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High-throughput screening for the
investigation of the biological effects of
millimetre-wave radiation
March 2003
Thesis presented for the degree Doctor of Philosophy
David T. Pooley
Department of Medical Physics and Bioengineering
University of Wales College of Medicine
Heath Park, Cardiff
United Kingdom
I
Summary
This thesis reports on the design and evaluation of a high-throughput
screening system for investigating the biological effects of microwave and
millimetre-wave radiation. The approach presented here differs
significantly from others with the deployment of a continuous-culture
device and sample handling technology that allows for the rapid
presentation of test samples in a uniform physiological state. The
millimetre-wave exposure cell was operated as a flow-through device. To
remove convolution-by-flow effects this was combined with a
segmentation scheme. A sensitive bioluminescence-based reporter
monitored the temporal evolution in each assay segment using optical
detector arrays mounted at three observation points: - pre-millimetre-
wave exposure, syn- exposure and post-exposure. With this approach,
the continuous monitoring of bioluminescence may be used on different
time scales to measure energy metabolism, gene expression and growth.
Exposed and control samples were monitored and any combination of
stimulus parameters, namely, radiation frequency, intensity and
polarization (or any modulation of these), which induced a response that
exceeded a noise threshold of the system could be further investigated
automatically in real-time. The device could screen systematically or use
feedback to automatically investigate a region of interest at increasing
resolution in parameter space. Environmental parameters such as
temperature and magnetic and electric fields were carefully controlled.
The instruments operation was characterized over the 26 and 40 GHz
frequency range and this gave confidence that the technique could be
applied to the entire mm-wave range. This sensitive “active” search
system may have application in industry, biomedical research and
environmental health.
II
Declaration
This work has not previously been accepted in substance for any degree
and is not being concurrently submitted in candidature for any degree.
Signed……………………………………………….(candidate)
Date………………………………………………….
STATEMENT 1
This thesis is the result of my own investigations, except where otherwise
stated. Other sources are acknowledged by footnotes giving explicit
references. A bibliography is appended.
Signed……………………………………………….(candidate)
Date………………………………………………….
STATEMENT 2
I hereby give consent for my thesis, if accepted, to be available for
photocopying and for inter-library loan, and for the title and the summary
to be made available to outside organisations.
Signed……………………………………………….(candidate)
Date………………………………………………….
III
Acknowledgements
I would like to thank everyone who has helped me complete this thesis. In
particular Colin Gibson and John Magee for supervising the project. I
would particularly like to thank John Magee for teaching me how to write
good scientific English. I would also like to give special thanks to William
Stuart for his help in the development of the software that, with his
assistance, operated impeccably. David Lloyd is thanked for his
assistance in relation to the continuous-culture system and for imparting
an enduring interest in bioluminescence and circadian and ultradian
biology. Brian Ellison is acknowledged for his assistance in ensuring that
the millimetre-wave technique was sound. John Spencer and the staff of
the micro-machining laboratory at the Rutherford Appleton Laboratory are
thanked for constructing the exposure cell. The European Space Agency
and Cardiff Partnership Fund are thanked for their financial assistance.
Most importantly, thanks are due to friends and family without whose
support this thesis would not have been completed.
IV
“I believe that this concept (i.e. quantum coherence) may have a much
wider range of application, in particular, in systems which are relatively
stable but not near thermal equilibrium, and which show an organised
collective behaviour which cannot be described in terms of an obvious
(static) spatial order”
H. Fröhlich, Proceedings of the first
International Conference on Theoretical
Physics and Biology, Versailles, 1967
V
Publications
Several papers based on the work contained within this thesis have been
published or are in the process of being published.
D. Pooley, B. Ellison, C. Gibson and W. Stewart. “A cell-culture exposure
system for millimetric wavelength radiation” Proceedings of the European
Conference on Microwave Technology, London, 2001
D. Pooley, C. Gibson, W. Stewart, J. Magee and B Ellison. “Biological
effects of millimeter-wave radiation: a high-throughput screening system”.
Review of Scientific Instruments, March 2003. (Also featured in March
2003 edition of the Virtual Journal of Biological Physics Research).
