Scientific integration of the Polish-Ukrainian borderland area in the field of monitoring and detoxification of harmful substances in environment.
Electrochemical biosensors on arginine
assay
Nataliya Stasyuk
Department of Analytical Biotechnology, ICB, NAS of Ukraine
What is biosensor?
Definition of a biosensor
• A biosensor:
• A device that uses specific biochemical
reactions mediated by isolated enzymes,
immunosystems, tissues, organelles or
whole cells to detect chemical compounds
usually by electrical, thermal or optical
signals. Source:
• PAC, 1992, 64, 148 (Glossary for chemists
of terms used in biotechnology.)
BIOSENSORS
Bioanalytical devices which are hybrids of
bioelement (biorecognition unit) and physicochemical transducer (signal converting unit).
4
Biocatalytic sensor
(enzyme- or cells-based)
The biocatalyst (a) converts the substrate to product.
This reaction is determined by the transducer (b) which converts
it to an electrical signal. The output from the transducer
is amplified (c), processed (d) and displayed (e).
5
Enzymes as sensors’
biorecognition elements
ADVANTAGES:
·
·
High selectivity
Fast response
ENZYMES
• Recombinant human arginase I (liver
isoform)
• Recombinant bacterial arginine
deiminase
7
Arginase in urea cycle
Arginase
Urease
2NH3 + CO2
8
Comparison of different types of developed biosensors on L-Arg.
Mode of signal
registration
Biocomponent
LOD, mM
Linear
range, mM
Response
time (95%),
min
Stability
, days
Reference
Potentiometric,
ASE*
Bacterial cells
0.05 – 1.0
Rechnitz et.al., 1977
Potentiometric,
NH3 gas sensor
Bacterial cells
0.008 – 1.0
Grobler et. al., 1982
Potentiometric,
NH3 gas sensor
U/A**
Potentiometric
U/A
Potentiometric,
ASE
U/A
Potentiometric
U/A
Potentiometric
ISE
U/A
Nikolelis and
Hadjiioannou, 1983
5.0
0.1-1.0
0.01
U/A
Potentiometric,
pH
Potentiometric, ISE
0.03-3.0
0.1-30
Ivnitski and Rishpon, 1993
1.5 - 4.0
21
0.01-1.0
U/A
Koncki et al., 1996
Komaba et al., 1998
0.025-0.31
10.0
Karacaoglu et.al, 2003
0.03-0.05
5.0-7.0
10-6-103
0.7-5.0
60
Kaur, http://
hdl.handle.net
Lvova et al., 2003
Potentiometric, ISE
U/A
0.1
0.12 - 40
1.5 – 5.0
15
Stasyuk et al., 2011
Conductometric
U/A
0.0005
0.01-4.0
2.0
45
Saiapina et al., 2012
Amperometric
U/A
0.038
0.07-0.6
0.17
3
Our work
Sensors and Actuators B 52 (1998) 78–83
Biological determination of Ag(I) ion and arginine
by using the composite film of electroinactive
polypyrrole and polyion complex
Schematic illustrations of bienzyme system for the detection of arginine.
Biosensors and bioelectronics 43 (1996) 667-674
Enzymatic analysis of arginine with the
SAW/conductance sensor system
Dezhong Liu, Aifeng Yin, Kai Ge, Kang Chen, Lihua Nie and Shouzhuo Yao
A specific and simple method for the determination of arginine
was developed by using a new type sensor, a surface acoustic
wave (SAW)/conductance sensor system. The assay was based
on two coupling reactions involving arginase (E.C. 3.5.3.1) and
urease (E.C. 3.5.1.5) with measurement of frequency shift that
resulted from the changes of conducting ions produced in the
reactions.
