Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором
Розроблено та оптимізовано методику визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором. Підібрано оптимальне співвідношення та концентрацію ферментів у біоселективному елементі біосенсора для найбільшої його стабільності та чутливості. Розглянуто різні варіанти ви...
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Пєшкова, В.М. Солдаткін, О.О. Дзядевич, С.В. 2019-06-20T04:19:44Z 2019-06-20T04:19:44Z 2007 Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором / В.М. Пєшкова, О.О. Солдаткін, С.В. Дзядевич // Біополімери і клітина. — 2007. — Т. 23, № 6. — С. 501-510. — Бібліогр.: 25 назв. — укр., англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000785 https://nasplib.isofts.kiev.ua/handle/123456789/157520 .577.15.543.555 Розроблено та оптимізовано методику визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором. Підібрано оптимальне співвідношення та концентрацію ферментів у біоселективному елементі біосенсора для найбільшої його стабільності та чутливості. Розглянуто різні варіанти виявлення сахарози в зразках за допомогою біосенсора та проведено визначення сахарози та глюкози в соках і солодких напоях. Запропоновану методику можна надалі використовувати у харчовій промисловості для контролю і оптимізації виробництва. The working procedure of sucrose and glucose conductometric biosensors with real samples of juices and sweet drinks has been presented. For the purpose of sensitivity and stability improvement of the sucrose biosensor, optimal enzyme proportions and concentrations in its bioselective element have been chosen. Different variants of determination of sucrose and glucose in real samples using the biosensor have been considered. The sucrose and glucose measurement has been carried out in juices and sweet drinks. The method suggested could be used in food industry for the production control and optimization. Разработана и оптимизирована методика определения сахарозы в соках и сладких напитках с помощью кондуктометрического ферментного биосенсора. Подобраны оптимальное соотношение и концентрации ферментов в биоселективном элементе биосенсора для наибольшей его стабильности и чувствительности. Рассмотрены разные варианты выявления сахарозы в образцах с помощью биосенсора и проведено определение сахарозы и глюкозы в соках и сладких напитках. Предложенную методику можно использовать в дальнейшем в пищевой промышленности для контроля и оптимизации производства. uk Інститут молекулярної біології і генетики НАН України Біополімери і клітина Молекулярна та клітинна біотехнології Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором Оптимизация методики определения сахарозы в соках и сладких напитках кондуктометрическим ферментным биосенсором Optimization of sucrose measurement working procedure in real samples using conductometric enzyme biosensor Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором |
| spellingShingle |
Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором Пєшкова, В.М. Солдаткін, О.О. Дзядевич, С.В. Молекулярна та клітинна біотехнології |
| title_short |
Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором |
| title_full |
Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором |
| title_fullStr |
Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором |
| title_full_unstemmed |
Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором |
| title_sort |
оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором |
| author |
Пєшкова, В.М. Солдаткін, О.О. Дзядевич, С.В. |
| author_facet |
Пєшкова, В.М. Солдаткін, О.О. Дзядевич, С.В. |
| topic |
Молекулярна та клітинна біотехнології |
| topic_facet |
Молекулярна та клітинна біотехнології |
| publishDate |
2007 |
| language |
Ukrainian |
| container_title |
Біополімери і клітина |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Оптимизация методики определения сахарозы в соках и сладких напитках кондуктометрическим ферментным биосенсором Optimization of sucrose measurement working procedure in real samples using conductometric enzyme biosensor |
| description |
Розроблено та оптимізовано методику визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором. Підібрано оптимальне співвідношення та концентрацію ферментів у біоселективному елементі біосенсора для найбільшої його стабільності та чутливості. Розглянуто різні варіанти виявлення сахарози в зразках за допомогою біосенсора та проведено визначення сахарози та глюкози в соках і солодких напоях. Запропоновану методику можна надалі використовувати у харчовій промисловості для контролю і оптимізації виробництва.
The working procedure of sucrose and glucose conductometric biosensors with real samples of juices and sweet drinks has been presented. For the purpose of sensitivity and stability improvement of the sucrose biosensor, optimal enzyme proportions and concentrations in its bioselective element have been chosen. Different variants of determination of sucrose and glucose in real samples using the biosensor have been considered. The sucrose and glucose measurement has been carried out in juices and sweet drinks. The method suggested could be used in food industry for the production control and optimization.
