Analysis of the intrinsic scatter and fluorescence profiles of marine algae can be used for general classification of
organisms based on cell size and fluorescence properties. We describe the design and fabrication of a Microflow
Cytometer on a chip for characterization of phytoplankton. The Microflow Cytometer measured distinct side-scatter and
fluorescence properties of Synechococcus sp., Nitzschia d., and Thalassiosira p. Measurements were confirmed using
the benchtop Accuri C6 flow cytometer. The Microflow Cytometer proved sensitive enough to detect and characterize
picoplankton with diameter approximately 1 mm and larger phytoplankton of up to 80 mm in length. The wide range in
size discrimination coupled with detection of intrinsic fluorescent pigments suggests that this Microflow Cytometer will
be able to distinguish different populations of phytoplankton on unmanned underwater vehicles. Reversing the
orientation of the grooves in the channel walls returns the sample stream to its original unsheathed position allowing
separation of the sample stream from the sheath streams and the recycling of the sheath fluid.
A rapid, automated, multi-analyte Microflow Cytometer is being developed as a portable, field-deployable sensor for onsite
diagnosis of biothreat agent exposure and environmental monitoring. The technology relies on a unique method for
ensheathing a sample stream in continuous flow past an interrogation region where optical fibers provide excitation and
collect emission. This approach efficiently focuses particles in the interrogation region of the fluidic channel, avoids
clogging and provides for subsequent separation of the core and sheath fluids in order to capture the target for
confirmatory assays and recycling of the sheath fluid. Fluorescently coded microspheres provide the capability for highly
multiplexed assays. Optical analysis at four different wavelengths identified six sets of the coded microspheres
recognizing Escherichia coli, Listeria, and Salmonella as well as cholera toxin, staphylococcal enterotoxin B (SEB), and
ricin, and assay results were compared with those of a commercial Luminex analysis system.
A multi-analyte diagnostic system based on a novel microflow cytometer is under development as a portable, fielddeployable
sensor for environmental monitoring and for rapid point-of-care and on-site diagnosis of exposure to
biothreat agents. The technology relies on a unique method for ensheathing a sample stream in continuous flow past an
illuminated interrogation region. This sheathing approach efficiently focuses particles in the interrogation region of the
fluidic channel and minimizes clogging by complex samples. Fluorescently coded microspheres provide the capability
for highly multiplexed assays. In this report, separation of six microsphere sets was demonstrated with determination of
immunoassays on three of the six sets; comparison to the commercial platform was made.
An array biosensor developed for performing simultaneous analysis of multiple samples for multiple analytes has been miniaturized and fully automated. The biochemical component of the multi-analyte biosensor consists of a patterned array of biological recognition elements ("capture" antibodies) immobilized on the surface of a planar waveguide. A fluorescence assay is performed on the patterned surface, yielding an array of fluorescent spots, the locations of which are used to identify what analyte is present. Signal transduction is accomplished by means of a diode laser for fluorescence excitation, optical filters and a CCD camera for image capture. A laptop computer controls the miniaturized fluidics system and image capture. Data analysis software has been developed to locate each spot and quantify the fluorescent signal in each spot. The array biosensor is capable of detecting a variety of analytes including toxins, bacteria and viruses and shows minimal interference from complex physiological sample matrices such whole blood and blood components, fecal matter, saliva, nasal secretions, and urine. Some results from recent field trials are presented.
An array biosensor developed for simultaneous analysis of multiple samples has been utilized to develop assays for toxins and pathogens in a variety of foods. The biochemical component of the multi-analyte biosensor consists of a patterned array of biological recognition elements immobilized on the surface of a planar waveguide. A fluorescence assay is performed on the patterned surface, yielding an array of fluorescent spots, the locations of which are used to identify what analyte is present. Signal transduction is accomplished by means of a diode laser for fluorescence excitation, optical filters and a CCD camera for image capture. A laptop computer controls the miniaturized fluidics system and image capture. Results for four mycotoxin competition assays in buffer and food samples are presented.
Array biosensors provide the capability of immobilizing multiple capture biomolecules onto a single surface and therefore offer the exciting prospect of multi-analyte detection. A miniaturized, fully automated, stand-alone biosensor is reported which can simultaneously test multiple samples for multiple analytes. This portable system (< 10 lbs) is particularly appropriate for on-site monitoring for food safety, infectious disease detection, and biological warfare defense. The surface-selective nature of this technology allows determination of binding constants and tracking of both specific and non-specific binding events as they occur. Thus, it provides an exciting new research tool for characterizing the interactions of biomolecules with surfaces or immobilized receptors in real time. This capability has important implications for development of new materials and sensors.
The array biosensor is capable of detecting and identifying multiple analytes in multiple samples simultaneously. Using fluorescence immunoassays on a planar waveguide and miniaturized fluidics, the sensor is automated and portable. Assays are sensitive and require 12 minutes to perform. Environmental contaminants in the sample fail to generate false positive or false negative results in tests performed to date. Measurements can be conducted in real time using spots as small as 80 micrometers . The waveguide can be coated with indium tin oxide (ITO) to create a charged field at the surface to further regulate the interaction of sample components with the surface.
