Euroanalisis poster1 1
1
María Cuartero Botía a , Joaquín A. Ortuño Sánchez-Pedreño a , Mª Soledad García García a and Francisco García Cánovas b a Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, Murcia E-30071, Spain b Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Murcia E-30100, Spain [email protected] NEW ASSAY OF THE ACETYLCHOLINESTERASE ACTIVITY BY POTENTIOMETRIC MONITORING OF ACETYLCHOLINE (C) 3 N + CH 2 CH 2 OCCH3CH 3 A C hE H 2 O (Acetylcholinesterase enzym e) (CH 3 ) 3 N + CH 2 CH 2 OH + HOCCH 3 O O Acetylcholine (AC h) Choline (C h) REACTION IN STUDY POTENTIOMETRIC MONITORING OF SUBSTRATE CONCENTRATION To monitor the kinetics of a hydrolysis reaction the detection system must satisfy some requirements: Sufficient sensitivity and stability Low background noise Low drift Rapid response Sufficient selectivity over the product of the reaction 1. DEVELOPMENT OF AN ACETYLCHOLINE-SELECTIVE ELECTRODE: Membrane: 100 mg PVC 200 mg of plasticizer 1.5 mg ionic additive NPOE best selectivity and sensitivity 0.5% of ionic additive best sensitivity and stability 3. KINETIC DATA ANALYSIS the ISE is able to follow the continuos decrease in ACh concentration Other response characteristics in 7.5 pH phosphate buffer medium: Slope 59.2 mV/dec; linear range 1x10 -6 – 1x10 -3 M; Detection limit 1.5x10 -7 M; drift in the linear range 0.05-1.1 mV/h; Excellent repeatability of the calibration parameters. log [C ]/M -6 -5 -4 -3 Potentialchange /m V 0 50 100 150 200 -6 -5 -4 -3 -6 -5 -4 -3 DOS NPOE TCP K + ACh Ch Na + H + K + K + ACh ACh Ch Ch Na + Na + H + H + Selection of the plasticizer -8 -6 -4 -2 P otentialchange /m V 0 50 100 150 200 log [C ]/M 0.5 % 1 % 0.3 % Selection of the amount of ionic additive tim e /s 0 5 10 15 20 25 [A C h]/M 3.8e-4 3.9e-4 4.0e-4 10 m lmin -1 5 m lmin -1 2.5 m lmin -1 ― theoretical values calculated from the dilution program Evaluation of the response time tim e /s 0 200 400 600 800 Potential /mV 160 180 200 220 240 0 200 400 600 800 A C concentration /M x 10 5 0.0 0.2 0.4 0.6 0.8 1.0 tim e /s 1 2 3 4 5 2. POTENTIOMETRIC MONITORING OF AChE-CATALYZED HYDROLYSIS OF ACh Conversion of the potential into ACh concentration using an ACh calibration graph 1. Potential signal of the 7.5 pH phosphate buffer medium; 2. Injection of an aliquot of ACh.; 3. Potential response of the ACh; 4. Injection of the AChE; 5. Potential decrease due to ACh decrease during the hydrolysis; 6. Calculation of the initial rate. Hydrolysis conditions: pH = 7.5 Tª = 28ºC 6 [AC h] 0 (M x10 4 ) 0 2 4 6 V (M s -1 x10 8 ) 0 2 4 6 8 U m l -1 0.0 0.1 0.2 0.3 0.4 V (M s -1 ) 0 1e-6 2e-6 3e-6 4e-6 5e-6 6e-6 Variation of [ACh] 0 Variation of AChE concentration [AChE]=5.4x10 -3 Uml -1 [ACh] 0 =4x10 -4 M CONCLUSIONS The developed ACh-selective electrode based on a NPOE plasticizer polymeric membrane and containing 0.5 % of ionic additive permits the enzymatic-hydrolysis of ACh to be monitored. When the data obtained for the variation of the initial substrate concentration are fitted to Michaelis-Menten equation, the values obtained for K m and V max were 4.7x10 -5 ± 0.3x10 -5 M and 8.3x10 -8 ± 0.2x10 -8 Ms -1 , respectively. A linear relationship was obtained between the initial rate and the AChE concentration in a concentration range of almost four decades. This regression might be used for the determination of AChE in problem samples.
