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RESEARCH PROFILES Two-color nanoparticles identify single molecules Although scientists have been able to detect single biomolecules for ∼15 years, the procedure usually requires pure samples and solvents. Moreover, the target needs to be derivatized to become fluorescent. “For real applications in chemistry and medicine, there is no way that you can modify the small number of molecules you want to detect,” says Shuming Nie at Emory University and the Georgia Institute of Technology. “And even if you could, the signal level from single dye molecules or fluorescent proteins is not that intense.” As described in the February 15 issue of Analytical Chemistry (pp 1061–1070), Nie and colleagues at Emory University; Georgia Tech; the Centers for Disease Control and Prevention; and the University of Georgia, Athens, have devised an immunoassay with two-color fluorescence correlation detection, which solves several major problems associated with single-molecule detection. The new assay can detect a few DNA or protein molecules or virus particles without purifying, amplifying, or modifying them. If a target is sandwiched between a red and a green nanoparticle, an intense and stable signal is produced. And if only red and green signals that are correlated in time (coincident photons) are counted, unbound probes are readily distinguished from probes bound to targets. “The practical applications I am envisioning are infectious-disease detection, biowarfare-agent detection, as well as early detection of cancer and cardiovascular disease,” Nie explains. To study protein detection, the researchers mixed a solution of tumor necrosis factor alpha with red and green nanoparticles conjugated to two different monoclonal antibodies of the protein. To detect oligonucleotides, they conjugated nanoparticles to complementary pieces of DNA. To show that viruses can also be counted, the researchers conjugated red
(b)
Probe volume ~3 fL
(a)
Correlated signal
No correlation
(a) When single nanoparticles move past a tightly focused laser beam, no signal is recorded. (b) When a red nanoparticle and a green nanoparticle move past the beam at the same time, a signal is recorded.
and green nanoparticles to two different monoclonal antibodies of respiratory syncytial virus (RSV). One antibody targeted F protein; the other targeted G protein. Having multiple F and G sites on its surface, the virus bound many nanoparticles; this produced very intense coincidence signals. In contrast, RSV deprived of its G sites generated little or no signal. Both luminescent quantum dots (semiconductor nanocrystals that fluoresce brightly) and energy-transfer nanoparticles (polymer nanoparticles embedded with a donor–acceptor dye pair that undergoes fluorescence resonance energy transfer) could be used, and they were much brighter and more stable than conventional dyes. Moreover, both the red and green nanoparticles could be excited with a single wavelength. “One laser is cheaper than two,” Nie says. “But more fundamentally, it is very difficult to focus two laser beams exactly in the same spot in three dimensions.” The researchers injected a few nanoliters of sample into a capillary tube that sat on the stage of an epifluorescence microscope. A laser beam passing through the microscope’s objective lens was focused tightly on a window in the tube. When nanoparticles passed by the window and fluoresced, photons traveled through the objective lens, through filters outside the microscope, and past two single-photon detectors. Nanoparticles that were not bound to target mol-
ecules produced random signals. But when a red nanoparticle and a green nanoparticle were both bound to a target molecule, they produced signals at the “same” time (within 3.5 µs). Counting coincident signals therefore revealed the number of targets in a given volume of solution in real time. The approach generated 1000 data points per second. Using serial dilutions, the researchers obtained accurate results for concentrations between 20–30 femtomolar and 2–3 picomolar. Statistical noise made it impossible to count