Разработана и оптимизирована методика определения сахарозы в соках и сладких напитках с помощью кондуктометрического ферментного биосенсора. Подобраны оптимальное соотношение и концентрации ферментов в биоселективном элементе биосенсора для наибольшей его стабильности и чувствительности. Рассмотрены разные варианты выявления сахарозы в образцах с помощью биосенсора и проведено определение сахарозы и глюкозы в соках и сладких напитках. Предложенную методику можно использовать в дальнейшем в пищевой промышленности для контроля и оптимизации производства.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/157520 |
| citation_txt |
Оптимізація методики визначення сахарози в соках і солодких напоях кондуктометричним ферментним біосенсором / В.М. Пєшкова, О.О. Солдаткін, С.В. Дзядевич // Біополімери і клітина. — 2007. — Т. 23, № 6. — С. 501-510. — Бібліогр.: 25 назв. — укр., англ. |
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2025-11-25T20:44:34Z |
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| fulltext |
Op ti mi za tion of su crose mea sure ment work ing
pro ce dure in real sam ples us ing conductometric en zyme
biosensor
V. N. Peshkova1,2, A. A. Soldatkin1, S. V. Dzyadevych1
1In sti tute of mo lec u lar bi ol ogy and ge net ics NAS of Ukraine
Academicain Zabolotnog str., 150, Kyiv, 03680 Ukraine
2Na tional Taras Shevchenko Uni ver sity of Kyiv,
64, Volodymyrska street, 01033 Kyiv, Ukraine.
victoir@ukr.net
The work ing pro ce dure of su crose and glu cose conductometric biosensors with real sam ples of juices and
sweet drinks has been pre sented. For the pur pose of sen si tiv ity and sta bil ity im prove ment of the su crose
bio sen sor, op ti mal en zyme pro por tions and con cen tra tions in its bioselective el e ment have been cho sen.
Dif fer ent vari ants of de ter mi na tion of su crose and glu cose in real sam ples us ing the bio sen sor have been
con sid ered. The su crose and glu cose mea sure ment has been car ried out in juices and sweet drinks. The
method sug gested could be used in food in dus try for the pro duc tion con trol and op ti mi za tion.
Keywords: conductometric en zyme bio sen sor, im mo bi lized en zymes, su crose, glu cose
In tro duc tion. Su crose, glu cose, and other car bo hy -
drates are the con stit u ent parts of many food prod ucts,
in clud ing fruit, veg e ta bles, and fruit drinks. Due to the
fact that the con tents of these car bo hy drates in fruit and
veg e ta bles is con nected di rectly with cul ti vat ing and
stor ing con di tions of the lat ter, and may also de ter mine
the de gree of their rip en ing and qual ity, there is a need
in well-tuned sys tem of their mon i tor ing [1]. Su crose,
a key com po nent of mo las ses, is used in food, phar ma -
ceu ti cal, and cos metic in dus try [2, 3] and in some pro -
cesses of biotransformation in bio tech no log i cal man u -
fac tur ing [4]. Mo las ses is also known to be the nat u ral
source of var i ous prod ucts, in par tic u lar, sugar [5]. The
pro cess of bio tech no log i cal pro duc tion (cul ti va tion,
fer men ta tion, etc.) re quires per ma nent con trol for both
prod uct out put, and qual ity and con tents. Per ma nent
mon i tor ing of bio tech no log i cal pro cesses is nec es sary
for deeper un der stand ing and op ti mi sa tion, as well as
for the con trol of their course.
Cur rent stan dard meth ods of ac cu rate su crose de -
ter mi na tion, in clud ing liq uid chro ma tog ra phy, chem i -
cal and op ti cal meth ods, used in food in dus try and med -
i cine, are disadvantageous in ne ces sity of highly skilled
per son nel and ex pen sive com pli cated equip ment [6].
Su crose con cen tra tion can also be de ter mined by iso to -
pic method and gas-liq uid chro ma tog ra phy but these
meth ods are not widely used due to their high price,
com pli cated pro ce dure, and re quir ing a lot of space [7].
One of the main dis ad van tages of the afore men tioned
ap proaches is quite com plex sam ple pre treat ment.
Polarimetric and re frac tor meth ods of de ter min ing su -
crose though faster, are less ac cu rate [6]. At the same
time they are sen si tive to a num ber of in ter fer ing com -
po nents, pres ent in the so lu tion [8]. There fore, de vel -
501
ISSN 0233-7657. Biopolymers and cell. 2007. vol. 23. N 6. Translated from Ukrainian.
Ó V. N. PESHKOVA, A.A. SOLDATKIN, S. V. DZYADEVYCH, 2007
op ment of more con ve nient, pre cise, se lec tive, fast, and
in ex pen sive method of de ter mi na tion of su crose con -
tent in var i ous al co holic and non-al co holic bev er ages
and food stuff is an is sue of the day.
