Electrochemical biosensors on arginine assay

Nataliya Stasyuk

 Department of Analytical Biotechnology, ICB, NAS of Ukraine

Scientific integration of the Polish-Ukrainian borderland area in the field of monitoring and detoxification of harmful substances in environment.Scientific integration of the Polish-Ukrainian borderland area in the field of monitoring and detoxification of harmful substances in environment.

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 physico-chemical 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

2NH3 + CO2

Urease

Arginase

8

Comparison of different types of developed biosensors on L-Arg. Mode of signal

registration

Biocomponent LOD, mM

Linearrange, mM

Response time (95%),

min

Stability, days

Reference

Potentiometric,ASE*

Bacterial cells0.05 – 1.0 Rechnitz et.al., 1977

Potentiometric, NH3 gas sensor

Bacterial cells0.008 – 1.0 Grobler et. al., 1982

Potentiometric, NH3 gas sensor

U/A**0.03-3.0 5.0

Nikolelis and Hadjiioannou, 1983

Potentiometric U/A 0.1-1.0 Ivnitski and Rishpon, 1993

Potentiometric,ASE

U/A0.01 0.1-30 1.5 - 4.0 21 Koncki et al., 1996

Potentiometric U/A 0.01-1.0 Komaba et al., 1998

Potentiometric,pH

U/A0.025-0.31 10.0 Karacaoglu et.al, 2003

PotentiometricISE

U/A 0.03-0.05 5.0-7.0 Lvova et al., 2003

Potentiometric, ISEU/A

10-6-103 0.7-5.0 60Kaur, http://hdl.handle.net

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

Schematic illustrations of bienzyme system for the detection of arginine.

Biological determination of Ag(I) ion and arginine by using the composite film of electroinactive

polypyrrole and polyion complex

Sensors and Actuators B 52 (1998) 78–83

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.

Biosensors and bioelectronics 43 (1996) 667-674

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

Measuring cell50 mM Hepes buffer, pH 7.5

Working – ASEelectrode

Reference electrodeAg/AgCl/3 M KCl

Potentiometric biosensor in a two-electrode configuration

A NEW BI-ENZYME POTENTIOMETRIC SENSOR FOR ARGININE ANALYSIS BASED ON

RECOMBINANT HUMAN ARGINASE I AND COMMERCIAL UREASE

Arginine

Ornithine

Urea

2 NH4+

Arginase I

Urease

The scheme of biosensor membrane

CO2

Am

mon

ium

sel

ecti

ve e

lect

rod

e

A B

C

Fig. Characterization of obtained AuNPs: SEM micrographs – before (A) and after arginase I immobilization (B); C - X-ray microanalysis.

Au-structures have been functionalized by their pretreatment using 16-mercaptohexa-decanoic acid followed by its activation using carbodiimide-pentaphenol-ester method and blocking non-reacted activated sites by aminoethoxyetanol.

Au + HS-(CH2)15-COOH → Au...S-(CH2)15-COOH

+

Au...S-(CH2)15-C(O)≈O-R

PFP CDI

+DIPEA: (iPr)2NEt

ActivationActivation

Basic catalyst

↓

↓FunctionalizedAu-Electrode

Blocking of un-reacted carboxylic groups with AEE (aminoethoxyethanol)

1818

General scheme of enzymes immobilization on gold General scheme of enzymes immobilization on gold surfacesurface

0

20

40

60

80

100

120

140

160

180

200

1.00.50.0-0.5-1.0-1.5-2.0

Y = A + B * XA 139.8B 45.2

R SD N P---------------------------------------------0.99679 3.94538 6 <0.0001---------------------------------------------

E,

mV

lg [mM NH4+]

The response of the bare ASE to ammonium ions.

Calibration curves for L-Arg determination with Arginase-based bi-

enzyme biosensor

0 10 20 30 40 50

0

20

40

60

80

100

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0

120

140

160

180

200

220

Е, m

V

lg [Arginine, mM]

Equation y = a + b*x

Weight No Weighting

Residual Sum of Squares

268.8963

Pearson's r 0.98538

Adj. R-Square 0.96774

Value Standard Error

?$OP:A=1 Intercept 147.52336 2.1491

?$OP:A=1 Slope 34.72863 2.00164

E

, mV

L-Arg, mM

Model Hyperbl

Equation y = P1*x/(P2 + x)

Reduced Chi-Sqr

5.74698

Adj. R-Square 0.99395

Value Standard Error

B P1 92.25827 1.95322

B P2 4.70971 0.38197

A0 10 20 30 40

0

10

20

30

40

50

60

70

80

90

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0100

110

120

130

140

150

160

170

180

Е, m

V

lg [Arginine, mM](B)

