The ability to detect high explosive compounds is a fundamental step in achieving the goal of creating devices capable of
'sniffing' out explosive devices. To detect high explosive compounds such as 2,4,6-trinitrotoluene (TNT), a molecularly
imprinted polymer (MIP) sensor was developed. This sensor consists of MIP microspheres prepared using methacrylic
acid as the functional monomer in a precipitation polymerization reaction. The MIP microspheres are then combined
with fluorescent semiconductor nanocrystals, or quantum dots, via a simple crosslinking procedure. To study the
sensor's ability to detect nitroaromatic analytes, the fluorescent-labeled MIP particles were exposed to aqueous 2,4-
dinitrotoluene (DNT), a nitroaromatic molecule very similar to TNT. Characterization of the MIP particles shows a
uniform size distribution, with an average diameter of approximately 615 nm. Imaging of the particles also shows that
spherical shapes are being produced by the precipitation polymerization reaction. Preliminary data indicate that the
sensor is capable of detecting nitroaromatic compounds in an aqueous solution. These results illustrate the future
application of the fluorescent-labeled MIP sensor for detecting high explosives, with the potential for use in detecting
vapors from explosive devices and in an array of environmental conditions.
Molecular recognition is an important aspect of any biosensor system. Due to increased stability in a variety of
environmental conditions, molecular imprinted polymer (MIP) technology is an attractive alternative to biological-based
recognition. This is particularly true in the case of improvised explosive device detection, in which the sensor must be
capable of detecting trace amounts of airborne nitroaromatic compounds. In an effort to create a sensor for detection of
explosive devices via nitroaromatic vapor, MIPs have been deployed as a molecular recognition tool in a fluorescence-based
optical biosensor. These devices are easily scalable to a very small size, and are also robust and durable. To
achieve such a sensor scheme, polymer microparticles synthesized using methacrylic acid monomer and imprinted with a
2,4-dinitrotoluene (DNT) template were fabricated. These microparticles were then conjugated with green CdSe/ZnS
quantum dots, creating fluorescent MIP microparticles. When exposed to the DNT template, rebinding occurred
between the DNT and the imprinted sites of the MIP microparticles. This brings the nitroaromatic DNT into close
proximity to the quantum dots, allowing the DNT to accept electrons from the fluorescent species, thereby quenching the
fluorescence of the quantum dot. Results indicate that this novel method for synthesizing fluorescent MIPs and their
integration into an optical biosensor produced observable fluorescence quenching upon exposure to DNT.
Fluorescence based biosensors have the ability to provide reliable pathogen detection. However, the performance could
be improved by enhancing the effective surface area of the biosensor. We report on a new nanofibrous fluorescencebased
biosensor, whereas a sol-gel platform mesh was constructed by utilizing electrospinning techniques. Furthermore,
incorporating cetyltrimethylammonium bromide (CTAB) and conducting pore-forming techniques resulted in a high
surface area material suitable for biosensor immobilization. The biosensor was designed to detect Helicobacter hepaticus
bacterium by sandwiching the pathogen between two antibodies, one labeled with Alexa Fluor 546 fluorescent dye and
the other with 20nm Au nanoparticles. In the presence of pathogen, the close proximity of Au nanoparticles quenched
the Alexa Fluor fluorescence, suggesting that the electrospun fiber platforms are suitable for sensing H. Hepaticus.
Additionally, sol-gel fibers used as biosensor platform have the added benefit of increased immobilization, as
fluorescence intensity from immobilized biosensors is 8.5x106 cps higher on fibers than on a flat, non-porous substrate.
We have been investigating a new nanomaterial-based optical platform for the immobilization of protease substrates for
the development of a biosensor to detect medically relevant enzymes. Metallic nanoparticles have been deposited onto
thin films and are being used for their optical properties. Two different peptide sequences have been designed as trypsin
substrates that are designed to be immobilized onto the surface of the thin films. The peptides were synthesized with a
fluorophore attached at the terminal end of the peptide to allow for fluorescence sensing. Fluorescent molecules in close
proximity to metallic elements will have their fluorescence signal quenched due to surface plasmon resonance (SPR)
effects. When the peptide is cleaved by trypsin, the fluorophore is separated, resulting in a detectable change in
fluorescence intensity.
These novel nanomaterial-based optical platforms have been fabricated using physical vapor deposition. Innovative
techniques have been invented using these machines to acquire nanoparticles in the range of a few nanometers on these
thin films. It is known that nanoparticles with dimensions less than their bandwidth display optical properties much
different from their bulk counterparts. We have immobilized the peptide substrates to the surface of the metallized thin
films so they are in close proximity with each other. Polydimethlysiloxane (PDMS) was molded to create small wells
and placed on the thin films. Fluorescent microscopy was used to image the wells as various concentrations of the
enzyme were introduced resulting in a recovery of green fluorescence from the fluorophore on the cleaved portion of the
peptide. Different size nanoparticles and different immobilization processes are being used to optimize the design of the
protease biosensor.