Enzyme-based semi-quantitative analysis by
PHENOL RED:
Predicted advantages of
nanoparticles
• Possibility to create a higher concentration
of biorecognition element on nanoparticles
surface
• Stabilization of the enzymes
• Ability for autoassembly
• Improving catalytic activity
• Ability for direct electron transfer from the
protein to the electrode surface
(nanobiosensors of the 3rd generation)
13
Direction of practical application of
nanobioparticles
• Directed drug delivery
• Separation of biomolecules and cells
• Development of nanomechanical
systems/machines
• Analytical biotechnologies (including
Nanobiosensorics)
14
Potentiometric biosensor in a two-electrode configuration
Working – ASE
electrode
Reference electrode
Ag/AgCl/3 M KCl
Measuring cell
50 mM Hepes
buffer, pH 7.5
2 NH4+
Urea
Arginine
Urease
Arginase I
CO2
Ornithine
The scheme of biosensor membrane
Ammonium selective electrode
A NEW BI-ENZYME POTENTIOMETRIC SENSOR
FOR ARGININE ANALYSIS BASED ON
RECOMBINANT HUMAN ARGINASE I AND
COMMERCIAL UREASE
B
A
C
Fig. Characterization of obtained AuNPs: SEM micrographs –
before (A) and after arginase I immobilization (B); C - X-ray
microanalysis.
General scheme of enzymes immobilization on gold
surface
Au + HS-(CH2)15-COOH → Au...S-(CH2)15-COOH
+
Activation
↓
PFP
+
Basic catalyst DIPEA: (iPr)2NEt
CDI
Blocking of un-reacted carboxylic groups with
↓ AEE (aminoethoxyethanol)
Au...S-(CH2)15-C(O)≈OR
Au-structures have been functionalized by their pretreatment
using 16-mercaptohexa-decanoic acid followed by its activation
using carbodiimide-pentaphenol-ester method and blocking non18
reacted activated sites by aminoethoxyetanol.
Functionalized
Au-Electrode
The response of the bare ASE to ammonium ions.
200
180
160
E, mV
140
120
Y=A+B*X
A
139.8
B
45.2
100
80
60
R
SD
N P
--------------------------------------------0.99679 3.94538 6 <0.0001
---------------------------------------------
40
20
0
-2.0
-1.5
-1.0
-0.5
0.0
+
lg [mM NH4 ]
0.5
1.0
Calibration curves for L-Arg
determination with Arginase-based bienzyme biosensor
Hyperbl
Equation
y = P1*x/(P2 + x)
Reduced
Chi-Sqr
5.74698
Adj. R-Square
0.99395
90
Value
y = P1*x/(P2 + x)
Reduced
Chi-Sqr
23.86637
0.96926
B
P1
92.25827
1.95322
B
P2
4.70971
0.38197
Value
80
Standard Error
?$OP:F=1
Imax
?$OP:F=1
Km
220
Equation
y = a + b*x
Weight
No Weighting
Residual Sum of
Squares
Е, mV
200
2.78209
1.12709
0.19069
40
60
268.8963
Pearson's r
0.98538
Adj. R-Square
0.96774
Value
?$OP:A=1
Intercept
?$OP:A=1
Slope
Standard Error
147.52336
2.1491
34.72863
2.00164
180
160
180
50
170
160
40
-1.0
0
-0.5
0.0
0.5
1.0
1.5
2.0
0
10
20
30
L-Arg, mM
40
68.7774
0.99499
Pearson's r
0.98889
Value
?$OP:A=1
Intercept
?$OP:A=1
Slope
Standard Error
135.45147
0.8667
26.22984
0.87854
140
130
110
100
-1.5
10
lg [Arginine, mM]
No Weighting
Residual Sum of
Squares
120
20
120
y = a + b*x
Weight
150
140
20
Equation
Adj. R-Square
30
A
Standard Error
74.72172
70
60
E, mV
Hyperbl
Equation
Е, mV
80
Model
Adj. R-Square
E, mV
100
Model
-1.0
(B)
-0.5
0.0
0.5
1.0
1.5
2.0
lg [Arginine, mM]
0
50
0
B
10
20
30
40
L-Arg, mM
The potentiometric response of bi-enzymatic electrode, based on
urease and different arginase forms integrated in 2 % calcium
alginate gel to the L-arginine logarithm concentration: A – free
arginase, E (51.1 U·mL-1) and B – enzyme, immobilized on NPs,
ENPs (35.5 U·mL-1). LOD: 10-4 M
130
120
110
100
90
80
70
60
50
40
30
20
10
0
-10
-20
-30
Е, (mV)
ratio, (%)
Amino acids
L-A
rgi
nin
Ca
e
na
va
nin
D,L
e
-Va
L-C line
ys
te
Cit ine
ru
L-O lline
rni
thi
D,
L-L ne
ys
L-I
in
so
leu e
c
L-P ine
rol
i
L-L ne
L-G ysin
e
lu
L-T tam
ine
ryp
top
ha
Gl
uta n
ma
te
E, mV and % ratio
The selectivity of Arginase-based
biosensor
Response of biosensor to different amino acids in
concentration 10 mM: black columns – E, mV;
grey – ratio, % to L-arginine signal.
