SlipChip Counts Molecules with Chemistry and a Cell Phone

In developing nations, rural areas, and even one's own home, limited access to expensive equipment and trained medical professionals can impede the diagnosis and treatment of disease. Many qualitative tests that provide a simple "yes" or "no" answer (like an at-home pregnancy test) have been optimized for use in these resource-limited settings. But few quantitative tests—those able to measure the precise concentration of biomolecules, not just their presence or absence—can be done outside of a laboratory or clinical setting. By leveraging their discovery of the robustness of "digital," or single-molecule quantitative assays, researchers at the California Institute of Technology (Caltech) have demonstrated a method for using a lab-on-a-chip device and a cell phone to determine a concentration of molecules, such as HIV RNA molecules, in a sample. This digital approach can consistently provide accurate quantitative information despite changes in timing, temperature, and lighting conditions, a capability not previously possible using traditional measurements.

In a study published on November 7 in the journal Analytical Chemistry, researchers in the laboratory of Rustem Ismagilov, Ethel Wilson Bowles and Robert Bowles Professor of Chemistry and Chemical Engineering, used HIV as the context for testing the robustness of digital assays. In order to assess the progression of HIV and recommend appropriate therapies, doctors must know the concentration of HIV RNA viruses in a patient's bloodstream, called a viral load. The problem is that the viral load tests used in the United States, such as those that rely on amplification of RNA via polymerase chain reaction (PCR), require bulky and expensive equipment, trained personnel, and access to infrastructure such as electricity, all of which are often not available in resource-limited settings. Furthermore, because it is difficult to control the environment in these settings, viral load tests must be "robust," or resilient to changes such as temperature and humidity fluctuations.

Many traditional approaches for measuring viral load involve converting a small quantity of RNA into DNA, which is then multiplied through DNA amplification—allowing researchers to see how much DNA is present in real time after each round of amplification, by monitoring the varying intensity of a fluorescent dye marking the DNA. These experiments—known as "kinetic" assays—result in a readout reflecting changes in intensity over time, called an amplification curve. To find the original concentration of the beginning bulk RNA sample, the amplification curve is then compared with standard curves representing known concentrations of RNA. Since assays, such as those for HIV, require many rounds of DNA amplification to collect a sufficiently bright fluorescent signal, small errors introduced by changes in environmental conditions can compound exponentially—meaning that these kinetic measurements are not robust enough to withstand changing conditions.

In this new study, the researchers hypothesized that they could use a digital amplification approach to create a robust quantitative technique. In digital amplification, a sample is split into enough small volumes such that each well contains either a single target molecule or no molecule at all. Ismagilov and his colleagues used a microfluidic device they previously invented, called SlipChip, to compartmentalize single molecules from a sample containing HIV RNA. SlipChip is made up of two credit card-sized plates stacked atop one another; the sample is first added to the interconnected channels of the SlipChip, and with a single "slip" of the top chip, the channels turn into individual wells.

In lieu of PCR, the researchers used a different amplification chemistry on this chip called digital reverse transcription-loop-mediated amplification (dRT-LAMP), which produces a bright fluorescent signal in the presence of a target molecule during the amplification process. The dRT-LAMP technique eliminates the need for continuous tracking of the intensity of fluorescence; instead, just one end-point readout measurement is used. The resulting patchwork of "positive" or "negative" wells on the device, in combination with statistical analysis, enables single molecules to be counted.

"In each well, you are performing a qualitative experiment; the result is like a pregnancy test: either yes or no, positive or negative, for the presence of an HIV RNA molecule," says David Selck, a graduate student in Ismagilov's lab and a first author on the study. "But by doing a couple of thousand qualitative experiments, you end up getting a numerical, quantitative result: the concentration of HIV RNA molecules in the sample. By calculating the concentration from the number of wells that contain fluorescence—and therefore HIV—you're leveraging the robustness of many qualitative 'yes or no' experiments to fulfill the need for a quantitative, numerical result," he says.

When the researchers compared quantification results from dRT-LAMP to those obtained by the real-time, kinetic version of this chemistry, RT-LAMP, they found that the digital format provided accurate results despite changes in temperature and time, while the kinetic format could not. This finding adds to a body of research that the laboratory has been developing on the robustness of converting analog signals (i.e., a readout reflecting a changing concentration over time) into a series of positive or negative digital signals. Another recent paper, published in the Journal of the American Chemical Society, explored a variation on this analog-to-digital conversion.

Ismagilov's group also tested a way to take an image of the fluorescence pattern in the wells of the SlipChip and, from that image, determine the viral load—without the use of expensive microscopes or trained staff. They turned to a nearly ubiquitous 21st-century technology: the smartphone.

