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Cellulose films have been proposed as a convenient substrate for producing flat and homogeneous surface coatings. Additionally, amino-labelled cellulose species, like aminopropyltrimethylsilylethercellulose (ATMSC), are excellent support matrices for covalent binding of biomolecules, with low probe density and prevention of non-specific adsorption of unbound analyte molecules. Due to ATMSC films fulfil important requirements as substrate for analyse techniques of surface-tethered proteins and nucleic acids, we consequently report a new preparation for DNA-functionalised surfaces. Single-stranded DNA molecules are covalently coupled to cellulose-coated glass cover slips to interact with complementary free Cy5-labelled oligonucleotides in solution. Hybridisation efficiencies at the new substrate and at standard surface coatings are determined by detection of the surface-generated fluorescence. In order to discriminate against the fluorescence from unbound oligonucleotides the detection volume was restricted to the surface by collecting supercritical angle fluorescence (SAF). Thus, it is demonstrated that cellulose films are utilised to investigate DNA-hybridisation reactions highly sensitive.

Imaging of single DNA molecules has enabled detailed studies of dilute polymer dynamics and rigorous testing of assumptions and predictions of molecular theories. It is of interest to extend these methods to the study of entangled polymers and to correlate molecular dynamics with rheology measurements. Progress in this direction has been hampered, however, by a lack of available DNA samples in sufficient quantities and covering a wide range of lengths. Here we describe the preparation of a suitable set of molecules ranging in length from ∼3 to 300 kilobase pairs. These constructs are replicated as plasmids or as fosmids or bacterial artificial chromosomes fitted with an inducible high-copy number origin of replication. DNA sequences were chosen to allow molecules to be linearized by single-cutting restriction enzymes. We show that these molecules can be imaged and characterized by fluorescence microscopy and can be prepared in sufficient quantities for bulk rheology measurements.

The conformation and dynamics of circular polymers is a subject of considerable theoretical and experimental interest. DNA is an important example because it occurs naturally in different topological states, including linear, relaxed circular, and supercoiled circular forms. A fundamental question is how the diffusion coefficients of isolated polymers scale with molecular length and how they vary for different topologies. Here, diffusion coefficients D for relaxed circular, supercoiled, and linear DNA molecules of length L ranging from ≈6 to 290 kbp were measured by tracking the Brownian motion of single molecules. A topology-independent scaling law D ∼ L −ν was observed with ν L = 0.571 ± 0.014, ν C = 0.589 ± 0.018, and ν S = 0.571 ± 0.057 for linear, relaxed circular, and supercoiled DNA, respectively, in good agreement with the scaling exponent of ν ≅ 0.588 predicted by renormalization group theory for polymers with significant excluded volume interactions. Our findings thus provide evidence in support of several theories that predict an effective diameter of DNA much greater than the Debye screening length. In addition, the measured ratio D Circular / D Linear = 1.32 ± 0.014 was closer to the value of 1.45 predicted by using renormalization group theory than the value of 1.18 predicted by classical Kirkwood hydrodynamic theory and agreed well with a value of 1.31 predicted when incorporating a recently proposed expression for the radius of gyration of circular polymers into the Zimm model.

We describe the rapid detection of single nucleotide polymorphisms (SNPs) by real-time observation of primer elongation. The enzymatic elongation of surface-bound primers is monitored by detecting the increase of surface-bound fluorescence caused by the incorporation of Cy5-labelled deoxycytidine 5'-triphosphate residues (Cy5-dCTPs) into the corresponding strand. In order to discriminate against the fluorescence from unbound Cy5-dCTPs, the detection volume was restricted to the surface by collecting supercritical-angle fluorescence. The efficiency of enzymatic double-stranded DNA synthesis is governed by the complementarity of the primer and template. An SNP in the sequence of the primer obstructs its elongation increasingly with decreased distance of the mismatch to the 3' end of the primer. By real-time fluorescence detection during primer elongation, SNPs can be detected within a few minutes, which is significantly faster than in experiments where the fluorescence is measured after completion of the reaction. We demonstrate the efficiency of the method by detecting an SNP in the ErbB2 gene that is involved in causing a higher risk of breast cancer.

Fluorescence Resonance Energy Transfer (FRET) is a powerful tool to determine distances between chromophores bound to macromolecules, since the efficiency of the energy transfer from an initially excited donor to an acceptor strongly depends on the distance between the two dye molecules. The structure of the noncovalent complex of double-strand DNA (dsDNA) with thiazol orange dimers (TOTO) allows FRET analysis of two intercalated chromophores. By intercalation of two different TOTO dyes we observe an energy transfer from TOTO-1 as donor and TOTO-3 as acceptor. In this manner we are able to determine the mean distance between two proximate TOTO molecules bound to dsDNA. Thus the maximum number of binding positions for this type of intercalation dyes in the dsDNA can be obtained. Furthermore the dependency of the acceptor emission on the donor concentration is analysed. The emission of TOTO-3 reaches a maximum when the acceptor-to-donor ratio is 1:10.

For a better understanding of the adsorption behaviour of biomolecules the surface tension parameters of surface-coatings for biological applications are determined by contact angle measurements. The correlation between surface tension parameters and chemical composition of thin films made of cellulose derivatives with varying functional groups and amino-functional thin films with varying backbone on glass cover slips is investigated. Calculations are based on two different theoretical methods, the Geometric mean (GM) approach according to Owens, Wendt, Rabel and Kalble and the Lifshitz-van der Waals acid/base (LW/AB) approach according to van Oss, Good and Chaudhury. The values obtained for the surface tension components from the different approaches are consistent. All films investigated reduce the surface tension of the cover slip and are monopolar with strong electron-donor capabilities. While the polarity of the films varies with the choice of the functional group at the cellulose backbone, we only found a minor effect when the backbone varies. For the cellulose derivatives the degree of substitution correlates well with the electron-donor/acceptor properties.

Recent trends in the development of microfluidic and biodiagnostic chips favor polymer materials over glass, primarily for optical and economical reasons. Therefore, existing chemical methods to prepare biomolecule microarrays on glass slides have to be adapted or replaced in order to suit polymer substrates. Here we present a strategy to immobilize DNA and antibodies on cyclic polyolefin slides, like Zeonor. This polymer represents a class of new polymeric materials with excellent optical and mechanical properties. By plasma and liquid chemical treatment followed by coating with polyelectrolytes, we have succeeded in immobilizing DNA onto the polymer substrate, yielding stable and versatile biosensor surfaces. We demonstrate the stability and usage of the coated Zeonor substrates not only in DNA chip technology but also in protein chip technology with DNA-directed immobilization of proteins.

We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP—Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTPs.

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-L-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7-14%. The proposed method is straightforward and can be applied to standardized microtiter plates.

Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction.

Utilising the strong affinity between nucleic acids and an intercalating pyrene derivate, a novel efficient method for unspecific immobilisation of double-stranded DNA on to solid support for applications in bioanalytic, biophysics and microbiology is presented.