The array biosensor has been developed for simultaneous analysis of multiple samples for multiple analytes. A patterned array of capture antibodies is immobilized on the surface of a planar waveguide and a sandwich immunoassay conducted using a cocktail of fluorescent tracer antibodies. Upon excitation of the fluorescent label using a 635 nm diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. The assays are fast, sensitive, and specific.
A fluorescence-based immunosensor has been developed for simultaneous analyses of multiple samples for 1 to 6 different antigens. A patterned array of recognition antibodies immobilized on the surface of a planar waveguide is used to 'capture' analyte present in samples. Bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. A new design for a fluidics distribution system is shown, as well as results from assays for physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D- dimer, a marker of sepsis and thrombotic disorders.
As biosensors become more sophisticated and commercially available, the general appreciation for their capabilities also increases. We now focus on multi-analyte sensors and address the problems inherent in discriminating multiple simultaneous signals without loss in assay speed, specificity or sensitivity. Furthermore, the goals of portability, simplicity and low cost have not diminished in importance. NRL is developing a multi-analyte sensor designed to be portable, inexpensive, and easy to use. To achieve these goals, we use a room temperature CCD, a diode laser, and a disposable waveguide. While our goals of using automated fluidics and automated image processing are not yet completely realized, we have fabricated a prototype biosensor which fits into a tackle box with a associated portable computer. Simple microscope slides are used as waveguides and precoated with arrays of immobilized antibodies. Fluorescence immunoassays are performed on these waveguides using as many as 6 samples at a time and assaying for up to 5 different analytes in each samples Sensitivities in the 1-10 ng/ml range have been achieved in 10-minute assays. Initial studies in clinical fluids indicate that assays can be run on samples such as whole blood, plasma, urine, saliva and nasal secretions.
A fluorescence-based immunosensor using a charge-coupled device (CCD) as a detector has been developed
for detecting multiple analytes within small (150 ml) sample volumes. Wells approximately 2 mm in diameter
were patterned onto glass coverslips using a photoactivated optical adhesive. In a model detection system, four different antigens were covalently attached to the bottoms of these wells, and the polymer subsequently removed to form the sensing surface. The coverslips were mounted over a scientific grade CCD operating at ambient temperature in inverted (multipin phasing) mode. A two-dimensional graded index of refraction (GRIN) lens array was used to focus the sensing surface onto the CCD. Solutions of fluorescently labeled antibodies were then placed on the coverslip, and the amount of antibody bound at each location on the coverslip was determined by quantitative image analysis. Concentrations as low as 50 ng/ml of Cy5-labeled
antibodies could be detected using the sensor. The small footprint and minimal system requirements of the design should facilitate its incorporation into portable biosensors.
Fiber optic biosensors using evanescent wave excitation of fluorescence have proven their ability to detect antigens rapidly in a variety of environmental and clinical samples. One problem associated with these biosensors is the fiber-to-fiber variability in measured signal. We have addressed this problem by labeling an immobilized anti-trinitrotoluene ((alpha) TNT) capture antibody with the fluorescent cyanine derivative Cy5.5 (emission (lambda) max equals 696 nm). The antigen (a TNT analog) was then labeled with fluorescent Cy5 (emission (lambda) max equals 668 nm). Both fluorophores were excited by 635 nm light, and their emission was collected using a fiber optic spectrometer. The fluorescence from the Cy5.5 labeled capture antibody served as a calibration signal for each fiber and was used to correct for differences in optics, fiber defects, and varying amounts of immobilized capture antibody. The calibration process could be used repeatedly following fiber regeneration. However, when each immobilized antibody was labeled with at least one Cy5.5 fluorophore, fluorescence resonance energy transfer (FRET) was observed between the Cy5-antigen donor and the Cy5.5-labeled acceptor. The extent of FRET affected the measured antigen and calibration signal, and these signals had to be adjusted accordingly. We describe the procedures to account for fluorescently labeled antibodies during extended biosensor use.
We have developed a fiber optic sensor for rapid and direct analysis of PCR-amplified DNA fragments with minimal sample processing and real-time data readout. To accomplish this, a novel DNA-recognition system was built onto the surface of fused silica fibers. DNA fragments, labeled with a fluorophore during amplification, are bound to and detected at the fiber surface by means of evanescent wave excitation/emission. Excess unincorporated fluorescent single-stranded oligonucleotide PCR primers make only a small contribution to the signal, as the modified fiber surface only efficiently binds double-stranded DNA with the proper PCR-incorporated terminal nucleotide sequence (5'-ATGACTCAT-3'). The surface- bound double-stranded DNA recognition element utilizes a genetically engineered dimeric sequence-specific DNA binding protein. Self-assembly into the proper conformation for binding DNA occurs by means of specific interactions of the active dimer with the Fc domains of a layer of IgG molecules (antibodies) covalently attached directly to the fiber surface. The modified fiber surface is regenerated between samples by stripping away bound DNA with high salt concentrations.
