The Nano Toolkit

Recently, nanotechnology has been revolutionizing many important areas in molecular biology, especially in the detection and manipulation of proteins and biological species at the molecular and cellular level. The combination of molecular biology and nanotechnology opens the possibility of detecting and manipulating atoms and molecules using nanodevices, with the potential for a wide variety of medical uses at the cellular level.

Today, the amount of research in biomedical science and engineering at the molecular level is growing exponentially because of the availability of new investigative nanotools based on protein nanotechnology. These new analytical tools are capable of probing the nanometer world and will make it possible to characterize the chemical and mechanical properties of cells, discover novel phenomena and processes, and provide science with a wide range of tools, including materials, devices, and systems with unique characteristics. The marriage of electronics, biomaterials, and molecular biology and nanotechnology is expected to revolutionize many areas of biology and medicine in the 21st century.

The combination of nanotechnology and molecular biology has already led to a new generation of devices for probing the cell machinery and elucidating molecular-level life processes heretofore invisible to human inquiry. Tracking biochemical processes within intracellular environments can now be performed in vivo with the use of fluorescent molecular nanoprobes and nanosensors (6). With powerful microscopic tools using near-field optics, scientists are now able to explore the biochemical processes and nanoscale structures of living cells at unprecedented resolutions. It is now possible to develop nanocarriers for targeted delivery of drugs that have their shells conjugated with antibodies for targeting antigens and fluorescent chromophores for in vivo tracking.

The development of metallic nanoprobes that can produce a surface-enhancement effect for ultrasensitive biochemical analysis is another area of active nanoscale research. Plasmonics refers to the research area dealing with enhanced electromagnetic properties of metallic nanostructures. The term is derived from plasmons, which are the quanta associated with longitudinal waves propagating in matter through the collective motion of large numbers of electrons. Incident light irradiating these surfaces excites conduction electrons in the metal and induces excitation of surface plasmons, which, in turn, leads to enormous electromagnetic enhancement for ultrasensitive detection of spectral signatures through surface-enhanced Raman scattering (SERS) (7) and surface-enhanced fluorescence. Figure 6 shows a scanning electron micrograph of SERS-active nanospheres (300-nm diameter coated with a 100-nm layer of silver). Nanoparticle-based SERS technology has enabled sensitive detection of a variety of compounds of medical interest. SERS nanoprobe technology has also been incorporated into the design of several fiberoptic probes for diagnostics (8). The development of a SERS gene probe technology based on solid surface-based technology has also been reported. One study demonstrated the selective detection of human immunodeficiency virus DNA and a cancer gene (9).

Optical nanosensors, which have dimensions on the nanometer size scale, have been developed to probe individual chemical species in specific locations

Fig. 6. Nanoprobes consisting of nanospheres coated with nanoshells of silver.

Fig. 7. Fiberoptics nanosensor for single-cell analysis. (Adapted from ref. 10.)

throughout a living cell (10). Figure 7 is a photograph of a fiberoptic nanoprobe with an enzyme substrate designed to detect caspase 9, a protein expressed during apoptosis in a single cell (11). An important advantage of the optical sensing modality is its capability to measure biological parameters in a noninvasive or minimally invasive manner owing to the extremely small size of the nanoprobe. Following measurements using the nanobiosensor, cells have been shown to survive and undergo mitosis (12). Biomedical nanosensors, both in vivo and ex vivo, will play an important role in the future of medicine. The capability to detect important biological molecules at ultratrace concentrations in vivo is central to many advanced diagnostic techniques. Early detection of diseases will be made possible by tracking down trace amounts of biomarkers in tissue.

The development of nanotechnology-based devices and techniques has allowed measurements of fundamental parameters of proteins and biological species at the molecular level. With "optical tweezer" techniques, small particles may be trapped by radiation pressure in the focal volume of a high-intensity, focused beam of light. This technique, also called "optical trapping," may be used to move small cells or subcellular organelles around at will by the use of a guided, focused beam (13). Ingenious optical-trapping systems have also been used to measure the force exerted by individual motor proteins (14). A bead coated with an immobilized, caged bioactive probe was inserted into tissue or even a cell and moved to a strategic location by an optical-trapping system. The cage could then be photolyzed by multiphoton uncaging in order to release and activate the bioactive probe. Optical tweezers can also be used to determine the precise mechanical properties of single molecules of collagen, an important tissue component and a critical factor in diagnosing cancer and the aging process (15). The optical tweezer method uses the momentum of focused laser beams to hold and stretch single collagen molecules bound to polystyrene beads. The collagen molecules are stretched through the beads using the optical laser tweezer system, and the deformation of the bound collagen molecules is measured as the relative displacement of the microbeads, which are examined by optical microscopy.

The study of biological applications of nanotechnology will be important to the future of biological research and medical science. Medical applications of nanomaterials will revolutionize health care in much the same way that materials science changed medicine 30 yr ago with the introduction of synthetic heart valves, nylon arteries, and artificial joints. The protein nanotechnologies discussed previously are just some examples of a new generation of nanotools that have the potential to detect, identify, and manipulate single proteins in vivo and drastically change our fundamental understanding of the life process itself. They could ultimately lead to the development of new modalities of early diagnostics and medical treatment and prevention beyond the cellular level to that of individual proteins, the building blocks of the life process.