Glucose binding protein (GBP) is a monomeric periplasmic protein. It is synthesized in the cytoplasm of
Escherichia coli which functions as a receptor for transport D-glucose. GBP binds glucose with high affinity. The
binding mechanism is based on a hinge motion due to the protein conformational change. This change was utilized as an
optical sensing mechanism by applying Fluorescence Resonance Energy Transfer (FRET). The wild-type GBP lacks
cysteine in its structure, but by introducing a single cysteine at a specific site by site-directed mutagenesis, this ensured
single-label attachment at specific sites with a fluorescent probe. The other sites were amino sites, which were labeled
with second fluorophore. The near IR FRET pair, Alexa Fluor 680 (AF680) and Alexa Fluor 750(AF750), was utilized.
The AF680 targeted the amine sites, which was the donor fluorophore, while the AF750 labeled the single cysteine site,
which was the acceptor fluorophore. The sensing system strategy was based on the fluorescence changes of the probe as
the protein undergoes a structural change upon binding. This biosensor had the ability to detect down to 10 uM
concentrations of glucose. Next the probes were uploaded into red blood cells via hypo osmotic dialysis. The sensor
responded to glucose while encapsulated with the red cells. These results showed the feasibility of an intracellular
glucose biosensor.
We report herein on the development of a FRET-based method to detect changes caused by viral protein-receptor binding. FRET fluorophore pairs (donor and acceptor fluorophores) were tagged to two specific receptors, both which bind to a viral protein. When the binding event occurs, the distance between the donor and acceptor FRET fluorophores is decreased, thus initiating the fluorescence resonance energy transfer (FRET). Since the binding event is unique to the viral protein, fluorescent change indicates the present of the virus. In this paper, the viral protein gp120, which is the featured protein on the surface of HIV-1, was detected. The receptors, CD4 and gp120-antibody which specifically bind to gp120, were conjugated to the FRET fluorophore pair, AMCA-NHS (succinimidyl-7-amino-4-methylcoumarin-3-acetic acid) and FITC (fluorescein isothiocyanate) respectively. Spectrofluorimetry was used to detect the fluorescent change between AMCA-NHS and FITC peak intensities when the receptors bind to the gp120. Specific binding gp120 and non-specific binding gp120 were used to test the selectivity of the sensor. The results indicated that FRET-conjugated receptors can efficiently detect the presence of gp120.
We report herein on the development of a dual receptor detection method for enhanced biosensor monitoring. The proposed scheme requires the integration of a chemical transducer system with two unique protein receptors that bind to a single biological agent. Optical transduction occurs because the two protein receptors are tagged with special molecular groups. When bound to a single biological agent, these fluorescently labeled proteins undergo a change in fluorescence. This `fluorescent switching' relies on the well-known mechanisms of fluorescent resonance energy transfer (FRET). The paper focuses on the investigation and optimization of the chemical transduction system (FRET). A number of FRET dye pairs were tested in a spectrofluorimeter, and promising FRET pairs (FITC/TRITC and DMACA/FITC) were further tagged to the protein, avidin and its ligand, biotin. Due to their affinities, the FRET-tagged biomolecules combine in solution, resulting in a stable, fluorescent signal from the acceptor FRET dye with a simultaneous decrease in fluorescent signal from the donor FRET dye. The results indicate that the determined FRET pairs can be utilized in the development of dual receptor sensors.
We report here on the development of a sol-gel based fiber optic sensor to monitor local blood pH. These sensors are being developed for invasive medical applications; that is, they will be coaxially threaded through a catheter beyond an occlusion in the vascular system. The fiber optic pH sensor design was based on the immobilization of a pH sensitive dye, seminaphthorhodamine-1 carboxylate, onto the tip or surface of an optical fiber using the sol-gel method. The fiber optic pH sensor was tested in phosphate buffer saline and human whole blood using a miniature fluorimeter system. Linear responses were obtained in blood in the pH range 6.7 to 8.0, which demonstrates potential for use for in vivo sensing.
Nd:YAG laser systems, coupled to silica fibers, have shown great benefits as surgical tools. Using the laser system with a bare silica fiber, laser surgeons can photocoagulate tissue to depths of 4 to 5 mm in a non-contact mode. In a contact mode, incision and cauterization of the nearby tissue can be achieved. Although these two capabilities provide powerful tools for hemostatic procedures, research performed at Iowa State University has shown that the silica fiber tips undergo extensive damage when in contact with tissue. Chemical and thermal degradation of the silica glass surface plays a key role. Damaged fibers do not transmit a significant fraction of the laser light launched down them. Instead, essentially all of the laser energy is converted to heat at the contact point. The tip can then be used only to incise tissue. We report here on the development and characterization of a new optical fiber that offers improved chemical resistance and also high temperature resistance. The new fibers were pulled from glass rods with a composition of 92.5 wt.% SiO2 and 7.5 wt.% TiO2 and then cladded with a fluorinated hard polymer. The new fibers effectively deliver energy even after the fiber comes into contact with tissue while the silica fiber tips undergo catastrophic damage. Also, preliminary clinical testing of the new fibers has demonstrated the stability of the fibers in contact with tissue during gynecological surgical procedures.
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