Cur rently, there is cer tain in for ma tion on de vel op -
ment of a num ber of su crose biosensors [6, 9–17].
Thus, [16] pres ents amperometric su crose bio sen sor for
on-line con trol of the pro cess of en zy matic
biotransformation of glu cose into fruc tose by
glucosoisomerase. Lit er a ture also con tains the pos si -
bil ity of ap pli ca tion of multi-en zy matic sen sors for si -
mul ta neous de ter mi na tion of sev eral sac cha rides, e.g.
su crose, malt ose, and glu cose, us ing potentiometric
sen sor [11].
The de vel op ment of su crose biosensors may in -
clude var i ous bi o log i cal ma te ri als and meth ods of im -
mo bi li sa tion of en zymes. Thus, in [18] au thors re port
the ap pli ca tion of the method of elec tro chem i cal poly -
meri sa tion of phenylenediamine on the sur face of elec -
trodes for im mo bi li sa tion of invertase, mutarotase, and
glu cose oxidase. The yeast cells, as an invertase re -
source, were co-im mo bi lized with glu cose oxidase and
used in [9], ox y gen con sump tion be ing a mea sure in
this case. The de vel op ment of novel method of im mo -
bi li sa tion of en zymes on the sur face of elec trodes, i.e.
when en zymes (glu cose oxidase, invertase, and per oxi -
dise) are bound to che late sepharose by dif fer ent metal
ions and lectin concanavalin A, is pre sented in [12].
This method of im mo bi li sa tion al lows elu tion and re -
cur rent im mo bi li sa tion of dif fer ent en zymes on trans -
ducer sur face as well as iden ti fi ca tion of dif fer ent sub -
strates in multi-com po nent so lu tion.
Thus, the ma jor ity of su crose biosensors de vel -
oped up-to-date are amperometric [6, 9–17]. Along
with their in her ent ad van tages, amperometric sen sors
have some es sen tial dis ad van tages com pared to
conductometric ones: i) the need for high ap plied po -
ten tial that re sults in er rors, as so ci ated with the pres -
ence of other electrooxidizing com po nents, e.g. ascor -
bic acid, in the tested so lu tions; ii) they re quire com -
pli cated and ex pen sive ref er ence elec trodes; iii) the
ne ces sity to work with high volt age, which en ables
Far a day pro cesses on the sur face of elec trodes. More -
over, amperometric biosensors are usu ally more ex -
pen sive in terms of mass pro duc tion than
conductometric ones.
Methods of conductometric bio sen sor ap pli ca tion
for su crose anal y sis are rather prom is ing, since they are
suf fi ciently sim pler, more con ve nient, ac cu rate, and al -
low meet ing im por tant scientific and in dus trial chal -
lenges [19, 20]. In the pre vi ous work we de vel oped lab -
o ra tory pro to type of su crose conductometric bio sen sor
[21]. But suc cess ful ap pli ca tion of conductometric bio -
sen sor for anal y sis of real sam ples re quires op ti mi sa -
tion of an a lyt i cal char ac ter is tics. There fore, our work
was aimed at the ad just ment of the de vel oped method
of de ter min ing su crose con tent in real sam ples us ing
conductometric bio sen sor.
Ma te ri als and Methods. Ma te ri als. The
frozen-dried prep a ra tions of en zymes used in the ex per -
i ments were as fol lows: glu cose oxidase (GOD) from
Penicillium vitale (ÅÑ 1.1.3.4.) with ac tiv ity of 130
U/mg from Diagnosticum (L’viv, Ukraine); invertase
(ÅÑ 3.2.1.26) from baker’s yeast with ac tiv ity of 355
U/mg from Sigma-Aldrich Chemie (Ger many);
mutarotase (ÅÑ 5.1.3.3.) with ac tiv ity of 100 U/mg
from Biozyme Laboràtories Ltd., (UK). Bo vine se rum
al bu min (BSA) (V frac tion) and 50% aque ous so lu tion
of glutaraldehyde (GA) were ob tained from
Sigma-Aldrich Chemie. Su crose was used as a sub strate
and an a lyzed sub stance, po tas sium-phos phate so lu tion
(ÊÍ2ÐO4-NàÎÍ), ðÍ 7.2 from Ìårck was used as a
buffer. Other non-or ganic com pounds were of an a lyt i -
cal grade.
