TAP CHI HOA HQC T.49(2) 172-176 T H A N G 4 N A M Oil
C6 DINH
K H A N G T H ^IgM
K H A N GVI RUT
B A N GPHU'aNG PHAP CONG HOA TRI CHO C A M B I ^ N M I ^ N DICH DIEN HOA
• • a m
Tran Quang Huy''\ Nguyen Thj Hong Hanh', Pham Van Chung', Phan Thj Nga', Mai Anh Tuan i_2
' Vi?n V? sinh djch te Trung uang
^Vi?n Ddo tgo qudc te ve Khoa hgc vgt li?u, Trur/ng Dgi hgc Bdch khoa Hd Ngi Den Tda sogn 19-5-2010
Abstract
In this study, we describe the immobilization methods of IgM antibody against Japanese Encephalitis (JE) virus for electrochemical immunosensors. These methods were experimented in order to optimize the efficiency, orientation of antibody as well as minimize the loss of its activity on the sensor surface over the time. Atomic force microscope, Fourier transform infrared spectrometer and fluorescence microscope were performed to survey the characterizations of the sensor surface after using different immobilizing methods of the antibody. The results showed that the silanized sensors using 5% glutaraldehyde as a cross linker between IgM antibody and 3-aminopropyl-triethoxy-silance (APTES) layer is the most advantageous method for electrochemical immunosensors application, it revealved that the signal detection is 2 to 3 fold higher than the the use of other methods. The detection of immunosensors was sensitive, with the quick response time, about 5 minutes and stability after 20 minutes for the detection of JE viral antigens in the sample diluted 60 times in comparison with the initial dilution used for MAC-ELISA tests.
Keywords: Covalent immobilization, IgM antibody, lEV antigens, immunosensor, biosensor.
I.MC)DAU
Cdm bien mien djch Id cam biln sinh hgc dugc phdt trien tren co sd tinh ddc hieu ciia phan irng khdng nguyen - khdng thi ciia cdc doi tugng sinh hgc. Trong dd, mdt trong hai phdn tir k'dng nguyen hodc khdng the dugc cd djnh tren bd chuyin doi tin hieu thich hgp Idm phdn tir dd, phan tu cdn Igi Id doi tugng dugc dd tim trong mdu phdn tich. Dua theo nguyen ly dd tim, cdm biln mien djch dugc phan thdnh cdc logi khdc nhau nhu cdm biln dign hda, quang hgc, tu hay khoi lugng J l , 2]. Vdi nhirng uu diem ndi bat ciia cdm biln miln djch nhu khd ndng phdt hien nhanh tgi chd, dg nhgy, dg ddc higu vd dg chgn Igc cao, kich thudc cdm biln nhd ggn, tign dyng, lugng mdu can phdn tich cd nong dg thap, khdng can chat ddnh dau, chi phi cho sinh pham vd hoa chat khdng cao nhu mdt so kit chan doan khac, cam biln mien djch ngdy cang thu hut dugc cac nhd khoa hgc tren the gidi quan tdm nghien ciru vd phdt triln irng dyng trong vigc dd tim mam benh [1].
Olivier Lazcka vd mgt so tdc gid cho rdng cam biln sinh hgc, trong dd cd cam biln mien djch cd triln vgng to Idn dl thay thi mgt phdn hay hodn todn cdc phuang phdp chan dodn truyin thong nhdm phdt hign, phdn tich cdc tdc nhdn gay benh trong tuong lai gdn [3]. Tuy nhien, dl ddm bdo dugc do nhgy cao vd tinh higu qua ciia cam biln miln djch, ngodi viec ndng cao cdng nghe chl tgo bg chuyin ddi tin higu
thich hgp thi vice cd djnh cdc phdn tir sinh hgc (khdng nguyen/khang the) len tren bl mat cdm biln vd ciing quan trgng nhdm dam bdo dugc tinh djnh hudng, hieu sudt gdn kit tren bl mat, do dn djnh ciia cdc phan tir sinh hgc sau khi cd djnh [2, 4]. Co djnh khdng the len be mgt cdm biln dien hda chu ylu dga tren cac phuang phap nhu: hap phy true tilp, dign hda, cgng hda trj, tg tdng hgp cac don Idp (SAM)...
