An Anopheles gambiae salivary gland promoter analysis in Drosophila melanogaster and Anopheles stephensi.

Regulatory regions driving gene expression in specific target organs of the African malaria vector Anopheles gambiae are of critical relevance for studies on Plasmodium-Anopheles interactions as well as to devise strategies for blocking malaria parasite development in the mosquito. In order to identify an appropriate salivary gland promoter we analysed the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4. An 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic Anopheles stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-rather than in the distal-lateral lobes of female glands. Surprisingly, a promoter fragment from the D7r4 gene conferred strong tissue-specific expression in Drosophila melanogaster but only low transcription levels in transgenic An. stephensi. These results imply a certain conservation of gland-specific control elements between the fruit fly and the mosquito suggesting that an increased degree of complexity, probably connected to the evolution of haematophagy, underlies the regulation of tissue-specific expression in mosquito female salivary glands.

Design and peptide-based validation of phage display antibodies for proteomic biochips.

To validate potential application of phage display-antibody arrays for high-throughput screening on a novel proteomics biochip, we examined the epitopes versus the full protein of glucose-6-phosphate-dehydrogenase (G6PD) from yeast. In a predictive approach, we used the Hopp-Woods method and compared the results with antibodies directed against the entire enzyme. In total, 16 peptides of a length of 11 amino acids each fulfilling the desired criteria were identified and synthesized. Subsequently, antibodies against G6PD were raised using a phage display library. Selective interaction of the antibodies with certain peptides facilitated the identification of epitopes predicted by the hydropathic profile. The setup was adapted to a novel biochip system based on surface-enhanced absorption for direct CCD-camera based screening.

Slide-format proteomic biochips based on surface-enhanced nanocluster-resonance.

The most fundamental properties of metal nanoclusters, namely the high local-field enhancement and nanoscale resonance behavior of the cluster electron plasma when exited by electromagnetic radiation, have been used to set up a variety of sensors transducing biorecognitive interactions into optical signals. This paper focuses on applications of resonant-cluster technology, which enabled us to monitor biorecognitive binding of a variety of proteins on a chip, thus constructing high-throughput interaction-screening devices. Decisive for this type of sensor is the nanometric distance from the local field surrounding a cluster to other parts of the sensor interacting with this field. In particular, the cluster-mirror or cluster-fluorophore distance gives rise to a variety of enhancement phenomena. Depending on the desired application this "resonance"- distance is approximately 5-400 nm. All types of sensor can be set up on photolithographically constructed microchips, but microscopic glass slides can also be employed; this also enables the use of standard devices for dotting and read out. Using slide based chips a standard format of 3,200 microdots (125 microm in diameter) was the basis of either microassays applying direct optical transduction via surfaceenhanced absorption or striking for more sensitivity via surface-enhanced fluorescence.

High-throughput assays on the chip based on metal nano-cluster resonance transducers.

High throughput transducers using metal cluster resonance technology are based on surface-enhancement of metal cluster light absorption. These devices can be used for detection of biorecognitive binding, as well as structural changes of nucleic acids, proteins or any other polymer. The optical property for the analytical application of metal cluster films is the so-called anomalous absorption. An absorbing film of clusters positioned 10--400 nm to a mirror surface reacts in a similar way to a reflection filter. At a certain distance of the absorbing layer to the mirror the reflected electromagnetic field has the same phase at the position of the absorbing cluster as the incident fields. This feedback mechanism strongly enhances the effective cluster absorption coefficient. The system is characterised by a narrow reflection minimum whose spectral position shifts sensitively with the interlayer thickness, because a given cluster-mirror distance and wavelength defines the optimum phase. Based on this principle a set of novel tools including biochips and micro arrays is presented, which enabled us to transduce binding, as well as changes of protein-, DNA- and polymer-conformation, quantitatively into an optical signal which can be observed directly as a colour change of a sensor-chip surface.

Nanofilms and nanoclusters: energy sources driving fluorophores of biochip bound labels.

Nanoclusters and nanofilms have the potential to amplify fluorescence and thus to enhance the signal of labeled biomolecules on biochip surfaces. Fluorescent molecules are bound at a certain distance to a resonant layer of a metal or a semiconductor or both, resulting in enhanced absorption and emission of the fluorophore within the electromagnetic near-field. This property makes the system highly useful for interaction studies, including those of DNA and proteins. Due to the amount of data, derived from various sequencing projects and from Proteomic interaction studies within the next years, microarrays (or biochips) will represent a central technology in every lab facilitating high-throughput screening and being easily interfaced with computer databases. However, most chips suffer from the disadvantage of insufficient signal-to-noise (background) ratio and are thus limited to molecules of medium-to-high abundance. Novel approaches are needed for identification of, e.g., low copy RNAs or regulatory proteins. Here we present a study, using novel surface enhanced chips in the standard glass-slide-formats. Applying surface-enhanced fluorescence (SEF), the chips turned out to be useful for interaction studies, such as DNA hybridization, thereby strongly enhancing the on-chip-signals. Compared to standard glass-slide-DNA chips, both the fluorescent signals as well as signal-to-noise ratio were considerably higher.