Nghiên cứu chế tạo và ứng dụng kit phân tích nhanh urea và urea biosensor

14- SO KHOA HOC CONG NGHC TP HO CHI MINH 000 CHUONG TRiNH PHAT TRIEN NONG NGHIEP — CONG NGHIEP THVC PHAM BA.0 CAO TONG KET DC TAI: "NGHIEN CeU CHE TAO VA eNG DUNG KIT Tile NHANH UREA VA UREA-BIOSENSOR" CFI() NI-HEM DE TAI: TS TRAN BICH LAM TruVng Dai hoc Bach khoa HCM Tp HCM 3/2009 /eV ei(/n •.**Sh' **** hue cSc @dna @iii etfe Qttim CIA Z0/11 blting S Bh0 10 fln;ti **** &Mg NrOinatttilk **** 77 killn an Man (ML /ha if 1114 ilin -0.11d (MU 11161 Da 1,1I,4't firth sit'p cla t90 diAt.,k1q_11 thui4111 tai 110611 thiArd, nhiem ( d/i//‘ rli/// r'%/ //I /2%/y (.44//y 1/ cho nhom -tht;(c hien de vbt r(ir eC,,y /ei t‘a'"/ • I/ glut (Themggitanit Sum(' Ofi !hi aim 76(mh aS o HI( ghanh ginn rbde 2iriiu Qmjnh grtin ghl as gltfityin mai (Nguyen grtin (Nguyen rai CNDT: Kl3eh -ettin MIJC LUC PHAN I TONG QUAN 1.1 Nguyen nhan c6 mat urea va cac phucsng phap xac dinh urea 1.1.1 Ve van de urea thkrc pham 1.1.2 Cac phuking phap xac dinh urea 1.2 Enzym urease 1.3 Kit thin nhanh va urea biosensor Trang 2 1.3.1 Wit so loai kit phCin tich urea 1.3.2 Biosensor PHAN 11 Nol DUNG VA PEILIONG PHAP NGHIEN C01.1 11 2.1 Nguyen vat lieu 2.2 NOi dung 1: Nghien eau the tao kit this urea 2.2.1 NOi dung 2.2.2 Plurang phap nghien ctru 2.3 NOi dung 2: This nghiem dng dung phan tich urea 2.4 NOi dung 3: Nghien cd'u che'tao bioreceptor 2.4.1 Thi nghiem 1: Nghien ecru co dinh urease tree be mat dien ckrc thay tinh 2.4.2 Thi nghiem 2: Nghien dru co dinh urease tren ming mong 11 12 12 13 14 14 15 2.4.3 Mc dinh cac thong s6 co dinh enzyme 16 2.4.4 Khao sat tinh chit Ong hoc cua enzym urease c6 dinh 16 2.5 NOi dung 4: Ch6 tao va ang dung urea biosensor 18 2.5.1 Kiem dinh kha nang phzit hien urea cua bioreceptor 18 2.5.2 Phu'Ong phap the tao biosensor 18 2.5.3 Gng dung urea biosensor phan tich urea thkfc phiim 18 PHAN III KET QUA VA THAO LUAN 3.1 Nghien eau sit dung urease the tat) kit this nhanh urea 3.1.1 Xac dinh tinh chat cua the pham urease 19 19 19 3.1.2 Nghien ct?u the tao kit Oat hien urea 19 3.2 ling dung kit thir phzit hien urea -1 3.2.1 Sit dung kit thit urea clang ling thily tinh 24 3.2 I Phat hien urea sifa 25 3.2.1.2 So sanh ket quzi phan tich sir clung kit thity tinh vc i ke:t qua 12 phan tich bang may quang ph6 25 3.2.1.3 26 Phat hien urea ca 3.2.1.4 Mau nadc mdm 28 3.2.2 Set dung kit this urea clang gidy chi thi 29 3.2.2.1 Khao sat thbi gian hien mau cU'a kit 29 3.2.2.2 Khzio sat thdi gian sit dung Oa kit 29 3.2.2.3 Khao sat kha nang phzit hien cila kit mau thkfc pheim 31 3.3 Nghien eau co dinh urease the tao bioreceptor 33 3.3.1 Nghien citu co/ dinh enzym urease len be mat Only tinh 33 3.3.2 Nghien ecru phtrang phap c6 dinh urease von ming PET 34 3.3.3 Khao sat die you to anh !luting den hieu qua c6 dinh enzyme urease len !ming PET 35 3.3.4 Hieu suit c6 dinh urease len ma ng PET 38 3.3.5 Khao sat cac you to anh huong den hieu qua c6 dinh enzyme urease len mang PET 38 3.3.5.1.Khao sat tinh chat dOng hoc dm enzyme urease co