New microfluidic device minimizes loss of high value samples

September 9, 2020

A major collaborative effort that has been developing over the last three years between Arizona State University and European scientists has resulted in a significant technical advance in X-ray crystallographic sample strategies.

The ASU contribution comes from the School of Molecular Sciences , Department of Physics and the Biodesign Institute Center for Applied Structural Discovery. Alexandra Ros, a professor in ASU’s School of Molecular Sciences and the Center for Applied Structural Discovery in the Biodesign Institute. Photo credit: Mary Zhu Download Full Image

The European X-ray Free Electron Laser (EuXFEL) is a research facility of superlatives: It generates ultrashort X-ray pulses — 27,000 times per second and with a brilliance that is a billion times higher than that of the best conventional X-ray radiation sources. After 10 years of construction, it opened for initial experiments in late 2017. The group of Alexandra Ros, professor in ASU’s School of Molecular Sciences, was awarded the second allocation of beam time amongst worldwide competitors.

Their results, published Sept. 9 in Nature Communications, validated a unique microfluidic droplet generator for reducing sample size as well as waste (which can be as high as 99%) in her team’s Serial Femtosecond Crystallography (SFX) experiments. Using this, they determined the crystal structure of the enzyme 3-Deoxy-d-manno-Octulosonate 8-Phosphate Synthase (KDO8PS) and revealed new detail in a previously undefined loop region of the enzyme that is a potential target for antibiotic studies.

“We are excited that this work, resulting from a huge collaborative effort, has been well received in the XFEL community,” Ros said. “We are further developing this method and are seeking synchronization of the microfluidic droplets with the pulses of XFELs. At this very moment, a small team of ASU students has just finished performing experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory in Menlo Park, California, to refine the method. There could not have been better timing for the publication of our work.”

SLAC has been the XFEL facility best known to U.S. scientists, where the now-famous work on crystallography of protein nanocrystals — by the ASU team led by professors John Spence and Petra Fromme — was carried out. SLAC and its companion in Europe, also at Hamburg, have been very successful and consequently, have become heavily overbooked. The coming-on-line of the new facility, with its giant 2.6-mile accelerator tunnel and atomic length scale resolution, has relieved some of the demand on the other facilities, while offering grand new possibilities in the physical sciences.

The ASU team with EuXFEL beam line scientists during one of the designated shifts. The experimental hutch is behind the team. From left: Nadia Zatsepin (ASU), Jorvani Cruz Villarreal (ASU), Patrik Vagovič (EuXFEL), Petra Fromme (ASU), Jose Meza Aguilar (ASU), Alexandra Ros (ASU), Jesse Coe (ASU), Ana Egatz Gomez (ASU), Gerrit Brehm (ASU/Georg August Univeristy Goettingen), Austin Eichelmeyer (ASU), Richard Kirian (ASU), Richard Bean (EuXFEL), Marc Messerschmidt (EuXFEL), Katherina Doerner (EuXFEL).

SFX is a promising technique for protein structure determination, where a liquid stream containing protein crystals is intersected with a high-intensity XFEL beam that is a billion times brighter than traditional synchrotron X-ray sources.

Although the crystals are destroyed by the intense XFEL beam immediately after they have diffracted, the diffraction information can, remarkably, still be recorded thanks to the state-of-the-art detectors. Powerful new data analysis methods have been developed, allowing a team to analyze these diffraction patterns and obtain electron density maps and detailed structural information of proteins.

The method is specifically appealing for hard-to-crystallize proteins, such as membrane proteins, as it yields high-resolution structural information from micro- and even nanocrystals, thus reducing the contribution of crystal defects and avoiding the tedious, if not impossible, growth of the large crystals demanded by traditional synchrotron-based crystallography.

While crystallography with XFELs has been a powerful technique for unraveling the structures of large protein complexes and also permitting time-resolved crystallography, this cutting-edge science nevertheless engenders a major problem. Because of the small “hit” rate it requires huge amounts of suspended protein, which although not irradiated, are cumbersome to retrieve for most protein samples. As much as 99% of the protein can be wasted.