Sen sor de sign. The conductometric trans duc ers
pro duced ac cord ing to our rec om men da tions and out -
lines in Lashkarev In sti tute of Semi con duc tor Phys ics
of Na tional Acad emy of Sci ences of Ukraine (Kyiv,
Ukraine) con sisted of two iden ti cal pairs of gold
interdigitated elec trodes made by gold vac uum evap o -
ra tion onto pyroceramic sub strate (5 õ 40 mm) (Fig. 1).
The sur face of sen si tive area of each elec trode pair was
about 1.0´1.5 mm. The width of each of interdigital
spaces and dig its was 20 mm.
Bioselective mem brane pro duc tion. The so lu tion
con sist ing of invertase, mutarotase, glu cose oxidase in
40 mM phos phate buffer, pH 7.4, with 20% glyc erol
was used to pro duce the work ing mem brane while the
same mix ture with BSA in stead of en zymes – for the
ref er ent mem brane. Prior de po si tion onto trans ducer
sur faces, the so lu tions for both work ing and ref er ent
mem branes were mixed with glutaraldehyde aque ous
502
PESHKOVA V. N., SOLDATKIN A. A., DZYADEVYCH S. V.
so lu tions of dif fer ent con cen tra tion in 1:1 ra tio. The so -
lu tions ob tained were im me di ately de pos ited onto the
trans ducer work ing part. The pro tein con tent was the
same in both mem branes. Be fore us age, the sen sors
dried dur ing dif fer ent time pe ri ods in the air at room
tem per a ture were then washed out in the work ing
buffer so lu tion.
Scheme of ex per i men tal mea sur ing set-up is shown
in Fig. 2. The al ter nat ing volt age with the fre quency of
100 kHz and am pli tude 10 mV was ap plied from the
low-fre quency sig nal gen er a tor GZ-118 (Ukraine) to
the dif fer en tial pair of interdigitated elec trodes placed
in an ex per i men tal ves sel with the so lu tion tested. The
sig nal ob tained at the elec trodes was trans ferred from
the 1 kOhm load re sis tance via dif fer en tial am pli fier
Unipan-233-6 onto lock-in nanovoltmeter Unipan-233
(Po land) where upon at the reg is ter ing ap pa ra tus. The
de pend ence of the out put sig nal on the sub strate con -
cen tra tion in the so lu tion was mea sured.
The mea sure ment pro ce dure. The mea sure ments
were car ried out in an open cell at room tem per a ture.
The 10 mM phos phate buffer, pH 6.3 was used at in -
ten sive stir ring. The sen sor was placed in 2 ml cell,
filled with phos phate buffer. To ob tain sta ble ini tial
sig nal (base line) the sen sor was steeped for some time
in buffer so lu tion. Then to ob tain the sig nal for sub -
strate of cer tain con cen tra tion, the cell was added an
aliquot of stan dard con cen trated out put sub strate so lu -
tion (su crose or glu cose) or an aliquot of juice. The
ex per i ments were per formed at least in three se ries.
The ef fect of non spe cific vari a tions of out put sig nal
ow ing to tem per a ture and pH changes and elec tric in -
ter fer ences was avoided by dif fer en tial mode of mea -
sure ment.
Re sults and Dis cus sion. The ba sic cas cade of en -
zy matic re ac tions for su crose de tec tion by
conductometric bio sen sor is as fol lows:
invertase
su crose + Í2ήb-D-fruc tose + a-D-glu cose (1)
mutarotase
a-D-glu cose®b-D-glu cose (2)
glu cose oxidase
b-D-glu cose + Î2®D-gluconolactone + Í2Î2 (3)
ß
D-gluconic acid + Í2Î D acid re sid uum + Í+ (4),
As a re sult, su crose is grad u ally de com posed with
invertase, mutarotase, and glu cose oxidase to hy dro gen
per ox ide and D-gluconolactone. In its turn, the lat ter is
spon ta ne ously hy dro lyzed to gluconic acid which dis -
so ci ates to the acid re sid uum and a pro ton, the so lu tion
con duc tiv ity be ing changed which can be reg is tered by
the conductometric trans ducer [22].
Work ing char ac ter is tics of conductometric bio sen -
sor are known to de pend on the con tent and the method
of im mo bi li sa tion of en zy matic mem brane. For in -
stance, sen sor re sponse to glu cose and su crose de pends
not only on the ac tiv ity of these en zymes and dif fu sive
pro cesses in bioselective mem brane but also on the ra -
tio of these en zymes in bio sen sor mem brane. There -
fore, the op ti mi za tion of en zy matic con tent of mem -
brane of su crose bio sen sor to the level of high est sen si -
tiv ity, when the re sponses for su crose and glu cose
matched, was stud ied in the first place.