[1, 4]. Phuang phap dign hda vd phuong phdp cgng hda trj thudng dugc sir dyng hon cd nhd nhirng lien ket tuang ddi ben virng giira cdc phdn tu cua khdng the tren be mat cam biln. Trong dd, co djnh diing phuang phdp cgng hda trj thdng qua cdc nhdm churc dugc tgo ra tren bl mgt cam biln nhu: amino -NH2, cacboxNl -COOH, andehyt -CHO, thiol -SH, hydro.xyl -OH [5].
Ngay nay, rat nhilu cdc benh truyin nhilm dang de dga nghiem trong tdi sire khde cgng dong. Khong che va ngdn chdn kjp thdi cdc tac nhan gay benh truyin nhilm ludn Id yeu cau cap thilt nhdm gidm thilu nguy co lien quan tdi sire khde vd nhirng thiet hgi vl mat kinh tl xa hdi. Chinh vi vay, phat hien nhanh, nhgy vd sang Igc mdm benh truyin nhilm la mdu chdt de ngdn chdn qud trinh lay nhilm, cd bien phdp cdch ly hay dilu trj kjp thdi [6].
Viem nao Nhdt Bdn id mdt tron^ nhirng benh nhiem vi rut cap tinh d he thong than kinh trung uong. Theo thong ke, ty Ig mdc do vi rut VNNB d Viet Nam trong nhirng nam gan day chiim khoang
TCHH, T. 49(2), 2011 Trdn Quang Huy vd cgng sir 30% trong tong sd cdc ca viem nao do vi riit, nguy
CO cao d tre em tir 1 den 15 tudi. Ty lg tit vong tir 0,3 din 60% phy thudc vdo thdi gian phdt hign benh sdm vd dieu trj kjp thdi [6, 7].
Hien nay, cd rat nhieu phuang phdp vd thiet bj chdn dodn phdng thi nghigm nhdm tim ra tdc nhdn gdy benh nhu: phdn lap vi riit, huyet thanh hgc, ELISA, PCR... Tuy nhien, cdc phuang phdp truyin thong ndy thudng mat hdng gid tdi hdng tuan dk bilt dugc kit qud [7, 8]. Do dd, phdt triln nhQ-ng bg kit chan dodn nhanh, chinh xdc tdc nhdn gay bgnh truyin nhiem ludn la yeu cdu cdp thiet doi vdi cdc nhd khoa hgc trong nude vd tren thi gidi.
Nhdm phdt trien bg cdm biln mien djch dign hoa dk phdt hign nhanh, sdng Igc vi nit VNNB tir cdc vy djch, chiing tdi sir dyng khdng thi IgM khdng vi rut VNNB tinh che tir huyet thanh bgnh nhdn trong giai dogn dau nhiem vi rut de tiln hdnh thir nghigm cdc phuang phdp cd djnh khdng the, tim ra phuang phdp thich hgp cho muc dich nghien cuu. Khdng the IgM Id mdt trong hai logi khdng the cd sd lugng nhieu nhdt trong huyet thanh sau IgG, ddc biet Id trong giai dogn ddu cua qud trinh nhiem. Hon nu-a, khdng the IgM CO 10 vj tri gdn khdng nguyen, cao gdp 5 ldn so vdi khdng the IgG [7].
2. PHUONG P H A P T H U C NGHIEM 2.1. Vat Ueu, hoa chat
Bd cdm bien dign hda vdi cdu hinh dien cue tren dudi dd dugc tdc gid vd nhdm nghien ciru thiet ke, che tgo thdnh cdng tgi Vien ITIMS, Trudng Dgi hgc Bdch Khoa Hd Ndi [9].
A
. /
-OH -OH
APTES
<CjH,0)jSi(CH^jNHj
Khdng thi IgM khdng vi riit VNNB tinh chl tir huyet thanh bgnh nhan, khdng nguyen vi riit VNNB cung cap bdi Phdng thi nghigm vi rut Arbo, Khoa Vi riit, Vign Vg sinh Djch te Trung uong.
