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Development of a miniaturized immunochemical flow-injection system for on-site analysis of selected nitroaromatics and pesticides in water [Elektronische Ressource] / Ioan Manuel Ciumasu


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Published 01 January 2006
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Technische Universität München
Wissenschaftszentrum Weihenstephan
Lehrstuhl für Ökologische Chemie und Umweltanalytik

Development of a miniaturized immunochemical flow-injection system for
on-site analysis of selected nitroaromatics and pesticides in water

Ioan Manuel Ciumasu

Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften (Dr. rer. nat.)

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr.rer.nat.habil. Wilfried Huber
Prüfer der Dissertation: 1. Univ.-Prof. Dr.rer.nat. Dr.h.c.(RO) Antonius Kettrup
2. Univ.-Prof. Dr.med.vet. Dr.med.vet.habil. Johann Bauer
3. Priv.-Doz. Dr.rer.nat. Petra Krämer

Die Dissertation wurde am 29.07.2005 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 09.11.2005 angenommen.

for my wife

Publications related to the PhD thesis
Related research articles:
1. Ioan M. Ciumasu, Petra M. Krämer, Cristina M. Weber, Günther Kolb, David Tiemann,
Stefan Windisch, Ines Frese, Antonius A. Kettrup. A new, versatile field immunosensor
for environmental pollutants. Development and proof of principle with TNT, diuron and
atrazine. Biosensors and Bioelectronics 21: 354-364.

2. Petra M. Krämer, Elisabeth Kremmer, Cristina M. Weber, Ioan M. Ciumasu, Stephan
Forster, Antonius A. Kettrup. Development of new rat monoclonal antibodies with
different selectivities and sensitivities for 2,4,6-trinitrotoluene (TNT) and other
nitroaromatic compounds. Analytical and Bioanalytical Chemistry 382: 1919-1933.

Related oral presentations:

3. I. Frese (oral presenter), G.Kolb, D. Tiemann, P.M. Krämer, I.M. Ciumasu, C.M. Weber,
2003. Development of an Optical Cell for an Automated Miniaturized Immunochemical
Device for On-site Screening of Pesticides Residues in Water. In: Proceedings of the
Oral presentation at "Sensor 2003" International Trade Fair and Conference, 13-15 May
2003, Nuremberg, Germany. Further information on the conference at :

4. G. Kolb (oral presenter), I. Frese, V. Hessel, I.M. Ciumasu, P.M. Krämer, H. Löwe, D.
Tiemann, 2004. An automated, portable immunochemical flow-injection system for on-
site analysis of environmentally hazardous chemicals. In: Proceedings of the Oral
Presentation at Lab Automation 2004, 1-5 February 2004, San Jose, CA, USA. Further
information on the conference at: http://labautomation.org/LA/LA04/conference

5. P.M. Krämer (oral presenter), I.M. Ciumasu, C.-M. Weber, A.A. Kettrup, D. Tiemann, I.
Frese, G. Kolb, 2004. Entwicklung eines neuen, tragbaren, automatisierten Einweg-
immunosensors für eine Vor-Ort-Auswahl von mit Umweltschadstoffen belasteten
Proben. Abschlussveranstaltung des Graduiertenkollegs 'Analytische Chemie', 2.–4.
Februar 2004, Blaubeuren.

Publications related to the PhD thesis
Related posters:

6. P.M. Krämer, I.M. Ciumasu, C.M. Weber, G. Kolb, I. Frese, B. Werner, A.A. Kettrup.
Development of an automated miniaturized immunochemical device for on-site
thscreening of pesticide residues in water. Poster at "The 10 IUPAC International
Congress on the Chemistry of Crop Protection", 4-9 August, 2002, Basel, Switzerland.
Congress proceedings, Main Topic 6 – Residues and Consumer Safety, Subtopic 6a –
Trends in Analytical Methods and Instrumentation, p 201;

7. I.M. Ciumasu, P.M. Krämer, C.M. Weber, G. Kolb, D. Tiemann, S. Windisch, I.Frese,
A.A. Kettrup. Single-use immunosensor for environmental pollutants. Proof of principle
for nitroaromatics and pesticide. The Eighth World Congress on Biosensors, 24-26 May
2004, Granada, Spain. Poster abstract (BS67) in the Congress Proceedings, Topic

8. P.M. Krämer, C.-M. Weber, I.M. Ciumasu, E. Kremmer, A.A. Kettrup. Development of
monoclonal antibodies for 2,4,6-trinitrotoluene and its metabolites 2-amino-4,6-
dinitrotoluene and 4-amino-2,6-dinitrotoluene for their use in immunosensor. The Eighth
World Congress on Biosensors, 24-26 may 2004, Granada, Spain. Poster abstract
(BS632) in the Congress Proceedings, Topic "Immunosensors".


This work was funded by the Bmbf (Ministry for Science and Education, Germany),
project number FKZ 02WU0102. Bmbf also funded the work of the Institut für Mikrotechnik
Mainz GmbH (IMM), the instrument manufacturer, project number FKZ 02WU0102.

