At a protein chip conference in San Diego two months ago, the FDA’s Emanuel Petricoin was sitting in the back of the room, checking his e-mail, and half-listening to what he expected would be a predictable progression of “the same old talks, over and over again.”
Then Eric Henderson took the stage. Henderson, the chief scientific officer and founder of BioForce Nanosciences in Ames, Iowa, demonstrated in his presentation how the company had used its NanoArrayers to fit 1,521 antibody spots within the impossibly small area of 85 square micrometers, and how it was using atomic force microscopy to detect binding interactions on the arrays.
“When Eric started talking, it completely grabbed my attention,” said Petricoin, who co-directs the NCI-FDA clinical proteomics program with Lance Liotta. “The examples he used were astounding.”
Petricoin approached Henderson after the talk and arranged to discuss the possibility of a collaboration. “It was very obvious after about five minutes,” Petricoin said, that the BioForce nanoarray technology would be a good fit for the work he and Liotta are doing.
Seven weeks later, BioForce and the NCI-FDA clinical proteomics program formally announced their collaboration, in which Liotta and Petricoin are adapting their reverse-phase protein microarray technology to the NanoArray platform. These microarrays include spots of cell lysate from specified tissues, which are interrogated using antibodies.
Liotta and Petricoin produce the reverse-phase protein array using laser capture microdissection (LCM), a technique that allows them to procure as few as several hundred cells of interest directly from specified regions of human or any tissue specimens. They then lyse the cells and spot the lysate onto the array.
The researchers are currently testing out different substrates, spot sizes, and lysates to see which yields the optimal signal-to-noise ratio, and said they have obtained some “promising early results.” Once they optimize the NanoArray platform for these reverse-phase protein arrays, Liotta and Petricoin plan to use BioForce’s NanoArrays for protein profiling of human tissue biopsy samples and monitoring therapy during clinical trials.
Why Smaller is Better
For Liotta and Petricoin, the size of the NanoArrays presented clear advantages in their clinical work. “Many companies talk about clinical proteomics discovery using arrays, but the dirty secret is, if you want to talk about patient clinical samples, you do not have the luxury of having a million cells to work with,” said Liotta. “If you can fit several hundred patients on a tiny spot, that is the same surface area of [an ordinary protein array spot] that could have a dramatic impact in sample analysis,” he added.
The NanoArrayer’s ultra-miniaturized microfabricated deposition tools allow the user to deposit as few as several hundred attoliters on each spot of the array. Consequently, researchers can put in hundreds of thousands of spots from a single sample and get a good coefficient of variation without losing space on the array. Liotta likes to spot onto the array different concentrations of a sample along a dilution curve in order to account for the dynamic range of proteins present in the cells and the different affinities of proteins for the antibody targets. Thus, this decrease in space per spot works particularly well, he said.
A second way that BioForce attracted Liotta and Petricoin’s attention was with its expertise in atomic force microscopy, or AFM. “Their experience in the AFM field, [enables us] to go into a non-tagging-based system where you don’t have a secondary label,” Liotta said, explaining that the secondary label is extremely problematic with antibody arrays. “Fluorescence or biotinylation often destroys the antibody’s ability to interact with a cell lysate.”
In BioForce’s AFM-based detection system, the AFM NanoReader, micrometer-scale probes scan the surface of the array with nanometer resolution, according to Henderson. The probe consists of a cantilever with a pointed tip that does the actual scanning, and a laser that bounces off of the top of this cantilever and delivers a signal to a detector. As the tip moves up and down around the surface, this angle of the laser deflection changes, and creates a pattern that approximates the topography of the surface.
Initially, Liotta and Petricoin are using traditional fluorescence- and chemiluminescence-based detection methods, but hope to move to AFM. If they can bring together LCM, with its ability to extract such precise small tumor samples, and NanoArrays, with their miniscule sample requirements, and AFM, with its capability of detecting binding between molecules without perturbing the sample, “we will be approaching the Holy Grail of systems biology,” Liotta said.
Tiny Arrays, Growing Company
BioForce, meanwhile, is working on additional collaborations as well as a financing and hiring effort.
Currently, Gulfstream Capital Group of Boca Raton, Fla., is facilitating its Series A round of funding within the US, and Société Générale Asset Management has also led financing globally. This round of funding is closing soon, but the company plans to initiate a second round of funding later in the year.
Additionally, the company has increased its headcount to 13, hiring four new people in the past month, including its CEO, Gary Alianell. Alianell, who was formerly chief operating officer at Madison, Wis., genomics startup EraGen Biosciences and also worked in management at Beckman Instruments for 10 years, is operating out of a Southern California base. “We are going through an expansion phase,” said Henderson. The company hopes to continue this expansion with additional hires on the business, science, and legal side.
This is a major change of pace for the company, which Henderson started in 1994 as an incubator for his nano-ideas, and which has survived mainly on NIH Small Business Innovation Research grants and NSF grants.
In this new phase, BioForce is focusing 80 percent of its resources on developing its NanoArrayer instrument and 20 percent on AFM, due to the more defined market for the NanoArrays. The company hopes to develop an alliance with an instrumentation company to build the NanoArrayers, but may manufacture them in the Ames, Iowa, facility. The company is now working through “a portfolio of about 10 different collaboration negotiations,” Henderson said.
The company also has an ongoing relationship with Bothell, Wash.-based Prolinx, using Prolinx’s Versalinx surface chemistry on the NanoArrays. With Versalinx, the surface probes are first conjugated to phenyldiboronic acid (PDBA) in solution, then this complex attaches salicylhydroxamic acid (SHA) that is already bound to the surface. Karin Hughes, Prolinx’s vice president of R&D, said the company’s technique of binding the probes through a two-step process eliminates the debris that often attaches to the surface of amide-bonded surface chemistry. “AFM is highly dependent on [the microarray] surface and how clean it is,” Hughes said.
Prolinx hopes to extend its collaboration with BioForce to the collaborative marketing of NanoArrays. “They are taking the protein array field one step further,” said Hughes. “As the protein array market continues to heat up, they will continue to heat up.”