ANALYTICAL SCIENCE IN SWITZERLAND 705 CHIMIA 5/ (1997) Nr. 10 (Ok.obcr) Chimia 51 (1997) 705-713 cific sites on mRNA and, therefore, © Neue Schweizerische Chemische Gesellschaft block the expression of cell proteins, lSSN 0009-4293 required novel methods in order to allow a fast detection of the parent compounds and the enzymatic degra- Advanced Analytical Methods dation products. Capillary Electro- phoresis and Optical Biosensors have for Pharmaceutical the potential to address these issues. - The discovery of new molecular mark- and Diagnostic Applications ers to diagnose specific diseases such as Fatty Acid Binding Protein (FABP) for acute myocardial infarction, or the Markus Ehrat*, Gert L. Duveneck, Gerhard M. Kresbach, Peter Oroszlan, and identification of RNA and DNA se- Aran Paulus quences to identify viral infections or genetic diseases require complicated assay procedures in order to achieve Abstract. Driving forces for the development of novel analytical technologies in the limits of quantitation in the range of 10 life-science industry are described. Technologies which either were developed in Bio- to 1000 molecules. With signal or tar- Analytical Research or brought to a reliability required for routine applications will be get amplification methods such as elucidated and, on the basis of practical examples, the impact of modern analytical Polymerase Chain Reaction and use of technologies on the industrial research and development will be discussed: Optical labeled branched DNA such limits of biosensors based on evanescent excitation of luminescence allow for real-time moni- quantitation can be achieved today. toring of the binding of active compounds to specific biomolecular recognition sites. However, many of these amplification Molecular imaging technologies have the potential to gain rapid access to physical methods are not suitable for fast diag- maps of genomic materials. Capillary electrophoresis or affinity gel electrophoresis are nostic point of care applications. Opti- well suited for the fast determination of oligonucleotide mixtures in nl amounts of cal biosensors based on evanescent samples. Integrated capillary electrophoresis on chips will allow to multiplex capillary excitation of fluoresence are consid- systems at low costs and results in high separation efficiencies. MALDI- TOF MS is an ered by us as a potential technology for easy to operate non-scanning mass spectrometric instrumentation for the analysis of ultrasensitive and fast detection of ol- high molecular weight biopolymers such as immunoglobulins. igonucleotides and DNNRNA strands. - The investigation of bioaffinity inter- actions is not only an impoltant task Introduction nologies allows not only the efficient iden- for the chemical optimization of the tification of molecular drug targets but potency of drug interaction with a re- Analytical technologies playa key role also opens novel ways for diagnosing in- ceptor but also for the detection of in the research and development process fectious and genetic diseases. In the last binding properties of drugs to blood of pharmaceutical products: in the research years activities in our department were proteins. This type of analytical infor- phase they are indispensable for the iden- focused on evaluating, adapting and fur- mation can be obtained in an easy and tification of the molecular targets, the se- ther developing advanced analytical tech- accurate way by the application of lection of a substance with a suitable bind- nologies and methodologies in those are- evanescent wave biosensors. ing affinity to the target or the char- as. Examples below shall identify the major - Biotechnology and molecular biology acterization of the molecular structure. In driving forces for such developments as in general, the human genome project the development phase analytical tasks well as their technical implementations. in particular had a tremendous impact are the identification of metabolites, the - Novel, more potent drug candidates on the development of advanced ana- parameters of drug-protein interactions, which are administered at lower doses lytical techniques. The high molecular and the determination of pharmacokinetic and, therefore, result in much lower weight of many biopolymers, the com- parameters. There typically large num- blood concentration levels set new plex mixtures in biotechnological pro- bers of animal and human samples have to standards in terms of the detection lim- cesses as well as the very small vol- be analyzed. In close context with pharma- it of analytical methods used for mon- umes in which minute concentrations ceutical applications is the development itoring their metabolic and pharma- of analytes have to be determined, re- of analytical technologies