Relative response, %
Storage stability of bi-enzyme
biosensor
120
110
100
90
80
70
60
50
40
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Time, days
Storage stability of two types of bi-enzymic ASE
electrodes based on E (black line) and ENPs (grey line).
Biosensor analysis of L-Arg in Real
sample – Tivortin
From instruction, mM
199,3
210
Biosensor, mM
200 ±0,01
A
127,14286 0,75142
B
45,91837 0,58913
Tivortin
------------------------------------------------------------
200
R
190
SD
N
P
E, mV
-----------------------------------------------------------0,99992
180
0,20203
3
170
0,00817
n=40
160
150
A
119,4316 1,36584
B
42,34309 1,18029
------------------------------------------------------------
140
n=80
R
130
SD
N
P
-----------------------------------------------------------0,99922
0,70943
4
120
0,2
0,4
0,6
0,8
1,0
1,2
lg [mM, arginine]
1,4
1,6
7,76081E-4
Biosensor analysis of L-Arg in Real sample –
Cytrarginine
From instruction, mM
475
Cytrarginine
A
147,85714
6,01133
B
32,65306
4,71306
------------------------------------------------------------
200
R
SD
N
P
-----------------------------------------------------------0,98974
1,61624
3
0,09126
190
E, mV
Biosensor, mM
477 ±0,01
180
n=100
170
160
A
149,20658
2,03504
B
24,08398
1,76306
------------------------------------------------------------
n=200
R
SD
N
P
-----------------------------------------------------------0,99468
1,08493
4
0,00532
150
0,2
0,4
0,6
0,8
1,0
1,2
lg [mM, arginine]
1,4
1,6
Biosensor analysis of L-Arg in Real sample –
Aminoplazmal 10% E
From instruction, mM
8
Biosensor, mM
8.5±0,02
Aminoplazmal 10% E
220
A
134
0
B
50
0
------------------------------------------------------------
210
E, mV
R
SD
N
P
------------------------------------------------------------
200
1
0
190
3
<0.0001
n=1
A
130,00249 3,21451
B
50,55705 2,77782
------------------------------------------------------------
180
R
N
0,99699
1,66964
160
0,6
P
------------------------------------------------------------
n=2
170
SD
0,8
1,0
1,2
1,4
lg [mM, arginine]
1,6
4
0,00301
Conclusions
• To improve the enzyme stability, the purified arginase
and nanosized carriers, namely, gold and silver
nanoparticles were synthesized;
• Sensitive potentiometric bi-enzyme biosensor based on
recombinant arginase I and commercial urease
immobilized on the surface of ammonium-selective
electrode was constructed and some characteristics of
the bioelectrode were estimated.
• The created laboratory prototype of arginine-selective
biosensor exhibits a good response performance to LArg with the linear range from 0.5 to 40 mM.
• The bi-enzyme electrode is characterized by a high
storage stability and selectivity for arginine assay in
real samples.
Amperometric sensor versus potentiometric one
• Potentiometric detection of Arg based on NH4+-electrode
is not sensitive (0.1-1.0 mM), while normal content of Arg
in blood is less than 0.1 mM (Stasyuk et al. // J. of Materials
Science and Engineering: A, 2011, (1), p. 819-827);
• Amperometric transduction of the signal is usually much
more sensitive.
