Optical fibers are wave-guides that can transmit light with minimal attenuation via total internal reflection. Optical fibers are composed of a high refractive index material (core) encased by a lower refractive index material (cladding) so that light incident on the core-cladding boundary is always internally reflected. (Figure1)
Using optical fiber bundles (made by Schott) consisting of 50,000 individually cladded 4.5 μm optical fibers, we generate high-density optical arrays for high sensitivity measurements. The core and cladding of the fibers are both made of silica but are doped differently. As a result, they have different etching properties such that the fiber cores etch faster than the cladding when submerged in an acidic solution. Taking advantage of this property, the optical fiber bundle can be acid etched to generate an array of 50,000 femtoliter sized wells (Figure 2). A solution trapped within the array of wells can be optically probed from the non-etched end of the fiber bundle.
The femtoliter microwell arrays can be used to simultaneously trap and analyze hundreds of individual enzyme molecules. Single molecule isolation is achieved by mixing a very dilute solution of enzyme with excess fluorogenic substrate, filling the etched wells, and then sealing the wells so that no leakage occurs. The enzyme concentration is diluted such that, on average there is 1 enzyme or less for every 10 wells. Using the Poisson equation for rare events, p(n) = e-xxn/n! , where x is the average number of enzyme molecules per well, the probabilities of finding 0, 1 and 2 enzymes in a well can be calculated to be, p(0)=0.904; p(1)=0.09; p(2)=0.0045. Therefore, while most wells will be empty, approximately 10% of the wells will contain single enzyme molecules. The probability of finding more than one enzyme per well, p(2), is very low. Since the enzymes are sealed with a fluorogenic substrate, the presence of an enzyme can be detected from the non-etched end of the fiber bundle by measuring the fluorescent product generated due to the enzyme- catalyzed reaction. Figure 3 shows a general scheme of single molecule isolation within the fiber microarray using a mechanical sealing method.
In our laboratory, we are developing single molecule assays for virus particles, proteins, and RNA biomarkers. The principle of these assays is presented in Figure 4. In contrast to conventional enzyme linked immunosorbant assays (ELISAs) where target antigens are captured in bulk by antibodies coated on the surface of a microwell plate, this digital ELISA utilizes paramagnetic beads to form enzyme labeled immunocomplexes at the single molecule level (Figure 4a). In very dilute solutions of target, the Poisson equation applies, where x is the average number of protein molecules per bead, and the probabilities of finding 0, 1 and 2 molecules on a bead can be calculated. Beads that carry 1 protein molecule will generate a detectable fluorescent product due to enzymatic signal amplification and are considered “on,” while those without a protein molecule will not generate any detectable signal and are considered “off.” Only wells with a bead that carries a labeled immunocomplex will produce fluorescent product. Unlike conventional ELISA, where the fluorescent product diffuses in a large volume of solution, in digital ELISA the reaction volume is confined to femtoliter wells, so the fluorescence product remains concentrated and detectable with a conventional microscope. The percentage of “on” beads is proportional to the analyte concentration.
Detection based on counting the number of beads that generate fluorescent product (“on” wells) over the beads with no signal (“off” wells) provides digital, or binary, detection. By capturing and counting single molecules, the method developed in our lab is very sensitive, with detection limits in the femtomolar to attomolar range. Moreover, when the digital readout (i.e. counting the number of beads that provide signal) is combined with the analog readout (i.e. measuring the intensity of the signal), the dynamic range can be extended by several orders of magnitude.3 This combined readout capability provides both high sensitivity and a wide dynamic range.
1. Zhang, H.; Nie, S.; Etson, C. M.; Wang, R. M.; and Walt, D. R.; Oil-sealed femtoliter fiber-optic arrays for single molecule analysis. Lab on a Chip, 2012, 12, 2229-2239.
2. Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. C., Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 2010, 28 (6), 595-9.
3. Rissin, D. M.; Fournier, D. R.; Piech, T.; Kan, C. W.; Campbell, T. G.; Song, L.; Chang, L.; Rivnak, A. J.; Patel, P. P.; Provuncher, G. K.; Ferrell, E. P.; Howes, S. C.; Pink, B. A.; Minnehan, K. A.; Wilson, D. H.; Duffy, D. C., Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range. Analytical Chemistry 2011, 83 (6), 2279-85.