Miniaturized Flow Injection Systems for Time Resolved Step-Scan FT-IR Spectroscopy of Fast Reacting Chemical Systems in Solution

This research project aims at the development of new analytical techniques capable to provide molecular specific information of the very first steps of chemical reactions in solution as a function of time. This information is of high importance in various fields of research such as catalyst-development and biochemistry and is not accessible with state-of-the-art techniques. Therefore, the proposed techniques will present valuable tools to look at fundamental processes on a molecular specific level and contribute to elucidate their mechanism on a molecular scale. In this project two methodologically different workpackages can be defined. Workpackage 1: Time resolved FT-IR spectroscopy of chemical reactions in solution: Modern time resolved (TR) FT-IR spectroscopy, including the step-scan (S 2 ) technique will be hyphenated to miniaturised flow injection like systems. The tasks of the m -flow systems will be rapid and reproducible initiation of the reaction under investigation. For this purpose new mixing principles such as electrophoretically induced mixing, miniaturised Moebius mixers and a combination of both will be investigated. These approaches are characterised by an electrically induced and hence triggerable mixing process, low reagent consumption and m -flow-cells for high quality FT-IR spectra acquisition in very small volumes. Therefore, TR-S2 -FT-IR spectroscopy will be, for the first time, applicable to chemical reactions in solution including irreversible ones, reaching a time resolution in the sub-ms range. The new techniques will be applied to investigate proteine folding as well as to identify reaction intermediates in catalyst research. Workpackage 2: Time resolved FT-IR spectroscopy of structural changes of immobilised biomolecules and bioligand interactions: The biomolecules will be immobilised either on a miniaturised, high sensitive total internal reflection (ITR) element (planar waveguide) or on microbeads made of e.g. latex or sephadex. In both cases the reactions under investigation will be initiated by supplying pulses of reagents to the biomolecules using fast flow injection like systems. In the first approach the ITR element acts as the surface for biomolecule immobilization and as the sensing part. The second approach employs microbeads as support matrix which can be highly accurately manipulated using the flow system. The micro-beads probed by transmission measurements using fibre optics or again using specially designed m -ITR elements. A measurement cycle will start with introduction of a few micro- beads into the specially designed flow cell. As the beads are retained at the place of IR-detection, spectral changes induced by short reagent pulses delivered by the flow injection system can be directly followed. A repeated analysis is possible by automatically exchanging the used beads by new, fresh ones again using the flow system. The main advantages of this approach are no need for regeneration of the immobilised biomolecules - fresh biomolecules are introduced with new beads - and a high variety of already derivatized commercially available beads. This techniques will be applied to investigate structural changes of enzymes due to interaction with its substrate and inhibitors as well as to investigate antigen-antibody interactions on a molecular specific basis. For the successful development of the planned techniques we will continue and extend our national and international co-operations in order to take advantage of already available know-how in an optimum way.

Within this project lab-on-a-chip systems with mid-IR spectroscopic detection have been developed. These miniaturized analysis systems are capable of initiating and monitoring arbitrary chemical reactions with a time resolution in the low millisecond time range. The chemical reactions to be monitored are initiated by fast, diffusive mixing of reagents in a mid-IR transparent flow cell. For this purpose a dedicated microstructure made of structured SU-8 polymer and a silver layer has been developed and optimized throughout this project. Mid-infrared spectra can be recorded before and during the course of reaction. The miniaturized format allows repeated experiments by low overall sample consumption. In this way both a high time resolution and good signal to noise can be achieved. Because of the molecular specific information contained in the recorded mid-IR spectra information on the reaction products as well on intermediates is directly accessible. Furthermore, the formation of hydrogen bonds and other types of molecular interaction can be detected. The developed system has, e.g., successfully been applied to elucidate the interaction between the antibiotic vancomycin and a tripeptide (precursor of a membrane protein of gram positive bacteria). It was found that the binding proceeds via a short lived intermediate (200 ms) and involves subsequent formation of different types of hydrogen bonds. In a similar way, other chemical systems including protein folding or bio-ligand interactions can now be studied with the lab-on-a- chip instrumentation developed during this project. To extract maximum chemical information from the recorded data sets (spectra as a function of time) modern methods of data analysis have been developed and applied. It is now possible to identify the number of different chemical species involved in the reaction, to extract their mid-IR spectrum from the data set and to determine their concentration profile over time. In a further part of the project capillary electrophoresis, which is capable of highly efficient separation of mixtures of different chemical substance, has been successfully combined with on-line mid-IR detection. For this purpose again a dedicated micro-chip has been developed. Using this new method it is e.g. possible to separate proteins and to directly determine their secondary structure.