The author feels grateful for the PhD studentship and the technical support provided
by TUM, especially the Faculty of Nutrition, Agriculture and Environment, Department of
Ecological Chemistry and Environmental Analysis as PhD student all along the research
project (2001 – 2004) and for the finalization of the PhD thesis.
The author thanks GSF–National Research Centre for Environment and Health,
Neuherberg, Germany, especially the Institute of Ecological Chemistry, for hosting this work
and for insuring excellent research conditions during the research project.

The author thanks to Univ. Professor Dr. Antonius A. Kettrup for effective leadership
and kind supervision of this PhD work.

The author thanks to the project leader, Dr. Petra M. Krämer, for fruitful and pleasant
collaboration, and for precious help in forging a professional personality.

The author thanks Dr. Ing. Günther Kolb, Dipl.-Ing. David Tiemann and Dr. Ines
Frese, from IMM (Institut für Mikrotechnik Mainz), for their close collaboration.
The author thanks Dr. Elisabeth Kremmer, from GSF – Institute for Molecular
Immunology, for her collaboration during our research project.
The author thanks Dipl.-Chem. Hartmut Thomas from WASAG DECON GmbH,
BioPlanta, Leipzig for amiable exchange of scientific info and experience.

The author expresses his gratitude for the friendly collaboration of all the technical
and scientific staff from the GSF Institute for Ecological Chemistry, especially Dr. Sigurd
Schulte-Hostede – provisional director of the institute, Mrs Cristina Mihaela Weber, Dr.
Mariana Neamtu, Dr. Monica Pantiru, Dipl.-Ing. Annette Franke, Mr. Geza Cocsis, Ms.
Sylvia Oberleitner, Ms. Sabine Röttmuller and Mr. Stephan Forster.

But most of all I thank my wife, Maria Cristina Antal, for being so lovely all these
years of geographical quasi-separation.

Abbreviations and acronyms

Ab Antibody / antibodies
AFM Atomic force microscopy
Ag Antigen / antigens
An Analyte
ARIS Apoenzyme reactivation immunoassay
BSA Bovine serum albumine
CE Capillary electrophoresis
CEC electro-chromatography
CFI Continuous flow immunosensor
CR Cross-reactivity
CV Cyclic voltametry
DCC 1,3-Dicyclohexyl carbodiimide
Dil. Dilution
DL Detection limit
DMF Dimethyl formamide
DNP-glycine N-(2,4-dinitrophenyl)-glycine
DNP- γ-AmBA 2,4-Dinitrophenyl- γ-aminobutyric acid
DNP- ε-AmCA ε-aminocaproic acid
DNT Dinitrotoluene
DOM Dissolved organic matter
ECD Electron capture detection
EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
ELISA Enzyme-linked immunosorbent assay
EPA Environmental protection agency (USA)
Eq. Equation no
Fab Fraction antigen binding (of the Ab molecule)
Fc Fraction constant (of the Ab molecule)
FIA Flow-injection analysis
Fig. Figure no
FP Field prototype (of the developed immunosensor)
FRET Fluorescence resonance energy transfer
Gam Goat anti-mouse IgG
GC Gas chromatography
GSF GSF – National Research Centre for Environment and
Health, Neuherberg, Germany

Abbreviations and acronyms
HRMS High resolution mass spectrometry
HPLC High performance liquid chromatography
HRP Horseradish peroxidase (E.C.
IgG Immunoglobuline
IC Ion chromatography
IC Analyte concentration producing 20% inhibition 20
IC oducing 50 50
ICP Inductively coupled plasma
IDLIF Indirect laser-induced fluorescence
Inc. Incubation
Ip Isoproturon
IMM Institut für Mikrotechnik Mainz
IÖC Institut für Ökologische Chemie - GSF
ITMS Ion trap mass spectrometry
k Association constant a
k Dissociation d
KLH Keyhole limpet hemocynine
LASER Light amplification by stimulated emission of radiation
LC Liquid chromatography
LP Laboratory prototype (of the developed immunosensor)
µ-TAS Micro-total-analysis-systems
mAb Monoclonal antibody
MECK Micellar electro-kinetic chromatography
MIMS Membrane induction mass spectroscopy
MP Skimmed milk, powder; used in solution
MPA 3-Mercaptopropanoic acid; mercaptan
MRL Maximum residues level
MS Mass spectrometry
MW Molecular weight
NHS N-hydroxysulfosuccinimide sodium salt
NMR Nuclear mass resonance
NPD Nitrogen-phosphorus detector
n Number of measurements
NT Nitrotoluene
OD Optical density (absorbance reader)
PAN Pesticide action network
PASA Parallel affinity sensor array