Op ti mal mutarotase con tent in en zy matic mem -
brane was se lected. For this pur pose, gels of dif fer ent
mutarotase con cen tra tion were pre pared in 40 mM
phos phate buffer (pH 7.4) with 20% glyc erol. GOD
503
OP TI MI ZA TION OF SU CROSE MEA SURE MENT WORK ING PRO CE DURE IN REAL SAM PLES
Fig.1 Gen eral view of a
conductometric pla nar
interdigitated elec trode
con cen tra tion was 5%. This con cen tra tion was se lected
ac cord ing to data pre sented in [23], where it was shown
to be op ti mal for glu cose de ter mi na tion, as these pa -
ram e ters are ideal for op ti mal cor re la tion be tween sen -
si tiv ity and dy namic range of bio sen sor work. The
mix ture for prep a ra tion of ref er ent mem brane dif fered
from that of work ing mem brane by BSA, which was
used in stead of en zyme, so, both mem branes con tained
the same amount of pro tein. Prior to de po si tion onto
trans ducer sur faces, the so lu tions for both work ing and
ref er ent mem branes were mixed with 2% GA aque ous
so lu tion in 1:1 ra tio. The so lu tions ob tained were im -
me di ately de pos ited onto the trans ducer work ing part.
The sen sors were dried for 2 hours at room tem per a ture.
Work ing cell was added invertase sur plus be fore the
mea sure ment. Sen sor re sponses to 1 mM glu cose and 1
mM su crose were reg is tered. Fig.3 a shows that at
mutarotase con cen tra tion of 4% and higher, su crose
bio sen sor re sponse to glu cose and su crose equal and
thus this value was used in fur ther re search.
The next stage was the se lec tion of op ti mal
invertase con cen tra tion as a part of en zy matic sen sor
mem brane. For this pur pose gels of dif fer ent invertase
con cen tra tion were pre pared, which con tained
mutarotase and GOD of con stant con cen tra tion 4 and
5% re spec tively. Fig.3, b shows that at 5% invertase
con tent in en zy matic mem brane, sen sor re sponse to
glu cose and su crose is the clos est to 1. The in crease in
invertase con cen tra tion in en zy matic mem brane re -
sulted in a slight in crease in sen sor re sponse to glucose
than to su crose. Thus, the op ti mal con cen tra tion of
invertase in en zy matic mem brane was de ter mined to be
5%. This invertase con cen tra tion was used for fur ther
prep a ra tion of en zy matic mem brane.
The aim of the re search was to de velop the mea -
sure ments pro to col of work of conductometric bio sen -
sor with real juice sam ples, thus we fo cused spe cif i cally
on this is sue. As en zy matic mem brane of su crose bio -
sen sor con tains GOD, the main er ror in su crose bio sen -
sor re sponse is caused by glu cose, pres ent in sam ples
(juices) along with other sac cha rides.
There are sev eral so lu tions to this prob lem:
– im mo bi li za tion of GOD in the ex ter nal
mem brane layer, which will de com pose glu cose, pres -
504
PESHKOVA V. N., SOLDATKIN A. A., DZYADEVYCH S. V.
Fig.2 Scheme of ex per i men tal
measuring set-up for conductometric
mea sure ments: 1 – gen er a tor, 2 –
elec trodes, 3 – load re sis tors, 4 – dif -
fer en tial am pli fier, 5 – lock-in
nanovoltmeter; 6 – reg is ter ing de vice
ent in the so lu tion, be fore it reaches the in ter nal en zy -
matic layer (sen si tive to su crose);
– ap pli ca tion of flow anal y sis, which is spe -
cific for GOD re ac tor to be lo cated in front of su crose
de tec tor, which will also re sult in de com po si tion of glu -
cose. Ba si cally this sys tem is sim i lar to the first one;
– ap pli ca tion of spe cific glu cose sen sor for
de ter min ing the con cen tra tion of free glu cose in the so -
lu tion and fur ther iden ti fi ca tion of the dif fer ence be -
tween sig nals of two en zy matic biosensors, the first of
which re sponds to both glu cose and su crose, while the
sec ond one – to glu cose only.
The lat ter method was se lected to de ter mine su -
crose con tent due to its be ing sim ple, con ve nient, and
re quir ing no ad di tional el e ments of sys tem or ma nip u -
la tions. Be sides, glu cose bio sen sor was used as con trol
sen sor, as pre vi ous works re vealed sig nif i cant re li abil -
ity of re sults of its ap pli ca tion [24], which also al lowed
us to use glu cose sen sor to ver ify the data, ob tained
with su crose sen sor.
