Submerged Explosives Detection Platforms using Immunosensing Technology

Mr. Scott P. Veitch (Author) Systems Engineer

SubChem Systems, Inc. Narragansett, RI, USA

VeitchSP@subchem.com

Dr. Anne W. Kusterbeck (Co-Author) Deputy Director

Center for Bio/Molecular Science and Engineering Washington, DC, USA

Anne.Kusterbeck@nrl.navy.mil

Mr. Robert J. Fratantonio (Co-Author) Systems Engineer

SubChem Systems, Inc. Narragansett, RI, USA

Bob@subchem.com

Mr. Peter J. Egli (Co-Author) Research Chemist

SubChem Systems, Inc. Narragansett, RI, USA Pete@subchem.com

Dr. Alfred K. Hanson (Co-Author)

President SubChem Systems, Inc. Narragansett, RI, USA Hanson@subchem.com

Mr. Paul T. Charles (Co-Author)

Research Chemist Center for Bio/Molecular Science and Engineering

Washington, DC, USA Paul.Charles@nrl.navy.mil

Dr. Jeffrey R. Deschamps (Co-Author)

Research Chemist Center for Bio/Molecular Science and Engineering

Washington, DC, USA Jeffrey.Deschamps@nrl.navy.mil

Dr. Andre A. Adams (Co-Author)

Research Chemist Center for Bio/Molecular Science and Engineering

Washington, DC, USA Andre.Adams.ctr@nrl.navy.mil

Abstract— One of the most difficult aspects of maintaining port and maritime security is the detection, localization, and classification of submerged explosive devices, biochemical agents, and contraband including narcotics. The Explosives Ordnance Disposal (EOD) community has expressed the need for improved methods of detection to augment current capabilities. SubChem Systems Inc. and the U.S. Naval Research Laboratory (NRL) has been working to transition displacement-based immunosensing technology to a commercial system with application towards submerged munitions and contraband detection, classification, and localization. Immunosensing is based upon antibody specificity for a desired target. Funded by the Office of Naval Research, the partners have demonstrated using this technology as a means of chemical detection onboard a custom designed payload for an autonomous underwater vehicle (AUV). The NAVY Small Business Innovative Research (SBIR) program enabled SubChem Systems to provide a concept adaptation of the AUV payload to a diver held version for use in high clutter, low visibility, environments that present an added danger to EOD divers.

Keywords-TNT, Explosives, AUV, EOD, Immunosensor, Chemical Sensing, Submerged Munitions

I. INTRODUCTION There are two common technologies employed for detection, classification and localization of submerged malicious objects. Imagery and Acoustic reflection are used to provide a rendered image or relief from a surface to discriminate an object from its surroundings. Both technologies are used widely in the EOD, research, and recreational communities and both have limitations. Subsurface lighting, water clarity, bio-fouling, and high clutter conditions can render imagery less effective and possibly completely ineffective. Acoustic reflection, designed to step beyond the range limitation and subsurface limited visibility barrier, can still be ineffective when bio-fouling and high clutter object burial is considered. When surveying by either by Unmanned Underwater Vehicle (UUV) or EOD diver, excessive marine growth can impede visual and acoustic detection and classification is extremely difficult as object surfaces can appear blended together. Moving the vessel of opportunity to waters with better visibility or removing the vessel from the water is likely not an economical or realistic option. Conventional submerged explosives and munitions can leach potential chemical signatures through their respective housings due in part to either decomposition over time (corrosive effects of the marine environment) or inadequate mechanical

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design considerations[6] (such as improvised explosive devices, IED). Intact sea-dumped munitions dating back as far as World War II can present trace levels of TNT in the marine environment up to parts per billion[6]. Plume studies have provided insight as to how the distribution in the water space can expand up to hundreds of meters from the source while still maintaining a trace signature. This is in part due to the non-Gaussian distribution where the explosive is thought to be in higher concentrations in a heterogeneous structure[6]. Tracing an explosive agent through its chemical composition can be performed to detect and localize a threat[6]. Given the potential permeability of the container, submersible chemical sensing with real-time in-situ sensing capabilities can provide additional information to aid detection in complex harsh environments and heavy marine growth situations. Immunosensing technology can provide a submerged platform in the marine environment with a fast response, low-power submersible chemical sensor for TNT detection as well as additional potential parameters including DNT, PETN, RDX, PCB, and biological agents like saxitoxin to sub parts per billion detection limits. This technology can be used to augment systems with traditional imagery and acoustic based instrumentation. Such applications may include, but are not limited to, hull inspection, mine counter measure operations, and port security surveillance.

