Introduction to bioelectronics: “Interfacing biology with electronics”

Biosensors & Bioelectronics 9 (1994) iii-xiii

Introduction to bioelectronics :"Interfacing biology with electronics"

I. GENERAL SCOPE

Bioelectronics deals with the coupling of biologi-cal function units to electronics . Bioelectronicdevices include those for biochemical sensing,information processing, as well as storage andactuating . Interfacing of biology with electronicsis the key issue and was hence taken as thesubtitle of the workshop . Because of its inherentinterdisciplinary approach, bioelectronics isclosely linked to many other fields in materialsand life sciences .

Continuing and growing interest in bioelectron-ics is basically driven by current trends in variousmicro- and nanotechnologies, and by progress inthe theoretical understanding of highly versatileand efficient biological systems .

The international scientific community basicallyagrees concerning the importance of tasks andsubtasks in current research and development(R&D) of bioelectronics .In Japan, for example, the co-ordination ofvarious R&D efforts is perfectly organised . Vari-ous activities have been co-ordinated there formore than 8 years . A major driving force concernsthe long-term goal to develop biodevices and, inparticular, bincomputers . The realisation of the

0956-5663/94, $07.00© 1994 Elsevier Science Ltd .

Wolfgang Gopel & Peter Heiduschka

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

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

latter is expected later than the year 2020 .The expected economic impact, however, ofbioelectronics on information technologies ingeneral is expected to exceed already within thenext 20 years any other field such as transport,telecommunications, or life sciences . One motiv-ation is the clearly visible limitation of currenttechnologies in the semiconductor industries tosolve complex problems in life sciences . In thiscontext, there is a general interest to exploit newapproaches for future information technologies .

Main stream approaches and concepts in Japanconcern

•

the self-organisation, self-assmbly, self-repair, self-error correction and self-reproduction as realised in and deducedfrom biological structures (Hotani, Nagay-ama, Yoshikawa),

•

biodevices based on protein engineeringand molecular computers making useof the knowledge and structures fromneuronal systems including brains (Isoda,Sasabe),

•

development of computer systemsapproaching human users by includingengineering aspects of anthropologicalphenomena like memory, personality,

nl

W. Gopel & P. Heiduschka

iv

learning pattern recognition (Matsumoto,Tsumoto, Okajima) and

•

molecular and bioelectronics devices withparticular 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 focusexplicitly towards bioelectronics . Important pro-gress has been made, however, by manyresearchers in a variety of related fields concern-ing, for instance, local probe techniques, synthesisof molecular and supramolecular materials, prep-aration of micro- and nano-structures, or proteinengineering. These European activities will nowbe described along six different lines .

II. ORGANISATION OF THE FOLLOWINGTOPICS

The broad field of "interfacing biology withelectronics" will be covered in this special issueby different contributions focusing first at generalconcepts concerning

•

"bioelectronics and supramolecular engin-eering" (part 1)

and then at selected topics concerning

•

materials preparation and synthesis (part 2),•

structures of layers, supramolecular, andmesoscopic 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 followingtable will now be characterised briefly .

Part 1 : Survey on bioelectronics andsupramolecular engineering

In an introduction to basic concepts, generalparadigms of the evolution of biological molecularstructures are treated by Kuhn in his paper"Reflections on biosystems motivating supramol-ecular engineering" . The key-and-lock sequencesleading to self-assembly of supramolecular struc-tures in nature and the principles of programmedenvironmental changes are identified here as the

Biosensors & Bioelectronics

most promising approaches for future supramol-ecular engineering which aim at the developmentof complex bioelectronics devices . Severalexamples 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 chemisorptionof selected molecules with functional groupsexhibiting receptor properties . Monolayerassemblies that make possible the light-inducedelectron transfer through the layer are of parti-cular interest in view of utilising photon-inducedprocesses similar to those occurring in the photo-synthetic reaction centre . In this context, bacterialphotosynthetic reaction centres are discussed asmodel systems for supramolecular assemblieswith respect to driving forces of their molecularevolution (Fig . 1) . The links between evolutionin the origin of life and the present requirementsfor engineering on the molecular and supramol-ecular level are discussed comparatively . Animportant message of this article is that effortsin this field should not only stick too closelyto paradigms of "man-made" engineering andconstruction principles at the risk of neglectingthe basic strategical approaches in nature, wherestructures and functions have been developed byvariation and selection .

