Biosensors & Bioelectronics 9 (1994) iii-xiii

Introduction to bioelectronics :
"Interfacing biology with electronics"

Wolfgang Gopel & Peter Heiduschka

University of Tubingen, Institute of Physical and Theoretical Chemistry, and Centre of Interfacial Analysis and
Sensors, Auf der Morgenstelle 8, D-72076 Tubingen, Germany

Tel : [49] 7071 296904. Fax [49] 7071 296910 . Email: goepel @mailserv .zdv.uni-tuebingen .de

I . GENERAL SCOPE latter is expected later than the year 2020 .

Bioelectronics deals with the coupling of biologi-
cal function units to electronics . Bioelectronic
devices include those for biochemical sensing,
information processing, as well as storage and
actuating . Interfacing of biology with electronics
is the key issue and was hence taken as the
subtitle of the workshop . Because of its inherent
interdisciplinary approach, bioelectronics is
closely linked to many other fields in materials
and life sciences .
Continuing and growing interest in bioelectron-
ics is basically driven by current trends in various
micro- and nanotechnologies, and by progress in
the theoretical understanding of highly versatile
and efficient biological systems .
The international scientific community basically
agrees concerning the importance of tasks and
subtasks in current research and development
(R&D) of bioelectronics .
In Japan, for example, the co-ordination of
various R&D efforts is perfectly organised . Vari-
ous activities have been co-ordinated there for
more than 8 years . A major driving force concerns
the long-term goal to develop biodevices and, in
particular, bincomputers . The realisation of the
The expected economic impact, however, of
bioelectronics on information technologies in
general is expected to exceed already within the
next 20 years any other field such as transport,
telecommunications, or life sciences . One motiv-
ation is the clearly visible limitation of current
technologies in the semiconductor industries to
solve complex problems in life sciences . In this
context, there is a general interest to exploit new
approaches for future information technologies .
Main stream approaches and concepts in Japan
concern
• the self-organisation, self-assmbly, self-
repair, self-error correction and self-
reproduction as realised in and deduced
from biological structures (Hotani, Nagay-
ama, Yoshikawa),
• biodevices based on protein engineering
and molecular computers making use
of the knowledge and structures from
neuronal systems including brains (Isoda,
Sasabe),
• development of computer systems
approaching human users by including
engineering aspects of anthropological
phenomena like memory, personality,