D. Pooley, C. Gibson, W. Stewart, D. Lloyd, M Rayner-Brandes and G
Jones. “The continuous culture of photobacterium”. Submitted.
Biosensors and Bioelectronics January 2003
VI
Contents
Summary...................................................................................................II
Declaration ...............................................................................................III
Publications............................................................................................. VI
Contents................................................................................................. VII
Figures .................................................................................................... XI
Glossary ................................................................................................ XIII
Chapter 1 .................................................. Error! Bookmark not defined.
Theory ....................................................... Error! Bookmark not defined.
1.1 Biological systems ...........................Error! Bookmark not defined.
1.1.1 Bond energies ...........................Error! Bookmark not defined.
1.1.2 Vibrations and conformational transitions in proteins ........Error!
Bookmark not defined.
1.2 Theoretical models ..........................Error! Bookmark not defined.
1.2.1 Quantum and classical electrodynamics ..Error! Bookmark not
defined.
1.2.2 Thermal masking.......................Error! Bookmark not defined.
1.3 Dielectric heating .............................Error! Bookmark not defined.
1.3.1 Absorption in a lossy dielectric .. Error! Bookmark not defined.
1.4 Nonthermal absorption mechanismsError! Bookmark not defined.
1.4.1 The Fröhlich-condensate .......... Error! Bookmark not defined.
1.4.2 Davydov soliton.........................Error! Bookmark not defined.
1.4.3 Bohr coupling ............................Error! Bookmark not defined.
Summary ............................................... Error! Bookmark not defined.
Chapter 2 .................................................. Error! Bookmark not defined.
Experimental evidence ..............................Error! Bookmark not defined.
2.1 Introduction...................................... Error! Bookmark not defined.
2.2 Physical effects................................ Error! Bookmark not defined.
2.2.1 Effects on cell-free enzyme systems........Error! Bookmark not
defined.
2.2.2 Enhanced diffusion effects ........ Error! Bookmark not defined.
2.2.3 Convection effects.....................Error! Bookmark not defined.
2.2.4 Exposure in the near and far field ............Error! Bookmark not
defined.
2.2.5 Fröhlich-Davydov effect in acetanilide......Error! Bookmark not
defined.
2.3 Effects of mm-wave radiation on Biological systems ...............Error!
Bookmark not defined.
2.3.1 Prokaryotes ...............................Error! Bookmark not defined.
2.3.2 DNA tropic effects .....................Error! Bookmark not defined.
2.3.3 Spectroscopic studies ............... Error! Bookmark not defined.
2.3.4 Miscellaneous effects................ Error! Bookmark not defined.
2.4 Lower eukaryotes ............................Error! Bookmark not defined.
2.4.1 Growth and division rate effects Error! Bookmark not defined.
2.5 Mammalian cells ..............................Error! Bookmark not defined.
2.6.1 Passive radiometry.................... Error! Bookmark not defined.
2.7 Discussion ....................................... Error! Bookmark not defined.
Summary ............................................... Error! Bookmark not defined.
Chapter 3 .................................................. Error! Bookmark not defined.
VII
Description of the Instrument ....................Error! Bookmark not defined.
3.1 Continuous-culture device ............... Error! Bookmark not defined.
3.2 Mixing chamber ...............................Error! Bookmark not defined.
3.3 Transit time...................................... Error! Bookmark not defined.
3.4 Flow segmentation...........................Error! Bookmark not defined.
3.5 Environmental chamber................... Error! Bookmark not defined.
3.5.1 Magnetic shielding .................... Error! Bookmark not defined.
3.5.2 Helmholtz coils ..........................Error! Bookmark not defined.
3.5.3 Temperature control.................. Error! Bookmark not defined.
3.6 Flow-through exposure cell.............. Error! Bookmark not defined.
3.6.1 Optical detection system ........... Error! Bookmark not defined.
3.7 Analysis Software ............................Error! Bookmark not defined.
3.7.1. Introduction ..............................Error! Bookmark not defined.
3.7.2 Channel processor .................... Error! Bookmark not defined.
3.7.3 Comparator ...............................Error! Bookmark not defined.
Chapter 4 .................................................. Error! Bookmark not defined.
