FIBER OPTIC NANOBIOSENSORS
Because of the complexity of biological systems and the number of possible interference to chemical nanosensors, the need for added specificity in cellular analyses can arise. To achieve this added specificity, fiber optic nanobiosensors are often employed. Like their larger counterparts, conventional fiber optic biosensors, biological receptor molecules (i.e., antibodies, enzymes, etc.) are used to provide added specificity. The different types of bio-receptor molecules that have been used for the fabrication of fiber optic nanobiosensors include antibodies, oli-gonucleotides, and enzymes, thereby allowing for the detection of a wide array of analytes.
Antibody-Based Fiber Optic Nanobiosensors
The first fiber optic nanobiosensor was reported by Alarie and VoDinh[28] in 1996. In this work, antibody-based nanobiosensors for the DNA adduct, benzo[a]pyrene tetrol (BPT), were developed and characterized. The fabrication of these first nanobiosensors was performed in a process very similar to that used in the development of fiber optic chemical sensors. Initially, a 600-p.m-diameter multimode fiber optic was tapered down to 40 nm at the tip. After tapering, a thick layer of silver was applied to the sides of the fiber in such a way as to prevent the coating of the fiber’s tip. Next, the uncoated fiber tip was silanized, and antibodies were attached via a covalent-binding procedure.[28] Following attachment of the antibodies, the fiber optic nanobiosensors were characterized in terms of antibody binding affinity as well as sensitivity and absolute detection limits. From these measurements, it was found that the antibodies had retained greater than 95% of their native binding affinity for BPT after being bound and that the absolute detection limit for BPT using these nanobiosen-sors was approximately 300 zmol (i.e., 300 x 10~21 mol).
Shortly after the development of this first antibody-based fiber optic nanobiosensor, several others were developed and applied to in vitro measurements within individual living cells.[5,29,46-48] In one such study, nano-biosensors for BPT were prepared as described above and used to obtain quantitative measurements of intracellular concentrations of BPT in the cytosol of two different cell lines: 1) rat liver epithelial cells, and 2) human mammary carcinoma cells.[29] Unlike previous intracellular analyses that employed relatively large cells (i.e., mouse oocytes and neurons), the cells analyzed in this study were spherical in shape and had diameters of approximately 10 mm, thereby demonstrating that fiber optic nanosensors and nanobiosensors could be used to analyze cells the size of typical mammalian somatic cells (i.e., 10-15 mm) without destroying them. In fact, this study also demonstrated that the insertion of a fiber optic nanosensor or fiber optic nanobiosensor into such a cell and the subsequent measurement seemed to have little effect on the cell’s normal function. This was demonstrated by inserting the fiber optic nanobiosensor into the cytoplasm of a cell beginning to undergo mitosis and monitoring cell division following incubation of the nanofiber in the cell for 5 min. Fig. 6 contains an image of the fiber optic nanosensor being inserted into this cell, as well as a second image showing the two daughter cells that it divided into after approximately 2 hr.
Molecular Beacon-Based Fiber Optic Nanobiosensors
To detect the presence of oligonucleotides (i.e., RNA, DNA, etc.) in intracellular environments, a class of fiber optic nanobiosensors that employ a relatively new type of bioreceptor molecules known as a molecular beacon has been developed.[49] Molecular beacons are hairpin-shaped oligonucleotide probes that rely on the complementarity of nucleic acids (i.e., adenine:thymine, cytosine:guano-sine, etc.) to form the basis for the molecular recognition of a specific oligonucleotide sequence. As shown in Fig. 7, molecular beacons form a stem-loop structure in the absence of the target sequence, in which a fluorophore [e.g., fluorescein isothiocyanate (FITC), etc.] on one end of the stem is in close proximity to a nonfluorescing, quenching moiety [e.g., dimethylaminophenylazobenzoic acid (DABCYL), etc.] on the other end.[50] In this state, when the fluorophore on the molecular beacon is excited, an energy transfer takes place between the excited fluo-rophore and the quencher, either by direct energy transfer or fluorescence resonance energy transfer (FRET), and minimal fluorescence emission occurs. However, when the loop sequence of the molecular beacon comes into contact with its complementary sequence, the stem begins to unzip, creating a spatial separation between the fluo-rophore and the quencher. This spatial separation in turn causes a dramatic increase in the fluorescence intensity of the molecular beacon, with some reports stating enhancements exceeding 200-fold.[51]
Fig. 6 Photograph of an individual cell that is probed with a fiber optic nanosensor as it begins to undergo mitosis, and a second photograph of that same cell approximately 2 hr later, having divided into two daughter cells.