0956-5663/94, $07 .00© 1994 Elsevier Science Ltd . nl


W. Gopel & P. Heiduschka
learning pattern recognition (Matsumoto,
Tsumoto, Okajima) and
• molecular and bioelectronics devices with
particular emphasis on adapting manufac-
turing technologies to prototype molecu-
lar sensors, biosensors, biomolecular func-
tion units, and arrays of oriented cells
(Aizawa, Karube) .
In Europe, a few dedicated activities only focus
explicitly towards bioelectronics . Important pro-
gress has been made, however, by many
researchers in a variety of related fields concern-
ing, for instance, local probe techniques, synthesis
of molecular and supramolecular materials, prep-
aration of micro- and nano-structures, or protein
engineering . These European activities will now
be described along six different lines .
II . ORGANISATION OF THE FOLLOWING
TOPICS
The broad field of "interfacing biology with
electronics" will be covered in this special issue
by different contributions focusing first at general
concepts concerning
• "bioelectronics and supramolecular engin-
eering" (part 1)
and then at selected topics concerning
• materials preparation and synthesis (part 2),
• structures of layers, supramolecular, and
mesoscopic systems (part 3),
• molecular recognition, signal transduction,
and sensors (part 4),
• analytical techniques (part 5), and
• applications (part 6) .
These topics and authors listed in the following
table will now be characterised briefly .
Part 1 : Survey on bioelectronics and
supramolecular engineering
In an introduction to basic concepts, general
paradigms of the evolution of biological molecular
structures are treated by Kuhn in his paper
"Reflections on biosystems motivating supramol-
ecular engineering" . The key-and-lock sequences
leading to self-assembly of supramolecular struc-
tures in nature and the principles of programmed
environmental changes are identified here as the
iv
Biosensors & Bioelectronics
most promising approaches for future supramol-
ecular engineering which aim at the development
of complex bioelectronics devices . Several
examples are given for strategies in supramolecu-
lar engineering by starting from cryptate mol-
ecules and monolayers formed by the Langmuir-
Blodgett (LB)-technique or by chemisorption
of selected molecules with functional groups
exhibiting receptor properties . Monolayer
assemblies that make possible the light-induced
electron transfer through the layer are of parti-
cular interest in view of utilising photon-induced
processes similar to those occurring in the photo-
synthetic reaction centre . In this context, bacterial
photosynthetic reaction centres are discussed as
model systems for supramolecular assemblies
with respect to driving forces of their molecular
evolution (Fig . 1) . The links between evolution
in the origin of life and the present requirements
for engineering on the molecular and supramol-
ecular level are discussed comparatively . An
important message of this article is that efforts
in this field should not only stick too closely
to paradigms of "man-made" engineering and
construction principles at the risk of neglecting
the basic strategical approaches in nature, where
structures and functions have been developed by
variation and selection .
A broad introduction to various aspects of
bioelectronics is given in the presentation of
Nicolini entitled "From protein engineering to
bioelectronics" . Particular emphasis is put on
materials, assembly techniques, as well as infor-
mation transfer between artificial structures and
biological function units . This survey covers in
particular the topics : neural chips, biomolecular
engineering, thin biofilm formation, unique
properties of protein films, and biomolecular
electronic devices .
More complex systems are discussed in the
following article by Breer entitled "Molecular
mechanisms of signal transduction in olfaction" .
Here, a survey is given about our current
knowledge concerning the molecular and cellular
processes in olfaction of vertebrates . A schematic
view of the proposed mechanisms for signal
transduction in olfaction is given in Fig . 2. The
chemical nature of the receptor molecules in
signal cascades and the first details about the
organisation of different receptors in olfactory
organs including their topographic localisation
presented in this paper give an excellent idea of
new aspects of biological signal transduction and
Biosensors & Bioelectronics "Interfacing biology with electronics"

TABLE 1 Survey on the different papers, their classification diamonds) and overlap (crosses) to
the different parts 1-6 .

Part 1 Part 2 Part 3 Part 4 Part 5 Part 6

First author Survey on Materials Structure of Molecular Analytical Applications
bioelectronic preparation, layers, recognition, techniques
s and synthesis, supra- signal
supramole- and molecular transduction
cular properties and and sensors
engineering mesoscopic
s stems
Adami et at

Barraud ~©
Bidan et at x
Breer x x
Brunori

~ Djlh:
Comtat et at ~~©© x
Connoll _~© x
Davide et at
Decher et at
De Rosa et aL
De Vena et aL
Duine -~'
En elbor hs ~
Erokhin et at r~~®
Gd el ©~~,aaaa
Hintsche et aL x
Kov'cs-V . et al.
Kuhn r~~aaa
-©©0 ~
McAdams et aL
IM g rM I M-~l
x
Nicolini ~ © x

Olthuis
Overbeck et at --©- x
Perham ~0©©
Pett x
Pfeiffer et aL -©®©
Rada et at
_ --
Rutten et a/. --~ x

~-j
mi, - -©
Torchut et at
Treloar et aL x
.- -- ~


W. Gopel & P. Heiduschka Biosensors & Bioelectronics

3 nm
Energy 1W a)

special pair of bacteriochlorophyll a (BChl a), P870

b)
BL by

QL 2+
\ ubiquinone, Q
Fe
Fig. 1. (a) Energy scheme of the electron transport chain in the bacterial photosynthetic reaction centre . The photo-
excited electron is removed from P870* via the bacteriochlorophyll BL and the bacteriopheophytin H L to the
menaquinone QL where it is stored for about 100 ps before being transferred to Q M and carried to the pool. Since
BL has the same energy level as P870*, electron oscillation occurs . The spatial distance between P870* and QL
must be large enough to avoid recombination by electron tunneling (3 nm), and the energy level of QL must be
sufficiently lower than that of P870* in order to avoid recombination by thermal activation (0.5 eV) . (b) Scheme
of the optimum spatial arrangement of the components of the reaction centre . The dotted line indicates the pathway
of the electron.