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Transcript of Euroanalisis poster1 1
María Cuartero Botía a, Joaquín A. Ortuño Sánchez-Pedreño a, Mª Soledad García García a and Francisco García Cánovas b aDepartment of Analytical Chemistry, Faculty of Chemistry, University of Murcia, Murcia E-30071, Spain
bDepartment of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Murcia E-30100, [email protected]
NEW ASSAY OF THE ACETYLCHOLINESTERASE ACTIVITY BY POTENTIOMETRIC MONITORING OF ACETYLCHOLINE
(C)3N+CH2CH2OCCH3CH3
AChE
H2O
(Acetylcholinesterase enzyme)
(CH3)3N+CH2CH2OH + HOCCH3
OO
Acetylcholine (ACh) Choline (Ch)
REACTION IN STUDY
POTENTIOMETRIC MONITORING OF SUBSTRATE CONCENTRATION
To monitor the kinetics of a hydrolysis reaction the detection system must satisfy some requirements:
Sufficient sensitivity and stability Low background noise
Low drift Rapid response
Sufficient selectivity over the product of the reaction
1. DEVELOPMENT OF AN ACETYLCHOLINE-SELECTIVE ELECTRODE:
Membrane:100 mg PVC
200 mg of plasticizer1.5 mg ionic additive
NPOE best selectivity and sensitivity 0.5% of ionic additive best sensitivity and stability
3. KINETIC DATA ANALYSIS
the ISE is able to follow the continuos decrease in ACh concentration
Other response characteristics in 7.5 pH phosphate buffer medium: Slope 59.2 mV/dec; linear range 1x10-6 – 1x10-3 M; Detection limit 1.5x10-7 M; drift in the linear range 0.05-1.1 mV/h; Excellent repeatability of the calibration parameters.
log [C] / M
-6 -5 -4 -3
Po
ten
tial
ch
ang
e / m
V
0
50
100
150
200
-6 -5 -4 -3 -6 -5 -4 -3
DOSNPOETCP
K+
ACh
Ch
Na+
H+
K+
K+
ACh ACh
Ch
Ch
Na+
Na+
H+
H+
Selection of the plasticizer
-8 -6 -4 -2
Po
ten
tial
ch
ang
e / m
V
0
50
100
150
200
log [C] / M
0.5 %
1 %
0.3 %
Selection of the amount of ionic additive
time / s
0 5 10 15 20 25[A
Ch
] / M
3.8e-4
3.9e-4
4.0e-4
10 ml min-1 5 ml min-12.5 ml min-1
― theoretical values calculated from the dilution program
Evaluation of the response time
time / s
0 200 400 600 800
Po
tent
ial /
mV
160
180
200
220
240
0 200 400 600 800
AC
con
cent
ratio
n /
M x
105
0.0
0.2
0.4
0.6
0.8
1.0
time / s
1
2
3 4
5
2. POTENTIOMETRIC MONITORING OF AChE-CATALYZED HYDROLYSIS OF ACh
Conversion of the potential into ACh concentration using an ACh calibration graph
1. Potential signal of the 7.5 pH phosphate buffer medium; 2.
Injection of an aliquot of ACh.; 3. Potential response of the
ACh; 4. Injection of the AChE; 5. Potential decrease due to
ACh decrease during the hydrolysis; 6. Calculation of the
initial rate.
Hydrolysis conditions:pH = 7.5Tª = 28ºC
6
[ACh]0 (Mx104)
0 2 4 6
V (
Ms-1
x108 )
0
2
4
6
8
U ml-1
0.0 0.1 0.2 0.3 0.4
V (
M s
-1)
0
1e-6
2e-6
3e-6
4e-6
5e-6
6e-6
Variation of [ACh]0 Variation of AChE concentration
[AChE]=5.4x10-3 Uml-1
[ACh]0=4x10-4 M
CONCLUSIONS The developed ACh-selective electrode based on a NPOE plasticizer polymeric membrane and containing 0.5 % of ionic additive permits the enzymatic-hydrolysis of ACh to be monitored. When the data obtained for the variation of the initial substrate concentration are fitted to Michaelis-Menten equation, the values obtained for Km and Vmax were 4.7x10-5 ± 0.3x10-5 M and 8.3x10-8 ± 0.2x10-8 Ms-1, respectively. A linear relationship was obtained between the initial rate and the AChE concentration in a concentration range of almost four decades. This regression might be used for the determination of AChE in problem samples.
