Medical brain notes 43

  QUANTUM SIZE EFFECT AND NANOCRYSTAL COLORS When materials are subdivided into sufficiently small fragments, quantum effects begin to influence their physical properties. The fluorescent nanoparticles discussed earlier are in fact  semiconductors  that are small enough to show such quantum effects.   Semiconductors are substances that conduct electricity under some conditions but not others. In  N-type  semiconductors (as in normal electric wires) the current consists of negatively charged electrons. In  P-type  semiconductors the current consists of  holes .   A hole is the absence of an electron from an atom. Although not physical particles, holes can move from atom to atom. Electrons and holes may combine and cancel out, a process that releases energy. Conversely, energy absorbed by certain semiconductors may generate an electron-hole pair whose two components may then move off in different directions.   Nanoparticle labels can ...

  NANOPARTICLES FOR DELIVERY OF DRUGS, DNA, OR RNA Because nanoparticles can be targeted to specific tissues, they can be used to deliver a variety of biologically active molecules, including both pharmaceuticals and genetic engineering constructs.   Large polymeric molecules such as DNA may themselves be compacted to form nanoparticles of around 50 to 200 nm in size. This involves addition of positively charged molecules (e.g., cationic lipids, polylysine) to neutralize the negative charge of the phosphate groups of the nucleic acid backbone. Other molecules may be added to promote selectivity for certain cells or tissues.   Alternatively, hollow nanoparticles  (nanoshells)  may obviously be used to carry other, smaller molecules. Such nanoshells must be made from biocompatible materials such as polyethyleneimine (PEI) or chitosan. The latter alternative seems popular at present, because it is both naturally derived and biodegradable. Chitin is a beta-1,4-linke...

  NANOPARTICLES IN CANCER THERAPY It is possible to destroy tumor cells by a variety of toxic chemicals or localized heating. In both cases a major issue is delivering the fatal reagent to the cancer cells and avoiding nearby healthy tissue. When using toxic chemical reagents, the reagent must be not only  delivered specifically to the target cells but also prevented from diffusing out of the cancer cells. Both related objectives may be achieved by using hollow nanoparticles to carry the reagent. Nanoparticles may be targeted to tumors by adding specific receptors or reactive groups to the outside of the nanoparticles. These are chosen to recognize proteins that are solely or predominantly displayed on the surface of cancer cells. It is hoped that such nanoparticles will be safe to give by mouth. Diffusion is more difficult to deal with, but may be limited to some extent by designing nanoparticles for slow release of the reagent. A clever alternative is to produce the toxic ag...

  ASSEMBLY OF NANOCRYSTALS BY MICROORGANISMS It has been known for many years that bacteria may accumulate a variety of metallic elements and may modify them chemically, usually by oxidation or reduction. For example, many bacteria   accumulate anions of selenium or tellurium and reduce them to elemental selenium or tellurium, which is then deposited as a precipitate either on the cell surface or internally. Certain species of the bacterium  Pseudomonas  that live in metal-contaminated areas and the fungus  Verticillium  can both generate silver nanocrystals. Recently, it has been found that when  Escherichia coli  is exposed to cadmium chloride and sodium sulfide, it precipitates cadmium sulfide as particles in the 2- to 5-nm size range. In other words, bacteria can “biosynthesize” semiconductor nanocrystals. Rather more sophisticated is the use of phage display to select peptides capable of organizing semiconductor nanowires. As described, phage...

  NANOTUBES Carbon  nanotubes  are cylinders made of pure carbon with diameters of 1 to 50 nanometers. However, they may be up to approximately 10 micrometers long. Pure elemental carbon exists as diamond or graphite. In diamond, each carbon is covalently linked to four others forming a 3-D tetrahedral lattice that is extremely strong. In contrast, graphite consists of flat sheets of carbon atoms that form a hexagonal pattern. In the sheets of graphite, each carbon atom is covalently bonded to three neighbors and the sheets can slide sideways over each other, because there are no covalent linkages between atoms in different sheets. To form a nanotube, a single sheet of graphite is rolled into a cylinder. The sheets may be rolled up straight or at an angle to the carbon lattice and may be of various diameters.   Depending on the diameter and the torsion, the nanotube may act as a metallic conductor or a semiconductor. Not surprisingly, nanotubes are now finding many u...

  ANTIBACTERIAL NANOCARPETS Nanocarpets  are formed by stacking a large number of nanotubes together, with their   cylindrical axes aligned vertically. Nanocarpets capable of changing color and of killing bacteria have been assembled from specially designed lipids that spontaneously assemble into a variety of nanostructures depending on the conditions. In water, nanotubes are formed. Partial rehydration of dried nanotubes generates a side-by-side array- the nanocarpet. The lipid consists of a long hydrocarbon chain (25 carbons) with a diacetylenic group in the nanotubes are about 100 nm in diameter by 1000 nm in length. The walls of the nanotubes consist of five bilayers of the lipid. Both the separate lipid molecules and the assembled nanocarpet kill bacteria. Like other long-chain amino compounds, they act as detergent molecules and disrupt the cell membrane. Consequently, the nanocarpet provides a surface lethal to bacteria. This property could be very useful if nanoca...

