New technologies for fabricating biological microarrays

B. J. Larson.
Ph.D. thesis, University of Wisconsin-Madison, August (2005).

Microarrays, composed of thousands of spots of different biomolecules attached to a solid substrate, have emerged as one of the most important tools in modern biological research. This dissertation contains the description of two technologies that we have developed to reduce the cost and improve the quality of spotted microarrays.

The first is a device, called a fluid microplotter, that uses ultrasonics to deposit spots with diameters of less than 5 microns. It consists of a dispenser, composed of a micropipette fastened to a piece of PZT piezoelectric, attached to a precision positioning system. A gentle pumping of fluid to the surface occurs when the micropipette is driven at specific frequencies. Spots or continuous lines can be deposited in this manner. The small fluid features conserve expensive and limited-quantity biological reagents. Additionally, the spots produced by the microplotter can be very regular, with coefficients of variability for their diameters of less than 5%.

We characterize the performance of the microplotter in depositing fluid and examine the theoretical underpinnings of its operation. We present an analytical expression for the diameter of a deposited spot as a function of droplet volume and wettability of a surface and compare it with experimental results. We also examine the resonant properties of the piezoelectric element used to drive the dispenser and relate that to the frequencies at which pumping occurs. Finally, we propose a mechanism to explain the pumping behavior within the microplotter dispenser.

The second technology we present is a process that uses a cold plasma and a subsequent in vacuo vapor-phase reaction to terminate a variety of oxide surfaces with epoxide chemical groups. These epoxide groups can react with amine-containing biomolecules, such as proteins and modified oligonucleotides, to form strong covalent linkages between the biomolecules and the treated surface. The use of a plasma activation step followed by an in vacuo vapor-phase reaction allows for the precise control of surface functional groups, rather than the mixture of functionalities normally produced. By maintaining the samples under vacuum throughout the process, adsorption of contaminants is effectively eliminated. This process modifies a range of different oxide surfaces, is fast, consumes a minimal amount of reagents, and produces attachment densities for bound biomolecules that are comparable to or better than commercially available substrates.

We show applications of these two technologies in the fabrication of protein microarrays, enhancement of MALDI mass spectrometry, deposition of polymer electronics, directed growth of carbon nanotubes, and the chemical modification of carbon-containing materials.

BradLarsonPhDThesis.pdf3.77 MB