II. METHODOLOGY

A. Immunosensing Several chemical sensing methods have been used on in-situ devices for detection of 2,4,6-trinitrotoluene (TNT) as a constituent of unexploded ordnance (UXO) originating from a malicious device. Such methods include solvent extraction, flow injection analysis, amplified fluorescent polymers, ion mobility spectroscopy, and scanning square wave voltammetry[7].

SubChem Systems Inc. and the NRL are pioneering a new type of chemical sensing for submerged unexploded ordnance (UXO) featuring immunosensing. Immunosensing describes the ability to leverage the specificity of an antibody to its potential target. Antibodies with specificity to the target of interest are first immobilized on the micro-fluidic channels of a substrate. Then the analog of the target that has been labeled is bound with a fluorophore that excites in the near-IR region. Sample flow is permitted to move across the micro-fluidic channels of the substrate. If the target constituent is present in the sample flow, the higher affinity of the target for the antibody displaces the fluorophore labeled analogs from the antibodies, and they will fluoresce when excited downstream by a near-IR light source. The higher the number of target molecules, the greater the fluorescent signal when subjected to excitation [5]. To incorporate immunosensing into instrumentation, the basis

of the methodology requires fluorescent labels and paired antibodies with an appropriate affinity for the target constituent. Monoclonal antibodies specific for TNT (mAb close 30-1) were developed for the NRL by Cell Essentials, Inc. (Boston, MA; http://www.cell-essentials.com/custom). Synthesis of a fluorescence dye immobilization of the TNT antibodies are performed as described previously[2]. Briefly, the substrate surfaces are coated with a tetraethylorthosilicate (TEOS) solution and allowed to incubate for 2 minutes, after which, the gel is removed[2]. The substrate is based cleaned with NaOH and rinsed with Milli-Q water, acid cleaned and treated with 3-mercaptopropyltrimethoxysilane. A solution of N-succinimidyl-4-maleimidobutyrate (GMBS) was then used as the link to bond the TNT antibody. Once the complex was completed it was then allowed to incubate at 4 °C over night[2]. Prior to substrate use, antibody bonding sites are saturated with the fluorophore analog, that is, the AlexaFluor-Cad-TNB conjugate to form the sensing complex for TNT[2].

B. Prototype Development SubChem Systems, Inc. and the NRL have been working to prototype an immunosensor design for in-situ seawater chemical analysis for the purpose of 2,4,6-Trinitrotoluene (TNT) detection. The goal of the design is to incorporate a versatile pressure compensated micro-fluidic system while employing the latest technological advancements of the NRL immunosensor device. The primary mechanism for the detection of TNT by immunosensing is through the use of the micro-fluidic Poly methyl methacrylate (PMMA) substrate called the Coupon. The Coupon is host to the TNT antibodies labeled with fluorescent dye. The coupon is a novel device designed and fabricated by researchers at NRL with fluidic system constraint design criteria provided by SubChem Systems. The coupon is fabricated by first micro-milling a brass embossing negative of the micro-fluidic paths, approximately 39 measuring 25mm long[1]. Once the negative is fabricated, the negative is heated and pressed onto a piece of PMMA substrate using a technique called “hot embossing”. This imprints the fluidic channels onto the PMMA substrate after release. Then the substrate is subjected to tri-solvent annealing[3] to thermally bond a PMMA coverslip. The end result is a sealed PMMA substrate incorporating the fine scale micro-fluidic path geometry. PMMA substrate was selected due to the versatility in surface finish options for antibody attachment, durability, and resistance to bio-fouling.

Figure 1: The NRL Immunosensor Coupon. Intake and dispense ports can be seen on the left and right. Four mounting holes were fabricated for face seal alignment at all corners.