27
Amperometry in a three-electrode configuration
Counter electrode
Working electrode
Measuring cell
Reference electrode
Ag/AgCl/3 M KCl
or SC electrode
100 mM phospate
buffer, pH 7.5
Principal scheme of L-Arg detection by
bi-enzyme/PANi-Nafion/Pt-electrode
Pt
PANi+RSO3Nafion - PANi
Urea +
2Н2О + Н+
L-arginine + Н2О
2NH4++
HCO3-
Arginase
PANi0RSO3Urea + L-ornithine
PANi+ and PANiº – an oxidized and reduced forms of PANi, respectively;
RSO3- - a skeleton of Nafion with the sulfonate groups.
Formation of PANi-Nafion film on 3 mm Pt
electrode
0.15
(A)
I, A
0.10
0.05
0.00
-0.05
-0.10
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
E, V
Cyclic voltammograms at 22 °C, scan rate of 50 mV∙s-1 vs Ag/AgCl (3M KCl) as
reference electrode, in electrolyte solution (0.2 M aniline in 0.5 M H2SO4 )
30
Structural characteristics of PANi film
Atomic Force Microscopy micrograph of the
PANi film formed on Pt electrode by 11 cycles
of electrodeposition.
PANi
A
The Gaussian distribution curve of the PANi
film thickness resulting from the AFM.
PANi
B
Pt
Pt
SEM images of PANi films on the surface of Pt electrode: freshly prepared film (A); after
3 days of storage (B).
31
Optimization of working parameters
for PANi-Nafion modified Pt electrode
I, A
10
1
2
3
5
0
-5
-10
-15
-0.4
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
E, V
Cyclic voltamperometric current responses of PANi-Nafion/Pt
electrode in PB as a control (1, black), on 0.5 mM NH4CI in PB
(2, red) and on 3.5 mM NH4CI in PB (3, blue).
Characterization of PANi-Nafion-modified Pt-electrodes
0.5
5
0.0
4
-0.5
0.01 mM 0.03 mM
1
(A)
0.15 mM
0.00
0.07 mM
-1.0
0.07 mM
0.25
I, A
Current (A)
A
0.3 mM
-0.25
-0.50
0.6 mM
-0.75
3
-1.5
1.2 mM
-1.00
0.14 mM
-1.25
-2.0
-2.5
+
0.3 mM NH4
-3.0
2.4 mM
0.5 A
-1.50
-1.75
3.2 mM
-2.00
2
-2.25
-3.5
900
1000
1100
1200
1300
1400
1500
1600
1700 -2.50
Time (s)
Chronamperometric response of PANiNafion-modified electrode (1-3) and
control (PANi-modified) electrode (4)
upon subsequent additions of NH4CI under
different potentials: -100 mV (1); - 200
mV (2, 4); – 300 mV (3).
1000
1200
1400
1600
1800
2000
2200
2400
2600
Time,s
Chronamperometric current
responses (inserted) upon
subsequent additions of
NH4CI
2800
Characteristics of PANi-Nafion/Pt electrodes (d=3.0 mm) vs the
Ag/AgCl electrode at - 200 mV, 22 °C, in 30 mM PB, pH 7.5.
Calibration curve for amperometric response of the urease-PANiNafion/Pt electrode (b) on ammonia ions and urea, respectively.
Insets: chronamperometric current responses upon subsequent
additions of NH4CI (a) and urea (b). Linearity: 0.03 – 0.3 mM urea.
