Tong Quan Tai Lieu Ve Biosensor

o an: Tong quan tai lieu ve Biosensor
CHNG 1
GII THIEU CHUNG VE BIOSENSOR
1.1. LCH S PHAT TRIEN CUA BIOSENSOR
Nam 1956, giao s Leland Clark Jnr ngi khai sinh ve khai
niem ien cc sinh hoc (Biosensor) a co cong bo ve ien
cc Oxy. Da tren kinh nghiem va tam huyet cua mnh, ong
a phat trien lnh vc phan tch giup co the o lng c
trong c the.
Nam 1962, tai Hoi ngh Khoa hoc Vien han lam New
York, ong a co bai dien thuyet: Lam the nao e cac
ien cc ien hoa (phng phap pH, cc pho, phep o ien
the, phep o o dan ien) thong minh hn. Trong o, ong
trnh bay cach lam ien cc ien hoa thong minh hn bang
cach them enzym vao may chuyen oi nh nhng chiec
banh sandwich c bao boc bi mot lp mang.
Nam 1975, y tng cua Clark tr thanh hien thc vi s
cong bo cua cong ty Yellow Springs Instrument (Ohio) ve may
phan tch glucose da tren viec o dong ien cua hydro
peroxide. ay la lan au tien cac phong th nghiem tren the
gii phan tch da tren mot Biosensor. Cung vao nam nay,
Biosensor tien them mot bc mi la khi Divis e xuat rang
vi khuan co the c dung nh mot yeu to sinh hoc trong
ien cc vi khuan e o ham lng ru.
Nam 1976, La Roche (Thuy S) gii thieu may phan tch
Lactat (Lactate Analyser LA640) trong o s dung tac nhan
trung gian hexacyanoferrat hoa tan e chuyen cac electron t
lactatdehydrogenase ti mot ien cc. Mac du khong thanh
cong tren thng trng nhng la bc ot pha quan trong
cho mot the he cua Biosensor c ng dung trong the thao
va chan oan lam sang.
Nam 1987, ien cc enzym screen-printed c cong bo
bi Medisense (Cambridge, USA) vi dung cu o co kch thc
nh mot chiec but cho phep giam sat lng glucose trong mau
tai nha. ien cc nay c thiet ke lai lam cho thong dung
hn va so lng ban cua Medisense a at ti 175 trieu ola
1

nam 1996 khi ho c Abbort mua lai. Hien nay Hang Boehringer,
Manheim va Bayer ang canh tranh nhau rat gay gat ve Biosensor
va lng ban ra cua ba cong ty chiem u the tren th trng
Biosensor cua the gii ti 85%.
1.2. KHAI NIEM VE BIOSENSOR

Biosensor thc chat la mot thiet b phan tch chuyen
mot tn hieu sinh hoc thanh mot tn hieu ien. au tien,
Biosensor se nhan dang hien tng va bien dch thanh mot
ac tnh co the nh lng c, sau o ac tnh nh lng
nay c chuyen oi thanh mot tn hieu ien bi mot bo
bien nang. Trong Biosensor, hien tng c nhan dang bi
mot he thong sinh hoc goi la c quan thu cam sinh hoc
(bioreceptor). He thong nay se tiep xuc trc tiep vi mau
phan tch gay ra phan ng va tao thanh hp chat nhay cam
cho Biosensor. C quan thu cam sinh hoc co ac tnh chon loc
ac biet oi vi chat phan tch.
Hnh 1.1. S o cau tao cua biosensor

Chu thch: Chat xuc tac sinh hoc (a) chuyen c chat (S) thanh
san pham (P). Bo bien nang (b) chuyen san pham phan ng
thanh tn hieu ien. Bo khuech ai (c) nham khuech ai tn hieu
ien cua bo bien nang. Bo vi x ly tn hieu (d). Man hnh
hien th (e).
1.3. AC IEM YEU CAU CUA BIOSENSOR

e nh lng, Biosensor phai ap ng yeu cau lien quan
en o lng: kha nang lap lai, kha nang tai s dung cao,

tnh chon loc, tnh nhay cam, vung tra li tuyen tnh va thi
gian ap ng tn hieu tot.
Cac phep o co o lap lai tot neu nh hai loat ket qua
thu c tng t nhau c thc hien bi cung ngi
phan tch, s dung cung biosensor trong cung mot mau
phan tch.
Phng phap o co kha nang tai s dung cao neu cac
ket qua trc co the at c khi tien hanh phan tch
lap lai lan hai.
Tnh chon loc cua biosensor the hien kha nang nhan ra
mot hp chat n trong hon hp cac cau t cua mau,
kha nang nay phu thuoc vao c quan thu cam sinh hoc
va bo bien nang. Mot biosensor co tnh chon loc cao neu
nh thanh phan tap chat cua mau thap.
Tnh nhay cam cua biosensor the hien s thay oi ve
lng th se gay ra s thay oi ve tn hieu tra li:
a = s m
Trong o a : o dao ong ve bien o cua dong ra
m : o dao ong ve bien o cua dong vao
s : o nhay cam cua biosensor, ac trng cho
mc o phu hp cua biosensor trong mot ng dung cu
the.
oi vi Biosensor o bang ien the th bien o cua tn
hieu tra li ty le thuan vi logarite cua nong o chat
phan tch. Theo nh luat Nernst: a = s (logc).
Vung tn hieu tuyen tnh thu c la mot ng cong
hieu chnh cua tn hieu tra li vi nong o khac nhau
cua chat phan tch. ng cong ch that s co y ngha
neu tien hanh hieu chnh ca hai loat nong o tang va
giam. ng cong hieu chnh goi la tot neu nh tn hieu
tra li on nh theo thi gian.

Thi gian ap ng kha dai bi ban chat cua
ng cong hieu chnh, no cho biet mot phng phap o
cho tn hieu tra li nhanh hay cham khi thay oi nong o.
1.4. NGUYEN LY HOAT ONG CUA BIOSENSOR

Cac chat can phan tch trong mau phan tch se i vao
trong ien cc. Mang ngoai (external membrane) cua biosensor
se cho cac chat can phan tch tham qua. Cac thanh phan
sinh hoc (enzym, te bao vi sinh vat, mo, c quan) se phan ng
vi chat can phan tch va tao ra cac ap ng ma cac bo
bien nang (transducer) co the phat hien c. Cac thanh
phan sinh hoc ay thc hien cac hoat ong sau:
Bien oi cac chat can phan tch thanh cac chat hoa
hoc khac thong qua cac phan ng sinh hoa (bieu dien
bang vong tron rong trong s o).
Giai phong ra cac san pham hoa hoc roi t ay tao ra
cac tac nhan kch thch.
Thay oi cac ac tnh nh quang hoc, ien hoc, c hoc.
Tao ra mot so cac ap ng khac nhau vi lng co the
o c.
Con co mot so mang khac gan bo phan transducer,
nhng mang nay co the co cac ac tnh tham khac nhau
so vi mang ben ngoai. Tn hieu ra cua ien cc thng
phu thuoc vao loai bien nang ma no s dung.
Hnh 1.2: Nguyen ly hoat ong chung cua mot Biosensor:

- Chat can phan tch
- Tac nhan kch thch (chat tao ra tn hieu)
Bang 1.1: Mot so ien cc enzym thng s dung
ien cc e
xac nh
Enzym
San pham
Glucose
Glucooxydase va catalase
H2O2, O2
Saccarose
Invertase, glucooxydase
-galactosidase,
glucooxydase
Alcoloxydase
Urease
Pennicillinase
H2O 2
Lactose
Ru
Ure
Pennicillin
H2O 2
H2O 2
NH4+, CO2
H+
CHNG 2
PHAN LOAI BIOSENSOR
2.1. IEN CC IEN HOA (Electrochemical biosensor)
2.1.1.
ien
biosensor)
cc
ien
the
(potentiometric

ien cc o ien the hoat ong da tren nguyen tac
xac nh s khac nhau ve ien the gia ien cc o (probe
electrode) va ien cc so sanh (reference electrode) (la ien cc
co ien the khong oi). S khac nhau ve ien the gia hai
ien cc la ham cua hoat o cac ion trong dung dch ien
phan n at ien cc (ieu kien hoat ong cua ien cc o
ien the la khong co dong ien trong mach o, v the ngi
ta goi no la ien cc co dong ien bang khong). ien the
nay c xac nh theo phng trnh Nerst:
E E0
Trong o:
RT
ln 1
nF
2
Eo: ien the oxy hoa-kh tieu chuan;

R: Hang so kh;
T: Nhiet o tuyet oi;
F: hang so Faraday;
N: so ien t trao oi cua c chat;
1, 2: Hoat o trong dung dch va trong lp mang
ien cc.
ien cc o (probe electrode):
ien cc o la ien cc co tham gia phan ng ien hoa
vi mot trong cac cau t cua can bang ien the. ien
cc o phai co the ien cc c thiet lap u nhanh va
co o chnh xac cao. Trong ien cc ien hoa, ien cc o
thng dung la ien cc mang va ien cc kh.
ien cc kh:
ien cc c cau tao bi kim loai tr nh Pt, Autiep
xuc ong thi vi kh va dung dch cha ion tng ng vi kh
nay. Tren th trng a co ien cc kh hydro, ien cc Oxy,
ien cc kh clo c s dung rong rai.
ien cc mang chon loc ion:
ien cc mang chon loc thong thng la cac ban pin co lp
mang phan cach dung dch can phan tch vi mot dung dch
6

chuan ben trong. Nguyen tac hoat ong cua ien cc
mang la da vao s xuat hien cua mot ai lng ien the
tren be mat cua mang phan cach ch khong phai do phan
ng ien hoa kem theo s van chuyen ion. Mang co tac
dung thuan nghch vi mot ion hay mot nhom ion trong dung
dch mot cach chon loc. ien cc thuy tinh la ien cc ien
hnh cua loai ien cc mang nay. Tat ca cac loai ien cc
mang eu phai ap ng cac yeu cau bat buoc sau ay:
1.
Mang phai khong c tan trong dung dch can
phan tch. e ap ng c ieu nay, mang phai c
cau tao t nhng chat co phan t lng ln nh thuy
tinh silicate hoac nha polymer.
2.
Mang chon loc phai co o dan ien. o dan
ien nay thng c tao ra nh vao s dch chuyen cua
cac ion hoa tr mot ben trong mang.
3.
Mang hoac mot vai phan t cha trong bo
khung cua mang phai co kha nang tac dung chon loc
vi ion can khao sat theo nguyen tac: trao oi ion, ket
tinh hay tao phc. Hai nguyen tac au pho bien hn
nguyen tac th ba.
ien cc mang thuy tinh:
ay la loai ien cc mang thong dung nhat. ien cc

gom mot ong thuy tinh, au cua ong la mot bau tron
cau tao bang thuy tinh mong (0.06-0.10mm). ay chnh la lp
mang thuy tinh co thanh phan xac nh va co kha nang
trao oi chon loc vi proton va cac cation khac.
ien cc mang thuy tinh o pH: Pho bien nhat la
thuy tinh cha 22%Na2O, 6%CaO va 72%SiO2 co kha nang trao
oi chon loc ion H+ en pH xap x 9. pH cao hn, lp mang
thuy tinh tr nen kem chon loc vi H + v co the trao oi vi
Na+ cung nh cac cation hoa tr I khac. Ngay nay ngi ta a
s dung loai thuy tinh trong o Na + va Ca2+ c thay the bi
Li+ va Ba2+ co kha nang xac nh chon loc H+ pH cao hn.
Phan cuoi cua ien cc la mot bau thuy tinh co thanh
mong va co thanh phan ac biet. Ben trong bau thuy tinh
cha dung dch H+ co nong o xac nh. Nhung vao dung

dch H+ la day dan Pt hay kim loai Ag phu AgCl. Toan bo bo
phan tren c at trong ong bao ve.
Khi nhung ien cc thuy tinh vao dung dch cha ion H +, hai
be mat cua mang thuy tinh tiep xuc vi H+ co phan ng
trao oi:
H+tt
H+dd + Na+dd
Na+dd
ngha la hai mat cua lp thuy tinh se tao ra hai lp gel

H2SiO3 (day 10-4-10-5 mm) do s hien ien cua H + trong thuy tinh.
V co hien tng trao oi ion xay ra hai mat cua mang
thuy tinh nen moi lp gel se xuat hien mot ien the co
gia tr phu thuoc vao hoat o cua H + trong dung dch va hoat
o cua H+ trong lp gel. Hay noi cach khac, hieu the mang E
qua lp thuy tinh se phu thuoc vao hoat o cua H + ben trong
bau, cua H + ngoai bau va hoat o H + trong hai lp gel.
Vi gia s rang hien tng trao oi ion xay ra hai mat
thuy tinh gan giong nhau va do co nh hoat o H + trong
bau thuy tinh nen hieu the mang E ch phu thuoc vao hoat
o cua H+ trong dung dch can khao sat.
Bang viec thay the thanh phan cua thuy tinh sao cho ien
the mang khong phu thuoc vao gia tr pH, thng s dung
Al2O3 va B2O3 ta co the tao ra cac ien cc mang thuy tinh
dung xac nh cac cation hoa tr I nh Na+, K+, NH4+
au do kh:
au do kh la cac thiet b o co tnh chon loc va o nhay
cao dung e xac nh cac kh hoa tan hoac cac ion co the
chuyen thanh kh hoa tan khi ieu chnh pH cua moi trng.
Bo phan chnh cua au do kh la mot lp mang xop mong
c lap vao phan cuoi cua au do va co the c thay
the mot cach de dang. Tam mang nay c goi la mang
tham kh, c che tao t cac polymer chu nc nh
polytetrafluoroethylene hoac polypropylene vi o xop khoang
70% (kch thc lo xop nho hn 1m). Lp mang nay se
phan tach dung dch can phan tch vi dung dch NaHCO 3 va
NaCl (neu thiet b dung e o CO2 hoa tan).
ien cc chuan:

