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Biomolecular complexes, nanocircles and aggregates of DNA and streptavidin (STV) are studied by dynamic scanning force microscopy. More structural details are observed with an improved dynamic mode assisted with a special feedback circuit (Q-control), as shown in the picture. Under otherwise identical conditions, these improvements indicate that the well cited models relating enlarged lateral size to the finite geometry of the SFM tip have to be modified for soft samples susceptible to tip-sample interactions.

The self-assembly of bis-biotinylated double-stranded DNA and the tetravalent biotin-binding protein streptavidin (STV) have been studied by non-denaturing gel electrophoresis and atomic force microscopy (AFM). The rapid self-assembly reproducibly generated populations of individual oligomeric complexes. Most strikingly, the oligomers predominantly contained bivalent STV molecules bridging two adjacent DNA fragments to form linear nanostructures. Trivalent STV branch points occurred with a lower frequency and the presence of tetravalent STV was scarce. However, valency distribution, size and the exchange dynamics of the supramolecular aggregates were highly sensitive to stoichiometric variations in the relative molar coupling ratio of bis-biotinylated DNA and STV. The largest aggregates were obtained from equimolar amounts while excess STV led to the formation of smaller oligomers appearing as fingerprint-like band patterns in electrophoresis. Excess DNA, however, induces a complete breakdown of the oligomers, likely a consequence of the instability of STV conjugates containing more than two biotinylated DNA fragments. It was demonstrated that the oligomers can further be functionalized, for instance by the coupling of biotinylated immunoglobulins. Both pure and also antibody-modified DNA-STV oligomers were used as reagents in immuno-PCR (IPCR), a highly sensitive detection method for proteins and other antigens. Employment of the supramolecular reagents led to an approximately 100-fold enhanced sensitivity compared to the conventional IPCR procedure.

Covalent DNA-streptavidin conjugates have been utilized for the reversible and site-selective immobilization of various biotinylated enzymes and antibodies by DNA-directed immobilization (DDI). Biotinylated alkaline phosphatase, beta-galactosidase, and horseradish peroxidase as well as biotinylated anti-mouse and anti-rabbit immunoglobulins have been coupled to the DNA-streptavidin adapters by simple, two-component incubation and the resulting preconjugates were allowed to hybridize to complementary, surface-bound capture oligonucleotides. Quantitative measurements on microplates indicate that DDI proceeds with a higher immobilization efficiency than conventional immobilization techniques, such as the binding of the biotinylated proteins to streptavidin-coated surfaces or direct physisorption. These findings can be attributed to the reversible formation of the rigid, double-stranded DNA spacer between the surface and the proteins. Moreover, BIAcore measurements demonstrate that DDI allows a reversible functionalization of sensor surfaces with reproducible amounts of proteins. Ultimately, the simultaneous immobilization of different compounds using microstructured oligonucleotide arrays as immobilization matrices demonstrate that DDI proceeds with site selectivity due to the unique specificity of Watson-Crick base pairing.

Immuno-polymerase chain reaction (immuno-PCR) allows the detection of protein amounts as low as a few hundred molecules. This enhanced sensitivity is useful for a variety of applications in analytical and biomedical sciences. Application of this technique as a routine method requires the rapid quantification of the PCR products, preferably as an automated readout by microplate-based assays. Here, three methods are compared for detecting such amplified products, i. e., direct staining with a fluorescent intercalating dye, an enzymatic assay utilizing doubly hapten-labeled products, and gel electrophoresis. The enzymatic assay, carried out with either chromogenic or fluorogenic substrate for enzymatic signal amplification, was found to be the most sensitive method. The optimized assay was tested in direct immuno-PCR assays for detecting immunoglobulins (IgG) from mouse and rabbit as well as in a sandwich immuno-PCR assay for detecting recombinant hepatitis B surface antigen (rec. HBsAg). Sensitivity limits were found to be as low as 15 fg (10(-19) mol) IgG, representing a 1000-fold enhancement compared to enzyme-linked immunosorbent assay detection, and about 70 fg (2 x 10(-18) mol) rec. HBsAg, improving the detection limit of currently available methods by a factor of about 700. The well-reproducible enzymatic amplification signal further enhances the sensitivity of immuno-PCR and should render the method suitable for routine laboratories.

Aus eins mach viele - Dendrimere binden mehr Sondenmolekule und bilden so homogenere Biochip-Oberflachen als lineare Linker. Fur den direkten Nachweis von Antikorpern aus Blutserum lassen sich auf dendritisch aktivierten Oberflachen Antigene verankern.

Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA-tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel-retardation assays.

As traditional "top-down" approaches to device fabrication are reaching their limits, "bottom-up" approaches based on the assembly of individual molecules are receiving increasing attention. Biomolecules such as DNA are particularly promising because of their specific recognition capabilities. In his Perspective, [Niemeyer][1] highlights the molecular lithography approach developed by [ Keren et al .][2] Combined with protein bioengineering, molecular lithography provides a new, powerful tool for using DNA in future nanometer-scale devices. [1]: http://www.sciencemag.org/cgi/content/full/297/5578/62 [2]: http://www.sciencemag.org/cgi/content/short/297/5578/72

The simultaneous identification and quantification of several different proteins is crucial for the understanding of cellular processes at the protein level, and for detecting changes in protein expression signatures due to disease, drugs or any biological perturbation. High-throughput, high sensitivity, nanotechnology-based analytical tools, with low requirements on the sample amount and hands-on processing time, are prerequisites for further developments in this field.We use atomic force microscopy nanografting (AFM-NG) to fabricate innovative nano-arrays for immuno-assay, with complete and efficient control on protein/antibody immobilization. By combining NG and DNA-directed immobilization (DDI), different protein-ssDNA conjugates are driven simultaneously on top of a nano-array of complementary ssDNA strands, previously prepared on a gold film surface functionalized with a protein repellent reference carpet. The integrity and biochemical functionality of the immobilized proteins were demonstrated as a proof of principle by AFM topography measurements and immunological assays. Different proteins were detected within a complex matrix of standardized human serum, demonstrating the specificity of the biomolecular recognition, and the absence of any nonspecific binding and cross-reactivity. Protein folding was tested by studying protein-ligand interactions.In a parallel approach we promote protein immobilization in controlled orientation, by means of nitrilotriacetic acid (NTA)-terminated thiols. NTA binds specifically to a polyhistidine tag of the protein (through Ni chelation), which can be genetically engineered in recombinant proteins in a known position. This immobilization is reversible and ensures surface re-usability. Technically, the know-how for both DNA and NTA nanografting based protein immobilization can be combined to exploit the extreme selectivity and parallelizability of the former with the versatility of the latter. Specifically, by using a ssDNA-NTA conjugate, the added to our protein nano-arrays the opportunity to immobilize in an oriented manner through the histidine tag an enormous variety of proteins.

We report on the synthesis and characterization of cadmiumsulfide quantum dots (CdS-QDs) with three different types of capping ligands, mercaptoacetic acid (CdS-MAA), cysteamine, and 2-mercaptoethanesulfonic acid (CdS-CM), or tiopronin (CdS-TPN). All three types of QDs generate reactive oxygen species (ROS), in particular, superoxide- and hydroxyl radicals, upon irradiation with UV light. The ROS were analyzed using electron paramagnetic resonance (EPR) spectroscopy. Moreover, the hydroxyl radical generation from the three different QDs was monitored using a terephthalate-based fluorimetric assay. The three QDs were then used as photocatalyst to trigger the peroxidase activity of horseradish peroxidase.

Cadmium sulfide quantum dots modified with the enzyme cytochrome P450 CYP152A1 were investigated to evaluate the potential of these nanohybrids as photoactivated catalysts. The quantum dots produce reactive oxygen radicals upon irradiation and these radicals are used by the CYP152A1 enzyme to catalyze the hydroxylation of myristic acid. We here explore the effects of the relative stoichiometry of enzyme and quantum dots on the activity of the nanohybrids as well as the stability of the nanohybrid and of the enzyme towards several damaging factors such as UV light, hydrogen peroxide, and heat. We found that the enzymatic activity is greatly decreased after long exposure to UV light. We also demonstrated the possibility of using pre-irradiated quantum dots to perform chemical transformations. Based on these findings, it was possible to optimize the nanohybrid composition as well as the reaction conditions in order to maintain the catalytic activity of the enzyme in the nanohybrids. Finally, we showed that it was possible to control the photocatalytic activity of the nanohybrids by simply switching on and off the UV light.

Die Immuno-PCR erganzt etablierte Verfahren der PCR-Nucleinsaureanalytik. Mit der neuen Methode konnen Proteine ahnlich empfindlich nachgewiesen werden, wie dies fur Nucleinsauren moglich ist.