Abbreviations and acronyms
PBS Phosphate buffered saline
PBST Phosphate buffered saline containing Tween 20
PMMA Polymethylmethacrylate; acrylic glass;
Plexiglas; Lucite (in USA)
POPs Persistent organic pollutants
Pre-inc. Pre-incubation of analyte before the enzyme-tracer
Prot A Protein A A/G A/G
Prot G G
PTFE Polytetrafluoroethene (plastics)
PVC Polyvinyl chloride
RDX Hexahydro-1,3,5-trinitro-1,3,5-triazine
READ Reversed electron attachment detection
RLU Relative light units
RP-HPLC Reverse-phase HPLC
RT Room temperature
SAM(s) Self-assembled monolayer(s)
SEM Scanning electron microscopy
SFA Segmented flow analysis
SPE Solid-phase extraction
SPMDs Semipermeable membrane devices
SPME microextraction
STM Scanning tunnelling microscopy
TAT 2,4,6-Triaminotoluene
TMB 3,3',5,5'-Tetramethylbenzidine
TNT 2,4,6-Trinitrotoluene
TNP–glycylglycine Trinitrophenyl-glycylglycine
TNP- α-AmBA yl- α-Aminobutyric acid
Tr Enzyme-tracer
UV Ultraviolet
w/v Weight per volume
(n)x (n) times
Y/m/d Year / month / day
-1Zero dose Reference; analyte (standard) concentration 0 µg l

List of tables and figures
List of tables

Table 1. Chemicals and standards
Table 2. Composition of the used buffers
Table 3.substrates for HRP
Table 4. Catching proteins / antibodies
Table 5. Anti-analyte monoclonal antibodies, enzyme-tracers, and enzymes for
producing enzyme-tracers
Table 6. Proteins, surfactants and buffers that were used as blocking solutions
Table 7. Materials and instruments
Table 8. Surfaces and volumes specific to each stage of the sensor development
Table 9. Cross-reactivities in TNT-ELISA, with the in-house produced enzyme-tracer
TNP-Glycylglycine–HRP, as compared with the literature (Zeck et al., 1999)
Table 10. Methods used for the analysis of explosives and their degradation products in
water and soil
Table 11. Nitroaromatic compounds used in ELISA, with molecular structures and
measured cross-reactivity (CR)
Table 12. Comparative table with standard curve parameters obtained with various
catching proteins in TNT-ELISA
Table 13. Automatic steps: Program for fluid handling and measurement of one chip
Table 14.ps: Program for preparation washing (for making the conduit air-
free) before starting any measurement and after finishing all the measurements
Table 15. Automatic steps: Program for substrate set-up before measurements
Table 16.ps: Program for the washing of the ground plate in between
measurements (to avoid inter-measurements contamination)

List of figures

Figure 1. Details (A) and portable box (B) of the temperature-controlled field prototype
Figure 2. Hypothetical standard curves (%Control)
Figure 3. Variable reaction speeds in the formation of Ab-Ag complexes towards
Figure 4. Hypothetical example for the relative importance of direct inhibition, facilitated
binding and feed-back inhibition of Tr, at a given moment of the incubation time
Figure 5. Hypothetical standard curves (%Control) with different relative importance of
facilitated binding and feed-back inhibition of Tr

List of tables and figures
Figure 6. Combined representation of the inhibition dynamic with competing antigens
(standard curve), and dissociation of Ab – An complexes formed before
washing and Tr incubation
Figure 7.
(standard curve), and dissociation of
washing and Tr incubation
Figure 8. Laboratory sensor prototype
Figure 9. Field sensor prototype (portable box)
Figure 10. Field prototype – user interface
Figure 11. Golden structures in experimental glass vials (batch ELISA set-up)
Figure 12. Golden structures in glass vials detailed view of two structure types: A. Final
version (Aspect 1); B. Earlier version (Aspect 0.5)
Figure 13. Golden structures detailed view of the final version
Figure 14. Single-use chips
Figure 15. Scheme of the incubation / measurement cell and of the detection principle
Figure 16. Standard curves, with TNT-ELISA, using the enzyme-tracers TNP-glycyl-
glycine–HRP and DNP- γ-AmBA–HRP
Figure 17. Standard curves, optimization with TNT-ELISA, using the Tr DNP- γ-AmBA–
Figure 18.using the enzyme-tracers TNP- α-AmBA–
HRP, and DNP- ε-AmCA–HRP
Figure 19. Stability of the current Tr (TNP-Glycylgycine – HRP) in time, at +3°C, and at -
28°C with the commercial SuperFreeze stabilizor
Figure 20. Standard curves, with 2,4,6-TNT and its main degradation products ( ) 2-
amino-4,6-DNT and 4-amino-2,6-DNT
Figure 21. Standard curves, with TNT-ELISA, using two different coating reagents and an
-1Ab concentration of 400 µg l
Figure 22.using two different coating reagents and an
-1Ab concentration of 285 µg l
Figure 23.using three different coating reagents and
-1an Ab concentration of 400 µg l
Figure 24. Standard curves – TNT-ELISA optimizations, using Protein A/G and two Ab
Figure 25. Standard curves, with TNT-ELISA, using three different Ab concentrations
which were applied directly on microtiter plate (no catching protein)
Figure 26. Standard curves, with fast TNT-ELISA, with three Ab concentrations which
were immobilized via Gam