Chronamperometric current response to L-Arg (A) and
calibration curve for amperometric response of the bienzyme electrode (B)
0.5
0.4
0.3
(A)
0.05 mM
0.12 mM
0.2 mM
0.25 mM
0.3 mM
-800
I, nA
I, A
0.6
-600
0.5 mM
0.2
0.1
(B)
Imax= 1280.78 ± 164.32 нА
KM
= 1.28 ± 0.29 мМ
-500
0.7 mM
0.1 A
0.0
-700
Model
Hyperbol
R^2
= 0.977
-400
1.2 mM
-0.1
1.8 mM
-0.2
-300
-200
-0.3
-100
-0.4
0
-0.5
600
700
800
900
1000
1100
Time, s
1200
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
[L-Arginine], mM
Sensitivity: 110 ± 1.3 nA∙mM-1∙mm-2, LOD: 3.8 ∙10-5 M
2.0
Characteristics of the developed L-Arg biosensor
Relative current responce / %
120
a
100
80
60
40
20
0
6.0
6.5
7.0
7.5
8.0
8.5
9.0
pH
Effect of pH influence
Response to 0.25 mM analyte.
The tested solutions contained
0.25 mM amino acids in 30 mM
phosphate buffer, pH 7.5
100
c
Relative responce / %
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
Time / hours
50
60
70
80
Storage stability tested with 25 mM
L-Arg in PB during 3 days.
Bioelectrode was kept in freezer at
4 °C in PB, supplemented with
10 mM CaCl2
Comparison of different methods for L-Arg assay
in real pharmaceuticals
І, А
-0.15
-0.30
1) Citrarginine
n 6000;
А =0.027 A
В =0.341 A/mМ
R =0.991
Ccalcul. 475.1 mМ
-0.10
2
-0.05
0.00
-0.35
1
-0.25
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Declared
by
producer
-0.40
1) "Aminoplazmal' 10 % Е"
n 50;
А =0.051 A
В =0.329 A/mМ
R =0.991
Ccalcul 7.7 mМ
-0.15
2
-0.10
-0.1
1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
2) "Tivortin"
n 625;
А =0.146 A
В =0.458A/mМ
R =-0.992
Ccalcul.= 199.3 mМ
A
-0.35
-0.30
-0.25
1) "Tivortin"
n 333.3;
А =0.229 A
В =0.379 A/mМ
R =0.988
Ccalcul. 201.4 mМ
2
-0.20
1
-0.15
0.0
0.1
0.2
0.3
0.4
0.5
0.6
[L-arginine], mМ
[L-arginine], mМ
[L-arginine], mМ
Sample
-0.45
-0.20
-0.05
0.05
-0.1
C
2) "Aminoplazmal' 10 % Е"
n 33.3;
А =0.086 A
В =0.363 A/mМ
R =0.979
Ccalcul 7.9 mМ
I, A
-0.20
B
I, A
-0.25
2) "Citrarginine"
n 3000;
А =0.042 A
В =0.257A/mМ
R =0.989
Ccalcul. 484.6 mМ
Concentration of L-Arg, mM
Amperometric biosensor
Potentiometric biosensor
Determined
CV*, %
Dif.**, %
Determined CV, %
Dif., %
“Тivortin”
199.3
200.3 ± 2.5
0.8
+ 0.5
200.7 ± 4.5
2.1
+ 0.7
“Citrarginine”
“Аminoplazm
al 10% Е”
475.0
479.9 ± 4.7
1.4
+ 1.0
447.2 ± 3.3
7.9
- 5.9
8.0
7.8 ± 0.3
2.2
- 2.5
8.5 ± 2.5
1.3
+ 6.3
Conclusions 2
1. An
amperometric
urease-arginase-biosensor
on
L-arginine has been developed and optimized for the
first time.
2. The constructed biosensor is characterized by a low
applied potential (−200 mV), fast response to the analyte
(10 s), broad linear dynamic range (0.05 to 0.6 mM),
high selectivity and sensitivity (110 ± 1.3 nA∙mM-1∙mm-2)
and a low limit of detection (3.8·10-5 M).
3. The proposed biosensor was successfully tested for
L-Arg assay in some commercial pharmaceuticals using
the multiple standard addition method.
