u g c sau khi loc n i a 5 l l n b i n g nubc c l t v i Ipc ICr d l thu hgt nano ICP tinh Fe304 cfl b l mgt l i c i c nhdm amino (amino-FesOa).
HQI NGHI KHOA H l ^ C Q N G NGH? S j ^ HOC T O A N Q U Q C ^ 4 3
Phat biin khing the IgY gan trbn be wphat nano tu tinh
Khang t h l IgY v i hgt hano tCr tinh FesQ^tflugc trdn vfl-i nhau VnmU'$2r.\ v l I h l lich dung iiIi«h.|3Pr.ei4ita d a so hgt nano tCr tinh FeaOi d u g c gan kel v d i ^^.g i h l IgY trSn be mgt. H f i ^ i t ^ p h d n t i n g g i n d u g 4 : f f i . - o ' - h f # i ^ t R 6 n g , frong thdi gian 60 phiil, lac nhe 50 rpm/phuL %/ic r i i a v i t i c h khang th& a § figt,- iJ^t v d i hgt nano tCr ^ ^ ^ - f ^ ^ l M g t i f t n j & n g ngoai. Thu phan cgn tCr v i k i l m tra i.'-? g i n cOa khang t h l ySi h'gl nano tO Hnh F e j ^ ' | ^ ^ ^ dr tren gel polyacrylamlde 12,6%. P h i i c h g p hgt n3.ri0 tfr tinh Fe304 gan 6upsm Wiing t h l gpi tat l i ^m^W' W ^ n g t h l (IgY- • FesOa).
Xac dinh kha ning t$p tmng vi khuan S, fyphimurium cba phO-fff!^ IgY-1=0304
hap ttiy quang hoc tgi b u d c sflng 600 nm, v i c l y t r i i c i c mau trfin fiJa thgch d l d e m s6 lugmg w ^ . fi^ m J u ndy s§
flugc SLF dvng de nghien c u u s y t | p trung vi k h u i n S. typhimurium bSftg each sCr dung hgt nafig ^ ^ t^kfig t h l IgY.
Xdc djnh m i i c do v i gidi hgn tgp frung vi kht^r* cfla p h i i c h g p IgY- Fe304.
K^T QUA
C h l tgo hgt nano tCr t f n h F e j O i vd chCpc nang h d a b e mat bang eac n h f l m a n i j n o
K i t qud n h i l u xg tia X cho i h l y v f t l i f u l i Fe304 v d i d u tnic spinel d i o {Hinh 1). Anh h i l n v l ^^Bifthhtittaz cho t h i y cdc hgt tCr cd kich thud'c kha ddng n h i t , kich t h u d c cua hat ICP ICr 10 ntn den 20 nm v i cfl tCr Xmi^^ emu/fl (Thach et
" al, 2008). Sau kht d u g c chiic nang hfla amino t r f n b l mat (amino- Fe304), hgt nano san s i n g g|jf J ^ f i f ^ g I h l IgY.
Hinh 1. K^t quS n h i l u x? tta X (A), dnh hiSn vi difn lii' t r u y i n qua (B), duirn^ cong tii- hda (C) cOa h^t mm tiy Iln1l HtOt G i n k h i n g the IgY dac hieu 5 . typhimurium vbi amino-FejOt
K h i n g I h l IgY da flnh sgch d u g c b i o qudn frong dung dich d f m phosphate pH 7,4, ndng flf khaag ihft | | 0,76 mg/ml.
.Khdng t h l nay d u g c l i m loang 10 l l n trotlg dung dich d f m phosphate pH 6.3 v d i s y cfl m$t g ^ l sSfnQ . Pa304. Tgi pH=6,3 s u lien ket giO'a khang t h l IgY v i h?t nano ICr tinh Fe304 l i lAl fihll. San pham p h i n Cfng ggfl ^\im ( h i IgY vdi hgt nano tCr tinh Fe304 d u a c p h i n tich bang d i f n di b i l n Unh protein Ifgn gel polyacrylamid (Hinh t ) .