Droplets are generated in the microfluidic T-junction, indicated by red arrows. The droplet carries crystals that will be delivered to the XFEL.

Herein lies the major technical advance made by Ros and her team. They have developed a 3D-printed microfluidic device, which is high-resolution, and generates aqueous-in-oil droplets of variable droplet segmentation that can be synchronized to the free electron laser pulses. This dramatically reduces the amount of purified protein needed for the European XFEL experiment from the currently typical (and almost inaccessible) 1 g requirement for full data set recording.

The researchers’ approach interleaves sample-laden liquid “slugs” within a sacrificial liquid, so that a fast-moving liquid microjet is maintained with sample present only during exposure to the femtosecond XFEL pulses (one millionth of one billionth of a second in duration).

The team of scientists has demonstrated droplet generation of the enzyme KDO8PS crystal suspensions with the microfluidic droplet generator and shown that the droplet generation frequency can be controlled by the rates of the aqueous and oil streams. The diffraction quality of the crystals of KDO8PS is similar both when injected in aqueous droplets surrounded by oil or by continuous injection with a gas dynamic virtual nozzle, with approximately 60% reduction in sample consumption achieved with droplet injection.

The determined structure revealed new detail in a previously undefined loop region of KDO8PS, a potential target for antibiotic studies. These results advocate for future routine integration of droplet generation by segmented oil flow at other XFELs around the world.

Co-first authors on this paper are Ros’ doctoral students Austin Echelmeier (graduated) and Jorvani Cruz Villarreal in the School of Molecular Sciences.

Jenny Green

Clinical associate professor, School of Molecular Sciences

480-965-1430 Jenny.Green@asu.edu

ASU Law students gain paid summer employment, invaluable career experience through innovative Smart State initiative

When summer employment opportunities were postponed or canceled for students of the Sandra Day O’Connor College of Law due to COVID-19, faculty quickly stepped in to provide unique, paid internships and externships. For Diana Bowman, ASU Law’s associate dean for international engagement and co-director for the Center for Smart Cities and Regions, that meant creating a real-world, practical ex...

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ASU Law students gain paid summer employment, invaluable career experience through innovative Smart State initiative

September 9, 2020

Bowman is ASU’s lead on The Connective, a collaboration of ASU, the Maricopa Association of Governments, the Greater Phoenix Economic Council, the Institute for Digital Progress and the Partnership for Economic Innovation. Working with the Arizona Commerce Authority (ACA), The Connective and ASU, Bowman and the team developed an intensive program that ultimately brought onboard 14 students. photo of smart city innovation ASU Law students had the opportunity to work on a special Smart City/Smart Regions program over the summer, gaining insight to take them into future careers. Download Full Image

“When I think about smart cities — and this is how I framed it for our students on Day One — what we are trying to do is improve quality of life for those who live and work in those environments,” Bowman said. “And there really is no other university like ASU that is as focused on the smart city concept and creating an evidence-based, best practice on what smart cities and regions are. So we really are on the cutting edge.”

The summer program had two key focus areas — The Connective’s smart cities and region consortium, and the Arizona Commerce Authority, with students able to work collectively across both. Bowman said the team first focused on helping students feel comfortable with the subject matter, and then provided a road map for the state of Arizona’s vision for smart regions and a smart state to empower the students and give them a sense of purpose.

Diana Bowman, ASU Law’s associate dean for international engagement and co-director for the Center for Smart Cities and Regions.

Equally important was the opportunity for students to build out their professional networks. “Every Friday, we brought in experts from our partner organizations, including Amazon Web Services, Sprint, Dell and Snowflake, so that our students were able to hear and learn directly from professionals working in the local smart region arena,” Bowman said.

