Tru tng
Mt cm bin sinh hc c nghin cu da trn vic s dng cc ZrO2 sol-gel
matrix cho enzyme c nh trong iu kin nh. iu ny
bioceramic zirconia alcogel c chun b bi cc tuyn ng alcohothermal
cun tiu thuyt vi mt mui v c gi r Zr (NO3) 4 5H2O
vi nhiu tnh nng hp dn bao gm mt din tch b mt ln (khong 460 m2
g-1) cng nh khi lng l chn lng v pht trin tt
mesoporosity kt cu, v ci nga peroxidase c chn l mt enzyme m hnh.
Cc kt qu ca in t truyn qua
knh hin vi (TEM) v BET o ca cht nn cho thy rng ma trn zirconia
nh chun b c mt li th
vi mi trng v din tch b mt ln c sn ti enzyme cao. Cc thng s nh
hng n c nhng cm by ca
enzyme v cc phn ng cm bin sinh hc c ti u ha. Kt qu l thit b cm
bin c nhy cao trng by ca 111 mM A-1 cho
hydrogen peroxide trn mt phm vi rng cc nng t 2,5 10-7 n 1,5 10-4
mol l-1, phn ng nhanh chng ca t hn
10 s v n nh tt hn 3 thng.
2002 Elsevier Science BV Tt c quyn c bo lu.
T kha: Nanoporous ZrO2 ma trn; Sol-gel; cm bin sinh hc
1. Gii Thiu
S pht trin ca vt liu mi c th ci thin
nng lc phn tch ca cc thit b cm bin l mt
Kha cnh quan trng ca cng ngh cm bin sinh hc. V c xp
vt liu sol-gel m ra mt con ng mi
ch to cc cm bin ha hc v cm bin sinh hc
v nhng thuc tnh hp dn ca vt liu sol-gel,
bao gm c s n gin ca vic chun b, du dng
xp, ng gi nhit thp, khng ng k
sng, c kh v n nh biodegradational
[1-12]. Nhn xt c trnh by trong cc nh nc-of-the
ngh thut ca cm bin sinh hc in ha s dng sol-gel
vt liu [4-6,13,14]. K t Braun et al. [1] chng minh
kh nng c nh m trong
sol-gel silica ma trn. iu ny lp ca mate- gm
* Tc gi. Fax: + 86-2165641740.
E-mail address: [email protected] (J. Kng).
rials c p dng hn na sn xut silica
phn tch d. Tuy nhin, cc nghin cu gn y dng nh
cho thy rng c rt nhiu s ch c tr tin s dng
ca vt liu dn in bioceramic oxit kim loi. Cc
c tnh vt l v ha hc ca oxit kim loi
c th d dng thao tc, cho d dng kim sot
cc, l chn lng phn b kch thc v tnh cht in t.
Glezer v Lev li dng cao
tin hnh vanadi pentaoxide sol-gel cho thit k
mt cm bin sinh hc glucose [15]. Topoglidis et al.
[16,17] bo co s c nh v bioelectrochemistry
ca cc protein trn TiO2 nanoporous v ZnO
b phim, tng ng. Cosnier et al. [18] iu tra mt
cm bin sinh hc glucose trong phim polypyrrole
electrodeposited
trn TiO2. Wang v Pamidi [19] bo co
cc in cc vng-men mi cho electroanalytical
cc ng dng bng cch thm cc enzyme cc sol-gel
gii php bng cch phn tn quyn lc bng kim loi. Nhiu
cm bin sinh hc amperometric mi da trn cun tiu thuyt sol-gel
hn hp m ra trin vng ln cho phn tch
ng dng.
Ngoi ra, xem xt s hp th protein trn
phim nanocrystalline ch yu l in v c kim sot
bng pH, ph protein v cc gii php
cng ion [16,17]. y, chng ti m rng nghin cu
mt ma trn hp dn, ZrO2 bng phng php sol-gel. Ca n
im ng in l khong 4,15 [20], m nn c
ph hp hn cho s hp th ca protein IEP cao.
Zirconia l mt nguyn liu quan trng m cng ngh
gn y thu ht c s quan tm ng k trong thc t
ng dng [21]. N l mt loi vt liu duy nht ca sc
tr ha hc v tnh nng biocompatibility;
hn na b mt ca n c c oxy ha v gim
ti sn, cng nh cc thuc tnh c tnh axit v c bn [22].
