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PASSIVE CHEM/BIO TAGS 16 assays; patterned mesophases for compartmentalizing detection devices; and quantum dots for lasing, detecting, and signaling. Current recognition systems in biology primarily involve DNA hybridization or antigen-antibody binding. These methods are effective but often require significant sample preparation time and may be messy, involving unstable secondary reagents. The chemistry is often complicated, and detection of each new target requires that a new chemical assay be developed. DNA assays take âmore time, reagents, and sample preparation than are generally acknowledged.â The recently developed fluorobodies, which are simple fluorescent tags that bind to a specific target and travel with it, offer a method for simpler and faster assays. Fluorobodies consist of antibody-binding loops fused with thermally stable green fluorescence protein (GFP). The recognition surface is generated and selected using phage display techniques to create large libraries, yielding high-affinity tags. These tags can bind, for instance, to unique glycolipids or glycoproteins on the cell surface of pathogens and thus identify them. (A comment at this point was that the GFP tags are monovalent, whereas most real antibodies are divalent, significantly increasing their affinity and specificity.) When mice eat the GFP, their skin fluoresces green. The latest development in this technology is split-GFP, whereby fluorescence occurs only after the binding to the target has occurred. By incorporating the fluorobodies into patterned mesophases (micrometer-level, not nanometer-level) on Si wafers, arrays of chemical and biological sensing elements can be established, making systematic, multiplexed analyses possible. The second focus of Trewhella's presentation was quantum dots (Q-dots)âsemiconductor nanocrystals whose fluorescence properties can be tuned by varying their size. When the Q-dots are stabilized by incorporating them in a sol-gel nanocomposite, they can be combined with optical microcavities to produce microlasing devices. This lasing property can be exploited in the interrogation of Q-dot arrays. One can also use the Q-dots as luminescent tags by crosslinking them with biological molecules that bind to specific sites. Trewhella foresees revolutions exploiting the nano-bio interface involving molecular recognition elements held in predesigned scaffolds to generate a signal and amplify it (e.g., using Q-dots), as well as the use of active biomolecules, such as motor proteins, to assemble and actuate nanomaterials and structures (e.g., nanowires with programmable interconnects). Asked whether one can hook Q-dots on DNA, Trewhella said that this is probably feasible, although no one has done it, probably because of the highly hydrophobic environment of DNA. PANEL 4 DISCUSSION Discussion began with a question about the relative merits of natural antibodies and artificial structures such as fluorobodies. The advantage of antibodies is that they are raised in vivo and therefore naturally avoid binding to targets they are not supposed to bind to. Fluorobodies would not be expected to show this specificity. Most tests of fluorobodies are against intended targets, not against all of the other molecules that might bind nonspecifically to the binding loop. Nevertheless, fluorobodies were developed to detect successfully folded proteins in the human genome project and appear to show the same specificity as antibodies. The binding loops of fluorobodies may not be identical to the corresponding antibodies, but they must be similar. The observation was made that Q-dots (at least those made from CdSe nanocrystals) are not covert, since the Cd signature is detectable, for example, with x-rays. Could an invisible, two-dimensional bar codeâfor example, one that uses upconverting phosphors that can be irradiated at 980 nm and emit in the green and blueâ be transferred to the skin of an uncooperative target? This would depend on how long the bar code takes to transfer and how long it can be expected to last. There was also speculation about feeding GFP to targets, thus creating people with fluorescent skin.
PASSIVE CHEM/BIO TAGS 17 With regard to active markers added to products, what is the mass fraction of marker? Markers are typically small compounds, generally present at the parts-per-billon level, although with deuterium-substituted isotopes, one can operate on the parts-per-trillion level. Isotag uses only stable isotope markers, in deference to public perceptions of the risks of radioactivity. Given variability in the natural abundance of isotopicsâfor example, deuterium is more abundant on the West Coast than on the East Coastâit was asked whether it would be possible to use geographical differences in isotope abundances to determine where a person had been. The answer is likely to be no, because people move around so much. Some work is being done to try to determine where people have been by analyzing the populations of bacteria that colonize their bodies, given that these populations reflect local environments. In a similar vein, it was asked how the anthrax spores mailed to various individuals 2 years ago could have been traced. One possibility was isotopic analysis not only of the spores themselves, but also of the admixed chemicals to determine where the spores had been grown. However, the biggest hope was DNA analysis of the strain. Unfortunately, DNA analysis revealed that the strain was garden variety anthrax used broadly to test vaccines. Another question was whether it would be possible to irradiate individuals with neutrons and create tags inside their bodies by neutron activation. Such tags were seen as likely to be short-lived (a few hours), although it was noted that palladium activates down to stable isotopes, which might be detectable for much longer times.
PASSIVE CHEM/BIO TAGS 18