Hlnh 2 (A) Phan I ch khang the IgY d^c h Su S typh mu um en kSt vM hat nano t u t nh Fs O b i n g « r ^ 4 if^a^ ^ ,^h p otflin trgn gel po yarcrylam d 12 6 nhu^m b;ic (B) MSu dung d ch vJ khuan Saimohblla khflng cd IgY F e * ^ , ^ ) ^ ^^ng t^ch vl
khuan Salmonella c6 bo sung tgY Fe)04 ,, Budng chgy 1: khdng thl'IgY; 2: lgY-Fe304,3: dung djch sau tach Kr; 4: dung t^^rO-a phan cgn tir; 5: thang &t%i{^f j , ^ i n • 'M
Hinh 2A l i k i t q u i d i f n dl cda khang I h l IgY (dufl'ng 1), IgY-FeaO^ ((Rfdng 2), p h l n dung djch ^ h * t ! 6 ' f e a ^ ' 5 ( h i 6i t 0 cdc hat ICP cfl g i n IgY l l n t h d n h i t (dudng 3). p h l n dung djch Ihu 6\s^ sau *}il lgc riia igY-Fe4ei|,((S£Mff|n 4) v i ttlfSi protein c h u i n (dudng 5). Cdc bdng pnstein b d u d n g 1 friing vdi c i c b d i i ^ ^ r o t a n d d u d n g ^ © j i j .^ '.^^ j j ^ y j t h l n f t j f f l IgY da g i n flianh cflng vao hat nano Fe304. Theo ly fliuylt, Wii cho d u r a a M i c d hgt nano'Kr ttrfe | a f e ^ ^ a 3 ^ u g c 1 ^ ndng hfla b l m i l b i n g nhflm chCrc amino (-NHz) flil nhdm n i y sg liSr* tsk tJOng hfla fri vfli n h d m e & A j b x y l ' ( - C O O H ) ^
HQI NGH! KHQA HQC G O N G N G H $ SINH HOC T O A N Q U Q C 2013
phdn tir khdnq t h l hinh ihAnh nen liSn k i t peptide. B d li-ccr d i l khing.flilJieD-kltJmlfaatnanoJLUiRh.-BaBq-niaHii!mM^>-- frudng ngoai tgo bdi m f t thanh nam cham, ta cd flie t i c h rieng lgY-Fe304. Do hgt nano d u a vao la d y n§n h i u h i t cdc khdng I h l IgY da l i f n k l l v d i c i c hat nano nen d u d n g chgy sd 3 khdng xuat h i f n bang protein nao. V i f c t i y n i a IgY- Fe304 n h i l u l l n lam mat di m f t Iugng nhd IgY n i n d u o n g chgy s d 4 x u l l h i f n mot bang m d . Nhu-ng khang t h l n i o chua tifn k i t d u g c vdi hgt nano ICr tlnh h o f c l i f n k i t khdng ben se d u g c Iogi bfl thflng qua pha Ipc n i a phan cgn tir.
Hlnh 2B_vi 2C minh hga cho sd l u g n g te b i o Salmonella giam dl bring pha djch noi sau l i c h t i r - d l i vdi m l u ddi chdrig ~ Mifl'ng cd'hgt tCr (Hlnh 2B) vd mdu dung djch vi k h u i n cfl bo sung lgY-Fe304. O m l u doi chiing cfl s6 l u p n g k h u i n lgc mpc t r f n dTa thgch r i t n h i l u (147 dgng khuan lgc), frong khi m l u c d lgY-Fe304 flil so l u g n g k h u i n lac da g l i m di dang k l (chl efln 19 k h u i n Igc). Dieu n i y chCrng tfl rang r i t n h i l u d c t l b i o Salmonella da t^ tdm b i t n f n s a u khi t i d i fri ngang cdc t l b i o d i di theo lgY-Fe304 tgo thanh c | n l i n g , do dd bong pha dich n l i sfl l u g n g t l bao vl k h u i n da tn giam.