N c bo co rng titania- v zirconia-sol-gel
in cc carbon composite c s dng lm cht nn
cho gim qu in p ca qu trnh oxy ha NADH
phn ng [23]. Nghin cu ng v Kuwana ca [24]
cho thy s phn tn ca nhiu oxit kim loi
nh SiO2, ZrO2 trn b mt in cc nng cao
mc xy ha ca hydroquinone, m do
n s hp th ca cc loi electroactive v
gia tc ca bc chuyn proton. V vy, trong
bioanalysis in, cn c nghin cu nh mt
ng c vin ha hn quan trng i vi cht xc tc v h tr
vt liu. Tuy nhin, vic s dng rt t tin
nguyn liu ban u c yu cu, trong gii hn mnh
ch to c th c ca mt cm bin sinh hc. Chng ta khng
nhn thc ca bt k bo co trc y nh vy thun tin hn
v phng php kinh t chun b ZrO2
ma trn cho c nh m. y, vic chun b,
c tnh v tin ch ca mt electroanalytical
cm bin sinh hc zirconia n nh sol-gel c ngun gc c
m t.
Trong bi bo ny, chng ti trnh by ln u tin mt cun tiu thuyt
enzyme cm bin sinh hc da trn mt ma trn ZrO2 mao
sn sng chun b bng cch th tc sol-gel s dng mt
mui v c gi r [Zr (NO3) 4 5H2O] l nguyn liu ban u
trong iu kin trung tnh. Kt qu cho thy
ng tng hp tiu thuyt ny dnh s chun b ca
zirconia vi nhiu tnh nng hp dn bao gm
din tch b mt ln cng nh khi lng l chn lng v mt
pht trin tt r kh trong phm vi mao, cung cp
mt cch tip cn kinh t mi cho vic c nh m.
N gi rng vic s dng cc ZrO2 sol-gel
c th thun li cho s pht trin ca cc cm bin sinh hc.
Kt qu l cm bin sinh hc trng by nhy cao
111 A mM-1, phn ng nhanh chng v n nh tt.
Mt so snh ca PGE / PTH / HRP zirconia sol-gel
cm bin sinh hc da trn cc cm bin PGE / ZrO2 da l
cng trnh by.
2. Thc nghim
2.1. Ho cht
Peroxidase ci nga c mua t Sigma
(HRP, EC1.11.1.7, A> 250 n v / mg), thionine (TH)
t Fluka, Zr (NO3) 4 5H2O v H2O2 (30% (w / v))
gii php c mua t Thng Hi Chemical
Cng ty Thuc th. Tt c cc ha cht khc l cc phn tch
lp v c s dng m khng cn tinh ch thm.
Tt c cc gii php c chun b vi s chng ct th hai
nc kh ion.
2.2. Dng c
Mt h thng ba in cc lm vic, vi mt a bch kim
l quy in cc, mt tham chiu cc calomel bo ha
in cc (SCE), v cc cm bin sinh hc lm vic
in cc. Cc php o c thc amperometric
ra trn CH 660A (CH Instrument in ha
Workstation, USA). Mt my khuy t v qun bar vi
mt iu khin nhit khng i cung cp cho cc i lu
giao thng vn ti.
Knh hin vi in t truyn qua (TEM) hnh nh
ca ZrO2 ma trn sol-gel c chp vi mt Hitachi
H-800, s dng mt in th gia tc 200 kV
(Hitachi, Tokyo, Nht Bn). Bin i Fourier hng ngoi
(FT-IR) ph o bng bt vin KBr cha
mng sol-gel hoc mng tyrosinase / sol-gel,
thu c trong khong 2000-500 cm-1
trn mt Avatar 360 ph k (Nicolet, M) lc
nhit phng. Din tch b mt ca cc mu
c o bng phng php BET bi N2 hp ph
v gii hp 77 K trong mt micrometrics
H thng ASAP2000.
2.3. Chun b zirconia gii php sol-gel
Theo chun b ca chng ti [25], thch hp
lng Zr (NO3) 4 5H2O c ha tan trong 50 ml tuyt i
ethanol thu c dung dch 0,3 M tip theo
a cc gii php vo mt ni hp trong 50 ml dung.
Sau khi nim phong, ni hp c duy tr
383 K trong 60 pht, sau ngui phng
nhit . Do A alcogel m thu c.
Cc gel ethanol c t nhit phng
180 pht. Mt ZrO2 n nh v ng nht sol-gel c
thu c (0,4 M) v c s dng nh mt tiu chun chng khon
gii php. Cng thc ny c th c thay i khi
thng s thc nghim nht nh c nghin cu.
