Meeting report

German researchers announce most up-to-dateresults on biosensorsReport on 3rd Biosensors Symposium 2003 (the German NationalMeeting on Biosensors, Biochips, and Bioarrays), held at Potsdam,Germany, 30 March–1 April 2003

Gunter Gauglitz*

1. Introduction

In 1999, Peter Luppa started a successful series of sym-posia on biosensors, biochips, and bioarrays, whichconvenes every two years. The intention was to orga-nize a national conference in Germany, and to scheduleit in the years between the worldwide biosensor con-gress. Young research scientists would get the opportu-nity to demonstrate progress and trends in the fast-developing ¢eld of biosensing in German-speakingcountries. In contrast to other bioanalytic and bio-sensor conferences, post-docs and PhD students were togive lectures, rather than research group leaders, whofrequently have the opportunity to lecture elsewhere.The two-and-a-half-day meeting was launched for

the ¢rst time in Munich in 1999; having proved suc-cessful, it was repeated 2001 in Tu« bingen, and this yearin Potsdam. More than 250 scientists from academiaand industry met at the Neue Palais in Potsdam, giving25 lectures and presenting more than 70 posters. Sincethe ¢eld is rather wide-ranging, the philosophy is tostart each session with a tutorial by post-docs and addshort talks by advanced PhD students. In the openingsession on the ¢rst evening, well-known research scien-tists gave introductory plenary lectures.The large number of participants demonstrated the

quality and the acceptance of this philosophy. An ever-increasing number of post-docs from other Europeancountries are joining the German-speaking scientists.Since the deadline for submitting abstracts is usually

shortly before the meeting itself, the lectures provideup-to-date research results of the groups, and give avery good and current survey on biosensor research inCentral Europe.

2. Reviews in the introductory session

Biomolecular motors, molecular imprinted polymers(MIPs) and trends in proteomics technologies are con-sidered to be topics of urgent interest. For this reason,Eberhard Unger (Jena) demonstrated on Sunday eve-ning the feasibility of biomolecular linear motors andactuators in nanometer dimensions. He focused on thekinesin-microtubules system, which under cell-free in-vitro conditions allows the generation of a force able totransport arti¢cial loads. His presentation showed themechanical cycles of the forces generated by the kine-sins. The demonstrated transport of these linear motorswill certainly ¢nd application in nano-actuators, nano-robotics, chemical reaction techniques in femto or attodimensions, or might be helpful in any kind of sensorsystem if motion or transport in a precision range ofnanometers has to be established. Such biological devi-ces might ¢nd application in the area of separation andanalytics in microvolumes (see also [1]).MIPs as biomimetic receptors for biosensors represent

another emerging ¢eld, as K. Haupt (Lund) demon-strated in the second lecture. During copolymerizationof synthetic polymers together with molecular targetmolecules, there is produced a molecular template,which follows the approaches of biomolecular recogni-tion structures. Such MIPs are intended to demonstrateantibody or receptor properties, combined with a stabi-lity that neither antibodies nor receptors will achieve.Production is less costly and easier than normal

Institut fur Physikalische und Theoretische Chemie, Auf der Morgenstelle 8,

D-72076 Tubingen, Germany

*Tel.: +49 7071 29 76927 or 74667; Fax: +49 7071 29 5490;

E-mail: Guenter.Gauglitz@ipc.uni-tuebingen.de

Trends in Analytical Chemistry, Vol. 22, No. 6, 2003 Meeting report

0165-9936/03/$ - see front matter # 2003 Published by Elsevier Science B.V. doi:10.1016/S0165-9936(03)00609-5 xi

immunology. A separation factor of a=5.0 for enantio-mers and 2.4 D pesticide monitoring was demonstratedas well as examinations to understand the quality of theMIPs using attenuated total re£ection spectroscopy(ATR) in a £ow cell [2].The third introductory lecture was given by M. Blu« g-

gel (Dortmund) on present developments in proteomicstechnologies. First he argued why analytical biosensingis essential in genomics and proteomics; he then com-pared present technologies and stressed the importanceof ¢nding new tools, especially in the area of mass spec-trometry. The development in MALDI-MS in combi-nation with automated sample preparation wassummarized with particular mention of the automationof sample preparation by robots as the 2D-gel-picker.Furthermore, he pointed out the necessity of bioinfor-matics to the development. Even in recent develop-ments, such as peptide ¢ngerprint algorithms or de-novosequence algorithms, there remains a lack of dataanalysis.