dinh 38 3.3.5.2 Kith() sat Anil twang dm nhiet dO den hoat tinh dm enzyme urease c6 dinh 40 3.3.5.3 KB() sat dO ben nhiet dm enzyme urease co dinh 41 3.3.5.4 Khao sat Anil Inning dia pH den hoat tinh enzyme urease co dinh 42 3.3.5.5 Khao sat anti Wang dm dung dich dem den hoat tinh enzyme urease c6 dinh 42 3.3.6.Khzio sat kha nang bao quart enzyme urease c6 dinh 43 3.3.7.Khao sat the dO bao quail enzyme urease c6 dinh 44 3.3.8.Khao sat kha nang tai sir dung dm enzyme urease c6 dinh 3.4 Nghien cdu the tao biosensor 3.4.1 Mc dinh giai flan phat hi0 urea ciia bioreceptor 3.4.2 Ole' Lao hiosensor 3.5 Sir dung hiosensor phan tich urea thyc phzi'm 3.5.1 Sit dung biosensor phan tich urea sirs tuai 3.5.2 Sit dung hiosensor phan tich urea ca 3.5.3 Sr I dung biosensor philn tich urea int& intim 3.5.4 Bmic du ughien ct?u sir dung urea biosensor phat hien kim loci Ong 46 47 47 48 48 48 50 52 52 3.5.4.1 So sanh khzi nang ire the hoat tinh urease c6 dinh cCia cac ion kim loci 52 3.5.4.2 Xzic dinh gieri han [He] ire the hoan Loan urease 53 PHAN IV KET LUAN VA KILN NGHI PHU LVC TAI LIEU THAM KHAO 55 56 63 DANH MUC BANG Bang 3.1: Tinh clidt cila the phSm urease Bang 3.2: Khoing pH chuydn mau va mau sac cua cac chdt chi thi Bang 3.3: Phan ring mau all kit alit urea clang 6ng thy tinh Bang 3.4: Mau thqc phSm phan tich urea Bang 3.5: Kel qua dinh lacing urea sita Wang kit thily tinh k6t hop pH Bang 3.6: K6t qua dinh lu'ong urea sita Wangphdong pluip quang ph6 so mini Bang 3.7: So sanh sur tu'Ong thich giCra hai phuOng, phap Bang 3.8: B6 sung nOi chain urea mau ca chi yang Bang 3.9: Ham lu'Ong urea cac mau ca dinh lu'Ong bang pIntong phap quang ph6 Bang 3.10: Ham lacing urea cac mau nude mdm dinh Wong bttng phudng pluip quang ph6 Bang 3.11: K6t qua kh5o sat hoat tinh ctia urease tren kit theo theii gian Bang 3.12: Ham liking urea mau Ca dinh Mpg Wing quang phCi Bang 3.13: Cac thong s6 dOng hoc cua enzyme urease tir va c6 dinh Bang 3.14: Hing s6 t6c vo hoat enzyme va chu ki ban huy cua enzyme urease tir va c6 dinh tren mang PET Bang 3.15: Hi ng s6 t6c vo hoat k va thai gian ban huY t1/2 cita enzyme urease co dinh cac dung dich bao quail va urease tir Bang 3.16: Kdt qua ham luvng urea cac mau stra )(tic dinh bang biosensor Bang 3.17: So sanh ke't qua urea sera bai biosensor va phicang phap quang phO' Bang 3.18: N6ng d6 urea cac luau cti theo *rang pilaf) biosensor Bang 3.19: So sanh ham lung urea cac matt ca Wing biosensor va quang ph6 Bang 3.20: N6ng d6 urea c6 cac mau mr6c mam theo *rang phap ApH Bang 3.21: Tin hieu irc the urease bOui kim loci naeng coil dung dich urea DANH MUC HiNH Hinh 1.1: Trung tam host dung va cd the )(de tac cua urease Hinh 1.2: Tinh thd urease &roc J.B.Sumner chie't tach va k6t tinh Hinh 1.3: Cdu true phan ter Urease tit Bacillus pasterrii Hinh 3.1: Tu'ong quan nong de urea WI ApH this bhng kit thuj, tinh Hinh 3.2: Su' bie'n d6i oh pH theo nong de urea, xac (lath thng urease cCi dinh (rung gel Hinh 3.3: Thang mat] ban dinh hiclng urea bting gidy chi thi Hinh 3.4: Cac