38
L-arginine-selective microbial amperometric
sensor based on recombinant yeast cells overproducing human liver arginase I
Pt
PANi+ RSO3 - + NH4+
Nafion - PANi
PANi0 RSO3- NH4+
2NH4++
+HCO3-
Urea +
2Н2О + Н+
L-arginine + H2O
Arginase
in the cell
Urea +
L-rnithine
Chronoamperometric current responses on L-Arg
0.15 mM
0,00
0.3 mM
0,02
1
0.6 mM
I (A)
0,04
1.2 mM
2.9 mM
0,06
3.4 mM
0,08
2
0,10
0,12
0,14
800
850
900
950
1000
1050
Time (s)
1100
1150
1200
Chronoamperometric current responses upon subsequent additions
of L-Arg aliquots of the developed cell-PANi–Nafion/Pt electrodes
for native (1) and permeabilized (2) cells. Conditions: - 200 mV vs
Ag/AgCl electrode in 30 mM phosphate buffer, pH 7.5 at 22 °C.
Calibration curves for amperometric response on L -Arg
of the developed p-cell-PANi–Nafion/Pt electrode (A, B).
120
A
60
100
50
40
60
І, nА
I (nА)
80
B
Y=A+B*X
A = 3.02 ± 0.3 nA
B = 98.5 ± 8.4 nA/mM
R = 0.993
Chi^2/DoF = 609.01
R^2 = 0.995
Imax = 111 ± 3.2 nA
40
20
20
Kapp
M = 0.500 ± 0.050 mM
0
30
10
0
0.0
0.5
1.0
1.5
2.0
2.5
L-Arg (mM)
3.0
3.5
0.0
0.1
0.2
0.3
0.4
L-Arg, mM
0.5
Conditions: - 200 mV vs Ag/AgCl electrode in 30 mM
Phosphate buffer, pH 7.5 at 22 °C .
0.6
Characteristics of the p-cells-based sensor
100
A
Relative response (%)
Relative response (%)
100
80
60
40
20
B
90
80
70
60
50
40
30
20
10
0
e
g
Ar anin
v
na
Ca
s
Ly
r
Se
o
Pr
s
Cy
l
e
Va llin
u
tr
Ci
r
n
Th Or
Ile
0
0
10
20
30
40
50
Time (hours)
60
70
80
A – selectivity; response to the tested solutions containing
0.15 mM corresponding L-amino acid in 30 mM Phosphate
buffer (PB), pH 7.5; B – storage stability at the +4°С in 30
mM PB, pH 7.5; response to 0.15 mM Arg.
Concentration of L-Arg (CArg) in food samples
determined by different analytical methods, mM
Method
Biosensor
[this paper]
Arginase-based enzymatic
fluorimetric
(E-Fl)
spectrophotometric
(E-Sp)
Referent
chemical
(R-Ch)
CArg
CV*
,%
CArg
CV,
%
CArg
CV, %
CArg
Wine
“Chardonnay”
(dry, white).
0.98 ± 0.11
11.2
1.05 ± 0.04
3.75
0.958 ± 0.05
4.80
0.966 ±
0.06
6.43
Wine “Moution
Cadet” (dry,white).
1.96 ± 0.05
0.51
1.97 ± 0.01
1.01
1.96 ± 0.05
2.01
1.95 ±
0.04
1.54
Wine “Massandra”
(sweet, red)
2.46 ± 0.06
1.22
2.55 ± 0.04
1.20
2.36± 0.06
1.70
ND**
-
Juice “Sadochok”
ND
-
2.055 ±
0.041
2.43
1.99± 0.03
2.01
2.21 ±
0.08
2.26
CV,
%
1
2,8
Reference methods, mM
2,6
2,4
2,2
2,0
1,8
1,6
Massandra 3
1 - E-Fl
A = 0.042 ± 0.04
B = 0.980 ± 0.03
R = 0.996
2
Moution
Cadet
2 - E-Sp
A = 0.030 ± 0.001
B = 0.97 ± 0.05
R = 0.998
1,4
3 - R-Ch
A = 0.018 ± 0.001
B = 1.00 ± 0.02
R= 1
1,2 Chardonnay
1,0
0,8
0,8
1,0
1,2
1,4
1,6
1,8
2,0
Microbial biosensor, mM
2,2
2,4
2,6
Correlations between the results of L-Arg estimation in wines by different
methods: 1 –enzymatic-fluorometric (E-Fl), 2 –enzymatic-spectrophotometric (ESp), and 3 – reference chemical (R-Ch,) relatively to the proposed cell-based
biosensor’s data. Some statistical data are presented on the graphs: parameters of
linear regression A and B (coefficients of the equation Y=A+BX), R - linear
regression coefficient.