Protein A gdn hgt nano vdng (PrA.Au, dudng kinh hgt vdng cd 5 nm), khdng the IgM khdng IgM ngudi gdn FITC, 3-aminopropyI-triethoxy-siIance (APTES), glutaraldehyde (G.A) dugc mua tir cdng ty Sigma, Hoa Ky.
Cdc hda chat khdc ddm bdo cdc yeu cdu phdn tich.
2.2. Phuong phap thuc nghigm
a) Cd dinh khdng the IgM khdng vi rut VNNB cho cdm bien mien dich
Trudc tien, cdm bien dugc ngdm trong dung djch KOH/MeOH (1:1, khdng pha loang) trong 30 pliut de logi bd chat bdn bdm dinh tren be mat vd tgo ra nhilu nhdm chuc hydroxyl -OH. Sau dd, sii- dung nude khir ion de rira sgch vd sdy khd cdm bien bang khi nito trudc khi silan hda vdi 5% APTES/MeOH trong 1 gid. Qud trinh silan hda nhdm tgo ra nhdm churc amino -NH2 de lien ke vdi khdng the tren be mat cdm bien (hinh 1). Mdt gigt axit axetic dugc them vdo dung djch trong qud trinh silan hda de djnh hudng nhdm amino -NH2 ra phia ngodi [5]. Rura be mat cdm bien 3 ldn bang nude khu ion, Idm khd vdi khi nito vd ii nhiet tgi 100 120°C trong 6 8 phut dl logi bd hodn todn nhirng phdn tir nude du thira tren be mat, de ngudi vd luu giir trong hop khd d nhiet do phdng cho qud trinh thi nghiem.
y
I
'y—O— ^i — (CH;)
(CH;)
— NH.
— NH.
Hinh 1: Qud trinh silan hod be mat cdm bien
Trong nghien ciru ndy, 3 phuang phdp cd djnh khdng the IgM da dugc thir nghiem de tim ra phuang phdp thich hgp nhdt cho cdm bien mien djch dien hoa [4, 5, 11]. Quy trinh thi nghiem diu dugc thuc hien d nhiet do phdng.
APTES - Khdng thi (APTES-IgM)
Cdm bien sau khi silan hda dugc u true tilp vdi 1 mg/ml khang thi IgM khdng vi riit VNNB trong 1 gid thong qua lien kit -NH2 cua APTES va nhdm -COOH cua khdng the, cdc vi tri khdng gdn kit ddc hieu tren bl mat cam biln dugc khda vdi 2%
BSA/PBS trong 30 phut, rira bdng PBS, pH 7,0 vd dl kho trong khdng khi.
APTES - G.A - Khdng thi (APTES - G.A - IgM) Cdm bien sau khi silan hda dugc nhiing trong
5% (v/v) G.A 30 phut de tgo lien ket cheo giira APTES vd khdng the thdng qua 2 nhdm chuc -CHO cua G.A, sau dd rua 3 ldn vdi nude khir ion trudc khi li vdi 1 mg/ml khdng the IgM khdng vi riit VNNB trong 1 gid, cdc vj tri khdng gan ket ddc higu tren be mat cdm biln dugc khda vdi 2%) BSA/PBS trong 30 phiit, rua bang PBS, pH 7.0, de khd trong khdng khi.
APTES - G.A - Protein A gdn Au - Khdng the (APTES-PrA.Au-IgM)
Cam bien da silan hda dugc nhiing trong 5%
(v/v) G.A trong 30 phiit tgo lien ket cheo vd rua 3 ldn vdi nude khu ion trudc khi u vdi 1 mg/ml protein A.Au/PBS trong 30 phiit, sau dd rua vdi PBS vd li vdi 1 mg/ml khdng thi IgM khdng vi rut VNNB trong 1 gid, cdc vi tri khdng gdn kit ddc hieu tren bl
TCHH, T. 49(2), 2011
mat cdm biln dugc khda vdi 2% BSA/PBS trong 30 phiit. Sau dd rira trong PBS, pH 7.0 vd dk khd trong khdng khi.