3. Session topics

3.1. Sensors for nucleic acids/biochipsThe tutorial on DNA and protein analysis on arrays ^technological and economic considerations byH. Eickho¡ (Berlin) ^ covered currently known DNA-chip and protein microarray technologies, which areemployed for high throughput analysis [3]. The presentapproaches are accompanied by unpredictable surfacee¡ects, low binding capabilities and/or denaturatedbiomolecules. For interaction studies involving pro-teins innovative chip designs are especially required tomonitor the interactions at the necessary detectivity.Even though market surveys predict a bright future forbiochips, the markets themselves do not show the pro-mised development.Technical applications of on-chip PCR, presented for

the ampli¢cation of bacteriophage M13 using £uores-cence-labeled probes, were the topic of the ¢rst shorttalk. In addition, application diagnostics was discussedby the Bier group (Bergholz Rehbru« cke).This PCR ampli¢cation was also demonstrated by the

Cammann group (Mu« nster) in the ¢eld of nutrition ana-lysis [4]. Evanescent ¢eld excitation is used for fast andtime-resolved monitoring of hybridization at hundredspots in an array. Parallel detection is achieved by aCCD camera.Instead of optical devices, a digital multielectrode

array was used in november AG (Erlangen) [5] to detectrelevant DNA sequences in blood samples with sub-sequent ampli¢cation by PCR. For the ¢rst time, medi-cally relevant biological data were presented in aninstrument that used redox recycling as the measure-ment principle.

The last talk of this session was presented by FRIZBiochem (Munich), Hartwich [6] introducing DNAchips with electric detection of hybridization by apply-ing an electrically detected displacement essay (EDDA).This method avoids £uorescence label methods andproblems with non-speci¢c adsorption. The speakerdemonstrated that redox-labeled signal oligonucleo-tides allow monitoring of the status of hybridization ofimmobilized label free capture DNA molecules on goldelectrodes. LADER (light addressable direct electricalreadout) was presented as a DNA detection formatbased on various electron transfer processes of single-and double-strand DNA. Of special interest was anapproach that causes the DNA chains to straighten byadding thiols in between.

3.2. MiniaturizationIn this session, the tutorial on aspects of miniaturizationand integration by M. Wegner (Tu« bingen) summarizedthe state of the art and brought out advantages and pro-blems of miniaturization [7]. Application of micro-system technologies allows to the construction of chipseven in nano-dimensions, improving £uidics, reducingdi¡usion layers and thereby reducing response timesand improving interaction processes. However, bio-compatibility of material, £ow processes and mixing areof increasing signi¢cance. The various new trends inimproved sample mixing were especially discussed.The systematic survey of the odds and ends of minia-

turization proved to be a very good introduction to thefollowing lectures as e.g. on the capabilities of TopSpottechnology for high-throughput production of micro-arrays given by M. Daub (Freiburg), who demonstratedthe possibility to spot 96 (or even 384) di¡erent samplesin parallel at volumes from 200 pl up to a few nl andspacings of few hundred micrometers [8].A very interesting application of laser scanning £uo-

rescence endoscopy inmedicine was given by the Seegergroup (Zu« rich). The development of new £uorophoresin photodynamic diagnostics was reported for markingbrain tumors and allowing laser ablation of cancer cellswith a confocal £uorescence endoscope in combinationwith an NMR tomograph. Control by this tomographand increased contrast during imaging of the malig-nant cells increases the chance of preventing a relapse.An innovative application of the scanning electro-

chemical microscope (SECM) was presented by theSchuhmann group (Bochum). Instead of an opticalread-out of DNA chips ultra-microelectrodes were usedto monitor local electrochemical properties of the chipsurface. Redox labels, intercalation and methods polar-izing the DNAmodi¢ed electrode were not used [9].