self organisation . They also give an excellent
insight to the current "state of the art" to
investigate and identify such structures and func-
tions by using a broad spectrum of biochemical
techniques .
Of key importance for any bioelectronic device
which interfaces biological function units with
electronics are hybrid systems treated in the
following article by Gopel on "Controlled signal
transduction across interfaces of `intelligent' mol-
ecular systems" . Starting from simple molecular
layer structures, four case studies are discussed

vi
Biosensors & Bioelectronics
input
(odorant molecules)
result
(smell)
Fig. 2. Proposed mechanism of signal transduction in
olfaction . After the binding of an odorant (L) to a
receptor (R), a specific G -protein (G) binds guanosine
triphosphate (GTP) and releases guanosine diphosphate
(GDP) . Subsequently, an olfactory adenylate cyclase
(AC) is activated by a dissociating part of G . Adenosine
triphosphate (ATP) is converted into the second messen-
ger cyclic adenosine monophosphate (cAMP) . The
latter reaches its highest level after 50 ms and opens an
olfactory cyclic nucleotide-gated ion channel (CNC) for
sodium ions . (Another possible second messsenger not
shown here is 1,4,5-triphosphate (IP 3) formed by
phospholipase C. The IP3 opens channels for calcium
ions.) The cAMP stimulates protein kinase A (PKA)
which is involved in switching off the cAMP-generating
cascade by phosphorylation of the receptor protein .
which concern biomolecular recognition in homo-
geneous matrices and in membranes by means
of synthetic (biomimetic) and natural function
units . In the outlook concepts are stressed to
develop hybrid devices. The example of an
"electronic nose" is chosen here to characterise
"Interfacing biology with electronics"
the current "state of the art" in fully synthetic
multisensor arrays (see Fig . 3) and, for compari-
son, to characterise future concepts which aim
at combing natural biological function units
as discuss d above by Breer with man-made
semiconductor devices .
Part 2: Materials preparation, synthesis, and
properties
Building blocks of biological function units in
general consist of proteins, fatty acids, carbo-
hydrates, nucleic acids, and other natural biologi-
cal materials .
In the context of bioelectronics, proteins, and
in particular enzymes, attract by far the most
attention. Redox proteins are of particular inter-
est because they can be coupled directly to
electrochemical transducers . Preparation, struc-
ture, and function of a variety of such enzymes
is discussed by Comtat, McNeil, Brunori,
Schuhmann, Canters, Laval, Hintsche, Koudelka-
Hep, Cass, Scheller, Olthuis, Treloar (for simpli-
fication, first authors only are mentioned here
and in the following to identify the paper) .
A detailed contribution on the broad variety
of "Dehydrogenases in natural and artificial
electron transfer" is given by Duine in the context
of selected concepts to realise cofactor-electrode
directed electron transfer .
In view of preparing long-term stable structures
an important approach is to characterise and
produce redox proteins from extreme thermo-
philic bacteria as discussed by Rossi or to use
lipids of archaea as discussed by de Rosa .
An important aspect to "fine-tune" the proper-
ties of these materials is their gene technological
optimisation as stressed in various contributions,
e .g. by Nicolini, Canters, Perham, and Brunori
with a selected interesting example illustrated in
Fig . 4 which is discussed by Canters .
An important key to predict details of protein-
folding is to identify these motifs or domains
preserved during evolution . In this context,
"Structural aspects of biomolecular recognition
and self-assembly" are discussed by Perham
with the aim of designing novel proteins and
performing protein manipulations which may be
described as "molecular lego" .
In contrast to this approach to start from
natural systems, the modelling of properties of
biological systems by synthesising biomimetic
materials appears to be of increasing interest.
vu


W. Gopel & P. Heiduschka Biosensors & Bioelectronics

comparison with
calibration data

analyte: electronics result:

gas or filter or catalyst sensors and & data feature pattern- chemical
liquid membrane or enzyme transducers pretreatment vector recognition composition

Fig. 3. Schematic presentation of the different components in an artificial "electronic noise" which may be correlated
with components of the olfactory system of vertebrates or which may be replaced in part by biomolecular analogues
in future hybrid devices .

native azurin second

path for
electrons?

