Nanowell technology advances the study of proteins

August 14, 2012

Proteins are the biomolecules that carry out the business of biology.  They provide structure to our cells and tissues including muscle, cartilage, ligaments, skin and hair.  Proteins are also the machines of life, performing the molecular activities that keep us functioning including metabolizing energy, transmitting signals, attacking invaders, digesting food, dividing cells and overseeing innumerable other cellular processes. They are critical in the maintenance of health and when they malfunction they lead to disease.

Joshua LaBaer and his colleagues at Arizona State University’s Biodesign Institute announce important improvements in protein microarray technology—a valuable tool for investigating the functions of the vast catalog of human proteins known as the proteome. 

Protein microarrays are tools that display thousands of proteins in high spatial density.  They allow investigators to probe the functions of thousands of proteins simultaneously.  However, protein microarrays are usually arranged on a flat surface, like a microscope slide.  This creates the possibility that reactants can diffuse from one reaction feature to the next.

The new nanotechnology platform uses etched nanowells that physically isolate each feature from all of the others, thereby preventing diffusion. This permits greater numbers of proteins to be analyzed at one time, at lower cost, and enables experiments that simply cannot be performed in a planar system. The improvements will be applied to the investigation of disease biomarkers—protein factors in blood that may be used to pinpoint diseases like cancer and diabetes while they are still in a pre-symptomatic state.

The group’s results recently appeared in the Journal of Proteome Research.

LaBaer’s approach addresses two fundamental challenges in protein microarray studies. The first has to do with the difficulties associated with the production, purification and storage of proteins. The second challenge (and central focus of this new work) is to improve the density of proteins that may be printed on a single microarray slide. The improved technique enhances the prospects for functional protein studies and is the highest density of individual proteins in nano-vessels demonstrated on a single slide.

“We envision broad applications for this platform, which enables running many simultaneous biochemical reactions without having to worry about local diffusion,” LaBaer says. “The piezoelectric printing can be used to address different reactants to the many different nanowells making the technology particularly powerful.  That is, by mixing and matching, a researcher can execute many-to-many screening experiments or test various proteins to see if they act as subunits in complex biochemical reactions without having to worry about cross-reactivity or diffusion. ”

LaBaer directs the Virginia C. Piper Center for Personalized Diagnostics. The state-of-the-art facility at the Biodesign Institute is devoted to examining the subtleties of protein structure and function, particularly as they relate to the origin and progression of human diseases.

The center houses vast repositories of protein expression-ready plasmids from some 950 organisms, including humans. Plasmids are circular pieces of DNA that contain individual genes that code for various proteins – they act like little genetic flash drives that allow researchers to produce the proteins they carry.  These plasmids are maintained in specialized robotic freezers at -80 degrees Celsius.

Through an international program for sharing genetic material—known as DNASU—more than 300,000 plasmid clones have been sent to laboratories in over 39 countries and 46 states. LaBaer’s group uses these tools to explore protein biomarkers for a variety of diseases including cancers of the breast, ovaries, prostate and lung.

Protein arrays typically consist of a glass slide or chip on which a library of proteins for study have been immobilized. High-throughput methods can then be applied to investigate protein behavior. But before proteins are suitable for microarray analyses, they typically must be purified—a messy and labor-intensive process. Purified proteins can degrade over time and have finicky requirements for storage and handling. These limitations encouraged LaBaer to approach the problem in a new way.

His efforts led to the development of the Nucleic Acid Programmable Protein Array (NAPPA). Rather than print purified proteins on the microarray slide, LaBaer instead prints the blueprints for these proteins, in the form of plasmid genes. A stable microarray can be prepared in this way and later coated with a specialized extract from cells that will convert the genes into proteins at the time of experiment.

The NAPPA technique has been used successfully for biomarker discovery studies, using planar slides each containing upwards of 2000 proteins. However, problems can arise at higher spot densities, due to diffusion. The higher the spot density in the microarray, the more serious the problem becomes.

An alternative method, outlined in the new study, is to replace planar slides with microarray slides meticulously etched with tiny nanowells, in which the selected biochemical reactants can be safely confined. The technique uses silicon micro fabrication technology to precisely etch thousands of nanowells onto slides. The group also developed a sophisticated liquid dispensing system to position and dispense genes and reagents into individual nanowells.

The higher density microarrays reduced the spatial separation of spotted proteins. Experiments with traditional planar slides showed that problems with diffusion of reactants occurred when center-to-center separation distances between spots on the microarray were less than 400nm. While minimal diffusion was observed at a spacing of 750nm, significant diffusion was noted when the spot separation was reduced to 375nm.

When the planar slides were replaced with a nanowell array, issues of diffusion and chemical cross-talk were largely eliminated. Each nanowell is sealable and therefore capable of fully isolating reaction events (protein expression and antibody capture) occurring simultaneously on the microarray. The etched nanowells, fabricated on silicon wafers, were each 75 microns deep and around 250 microns in diameter.

The extreme precision necessary to produce the high-density arrays required a new method of piezoelectric printing, allowing an array density of 8000 proteins per slide—the highest protein density thus far reported. For these experiments, 287 randomly selected genes, 193 from Vibrio cholera and 96 from human were used.

The team also produced proof-of-concept ultra high-density arrays containing 24,000 proteins per slide, again demonstrating largely diffusion-free results when the reactants were dispensed into the air-tight microchambers provided by the nanowells.

LaBaer says that the use of new ultra high-density protein microarrays introduces for the first time the possibility of proteome-wide screening. The addition of NAPPA eliminates issues of protein storage, purification and expression, making the combined technology an attractive, high-throughput avenue for accelerated protein research.


Written by: Richard Harth
Science Writer: Biodeisgn Institute
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