3.3. Biomimetic sensorsIn the tutorial on nucleic acids as bio-noses by F. Klein-jung (Berlin), the process of SELEX (systematic evolution

Meeting report Trends in Analytical Chemistry, Vol. 22, No. 6, 2003

xii http://www.elsevier.com/locate/trac

of ligands by exponential enrichment) was introduced.It is an in-vitro selection and ampli¢cation techniquethat allows the selection of oligonucleotides (apta-meres) that bind preferentially to a given target mole-cule. This is done out of a large pool of oligomeres withvariable sequences (synthetic random oligonucleotidelibrary) [10]. The computer-supported method pro-duces not only aptameres but also spiegelmeres withinteresting properties (primary, secondary, tertiary),which have higher stability compared to aptameres andexhibit ideal properties as recognition elements inbio-noses.The Strehlitz group (Leipzig) reported interesting

examples of the production of such aptameres in biomo-lecular interactions for the development of new bio-sensors. The SELEX process was implemented using£uorescence-labeled ssDNA and magnetic streptavidinbeads as target molecules. Besides speci¢c target mole-cules, such as nucleotides, amino acid, polysaccharides,antibiotics, peptides or proteins, even complex targetstructures, such as viruses, spores or even cells, can beused.The Scheller group (Potsdam) reported on a new pro-

cess for preparing MIPs [11], cross-linking vinylimid-azole monomers in order to produce an analogue totransition state of esterolytic reactions. The demon-strated imprint catalytic activity was applied to thecleavage of phenolic esters and carbamates.G. Tovar (Stuttgart) reported on MIP nanospheres as

fully synthetic receptors for biosensor application insolution and at surfaces [12].The Wolfbeis group (Regensburg) [13] reported a

new application of the spreader-bar technique, notimprinting the volume but rather the surface. The ¢rstpositive results of enantioselectivity of such an arti¢cialreceptor were presented in the case of bisnaphthyl-2,2-diamine. Chiroselective coe⁄cients were determinedusing capacitive measurements and surface plasmonresonance (SPR) transducers.

3.4. Protein engineering/enzymatic sensorsT. Bachmann (Stuttgart) gave a tutorial on the designof acetylcholinesterase for application in biosensors.Besides a survey on the application of this acetylcholinesystem in biosensing in the recent years, there was alsodiscussion of bioinformatics for understanding thestructure, function, and interaction of acetyl-cholinesterase and for improving its reactivity in orderto supply new enzyme varieties. The di¡erent processes,expression and puri¢cation, as well as the analyticalapplication, were surveyed [14].The Schuhmanngroup demonstrated the high quality

of electrochemical disposable polymers with covalentlybound redox relays [15]. Combinatorial synthesis of thepolymers supplied samples with a variety of hydro-philic, hydrophobic and steric properties. Electro-

chemistry was used to monitor the polymerizationprocess in the wells of microtiter plates and to char-acterize these libraries. This redox polymer library canbe used to optimize reagentless glucose biosensors.The Urban group (Freiburg) [16] presented experi-

ments to improve the stability of proteins in biosensormembranes and the hydrogels.The Bilitewski group (Braunschweig) [17] talked

about a coupling of electrophoretic and enzymatic prin-ciples in micro£uidic chips demonstrated in case of sac-charide analytics.At the end of the session, a very interesting new possi-

bility for detecting superoxide radicals was proposed bythe groups of Lisdat and Scheller (Potsdam) [18], whopresented multilayer electrodes with multilayers ofcytochrome C and sulfonated polyaniline (PASA). Thisapproach greatly increased the number of layers of pro-teins on the electrode, in comparison with monolayerelectrodes. The authors proved for the ¢rst time that,even in an 8-layer system, electron transfer took place,when a value higher than 2 had not yet been publishedin the literature. The measurement of radical concen-tration in vivo and in vitro is of great interest in medi-cine, nutrition and production of antioxidantscontained in cosmetics. This approach allows fastquanti¢cation of radical concentration and monitoringof their dependence on the amount of antioxidantpresent.