The researchers placed the SlipChip in a makeshift darkroom (a shoebox with a hole in the top) and then photographed its wells using a smartphone outfitted with a special filter attachment—so that the smartphone flash would be able to "excite" the fluorescent DNA dye, and the smartphone camera could capture an image of the fluorescence. The resulting images were uploaded to Microsoft SkyDrive, a cloud-based server, where custom software—designed by the researchers—determined the viral load concentration and sent the results back in an email. These capabilities allow the digital approach to perform reliably with automated processing, regardless of how poor the imaging conditions may be. As an example of its simplicity, a 5-year-old child was able to use this cell phone imaging method to obtain quantitative results using strands of RNA extracted from a noninfectious virus (a video of this demonstration is available on the Ismagilov lab's YouTube channel).

"We were surprised that this cell phone method worked, because both cell phone imaging and automated processing are error prone," Ismagilov says. "Because digital assays involve simply distinguishing positives from negatives, we found that even these error-prone approaches can be used to count single molecules reliably."

The fact that this method is robust not only to changes in time and temperature but also is amenable to cell phone imaging and automated processing makes it a promising technology for limited-resource settings. "We believe that our findings of the robustness of digital amplification could signal a major paradigm shift in how quantitative measurements are obtained at home, in the field, and in developing countries," Ismagilov says.

The researchers stress that there is still room for improvement, however. "While in this study we were examining robustness and used purified RNA, the next generation of devices will isolate HIV RNA molecules directly from patients' blood," says Bing Sun, a graduate student in Ismagilov's lab and a first author on the study. "We will also adapt the devices for other viruses, such as hepatitis C. By combining these improvements with the cell phone imaging method, we plan to create something that could actually be used in the real world," Sun adds.

The paper is titled "Increased Robustness of Single-Molecule Counting with Microfluidics, Digital Isothermal Amplification, and a Mobile Phone versus Real-Time Kinetic Measurements." In addition to Selck, Sun, and Ismagilov, the paper is coauthored by Mikhail A. Karymov, an associate scientist at Caltech. The work was funded by the Defense Advanced Research Projects Agency award number HR0011-11-2-0006, and by the National Institutes of Health award numbers R01EB012946 and 5DP1OD003584. Microfluid technologies developed by Ismagilov's group have been licensed to Emerald BioStructures, Randance Technologies, and SlipChip LLC.

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Tuesday, December 10, 2013
Noyes 153 (J. Holmes Sturdivant Lecture Hall) – Arthur Amos Noyes Laboratory of Chemical Physics

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Friday, January 10, 2014
Center for Student Services 360 (Workshop Space) – Center for Student Services

Undergraduate Teaching Assistant Orientation

Caltech Names Thomas F. Rosenbaum as New President

To: The Caltech Community

From: Fiona Harrison, Benjamin M. Rosen Professor of Physics and Astronomy, and Chair, Faculty Search Committee; and David Lee, Chair, Board of Trustees, and Chair, Trustee Selection Committee

Today it is our great privilege to announce the appointment of Thomas F. Rosenbaum as the ninth president of the California Institute of Technology.

Dr. Rosenbaum, 58, is currently the John T. Wilson Distinguished Service Professor of Physics at the University of Chicago, where he has served as the university's provost for the past seven years. As a distinguished physicist and expert on condensed matter physics, Dr. Rosenbaum has explored the quantum mechanical nature of materials, making major contributions to the understanding of matter near absolute zero, where such quantum mechanical effects dominate. His experiments in quantum phase transitions in matter are recognized as having played a key role in placing these transitions on a theoretical level equivalent to that which has been developed for classical systems.

But Dr. Rosenbaum's scientific achievements were not solely what captured and held the attention of those involved in the presidential search. We on the search committee were impressed by Dr. Rosenbaum's deep dedication, as Chicago's provost, to both undergraduate and graduate education—both critical parts of Caltech's mission. He has had responsibility for an unusually broad range of institutions and intellectual endeavors. Among his achievements as provost was the establishment of the Institute for Molecular Engineering in 2011, the University of Chicago's very first engineering program, in collaboration with Argonne National Lab.

We also believe that Dr. Rosenbaum's focus on strengthening the intellectual ties between the University of Chicago and Argonne National Lab will serve him well in furthering the Caltech-JPL relationship.

As provost, Dr. Rosenbaum was also instrumental in establishing collaborative educational programs serving communities around Chicago's Hyde Park campus, including the university's founding of a four-campus charter school that was originally designed to further fundamental research in education but which has also achieved extraordinary college placement results for disadvantaged Chicago youths.

