April 1, 2006 (Vol. 26, No. 7)
Refined Tools for Real-Time Single Particle Tracing and Dissection of Signaling Pathways
Nanotechnology is the creation and utilization of materials, devices, and systems through the control of matter on the nanometer-length scale, i.e., at the level of atoms, molecules, and supramolecular structures. It is the popular term for the construction and utilization of functional structures with at least one characteristic dimension measured in nanometers (one billionth of a meter, 10-9 m).
Nanoparticles possess a remarkable self-ordering and assembly behavior that is different from larger particles. Given the inherent nanoscale functional components of living cells, it was inevitable that nanotechnology would be applied in biotechnology, giving rise to the term nanobiotechnology. In nanoscale, a base pair in human genome is 0.4 nm, whereas proteins vary in size between 1󈞀 nm. The single cell is an ideal sensor for detecting various chemical and biochemical processes. The ability to work with an individual cell using nanotechnology is very promising.
The trend in miniaturization of technologies has continued from microfluidics to nanofluidics, from microarrays to nanoarrays and nanochips from biochips. Numerous nanodevices and nanosystems for sequencing single molecules of DNA are feasible. Nanobiotechnology will play an important role in the study of systems biology—also referred to as pathway, network, or integrative biology—in which proteomics plays an important role.
Nanobiotechnology will provide refined tools for the study of genomics and proteomics, real-time single particle tracing in living cells, and dissection of signaling pathways.
Processes in the Living Cell
Nanotechnology holds promise for the analysis of complex processes inside living cells. A method for nanomanipulation in living cells is the use of magnetic nanoparticles that are microinjected into the nucleus of living cells. Such particles can be functionalized by the covalent attachment of selected molecules, e.g., specific proteins. Magnetic tweezers, in combination with high-resolution microscopy, enables one to manipulate such nanoparticles inside living cells, thereby changing local genome structure. Nanoprobes in the nucleus could be used to monitor changes in chromosome arrangement associated with changes in gene expression.
Genetically encoded nanosensors can be used for monitoring metabolism in living cells. Quantum dots disguised by protein coating can penetrate cells, as cells mistake them for proteins. These coated particles can be used to track the proteins in a live cell and conduct a range of studies at the molecular level.
Molecular motors with nanometer-scaled conformational changes play an important role in cell function. Protein machines, as well as nucleic acids can act as nanomolecular motors.
Studies in genetics, genomics, and cell biology have provided a foundation on which to build an understanding of genome biology. Extending this knowledge will require merging these approaches with additional disciplines and new technologies, such as nanotechnology. Nanobiotechnology has provided novel approaches to DNA extraction and amplification, as well as reduced the time required for these processes to seconds.
Microfluidic devices enable polymorphism detection through rapid fragment separation using capillary electrophoresis and HPLC, together with mixing and transport of reagents and biomolecules in integrated systems. Development of a variety of SNP genotyping platforms is already well advanced through research in the field of nanobiotechnology.
Nanosphere’s (www.nanosphere-inc.com) nanoparticle technology enables a microarray-based method for multiplex SNP genotyping in total human genomic DNA without the need for target amplification. This direct SNP genotyping method requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes, says the company.
Nanomaterials are sensitive chemical and biological sensors and form the basis of numerous nanobiosensors used for research in genomics and applications in molecular diagnostics. Several technologies have been described for the use of nanobiosensors for SNP genotyping and mutation detection(1).
Novel nanotechnology approaches to DNA extraction and amplification have reduced the time required for these processes to seconds and amounts of samples required to nanoliter. Currently it is difficult for scientists to study a single live cell and find what gene it is expressing. However, this is possible by injecting an amplified molecular nanoprobe into the cell and it can be applied for early detection of disease.
Application of nanotechnologies in proteomics has been termed nanoproteomics, which is an extension of the scope of proteomics. Low abundant proteins and proteins that can only be isolated from limited source material can be subjected to nanoscale protein analysis—nano-capture of specific proteins and complexes, and optimization of all subsequent sample handling steps, leading to mass analysis of peptide fragments.