3.5. Receptors and drug screeningThe tutorial on biosensors for screening new drugsusing pharmacologically active substances was givenby M. Keusgen (Bonn) [19]. The report dealt withworldwide activities for screening plants and micro-organisms for substances to get new lead structures forthe development of new drugs. The present method ofscreening could be enhanced using biochips and follow-ing two strategies. In the case of ‘‘structural screen-ing’’, substances having a potential biological e¡ectwere examined for relevant structures. The examples ofsaponine and polyphenols (£avanoide) were discussedusing enzyme or antibody-based sensors. The otherapproach (functional screening) proposed uses a vari-ety of enzymes, receptors and cells, which are notscreened according to structural elements but ratherbiological e¡ects. Results were demonstrated for thecase of cysteine sulfoxides and screening for the dopa-min receptor agonist and antagonist using SPR. Minia-turization was considered necessary to obtain as manyparameters as possible in parallel. For the future, therewas focus on total cell biosensors.After this tutorial, the Steinem group (Regensburg)

reported on the preparation, stability, and ion-channelinsertion of solid supported membranes. To combinetheir stability with the quality of black lipid membranes,they were suspended on di¡erent porous substrates.

Trends in Analytical Chemistry, Vol. 22, No. 6, 2003 Meeting report

http://www.elsevier.com/locate/trac xiii

Tools of surface analysis in combination with transduc-tion principles were used for either alumina or siliconporous layers with pore sizes adjusted between 20 nmand 200 nm in the case of alumina and 500 nm to 3 mmin the case of porous silicon. The quality of the lipidbilayers for charge molecules was investigated by mon-itoring di¡usion of the membrane-impermeable £uor-escent dyes.Two SPR developments were presented in the area of

measuring ion channels and in drug screening. The¢rst was given by the Salzer group in Dresden [20] withthe intention of developing an imaging system for anarray of ion channels in thin polymer ¢lms with micro-pores. These were generated by electron lithographytechnique and were supposed to be used for label-freedetection using this imaging transducer. In modelexperiments, a good spatial resolution was measured.However, biological samples could not yet be measured.SPR was also used by the Keusgen group (Bonn))

with a set-up of eight parallel channels. Receptor/ligand interactions were intended to be measured bythis method. A typical application is the dopamin recep-tor, belonging to the group of G-protein coupled recep-tors, which play an essential part in the signal-transduction process in humans. Binding kinetics weredetermined for living cells as well as membrane pre-parations. Discrimination between non-speci¢c andspeci¢c interaction was possible. Thus, this methodpotential in the area of e⁄cient drug screening.

3.6. Protein chipsThe ¢nal tutorial on protein-array generation andrecent applications was given by D.J. Cahill (Berlin) anddemonstrated the high level of research at the Max-Planck Institute. The development and application ofhigh-density array technology and automation to gen-erate high-content, high-density protein and antibodyarrays were demonstrated. The protein chips allowedmeasurement of sera from patients with autoimmunedisease as well as chip-based assays for possible applica-tions in diagnostics. Improving expression vectors andsystems to express thousands of proteins in Pichia pas-toris and in Saccharomyces cerevisiae appropriate forhigh throughput were developed. Recombinant pro-teins are proposed for use in proteomics, and a ¢rstapplication could be demonstrated. Peptide librarieswere characterized. Phage display antibody was exam-ined using the protein array, and, ¢nally, pro¢ling ofantibodies in sera was established [21^23].K. Kro« ger (Tu« bingen) demonstrated the ¢rst 96-spot

array with direct optical read-out with a label-freere£ectrometric method in nanotiter plate format [24].This method was miniaturized and parallelized and cannow read more than 96 spots in an area of 12^18 mmwith spots being dispensed by the TopSpot process. Ananowell structurewas established by laser lithography