Because of their inherent sensitivity and specificity, molecular beacons have recently been used as bioreceptor molecules in the fabrication of fiber optic nanobiosensors for single-stranded DNA and RNA.[52,53] In these works, Liu and Tan and Liu et al. attached molecular beacons to the silica core of a fiber optic nanosensor through biotin-avidin linkages. By binding the molecular beacons to the tapered tip of the fiber optic in this method, a strong attachment was achieved, while not degrading the activity of the beacon.
Enzyme-Based Fiber Optic Nanobiosensors
In addition to the use of molecular beacons and antibodies as bioreceptor molecules, fiber optic nanobiosensors that employ enzymes for molecular recognition have also been developed.[54,55] Using enzymes as bioreceptors not only provides nanobiosensors with a high degree of specificity, but their catalytic activity can amplify the species being measured, allowing for sensitive analyses. One such enzymatic-based fiber optic nanobiosensor was developed for the indirect detection of glucose.[55] In this work, Rosenzweig and Kopelman immobilized the enzyme, glucose oxidase, and the oxygen-sensitive indicator, tris(1,10-phenanthroline) ruthenium chloride, in an acryl-amide polymer on the tapered end of a nanofiber via a photo-polymerization process. Therefore when the nano-biosensor is in the presence of glucose, the enzyme catalyzes the oxidation of glucose into gluconic acid, consuming oxygen. The resulting changes in oxygen levels are then measured via the oxygen-sensitive indicator dye. By using an enzymatic receptor, these nanobiosensors were capable of absolute detection limits of approximately 10"15 mol and a sensitivity five to six orders of magnitude greater than current glucose optodes.[55]
Fig. 7 Cartoon depicting the mechanism of action of a molecular beacon.
Another example of an enzymatic nanobiosensor was developed by Tan et al. for the indirect measurement of the neurotransmitter, glutamate. In this work, glutamate dehydrogenase is bound to the tip of the fiber optic nanobiosensor to achieve molecular recognition. When the sensor is in the presence of glutamate, the nonfluo-rescent species, NAD, is reduced into the autofluorescent species, NADH, which can then be monitored via fluorescence spectroscopy. By relating the intensity of the NADH fluorescence to glutamate concentration, sensitive analyses were performed. Following the construction of these nanobiosensors, their usefulness was evaluated by continuously monitoring the release of glutamate from individual neurons during stimulation.
FIBER OPTIC NANOIMAGING SENSORS
Although fiber optic nanosensors and nanobiosensors have proven to be useful tools for the spatially localized, quantitative analysis of chemical species in cells, only one sensor can be inserted into a typical mammalian somatic cell at a time, without causing significant damage.[56] This inability to monitor multiple locations simultaneously can dramatically limit the applicability of fiber optic nano-sensors and nanobiosensors to many types of cellular analyses. To overcome this limitation, a couple of research groups have recently begun to develop nanoscale fiber optic imaging probes and sensors comprised of thousands of individual fibers arranged in a coherent bundle.[57,58] These nanoimaging sensors represent the latest advance in fiber optic-based nanosensors, and are capable of obtaining measurements at thousands of different locations simultaneously with nanometer-scale spatial resolution.
Fiber Optic Nanoimaging Probe Fabrication
Similar to single-fiber-based nanosensors and nanobio-sensors, nanoimaging probes can be fabricated either through a chemical etching process, or a heated pulling process. In the chemical etching process, commercially available coherent imaging bundles with 30,000 individual fibers are etched in HF to form an array of silicon tips.[58] Following formation of the array of silicon tips, a layer of gold is sputtered over the entire surface to prevent light from escaping from the tapered sides of each fiber element. After deposition of the gold overlayer, a polymer is electrochemically deposited over the gold and heat- cured to expose the gold at the tips of the individual fiber elements. Finally, the exposed gold surface on each of the individual fiber element tips is dissolved to produce free silica surfaces for spectroscopic probing.