bade dinh tinh skt c6 mat urea mau thil 11111113.5: De) thi tttdng quan giaa ApH va nong de urea b6 sung salt Hinh 3.6: Do thi arcing quan gitaa do? hdp thu va nong de urea b6 sung (rung ca Hinh 3.7: Dien bie'n hoot tinh urease tri.'n kit theo th6i gian Hinh 3.8: St:i' dung kit gidy chi thi dd Oat hien urea sib Hinh 3.9: SO' dung kit de phat hien urea rittac mom Minh 3.10: Bieu di so sanh hieu qua c6 dinh enzym cua pi-arcing plia p 11111113.11: Anh llirelig dm be day mang chitosan den hoot tinh urease co dinh 11111113.12: A1111 Iltrang dm pH dung dich urease den hoot tinh urease ce dinh I 3: AnIi hirang ctia thai gian ngam mang dung dich urease den hoot tinh urease cO &h Hinh 3.14: Anh huarng dm ming de glutaraldehyde ngam mang den hoot Link urease co dinh Hinh 3.1 5: Anh hubrig cua thai gian ngam glutaraldehy den host tinh urease co dinh Hinh 3.16: Anh twang dm Wing co chat den hoot tinh enzyme urease !link 3.17: Anh lliromg ciao nhiet de den hoot tinh enzyme urease ce dinh Hinh 3.18: Klido sat de ben nhiet dm enzyme urease Hinh 3.19: Khoo sat ink huang cua pH dung dich phan (mg den enzyme urease Ilink 3.20: Anh twang dia cac loai dem kliac den host Link enzyme urease Hinh 3.21: Khoo sit kha nang boo quan dm enzyme urease Hinh 3.22: Di) ben boo (pan cua enzyme urease c6 dinh vi urease ta Hinh 3.23: Khoo sit so Ian tai sir clang dm enzyme urease co dinh Hinh 3.24: Dci thi bidu dien ttfOng quan giiaa nong dig urea va ApH Hinh 3.25: Turing quan ApH va nong de urea be sung mItu sera tuai Hinh 3.26: Dirimg hieu chink gitia plurcmg plilip (Y) va phirang phap quang ph6 (X) Hinh 3.27: Hieu ang ire the urease ce dinh tri 'n mang PET cua ion F1g2' BAO CAO NGHIN THU Ten de tai: Nghien ciru chi tao vcr ling dung kit thik nhanh urea vet urea-biosensor Chii nhiem de tai: TS Tear' Bich Lam Cu quan chii tri: Tru.iyng Dai hoc Bach khoa HCM Thoi gian thyc hien de tai: 11/2006-11/2008 Myc tieti: - Ling dung tutu cila enzym hoc linh vu'c phan tich nhanh - Tien tdi thay the cac b'd kit phan tich urea dirdc nhap tit nu'dc ngoai va giam al' dung cac phu'dng pilaf) phan tich Mi hoi cac thie't bi va hoa chdt d'it ti6n - Gop phan cung cap cong cu kidm tra va quin lY ve sinh an toan thu'c phdm Nei dung: 1- Nghien cdu chd tao kit thir nhanh urea 2- Nghien dill chd tao urea- biosensor 3- ting dung kk qua nghien cdu phan tich thuc pham Ket qui thu du'cic: Da" the tao dirge loci kit this dang gidy chi thi urea tien dung va urea biosensor sir dung dien chic pH có thd phat hien urea thu'c phgrn a gidi han quy Binh 50ppm Cac sin phdm la kit this nhanh urea va urea biosensor da dildc giii SC; KHCN HCM bay gidi thieu tai H"di chd KHCN to chdc tai Cdn Thd thang 12/ 2008 Da hoan hdong Mn tot nghiep va h8 trd kinh phi till:1'c hien dd tai Cao hoc va de tai Dai hoc vdi cac not dung tren NHAN I TONG QUAN 1.1 NGUYEN NHAN CO MAT UREA VA CAC PHeCiNG PHAP XAC DINH UREA 1.1.1 VE VAN DE UREA TRONG THIX PHAM Urea hay carbamide la san phAm sinh hoc dm qua trinh chuyen hoa axit amin, sinh bai chu trinh ornithine goi