A team from the Technical University of Denmark and the Copenhagen University Hospital recently published research results obtained with a microfluidic device known as a Multi-Stringency Array Washer (MSAW). According to the researchers, the MSAW facilitates probe optimization and, therefore, leads to increases in both microarray sensitivity and specificity. Using the MSAW also negates the need for extensive assay optimization and time-consuming thermal gradient/melting curve approaches, as reported at Select Bioscience’s “Microarray World Congress” held recently in San Francisco.
“Because microarrays are vast multiplex assays, potentially containing thousands of different probes, assay design relies on researchers’ ability to predict properties such as the melting temperature of every probe on the array,” explained Martin Dufva, Ph.D., associate professor at the Technical University of Denmark.
“Thermal gradients and their resulting melting curve data can be used to help produce meaningful and clear results from microarrays. The use of temporal or spatial temperature gradients to create melting curves has a number of drawbacks, however, including complexity of array fabrication, array size limitations, sample throughput, heat dissipation, and incompatibility with scanner instrumentation.”
The Danish researchers suggested that differential stringencies created by buffers of varying ionic strength represent a more useful, easily implementable, and flexible alternative to thermal gradients for assay development. The MSAW, which was developed to apply this concept, flows buffers of different ionic strength over different regions of a single microarray.
“We have published studies in which the MSAW was used with a microarray designed to genotype small variations in the human beta-globin gene, under different ionic strengths,” Dr. Dufva continued. “The results confirmed that an ionic gradient functions much like a temperature gradient, resulting in dissociation curves similar to those produced by discrete temperatures.
“Importantly, and in contrast to thermal-gradient technologies, the MSAW only requires washing for a fixed period, scanning is carried out at one temperature using dried slides, and there is no need for multiple exposures that can cause photobleaching. And, in contrast to multithermal technologies, the MSAW is adaptable to commercial high-density arrays as well as custom-made slide layouts.”
The MSAW developed by Dr. Dufva’s team is, in fact, a reincarnation of a multi-thermal array washer they previously developed. Whereas the multithermal array washer created eight individually controlled heating zones, each corresponding to a subarray, the MSAW creates eight zones that allow post-hybridization washing of each of the subarrays at different ionic strengths.
The MSAW device consists of a solid support onto which an elastomeric layer containing microfluidic chambers and channels is mounted. The chambers separate the slide into distinct regions, each of which can be washed under a different stringency condition. The glass slide containing the microarray is positioned via alignment features within the device and the assembly is sealed using a lid that applies pressure.
“It’s a convenient tool for assay development because we can test probe function empirically on a single slide, rather than having to run multiple slides at different stringency conditions or temperatures,” Dr. Dufva pointed out. “In addition to its use in probe design, the approach can also form part of the assay itself, allowing researchers to use combinations of probes on one slide that would generally not be feasible if assayed under a single stringency.”
The team has used the MSAW in a multiparametric study evaluating the hybridization of DNA to probes with different GC content and with different probe and spacer lengths. The results corroborate previous theories suggesting that surface effects have a major impact on melting temperature and hybridization. Their findings suggest that such surface effects reduce the melting temperature of hybrids significantly, and that probes closer to the surface need to be of higher affinity.
The data also pointed to a variation in signal between surface-proximal and surface-distal probes, with the signal from a proximally placed probe only equating that from a distally placed probe when washed at a 30-fold higher sodium ion concentration.
The upshot, according to the researchers, is that surface effects impact probe binding to a far greater extent than previously assumed. In order to achieve maximum hybridization to the complete oligo, a 60-mer polynucleotide probe, for example, should be designed as three separate sections—proximal, central, and distal. Probe segments nearer to the surface need to exhibit higher melting temperatures, whereas central and distal segments should display relatively low melting temperatures and binding strengths in order to avoid hairpins or partial hybridization.
“We are continuing to publish research demonstrating the utility of ionic gradients in the design of probes and assays,” Dr. Dufva stressed. “Additionally, our studies suggest that questions should be raised with respect to the quality of data obtained from current commercial arrays. In part, this is because the methods traditionally used to design and predict probe properties, such as nearest neighbor algorithm, may not as accurately estimate binding strength, specificity, and/or stability for immobilized probes, as previously believed.”