Biomedical Nanotechnology - Neelina H. Malsch

Ease of use — Because of the wide variety of individuals who need access to biodefense information, systems that are easy to use and do not require significant operator training or intervention are preferred. Ideally, such systems should be passive and their methods of use obvious to individual users. Results should also be easy to interpret.

Because of the potential utility of nanotechnology in the sensing field, recent advances in this area have the potential to significantly contribute to improved biodefense capabilities. Increasing control at the nanoscale level produces opportunities to develop smaller, more specific, power-efficient, and cheaper sensors for chemical and biological agents. The miniaturization of these capabilities increases their potential utility in sensor network applications and has begun to make it possible to integrate them into equipment easily taken into the field by individuals involved in biodefense. Table 5.1 summarizes the general sensor categories discussed in this chapter.

III. NANO-ENABLED SENSORS FOR MONITORING EXPOSURES

Mazzola (2003) reviewed several nanotechnology applications for biotechnology and their anticipated application timelines. The earliest products most applicable to biodefense are microfluidics and nanoscale material manipulations for making sensors. In addition to protecting against threats from agents known to have potential in biological warfare (BW) and terrorism, sensors will have applications to civil problems, for example in providing alerts and characterizing environmental contamination.

These microfluidic and early manipulation products will be followed by composite materials (peptide–lipid assemblies and fabrics) and biosensors (carbon nanotube arrays). Nanotechnology for drug delivery and tissue engineering appears several years from use but is approaching the clinical testing phase today. Nanotechnology that includes the integration of nanoelectric devices such as implantable sensors combined with response systems for drug delivery has the longest timeline to development.

Other sensor technologies are being applied to environmental monitoring for defense against terrorist attacks on water and agricultural supplies or products. For example, Sensicore reports the use of a polyurethane-based sensor membrane with ion-specific binding channels for electrolytes (Yoon et al., 2000) for basic water quality indicators (chlorine, pH, alkalinity, conductivity) and amperometric sensors utilizing permeable polymers for the detection of trace metals such as arsenic, lead, and mercury. The goal is to produce a field-usable device.

Doranz (2003) reported the application by Integral Molecular, Inc., of lipoparticle technology for the detection of biodefense pathogens. Lipoparticles are nanometerscale spheres surrounded by lipid bilayers embedded with conformally intact integral membrane protein receptors of interest. Bindings to the receptors are then read via an optical biosensor mechanism (see Hoffman et al., 2000).*

* See also http://www.integralmolecular.com/lipoparticles.htm (last accessed 12/3/03).

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U.S. Defense Threat Reduction Agency has demonstrated the use of such microsensors to detect chemical warfare agent simulants.

One general class of sensors based on mass measurements for chemical vapors has been suggested in terms of frequency encoding for use with resonant mass sensors (Guan, 2003). It makes use of frequency changes caused by the absorption of molecules on a sensor surface, and the subsequent measurement and application of a Fourier transform method to detect the frequency shift. One objective is to miniaturize the electronics and reduce the size of instrumentation in an effort to make the equipment portable. Such methods have been demonstrated for multisensor detectors. Biosensors serve as natural conduits for detecting bioagents and provide for biodefense detection and identification needs. Nanotechnology can tailor and miniaturize biosensors and furnish additional phenomenology and sensor architectures for detecting threats.

1. Biosensors

The use of organisms, subcellular structures, and biomolecules in sensors is well known. Many types of biological materials or molecules can act as receptor components, e.g., antibodies. These must be combined with detectors such as gas-sensing electrodes. A detector may be miniaturized but remain far from being a nanotechnology component even though its scale has been reduced. It is often advantageous to use a fluid environment for supporting these components in a detection system, and many examples will be discussed in the section on monitoring contact exposures. Biosensors may also apply to gas phase detection.

An example is the piezoelectric immunosensor. This device measures changes in mass on a crystal surface by measuring changes in the resonant frequency of a piezoelectric crystal (Kumar, 2000). The change is proportional to the sampling time and the concentration of analyte in a fluid flowing past the surface. A highly selective receptor is required. Kumar described the application of this class of sensor for detecting tuberculosis and other mycobacterial antigens.

It is often valuable for human health considerations to minimize detection and identification times. Development of real-time biosensors could also aid in responses required to characterize a terrorist attack on populations, agriculture, or food supplies, and minimize the harm done. In the case of a waste site, sensors can be employed at borders and neighboring sites or on samples retrieved from these locations to help manage outflows of effluents (or groundwater for contact sensors such as those discussed in the following section).