Sev eral pos si ble vari ants of de ter min ing su crose in
real sam ples were stud ied. First of all we stud ied the
case when re sponses to su crose and glu cose sen sors for
cer tain glu cose con cen tra tion did not match, which
505
OP TI MI ZA TION OF SU CROSE MEA SURE MENT WORK ING PRO CE DURE IN REAL SAM PLES
Fig.3 De pend ence of re sponse ra tio
to glu cose and su crose on mutarotase
(a) and invertase (b) con tent in en zy -
matic mem brane; mea sure ments
were per formed in 10 mM phos phate
buffer, pH 6.3, sub strate con cen tra -
tion 1 mM
hap pens quite of ten. This was nec es sary for the de vel -
op ment of method suit able for fur ther au tom a ti za tion
and real-time mea sur ing. Glu cose and su crose sen sors
were cal i brated prior to mea sure ment pro ce dure
(Fig.4).
Cal i bra tion curves for glu cose and su crose
biosensors cor re spond to y = k1x and y = k2x. Cor re la -
tion ra tio for these two func tions (K) was cal cu lated as:
K = k2x/k1x = k2/k1 (5)
Ob tain ing glu cose sen sor re sponse to cer tain con -
cen tra tion of glu cose and mul ti ply ing it for cor re la tion
ra tio K al lowed ob tain ing the value, cor re spond ing to
the value of re sponse, pro duced by su crose sen sor, to
the same glu cose con cen tra tion.
The data of Fig.4 and for mula 5 re sult in:
Ss+g = Ss + Sg; (6)
Sg = Gg·K, Ssi+g = Gsi+g·K (7)
Ss = Ss+g – Gg·K (8)
Sub sti tu tion of Ss into cal i bra tion curve (for S[S]) al -
lows de ter min ing su crose con cen tra tion in juice (c).
Cal i bra tion curve of su crose bio sen sor to su crose (S[S])
cor re sponds to y = k3x.
Thus,
c=Ss/k3 =(Ss+g–Gg·K)/k3(ex per i ment) (9)
c = (Gsi+g – Gg)/k1 (con trol) (10)
If the sen sor re sponses to the same con cen tra tion of
glu cose and su crose match, i.e. K = 1, then equa tion (9)
is sim pli fied to:
c = Ss/k3 = (Ss+g – Gg)/k3 (11)
Let us il lus trate the men tioned above method of de -
ter mi na tion of su crose con cen tra tion by ex per i ments in
juices. For this pur pose we car ried out a se ries of ex per -
i ments with cer tain con cen tra tion of sub strates. Ex per i -
men tal er ror did not ex ceed 4%. Cal i bra tion curve of
glu cose conductometric bio sen sor was lin ear in the
range of 0.001 – 1.5–2.5 mM of glu cose and in the
range of 0.001 – 2.5–10 mM of su crose, de pend ing on
the ex per i men tal con di tions [21]. Higher glu cose and
su crose con cen tra tions re sulted in sat u ra tion of bio sen -
sor re sponse. Con cen tra tion of glu cose and su crose in
juices is known to vary from 15 to 700 mM. Thus, the
sam ple has to be di luted for mea sur ing due to nar row
dy namic range of biosensors.
Prior to mea sure ment pro ce dure, glu cose sen sor
was emerged into the mea sur ing cell (2 ml) with 10 mM
506
PESHKOVA V. N., SOLDATKIN A. A., DZYADEVYCH S. V.
Fig.4 Sche matic rep re sen ta tion of dependences of su -
crose bio sen sor re sponses on su crose (S[S]) and glu -
cose (S[g]) con cen tra tions and of glu cose bio sen sor re -
sponses on glu cose con cen tra tion (G[g]) in the
so lu tion; S – su crose sen sor; G – glu cose sen sor; s –
su crose; si– glu cose, de com posed from su crose; g –
glu cose; Gg – glu cose sen sor re sponse af ter in tro duc -
tion of aliquot of juice into the mea sur ing cell; Gsi+g –
glu cose sen sor re sponse af ter the introduction of
aliquot of juice con tain ing invertase, which de com -
poses su crose to glu cose; Ss+g – sen sor re sponse to the
in tro duc tion of aliquot of juice into the mea sur ing
cell; Ss+g = Ss (re sponse to su crose) + Sg (re sponse to
glu cose, pres ent in the so lu tion)
phos phate buffer, pH 6.3, and sus tained for sev eral
min utes in or der to ob tain the base line. Next, juice
aliquot was added (1 ml) to ob tain 2 000-di lu tion in the
buffer so lu tion. Re sponse of glu cose sen sor (Gg) was
mul ti plied by K, pre vi ously cal cu lated from cal i bra tion
curves for glu cose from glu cose and su crose biosensors
(5). The value of Sg was ob tained, which is the sen sor
re sponse to only glu cose in juice sam ple. We also ob -
tained the value of glu cose con cen tra tion from cal i bra -
tion chart. Then, we used su crose bio sen sor and ob -
tained the re sponse (Ss+g) (sim i larly to glu cose sen sor)
to the in tro duc tion of the same aliquot of juice (1 ml).
