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Startup Diassess Creating Multiplex POC Device for Both Protein and Nucleic Acid Detection

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Working with a microfluidic system first developed at the University of California, Berkeley, newly formed company Diassess is planning to use a $148,000 Small Business Innovation Research grant from the National Institutes of Health to demonstrate a single-cartridge, self-powered, sample-to-answer, point of care device for multiplex detection of both protein and nucleic acid biomarkers.

According to the abstract for the SBIR award, the company plans to use the funding to support a proof-of-concept demonstration of the SIMBAS, or Self-powered Integrated Microfluidic Blood Analysis System, originally developed in the lab of Luke Lee at UC Berkeley.

Diassess cofounder, Ivan Dimov, a former student of Lee's who is the project's principal investigator, told PCR Insider that he intends to use the SBIR award to demonstrate that the SIMBAS platform works for detection of both nucleic acid and protein biomarkers in blood, using HIV and latent tuberculosis as the initial targets for the proof of concept.

If this goes well, he said, Diassess plans to move forward with a second phase project to optimize the selectivity and sensitivity of the device and its robustness under harsh environmental conditions. The company would then move to develop manufacturing processes leading to a marketable device, according to the grant abstract.

SIMBAS uses self-powered degas-driven flow to allow operation under varied environmental conditions without any external tubing or power supply.

Dimov and his Berkeley colleagues published a description of SIMBAS in Lab on a Chip in 2011, showing that the device efficiently extracts blood plasma from less than 5 mL of whole blood and can perform "multiple protein binding assays with high sensitivity without any external pumping mechanisms."

Dimov, Lee, and several European collaborators are all listed as inventors on a patent application for the approach, described as a "microfluidic multiplexed cellular and molecular analysis device and method."

SIMBAS, as described by the Berkeley team in its 2011 demonstration paper, is a two-step blood analysis system, which propels fluid through microfluidic channels in a polydimethylsiloxane substrate material to separate plasma from blood cells. Trenches patterned underneath the channels separate red and white blood cells.

"We are able to move the fluids around and control them in a self-powered way, and do more difficult sample prep like separation of cells and fractionation of sample without requiring any external systems," Dimov said.

"It fits on the palm of your hand and that's all you need to diagnose 10 to 20 or more conditions in one shot with very little sample and very little reagents."

In the 2011 study, the researchers used blood spiked with biotin to test the SIMBAS device's sample-prep ability. Moving forward with Diassess, Dimov said the first goal will be to demonstrate that the chip can be used to detect the presence of disease, and more specifically both protein and nucleic acid markers.

Dimov said the company has decided to use tuberculosis and HIV as test cases. "The key is to demonstrate we can do nucleic acid analysis together with protein analysis, which hasn't really been combined before," he said. "Both of these can be critical to diagnosing a disease … and being able to combine both can give you a greater overview of the disease going on. So we thought it was important to focus on this capability to prove the concept," he said.

To do this, Dimov said, the SIMBAS platform will be used to direct sample fractions to an array of different fluidic chambers equipped with chemistries for either immunoassay detection of proteins or loop-mediated isothermal amplification, or LAMP, for nucleic acid detection.

"You will have an array of chambers, and each will have different chemistry to detect different things. So, the different reactions don't interact with each other but you can read them simultaneously," Dimov said.

One advantage of using LAMP, he added, is that it allows for direct visual detection. "It has this characteristic that during the amplification reaction it generates lots of biophosphate ions, and if you put a fluorescent indicator to sense those, you can generate a large fluorescence change during the reaction, which you can see with your own eye," he said.

"You don't need a reader for it," said Dimov, "But we are thinking about adapting it to a camera phone to quantify it and read it out."

Proteins, meanwhile, can be read out through "a standard immunoassay," he said. But the group is also working on making the readout standard for both proteins and nucleic acids.

"Antibodies will detect the presence of the antigen, but instead of having a dye or something to show the antibodies are there, we will have a nucleic acid target on it and will do an amplification reaction," he said.

Other groups have taken a number of approaches to using LAMP for point-of-care assays — such as an HIV viral load test being developed at the University of Pennsylvania (PCR Insider 6/7/2012), or a PATH project to develop a LAMP-based device for malaria and other diseases (PCR Insider 6/9/2011) — but most have been single assays, not multiplex microfluidic systems.

Dimov noted that the Lee lab is also continuing to work with the SIMBAS approach with grants from the Bill and Melinda Gates Foundation (PCR Insider 12/22/2011).

Dimov said he and Diassess cofounder Benjamin Ross set out as a new company to "take the concept forward and focus it on really getting a sort of viable product out rather than developing new science."

By the end of the one-year Phase I SBIR project, Dimov said he hopes to have demonstrated the clinical potential of the chip. A Phase II effort would then involve refining what diseases the group wishes to target with an initial clinical device, and moving forward to validate it and create a commercial prototype.

"We are not certain yet, from a clinical application point of view, what diseases are the most relevant," Dimov said. "So, we will focus on specific diseases, and on generating a clinical prototype."

Dimov said that the company is also exploring integrating another method also developed with the Luke Lee lab and published earlier this year in Lab on a Chip, called digital loop-mediated DNA amplification (dLAMP), which uses LAMP to quantify nucleic acids present in a sample.

"The basic idea is that you can quantify very precisely the amount of nucleic acid in a sample just by counting how many positive reactions there are," Dimov explained.

"If instead of having many, many markers you focus just on one or two, and you want very high precision, you can envision a chip where you have an array with all of the same targets, but by counting how many of these chambers light up, you can know with high precision the amount of material that was there," he said.

"There are some diseases that require that because only when you go over a certain [concentration] threshold it's relevant, but below that its not."

Dimov said Diassess is "playing with all those concepts and fine tuning."

"Hopefully, we will have something out and demonstrated really soon," he said.

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