Anal. Chem.

The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240 000 molecules) of analyte

Diversity visualization by endonuclease: a rapid assay to monitor diverse nucleotide libraries

Many experiments require a fast and cost-effective method to monitor nucleic acid sequence diversity. Here we describe a method called diversity visualization by endonuclease (DiVE) that allows rapid visualization of sequence diversity of polymerase chain reaction (PCR) products based on DNA hybridization kinetics coupled with the activity of a single-strand specific nuclease. The assay involves only a limited number of steps and can be performed in less than 4h, including the initial PCR. After PCR, the homoduplex double-stranded DNA (dsDNA) is denatured and reannealed under stringent conditions. During the reannealing process, incubation with S1 nuclease removes single-stranded loops of formed heteroduplexes and the resulting digest is visualized on agarose gel. The sequence diversity is inversely proportional to the band intensities of S1 nuclease surviving dsDNA molecules of expected size. As an example, we employed DiVE to monitor the diversity of panning rounds from a single-framework, semisynthetic single-chain antibody fragment (scFv) phage display library. The results are in good agreement with the observed decrease in diversity in phage display panning rounds toward the selection of monoclonal scFv. We conclude that the DiVE assay allows rapid and cost-effective monitoring of diversities of various nucleotide libraries and proves to be particularly suitable for scaffold-based randomized libraries

Pushing the detection limits: the evanescent field in surface plasmon resonance and analyte-induced folding observation of long human telomeric repeats

Conventional analysis of molecular interactions by surface plasmon resonance is achieved by the observation of optical density changes due to analyte binding to the ligand on the surface. Low molecular weight interaction partners are normally not detected. However, if a macromolecule such as DNA can extend beyond the evanescent field and analyte interaction results in a large-scale contraction, then the refractive index changes due to the increasing amount of macromolecules close to the surface. In our proof-of-principle experiment we could observe the direct folding of long, human telomeric repeats induced by the small analyte potassium using surface plasmon resonance spectroscopy. This work demonstrates the feasibility of new evanescent field-based biosensors that can specifically observe small molecule interactions

A streamlined protocol for emulsion polymerase chain reaction and subsequent purification

Compartmentalization of polymerase chain reaction (PCR) reduces artifacts, especially when complex libraries are amplified. It allows clonal amplification of templates from complex mixtures in a bias-free manner. Here we describe a rapid, straightforward, and easy protocol for PCR in a water-in-oil emulsion (ePCR) including sample recovery by DNA purification. Furthermore, no special laboratory equipment is needed and inexpensive components are used. Therefore, our flexible protocol allows ePCR to be readily implemented in daily routine experiments for a broad range of applications