An operational issue for any biodefense sensor system is that even inexpensive sensors have practical limitations on what sites and areas they can cover if they must be widely distributed to be effective. In particular, caution must be exercised to recognize that small size, reasonable cost, and detection sensitivity do not necessarily mean that an impregnable field of sensors can be dispersed in an area to catch a potential threat. The gas throughput and diffusion of agents to the volume actually sampled may impose practical limits on detectability.

Many pressing problems could benefit from the continued development of biosensors. Future challenges will include the need to rapidly identify dangerous toxins

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single-layer organic shells. Importantly, they were found to be capable of detecting many classes of vapors including those of a number of explosives.

SAW devices use a piezoelectric substance upon which an acoustic wave is generated by an electric field, travels along the surface, and is affected by any vapors present. This interaction may be detected and analyzed. These types of detectors have been the subjects of numerous studies and may be used to search for explosives and hazardous materials.

Yinon concludes that the many types of detectors now in development may apply across a broad set of problems. The threat of terrorism with conventional weapons makes the development of small, inexpensive explosives detectors that can be massproduced of high interest. The same classes of sensors that protect against chemical and biological agents may be used for detecting explosive molecules. Protection of aircraft against smuggled bombs is another clear example where detectors designed for picking up explosives are of interest. An important question for further consideration is whether detectors at checkpoints could reduce the incidence of suicide bombers who proceed through public areas. The manner in which the sensors are deployed as well as their technical limitations must be considered for feasibility of use in any scenario.

The detection and identification of pathogens and biological agents in general can also be performed with an electronic nose, as indicated by the previous discussion of biosensors. This has been demonstrated with discrimination among anaerobic bacteria grown in vitro (Pavlou et al., 2002). Multisensor arrays containing conducting polymers were used to detect gases. Again, this detection method employed neural nets. Other techniques are, of course, also applicable to detecting organisms and may have their own advantages (e.g., mass spectrometry can be used to identify species).

Conducting polymers have been used to discriminate wine aromas (Guadarrama et al., 2001) and this example suggests nanosensors may eventually lead to food characterization — i.e., determining when a food has gone bad. Preservation is a more ambitious goal and requires sensing to detect processes before spoilage.

MEMS-based noses use cantilevers that respond when exposed to gaseous analytes. One such array uses the swelling of one or more polymer coatings as an indicator for detection using optical measurements of deflection (Baller et al., 2000). Experiments with these devices investigated the detection of organic molecules including methanol, toluene, and ethanol and will be further discussed later.

Biological threats and inorganic toxins (or explosives in the case of terrorism) are not the only hazards that may be detected with devices that operate at the nanoscale. It is also possible to detect the presence of radioactive materials with cantilevers that are kept at a distance of a few nanometers from an insulated metal surface (Thundat and Brown, 2002). Alpha particles can be detected as a result of charge accumulation or frequency shift. This method can be very sensitive. Single alpha particles have been detected. The time required for detection can be improved with the use of large-area detectors. The technique could be useful in detecting many types of low level activity by sampling gas, but alpha particles have very short ranges and will not penetrate far, so the method will not detect radioactivity that is well contained or shielded by soil or other factors. Vapors from a suspected contaminated material are needed or a detector must be demonstrated to be capable of measuring other, more penetrating particles such as gamma rays.

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One method for exploiting fiberoptics is to bind antibodies to the fiber, allow antigens to be captured, introduce antibodies with fluorophores, and sample these with a laser reflected through the fiber (Lim, 2003). This method may be useful for environmental monitoring for pathogens because a highly sensitive and specific method reduces the time required to cultivate, separate, and identify organisms and other biological agents. Lim reports that very good sensitivity and selectivity can be achieved, for example, detection of Bacillus anthracis at 105 colony-forming units/ml, cholera toxin at 100 pg/ml, and TNT at 10 ng/ml. In addition to high sensitivity, Lim points out that biosensors can reduce the time required to detect these hazards from hours or days to minutes. This capability would enable detection of contaminants in the field.

Binding of fluorophore molecules (placing functional groups on a structure) is an example of a type of approach to nanotechnology. Investigations of the properties of such molecules as DNA and proteins further demonstrate the connection of biotechnology and nanotechnology. Biological structures and molecules as detection devices can be integrated with many devices other than fiberoptics. For example, microbeams can be fabricated with nanostructured surfaces that detect proteins (Dutta, 2342). Microbeams and additional sensor enablers will be discussed in the following sections.