This successful conclusion to our eight-month presidential search was result of the hard work of the nine-member Faculty Search Committee, chaired by Fiona Harrison, and the 10-member Trustee Selection Committee, chaired by David Lee. We are grateful both to the trustees and faculty on our two committees who made our job so very easy as well as to those faculty, students, staff, and alumni who provided us with input and wisdom as we scoured the country for just the right person for our Caltech.

"Tom embodies all the qualities the faculty committee hoped to find in our next president," Harrison says. "He is a first-rate scholar and someone who understands at a deep level the commitment to fundamental inquiry that characterizes Caltech. He is also the kind of ambitious leader who will develop the faculty's ideas into the sorts of innovative ventures that will maintain Caltech's position of prominence in the next generation of science and technology."

"The combination of deep management experience and visionary leadership Tom brings will serve Caltech extremely well in the coming years," Lee adds. "The Board is excited about collaborating closely with Tom to propel the Institute to new levels of scientific leadership."

"The Caltech community's palpable and deep commitment to the Institute came through in all my conversations, and it forms the basis for Caltech's and JPL's lasting impact," Dr. Rosenbaum says. "It will be a privilege to work closely with faculty, students, staff, and trustees to explore new opportunities, building on Caltech's storied accomplishments."

Dr. Rosenbaum received his bachelor's degree in physics with honors from Harvard University in 1977, and both an MA and PhD in physics from Princeton University in 1979 and 1982, respectively. He did research at Bell Laboratories and at IBM Watson Research Center before joining the University of Chicago's faculty in 1983. Dr. Rosenbaum directed the university's Materials Research Laboratory from 1991 to 1994 and its interdisciplinary James Franck Institute from 1995 to 2001 before serving as vice president for research and for Argonne National Laboratory from 2002 to 2006. He was named the university's provost in 2007. His honors include an Alfred P. Sloan Research Fellowship, a Presidential Young Investigator Award, and the William McMillan Award for "outstanding contributions to condensed matter physics." Dr. Rosenbaum is an elected fellow of the American Physical Society, the American Association for the Advancement of Science, and the American Academy of Arts and Sciences.

Joining the Caltech faculty will be Dr. Rosenbaum's spouse, Katherine T. Faber, the Walter P. Murphy Professor of Materials Science and Engineering at Northwestern University. Dr. Faber's research focuses on understanding stress fractures in ceramics, as well as on the fabrication of ceramic materials with controlled porosity, which are important as thermal and environmental barrier coatings for engine components. Dr. Faber is also the codirector of the Northwestern University-Art Institute of Chicago Center for Scientific Studies in the Arts (NU-ACCESS), which employs advanced materials science techniques for art history and restoration. Dr. Rosenbaum and Dr. Faber have two sons, Daniel, who graduated from the University of Chicago in 2012, and Michael, who is currently a junior there.

Dr. Rosenbaum will succeed Jean-Lou Chameau, who served the Institute from 2006 to 2013, and will take over the helm from interim president and provost Ed Stolper on July 1, 2014. The board, the search committee, and, indeed, the entire Institute owes Dr. Stolper a debt of gratitude for his unwavering commitment to Caltech, and for seamlessly continuing the Institute's forward momentum through his interim presidency.

As you meet Dr. Rosenbaum today and over the coming months, and learn more about his vision for Caltech's future, we believe that you will quickly come to see why he is so well suited to guide Caltech as we continue to pursue bold investigations in science and engineering, to ready the next generation of scientific and thought leaders, and to benefit humankind through research that is integrated with education.

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Caltech Nobelist Zewail Named to UN Scientific Advisory Board

Ahmed Zewail, Linus Pauling Professor of Chemistry and professor of physics, has been selected as one of 26 members of a new Scientific Advisory Board established by the United Nations secretary-general.

The board, which will meet twice per year, will provide advice on science, technology, and innovation concerning sustainable development to the secretary-general and the heads of UN organizations. The creation of this new board, which was formally announced September 24 at the UN's first High-Level Political Forum on Sustainable Development, was the result of a recommendation from the report of the High-Level Panel on Global Sustainability in January 2012.

Made up of scientists from various fields in the natural, social, and human sciences, the board will have the main objective of improving the linkage between science and policy and of ensuring that up-to-date and rigorous science is reflected in policy discussions within the UN. In addition, board members will also advise on issues related to the public visibility and public understanding of science. The United Nations Educational Scientific and Cultural Organization (UNESCO) will serve as the secretariat of the board.

