A team led by researchers at the University of Freiburg has developed a microfluidic chip for applying proximity ligation assays to cell signaling studies.
The chip, detailed in a paper published last month in Molecular & Cellular Proteomics, combines a microfluidic cell-culturing platform with a chip-based PLA assay system to allow for more streamlined and highly multiplexed PLA-based cell signaling studies.
Using the platform, the researchers characterized Akt signaling upon stimulation with platelet-derived growth factor, monitoring the phosphorylation state of six pathway proteins and the cellular localization of another across 128 different cell culture microenvironments.
Owned by Swedish biotech firm Olink Bioscience and sold via license agreements by companies such as Sigma Life Science, the PLA uses pairs of antibodies attached to unique DNA sequences to detect proteins of interest. When the antibodies bind their targets, the attached DNA strands are brought into proximity and ligate, forming a new DNA amplicon that can then be quantified using real-time PCR. The quantity of the DNA corresponds to the quantity of the target protein.
Because the DNA labels can be constructed to hybridize only to their specific partner, PLA eliminates the common immunoassay problem of antibody cross-reactivity. Compared to conventional immunoassay methods like ELISA, PLA offers higher multiplexing capabilities as well as lower sample demands. The technique also offers very high sensitivity, allowing for the study of low-abundance proteins like transcription factors, noted University of Freiburg researcher Matthias Meier, one of the leaders of the MCP study.
One drawback of PLA, however, "is that it is a hassle to work with," Meier told ProteoMonitor.
"There are more than 25 chemical steps that you have to perform," he said. "And if you have a technician performing the assays [by hand] on a glass slide, [the process] is too long, with too many flush steps, too many buffer conditions, too many variables within the workflow."
By packaging the assay in a micofluidic platform, however, the researchers are able to streamline and automate the workflow, Meier said. "You can clearly define each step, program it, set the temperature, and so it is a very neat solution for the PLA technology."
Using the microfluidics platform, the researchers can cut the time needed to run the assay roughly in half, he said, from around 24 hours to 12. More significant than this gain in time, however, is the improvement in reproducibility achieved by reducing the amount of washing and pipetting researchers must do by hand. According to Meier, in work following up on the MCP study, he and his colleagues have demonstrated that use of the microfluidic platform reduced the PLA's variability by roughly two orders of magnitude.
This improved reproducibility is key for applications like cell signaling work, Meier said, noting that the technique's laboriousness and variability has made it poorly suited for comparing large numbers of samples against each other. With their microfluidics system, on the other hand, the researchers were able to perform experiments on 128 cell cultures simultaneously, monitoring multiple proteins or post-translational modifications in a variety of cell types and in response to various forms of stimulation.
Meier and his colleagues are not the first to apply microfluidics to PLA. In August, Olink announced a deal with Fluidigm to offer its Proseek Multiplex PLA product on that company's BioMark HD microfluidic real-time PCR platform (PM 8/2/2013).
Use of the BioMark HD platform allows researchers to measure in a single run the levels of up to 92 proteins in as many as 96 samples. Olink has set up three facilities across Europe that will run samples on the ProSeek Multiplex panels as a service for researchers without access to the BioMark HD platform, and the company plans in the near future to open several such facilities in the US, as well.
The Olink-Fluidigm collaboration differs from the work of Meier and his colleagues in that it is focused on solution-based PLA measurements. The University of Freiburg team, on the other hand, is applying microfluidics to cell culture work, which, Meier noted, allows them to capture protein localization information in addition to expression data.
The researchers, he said, are currently working on a next-generation version of the chip with capacity for 1,024 separate cell cultures. They hope also to increase the number of proteins they can measure per cell to 24, up from the seven analytes per cell they measured in the MCP study.
Meier said that he was open to possible collaborations with Olink on the device and "absolutely" interested in commercializing the platform down the road, however, he noted that for the time being much of the technology used in the chip was covered by patents owned by Fluidigm.
A number of these patents, however, are scheduled to expire in the next several years, Meier said. According to an S-1 filed by Fluidigm in 2010 with the US Securities and Exchange Commission, its patents covering the foundational work done by California Institute of Technology researcher and company founder Stephen Quake are scheduled to expire between 2017 and 2025, with additional patents expiring between 2012 and 2029.
Meier said that following expiration of these key patents, he and his colleagues plan to "see what we can do, if we can go to market with" their invention.
More than platform development, though, the researchers are interested in applying the technology to their biological interests, he said. "We are a signaling group, so the main goal is to see if we can use these microfluidic advances to study, for instance, how cells respond to growth factors – mainly factors that are increasing cell growth in cancer states."