As su crose sen sor is sen si tive to both glu cose and su -
crose, which are al ways pres ent in juices, sig nal Ss+g is
the sum of two sig nals, namely to glu cose (Sg) and su -
crose (Ss). Sg–Ss+g equals to Ss, i.e. sen sor re sponse to
su crose only (Fig.4). Next we de ter mined su crose con -
cen tra tion in juice sam ples us ing cal i bra tion curve of
su crose sen sor.
The data ob tained were ver i fied by glu cose sen sor
only. For this pur pose, the juice sam ple was added
invertase, cat a lyz ing the de com po si tion of su crose to
glu cose and fruc tose. De com po si tion of one su crose
mol e cule gives one glu cose mol e cule. This mix ture
was sus tained for 2 hours for spon ta ne ous trans for ma -
tion of a-D-glu cose to b-D-glu cose. Next we ob tained
sig nal Ssi+g (re sponse to glu cose and de com posed su -
crose) us ing glu cose bio sen sor, i.e. re sponse to the in -
tro duc tion of 1 ml of juice con tain ing invertase, and
sub tracted ear lier ob tained value of Sg (re sponse to glu -
cose). Ex trap o la tion of ob tained seg ment onto x-co or -
di nate al lows de fin ing the value of su crose con cen tra -
tion in juice, which was later com pared with that ob -
tained us ing su crose bio sen sor.
Ex per i men tal re sults re as sured us in ap pro pri ate -
ness of the ap pli ca tion of the de vel oped method for sub -
se quent anal y sis of su crose con cen tra tion in juices.
The lev els of su crose and glu cose con cen tra tion in
juices were de ter mined us ing the method of stan dard
ad di tions [25]. For this pur pose we analyzed the vari -
ant of su crose de ter mi na tion us ing glu cose and su crose
sen sors with equal re sponses to glu cose. We started
with ob tain ing glu cose sen sor re sponse to 1 ml of juice
and sev eral al ter nate in tro duc tions of 0.25 mM of glu -
cose into the cell. Then we built the lin ear de pend ence
of change in con duc tiv ity on sub strate con cen tra tion
(Fig.5, a), cross-point of which cor re sponded to glu -
cose con cen tra tion in sam ples, di luted 2 000 be fore -
hand.
Then, were reg is tered the re sponse of su crose sen -
sor to 1 ml of juice, which con sisted of gen eral sen sor
re sponse to glu cose and su crose. This value was sub -
tracted the value of glu cose sensor re sponse to glu cose.
The value ob tained was pro jected onto y-co or di nate
507
OP TI MI ZA TION OF SU CROSE MEA SURE MENT WORK ING PRO CE DURE IN REAL SAM PLES
Fig.5 De ter mi na tion of glu cose con cen tra tion in sam ples by glu cose sen sor (a) and su crose con cen tra tion by su crose bio sen sor (b) us ing the
method of stan dard ad di tions; mea sure ments were performed in 10 mM phos phate buffer, pH 6.3
and, us ing the same method (method of stan dard ad di -
tions), the value of su crose con cen tra tion in juice was
ob tained (Fig.5, b).
The re sults were ver i fied by con trol method, us ing
only glu cose sen sor. For this pur pose, the juice sam -
ple was added invertase, cat a lyz ing the de com po si tion
of su crose to glu cose and fruc tose. De com po si tion of
one su crose mol e cule gives one glu cose mol e cule.
This mix ture was sus tained for 2 hours for spon ta ne -
ous trans for ma tion of a-D-glu cose to b-D-glu cose.
Next we ob tained re sponse of glu cose bio sen sor to 1
ml of juice, which con sisted of gen eral bio sen sor
respose to al ready pres ent glu cose and su crose, de -
com posed to glu cose. Re sponse value was sub tracted
the re sponse value to glu cose by ear lier de vel oped
glu cose sen sor. Ex trap o la tion of ob tained seg ment
onto x-co or di nate al lows de fin ing the value of su crose
con cen tra tion in juice us ing the method of stan dard
ad di tions, which was later com pared with that ob -
tained us ing su crose bio sen sor.