In addition to chemical etching, nanoimaging probes have also been fabricated using a heated pulling pro-cess.[57] However, unlike the nanoimaging array created via chemical etching, the heated pulling process produces an array having a flat surface. This can be seen in Fig. 8A, which shows a side-on view of a fiber optic imaging bundle that has been tapered in a micropipette puller. To produce this flat surface, which is necessary for imaging, the delay between the final pull in the micropipette puller and the time at which the heating laser turned off is increased, thus allowing the fiber to cool off slightly and be broken, with all of the individual fiber elements being of the same length and diameter. By changing the heating temperature and the initial pull strength of the micropi-pette puller, fibers can be accurately and reproducibly tapered to all different diameters. Fig. 8 shows a fiber that has been tapered to have individual fiber element diameters of 800 nm on one end (Fig. 8B) and 4-p.m diameters on the untapered end (Fig. 8C).
Using these two different fabrication techniques, fiber optic nanoimaging probes and sensors that will not only allow for the quantitative measurement of individual chemical species, but also the simultaneous measurement at many different locations are being developed. Although these fiber optic imaging bundles are too large to be inserted into individual living cells, they can be used to monitor cellular membranes without damage.
Fig. 8 Photographs of a fiber optic nanoimaging probe that has been tapered using a heated pulling process. Panel A shows a side-on view of the fiber demonstrating the flat tip after pulling. Panels B and C are end-on views of the tapered end and the nontapered end of the fiber, respectively.
NANOPARTICLE-BASED OPTICAL NANOSENSORS
Although fiber optic-based nanosensors have had a large impact on the fields of cellular biology and biochemistry, significant advances and variations in optical nanosensors are constantly being made, from the use of more selective bioreceptors to the development of different types of optical-based nanobiosensors. One such advance in the last several years has been the development of nanopar-ticle-based optochemical sensors, with nanometer-scale sizes in all three dimensions. Because of the small sizes of these sensors, a large number of them can be implanted within an individual cell at one time, allowing for the monitoring of many locations simultaneously. Although many different nanoparticle-based sensors are currently being developed, three main classes have already shown a great deal of promise for intracellular analyses. These three classes are quantum dot-based nanobiosensors, polymer-encapsulated nanosensors known as PEBBLEs, and phospholipid-based nanosensors. Each of these different classes of nanoparticle-based sensors is described in more detail below.
Quantum Dot-Based Nanosensors
The first class of nanoparticle-based optical nanosensor that was developed involved attaching nanometer-scale semiconductor particles, known as quantum dots, to various biological receptor molecules including antibodies, oligonucleotides, and enzymes.[59-63] The quantum dots used in these nanosensors comprised ZnS particles capped with Cd-Se. By using quantum dots instead of conventional fluorescent dyes, these sensors exhibit much more intense emission as well as a much greater degree of photostability. These properties are very important when trying to monitor changes in chemical or biochemical species concentrations over time because most fluorescent dyes exhibit rapid and significant photobleaching with the small amount used in cellular analyses. Unfortunately, because quantum dot-based nanobiosensors exhibit luminescence emission whether bound to the analyte or not, there are limits to their applicability to cellular analyses. Additionally, a biocompatibility issue also exists for these sensors because the materials used to fabricate the quantum dots (e.g., Cd-Se, etc.) are toxic to cells. Because of their toxicity, there is a significant effort currently underway in many research groups to develop more biocompatible quantum dot-based nanobiosensors to perform long-term monitoring of cellular reactions or processes.