Equation y = a + b*x

Weight No Weighting

Residual Sum of Squares

68.7774

Pearson's r 0.99499

Adj. R-Square 0.98889

Value Standard Error

?$OP:A=1 Intercept 135.45147 0.8667

?$OP:A=1 Slope 26.22984 0.87854

E

, mV

L-Arg, mM

Model Hyperbl

Equation y = P1*x/(P2 + x)

Reduced Chi-Sqr

23.86637

Adj. R-Square 0.96926

Value Standard Error

?$OP:F=1 Imax 74.72172 2.78209

?$OP:F=1 Km 1.12709 0.19069

B

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

The selectivity of Arginase-based biosensor

L-A

rgin

ine

Can

avan

ine

D,L

-Val

ine

L-C

yste

ine

Citr

ullin

eL-

Orn

ithin

eD

, L-L

ysin

eL-

Isol

euci

neL-

Prol

ine

L-Ly

sine

L-G

luta

min

eL-

Tryp

toph

anG

luta

mat

e

-30-20-10

0102030405060708090

100110120130

Amino acids

Е, (mV) ratio, (%)

E,

mV

an

d %

ra

tio

Response of biosensor to different amino acids in

concentration 10 mM: black columns – E, mV; grey – ratio, % to L-arginine signal.

Storage stability of bi-enzyme biosensor

0 1 2 3 4 5 6 7 8 9 1011121314151617180

10

20

30

40

50

60

70

80

90

100

110

120

Time, days

Rel

ativ

e re

spo

nse

, %

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

0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6120

130

140

150

160

170

180

190

200

210

n=80

n=40

A 119,4316 1,36584

B 42,34309 1,18029

------------------------------------------------------------

R SD N P

------------------------------------------------------------

0,99922 0,70943 4 7,76081E-4

A 127,142860,75142

B 45,91837 0,58913

------------------------------------------------------------

R SD N P

------------------------------------------------------------

0,99992 0,20203 3 0,00817

Tivortin

E,

mV

lg [mM, arginine]

From instruction, mM Biosensor, mM

199,3 200 ±0,01

Biosensor analysis of L-Arg in Real sample – Cytrarginine

0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6150

160

170

180

190

200

n=200

n=100A 149,20658 2,03504B 24,08398 1,76306------------------------------------------------------------

R SD N P------------------------------------------------------------0,99468 1,08493 4 0,00532

A 147,85714 6,01133B 32,65306 4,71306------------------------------------------------------------

R SD N P------------------------------------------------------------0,98974 1,61624 3 0,09126

E,

mV

lg [mM, arginine]

Cytrarginine

From instruction, mM Biosensor, mM

475 477 ±0,01

Biosensor analysis of L-Arg in Real sample – Aminoplazmal 10% E

0,6 0,8 1,0 1,2 1,4 1,6160

170

180

190

200

210

220

A 130,002493,21451

B 50,55705 2,77782

------------------------------------------------------------

R SD N P

------------------------------------------------------------

0,99699 1,66964 4 0,00301

A 134 0

B 50 0

------------------------------------------------------------

R SD N P

------------------------------------------------------------

1 0 3 <0.0001

Aminoplazmal 10% E

E,

mV

lg [mM, arginine]

n=2

n=1

From instruction, mM Biosensor, mM

8 8.5±0,02

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 L-Arg 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

Measuring cell 100 mM phospate buffer, pH 7.5

Working electrode

Counter electrodeReference electrodeAg/AgCl/3 M KClor SC electrode

Amperometry in a three-electrode configuration

PANi+ and PANiº – an oxidized and reduced forms of PANi, respectively; RSO3- - a skeleton of Nafion with the sulfonate groups.

Principal scheme of L-Arg detection by bi-enzyme/PANi-Nafion/Pt-electrode

PtPANi+RSO3

-

Nafion - PANi

PANi0RSO3-

2NH4++

HCO3-

Urea + L-ornithine

L-arginine + Н2О

Arginase

Urease

Urea + 2Н2О + Н+

30

Formation of PANi-Nafion film on 3 mm Pt electrode

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 )

31

Structural characteristics of PANi film

Atomic Force Microscopy micrograph of the PANi film formed on Pt electrode by 11 cycles of electrodeposition.

The Gaussian distribution curve of the PANi film thickness resulting from the AFM.

SEM images of PANi films on the surface of Pt electrode: freshly prepared film (A); after 3 days of storage (B).

PANi

PtPt

PANi

A B

-0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4-15

-10

-5

0

5

10

E, V

I, A

1 2 3

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).

Optimization of working parameters for PANi-Nafion modified Pt electrode

Characterization of PANi-Nafion-modified Pt-electrodes

900 1000 1100 1200 1300 1400 1500 1600 1700-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

Time (s)

Cu

rren

t (

A)

5

4

3

2

0.07 mM

0.14 mM

0.3 mM NH+4

A

1

Chronamperometric response of PANi-Nafion-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).