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Incorporated into the coupon is a face sealing design to mount to the external side of the immunosensor housing. As the coupon is placed onto the external side of the prototype end cap, four alignment pins guide the coupon in place providing concentricity to the intake and dispense ports. The coupon is then clamped to the end cap using an aluminum clamp and thumb screws. Therefore, the coupon can be replaced in the field once expended and it is hydrostatically compensated. Though the two pieces of the coupon are bonded, hydrostatic pressure can stress and fracture the coverslip. Placing the coupon external to the housing compensates the device and enables access during deployment. Swapping the Coupon in the field also enables the immunosensor design to be versatile in the selection of the target analyte without any reconfiguration of platform or sensor. The electronics housed in the immunosensor were specifically engineered to be universal when considering the NRL immunosensing methodology. By replacing only the external coupon, the immunosensor can detect a multitude of other constituents including DNT, PETN, RDX, and biological agents like saxitoxin to sub parts per billion detection limits[4]. The prototype design houses a main sample pump, a calibration pump (plume pump) along with a buffer pump. A novel micro-fluidic design is used to mix the buffer with the sample flow while the solution is moved through the coupon. As the fluid crosses the micro-fluidic channels, fluoropore labeled anti-bodies are sheared off the coupon channel given the presence of the target analyte. Once sheared, a laser based light source is used to measure the potential of the fluorescence from the analyte. The design lends itself to be very low power. Unlike other flow through submersible instruments that require heat, the immunosensing technology can operate regardless of ambient temperature. Since the major power components are only three pumps and a light source, the power draw is on the order of less than 3W while sampling. When not sampling, the power budget can be less than 120uW. The prototype is designed to be a 12VDC nominal device. The instrument can be operated in any number of ways. Depending on the mission or sampling requirement, the instrument can be operated in both a tethered and untethered capacity either with a power source or on battery. Incorporated into the immunosensor prototype is a calibration pump, also referred to as the plume pump. It is not an integral part of the fluidic system but a closed circuit. The calibration pump circuit is merely designed to draw a calibration standard from an external source and dispense the product into the sample line of the instrument. Alternatively, the calibration standard dispense line can be placed upstream of the filtered inlet of the device as opposed to a junction on the inlet tubing. The flow rate of this calibration pump, like the other two pumps, is user programmable and can be self triggered for an in-situ calibration when autonomously sampling.

Figure 2: The SubChem Systems / NRL Immunosensor prototype (Right) with field programming and data collection deck box (Left).

C. Platforms incorporating immunosensing The Hydroid REMUS AUV was used for testing the immunosensing prototype payload. Designing per this vehicle is advantageous due to its wide acceptance in the DOD community, ease of use, and engineering capability. The REMUS100 used was upgraded with the Hydroid RECON interface for adaptive mission control. Originally designed for the REMUS100 AUV in 2009, the immunosensor prototype has a 7.5inch outer diameter, features the universal V-band design used by Hydroid, and is trimmed to have neutrally buoyancy. Though the existing immunosensor prototype has a lag time of approximately 15~30 seconds, it maintains a continuous sample flow. This is preferred for use on a survey platform such as the REMUS100. SubChem Systems engineers have designed, fabricated and fielded an external control payload which houses an adaptive mission controller to re-task the REMUS100 and a Benthos Acoustic modem for real-time data telemetry, command, and control. This payload section has been used by the company to integrate third party payloads. Data collected and commands to be issued are relayed via a topside controller with an identical Benthos Acoustic modem and 3G cellular access to the vessel. The immunosensor was designed for compatibility with the external control payload. Operators could command and control the REMUS100 giving it an alternate mission at will. A sample injector was incorporated into the REMUS100 conical nose to generate samples of UXO simulant during the REMUS100 deployments. The calibration pump would draw from a pre-concentrated reservoir of NESTT (Non-hazardous Explosives for Security and Training) to simulate a TNT plume signal ahead of the immunosensor filter on command. The calibration pump pushed fluid through a custom designed sample injector (Figure 3, grey piece). Sample flow through the sample injector was generated by the forward velocity of the REMUS100. Using the onboard real time acoustic doppler

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current profile velocity measurements from the REMUS100 coupled with knowledge of the manifold flow path geometry, operators were able to determine the concentration of the sample as a function of the volume flow rate of the pump. Keeping the REMUS100 velocity fixed at 1 m/s forward velocity and using the external control payload interface, operators were able to trigger different flow rates of 1 to 9 ml/min from the calibration pump. This flow rate range generated a simulated plume of 20ppb to 175ppb ahead of the immunosensor to assess capability while underway.

