The next generation of DNA microarrays
Polishing substrate surfaces may be key to miniaturization
As the research community races to unravel the mysteries of the human genome, new technologies are emerging to make the job easier. DNA microarrays have enabled significant advances in the field by allowing high-throughput studies of gene expression, but these often depend on cumbersome excitation sources, optics and detectors. Investigators are interested in developing smaller, more manageable devices -- and, in particular, in those that incorporate microarray technology with microelectronic circuitry to control interrogation of probes within the array.
Complementary metal oxide semiconductor (CMOS) technology holds promise as a possible solution. Deposited layers and patterned structures -- an array of microelectrodes -- can be “postprocessed” directly onto CMOS circuitry, enabling fabrication of the types of arrays sought by investigators. For example, CMOS technology enables larger numbers of electrodes to be interrogated and provides opportunities for more advanced sensor/actuator integration.
Deposition of thin films is essential to fabrication of such integrated circuits. For instance, use of silicon nitride as a final passivation layer creates an impervious barrier to diffusion of moisture and sodium ions. However, this is where things get challenging. It is necessary to control the temperature during deposition, so as not to detrimentally affect the composition or function of earlier deposited layers. Plasma-enhanced chemical vapor deposition recommends itself for this stage of microfabrication, as the technique operates at low temperatures and produces films with good passivation qualities. However, previous studies have reported difficulties when attaching biomolecules to these surfaces.
Researchers assessed the performance of silicon nitride layers in microarray experiments following chemical mechanical polishing of the surfaces. Shown here are the test samples in hybridization chambers.
Researchers with the University of Edinburgh in the UK may have come up with a solution. As reported in Langmuir on Dec. 19, they assessed the performance of silicon nitride layers in microarray experiments following chemical mechanical polishing of the surfaces. The study showed that this planarization significantly improved the silicon nitride surfaces with respect to attaching biomolecules.
The technique begins with deposition of silicon nitride films on integrated circuit-grade silicon substrates. To polish the surfaces, the researchers fed an abrasive, corrosive slurry onto a rotating polishing pad and forced wafers onto the pad. The slurry reacted with and weakened material on the wafer, and the polishing pad removed it. They then prepared the substrates for the microarray experiments.
They tested the efficacy of this method by assessing RNA extracted from mouse macrophage cells infected with murine cytomegalovirus, using various silicon nitride surfaces as DNA microarray substrates. For each of these, they evaluated the binding performance for positive control genes, negative control genes and the genes of interest. They labeled the RNA with cyanine 5, hybridized it with arrays spotted onto each of the slides they had prepared and scanned the slides with a ScanArray 5000 from Packard BioChip (now PerkinElmer) of Billerica, Mass.
They found that the fluorescence intensity from the negative control spots was low in all of the experiments, underscoring the relatively low levels of nonspecific binding of the labeled RNA for all of the surfaces. The signal from the positive control genes was significantly higher than from the negative control genes on the same surfaces. And with polishing, the positive control genes exhibited a 2.7- to 4.0-fold increase in fluorescence.
The genes of interest exhibited a similar pattern of expression across the surfaces, with polishing causing a 2.6- to 2.8-fold increase in fluorescence intensity. The higher intensity allows greater discrimination of low levels of gene expression. This suggests that, when combined with chemical mechanical polishing, plasma-enhanced chemical vapor deposition may be a viable means of developing DNA microarrays.
Chemical mechanical polishing of substrate surfaces could advance the use of integrated circuits in next-generation DNA microarrays. Polishing proved to enhance the images of positive control spots (left) and spots corresponding to the gene of interest (right) on surfaces prepared with low-pressure and plasma-enhanced chemical vapor deposition (LPCVD and PECVD, respectively). Images of spots on a glass slide are shown for comparison. Reprinted with permission of Langmuir.
The team -- including researchers from the Institute for Integrated Micro and Nano Systems and the Scottish Centre for Genomic Technology and Informatics, both at the University of Edinburgh -- has been working for several years to develop silicon fabrication techniques and MEMS structures to advance integrated biosensor arrays as part of the UK Department of Trade and Industry’s “Harnessing Genomics” program. To this end, optimization of integrated circuit-compatible surfaces for biomolecule attachment is of “fundamental importance,” according to Jonathan G. Terry at the Institute for Integrated Micro and Nano Systems, an author of the report.
Having achieved this, they plan to continue developing the technology for next-generation microarrays, which will contribute to further advancements in gene expression studies. “This work has been an initial step towards our ultimate goal of developing a fully integrated biosensing system incorporating on-chip excitation sources, image sensors, and control and reporting mechanisms,” Terry said. Ultimately, this could lead to significant improvements in the fields of biosensing and lab on a chip.
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