2.1 Introduction
2.2 Instrumentation
2.3 Sample Requirements
2.4 Imaging
2.5 Case Studies
2.6 SEM at Binghamton
2.7 References

4.1
4.2
4.3
4.4
4.5
4.6
4.7

6.1 Intoduction
6.2 STM
6.3 AFM
6.4 Case Study
6.5 SPM at BU
6.6 References

7.1 Thermal Analysis of Materials
7.2 Preparation of Nanofibers
7.3 Nanoindentation Analysis of Materials
7.4
7.5
7.6
7.7

3.1
3.2
3.3
3.4
3.5
3.6
3.7

5.1
5.2
5.3
5.4
5.5
5.6
5.7

1.3 Nanobiotechnology

Advancements in science and technology have allowed for the broadening of horizons and the miniaturization of amazingly complex devices. No longer is the brink of technology on another planet or somewhere deep in space with a giant, multi-billion dollar international space station, but deep within our bodies and the bodies of other organisms. Soon we will be able to construct devices that can help us in our day-to-day lives yet be so small that we will never even know that they are there.

Biological nanotechnology, or nanobiotechnology, is the incorporation of nano-scale machines into biological organisms for the ultimate purpose of improving the organism’s quality-of-life. To date there are a few methods for synthesizing nano-devices that have the potential for being used in an organism without risk of being rejected as antigens. Any nano-device needs a motor to power the device and a power supply for that motor. A motor is any object or device that can contain moving or fixed parts that converts one form of energy to another. The entire process that the energy undergoes during conversion from one form to another inside the motor is considered a mechanism.

Currently, research is being done into using the ATPase rotor-pump inside cells and mitochondria to power nanobiosystems. Other research involves the identification of pathogens in the human body and the destruction of these detrimental cells, drug delivery to sick cells, and the treatment and prevention of cancer using nanodevices

1.3.1 Nanomotors inside the body

“NEMS, or nano-electro-mechanical systems” (Montemagno 225) are biologically-based nanosystems that are powered by biological motors and chemical energy sources. F1 ATPase is a biological pump that exists in mitochondria which phosphorylates ADT into ATP, providing an energy supply for the cell as shown in Figure 1.

Figure 1

“The force generated by this motor protein is >100 pico-Newtons, which is among the greatest of any known molecular motor” (Montemagno 225). Thus, this protein has the potential to be an almost perfect nano-motor to power nano-devices.

For the nano-devices to be powered by the ATPase, modifications have to be made to the ATPase so it will provide energy for the mitochondria and the cell as well as the nano-devices, including “[M]utations in the g initiation codon from GTG (Valine) to ATG (Methionine), and Stop Codons from TAG to TAA” (Montemagno 226). Figure 2 depicts the structures of Valin and Methionin below.

Figure 2

Still further modification is needed for the attachment and motion of the specific biomolecular motors. This is accomplished by attaching a Histidine containing synthetic peptide, a Histidine tag, to the g subunit, which creates unique Cysteine residues. These special residues can allow for the attachment of both inorganic and organic compounds using thiol chemistry. This modification is accomplished using thermophilic bacterium in vitro. This process is outlined in Figure 3, the diagram above.

Figure 3

This setup using ATPase, a naturally occurring proton pump, accomplishes three goals: First, it provides easy manipulation of the codon sequence to supply power for nano-devices. Secondly, it allows for the possible production of large quantities of proteins. Finally, the setup imparts the ability to attach “handles” such as the Cysteine residues for the attachment of nano-devices and nano-motors. Still the question remains: how does one build a nano-device, now that we know how to power it?

One answer is structural DNA nanotechnology. This term describes the “construction of nano-sized molecules from the basic DNA components” (Seeman 7260). Molecules are assembled via ‘sticky end’ interaction and bonding among strands of DNA by use of RFLPs. Figure 4 shows the interaction of sticky ends after the DNA has been cut via RFLP, EcoR1. In structural DNA nanotechnology, instead of just working with double helix shaped DNA, the RFLPs are made to interact with the DNA to produce three-dimensional structures and molecules that interact with each other and cell membranes based on polarity and topological structure.

Figure 4

Reciprocal exchange among DNA strands leads to more and more complex and precise series of DNA strands until you get a strand that can generate the molecule or shape that you desire. Figure 5A shows two different DNA strands interacting with each other. Figure 5B shows the interaction of the end product with another strand producing a still more complex double helical structure.