Continuous culture of Photobacterium ...... Error! Bookmark not defined.
4.1 Introduction...................................... Error! Bookmark not defined.
4.1.1 Whole cell biosensors: bacterial bioluminescence ............Error!
Bookmark not defined.
4.1.2 Bioluminescence as a oxygen indicator ...Error! Bookmark not
defined.
4.1.3 Assay of toxic compounds by inhibition of bioluminescence
........................................................... Error! Bookmark not defined.
4.2 Design and construction of the continuous-culture device.......Error!
Bookmark not defined.
4.2.1 The culture vessel .....................Error! Bookmark not defined.
4.2.2 The light / turbidity monitoring system......Error! Bookmark not
defined.
4.2.3 Control system ..........................Error! Bookmark not defined.
4.3 Bioluminescence and the Fröhlich condensate: a conjecture ..Error!
Bookmark not defined.
Chapter 5 .................................................. Error! Bookmark not defined.
The exposure cell...................................... Error! Bookmark not defined.
5.1 Introduction...................................... Error! Bookmark not defined.
5.1.1 Design and construction of the cell ..........Error! Bookmark not
defined.
5.1.2 Selection of sample tube material ............Error! Bookmark not
defined.
5.1.3 Network analysis and simulation ..............Error! Bookmark not
defined.
5.1.4 Location of collimators .............. Error! Bookmark not defined.
Chapter 6 .................................................. Error! Bookmark not defined.
Results ...................................................... Error! Bookmark not defined.
6.1 Continuous culture system .............. Error! Bookmark not defined.
6.1.1 Operating the culture device as a turbidostat. Error! Bookmark
not defined.
6.1.2 Optimized long-term maintenance of photobacterium.......Error!
Bookmark not defined.
6.1.3 Oxygen......................................Error! Bookmark not defined.
VIII
6.1.4 Effect of chemical toxicants of photobacterium .................Error!
Bookmark not defined.
6.2 Calibration and measurement of the millimetre-wave exposure cell
.............................................................. Error! Bookmark not defined.
6.2.1 Return and transmission losses of different tubing materials
........................................................... Error! Bookmark not defined.
6.2.2 Return and transmission losses of different sample materials
........................................................... Error! Bookmark not defined.
6.2.3 Transmission losses of different tubing bore .. Error! Bookmark
not defined.
6.3 Performance characteristics of the apparatus Error! Bookmark not
defined.
6.3.1 Preliminary experiments............ Error! Bookmark not defined.
6.3.2 Segmented flow and its measurement using luminometers
........................................................... Error! Bookmark not defined.
6.3.3 Example spreadsheet ............... Error! Bookmark not defined.
6.4 Operation of the apparatus .............. Error! Bookmark not defined.
6.4.1 Running the apparatus.............. Error! Bookmark not defined.
Summary ............................................... Error! Bookmark not defined.
Chapter 7 .................................................. Error! Bookmark not defined.
Discussion................................................. Error! Bookmark not defined.
Conclusions............................................... Error! Bookmark not defined.
References................................................ Error! Bookmark not defined.
Appendix A................................................ Error! Bookmark not defined.
Appendix B................................................ Error! Bookmark not defined.
Appendix C................................................ Error! Bookmark not defined.
Appendix D................................................ Error! Bookmark not defined.
Appendix E................................................ Error! Bookmark not defined.
Appendix F ................................................ Error! Bookmark not defined.
Appendix G ............................................... Error! Bookmark not defined.
IX
X
Figures
Fig. 1 Schematic of a Prokaryotic cell.
Fig. 2 The coupling between the energy bath thermal modes and
those in biological systems.
