Molecular Binding At Nanoprobe
biochemical mechanisms for recognition. The main purpose of the recognition system is to provide the sensor with a high degree of selectivity for the analyte to be measured.
Several transduction methods are used in biosensors. The three main methods are based on optical detection, electrochemical detection, and mass-based detection. Other detection methods include voltaic and magnetic. New types of biosensor transducers are continually being developed. Each of the three main classes contains many different subclasses, creating a large number of possible transduction methods or combinations of methods. This chapter focuses on optical transduction methods. Figure 1 illustrates the conceptual principle of the biosensing process using a bioreceptor probe and an optical detection method.
Recent advances in nanotechnology leading to the development of submicron optical fibers have opened new horizons for intracellular measurements. Typically, the tip diameter of the optical fiber used in these sensors ranges between 20 and 100 nm. These sensors are based on the same basic principles as more conventional optical biosensors, except for the excitation process. Because the diameter of the optical fiber's tip is significantly less than the wavelength of light used for excitation of the analyte, photons cannot escape from the tip of the fiber to be absorbed by the species of interest, as is the case in larger fiberoptic sensors. Instead, in a fiberoptic nanosensor, after the photons have traveled as far down the fiber as possible, excitons or evanescent fields continue to travel through the remainder of the tip, providing excitation for the fluorescent species of interest present in the biosensing layer. An additional feature of evanescent excitation is that only species that are in extremely close proximity to the fiber's nanoprobe can be excited, thereby precluding the excitation of interfering fluorescent species elsewhere on the sample. Thus, any signal collected is from molecular species extremely close to the fiber (within the 100-nm near-field environment). This feature allows for an unprecedented level of localization.
The key to specificity with biosensor technologies lies in the bioreceptor molecules used. Bioreceptors are responsible for binding the analyte of interest to the sensor for measurement. Bioreceptors can take many forms and are as numerous as the different analytes that have been monitored using biosensors. However, bioreceptors can generally be classified into five major categories: antibodies/antigens, enzymes, nucleic acids/DNA, cellular structures/cells, and biomimetics. This chapter deals with biosensor systems using antibody probes, often called immunosensors, with optical detection. Another type of probe that involves an enzyme substrate is discussed in Subheading 4.
Immunological reactions involving the antigen-antibody binding reaction provide the basis for the specificity of immunoassays. Antibodies are complex biomolecules, made up of hundreds of individual amino acids arranged in a highly ordered sequence. Antibodies are produced by immune system cells when such cells are exposed to substances or molecules called antigens. The antibodies called forth following antigen exposure have recognition or binding sites for specific molecular structures (or substructures) of the antigen. The way in which an antigen and an antigen-specific antibody interact is analogous to a lock-and-key fit, in which specific configurations of a unique key enable it to open a lock. In the same way, an antigen-specific antibody fits its unique antigen in a highly specific manner, so that the three-dimensional (3D) structures of antigen and antibody molecules are complementary. Owing to this 3D shape fitting, and the diversity inherent in individual antibody makeup, it is possible to find an antibody that can recognize and bind to any one of a large variety of molecular shapes.
This unique property of antibodies is the key to their usefulness in immunosensors; their ability to recognize molecular structures allows one to develop antibodies that bind specifically to chemicals, biomolecules, microorganism components, and so on. One can then use such antibodies as specific probes to recognize and bind to an analyte of interest that is present, even in extremely small amounts, within a large number of other chemical substances. Since the first development of a remote fiberoptics immunosensor for in situ detection of the chemical carcinogen benzo[a]pyrene (BaP) (2), antibodies have become commonly used as bioreceptors in biosensors.
3. Materials and Methods
This section describes the protocols and instruments used in the fabrication of fiberoptics nanoprobes. Since fiberoptic nanoprobes are not commercially available, investigators must fabricate them in their own laboratories. Two methods are generally used for preparing the nanofiber tips. The so-called heat-and-pull method is the most commonly used. This method consists of local heating of a glass fiber using a laser or a filament and subsequently pulling the fiber apart. The shape of the nanofiber tips obtained depends on controllable experimental parameters such as the temperature and the timing of the procedure. The second method, often referred to as Turner's method, involves chemical etching of glass fibers. In a variation of the standard etching scheme, the taper is formed inside the polymer cladding of the glass fibers.
The experimental procedures for the fabrication of nanofibers using the heat-and-pull procedure, used by our laboratory, is schematically shown in Fig. 2 (21). The heat-and-pull procedure consists of pulling a larger silica optical fiber to produce the tapered nanotip fiber using a special fiber-pulling device (Sutter Instruments P-2000). This method yields fibers with submicron diameters. One end of a 600-^m silica/silica fiber is polished to a 0.3-^m finish with an Ultratec fiber polisher. The other end of the optical fiber is then pulled to a submicron length using a fiber puller. A scanning electron microscopy photograph of one of the fiber probes fabricated for studies is shown in Fig. 3. The distal end of the nanofiber is approx 30 nm.
To prevent leakage of the excitation light on the tapered side of the fiber, the sidewall of the tapered end is then coated with a thin layer (100 nm thick) of
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