2.4. Xy dng cc cm bin sinh hc H2O2
A 4 mm graphite nhit phn c in cc (PGE) l
s dng nh cc in cc c s cho vic sa i sol-gel-
H2O2 cm bin sinh hc. Cc PGE c nh bng bng 0.3, 0.1,
0,05 m ht alumina, ra k bng
acetone deionied v nc i ct trong mt
tm siu m, v lm kh trong khng kh trc khi s dng.
Th nht, cc electropolymerization ca thionine l
thc hin theo hai bc sau nghin cu trc y ca chng ti.
Gi PGE di mt tim nng khng i
1,5 V trong 5 pht v k thut qut chu k gia
-0,45 V v 0,05 V 100 mV s-1 pH 6,5 phosphate
dung dch m cha 0,1 mM thionine.
Tt c cc gii php xt nghim u l trit kh oxy
bi bt kh nit tinh khit, v mt dng chy lin tc
nit c duy tr qua cc gii php trong thi gian
th nghim, tr khi c quy nh khc.
Sau , c nh enzyme HRP trong ZrO2
ma trn sol-gel c thc hin bng cch b sung 10 l
ca HRP (10 mg ml-1) hon ton vi 10 l ZrO2
sol-gel. Vi mt micropipet, 10 l phn c nh l mt ng
keo c lng ng trn b mt PGE v sy
di 4 C trong khng kh. Sau , cc cm bin sinh hc c chun b
(PGE / PTH / HRP-sol-gel). Cc cm bin c gi trong
khng kh 4 C gia cc php o. Khi so snh,
PGE / ZrO2 in m khng cn enzyme cng l
chun b.
2.5. Th tc o lng
K thut qut th vng v cc php o amperometric
c thc hin vi 660 CHI in
my trm (CHI, Cordiva, USA). Ba in cc
H thng c s dng pht hin. Tt c cc th nghim
gii php l trit kh oxy bng bt
nit thng qua cc gii php trn tt c cc th nghim.
Trong cc th nghim tim nng lin tc, lin tip
b sung cc gii php chng khon H2O2 vi phosphate
m c thc hin v cc d liu hin thi gian l
ghi nhn nh l mt hm ca thi gian sau khi thm
H2O2.
3. Kt qu v tho lun
3.1. S tng tc gia ZrO2 sol-gel v HRP
ZrO2 sol-gel c cu trc xp ca ma trn
cho vic nhng cc enzym. iu tra cc vi cu trc
ca as-tng hp ZrO2 ma trn v
s bt ng ca enzyme trong ma trn, truyn
electron hnh nh knh hin vi c th nghim.
T hnh. 1, c th thy rng ma trn l rt cao
xp trong t nhin, trong bao gm cc cm
cc ht lin kt ngang khong 5 nm. Nhng hnh nh TEM ca
ZrO2 ma trn cng trng by mt b ng phc ba chiu
cu trc xp, trong cung cp mt ngha
tng b mt in cc c hiu qu cho enzyme
ti v chun b ti tt. Cc
din tch b mt v th tch l rng ca ma trn, o
s dng cc phng php BET bi N2 hp ph v
gii hp, khong 460 m2 g-1 v 2,9 cm3 g-1,
tng ng.
S tng tc gia ZrO2 ma trn v HRP
c nghin cu bng ph hng ngoi. Cc ma trn c hp ph
ban nhc ti 781 v 1054 cm-1, c im ca
khung rung ng linh hot khng i xng v i xng
hnh vi in ha ca in cc bin i
chu nh hng ca pH, v gi tr E0 gim
55 mV / pH gia pH 6 v 8.5 cho cc polyme
thionine, m c ng ca gi tr ca Nernstain
59 mV cho hai electron, qu trnh hai proton.
Ngoi ra, chiu dy, pH v HRP /
Nng ZrO2 l nhng yu t quan trng trn trong
Fluence cc thuc tnh ca cu trc ma trn v
hiu sut ca cc cm bin sinh hc. dy ca
phim sol-gel v phn ng ca enzyme c nh
b nh hng bi thay i t l c phiu ZrO2
chun sol-gel nc v ZrO2 / HRP. Kt qu
trong hnh. 3 cho thy s nhy cm v phn ng nng ng
phm vi ca cc cm bin sinh hc tng ln khi t l
ca ZrO2 / H2O nh hn 1: 2. Nu t l l
thp hn 1: 8, phn ng ca cc cm bin sinh hc dn
gim. Nh vy, t l ti u c c nh
1: 8 cho HRP vn, v b mt ca
cc in cc enzyme sol-gel bin i dng nh l
mn mng v sn chc trong iu kin nh vy. Sau , cc hiu ng
ca HRP (10 mg ml-1) / ZrO2 t l trn cm bin sinh hc
Phn ng l tip tc nghin cu v kt qu cho thy
rng t l ti u c c nh mc 1: 1 cho vic chun b
cc cm bin sinh hc.