References

1. Feynman, R. (1960) There's plenty of room at the bottom: an invitation to enter a new field of physics. Eng. Sci. February Issue.

2. Drexler, E. K. (1986) Engines of Creation, Anchor Books, New York.

3 Cosman, M., Lightstone, F. C., Krishnan, V. V., Zeller, L., Prieto, M. C., Roe, D. C., and Balhorn, R. (2002) Screening mixtures of small molecules for binding to multiple sites on the surface of tetanus toxin C fragment by bioaffinity NMR. Chem. Res. Toxicol. 15, 1218-1228.

4 Tuzun, R. E., Noid, D. W., and Sumpter, B. G. (1995) The dynamics of molecular bearings. Nanotechnology 6, 64-74.

5 Noid, D. W., Tuzun, R. E., and Sumpter, B. G. (1997) On the importance of quantum mechanics for nanotechnology. Nanotechnology 8, 119-125.

6 Vo-Dinh, T. (ed.) (2003) Biomedical Photonics Handbook, CRC Press, Boca Raton, FL.

7. Vo-Dinh, T. (1998) Surface-enhanced Raman spectroscopy using metallic nanostructures. Trends Anal. Chem. 17, 557-582.

8! Isola, N., Stokes, D. L., and Vo-Dinh, T. (1998) Surface-enhanced Raman gene probes for HIV detection. Anal. Chem. 70, 1352-1356.

9 Vo-Dinh, T., Stokes, D. L., Griffin, G. D., Volkan, M., Kim, U. J., and Simon, M. I. (1999) Surface-enhanced Raman scattering (SERS) method and instrumentation for genomics and biomedical analysis. J. Raman Spectrosc. 30, 785-793.

10 Vo-Dinh, T., Alarie, J. P., Cullum, B., and Griffin, G. D. (2000) Antibody-based nanoprobe for measurements in a single cell. Nat. Biotechnol. 18, 764-767.

11. Vo-Dinh, T. (2003) Nanosensors: probing the sanctuary of individual living cells. J. Cell. Biochem. 39(Suppl.), 154-161.

12. Vo-Dinh, T., Cullum, B. M., and Stokes, D. L. (2001) Nanosensors and biochips: frontiers in biomolecular diagnostics. Sens. Actuators B Chem. 74(1-3), 2-11.

13. Askin, A., Dziedzic, J. M., and Yamane, T. (1987) Optical trapping and manipulation of single cells using infrared laser beam. Nature 330, 769-771.

14 Kojima, H., Muto, E., Higuchi, H., and Yanagido, T. (1997) Mechanics of single kinesin molecules measured by optical trapping nanometry. Biophys. J. 73(4), 2012-2022.

15. Luo, Z. P., Bolander, M. E., and An, K. N. (1997) A method for determination of stiffness of collagen molecules. Biochem. Biophys. Res. Commun. 232(1), 251-254.

Kinetics and Mechanisms of Protein Crystallization at the Molecular Level

Peter G. Vekilov

Summary

This chapter focuses on the processes by which a protein molecule in a supersaturated solution joins a protein crystal. The pair of proteins ferritin/apoferritin is used as an example. The most commonly used technique in such investigations has been atomic force microscopy. I discuss the modifications and tests of the atomic force microscope necessary to obtain real-time, in situ molecular resolution imaging. Then, I review tests that establish a quantitative correspondence between the continuous models of crystal growth and the discrete nature of the processes at the molecular level. I address the issue of whether the incorporation of a molecule from a solution into a growth site on the crystal surface is limited by the slow rate of decay of an Eyring-type transition state. The conclusion is that, on the contrary, a scenario, envisioned by Smoluchowski and Debye, is followed in which the kinetics is only limited by the rate diffusion over the free-energy barrier of interaction between two molecules, or an incoming molecule and a surface. Review of the data for many other protein and nonprotein systems suggests that this conclusion is valid not only for the crystallization of ferritin/apoferritin, but also for many other protein and small-molecule crystallization systems. Finally, I review results establishing that the pathway that a molecule from a solution takes on its way to an incorporation site on the crystal surface is indirect: it includes adsorption on the surface and two-dimensional diffusion toward the incorporation site. These results will likely contribute to the understanding at the molecular level not only of the processes of crystallization of proteins and small molecules, but also of the deposition of layers of proteins and other soft materials on substrates, and of other processes of self-assembly in solution.

Key Words: Crystallization; solution thermodynamics; atomic force microscopy; diffusion-limited kinetics; transition states; activation energy; growth mechanisms; surface diffusion; direct incorporation.

From: Methods in Molecular Biology, vol. 300: Protein Nanotechnology, Protocols, Instrumentation, and Applications Edited by: T. Vo-Dinh © Humana Press Inc., Totowa, NJ

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