Fig. 3 Survey of epigenetic effects of millimetre-wave radiation
Fig. 4 Survey of growth rate effects of millimeter-wave radiation
Fig. 5 Overview of the main features of the apparatus
Fig. 6 Photograph showing continuous-culture device and
exposure chamber
Fig. 7 Vector network analyser with associated connections to the
exposure cell environmental chamber
Fig. 8 Inhibition of bioluminescence in Vibrio fischeri at different
concentrations of phenol
Fig. 9 Schematic cross-section of continuous-culture device and
mixing chamber
Fig. 10 Photograph of the continuous-culture device
Fig. 11 Software-based control panel for the continuous-culture
device and mixing chamber.
Fig. 12 Schematic of assembled fundamental-mode exposure cell
Fig. 13 Exposure cell prior to assembly
Fig. 14 Schematic cross-section of the exposure cell
Fig. 15 Response of bioluminescence to the introduction of fresh
growth medium
Fig. 16 Growth in the continuous-culture system
Fig. 17. Taken directly from the Labview control panel: continuous-
culture device activity over a 25 h period
Fig. 18 The return and transmission losses of an empty and loaded
cuvette
Fig. 19. Cuvette loaded with saline and Photobacterium growth
media
Fig. 20 Effect of increasing internal bore on transmission loss (S21).
Fig. 21 Simulated and measured S11 and S21 Parameters
XI
Fig. 22 Electric field distribution with a 4 % saline sample in the
tube
Fig. 23 Simulated and measured SAR with a 1 mW input power
Fig. 24 Channel processor control panel
Fig. 25 Output graph from channel comparator software
XII
Glossary
ATP
A nucleotide derived from adenosine. It is the major source of energy for
cellular reactions.
Average SAR
Volume-averaged specific absorption rate
Bioluminescence
Luminescence (light emission) produced by physiological processes (as
in the firefly).
Bose-Einstein condensation (BEC)
A phenomenon wherein the bosons making up a substance merge into
the lowest energy level, into a shared quantum state. More generally, it
refers to the tendency of bosons to occupy the same energy level.
Boson
Bosons are sub-atomic particles that have integral spin. They include
mesons (e.g., pions and kaons) and nuclei of even mass number (e.g.,
helium-4).
Cell membrane
A thin membrane around the cytoplasm of a cell; controls passage of
substances in and out of the cell
Chemostat
Device used for the continuous-culture of cells.
Chromosome
A threadlike body in the cell nucleus that carries the genes in a linear
order
XIII
Circadian biology
Of or relating to biological processes occurring at 24-hour intervals
Classical coherence
Order introduced through a phase transition for example boiling.
Conformation (macromolecular conformation)
Non-oscillatory change in shape
Continuous-culture device
Device use to culture cells in constant physiological state over extended
periods
Cytoplasm
The contents of a cell external to the nuclear membrane
DNA
A nucleic acid found in the nucleus of a cell and consisting of a polymer
formed from nucleotides and shaped like a double helix; associated with
the transmission of genetic information
Dielectric
A material with extremely low electrical conductivity
Electron transport chain
A complex chain of events and reactions and the basis of the cell's ability
to derive ATP from metabolic oxidation.
Enzymes
Any of several complex proteins that are produced by cells and act as
catalysts in specific biochemical reactions
XIV
Eukaryote
An organism with cells characteristic of all life forms except primitive
microorganisms such as bacteria; i.e. an organism with 'good' or
membrane-bound nuclei in its cells
Exponential phase culture
Culture that is not nutrient-limited
Extremely high frequency (EHF)
Electromagnetic radiation with frequency between 30 and 300 GHz also
referred to as millimetre-wave radiation.
Fröhlich (Bose-Einstein-type) condensation
Theoretically formed in biological systems at physiological temperatures
and akin to Bose-Einstein condensation; a macroscopic quantum effect.
Hydrogen bond
A chemical bond consisting of a hydrogen atom between two
electronegative atoms (e.g., oxygen or nitrogen) with one side be a
covalent bond and the other being an ionic bond.
Local SAR
Spatial distribution of energy
Luciferase
The enzyme system required for light emission
Lux operon
Genes related to bioluminescence
Lysogeny
Type of life cycle that takes place in a phage (bacterial virus) following its
infection of certain types of bacteria
Millimetre-wave or mm-wave
Electromagnetic radiation with frequency between 30 and 300 GHz
sometimes referred to as “EHF”.