  DETECTION OF VIRUSES BY NANOWIRES Nanowires  are what their name suggests. They have nanoscale diameters but may be several   microns long. They may be metallic and act as electrical conductors or they may be made from semiconductor materials.   Biosensors can be made using silicon semiconductor nanowires. These may be coated with antibodies that bind to a specific virus. Binding of the virus to the antibody triggers a change in conductance of the nanowire. For a p-type silicon nanowire, the conductance decreases when the surface charge on the virus particle is positive and, conversely, increases if the virus surface is negative. Single viruses may be detected by this approach (Fig. 7.14). It is also possible to attach single-stranded DNA to the nanowire. In this case, conductance changes are triggered by binding of the complementary single strand. Possible future applications include both clinical testing and sensors for monitoring food, water, and air for public ...

  ION CHANNEL NANOSENSORS Somewhat more complex than nanotubes and nanowires are  nanoscale ion channels  that are assembled into membranes.   These channels are designed so that they can be controlled to permit the movement of ions under only certain conditions. The ion flow generates an electrical current that is detected, amplified, and displayed by appropriate electronic apparatus.   Ion channels can be used as biosensors by attaching a binding site for the target molecule at the entry to the channel. Attached antibodies are often used for the binding sites as described (see Fig. 23.16). The simplest arrangement results in the channel being open in the absence of the target molecule and shut when it is detected. A drop in ion flow therefore signals detection of the target molecule.   At present, such ion channels are being developed using modified biological components. The ion channel itself can be made using the peptide antibiotic gramicidin A (made b...

  NANOENGINEERING OF DNA In “classical” genetic engineering, the sequence of DNA is deliberately altered in order to generate new combinations of genetic information. Even when major rearrangements are  made, in order to function as genetic information, the DNA must remain as a base-paired double-stranded helix with an overall linear structure.   In nanoengineering, the objective is to make structures using DNA merely as a structural element, rather than to manipulate genetic information. DNA is attractive because the double helix is a convenient structural module. Moreover, its natural base-pairing properties can be used to link separate DNA molecules together. However, a critical requirement for assembling 3D structures is branched DNA. Although branched structures do form in biological situations (especially the Holliday junction involved in crossing over during recombination), they are not permanent or stable.   Cross-shaped DNA can be generated by mixing four ca...

  DNA MECHANICAL NANODEVICES A rather more futuristic use for 3-D DNA structures is as frameworks for mechanical nanodevices. The essential components are some sort of moving parts. Several experimental prototype “DNA machines” have been designed or constructed that illustrate the concept. These all involve reversible changes in conformation of a DNA structure driven by changes in base-pairing. Such changes may be caused either by changing the physical conditions (heat, salt, etc.) or by adding segments of single-stranded DNA (ssDNA) that base-pair to some region of the DNA machine, as illustrated in Fig. 7.18. If ssDNA is used, then another single strand, complementary to the first, is added to convert the machine back to its original conformation. The result is a mechanical cycle that could in principle be used to perform some sort of task. The two ssDNA molecules may be regarded as “fuel,” and the final waste product is a double-stranded DNA consisting of the two paired ssDNA fu...

  CONTROLLED DENATURATION OF DNA BY GOLD NANOPARTICLES DNA hybridization is widely used to detect target sequences, both in the laboratory and in clinical diagnosis. Before hybridization can occur, the DNA double helix must be denatured into single strands. This is accomplished by the heating of bulk DNA. However, newly emerging nanotechnology may allow specific individual DNA molecules to be dissociated when required. Nanoparticles of about 1.4 nm and containing fewer than 100 atoms of gold are attached to double-stranded DNA. When the structure is exposed to radio waves (generated by an alternating magnetic field), the gold acts as an antenna. It absorbs energy and heats the DNA molecule to which it is attached. This melts the DNA double helix and converts it to single strands. Heating extends over a zone of about 10 nm so  surrounding molecules are unaffected. The heat is dissipated in less than 50 picoseconds, so the DNA may be rapidly switched between the double- and sing...

  CONTROLLED CHANGE OF PROTEIN SHAPE BY DNA Allosteric proteins change shape in response to the binding of signal molecules (allosteric effectors) at a specific site. The essence of allosteric control is that the shape change is transmitted through the protein and affects the conformation of distant regions of the protein. In allosteric enzymes, binding of an allosteric effector at a distant site alters the conformation of the active site and may change its affinity for the substrate. In this way, some enzymes are switched on and off in response to signal molecules. For example, phosphofructokinase is switched on by the buildup of AMP, which signals that energy is in short supply. The response increases flow into the glycolytic pathway. Similarly, many DNA binding proteins, such as repressors and activators, also change shape on binding small signal molecules. It is possible to change the shape of a protein artificially by mechanical force. This has been demonstrated by attaching a...