Xic dinh kha nSng lim gliu vl khuan S. typhimurium bang hat iigV-FeaO* ' '
Sau khi da c f l khdng t h l liSn k i t v d i hgt nano tCr tfnh Fe304, dung djch chira lgY-Fe304 d u g c frfn v f l i dung djch chda vi k h u i n (pH=6,3),nhif t d f khoang 4° C d l tgm t h a i k h i n g ^ 1 d u g c s y tang t n j d n g ciia vi k h u i n trong m d i thfl'i glan ngan. Sau 60 phdt hgt lgY-Fe304 se g i n v d i vi k h u i n . Hgt nano va vl k h u i n d u g c t i c h khoi dung djch bang tCr frudng ngodi. P h l n dung djch n l i d u g c fliu lai. C l y t r i i 100 pl dung dich noi l i n i^a thgch LB, 0 dia nudi d y d 37° C qua dem. s d l u g n g k h u i n Igc p h i t t r i l n fren c i c dfa thgc^ d u g c d i m v i ghi Igi frong B i n g 1 .
B i n g 1. Si^ giam s6 Iirgng vi k h u i n S. tyf^imurfum trong dung d c h sau t^ch ti>
Miu ngNSn cCru - D&i chiing IgY-FeiO. % bi l6m b i t 10'cfu/ml (147±3) "10 (19± I ) " 10 67 10'cfu/ml (300±7)>'10 ( 9 0 ± 2 ) > ' i o 70
Ghi chO: Miu d6l chQng li hd tir khdng gin khAng thS
Bdng 1 cho thay so l u g n g vi l ^ u l n trong dich n l i cfla thi n g h i f m hgt tCr gan k h i n g t h l da g i i m di d i n g ke chCrng td cdc vi k h u i n da d u g c t i c h cung vdi c i c hgt nano tCr t i n h . T h i n g h i f m d u g c fliyc h i f n t u a n g t y n h u p h l n t r i n , p h l n c | n tCr thu d u g c d u g c hda loang v i o 1 ml dung djch P B S . . d y t r i i 50 ^1 (1/20) dung djch sau hda loang len dta thgch LB, it 37°
C qua d i m . S6 lugng k h u i n Igc mpc trfln c i c dTa LB d u g c d i m va tong hgp d Bang 2.
Bang 2. So Itrgng vi khuin S. typhimurium trong c|n tir
M l u (cm/ml)10 100 1000
D6i chiing -
10 ± 1 95 ± 1 3 . 1020 ±30
Amino-FejOi S6 lugng
1 ± 1 7 ± 4 SO ± 2 0
%
6 7 9
IgY-FejOi So luong
9 ± 1 81 ± 6 ' 740 ±40
%
87 86 73
Ghi chd: miu d6i chirng chl cd dung djch vi khuin Idling cdhattirdi tmng cdng diiu Id^n thl nghidm, hd tir da dugc chUc nSng hoa amino, hat tir - khimg Ihi IA h§t tir da gin khing thS IgY
C i c so l i f u frinh b i y .trong Bang 2 cho t h i y r i n g , kha nang gdn k i t cfla hgt nano ICP Hnh v d i vi k h u i n phy thupc v i o ndng dp vi k h u i n cd frong m l u . Vdi amino-Fe304, do khflng gdn khang the dgc h i f u IgY nSn chCing v l n cd t h l gan k i t
• vdi vi k h u i n Safmpne//a nhS vao cdc nhdm"chCpc - N H 2 freri b^ mat cua hgt nano Hi tinh. Cdc hgt nano n i y c d kha nang lign k l l v d i c i c protein cfl the l i khang nguySn hogc khang t h l , do vgy nfl cd t h l hen k i t d u g c vdi t l b i o vi k h u i n . T u y nhign, s y l i f n k f t n i y khflng ben v i khflng d#c h i f u nen H I f g i n k i t flilp (nhd fian 10%). O d i l u k i f n fliich hgp, IgY- Fe304 cd t h l g i n k l l dgc h i f u v d i vl Jthuin Salmonella