PEBBLEs
Sensors
A second class of nanoparticle-based optical nanosensors that has already had a large impact on the fields of cellular biology and biochemistry was developed by Sasaki et al.,[64] Clark et al.,[65,66,68] Sumner et al.,[67] and Xu et al.[69,70] in the last several years. These sensors, known as PEBBLEs (probes encapsulated by biologically localized embedding), are comprised of fluorescent indicator dyes sensitive to ionic species (i.e., H+, Ca2+, etc.) that are embedded in 20-nm-diameter or 200-nm-diameter polymer or sol-gel spheres.[65,66] By encapsulating the fluorescent indicator dyes in a polymer matrix, they are protected from cellular degradation by proteins, while still allowing ions to pass and react with them. In addition, this polymer coating also protects the cell from the toxic effects of the dye.
Since they were first developed in 1996, PEBBLEs have been applied to the measurement of many different species (pH, Ca2+, NO, O2, and Zn2+) within individual cells.[65-70] In these analyses, large quantities of PEBBLEs are inserted in a cell, either by a gene gun or a similar device, to ensure that sensors are present at every location in which a measurement might be desired. Once the sensors have been injected into the cell, the entire cell is illuminated and the fluorescence signal from the indicator dye is measured over the autofluorescent background of the cell.
Because of the potential for intense autofluorescence from the cell, depending on the excitation wavelength used, the detection limits of such analyses can be relatively high. To overcome this problem, Anker et al.[71] and Anker and Kopelman[72] have recently developed magnetically modulated variations of these sensors, known as MagMOONs (magnetically modulated optical nano-probes). These MagMOONs are created around aspheric magnetic nanoparticles that can be rotated in the presence of a rotating magnetic field. As the particles begin to rotate, the optical emission from the sensor is modulated. During demodulation, the emission from the sensors can be separated from the continuous autofluorescence background signals, thereby dramatically improving the detection limit of such sensors.
Phospholipid Sensors
The third major class of nanoparticle-based sensors that has already demonstrated a significant impact on cellular analyses is phospholipid-based sensors. Like the PEBBLEs described above, these nanosensors also employ an encapsulation technique to ensure biocompatibility with the cell being investigated.1-73-78-1 Within this class of nanosensors, two distinct subclasses exist: liposome sensors,[77,78] and lipobead sensors.[73,75] The first of these subclasses, liposome-based nanosensors, employs fluorescent indicator dyes encapsulated in the internal aqueous compartment of a liposome. This allows the dye, which is sensitive to a particular analyte, to retain its solution-based characteristics (i.e., spectral emission profile, Stokes shift, response time, etc.) while preventing toxic dye molecules from diffusing throughout the cell. Currently, liposome nanosensors have been developed and applied to the measurement of molecular oxygen[78] and pH[77] in various cellular environments, demonstrating a high degree of sensitivity as well as specificity.
Recently, a variation of these liposome-encapsulated nanosensors was developed by Ji et al.,[73] McNamara et al.,[75] McNamara and Rosenzweig,[79] and DeCoster et al.[80] in which the fluorescent indicator dye molecules were immobilized onto a polystyrene nanoparticle prior to being encapsulated in a phospholipid membrane. This second subclass of phospholipid-based nanosensors, known as lipobead nanosensors, is more stable and less susceptible to biological degradation than liposome-based sensors. An additional advantage of these lipobead nano-sensors over liposome nanosensors or PEBBLEs is that the fluorescent indicator dyes can be partially embedded in the phospholipid membrane, allowing the measurement of a much larger number of chemical species, because the analyte does not need to diffuse through a protective coating before interacting with the dye.[73,75]
CONCLUSION
With interest in nanotechnology and its practical use rising, the development of optical nanosensors for microscopic analyses has increased dramatically over the last decade. Since the development of the first fiber optic nanoprobes for near-field scanning optical microscopy, optical nanosensors have evolved into many different forms, each having its own distinct advantages for a particular type of analysis. Furthermore, these optical nanosensors and nanobiosensors have begun to demonstrate their ability to obtain reliable and useful measurements of chemicals species within cellular and even subcellular environments. Based on the rapid impact that optical nanosensor and nanobiosensor technologies have already had on cellular biology and biomedical diagnostics, future developments (i.e., smaller, less invasive sensors; more biocompatible sensors; etc.) should revolutionize the fields of healthcare and pharmaceutical development by providing a much greater understanding of basic cellular reaction pathways for various biological functions and diseases.
