November 17, 2021

Nested Nanowells Speed Single Cell Studies

With the N2 chip, researchers can analyze more than 1,000 proteins in 108 single cells in one day

Analytical chemist holds a nested nanoPOTS chip

Analytical chemist Ying Zhu holds a nested nanoPOTS chip, the next generation of technology developed at Pacific Northwest National Laboratory to prepare single cells for proteomics.

(Photo by Andrea Starr | Pacific Northwest National Laboratory)

Researchers tracking the behavior of cancerous tumor cells have a new tool in their arsenal that can process 10 times the number of cells in one day. A new nested nanoPOTS chip, developed at Pacific Northwest National Laboratory (PNNL), was reported October 29 in the journal Nature Communications.

Analytical chemist Ying Zhu and his colleagues first described the nanoPOTS technology in 2018. NanoPOTS stands for Nanodroplet Processing in One pot for Trace Samples, and it is a method for analyzing hundreds of proteins in individual cells at the same time.

“Individual cells work in concert. The ability to analyze proteins in each cell is key to getting detailed information on the biological role of each cell,” Zhu said. “From there, we can start to map how cells work together in tissue and organs.” Zhu has worked on a team with colleagues at the Environmental Molecular Sciences Laboratory, or EMSL, a Department of Energy Office of Science user facility located at PNNL, to use nanoPOTS to study proteins in cells of mouse uterine tissue.

Nine nanowells per nest

The challenge for the single-cell proteomics method is handling the tiny amounts of protein content within a single cell. Every protein matters during sample preparation and analysis.

Each sample is then analyzed using a precise molecular identification technique called mass spectrometry. This approach uses extremely tiny samples: more than 250 single-cell samples from a nanoPOTS chip can fit in a drop of water.

The original nanoPOTS technology confined samples in individual nanowells organized in a grid on the chip. This approach reduced sample loss by more than 99 percent compared to other technologies at the time.

As described in Nature Communications, the design of the new nanoPOTS chip, called N2, greatly increases the number of wells per chip to 243 nanowells on one chip. Groups of nine nanowells are nested in each of 27 clusters across the chip.

nanoPOTS N2 compared side by side with previous version
The original nanoPOTS chip (left) had 27 individual nanowells organized on the surface. The new nested nanoPOTS chip (right) has an array of 27 nested areas, each containing nine nanowells, on the surface. (Photo by Andrea Starr | Pacific Northwest National Laboratory)

With the N2 chip, Zhu and his colleagues analyzed about 100 individual mouse cells derived from the lung, immune system, and axillary lymph node vessel. They quantified about 1,500 proteins in each single cell and used this information to classify cells based on protein abundance.

“We are also working to make this technology easy for other laboratories to use,” Zhu said. The N2 chip can be manufactured in a standard clean room, and we used a commercial single-cell isolation system for liquid handling rather than a custom-built system as before.”

PNNL recently licensed the nanoPOTS technologies to the biotechnology companies SCIENION and Cellenion. Cellenion makes the single-cell isolation system that Zhu and his colleagues used with the N2 chip.

“The goal of this partnership is to combine commercial precision liquid handling systems with the nanoPOTS platform to develop an effective system for sample preparation for single-cell mass-spectrometry-based proteomics,” said PNNL commercialization manager Jennifer Lee.

The development of the N2 chip was supported by internal grants from PNNL and EMSL, as well as the National Institutes of Health.

Other PNNL authors on the paper include: Sarah Williams, Lye Meng Markillie, Song Feng, Chia-Feng Tsai, Victor Aguilera-Vazquez, Ronald Moore, Dehong Hu, Hardeep Mehta, Tao Liu, Joshua Adkins, Richard D. Smith, Geremy Clair, and Ljiljana Paša-Tolic. Jongmin Woo, now at the University of North Carolina at Greensboro, made significant contributions to this work while he was at PNNL.

Visit the Available Technologies page to learn more about nanoPOTS licensing opportunities, and read more about the new nested nanoPOTS chip on the EMSL website.


About PNNL

Pacific Northwest National Laboratory draws on its distinguishing strengths in chemistry, Earth sciences, biology and data science to advance scientific knowledge and address challenges in sustainable energy and national security. Founded in 1965, PNNL is operated by Battelle for the Department of Energy’s Office of Science, which is the single largest supporter of basic research in the physical sciences in the United States. DOE’s Office of Science is working to address some of the most pressing challenges of our time. For more information, visit For more information on PNNL, visit PNNL's News Center. Follow us on Twitter, Facebook, LinkedIn and Instagram.