Figure 5A & B

This type of reciprocal exchange amongst double helixes produces branched junctions in the molecular structure that are flanked by four other double helixes. That allows for an extreme amount of biological diversity, as the formula is exponential; “reciprocal exchange has been found and done between three, four and five helixes and there is no obvious limit to the number of branched junctions that may flank such a structure.” (Seeman 7261). Figure 6A shows a cube-like structure of DNA developed by reciprocal exchange and branched junction formation with sticky ends of DNA being used to close helical axis of the cube. Figure 6B shows a fourteen-Catenane structure that consists of six double helical strands combining to form the square faces and double helical strands that, together, form the hexagonal faces.

Figure 6A & B

This interaction of DNA strands shows the variability and potential in nano-creation with DNA so that the body does not develop an immune response or consider the nano-device an antigen. Synthesis of such nano-machines is currently in progress at many laboratories, including Rutgers, which is developing a viral protein nano-motor seen on the right in Figure 7. The peninsula-shaped objects off the structure would be used to attach to the His or Cys residues on the ATPase and would be used to power the motor throughout the body.

Figure 7

Theoretically, such motors could be dispersed throught some liquid medium and then injected, implanted, or swallowed into the body. Once the motors are inside, they should be programmed to perform specific tasks such as recognizing problematic cells and then either repairing or destroying them. Various methods will be discussed pertaining to these functions.

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1.3.2 Applications

Biosensors. Biosensors are the key to identifying pathogens. According to Ashok Kumar, a biosensor is “…an analytical tool consisting of biologically active material used in close conjunction with a device that will convert a biochemical signal into a quantifiable electrical signal.” Two parts define a biosensor: a receptor and a detector (see Figure 8). While the receptor is responsible for the selectivity of the sensor, only joining with antigens having the designated frequency, the detector translates this change and sends the results via electronic signal.

Figure 8

Diverse biosensors have been developed utilizing cantilevers, optical fibers, and stressed antibodies to detect viruses and other kinds of disease. The simplest and probably most accurate method of identification is the cantilever method. A cantilever, made of silicon, looks somewhat like the diving board of a swimming pool (see Figure 9). Like a diving board, the cantilever has a fixed resonance frequency, dependent on its mass. It is very sensitive to additional mass placed upon it in the form of adsorbed molecules. The heavier the total weight is upon the cantilever, the lower its frequency. Cantilevers typically measure four microns long, one micron wide, and about thirty nanometers thick (Gupta). Consequently, the cantilevers are extremely sensitive and will bend under the weight of something as small as a single virus particle -- a particle that can be as few as twenty nanometers in length.

Figure 9

Once a particle lands on the cantilever, there are several ways to determine how much the cantilever is bent. The most common method is called optical beam deflection (Figure 10), in which a laser diode is shone upon the free end of the cantilever, and then the reflected beam is analyzed by a position-sensitive detector. Since different particles generally have unique masses, a biosensor can identify them by comparing the mass upon the cantilever to a large database of particle masses located within the nano-electro-mechanical device.

Figure 10

Optical fibers can also be used in biosensors. The basic idea behind this is that an antibody is attached to the end of a fiber optic cable, and when the cable and antibody make contact with the antigen (if indeed it exists within the sample), a reaction occurs that gives off visible light, which is then channeled through the cable to a device which can measure its intensity and report back the results. This method also has environmental applications, such as detecting cancer-causing agents in groundwater. That particular application was successfully accomplished in 1987 to detect the carcinogen benzopyrene (Jacobson). The biggest benefit of this method is that there is no possibility for electrical interference.

Finally, the stressed-antibody technique is another method for identifying pathogens. It is conceivably possible to measure the physical stress placed upon the antibody during the reaction with the antigen. This can be done through use of piezoelectric crystals such as quartz (see Figure 11), the resonant frequency of which changes based on the pressure of mass at the crystal’s surface (Kumar, Biosensors).

Figure 11
quartz crystal, a piezoelectric material from
http://dph1701.tripod.com/geology/properties/luster.html

While biosensors are useful in their own right, having the potential to inform doctors and other professional medical workers of a patient’s diagnosis, the most useful biosensors should have the ability to treat diseases as well. This would involve either delivering medication to problem cells or destroying the targeted cells.

Drug Delivery. The benefits of drug delivery at the cellular level are enormous. By only targeting the afflicted cells, less of the drug is needed, reducing side effects and making the drug less expensive. Also, since the drug only needs to go to certain targets instead of the whole body, it works faster to relieve the patient. Special macromolecules have been used in the past to carry the drug, but there are special benefits to using micro- and nano-particles. The smaller the drug-carrying unit, the more it tends to concentrate itself in enflamed areas (Kumar, Nano). Using nanobiotechnology, drug delivery can be accomplished by encapsulating the drug inside a membrane with channels that open and close according to outside stimuli (see Figure 12). According to Ashok Kumar, these channels can also be “modified by genetic engineering to enhance the permeation of a specific substrate…[such that it acts] as a prefilter, keep[ing] off other substrates, and thus enhanc[ing] the sensitivity of the enzyme.”