A new detection technique called Multi Photon Detection is in development at BioTraces (www.biotraces.com) and enables quantification of sub-zeptomole amounts of proteins. It can be used for diagnostic proteomics, particularly for cytokines and other low abundance proteins. BioTrace is developing protein biochips to detect as low as 5-fg/mL (0.2 attomole/mL) concentration of proteins. Thus, this innovative type of the P-chips might permit about 1,000 fold better sensitivity than current protein biochips, according to the company.
In nanoflow LC (nanoLC), chromatographic separations are performed using flow rates in the range of low nanoliter per minute that results in high analytical sensitivity. NanoLC, in combination with tandem mass spectrometry (MS), was first used to analyze peptides and as an alternative to other mass spectrometric methods to identify gel-separated proteins. Protein identification using nanoflow liquid chromatography-mass spectrometry-MS (LC-MS-MS) provides reliable sequencing information for low femtomole level of protein digests.
Single-walled carbon nanotubes are being used for investigating surface-protein and protein-protein binding and developing highly specific electronic biomolecule detectors.
Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins, is overcome by immobilization of polyethylene oxide chains. A general approach is then advanced to enable the selective recognition and binding of target proteins by conjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. These arrays are attractive because no labeling is required, and all aspects of the assay can be carried out in solution phase.
This scheme, combined with the sensitivity of nanotube electronic devices, enables highly specific electronic sensors for detecting clinically important biomolecules, such as antibodies associated with human autoimmune diseases. Interfacing novel nanomaterials with biological systems could therefore lead to important applications in proteomics and disease diagnosis.
Atomic Force Microscopy
As a large fraction of proteins are likely to be membrane-bound, technical improvements are needed in the analysis of membrane proteins. Membrane proteins can be visualized by labeling with gold nanoparticles in cells and using photothermal interference contrast method.
In contrast to other methods, specimens prepared for atomic force microscopy (AFM) remain in a plastic state that enables direct observation of the dynamic molecular response, creating unique opportunities for studying the structure–function relationships of proteins and their functionally relevant assemblies.
While electron crystallography provides atomic scale 3-D density maps of membrane protein crystals, AFM gives insight into the surface structure and dynamics at subnanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed.
AFM application in imaging and nanomanipulation include the extraction of chromosomal DNA for genetic analysis, the disruption of antibody-antigen bonds, the dissection of biological membranes, the nanodissection of protein complexes, and the controlled modulation of protein conformations.
Researchers at the Georgia Institute of Technology have created FIRAT (Force sensing Integrated Readout and Active Tip), a sensitive AFM technology that is capable of high-speed imaging, 100 times faster than current AFM technology. FIRAT can capture additional measurements not possible before with AFM, including parallel molecular assays for drug screening and discovery.
Proteins are switched on and off in living cells by a mechanism called allosteric control; proteins are regulated by other molecules that bind to their surface, inducing a change of conformation, or distortion in the shape, of the protein, making the protein either active or inactive.
Scientists at UCLA have made an artificial nanoscale mechanism of allosteric control based on mechanical tension. They chemically string a short piece of DNA around the protein. By inserting a molecular spring on the protein, they can control the stiffness of the spring externally. By gluing together two disparate pieces of the cell’s molecular machinery, a protein and a piece of DNA, they have created a spring-loaded protein that can be turned on and off. This research has the potential to start a new approach to protein engineering.
This could lead to a new generation of targeted smart drugs that are active only in cells where a certain gene is expressed, or a certain DNA sequence is present; such drugs would have reduced side effects.
Genomics and Proteomics
The role of bioinformatics in analyzing data from genomics and proteomics is considered indispensable. As nanotechnology enables greater sensing and collecting of data, the information flow could become measured in petabytes, or quadrillion bytes of information. Accelrys (www.accelrys.com) says its computational nanotechnology modeling using quantum mechanics, classical atomistic methods, and/or mesoscale simulations enable scientists to visualize and predict behavior emerging at length scales of up to 100 nm.
Use of nanobiotechnology in genomics and proteomics forms the basis for applications in molecular diagnostics, drug discovery, drug delivery, gene therapy, RNA interference, and eventually in nanomedicine(2). By refining molecular diagnostics, including applications at point-of-care, and linking diagnostics to therapeutics, nanobiotechnology will facilitate the development of personalized medicine.