Analyzing single protein molecules using optical methods.

Studies on single protein molecules have advanced from mere proofs of principle to insightful investigations of otherwise inaccessible biological phenomena. Recent studies predict a tremendous number of possible future applications. The long-term vision of biologists to watch single molecular processes in real time by peering into a cell with three-dimensional resolution might finally be realized. Another fascinating perspective is the identification and selection of single favorable variants from complex libraries of diverse biomolecules.

Dual-color fluorescence cross-correlation spectroscopy for monitoring the kinetics of enzyme-catalyzed reactions.

Dual-color fluorescence correlation spectroscopy is a biophysical technique that enables precise and sensitive analyzes of molecular interactions. It is unique in its ability to analyze reactions in real time at nanomolar substrate concentrations and below, especially when applied to the monitoring of enzyme-catalyzed reactions. Furthermore, it offers a wide range of accessible reactions, restricted only by the prerequisite that a chemical bond or a physical interaction between two spectrally distinguishable fluorophores is established or broken. Recently, the optical setup of dual-color fluorescence correlation spectroscopy has been extended toward two-photon excitation, resulting in several advantages compared with standard excitation, such as lower fluorescence background, an even larger spectrum of potential fluorescence dyes to be used, as well as a more stable and simplified optical setup. So far, the method has been successfully employed to analyze the kinetics of nucleic acid and peptide modifications catalyzed by nucleases, polymerases, and proteases.

Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening.

Fluorescence-based assay technologies play an increasing role in high-throughput screening. They can be classified into different categories: fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy. In this work we present an alternative analytical technique for high-throughput screening, which we call confocal fluorescence coincidence analysis. Confocal fluorescence coincidence analysis extracts fluorescence fluctuations that occur coincidently in two different spectral ranges from a tiny observation volume of below 1 fl. This procedure makes it possible to monitor whether an association between molecular fragments that are labeled with different fluorophores is established or broken. Therefore, it provides access to the characterization of a variety of cleavage and ligation reactions in biochemistry. Confocal fluorescence coincidence analysis is a very sensitive and ultrafast technique with readout times of 100 ms and below. This feature is demonstrated by means of a homogeneous assay for restriction endonuclease EcoRI. The presented achievements break ground for throughput rates as high as 10(6) samples per day with using only small amounts of sample substance and therefore constitute a solid base for screening applications in drug discovery and evolutionary biotechnology.

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy.

A method for sensitively monitoring enzyme kinetics and activities by using dual-color fluorescence cross-correlation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two fluorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored on-line at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a KM of 14 +/- 1 nM and a kcat of 4.6 +/- 0.2 min-1. In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.

Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: high throughput screening for enzyme activity.

Dual-color fluorescence cross-correlation spectroscopy (dual-color FCS) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies. In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary biotechnology is described. This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depend on the characterization of diffusion parameters that is the prerequisite for conventional fluorescence correlation spectroscopy. Dual-color FCS parameters were optimized to achieve the shortest analysis times. A simulated HTS with homogeneous assays for different restriction endonucleases (EcoRI, BamHI, SspI, and HindIII) achieved precise yes-or-no decisions within analysis times of about 1 s per sample. RAPID FCS combines these short analysis times with the development of fast and flexible assays resulting in sensitive, homogeneous fluorescence-based assays, where a chemical linkage between different fluorophores is either cleaved or formed, or where differently labeled molecules interact by noncovalent binding. In principle, assay volumes can be reduced to submicroliters without decreasing the signal strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology. RAPID FCS can accurately probe 10(4) to 10(5) samples per day, and possibly more. In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where rare and specific binding or catalytic properties have to be screened in large numbers of samples.

Principles and methods of evolutionary biotechnology.

Evolutionary biotechnology applies the principles of molecular evolution to biotechnology, leading to novel techniques for the creation of biomolecules with a great variety of functions for technical and medical purposes. Several basic principles for the application of evolutionary strategies can be derived from a comprehensive theory of molecular evolution. Prerequisites for evolutionary biotechnology are summarized with respect to the different classes of biomolecules and a few, selected applications are described in detail. Concepts for the technical implementation of evolutionary strategies are presented which allow automatized, high throughput processes.

Development and evaluation of a dipstick immunoassay format for the determination of atrazine residues on-site.

On the basis of a semi-quantitative dipstick immunoassay (IA) for atrazine with visual detection (Giersch, T., J. Agric. Food Chem., 1993, 41, 1006), a quantitative format suitable as a field assay for the analysis of pesticide residues in water and liquid food samples on-site is described. For antibody immobilization, different membranes and immobilization techniques were investigated. The measuring range for atrazine was 0.3-10 micrograms l-1 using reflectance detection. The total assay time was about 25 min with dipsticks previously coated with antibody. Atrazine-spiked water and liquid food samples were selected for assay evaluation. The samples could be measured directly without the need for any prior enrichment or clean-up steps. A satisfactory agreement was found between the results of the dipstick IA and HPLC or GC measurements of both the original and spiked samples.