Nanochannels for the identification of single molecules
A device that can simply and quickly identify an extremely low concentration of target molecules amid a high concentration of normal molecules is in high demand in many fields. For instance, the detection of early-stage cancer is vital to improve the patient prognosis. Currently, surgery is the most effective tumor treatment, but its success is highly dependent on the stage of the cancer development. To allow early treatment, having an efficient way to detect an extremely low concentration of tumor markers such as p21Cip1/Waf1, FOXO3a, and MDM2 in a high concentration of normal proteins—such as in blood or other body fluids—is critical.1 However, the sensitivity and accuracy of current techniques that detect these molecules are relatively low.
Liquid chromatography, electrophoresis, and mass spectrometry—integrated with microfluidic devices—are currently used for molecule identification.2,3 Here, molecules are separated and identified based on their mass-to-charge ratio. Although these techniques are well developed, the sensitivity is far below the single-molecular level. Normally, more than 107 molecules are required for identification, and the signals are averaged over all molecules in a nanoliter detection volume.
To fulfill the need for a genuinely single-molecular detector, we have developed nanochannel devices that allow an ultra-sensitive and rapid single-molecule level diagnostics by monitoring photon-burst signals from individual molecules. The nanochannels' diameters(15 – 100nm) are comparable to the size of molecules. They have been fabricated using both top-down and bottom-up processes: in the former, electron-beam lithography, reactive-ion etching, and glass bonding were used to make the nanochannel devices. Figure 1(a) shows a cross-sectional scanning-electron microscope (SEM) image of nanochannel made by this process. The bottom-up process involved a scanned, coaxial, electrospinning method.4 A coaxial jet, composed of motor oil as the core and silica sol-gel solution as the shell, was extruded through a coaxial source and deposited on the rotating collector as oriented coaxial nanofibers. The cross-sectional SEM image of nanochannel by the bottom-up approach is shown in Figure 1(b). Target molecules were electrokinetically infused into the nanochannel and the excitation laser focused into the center of the nanochannel. From the laser spot size (0.3 – 1.5μm) and the molecule confinement via the nanochannel, the detection volume was estimated to be in the low-attoliter to zeptoliter range. This small detection volume theoretically provides single-molecular excitation under physiological concentrations (μM). Thus, a single protein, bound to a specific fluorescent antibody, could be identified among millions of normal molecules by statistically analyzing the photon-burst signals. These are determined by the individual electrokinetic molecule transport, as well as quantum efficiency. The single-molecule photon-burst signal is shown in Figure 1(c). Compared to traditional assays—which take a long time to process, have low sensitivity, require a large amount of protein and are easily contaminated—nanochannel analysis can detect single proteins in a much more sensitive manner: even more sensitive than mass spectrometry.
In summary, we have developed a nanochannel device that has zeptoliter detection volume and allows for single-molecule level diagnostics. We will continue to utilize and refine this device for the identification of single molecules from photon-burst signals.