XV
Organelle
A specialized and usually spatially localised part of a cell; analogous to
an organ
Phage
Bacterial virus
Phonon
A quantum of lattice vibrational energy that in analogy to a photon, can be
viewed as a wave packet with particle-like properties.
Prokaryote
A unicellular organism having cells lacking membrane-bound nuclei;
bacteria are the prime example but also included are blue-green algae
and actinomycetes and mycoplasma.
Radiometry and passive radiometry
A device used to detect and measure radiant energy (In this
electromagnetic radiation in the microwave and millimetre-wave spectral
range).
Soliton
A non-linear wave that propagates as a coherent entity
Specific Absorption Rate or SAR
Absorbed power W kg-1
Stationary phase culture
Nutrient limited high density cell culture
Ultradian biology
Oscillatory biological processes with a periodicity less than 24 hr
Vibrational modes
A regular periodic variation in value about a mean
XVI
Background
Over the last thirty years a number of insights into the biological effects of
interaction with mm-wave radiation have been presented, although their
interpretation remains controversial (Gos et al. 1997b). In many cases,
these publications relate to incident power exposures at levels below that
recommended for human exposure (< 10 mW cm-2) (Polk 1995b).
Induced effects are often reported to have nonlinear power and frequency
dependencies inconsistent with induction through heating (Polk 1995b).
However, these may be related to what has been termed complex
behaviour in biological systems.
Theoretical models such as the Fröhlich condensate (Fonseca et al.
2000; Frohlich 1982a; Mesquita, Vasconcellos, & Luzzi 1998b) or the
Davydov soliton (Scott, Williams, & Lloyd 1983) are discussed in past
papers in connection with experimental observations, although these
have largely proved inconclusive with poor reproducibility and several
confounding factors were identified (Furia, Hill, & Gandhi 1986b; Gos,
Eicher, Kohli, & Heyer 1997b). Spectroscopic approaches to determine
the existence of Fröhlich-condensate-type vibrational modes have been
unsatisfactory, since their frequency coincides with the Brillouin zone
boundary (Mesquita, Vasconcellos, & Luzzi 1998b).
The potential for human exposure to mm-wave radiation at frequencies
and power levels that have been associated with biological effects will
increase significantly in the coming decades. In part, this is due to the
recognition of the significant commercial potential that this region offers
for telecommunication systems, automobile collision avoidance radar,
aviation radar and weather monitoring and other applications. These are
all free-space transmission technologies in which human exposure is
inherent and will greatly exceed background levels. In addition,
millimetre-wave radiation is also used therapeutically in some countries.
This is in contrast to the situation in 1986 (Furia 1986), where the scope
XVII
for human exposure was limited to satellite communications systems and
military equipment.
The existence of non-thermal interactions would be highly significant as
the current public-health exposure guidelines are thermally based, and
non-thermal interactions could represent a potential avenue for the
selective interaction with biological systems. This would have the
potential for considerable social and economic impact.
Experiments involving millimetre-wave radiation and biological systems
are, by necessity, complex and time-consuming. It was also recognized at
the beginning of the project that historically, attempts to replicate
millimetre-wave induced effects have met with failure. Rather than
attempting to replicate prior experimental studies per se, the approach
here is to develop a methodology that is flexible, providing the basis for
experimentation on both eukaryotic and prokaryotic cultures and over a
wide spectral range.
The thesis has been written so that it is accessible to a general scientific
audience and specialists in radio frequency engineering and biology. A
glossary is provided and words that appear in the glossary are indicated
with bold type in the text.
XVIII
Aims: -
Design and construct an instrument that is flexible and which can: -
(a) be deployed in the investigation of a wide variety of culturable
cell types.
(b) employ a sensitive and higher integrative reporter to detect
biological effects.
(c) systematically screen parameter space in reasonable time
scales.
(d) attain high level of physiological reproducibility.
(e) incorporate feedback into the analysis and control system.
(f) be used to determine the existence or otherwise of non-
thermal interactions in biological systems with confidence.
XIX
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