Hien vi luc nguyen tir (AFM)
Kinh hien vi lgc nguyen tu (Multimode, Vecco, Hoa Ky) dugc sir dyng dl quan sdt sg thay doi tren bl mdt cdm biln sau mdi phuang phdp co djnh khdng thi khdc nhau. Dg map md tren dnh thu dugc phdn dnh khd ndng gdn kit ciia khdng thi tren bl mdt cdm biln. Phep do dugc thgc tgi Vign Vat ly ky thugt, Trudng Dgi hgc Bdch khoa Hd Ngi.
Phd hap thu hdng ngogi FTIR
Phep do phd hong ngogi FTIR (Nicolet 6700, Thermo, I loa Ky) dd dugc thgc hign nhdm phdn tich vd xdc nhgn sg tdn tgi ciia khdng the tren be mdt cdm bien thdng qua phd hdng ngogi bj hap thy bdi dao dgng ciia cdc lien kit hod hgc tir nhirng phdn tir cd tren be mdt cdm bien. Ket qud dugc so sdnh vdi cdc nghien CU'U da cdng bo trudc ddy.
Hien vi huynh quang
Ky thudt huynh quang dugc dp dyng dk xdc nhgn lugng khdng the gdn ket tren be mat cdm bien thdng qua thi nghiem tren philn silic. Cdc budc cd djnh khdng the IgM khdng vi riit VNNB len be mat phien silic dugc thuc hign tuong tu qui trinh ciia cdc phuong phdp cd djnh neu tren. Sau dd ii vdi khdng the IgM kl dng ngudi gdn FITC (pha loang theo ty lg 1:50, u 40 phiit d nhigt dg phdng), rOra bang PBS rdi de khd tu nhien vd quan sdt dudi kinh hien vi huynh quang Elipse 90i (Nikon, Nhdt Ban).
H? khuech dgi Lock in
Tin higu dd tim khdng nguyen vi rut VNNB dugc do bdng hg khulch dgi Lock-in (SR 830, Stanford Research System), cdc phep do thgc hign d
Cd dinh khdng thi x iM...
nhigt dg phdng vdi cdc thdng so ddu vdo a ngc thiet Igp: dign thi 100 mV. tan so 10 kHz.
3. KET QUA VA T H A O LUAN
3.L Hinh anh AFM ve syr thay doi hinh thai be mdt cam bien
Trudc khi co djnh khdng the, bl mat cam bien dugc rira sgch theo quy trinh de logi bd hoan todn chat ban bdm dinh tren bl mdt, dugc kiem tra bdng kinh hiln vi soi noi (SMZ800, Nikon) va hiln vi dign tir qu^t cd dg phan gidi cao (S-4800, Hitachi) (dir ligu khdng dua ra). Sau dd, cdm bien dugc c6 djnh vdi khdng thi IgM khang vi rut VNNB bdng cdc phuang phdp khdc nhau. Nhu quan sat tren hinh 2 A, B vd C thay rdng, dg map md cua cdc hinh dnh hodn todn khdc nhau. Tren hinh 2A, be mat tuong doi phdng, xuat hign cdc hat cd kich thudc deu nhau dudi Inm nhd len, dd dugc cho Id cdc hgt tinh the Si02 thdnh phan, ngodi ra, mgt sd vj tri nhd cao ban dugc xem nhu sg cd mdt cua nhirng "ddm" nho khdng thi IgM lien kit vdi be mat cam biln. Khi sOr dyng G.A lam chdt trung gian lien ket vdi khdng the vd ldp APTES, be mat xudt hien nhirng chd Idi Idm ban, ddc bigt Id nhieu vj tri hgt nhd cao hdn len so vdi bl mat cdc hgt khdc xung quanh dugc cho la nhQ-ng "ddm" khdng the tap trung Igi, vdi sd lugng nhieu ban phuang phdp khdng sir dung G.A (hinh 2B). Ddc biet, khi sir dung PrA.Au, cdc hgt nano vdng cd 5 nm lam tdm djch chuyen dien tiir giua cdc phdn tir sinh hgc vd dign cue cung nhu giQ-a cdc dien cgc, cdn PrA chi ddng vai trd la chat on djnh bl mat cho cdc hgt vdng. Tren hinh 2C cho thay xudt hien rdt nhieu cdc dinh nhd chieu cao hon 5 nm dd dugc cho Id do tdc dgng ciia hgt vdng vd cdc phdn tu PrA.