Trong hau het cac ng dung ien hoa, ngoai ien cc o ta
phai s dung them mot ien cc co ien the xac nh va
khong oi. ien cc nay c goi la ien cc chuan hay
ien cc so sanh. ien cc chuan phai khong tham gia phan
ng vi bat ky thanh phan nao trong dung dch can khao
sat, phai thuan nghch va tuan theo phng trnh Nerst, phai
co ien the khong oi theo thi gian va co the lay lai gia
tr the ban au sau khi co dong ien nho chay qua ien cc
chuan thng la ien cc kim loai loai hai (kim loai M phu mot
lp hp chat t tan MA cua kim loai o va c nhung vao
trong dung dch muoi co cha anion A- co nong o xac nh).
ien cc calomel:
ien cc calomel c tao thanh bi kim loai Hg tiep xuc vi

dung dch cha muoi Hg2Cl2 bao hoa va KCl co nong o xac
nh (bao hoa hay 1M).
ien cc Ag/AgCl:
Ngay nay ien cc Ag/AgCl c s dung rong rai e lam

ien cc chuan thay cho ien cc calomel. Nong o dung dch
KCl s dung la bao hoa hay 3.5M
Cac ien cc chuan thng co mot lo nho gan au pha
tren. Lo nay thng co mot nut ay kn khi khong s dung
e chong lai s bay hi cua dung dch pha trong.
Hnh 2.1: Cau tao cua mot biosensor o ien the n

gian
Chu thch:
e) ien the gia ien cc

o va ien cc so sanh
a) Mang ban tham
g) Dung dch HCl
b) Thanh phan sinh hoc
f) ien cc o Ag/AgCl
c) Mang thuy tinh
h) ien cc so sanh
d) ien cc thuy tinh o pH
ng dung: ien cc o ien the c dung e xac nh

glucose vi s co nh enzym glucooxydase len ien cc:
D-glucose
Glucooxydase
D-gluconat
H+
o lng H+ tao thanh (o pH sau khi giai phong H +) se xac

nh ham lng D -glucose.
Xac nh Ure vi s co mat cua enzym Urease:
NH2CONH2
NH2CONH2 +
H2O
pH=9
2NH3
H2O + 2H+
10
urease
+ HCO3- + H+
2NH4+
CO2
Chu thch:
c Cap bao ve
e Dung dch ien phan
m mang k nc
r ien cc so sanh
Hnh 2.2: Cau tao ien cc pCO2

Xac nh lysin:
lysin
2.1.2.
ien
biosensor)
lysindecarbocylase
cc
cadalverin
dong
ien
CO2
(Amperometric
ien cc o dong ien hoat ong da vao dong ien

chay qua mach o khi at mot hieu ien the gia hai ien cc
(ien cc o va ien cc so sanh). Mat o cua cac hat tch
ien ty le thuan vi cng o dong ien chay gia hai
ien cc.
Cng o dong ien chay gia hai ien cc la ham
cua mat o cac hat tch ien trong dung dch va ien the
at gia hai ien cc. Trong a so trng hp, ngi ta thng
tien hanh oxy hoa hoac kh mot loai hat tch ien tren cc
o.
Neu at vao ien cc o mot ien the E bien thien so vi
ien cc so sanh va ve ng cong I = f(E), chieu cao I cua
bac gii han khuech tan se ty le vi nong o cua hat b
oxy hoa hoac b kh tren ien cc o.
11
Hnh 2.3: o th I = f(E)

Chu thch:
1- E qua nho e s oxy hoa co the xay ra
2- van toc oxy hoa ien hoa va cng o dong

ien se tang cng vi s tang hieu ien the
3- cng o khong phu thuoc vao hieu ien the
va I = K.Cred
Hien nay cac ien cc o dong ien ang c s dung
rong rai la ien cc oxy va ien cc hydroperoxyde. Oxy va
hydroperoxyde (H2O2) eu la nhng chat c sinh ra trong mot
so phan ng co enzym xuc tac, cung nh trong phan ng
co cac chat trung gian oxy hoa-kh nhan tao: ferrocyanua, ion
N-metyl-phenazini (NMP) va benzoqiunon va chung co the xac
nh nh o dong ien.
Cac ien cc o dong ien thng s dung cac enzym
oxydoreductase e gan vao mang. Oxy, NAD +, NADP+ c dung
nh chat nhan ien t e phuc hoi enzym sau khi tham gia
phan ng xuc tac c chat. Cac enzym s dung Oxy nh
oxydase va enzym s dung NAD+, NADP+ c goi la
dehydrogenase hay reductase. Trong hai nhom enzym nay th
oxydase c dung nhieu nhat. Cac ien cc o dong ien
c s dung e xac nh cac c chat cua enzym nh glucose,
monosacharide, hypoxanthyl, lactate, acid amin, sulfite, salicylate,
oxalate va pyruvate.
ien cc enzym e o nong o glucose co cau tao gom:
mot anod Pt va mot catod Ag phu AgCl tiep xuc vi mang a
co nh glucoseoxydase (GOD). Ca hai eu c at trong dung
dch ien phan KCL. ien ap phan cc 0.68 Volt c at vao
gia hai ien cc. Di tac ong cua ien ap phan cc nay
H2O2 c giai phong ra se b oxy hoa mat phan cach
gia anod va mang theo phan ng:
12

GOD
Glucose + O2
H2O2
Acid gluconic
H2O2
+ 2H+ + 2e-
O2
Dong ien o c ty le vi lng H 2O2 b oxy hoa va do o

ty le vi lng glucose trong moi trng can o.
Hnh 2.4: S o nguyen tac cua ien cc o dong ien

Cung nh o glucose, e o lng lactate, dung ien cc
co gan lactatoxydase (LOD) t Pediscoccuspecies xuc tac phan
ng:
Lactate + O2
lactatoxydase
pyruvat + H2O2
e o lng cholesterol th gan enzym cholesteroloxydase

(ChOD) t Rhodococcus eryththropolis, xuc tac phan ng:
Cholesterol + O2
ChOD Chlest-4-ene-3-one + H O
2 2
e o lng lactose th gan enzym -galactosidase (-GAL) t

E.coli va glucoozydase (GOD) t A.niger, xuc tac cac phan ng:
lactose
-D-glucose
+ O2
H2O
+
-GAL
H2O
-D-glucose +
GOD acid gluconic

+
D-galactose
H2O2
Do qua trnh chuyen khoi H 2O2 en ien cc anod ma

van toc phan ng anod thng b gii han. Khi o, cng
o dong ien anod ty le thuan vi nong o H 2O2 trong
moi trng tiep xuc vi anod.
13

Cung co the xac nh lng glucose qua o nong o Oxy
hoa tan trong dung dch nh ien cc Oxy hay ien cc Clark,
co cau tao gom: hai ien cc co kha nang phan cc, mot
catod bang Pt va mot anod bang Ag phu AgCl. Ca hai ien cc
c at vao chat ien phan la KCl. He thong ien cc nay
c ngan cach vi moi trng nghien cu bang mot mang
tham kh k nc bang teflon hay polypropylen) cho Oxy tham qua.
Di tac dung cua ien ap phan cc 0.65V at vao gia hai
ien cc, Oxy khuech tan qua mang se b kh tai catod theo
phan ng:
O2 + 4e- + 4H+
2H2O
Hnh 2.5: Cau tao cua ien cc Oxy xac nh nong o

Glucose
Dong ien chay gia hai ien cc do phan ng ien hoa ty
le vi lng O2 b kh va do o ty le vi lng O 2 va lng
glucose. Dong ien c o sau khi khuech ai va c ch th
trc tiep bang so hoac bieu dien di dang nong o Oxy
hay ap suat rieng phan cua Oxy.
Phng phap o dong ien nay co nhng ac iem
sau:
Phan ng Enzym se phu thuoc vao van toc khuech
tan cua c chat qua mang
Khi phan ng oxy hoa kh xay ra, gradient nong
o cua c chat giam dan, do o van toc chuyen khoi
14

cham va co the dong ien b kh, e duy tr dong
ien khong oi can phai gi vng vung tiep xuc cang
nho cang tot (tc la can ieu che cac vi ien cc co
ng knh rat nho, en vai m).
Van toc phan ng oxy hoa kh phu thuoc vao
nong o Oxy hoa tan. e han che anh hng cua nong
o Oxy hoa tan trong dung dch en ket qua phan tch th
ngi ta khong s dung oxy lam chat nhan ien t ma
s dung cac chat trung gian e van chuyen ien t
en be mat ien cc. Cac chat van chuyen ien t
trung gian co the la ion Fe 3+, N-methylphenazinium (NMP)
hoac tetracyanoquinodimethane (TCNQ), hexacyanoferate.
2.2. IEN CC O NHIET (Calorimetric biosensor)

Cac phan ng gia c chat va chat xuc tac sinh hoc
thng giai phong ra nhiet nang va ngi ta co the o lng
nhiet giai phong ra o bang mot thiet b nhiet (ien cc
o nhiet), t o se tnh c lng c chat ban au. Khi nhiet
lng toa ra cang ln th o nhay phat hien cang ln. Do o
e tang nhiet lng toa ra, co the co nh ong thi 2
hoac 3 enzym (cung co the len ti 4-5 enzym).
Bang 2.1: Enthanpi cua mot so phan ng co enzym xuc tac:
C chat
Enzym xuc tac
H2O 2
Catalase
Cholesterol
Cholesterol oxydase
52.9
Glucose
Glucooxydase
80.0
Glucose
Hexokinase
27.6
Ure
Urease
6.6
Uric acide
Uricase
49.1
Ester
Chymotrypsin
4-16
Pennicilin G
Pennicillinase
67
Tinh bot
Amylase
-H, kJ/mol
100.4
15

Saccharose
Invertase
20
S dung ong thi enzym glucooxydase oxy hoa ng

glucose va enzym catalase e oxy hoa peroxide. Hai phan ng
nay xay ra cung mot luc do o lng nhiet toa ra nhieu hn.
GOD
Glucose + O2
H2O2
catalase
O2
Acid gluconic
H2O2
+ 2H+ + 2e-
Qua nghien cu ta thay c 0.1M c chat chuyen hoa hoan

toan thanh san pham se tao ra 100 kJ/mol. Cac thiet b nay
phai luon c at trong he thong cach nhiet e gi moi
trng on nh nhiet o.
2.2.1.
ien cc enzym cap nhiet
Trong loai ien cc nay, bo bien nang la cap nhiet ien.

Tren cap nhiet, ngi ta phu mot lp mang cha enzym.
Cap nhiet co cau tao gom hai day dan noi vi nhau nh
moi han. Sc ien ong E cua mach phu thuoc vao ban
chat cua day dan va vao nhiet o T 1, T2. Thong thng
nhiet o cua mot moi han c gi gia tr khong oi
va biet trc, goi la nhiet o chuan T 1 = Tref. Nhiet o cua
moi han th hai khi at trong moi trng nghien cu se
nhay cam vi s thay oi nhiet o cua moi trng gay ra do
phan ng xuc tac bi enzym.
u iem: Kch thc cap nhiet nho nen co the o nhiet
o tng thi iem cua oi tng can nghien cu va
tang van toc hoi ap tn hieu (do nhiet dung nho). Tren c
s nay, Bataillard a a ra cai tien la s dung mot cap
nhiet ban dan (intergrated silicon) e nh lng glucose, ure va
penicillin. Do tnh chat cua cap nhiet va lp tiep xuc enzymcap nhiet co tnh truyen nhiet cao cho phep khong phai kiem
soat chat che nhiet o cua moi trng xung quanh, do o
co the thu nho kch thc cua thiet b e co the cay vao
di da o trc tiep nong o cac chat trong mau.
16
Hnh 2.6: S o ien cc enzym-cap nhiet
2.2.2.
ien cc enzym- nhiet ien tr:
Loai ien cc nay con co the goi la ien cc ao nhiet

v buong o nhiet hoan toan b co lap vi moi trng xung
quanh nh lp ao nhiet, qua trnh nay gan giong vi qua
trnh oan nhiet. Loai thiet b nay co cau tao n gian va
c s dung rong rai nhat.
Hnh 2.7: S o ien cc enzym nhiet ien tr.

Chu thch:
mao dan
nhiet
nhiet
b-
alp
vo
Ong
so sanh
ao
e- nhiet ien tr
f- cot enzym (1mL)
g- nhiet ien tr
c- Thiet b trao oi
o
h- ong mao dan
d- lp cach nhiet
i- Nguon ien
17

C chat c i vao thiet b trao oi nhiet roi qua cot
enzym e phan ng vi enzym va giai phong ra nhiet, nhiet
o giai phong ra se c o bi nhiet ien tr so sanh. Ket
qua o c sau o i ra qua bo x ly e thu nhan tn hieu.
Moi quan he gia ien tr va nhiet o c xac nh theo
phng trnh:
Hay:
Trong o R1: ien tr cua nhiet ien tr so sanh, R 2: ien tr

cua nhiet ien tr o, T1: nhiet o cua mau sau khi qua bo
phan gia nhiet, T2: nhiet o cua mau sau khi qua cot enzym, B:
Hang so nhiet o cua nhiet ien tr.
Khi s thay oi nhiet o rat nho, B(1/T1-1/T2) nho hn 1 th khi
o eB(1/T1-1/T2) ~ 1 + B(1/T1-1/T2) va do o ta co:
Khi T1~T2 th bieu thc tren tr thanh:
o giam tng oi ien tr (R/R) cua nhiet ien tr th ty

le thuan vi o tang nhiet o T. Hang so ty le (-B/T12) la
-4%oC-1. Do o da vao s thay oi nhiet o ma co the xac
nh c chat can phan tch.
Ngoai cach che tao cot vat lieu sinh hoc nh tren, ngi ta
con co the bao ben ngoai nhiet ien tr mot lp vo silicon
va at mang co nh enzym tren, nh o thu nho c
kch thc thiet b.
18
Hnh 2.8: ien cc Enzym-nhiet ien tr co vo boc

silicon
Cung co the tch hp nhieu nhiet ien tr tao ra ien cc
nhiet nh lng ong thi nhieu chat. Cac nghien cu ve
loai ien cc nhiet o cho thay ngoai nhng ng dung trong
cac lnh vc y te, moi trng, thc pham, chung con co the
co nhng ng dung ac hieu nh:
-
Xac nh cac hang so K i, Km, Vm cua enzym co nh

