Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays.

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 µl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-ß-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique.

Lack of insertional-deletional polymorphism in a collection of Mycobacterium ulcerans isolates from Ghanaian Buruli ulcer patients.

Mycobacterium ulcerans causes the devastating infectious skin disease Buruli ulcer and has a monomorphic population structure. The resolution of conventional genetic fingerprinting methods is therefore not sufficient for microepidemiological studies aiming to characterize transmission pathways. In a previous comparative genomic hybridization analysis with a microarray covering part of the M. ulcerans genome, we have found extensive insertional-deletional sequence polymorphisms among M. ulcerans isolates of diverse geographic origins that allowed us to distinguish between strains coming from different continents. Since large numbers of insertion sequences are spread over the genome of African M. ulcerans strains, we reasoned that these may drive large sequence polymorphisms in otherwise clonal local mycobacterial populations. In this study, we used a printed DNA microarray covering the whole genome of the Ghanaian M. ulcerans reference strain Agy99 for comparative genomic hybridization. The assay identified multiple regions of difference when DNA of a Japanese M. ulcerans strain was analyzed. In contrast, not a single insertional-deletional genomic variation was found within a panel of disease isolates coming from an area of Ghana where Buruli ulcer is endemic. These results indicate that, despite the expectations deduced from other mycobacterial pathogens, only analyses of single nucleotide polymorphisms will have the potential to differentiate local populations of M. ulcerans.

Fast and reliable protein microarray production by a new drop-in-drop technique.

In contrast to DNA microarrays, production of protein microarrays is an immense technological challenge due to high complexity and diversity of proteins. In this paper we investigate three essential aspects of protein microarray fabrication based on the highly parallel and non-contact TopSpot technology: evaporation of probes during long lasting production times, optimization of protein immobilization and improvement of protein microarray reproducibility. Evaporation out of the printhead reservoirs was reduced to a minimum by sealing the reservoirs with gas permeable foils or PDMS frames. This led to dramatically lowered setup times through the possibility of long-term, ready-to-print storage of filled printheads. To optimize immobilization efficiency 128 printing buffers were tested by printing two different proteins onto seven different microarray slide types. This way we were able to reduce the CV of spot diameter on the microarray slide below 1.14%. To remarkably increase protein immobilization efficiency on microarray slides the commonly used EDC-NHS system (a laboratory method for immobilization of proteins) was miniaturized by using a new drop-in-drop printing technique. Additionally the very fast UV cross-linking was used to immobilize antibodies. The optimized system was used to produce antibody microarrays and with it microarray ELISA experiments were performed successfully.

Highly parallel dispensing of chemical and biological reagents.

We present a technology for the highly parallel dispensing of a multitude of reagents. It allows one to dispense up to 96 different reagents simultaneously in a fixed array, in a volume range of 100 pL up to several nL. The pitch of the dispensed droplets can be as small as 500 microm. All channels are fired simultaneously, giving an unprecedented throughput. The system was originally developed for the high-throughput fabrication of microarrays, but can easily be adopted for other applications such as highly parallel filling of nanotiterplates. Based on our standard configuration we achieved droplets with 125- micro m in-flight diameter (1.2 nL) with a CV of <1%.

Non-contact production of oligonucleotide microarrays using the highly integrated TopSpot nanoliter dispenser.

For the first time we report on the production of oligonucleotide microarrays using a highly parallel and highly integrated, pressure driven TopSpot nanoliter dispenser. The system enables non-contact printing of different media like oligonucleotides, DNA or protein solutions. We optimized the printing buffer needed for oligonucleotides microarrays production with respect to two major aspects: microfluidical optimum for droplet dispensing and biochemical coupling efficiency on different commercially available microarray slides. Coefficient of variations (CVs) of generated spot diameters were measured to be smaller than 1% within one single dispensing nozzle and smaller than 1.5% within all 24 parallel nozzles of the printhead for all printing buffers used. No carry-over and no cross-talk was found, in extensive experiments with oligonucleotides. Optimized printing buffer compositions and concentrations for oligonucleotide microarrays were found, as well as optimized coupling protocols. Furthermore, buffers and protocols were adapted to a host of different microarray slides used. With this system, prime critical points of microarray production are solved, leading to high quality high throughput microarray fabrication.

A highly parallel nanoliter dispenser for microarray fabrication.

We report about the correlation between satellite free droplet release and liquid viscosity in a highly parallel, pressure driven nanoliter dispenser. In extensive studies, we found that for liquids of different viscosities the duration of the pressure pulse is the predominant effect compared to pressure amplitude. This result is of essential importance when actuation parameters have to be adopted for different media like oligonucleotide, DNA or protein solutions as it is the case for the non-contact high throughput fabrication of microarrays (Ducree et al., 2000). Experiments with oligonucleotides as well as with different proteins showed ascertained carry-over and cross-contamination free printing of DNA and protein microarrays. With it a prime critical point of microarray production is solved, leading to high quality whilst high throughput microarray fabrication. For oligonucleotides printing, we found CVs to be better than 1% within one single dispensing channel and 1.5% within all 24 channels of a 24 channel printhead for each used printing buffer. By optimizing the protein printing buffer the CVs for protein printing were reduced to about 1% within all 24 channels. As a serious practical application test oligonucleotides microarrays were produced using our nanoliter dispenser system. With it a full DNA hybridization experiment was performed. Clear positive signals one hand and no signals in the negative controls on the other hand showed that our system is suited for microarray production.