Figure 3: The REMUS100 conical nose with integrated sample injector (bottom center of the nose). Though originally designed for the Hydroid REMUS100, an alternative application is for a diver held unit. SubChem Systems received funding through the NAVY SBIR program in 2009. The purpose of the funding was to assess the relevance of several different UXO detection methods and to provide a concept diver held chemical sensor for UXO. The chemical sensor had to meet the constraints of not only being operated by a diver but had to be readily integrated to the diver held imaging and navigation system, hull unmanned vehicle localization system, and REMUS100 Swordfish variant currently being developed for the NAVY EOD. SubChem Systems leveraged existing infrastructure built into customer and prototype instrumentation to provide a reconfigurable sensor design for fixed and mobile platforms with operating depths down to 100 meters. The aft end of the immunosensor was originally designed to accommodate the SubChem Systems REMUS100 external controller payload section. Removing this section, engineers designed an end cap replacement for operation as a standalone battery operated device or device on an autonomous platform. Likewise, engineers designed a proof of concept diver interface end cap featuring a submersible LCD screen (Figure 4), helmet heads up display interface, lanyard attachment handles, and thumb toggle switches for operation. Engineers were able to provide sponsors with a bench demonstration of the proof of concept LCD interface for the diver. The proposed diver held instrument was merely a modification to the existing instrument design of the immunosensor prototype currently in development. Therefore, the entire immunosensor design was leveraged and could be field

reconfigurable. The overall dimensions were on par with the existing prototype.

Figure 4: Proof of concept LCD screen layout (left) for diver operation of the immunosensor. Actual screen shot (right). The versatility in the immunosensor prototype design, illustrated in Figures 5 and 6, show how easily it could be reconfigured to satisfy integration to multiple EOD systems. The immunosensor prototype utilizes the Hydroid V-band design to interlock the sensor to the REMUS100. Therefore, engineers provided proof of concept attachments to clamp in place of the REMUS100 vehicle.

Figure 5: An illustration of the proof of concept aft end diver interface to the immunosensor.

Figure 6: An illustration of the modularity of the concept design.

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During fiscal year 2011, SubChem Systems has been involved in the design and fabrication of two fixed moored subsea instrumented frames. These frames are designed to integrate a suite of water quality instruments to which their data is transmitted topside to a web-enabled controller. Operators have the ability to program the sampling rate for each individual sensor remotely. Once the frame is deployed, operators are able to communicate tethered and/or wirelessly with each sensor as well as log into the web interface for real time plotting and raw data retrieval. The submersible instrument multiplexor was designed to infuse the immunosensor data as well. Data from the immunosensor can be available on the internet to all participating parties for the effort including via smart phone and 3G cellular connection. The capability for intermittent sampling, with the immunosensor, could facilitate a long term UXO monitoring platform for munitions dump site monitoring.

III. FIELD TESTING ONBOARD THE REMUS100 On August 10 and 11, 2010, Researchers from SubChem Systems Inc. and the NRL performed a demonstration to field test the immunosensor prototype. This field demonstration utilized the URI REMUS100 also equipped with SubChem Systems Inc. external payload controller. A research vessel was tender to the URI REMUS100 with a 3G cellular connection to allow remote users’ access to the real time data from the REMUS100 and immunosensor. The REMUS100 was programmed to perform typical survey patterns, each 2 hours in duration, in the Narragansett Bay west passage in Rhode Island. The average depth in this location was 10 meters. The REMUS100 was programmed to traverse the shoreline in a “mowing” survey pattern at a fixed depth of 3m at a velocity of 1 m/s. At several points during the missions, operators on the tending vessel sent a command to turn on the immunosensor calibration pump.

Figure 7: Time series plot of the raw data from the immunosensor while onboard the REMUS100 during one of the missions of August 10, 2010 (top). Post processed immunosensor data with exponential decay and outliers removed along with linear trend removed (bottom).

Illustrated in Figure 7 is the time series plot of the immunosensor response to the simulated plume of TNT vs. time. When a coupon was new or recharged, there was a “bleed off” time associated with weekly bonded analogs. During the first 300 seconds illustrated in figure 7 (top), there was an exponential decay from 16000 counts to a baseline of approximately 3000 counts. Once the baseline was reached, there was a stable linear trend in the signal response from the coupon. Once data from the vehicle and immunosensor was retrieved, post processing was used to detrend the data to remove the exponential decay, linear trend, and occasional outlier due to error in data parsing. The resulting data is displayed in Figure 7 (bottom). Over the course of the August 10 and 11, 2010 field deployment, three missions were completed overall. At the start of each mission, a newly charged coupon was placed on the immunosensor while aboard the research vessel prior to the REMUS100 launch. The turn-around time associated with exchanging the coupon was approximately 5 minutes, completed within the time it took to download the REMUS100 data set.