Fig. 4. Azurin is a blue copper protein with a Cue+-ion in the centre which is co-ordinated with two histidines,
one cysteine and one methionine . By mutation, the His"' can be replaced by Glyl" resulting in opening the protein
shell. An imidazole ring can now be introduced and linked with an electrode by a spacer (e.g., by a "molecular
wire") . For the mediation with other redox proteins, the spacer has to be sufficiently long, or a second path for
electrons has to be created by protein engineering .

An illustrative example is discussed by Laval, role : An approach for the electrocontrolled

synthetic receptor compounds are discussed by
Gopel and Barraud .
A promising concept to mimic in particular
different key-lock recognition structures is to
synthesise suitable supramolecular units some of
which are discussed by Kuhn, Perham, Gopel
and Barraud . The related use of monomers,
oligomers, and polymers in bioelectronic devices
is stressed by Aizawa, Schuhmann, Barraud,
Gopel, and Decher . In this context, the "Incor-
poration of sulfonated cyclodextrins into polypyr-
delivering of neutral drugs" is discussed by Bidan .
The use of inorganic materials as substrates
for devices is stressed in a variety of contributions
including those of McAdams, Gopel, Hintsche,
Koudelka-Hep, Rutten, and Hammerle .
Whole cells to be used in future devices are
discussed by Clark in the paper "Cell behaviour
on micropatterned surfaces" . "A thin film
microelectrode array for monitoring extracellular
neuronal activity in vitro" is reported by Nisch .
Biosensors & Bioelectronics "Interfacing biology with electronics"

Part 3: Structure of layers, supramolecular and
mesoscopic systems
Structural aspects in the controlled design of
monolayers and multilayers are discussed in a
general context by various authors including in
particular Nicolini, Kuhn, and Gopel . More
specific aspects include "Engineering supramol-
ecular artificial devices for specific functions"
prepared by the Langmuir-Blodgett (LB) tech-
nique as discussed by Barraud . Of special interest
are the two examples given in this article,
i.e . the biomimetic "dioxygen trap" and the
monomolecular d .c . conducting layers . "Long
range order and textures in lipid monolayers" is
presented by Mobius . An attempt to apply the
LB technique for the formation of protein layers
is described by Erokhin . A new example to use
lipid layers for the incorporation of enzymes and
for the transport of mediators as given by Laval
is characterised schematically in Fig . 5.
A new approach for the controlled formation
of layer structures on various different substrates
is described in the contribution "New nano-
composite films for biosensors : Layer-by-layer
adsorbed films of polyelectrolytes, proteins or
DNA" by Decher .
Examples for mesoscopic systems are "Microtu-
bules : Dissipative structures formed by self-
assembly" in living cells, presented by Engel-
borghs. These microtubules show two important
characteristics of many biological structures,
namely the self-assembly of the functional struc-
ture and the dissipative dynamic behaviour, i .e.
continuous energy consumption to maintain and/
or change a given state .
Part 4: Molecular recognition, signal
transduction and sensors
Biomolecular recognition structures are discussed
in a variety of different contributions, including
in particular those concerning biosensors (see
below), and additional topics stressed in the
articles by Breer, Perham, and Lundstrom . Model
systems for controlled signal transduction with
main emphasis on the biomimetic approach are
discussed by Gopel .
"Electronically modulated biological functions
of molecular interfaced enzymes and living cells"
are stressed by Aizawa . The conducting polymer
polypyrrole is used as molecular interface between
the enzymes fructose dehydrogenase and pyruvate
oxidase and the electrode as well as between
yeast cells and the electrode . The enzyme activity
and the gene expression of the cells could both
be modulated electronically in this arrangement .
One important goal of a variety of papers
concerns the electron transfer between redox
proteins and an electrode as illustrated

