DNA Millichips as a Low-Cost Platform for Gene Expression Analysis

Heinrich, K.; Wolfer, Jamison; Hong, DongGee; LeBlanc, Melissa; Sussman, Michael R.

doi:10.1104/pp.112.195230

Cited by 6 publications

(12 citation statements)

References 27 publications

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“…In a practical context, any decrease in S/N ratios stemming from the relatively small increases in background intensity observed here could be additionally offset by the potential utility of having flexible, plastic-backed arrays of oligonucleotides that can be manipulated, either during or after fabrication, in ways that are difficult using arrays fabricated on glass. 69 For example, these arrays could be bent, cut with scissors, and readily separated into smaller pieces without observable cracking or peeling of the polymer multilayers (see Figure S1 of the Supporting Information). …”

Section: Resultsmentioning

confidence: 99%

Fabrication of Oligonucleotide and Protein Arrays on Rigid and Flexible Substrates Coated with Reactive Polymer Multilayers

Broderick

Carter

Lockett

et al. 2012

ACS Appl. Mater. Interfaces

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We report a top-down approach to the fabrication of oligonucleotide and protein arrays on surfaces coated with ultrathin, amine-reactive polymer multilayers fabricated by the covalent ‘layer-by-layer’ (LbL) assembly of polyethyleneimine (PEI) and the amine-reactive, azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA). Manual spotting of amine-terminated oligonucleotide probe sequences on planar glass slides coated with PEI/PVDMA multilayers (~35 nm thick) yielded arrays of immobilized probes that hybridized fluorescently labeled complementary sequences with high signal intensities, high signal-to-noise ratios, and high sequence specificity. Treatment of residual azlactone functionality with the nonfouling small-molecule amine d-glucamine resulted in regions between the features of these arrays that resisted adsorption of protein and permitted hybridization in complex media containing up to 10 mg/mL protein. The residual azlactone groups in these films were also exploited to immobilize proteins on film-coated surfaces and fabricate functional arrays of proteins and enzymes. The ability to deposit PEI/PVDMA multilayers on substrates of arbitrary size, shape, and composition permitted the fabrication of arrays of oligonucleotides on the surfaces of multilayer-coated sheets of poly(ethylene terephthalate) and heat-shrinkable polymer film. Arrays fabricated on these flexible plastic substrates can be bent, cut, resized, and manipulated physically in ways that are difficult using more conventional rigid substrates. This approach could thus contribute to the development of new assay formats and new applications of biomolecule arrays. The methods described here are straightforward to implement and do not require access to specialized equipment, and should also be compatible with automated liquidhandling methods used to fabricate higher-density arrays of oligonucleotides and proteins on more traditional surfaces.

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Section: Resultsmentioning

confidence: 99%

Fabrication of Oligonucleotide and Protein Arrays on Rigid and Flexible Substrates Coated with Reactive Polymer Multilayers

Broderick

Carter

Lockett

et al. 2012

ACS Appl. Mater. Interfaces

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“…We then explore the use of PET-based arrays as a second-generation substrate for millichip fabrication. 20 …”

mentioning

confidence: 99%

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates

Holden

Carter

et al. 2015

Anal. Chem.

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The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with crosslinked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying MAS procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 × 14 μm and feature densities in excess of 125 000/cm2, similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily sub-divided into smaller arrays (or ‘millichips’) using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new potential applications for these important bioanalytical tools.

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“…The ability to reuse microarrays for multiple experiments not only decreases the costs associated with making or purchasing arrays [recently estimated at $450 per array (99)], but also allows for direct comparison of biological replicates or other samples on the same array, eliminating potential artifacts due to array variability. Table 1 summarizes the hybridization density and stability of arrays prepared on carbon substrates; this information was obtained from papers in which both spotted and in situ synthesized oligonucleotide arrays were prepared and analyzed.…”

Section: Carbon Substrates: Array Fabrication and Analysismentioning

confidence: 99%

Carbon Substrates: A Stable Foundation for Biomolecular Arrays

Lockett

Smith²

2015

Annual Rev. Anal. Chem.

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Since their advent in the early 1990s, microarray technologies have developed into a powerful and ubiquitous platform for biomolecular analysis. Microarrays consist of three major elements: the substrate upon which they are constructed, the chemistry employed to attach biomolecules, and the biomolecules themselves. Although glass substrates and silane-based attachment chemistries are used for the vast majority of current microarray platforms, these materials suffer from severe limitations in stability, due to hydrolysis of both the substrate material itself and of the silyl ether linkages employed for attachment. These limitations in stability compromise assay performance and render impossible many potential microarray applications. We describe here a suite of alternative carbon-based substrates and associated attachment chemistries for microarray fabrication. The substrates themselves, as well as the carbon-carbon bond-based attachment chemistries, offer greatly increased chemical stability, enabling a myriad of novel applications.

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DNA Millichips as a Low-Cost Platform for Gene Expression Analysis

Cited by 6 publications

References 27 publications

Fabrication of Oligonucleotide and Protein Arrays on Rigid and Flexible Substrates Coated with Reactive Polymer Multilayers

Fabrication of Oligonucleotide and Protein Arrays on Rigid and Flexible Substrates Coated with Reactive Polymer Multilayers

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates

Carbon Substrates: A Stable Foundation for Biomolecular Arrays

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