Hon nua, cd the mgt lugng khdng thi IgM nhat djnh gdn di lgc vdi cdc hgt vdng hogc lien kit true tilp vdi G.A. Do do, cdc hinh anh AFM cd thi thdy rd net su biln ddi bl mgt khi co djnh khang thi vdi cac phuong phdp khdc nhau.
Hinh 2: Hinh dnh AFM cua bl mat cam biln sir dyng cdc phuong phap co djnh khang the khac nhau. A ) B I mat cam biln dugc cd djnh khang thi true tilp tren ldp APTES. B) B I mat cam bien sau khi cd djnh khang the thdng qua lien kit cheo G.A vdi Idp APTES. C- B I mat cam bien siir dyng Protein A.Au va gdn khang the
sau khi ngam trong 5% G.A.
TCHH,-r. 49(277zan
3.2. Pho hap thu hong ngoai FTIR
Dg nhgy cua cam bien mien djch dign hda phy thugc rat ldn vdo higu sudt gdn ket vd su djnh hudng khdng thi tren be mat cdm biln. De xdc djnh so lugng khdng the IgM ton tgi tren be mat cam bien khi su dyng cdc phuong phdp co djnh khdc nhau, chiing tdi dd sir dyng thiet bj do phd hdng ngogi dl xdc djnh cdc mode dao ddng ddc trung cho cdc phan tir tren be mat cdm bien. Hinh 3 cho thdy, cdc dinh pho hdp thy thu dugc trong viing sd sdng 1800 -
1300 cm' ddc trung cho cdc viing vd mode dao dgng ciia protein (khdng thi). Vdi 3 phuong phdp khdc nhau deu xudt hien cdc dinh phd hap phu ddc trung cho protein, dd Id viing amit 1 trong khodng so sdng
1640 cm'', vdi cdc dao ddng chu yeu cua lien ket
Trdn Quang Huy vd cdng su
C=0, viing amide II trong khoang so sdng 1550 cm'' vdi cdc dao dgng ciia lien kit C-N vd N-H. Dinh phd tgi 1420 cm"' cung dugc cho Id dao dgng ciia C=0.
Tuy nhien, cudng dg hap thy mgnh nhdt Id ciia phuong phap chi su dyng G.A Idm phan tu trung gian, ddng nghTa vdi sg cd mat khdng the nhieu nhat tren be mdt cdm bien dugc ghi nhgn. Ngodi ra, cdc dinh pho tren khdng xudt hign doi vdi be mat cdm bien dugc silan hda nhung chua co djnh khdng the.
Ngugc Igi, dinh pho tgi 1590 cm'' ddc trung cho nhdm -NH2 cd thi tim thay tren bl mgt nay md hdu nhu khdng ghi nhgn dugc hogc dinh hap phy rat yeu tren cdc bl mgt khdc khi da co djnh khdng the.
NhQ-ng kit qud thu dugc phii hgp vdi mgt so nghien ciru ve pho hap thy hong ngogi ciia protein dugc cdng bo trudc ddy [10, 12].
(a) APTES-IgM (b)A(TES-G.A-lgM (c) APTES-G.A-PrAJlu-lgM (d) APTES
IMO 1550 Wavenumbers (cm-1)
Hinh 3: Phd FTIR xdc nhdn su tdn tgi cua khdng the IgM tren be mat cdm bien sir dyng cdc phuang phdp co djnh khdc nhau
3.3. Hien vi huynh quang
Bdng phuang phdp quan sdt su phdt quang ciia FITC cua khdng the khdng ngudi dudi kinh hien vi huynh quang, xdc nhgn gdn ket khdng thi IgM tren phien silic, chiing tdi nhdn thdy rdng, vdi phuang phdp cd djnh APTES - G.A - IgM, cdc ddm huynh quang mdu Id mg xudt hien day ddc vd ddng deu ban so vdi su dyng cdc phuong phdp cd djnh khdc, dom phdt quang xuat hign tren khap be mat phien silic khu vuc thuc hign phdn irng (hlnh 4). Dieu dd ghi nhdn su gdn ket tdt cua khdng the tren phien silic, Cling cd them bang chiirng churng td su gdn ket cd higu qud cua khdng thi IgM khdng vi riit VNNB tuong tu nhu tren be mat cdm bien mien djch.