pH ca qu trnh ch to sol-gel might
nh hng n hiu sut ca cc cm bin sinh hc. Cc s khng
gi tr in tch im ca khon h tr c tm quan trng
trong bt ng ca enzyme t ti cc gi tr pH
trn ZPC b mt ca ma trn s
c tch in m, v ngc li, ti cc gi tr pH
di ZPC b mt s c tch cc
tnh ph. N c bo co rng isoelectronic
im ca ci nga peroxidase l 8,9, trong khi ZrO2
c mt php o IEP 4,18 [20]. Th nghim ca chng ti
cho thy rng phn ng ti u ca
cm bin sinh hc thu c khong pH 6-7 cho immobilizing
HRP, m l ph hp tt vi cc
hot ng ti u ca HRP.
3.3. Phn ng amperometric ca cm bin sinh hc
Hnh. 4 cho thy voltammogrames cyclic ca
H2O2 cm bin sinh hc trong m phosphate kh oxy
gii php m khng H2O2 (a) v vi H2O2 (b). Trong
s vng mt ca H2O2, cc cm bin sinh hc khng c phn ng
v ch c nhng hnh vi in ho tiu biu
ca thionine electropolymerized c quan st.
Ngoi ra H2O2 trong dung dch m, gim
hin nay tng ln v khng tng trong qu trnh oxy ha
hin nay, trong ch ra rng mt phn ng xc tc
xy ra trn cc cm bin sinh hc. Kt qu ny cho thy
rng thionine hiu qu c th a n gia cc electron
trung tm oxi ha kh ca HRP v cc in cc.
Qu trnh chuyn electron gia H2O2
v cc cm bin sinh hc c minh ha trong nhng iu sau y
paragrap
Lc u, HRP gim H2O2 khuch tn t gii php
vo mng t bo v -xy ha HRP HRP-I
H2O2 + HRP HRP-I + H2O
Vic gim HRP-I HRP bao gm hai ring bit
bc
HRP-I + PTHH HRP-II + PTH
HRP-II + PTH HRP + PTH +
ni HRP-I v HRP-II l hai trung gian khng n nh.
PTHH v PTH + i din cho gim v b oxy ha
hnh thc ca poly (phenazine methosulfate). Sau
oxy ha PTH + c th c ti ch c gim n PTHH
ti cc in cc, mang li mt s gim hin ti
PTH + + H + + 2e PTHH
S ph thuc pH ca electrocatalytic hin ti
cho cc cm bin sinh hc quyt nh hai kha cnh: mt l
hiu ng pH ca hot ng ca HRP; cc khc l
nhng tim nng nh cao ca thionine. Xem xt nhng
hai kha cnh, nh hng ca pH trn cm bin sinh hc l
th nghim v mt pH ti u 6,8 c chn trong ny
lm vic (Fig. 5), mt s ng thun vi iu ha tan
HRP bo co trc [27]. Do , ZrO2
ma trn khng thay i gi tr pH ti u cho cc
phn ng bioelectrocatalytic ca HRP c nh
H2O2.
S ph thuc tim nng ca amperometric
tn hiu v nn hin ti ca cc cm bin sinh hc l
Hnh. 6. Mt ng cong phn ng hin thi gian in hnh cho vic thm lin tc
25 l, 1 mmol l-1 H2O2 n 10 ml dung dch m gi di khuy
cho mt enzyme in cc c ch to trong iu kin ti u. Hot ng tim nng
-0.2 V vs. SCE; ng cong chn l mt chun ca
cm bin sinh hc.
th nghim. Kt qu cho thy khu phn cao nht ca
tn hiu-to-background t c mc -0.2 V.
nhiu tim nng hot ng gim thiu thp
t loi electroactive hp khc.