la chu trinh urea xay a gan va bai tiet qua than, vao nu& tieu Ming dO urea nuac tieu khoang 12-25g/I, &Km bai tiet mOt khoang 25g Nhung urea khong chi cc') nuac tieu Ham lacing urea huyet tuang va dich I9c cau than co tai 53g/1801itingay, so co tai 28g dugc tai hAp thu va chi 25g bi dao thai VI urea la san phdm chinh cu6i cling ctia sat trao ddi protein co the dOng vat, la &rang chu yeu bait tiet ammonia nen y hoc, viec xac dinh urea bOnh phdm nhhm danh gia tInh tang hoat Ong ctia than Bang II thuat nguyen tir danh d'Au, dung 14C, da nghien cuu qua trinh chuy0n hoa urea vao ca the [1] Trir dOng vat nhai lai co the sa dung urea lam ngu6n chat dam gian tiep nhah0 vi sinh vat phong phiu song da co, doi vai cac Ong vat khac urea khong co gia tri dinh dung Hi0n mOt s6 thuc phim nu'dc da phat hiOn ham Wang urea kha cao gay tam lei bat an cho tieu dUng CO nhieu nguyen nhan din den sat c6 mat ctia urea thpt phdm: urea nOi sinh bai sat phan giai protein va axit amin xay qua trinh chi bien hay bao quan san phdm giau protein, phin eng thtly phan arginine, urea da dutdc sub dung bd sung vao moi trtging len men, &roc dung thgc an chAn nuoi bo sga, ngu' don dung de u'dp bao quan thdy san danh bit xa bpi, hoac c6 the gian lan thuting mai sg dung urea nham tang dO dam tong cua san phdm Ngoai urea xua't hiOn ngu8n nu'dc o nhiem va that ph'im 1:1 nhiem phan Thlic phdm bi nhiem phan la nguyen nhan gay mot s6 benh va dich bOnh lien quan den dutng ruOt, viem gan A, viem gan E Ve dOc tInh ctia urea, thi nghi0m ten dOng vat (heo, chi.* cling) nghien ciiu chuyen hoa dm urea (danh d'au 15N) thac An, thay rang, a heo, sau 48 gia co 52% urea duce bai tiet qua nu& tieu, 48 gia sau naa tiep tic CO 1,9% ducfc bai at theo nuac au LD50 doi vai chuOt la 11500-13000mg/kg th0 tong Hi0n chu'a cO quy dinh ctia Uy ban chuyen gia clia WHO va FAO (JECFA) ve gidi Ilan ham lu'Ing urea thtic phdm Rieng to chgc FDA (mg) 1993 va 2003 xac dinh urea khong phai la chAt dOc [1] Mac du qy vies kiem soot urea thpc phdm la can thiet Trong thang 8/2005, theo tong b6ctia vin Pasteur Nha Trong co 19/45 mAu ntidc mdm ki'em tra co nhiem urea vdi ham luting > 0,1g/1 [84] Nam 2008 Hiep h6i ntrOc chim HCM de nghi giai Ilan ham luting urea san phdm nudc mdm la 0,05g/1 (50ppm) 14/ can kiem sodt urea thuc phdm thir nhit la vi skf có mat cua urea lam tang chi s6 nitd tong an den viec xac dinh sai ham luting protein va gia tri dinh throng that cila thgc phdm Thu' hai, viec phan tich phat hien urea thuc phdm la gop phin lam giam thieu nguy cd gay dich benh va giam sat chit luting thuc phdm TM ba, kiem nghiem urea sea (Milk urea nitrogen, MUN) la pinking phap kidm sodt chec10 dinh du'dng thich hop cho ba sofa nham dam bao ham ltiOng urea stia Ichong vu'Ot qua gidi han 13mg/100m1.[144] Thkrc phim nao có the china ure? Ngoai they san, nu& mim va sira nhu'da de cap d tren thi rugu yang cung,cdn co ethyl carbamate la an xuat dm urea, phat sinh yang lau nam va la mot chat tien to gay ung thkr, nen Canada quy dinh ham luting urea nxou phai