3.4. Ket qua dd tim khang nguyen vi rut VNNB Dg nhgy cua cam bien mien djch su dung cdc
phuang phdp cd djnh khdc nhau dugc xdc djnh thong qua phep do dgc de phdt hign khdng nguyen vi rut VNNB trong mdu phdn tich, tren he khuyech dgi Lock in RS830. Ket qud lap Igi thi nghiem cho thdy rdng, tin higu dd tim khdng nguyen vi rut VNNB ciia cdm bien mien djch dign hda xudt hien ngay sau 5 phiit vd dn djnh sau khodng 20 phiit tinh tir khi nhiing cam bien vdo dung djch chua khdng nguyen.
Khd ndng phdt hign ndng dg mau nhd hon 60 lan so vdi pha loang khdng nguyen chuan cua ky thudt MAC-ELISA sdn xudt tgi Viet Nam. Ddc biet, tin higu dd tim khdng nguyen d nhQ-ng cdm bien su dyng phuong phap cd djnh APTES - G.A - IgM cao ban 2 den 3 ldn so vdi tin higu tir cdc cam bien khdc (hinh 5). Ngodi ra, nhu-ng thi nghiem ddi chung, do dac trong nude khir ion va dung djch PBS xl khdng chua mau, ket qua cho thdy tin hieu dau ra ciia cdc logi cam bien nay hau nhu khdng thay ddi hodc thay ddi khdng dang ke.
TCHH,T 49(2), 2011 Cd dinh khdng thi-^M-
Hinh 4: Khdng the IgM gdn ket higu qud tren be m§t phien silic thdng qua sg phdt quang ciia khdng the
khdng ngudi gdn FITC, su dyng phuang phdp APTES-G.A IgM
hda. Kit qud phan tich cho thay rdng phuang phap APTES - G.A - IgM cho kit qua tot nh. tin higu dd tim khdng nguyen ciia cam biln sir dyng phuong phdp ndy cao ban 2 din 3 Ian so vdi sOr dyng cac phuang phdp co djnh khdc. Ngodi ra, tin hieu ddp irng cung xuat hign ngay sau 5 phut vd on djnh sau 20 phut ke tu khi cho cdm bien tiep xiic vdi dung djch mdu pha loang 60 lan so vdi dung djch pha loang chuan sir dyng cho phuong phdp MAC-ELISA sdn xuat tgi Vigt Nam. Nhung kit qud ndy Id ca sd dl irng dyng phdt trien cdc bg cam bien mien djch dign hda nhdm phat hign nhanh vi rut viem nao Nhat Ban trong cdc vy djch d Vigt Nam.
Loi cam o'n: Cdng trinh ndy dugrc hodn thdnh nhd sw hd trgr cua Quy phdt trien Khoa hgc vd Cdng ngh? Quoc gia, de tdi md sd 106.16.181.09.
TAI LIEU THAM KHAO
10 0-,
7 5
—•—APTES-IgM - • — A P T E S - G.A - IgM -T— APTES -G A - PrA.Au - lgM_
^ * - P B S . • ' T=27°C
D 6 = 1 mg/ml
Olch = JEV ^
-fhc)! gian (phiit)
Hinh 5: Tin higu dd tim khdng nguyen vi rut VNNB cua cdm bien mien djch sir dyng cdc phuang phdp co
djnh khdng thi IgM khdc nhau 4. KET LUAN
Trong nghien ciru ndy, chung tdi dd thgc hign ba phuong phdp khdc nhau dl co djnh khdng thi IgM khdng vi rut VNNB cho cdm biln miln djch dign
2.
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Lien he: Tran Quang Huy
Vign Ddo tgo quoc t l vl Khoa hgc vat ligu Trudng Dai hgc Bach khoa Hd Ngi
Sdl,DgiCoViet, HdNgi Email: [email protected]