"I am pleased to be a member of this United Nations Scientific Advisory Board. Its objectives coincide with my vision to promote science in education and science in diplomacy," Zewail says. "The UN is an excellent platform for an outreach to all nations."

In 1999, Professor Zewail was awarded the Nobel Prize in Chemistry for developing the field of femtochemistry, which uses ultrashort laser flashes to enable the study of chemical reactions in real time at the scale of quadrillionths of a second. He and his group later developed a technique called four-dimensional electron microscopy for the direct imaging of matter in the three dimensions of space and in time with applications spanning the physical and biological sciences.

He is currently director of the Center for Physical Biology at Caltech funded by the Moore Foundation.

Zewail's new involvement with the UN reflects his long-standing interest in global affairs, particularly as they relate to science, education, and world peace. His commentaries on such global issues have been presented in numerous articles, a number of books, and public addresses all over the world. Since the 2011 revolution in Egypt—his native country—Zewail has also played a critical role in that nation's events.

In addition to his international interests, Zewail has been involved in domestic science policy. In 2009, he was appointed to President Obama's Council of Advisors on Science and Technology, and he was named the first U.S. Science Envoy to the Middle East as part of a program sponsored by the White House and the State Department to foster science and technology collaborations between the United States and nations throughout the Middle East, North Africa, and South and Southeast Asia.

Among other honors, Zewail has received the Albert Einstein World Award of Science, the Benjamin Franklin Medal, the Robert A. Welch Award, the Leonardo da Vinci Award, the Wolf Prize, the Priestley Medal, and the King Faisal International Prize. He is a recipient of the Grand Collar of the Order of the Nile, Egypt's highest state honor, and has been featured on postage stamps issued to honor his contributions to science and humanity. Zewail holds honorary degrees from 40 universities around the world and is an elected member of many professional academies and societies, including the National Academy of Sciences, the American Philosophical Society, The Royal Society of London, and the Swedish, Russian, Chinese, and French academies. In recognition of his contributions as a world leader in science and public service, he has received the Top American Leaders award given jointly by the Washington Post and Harvard Kennedy School's Center for Public Leadership.

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Caltech Named World's Top University in Times Higher Education Global Ranking

For the third year in a row, the California Institute of Technology has been rated the world's number one university in the Times Higher Education global ranking of the top 200 universities.

Harvard University, Oxford University, Stanford University, and the Massachusetts Institute of Technology round out the top five schools in the 2013–2014 rankings.

Times Higher Education compiled the listing using the same methodology as in the 2011–2012 and 2012–2013 surveys. Thirteen performance indicators representing research (worth 30 percent of a school's overall ranking score), teaching (30 percent), citations (30 percent), international outlook (which includes the total numbers of international students and faculty and the ratio of scholarly papers with international collaborators, 7.5 percent), and industry income (a measure of innovation, 2.5 percent) make up the data. The data were collected, analyzed, and verified by Thomson Reuters.

The Times Higher Education site has the full list of the world's top 400 schools and all of the performance indicators.

Kathy Svitil
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Friday, October 4, 2013

Undergraduate Teaching Assistant Orientation

Caltech Researchers Synthesize Catalyst Important In Nitrogen Fixation

Inspired by an enzyme in soil microorganisms, researchers develop first synthetic iron-based catalyst for the conversion of nitrogen to ammonia.

As farming strategies have evolved to provide food for the world's growing population, the manufacture of nitrogen fertilizers through the conversion of atmospheric nitrogen to ammonia has taken on increased importance.

The industrial technique used to make these fertilizers employs a chemical reaction that mirrors that of a natural process—nitrogen fixation. Unfortunately, vast amounts of energy, in the form of high heat and pressure, are required to drive the reaction. Now, inspired by the natural processes that take place in nitrogen-fixing microorganisms, researchers at Caltech have synthesized an iron-based catalyst that allows for nitrogen fixation under much milder conditions.

In the early 20th century, scientists discovered a way to artificially produce ammonia for the manufacture of commercial fertilizers, through a nitrogen fixation technique called the Haber-Bosch process. Today, this process is used industrially to produce more than 130 million tons of ammonia annually. Microorganisms in the soil that live near the roots of certain plants can produce a similar amount of ammonia each year—but instead of using high heat and pressure, they benefit from enzyme catalysts, called nitrogenases, that convert nitrogen from the air into ammonia at room temperature and atmospheric pressure.

In work described in the September 5 issue of Nature, Caltech graduate students John Anderson and Jon Rittle, under the supervision of their research adviser Jonas Peters, Bren Professor of Chemistry and executive officer for chemistry, have developed the first molecular iron complex that catalyzes nitrogen fixation, modeling the natural enzymes found in nitrogen-fixing soil organisms. The research may eventually lead to the development of more environmentally friendly methods of ammonia production.