(Stefuca, 1990)
nh lng ADP, ATP bang cach s dung puruvatkinase va

hexokinase ong thi co nh tren aminopropyl CPG
(Kirstein, 1989).
2.3. IEN CC O QUANG (Optical Biosensor)

ien cc o quang c che tao da vao cac tnh chat
vat ly cua anh sang e phat hien ra s bien oi nho trong
mau phan tch.
Nguyen tac hoat ong: Dung dch phan tch c tiep
xuc vi mang va khi phan ng enzym xay ra th se co hien
tng anh sang b hap thu hoac phat ra anh sang mau.
Ngi ta co the xac nh c ham lng cac chat cua
mau phan tch thong qua viec o o hap thu anh sang hoac
s phat quang anh sang cua cac chat tao thanh do phan
ng xuc tac bi enzym.
C s cua kieu ien cc quang la s ket hp cac si dan
anh sang vi phep trac pho quang, phep trac huynh quang,
19

hoac phep o phan xa quang. No co kha nang ch bao
nhng thay oi cua cac thong so quang hoc, chang han nh
s hap thu anh sang, chieu dai bc song hoac ch so
phan xa trong moi trng o bao quanh so dan. Nhng thiet
b nay gan vao hoac la si n hoac la chum si kep e
anh sang ti va chum tia sang c o.
2.3.1.
ien cc o phan xa quang:
ien cc nay hoat ong theo nguyen tac: nang suat phan xa
t mat phan xa tuan theo phng trnh:
R R0
I
,%
I0
Trong o R- nang suat phan xa, R0-hang so phan xa cua

moi trng chuan, I-cng o cua tia ti, I o- cng o cua
tia phan xa.
Moi quan he gia R vi nong o chat hap thu mau theo
phng trnh:
C
K 1 R
2R
Trong o C-nong o chat hap thu, K-hang so hap thu,

hang so tan sac
Ky thuat nay c dung e nh lng glucose trong mau nh
thuoc hien mau MBTH (3-methyl-2-benzotiazolinon) va DMAB (3dimethyl-aminobenzoic acid). Hai enzym Glucooxydase (GOD) va
peroxydase c co nh bang cach hap thu len be mat tam
nen a phu thuoc nhuom. Khi mau phan tch tiep xuc vi
tam nen th phan ng xay ra nh sau:
Glucosemau
H2O2
O2
MTBH
GOD
+
Acid Gluconic +
DMAB
peroxydase
H2O2
MTBH-DMAB (ma
u xanh)
neu H2O2 co nong o cao th phai dung enzym catalase. Mau

phan tch sau o c em o nang suat phan xa bc
song thch hp va t o se tnh c ham lng glucose.
Vi Biosensor da tren nen tang cua s phan xa toan phan
(total internal reflection TIR) th be mat day dan c phu
20

khang the, se tiep xuc vi mau phan tch. Khi song anh
sang i en lp mang nhay th ch co mot phan nho song
anh sang b hap thu vao trong moi trng, con tat ca
chung b phan xa lai nhieu lan va dan dan nguon sang b
suy giam. Da vao lng anh sang phan xa, ta co the tnh
c ham lng mau phan tch.
Hnh 2.9: Biosensor da tren hien tng phan xa toan

phan
2.3.2.
ien cc o phat quang:
Nguyen tac hoat ong: C chat tac dung vi enzym co

nh e tao thanh san pham co kha nang phat ra anh
sang mau. o anh sang mau o mot bc song nhat
nh se suy ra c nong o c chat.
Da vao ac tnh nay, ngi ta thng s dung no e xac
nh cac vi khuan trong thc pham. Khi cac vi khuan tac dung
ac hieu vi ATP va D-luciferin di tac dung cua enzym
luciferase se tao thanh oxyluciferin, AMP. Pyrophosphate, CO 2 va
anh sang vang o c bc song 562nm. T o xac nh
lng vi khuan co mat trong thc pham.
2.4. IEN CC IEN AP (Piezoelectric biosensor):

Nguyen ly cua loai ien cc nay da vao s dao ong
cua cac tinh the. Cac chat ien moi t nhien nh thach anh,
tuamalin, hoac nhan tao nh liti sulfat, thach anh tong hpdao
ong khi chu tac ong cua t trng. Tan so dao ong cua
21

tinh the phu thuoc vao o day va s bien dang cua tinh
the, moi tinh the co mot tan so dao ong ac trng. Tan
so dao ong nay thay oi theo s hap thu hay nha hap thu
t be mat tinh the, thong thng no se tang khi co tap
chat hap thu len tren be mat cua tinh the. o tan so dao
ong se biet c nong o cua c chat can o.
Tan so dao ong c tnh theo cong thc:
trong o: f: Chenh lech tan so dao ong (Hz), m: chenh lech

khoi lng cua chat hap thu (g), K: hang so ac trng cho s
dao ong cua tinh the, phu thuoc vao be day va o nt
cua tinh the, A: dien tch be mat hap thu (cm2).
ien cc ien ap c dung e o amoniac, methane, lu
huynh dioxit va c chat phosphate hu c. Ta co the dung no
e xac nh nong o formaldehyde nh formaldehyde
dehydrogenase hay xac nh d lng thuoc tr sau vo c
phospho von km ham enzym xholinesterase.
u iem: Cho thi gian ap ng nhanh, nho gon va re tien
Nhc iem: Khong dung phan tch mau dang long, de b
h hong do o am khong kh.
Hnh 2.10: S o cau tao cua ien cc ien ap

22
2.5. IEN CC MIEN DCH (Immunobiosensor):

ien cc mien dch hoat ong da tren tnh chat khang
nguyen khang the. Tc la cac khang the phan ng ac
hieu vi cac khang nguyen sinh ra no.
Moi khang the la mot protein hnh ch Y, gom 4 chuoi
polypeptide, trong o co hai chuoi nang va hai chuoi nhe lien
ket vi nhau bi cac cau disulfite. Mot phan cau truc cua
cac chuoi la co nh, nhng cac phan au mut cua hai
nhanh ch Y lai bien oi va tao nen cac v tr ket hp co
kha nang phan ng vi cac chat hoa hoc khac goi la
khang nguyen. Khang nguyen la cac chat la co ban chat
protein hay hydrat cacbon.
Phan ng khang nguyen khang the c xac nh bang
nhieu cach, trong o co ky thuat ELISA (enzym linked
immunosorben assay: ky thuat hap thu mien dch co gan
enzym). Nguyen tac cua ky thuat ELISA nh sau:
Khang the ac hieu vi khang nguyen c co nh tren
be mat cua mot ong. Mot hon hp gom mot lng a biet
phc ket enzym-khang nguyen va mot lng cha biet khang
nguyen cua mau c at vao ong. Sau mot thi gian thch
hp khang the, phc enzym-khang nguyen va khang nguyen
t do co the gan ket hoac van trang thai t do, phu
thuoc vao nong o cua chung. Cac khang nguyen t do
c ra troi va loai bo. Lng phc ket enzym-khang
nguyen gan vi khang the c xac nh bang van toc
phan ng enzym.
23
Hnh 2.11: Nguyen ly hoat ong cua ky thuat ELISA

ELISA c s dung e phat hien va khuech ai mot
phan ng khang nguyen-khang the. Lng khang nguyen
lien ket vi enzym gan vi khang the co nh c xac
nh bang no o tng oi gia khang nguyen lien ket va
khang nguyen t do va c nh lng bang ty le cua
phan ng enzym. e co the ap ng nhanh chong, ngi ta
s dung cac enzym co kha nang s dung lai nhieu lan. o
nhay cua phng phap nay cung co the tang len bang
cach s dung cac phan ng co xuc tac enzym. Cac phan
ng nay cho ap ng nhanh hn: chang han tao ra cac san
pham phat quang, hoac huynh quang, hoac co mau am hn.
Ky thuat nay hien nay c s dung rong rai trong cac
phong phan tch th nghiem.
24

Gan ay e tang pham vi ng dung, van toc va o nhay,
ky thuat ELISA c ket hp vi cac bo Biosensor hnh thanh
phng phap s dung ien cc mien dch (immunosensor). Cac
ien cc mien dch nay rat thch hp khi s dung vi ien
cc ien ap hoac cac ien cc o ien the.
Hnh 2.12: Nguyen ly hoat ong cua ien cc mien

dch
(I): ien cc sinh hoc dung ay thay the cho he thong
giam nh mau truyen thong.
a) Ong c phu khang nguyen co nh. Mot lng d
phc ket enzym-khang the c at vao trong ong va xay
ra s gan ket;
25

b) sau mot thi gian thch hp, nhng phan t nao khong
c gan ket se b ra troi;
c) dung dch cha khang nguyen phan tch c a vao
ong, phu thuoc vao nong o khang the ma xay ra s gan
ket va loai mot so phc ket enzym-khang the. Lng phc
ket enzym-khang the loai ra c xac nh bang tn hieu ap
ng t bo ien cc sinh hoc.
(II): ien cc sinh hoc nay da vao giam nh trc tiep cac
khang nguyen gan be mat c phu khang the.
a) ien cc sinh hoc c phu khang the (co nh), ac
hieu vi khang nguyen phan tch. Bo chuyen oi c
ngam trong dung dch cha mot hon hp gom mot lng
a biet phc ket enzym-khang nguyen va nong o
cha biet khang nguyen phan tch;
b) sau mot thi gian thch hp, khang nguyen va phc ket
enzym-khang the c gan vi khang the;
c) nhng phan t nao khong c gan vi khang the b
ra troi va loai bo. Lng phc enzym-khang nguyen
gan vi khang the c xac nh trc tiep t tn hieu
chuyen oi.
2.6. IEN CC VI SINH VAT (microbial biosensor)

ien cc vi sinh vat c che tao e phat hien cac chat
hoa hoc, da vao s ho hap va trao oi chat cua vi sinh
vat.
ien cc nay gom hai loai:
ien cc dung e o s thay oi hoat lc ho hap cua vi
sinh vat co nh vi mot thiet b o ien the.
ien cc dung e o cac chat sinh ra t vi sinh vat va
phan ng de dang vi ien cc.
Cac vi khuan hieu kh s dung Oxy va san sinh nang lng.
Nh viec o lng Oxy tieu thu thong qua ien cc O 2, ta co
the xac nh va anh gia c hoat lc ho hap cua vi
khuan. Oxy tham qua mang teflon va b kh tren tren ien
cc platin. Neu cac thanh phan cua dung dch co anh hng
26

en hoat lc ho hap th nong o cua no co the c nh
lng thong qua viec xac nh nong o Oxy.
Ngi ta thng dung ien cc nay e xac nh ch so
BOD (Biochemical Oxygen Demand nhu cau Oxy sinh hoa) ( la
lng Oxy do vi sinh vat tieu thu e oxy hoa sinh hoc cac chat
hu c trong bong toi ieu kien chuan ve nhiet o va
thi gian). Ch so BOD la thc o lng chat hu c gay o
nhiem nc va no c s dung e anh gia chat lng
nc. Cac chat gay o nhiem co the b phan huy bi vi
sinh vat nh tieu thu Oxy. V vay mc o o nhiem co the
c xac nh thong qua viec o lng Oxy.
ien cc c che tao bang cac co nh trichosporon
cutaneun vao mang cellulose va gan vi ien cc Oxy.
Nguyen ly hoat ong cua ien cc o BOD: Oxy
khuech tan t dung dch bao hoa khong kh qua mang tham
tch i vao mang co cha cac te bao vi sinh vat roi qua
mang teflon va cuoi cung chung b kh tren be mat catod Pt
cua ien cc oxy. Dong ien cua ien cc Oxy dan dan on
nh do van toc khuech tan cua Oxy qua mang can bang vi
van toc tieu thu Oxy do ho hap cua vi sinh vat co nh tren
mang. Ngi ta goi dong ien nay la dong ien c ban
(base current). Neu ta cho chat ong hoa hu c vao dung dch
o th kha nang phan tan oxy hoa tan trong dung dch tang do
o vi sinh vat se s dung oxy cho ho hap tot hn. Ket qua
la van toc ho hap tang dan con nong o oxy hoa tan
giam dan, dan en dong giam dan cho ti khi at en trang
thai on nh mi va ngi ta goi dong ien nay la dong
nh (pick current). S khac nhau gia dong c ban va dong
nh c goi la dong ra (output), tn hieu cua dong ra ty le
thuan vi nong o cua cac chat hu c b phan huy sinh
hoc cua mau o. V vay ta co the biet c nong o BOD
da tren ien cc vi sinh vat.
27
CHNG 3
PHNG PHAP CHE TAO BIOSENSOR
Viec che tao Biosensor bao gom ba bc:
Chon la bo thu cam sinh hoc
Chon la bo bien nang
Co nh thanh phan sinh hoc len bo bien nang.
3.1. CHON LA BO THU CAM SINH HOC (Bioreceptor):

Bo thu cam sinh hoc ong mot vai tro la thiet b nhan
dang sinh hoc. Khi co mat chat can kiem tra, bo thu cam sinh
hoc phai tao ra mot hieu ng hoa ly co kha nang phat
hien bi bo bien nang. ieu nay lien quan nhieu qua trnh
chang han nh s xuc tac sinh hoc, s cap oi mien dch hay
s nhan cam hoa hoc.
3.1.1. Enzym:
Chat xuc tac sinh hoc thng c s dung la enzym v
co san tren th trng, chang han nh glucose oxidase (enzym
oxy hoa glucose) va urease (Enzym thuy phan ure). Cac enzym
nay c thu nhan t nhieu nguon sinh hoc khac nhau va
c s dung mot mnh hay ket hp vi cofactor cua chung
nh NAD+ va NADP+.
S s dung cac enzym thng mai co nhieu thuan li
chang han nh kha nang san xuat hang loat vi nhng ac
iem, thi gian song biet trc va san co. V vay cac enzym
thng mai ong mot vai tro quan trong trong cac Biosensor
hien ang co mat tren th trng. Nhng ben canh o chung
co nhng bat li nh t ben va khi hoat ong can co cofactor
ket hp, ong thi mot enzym n le th khong xuc tac hoan
toan cho mot chuoi phan ng, v vay trong biosensor thng
ket hp nhieu enzym vi nhau theo mot trnh t hp ly e
am bao s hoat ong toi u cua Biosensor.
3.1.2. Vi sinh vat:
C the vi sinh vat co ay u cac enzym va cofactor can

thiet trong mot moi trng a c toi u hoa. Trong moi
trng thch hp, Vi sinh vat se san sinh va en bu bat c
s mat mat hoat tnh cua Enzym theo thi gian.
3.1.3.
Mo va c quan:
Co the tan dung cac phan mo thc vat hay mo ong

vat lam nguon nguyen lieu cung cap enzym v chung co kha
nang gan ket cao, co mot cau truc u manh e gan ket
trc tiep len bo bien nang ma khong can en ky thuat co
nh protein.
Mo thc vat ong vat:
Thc vat la nguon enzym hu dung cho hoa hoc phan tch.
Bang viec s dung bo bien nang thch hp, co the che tao
Biosensor co tnh nang on nh cao v cac enzym van c duy
tr hoat tnh trong mo thc vat. Tng t nh vay, mo ong vat
c xem nh la mot bo thu cam sinh hoc hu dung cho s
phat hien chon loc L-amiono acid ma khong b anh hng
ang ke bi D-amino acid. e tang kha nang chon loc co
the ket hp mot chat khang khuan e tranh s nhiem
khuan, chang han nh them 0.02% NaOH vao ien cc
glutamine.
Bo phan c quan:
Cac xuc tac sinh hoc cung c tm thay trong cac c quan
te bao nh lysosome, luc lap, the hat va vi the v chung cha
nhieu he thong enzym c dung trong biosensor. Chang han
nh vi the gan co he thong enzym monooxidase vi cytochrome
P 450 xuc tac cho phan ng oxy hoa nhieu acid beo, hormone
steroids Cac c quan te bao c gan trc tiep len bo bien
nang o dong ien trong thiet b Biosensor.
3.1.4.
Tac nhan mien dch:
Khang nguyen va khang the cung c s dung lam

bo thu cam sinh hoc. Tnh chon loc cua Biosensor c quyet
nh bi khang the va tnh nhay cam c quyet nh bi
enzym ket hp. Khang the c co nh vao bo bien nang
nh vao ky thuat Mien dch hoc Enzym (EIA). Hoac thay enzym
bang chat mang ion ong vai tro nh chat trung gian cua cac
qua trnh ien hoa mien dch. Khang nguyen thch hp vi

khang the c ket hp vi chat mang ion tao ra s tiep hp
c phat hien bi bo bien nang ien hoa.
3.1.5.
Bo thu cam hoa hoc:
Mang te bao cung c s dung do kha nang kch thch

hoa hoc gay ra s thay oi cau tao. Cac te bao than kinh
cung c dung trong phat hien thuoc, oc to hay cac chat
khac.
3.2. CHON LA BO BIEN NANG:
Bang 3.1: Hien trang nghien cu cua Biosensor vi bo bien
nang tng ng:
Bo bien
nang
o
dong
Te
ien
bao
ien
o
hoa
ien
the
Ban dan
(IFET)
o nhiet
(Thermistor)
o quang (si
quang hoc)
Ap ien
C hoa hoc
Chu thch:
tien
Biosensor
Enzym
Te
bao, vi
sinh vat
Tac
nhan
mien
dch
Mo thc
vatong
vat
xxx
xx
xx
xxx
xx
xx
xx
x
x
Bo thu
cam
hoa hoc
x: ang nghien cu c ban
xx: a nghien cu va phat trien nhng mau au

xxx: Nhng thiet b co mat tren th trng
Phu thuoc vao kieu phan ng va tng ng dung cu the

cua Biosensor ma chon bo bien nang phu hp.
Neu no c dung trong sinh hoc th can phai ap ng
cac tieu chuan tng ng nh kha nang tach protein, lipid
hay te bao.
Neu no c dung trong invivo th can phai giam kch
thc toi thieu va oi hoi hnh dang cua no phai phu
hp e khong pha huy mo thc vat-ong vat, ong
thi cung co kha nang loai bo oc to, kim loai hoac
cac thanh phan a phan t ra khoi bo bien nang.
Van e tac ong hoa hoc cung anh hng en s chon la
bo bien nang. Co the ket hp enzym urese vao cac ien cc
pH, pCO2, hay pNH3. Bo bien nang tot nhat cho viec xac nh
ure trong mot mau sinh hoc la cac ien cc pCO 2, pNH3 bi
chung co mot mang tham thau e loai bo tat ca nhng
anh hng cua cation va anion.
3.3. S CO NH THANH PHAN SINH HOC LEN BO