len tfli 8 7 % . H i f u s u i t gan k i t lot n h i t dgt d u g c khi v i k h u i n c d n i n g d f ICP 10 d i n 100 cfu/ml. O m l u cfl ndng d f vi k h u i n cao h a n , tuy hieu s u i t gan k i t i h i p nliung s6 l u g n g vi khuan d u g c tach Kr n h i l u . D i l u n i y c d ttil do s6 hgt hgt nano tCr linh Fe304 g i n k h i n g the ch? dCi dk gdn-kit v d i mpt s6 l u g n g vi k h u i n n h i t ^ n h . N g u g c Igi d l i vdi c i c m l u c d ndng d p vi k h u i n q u i ttiip ttii k h i nang b l l g f p vi k h u i n cfla hgt hgt nano tir tfnh Fe304 khfl x i y ra. Trong nhO'ng h r f d n g h g p nay, n g u o i ta thufl'ng nufli vi k h u i n trong m f t ttidi gian ngan d l chiing S n g sinh v l s l l u g n g . B i n g c i d i n i y chCing ta cd the l i m ^ n g d f nhgy v i d f chinh xdccOa cac thir n g h i f m .
K t T L U A N
Chflng tdi da c h l tgo d u g c hgi nano tip Hnh Fe304 cfl kich ttiudc ICF 10 d i n 20 nm b i n g phuang phap d i n g k i t tQa. Hat nano s i l ft> sau khi d u g c amin hda b l mgt bang APTS d a lien k i t tflt vfli phan li> khdng the IgY k h i n g d i e h i f u S, typhimurium. H i f u s u i t t f l m b l l vi k h u i n S. typhimurium cua lgY-Fe304 len den 87%. Kel qua nay m d ra t r i l n vpng clio v l f c ling dyng hgt nano tCr tinh Fe304 d l t f p frung m f t so tac nhan gay b f n h d n chan dodn nhung cd n i n g dp t h i p Irong m l u m d khflng d n p h i i t r i i qua glai dogn ldm g i i u vl k h u i n bang nufli d y , do v f y cd t h l nit ngan Ihdi gian c h i n d o i n v i fliu d u g c k l l qud chlnh x i c .
Utidvatm
Cdng trinh nay dirpc Aifc hlpi vbisvhdtrgvetai chinh ricdudn VS/BT3 Unigc chuang trinh hgp tdc Vift Nam-TTnffDiin
T A I u e u T H A M K H A OPmsak-Sochaczewski E., Luong J H.T. (1989) Utilization of two improved enzyme Immunoassays based on avidin-bioBn Interaclion for
HOI NGHI KHQA HQC C Q N G NGH? SINH HQC T O A N Q U Q C 2013 the detection of Salmoneila, Nalhnd Research Council Proceedings, Canada 89:321-327.
H food samples using Dynabeada' anti-Sa/nwne//a and a conventional
Jain K. (2003) NanodignosQcs: application of nanotechnology in molecular diagnostic. Expert. Rev. Mol. Diagn., 3{2): 153-161.
Emerich D., Thanos C. (2003) Nanolechnology and medicine. Expert. Opin. Biol. Ther. 3(4}: 6SS-G63.
Jianrong C , Yuqing M , Nongyue H., Xiaohua W. and Sijiao L. (2004) Nanolechnology and biosensors. Biotechnd. Adv. 22(7): 505-518.
ENRICHMENT OF SALMONELLA TYPHIMURIUM BY USING MAGNETIC NANOPARTICLES FejOi CONJUGATED WITH IgY
Nguyen Thl T r u n g \ Nguyen Hoang H a i ^ T r U o n g Nam H a l ' ' 'institute of Biotechnology, Vietnam Academy of Science and Technology
^University of Natural Sdences, Vietnam National University, Hanoi