in a polymer plate, which was glued to the opticaltransducer glass slide. Taking into account the di¡u-sion processes and avoiding evaporization by a coolingsystem, this miniaturized re£ectrometric interferencespectroscopy allows time-dependent kinetic monitoringof the biomolecular interaction process in nanolitercavities.A surface acoustic wave (SAW) sensor as a tool for

direct detection of mass changes at a surface was pre-sented as a biosensor for medical diagnostics by theRapp group (Karlsruhe) [25]. Mass-sensitive measure-ment using this SAW allows measurement of the inter-action of mouse-Ig with immobilized monoclonal anti-mouse-Ig antibodies with a limit of detection (LOD)lower than 7 pg/mm2. Combination of this SAW devicewith £ow injection analysis demonstrates an interest-ing device for multisample measurements.Weller’s group (Munich) [26] used the PASA (paral-

lel a⁄nity sensor array) system to set up more than 50spots integrated in a £ow-through cell with approxi-mately 120 ml volume. The detection is based on a lumi-nol process and allows parallel determination of quite anumber of antibiotics in milk below the LOD required bylaw. No sample preparation is necessary. The test is ¢n-ished within 8 minutes. This instrumentation providesa ¢rst real-world application of biochip arrays.The published association rate constants for binding

processes in homogeneous phase di¡er from those atheterogeneous phase because of steric problems anddi¡usion-limited processes. M. Kumpf (Tu« bingen)demonstrated a new approach that combines experimentand simulation by way of time-resolved measurement ofthe binding process in homogeneous phase. This is donevia the concentration of non-blocked receptor insolution,which is determined bymass-transport-limitedmeasurements of the interaction of the non-blockedreceptors with immobilized catcher molecules.

4. Summary and outlook

The overwhelming majority of the presentations andthe poster papers demonstrated the high quality ofbiosensor and biochip research in German-speakingcountries, allowing participants to get up-to-date infor-mation, since laboratories were reporting their currentresearch and were even giving the ¢rst results of theirinnovative approaches. As a result, all the participantsfeel that it is essential to be updated in such a meeting,which can provide the latest information faster thanany printed media. It is the opportunity to discussresults shown on the posters directly with the authorsand to hold discussions over several days that make thismeeting so successful.In addition, the scienti¢c board awarded three prizes

for the best poster presentations, two donated by the

Meeting report Trends in Analytical Chemistry, Vol. 22, No. 6, 2003

xiv http://www.elsevier.com/locate/trac

ABC journal. First prize went to G. Proll (Tu« bingen) forhis poster on £uorescence nanoparticles; second prizeto J. Halamek (Potsdam) [27] for his poster on new prin-ciples of direct real-time monitoring of interaction ofcholinesterase and its inhibitors by piezoelectric bio-sensors; and, third prize to N. Gajovic-Eichelmann(Bergholz-Rehbru« cke) [28] for the formation of linearnanostructures using DNA.All participants felt the meeting to be very successful

and are looking forward to meeting 2005 in Regens-burg for the next symposium, which is being organizedby O. Wolfbeis close in time and location to ANAKON2005.

References

[1] K.J. Bo« hm, E. Unger, in: H. Singh-Nalwa (Editor), Encyclopediaof Nanoscience and Nanotechnology, American Scienti¢c Pub-lishers, Stevenson Ranch, California, USA, 2003, in press.

[2] K. Haupt, Chem. Commun. 2 (2002) 171.[3] H. Eickho¡, U. Schneider, E. Nordo¡, L. Nyarsik, G. Zehetner,

W. Nietfried, G. Lehrach, Technology development for DNAchips, in: E.V. Grigerenko (Ed.), DNA Arrays: Technologies andExperimental Strategies, CRC Press, Boca Raton, Florida, USA,2001, pp. 1^9.

[4] C. Peter, M. Meusel, F. Grawe, A. Katerkamp, K. Cammann,T. Boerchers, Fresenius’ J. Anal. Chem. 371 (2001) 120.