BIEN NANG:
Viec co nh cac thanh to sinh hoc, nhat la enzym
thng em lai nhieu li nhuan hn trong viec ng nh:
Lam cho enzym trong nhieu trng hp ben hn.
Tach phc enzym chat mang ra khoi mau de dang.
Hoat o enzym gi c on nh trong mot thi gian dai.
Tuy nhien, viec s dung enzym co nh cung co nhng han
che nhat nh nh:
S chuyen khoi b han che.
Co the mat hoat tnh khi co nh.
Khong co hieu qua oi vi c chat ran.
Mat tnh thch nghi hnh the.
Nhng nhng han che tren la khong ang ke so vi nhng
li ch ma enzym co nh mang lai. Do vay ngay cang co
nhieu nghien cu mi cung nh cac cong nghe mi e co

nh enzym.
Ngi ta thng co nh cac thanh phan sinh hoc len mot
chat mang ran bang nhieu cach. Trong ky thuat co nh, can
am bao nhng yeu cau nhat nh, nhat la khi cac ien
cc sinh hoc (biosensor) c a vao s dung trong thc te:
Cac cau t sinh hoc phai gi c hoat o khi gan tren
be mat biosensor.
Mang sinh hoc phai c gan chat vi be mat cam
bien va van gi c cau truc va chc nang.
Mang sinh hoc a c co nh phai on nh va ben
trong mot thi gian dai.
Vat lieu sinh hoc can co tnh ac trng rieng oi vi
tng cau t sinh hoc.
e co nh cac thanh phan sinh hoc trong Biosensor, ngi
ta thng s dung cac ky thuat nh hap thu vat ly, bao goi
trong khuon gel hoac trong polymer, lien ket ong hoa tr vi
chat mang va lien ket cheo cac protein.
Hnh 3.1: Mot so phng phap co nh thng

dung trong ien cc sinh hoc
3.3.1.
S co nh Enzym:
a. S co nh bang hap thu vat ly:

ien cc Enzym au tien c che tao bi Updike va
Hicks bang cach co nh Enzym Glucose oxidase trong mot lp
gel polyacrylamide, sau o Enzym nay c gan len mang
Plastic cua ien cc oxy. Hoac co the co nh mot enzym len
mot thanh phan nhay cam cua mot ien cc bang mang
tham thau ngan can s khuech tan protein. Guibault va Shu
a che tao mot ien cc enzym nhay cam vi ure bang cach
trai mot dung dch huyen phu cua enzym urease len be mat
cua ien cc co mang nilon va bao boc kht hoan toan bang
mang tham thau, sau o ien cc enzym nay c ra sach
vi nc va san sang e s dung.
Nguyen tac cua phng phap hap phu vat ly nh sau:
Hap phu enzym len chat mang nh lc tng tac yeu
gia chat mang va protein nh lc Valderwaalls, lien ket
hydro va lien ket k nc. Khi chat mang khong co lo
xop, enzym bam tren be mat chat mang. Khi chat mang
co lo xop, enzym chui vao trong cac lo xop cua chat
mang.
Neu chat mang co cha ien tch, lien ket gia chat
mang va enzym la lien ket ion (Lien ket nay ben hn so
vi hap phu).
Mot so chat mang thng s dung e co nh enzym bang
phng phap hap phu hay lien ket ion:
Chat mang hu c: than hoat tnh, cellulose, tinh bot,
dextran, collagen, albumin, agarose, chitin.
Chat mang vo c: silic, thuy tinh xop, oxide cua kim loai.
Chat trao oi ion: amberlit, DEAE sephadex
DEAE celllulose, CM cellulose.
CM sephadex,
Polymer tong hp: polyamide, polyacrylamide, polystyrol, nilon,

polyvinyl.
Phng phap ieu che: Cho enzym va chat mang tiep xuc vi
nhau (khuay tron), sau o ra e loai bo nhng phan t b
gan yeu len chat mang.
Cac yeu to anh hng en lng enzym co nh c

va o ben cua lien ket co nh:
Nong o protein enzym: lng enzym co nh len chat
mang thng ty le thuan vi nong o cua no mot
gii han nhat nh.
pH: pH moi trng phu thuoc vao so lng va ban chat
cua cac nhom tch ien chat mang cung nh protein
enzym. S thay oi pH thng anh hng ln en lng
enzym co nh c bang lien ket ion. ong thi s thay
oi pH ot ngot thng dan en s nha hap phu cua
enzym.
Lc ion cua moi trng: s co mat cua cac muoi tch
ien trai dau vi chat mang co the keo theo s ket
tua cuc bo cua protein trong dung dch. Tuy nhien, s co
mat cua muoi cung co the lam tang o hoa tan cua
protein do o anh hng xau en hieu qua co nh.
Nhiet o: nhiet o tang lam duoi mach protein enzym, do
o lam tang lien ket protein enzym vi chat mang, nhng
cung lam mat hoat tnh cua enzym.
Khoi lng phan t va ban chat cua chat mang: enzym
co khoi lng phan t cang nho th hap phu cang cao.
Nhng chat mang co cha nhieu nhom hao nc hap
phu tot hn va ben hn.
Tuy nhien s co nh vat ly ngay nay t c s dung do
s co mat cua enzym trong dung dch khong gi c hoat tnh
trong thi gian dai. Do o s co nh hoa hoc, s dung cac
lien ket ong hoa tr se am bao o ben cua enzym trong
mot thi gian kha dai.
b. S co nh bang lien ket ngang (cross linking
immobilization):
S tao lien ket ngang la qua trnh s dung mot tac
nhan a chc e tao cau noi gia cac nhom xuc tac sinh
hoc khac nhau hay protein khac nhau e tao ra mot hp chat
co trong lng phan t ln rat nhieu va khong co tnh hoa
tan.
Co the tao lien ket ngang cac phan t cua cung enzym
hay co ket hai hay nhieu protein vi nhau (enzym vi enzym hay
enzym vi protein hoac nhieu enzym tren protein mang chang
han nh albumin trong huyet thanh cua bo (BSA). Ngay ca c
quan hay cac te bao cung co the ket dnh c bang viec
tao ra cac lien ket ngang.
Glutaraldehyde la tac nhan tao lien ket ngang thng
dung. Tac nhan co hai nhom chc aldehyde cac au mach
nay se phan ng vi nhom amine tren phan t protein hay
enzym va tao ra cac hp chat mang tnh base:
Cung co the thay Glutaraldehyde bang tac nhan hai chc

khac nh hexamethylene diisocyanate O=C=N-(CH 2)6-N=C=O lam
tac nhan tao lien ket ngang.
Co 4 phng phap chnh e co nh enzym bang lien ket
ngang:
Phng phap ngam (Immersion method)
ng dung: Phng phap ngam nay dung e co nh
enzym urease len ien cc thuy tinh hay ien cc pH-nhay cam
vi cac cation hoa tr I.
Nguyen tac: Chuan b dung dch co nh: Lay 600 n v
I.U enzym Urease hoa tan trong 1ml em phosphate 0.02M, pH =
6.8. Them vao 1 ml dung dch albumine huyet thanh bo 17.5%,
sau o them 0.07 ml dung dch glutaraldehyde 25% e thu c
nong o cuoi cung 0.8%. Khuay dung dch trong 2 phut.
Chuan b be mat hoat ong: au tien ien cc cation
c ra bang nc cat, sau o lau kho bang giay loc.
Nhung ngap bau ien cc trong dung dch co nh tren, xoay
ien cc nhe nhang xung quanh truc cua ien cc trong 15
phut e tao mot lp dung dch co nh ong nhat. Mot
vong O c gan kht vao e gi cho mang enzym nay bam

chat len bau ien cc. Sau o ra ien cc bang nc cat,
dung dch glycine, va cuoi cung bang nc cat e tach ra
hay trung hoa tac nhan tao lien ket ngang.
Hnh 3.2: Nguyen tac cua phng phap ngam

u iem: Phng phap thc hien n gian, thch hp cho
viec co nh cac Enzym len a so cac bo bien nang, ac
biet bo bien nang nho.
Phng phap
method):
ket
hp
trc
tiep
(Direct
binding
Nguyen tac: Nho 10 l dung dch cha enzym len tren nh

cua mot ien cc, sau o nho 10l dung dch cha tac nhan
tao lien ket glutaraldehyde.
Phng phap nay cung c dung e co nh enzym len
mang k nc cua cac ien cc cam ng kh nh mang
fluorocarbon hay polytetrafluoroethylene.
Hnh 3.3: Phng phap ket hp trc tiep

u iem: Tiet kiem enzym, dung e co nh cac enzym
co gia thanh cao.
Phng phap s dung bnh phun (Use of aerosol):
Nguyen tac: au nhay cam cua mot ien cc c ra
va ngam trong dung dch enzym khoang 20 phut e am bao
s hap thu enzym len ien cc enzym. ien cc nay sau o
c say kho 4oC va xoay. Glutaraldehyde c bm len tren
ien cc t mot khoang cach u ln e ngan can tao giot.
Sau khi tao lien ket ngang, ien cc enzym c ra vi nc,
san sang cho viec s dung.
u iem: be day lp enzym cc ky mong (1-2m) va
cac biosensor tao ra co thi gian ap ng cc ngan (5-10s).
ng dung: Phng phap nay dung e co nh enzym
pennicilinase, urease va acetylcholinesterase, co nh cac protein
len nhieu chat mang.
Hnh 3.4: Phng phap s dung bnh phun

S dung mang membrane:
Cac mang membrane tang cng-gia co (Re-inforced
membranes): Enzym c co nh len mot dien tch rong cua
mang nylon bang nhng da nho cha membrane enzym co
cung me va s co nh giong nhau. Sau o cac mang nay
c kep vao trong bo bien nang. Mang membran tang
cng-gia co nay co phan si do o cai thien tnh chat c
hoc cua mang enzym.
Cac mang membrane gan nhom chc san (Prefunctionalized
membrane) dung cac mang membrane xop san co tren th
trng. Nhng mang nay co cac nhom chc lien ket co
kha nang phan ng vi v tr t do cua enzym. Cac mang
nay sau o em ngam trong dung dch cha enzym a chon e
thu c mot mang enzym vi nhng tnh chat c hoc hu
dung.
3.1.3.
S co nh cofactor:
Cofactor c co nh len chat mang ong thi cung luc vi

enzym bang phng phap hap thu vat ly, hay bang lien ket
ngang.
3.2. Co nh Vi sinh vat
Vi sinh vat co kch thc ln hn enzym do o de dang co
nh bang phng phap vat ly. Chung co the c co nh
bang gel agar hay polyacrylamide hoac mang tham thau va
mang loc membrane nh millipore 0.22 m che tao t ester
cellulosic. Toan bo te bao vi sinh vat c co nh trong huyen
phu collagen a c x ly bi glutaraldehyde. Mang collagen

cung c s dung nh mot chat mang vat ly.
Mang k nc va vi sinh vat co the c gan ong thi
len cac ien cc khuech tan kh chang han nh ien cc
pNH3, pO2, pCO2. Vi sinh vat c nhot trong cac lo mang loc
cellulose acetate, sau o ra cac te bao vi sinh vat t do tren
be mat va at mang vi sinh vat tren vao ien cc dung
mang tham thau va ngam trong dung dch em e loai bo
cac thanh phan dinh dng anh hng ti phep o. Cac
ien cc vi sinh vat c bao quan nhiet o 4 0C trong dung
dch em phosphate.
Hnh 3.5: ien cc vi khuan o dong ien