L-arginine selective biosensor based on the
arginine deiminase (ADI)
Pt
PANi+ RSO3 - + NH4+
Nafione - PANi
PANi0 RSO3- NH4+
L-arginine + H 2O
ADI
NH4+ + L-citrulline
-2,5
Results of Y. BORETSKY
-2,0
-1,5
I, mkA
Argininedeiminase M. hominis from
the recombinant yeast strain E. coli.
-1,0
-0,5
0,0
800
850
900
950
1000
1050
1100
1150
Time, s
-2,5
-2,0
I, mkA
-1,5
-1,0
Chi^2/DoF = 0.0317
R^2 = 0.952
Imax = -2.74 ±0.295 mkA
Km = 5.77 ±1.69 mkA/mM
-0,5
Electrophoregram of purified preparate of ADI:
1. total protein of inductive cells
2. Fraction of “inclusion bodies”
3. Peak of elution from the QAE-Sepharose
4. Peak elution from the Phenyl-Sepharose.
5. Markers of molecular weight.
0,0
0,0
2,5
5,0
7,5 10,0 12,5 15,0 17,5 20,0 22,5
[Arginine], mM
Chronoamperometric response and
calibration graph on L-Arg
Electrochemical characteristics of PANi-Nafion/Pt electrode
Results of Y. KORPAN
300
60
1st cycle
2nd - 6th cycles
7th cycle
200
D
40
C
100
A
20
B
Current, A
I, A
0
-100
E
-200
-300
0
-20
20 mM PB, pH 7.4
+ 0,2 mM NH4Cl
B'
A'
+ 0,4 mM NH4Cl
-40
-400
+ 2,4 mM NH4Cl
C'
-500
-0,6
-60
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
1,0
1,2
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
Potential, V
V
Formation of PANi-Nafion film on 3
mm Pt electrode
Cyclic voltammometric current responses
of PANi-Nafion/Pt electrode in PB on NH4CI
3,0
2,5
I, A
2,0
1,5
1,0
0,5
0,0
0
100
200
300
400
500
NH4Cl, M
Chronamperometric current responses (inserted) upon subsequent additions of NH4CI
Characteristics of amperometric biosensor based on ADI
Results of Y. KORPAN
3,0
2,5
I, A
2,0
1,5
1,0
0,5
0,0
0
100
200
300
400
500
L-Arg, M
Calibration graph and chronoamperometric current response
onto subsequent addition of L-Arg at potential – 350 mV,
22 °C, 20 мМ PB, рН 7,4.
Linearity of amperometric current response in the range from
0,07 – 0,6 mM L-Arg (R=0,999)
CONTRIBUTORS
Institute of Cell biology, NAS of Ukraine, Lviv (Ukraine):
Prof. A. SIBIRNY
Prof. M. GONCHAR
Dr. Sci. Y. BORETSKY
PhD. G. GAYDA
PhD. O. SMUTOK
PhD. L. FAYURA
R. SERKIZ
Institute of Molecular Biology and Genetics, NAS of
Ukraine, Kiev (Ukraine):
PhD. Y. KORPAN
ACKNOWLEDGEMENTS
• This work was financially supported by Scientific
integration of the Polish-Ukrainian borderland area in
the field of monitoring and detoxification of harmful
substances in environment (cross-border project PLBY-UA 2007-2013, cofinanced by the European
Union), NAS of Ukraine (Project 13/2014, program
“Sensors for Medical, Environmental, Industrial, and
Technological Needs”), by NATO (Project CBP.
NUKR.SFP 984173), by Individual grants for young
scientists of FEMS (Stasyuk-2013) and OPTEC
company (Stasyuk-2014).
Thank you for your attention!