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Protein-based capacitive biosensors for the detection of heavy-metal
ions [pic]
Alessia Mortari
Department of Analytical Chemistry
Doctoral Thesis
5th September 2008
Akademisk avhandling som för avläggande av teknologie doktorsexamen vid
tekniska fakulteten vid Lunds Universitet kommer att offentligen försvaras
fredagen den 5 september 2008, kl. 10.30 hörsal B, på Kemicentrum,
Getingevägen 60, Lund. Academic thesis which, by due permission of the Faculty of Engineering at
Lund University, will be publicly defended on Friday the 5th of September
2008, at 10.30 a.m., lecture hall B, at the Centre of Chemistry and
Chemical Engineering, Getingevägen 60, Lund. Faculty opponent: Professor Wolfgang Schuhmann, Department of Analytical
Chemistry, Gebäude NC/788, D-44780 Ruhr-Universität Bochum, Bochum,
Germany.
Doctoral Dissertation 2008
Department of Analytical Chemistry
Lund University
Sweden
© Alessia Mortari
ISBN-nr 978-91-7422-201-2
Printed by Media-Tryck (Lund, 2008) You have brains in your head.
You have feet in your shoes.
You can steer yourself
any direction you choose.
You're on your own. And you know what you know.
And YOU are the guy who'll decide where to go. Dr. Seuss |Organization |Document name |
|Lund University |Doctoral thesis |
|Department of Analytical Chemistry | |
| |Date of issue |
| |June 2008 |
|Author(s) |Sponsoring organization |
|Alessia Mortari | |
|Title and subtitle |
|Protein-based capacitive biosensors for the detection of heavy metal ions |
|Abstract |
|Some metal ions, such as copper and zinc, are essential nutrients and |
|catalysts in biochemical reactions. Other metal ions, e.g. cadmium and |
|mercury, are highly toxic elements. |
|Heavy metals detection has proven difficult with classical as well as |
|experimental analytical methods. Novel techniques are required for the |
|measurement of bioavailable toxic elements and for detecting small ligands |
|binding, often weak and transient, yet vital to most cellular processes. |
|The here-discussed biosensors were developed for the measurement of |
|bioavailable concentration of toxic metals and to investigate the |
|biochemical characteristics of proteins of biomedical interest. |
|Heavy metal ions binding proteins, e.g. SmtA and S100A12, were used as |
|bio-recognition element. |
|Electrochemical capacitance was used to measure the protein-heavy metal ion|
|interaction. |
|The aim of the thesis is to review the research and the state-of-the-art of|
|the protein-based capacitive biosensors for the detection of heavy metal |
|ions. It covers the main aspects of the biosensor theory, research and |
|development, including the detection principle, with particular attention |
|to the transducer methods and gold electrodes, heavy metal ions |
|coordinating proteins, immobilisation methods and the experimental |
|biosensors applications. |
|Key words |
|Biosensor, capacitance, electrode, protein, heavy metal ions |
|Classification system and/or index terms (if any) |
|Supplementary bibliographical information |Language |
| |English |
|ISSN and key title |ISBN |
| |978-91-7422-201-2 |
|Recipient's notes |Number of pages |Price |
| |Security classification |
Distribution by (name and address)
I, the undersigned, being the copyright owner of the abstract of the above-
mentioned dissertation, hereby grant to all reference sources permission to
publish and disseminate the abstract of the above-mentioned dissertation.
Signature _______________________ Date ____2008-06-12____ Contents
Summary of abbreviations and symbols viii
List of papers ix Introduction 1
13 Biosensors 3 1. Bio-recognition element 3
2. Transducer 5
3. Immobilisation method 5
4. Analyte 6
5. Performance factors 6 Capacitive transducers for biosensor 8
15 Capacitance theory 8
16 Historical introduction 8
17 Electrochemical Double Layer 8
18 EDL and capacitive biosensor: the detection principle 11
19 Capacitance transducers 12
20 Square Wave Potential Step 12
21 Impedance Spectroscopy 22
22 Cyclic Voltammetry 25
23 Square Wave Current Step 26
Working electrodes and immobilisation method 28
25 Gold electrodes 28 6. Bar gold electrode 28
7. Sputtered gold electrode 32
8. Mesoporous gold electrodes 33 26 Self- Assembled Monolayer 36 9. Stability and properties 36
10. Protein coupling 38
11. Thioctic acid SAM and heavy metal ions interaction 38 Heavy metal binding proteins as biological sensing element 40
28 Phytochelatin 40
29 GST-SmtA 41
30 MerP and chimerical MerP*s 42
31 S100A12 42
Applications 44
33 Biosensor for environmental analysis 44
34 Biosensors for biomedical investigation 45
Conclusions and future perspectives 49
Summary of the appended Papers 51
Acknowledgements 53
References 55 Summary of abbreviations and symbols
BSA Bovine Serum Albumine
CV Cyclic Voltammetry
EDC 1-ethyl-3-(3-diamino)propyl-carbodiimide
EDL Electrochemical Double Layer
GCDL Gouy-Chapman Diffuse Layer
GST Glutatione-S-transferase
HMA Heavy metal associated motif
IHP Inner Helmholtz Plane
IS Impedance Spectroscopy
OHP Outer Helmholtz Plane
RC Resistance and Capacitance connected in series
SAM Self-Assembled Monolayer
SPR Surface Plasma Resonance
SWCS Square Wave Current Step
SWPS Square Wave Potential Step
TA Thioctic acid C Capacitance
CDL GCDL capacitance
CH Helmholtz layer capacitance
[pic] Biosensor Helmholtz layer capacitance
CTOT Total Capacitance
[pic] Biosensor total capacitance
[pic] Bulk ion concentration
d Inter-plate distance
e Value of unit charge
E Applied electrical potential
E" Potential distribution
EA Effect observed at analyte concentration [A]
Emax Maximum obtainable effect
f Frequency
G Conductance
I Current
j [pic]
K Constant
KA Affinity
Q Surface charge
Rs Solution resistance
S Surface
t Time
T Temperature
v Scan rate
Y Reactance
Z Impedance
( Intrinsic activity
(0 Free space permeability
( Solution dielectric constant
( Phase angle
( Fringing effect correction factor
(e Charge density
( Electrical conductivity
( Radial frequency
List of Papers
This thesis is based on the following articles. The articles can be
found at the end of the booklet, referred to in the text by their
roman number.
I Novel synthetic phytochelatin-based biosensor for monitoring of heavy
metal ions I. Bontidean, J. Ahlqvist, A. Mulchandani, W. Chen, W. Bae,
R. K. Mehra, A. Mortari and E. Csöregi, Biosensors and Bioelectronics
(2003) 18, 547-553. II Biosensors for detection of merc