A broad introduction to various aspects ofbioelectronics is given in the presentation ofNicolini entitled "From protein engineering tobioelectronics" . Particular emphasis is put onmaterials, assembly techniques, as well as infor-mation transfer between artificial structures andbiological function units . This survey covers inparticular the topics : neural chips, biomolecularengineering, thin biofilm formation, uniqueproperties of protein films, and biomolecularelectronic devices .

More complex systems are discussed in thefollowing article by Breer entitled "Molecularmechanisms of signal transduction in olfaction" .Here, a survey is given about our currentknowledge concerning the molecular and cellularprocesses in olfaction of vertebrates . A schematicview of the proposed mechanisms for signaltransduction in olfaction is given in Fig . 2. Thechemical nature of the receptor molecules insignal cascades and the first details about theorganisation of different receptors in olfactoryorgans including their topographic localisationpresented in this paper give an excellent idea ofnew aspects of biological signal transduction and

Biosensors & Bioelectronics

TABLE 1 Survey on the different papers, their classificationthe different parts 1-6 .

"Interfacing biology with electronics"

diamonds) and overlap (crosses) to

First authorPart 1Survey on

bioelectronics and

supramole-cular

engineering

Part 2Materials

preparation,synthesis,

andproperties

Part 3Structure of

layers,supra-

molecularand

mesoscopics stems

Part 4Molecular

recognition,signal

transductionand sensors

Part 5Analyticaltechniques

Part 6Applications

Adami et at

Barraud ~©Bidan et at x

Breer x xBrunori

~ Djlh:Comtat et at ~~©© xConnoll _~© x

Davide et atDecher et atDe Rosa et aLDe Vena et aL

Duine -~'En elbor hs ~Erokhin et at r~~®

Gd el ©~~,aaaaHintsche et aL xKov'cs-V . et al.

Kuhn r~~aaa-©©0 ~

McAdams et aLIM g rM I M-~l

xNicolini ~ © x

OlthuisOverbeck et at --©- x

Perham ~0©©Pett x

Pfeiffer et aL -©®©Rada et at_ --

Rutten et a/. --~ x

~-jmi, - -©Torchut et atTreloar et aL x

.- -- ~

W. Gopel & P. Heiduschka

Energy

BL

QL Q

special pair of bacteriochlorophyll a (BChl a), P870

Fe 2+

\ ubiquinone,

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 HL to themenaquinone QL where it is stored for about 100 ps before being transferred to Q M and carried to the pool. SinceBL has the same energy level as P870*, electron oscillation occurs . The spatial distance between P870* and QLmust be large enough to avoid recombination by electron tunneling (3 nm), and the energy level of QL must besufficiently lower than that of P870* in order to avoid recombination by thermal activation (0.5 eV) . (b) Schemeof 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 excellentinsight to the current "state of the art" toinvestigate and identify such structures and func-tions by using a broad spectrum of biochemicaltechniques .

Of key importance for any bioelectronic device

vi

3 nm 1W

by

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a)

b)

which interfaces biological function units withelectronics are hybrid systems treated in thefollowing article by Gopel on "Controlled signaltransduction across interfaces of `intelligent' mol-ecular systems" . Starting from simple molecularlayer structures, four case studies are discussed

Biosensors & Bioelectronicsinput

(odorant molecules)

result

(smell)Fig. 2. Proposed mechanism of signal transduction inolfaction . After the binding of an odorant (L) to areceptor (R), a specific G -protein (G) binds guanosinetriphosphate (GTP) and releases guanosine diphosphate(GDP) . Subsequently, an olfactory adenylate cyclase(AC) is activated by a dissociating part of G. Adenosinetriphosphate (ATP) is converted into the second messen-ger cyclic adenosine monophosphate (cAMP) . Thelatter reaches its highest level after 50 ms and opens anolfactory cyclic nucleotide-gated ion channel (CNC) forsodium ions. (Another possible second messsenger notshown here is 1,4,5-triphosphate (IP 3) formed byphospholipase C. The IP3 opens channels for calciumions.) The cAMP stimulates protein kinase A (PKA)which is involved in switching off the cAMP-generatingcascade by phosphorylation of the receptor protein .