Arizona Commerce Authority’s Dominic Papa, vice president of Smart State Initiatives, said the students’ contributions were invaluable. Papa is also an ASU Law 2020 JD in this newly created ACA position that was an outgrowth of initiatives he worked on with Bowman while a student.

“To have some of the best and brightest young legal minds help us craft a strategy of what a smart state should look like — to bring their innovative ideas and kind of push us to think differently was a huge benefit,” Papa said. “What we’re really trying to do is disrupt government by thinking differently and solving complex challenges — so we need outside thinkers who haven’t had government experience to really challenge traditional thinking and get us outside of our bubble.”

Dominic Papa, Arizona Commerce Authority’s vice president of Smart State Initiatives and ASU Law 2020 JD.

ASU Law students’ legal and detailed research skills gained in the classroom gave the ACA and The Connective leaders a fresh way of thinking, Papa added. “What the students did extremely well was help us better understand complex issues like autonomous vehicles, drones and 5G technology through concise briefs that enabled us to make rapid decisions. We were better positioned to handle complex, difficult challenges very quickly,” he said.

Riggs Brown, in his final year at ASU Law, worked on regional procurement processes and a “regulatory sandbox” to demonstrate innovation in a controlled environment. He said the experience was a perfect match for his passion for intellectual property and innovation in the law.

“There are all these new things that are happening in the world, and I come from a business background and feel like the law hasn’t caught up,” Brown said. “So when I had this opportunity to see where cutting-edge innovation is happening, I leapt on it.”

ASU Law student Riggs Brown.

Brown spent the first four weeks on procurement. “So much of the smart city and smart region work is about the need for collaboration and to be on the same page. And in procurement, every city has a different process and needs — so we had to create a baseline. The recognition that we all come from different places and diverse backgrounds, and that innovation and growth will only happen when we all come to the table, was really engaging.”

For fellow ASU Law senior Lori Rutten, her focus was data governance, making it a very unique experience given that it was a completely new concept to her — something she hadn’t previously explored or taken an interest in. “I am very appreciative for Dr. Bowman and The Connective to take on a student like me who didn’t have any experience in that field of law … but they still brought me on, and rallied around me and were extremely supportive of my learning process.”

Like Brown, Rutten said the experience helped her to really understand how important it is for governments and private entities to work together to achieve a better city, state and country.

ASU Law student Lori Rutten

“If everyone could learn to work together we could achieve so much more,” she said. “I always knew that but this helped me see it in a different light. Through the project, I was able to learn a lot about the importance of data governance being implemented into the different municipalities, and it’s amazing what it can achieve … so much more than I ever could have imagined, because it can benefit everyone — government, private and public companies and residents.”

Brown added that the experience showed him how many other doors will open with this type of legal experience. “Working as part of The Connective gave me the opportunity to see the intersection of innovation, business, science and the law in a way that is happening right now. While a lot of other schools look at this as an area to write papers or think about in a hypothetical way, at ASU Law we were actually impacting decisions being made. Dr. Bowman encouraged us to be the drivers of our own experience … allowing us to build on the things we were curious about and build on the things we wanted to be better at too.”

Papa said the ACA is extremely grateful for ASU Law’s generous contributions that enabled the summer program. “As an economic development organization for the state — sitting at the intersection of industry, government and academia, and being able to partner with ASU and ASU Law — is tremendous,” Papa said. “We are working together to build the next generation of legal professionals in the workforce and looking forward to much more to come.”

Added Bowman, “The summer provided the students with a unique skill set that they can take into their future and that will give them an advantage over other students in the job market. When you can go into a job interview and talk about how you, as a law student, helped build out a smart region in Arizona — that is really tangible. And so I am very excited about how this will propel our students into whatever careers they choose.”

The students’ efforts will be part of this year’s Smart Cities/Region Summit, to be co-hosted by ASU Law, ASU’s University Technology Office, the Arizona Commerce Authority and The Connective. The students also will be participating in a data exchange workshop being hosted by the Amazon Web Services Cloud Innovation Center in September.