Chemical methods of identifying these target molecules include techniques based on molecular mass, composition, functional groups, relative affinity for various surfaces, or other chemical properties. These methods may be complementary to alternative techniques for identifying the same classes of molecules discussed in other chapters (e.g., immunoassays for proteins, discussed in Chapter 7).
Several centuries of effort by many tens of thousands of chemists have gone into developing the equipment in a modern analytical chemistry laboratory. While the full complement of technologies could be useful for bioagent detection or identification, a review of the techniques most often used indicates that only two or three technologies stand out as the most promising candidates for routine rapid bioagent detection. They are discussed below.
Perhaps the most fundamental characteristic of a chemical component is its intrinsic molecular weight or mass. Mass spectrometers characterize molecules by taking advantage of differences in their mass. A simple metabolite may have a mass of several hundred daltons, while some of the large proteins have a mass well over a million daltons. Mass spectrometers can be among the most complex instruments in a chemical laboratory, yet they are highly versatile and are heavily used for many chemical analyses. (See Box 8.1 for a brief primer on mass spectrometers.) Over the past decade, many investigators have explored the application of mass spectrometry to the identification of biothreat agents.
Among the most definitive molecular discriminators of potential biothreat agents are proteins and distinctive lipid, peptide, and polysaccharide components of bacterial membranes and cell walls. Of the several thousand different proteins in a typical bacterium, some may be unique to a particular strain or subspecies and would therefore have excellent potential for the identification of the strain. Other proteins may be common to multiple species of dangerous microorganisms and thus serve as useful flags for the presence of these organisms.
Because of their relatively high cost and complexity, mass spectrometers are better suited to the identification of biological agents rather than their detection. Several different approaches have been taken to the identification of proteins and other complex cell components by mass spectrometry. One, often termed "biomarker fingerprinting," involves generating a mass spectrum of a prepared sample and attempting to match it against a previously collected library of mass spectra of known organisms. This method is the one most often proposed for bioagent identification by mass spectrometry. In this approach, it is not necessary to know the identity or function of the proteins or other complex molecules that are responsible for the signal. Like a fingerprint, it is the pattern of signals that is identified and compared with a list of potential suspects.
The separation of molecules based on their mass is, in principle at least, fairly straightforward. If each molecule in a mixture is given a push in the same direction, the lighter ones will move faster and the heavier ones slower, effecting a separation in space. Alternatively, a force can be applied to a mixture of moving molecules. The lighter ones will be more strongly affected by the force and the heavier ones less affected, due to their inertia, again resulting in a separation. Mass spectrometers of all varieties function on these simple principles and operate in a vacuum to avoid collisions of the target molecules with the nitrogen and oxygen molecules in air.
Mass spectrometers initially put an electric charge on the molecules to form ions. These ions can then be manipulated by an electric or magnetic field, providing the force needed to accelerate them and steer them into a desired path. In a time-of-flight (TOF) mass spectrometer, the same charge is put on all the molecules, and they are accelerated in a straight line so that they fly down an evacuated tube. By measuring how long they take to reach a detector at a fixed position, and taking into account the length of the flight tube and the charge on the ions, one can obtain a mass spectrum that plots the