which concern biomolecular recognition in homo-geneous matrices and in membranes by meansof synthetic (biomimetic) and natural functionunits . In the outlook concepts are stressed todevelop 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 syntheticmultisensor arrays (see Fig . 3) and, for compari-son, to characterise future concepts which aimat combing natural biological function unitsas discuss d above by Breer with man-madesemiconductor devices .

Part 2: Materials preparation, synthesis, andproperties

Building blocks of biological function units ingeneral consist of proteins, fatty acids, carbo-hydrates, nucleic acids, and other natural biologi-cal materials .

In the context of bioelectronics, proteins, andin particular enzymes, attract by far the mostattention. Redox proteins are of particular inter-est because they can be coupled directly toelectrochemical transducers . Preparation, struc-ture, and function of a variety of such enzymesis discussed by Comtat, McNeil, Brunori,Schuhmann, Canters, Laval, Hintsche, Koudelka-Hep, Cass, Scheller, Olthuis, Treloar (for simpli-fication, first authors only are mentioned hereand in the following to identify the paper) .

A detailed contribution on the broad varietyof "Dehydrogenases in natural and artificialelectron transfer" is given by Duine in the contextof selected concepts to realise cofactor-electrodedirected electron transfer .

In view of preparing long-term stable structuresan important approach is to characterise andproduce redox proteins from extreme thermo-philic bacteria as discussed by Rossi or to uselipids of archaea as discussed by de Rosa .

An important aspect to "fine-tune" the proper-ties of these materials is their gene technologicaloptimisation as stressed in various contributions,e .g. by Nicolini, Canters, Perham, and Brunoriwith a selected interesting example illustrated inFig . 4 which is discussed by Canters .

An important key to predict details of protein-folding is to identify these motifs or domainspreserved during evolution . In this context,"Structural aspects of biomolecular recognitionand self-assembly" are discussed by Perhamwith the aim of designing novel proteins andperforming protein manipulations which may bedescribed as "molecular lego" .

In contrast to this approach to start fromnatural systems, the modelling of properties ofbiological systems by synthesising biomimeticmaterials appears to be of increasing interest.

vu

W. Gopel & P. Heiduschka

native azurin

analyte :gas or

filter or

catalystliquid

membrane

or enzyme

An illustrative example is discussed by Laval,synthetic receptor compounds are discussed byGopel and Barraud .

A promising concept to mimic in particulardifferent key-lock recognition structures is tosynthesise suitable supramolecular units some ofwhich are discussed by Kuhn, Perham, Gopeland Barraud . The related use of monomers,oligomers, and polymers in bioelectronic devicesis stressed by Aizawa, Schuhmann, Barraud,Gopel, and Decher. In this context, the "Incor-poration of sulfonated cyclodextrins into polypyr-

comparison withcalibration data

electronicssensors and

& data

feature

pattern-transducers pretreatment vector

recognition

Biosensors & Bioelectronics

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

in future hybrid devices .

secondpath forelectrons?

result:chemical

composition

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 proteinshell. An imidazole ring can now be introduced and linked with an electrode by a spacer (e.g., by a "molecularwire") . 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 .

role: An approach for the electrocontrolleddelivering of neutral drugs" is discussed by Bidan.

The use of inorganic materials as substratesfor devices is stressed in a variety of contributionsincluding those of McAdams, Gopel, Hintsche,Koudelka-Hep, Rutten, and Hammerle .

Whole cells to be used in future devices arediscussed by Clark in the paper "Cell behaviouron micropatterned surfaces" . "A thin filmmicroelectrode array for monitoring extracellularneuronal activity in vitro" is reported by Nisch .