Natural nitrogenase enzymes, which prime inert atmospheric nitrogen for fixation through the addition of electrons and protons, generally contain two metals, molybdenum and iron. Over decades of research, this duality has caused a number of debates about which metal was actually responsible for nitrogenase's catalytic activity. Since a few research groups had modest success in synthesizing molybdenum-based molecular catalysts, many in the field believed that the debate had been settled. The discovery by Peters' group that synthetic iron complexes are also capable of this type of catalytic activity will reopen the discussion.

This finding, along with a wealth of data from structural biologists, biochemists, and spectroscopists, suggests that it may be iron—and not molybdenum—that is the key player in the nitrogen fixation in natural enzymes. The iron catalyst discovered by Peters and his colleagues may also help unravel the mystery of how these enzymes perform this reaction at the molecular level.

"We've pursued this type of synthetic iron catalyst for about a decade, and have banged our heads against plenty of walls in the process. So have a lot of other very talented folks in my field, and some for much longer than a decade," Peters says.

The finding is a first for the field, but Peters says that their current iron-based catalyst has limitations—the Haber-Bosch process is still the industrial standard. "Now that we finally have an example that actually works, everyone wants to know: 'Can it be used to make ammonia more efficiently?' The simple answer, for now, is no. While we're delighted to finally have our hands on an iron fixation catalyst, it's pretty inefficient and dies quickly. But," he adds, "this catalyst is a really important advance for us; there is so much we will now be able to learn from it that we couldn't before."

Funding for the research outlined in the Nature paper, titled "Catalytic conversion of nitrogen to ammonia by an iron model complex," was provided by the National Institutes of Health and the Gordon and Betty Moore Foundation.

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Arnold Appointed New Director of Rosen Bioengineering Center

Now in its sixth year of exploring the intersection between biology and engineering, the Donna and Benjamin M. Rosen Bioengineering Center has chosen Caltech professor Frances Arnold as its new director. Arnold, the Dick and Barbara Dickinson Professor of Chemical Engineering, Bioengineering and Biochemistry began her tenure as director on June 1.

A recipient of the 2011 National Medal of Technology and Innovation, Arnold pioneered methods of "directed evolution" – processes now widely used to create biological catalysts that are important in the production of fuels from renewable resources. She was selected for the directorship because "of her demonstrated leadership in the field of bioengineering," says Stephen Mayo, William K. Bowes Jr. Foundation Chair of the Division of Biology and Biological Engineering.

The Rosen Center supports bioengineering research through the funding of fellows and faculty from many disciplines, including applied physics, chemical engineering, synthetic biology, and computer science.

"Bioengineering is an incredibly exciting field right now," Arnold says. "Solutions to some of the biggest problems in science, medicine, and sustainability will come from the interface between biology and engineering, and Caltech is well positioned to be at the forefront. The Rosen Center will help make that happen with innovative programs for bioengineering research and education."

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Tom Miller Wins Teacher-Scholar Award

The Camille and Henry Dreyfus Foundation has recognized Thomas F. Miller, professor of chemistry at the California Institute of Technology (Caltech), with a 2013 Camille Dreyfus Teacher-Scholar Award.

The award provides a $75,000 unrestricted research grant to "support the research and teaching careers of talented young faculty in the chemical sciences," according to the foundation.

"I am very grateful to my colleagues in the Chemistry and Chemical Engineering Division here at Caltech for nominating me for this award," Miller says. "I am also thankful to the Dreyfus Foundation for its generous grant, which will greatly benefit my research efforts."

Miller is an expert in developing theoretical and computational methods to understand a variety of molecular processes including enzyme catalysis, solar energy conversion, dendrite formation in lithium batteries, and the transport of proteins across cell membranes.

The Camille Dreyfus Teacher-Scholar Awards program is open to institutions in the United States that offer a bachelor's degree or higher in the chemical sciences, biochemistry, materials chemistry, and chemical engineering. Academic institutions may nominate one researcher per year for the award.

Miller earned a bachelor of science degree from Texas A&M University in 2000 and a PhD at the University of Oxford in 2005. He became an assistant professor at Caltech in 2008 and in 2013 was named professor of chemistry. He is the recipient of a Dreyfus New Faculty Award, Sloan Research Fellowship, National Science Foundation CAREER Award, American Chemical Society Hewlett-Packard Outstanding Junior Faculty Award, and an Associated Students of Caltech Teaching Award.

Brian Bell
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