Researchers have developed a microfluidic system for counting individual protein molecules in cells. The method captures individual cells, lyses them, labels their contents and detects them individually with fluorescence microscopy. Developed by Richard N. Zare of Stanford University in California and his colleagues there and at Carnegie Institution, also in Stanford, the technique avoids the pitfalls of ensemble imaging and is particularly useful for studies where there are fewer than 1000 molecules of a particular protein of interest.Although single-molecule analysis can be done in vivo, Zare explained that it is limited. “These techniques restrict analysis to one or perhaps a few species at a time because of the need to resolve fluorescence from different probes. Moreover, these applications are limited to cases where the cell environment does not appreciably affect the fluorescence intensity of the reporter molecule or where endogenous fluorescence does not seriously interfere with the measurements.” The microfluidic chip forms the basis of the system. Using standard microfabrication techniques in polydimethysiloxane (PDMS), the researchers built an intricate system of channels and valves that allowed them to conduct the analysis one cell at a time. In the system, a cell in buffer is injected and captured between a standard two-way valve and a three-state valve developed in Zare’s lab. The three-state valve has a partially open position that enables trapping of the cell in a reaction chamber, while buffers or other solutions can be added or removed. The three-state valve is a key part of a novel microfluidics system because it allows researchers to capture a cell and hold it in place while delivering solutions to rupture the cell membrane and label target molecules within the cell.Once a cell was trapped, the investigators ruptured the cell wall and used antibody labels to tag the molecules of interest. When the valve was released, electrophoresis methods separated molecules and moved them down a channel to the molecule-counting section of the chip. The molecule-counting part uses a modified confocal setup. A standard setup creates an illumination spot roughly 500 nm wide, which is not sufficient illumination in a 100-μm-wide channel. Therefore, Zare and his colleagues reconfigured the setup to use a cylindrical lens, creating a line or curtain of illumination that spanned the width of the channel that excited fluorescence in labeled or autofluorescent molecules passing through. “[Currently] we detect about 60 percent of the molecules, which means we could detect with confidence something like three, plus or minus one, molecules,” Zare explained. Autofluorescence from the PDMS that makes up the chip is interfering, he added. “In the future, I anticipate that we can count almost all the molecules that pass through our detection curtain.” The group has done preliminary testing on the cyanobacterium Synechococcus, a photosynthetic microbe that uses a molecule called phycobiliprotein as a light-harvesting complex. The bacterium is a model system for studying photosynthesis and carbon fixation. In addition, phycobiliprotein is autofluorescent, making it ideal for testing the system. Zare said that he will continue to use the system to study Synechococcus. “Single-cell analysis promises to provide new perspectives on biology. It resolves the heterogeneity present within a sample population. This heterogeneity can provide useful information with regard to important cellular processes, especially if a significant number of cells display a marked deviation from the assumption of the average cell.”Science, Jan. 5, 2007, pp. 81-84.