Biosensors & Bioelectronics

Part 3: Structure of layers, supramolecular andmesoscopic systems

Structural aspects in the controlled design ofmonolayers and multilayers are discussed in ageneral context by various authors including inparticular Nicolini, Kuhn, and Gopel . Morespecific aspects include "Engineering supramol-ecular artificial devices for specific functions"prepared by the Langmuir-Blodgett (LB) tech-nique as discussed by Barraud . Of special interestare the two examples given in this article,i.e . the biomimetic "dioxygen trap" and themonomolecular d .c . conducting layers . "Longrange order and textures in lipid monolayers" ispresented by Mobius . An attempt to apply theLB technique for the formation of protein layersis described by Erokhin . A new example to uselipid layers for the incorporation of enzymes andfor the transport of mediators as given by Lavalis characterised schematically in Fig . 5 .A new approach for the controlled formation

of layer structures on various different substratesis described in the contribution "New nano-composite films for biosensors : Layer-by-layeradsorbed films of polyelectrolytes, proteins orDNA" by Decher .

Examples for mesoscopic systems are "Microtu-bules : Dissipative structures formed by self-assembly" in living cells, presented by Engel-

H3C-~ C ,COO

pyruvate

DMPC +ubiquinone

OTS

A1 2 0 3

borghs. These microtubules show two importantcharacteristics 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, signaltransduction and sensors

Biomolecular recognition structures are discussedin a variety of different contributions, includingin particular those concerning biosensors (seebelow), and additional topics stressed in thearticles by Breer, Perham, and Lundstrom . Modelsystems for controlled signal transduction withmain emphasis on the biomimetic approach arediscussed by Gopel .

"Electronically modulated biological functionsof molecular interfaced enzymes and living cells"are stressed by Aizawa . The conducting polymerpolypyrrole is used as molecular interface betweenthe enzymes fructose dehydrogenase and pyruvateoxidase and the electrode as well as betweenyeast cells and the electrode . The enzyme activityand the gene expression of the cells could bothbe modulated electronically in this arrangement .

One important goal of a variety of papersconcerns the electron transfer between redoxproteins and an electrode as illustrated

H3C-~ 000- + C02

"Interfacing biology with electronics"

Fig. 5. After modification of aluminium oxide (A1 203) with a layer of octadecyltrichlorosilane (OTS), a lipid layerwas formed by fusion of vesicles of dimyristoylphosphatidyl-choline (DMPC) and ubiquinone . Pyruvate oxidasefrom E . coli was incorporated in the layer. In the course of the oxidation of pyruvate, acetate is formed andubiquinone 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 contributionsconcern "Electron transfer between metals andbiomolecules in the conception of new bioelectro-chemical biosensors" as treated by Comtat,"Electron transfer between modified enzymesand conducting-polymer modified electrodes"discussed by Schuhmann, and "Direct electrontransfer bioelectronic interfaces" as presented byMcNeil. In the latter paper, cytochrome c is usedwhich draws increasing attention as a mediatorfor bioelectronic applications (Fig . 7) .

Of particular importance for the short termrealization of any biolectronic device in practicalapplications are sensors and biosensors with mainemphasis on electrochemical sensors . Designconcepts to develop new sensors are discussedby a few specialised papers, including the "Con-struction of nanoband electrode arrays, signalprocessing and application to biosensing devices"by Hintsche, "Electrochemical and photolitho-graphical techniques for the modification ofmicroelectrodes" by Koudelka-Hep, "3D multimicro electrode systems for neuromuscular signalinterfacing and control" by Rutten, as well asthe application of novel concepts of "Signalprocessing in biosensors" performed by multi-enzyme systems by Scheller (see Fig . 8) . Usingthe system myokinase/pyruvate kinase/pyruvateoxidase, an exponential amplification of ADPcould be performed with an amplification factorof 800 . Other biosensors are discussed by Treloarand Sartore . Thin film sensors for gas sensingare presented by Petty . Practical and economicalaspects of biosensors are stressed in a critical,but realistic survey by Connolly .

An affinity sensor based on the optical methodof surface plasmon resonance (SPR) is discussedin the paper "Real time biospecific interactionanalysis" by Lundstrom . The use of "large-scalebiomolecular function units" is described inarticles by Clark and in the paper on "Analysisof natural neural networks" by Hammerle . Inboth approaches, nerve cells are investigated,whereas the above-mentioned approach discussedby Aizawa deals with the immobilisation of yeastcells .