H3C-~ C ,COO
H3C-~ 000- + C0 2
pyruvate

DMPC +
ubiquinone
OTS

A1 2 0 3

Fig. 5 . After modification of aluminium oxide (A1 203) with a layer of octadecyltrichlorosilane (OTS), a lipid layer
was formed by fusion of vesicles of dimyristoylphosphatidyl-choline (DMPC) and ubiquinone . Pyruvate oxidase
from E . coli was incorporated in the layer. In the course of the oxidation of pyruvate, acetate is formed and
ubiquinone is reduced . Ubiquinol diffuses laterally to the gold electrode . Here it is oxidised and deetected
electrochemically.

ix
W. Gopel & P. Heiduschka
schematically in Fig. 6 . Specific contributions
concern "Electron transfer between metals and
biomolecules in the conception of new bioelectro-
chemical biosensors" as treated by Comtat,
"Electron transfer between modified enzymes
and conducting-polymer modified electrodes"
discussed by Schuhmann, and "Direct electron
transfer bioelectronic interfaces" as presented by
McNeil . In the latter paper, cytochrome c is used
which draws increasing attention as a mediator
for bioelectronic applications (Fig . 7) .
Of particular importance for the short term
realization of any biolectronic device in practical
applications are sensors and biosensors with main
emphasis on electrochemical sensors . Design
concepts to develop new sensors are discussed
by a few specialised papers, including the "Con-
struction of nanoband electrode arrays, signal
processing and application to biosensing devices"
by Hintsche, "Electrochemical and photolitho-
graphical techniques for the modification of
microelectrodes" by Koudelka-Hep, "3D multi
micro electrode systems for neuromuscular signal
interfacing and control" by Rutten, as well as
the application of novel concepts of "Signal
processing in biosensors" performed by multi-
enzyme systems by Scheller (see Fig . 8) . Using
the system myokinase/pyruvate kinase/pyruvate
oxidase, an exponential amplification of ADP
could be performed with an amplification factor
of 800 . Other biosensors are discussed by Treloar
and Sartore . Thin film sensors for gas sensing
are presented by Petty . Practical and economical
aspects of biosensors are stressed in a critical,
but realistic survey by Connolly .
An affinity sensor based on the optical method
of surface plasmon resonance (SPR) is discussed
in the paper "Real time biospecific interaction
analysis" by Lundstrom . The use of "large-scale
biomolecular function units" is described in
articles by Clark and in the paper on "Analysis
of natural neural networks" by Hammerle . In
both approaches, nerve cells are investigated,
whereas the above-mentioned approach discussed
by Aizawa deals with the immobilisation of yeast
cells .
Part 5: Analytical techniques
In research and development of bioelectronic
devices, a broad variety of classical and novel
analytical techniques is currently applied and
their results, without further details on the
x
Biosensors & Bioelectronics
experimental set-up, are discussed in the different
contributions listed above .
Methods which characterise interfaces (if at all
possible down to the atomic scale) are of increas-
ing importance . These techniques include in
particular scanning tunneling microscopy (STM)
and related scanning probe techniques ("SXM"
Fig . 9), the established techniques of 2-dimen-
sional Fourier-transform NMR (2D-FT-NMR),
EPR, surface plasmon resonance (SPR), X-
ray crystallography, Raman spectroscopy, optical
spectroscopies between the IR and the UV range,
monitoring of dichroic ratios, optical microscopy,
spectroelectrochemistry, the broad spectrum of
different electrochemical techniques, impedance
spectroscopy (IS), and ellipsometry .
The use of IS for the investigation of "The
linear and nonlinear electrical properties of the
electrode-electrolyte interface" is stressed by
McAdams . The "Characterisation of proteins by
means of their buffer capacity, measured with
an ISFET-based coulometric sensor-actuator sys-
tem" is discussed by Olthuis .
The analytical techniques to investigate inter-
faces include also the surface analytical spec-
trometers which utilise electrons, photons, ions
and atoms as probes . Examples are X-ray photo-
emission spectroscopy (XPS), ultraviolet photo-
emission spectroscopy (UPS), electron energy loss
spectroscopy (ELS), scanning Auger microscopy
(SAM), scanning electron microscopy (SEM),
and secondary ion mass spectroscopy (SIMS) .
A short overview of the techniques is given in
the paper by Gopel . The local probe techniques
(SXM) are discussed in detail by Michel in the
paper "Local probe investigation of molecular
material" .
Specific optical microscopies are discussed in
two contributions . "Microscopic investigations of
the interaction of proteins with surfaces" are
outlined by Cass with the aim of investigating
the lateral distribution of glucose oxidase in thin
polyphenol layers . The orientation of hydro-
carbon chains in LB monolayers of lipids is
determined by Mobius using Brewster angle
microscopy.
Part 6: Applications
The often-investigated bacteriorhodopsin is a
"model system" for a biomolecular function unit
with applications for information storage devices .
It may be isolated in the form of stable purple
 b

