Port and harbor security has rapidly become a point of interest and concern with the emergence of new improvised
explosive devices (IEDs). The ability to provide physical surveillance and identification of IEDs and unexploded
ordnances (UXO) at these entry points has led to an increased effort in the development of unmanned underwater
vehicles (UUVs) equipped with sensing devices. Traditional sensors used to identify and locate potential threats are
side scan sonar/acoustic methods and magnetometers. At the Naval Research Laboratory (NRL), we have
developed an immunosensor capable of detecting trace levels of explosives that has been integrated into a REMUS
payload for use in the marine environment. Laboratory tests using a modified PMMA microfluidic device with
immobilized monoclonal antibodies specific for TNT and RDX have been conducted yielding detection levels in the
low parts-per-billion (ppb) range. New designs and engineered improvements in microfluidic devices, fluorescence
signal probes, and UUV internal fluidic and optical components have been investigated and integrated into the
unmanned underwater prototype. Results from laboratory and recent field demonstrations using the prototype UUV
immunosensor will be discussed. The immunosensor in combination with acoustic and other sensors could serve as a
complementary characterization tool for the detection of IEDs, UXOs and other potential chemical or biological
Increased emphasis on maritime domain awareness and port security has led to the development of unmanned
underwater vehicles (UUVs) capable of extended missions. These systems rely most frequently on well-developed
side scan sonar and acoustic methods to locate potential targets. The Naval Research Laboratory
(NRL) is developing biosensors for underwater explosives detection that complement acoustic sensors and
can be used as UUV payloads to monitor areas for port and harbor security or in detection of underwater
unexploded ordnance (UXO) and biochemical threats. The prototype sensor has recently been demonstrated
to detect explosives in seawater at trace levels when run in a continuous sampling mode. To overcome
ongoing issues with sample preparation and facilitate rapid detection at trace levels in a marine environment,
we have been developing new mesoporous materials for in-line preconcentration of explosives and other
small molecules, engineering microfluidic components to improve the signal, and testing alternative signal
transduction methods. Additional work is being done to optimize the optical components and sensor response
time. Highlights of these current studies and our ongoing efforts to integrate the biosensor with existing
detection technologies to reduce false positives are described. In addition, we present the results of field tests
that demonstrate the prototype biosensor performance as a UUV payload.
The aim of developing bio-inspired sensing systems is to try and emulate the amazing sensitivity and specificity observed in the natural world. These capabilities have evolved, often for specific tasks, which provide the organism with an advantage in its fight to survive and prosper. Capabilities cover a wide range of sensing functions including vision, temperature, hearing, touch, taste and smell. For some functions, the capabilities of natural systems are still greater than that achieved by traditional engineering solutions; a good example being a dog's sense of smell. Furthermore, attempting to emulate aspects of biological optics, processing and guidance may lead to more simple and effective devices. A bio-inspired sensing system is much more than the sensory mechanism. A system will need to collect samples, especially if pathogens or chemicals are of interest. Other functions could include the provision of power, surfaces and receptors, structure, locomotion and control. In fact it is possible to conceive of a complete bio-inspired system concept which is likely to be radically different from more conventional approaches. This concept will be described and individual component technologies considered.
Emerging biosensor approaches may prove useful in reducing false positives and improving detection probabilities for unexploded ordnance (UXO) and underwater explosives. NRL researchers previously developed a biosensor that was field-tested and validated for use in environmental remediation to detect explosives in groundwater. The sensor relies on the selective recognition by antibodies of target analytes, including the common explosives TNT and RDX. Laboratory work has demonstrated that sensors based on these displacement immunoassay formats can detect explosives at the part-per-trillion level in seawater. More recently, participating in an Office of Naval Research program on Chemical Sensing in the Marine Environment (CSME), tests were conducted in controlled underwater experiments at San Clemente, CA and Duck, NC. Simulated UXO targets, autonomous underwater vehicles (AUV) and multiple sensor approaches were used to demonstrate the feasibility of underwater chemical sensing. Efforts are now underway to integrate the biosensor into an underwater platform as part of a broader sensor system. We will describe results of these studies and outline possible operational scenarios for applications in harbor security.