Part 5: Analytical techniques

In research and development of bioelectronicdevices, a broad variety of classical and novelanalytical techniques is currently applied andtheir results, without further details on the

x

experimental set-up, are discussed in the differentcontributions listed above .

Methods which characterise interfaces (if at allpossible down to the atomic scale) are of increas-ing importance . These techniques include inparticular 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, opticalspectroscopies between the IR and the UV range,monitoring of dichroic ratios, optical microscopy,spectroelectrochemistry, the broad spectrum ofdifferent electrochemical techniques, impedancespectroscopy (IS), and ellipsometry .

The use of IS for the investigation of "Thelinear and nonlinear electrical properties of theelectrode-electrolyte interface" is stressed byMcAdams . The "Characterisation of proteins bymeans of their buffer capacity, measured withan 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, ionsand atoms as probes . Examples are X-ray photo-emission spectroscopy (XPS), ultraviolet photo-emission spectroscopy (UPS), electron energy lossspectroscopy (ELS), scanning Auger microscopy(SAM), scanning electron microscopy (SEM),and secondary ion mass spectroscopy (SIMS) .

A short overview of the techniques is given inthe paper by Gopel . The local probe techniques(SXM) are discussed in detail by Michel in thepaper "Local probe investigation of molecularmaterial" .

Specific optical microscopies are discussed intwo contributions . "Microscopic investigations ofthe interaction of proteins with surfaces" areoutlined by Cass with the aim of investigatingthe lateral distribution of glucose oxidase in thinpolyphenol layers. The orientation of hydro-carbon chains in LB monolayers of lipids isdetermined by Mobius using Brewster anglemicroscopy.

Part 6: Applications

The often-investigated bacteriorhodopsin is a"model system" for a biomolecular function unitwith applications for information storage devices .It may be isolated in the form of stable purple

Biosensors & Bioelectronics

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 "wipemechanism"); (c) mediator in a polymer network (charge transfer by "electron relays") ; (d) protein modified bymediators; (e) organic conducting salts (e.g . TTF-TCNQ); (f) composite electrodes with mediator embedding theenzyme, (e.g. "carbon paste electrode") ; (g) conducting polymers as "molecular wires"; and (h) direct electrontransfer 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

membranes, it can be modified gene technologi-cally and it is already used for optical informationprocessing. In the general article of Nicolinireference is given to the current literature onthis topic .

The most promising short-term applicationsconcern biosensors for clinical analysis andenvironmental control as discussed by Scheller,Koudelka-Hep, Connolly, McNeil, Hintsche, andSartore . A critical market analysis for the differentconcepts and types of biosensors is given byConnolly in her contribution "Biosensors forclinical diagnostics - opportunities and perform-ance requirements" . Extracorporal sensors for

Biosensors & Bioelectronics

Fig. 7. Heme group of the natural mediator cytochrome c attached to the protein shell by cysteine (Cys) residues .

the human skin are discussed in the paper"Bioelectric cutaneous and microcirculation sen-sors for the study of vigilance and emotionalresponse during tasks and tests" by Dittmar .Other applications concern formal aspects ofinformation theory including "Neural networkarchitectures for industrial applications" by Mas-tretta and the "Co-operative classifiers for highquality hand-printed character recognition" byBaccarani. "Self-organising sensory maps inodour classification mimicking", which areimportant for the development of artificial olfac-tory systems are stressed by Davide .

Biosensors & Bioelectronics

C2

C1

detectableProduct

P

"Interfacing biology with electronics"

electrode

Fig. 8. Scheme of an amplification mechanism for biosensing . The analyte A acting as the substrate S, is convertedby the enzyme E l together with the cofactor C, resulting in the products P* and S2. The S2 is a substrate for theenzyme E2 chosen in an appropriate way to obtain again the substrate S, for the enzyme E l. By this way, moreC, can be converted. This results in a higher signal of P* at the electrode . If the enzymatic reactions provide onemolecule 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 interactionof 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