Fig. 6. Different mechanisms of electron transfer between enzymes (grey u-shaped structures) and electrodes : (a)
Mediator in solution (charge transfer by free particles (upper part), or by particles incorporated in free micelles
(lower right part)) ; (b) mediator immobilised at the electrode or the enzyme (charge transfer by the "wipe
mechanism") ; (c) mediator in a polymer network (charge transfer by "electron relays") ; (d) protein modified by
mediators ; (e) organic conducting salts (e .g. TTF-TCNQ) ; (f) composite electrodes with mediator embedding the
enzyme, (e.g. "carbon paste electrode") ; (g) conducting polymers as "molecular wires"; and (h) direct electron
transfer between protein (optimised by mutation) and electrode . M is the active centre containing a metal, and C
denotes another complex for electron transport introduced by a mutation .
W. Gopel & P. Heiduschka
Fig. 7. Heme group of the natural mediator cytochrome c attached to the protein shell by cysteine (Cys) residues .
membranes, it can be modified gene technologi-
cally and it is already used for optical information
processing . In the general article of Nicolini
reference is given to the current literature on
this topic .
The most promising short-term applications
concern biosensors for clinical analysis and
environmental control as discussed by Scheller,
Koudelka-Hep, Connolly, McNeil, Hintsche, and
Sartore . A critical market analysis for the different
concepts and types of biosensors is given by
Connolly in her contribution "Biosensors for
clinical diagnostics - opportunities and perform-
ance requirements" . Extracorporal sensors for
Biosensors & Bioelectronics
the human skin are discussed in the paper
"Bioelectric cutaneous and microcirculation sen-
sors for the study of vigilance and emotional
response during tasks and tests" by Dittmar .
Other applications concern formal aspects of
information theory including "Neural network
architectures for industrial applications" by Mas-
tretta and the "Co-operative classifiers for high
quality hand-printed character recognition" by
Baccarani. "Self-organising sensory maps in
odour classification mimicking", which are
important for the development of artificial olfac-
tory systems are stressed by Davide .
Biosensors & Bioelectronics "Interfacing biology with electronics"

C2

detectable
Product

C1 electrode
Fig. 8. Scheme of an amplification mechanism for biosensing . The analyte A acting as the substrate S, is converted
by the enzyme E l together with the cofactor C, resulting in the products P* and S2. The S2 is a substrate for the
enzyme E2 chosen in an appropriate way to obtain again the substrate S, for the enzyme E l. By this way, more
C, can be converted. This results in a higher signal of P* at the electrode . If the enzymatic reactions provide one
molecule of each product per reaction, the amplification is linear . If two molecules of S, or S2 are produced, the
amplification is exponential, as long as the cofactors are present in excess amounts .

Fig. 9. Principle of local probe microscopies : A small tip is scanned across the surface of interest. A local interaction
of choice is recorded . This results in an image representing the surface with respect to the kind of interaction, i.e.
the tunneling current (STM), the force, (AFM), capacitance (SCM), optical reflection (SNOM), etc.

Rill