One of the wonders of biology is how trillions of living parts in a human body work together constantly to produce reliable and predictable events, like the beating of the heart, the taking of a breath and the processing of light from the eye into words like these, translated by the brain into information. Equally remarkable - and of great importance to medicine - is that each of these cells has its own distinctive character and its own way of reacting to its environment. A dose of radiation, changes in nutrition and other events can have sharply different effects on individual cells, even in a tissue or tumor where all are labeled the same.
To understand human biology fully, then, it is necessary to look at the metabolic processes of cells one by one. Such single-cell analysis was beyond the reach of technology until recently. Scientists and engineers, however, are learning how to sort cells using microfluidics and to examine the RNA and DNA of each. And at the California Institute of Technology's Kavli Nanoscience Institute (KNI), researchers have found a way to integrate the whole process on a device about one-inch square, putting single-cell analysis on a single chip.
Making Sense of a Thousand Moving Cells
Single Cell Analysis on a Chip. The chip works by first introducing 30 cells into 30 lysing chambers, ensuring only one cell per chamber. RNA molecules of interest are captured and concentrated in sieve valves. Finally, the RNA is converted into complementary DNA and then amplified before fluorescence intensity measurements are used to provide real-time PCR information that can be used to derive the original RNA concentrations in the cells. (Courtesy, KNI)
As KNI Co-Director Axel Scherer points out, cells are continually active and are not doing the same thing at the same time, and earlier technology missed much of this activity by analyzing cells in batches. “In the old days, you would take a thousand cells and lyse them [burst them open],” he says. “The problem is that a thousand cells would be different, especially if they are stem cells. So you would get an average, not seeing what individuals cells are doing.”
Scherer, the Bernard A. Neches Professor of Electrical Engineering, Applied Physics and Physics at Caltech, says the new technology can measure and compare the genomic material of 30 cells. It isolates the cells, extracts their RNA and then converts it to corresponding DNA fragments, which are then amplified by a polymerase chain reaction (PCR) process. Among other things, scientists using this system can see that individual cells under identical conditions produce different levels of messenger RNA, the molecule the carries instructions for the cell to produce certain proteins. “We found that you could get a large change, on the order of 50%, in the concentration of RNA,” Scherer says.
The chip designed by Scherer and his colleagues at KNI weds advanced microfluidic technology with a rapid heating and cooling cycle to optimize the PCR process. Its front end is a loading circuit that directs 30 cells into as many separate chambers, where each is lysed to release its RNA. The RNA then is converted to chemically corresponding regions of DNA, which are then copied (up to a billion times) through 30 heating and cooling cycles in the PCR process. The final step is measurement of the resulting DNA – in effect, a genomic fingerprint of each cell.
Microfluidics is not a new technology, but Scherer says this single-cell analysis system improves on it at least a couple of ways. One is the channel design, which is three-dimensional (not simply laid out on a flat surface) and employs different channel geometries depending on the type and speed of chemical reaction needed at any given point in the process. “These are not trivial chips,” says Scherer. The other advance is the linking of microfluidics with PCR temperature cycling. “I know a lot of people who are doing fluidics on small scale, I call that plumbing,” he says. “We add heating and cooling.”
Creating a Nano-Laboratory
Axel Scherer (left) in his lab with Doug Smith, editor of Caltech’s science and engineering magazine. (Courtesy, KNI)
The potential uses for this device are many. It could aid in profiling pathogens to develop new vaccines or antibiotics. Scherer says it could shed light on the very early stages of cancer, giving scientists clues as to when and why individual cells turn malignant. Scherer’s main collaborator on the single-cell analysis project, Scott Fraser, hopes “to dissect genetic regulatory networks that guide the normal development and function of cells.” Fraser, the Anna L. Rosen Professor of Biology at Caltech, says he wants to “follow the cellular logic” -- from signaling factors to the reception of the signal to the activation and/or expression of DNA binding factors that turn genes on and off, and finally to the activation of genes – all in a particular cell. “Most analyses are happy with homogenates of many thousands of cells, which won't work for us,” he says.
Beyond the study of cells, the single-cell project also advances a fundamental mission at the Institute – to bring nanotechnology from the lab into the mainstream. The KNI, founded in 2004, draws on a well-stocked toolkit of nanoscale technologies that have been developed at Caltech over the past decade or more. The microfluidics and rapid-fire temperature cycling used in the single-cell analysis chip are just two examples. Fraser says researchers at KNI “have refined a number of different microfluidic approaches, several different optical sensors for defined macromolecules, electrochemical sensors of at least three different sorts, mass spectrometry of three different approaches and cell calorimetry.”
Now, says Scherer, the task is to take these technologies “and put them together into functional systems.” The single-cell analysis chip is one such system. Others being developed at the KNI include a portable, battery-powered PCR device for identifying pathogens in blood or urine samples, and a disposable spectrometer using cheap, tiny lasers. As Fraser puts it, “I view the KNI as one of our best chances toward making nanotechnology sufficiently robust to be used rather than just demonstrated.”