[5] E. Palecek, J. Hassmann, Electrochemical method for determina-tion of nucleic acids. (November Aktiengesellschaft Gesell-schaft fu« rMolekulareMedizin,Germany), PCT Int.Appl., 2002.

[6] G. Hartwich, P. Frischmann, U. Haker, H. Wieder, Method andkit for displacement assays that detect ligate-ligand associationevents especially nucleic acid hybridization. (Friz BiochemGmbH, Germany), PCT Int. Appl., 2003.

[7] D.R. Reyes, D. Iossi¢dis, P. Auroux, A. Manz, Anal. Chem. 74(2002) 2623.

[8] B.D. Heij et al., A tunable and highly perled picoliter dispenser,Proc. MEMS 2002.

[9] Ch. Kurzawa, M. Mosbach, E. Bonsen, W. Schuhmann, Proc.Electrochem. Soc. (2001) 2001-18 (Chemical and BiologicalSensors and Analytical Methods II), 304.

[10] A. Vater, S. Klussmann, Curr. Opin. Drug Discovery Dev. 6(2003) 253.

[11] F. Scheller, U. Wollenberger, A. Warsinke, F. Lisdat, Curr.Opin. Biotechnol. 12 (2001) 35.

[12] A. Weber, N. Dettling, H. Gunner, G.E.M. Tovar, Macromol.Repet. Commun. 23 (2003) 824.

[13] T. Hirsch, H. Kettenberger, O.S. Wolfbeis, V.M. Mirsky, Chem.Comm. 3 (2003) 432.

[14] H. Schulze, S. Vorlova, F. Villatte, T. Bachmann, R.D. Schmid,Biosens. Bioelectron. 18 (2003) 201.

[15] C. Kurzawa, A. Hengstenberg, W. Schuhmann, Anal. Chem.74 (2002) 355.

[16] G. Urban, Sens. Update 8 (2001) 189.[17] T. Richter, L. Shultz-Lockyear, R. Oleschuk, U. Bilitewski,

D. Harrison, Sens. Actuators, B B81 (2002) 369.[18] S. Ignatov, D. Shishniashvili, F.W. Scheller, Biosens. Bioelec-

tron. 17 (2002) 191.[19] M. Keusgen, Naturwissenschaften 89 (2002) 433.[20] G. Steiner, V. Sablinskas, A. Hubner, C. Kuhne, R. Salzer,

J. Mol. Struct. 509 (1999) 265.[21] P. Angenendt, J. Glo« kler, D. Murphy, H. Lehrach, D.J. Cahill,

Anal. Biochem. 309 (2002) 253.[22] W. Arap, et al., Nat. Med. (NY) 8 (2002) 121.[23] J.D. Hoheisel, D. Cahill, Curr. Opin. Chem. Biol. 6 (2002) 11.[24] K. Kroeger, J. Bauer, B. Fleckenstein, J. Rademann, G. Jung,

G. Gauglitz, Biosens. Bioelectron. 17 (2002) 937.[25] N. Barie, M. Rapp, Biosens. Bioelectron. 16 (2001) 979.[26] E. Martlbauer, A. Strasser, R. Dietrich, K.-J. Zaadhof,

M. Weller, B. Knecht, R. Niessner, Miniaturized biosensors forprotection of the quality of milk products, Diskussionstagung-Forschungskreis der Ernaehrungsindustrie e.V., 2002, 60th(Forschung im Dienste der Lebensmittelqualitaet), pp. 55-64.

[27] J. Halamek, A. Makower, P. Skladal, F.W. Scheller, Biosens.Bioelectron. 17 (2002) 1045.

[28] F.F. Bier, N. Gajovic-Eichelmann, R. Hoelzel, AIP ConferenceProceedings (DNA-Based Molecular Construction) 640 (2002)51.

Trends in Analytical Chemistry, Vol. 22, No. 6, 2003 Meeting report

http://www.elsevier.com/locate/trac xv