Chronamperometric current responses (inserted) upon subsequent additions of NH4CI

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-PANi-Nafion/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 bi-

enzyme electrode (B)

Sensitivity: 110 ± 1.3 nA∙mM-1∙mm-2, LOD: 3.8 ∙10-5 M

Response to 0.25 mM analyte. The tested solutions contained 0.25 mM amino acids in 30 mM phosphate buffer, pH 7.5

Characteristics of the developed L-Arg biosensor

6.0 6.5 7.0 7.5 8.0 8.5 9.0

0

20

40

60

80

100

120

pH

Rel

ativ

e cu

rren

t re

spon

ce /

%

a

0 10 20 30 40 50 60 70 800

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e re

spon

ce /

%

Time / hours

c

Effect of pH influence

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

Sample

Concentration of L-Arg, mMDeclared

by producer

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” 475.0 479.9 ± 4.7 1.4 + 1.0 447.2 ± 3.3 7.9 - 5.9

“Аminoplazmal 10% Е”

8.0 7.8 ± 0.3 2.2 - 2.5 8.5 ± 2.5 1.3 + 6.3

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.

Conclusions 2

38

L-arginine-selective microbial amperometric sensor based on recombinant yeast cells over-

producing human liver arginase I

Pt

PANi+ RSO3 - + NH4

+

Nafion - PANi

PANi0 RSO3- NH4

+

2NH4++

+HCO3-

Urea + L-rnithine

L-arginine + H2O

Arginase in the cell

Urease

Urea + 2Н2О + Н+

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.

Chronoamperometric current responses on L-Arg

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).

0 10 20 30 40 50 60 70 800

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e re

spon

se (

%)

Time (hours)

B

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.

Characteristics of the p-cells-based sensor

Concentration of L-Arg (CArg) in food samples determined by different analytical methods, mM

Method Biosensor [this paper]

Arginase-based enzymatic Referent chemical(R-Ch)

fluorimetric (E-Fl)

spectrophotometric(E-Sp)

CArg CV*

, %CArg CV,

%CArg CV, % CArg CV,

%

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 2.011.95 ± 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 2.012.21 ± 0.08 2.26

0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

2,2

2,4

2,6

2,8

Moution Cadet

Massandra

23

Microbial biosensor, mM

3 - R-Ch A = 0.018 ± 0.001 B = 1.00 ± 0.02 R = 1

1 - E-Fl A = 0.042 ± 0.04 B = 0.980 ± 0.03 R = 0.996

2 - E-Sp A = 0.030 ± 0.001 B = 0.97 ± 0.05 R = 0.998

Ref

eren

ce m

eth

ods,

mM

1

Chardonnay

Correlations between the results of L-Arg estimation in wines by different methods: 1 –enzymatic-fluorometric (E-Fl), 2 –enzymatic-spectrophotometric (E-Sp), 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)

0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5

0,0

-0,5

-1,0

-1,5

-2,0

-2,5

Chi^2/DoF = 0.0317R^2 = 0.952Imax = -2.74 ±0.295 mkAKm = 5.77 ±1.69 mkA/mM

I, m

kA[Arginine], mM

800 850 900 950 1000 1050 1100 1150

0,0

-0,5

-1,0

-1,5

-2,0

-2,5

I, m

kA

Time, s

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.

Chronoamperometric response and calibration graph on L-Arg

Argininedeiminase M. hominis from the recombinant yeast strain E. coli.Results of Y. BORETSKY

-0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2-500

-400

-300

-200

-100

0

100

200

300

I,

A

V

1st cycle 2nd - 6th cycles 7th cycle

A B

C

D

C'

B'A'

E

Formation of PANi-Nafion film on 3 mm Pt electrode

-0.4 -0.2 0.0 0.2 0.4 0.6 0.8-60

-40

-20

0

20

40

60

20 mM PB, pH 7.4 + 0,2 mM NH

4Cl

+ 0,4 mM NH4Cl

+ 2,4 mM NH4Cl

Cu

rre

nt, A

Potential, V

Cyclic voltammometric current responses of PANi-Nafion/Pt electrode in PB on NH4CI

Chronamperometric current responses (inserted) upon subsequent additions of NH4CI

Electrochemical characteristics of PANi-Nafion/Pt electrodeResults of Y. KORPAN

0 100 200 300 400 500

0,0

0,5

1,0

1,5

2,0

2,5

3,0

I,

A

NH4Cl, M

0 100 200 300 400 500

0,0

0,5

1,0

1,5

2,0

2,5

3,0

I,

A

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)

Characteristics of amperometric biosensor based on ADIResults of Y. KORPAN

CONTRIBUTORS Institute of Cell biology, NAS of Ukraine, Lviv (Ukraine):

Prof. A. SIBIRNYProf. M. GONCHAR Dr. Sci. Y. BORETSKYPhD. G. GAYDAPhD. O. SMUTOKPhD. L. FAYURAR. 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 PL- BY-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!