Hnh. 6 hin th mt phn ng c trng ca amperometric
cc cm bin sinh hc sau khi b sung phn c k tip
H2O2. Gim quy nh hin nay t l thun vi
nng H2O2 c quan st. Hin
t hn 10 giy t 95% gi tr trng thi n nh,
l do s khuch tn nhanh chng ca nn
trong b phim xp. Cc cm bin sinh hc trng by mt
phm vi hiu chun tuyn tnh t 2,5 10-7 n 1,5
10-4 mol l-1 vi nhy 111 mM A-1 v
mt phng trnh hi quy: I (A) = 0,081 + 0.111C (M)
(Trong hnh. 7) v ng nhp I (A) = 2,24 + 0,87 log C (M) (trong
Hnh. 6 chn ng cong), tng ng. Gii hn pht hin
l 1,0 10-7 mol l-1 ti mt t l tn hiu-to-noise ca
3. Nh mt nhy cao c th c quy cho s kin
vi mi trng ca ma trn ZrO2, m
c i lc chung ca vic np protein v c th
n nh hot ng sinh hc ca n n mt mc ln.
chng minh cc chc nng xc tc ca
ZrO2 H2O2, c im ca amperometric
cc ZrO2-HRP / PGE v ZrO2 / PGE c so snh.
T hnh. 7 v ng cong chn, s nhy cm ca cc cu
l ln hn so vi sau ny khong 10 ln.
Cc ZrO2 / PGE in trng by mt lot tuyn tnh t
2.0 10-4 n 3,5 10-2 mol l-1 vi mt gii hn pht hin
1,0 10-6 mol l-1.
Cc cm bin sinh hc pht trin da trn ma trn ZrO2
hin th lp li tt. Cc chun tng i
lch xc nh bng xt nghim 20 lin tip ca
mt l-1 mu H2O2 5,0 10-5 mol l 2,7%. Nh
mt lot su cm bin sinh hc, trong thc hin mt cch c lp
trong cng iu kin, lch chun tng i
7,5% thu c cho cc c nhn hin ti
phn ng vi 5,0 10-5 mol l-1. Ngoi ra, chnh xc
ca cm bin c nh gi bng cch xc nh
s phc hi ca hydrogen peroxide sau khi b sung tiu chun
cc nng khc nhau, t 8,5 10-6 ti
7,5 10-4 M trong cc mu sa tit trng. Cc cm bin sinh hc
cho thy kt qu kh quan vi s phc hi trung bnh
100,3%.
S n nh hot ng c iu tra bi lin tip
s o ca mt 7,5 10-5 mol l-1 H2O2
mu. Sau 160 php o trong 5 h, cc cm bin sinh hc
gi li khong 82% hot ng ban u ca n, v khng sng
ca HRP / ZrO2 sol-gel mng c quan st
trong cc gii php trong cc php o lin tc ny.
Ngoi ra, s n nh lu di ca ZrO2
cm bin sinh hc sol-gel c ngun gc c nghin cu. Cc phn ng
cc cm bin sinh hc c th nghim ngy qua ngy trong sut
2 tun u tin, sau cc th nghim l
nh k c thc hin trong khong thi gian 3 thng
v lu tr kh ti 4 C. Kt qu cho thy rng
phn ng gim khong 4,5% ca s nhy cm ban u ca n
sau 2 tun v vn cn 75% sau 3 thng
s dng, trong khi phm vi tuyn tnh nng ng ca cc cm bin sinh hc
gim dn theo thi gian bo qun. iu ny
n nh lu di l do s tng tc
gia HRP v ma trn. Cc amin v carboxyl
nhm cc enzyme c th tng tc vi mt s
cc trang web c th ca cc cht nn ZrO2, ngn chn
cc enzyme t r r ra khi ZrO2 sol-gel
mng.
4. Kt lun
Cc cm bin sinh hc da trn bt ng ca HRP
trong mt ma trn ZrO2 sol-gel trn mt electropolymerized
thionine in cung cp c im li th bin i,
bao gm nhy cao v di hn
n nh. Mt qu trnh alcohothermal mi l v d dng c
c pht trin chun b zirconia n nh sol-gel
s dng mt mui v c gi r nh cc nguyn liu ban u
vi mt s c tnh mong mun bao gm mt lng ln
din tch b mt v xp rt pht trin cc
phm vi mao, cung cp mt vi mi trng thun li
cho enzyme rng buc. Kt qu cho thy
zirconia sol-gel l mt ma trn hp dn cho enzyme
c nh v cc php o electroanalytical.
Khng mt enzyme c quan st thy trong cc sol-gel
mng t bo v hot ng ca enzyme c duy tr. N
c th c m rng n cc protein khc ch to cc
cm bin sinh hc.
