With the advent of the Vision for Space Exploration, there is an immediate need for sensitive biosensors that can be used for spacecraft environmental monitoring (air and water). Optical biosensors have significant potential in this regard, since a given target species (antigen) can be captured by a bound antibody and this capture event can be detected via fluorescence emission. Recent efforts at the NASA Glenn Research Center have focused on increasing the sensitivity of a certain class of optical biosensors, namely enzyme-linked immunosorbent assay (ELISA)-based biosensors, for space exploration applications.
ELISA-based biosensors have proven to be accurate in detecting a wide variety of antigens (ref. 1). In the sandwich ELISA biosensor (see the sketch), a given antibody (the capture antibody) is bound to the substrate. In the presence of an antigen, a complex is formed. If a second fluorescently labeled antigen-specific antibody is introduced and binds to the antigen, the sandwich is complete and the target antigen can be identified by fluorescence detection. The sensitivity of such capture-based biosensors can be dramatically improved by increasing the substrate surface-area-to-volume ratio (ref. 2).
ELISA sensing mechanism.
Because of their high surface-area-to-volume ratio, porous glasses are prime candidates as biosensor substrates. Two particular candidate materials are Vycor porous glass (Corning Incorporated, Vycor glass code 7930), which has a 4-nm pore size and a surface-area-to-volume ratio of 375 m-1, and controlled-porosity glass (CPG), which has surface-area-to-volume ratios of 16 and 8 m-1, respectively, for 50-nm and 100-nm pore-size samples. Before these substrates can be utilized effectively, however, the effect of the nano-scale pore structure on molecular diffusion needs to be understood.
Accordingly, two-photon excitation fluorescence correlation spectroscopy was used to examine the microscale diffusion of biomolecules within the candidate materials. The experimental correlation data were fit to theoretical functional forms, and the diffusion timescales were calculated. These results within the substrate were then compared with those in bulk solution to determine the effect of the confining geometry on the diffusion timescales.
Fluorescence correlation data of Rhodamine 6G in methyl alcohol (MeOH).
To quantify the effects of confinement, we examined the diffusion of a simple fluorophore, Rhodamine 6G, in the Vycor sample. Results are illustrated in the preceding graph for the diffusion of Rhodamine 6G in both Vycor and bulk solution. The correlation data fit the three-dimensional diffusion model very well. From the data, it was determined that the diffusion timescale within Vycor is 13 to 14 times slower than in the bulk solution. In addition, the diffusion of fluorescein isothiocyanate (FITC)-labeled antibodies (a complex molecule) was also studied. Studies were only performed in the CPG, since the pore size (4 nm) of the Vycor substrate was prohibitively small. In this case, the diffusion timescale Dt in the 50-nm CPG was determined to be 16 times slower than in the bulk solution (see the following graph), whereas the timescale in the 100-nm CPG was roughly 8 times slower than in the bulk solution.
Fluorescence correlation data of FITC-labeled antibody in bulk solution and within the 50-nm pore size CPG; Dt(bulk) ~16Dt(50-nm CPG).
These studies have examined the diffusion of simple and complex molecules in nanoporous substrate materials, demonstrating that the confined geometry results in longer diffusion times (slower diffusion) in comparison to that in bulk solution. However, the reduction in diffusion speed is not so great that the throughput of an operational sensor would be compromised. Considering the significant increase in sensing surface area (and the resulting increase in detection sensitivity), ELISA-based biosensors utilizing nanoporous glass substrates hold great promise for exploration.
Glenn contacts:
Marius Asipauskas, 216-433-8778, Marius.Asipauskas-1@nasa.gov
Dr. Gregory A. Zimmerli, 216-433-6577, Greg.Zimmerli@nasa.gov
Dr. David G. Fischer, 216-433-6379, DGFischer@nasa.gov
Authors:
Marius Asipauskas, Dr. Gregory A. Zimmerli, and Dr. David G. Fischer
Headquarters program office:
Exploration Systems Mission Directorate
Programs/projects:
Human Research Program
Last updated: November 1, 2007
Responsible NASA Official:
Gynelle.C.Steele@nasa.gov
216-433-8258
Point of contact for NASA Glenn's Research & Technology reports:
Cynthia.L.Dreibelbis@nasa.gov
216-433-2912
SGT, Inc.
Web page curator:
Nancy.L.Obryan@nasa.gov
216-433-5793
Wyle Information Systems, LLC
