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Nanochannels for the identification of single molecules

Nanochannels with diameters from 15nm to 100nm were fabricated and used to isolate the fluorescence signal from a single molecule.
28 April 2006, SPIE Newsroom. DOI: 10.1117/2.1200603.0109

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.

Figure 1. These cross-sectional scanning-electron micrographs show nanochannels made by top-down (a) and bottom-up (b) processes. (c) The photon counts show a peak when a target molecule is present. The figure gives the counts during 10μs bins as a function of time of 2ms for a 5μM solution of 5-Iodoacetamidofluorescein excited by laser light at 800nm. The scale bars in both images are 100nm long.

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.

Jun Kameoka
Electrical Engineering, Texas A&M University
College Station, Texas
Molecular and Cellular Oncology, MD Anderson Cancer Center
Houston, Texas
Prof. Kameoka is currently an assistant professor at Texas A&M University. He has been working on nanofluidics, nanosensor and nanomaterials over five years
Nick Jing
Texas A&M University
College Station, Texas 
Miao Wang
Texas A&M University
College Station, Texas