In pursuit of highly miniaturized sensors for detecting biological molecules and chemical agents at very low concentrations, a number of nanoscale detection techniques have the potential to achieve nearly single-molecule detection. One family of technologies includes an entire sensor built at the nanoscale. These technologies have as their bases individual nanostructures such as nanotubes, nanowires, nanoparticles, or microcantilever systems.

1. Microcantilevers for Biosensing

Scientists have been using micromachined cantilevers as force probes in atomic force microscopy (AFM) for several years. The extreme sensitivity of the probes has prompted research into incorporating these structures into biosensors. Recent studies report success in detecting the binding of biological analytes on the surfaces of the microcantilevers that cause tiny conformational changes within the microcantilever structure. Depending on the type of analyte for which the device is designed, the magnitude of the conformational change, and the sensitivity and specificity of the microcantilever structure, these systems are useful in various kinds of biosensors that may be critical tools in biodefense efforts.

Microcantilevers are tiny plates or leaf structures, usually measuring 0.2 to 1.0 μm thick, 20 to 100 μm wide, and 100 to 500 μm long. One end is connected to a support. In order to make them useful as biosensors, the plates, usually made of silicon, are coated on one side with a different material that can then be functionalized in various ways. Ideally, when exposed to the analyte of interest, the functionalized side will undergo stress-induced conformational changes while the other side remains unaffected, resulting in plate deflection.

Three mechanisms of analyte-induced stresses have been described (Sepaniak et al., 2002): compressive stresses caused by the physical expansion of the

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Recent studies have taken advantage of spectroscopic techniques including the control of fluorescence intensity (Dubertret et al., 2001) and spectrum fingerprinting (Cao et al., 2002; Gerion et al., 2002). Other approaches have involved measuring electrochemical changes resulting from hybridization or enzyme activity (Park et al., 2002; Chakrabarti and Klibanov, 2003; Xiao et al., 2003). In each case, the binding of the complementary molecule induces a measurable change in the nanoparticle that is then detected.

Using nanoparticles or nanocrystals in biosensors can enhance the speed, portability, sensitivity, and selectivity of the process. For instance, the need for a laboratory to process samples through the sensor may be removed with the development of dry reagent systems (Glynou et al., 2003). The instability that often plagues DNA hybridization techniques can be alleviated through the use of DNA analogues (Chakrabarti and Klibanov, 2003). Unlike the microcantilever systems, this type of sensor is not often used to detect or measure the extent of nucleotide base mispairings; however, in some cases, this level of sensitivity is achieved through techniques that exploit the decreasing stability of imperfectly hybridized oligonucleotides (Dubertret et al., 2001; Park et al., 2002).

3. Functionalized Nanotubes and Nanowires

Other recently developed tools useful in the miniaturization of biosensing devices include nanowires and nanotubes. Similar to both microcantilevers and nanoparticles, nanotubes and nanowires can be integrated into a biosensor through functionalized coatings and built-in detection mechanisms. Nanotubes are particularly promising because of their durability and extreme sensitivity to electronic transport and voltage caused by interjunction temperature differences (Baughman et al., 2002). Their shape and durability make them ideal candidates for integration into portable self-contained sensing devices (chips or other immobilized arrays) that will perhaps make on-site applications away from the laboratory possible.

These qualities have been used in several studies demonstrating successful biosensors for DNA, proteins, and enzymes. It has been shown that DNA (Williams et al., 2002) and proteins (Besteman et al., 2003; Star et al., 2003) retain their biological activities when covalently bound to nanotubes. Subsequent analyte binding to these active molecules results in detectable changes in the nanotubes that can be measured at extremely high sensitivity, in some cases even allowing the detection of a singlemolecule redox reaction (Besteman et al., 2003). The tiny electrochemical or physical changes on the surface of the tube or wire resulting from the biological reaction of interest cause measurable changes in its conductance or resistance that can then be easily detected and quantified (Kong et al., 2000; Besteman et al., 2003). Nanotubebased DNA sensors can be made even more stable when DNA analogues are used (Williams et al., 2002) and protein sensors gain even greater specificity when shorter nanotubes are used (Besteman et al., 2003).

Mere detection of a biological material is not sufficient. One issue with detection technologies is to determine whether the organism is alive so that a terrorist’s use of dead anthrax spores, for example, does not trigger a response that gives a false indication of the threat. Depending on the sensing mechanism, sensors at the

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