Cung co the co nh mot enzym va mot te bao vi sinh
vat len cung bo bien nang. Vi sinh vat c co nh bang
phng phap vat ly va enzym c co nh bang phng
phap lien ket ong hoa tr e tranh s nha protein.
Vi khuan acetic thuoc loai Acetobacter xylinum co kha
nang tao ra mang mong trong moi trng tnh sau khi x ly
vi glutaraldehyde va co nhng tnh chat c hoc nhat nh th
co the co nh trc tiep vao ien cc Oxy ma khong can
dung mang tham thau.
3.3.2.
Co nh cac tac nhan mien dch:
Nhieu ien cc mien dch c tao ra bang cac co nh cac

tac nhan mien dch len cac bo bien nang khac nhau.
a) Co
nh
antibodies):
khang
the
(Immobillization
of
Muc ch: e phat hien ra khang nguyen tng ng tao ra

khang the. Do khang the co cau truc la protein nen phng
phap co nh khang the tng t nh phng phap co nh
enzym: dung phng phap co nh trc tiep hay s dung mang
membrane.
Co nh trc tiep len bo bien nang:
Cac khang the co the c gan len mot ien cc carbon
bang lien ket cong hoa tr. Cac nhom COOH c tao ra tren
ien cc carbon bang phan ng ien hoa trong moi trng
acid nitric HNO3 va kali bicromat K2Cr2O7 va gan vi khang the
bang mot tac nhan tao lien ket ngang nh carbodiimide.
Hoac co the co nh bang phng phap hap phu khang
the len cac lp kim loai vang hay bac lang tren thuy tinh.
Sau o dung phng phap do cong hng gen nguyen sinh be
mat.
S co nh bang mang membrane:
Cac khang the cung co the c co nh len cac mang
membrane theo trnh t nh sau: au tien mang bromoacetylcellulose c ngam trong dung dch cha hexamethylene
diamine, sau o ngam trong dung dch diepoxy butadiene, dung
dch nay tao ra cac nhom se phan ng vi khang the, cuoi
cung mang nay vi khang the a uc co nh c ra
vi dung dch ethanolamine e loai bo nhng nhom epoxy
khong phan ng.
Khang the cung co the c co nh trong cac mang nylon
hoac hap thu vat ly bang cac mang collagen va
polyvinylbutyral, sau o c lap vao ien cc ISFET. Mang nay
c hoat hoa vi glutaraldehyde trc khi ket hp vi khang
the. Cac khang the cung co the c co nh len si quang
hoc.
b) S co nh khang nguyen:
Khang nguyen la cac hp chat bao gom protein,
hydratcarbon,chung c co nh bang nhng cach khac
nhau, khong nh khang the va enzym. Yeu to quyet nh tnh

khang nguyen dinitrophenol (DNP) co the c ket hp vi
mot chat mang ion dibenzo-18-crown-6 (DB18C6) va sau o c
co nh trong mang polymer. au tien, ether nay c chuyen
thanh dan xuat dang trans-dinitro va sau o thanh dan xuat
arylaminey thch hp cho viec ket hp vi fluorodinitrobenzene.
Phc hp nay c hoa tan trong tetrahydrosulfan va them
dibutylsebacate, sau o ket hp phc hp nay trong mang
polyvinylchloride (PVC). Cuoi cung mang khang nguyen c
chia thanh nhng a nho c gan len tren nh cua ien
cc. Cac biosensor tao ra nhay cam trc tiep vi cac khang
the. Tuy nhien, ien cc tao ra bang cach nay van co nhc
iem o la DNP co mot ai lc oi vi ion K + cua dung dch
chat ien phan ben trong ien cc, do o cho ket qua khong
chnh xac. e han che ieu nay, mot ien cc ran vi nha
epoxy nhoi a granite c che tao. au tien, ngi ta o nay
nha thong vao ben trong ong PVC cha mot da bang Pt
cung ng knh vi ong, sau o o dung dch tetrahydrofuran,
10 mg/ml DNP-khang nguyen va 15% triocyl acetate vao trong lo
khoan cua nha thong.
Hnh 3.6: S co nh khang the len mang membrane

bromo-acetylcellulose
Hnh 3.7: ien cc khang nguyen dung PVC ran vi

DNP
c) S gan enzym:
Khi mot tac nhan mien dch c co nh len bo bien

nang hay len mang membrane th s ket hp mien dch gia
khang nguyen va khang the xay ra nhng kho c phat
hien bi bo bien nang, thong thng phan ng mien dch
co the c do khi bo sung vao enzym e xuc tac qua trnh
phan ng. Khang nguyen hay khang the c gan vi mot
enzym s dung ky thuat EIA (mien dch enzym) hay ELISA. Enzym
c gan vi tac nhan mien dch bang tac nhan tao lien
ket ngang hai chc glutaraldehyde.
Tuy thuoc vao tng loai bo bien nang ma gan khang
nguyen hay khang the vi enzym cho phu hp: Neu s dung
bo bien nang la ien cc pO2 th khang nguyen c gan
vi enzym glucose oxidase hoac catalase. Khi bo bien nang la
ien cc pNH3 khang nguyen c gan vi enzym urease.
3.3.3.
Co nh mo, c quan va chemoreceptor:

a) C quan va mo thc vat ong vat:
au tien, mot lat mo thc vat hay ong vat c cat

ra, va chen vao gia hai mang ban tham roi gan chung
len bo bien nang. Thng dung ien cc pO 2, pCO2, hay pNH3.
Mo ong vat co the la gan bo, gan tho, bap tht tho hay c
ruot, hay thanh phan te bao. Cac c quan nay c gan vao
bo bien nang se lam cho Biosensor chon loc hn bi v chung
cha nhng enzym ac biet.
Biosensor gan mo thc vat ong vat nhn chung co thi
gian ap ng dai hn Biosensor gan enzym. Thi gian ap ng
nhanh cua Biosensor nay co the at c neu nh gan cac
mo nay trong bot than dang paste nham tao ra s tiep xuc
ln hn gia chat xuc tac sinh hoc va thanh phan cam
ng: Cat nhng lat mo va nhoi vao trong ho va, sau o
tron vi dau khoang va bot than ch e tao hon hp nhao
va cuoi cung trai eu len au ien cc.
b) Chemoreceptor:
Receptor la rau nho cua loai cua xanh Callinectes sapidus
co si than kinh c s dung nh bo bien nang e truyen
dan xung ong than kinh gay ra bi khu giac hay v giac,
chung c co nh trc tiep vao ien cc do Pt cua ien
cc o ien the. ien cc so sanh Ag/AgCl c nhung vao
dung dch muoi, va se tao ra s chenh lech ien the so vi

ien cc do. Bang viec dung cac lieu tac nhan kch thch
khac nhau vao trong dong chat mang se gay ra nhng thay
oi ve cau tao cua receptor va ket qua tao ra mot s thay
oi ve kha nang tham cua mang kch thch hay s thay oi
hoat tnh enzym c gan len mang. S phan cc cua si truc
axon se lam tang ien the o c.
Biosensor da tren cac receptor than kinh co the dung e
phat hien oc to hay cac chat oc hoa hoc a s dung
trong chien tranh. S dung receptor acetylcholine co the xac
nh acetylcholine bang cach do tr khang ac trng so vi
cac bo truyen than kinh khac, receptor nay c gan enzym
acetylcholinesterase nhay cam vi hp chat phosphor hu c va
carbamate, receptor c co nh len bo bien nang thch hp
bang cach ngam au nhay cam cua ien cc vao trong dung
dch liposome cha receptor.
CHNG 4
IEN CC ENZYM
4.1. NGUYEN TAC HOAT ONG:
ien cc enzym hoat ong nh sau:
Chuyen c chat t trong dung dch vao trong mang enzym
Khuech tan c chat ben trong mang enzym, kem theo
qua trnh bien oi c chat thanh san pham nh phan
ng co enzym xuc tac.
Chuyen san pham ve pha bo bien nang
Chuyen oi nong o san pham be mat phan cach
gia bo bien nang va san pham thanh tn hieu ien
nh bo bien nang.
Hnh 4.1: Nguyen tac hoat ong cua ien cc

enzym
S : c chat
P: san pham
Lp mang enzym cang mong th s can bang nong o c

chat ben trong va ben ngoai lp mang enzym cang nhanh
at c. Dung dch mau can c khuay ao ong eu e

am bao c chat la ong nhat.
4.2. C S LY THUYET:
S xuc tac cua enzym thng c thc hien bang cach tao
ra t nhat mot phc hp tam thi gia enzym va c chat. Mo
hnh n gian nhat la mo hnh cua Michaelis. Mo hnh nay
tha nhan rang phan ng ch xay ra vi mot c chat va
tao ra ch mot san pham:
E + S
k+1
ES
k-1
d [ P]
d[S ]
k+2
+ P
[S ]
Toc o phan ng: V dt dt Vm K [ S ]

m
Vi Km la hang so Michaelis, K m
(1)
(2)
k 1 k 2
,
k 1
Vm la toc o phan ng cc ai khi [S] >> K m, Vm = k+2 [E0]

[E0]: nong o enzym ban au.
[S]: nong o c chat
[P]: nong o san pham.
oi vi enzym a biet, toc o phan ng V la ham cua ty
[S ]
so [S]/Km. Khi [ S ] / K m 0 , th V Vm K . Khi [ S ] / K m th V Vm .

m
Ben trong lp mang enzym, toc o phan ng bao gom ca
toc o khuech tan nhieu c chat trong dung dch:
Vm [ S ]
[ S ]
2 [S ]
DS
0
2
t
K m [S ]
x
Vm [ S ]
[ P ]
2 [ P]
DP
0
2
t
K m [S ]
x
(3)
(4)
Vi t: thi gian phan ng,

DS, DP la he so khuech tan cua c va san pham ben
trong lp mang enzym
x: khoang cach gia mot iem cho trc trong mang
enzym va be mat ngoai cua ien cc.
4.2.1.
nhanh:
S tra li tn hieu trang thai
Phng trnh (3) va (4) cho biet nong o cua c chat va

san pham be mat bo bien nang phu thuoc vao hang so
Michaelis Km, van toc phan ng cc ai V m cua lp mang
enzym, be day cua lp mang va he so khuech tan D S, DP.
Gia thiet rang nong o cua san pham be mat ngoai
cua ien cc bang khong (do s khuech tan ch xay ra
trong lp mang enzym. Nhiet o, V m, Km, DS, DP xem nh khong
oi trong suot toan bo lp mang. ong thi gia nh rang
bo bien nang khong lam that thoat san pham hay c chat.
Ta co: s
V e2
[S ]
[ P]
t
x
,p
, m , t ' 2
, x'
Km
Km
K m DS
e
e / DS
Vi e: be day cua lp mang enzym.

Khi o phng trnh (3) va (4) tr thanh:
s 2 s
s
0
2
t ' x '
1 s
(3)
p DP 2 s
s
0
2
t ' DS x'
1 s
(4)
giai oan nay, phan ng xay ra nhanh chong, co s

ket hp gia c chat va enzym e tao ra phc hp enzym - c
chat.
Nong o c chat va nong o san pham trong mot n

v the tch dung dch la Km, thi gian khuech tan cua c chat
qua be day cua lp mang la e2/DS. thi gian t = 0, nong
o c chat va nong o san pham trong mang bang 0. Nong
o c chat trong dung dch luon luon gi khong oi va bang
Km.
oi vi mang enzym:
Vm = 1,92.10-6 mol.cm-3.s-1
Km = 2,1.10-5 mol.cm-3
e = 2,5.10-3 cm
DS = DP = 1,2.10-5 cm2.s-1.
Thi gian e at ti trang thai on nh: t = 0.7. e 2/DS = 36
giay.
Ty so e2/DS xac nh thi gian ap ng cua Biosensor bi v
no bieu dien thi gian ma lp mang enzym at en trang
thai on nh. Co the giam thi gian nay bang cach tang
tnh tham cua mang oi vi c chat (tang D S) hay giam be
day cua lp mang (giam e), trong o giam e la phng phap
hieu qua nhat. Nhng giam e se anh hng en tnh chat
vat ly cua mang enzym. Ngoai ra thi gian ap ng cua
Biosensor luon ln hn thi gian ap ng cua bo bien nang.
4.2.2.
S tra li tn hieu trang thai on nh:
Trong giai oan on nh, thi gian ap ng cua Biosensor

phu thuoc rat nhieu vao ty so [S]/K m. Khi [S]/Km ln, toc o
phan ng tien ti gia tr cc ai V m, ty le thuan vi nong
o enzym co nh. Sau o toc o phan ng oc lap vi
nong o c chat va nong o san pham be mat tiep
xuc vi bo bien nang khong oi khi nong o c chat ln.
Hnh 4.2: o th bieu dien nong o san pham theo

nong o c chat ng vi cac gia tr khac nhau.
ng cong bieu dien moi quan he phu thuoc gia nong o
c chat va nong o san pham co ac iem:
Tuyen tnh nong o c chat nho hn 0.1K m, khi tang
th vung tuyen tnh cung tang theo
o doc cua ng tuyen tnh khong phu thuoc vao gia
tr .
Khi u ln, ng tuyen tnh nay dan at en ng
thang tai iem co nong o [P]e = [S]o.
Vung tuyen tnh nay phu thuoc rat nhieu vao chat
lng cua enzym s dung.
Vung tuyen tnh nay tng ng vi ong hoc enzym bac
mot theo nong o c chat, ngc lai vung co nong o
cao hn th tng ng vi ong hoc bac khong.
a) ong hoc phan ng enzym bac I:
ong hoc enzym xay ra khi [S] << K m, khi o, phng trnh (3) va
(4) at en trang thai on nh:
DS
2 [ S ] Vm
[S ]
Km
x 2
[ S ] [ P ]
0:
t
t
(6)
DP
V
2 [ P]
m [S ]
2
Km
x
(7)
Nong o san pham bo bien nang c tnh theo cong

thc:
1
[ P]e [ S ]o 1
cosh(e)
(8), trong o = Vm/(DSKm) va cosh(e) la
ham cos hyperbolic cua (e).

Nong o san pham [P]e ty le thuan vi nong o c chat
[S]o
b) ong hoc bac 0:
ong hoc bac 0 xay ra khi [S] >>K m trang thai on nh va
phng trnh (3) va (4) tr thanh:
2 [S ]
DS
Vm
x 2
DP
2 [ P]
Vm
x 2
(10)
(11)
khi o [P]e = Vme2/2DP, nong o c chat c do bi bo bien

nang th oc lap vi ca nong o c chat va K m, khi nong o
c chat [S]>>Km th biosensor khong con kha nang xac nh c
chat nhng co the xac nh chat c che, chat nay anh
hng en toc o phan ng cc ai Vm.
4.2.3.
Chat c che enzym:
Chat c che (chat km ham) enzym la nhng hp chat

hoa hoc co hnh dang va cau truc tng t nh c chat, se
lam cham qua trnh phan ng enzym.
Chat c che c chia lam ba loai:
Chat km ham canh tranh (Competitive inhibitors)
Chat
competitive inhibitors)
inhibitors)
km
ham
khong
canh
tranh
(Non
Chat km ham phi canh tranh (Uncompetitive
Hnh 4.3: S o cac kieu km ham phan ng enzym

a) Chat km ham canh tranh: ( Competitive inhibitors)
E
+
I
ES
KI
EI
Trong o KI la hang so phan ly cua phc hp EI. Toc o

phan ng k hi co mat chat c che, VI c xac nh:
V I Vm
[S ]
[I ]
K m 1
[ S ]
KI
at i = [I]/KI va s = [S]/Km th khi o VI Vm
s
.
1 i s
Mc o km ham cua phan ng c ac trng bi ai lng

1
VI
Ma V = Vm [s / (1+s)] nen
i
. Do o co the giam o km
1 i s
ham neu nong o c chat tang. S km ham canh tranh co

the b loai bo neu nh d nong o c chat.
b) Chat km ham phi canh tranh (uncompetitive
inhibitors)
Chat km ham phi canh tranh ch tng tac vi phc hp ES
theo phng trnh phan ng:
E
ES
+
I
KI
ESI
trang thai on nh, ta thu c:

VI
Vm
[S ]
s
Vm
[I ]
Km
1 s si
1
1
[
I
]
KI
1
KI
Do o o km ham c tnh theo cong thc:
si
, gia
1 s si
tang theo nong o c chat [S] khi cung nong o chat km

ham [I].
c) Chat km ham khong
competitive inhibitors)
canh
tranh
(non
Chat km ham khong canh tranh ket hp vi ca enzym

va phc hp ES tai mot v tr khac vi tam hoat ong cua
enzym.
E
+
I
K'I
KI
EI
ES
+
I
ESI
E + P
Ket qua tao ra 3 phc hp ES, EI, ESI ma ch co ES tao ra san

pham cua phan ng. Chap nhan rang tng tac qua lai gia
enzym va chat canh tranh khong b anh hng bi c chat, do
o KI ~KI, khi o toc o phan ng km ham enzym la:
VI
Khi o
Vm
[S ]
s
Vm
[I ] K m [S ]
1 i s is
1
KI
i
, oc lap vi nong o c chat. Do o loai chat
1 i
km ham nay khong c loai bo bi s d tha c chat.