  BIOMOLECULAR MOTORS A major aim of nanotechnology is to develop molecular-scale machinery that can carry out the programmed synthesis (or rearrangement) of single molecules (or even atoms), or other similar nanoscale tasks. The term (nano) assembler  refers to a nanomachine that can build nanoscale structures, molecule by molecule or atom by atom. And the term (nano) replicator  refers to a nanomachine able to build copies of itself when provided with raw materials and energy. This, of course, sounds remarkably like a living cell. Indeed, the organelles of living cells may be regarded as nanomachines and have provided both inspiration and components for nanotechnologists. To operate, nanomachines will need energy, which will be provided by “molecular motors.” At present such devices are still in development. It has been suggested that biological structures might be used for this purpose. Examples include the ATP synthase, the flagellar motor of bacterial cells, various ...

  Genomics and Gene Expression INTRODUCTION   Sequencing the entire human genome was a daunting task conceived by an initiative from the Department of Energy in 1986. The goal was to have a high-quality reference set of sequence information from each human chromosome. The initiative was strengthened when the National Institutes of Health (NIH) joined the effort in 1990. During the 1990s, many other collaborators around the world joined the effort. Finally, in June 2000, the first working draft of the human genome was announced. The sequence was refined, and a final high-quality sequence was finished in April 2003. The Human Genome Project has also involved sequencing entire genomes of model organisms such as mouse and  Drosophila , enhancing computational methods for sequence data analysis, comparing the function of genes among different organisms, and studying human variation.   The availability of genome sequences has revolutionized many areas of biology from the c...

  GENETIC MAPPING TECHNIQUES   Genomes are sequenced by making libraries of genomic DNA segments and then sequencing each of the segments. These stretches must then be compiled into the final sequence. To structure the sequence data into a draft genome, the Human Genome Project started by compiling a working genome map.  Genome maps  provide various landmarks for use when putting together sequence data. There are two different categories for genome maps,  genetic   maps  and  physical maps . Genetic maps are based on the relative order of genetic markers,   but the actual distance between the markers is hard to determine. Physical maps are more precise and give the distance between markers in base pairs.   Traditional genetic maps are based on the recombination frequency between genes.   In eukaryotic cells, recombination occurs between homologous pairs of chromosomes during meiosis. If two genes are close together on the same chrom...

  PHYSICAL MAPS USE SEQUENCE DATA Genetic markers such as SNPs, VNTRs, RFLPs, and microsatellites are useful, but for large genomes like the human genome, these still do not provide enough markers. The map builders needed other types of markers, such as  sequence tagged sites (STSs)  (Fig. 8.4). These are simply short sequences of 100–500 base pairs that are unique and can be detected by PCR. A specialized type of STS is the  expressed sequence tag (EST),  so called because it was identified in a cDNA library. This means that the EST is expressed as mRNA. These small pieces of sequence data are just portions of larger genes; therefore, many different ESTs may be found for one single gene.   Mapping physical markers resembles linkage analysis for genes in the sense that the closer they are, the more likely they will remain together. However, linkage for physical markers  is determined by restriction enzyme digestion (Fig. 8.5). Either entire genomes or ...

  RADIATION HYBRID AND CYTOGENETIC MAPPING Library clones can sometimes be unreliable because large cloned segments may actually consist of two fragments of DNA, from different parts of the genome, inserted into the same vector.  Radiation hybrid mapping  overcomes these limitations by examining STSs or ESTs on original chromosomal fragments (Fig. 8.7). To generate these, cultured human cells are treated with x-rays or  γ -rays to fragment the chromosomes. The radiation dosage controls how often the chromosome breaks, and thus the average length of the fragments. The human cells possess a marker enzyme that allows them to grow on selective media. After irradiation, the human cells are fused to cultured hamster cells using polyethylene glycol or Sendai virus. The hamster cells do not have the selective marker. Consequently, only those hamster cells that fuse with human cells survive. The fragments of human chromosomes become part of the hamster nucleus, and the indivi...

  SEQUENCING ENTIRE GENOMES Sequencing the entire genome from one organism can be accomplished in different ways.  Chromosome walking  allows the researcher to identify and sequence one clone and   then, using those data, to find overlapping clones (Fig. 8.9). After those are identified  and sequenced, more overlapping clones are identified. The process goes in order either up or down the chromosome, compiling the sequence piece by piece. Usually the first clone is located relative to a particular marker, such as an STS  or RFLP. Chromosome walking is often used to characterize genes responsible for a particular disease. Analysis of DNA from people with the disease may have revealed a particular RFLP that is always present in those with the disease, but absent in unaffected people. This RFLP can be identified in a library clone. Then chromosome walking both upstream and downstream of the RFLP will, it is hoped, provide the whole gene sequence. Although chro...