Explosives are one of many hazardous waste problems of concern to the Department of Defense. Defective storage facilities or byproducts of weapons manufacture have led to contamination of soil and water with explosives. Most explosives are toxic, thus posing an ecological and human health hazard. The ability to do on-site or down-stream detection of explosives will be invaluable for site characterization and remediation by saving both time and money. The evanescent wave fiber optic biosensor that was developed at NRL has been modified for the detection of trinitrotoluene (TNT), by developing a competitive immunoassay on the surface of an optical probe. A fluorescently labelled analog of TNT, trinitrobenzenesulfonic acid (TNB), was used as the competitor. Enzyme-linked immunosorbent assays were performed to determine the best fluorescently labeled competitor available to be able to achieve high sensitivity in the fiber optic assay. For the competition assay, 7.5 ng/ml Cyanine 5-ethylenediamine-labelled TNB (Cy5-EDA-TNB) was exposed to an antibody-coated optical fiber generating specific signal above background that corresponds to the 100% or reference signal. Inhibition of this signal was observed in the presence of TNT with the percent inhibition proportional to the TNT concentration in the sample. Detection sensitivities in aqueous solutions containing 10 ng/ml TNT (8 ppb) have been achieved using this system.
Cyanine fluorescent dyes which can be coupled to proteins have recently become available. They are excited in the 500-700 nm range and have large extinction coefficients which make them good candidates for sensitive immunosensor applications. We evaluated three of these dyes (Cy3, Cy5, and Cy5.5) in a direct fluoroimmunoassay on evanescent wave fiber optic biosensors. The biosensors differed in laser excitation sources and emission filters in order to accommodate dye requirements. The assay consisted of rabbit anti-goat IgG immobilized fiber probes being exposed to fluorophorelabeled goat IgG (gIgG). Upon binding of the dye-conjugated protein to the fiber, a signal was generated. When using the 514 nm laser device, Cy3-gIgG gave a substantially larger signal than TRITC-gIgG. On the 650 nm laser device, Cy5-gIgG provided very good signal, while that for Cy5.5 was moderate. Cy5.5 produced an excellent response on the 670 nm laser device. Use of these dyes provides a mechanism for improving the fiber optic biosensor by changing the excitation/emission region to an area of low background for clinical and environmental samples.
Based upon a biosensor design which utilizes standard fluorescent dyes (FITC, TRITC), a new device has been developed which incorporates a laser diode light source to excite novel near infrared (NIR) dyes. The purpose of switching to the NIR regime is to decrease the background fluorescence of biological samples and to decrease the size and power requirements of the biosensor. New dyes which fluoresce in the NIR have been conjugated to protein antigen and immunoassays performed. Assay results using excitation at 780 nm are shown.
We have developed an evanescent wave fiber optic biosensor which uses long fibers to facilitate the analysis of environmental and clinical samples for hazardous materials. The use of antibody/antigen binding with fluorescence-based sensing in the evanescent wave yields a sensor that is unique, adaptable, and sensitive. The variety of substances that could be detected is limited only by their antigenicity. Sensing in the real world poses several challenges that must be met. We have focused on the development of several aspects of the sensing system to transition this sensor into a field deployable device. Recent developments presented here include optimized fiber optic probe tapering, a flow chamber to facilitate sampling, and probe regeneration for repetitive analysis. Preliminary experiments assessing the potential to detect analytes in biological and environmental fluids are also presented.
A fiber optic biosensor has been developed which monitors fluorescence to detect antibody/antigen binding within the evanescent wave. The sensing region is formed by removal of cladding from the core along the distal end of a step-index optical fiber and attaching the antibody. Reducing the radius by tapering the probe overcomes the mismatch in V-number which arises between the declad, immersed probe, and the clad fiber. Ray tracing analysis of tapered probes and of combination taper probes demonstrates that the evanescent wave penetration depth increases along the length of the taper, creating a probe with increased excitation light available at the surface for stimulating fluorescence.
To develop an improved fiber optic biosensor both the optical component selection and the signal coupling efficiency were investigated. The emission filter and fiber connectors were carefully chosen to reduce their contribution to noise in the system. We used long, fused silica fibers that had several centimeters of cladding removed along the distal end. This exposed core is coated with the recognition molecules that bind analyte-fluorophore complexes from the sample solution. A fluorescent signal generated in the evanescent wave region of the unclad, immersed portion of the probe is lost as it enters the cladded portion of the fiber because of a V-number mismatch. To minimize the mismatch, the core radius is reduced along the uncladded region to form a continuous taper. An assay using the tapered fiber and the described optical configuration is presented that demonstrates instantaneous signal generation in response to nanogram amounts of a toxic material.
A fiber optic-based biosensor which integrates a novel array of optical and electrical components, together with long fused silica fibers and proteins for detection of analyte in solution has been put into limited production. The optical fiber core near the distal end is tapered and coated with either antibodies or DNA binding proteins. Assays are performed by flowing a solution containing the fluorescently tagged ligand molecules over the coated fiber. Within seconds, analyte recognition occurs and a fluorescence signal is transmitted back up the fiber. Applications for the biosensor include clinical diagnostics, pollution control, and environmental monitoring. Ten of the laboratory devices described in this article have been constructed and are being used for assay development. In addition, the prototype of a truly portable device has recently been built and is now being tested.