[I ]
Dung ai lng r 1 K e ac trng cho phan ng km ham

I
enzym
Khi o dung KP la hang so toc o phan ng km ham, V P la
toc o phan ng km ham cc ai.
KP = r.Km va VP = Vm (phan ng km ham canh tranh)
KP = Km / r va VP = Vm /r (phan ng km ham phi canh tranh)
KP = Km va VP = r.Vm (phan ng km ham khong canh tranh).
d) ieu khien phan ng km ham:
Khi enzym c co nh len chat mang ran, phan ng xay ra
trong pha d the do tam hoat ong cua enzym nam trong pha
ran cua chat mang va c chat nam trong pha long cua dung
dch. Nong o c chat khac nhau tai moi iem trong chat
mang, do o toc o phan ng enzym khong giong nhau. Nong
o c chat trong lp mang enzym luon thap hn so vi trong
dung dch.
Nh vay e loai bo anh hng cua chat km ham en phan
ng enzym, can co s d tha nong o c chat, nong o
enzym cao va o tinh sach cung nh tnh ac hieu cua enzym.
e) S ap ng tnh hieu cua ien cc enzym khi
co mat cac chat km ham:
o th log log mo ta moi quan he gia nong o san pham
theo nong o c chat theo cac kieu kiem ham khac nhau:
o th a) : Km ham canh tranh.

Toc o phan ng khi co mat chat km ham canh tranh oc
lap vi nong o chat km ham thuoc vung co ong hoc
bac 0 va c mo ta theo phng trnh:
Vm .e 2
log[ P] e Log
2D
Chat km ham canh tranh ch phu thuoc vao gia tr K m ma

khong anh hng gia tr cc ai [P] e. Ngc lai, vung tuyen
tnh co ong hoc bac I, theo phng trnh:
log[ P ] e log[S ] 0 log 1

cosh(e)
vi (e)2 = = Vm.e2/ KmD.
Cac chat km ham canh tranh lam tang gia tr K m, giam va

do o lam giam log[P]e.
o th b): km ham phi canh tranh
Khi co mat chat km ham phi canh tranh th khong lam thay
oi ty le Vm/Km, do o vung tuyen tnh co ng cong ap
ng la nh nhau oi vi moi gia tr r. Ngc lai, nhng chat
nay lam giam vung ong hoc bac 0 qua viec giam gia tr V m
xuong Vm/r.
Hnh 4.4: S tra li tn hieu cua ien cc enzym theo

cac kieu km ham khac nhau
o th c): km ham khong canh tranh:
Chat km ham nay ch anh hng en gia tr (giam)
thong qua Vm, do o vung tuyen tnh va vung ong hoc bac 0
moi gia tr r la khac nhau.
Nh vay qua viec quan sat ng cong tra li cua ien

cc o ien the se xac nh c kieu km ham phan ng
enzym
4.3. CAC YEU TO ANH HNG EN S TRA LI

CUA IEN CC ENZYM
4.3.1.
Thi gian ap ng:
Thi gian ap ng la thi gian at c trang thai on

nh cua biosensor, cung la thi gian at c trang thai ma
tai o nong o c chat va nong o san pham la hang so.
Hnh 4.5: S tra li tn hieu cua ien cc

acetylcholinesterase cac nong o c chat khac nhau
Gia tr nay rat kho anh gia chnh xac do phu thuoc
vao nhieu yeu to nh bo bien nang, s ao tron, phng
phap co nh enzym. Neu s dung mang enzym urease co
nh len ien cc cation hoa tr I th cho thi gian tra li di
1 phut. Nhng neu co nh no len ien cc pCO 2 cho thi gian
dai hn 2-3 phut. o la do khi dung ien cc pCO 2 th xay ra
s khuech tan CO2 qua mang tham kh. S dung phng phap
co nh bang lien ket ngang se tao ra mang enzym cc mong
1 2m cho thi gian tra li ch trong vai giay en vai phut.
Khuay ao mau e tang kha nang khuech tan kh cung
giam c thi gian ap ng.
4.3.2.
Cac yeu to anh hng en s ap ng tn
hieu cua biosensor:
o ben cua ien cc enzym phu thuoc rat nhieu vao cac
thong so vat ly va hoa hoc.
Vat ly: o day cua mang enzym: mang cang mong th
o ben cang tang
Nong o enzym cang ln th thi gian tra li cang
toi u
Be mat bo bien nang
Hoa hoc: phng phap co nh enzym: enzym co nh
len mang tham thau t ben hn enzym co nh trong
mang gel. Enzym co nh bang lien ket ngang cho ien
cc enzym co o ben nhat
a) Anh hng cua pH:
V pH anh hng en hoat tnh cua enzym va pH anh
hng en s phan ly san pham cua phan ng enzym c
phat hien hien bi bo bien nang nen pH cung anh hng
en s tra li tn hieu cua ien cc enzym.
Hoat tnh enzym giam co the ieu chnh bang cach
tang nong o enzym trong lp mang, khi o c chat b bien
oi mc o ln nhat nen ien cc enzym t phu thuoc
vao pH.
Hnh 4.6: S phu thuoc cua tn hieu tra li vao pH cua

ien cc AchE 25oC
b) Anh hng cua nhiet o:
Khi nhiet o tang se lam cho hoat lc xuc tac cua enzym e
tang do o toc o phan ng cung tang, nhng nhiet o cao
lam bien tnh enzym.
Tang nhiet o se lam tang toc o khuech tan cua cac
chat hoa hoc khac nhau trong mang enzym do o lam giam
thi gian tra li tn hieu.
Trong vung nhiet o t 10 40 oC: tn hieu tra li tng oi on
nh, tren 40oC, hoat tnh enzym b bien tnh do o tn hieu tra
li se giam nhanh chong, nhiet o di 10oC hoat tnh enzym
b yeu nen tn hieu tra li thap.
Do o ien cc nen hoat ong nhiet o khoang 30 oC
la toi u.
Hnh 4.7: S phu thuoc tn hieu tra li cua ien cc

AchE theo nhiet o nong o Acetylcholin 10-2M
c) Anh hng cua cac hp chat khac:
Anh hng ti bo bien nang:
Mot bo bien nang co the c dung e xac nh nhieu
chat khac nhau nh co the dung ien cc pNH 4 trong o nong
o ure, nhng no se phat hien cac ion NH 4+, Li+, Na+, K+. Do o
lam anh hng en tnh chnh xac cua phep o.
V vay, tuy san pham tao thanh ma dung bo bien nang
nhay cam ac biet vi san pham cho phu hp. Trong trng
hp tren nen dung ien cc pCO 2 e loai bo bt anh hng
cua cac hp chat ion.
Anh hng en hoat tnh enzym:
Trong mau thng co chat c che cung nh chat kch thch
phan ng enzym, khi o chung se km ham hay gia tang toc
o phan ng, do o anh hng en tn hieu tra li cua
ien cc enzym. Neu trong mau co mat chat c che th co
the loai bo chung bang cach tang nong o enzym trong
mang enzym.
Khi xac nh nong o ure trong mau co mat chat c che
NaF, neu ien cc dung 5 n v hoat o urease trong 1 mm 3 th
se khong b anh hng en s tra li tn hieu cua ien cc
bi nong o NaF ti 5.10-2M, con neu dung 0.5 n v hoat

o tren 1 mm3 th tn hieu tra li b anh hng {NaF] =
3.10-3M.
Hnh 4.8 Nong o c chat va nong o san pham theo

nong o enzym trong lp mang enzym
o th a): Lp enzym co nh co nong o thap:
Nong o c chat giam khi khuech tan vao trong bo bien
nang, nong o nay co gia tr [S]e be mat bo bien nang,
san pham khuech tan t lp mang enzym vao be mat bo
bien nang [P]e luon nho hn nong o c chat ban au [S] o.
trang thai on nh, tai moi iem trong mang enzym ta luon co
[S] + [P] = [S]0.
o th b): Lp mang enzym co nong o cao:
Khi nong o c chat cao, toc o bien oi c chat nhanh, do
o phan ng ch xay ra mot phan cua mang enzym.
Cuoi mang, nong o c chat [S] e = 0, vung nay goi la
deadcore. Xung quanh vung nay, enzym co hoat tnh khac so
vi hoat tnh enzym co nh, nong o san pham tien ti gia
tr [P]e va gia tr nay bang vi [S] 0 trong mau . Do o khong
co s anh hng cua chat c che trong vung nay nen co
the loai bo anh hng cua chat km ham bang viec dung
nong o enzym cao trong mang.
Hoat tnh cua enzym cung b anh hng neu dung enzym
khong ac hieu. Cho nen can s dung cac enzym co tnh
ac hieu cao vi c chat cung nh tinh khiet khi co nh len
mang enzym.
Anh hng en c chat:
Khi co mat cac chat khac, no se lam giam kha nang
khuech tan cua c chat vao trong mang enzym, cung nh km
ham qua trnh phan ng enzym, do o can loai bo cac chat
nay ra khoi mau trc khi phan tch, hoac s dung bo bien
nang ac biet nhay cam vi chat can phan tch.
Bang 4.1: Mot so ien cc enzym o ien the

Chat
phan
tch
Enzym (E)
Ky thuat co nh
Bo bien
nang
E + polyacrylamide
pNH4
E + BSA + GA
pNH4
E + polyacrylamide
Ure
Glucose
Acid amin
Urease
Glucose
oxidase
L amino acid
oxidase
E + ong polymer
methacrylamide
acrylamide
E + BSA + GA
(E) hoa tan/mang
tham thau
E + BSA + GA
E + BSA + GA
E + GA
(E) hoa tan/ mang
cellophane
E + BSA + GA
Thi gian
ap ng
25 60
giay
30 giay 1
phut
30 40
giay
pNH4
(nonactine)
pH
(thuy
5 10 giay
tinh)
PH (
1 1.5 phut
Vung
tuyen tnh
(M)
5.10-5 10-2
10-4 10-2
10-5 2.10-2
5.10-5 5.10-4
2.10-4 2.10-3
pCO2
1 5 phut
10-4 10-1
pCO2
pCO2
pNH3
2 3 phut
1 2 phut
1.5 2 phut
3.10-4 10-2
3.10-4 10-2
10-4 10-2
pNH3
5 8 phut
5.10-4 7.10-2
pNH3
2 3 phut
26 38
giay
3.10-4 10-2
E / nylon
pNH3
0.2 10 mg/dl
E + BSA + GA
Pt Ir
1 150 mg/dl
E + polyacrylamide
pNH4
3 5 phut
2.10-4 2.10-3
Glutamin
Tyrosin
Lysin
Phenyl
alanin
Methionin
Histidin
Aspartam
Amygladi
n
Penicillin
Adenosin
Acetylchol
in
Nicotine
D amino acid
oxidase
L amino acid
oxidase
Glutaminase
Tyrosine
decarboxylase
Lysine
decarboxylase
Phenylalanin
ammonia lyase
Methionin lyase
Histidin
ammonia lyase
Carboxypeptida
se A, Laspartase
E + polyacrylamide
pNH4
E + BSA + GA
pNH4
E / nylon / cellophan
E / mang tham thau
1 2 phut
pNH4
pNH3
30 giay-1
phut
1 2 phut
4 5 phut
10-4 10-1
10-42.10-3
E / giay tham thau
pCO2
1 5 phut
3.10-45.10-3
E + BSA + GA
pCO2
5 7 phut
10-43.10-3
E / vi lo
pNH3
12 phut
10-46.10-4
E + BSA + GA
pNH3
3 phut
10-5 10-2
E + BSA + GA
pNH3
3 8 phut
10-5 10-2
4 8 phut
4,25.10-4
8,1.10-3
E + BSA + GA
pNH3
- glucosidase
E + polyacrylamide
pCN
Penicillinase
E + polyacrylamide
E + BSA/mang tham
thau
pH
10 30
phut
2 3 phut
pNH3
7 - 12 phut
Adenosine
deaminase
Acetylcholin
esterase
10-4 - 5.10-3
10-4 10-2
4.10-510-3
4.10-510-3
7.10-7-10-2
E + BSA + GA/collagen
pNH3
5 14 phut
E + GA
pH
1 phut
3,5.10-5
1,6.10-2
10-4 3.10-3
E + polyacrylamide
pH
1 3 phut
2.10-5 10-3
E + BSA + GA
pH
2 3 phut
10-3 10-1
Neostigmi
n
Physotigm
in
Fluoride
Ag+
Hg2+
Thuoc
tr sau
(TTS)
H2O 2
Arginase
Urease
Acetylcholin
esterase
Urease
E + BSA + GA
pH
2 3 phut
5.10-7 5.10-5
E + BSA + GA
pH
2 3 phut
3.10-8 3.10-6
E + BSA + GA
pCO2
2 5 phut
3.10-4 10-2
E + BSA + GA
pH
2 3 phut
10-4 2.10-2
E + BSA + GA
E + GA
pCO2
pCO2
1 2 phut
1 2 phut
10-8 3.10-7
2.10-8 2.10-6
Cholinesterase
E + BSA + GA
pH
2 3 phut
10-8 10-5
(tuy loai TTS)
Peroxidase
urease
E + BSA + GA/collagen
E + polyacrylamide
pF
pNH4
< 10 phut
8.10-5 10-2
1.6 16 UI
Bang 4.2: Mot so ien cc enzym o dong ien:

Chat
phan
tch
Glucose
Enzym (E)
Ky thuat co nh
Bo
bien
nang
Glucose
oxidase
E + polyacrylamide
pO2
E + BSA + GA
E/nylon
E+
polyacrylamide/milipore
E + gelatin + GA
E + polyacrylamide
E/collagen
E + BSA + GA
Thi gian
ap ng
Vung
tuyen tnh
(M)
pO2
pO2
30 giay 3
phut
1 3 phut
26 38 giay
0 2.10-2
1 20mg/dl
pO2
1 2 phut
0 200 mg/l
pO2
Pt
Pt
Pt
2 3 phut
1 phut
2 3 phut
10 giay
0 2 g/l
0 1.5.10-2
10-7 2.10-3
10-7 2.10-3
0 0.5 g/l
Glucose
dehydrogenase
Alcohol
dehydrogenase
ethanol
Tinh bot
Cholin
Alcohol oxidase
catalase
a-amylase
glucose
oxidase
Cholin oxidase
E + silan + GA
E + p-benzoqiunon +
BSA/cellulose
E + silan + GA/silicon
E + acrylamide/mang
tham thau/ mang
silicon
E + BSA + GA
E /mang ban tham
E/cellophan
E + ferrocene graphite
E + NMP+ TCNQ-
SnO2
10 phut
10-4 10-2
Pt
0.5 - 3 phut
10-6 3.10-3
Pt
2 3 phut
0 6 g/l
Pt
1 10 phut
0 2,5.10-2
Pt
Pt
Pt
Graphite
Pt
1 2 phut
6 phut
3 4 phut
1 1.5 phut
-
0 2.7.10-2
10-5 7.10-3
5.10-4 6.10-3
10-2 3.10-2
0 2.6.10-3
E + BSA + GA
pO2
3 15 phut
8.10-4 10-3
E/mang tham thau
Pt
5 10 phut
E /collagen
Pt
30 phut
E + GA
pO2
1-2 phut
0 25 mg/l
E + BSA + GA
Pt
2 3 phut
E/nylon
pO2
1 - 2 phut
2.10-52.10-4
CHNG 5
NG DUNG CUA BIOSENSOR
5.1. NG DUNG TRONG CONG NGHE THC PHAM:
Trong nganh thc pham, viec kiem tra chat lng thc pham
da tren cac phng phap truyen thong thng mat nhieu
thi gian t vai gi en vai ngay. Nhng vi ky thuat dung
biosensor a mang lai cho nganh cong nghiep thc pham mot
thiet b theo doi va o lng nhanh, o nhay cao, s dung
thuan li hn so vi nhng phng phap truyen thong.
Bang 5.1: Mot so ng dung cua biosensor trong cong nghiep
thc pham:
Hp chat
can o
Cac acid
amin
Cac hp
chat amin,
amid, d
vong
Hydratcarbon
Acid hu c
Cac ru
va cac
phenol
Cac hp
chat khac
ng dung phat hien (nh tnh va nh

lng)
Alanin, arginin, asparagin, acid aspartic, cystein,
glutamin, acid glutamic, glutathiol, histidin, leucin,
lysin, methionin, N-acetyl methionin, phenylalanin,
sarcosin, serin, tyrosin, tryptophan, valin
Aminopyrin, anilin, amin thm, acetyl cholin, cholin,
phosphatidyl-cholin, creatin, guanidin, guanosin,
penicilline, spermin, creatin, acid uric, ure, xanthyl,
hypoxanthin.
Amygdalin, galactose, glucose, glucose-6-phosphat,
lactose, maltose, sacharose, tinh bot.
Acid acetic, acid formic, acid gluconic, acid isocitric,
acid ascorbic, acid lactic, acid malic, acid oxalic, acid
pyruvic, acid succinic, acid nitrylacetic.
Acetaldehyde, bilirubin, catechol, cholesterol,
cholesterol ester, ethanol, glycerol, glycerol ester,
methanol, phenol.
Chat khang sinh, o ti cua ca tht, vitamin
a) o nong o glutamin va nong o glutamat
Glutamat trong thc pham khong ch anh hng en mui v

cua thc pham ma con lien quan en kha canh an toan ve
sinh thc pham. Cac loai o hop tht ca , o hop nc xup,
dau giam, nc sot c bo sung mot lng mono natri
glutamat co tac dung tang mui v, nhng mot lng ln
glutamat co the gay ra nhng dau hieu benh ly nghiem
trong anh hng sc khoe con ngi nh: tim ap nhanh, au
da day.
e nh lng glutamat, s dung ien cc o dong ien co
gan mang enzym co nh glutamat oxydase. Glutamat oxydase
cung co the c ong co nh vi glutaminase e o nong
o glutamin da tren phan ng:
glutamin
glutamat
glutaminase
glutamat oxydase
glutamat
NH3
- ketoglutarat + NH3 + H2O2
Viec o nong o glutamin co y ngha rat quan trong bi

o la ch tieu anh gia nguon nit cua moi trng nuoi
cay vi sinh vat cong nghiep.
Glutamat oxydase co hoat tnh toi u tai pH = 7.8, con
glutaminase t E.coli lai hoat ong tai vung acid. Neu thay the
bang glutaminase trung tnh t Bacillus th ca hai enzym nay eu
co hoat tnh toi u tai pH = 7 va co tnh chu nhiet tot. Mang
enzym tao thanh rat on nh va co hoat tnh tot c s
dung trong thng mai.
Mang enzym cho ket qua o at tuyen tnh en 8mM
glutamin, khong ap ng vi cac chat khac (tr glutamat),
va gi c trang thai kho trong thi gian t nhat 6 thang
4oC, thi gian ap ng 30 giay nhanh hn rat nhieu so phng
phap sac ly long cao ap HPLC.
b) o nong o cholin:
Trong c the, cholin la thanh phan cua phospholipid cau tao
nen mang te bao va mang cua nhieu bao quan trong te
bao, mat khac cholin con la tien chat e tong hp nen
acetylcholin (chat co vai tro chuyen s kch thch than kinh),
ong thi cung la nguon cac nhom methyl trong c the.
Cholin thng c bo sung vao thc an cho tre em va cac

san pham dinh dng cho ngi ln.
Viec xac nh nong o cholin co the thc hien bang
phng phap theo doi s phat trien cua vi sinh vat tren moi
trng c chat, bang phng phap quang pho hay sac ky. Tuy
nhien, cac phng phap nay thng t chnh xac, thi gian dai
va gia thanh cao. Vi s s dung ien cc enzym co gan
cholin oxydase c che tao da tren c s phan ng oxy hoa
se cho thi gian nhanh, chnh xac hn rat nhieu.
Phan ng oxy hoa xay ra trong ien cc:
cholin + O2
cholin oxydase
betain aldehyde + O2
H2O2 +
H2O2
betain aldehyde
+
betain
Gia tr cua phep o at tuyen tnh trong khoang t 5mg/l

en t nhat la 160 mg/l khi s dung dung dch cholin hydroxyde
nong 111mg/l lam chuan. Thi gian thu c ket qua ch sau
30 giay va gia re.
c) o ham lng con:
Ham lng con c xac nh bang cach s dung alcohol
oxydase:
ru
+ O2
alcohol oxydase
H2O2
+ aldehyde
Tuy nhien mot han che cua ien cc nay la tnh khong
ben cua alcohol oxydase dang kho, do o can co bien
phap bao quan thch hp e on nh hoat tnh enzym.
d) Phat hien s co mat cua vi khuan co mat trong
thc pham:
Dung ien cc sinh hoc phat quang e phat hien s co
mat cua vi khuan trong thc pham khi vi khuan nay tac dung
ac hieu vi ATP va D-luciferin e giai phong ra anh sang

phat quang nh tac dung xuc tac cua enzym luciferase:
ATP
D-luciferin
luciferase
oxy luciferin + AMP + CO2 + (562nm) a

nh sa
ng va
ng
e) ien cc mien dch o ham lng albumin, insulin:

ien cc mien dch ket hp vi ien cc o dong ien
e xac nh insulin, albumin:
Cho khang the khang insulin a biet (lay t ln) len tren
mot lp mang a gan trong ien cc Oxy e cac khang the
co the bam tren lp mang o, sau o cho insulin can kiem
tra len mang va cho tiep khang nguyen (insulin) a gan
catalase va bo sung c chat la H2O2, phan ng nh sau:
H2O2
catalase - insulin
H2O
1/2 O2
Ham lng O2 tao thanh c nh lng nh ien cc O 2

t o xac nh c ham lng insulin.
ien cc mien dch ket hp vi ien cc o quang:
Hnh 7.1 mo ta ien cc mien dch s dung bo chuyen oi
quang hoc, trong o bien oi sinh hoc tao ra ap ng quang
thong qua mot mang ien cc (sensor chip).
Khi mau cha khang nguyen i qua se phan ng ac hieu
vi khang the tren be mat sensor chip va do o lam thay
oi bc song va goc phan xa cua anh sang chieu vao
mat ben kia cua sensor chip. o cac thong so quang hoc se
cho ta nong o khang nguyen co trong mau.
f) Kiem tra ham lng cloramphenicol trong san pham
thuy san:
Dung ien cc mien dch da tren nguyen tac ELISA e
kiem tra ham lng cloramphenicol trong thuy san.
Cac gieng c phu lp khang the khang cloramphenicol

(cloramphenicol chuan hoac dung dch chuan). Cloramphenicol
gan enzym va khang the khang cloramphenicol c bo sung
vao gieng. Cloramphenicol t do va cloramphenicol gan ket
enzym se canh tranh cac v tr ket gan cua khang the khang
cloramphenicol (phan ng mien dch enzym canh tranh). ong
thi cac khang the c bo sung vao c co nh lai bi
cac khang the tren thanh gieng. Cloramphenicol ket gan
enzym b d th se c loai bo bang cach ra. Bo sung vao
gieng c chat cua enzym (ure peroxyde) va chat tao mau
(tetramethyl benzidin) va u. Enzym gan ket se chuyen hoa
chat tao sac von khong mau thanh san pham co mau
xanh. Khi bo sung chat ket thuc phan ng se chuyen t
mau xanh sang mau vang. o cng o mau tren may o
mau quang ien bc song 450 nm, th nhan thay cng
o mau ty le nghch vi ham lng cloramphenicol co trong
mau.
Giai oan u c thc hien trong may u on nh nhiet o.
Ket qua phan ng c oc bang may oc theo nguyen tac
so mau va so sanh vi o th chuan e xac nh nong o
khang sinh. Phng phap nay co the phat hien cloramphenicol
nong o 0.05ppb.
Loai thiet b nay con c s dung e kiem tra mot so
chat khac bang cac loai chat chuan khac nhau (cac kit
chuan) vi gii han phat hien rat thap.
Bang 5.2: Mot so cac kit chuan s dung va gii han phat
hien:
Loai kit
cloramphenicol
- mau sa
- mau tht, trng
- mau tom
clenbuterol
- mau nc tieu
- mau tht
diethylstilbestrol
Histamin
- ca tuyet
- bot ca
Gii han phat hien

0.15 ppb
0.10 ppb
0.05 ppb
0.10 ppb
0.04 ppb
0.02 ppb
20 ppb
125 ppb
g) Xac nh ham lng mycotoxin trong ngu coc va

thc pham
Mycoloxin la oc to sinh ra trong qua trnh phat trien cua
nam moc co mat trong cac san pham nh: lac, ngu coc, hoa
qua.chung cung co the xuat hien trong thc an gia suc. Gia
suc khi an cac thc an nay, oc to mycotoxin co the i qua
qua trnh trao oi chat roi co mat trong cac san pham nh
trng, sa, tht.
Co nhieu phng phap xac nh mycotoxin trong o phng
phap xac nh bang ien cc mien dch ang c s dung
rong rai. Phng phap nay da tren nguyen tac lien ket
ac hieu khang nguyen khang the. Mot nong o co nh
cua khang the c tron vi mau can phan tch co cha
ham lng mycotoxin cha biet, khang the va mycotoxin se tao
thanh phc. ien cc se xac nh nong o khang the t do
(khong tao phc vi mycotoxin). Da vao o th mycotoxin
chuan co the nh lng c mycotoxin co trong mau can
phan tch.
5.2. NG DUNG TRONG MOI TRNG
Cac ien cc enzym s dung trong phan tch moi trng
co the chia thanh ba nhom da vao ac iem cua mot
phan ng enzym:
Nhom th nhat : cac chat c nh lng la c chat
cua phan ng enzym.
Nhom th hai: cac chat c nh lng la cac chat
km ham hoat tnh xuc tac cua enzym. V du, cac hp
chat c phospho va cacbonat.
Nhom th ba: nong o cac ion kim loai dc phat
hien nh vao viec chung lien ket vi phan
apoenzym do o phuc hoi hoat tnh cua enzym.
a) nh lng ion kim loai nang
Cac enzym thuoc nhom metalloenzym can co cac ion kim
loai tham gia vao trung tam hoat ong e duy tr c hoat tnh
xuc tac cua enzym, do 1o khi cac ion kim loai nay b tach ra
khoi enzym ( v du, do tac dung cua tac nhan tao phc) th se
lam mat hoat tnh xuc tac cua enzym.hoat tnh xuc tac cua
enzymco the phuc hoi khi cho enzym tiep xuc vi moi trng
cha ion kim loai th ion kim loai se thu hut lai vao trung tam
hoat ong cua enzym. Do o co the nh lng c nong o
kim loai nang trong mau thong qua viec xac nh hoat ong
xuc tac cua enzym tao thanh sau khi cho phan apoenzym a
c co nh tiep xuc vi mau.
u iem cua phng phap nay la co the phat hien cac
kim loai nong o rat nho ( mol) va co the ph1t hien
c bat ky mot kim loai nao co mat trong nhom
metalloenzym.
Bang 5.3: gii thieu mot so ien cc xac nh ion kim loai
Kim
loai
Hg (II)
Zn (II)
Cu (II)
Cu (II)
Enzym
ien cc
Urease
phosphatase kiem
apoenzym:
phosphatase
Apoenzym:
galactooxydase
Apoenzym:
tyrosinase
ien cc NH4+
PH ISFET
ien cc nhiet
ien tr
ien cc
Pt/Ag/AgCl
Nong o
(mmol/l)
0 150 nm/l
0.1 1 (sai so
2.3%)
0.01 1
0.1 1 (sai so
7%)
ien cc O2
< 0.5
b) nh lng phenol:
Viec phan tch nong o cac hp chat phenol da tren phan
ng:
Phenol
O2 + 2H+
tyrosinase (polyphenol oxydase
tyrosinase
o - diphenol +
O2
o - quinon
Fe(CN)64- tyrosinase
o- diphenol
o - quinon
o- diphenol
+ Fe(CN)63-
Bang 5.4: ien cc dung tyrosinase:
ien
cc
ien
cc
graphit
vi
TCNQ
ien
cc O2
(Clark)
ien
cc O2
(Clark)
Nong o phat hien
Sai so
0.23 0.65
Thi
gian
12 35
s
1 46 (trong hexan)
4.4% (trong hexan)
<2
5 190 (trong dung dch 7.2% (trong dung dch phut
em)
em)
0.1 5 (trong dung dch
3 phut
em va cloroform
c) nh lng phosphat, nitrit, nitrat, sulfat