Thus, pre sented method of su crose de ter mi na tion
in real sam ples was shown to be op ti mal, sim ple and
con ve nient for fur ther de ter mi na tion of su crose and
glu cose con cen tra tions in juices and sweet drinks.
The re sults of these mea sure ments are pre sented in
Fig.6. Ex per i men tal er ror did not ex ceed 10%.
Con clu sions. Op ti mal con cen tra tions of en zymes
in bioselective el e ment of su crose conductometric
biosensors were se lected to achieve its max i mal sta bil -
ity and sen si tiv ity, cor re spond ing to 5% glu cose
oxidase, 4% mutarotase, and 5% invertase. The work -
ing pro ce dure of conductometric su crose and glu cose
biosensors in real sam ples of juices and sweet drinks
was tested. Dif fer ent vari ants of su crose de ter mi na -
tion in juice and sweet drinks sam ples by biosensoric
method were ex am ined. The for mula of re cal cu la tion
of data was pre sented. The work pres ents the re sults
of su crose and glu cose de ter mi na tion us ing biosensors
in juices and sweet drinks. Ex per i men tal er ror did not
ex ceed 10%. The ap pli ca tion of de vel oped su crose
and glu cose biosensors in food in dus try for con trol
and op ti mi za tion of bio tech no log i cal pro cesses has
been pro posed.
The work was fi nan cially sup ported by Na tional
Acad emy of Sci ences of Ukraine within com plex sci -
ence and tech nol ogy pro gram Sen sor Sys tems for Med i -
cal, Eco log i cal, In dus trial, and Tech ni cal pur poses.
508
PESHKOVA V. N., SOLDATKIN A. A., DZYADEVYCH S. V.
Fig.6 De ter mi na tion of glu cose
and su crose con cen tra tions in sam -
ples by su crose and glu cose sen -
sors: 1 – pine ap ple nec tar
Sokovita, 2 – or ange nec tar
Sokovita, 3 – apri cot juice Biola, 4
– or ange juice Rich, 5 – ap ple
juice, pro duced by Odessa Baby
Food Plant; 6 – Coca Cola; 7 –
Fanta; 8 – Dushes (a – glu cose, b –
su crose, c – su crose con trol)
Â. Í. Ïåøêîâà, À. À. Ñîëäàòêèí, Ñ. Â. Äçÿäåâè÷
Îïòèìèçàöèÿ ìåòîäèêè îïðåäåëåíèÿ ñàõàðîçû â ñîêàõ è
ñëàäêèõ íàïèòêàõ êîíäóêòîìåòðè÷åñêèì ôåðìåíòíûì
áèîñåíñîðîì
Ðåçþìå
Ðàçðàáîòàíà è îïòèìèçèðîâàíà ìåòîäèêà îïðåäåëåíèÿ
ñàõàðîçû â ñîêàõ è ñëàäêèõ íàïèòêàõ ñ ïîìîùüþ
êîíäóêòîìåòðè÷åñêîãî ôåðìåíòíîãî áèîñåíñîðà. Ïîäîáðàíû
îïòèìàëüíîå ñîîòíîøåíèå è êîíöåíòðàöèè ôåðìåíòîâ â
áèîñåëåêòèâíîì ýëåìåíòå áèîñåíñîðà äëÿ íàèáîëüøåé åãî
ñòàáèëüíîñòè è ÷óâñòâèòåëüíîñòè. Ðàññìîòðåíû ðàçíûå
âàðèàíòû âûÿâëåíèÿ ñàõàðîçû â îáðàçöàõ ñ ïîìîùüþ
áèîñåíñîðà è ïðîâåäåíî îïðåäåëåíèå ñàõàðîçû è ãëþêîçû â
ñîêàõ è ñëàäêèõ íàïèòêàõ. Ïðåäëîæåííóþ ìåòîäèêó ìîæíî
èñïîëüçîâàòü â äàëüíåéøåì â ïèùåâîé ïðîìûøëåííîñòè äëÿ
êîíòðîëÿ è îïòèìèçàöèè ïðîèçâîäñòâà.
Êëþ÷åâûå ñëîâà: êîíäóêòîìåòðè÷åñêèé ôåðìåíòíûé
áèîñåíñîð, èììîáèëèçîâàííûå ôåðìåíòû, ñàõàðîçà, ãëþêîçà.
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