Figure 12

Cell Destruction. One method of destroying cells that is still being researched involves magnetizable beads. Basically, these beads are attached to the target cells using an antigen-antibody reaction, and then are aggregated by a magnet. Finally, a magnetic pulse is sent through the compounds, causing the beads to rupture the target cells (see Figure 13). This method of destruction is far superior to methods used now to kill cancer cells, like chemotherapy. While chemotherapy targets all cells, both the cancerous and the beneficial, the bead method targets only those that respond to the antigen-antibody reaction.

Figure 13

Another method of targeted cell destruction is called laser-beam-triggered microcavitation. Short bursts of laser light are used to heat the target particles, which causes them to reach a temperature over 100°C. This causes the fluid surrounding the particles to vaporize, which leads to another process called microcavitation, or the creation of temporary small bubbles by a moving source. Small bubbles created in the cytoplasm rapidly expand and collapse, causing local cell damage and often, cell death (Leszczynski, 399).

Cancer Treatment. Most animal cells have diameters of 10,000 to 20,000 nanometers. Since nanoscale devices have at least one dimension less than 100 nm, they will easily be able to enter cells and the organelles inside in order to interact with DNA. Specifically, nanodevices could be used to detect cancerous cells.

One such device that could locate cancerous cells at a very early stage is the cantilever. Cantilevers on a nano scale could be engineered to attach to cancer-associated molecules. This could be done by binding to certain DNA sequences or proteins that have been linked to cancer. After the cantilevers are attached, any changes in the surface tension of the DNA or protein would cause the cantilever to bend. By monitoring the cantilevers it is possible to pinpoint exactly which cells are cancerous, even if there are very few of them, i.e. at a very early stage. Also, other devices could be designed to actually read the genetic code of a cell and search for errors in the DNA that may contribute to cancer. It is believed that devices with nanopores, holes through which only one DNA strand can pass at a time, will make this process of DNA sequencing more efficient. As a strand passes through a nanopore (see Figure 14), the shape and electrical properties of each base can be monitored, thus enabling scientists to decode the DNA one base at a time in their search for errors.

Figure 14

Yet another way that nanodevices could be useful in the detection of cancer comes in the use of quantum dots (Figure 15). Quantum dots are tiny crystals that glow under ultraviolet light. The wavelength emitted by the dot depends on the size of the crystal. Thus, by manipulating their size, crystals can be manufactured to emit any desired wavelength. These crystals could be incorporated into latex beads designed to bind to specific DNA sequences associated with cancer, and would act as a dye when under UV light, thus pinpointing the exact location of the faulty DNA sequence. Since many crystals can be inserted into a single latex bead, the combination of crystals could create a spectrum as unique as a bar code,and can be used to identify specific regions of DNA.

Figure 15

Beyond the detection of cancer, nanodevices can also be used to treat cancerous cells. Because nanodevices are so small, they can target specific cells, leaving neighboring non-cancerous cells untouched (Figure 16).

Figure 16

Eventually, scientists hope to design a nanodevice that circulates through the body, detects specific cancerous cells, assists with imaging the cells, delivers a toxin to destroy the cells, and monitors the effectiveness of the toxin. Currently, research is being done using a nanoparticle called a dendrimer, shown in Figure 17.

Figure 17

Dendrimers have a very branched shape, giving them large amounts of surface area to which scientists can attach all of the necessary components to fulfill the description above. Another nanodevice that could be used to destroy cancerous cells is a nanoshell. Nanoshells are tiny beads coated with gold, a simulation of this is shown in Figure 18. By changing the thickness of the gold coating, scientists can design the shells to absorb a specific wavelength of light. It has been found that the shells that are the most useful absorb near-infrared light, which is able to travel through several centimeters of human tissue without harming it. When nanoshells absorb this light, an intense heat is created that is lethal to cells. Nanoshells can be linked to antibodies that seek out cancerous cells. Then, when near-UV light is applied the heat generated would destroy the cancerous cells. When attempted in laboratory cultures, this technique destroyed the target cells while leaving neighboring cells unharmed.

Figure 18

These ideas are in various stages of research and development. Researchers hope to have quantum dots, nanopores, and other detection devices ready for clinical use within 5-15 years. Devices that both detect and destroy are more complicated, and thus may take as long as 20 years to design and perfect.

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