Figure 8: Corresponding August 10, 2010, contour plot of the processed immunosensor response with REMUS100 positioning. REMUS100 trajectory overlay is the calibration pump flow rate setting.

IV. CONCLUSION One of the key benefits to utilizing immunosensors as a means of chemical sensing is the reality that the fluorophore labeled molecules present on the coupon are designed to be displaced from the device only if the target analyte is present in the sample flow. There are a finite number of antibodies with target labeled analogs, but without the presence of the target, the coupon is not expended. Therefore, the longevity of the coupon is a function of the shelf-life of the antibodies and the

The Office of Naval Research, ONR (ONR) The Navy Small Business Innovation Research (SBIR)

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concentration of the target analyte in the sample flow. On a deployment, it is possible that a coupon could last for multiple missions if no target analyte was present. The immunosensing method employed and the instrumentation designed around it requires no heating and no reagents making this methodology favorable for rapid response deployments on battery operated platforms. Researchers and incident responding personnel could feasibly have a pack of coupons available for a variety of analytes. Furthermore, researchers at NRL have demonstrated the feasibility of immobilizing multiple antibodies on a single device for multiplexed detection[8]. This may not yield the concentration for any one specific analyte, but it would present the operator with a digital response whereby a threat was detected or not. The method of fabricating the coupon can be easily adapted for mass production. Therefore, it is feasible that if the complexity of the coupon fluidic preparation was removed from the user’s responsibility whereby charged coupon packs were to be supplied, this technology could be adopted, commercialized, and considered a consumable. The marriage of the coupon design and the associated instrumentation of the immunosensor bring a straight forward field ready sensor for an operator. Looking forward from this stage, the authors will be seeking the means to move this technology forward into such areas as monitoring munitions dump sites. In parallel, the authors are looking to make additional improvements to the sensor design. Efforts will be focused on further miniaturizing the instrumentation leveraging SubChem Systems recent developments in micro-fluidic manifold design. Efforts will also focus on further characterizing the NRL coupon.

ACKNOWLEDGMENT The authors would like to acknowledge the University of Rhode Island for the continued support in the use of the URI REMUS100 autonomous underwater vehicle and research vessel accommodations. The authors would also like to thank the Office of Naval Research for supporting this development.

REFERENCES

[1] Adams, A. A., Charles, P. T., Veitch S. P., Hanson, A. K., Deschamps J. R., Kusterbeck, A. W., “REMUS100 AUV with an integrated microfluidic system for Explosives Detection,” Biosensors and Bioelectronics, unpublished.

[2] Charles, P. T., Adams, A. A., Deschamps, J. R., Veitch, S. P., Hanson, A. K., Kusterbeck, A. W., “Explosives detection in the marine environment using UUV-modified immunosensor,” SPIE, in press.

[3] Charles, P. T., Adams, A. A., Howell, P. B., Trammell, S. A., Deschamps, J. R. and Kusterbeck, A. W., “Fluorescence-based sensing of 2,4,6-Trinitrotoluene (TNT) Using a Multi-channeled Poly(methyl methacrylate) (PMMA) Microimmunosensor,” Sensors vol. 10 (1), pp.876-889, 2010.

[4] Kusterbeck, A. W., Blake, D. A., “Flow immunosensors,” Optical Biosensors (Second Edition): Today and Tomorrow, Elsevier, B.V., pp.243-285, 2008.

[5] Kusterbeck, A. W. and Charles, P. T., “Field demonstration of a portable flow immunosensor,” Field Analytical Chemistry & Technology, vol. 2, pp.341–350, 1998.

[6] Fisher, M., Dock, Matthew., “Detection of trace explosive signatures in the marine environment,” Trace Chemical Sensing of Explosives (ed Woodfin, R. L), John Wiley and Sons, Inc., pp.133-150, 2007.

[7] Singh, S., “Sensors-An effective approach for the detection of explosives,” Journal of Hazardous Materials, vol. 144, Elsevier, B.V., pp.15-28, 2007.

[8] Narang, U., Gauger, P. R., Kusterbeck, A. W., Ligler, F. S.,”Multianalyte detection using a capillary-based flow immunosensor,” Analytical Biochemistry, Academic Press, pp.13-19, 1998.

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