A continuous flow fluorescence based immunosensor has been developed at the Naval Research Laboratory as an inexpensive, field portable device to detect environmental pollutants. Detection of environmental pollutants such as explosives [e.g. trinitrotoluene (TNT) and hexahydro-1,3,5 trinitro- 1,3,5-triazine (RDX)[ and polychlorinated biphenyls (PCBs) have been achieved at low level concentrations. The continuous flow immunosensor (CFI) employs antibodies as recognition elements for specific antigens. Antibodies specific for the environmental pollutants of interest are covalently immobilized on a solid support matrix. Subsequent saturation of the antibody-support complex with a fluorescence analog (i.e. cyanine dye) of the pollutant completes the sensor matrix. The derivatized matrix is prepacked into a micro column with a continuous flow stream of buffer that removes nonspecifically bound fluorescent analog. After a stable baseline is obtained sample injections of the desired pollutant (PCBs, TNT, RDX, etc.) into the flow stream displaces the fluorescence analog from the immobilized antibody on the solid support. A signal response over background from the displaced fluorescence analog is measured and integrated by an in-line fluorometer. Dose response curves reveal the lowest limit of detection for TNT and RDX is 20 ppb (parts-per-billion). Detection limits for PCBs is slightly higher at 1.0 ppm (part-per-million). Results from field trials conducted at two military bases, Umatilla Army Depot (Hermiston, Ore.) and Site F and A at Naval SUBASE Bangor (Bangor, Wash.) demonstrated the capabilities of the immunosensor in performing on-site field analysis in groundwater and soil leachate matrices.
To assist in airport surveillance efforts, a biosensor based on antibody recognition of individual explosives and drugs has been developed at the Naval Research Laboratory. Analysis of samples containing ng/mL levels of the material are completed in under one minute. Immunoassays for the explosives and the five major drugs of abuse are currently available. The intrinsic nature of antigen-antibody binding also provides the unit with an inherently high degree of selectivity. A portable version of the biosensor that can be run by non-technical personnel is also being engineered. The device, including pumps and fluorometer, will be housed on a modified PCMCIA cartridge fitted into a laptop computer. To run assays, a disposable coupon containing the antibody/fluorescent-antigen complex is inserted into the unit and samples are introduced via a sampling port. Results can be viewed in real time or stored on the computer for later data retrieval and analysis.
Advances in long wavelength fluorophore development have reduced interference from the naturally occurring background fluorescence often present in environmental samples, permitting significant progress in explosive and environmental sensing. The Center for Bio/Molecular Science and Engineering at the Naval Research Laboratory has developed a fluorometric, antibody-based, continuous flow immunosensor which can detect nanogram quantities of small molecular weight molecules such as trinitrotoluene and pentaerythritol tetranitrate. In the flow immunosensor, antibodies are immobilized onto a solid support matrix, exposed to a fluorescently tagged analyte, and placed into a small disposable column connected to an aqueous flow stream. Upon sample introduction, an amount of the fluorescently labelled analyte is displaced that is proportional to the concentration of unlabelled analyte present in the sample. The intrinsic nature of anti-body antigen binding also provides the unit with an inherently high degree of sensitivity and specificity. A positive signal can be generated in under 2 minutes, allowing quick analysis of numerous samples with minimal data handling. Development of a single column assay for the detection of both explosives has decreased both detection time and cost per assay. Cross reactivity studies and studies investigating interference from intrinsic fluorescence in several media have also been conducted.
An antibody-based biosensor has been developed at the Naval Research Laboratory which is capable of detecting both drugs and explosives present at low levels in an aqueous sample. In the flow immunosensor, antibodies are immobilized onto a solid substrate, allowed to bind a fluorescently labeled signal molecule, placed in a small column and attached to a buffer flow. Upon sample introduction, an amount of the fluorescent signal molecule is released that is proportional to the concentration of applied sample. The response time of the sensor is under a minute, and multiple samples can be analyzed without the need for additional reagents. Quantitative assays are being developed for a variety of compounds, including TNT, DNT, PETN, and cocaine. The laboratory prototype has been used to study how choice of fluorophore, antibody density, and flow rate affect the signal intensity and column lifetime. A self-contained commercial instrument which can analyze up to seven different compounds from a single sample is currently being engineered under a Cooperative Research and Development Agreement.