Cac hp chat nay ngay cang co mat nhieu trong moi
trng, ac biet la trong nc uong. S gia tang nguon nit,
phospho trong nc co the gay ra hien tng ph dng, gay
mat can bang sinh thai (theo tieu chuan EC, nong o cho
phep cua phospho la: 5mg/l P 2 O5 ). Nong o cua phospho
thong thng c xac nhbang phng phap o mau. Tuy
nhien, nong o cua phospho trong nc co the c nh
lng bang cac ien cc enzym.
Theo Kubo(1991), nong o phosphat co the xac nh bang
cach s dung ien cc pyruvatoxydase t pediococcus xuc tac
vi oxy hoa pyruvat trong ieu kien co mat phosphat va O2 e
tao thanh acetylphosphat, hydro peroxyt va CO 2. Lng O2 tieu
thu c dung e dnh lng phosphat:
CH3COCOOH
H3PO4
1/2 O2
CH3COP + H2O2 + CO2
Cac hp chat cha nitrat, nitrit co the nh lng theo phan

ng oxy hoa kh xuc tac bi nitratreductase va nitritreductase:
NO3-
2H+
NO2-
H+
NO2-
H2 O
NH4+
2H2O
OHNH3
Bang 5.5: nh lng nitrit, nitrat, sulfat

ien cc
ch th
Chat
Enzym
Nitrit
Nitrit reductase
Nitrat reductase
va nitrit
reductase
Arylsulfatase
Nitrate
Sufate
ien cc NH3
Gii han
phat hien
(mmol/l)
50
ien cc NH4+
ien cc Pt
50
100
Bang 5.6: ien cc enzym nh lng phosphate:
Enzym
Phosphatase va
glucooxydase
Nucleosiphosphori
lase va
xanthinoxydase
Nucleosiphosphori
lase va
xanthinoxydase
Pyruvat oxydase
Nong
o
(mol/
l)
Sai
so
(%)
ien
cc
ch
th
Thi gian
ap ng
(phut)
Tnh on
nh
0.01
5.9
H2O 2
5 10
> 100 th
nghiem
(TN)/ 3
thang
0.3 1
mM
10
O2
> 70 TN/ 1
thang
10 250
5.9
H2O 2
30 TN
7 ngay
(kha
nang
ap ng
giam
50%)
12 80
5.9
O2
Nucleosiphosphori
lase va
0.5 100
xanthinoxydase
O2
> 8 ngay/
300 ngay
1.5
d) nh lng cac chat oc

Cac loai thuoc tr sau phospho co oc tnh rat cao, ngoai
tac dung oc s cap, chung con lam bien oi cac qua trnh
trao oi chat cua te bao bang cach tac dung len cac
enzym thiet yeu cua te bao nh esterase, oxydase,
phosphorylase, dehydrogenase. Theo tieu chuan EC, nong o toi
a cho phep cua d lng cac chat nay trong nc uong la
0.1 g/l oi vi moi loai rieng biet va 0.5 g/l oi vi hon
hp. Viec xac nh cac chat oc nay c thc hien bang
sac ky long cao ap (HPLC) hoac sac ky khoi pho (GC/MS)
nhng thiet b at tien, phng phap phc tap va trnh o ky
thuat cao.
S dung ien cc co gan enzym cholinesterase da vao ac
iem cac chat oc nay se km ham enzym nay em lai
nhieu thuan li trong phan tch.
Qua trnh phan ng thuy phan cholin nh cholinesterase nh
sau:
CH3COS(CH2)2N+(CH3)3
Acetyltiocholin
HS(CH2)2N+(CH3)3
Acetylcholinesterase
CH3COOAcetate
HS(CH2)2N+(CH3)3
Tiocholin
S-(CH2)2N+(CH3)3
H+
Phan ng oxy hoa tao thanh H +, tren c s o co the dung

ien cc pH hay dung phan ng oxy hoa tiocholin tai ien cc
Pt e xac nh hoat o enzym.
e) Ky thuat ELISA trong xac nh d lng thuoc tr
sau
Ky thuat ELISA e xac nh d lng tr sau gom hai phng
phap cd ELISA (competitive direct ELISA ELISA canh tranh trc
tiep) va phng phap ci ELISA (competive indirect ELISA ELISA
canh tranh gian tiep).
Trong cd ELISA, khang the c co nh tren be mat cac

gieng gia the ran, cho mau thuoc tr sau co ham lng
nhat nh a c gan enzym va thuoc tr sau can
phan tch vao cac gieng nay, chung se canh tranh e
bam vao khang the. Sau mot thi gian phan ng cac
chat con d se b ra troi. Cuoi cung, c chat nay se b
enzym xuc tac va tao ra cac san pham co mau sac.
Cng o mau ty le nghch vi lng thuoc tr sau can
xac nh trong mau. Neu thuoc tr sau nhieu th lng
cong hp enzym do tnh canh tranh kem hn se b gi lai t,
mau yeu va ngc lai.
Trong ky thuat ci ELISA, cac gieng c phu mot lp
thuoc tr sau lien ket vi protein, con thuoc tr sau
can xac nh c them vao vi mot lng khang the
co nh. e mot thi gian, thuoc tr sau can phan tch
(khang nguyen) va thuoc tr sau lien ket se canh tranh
bam vao cac khang the, lng thuoc tr sau cang
nhieu th lng khang the ban au b gi lai bang cach
cho c chat vao, chuyen hoa tao ra cac san pham co
mau. Cng o mau ty le nghch vi lng chat can
xac nh trong mau ban au.
Phng phap nay co u iem la gia thanh re, de t ong
hoa, nhng nhc iem la trong mot thi gian ch phat hien
c mot loai thuoc tr sau.
f) ien cc em vi sinh vat:
Vi sinh vat c em bang th nghiem vi sinh vat, nhng thng
ton nhieu thi gian, ac biet khi xac nh vi sinh vat gay
benh nh Samonella va Listeria sp. Khi xac nh Samonella, trc
tien can 24 gi e lam moi trng, lam cho vi sinh vat can
xac nh t trang thai tnh trong thc pham sang trang thai
hoat ong. Sau o canh trng c u e Samonella phat
trien. Sau 24 gi phat trien, cay moi trng co cha vi sinh
vat sang a thach, mot hoac hai ngay sau xac nh lng
Samonella. Ket qua tm c cac loai khang the nh somatic (O)
hay flagella (H). Phng phap nay chnh xac nhng thi gian lau
gan 5 ngay, ket qua a ra ch mang tnh nh tnh.
Cac dung cu phan tch nhanh hn thuoc ky thuat ELISA a
c s dung e xac nh Samonella, Staphylococcus va cac
noi oc to: mau thc pham c x ly bang enzym e at

ti mot kch thc nhat nh, sau o c loc tren mang loc
ke o k nc HGMF (hydrophobic grid membrane filter) e chuan
b cho bc xac nh tiep theo.
Hnh 7.2: Biosensor phat hien E.coli

5.3. NG DUNG TRONG Y HOC:
ien cc enzym co rat nhieu ng dung trong viec phat hien
va chan oan benh tat. Ngay nay no con la cong cu hu
hieu e cham soc sc khoe tai nha vi rat nhieu tnh
nang tot.
a) o nong o glucose:
Glucose la nguyen nhan chnh gay ra benh tieu ng. Benh
nay se lam ngi benh mat kha nang kiem soat nong o
glucose trong mau dan en s tang qua ng huyet, co
the gay t vong neu qua t glucose en nao. Viec ieu tr
benh tieu ng oi hoi ngi benh phai c theo doi
thng xuyen ham lng glucose trong mau, do o yeu cau
at ra la phai phat trien mot thiet b cho phep ngi benh
co the t theo doi c nha. Biosensor vi kha nang chnh
xac, co kha nang tai s dung va ap ng c cac yeu
cau tren.
Biosensor o nong o glucose hien nay co mat tren th trng
hoat ong theo nguyen tac nh ien cc o dong ien, ien
cc o quang nhng eu da tren phan ng xuc tac oxy hoa
vi s co mat cua GOD (glucooxydase). Trong o ang chu y
la loai thiet b cam tay vi loai chip che tao tren c s cua
ien cc chon loc ion va ien cc o dong ien, kch thc
nho, de mang theo, tien s dung.
b) o nong o ure
Viec kiem tra nong o ure trong mau, ac biet oi vi
nhng ngi mac benh than co y ngha quan trong.
Thiet b nay hoat ong tren nguyen tac ien cc o ien
the, ure b thuy phan di tac dung cua xuc tac urease:
(NH2)2CO
3H2O
GOD
CO2 + 2OH-
2NH4+
Nong o NH4+ ty le vi nong o ure c o bang cach

dung mot lp mang tham chon loc ion NH4+.
Hnh 7.3 : Mot so Biosensor

thng mai
CHNG 6
KET LUAN
Biosensor a phat trien vt bac nhanh chong trong suot
nhieu nam qua. Nh vao cac phng phap ket hp mi cua
cac bo thu cam vi nhieu bo bien nang . ac iem, tnh
chat cua cac biosensor nay a c cai thien va tnh xac
thc cang ngay cang cao a tao ra nhieu ng dung mi.
ng dung chnh cua Biosensor la trong lnh vc y khoa
(cham soc sc khoe benh nhan). Biosensor ac biet thch hp
cho phan tch cac mau trong moi trng sinh hoc phc tap ma
khong can tac nhan phan ng hoa hoc. Biosensor co the
c dung in-vivo bi v no tao ra mot tn hieu lien tuc, co

the ieu khien nong o cac chat tao thanh trong qua trnh
trao oi chat trong mot thi gian nhat nh nen c ng
dung quan trong trong kiem soat nong o ng trong mau
ngi benh.
Cac ng dung cua Biosensor trong cong nghe thc pham
cung rat phat trien. ot pha quan trong la kha nang kh
trung trong cac qua trnh len men. Biosensor cung ong vai tro
quan trong trong kiem soat trc tiep chat lng thc pham.
Moi trng cung can co s kiem soat chat lng lien
tuc ma cac ky thuat hoa ly hien tai th gii han ac biet
khi kiem tra o oc hai cua moi trng. Biosensor ra i a
ap ng c nhng nhu cau o.
Nghien cu hien tai tap trung vao viec cai thien tnh
nhay cam cung nh tnh chon loc cua biosensor. Nhieu cong ty
Nhat Ban a phat trien va san xuat nhieu loai biosensor.
Nam 2000, th trng ve Biosensor a c thanh lap va to
chc 2 nam mot lan. Tai ay tap trung cac nghien cu phat
trien biosensor trong nhieu lnh vc khac nhau cua cac chuyen
gia au nganh va co giai thng trao tang. ieu nay a
khang nh s phat trien nhanh chong cua Biosensor.
TAI LIEU THAM KHAO

[1]. TRAN MINH CANH, Biosensor, Chapman & Hall
[2]. PGS.TS ANG TH THU, PGS. LE NGOC TU, TS. TO KIM ANH,
PGS.TS PHAM THU THUY, T.S. NGUYEN XUAN NAM, Cong
nghe Enzym, Nha xuat ban Khoa hoc Ky thuat Ha Noi
[3]. TRAN MINH CANH, BROUN G., Construction and study of
electrodes using cross linked enzymes
[4]. UPDIKE S.J., HICKS G.P., The enzyme electrode, Nature

[5]. YODAK., Methods in Enzymology, Vol 137
[6]. http://www.lsbu.ac.uk/biology/enztech/biosensors.html
[7]. http://www.lsbu.ac.uk/biology/enztech/calorimetric.html
[8]. http://www.lsbu.ac.uk/biology/enztech/potentiometric.html
[9]. http://www.lsbu.ac.uk/biology/enztech/amperometric.html
[10].http://www.lsbu.ac.uk/biology/enztech/optical.html
[11].
http://www.lsbu.ac.uk/biology/enztech/piezo.html
[12].http://www.lsbu.ac.uk/biology/enztech/immuno.html
[13].
http://www.biodot.com/products/products_biosensor.htm
[14].http://www.biosensors-congress.elsevier.com/

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o an: Tong quan tai lieu ve Biosensor

o an: Tong quan tai lieu ve Biosensor

1.2. KHAI NIEM VE BIOSENSOR

Hnh 1.1. S o cau tao cua biosensor

1.3. AC IEM YEU CAU CUA BIOSENSOR

o an: Tong quan tai lieu ve Biosensor

o an: Tong quan tai lieu ve Biosensor

1.4. NGUYEN LY HOAT ONG CUA BIOSENSOR

o an: Tong quan tai lieu ve Biosensor

Hnh 1.2: Nguyen ly hoat ong chung cua mot Biosensor:

o an: Tong quan tai lieu ve Biosensor

Eo: ien the oxy hoa-kh tieu chuan;

o an: Tong quan tai lieu ve Biosensor

ien cc mang thuy tinh:

ay la loai ien cc mang thong dung nhat. ien cc

o an: Tong quan tai lieu ve Biosensor

ngha la hai mat cua lp thuy tinh se tao ra hai lp gel

o an: Tong quan tai lieu ve Biosensor

ien cc calomel c tao thanh bi kim loai Hg tiep xuc vi

Ngay nay ien cc Ag/AgCl c s dung rong rai e lam

o an: Tong quan tai lieu ve Biosensor

Hnh 2.1: Cau tao cua mot biosensor o ien the n

e) ien the gia ien cc

a) Mang ban tham

g) Dung dch HCl

b) Thanh phan sinh hoc

c) Mang thuy tinh

d) ien cc thuy tinh o pH

ng dung: ien cc o ien the c dung e xac nh

o lng H+ tao thanh (o pH sau khi giai phong H +) se xac

o an: Tong quan tai lieu ve Biosensor

Hnh 2.2: Cau tao ien cc pCO2

ien cc o dong ien hoat ong da vao dong ien

o an: Tong quan tai lieu ve Biosensor

Hnh 2.3: o th I = f(E)

1- E qua nho e s oxy hoa co the xay ra

2- van toc oxy hoa ien hoa va cng o dong

o an: Tong quan tai lieu ve Biosensor

Dong ien o c ty le vi lng H 2O2 b oxy hoa va do o

Hnh 2.4: S o nguyen tac cua ien cc o dong ien

e o lng cholesterol th gan enzym cholesteroloxydase

e o lng lactose th gan enzym -galactosidase (-GAL) t

GOD acid gluconic

Do qua trnh chuyen khoi H 2O2 en ien cc anod ma

o an: Tong quan tai lieu ve Biosensor

Hnh 2.5: Cau tao cua ien cc Oxy xac nh nong o

o an: Tong quan tai lieu ve Biosensor

2.2. IEN CC O NHIET (Calorimetric biosensor)

Enzym xuc tac

o an: Tong quan tai lieu ve Biosensor

S dung ong thi enzym glucooxydase oxy hoa ng

Qua nghien cu ta thay c 0.1M c chat chuyen hoa hoan

ien cc enzym cap nhiet

Trong loai ien cc nay, bo bien nang la cap nhiet ien.

o an: Tong quan tai lieu ve Biosensor

Hnh 2.6: S o ien cc enzym-cap nhiet

ien cc enzym- nhiet ien tr:

Loai ien cc nay con co the goi la ien cc ao nhiet

Hnh 2.7: S o ien cc enzym nhiet ien tr.

o an: Tong quan tai lieu ve Biosensor

Trong o R1: ien tr cua nhiet ien tr so sanh, R 2: ien tr

Khi T1~T2 th bieu thc tren tr thanh:

o giam tng oi ien tr (R/R) cua nhiet ien tr th ty