for human diag- cokinetic behavior. For example, the quired a reevaluation of existing ana- nostics. The enormous progress made in daily dose of the new aromatase inhib- lytical methods. For the determination molecular biology within the past years itor Letrozole (Femara®) is only 2 mg of biopolymers in complex samples it together with advanced analytical tech- compared to 250 mg for aminogluteth- was Matrix Assisted Laser Desorption imide (Orimeten®) which results in Ionization Time of Flight Mass Spec- blood concentrations in the nanomolar trometry (MALDI- TOFMS) which did range for Femara compared to micro- anow a fas't analysis without a tedious *Carrespandence: Dr. M. Ehrat molar for Orimeten. sample preparation method. We will Navartis Pharma AG - Novel chemical classes of drug com- demonstrate how MALDI- TOF MS in BioAnalytical Research pounds often require more powerful combination with a simple sample prep- K-127.156 CH-4002 Basel separation and detection methods. The aration method was applied to monitor Tel.: +41 6\ 6966069 introduction of antisense oligonucle- the antibody production during a cell Fax: +41616964504 otides, typically oligonucleotides with cultivation. Approaches to establish E-Mail: [email protected] 20 to 25 bases which hybridize to spe- functional physical maps, e.g., to in- ANALYTICAL SCIENCE IN SWITZERLAND 706 CHI MIA 5/ (1997) Nr. 10 (Oktober) vestigate the localization of genes and mate senSItIVIty, a threefold selectivity Planar waveguides provide different to monitor the expression of mRNA approach was incorporated: possibilities of detection configurations. are currently under development. 1) The evanescent wave sensing princi- Both the fluorescence that is excited in the - Novel approaches in drug research such ple for spatial selectivity, evanescent field, but isotropically emitted as combinatorial chemistry, random 2) the immobilized biological recogni- ('volume detection') and the portion of screening or the assessment of genetic tion elements for (bio)chemical selec- fluorescence light, that is coupled back information pose increasing demands tivity, and into the waveguide, can be detected. With on analytical technologies in terms of 3) the luminescent labeling for amplifi- a second grating the guided emission light small sample volumes and high sam- cation and specific signal generation. can be coupled out of the waveguide (' grat- ple throughput. Miniaturization, mul- The evanescent field penetrates only ing detection') and directed towards the tiplexing and automation allow to reach some 100 nm from the sensor surface into detection system (Fig. 1). these goals. We will introduce the con- the solution and thus allows a real-time cept of Integrated Capillary Electro- monitoring of binding kinetics. As in the Immuno and Oligonucleotide Assays phoresis, demonstrate how such chips well accepted and widely used solid-phase Target analyte classes, in general, can can be obtained at low costs and - bioassays, the immobilized biorecogni- be any molecules specifically binding to using lambda phage digests- how DNA tion elements on the transducer surface their immobilized affinity partners by fragments can be separated at picomo- can be used to selectively capture the (bio)specific recognition or leading to a lar concentrations on 20 mm short chan- analytes.The fluorescent label of the trac- specific change of a physical-chemical nels. er, added at known concentrations, allows property of the recognition element. Until for an easier differentiation between spe- now, the list of affinity systems adapted to cific and nonspecific binding events, pro- the FOBIA system reflects a large part of Optical Biosensors vides superior sensitivity and makes the the spectrum of known biochemical affin- sensors signal independant of the molecu- ity interactions (antigen-antibody, recep- Outline of Technology lar weight of the analytes. tor-ligand, complementary DNAJRNA, ol- The demand in life sciences and hu- igonucleotide-strands, protein-drug inter- man diagnostics for analytical methods Transducer Layout and Optical action) and could be extended to synthetic adequate to measure biospecific interac- Configuration affinity systems as well. Due to the inher- tions at extremely low concentrations, in The transducer consists of a thin ent extremely high sensitivity and real- small sample volumes and with a mini- waveguiding layer « 200 nm) with a high time measuring capability of FOBIA, its mum of sample preparation is the driving refractive index (> 2.2) on a glass or plas- application in new target areas, like trac- force for the development of a novel gen- tic substrate. Excitation light is coupled ing RNA/DNA targets in biological sam- eration of biochemical sensors [1]. FO- into