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Biomedical Optics & Medical Imaging

Manipulating biomolecules in space and time

Polymer layers for microarrays patterned by laser ablation adsorb biomolecules only in specified regions and improve solid-phase transfection of cells when a voltage is applied.
5 February 2007, SPIE Newsroom. DOI: 10.1117/2.1200702.0650

Complex biological systems demand high-throughput, cost-effective, high-sensitivity analysis techniques. For example, the determination of gene function, or genomics, promises to help unravel and treat many genetic disorders, such as cancer. For the past decade, researchers have monitored gene function using microarrays, in which various DNA fragments or proteins are arranged on a plate to simultaneously detect thousands of different biomolecules extracted from cells.

To allow genomic analysis within living cells, microarrays of cells have also been investigated. Of particular interest are transfected cell microarrays (TCMs),1,2 first developed by Ziauddin and co workers.3 Here, an array of cell clusters is formed, such that each is transformed with a different DNA sequence. In brief, spots of various DNA fragments of interest are first arrayed on a glass slide at addressable locations. Cells are then seeded over the array, and those that attach on top of particular DNA spots take up and express that DNA, a process termed ‘solid-phase transfection.’

Creating these devices requires surfaces that control the behavior of cells and of the biomolecules they use to interact with surfaces, so that adjacent DNA spots and cell clusters are effectively separated to prevent cross-talk. Furthermore, efficiently transporting the DNA sequences of interest to, and inserting them inside, the cells remains a challenge. For this purpose we have developed a surface that is able to manipulate biomolecules in both space and time, for application as a TCM.4

Our process is illustrated in Figure 1. The surface is constructed by first depositing an allylamine plasma polymer (ALAPP) that contains amine functionality. An aldehyde-functionalized poly(ethylene glycol) (PEG) chain is then grafted onto the surface by reductive amination. Finally, laser ablation of this surface through a mask removes the PEG layer—which resists adsorption of proteins and DNA—in specific regions, re-exposing the underlying ALAPP layer, which readily adsorbs biomolecules. Thus, the patterned surface spatially controls biomolecule adsorption.


Figure 1. Schematic of the formation of a transfected-cell microarray (TCM). A boron-doped, p++ silicon substrate was modified sequentially by plasma polymerization and poly-(ethylene glycol) (PEG) grafting. Laser ablation was used to expose allylamine-plasma-polymer (ALAPP) wells within the PEG surface. A robot was then used to spot DNA into these wells. Cells seeded onto this surface grew exclusively within the wells, over the DNA spots. Application of a negative voltage triggered the release of DNA from the surface, making it available for uptake by adhered cells. Transfected cells are depicted as green. The schematic is not drawn to scale. Reprinted from Reference 4.

In addition, we usually construct this surface on highly doped silicon, so that it can act as an electrode for subsequent electrochemistry. This can be used for the temporal control of adsorption and desorption of polyanionic DNA, as previously demonstrated by Wang and co-workers.5

Spatial control of both protein and DNA,6 as well as site-directed cell attachment,7 have been successfully demonstrated on this surface. These have been combined to form a TCM, where each cell colony is chemically separated by the PEG layer, with the cells attaching to only the ALAPP regions.4 A fluorescence image of a TCM formed using this strategy is shown in Figure 2. When the DNA was pre-adsorbed before cell seeding, it was taken up and expressed by the cells. Driving the DNA away from the surface by applying a negative voltage6 increased the efficiency of this process from 10% to 30%, as determined by the number of cells that expressed DNA they took up. For this demonstration, we used a DNA fragment that encodes for the expression of a green fluorescent protein as a reporter for its uptake.


Figure 2. Fluorescence-microscopy image of a TCM with HEK-293 cells on an ALAPP/PEG patterned surface. Cells were stained with Hoechst 33342. The inset shows an overlay of the dye fluorescence and that due to the expression of green fluorescent protein. Scale bar is 150µm. Reprinted from Reference 4.

The further development of high-throughput genomic studies and biomaterials requires advanced surfaces that can manipulate biomolecules in both space and time. Our patterned surface exhibits good spatial control of both protein and DNA and shows promise for such a biodevice. Combining this patterned surface with electrochemistry led to the patterned and switchable adsorption and desorption of DNA that, when used as a TCM, improved the efficiency of DNA uptake and expression to attached cells. We plan to use this approach with other DNA fragments and cell lines to see how widely it can be applied.


Andrew Hook
School of Chemistry, Physics and Earth Sciences
Flinders University
Bedford Park, South Australia, Australia
Molecular and Health Technologies
CSIRO
Clayton, Australia
 
Helmut Thissen
Molecular and Health Technologies
CSIRO
Clayton, Australia
 
Nicolas Voelcker
School of Chemistry, Physics and Earth Sciences
Flinders University
Bedford Park, Australia